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. Author manuscript; available in PMC: 2025 Dec 1.
Published in final edited form as: Vascul Pharmacol. 2024 Nov 12;157:107440. doi: 10.1016/j.vph.2024.107440

Prolonged L-NAME exposure changes the vasodilator factor from NO to H2O2 in human arterioles in response to A23187

Natalya S Zinkevich a,b,c,*, Kostiantyn Drachuk b,c,*, David X Zhang b,c,*
PMCID: PMC11624973  NIHMSID: NIHMS2036088  PMID: 39537001

Abstract

The Ca2+ ionophore A23187 induces endothelium-dependent and non-receptor-mediated vasodilation in human adipose arterioles (HAAs). The purpose of this study was to determine the mechanism of A23187-induced dilation in HAAs from patients with and without coronary artery disease (CAD). HAAs were freshly isolated from adipose tissues obtained from non-CAD (n=25) and CAD (n=14) patients, and vascular reactivity was studied by videomicroscopy. No difference in baseline dose response to A23187 was observed between non-CAD and CAD subjects. However, acute (30 min) incubation with N(omega)-nitro-l-arginine methyl ester (L-NAME), NO synthase inhibitor strongly reduced A23187-induced dilation in non-CAD arterioles, while catalase, an H2O2 scavenger, largely abolished dilation in CAD. Surprising, prolonged (90 min) incubation with L-NAME restored A23187 response in non-CAD subjects, which was subsequently inhibited by catalase. The action of prolonged L-NAME exposure was not reversible after washing with Krebs while the effect of acute L-NAME exposure was largely reversible. To further determine the role of mitochondria-derived ROS in A23187-induced dilation, arterioles were treated with rotenone, an inhibitor of complex I of the electron transport chain. Rotenone abolished A23187 response in CAD patients and in non-CAD arterioles after prolonged L-NAME, but not in non-CAD controls. These data indicate that NO contributes to A23187-induced dilation in HAAs from non-CAD patients and H2O2 contributes to the dilation in CAD patients. Prolonged L-NAME exposure induces a NO-H2O2 switch in the mechanism of dilation in non-CAD subjects. Moreover, the effect of prolonged L-NAME exposure is not readily reversible, while the action of acute L-NAME exposure is reversible.

Keywords: A23187, endothelium-dependent vasodilation, microcirculation, nitric oxide, reactive oxygen species

Graphical Abstract

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1. Introduction

Endothelial cells line the lumen of blood vessels and release vasoactive substances, such as nitric oxide (NO), prostaglandins (PGI2), hydrogen peroxide (H2O2) or other soluble factors previously termed endothelium-derived hyperpolarizing factor(s) (EDHF), in response to shear stress and chemical stimuli such as acetylcholine (ACh) [1]. These substances diffuse to vascular smooth muscle cells (VSMC) eliciting vasodilation and controlling blood flow in resistance arteries and arterioles. Previous studies from our group demonstrated that different endothelial-derived factors mediate vasorelaxation in response to flow in human coronary or adipose arterioles with age and onset of coronary artery disease (CAD), revealing a novel mechanism of vascular regulation in the human microcirculation [2, 3].

The stimulus-induced release of endothelial factors often utilizes a common pathway that involves an increase in the intracellular Ca2+ within endothelial cells [4]. Accordingly, highly selective Ca2+ ionophore A23187 has been shown to promote endothelium-dependent relaxation by increasing intracellular calcium levels in various animal models including rabbit [5], canine [6], and rat artery rings under physiological condition [7]. The concentrations of A23187 seem critical for obtaining consistent relaxation responses. For example, Shi et al. reported that while doses up to 10−7M of A23187 promote vasorelaxation, higher concentrations (0.3–1 μM) of A23187 elicit endothelium-dependent vasoconstriction in rat femoral arteries, which was more pronounced in streptozotocin-induced diabetic rats [8]. This endothelium-dependent response to higher concentrations of A23187 was prevented by selective COX-1 inhibitor indomethacin, which aligns well with the finding that the level of COX-1 was significantly elevated in streptozotocin-treated rats as compared to untreated control [8].

Preliminary data from our group obtained on human adipose arterioles (HAAs) from subjects without CAD suggest that A23187 dilation is mediated by NO [9]. We have also identified a non-NO/PGI2 component, likely H2O2, mediating A23187 concentration-dependent responses in HAAs from CAD subjects. The mechanism responsible for the switch from NO- to H2O2-dependent A23187 responses remains elusive. There is evidence that NO-mediated S-nitrosylation of the mitochondrial respiratory chain leads to the scavenging of mitochondrial reactive oxygen species (ROS). It may also inhibit ROS production from mitochondria [10]. Interestingly, S-nitrosylation protects cells from redox stress only when ROS levels are low, as higher levels of ROS result in peroxynitrite (ONOO−) formation [11] which leads to endothelial NO synthase (eNOS) uncoupling with consequent production of highly reactive superoxide anion (O2−) that reacts with NO resulting in the accumulation of ONOO− [12]. eNOS uncoupling is thought to be responsible for the development of endothelial dysfunction and many vascular pathologies including CAD [13].

We hypothesized that basal NO inhibits H2O2-mediated component of dilation in non-CAD arterioles and thus prolonged exposure of non-CAD arterioles with eNOS inhibitor, N(omega)-nitro-l-arginine methyl ester (L-NAME) can produce a switch from NO- to H2O2-dependent A23187 concentration-dependent responses like those observed in the microvasculature of subjects with CAD. We also hypothesized that mitochondrial ROS mediate A23187 responses in HAAs after prolonged L-NAME exposure. Therefore, the objectives of this study were three-fold: firstly, to determine the role of NO and H2O2 in A23187 concentration-dependent dilation in the presence and absence of CAD; secondly, to determine the effect and reversibility of prolonged (90 min) L-NAME exposure on A23187 dilation to elucidate whether prolonged incubation with L-NAME results in a transition from NO-dependent to H2O2-dependent vasodilation in HAAs and whether the effect of acute (30 min) or prolonged (90 min) L-NAME is reversible; and thirdly, to test whether mitochondrial ROS play a role in H2O2-dependent vasodilation.

2. Materials and methods

2.1. Human tissue acquisition.

The work was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans and their tissues. Informed consent to use their tissue was obtained from male and female patients before their scheduled surgery. De-identified and discarded adipose tissue samples were collected on the day of the surgery and placed into ice-cold HEPES buffer as previously described [14]. Institutional Review Board (IRB) approval with the waiver of consent was approved by the Medical College of Wisconsin/Froedtert Hospital IRB. Patients with CAD were identified by chart documented diagnosis, while non-CAD individuals had no prior diagnosis of CAD.

2.2. Vascular reactivity.

HAAs with a maximal diameter over 100 μm and under 250 μm freshly isolated from adult subjects were cannulated and pressurized at 60 mmHg in Krebs – physiological salt solution (PSS) composed of (in mM): 123 NaCl, 4.7 KCl, 1.2 MgSO4, 2.5 CaCl2, 19 NaHCO3, 1.2 KH2PO4, 0.026 EDTA, and 11 glucose, gassed with 21% O2 – 5% CO2 and kept at 37°C to maintain pH=7.4. Changes in internal vessel diameter were detected by a videomicroscopy system as reported previously [15].

Vessels were preconstricted with endothelin-1 (ET-1, 0.05–0.3 nM) by 30–50% of the baseline internal diameter. Two dose-dependent response curves to endothelium-dependent vasodilator A23187 (log10−11 – 10−7 M) were performed on each arteriole in the absence and presence of inhibitors, including acute L-NAME (10−4 M), catalase (500U/ml), rotenone (10−6 mol/L), or vehicle added to the organ bath 30 minutes prior to constriction with ET-1. To observe the prolonged effects of L-NAME incubation, the inhibitor was added to the organ bath 90 minutes prior to constriction with ET-1. The L-NAME washing protocol involved the replacement of the old solution in the organ bath with fresh Krebs buffer. This wash was repeated at least five times to eliminate traces of the inhibitor, after which the vessel was allowed to equilibrate at 60 mmHg for 30 min. At the end of each experiment, papaverine (10−4 mol/L) was added to the bath to obtain maximal dilation.

2.3. Reagents.

Endothelin-1 (ET-1), A23187, N(omega)-nitro-l-arginine methyl ester (L-NAME), catalase, rotenone, and papaverine were purchased from Sigma-Aldrich. Stock solutions (1000× or higher) were prepared in DMSO or distilled water. ET-1 stock was prepared in saline with 1% bovine serum albumin. All concentrations depict the final concentrations in the organ bath.

2.4. Statistical Analysis.

Data are presented as means±SEM. For all dose-response curves, differences between groups at each dose were assessed using a two-way (factor) repeated measures ANOVA (factor 1, doses; factor 2, different treatments), followed by the Student-Newman-Keuls multiple comparison test. SigmaPlot (version 15.0) was used to conduct statistical analysis. P values of <0.05 were considered statistically significant and depicted in figures as *P<0.05.

3. Results

3.1. Patient and vascular characteristics.

A total of 54 adipose arterioles dissected from 25 non-CAD and 14 CAD subjects were utilized for this study. Several vessels were dissected from each patient/tissue, with two dose-response curves performed on each vessel unless otherwise indicated. Patient demographics are summarized in Table 1. Vascular characteristics are shown in Table 2. No statistical significance for baseline internal diameter, maximal diameter, or percent tone was found between non-CAD and CAD groups. HAAs from non-CAD (maximal dilation at 10−7 M: 74±3%, n=20, Fig. 1A) and CAD (maximal dilation at 10−7 M: 69±2%, n=15, Fig. 1A) control groups demonstrated similar vascular responses to A23187.

Table 1.

Patient demographics

non-CAD CAD
Sex
 Male (n) 3 11
 Female (n) 22 3
Age (year) (mean ± SEM) 45 ± 3 73± 2
Body Mass Index (mean ± SEM) 30.9 ± 1.4 28.9 ±1.5
History/Risk factors
 Hypertension (n) 2 10
 Hyperlipidemia (n) 1 9
 Smoking (n) 0 1
 Atrial fibrillation (n) 0 0
 Diabetes (n) 0 3
 Tumor (n) 5 4
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Values for age and body mass index (BMI) are shown as mean ± SEM; n indicates the number of patients.

Table 2.

Vascular characteristics

non-CAD CAD
Baseline ID 132.0 ± 7.1 144.6 ± 8.1
Max Diameter 135.4 ± 7.4 144.8 ± 8.1
% Tone 1.7 ± 0.4 0.1 ± 0.1
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Data presented as mean ± SEM; ID indicates the internal diameter; % tone characterizes the developed tone during pressurizing at 60 mmHg preceding preconstriction with endothelin-1.

Figure 1. The role of NO and H2O2 in A23187-induced dilation in human adipose arterioles from non-CAD and CAD patients.

Figure 1.

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A) A23187 dose-dependent responses in untreated non-CAD (n=20) and CAD (n=15) HAAs are not significantly different. B-E) The effect of acute L-NAME exposure (30 min) and catalase on A23187-induced dilation in HAAs from non-CAD and CAD subjects. B) An acute exposure to NO synthase inhibitor L-NAME markedly reduced A23187 dose-dependent responses in non-CAD HAAs as compared to untreated control (n=5, *P<0.05). C) A H2O2 scavenger catalase had no effect on A23187-induced dilation in non-CAD HAAs (n=5). D) An acute exposure to NO synthase inhibitor L-NAME had no effect on A23187 dose-dependent responses in CAD HAAs (n=5). E) A H2O2 scavenger catalase abolished A23187-induced dilation in CAD HAAs (n=5; *P<0.05).

3.2. The role of nitric oxide and hydrogen peroxide in A23187 concentration-dependent dilation of adipose arterioles from non-CAD and CAD subjects.

To address the first objective of our study, we examined the role of NO and H2O2 in A23187-induced dilation before and after acute incubation with L-NAME. In non-CAD arterioles A23187 elicited concentration-dependent dilation (maximal dilation at 10−7 M: 68±5%), and this dilation was largely abolished after acute exposure to L-NAME (maximal dilation at 10−7M: 21±7%, Fig. 1B, n=5, *P<0.05), but was not affected by catalase (maximal dilation at 10−7M: 69±10%, Fig. 1C, n=5). HAAs obtained from CAD patients responded to A23187 in a similar dose-dependent fashion (maximal dilation at 10−7 M: 68±3%), but this dilation was not affected by acute L-NAME exposure (maximal dilation at 10−7 M: 65±3%, Fig. 1D, n=5). Instead, the dilation was reduced by catalase (maximal dilation at 10−7 M: 26±5%, Fig. 1E, n=5, *P<0.05). The vascular responses depicted in Figure 1 support our hypothesis that A23187 concentration-dependent dilation in non-CAD HAAs is primarily NO-dependent, while H2O2 mediates A23187-dependent vasoreactivity in CAD arterioles.

3.3. Vascular responses of non-CAD arterioles after prolonged L-NAME exposure resemble those of CAD.

Prolonged L-NAME incubation for 90 min restored A23187-dependent dilation (maximal dilation at 10−7M: 80±6%, Fig. 2A, n=5), which was subsequently inhibited by hydrogen peroxide scavenger catalase (maximal dilation at 10−7M: 14±4%, Fig. 2B, n=5; *P<0.05). Comparison of Figures 2B and 1E reveals similar vascular responses suggesting a switch from NO to H2O2 dilation after prolonged exposure to L-NAME.

Figure 2. The effect of prolonged L-NAME exposure on mechanisms of A23187-induced dilation in HAAs from non-CAD subjects.

Figure 2.

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A) A prolonged (90 min) exposure to NO synthase inhibitor L-NAME no longer inhibited A23187 dose-dependent responses (n=5). B) In the presence of prolonged L-NAME, H2O2 scavenger catalase abolished A23187-induced dilation (n=5; *P<0.05).

3.4. Acute but not prolonged exposure to L-NAME is reversible.

To test whether acute L-NAME inhibition can be reversed, we ran the first dose-response curve after preincubation with L-NAME for 30 min, then washed arterioles with Krebs and ran the second dose-response 30 min after washout of the inhibitor L-NAME. As shown in Fig. 3A, the wash with Krebs has restored A23187 dose-dependent responses (maximal dilation at 10−7M: 70±4%, n=5; *P<0.05), suggesting that acute eNOS inhibition can be reversed.

Figure 3. The reversibility of acute and prolonged L-NAME treatments on A23187-induced dilation in non-CAD HAAs.

Figure 3.

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The vehicle and L-NAME response curves have been reversed to L-NAME and vehicle (or vehicle + catalase) to determine whether acute/prolonged L-NAME effects are reversible. A) A23187 dose-dependent responses in HAAs were restored 30 min after washing out acute (30 min) exposure of arterioles to NO synthase inhibitor L-NAME (n=5, *P<0.05). B) H2O2 scavenger catalase enhanced A23187-dependent responses 30 min after washing out acute L-NAME, even though dilation was not fully restored (n=5; *P<0.05). C) H2O2 scavenger catalase abolished A23187-induced dilation 30 min after washing out prolonged (90 min) L-NAME, (n=5; *P<0.05).

Then we compared the effects of catalase after washing out acute and prolonged L-NAME exposure. H2O2 scavenger heightened dose-dependent responses to A23187 after washing out acute L-NAME treatment (maximal dilation at 10−7M: 40±14%, Fig. 3B, n=5; *P<0.05), and largely abolished A23187-dependent dilation after washing out prolonged L-NAME exposure (maximal dilation at 10−7M: 7±4%, Fig. 3C, n=5; *P<0.05). These results indicate that the effect of prolonged L-NAME treatment is not readily reversible and therefore H2O2 remains the main mediator of A23187-induced dilation.

3.5. Role of mitochondrial ROS in A23187 dose-dependent responses.

To determine whether mitochondrial ROS are involved in the dilator response to A23187 we used rotenone, an inhibitor of mitochondrial electron transport chain (ETC). Rotenone produced no effect on A23187-dependent responses in non-CAD subjects (maximal dilation at 10−7M: 68±6%, Fig. 4A, n=5) but prevented A23187-induced dilation in CAD individuals (maximal dilation at 10−7M: −6±9%, Fig. 4B, n=5; *P<0.05) suggesting the involvement of mitochondrial-derived hydrogen peroxide in CAD arterioles. After prolonged L-NAME exposure, however, HAAs extracted from non-CAD subjects demonstrated CAD-like responses to rotenone (maximal dilation at 10−7M: 20±3%, Fig. 4C, n=5; *P<0.05). HAAs incubated with rotenone did not constrict well to endothelin-1, which prevented us from running two dose-response curves on the same vessel. Fig. 4C depicts data from experiments where separate vessels were used for individual dose-response curves.

Figure 4. The effect of rotenone on A23187-induced dilation in HAAs from non-CAD subjects in the absence and presence of prolonged L-NAME exposure and from CAD subjects.

Figure 4.

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A) In non-CAD arterioles, the mitochondrial electron transport chain (ETC) inhibitor rotenone had no effect on A23187 dose-dependent responses (n=5). B) In CAD arterioles rotenone completely inhibited A23187 dose-dependent responses (n=5; *P<0.05). C) In the presence of prolonged L-NAME, rotenone largely abolished A23187-induced dilation, suggesting a role of mitochondrial ROS in mediating the vasodilator response (n=5; *P<0.05).

4. Discussion

There are four major findings of this study. First, HAAs from non-CAD patients demonstrate similar A23187-induced dilation compared to CAD patients. Second, in non-CAD subjects, A23187 response is mediated by NO, whereas H2O2 mediates the dilation in CAD subjects, and mitochondria are involved in H2O2 origination. Third, prolonged exposure (90 min incubation) of L-NAME switches the mechanism of the dilation in non-CAD patients from NO to H2O2. Fourth, the effect of prolonged exposure of L-NAME on A23187 response is not readily reversible 30 min after washing out L-NAME, while the influence of acute exposure (30 min incubation) is reversible. Together, these results reveal a novel mechanism of A23187-induced dilation in HAAs from non-CAD and CAD patients and demonstrate a pathway to induce NO-H2O2 switch in non-CAD subjects by prolonged L-NAME exposure. A schematic diagram of the suggested mechanisms is illustrated in the graphic abstract.

4.1. NO-mediated dilation in non-CAD subjects

The endothelium plays a major role in vascular homeostasis by releasing vasoactive substances including NO, PGI2, and H2O2 [1]. The contribution of these factors in vasorelaxation changes with age [2] and in the presence of CAD [3]. Vascular reactivity in human resistance circulation has been extensively studied since the 1990s. Our data coincide with historical data reporting maximal dilation to A23187 in human coronary at 10−7 M: 77±9% [16], and further revealed that NO is the main vasodilator factor involved in A23187-induced dilation in non-CAD arterioles. Mechanistically, endothelium-dependent vasodilating compound A23187 moves calcium across the membrane independent of channel or receptor stimulation [17]. Increases in intracellular calcium results in eNOS activation through the calcium regulatory protein calmodulin (CaM) and phosphorylation [18] leading to nitric oxide release and smooth muscle relaxation. In rat renal hypertensive arteries [7] and streptozotocin-induced diabetic rats [8] lack of NO results in impaired A23187 responses.

4.2. H2O2-mediated dilation in the presence of CAD

Contrary to data obtained on animal models mimicking pathological conditions in humans, we have not observed significant differences in A23187 dose-dependent responses in non-CAD and CAD HAAs (Fig. 1A). Instead, we observed that a non-NO, most likely H2O2 component mediates A23187 responses in the presence of CAD. Our data coincide with historical data from Dr. Gutterman’s group where most human tissue samples used in the studies were collected from individuals with CAD [16]. These previous studies revealed the largely preserved vasodilatory responses to flow and other endothelial agonists such as bradykinin within non-CAD older and CAD adults in adipose [19] and coronary arterioles [2].

It is a well-known fact that reactive oxygen species (ROS) contribute to endothelial dysfunction and a variety of cardiovascular pathologies including CAD [13]. On the other hand, ROS such as H2O2 can act as signaling molecules in endothelial cells [20], although the precise mechanism by which they contribute to vasodilation is not fully elucidated. For example, our recent studies demonstrated that H2O2 released by NOX4 regulates the activity of Ca2+-permeable TRPV4 channel by Ser824 phosphorylation [21] in endothelial cells, and established the contribution of NOX4 to endothelial Ca2+ entry and ACh-induced dilation in arterioles obtained from non-CAD subjects [14]. The role of ROS in stimulation of NO release in endothelial cells has also been extensively studied. H2O2 activation of eNOS with subsequent NO release occurs via receptor-mediated pathway and via non-receptor mediated mechanism. In cultured endothelial cells, angiotensin II-stimulated NO release was abolished by hydrogen peroxide scavenger, PEG-catalase, or the AT1 receptor antagonist, Losartan, while A23187-mediated NO production was not inhibited by catalase [22]. Interestingly, when endothelial cells were pretreated with H2O2, calcium responses to A23187 were enhanced, which was largely inhibited by catalase [23]. In the present study, we mainly used catalase (acting as an extracellular H2O2 scavenger by adding it to the vessel bath) to determine the role of H2O2 as a transferable relaxing factor in A23187-induced dilation. Future studies will be required to examine other possible intracellular roles of ROS in endothelium-mediated responses.

A possible explanation of NO-H2O2 switch in CAD arterioles is given in the study conducted by Marelli, demonstrating the potentiation of EDHF-mediated component of A23187-induced dilation in rat cerebral arteries after ischemia/reperfusion (I/R) injury [24]. In endothelial cells, the Ca2+ threshold for EDHF-mediated dilation is higher compared to NO-mediated component (340 vs 220 nm/L respectively). Thus, under physiological conditions, NO-mediated response precedes EDHF-mediated response. A23187 increased endothelial Ca2+ response after I/R [24]. We suggest that under pathological conditions (such as I/R in Marelli study or CAD in our study) A23187 can somehow more intensively stimulate Ca2+ release from internal stores and influx from extracellular space leading to EDHF threshold overcoming.

4.3. The switch from NO to H2O2-mediated dilation after prolonged L-NAME

The switch from NO to H2O2-dependent flow-induced dilation (FID) during CAD, the involvement of mitochondrial ROS [25] and the phenomena of crosstalk between NOX and mitochondrial ROS in mediating flow responses, termed ROS-induced ROS release (RIRR) have been of interest to our group [19]. Previous studies have proposed several mechanisms that contribute to the switch from NO to H2O2 as the mediator of FID in human arterioles [26]. For example, a study conducted by Freed et al. in 2014 identified a role of ceramide, a bioactive sphingolipid produced by endothelial cells, in eliciting the switch from NO to H2O2-mediated FID [27]. Overnight ceramide exposure, but not acute ceramide exposure, evoked a shift in vasodilator from NO to H2O2 in non-CAD arterioles, which is potentially mediated through the ability of chronically accumulated ceramide to decrease eNOS phosphorylation/activation [28] and thereby attenuate a negative feedback inhibition of H2O2 formation by NO [29]. Recently, another study from Freed’s group suggests that S1P, a metabolite of ceramide is required to maintain NO-mediated dilation in non-CAD patients and H2O2-mediated dilation in CAD subjects [30].

In the present study, acute (30 min) L-NAME incubation of non-CAD HAAs resulted in robust inhibition of A23187 vasorelaxation (Figure 1B), supporting our initial hypothesis of NO-dependent mechanism of A23187 vasoreactivity in healthy arterioles. Prolonged L-NAME treatment restored A23187 dose-dependent responses (Figure 2A) that were inhibited by catalase suggesting the switch from NO- to H2O2-dependent relaxation mechanism (Figure 2B). Furthermore, our data revealed that acute eNOS inhibition is reversible after the wash (Figures 3A, and 3B), while prolonged is not (Figure 3C). Together, this is the first study demonstrating that prolonged (90 min) L-NAME exposure changes responses in non-CAD HAAs (Figure 2) to those observed in HAAs extracted from CAD subjects (Figure 1). Although not a physiological agonist, it is intriguing that A23187-induced responses somewhat mimic FID in terms of the mediators of dilation involved in non-CAD and CAD arterioles. Since prolonged L-NAME incubation in the culture medium has been reported for flow-induced dilation in a previous study [2], we plan to assess in the future whether some common mechanisms, such as ceramide-mediated signaling, are involved in vascular responses of human arterioles to A23187 and flow stimuli.

It has been long recognized that EDHF can serve as a “second line of defense” in preserving the dilation, especially in arterioles, when the NO pathway is compromised. In 1996 Bauersachs et al. revealed that exogenously added NO abolishes EDHF-induced dilation in rabbit carotid and porcine coronary arteries [31]. A subsequent study by Nishikawa et al. demonstrated that dose-dependent arteriolar vasorelaxation to bradykinin is mediated by NO after inhibition of EDHF and prostaglandin synthesis, which is switched to EDHF through a compensatory dilator derived from cytochrome P-450 (likely epoxyeicosatrienoic acid) in the presence of NO and prostaglandin inhibitors [32]. Interestingly, we observed the change of vasodilator factor from NO to H2O2 in human arterioles in response to A23187 only after prolonged L-NAME treatment (90 min) but not after acute L-NAME treatment (30 min), indicating a potentially different signaling mechanism involved in the compensation of impaired NO-mediated dilation by H2O2.

Based on our knowledge, most studies investigating the effect of L-NAME on endothelium-dependent responses to A23187 used the incubation time of 30 min [8] which is consistent with the incubation time used by our group, or did not specifically state how long the incubation time with L-NAME was [33]. According to the study by Hutcheson et al. conducted in rabbit superior mesenteric arteries, L-NAME incubation with or without PGI2 inhibitor indomethacin resulted in a minor inhibition of A23187 dose-dependent relaxation, suggesting the involvement of EDHF in this vascular bed [33]. To explain why earlier studies focused on A23187 dilation did not use ROS inhibitors, such as catalase, we want to acknowledge that H2O2 has been identified as an EDHF only in subsequent years (during 2000s) [34]. Previously Tand et al. demonstrated that endothelial cells of the rat thoracic aorta, obtained from both normotensive and hypertensive rats in the presence of L-NAME respond to A23187 by elevating ROS [35], which coincides with our results.

4.4. The role of mitochondrial ROS in H2O2-mediated dilation

Calcium release often coincides with ROS formation and mitochondria are an important source of free radicals [36 37]. In a study conducted by Przygodzki et al. on cultured cardiomyocytes A23187 exposure resulted in enhanced mitochondrial ROS production. This A23187-induced ROS release was not impacted by L-NAME and not linked to cell death [38]. We tested the role of mitochondria-derived ROS in A23187-induced dilation by using ETC inhibitor rotenone. Our data shown in Figure 4 demonstrates that mitochondrial ROS play a role in A23187 dose-dependent responses in the presence of CAD (Figure 4B) and after prolonged exposure to L-NAME in non-CAD HAAs (Figure 4C). No effect of mitochondrial ROS was observed in non-CAD HAAs not pretreated with L-NAME (Figure 4A). Dikalov et al. suggested that physiological nitric oxide levels may have an antioxidant effect by reducing mitochondrial superoxide and hydrogen peroxide production [39].

The role of mitochondrial ROS in health and disease has been intriguing scientists for decades, and the use of mitochondrial ETC inhibitors is instrumental in identifying the sites and mechanisms of ROS production in both the isolated mitochondrial preparations and in intact cells [40, 41]. As an inhibitor of complex I, rotenone has been shown to inhibit ROS production from complex III, whereas it can either inhibit or stimulate mitochondrial ROS production from complex I depending on specific substrate availability and the respiratory states such as forward- or reverse electron transport. For example, Guaras et al. reported the substrate-sensitivity of mitochondrial electron transport chain inhibition: fatty acid-induced ROS release was inhibited by rotenone, whereas rotenone elicited ROS production in galactose-fed cells [42]. Based on more recent studies of mitochondrial reverse electron transport, Scalio et al. outlined the importance of the site at which ROS is generated in determining the overall effect of ETC inhibitors such as rotenone [41]. Onukwufor et al. further discussed the effects of pharmacological inhibitors on ROS production, stating that the inhibitory effect of rotenone relies on reverse, but not on forward electron transport [43]. This proposal is supported by a recent study conducted by Fukushima et al., which attributed 90% of ROS generated during ischemia reperfusion injury to reverse electron transport triggered at the Q site at the complex I. Interestingly, this influx of ROS was blocked by rotenone [44]. Due to technical difficulty, the exact mechanism of rotenone on mitochondrial ROS production in human arterioles were not explored in the present study. Nevertheless, there are many questions remain unanswered, e.g., whether mitochondrial ROS produced following prolonged L-NAME incubation are derived from reverse or forward electron transport, or from complex I or complex III.

Future studies will explore the interactions between eNOS and mitochondria or other ROS-producing enzymes in endothelial cells by examining NO-mediated protein modifications that accompany the process. For example, we will determine whether the switch from NO to H2O2 observed at the onset of CAD and by prolonged L-NAME incubation in non-CAD arterioles involves activation of other ROS-releasing proteins such as NOXs. Our recent studies demonstrated that H2O2 released by NOX4 regulates the activity of Ca2+-permeable TRPV4 channel by Ser824 phosphorylation [19] indicated colocalization of TRPV4 and NOX4 in endothelial cells, and established contribution of TRPV4 and NOX4 to ACh-induced dilation in arterioles obtained from non-CAD subjects [14]. Our future studies will test the involvement of NOXs in A23187-induced dilation and investigate whether communication between NOX and mitochondria plays a role in mediating A23187 dose-dependent responses in the presence of disease.

5. Study Limitations

This study utilized discarded human surgical samples; therefore, we had no healthy control group as all subjects underwent surgery due to underlying health reasons. To address this limitation, our non-CAD patients include subjects that have ≤ 1 risk factor for CAD. In addition, and in most of our experiments we used each vessel as its own control where two dose-response curves with and without an inhibitor were performed. To comply with IRB requirements, we were collecting only subset of relevant cardiovascular risk factors, which precluded us from obtaining comprehensive information pertaining to subjects’ health history. The sample size used per individual study is small (n=5), however, studies conducted by our group have been successfully utilizing similar sample sizes to compare relationships between the treatment groups for many years [45].

Other potential limitations of this study involve age differences between non-CAD and CAD subjects. In our study [2] assessing age-related differences in flow-induced dilation between older (>55 years old) and younger adults, we found a modest, but significant reduction in FID in older subjects, regardless of disease state. We have not observed significant differences in the magnitude of A23187-induced dilation among CAD and non-CAD groups in our study (Fig. 1A). Our group has been using rotenone to inhibit mitochondrial ROS production in functional vessel studies with consistent results [25, 27, 46, 47]. Therefore, we omitted the use of fluorescent probe MitoPY1 to measure mitochondrial H2O2 as previously demonstrated by our group [19, 25].

6. Perspectives and Significance

The present study provides evidence that different vasodilatory mechanisms (i.e., NO vs H2O2) mediate A23187 dose-dependent responses in non-CAD and CAD arterioles. In the absence of NO, A23187-mediated calcium increase is linked to mitochondrial ROS production, thus preserving vasorelaxation in the presence of CAD. A better understanding of signaling mechanisms involved in the process may help to identify new therapeutic targets to treat CAD or other cardiovascular pathologies.

Another unique aspect of our study is the demonstration of the switch from NO to H2O2-mediated A23187 dose-dependent vasorelaxation following prolonged L-NAME incubation as well as relative irreversibility of prolonged L-NAME incubation. Based on our knowledge, this is the first study to compare the effects of acute (30 min) and prolonged (90 min) L-NAME incubations and report the differences in vasoreactivity. Our findings will help other research groups to improve experimental design and data interpretation and reproducibility.

A limitation of vascular studies is that animal models are often used for understanding human pathologies and, when human samples are being used, the underlying conditions may not be evaluated with great accuracy and thus it is difficult to control for heterogeneity among subjects. From this perspective, our findings not only enhance our understanding of CAD pathogenesis but also present an experimental model permitting conversion from non-CAD to CAD-like mechanism of dilation. This model will allow comparison of data obtained from the same non-CAD discarded tissue, which should reduce heterogeneity and improve the efficiency of the usage of human surgical samples.

Highlights.

  • A23187-induced dilation of human arterioles is similar in non-CAD and CAD patients

  • NO mediates A23187 response in non-CAD, while H2O2 – in CAD

  • Prolonged exposure (90 min incubation) of L-NAME induces NO-H2O2 switch in non-CAD

  • The effect of prolonged L-NAME exposure is not reversible

  • Mitochondria are likely involved in H2O2-mediated pathway of A23187-induced dilation

Acknowledgements

We want to thank David D. Gutterman for helpful comments on this manuscript.

Funding

This work was supported by the National Heart, Lung, and Blood Institute Grant RO1-HL 096647 (to D.X.Z.).

Declaration of interests

David X Zhang reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

HAAs

human adipose arterioles

CAD

coronary artery disease

L-NAME

N(omega)-nitro-l-arginine methyl ester

NO

nitric oxide

PGI2

prostaglandins

H2O2

hydrogen peroxide

EDHF

endothelium-derived hyperpolarizing factor

ACh

acetylcholine

VSMC

vascular smooth muscle cells

ROS

reactive oxygen species

ONOO−

peroxynitrite

eNOS

endothelial NO synthase

O2−

superoxide anion

ET-1

endothelin-1

ETC

electron transport chain

Footnotes

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CRediT authorship contribution statement

Natalya S. Zinkevich: Conceptualization, Methodology, Investigation, Writing - Original Draft. Kostiantyn Drachuk: Formal analysis, Investigation, Writing - Review & Editing, Visualization. David X. Zhang: Conceptualization, Resources, Writing - Review & Editing, Supervision, Project administration, Funding acquisition.

Declaration of Competing Interest

The authors have no conflict of interest to disclose.

Data availability

Data will be made available on request.

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Data Availability Statement

Data will be made available on request.

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