Cyanide Beyond Toxicity: A Systematic Review of Its Effects on Vascular Function

ABSTRACT

Cyanide is widely recognized for its potent toxicity, yet evidence shows that concentrations below 1 μM may enhance cytochrome c oxidase activity and have a regulatory function. Recent findings also demonstrate that mammalian cells, including endothelial cells, produce cyanide endogenously, where it can modulate mitochondrial bioenergetics. However, the vascular implications of this endogenous production remain unexplored. The review addresses this gap and evaluates the vascular effects of glycine, a proposed substrate for endogenous cyanide synthesis. This systematic review was conducted in accordance with PRISMA 2020 guidelines. Seventy‐eight studies were included. Eligible studies with quantifiable vascular outcomes were screened and synthesized. Exogenous cyanide elicited vascular responses through mitochondrial inhibition, modulation of calcium signalling and interference with the soluble guanylyl cyclase/cyclic guanosine monophosphate pathway. Subchronic low‐dose in vivo cyanide exposure reduced contractions and enhanced relaxation in endothelium‐denuded aortic rings. Collectively, evidence indicates a biphasic pattern: high concentrations are cytotoxic, whereas low concentrations may exert protective/regulatory effects. Low‐dose cyanide may have therapeutic potential in managing vascular disorders associated with endothelial dysfunction. Determining an effective and safe dosage range is crucial, and further studies are needed to clarify the role of endogenous cyanide in regulating vascular function.

Plain Language Summary

Cyanide is well known as a poison, but recent research suggests that small concentrations of cyanide have regulatory roles in physiological processes. In this review, we investigated how cyanide affects blood vessel function and the cells that regulate it. We found that while high concentrations are harmful, low concentrations may help blood vessels relax and improve their function. These effects might even have health benefits. Our study also highlights the need for future research to investigate how vascular tissues may naturally produce cyanide and its impact on vascular function.

Abbreviations

6‐CYD

6‐cyanodopamine

ACh

acetylcholine

BK_Ca

big‐conductance K⁺ channels

CC

carotid compliance

CCOx

cytochrome c oxidase

cGMP

cyclic guanosine monophosphate

CN

cyanide

CN⁻

cyanide anion

CN‐COOH

cyanocarboxylic acid

CO

carbon monoxide

DMOG

dimethyloxalylglycine

EC₅₀

half maximal effective concentration

EFS

electric field stimulation

EPO

erythropoietin

ETC

electron transport chain

Fe²⁺

ferrous iron

Fe³⁺

ferric iron

GC

guanylate cyclase

GSH

reduced glutathione

GSS2

glutathione synthetase 2

GSSG

oxidized glutathione

GTN

glyceryl trinitrate

H₂NOH

hydroxylamine

H₂S

hydrogen sulfphide

HCN

hydrogen cyanide

HIF‐1α

hypoxia‐inducible factor‐1α

HOCl

hypochlorite

HPV

hypoxic pulmonary vasoconstriction

I _Cl(Ca)

Ca²⁺‐activated Cl⁻ currents

IP₃

inositol 1,4,5‐trisphosphate

K_ATP

ATP‐sensitive potassium channel

KCN

potassium cyanide

LC₂₀

20‐kDa myosin light chain

LC–MS/MS

liquid chromatography with tandem mass spectrometry

LD₅₀

median lethal dose

L‐NNA

NG‐nitro‐L‐arginine

LWR

lumen width/wall thickness ratio

MPO

myeloperoxidase

NA

noradrenaline

NaCN

sodium cyanide

NO

nitric oxide

NOAELs

no‐observed‐adverse‐effect levels

NOS

NO synthase

NMDA

N‐methyl‐D‐aspartate receptor

NMR

nuclear magnetic resonance

NSC

non‐selective cation channels

PA

pulmonary artery

PAP

pulmonary artery pressure

PhE

phenylephrine

PLC

phospholipase C

RIEVD

rat‐isolated epididymal vas deferens

ROS

reactive oxygen species

sGC

soluble guanylate cyclase

SF

sucrose‐fed

SHR

spontaneously hypertensive rats

SNP

sodium nitroprusside

SOD

superoxide dismutase

SP

substance P

STICs

spontaneous transient inward currents

TB

thrombin

THC

trihistidyl‐cobinamide

TRP3

transient receptor potential channel 3

TST

thiosulfate sulfurtransferase

VEGF

vascular endothelial growth factor

VSCC

voltage‐sensitive Ca²⁺ channels

WKY

Wistar‐Kyoto rats

α₃/Cu_B

heme α₃ paired with copper B

ΔΨm

mitochondrial membrane potential

1. Introduction

Cyanide is traditionally recognized as an environmental toxin, poison and cytotoxic agent. The term cyanide (CN) encompasses both the cyanide anion (CN⁻) and undissociated hydrogen cyanide (HCN). Cyanide is classified as a weak acid (pKa = 9.22, 25°C). At physiological pH, cyanide predominantly exists as HCN, which is the volatile and more toxic form. Due to its small molecular size and solubility characteristics, cyanide can readily cross cell membranes, facilitating rapid tissue distribution and cellular impact [1]. Cyanide's toxic effects are primarily attributed to its inhibition of mitochondrial Complex IV (cytochrome c oxidase, CCOx), the terminal enzyme in the mitochondrial electron transport chain (ETC). This inhibition disrupts cellular respiration, triggering anaerobic metabolism (glycolysis) and leading to hyperlactatemia [2]. Cyanide's inhibition of mitochondrial ETC reduces the amount of oxygen consumed and increases reactive oxygen species (ROS), contributing further to cyanide's toxicity [3].

Randi et al. published the first report, demonstrating the two‐stage effect of cyanide on CCOx, dependent on its concentration. They highlighted the stimulatory effect of low cyanide concentrations on CCOx and suggested that cyanide at endogenous levels plays a regulatory role [4]. Zuhra and Szabo pointed out that cyanide meets many criteria for being classified as a mammalian gasotransmitter, sharing several characteristics with nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H₂S). Additionally, they proposed that cyanide shows regulatory effects up to a concentration of 1 μM, while concentrations above 10 μM induce apoptosis followed by necrosis [5]. Exogenous exposure to cyanide has long been considered the primary cause of elevated cyanide levels in the bloodstream. There are numerous sources of cyanide intoxication, including cigarette smoking, fires, insecticides, meat frying and grilling, and industrial accidents that lead to the release of cyanide into water supplies [6]. Additionally, cyanide is extensively employed in gold and other precious metal mining, primarily for leaching. However, it carries significant risks, such as the release of cyanide into open ponds, leading to biological exposure [7]. On the other hand, many edible plants naturally produce cyanogenic glycosides (e.g., linamarin, prunasin, amygdalin and lotaustralin) as plant toxins. The release of cyanide occurs when these plants are chewed or digested. Cassava ( Manihot esculenta ), a major carbohydrate source and staple food in Africa, Southeast Asia and the Americas, is a notable example. Despite its nutritional importance, all parts of the cassava plant contain high levels of cyanogenic glycosides, which can lead to cyanide intoxication. Bitter almonds, apple seeds, cherry kernels and bitter apricot kernels are other sources with high concentrations of cyanogenic glycosides that may result in cyanide intoxication [6, 8].

Zuhra et al. recently demonstrated that cyanide is endogenously produced in mammalian cells and tissues, including endothelial cells [9]. Their findings revealed that optimal levels of endogenously produced cyanide stimulate mitochondrial bioenergetics, cellular metabolism and cell proliferation. Conversely, impaired endogenous cyanide production was shown to disrupt cellular bioenergetics. Furthermore, they demonstrated that low concentration/dose cyanide treatment exhibits cytoprotective effects both in vitro and in vivo. In this study, glycine was evaluated as a potential substrate involved in the endogenous synthesis of cyanide, while the activity of thiosulfate sulfurtransferase (TST, also known as rhodanese) was shown to regulate cyanide elimination [9].

Given the well‐established roles of other gasotransmitters in vascular homeostasis and the regulation of vascular contractility, the vasculature could serve as a significant target for endogenous cyanide, which has been proposed as a gasotransmitter. If one considers examples from other gasotransmitters: NO is synthesized in endothelial cells and acts as a potent vasodilator through cyclic guanosine monophosphate (cGMP)‐dependent relaxation of vascular smooth muscle; H₂S is produced in both endothelial and smooth muscle cells and induces vascular relaxation by opening smooth muscle voltage‐gated type 7 potassium channels (K_V7) and ATP‐sensitive potassium channels (K_ATP); and CO is likewise formed in both endothelial and smooth muscle cells, promoting vasodilation by increasing the activity of big‐conductance K⁺ channels (BK_Ca). These gasotransmitters also interact with each other to regulate vascular tone [10, 11]. While the role of endogenously synthesized cyanide in vascular regulation remains unexplored, the effects of exogenous cyanide have been studied for many years [12, 13, 14]. Building on recent findings that cyanide is endogenously produced in vascular endothelial cells and can exert regulatory functions at concentrations below 1 μM in cell culture [9], this review aims to evaluate its potential role in vascular function by synthesizing current evidence on the effects of exogenous cyanide, elucidating its mechanisms of action and addressing key inconsistencies in the literature.

2. Methods

2.1. Search Strategy

This systematic review was conducted in accordance with PRISMA 2020 guidelines [15]. Screening and data extraction were performed by one reviewer (E.A‐A.) and verified by a second reviewer (U.S.). Disagreement between the reviewers was resolved through discussion. Reviewers were not blinded to the journal titles and authors. Microsoft Excel was used as the data management tool. No automation tools were used.

The articles included in the current review were retrieved from PubMed and ScienceDirect databases. The searches were conducted using a combination of keywords that appeared in the title or abstract, specifically ‘cyanide and vascular’, ‘cyanide and vasculature’, ‘cyanide and relaxation’, ‘cyanide and contraction’, ‘cyanide and endothelium’, ‘cyanide and dilatation’, ‘cyanide and organ bath’, ‘cyanide and vasodilator’, ‘cyanide and smooth muscle’, ‘cyanide and endothelial cells’ and ‘cyanide and guanylate cyclase’. The search was finalized on 13 January 2025, and only studies reporting reliable, quantifiable outcomes were considered for inclusion. Studies that did not meet the inclusion criteria or align with the theme of the review were excluded from the analysis (Figure 1).

FIGURE 1.

PRISMA flow chart of the systematic review process.

2.2. Outcomes and Other Variables

Primary outcomes included vascular contractile and relaxant responses to cyanide, as well as changes in molecular targets such as Ca²⁺ signalling and cGMP levels. Additional variables extracted included species, tissue type, cyanide concentration, exposure duration and experimental conditions.

2.3. Effect Measures

Effect measures such as percentage change in contraction/relaxation, half‐maximal effective concentration (EC₅₀) values and maximum effective concentration (EC_max) values were extracted as reported by the original studies. No pooled quantitative effect estimates were calculated.

2.4. Certainty of Evidence

A formal GRADE assessment was not performed because of heterogeneity in study designs and outcomes. Overall certainty of the evidence is considered low, and findings should be interpreted cautiously.

2.5. Risk of Bias Assessment and Reporting

As all the included studies were experimental (in vitro, ex vivo or animal studies), a formal risk of bias tool was not applied. Instead, methodological limitations such as small sample sizes, lack of blinding and incomplete reporting were considered narratively. Due to the limited number of experimental studies and the absence of preregistered protocols, the risk of reporting bias could not be formally assessed.

2.6. Limitations of the Review Process

This review was limited to two databases (PubMed and ScienceDirect) and English‐language publications. Grey literature was not searched. Only studies reporting quantifiable vascular outcomes were included.

2.7. Registration and Protocol

The review was not prospectively registered, and no protocol was prepared.

3. Endogenous Production and Elimination of Cyanide

Cyanide and its metabolites have been detected in the biological fluids (e.g., plasma, saliva) of healthy mammals, with concentrations found to be elevated in smokers [16]. Using capillary gas chromatography, a highly sensitive method that excludes thiocyanate, the healthy volunteers' basal circulating cyanide level was determined to be 8.41 ± 3.09 ng/mL (n = 6) [17]. Basal cyanide production has also been identified in brain cell lines (PC12, rat pheochromocytoma cells), rat/hamster brains, mouse tissues and human cell lines [9, 18, 19]. The basal cyanide level in mouse blood was measured as 585 nM ± 73 nM in male mice and 364 nM ± 31 nM in female mice, and using the same method, the blood cyanide concentration was quantified as 540 ± 10 nM (n = 45) in healthy, non‐smoking human volunteers [9, 16].

Cyanide production has been measured using various methods, including electrochemical, liquid chromatography with tandem mass spectrometry (LC–MS/MS) and spectrophotometric techniques. The highest basal levels were found in the liver, with glycine stimulating production in the liver and spleen of mice [9]. HCN scavengers (antidotes) trihistidyl‐cobinamide (THC) and dicobalt edetate confirmed the specificity by reducing cyanide levels. Among 19 tested amino acids, only glycine stimulated production. In human primary hepatocytes and HepG2 cells, confocal microscopy showed detectable cyanide levels that increased with glycine and decreased with THC. Cells in serine/glycine‐free medium had reduced cyanide production, restored by re‐adding glycine. Cyanide was also detected in various human cell lines, showing similar patterns; notably, basal and glycine‐induced endogenous cyanide production has been reported in human umbilical vein endothelial cells (HUVECs) [9]. Even though the authors categorized cyanide production as basal and glycine (10 mM)‐induced, defining it as basal may be inaccurate because the standard culture medium contained glycine (400 μM), and production declined in the absence of glycine.

Lysosomal integrity and acidic pH are considered essential for cyanide production, and it is proposed that the following mechanism is for endogenous cyanide [9]. Thus, lysosomal peroxidases such as myeloperoxidase (MPO) use hydrogen peroxide (H₂O₂, produced by superoxide dismutase [SOD]) and chloride to generate hypochlorite (HOCl). In the acidic lysosomal environment, HOCl reacts with glycine to form N‐monochloroglycine, which converts to N,N‐dichloroglycine via acid catalysis. This molecule decomposes to cyanocarboxylic acid (CN‐COOH), releasing hydrogen cyanide and CO₂. Due to its gaseous nature, cyanide can diffuse out of the cell, potentially acting as a mammalian gasotransmitter [9].

In mammals, cyanide is eliminated through a displacement/transfer catabolic pathway involving enzymes such as TST, 3‐mercaptopyruvate sulfurtransferase and sulphide oxidoreductase [5]. Overexpression of TST in HepG2 cells enhanced their ability to degrade externally added cyanide and reduced endogenous cyanide concentrations. Conversely, the knockdown of the TST gene decreased the potassium cyanide (KCN) degradation rate, leading to an accumulation of endogenous cyanide [9].

Interestingly, 6‐cyanodopamine (6‐CYD), the first identified endogenous mediator containing cyanide, is produced in human platelets [20], rabbit isolated atria and ventricles [21] and rat isolated vas deferens (epithelium) [22]. Additionally, it has been detected in the plasma of patients with chronic kidney disease [23]. This suggests that cyanide may play diverse roles in biological systems by being incorporated into the structures of endogenous mediators.

4. Cytotoxic and Regulatory Effects of Cyanide

In mammals, CCOx comprises 13 subunits, with subunits I, II and III playing roles in proton pumping and the regulation of mitochondrial membrane potential (ΔΨm). The catalytic oxygen‐binding site, heme α₃ paired with copper B (α₃/Cu_B), is in subunit I. Cyanide's toxicity primarily results from its inhibition of oxygen binding at the α₃/Cu_B site, a target of gasotransmitters (Figure 2A) [24]. The inhibition of CCOx through post‐translational modifications, such as phosphorylation and S‐glutathionylation, helps maintain ΔΨm at healthy levels sufficient for ATP synthesis. These modified forms of CCOx exhibit lower activity compared with their native form [4, 25]. Additionally, when ATP/ADP ratios are high, ATP binds to the ATP‐binding site located in subunit IV of CCOx, leading to allosteric inhibition of the enzyme [26]. Through all these inhibitory mechanisms, energy metabolism is maintained.

FIGURE 2.

Effects of low and high cyanide concentrations on CCOx (A) and the proposed mechanism of cyanide's inhibitory effect on glutathionylation of CCOx (B). (A) Low cyanide concentrations enhance CCOx activity and mitochondrial respiration, whereas high concentrations inhibit CCOx (B). Proposed mechanism of cyanide‐induced deglutathionylation of CCOx. Cyanide displaces glutathione from cysteine residues via nucleophilic substitution, restoring free cysteines and increasing enzymatic activity. In this figure, low concentrations refer to 0.1 nM–1 μM, whereas higher concentrations refer to 10 μM and above. ADP, adenosine diphosphate; ATP, adenosine triphosphate; CCOx, cytochrome c oxidase; CN⁻, cyanide; Cyt c, cytochrome c. Created in BioRender. Alan Albayrak, E. (2025) https://BioRender.com/a99u470.

Randi et al. demonstrated that cyanide, at concentrations ranging from 0.1 nM to 1 μM, inhibits the constitutive inhibitory glutathionylation of CCOx, resulting in enhanced enzymatic activity. In contrast, exposure to 10 μM cyanide decreases CCOx activity (Figure 2A). In HepG2 cells, cyanide at 0.1 nM increased the oxygen consumption rate and ATP/ADP ratio, whereas a higher concentration (10 μM) reduced them. Cyanide stimulated cell proliferation in various human cell lines at 0.1 nM, whereas at 10 μM it inhibited proliferation. Neither concentration altered mitochondrial complex expression. Western blot analysis showed that cyanide at 0.1 nM reduced the basal glutathionylation of subunits I and III, increasing free cysteines and enzymatic activity. Spectral changes in CCOx were observed at both 0.1 nM and 10 μM [4]. Supernatants from cyanide‐producing Pseudomonas aeruginosa diluted to low concentrations stimulated cellular bioenergetics in HepG2 cells, while concentrated supernatants were inhibitory. These effects were absent with supernatants from cyanide‐deficient mutant strains. Additionally, the expression of six enzymes responsible for cyanide production was demonstrated in HepG2 cells. Cyanide exposure (0.1 nM–10 μM) exhibited biphasic regulation of MPO, characterized by upregulation at 0.1 nM–1 μM and downregulation at 10 μM [4].

Giamogante et al. published a controversy regarding the findings of Randi et al. They highlighted that the differences in band densities showing the effect of LCC on glutathionylation of subunits I and III in Randi et al.'s study were barely visible and that cysteine residues measured were influenced by reducing solvent before cyanide treatment. They argued that the spectral changes observed by Randi et al. align with cyanide binding at the α₃/Cu_B site rather than disulphides, as 1 nM cyanide produced a spectrum identical to the control [27]. Additionally, they demonstrated that cyanide (1 nM) did not affect the respiration of mouse liver mitochondria and showed that cyanide's binding rate to the α₃/Cu_B site is much faster than its interaction with Ellman's reagent. Overall, they concluded that under the conditions of Randi et al.'s study, the cyanide reaction would take weeks to complete, indicating that the effect is due to binding at the α₃/Cu_B site rather than deglutathionylation [27].

Zuhra and Szabo published a response to Giamogante et al., highlighting that isolated mitochondria might function differently compared with whole cells. They argued that in vitro reaction measurements between CCOx and cyanide were not relevant to their study. Furthermore, they tested the effect of cyanide (100 μM) on bovine serum albumin using Ellman's reagent and observed that the characteristic band disappeared after cyanide was added. They demonstrated that oxidized glutathione reduced free cysteines in CCOx, which was partially reversed by cyanide (0.1 nM). The authors proposed a nucleophilic displacement reaction, where the disulphide cysteine residue bound to glutathione reacts with cyanide, resulting in a free cysteine residue and CN‐glutathione release (Figure 2B) [28].

The literature includes studies showing that hypoxia and hypoxia‐induced ROS production initially reduce glutathione levels in various cells and tissues [29, 30, 31]. In contrast, a study has demonstrated that glutathione levels can adaptively increase in response to hypoxia [32]. These studies suggest that the fluctuations in glutathione levels observed in the early stages of hypoxia may also occur with cyanide exposure. Thus, it is essential to consider that altered glutathione levels may impact the process by which cyanide inhibits CCOx glutathionylation.

Cyanide at 1–10 mM concentrations and 10–15 mg/kg doses (sc) has been shown to activate voltage‐sensitive Ca²⁺ channels (VSCC), increasing cytosolic Ca²⁺ influx and total Ca²⁺ accumulation in PC12 cells and mice [33, 34]. In cultured carotid body glomus cells from newborn rabbits, both anoxia and cyanide elevated cellular Ca²⁺ levels, which were inhibited by VSCC blockers [35]. In PC12 cells, cyanide (1–10 mM) induced morphological changes, including mitochondrial swelling and depletion of secretory granules, effects that were blocked by VSCC inhibitors [36].

N‐methyl‐D‐aspartate (NMDA) receptors are ionotropic glutamate receptors that mediate Ca²⁺ influx during excitatory neurotransmission and play multiple physiological roles. Cyanide (10–100 μM) can directly target NMDA receptors and regulate intracellular [Ca²⁺] ([Ca²⁺]_i) in neuronal cells [5]. This effect of cyanide may be physiologically relevant, for example, by stimulating neurotransmitter release at non‐cytotoxic concentrations.

Cyanide has been shown to affect the phospholipase C (PLC) signalling pathway, which triggers the release of Ca²⁺ from intracellular stores through the action of inositol 1,4,5‐trisphosphate (IP₃) [37]. Cyanide at 1–10 μM concentrations rapidly elevated intracellular IP₃ levels in PC12 cells. A PLC inhibitor and a Ca²⁺‐free medium inhibited this response. Additionally, the cyanide‐induced increase in IP₃ levels was partially reduced in the presence of VSCC inhibitors. These findings indicate that the cyanide‐induced rise in IP₃ is associated with PLC activation, extracellular Ca²⁺ levels and VSCC channels [37].

Taken together, studies on brain cells and animal brains have demonstrated the Ca²⁺‐dependent effects of cyanide. While cyanide at 1–10 mM induces Ca²⁺ overload and exerts toxic effects, exposure at 1–10 μM initiates signalling cascades, such as IP₃ elevation, without causing toxicity.

Cyanide at 0.1 and 1 μM increased mitochondrial oxygen consumption and slightly elevated ROS generation without affecting cell viability in rat brain endothelial cells (RBE4) [38]. These concentrations stabilized and activated hypoxia‐inducible factor‐1α (HIF‐1α), leading to increased levels of erythropoietin (EPO), vascular endothelial growth factor (VEGF) and endothelial NO synthase (eNOS), resulting in an adaptive response. Cyanide‐induced preconditioning protected RBE4 cells from high glucose‐induced damage. In NT2 neuron‐like cells with functional mitochondria, cyanide at 0.1 and 1 μM increased ROS levels and protected against high glucose‐induced cell loss and caspase‐3 activation. However, in NT2 ρ0 cells lacking mitochondrial DNA, cyanide did not induce ROS production or protection against glucotoxicity. These findings suggest that low‐dose cyanide induces mitochondrial preconditioning, triggering protective responses via mitochondrial ROS and HIF‐1α signalling [38].

Zuhra et al. demonstrated that the cyanogenic compounds mandelonitrile, linamarin and amygdalin, known for their toxic properties, elevated cyanide levels in HepG2 cells when applied at concentrations of 1–100 μM [9]. Within this range, the authors suggested that these compounds generate cyanide at a rate of 0.1–0.2 nmol/mg protein/h, which is comparable to endogenous cyanide generation in HepG2 cells. However, they did not clarify whether ‘endogenous generation’ referred to glycine‐stimulated or basal conditions. In the same study, the application of these compounds at a higher concentration (300 μM) promoted cell proliferation and protected against hypoxia‐ and hypoxia/reoxygenation‐induced cell injury [9]. Importantly, the authors did not provide data demonstrating an increase in cyanide levels at 300 μM, the concentration at which these cellular effects were observed. In addition, amygdalin at doses of 10–30 mg/kg was reported to exert protective effects in a mouse model of myocardial ischaemia–reperfusion. The authors estimated that the cyanide concentrations resulting from these therapeutic doses would be in the range of 200 nM–1 μM [9].

The blood cyanide concentration in mice was 585 nM ± 73 nM in male mice and 364 nM ± 31 nM in female mice, and the administration of a subtoxic dose of KCN (0.1 mg/kg), glycine (100 mg/kg) and amygdalin (10 mg/kg) increased blood cyanide levels to 1–1.5 μM in male mice [9]. In a mouse myocardial ischaemia/reperfusion model, amygdalin (3–100 mg/kg) and glycine (300 mg/kg) prevented enlargement of infarct size. Similarly, in a mouse haemorrhagic shock model, amygdalin (10 mg/kg) protected against hepatic and pulmonary injury [9]. These findings underscore the protective effects of subtoxic cyanide doses and suggest that consuming limited amounts of cyanogenic compounds could be a therapeutic approach. However, its effects on other organs and systems have not been explored, and the impact of cyanide following injury or disease onset remains unknown. Therefore, it is crucial to investigate how cyanide influences cellular function when administered after injury, particularly given its toxic properties.

Zuhra et al. also showed that fibroblasts isolated from patients with glycine encephalopathy, a severe condition caused by mutations in genes encoding the glycine cleavage system, had higher glycine and cyanide levels than controls [9]. ATP generation, cell viability and proliferation rates were decreased in patients' fibroblasts [9]. Treatment with the lysosomal alkalinizer (hydroxychloroquine) reduced cyanide levels and improved bioenergetics. These findings indicate that endogenous cyanide production in glycine encephalopathy reaches cytotoxic levels and can be managed by regulating its synthesis [9]. Overall, dysregulation of endogenous cyanide, whether elevated above or reduced below optimal levels, compromises cellular bioenergetics, highlighting the importance of maintaining cyanide within an optimal range for cellular function.

5. Vascular Effects of Cyanide

Most studies examining cyanide's effects on vascular tissues have not focused on elucidating its specific mechanisms of action but have aimed to investigate the vascular impacts resulting from the inhibition of cellular respiration. Since the recent discovery of endogenous cyanide production and its regulatory effects, the role of cyanide in vascular function has not yet been explored. This section presents the vascular effects of exogenously applied cyanide at different concentrations on vascular tissues, smooth muscles and endothelial cells, categorized according to underlying mechanisms (Table 1, Figure 3). It should be noted that in vascular reactivity studies, cyanide is applied directly to vascular tissues; therefore, the effective concentrations are often considerably higher than those used in cell culture experiments, where much lower levels can exert biological effects.

TABLE 1.

Vascular effects of cyanide (ordered in increasing concentration).

Tissue type/cell line	Cyanide concentration	Observed effects	Proposed mechanisms	References
Rabbit, ferret and dog aorta	1 nM–10 μM	↑ Basal tension in rabbits	● Concentration and species‐dependent responses ● May increase calcium influx or intracellular levels (?)	[47]
	100 μM–1 mM	Relaxation in rabbits
	One concentration (1–10 mM)	Small contractions
	1 mM	NA‐induced contractions: Rabbit: ↓, Ferret: ↓, Dog: ↔ ↑ NA contraction rate
Rat PASMCs	1 μM	● ↔ Hypoxia‐induced increase in cytosolic oxidation ● ↑ [Ca2+]i during hypoxia	Complex IV plays no significant role in the cytosolic oxidant signal	[87]
Rat PA	4–8 μM	↓ SNP‐induced relaxations in precontracted tissues	Inhibits the enzyme that converts SNP to NO (?)	[68]
	7–50 μM	↔ Relaxations induced by H2NOH, sodium azide, GTN, NO2 − or HbNO
Rat PA myocytes	10 μM	● ↑ [Ca2+]i like hypoxia ● ↔ Hypoxia‐induced Ca2+ increase With myxothiazol: ↓ Cyanide‐induced Ca2+ → involves ETC before complex III With catalase overexpression: ↓ Ca2+ response → H2O2‐dependent mechanism	H2O2 production is necessary for cyanide‐induced Ca2+ increase	[85]
Rabbit aorta	14 μM	↓ SNP‐induced relaxations	Decreases cGMP levels	[69]
	40 μM	● ↓ SNP‐induced increase in cGMP levels ● ↔ NO2 −‐induced relaxations or cGMP levels
	100 μM	↔ NA‐induced contraction
	100–600 μM	↓ N3 −‐ and H2NOH‐induced relaxations
	200–240 μM	↔ Relaxations induced by HbNO, NO, NO2 −, GTN, adenosine, papaverine
Rat lung	50 μM	Pulmonary contraction	Inhibition of cellular respiration	[48]
Pig lung	50 μM, 100 μM	● ↑ PAP ● ↔ ATP levels and adenylate charge	Involvement of localized energy depletion in specific compartments	[49]
	1 mM	● Restored PAP to control levels ● ↓ ATP levels and adenylate charge
Rat portal vein	50 μM–4 mM	● Relaxation ● ↓ O2 consumption ● ↑ Lactate production	Inhibition of cellular respiration	[50]
Rabbit aorta	80 μM–3 mM	● OBPO2 = 250 mmHg → ↓ VO2 ● OBPO2 > 180 mmHg → full relaxation ● OBPO2 = 50–180 mmHg → Triphasic response: relaxation–contraction–relaxation ● OBPO2 < 7 mmHg → Minimal/no effect ● Cyanide after NA → VO2 ↓, partial relaxation (less than hypoxia) ● Cyanide after K+ → VO2 ↓, relaxation similar to hypoxia ● Repeated cyanide after VO2 inhibition → additional relaxation (oxygen‐independent)	● VO2 and contraction/relaxation responses dependent on OBPO2 ● Mechanisms independent of VO2 and hypoxia	[46]
Rat aorta	100 μM	● 5–60 min → max nitroprusside inhibition at 5 min ● ↓cGMP increases by KCl, BaCl2 and NA in the presence and absence of SNP ● ↓ Relaxation responses to sodium azide, hydroxylamine, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine, nitroglycerin, ACh, isoproterenol ● ↔ 8‐bromocyclic GMP‐induced relaxation	Inhibits GC activation by binding to the NO site on the heme	[12]
	100 μM and 1 mM	↓ SNP‐induced GC activation
	1 mM	↑ basal GC activity
Bovine PA	100 μM	Cyanide: ● ↓ SNP‐induced relaxation and cGMP ● ↔ Responses induced by other cGMP‐dependent relaxants ● ↔ Basal cGMP or tension Methylene blue (10–100 μM): ● ↓ Relaxations and cGMP induced by SNP, GTN, SNAP and ACh ● ↑ Contractions to PhE, K+, U46619 ● ↓ Basal cGMP levels ● Contractions	Not an sGC inhibitor, may chemically antagonize SNP	[72]
Rat PA	100 μM	Initial relaxation, contraction and final relaxation, similar to hypoxia	Inhibition of H2O2‐induced GC activation (?)	[52]
Human umbilical artery	100 μM	● A leftward shift in the concentration‐response curve of serotonin ● ↑ NA‐ and adrenaline‐induced contractions ● ↔ Basal tension	No specific mechanisms proposed	[51]
Human umbilical vein	100 μM	● ↔ Serotonin‐, NA‐ and adrenaline‐induced contractions ● ↔ Basal tension
Rat aorta	100 μM	● ↓ SNP‐, ACh‐ and atriopeptin II‐induced relaxations ● ↓ SNP‐ and ACh‐induced increase in cGMP levels	Binds to the heme moiety of sGC	[70]
Rat aorta, PA and mesenteric artery	100 μM	● Induced the second and third phases of the hypoxic response ● Restored hypoxia‐induced decrease in relaxations to ACh and DETA‐NO ● L‐NNA and ODQ caused contractions in the presence of cyanide	Disrupts NO‐CCOx interactions and enhances NO‐mediated relaxation via sGC	[71]
Rat small PA	100 μM	● Like insulin; ↔ PGF2α‐induced Phase 1 contractions but ↑ Phase 2 contractions under hypoxia ● ↔ Hypoxia‐induced increase in [Ca2+]i ● ↔ High K+‐induced contraction and basal tone in normoxia	Potentiate glucose‐dependent Ca2+ sensitization (?)	[89]
Rat portal vein smooth muscle cells	200–250 μM	● ↓ Force and the duration of spontaneous bursts of action potentials ● ↓ Force > ↓ electrical activity	Membrane‐independent mechanism	[100]
	1 mM	↓ High K+‐induced contraction, minimal effect on depolarization
	2 mM	● Abolished spontaneous activity ● Slight depolarization followed by repolarization ● Mild hyperpolarization (prolonged exposure) ● 4‐AP and TEA did not inhibit hyperpolarization
Rat portal vein	100–500 μM	● ↓ Electrically stimulated force, O2 consumption and maximal shortening velocity (V max) ● ↑ Lactate production	Inhibition of cellular respiration	[53]
Rat portal vein	100 μM–1 mM	● ↓ Spontaneous force/activity (like cromakalim) ● ↑ Basal [Ca2+]i, ↓ phasic [Ca2+]i transients With glibenclamide (K ATP blocker): ● ↓ Relaxations in spontaneous tissue ● ↔ High‐K+ precontracted tissue ● ↓ Cyanide‐induced hyperpolarization ● ↑ Maintenance of electrical and phasic Ca2+ activity ● ↔ Basal [Ca2+]i before/after cyanide	KATP channel activation and hyperpolarization	[98]
	2 mM	Hyperpolarization in 3‐hydroxybutyrate medium (without glucose)‐ inhibited by glibenclamide
Rabbit aorta	100 μM	● ↓ SNP‐induced relaxation ● ↔ NA‐induced contractions ● ↔ Relaxations induced by other direct vasodilators	● Inhibition of cellular respiration ● Specific antagonism of SNP‐induced relaxation	[73]
	300 μM	↓ NA‐induced contractions
	100 μM–1 mM	After SNP → ↑ tension
Rabbit aorta	100 μM, 1 mM	↓ KCl‐induced contraction (10–120 mM)	● Cyanide may enter smooth muscle via anion channels	[95]
	1 mM	↓ NA‐induced contraction (not affected by ouabain/verapamil)
	10 mM	● ↑ 10–30 mM KCl contractions ● ↓ ≥ 40 mM KCl contractions (verapamil‐like) Cyanide‐induced contractions (10 mM): ● ↑ By ouabain and verapamil ● ↔ By serotonin antagonist, phentolamine, pyrilamine, atropine ● ↓ By DIDS and chlorpromazine (anion channel blockers)
Rat portal vein	200 μM	● ↓ Contractions induced by Ca2+ addition to high K+ solution ● ↓ Contractions induced by high K+ in Ca2+‐containing solution Effect on LC 20 phosphorylation: ● ↔ During relaxation ● ↓ At plateau of K+‐induced contraction Effect on force and velocity: ● ↓ Force (in both control and stimulated veins) ● Slight ↓ in V max	Cyanide may act via multiple mechanisms, including impaired respiration and reduced LC20 phosphorylation	[13]
Rat thoracic aorta	320 μM	● ↓ Vascular ATP levels at 10 s and 3 min ● ↓ Vascular tone at 3 min	● No specific mechanisms proposed ● May involve ATP‐independent mechanisms	[55]
Rat tail artery	400–800 μM	● ↓ NA‐mediated contractions ● 400 μM → ↓ arterial wall ATP levels by ~50% ● ↔ [Ca2+]i ● 400 μM → effect reversible ● 800 μM → effect irreversible	● Inhibition of cellular respiration ● Ca2+‐independent mechanisms	[93]
Rat deep femoral artery	500 μM	↓ Hypoxia‐induced increase in PhE contractions	Inhibition of cellular respiration	[54]
Rat hindlimb vessel	1 mM	● ↔ Serotonin‐ and high concentrations of NA (> 50 nM)‐induced contractions ● ↔ Arterial PO2 ● ↓ Low concentrations of NA (< 50 nM)‐induced contraction ● ↓ Basal VO2 in response to serotonin and NA ● Slightly ↑ venous PO2 and perfusion pressure	No specific mechanisms proposed	[57]
Human placental veins	1 mM	● ↓ Lactate‐ and H2O2‐induced relaxations ● ↔ Relaxations to forskolin and GTN	Inhibits sGC activated by lactate‐induced H2O2 production (?)	[74]
PASMCs, Xenopus oocytes, systemic (mesenteric) artery myocytes	1 mM, 10 mM	● ↑ Cl− currents and STICs in PASMCs ● ↑ Sustained Ca2+ currents in oocytes ● CCCP and oligomycin or CCCP and thapsigargin → ↓ cyanide‐induced Ca2+ release ● ↑ Ca2+ sparks (confocal imaging) ● Stimulated Ca2+ release from intracellular stores ● Mitochondrial Ca2+ depletion → ↓ I Cl(Ca) ● Reversal potential shifted by replacing NaCl or using niflumic acid → confirms anion‐selective current ● Hypoxia → no Ca2+ release in systemic arteries ● Cyanide → induced Ca2+ release in systemic arteries	Mitochondria and SR, including RyRs and IP3Rs	[105]
Rat mesenteric artery	2 mM	● Small arteries (3rd/4th branch): ~81% relaxation (like N2), t1/2 ≈ 29 s (faster than N2) ● Medium arteries (2nd/3rd): ~69% relaxation (smaller than N2) ● Large arteries (1st): ~52% relaxation (smaller than N2) ● Endothelial removal did not affect responses (cyanide or N2) ● pO2 levels: Unchanged after cyanide	Vessel size‐dependent; may involve O2‐sensitive ion channels or non‐metabolic O2 sensing	[60]
Human umbilical vein endothelial cell line (Ea.hy926)	2–5 mM	● ↑ Ca2+ influx (dependent on extracellular Ca2+ and membrane potential) ● Reversibly ↑ ATP/bradykinin‐induced Ca2+ rise ● NMDG+ replacement → abolished inward current, ↔ outward current ● BKCa was not activated by cyanide alone ● ↑ BKCa current if preactivated	●Increases BKCa sensitivity by inhibiting metabolism ●Activates Ca2+‐permeable NSC channels, TRPV3 (?)	[97]
Primary rat PASMCs	2–10 mM	● Caffeine → ↓ cyanide‐induced Ca2+ increase ● Cyanide → prolonged caffeine‐induced I Cl(Ca)	Induces I Cl(Ca)	[104]
Porcine coronary artery	5 mM	● ↓ Hypoxia‐ and reoxygenation‐induced relaxations ● ↓ Force in K+‐precontraction ● More pronounced effects in endothelium‐denuded arteries	● No specific mechanisms proposed ● May involve endothelium‐dependent mechanisms	[42]

Abbreviations: ↑, increased/induced; ↓, decreased/inhibited; ↔ no effect; 4‐AP, 4‐aminopyridine; ACh, acetylcholine; ADP, adenosine diphosphate; ATP, adenosine triphosphate; BaCl₂, barium chloride; BK_Ca, big‐conductance Ca²⁺‐activated potassium channel; CCCP, carbonyl cyanide m‐chlorophenylhydrazone; [Ca²⁺]_i, intracellular calcium concentration; CN⁻, cyanide; cGMP, cyclic guanosine monophosphate; DIDS, 4,4′‐diisothiocyanato‐2,2′‐stilbene‐disulfonic acid; DETA‐NO, diethylenetriamine nitric oxide adduct; GC, guanylyl cyclase; GSH, reduced glutathione; GSSG, oxidized glutathione; GTN, glyceryl trinitrate (nitroglycerin); HbNO, nitrosyl haemoglobin; H₂NOH, hydroxylamine; IP₃R, inositol 1,4,5‐trisphosphate receptor; K_ATP, ATP‐sensitive potassium channel; LC₂₀, 20‐kDa regulatory myosin light chain; L‐NNA, NG‐nitro‐L‐arginine; NA, noradrenaline; NMDG⁺, N‐methyl‐D‐glucamine; NO₂ ⁻, nitrite; NOS, nitric oxide synthase; NSC, non‐selective cation; OBPO₂, organ bath partial oxygen pressure; PA, pulmonary artery; PAP, pulmonary arterial pressure; PASMCs, pulmonary arterial smooth muscle cells; PhE, phenylephrine; PKG, protein kinase G; PO₂, partial pressure of oxygen; ROS, reactive oxygen species; RyR, ryanodine receptor; SNP, sodium nitroprusside; SNAP, S‐nitroso‐N‐acetylpenicillamine; sGC, soluble guanylyl cyclase; SOD, superoxide dismutase; SR, sarcoplasmic reticulum; STIC, spontaneous transient inward current; TEA, tetraethylammonium; TRP, transient receptor potential; TRPV3, transient receptor potential vanilloid 3; VO₂, oxygen uptake; V _max, maximal shortening velocity.

FIGURE 3.

(A) An overview of the mechanisms and vascular targets of cyanide and (B) concentration‐dependent vascular responses. (A) In endothelial cells, CN⁻ enhances Ca²⁺ influx via non‐selective cation channels, increases K⁺ efflux, stimulates NO production through Ca²⁺/CaM‐dependent activation of NOS and promotes vasorelaxation via the NO–sGC–cGMP–PKG pathway. In smooth muscle cells, CN⁻ enters the cell via anion channels, modulates ion channel activity (K_ATP, VSCC, Cl⁻), inhibits MLCK, increases ROS and disrupts intracellular Ca²⁺ homeostasis through RyR, IP₃R and mitochondrial Ca²⁺. (B) In ex vivo experiments across different animal species and vascular tissues, cyanide elicited concentration‐dependent vascular responses, including contraction, relaxation or multiphasic effects. However, no effect was observed in some vascular tissues. Cyanide's ability to attenuate contractile responses was classified as relaxation, whereas its attenuation of relaxant responses was considered a form of contraction. ADP, adenosine diphosphate; ATP, adenosine triphosphate; CaM, calmodulin; cGMP, cyclic guanosine monophosphate; CN⁻, cyanide; IP₃R, inositol 1,4,5‐trisphosphate receptor; K_ATP, ATP‐sensitive K⁺ channel; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; NOS, nitric oxide synthase; NSCC, non‐selective cation channel; PKG, protein kinase G; ROS, reactive oxygen species; RyR, ryanodine receptor; sGC, soluble guanylyl cyclase; TRP3, transient receptor potential channel 3; VSCC, voltage‐sensitive calcium channel. Created in BioRender. Alan Albayrak, E. (2025) https://BioRender.com/qditpks.

In a study aimed at investigating the distribution of cyanide within tissues, non‐toxic doses of S35‐thiocyanate (0.8 mg/kg) and C14‐labelled cyanide (0.3 mg/kg) were intravenously injected into mice via the tail vein. The study reported that one of the areas showing higher cyanide radioactivity was the walls of large vessels compared with the blood, indicating that the vasculature is a significant target for cyanide [39].

Ahlemeyer and Johannsen demonstrated that cyanide (1 mM) reduced viability and increased lactate dehydrogenase release in cerebral endothelial cells but did not affect non‐cerebral cells, such as HUVECs [40]. These results suggest that vascular endothelial cells may exhibit resistance to cyanide, thereby allowing potential regulatory effects of cyanide to manifest without triggering toxic responses. In rabbit corneal endothelial cells, cyanide (1 mM) induced minor morphological changes over a period of 7 days [41]. Cyanide elevated the metabolic ratio, and cells survived by producing ATP through anaerobic glycolysis. However, glycolysis inhibition with iodoacetamide led to cell death, highlighting its critical role under cyanide‐induced mitochondrial inhibition [41]. Exposure to cyanide at 0.1 and 1 μM increases eNOS expression in brain endothelial cells (see above) [38], suggesting that comparable low concentrations may also modulate eNOS in vascular endothelial cells and thereby contribute to vascular function regulation. This intriguing possibility remains unexplored and warrants further investigation.

Cyanide (5 mM) completely blocked reoxygenation‐induced relaxations in hypoxic porcine coronary arteries, whereas other mitochondrial inhibitors (e.g., antimycin A, rotenone) had either no effect or only partial effects [42]. This suggests that cyanide influences mechanisms beyond CCOx. However, it should be noted that this effect may be attributed to the significantly higher concentration of cyanide used (5 mM) compared with other inhibitors (1 μM–1 mM). Additionally, cyanide more strongly reduced force and inhibited hypoxia/reoxygenation‐induced relaxations in endothelium‐denuded arteries than in endothelium‐intact arteries [42]. These findings suggest that the endothelium, possibly through NO production, plays a crucial role in mediating the effects of cyanide.

Cyanide inhibited respiration in porcine carotid artery (EC₅₀ = 0.11 ± 0.022 mM) and guinea pig stomach (EC₅₀ = 0.14 ± 0.024 mM) in HEPES–glucose buffer [43]. HEPES–pyruvate partially prevented this inhibition, with greater protection in the carotid than in the stomach, but this effect was lost in the bicarbonate buffer. Pyruvate also normalized ATP levels and pH after cyanide (1 mM) exposure in stomach tissue [43]. These findings suggest that cyanide toxicity on smooth muscle oxidative metabolism depends on tissue type and is influenced by the buffer system (HEPES/bicarbonate) and metabolic substrates (pyruvate/glucose). Additionally, increased lactic acid production from anaerobic respiration under cyanide exposure may lower pH, potentially impacting vascular function. However, Aalkjaer and Lombard demonstrated that hypoxia‐induced smooth muscle acidosis does not contribute to hypoxia‐induced vasodilation in cerebral and mesenteric small arteries [44].

Robinson et al. investigated the rate of cyanide loss in a Krebs–Henseleit solution in the presence/absence of foxhound femoral artery strips [45]. The study showed that cyanide at 300 μM was consumed more rapidly than at 30 μM. Reducing aeration rates decreased cyanide loss. In solutions containing tissue, the half‐life of cyanide (300 μM) was shorter (54 min) compared with solutions without tissue (98 min). The primary cause of cyanide loss was attributed to HCN evaporation [45].

Altering the organ bath oxygen pressure (OBPO₂) and adding cyanide were used to study the relationship between oxygen uptake (VO₂) and vascular mechanical tension in rabbit aorta strips [46]. Oxygen pressure was measured using an O₂ electrode, and VO₂ rate was calculated. The effects of cyanide on VO₂ and vascular responses were found to vary depending on the OBPO₂ levels (Table 1) [46]. These findings emphasize the importance of controlling aeration in organ baths when working with cyanide, as both the form of the cyanide response and the cyanide loss are highly dependent on PO₂ and the bubbling rate. The absence of PO₂ measurements in the studies raises the possibility that differences in oxygen levels may contribute to the conflicting results.

A study conducted on aortic strips from rabbits, ferrets and dogs reported that cyanide elicits concentration‐ and species‐dependent responses in vascular smooth muscle (Table 1) [47]. However, cyanide incubation increased the rate of noradrenaline (NA)‐induced contraction responses across all species, regardless of changes in contraction level. The authors attributed this effect to increased Ca²⁺ influx or levels, though they did not measure Ca²⁺ directly [47]. This study highlighted that cyanide may directly impact contraction mechanisms or alter vascular responses by inhibiting cellular respiration.

Referencing the following study is important due to its relevance to the effects of the endogenous mediator 6‐CYD, which contains cyanide, on smooth muscle contractions. 6‐CYD was shown to be produced in the epithelium of the vas deferens in rats [22]. 6‐CYD caused basal contractions detectable at a concentration of 1 mM in rat‐isolated epididymal vas deferens (RIEVD). Moreover, 6‐CYD (0.1–10 nM) enhanced electric field stimulation (EFS)‐induced contractions. Pre‐incubation with 6‐CYD at 0.1 nM did not alter NA‐induced contractions, while concentrations of 1, 10 and 100 nM significantly increased these contractions. Additionally, 6‐CYD enhanced adrenaline‐ and dopamine‐induced contractions, with the effect observed at concentrations starting from 10 to 100 nM, respectively [22]. This study suggests that cyanide, by being incorporated into the structure of a newly discovered group of 6‐cyano‐catecholamines, may influence vascular smooth muscle contractility. However, further studies are necessary to elucidate this phenomenon.

5.1. Impact of Metabolic Inhibition on Vascular Responses to Cyanide

Gottschall et al. demonstrated that cyanide (50 μM) caused pulmonary contraction similar to hypoxia in isolated perfused rat lungs [48]. Cyanide (0.05–0.1 mM) increased pulmonary artery (PA) pressure (PAP) in a concentration‐dependent manner in isolated pig lungs, while 1 mM cyanide restored PAP to control levels, mimicking progressive hypoxia. ATP and adenylate charge decreased at 1 mM but were unaffected at lower concentrations. Similar reductions occurred with hypoxia/anoxia in degassed, unventilated lungs. The authors suggested that pulmonary vasoconstrictor responses to hypoxia and cyanide occurred independently of the whole‐lung energy state and might be related to decreased energy state in specific lung compartments [49].

Lövgren and Hellstrand showed that cyanide (0.05–4 mM) induced relaxation, decreased O₂ consumption and increased lactate production in rat portal veins in a concentration‐dependent manner, closely resembling the effects of hypoxia. Moreover, the relationship between ATP turnover and mean spontaneous mechanical activity was similar in the presence of cyanide and hypoxia [50].

Cyanide (0.1 μM) caused a leftward shift in the concentration–response curve of serotonin, and it enhanced NA‐ and adrenaline‐induced contractions in human umbilical artery strips but not in human umbilical veins. Additionally, cyanide did not change basal tension in human umbilical vessels [51].

Cyanide (0.1 mM) responded similarly to hypoxia in precontracted arteries [52]. However, cyanide abolished the initial hypoxia‐induced relaxation and the subsequent contraction, while the final prolonged relaxation remained unaffected. The authors suggested that cyanide disrupts mitochondrial energy metabolism, crucial for maintaining vascular tone, and that the inhibition of hypoxia‐induced initial relaxation and transient contraction by cyanide could be related to the removal of relaxation mediated by guanylate cyclase (GC) activation through H₂O₂ [52]. However, this study did not directly investigate the proposed mechanism, highlighting the need for further research to address it.

Ekmehag and colleagues used cyanide (0.1–0.5 mM) to model hypoxic conditions. They observed that cyanide reversibly decreased electrically stimulated force, O₂ consumption and maximal shortening velocity (V _max), while increasing lactate production in the rat portal vein [53]. Cyanide (0.5 mM) inhibited the hypoxia‐induced enhancement of phenylephrine (PhE) contractions in the rat deep femoral artery, suggesting a potential role for mitochondria as oxygen sensors in vascular regulation [54].

In rat thoracic aortic rings, cyanide (320 μM) rapidly reduced vascular ATP levels at 10 s and 3 min, while a decrease in vascular tone was observed only at 3 min [55]. Although the authors did not explicitly discuss this aspect, these findings imply that cyanide may influence vascular tone independently of its effects on ATP levels. Besides its effects on vascular function, unlike hypoxia, cyanide (500 μM) did not affect ACh‐ or histamine‐induced contractions in opossum colonic tissue, indicating distinct mechanisms [56].

At a higher concentration (1 mM), similar to hypoxia, cyanide did not alter serotonin‐induced contraction but inhibited low concentrations of NA (< 50 nM)‐induced contraction in the rat hindlimb vessels [57]. In contrast, contractions induced by high concentrations of NA (> 50 nM) remained unaffected by either cyanide or hypoxia. Cyanide infusion (1 mM) decreased basal O₂ uptake without altering arterial PO₂ and slightly increased venous PO₂ and perfusion pressure [57]. Consistently, cyanide (3 mM) induced relaxation in KCl‐contracted rat detrusor muscle strips, an effect that was inhibited by both chronic obstruction (urethral ligation for 6 weeks) and the HIF‐inducer dimethyloxalylglycine (DMOG), suggesting a potential interaction between hypoxia‐related pathways and cyanide‐induced smooth muscle relaxation [58].

Cyanide (1 mM) induced a transient pressor response, followed by reduced vascular responses to hypoxia and angiotensin II in isolated blood‐ and plasma‐perfused rat lungs. Because blood flow remained constant and pulmonary oedema did not develop, this response was attributed to the vasoconstrictive effect of cyanide. Additionally, lactate accumulation increased in the perfusate of cyanide‐treated lungs [59]. However, the small sample size in this study limits the statistical significance of the findings (n = 1–3).

Cyanide (2 mM) induced rapid, endothelium‐independent relaxation in precontracted small mesenteric arteries, with the magnitude and speed of response varying by vessel size—higher than that of hypoxia in smaller vessels but less than that of hypoxia in medium and large vessels (Table 1). The authors attributed this to possible differences in O₂‐sensitive ion channel distribution or an additional O₂‐sensing step not directly linked to cellular metabolism in the mesenteric vascular bed [60].

Cyanide (5 mM) induced relaxation in KCl‐precontracted porcine coronary arteries, similar to hypoxia. Unlike hypoxia, cyanide did not affect the rapid component of endothelium‐dependent relaxations induced by substance P (SP), thrombin (TB) and the Ca²⁺ ionophore A‐23187. Cyanide (1 mM) abolished coronary artery respiration, but this inhibition was not involved in the rapid phase of endothelium‐dependent relaxation [61]. However, Griffith et al. showed that other mitochondrial respiratory inhibitors (e.g., rotenone, antimycin) reversed acetylcholine (ACh)‐ and A‐23187‐induced plateau relaxation responses in precontracted rabbit aorta strips and reduced endothelium‐derived relaxing factor release [62]. This discrepancy may be related to cyanide's non‐mitochondrial effects and variations in species, tissues or the type of relaxing agent used.

5.2. GC and cGMP Pathways in Cyanide‐Induced Vascular Responses

Sodium nitroprusside (SNP) is composed of a ferrous iron (Fe²⁺) molecule complexed with five cyanide groups and a nitrosyl group. It interacts with haemoglobin, releasing cyanide and NO, which has been associated with cyanide toxicity [63]. Cyanide is released in greater amounts than NO during the reduction of SNP. In the presence of cyanide (5–10 mM), NO release from SNP was blocked in vascular tissue (rabbit aorta), plasma and haemoproteins. This suggests that cyanide accumulation during SNP administration inhibits NO release, reducing its vasodilator effects and potentially necessitating higher concentrations of SNP [64]. Therefore, resistance/tachyphylaxis to the hypotensive effects of SNP observed in some patients has been the subject of many studies, with suggestions that it may be related to cyanide levels. In a study conducted for this purpose, after blood pressure dropped and returned to baseline following intravenous SNP infusion in dogs, cyanide was infused (0.0125 mg/kg/min), followed by a second SNP infusion. Although cyanide administration increased plasma and tissue cyanide levels, it did not alter the effects of SNP on blood pressure or heart rate, indicating that cyanide does not play a role in SNP resistance [65].

NO activates soluble GC (sGC) by binding to the ferrous (Fe²⁺) heme, forming a 5‐coordinate high‐spin ferrous nitrosyl complex, representing the active form of the enzyme. Cyanide, identified as the first ionic ligand to bind to the heme of sGC, interacts with the ferric (Fe³⁺) heme with a slow on‐rate and the ferrous (Fe²⁺) heme with a fast off‐rate, resulting in the formation of a 6‐coordinate low‐spin complex. This complex did not activate the enzyme and has been interpreted as a potential structural change in the protein associated with the oxidation of the heme iron [66, 67].

SNP‐induced relaxations were partially inhibited by the addition of cyanide (4–8, 7–70 and 8–48 μM, respectively) in precontracted rat PA strips, guinea pig ileum and rabbit gall bladder, but cyanide did not affect relaxations induced by other nitrovasodilators such as glyceryl trinitrate (GTN), NO₂ ⁻ and HbNO (Table 1) [68]. They suggested that cyanide may inhibit the enzyme responsible for converting SNP to NO, although the mechanism for why cyanide did not affect other nitrovasodilators remains unclear [68].

Kruszyna et al. demonstrated that cyanide (14–600 μM) inhibited the vasorelaxant effects of SNP, N₃ ⁻ and hydroxylamine (H₂NOH) in NA‐contracted rabbit aorta strips, while not affecting NA‐induced contractions or relaxations induced by HbNO, NO gas, NO₂ ⁻, GTN, adenosine or papaverine (Table 1) [69]. Furthermore, cyanide (40 μM) inhibited SNP‐induced increase in cGMP levels, but it did not affect NO₂ ⁻‐induced relaxations or cGMP levels [69].

Rapoport et al. investigated how varying cyanide (100 μM) incubation times influence nitroprusside‐induced relaxation in KCl‐contracted rat aorta strips, with maximum inhibition observed at 5 min incubation (Table 1) [12]. Cyanide (0.1 mM) prevented increases in cGMP levels induced by KCl, BaCl₂ and NA. Pretreatment with cyanide inhibited relaxation responses to other nitrovasodilators, ACh and isoproterenol but did not affect 8‐bromocyclic GMP‐induced relaxation. Cyanide also inhibited nitrovasodilator‐ and ACh‐induced increases in cGMP levels. While a relatively higher concentration of cyanide (1 mM) increased basal GC activity, it (100 μM, 1 mM) inhibited SNP‐induced enzyme activation [12]. Rapoport et al. also showed that cyanide (100 μM) inhibited SNP‐, ACh‐ and atriopeptin II‐induced relaxations and the SNP‐ and ACh‐induced increase in cGMP levels in rat aorta [70]. However, cyanide did not affect the cGMP levels elevated by atriopeptin II [70]. These studies suggested that these findings might be attributed to cyanide binding to the NO site on the heme moiety of sGC, thereby preventing its interaction with free radicals and inhibiting its activation [12, 70]. On the other hand, the inhibition of atriopeptin II‐induced relaxation by cyanide was interpreted as potentially resulting from a nonspecific effect of cyanide [70].

Cyanide (0.1 mM) elicited responses similar to the second (vasodilation) and third (vasoconstriction) phases of the hypoxic response in PhE‐contracted rat aorta, PA and mesenteric artery; however, the initial contractile phase of hypoxia was not observed [71]. Consistently, the hypoxic response's first phase was abolished in the cyanide's presence. The reduced relaxation responses to ACh and DETA‐NO during hypoxia were restored when cyanide was applied. While the NOS inhibitor NG‐nitro‐L‐arginine (L‐NNA) or the sGC inhibitor ODQ had no effect under hypoxia, both agents induced contraction in the presence of cyanide. Comparable results were obtained with other mitochondrial inhibitors. These findings indicate that NO fails to activate sGC in hypoxic vessels but retains this ability in the presence of cyanide, suggesting cyanide disrupts NO‐CCOx interactions, thereby enhancing NO‐mediated relaxation via sGC [71].

Ignarro et al. demonstrated that cyanide (100 μM) selectively inhibited SNP‐induced relaxations and cGMP accumulation in bovine PA rings, without affecting basal cGMP levels or vascular tone [72]. In contrast, methylene blue (10–100 μM) inhibited relaxations and cGMP increases induced by various vasodilators, including SNP, GTN and ACh, enhanced contractile responses and reduced basal cGMP levels (Table 1). They suggested that cyanide is not a potent sGC inhibitor and that cyanide's inhibition of SNP responses may be due to its chemical inactivation of SNP [72].

Cyanide (100–300 μM) antagonized the vasodilatory effects of SNP in NA‐contracted rabbit aorta strips, increasing tension when applied after SNP‐induced relaxation (Table 1) [73]. This antagonism was specific to SNP, as cyanide did not affect the relaxations induced by other vasodilators such as adenosine, GTN and papaverine. Notably, cyanide did not exhibit toxic effects, as tissue function was reported to return after washing [73].

Omar et al. showed that cyanide (1 mM) and hypoxia significantly inhibited lactate‐induced relaxation in human placental veins, while relaxations induced by forskolin and GTN remained unaffected [74]. They reported that vascular responses to lactate were independent of pH. Furthermore, H₂O₂‐induced relaxation was decreased in the presence of cyanide and MB presence but was not changed by hypoxia. The authors proposed that cyanide may inhibit sGC activated by lactate‐induced H₂O₂ production [74]. However, they did not directly investigate the lactate–H₂O₂–sGC relationship but inferred it from separate experiments.

Interestingly, it has been reported that SNP's inhibitory effect on platelet aggregation was enhanced in the presence of HUVECs, and this effect of SNP was reversed by cyanide (2 mM) [75]. Although the authors did not explain these findings, this suggests that cyanide can interfere with the functional effects of SNP in the presence of endothelial cells. In line with this, cyanide (100 μM–3 mM) has been shown to increase basal GC activity in rat liver, lung, stomach, colon and kidney tissues, though its effect on vascular smooth muscle was not examined [76]. However, how cyanide stimulates basal GC activity while inhibiting SNP‐induced GC activity remains unexplored.

The in vivo administration of cyanide (3 mg/kg, i.p.) in mice increased brain cGMP levels in association with hypoxia [77]. However, in rats, cyanide (5–20 mg/kg, i.p.) decreased cGMP levels in the striatum while elevating them in the cerebellum [78]. Furthermore, the convulsions induced by in vivo cyanide (8 mg/kg, s.c.) in mice were inhibited by L‐NNA and MB, suggesting a possible role for NO‐dependent sGC activation in cyanide‐induced convulsions [79]. However, neither cGMP levels nor sGC activity was measured in this study.

Overall, Rapoport et al. were the only researchers to demonstrate the inhibitory effects of cyanide on cGMP formation and relaxation by nitrovasodilators other than SNP in rat aorta. Other studies on different tissues could not replicate these findings. This discrepancy may be attributed to vessel‐specific and experimental condition‐dependent effects. Furthermore, there are different forms of sGC, including NO‐sensitive (Fe²⁺), NO‐insensitive (Fe³⁺) and hem‐free forms. Only the first form is sensitive to NO and SNP. The balance between these forms shifts from NO‐sensitive to NO‐insensitive when ROS levels increase [80]. Additionally, ROS, such as H₂O₂, which can be elevated by cyanide, may enhance the activity and expression of the sGC, contributing to regulating vascular function [3, 38, 81]. Moreover, H₂O₂ plays a significant role in cyanide production (see above) [9]. Considering the complexity of the cyanide–oxidative stress–sGC relationship, it is unsurprising that conflicting results have been reported regarding cyanide's impact on sGC. On the other hand, nuclear magnetic resonance (NMR) studies have reported that the cyanide ion binds to Cu, Zn SOD more strongly than other ions and exhibits inhibitory properties [82]. SOD exists in two forms: cyanide‐sensitive (Cu, Zn) and cyanide‐insensitive (Mn), making cyanide a valuable tool for isolating and investigating the targeted form [83]. However, cyanide (1 mM) did not affect the baseline or reoxygenation‐stimulated release of H₂O₂ in bovine PA endothelial cells [84]. Therefore, further comprehensive studies, including the measurement of ROS levels, are needed to better understand cyanide's effects on sGC.

5.3. Roles of Ion Channels, Ca²⁺ Levels and Phosphorylation in Vascular Effects of Cyanide

Waypa et al. demonstrated that ROS generated at the proximal ETC increased [Ca²⁺]_i in rat pulmonary arterial (PA) myocytes, contributing to PA contraction. Cyanide (10 μM) mimicked hypoxia by elevating [Ca²⁺]_i via an H₂O₂‐dependent mechanism involving the ETC upstream of complex III, as evidenced by reduced responses in the presence of myxothiazol and catalase overexpression (Table 1) [85].

This research group also reported that hypoxia increased [Ca²⁺]_i by inducing ROS signalling from the mitochondria in rat PA smooth muscle cells (PASMCs) [86]. Cyanide (1 μM) did not prevent the increase in cytosolic oxidation caused by hypoxia and further augmented [Ca²⁺]_i during hypoxia, whereas myxothiazol exhibited protective effects. The authors propose that electron flux into complex III is essential for generating the cytosolic oxidant signal, while electron transport through complex IV plays no significant role [87]. Additionally, they demonstrated that cyanide (10 μM) induced a transient (< 10 min) increase in PAP during normoxia, which was inhibited by ebselen (an antioxidant) or myxothiazol [86]. Moreover, cyanide (10 μM) further increased PAP augmented by hypoxia without affecting the response to U46619 in isolated rat lungs, so it failed to abolish hypoxic pulmonary vasoconstriction (HPV) [86]. Similarly, another study demonstrated that hypoxia and proximal ETC inhibitors increased PAP and reduced ROS levels in isolated rat lungs under hypoxia and inhibited outward K⁺ current in isolated pulmonary vascular smooth muscle cells, effectively abolishing HPV [88]. In contrast, the distal ETC inhibitor cyanide (15, 31 and 154 μM) increased ROS levels and PAP in a concentration‐dependent manner, without altering K⁺ currents or inhibiting HPV. The increase in ROS induced by cyanide was found to be independent of SOD inhibition, indicating that cyanide elevates ROS levels by inhibiting ETC [88].

Cyanide (100 μM), similar to insulin, did not affect Phase 1 but enhanced Phase 2 contractions induced by PGF₂α under hypoxia in rat small PAs. This phase correlates with glucose concentration without altering the hypoxia‐induced increase in [Ca²⁺]_i. Additionally, cyanide did not affect depolarization‐induced (high K⁺) contraction or basal tone under normoxic conditions. The authors proposed that cyanide may potentiate glucose‐dependent Ca²⁺ sensitization; however, further studies are required to validate this mechanism [89].

Cyanide (0.2 mM) inhibited contractions induced by both Ca²⁺ addition to depolarized solutions and by high K⁺ in Ca²⁺‐containing solutions in rat portal veins. Phosphorylation of the 20‐kDa regulatory myosin light chain (LC₂₀) was reduced at the plateau of high K⁺‐induced contraction, suggesting that cyanide‐induced inhibition of respiration leads to relaxation via decreased LC₂₀ phosphorylation (Table 1) [13]. Cyanide significantly decreased force in both control and electrically stimulated veins, but it caused a much less pronounced reduction in V _max. The authors attributed this observation to cyanide's potential to affect multiple mechanisms simultaneously [13].

Sward et al. showed that cyanide (0.4 mM) increased intracellular Ca²⁺ concentrations ([Ca²⁺]_i) but shortened the duration of Ca²⁺ transients, reducing spontaneous contractions in rat portal veins. Cyanide (0.2 mM) inhibited K⁺‐induced contraction without altering Ca²⁺ levels. Decreased ATP production was suggested to correlate with reduced LC₂₀ phosphorylation [90]. However, the same research group also reported that cyanide (3 mM) and other mitochondrial inhibitors reduced α₁‐adrenoceptor‐stimulated force without affecting global arterial wall [Ca²⁺]_i or LC₂₀ phosphorylation in rat tail artery. Confocal imaging revealed that these mitochondrial inhibitors increased the frequency but reduced the amplitude of α₁‐induced cellular Ca²⁺ waves, altering the intracellular Ca²⁺ wave pattern. They suggested that contraction may be influenced more by localized intracellular Ca²⁺ dynamics than by overall Ca²⁺ levels or phosphorylation [91].

Cyanide (0.5–1 mM) did not affect inward Ca²⁺ currents, elicited by depolarization pulses, in smooth muscle cells of the guinea pig portal vein and increased the rundown rate in four cells when patch electrodes contained 5 mM ATP (n = 3). However, cyanide (0.5 mM) significantly decreased inward currents when electrodes contained 0.3 mM (n = 11) or 0 mM (n = 7) ATP. Notably, full recovery was not observed [92]. These results suggest that the ATP levels or glycolytic activity in vascular smooth muscle cells may critically influence the effects of cyanide on Ca²⁺ levels. However, differences in sample sizes should be considered.

Cyanide (0.4–0.8 mM) inhibited NA‐induced contractions in the rat tail artery concentration‐dependently. The effect of 0.4 mM was reversible, while 0.8 mM was not. While [Ca²⁺]_i was not affected by 0.4 mM cyanide, ATP levels were reversibly reduced [93]. However, cyanide at 10 nM–100 μM neither induced relaxation nor contraction in the small arteries of the rat tail [94].

Cyanide (1 mM) reduced NA‐ and KCl‐induced contractions in rabbit aorta strips, with a higher concentration (10 mM) enhancing low concentrations of KCl‐induced responses but inhibiting responses to higher KCl levels, mimicking verapamil [95]. Cyanide‐induced contractions were potentiated by ouabain and verapamil, unaffected by various receptor antagonists, and attenuated by anion channel blockers, suggesting cyanide may enter vascular smooth muscle cells through anionic channels (Table 1) [95]. Indeed, Wisler et al. investigated the mechanisms of cyanide (CN⁻) transport into guinea pig mitochondria using kinetic measurements, revealing that at high concentrations (> 10 μM), passive diffusion predominates. In contrast, at low cyanide concentrations, active or facilitated transport becomes the primary mechanism. They suggested that it may be possible to inhibit cyanide entry into cells using anion channel blockers [96].

Cyanide (2–5 mM) increased [Ca²⁺]_i in the human umbilical vein endothelial cell line (Ea.hy926) via a mechanism dependent on extracellular Ca²⁺ and membrane potential, likely involving non‐selective cation (NSC) channels [97]. It enhanced agonist‐induced Ca²⁺ signals and sensitized preactivated BK_Ca channels but did not activate them directly in Ea.hy926 cells. These findings suggest cyanide promotes Ca²⁺ influx through Ca²⁺‐permeable NSC channels resembling transient receptor potential channel 3 (TRP3) (Table 1) [97].

Cyanide (0.1–2 mM) decreased spontaneous contractile activity and induced relaxation in rat portal vein, similar to cromakalim (a K_ATP channel opener), while glibenclamide (a K_ATP channel blocker) inhibited the effects of both cyanide and cromakalim [98]. However, glibenclamide did not affect cyanide‐induced relaxations in precontracted tissues. Cyanide increased basal [Ca²⁺]_i and reduced Ca²⁺ transients during phasic contractions, while glibenclamide improved the maintenance of these transients and electrical activity. Additionally, cyanide‐induced hyperpolarization in 3‐hydroxybutyrate medium was reversed by glibenclamide (Table 1) [98]. Consistently, smooth muscle cells isolated from the rabbit portal vein demonstrated the induction of a glibenclamide‐sensitive K⁺ current when subjected to metabolic poisoning with a combination of cyanide (2 mM) and 2‐deoxy‐D‐glucose [99]. These results indicate that K_ATP channel activation and hyperpolarization play roles in cyanide‐induced vascular responses. However, it should not be overlooked that this may be related to the decrease in ATP levels caused by cyanide's inhibition of cellular metabolism.

Cyanide (0.20–2 mM) shortened action potential bursts and reduced force in rat portal vein smooth muscle, with a greater effect on contraction than on electrical activity [100]. Higher concentrations (2 mM) abolished spontaneous activity and induced mild, variable changes in membrane potential, including slight hyperpolarization. These effects were largely resistant to K⁺ channel blockers. Furthermore, cyanide (1 mM) reduced high K⁺‐induced contraction, while depolarization was only slightly diminished, suggesting that cyanide may reduce force through membrane‐independent mechanisms (Table 1) [100]. However, it should be noted that the sample sizes (n values) are not specified in all protocols of this study.

In addition to vascular structures, the role of K_ATP channels in the effects of cyanide has also been explored in different smooth muscles. Cyanide (1–5 mM) activated a current due to the opening of glibenclamide‐sensitive K⁺ channels in pig urethra [101]. Additionally, cyanide (2 mM) significantly reduced spontaneous contractions in rat uteri, which were restored by high‐K⁺‐induced depolarization [102]. Cyanide also increased K⁺ efflux, causing hyperpolarization and suppressing excitation. Glibenclamide partially inhibited K⁺ efflux but did not affect cyanide's impact on contractions, indicating partial involvement of K_ATP channels. The glibenclamide‐insensitive K⁺ efflux persisted in a Ca²⁺‐free medium and was unaffected by butyrate, suggesting that neither Ca²⁺‐activated K⁺ channels nor hypoxia‐induced acidification contributed to the response [102].

Cyanide (1 mM) reduced spontaneous mechanical and electrical activity in guinea pig stomach smooth muscle [103]. ³¹P NMR showed ATP levels remained sufficient to induce contraction in the presence of cyanide, suggesting K_ATP channels remained closed [103]. However, this conclusion was based on metabolic data, without direct testing of K_ATP channel involvement.

Wang et al. demonstrated that cyanide (2–10 mM) induced depolarizing Ca²⁺‐activated Cl⁻ currents (I _Cl(Ca)) in primary rat PASMCs [104]. This activation is necessary for producing membrane depolarization, which leads to further opening of VSCC in smooth muscle in response to NA. Cyanide increased [Ca²⁺]_i, and this increase was inhibited by caffeine. While cyanide prolonged caffeine‐induced I _Cl(Ca), it did not affect caffeine‐induced I _K(Ca) [104]. In a subsequent study, cyanide (1 mM) increased transient inward Cl⁻ currents in electrically quiescent PASMCs and the frequency and amplitude of spontaneous transient inward currents (STICs) in voltage‐clamped myocytes. Cyanide (1 mM) also increased Ca²⁺ sparks in PASMCs [105]. In Xenopus oocytes lacking ryanodine receptors, cyanide (10 mM) induced a sustained Ca²⁺ current via IP₃R‐mediated intracellular Ca²⁺ release. When mitochondrial Ca²⁺ was depleted, cyanide‐induced I _Cl(Ca) was reduced. The anion selectivity of the cyanide‐induced current and its activation of I _Cl(Ca) were confirmed through ion substitution and chloride channel blockade. Notably, cyanide—but not hypoxia—induced Ca²⁺ release in mesenteric artery myocytes (Table 1) [105]. Overall, both mitochondria and SR, including RyRs and IP₃Rs, may be potential targets of cyanide, and cyanide‐induced metabolic inhibition may result in tissue‐specific responses distinct from those produced by hypoxia.

The effects of cyanide on Ca²⁺‐related contractile responses have also been investigated in various smooth muscle preparations. Taggart et al. reported that cyanide (2 mM) disrupted the maintenance of high K⁺‐induced contractions in rat uterine smooth muscle despite increasing [Ca²⁺]_i and preserving LC₂₀ phosphorylation. These findings suggest that cyanide alters the coupling between Ca²⁺, LC₂₀ phosphorylation and force generation, although the precise mechanisms remain unclear [106].

Cyanide (2 mM) inhibited the high K⁺‐induced increase in force; however, it elevated [Ca²⁺]_i in longitudinal smooth muscle isolated from healthy and diabetic rat urinary bladders [107]. Cyanide (2 mM) decreased electrically evoked contractions and intracellular Ca²⁺ transients in adult and neonatal rats and guinea pig ureters, although neonatal guinea pig ureters showed recovery of both parameters after an initial decline. These differences were not observed in high K⁺‐induced contractions, as cyanide reduced the contractile response but did not alter Ca²⁺ levels. The authors concluded that cyanide responses vary across species and developmental stages [108].

Cyanide (2 mM) abolished phasic contractions and Ca²⁺ transients in human myometrial strips from pregnant women, while increasing basal [Ca²⁺]_i and reducing basal and oxytocin‐induced force [109]. Although Ca²⁺ levels were elevated, cyanide suppressed contractile responses to oxytocin and carbachol, and these effects were not reversed by oxytocin or the Ca²⁺ channel agonist Bay K8644. In depolarized strips, cyanide continuously increased the Ca²⁺ signal while causing a transient force reduction [109].

Earley and Wray demonstrated that cyanide reversibly inhibited spontaneous uterine contractions in non‐pregnant rats, similar to hypoxia, while having a milder effect on carbachol‐ and oxytocin‐induced contractions [110]. Cyanide also inhibited high K⁺‐induced contractions, suggesting it may block Ca²⁺ entry through voltage‐gated channels during depolarization. Although cyanide‐induced acidification may contribute to its effects, the role of pH was not directly examined in this study [110].

Cyanide (2 mM) reversibly decreased pH, phasic contractions and Ca²⁺ transients following an initial brief transient increase in guinea pig ureter strips [111]. Cyanide also reduced high K⁺‐evoked tonic contractions but did not affect [Ca²⁺]_i. The authors suggested that cyanide might induce desensitization of myofilaments to Ca²⁺ [111]. However, further research is needed to confirm this mechanism.

Synthesizing these findings (Table 1, Figure 3) reveals that cyanide at 1 nM–10 μM tends to increase basal tension, whereas at 50 μM–5 mM, its effects shift towards reduced contraction or relaxation. However, over a broad concentration range (7 μM–1 mM), cyanide has been shown to inhibit SNP‐induced relaxations in various smooth muscle preparations. However, the mechanism, potentially involving sGC inhibition, remains debated. Cyanide has been shown to induce contraction at 1–10 mM. In PASMCs and human umbilical vein endothelial cells, cyanide (1–10 mM) increased [Ca²⁺]_i, I _Cl(Ca) and enhanced BK_Ca channel sensitivity. However, these effects have been limited to cell culture studies, with no direct investigation of contractile responses. Importantly, when evaluating the vascular effects of cyanide, the impact of ROS generation on molecular targets should not be overlooked, as H₂O₂ is known to play a role in cyanide formation, sGC activity and an increase in [Ca²⁺]_i. The effects described so far pertain to exogenously applied cyanide on vascular tissues. To date, no studies have investigated whether endogenous cyanide exerts any vascular effects.

In experimental studies with sublethal doses, the maximum blood cyanide concentrations observed in vivo are in the same range as those required to elicit vasorelaxant effects in vascular tissues ex vivo. For example, in rats given subcutaneous KCN (2, 4 and 6 mg/kg), the maximum cyanide blood concentrations reached 51.7, 59.8 and 68.8 μM, respectively. Similarly, sublethal KCN doses administered intravenously to pigs (1.7 mg/kg) and rabbits (2.5 mg/kg) resulted in maximum blood concentrations of 30.2 and 14.7 μM, respectively [112]. In another study, oral administration of 3 mg/kg KCN to rats, pigs and goats did not cause any signs of cyanide intoxication, while the maximum plasma cyanide concentrations were measured at 89.0, 57.5 and 93.5 μM, respectively [113]. These data demonstrate that the pharmacological relaxant effects of cyanide occur at concentrations (Figure 3B) that, in vivo, are reached by sublethal dosing but without causing signs of intoxication.

5.4. In Vivo Vascular Effects of Cyanide

Before discussing the vascular effects of cyanide in vivo, it is essential to outline the dose ranges at which cyanide exerts toxic effects. Establishing these thresholds provides an important perspective on the ‘dose distance to toxicity’, i.e., the margin between doses producing regulatory effects and those causing overt toxicity. Based on toxicological studies in experimental animals (Table 2), oral no‐observed‐adverse‐effect levels (NOAELs) generally fall within the range of ~1–30 mg/kg/day, while oral median lethal doses (LD₅₀) range between 4 and 40 mg/kg. For parenteral administration, LD₅₀ values have been reported at ~5–6 mg/kg. It should be noted, however, that reported thresholds vary depending on the compound tested (NaCN or KCN), as NaCN contains ~53% CN⁻, whereas KCN contains ~40% CN⁻. Additionally, the route of administration significantly influences toxicity, with NOAEL and LD₅₀ values generally lower after parenteral exposure compared with oral administration.

TABLE 2.

Reported NOAEL and LD₅₀ values for cyanide in laboratory animals.

Species	Compound	Route	NOAEL (mg/kg/day)	LD50 (mg/kg)	References
Mouse	NaCN	Oral	8.6–28.8	—	[121]
	KCN	Oral	—	9.9–11.9	[122]
	NaCN	Intraperitoneal	—	5.8	[123]
	KCN	Intraperitoneal	—	5.7	[124]
Rat	NaCN	Oral	4.5–12.5	—	[121]
	NaCN	Oral	21.7	—	[125]
	NaCN	Oral	—	49.1	[126]
Rabbit	NaCN	Oral	15	—	[121]
	KCN	Oral	20	—	[121]
	KCN	Subcutaneous	—	6	[124]
Sheep	NaCN	Oral	—	3.7	[127]

Cyanide (sodium cyanide, NaCN, 100–200 μg/kg, intra‐arterial) caused significant vasodilation and increased uterine blood flow, intrauterine pressure and conductance in non‐pregnant ewes. These effects were unaffected by propranolol, indicating a β‐adrenergic‐independent mechanism [114].

Ozolua et al. investigated how subchronic cyanide (KCN, 0.38 mg/kg/day, 25 days, oral) affected vascular responses in rabbit aorta rings. KCN did not alter general toxicological parameters [14]. It reduced NA‐ and Ca²⁺‐induced contractions and enhanced the relaxant effect of ACh in endothelium‐denuded aortic rings, but not endothelium‐intact rings, suggesting a possible competition between NO and cyanide at cellular targets. The reduced contractions were attributed to a decrease in [Ca²⁺]_i, potentially due to impaired energy‐dependent transporters, such as the Na⁺/Ca²⁺ exchanger. The authors suggested that the consumption of cassava‐based foods is unlikely to have a significant impact on vascular function in individuals with intact endothelial function [14]. These in vivo findings contrast with in vitro studies, which demonstrate reduced vascular contraction responses in an intact endothelium. However, in this study, cyanide was administered at a low dose (0.38 mg/kg/day), and blood cyanide concentrations were not measured. Therefore, it can be assumed that the concentration was much lower than that used in in vitro studies and likely within the regulatory range defined by Zuhra and Szabo [5].

Cyanide has been widely used to mimic hypoxia by stimulating peripheral chemoreceptors. In dogs, cyanide (NaCN, 0.05–0.4 mg/kg) stimulated carotid and aortic chemoreceptors, causing vasoconstriction in the gracilis muscle via sympathetic constrictor fibres and vasodilation in the paw via sympathetic dilator fibres [115]. This vasodilation was independent of ACh, histamine, bradykinin and beta‐adrenergic receptor activation. Reflex responses were abolished by bilateral denervation of carotid sinuses and vagotomy and were unrelated to baroreceptor activation. Additionally, carotid injection of cyanide led to a rapid drop in arterial pressure, while aortic infusion had no significant effect, although both routes induced bradycardia [115].

Cyanide (NaCN, 50–300 μg) injected into the fetal inferior vena cava elicited responses similar to those observed during hypoxia in lambs [116]. Without carotid innervation, neither cyanide nor hypoxia altered heart rate or hind‐limb vascular resistance. Conversely, in the absence of aortic innervation, cyanide decreased heart rate and increased hind‐limb vascular resistance, resembling the response seen in normal fetal lambs [116]. These findings suggest that carotid chemoreceptors, rather than aortic chemoreceptors, play a key role in mediating fetal cardiovascular responses to cyanide and hypoxia.

Stimulation of the carotid bodies by injecting cyanide (NaCN, 49–196 μg/kg) into the carotid artery did not affect blood pressure but induced bradycardia and increased femoral vascular resistance in pig‐tailed macaque monkeys [117]. However, intravenous cyanide (NaCN, 200 μg) promptly increased mean arterial pressure in intact lambs [118]. This hypertensive response was abolished by carotid denervation and vagotomy, revealing a tendency towards hypotension in the absence of chemoreceptor input [118].

Intravenous cyanide injection (KCN, 26 μg/g) in conscious mice induced a biphasic response in blood pressure via peripheral chemoreceptor activation [119]. The initial hypertensive phase was mediated by increased sympathetic vasoconstrictor activity, followed by a hypotensive phase partly due to parasympathetically induced bradycardia [119]. These findings suggest that vagal and sympathetic inputs to the heart and vasculature mediate the cardiovascular responses to cyanide. However, stimulating chemoreceptors with cyanide can elicit differential or even opposite responses in individual vascular beds and segments.

Taken together, these studies indicate that most in vivo experiments have employed cyanide doses within a sub‐toxic range, allowing detection of regulatory or mechanistic vascular effects rather than overt toxicity. By contrast, in another study, rats were administered a high cyanide dose (KCN, 30 mg/kg/day, orally) for 10 days (treatment phase), followed by a 10‐day withdrawal period during which treatment was discontinued [120]. This dose exceeded the reported NOAEL range for rats (4.5–21.7 mg/kg/day) (Table 2). In this study, cyanide led to a decrease in the lumen width/wall thickness ratio (LWR) in the middle cerebral artery, indicating augmented vascular resistance, and a significant increase in the diameter of the common carotid artery. After the withdrawal phase, the cyanide group exhibited further increases in vascular resistance and a reduction in LWR. These findings demonstrate that high‐dose cyanide‐induced ischaemia increases vascular resistance [120].

Finally, while most available in vivo studies have been performed in healthy animals, it remains unclear whether the vascular effects of cyanide would differ in advanced disease states such as diabetes, hypertension or atherosclerosis. Given the established role of endothelial dysfunction in these conditions, it is conceivable that the vascular impact of cyanide could be altered, and this represents an important direction for future investigation.

5.5. The Effects of Cyanide on Arterial Compliance

Carotid compliance (CC), measured by the change in volume within the artery in response to transmural pressure increases, was lower in spontaneously hypertensive rats (SHRs) compared with Wistar‐Kyoto rats (WKY), indicating a stiffer arterial wall in hypertension. Local intracarotid application of cyanide (100 mg/L) increased CC in SHR and WKY for up to transmural pressures of 125 and 100 mmHg, respectively. However, CC remained lower in the hypertensive than in the normotensive rats [128, 129]. In another study, cyanide (100 mg/L) application increased the CC of both WKY rats and SHR, with a greater effect observed in SHR [130, 131].

The acute local intracarotid injection of cyanide (100 mg/L) increased CC for pressure steps up to 125 mmHg in both Dahl salt‐sensitive and salt‐resistant rats, each tested under 0.4% and 7% NaCl diets, as well as in indapamide‐treated rats [132]. In aged normotensive rats, endothelial denudation did not alter the isobaric diameter of the carotid artery, which increased in parallel with the transmural pressure level. However, cyanide (100 mg/mL) directly affected the smooth muscle, increasing the arterial diameter and enhancing CC [133]. It should be noted that cyanide did not change compliance at pressure levels above 125 mmHg in all the studies mentioned above. Overall, chronic cyanide treatment may enhance the elastic properties of the arterial wall, which could be beneficial in diseases that compromise arterial wall integrity.

6. Vascular Effects of Glycine

Building on the findings of Zuhra et al., which demonstrate that mammalian cyanide production requires glycine [9], we investigated the effects of glycine on the vasculature (Table 3) in comparison to those of cyanide. Glycine, in addition to being an amino acid, is a bioactive molecule with antioxidant and anti‐inflammatory properties, playing a vital role in vascular health. Glycine, an inhibitory neurotransmitter acting via glycine‐gated Cl⁻ channels, was shown to reduce the VEGF‐induced increase in [Ca²⁺]_i in bovine endothelial cells (CPA) in a concentration‐dependent manner, with a maximum effective concentration (EC_max) of 1 mM. This effect was blocked by strychnine and was absent in Cl⁻‐free medium. Additionally, glycine (2–6 mM) inhibited the proliferation and migration of serum‐stimulated endothelial cells [134]. Glycine (1–10 mM) also decreased VEGF deprivation‐induced apoptosis in liver sinusoidal endothelial cells. This protective effect was inhibited by strychnine, underscoring the involvement of glycine‐gated Cl⁻ channels. Moreover, glycine (10 mM) prevented the significant reduction in Bcl‐2 (antiapoptotic) expression caused by VEGF deprivation [135]. These findings suggest that glycine exerts protective effects against apoptotic endothelial cell death.

TABLE 3.

Vascular effects of glycine.

Tissue/animal model	Concentration/dose	Observed effects	Proposed mechanisms	References
Rat, rabbit, Leghorn aorta	10–100 mM	● Contractions ● Indomethacin (COX inhibitor) completely blocked contractions ● Partial contribution from hyperosmolarity Effect of endothelium removal: ● ↓ contractions in rat & rabbit ● ↔ in Leghorns	Prostaglandin‐mediated (COX‐dependent) Rat and rabbit: Endothelium‐dependent Leghorns: Endothelium‐independent	[139]
Rat cerebral arterioles (parietal cortex)	1 M glycine, 0.1 mL applied topically (Final dose: 40 mg/kg)	● ↑ Arteriolar diameter to 150%–250% ● Onset: 1–3 min ● Duration: 5–10 min ● Reproducible	No specific mechanisms were investigated	[144]
Rat mesenteric arterioles	1 M glycine, 0.1 mL applied topically (Final dose: 40 mg/kg)	● ↑ Arteriolar diameter to 50%–80% ● Onset: 10–15 s ● Duration: 5–10 min ● Before and after histamine → protective and therapeutic against histamine‐induced microcirculatory disturbances	No specific mechanisms were investigated	[140]
Protein‐restricted pregnant rats (mesenteric artery and thoracic aorta)	3% dietary glycine	Mesenteric artery: ● Protein restriction → ↓ ACh‐induced vasodilation and NO release ● Glycine supplementation → restored/improved these responses Thoracic aorta: ↔ No significant changes with protein restriction or glycine eNOS expression: ↔ Across groups	● No specific mechanisms proposed ● May involve NO synthesis/pathway activation	[141]
Aortic rings from adult (5 months) and aged (15 months) rats	0.1 g/kg in drinking water, for 60 days	Effect on aged rats: ● ↓ PhE‐induced contraction ● ↑ ACh‐induced relaxation ● ↑ eNOS expression Effect on adult rats/denuded rings: ● ↔ No effect With L‐NAME (NOS inhibitor): ● PhE curve shifted left in both age groups ● Shift was greater in aged rats + glycine With SOD (superoxide scavenger): ● ↔ No effect on adult rats ● Rightward shift in aged rats → enhanced by glycine	● Endothelium‐dependent ● Improves NO‐mediated vasodilation ● Reduces the contribution of superoxide to vascular contraction	[142]
Sucrose‐fed (SF) rats	1% in drinking water, 4 weeks	● ↑ BP in SF rats → restored to control levels with glycine ● ↑ NO metabolites and eNOS expression ● ↑ ACh‐induced relaxation ● ↔ SNP‐induced relaxation ● ↓ GSH/GSSG ratio, GSS2, Zn/Cu‐SOD, Mn‐SOD in SF rats → all restored by glycine	● Improves endothelial NO signalling ● Enhances antioxidant capacity and glutathione balance	[143]

Abbreviations: ↑, increased/induced; ↓, decreased/inhibited; ↔ no effect; ACh, acetylcholine; BP, blood pressure; COX, cyclooxygenase; eNOS, endothelial nitric oxide synthase; GSH, reduced glutathione; GSS2, glutathione synthetase 2; GSSG, oxidized glutathione; L‐NAME, NG‐nitro‐L‐arginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase; PhE, phenylephrine; SF, sucrose‐fed; SNP, sodium nitroprusside; SOD, superoxide dismutase.

Endothelial cell hyperpolarization enhances Ca²⁺ influx, activating NOS and increasing NO levels while inhibiting NADPH oxidase and reducing superoxide production. McCarty et al. suggested that glycine‐induced hyperpolarization may promote endothelial health and exert anti‐atherogenic effects through these mechanisms [136]. Although supported by many studies, further research is required to confirm this hypothesis.

Tsuji‐Tamura et al. reported that glycine exerted a concentration‐dependent biphasic effect on vascular development in zebrafish embryos [137]. While 10 and 100 mM glycine enhanced survival and upregulated vegfaa and nos2a expression, 400 mM glycine reduced angiogenesis, suppressed nos2a and caused mortality. These effects were blocked by glycine transporter and glycine receptor inhibitors [137]. Moreover, glycine‐induced biphasic effects on angiogenesis in zebrafish embryos were shown to be modulated by PI₃K/Akt/mTOR signalling, with low‐concentration glycine (10 mM)‐induced angiogenesis being inhibited, and high‐concentration glycine (400 mM)‐induced anti‐angiogenesis being amplified by PI₃K/Akt/mTOR inhibition [138].

As discussed above, cyanide exhibits a biphasic effect like glycine, stimulating mitochondrial bioenergetics and cellular metabolism at low concentrations and demonstrating cytoprotective effects while being cytotoxic at high concentrations in liver cells [4, 9]. Additionally, cyanogenic compounds have been reported to exert protective effects against hypoxia‐induced cell injury by releasing low levels of cyanide [9]. Considering that 10 mM glycine stimulates cyanide production in liver cells and exhibits cytoprotective effects [9], it can be proposed that low levels of cyanide may contribute to the protective effects of glycine on endothelial cells. Furthermore, the harmful effects of high concentrations of glycine could potentially be attributed to the excessive production of cyanide.

Glycine (10–100 mM) induced concentration‐dependent vascular contractions in rat, rabbit and Leghorn aorta, with the response being endothelium‐dependent in rats and rabbits, but endothelium‐independent in Leghorns. These contractions were partly attributed to hyperosmolarity at higher concentrations and inhibited by indomethacin, suggesting a prostaglandin‐mediated mechanism (Table 3) [139]. Similarly, the effects of cyanide on vascular responses vary depending on the animal species [47]. Cyanide (1 nM–10 μM), like glycine, has been shown to induce contraction in rabbit aorta; however, the underlying mechanisms of cyanide‐induced contractions remain unclear. Cyanide at 100 μM and 1 mM has been observed to cause relaxation [47]. Studies discussed later also demonstrate the relaxant effects of in vivo glycine [140, 141, 142, 143]. Thus, the responses induced by glycine, similar to cyanide, appear to depend on its concentration and the specific tissue type. This supports the hypothesis that cyanide may play a role in glycine‐induced responses.

Topical application of glycine (40 mg/kg) induced rapid and marked vasodilation in both cerebral (150%–250%) and mesenteric (50%–80%) arterioles in rats, with effects lasting 5–10 min and proving reproducible (Table 3). Glycine also showed protective and therapeutic effects against histamine‐induced microcirculatory disturbances, although its vasodilatory mechanism remains unidentified [140, 144].

Brawley et al. showed that 3% dietary glycine supplementation improved ACh‐induced vasodilation and NO release in mesenteric arteries of protein‐restricted pregnant rats. No significant effects were observed in thoracic aorta responses and eNOS expression [141].

Gómez‐Zamudio et al. demonstrated that glycine (0.1 g/kg, 60 days, in water) enhanced ACh‐induced relaxation and reduced PhE‐induced contraction in endothelium‐intact aortic rings of aged rats, but not in adults or endothelium‐denuded vessels. In aged rats, glycine enhanced the effect of L‐NAME on vasoconstrictive PhE responses and amplified the SOD‐induced rightward shift in PhE responses (Table 3) [142]. These findings suggest that glycine treatment enhances endothelial regulation through the release of NO and reduces the role of the superoxide anion in vascular contraction.

Glycine supplementation (1%, in water, 4 weeks) normalized elevated blood pressure in sucrose‐fed (SF) rats and improved endothelial function by increasing eNOS expression and NO levels in the aortas. It also restored antioxidants (reduced glutathione [GSH]/oxidized glutathione [GSSG] ratio, glutathione synthetase 2 [GSS2], Zn/Cu‐SOD and Mn‐SOD) and enhanced ACh‐induced, but not SNP‐induced, vasorelaxation (Table 3) [143]. These findings suggest that glycine ameliorates vascular dysfunction and oxidative stress and restores glutathione in vascular tissues.

In conclusion, glycine administration enhances ACh‐induced relaxations and NO production, which are impaired by different conditions, and inhibits PhE‐induced contractions. As discussed above, exogenous cyanide has been demonstrated to induce relaxation and decrease contractions in various arteries and smooth muscle preparations in vitro [47, 61]. Moreover, similar to glycine studies, in vivo subchronic cyanide administration has been shown to reduce contraction responses and increase ACh‐induced relaxations in endothelium‐denuded aortas [14]. Glycine, reported as a potential substrate for cyanide, induces vascular responses resembling those of exogenous cyanide, suggesting the possibility of cyanide production within the vasculature. However, further studies are needed to identify the specific vascular cells responsible for cyanide production.

7. Conclusions and Future Perspectives

The importance of ATP in maintaining vascular function is well established, and high concentrations of cyanide have been shown to cause relaxation through ATP depletion. However, numerous studies have indicated that cyanide elicits distinct vascular responses compared with hypoxia, suggesting that it may have specific cellular targets. Advanced research has highlighted that cyanide can directly target sGC activity, myosin light chain phosphorylation, calcium and other ion channels and indirectly affect these targets through cyanide‐induced ROS production. Considering the vascular beneficial effects of cyanide at low doses, it can be suggested that controlled elevation of plasma cyanide levels by consuming cyanogenic products such as apple seeds, cherry kernels and bitter apricot kernels may benefit endothelial function.

Notably, glycine, recently identified as a substrate of cyanide, shares mechanisms of action similar to cyanide in the vasculature. With the emerging concept of cyanide as the potential fourth gasotransmitter, future studies should explore the production of endogenous cyanide in the vasculature and its effects on vascular function. This includes investigations involving endogenous substrates (e.g., glycine) and synthesis inhibitors coupled with measurements of cyanide concentration. Comprehensive studies in this domain could reveal more profound insights into cyanide's physiological and pathophysiological roles in the cardiovascular system and guide the development of therapeutic strategies for managing its effects.

Author Contributions

E.A‐A. and U.S. conceptualized the systematic review. E.A‐A. carried out the literature screening. E.A‐A. and U.S. wrote the manuscript. Both authors reviewed the manuscript and approved its submission.

Conflicts of Interest

The authors declare no conflicts of interest. At the time of publication, Ulf Simonsen was a member of the editorial board at BCPT and was, according to BCPT policy, completely excluded from the peer‐review process and editorial handling of the present manuscript.

Alan‐Albayrak E. and Simonsen U., “Cyanide Beyond Toxicity: A Systematic Review of Its Effects on Vascular Function,” Basic & Clinical Pharmacology & Toxicology 137, no. 5 (2025): e70124, 10.1111/bcpt.70124.

Data Availability Statement

Data sharing does not apply to this article as no datasets were generated or analysed during the current study.