NONO2P, a nitric oxide donor, induces relaxation in coronary artery, negative inotropism and hypotensive effect in rats

Abstract

Reduced NO synthesis and/or bioavailability is related with many cardiovascular diseases, such as coronary artery disease and hypertension. This study aimed to evaluate the effects of cis-[Ru(NO)(NO2)(phen)2](PF6)2-(NONO2P) on blood pressure in normotensive and hypertensive rats. Specifically, we wanted to investigate its action on the atrial contractility, mesenteric and coronary arteries function. Male Wistar and spontaneously hypertensive rats (SHR) (13–18 weeks old) were used to assess the NONO2P effects on blood pressure and heart rate. Superior mesenteric and coronary arteries, and atria were isolated for recording to analyze force changes. Cultured endothelial cells were used to measure intracellular reactive oxygen species (ROS) generation using fluorescent dye (dihydroethidium, DHE). Acute administration of NONO2P induced hypotension in non-anesthetized normotensive and hypertensive rats. Moreover, NONO2P caused a negative inotropic effect without altering cardiac rhythmicity. Further, NONO2P displays a vasorelaxant effect on different blood vessels (mesenteric and coronary arteries). For comparison purposes, we observed that NONO2P and NTG presented with a similar potency and maximum response values in inducing relaxation in coronary arteries. On the other hand, mesenteric arteries were more sensitive to both donors, NONO2P and NTG, than the coronary artery. In addition, exposure to NONO2P induced tolerance and increased ROS levels. This is the first evidence that NONO2P induces hypotension, negative cardiac inotropism and coronary artery relaxation. In addition, pre-exposure to NONO2P induces vascular tolerance. Overall, these results may shed light on the potential therapeutic use of NONO2P, particularly in treating angina and hypertensive crises.

1. Introduction

Nitric oxide (NO) is a simple and highly reactive diatomic molecule that has been demonstrated to participate in various critical biological activities such as vascular smooth muscle (VSM) relaxation and platelet aggregation [1]. The primary signaling mechanism involved in NO-induced vascular relaxation appears to include the activation of soluble guanylate cyclase (sGC), followed by cyclic guanosine monophosphate (cGMP) production and protein kinase G activation. Moreover, direct stimulation of potassium (K⁺) channels can cause vascular relaxation to NO via cGMP-independent activation in VSM [2]. However, reduced NO bioavailability has been reported in cardiovascular diseases such as coronary artery disease and hypertension. As a result, the use of NO donors could be a treatment option for NO deficiency [1].

The sodium nitroprusside salt (SNP) {Na₂[Fe(CN)₅NO]} is a compound that is frequently used as a NO-delivery agent in pharmacological and clinical research. SNP is utilized in clinical practice for a variety of conditions, such as hypertensive crisis, acute decompensated heart failure, and perioperative hypertension related to cardiac surgery [3]. However, SNP is potentially harmful due to side effects related to cyanide (CN⁻) formation during the reduction process, and this is a major obstacle to its therapeutic use [4].

Another limitation of other NO donors is the development of tolerance, a complex multifactorial process involving numerous vascular and extravascular modifications that can impair the NO donor hemodynamic effects and promote endothelial dysfunction. Tolerance to nitrovasodilators often develops between 12 and 48 h after continuous NO donor treatment, and subsequently, higher doses of the NO donor are required to maintain vasodilator effects, limiting their suitability for long-term therapy [5]. Several mechanisms appear to be involved in the development of vascular tolerance to nitroglycerin (NTG), such as (1) reduced NTG biotransformation, (2) enhanced vascular superoxide production, (3) desensitization of the sGC, (4) raised phosphodiesterase activity, and (5) elevated sensitivity to vasoconstrictors [6]. Therefore, a significant objective for scientists in the field related to this subject is to synthesize molecule(s) that can function as NO carriers, releasing NO in a controlled manner over the span of minutes. This controlled release is crucial as it may mitigate reflex side effects on the cardiovascular system often associated with NO donors such as SNP. Moreover, desirable NO donors that do not develop vascular tolerance and do not exhibit cytotoxicity have also been pursued. In this way, ruthenium-coordinated NO donors demonstrated some advantages, including thermal stability, physiological fluid stability, and slow NO-releasing properties [7]. We recently demonstrated that NONO2P, cis-[Ru(NO)(NO₂)(phen)₂](PF₆)₂, exhibits vasorelaxation activity in mesenteric arteries from Wistar rats, and this effect was attributed to the compound’s ability to produce NO [8,9]. Despite being found to be an ex vivo vasorelaxant factor, the chemical NONO2P has never been tested in vivo. This would be important to determine its hypotension potential when compared to SNP. Therefore, in the present study, we evaluated the effects of NONO2P on blood pressure in normotensive and hypertensive rats. We also investigated NONO2P action on the atrial contractility, mesenteric and coronary arteries function, and reactive oxygen species (ROS) production in cultured endothelial cells.

2. Material and methods

2.1. Animals

Ex vivo experiments were performed using tissue from male Wistar rats. In vivo experiments were performed in both Wistar and spontaneously hypertensive rats (SHR) (13–18 weeks-old). The animals were kept in a controlled environment with a constant temperature of 21° C, a 12-hour light–dark cycle (6 am to 6 pm), and unrestricted access to food and water at the Neuroscience Laboratory’s animal facility at the Institute of Health Sciences at the Federal University of Bahia. The experiments were carried out in compliance with the guidelines for the care and use of laboratory rats adopted by the National Council for Animal Experiments Control (CONCEA–Brazil). It was authorized by the Institute of Health Sciences’ Animal Use Ethics Committee at the Federal University of Bahia (CEUA/UFBA n° 4169290420).

2.2. Drugs and solutions

The drugs used in this study included (−)-phenylephrine hydrochloride (PE), acetylcholine chloride (ACh), cremophor, SNP, serotonin (5-HT), dihydroethidium (DHE), and NTG, all purchased from Sigma Chemical Co. (Saint Louis, MO, USA); ketamine and xylazine, obtained from Syntec (Tamboré, SP, Brazil); and heparin, sourced from Blau Farmacêutica (Cotia, SP, Brazil). According to the reaction reported by John B. Godwin and Thomas J. Meyer [10], the NONO2P was produced. For ex vivo assays, the NONO2P solutions were first prepared by dissolving NONO2P in ethanol (Êxodo científica, Sumaré, SP, Brazil), and they were then immediately diluted with distilled water. Tests on control preparations showed no relaxant effect at the final concentration of ethanol in both the organ bath (0.003 % ethanol) and the myograph (0.1 % ethanol). For in vivo assays, NONO2P was dissolved in cremophor (2:1) and NaCl 0.9 % (DIPA, Salvador, BA, Brazil) to reach the appropriate concentrations. Mesenteric arteries experiments were performed in Tyrode’s physiological solution (in mM): NaCl, 158.3; KCl, 4.0; CaCl₂, 2.0; MgCl₂, 1.05; NaH₂PO₄, 0.42; NaHCO₃, 10.0; and glucose, 5.6. Coronary arteries and atrial tissues experiments were performed in Krebs-bicarbonate solution (mM): NaCl, 117.0; KCl, 5.36; CaCl₂, 2.5; KH₂PO₄, 1.03; MgSO₄, 0.57; NaHCO₃, 25.0; and glucose, 11.1. All chemicals used for preparing Tyrode’s physiological solution and Krebs-bicarbonate solution were purchased from Êxodo Científica (Sumaré, SP, Brazil) or Vetec (Duque de Caxias, RJ, Brazil).

2.3. Effects of NONO2P and SNP on blood pressure (BP) and heart rate (HR)

This experiment was performed to investigate the effects of NONO2P on BP and HR in male Wistar (normotensive) and SHR (hypertensive) rats, as previously described by Moraes and colleagues [11]. Further, we also use SNP for comparison purposes. In order to measure the pulsatile arterial pressure (PAP), a polyethylene catheter was inserted into the abdominal aorta through the left femoral artery in anesthetized rats with ketamine/xylazine (75 mg/kg and 20 mg/kg i.p., respectively). For intravenous drug administration, a second catheter was placed into the inferior vena cava through the left femoral vein. Twenty-four hours after this surgical procedure, and complete recovery of animals, experiments were conducted in conscious, free-moving conditions on non-anesthetized rats. For this, cardiovascular parameters were recorded, and after 30 min of a stabilized measurement, SNP (35 μg/kg, i.v.) was administered as a positive control drug [12]. Next, NONO2P in a dose-dependent manner (0.1, 0.5, 1, 5 and 10 mg/kg, randomly) was given. The arterial catheter, connected to a BP transducer (AD Instruments, Colorado Springs, CO, USA), was used to continuously monitor the PAP for approximately 3 h, and the effect of each dose was observed for 10 min. The PAP was used to determine systolic blood pressure (SBP), diastolic blood pressure (DBP) and HR, and calculate the mean arterial pressure (MAP).

2.4. Influence of NONO2P on atrial contraction

The right atrium’s rhythmic and spontaneous contractions were measured, as previously described by Simões and colleagues [13]. For this, Wistar rats were sacrificed in a CO₂ chamber, hearts were rapidly removed, and the isolated left and right atria were kept in an organ bath containing Krebs-bicarbonate, at 37° C and gassed with a carbogenic mixture (95 % O₂ and 5 % CO₂). The resting tension of each atrium was adjusted to 5.0 mN and the tissues were equilibrated for at least 60 min before experiments. A force transducer (FORT-10; WPI, Sarasota, FL, USA) connected to an amplifier-recorder (Miobath-4; WPI, FL, Sarasota, USA) recorded the isometric tension. In order to ensure that all cardiomyocytes were excited during the experiment, the left atrium was electrically stimulated with square voltage pulses at a frequency of 3 Hz for 3 ms to 1.5 times the excitation threshold voltage of each cardiac tissue with an AVS stimulator (AVS Projetos, São Carlos, SP, Brazil). NONO2P was cumulatively added in 4 different concentrations (1 ρM, 0.1 nM, 10 nM, and 1 μM).

2.5. Vascular studies using mesenteric artery

As previously described by Alves and colleagues [14], the superior mesenteric arteries were quickly dissected, cleaned of connective tissue and fat, cut into rings (2 mm), and mounted in organ bath chamber. The chambers were filled with physiological Tyrode’s solution, maintained at 37° C, and continuously aerated with 95 % O₂ and 5 % CO₂. Rings kept stable for 60 min at 7.5 mN of rest tension. The superior mesenteric artery ring’s isometric tension was measured using a force transducer (Insight, Ribeiro Preto, SP, Brazil) coupled to a signal amplifier (Insight, Ribeiro Preto, SP, Brazil) and a computer equipped with data acquisition software. The presence of functional endothelium was determined by the ability of acetylcholine (ACh; 1 μM) to induce at least a 90 % relaxation response in rings of the mesenteric artery that had been contracted by phenylephrine (PE, 1 μM). NONO2P and NTG concentration-effect curves were obtained in endothelium-intact mesenteric rings contracted with PE (1 μM). Different concentrations of NONO2P (0.1 ρM to 3 μM) and NTG (0.1 ρM to 300 μM) were cumulatively introduced to the organ bath during the tonic phase of the contraction, and their results were compared.

2.6. Vascular studies using coronary artery

Intramyocardial first and second order branches of the left anterior descending coronary artery (LAD) were carefully dissected and cleaned from surrounding cardiac tissue, divided into 2 mm-long segments, as previously reported [15]. Following the procedure described by Mulvany and Halpern [16], two steel wires with a diameter of 40 μm were briefly inserted into the segment’s lumen and mounted in a Multi Myograph System (model 620 M; Danish MyoTechnology, Aarhus, Denmark) containing Krebs-bicarbonate. The bathing solution was kept at 37° C while being aerated with 95 % O₂ and 5 % CO₂. Vascular segments were stretched to their optimal lumen diameter to induce active tension. This was calculated based on the internal circumference/wall tension ratio of the segments by setting the interior circumference, L0, to 90 % of the tension that the vessels would experience if they were exposed to an amount of pressure that is equivalent to a transmural pressure of 100 mmHg (L100). After the vessels had stabilized, the contractility of the vessels was assessed by stimulating them with a high-K⁺ solution (80 mM potassium chloride, KCl), and the viability of the endothelium was assessed by examining the ability of ACh (3 μM) to produce relaxation during 5-HT (1 μM)-induced contraction [17,18]. The functional endothelium was considered intact if arterial rings relaxed more than 65 % to ACh. In another group of arteries, human hair was lightly rubbed against the intima of vessels to remove endothelial cells. The functional endothelium was considered eliminated if the coronary rings relaxed less than 10 % to ACh [19]. The vessels were pre-contracted with 5-HT (1 μM) after a 20-minute equilibration period. When a constant tone level was obtained, concentration-effect curves were produced by adding NONO2P cumulatively. The responses to NONO2P were examined in endothelium-intact and –denuded rings to determine the role of the functional endothelium in the relaxation caused by NONO2P. In addition, NTG concentration-effect curves were obtained in endothelium-intact rings precontracted with 5-HT (1 μM) and compared with NONO2P’s responses.

2.7. Investigation of vascular tolerance induced by NONO2P in mesenteric artery

According to the protocol reported by Paulo and colleagues [20], experiments to study the development of tolerance were performed by incubating the tissues with NONO2P (3 nM) for 30 or 60 min. After 80 min of washing, the rings were contracted with PE (1 μM). During the tonic phase of the contraction, a cumulative concentration-effect curve to NONO2P (0.1 ρM to 3 μM) was constructed. The concentration-effect curves to NONO2P were compared with those obtained using preparations that had not previously been exposed to NONO2P.

2.8. Intracellular ROS measurement on cultured endothelial cells

This experiment was carried out to investigate if NONO2P could stimulate ROS production in C57BL/6 mouse intestinal mesenteric endothelial cells (AcceGen Biotech, Fairfield, NJ, USA, Cat.# ABC-TC3197). The intracellular ROS levels were evaluated on mouse endothelial cells (passage 5) using the non-selective ROS dye, DHE. Briefly, 200,000 cells were seeded on a 24-well plate with a complete endothelial cell medium containing a growth factor supplement kit with 5 % fetal bovine serum and 1 % antibiotic–antimycotic solution (Cell Biologics, Chicago, IL, USA, M1168). After 24 h, the cells were confluent and the cell media was removed and replaced by Hank’s 1x balanced salt solution with calcium chloride and magnesium chloride (HBSS, HyClone Cytiva, Logan, UT, USA, #SH30030.02) and incubated with DHE (10 μM; Sigma, Saint Louis, MO, USA, #D7008) for 30 min at 37° C and protected from light, in the absence or presence of the vehicle (ethanol, 5 min) or the NONO2P (3 nM or 1 μM, 5 min or 30 min). The negative control was represented by cells without DHE incubation. Images were acquired on a Zeiss microscope with a 10x objective, using a red filter. The cells were analyzed by ImageJ Software as similar to 40 areas (w = 63 × h = 66, 16 bits) of fluorescent positive cells.

2.9. Data analysis

The data were expressed as means ± standard error of the mean (S.E. M.), and n represents the number of observations or the sample size. GraphPad Prism© software, version 9 (GraphPad Software Inc., La Jolla, California, USA), was utilized for all analyses. The maximal effect (E_max) correlates to the maximum relaxation of the tested drug, while the pharmacological potency, expressed as pEC50, to the negative logarithm of the concentration of a drug that produces 50 % of maximal response (− log EC₅₀). Relaxation was calculated as the percentage of relaxation relative to the maximal contraction induced by PE or 5-HT, which is considered 0 % relaxation. When the artery returns to its baseline tension, it was considered 100 % relaxation. The data were analyzed using nonlinear regression (curve fitting) with a model based on the equation: log(agonist) versus response – variable slope (four parameters) or a biphasic model. The biphasic model is the ideal choice for analyzing mesenteric concentration-effect curves of NONO2P when compared to “log (agonist) versus response – Variable slope (four parameters)” model, as determined by the extra-sum-of-squares F test and AIC. However, both models fit the concentration–response curve of NONO2P. In this way, for calculating the pEC50 values, we opted for the “log (agonist) versus response” model. We made this decision to allow comparison of pEC50 values between NTG and NONO2P. The hypothesis of a difference between the means was tested using the unpaired t-test (to compare two means) or ANOVA followed by Bonferroni’s post-test (to compare three or more means). The significance level (α) used in the hypothesis tests was 0.05, with a type 2 error probability less than 0.05 (p < 0.05).

3. Results

3.1. NONO2P decreased blood pressure in Wistar rats and SHR

The MAP in SHR rats was higher compared to that in Wistar rats (Wistar MAP: 101.89 ± 4.79 mmHg; SHR MAP: 136.63 ± 7.55 mmHg; n = 6, Fig. 1A). NONO2P administration reduced MAP in Wistar at the following doses: 5 and 10 mg/kg (Average decrease in MAP: 22.91 ± 4.73 %; 27.52 ± 5.29 %, respectively, n = 6) (Fig. 1B). However, heart rate was increased at the dose of 10 mg/kg (Average increase in HR:15.41 ± 5.91 %) in Wistar rats. Similar effects were induced by SNP (35 μg/Kg) (Average decrease in MAP: 35.74 ± 2.75 %; Average increase in HR: 24.23 ± 6.54 %, n = 6) (Fig. 1D). In SHR, 5 and 10 mg/kg doses induced antihypertensive effects associated with an increase in heart rate (Average decrease in MAP: 10.20 ± 1.69 %; 14.87 ± 3.89 %; Average increase in HR: 6.98 ± 1.40 %; 8.11 ± 0.51 %, respectively, n = 6). SNP (35 μg/Kg) (Average decrease in MAP: 29.58 ± 4.10 %; Average increase in HR:18.05 ± 5.46 %, n = 6) reduced blood pressure more than NONO2P in SHR rats (Fig. 1C and E). We observed that after the infusion of NONO2P (10 mg/kg), the MAP in SHR (SHR MAP after NONO2P treatment: 114.86 ± 3.73 mmHg; n = 6) returned to levels similar to those observed in Wistar rats (Fig. 1A).

Fig. 1.

Effects of intravenous administration of NONO2P on the MAP and HR in Wistar rats and SHR. Bar graphs showing MAP (mmHg) in Wistar rats, SHR, and SHR after NONO2P treatment (A, n = 6). Bar graphs showing average MAP reduction (mmHg) (%) in Wistar rats (B, n = 6). Bar graphs showing average MAP reduction (mmHg) (%) in SHR (C, n = 6). Average HR increase (%) in Wistar rats (D). Average HR increase (%) in SHR (E). Values are expressed as mean ± S.EM. *p < 0.05; **p < 0.01; ***p < 0.001, NONO2P versus vehicle (negative control); ^#p < 0.05; ^##p < 0.01; ^###p < 0.001 NONO2P versus SNP (positive control). Statistical analysis was performed using one-way ANOVA followed by the Bonferroni post-test.

3.2. Effect of NONO2P on the isolated atria

Based on the right atrial spontaneous beat rate, the effect of NONO2P on chronotropism was examined. The addition of cumulative concentrations of NONO2P (1 ρM, 0.1 nM, 10 nM, and 1 μM) did not result in a significant negative chronotropic effect (100.71 ± 0.71 %, 101.43 ± 0.87 %, 101.43 ± 0.87 %, and 100.71 ± 0.71 %, respectively) compared to control (100 ± 0) (Fig. 2A and C). To analyze the effect of NONO2P on the strength of cardiac contraction (inotropic effect), cumulative concentrations were added to the left atrium preparation. The concentrations of 1 ρM and 0.1 nM did not reduce cardiac force (93.35 ± 5.90 %, 81.47 ± 10.45 %, respectively). However, the high concentrations of NONO2P, 10 nM and 1 μM, demonstrated a negative inotropic effect (70.23 ± 8.61 %, 55.98 ± 9.43 %, respectively) compared to the control (100 ± 0) (Fig. 2B and D).

Fig. 2.

Effect of NONO2P on isolated right and left rat atria. Representative tracings showing the effect of NONO2P on the right atrial rate (A). Representative tracings showing the effect of NONO2P on left atrial contractile force (B). Bar graph showing the chronotropic effect of NONO2P (1 ρM, 0.1 nM, 10 nM, and 1 μM) on isolated right rat atria (C). Bar graph showing the negative inotropic effect of NONO2P on isolated left rat atria (D). Results are expressed as mean ± S.E.M. (n = 5). *p < 0.05; **p < 0.01 NONO2P versus control. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni post-test.

3.3. NONO2P induces endothelium-dependent and –independent relaxation in coronary artery

As shown in Fig. 3, NONO2P (1 nM to 100 μM) induced a concentration-dependent relaxation in endothelium-intact (E_max: 91.51 ± 6.72 %; pEC₅₀: 5.49 ± 0.07, n = 6) or endothelium-denuded (E_max: 100.97 ± 4.21 %; pEC₅₀: 6.16 ± 0.07, n = 5) coronary arteries rings, pre-contracted with 5-HT (1 μM). The relaxation induced by NONO2P was both endothelium-dependent and –independent in rat coronary rings. Arteries without endothelium exhibited a more potent relaxation response to NONO2P compared to arteries with endothelium.

Fig. 3.

NONO2P induces vasorelaxation in coronary artery from Wistar rats. Concentration-effect curve showing the relaxant effect of NONO2P on the isolated coronary artery rings with endothelium-intact and –denuded pre-contracted with 5-HT. Results are expressed as mean ± S.E.M. Statistical analysis was performed using unpaired Student’s t tests; *p < 0.05; **p < 0.01, endothelium-intact (n = 6) versus endothelium-denuded (n = 5) group.

3.4. Comparison of the effects of NTG’s and NONO2P’s on mesenteric and coronary arteries

Our data demonstrated that NONO2P (pEC₅₀: 8.47 ± 0.12, n = 9) was more potent than NTG (pEC₅₀: 7.02 ± 0.10, n = 6) in inducing relaxation in mesenteric arteries, and these NO donors showed distinct profiles of action. Interestingly, NONO2P caused similar E_max to NTG in mesenteric rings with endothelium-intact (E_max NONO2P: 106.91 ± 2.65 %, n = 9; E_max NTG: 102.66 ± 2.29 %, n = 6). However, E_max was achieved by NONO2P at 1 μM, whereas NTG was at 100 μM. When comparing the effect induced by NONO2P and NTG in each individual concentration tested, the relaxation induced by NONO2P was higher at concentrations of 30 ρM, 0.3 nM, 1 nM, and from the concentration of 100 nM to 3 μM compared with NTG (Fig. 4A).

Fig. 4.

Vasorelaxant effect of NONO2P and NTG in superior mesenteric and coronary artery rings from Wistar rats. The concentration–response curve showing the effect of NONO2P or NTG in isolated mesenteric (A) and coronary (B) artery rings in the presence of functional vascular endothelium pre-contracted with PE or 5-HT, respectively. The concentration–response curve shows the effect of NONO2P (C) and NTG (D) in mesenteric and coronary artery in the presence of functional vascular endothelium pre-contracted with PE or 5-HT, respectively. Values are expressed as mean ± S.E.M. Statistical analysis was performed using unpaired Student’s t tests, *p < 0.05; **p < 0.01; ***p < 0.001, NONO2P versus NTG group or NO donor in mesenteric versus NO donor in coronary group. The relaxation curve of NONO2P in mesenteric arteries with functional vascular endothelium has been previously published [8,9].

The vasorelaxation induced by NONO2P in coronary artery rings with endothelium-intact (E_max = 91.51 ± 6.72, pEC₅₀: 5.49 ± 0.07, n = 6) was similar compared with NTG (E_max = 106.99 ± 4.63, pEC₅₀: 5.44 ± 0.09, n = 5). Interestingly, these two donors also share the same potency in coronary arteries. However, the relaxation caused by NONO2P was less sensitive (or smaller) at the 0.3 μM concentration when compared with NTG (Fig. 4B).

Our findings show that both donors, NONO2P and NTG, have a less vasorelaxant effect on the coronary artery when compared to the mesenteric artery (Fig. 4C and 4D). The maximum vasorelaxant effect of NTG was the same between mesenteric (E_max NTG: 102.66 ± 2.29 %, n = 6) and coronary arteries (E_max = 106.99 ± 4.63, n = 5). However, the maximum vasorelaxant effect of NONO2P was lower in the coronary artery (E_max = 91.51 ± 6.72, n = 6) compared to the mesenteric artery (E_max: 106.91 ± 2.65 %, n = 9).

3.5. Vascular tolerance induced by NONO2P in mesenteric artery

A significant rightward shift was evident in a concentration-effect curve to NONO2P after pre-exposure of mesenteric rings with this NO donor at 3 nM for 30 (Fig. 5A) and 60 min (Fig. 5B), reducing the NONO2P-induced relaxation as previously observed (Fig. 4A). However, the tolerance was more evident in a shorter period (30) min when compared to a longer period of pre-incubation with this compound (Fig. 5 A and B).

Fig. 5.

NONO2P induced vascular tolerance. Concentration-effect curve to NONO2P were obtained in endothelium-intact mesenteric rings in the absence (control) or after pre-exposure to NONO2P for 30 min (A) 60 min (B). Values are expressed as mean ± S.E.M. Statistical analysis was performed using unpaired Student’s t tests, *p < 0.05; **p < 0.01; *** p < 0.001, Control versus pre-exposure to NONO2P for 30 (n = 6) or 60 min (n = 6). The relaxation curve of NONO2P in mesenteric arteries with functional vascular endothelium has been previously published [8,9].

3.6. NONO2P induces ROS production in cultured endothelial cells

The levels of intracellular ROS in endothelial cells were measured both in the absence or presence of NONO2P in a time and concentration-dependent manner. The ROS levels were significantly raised in the presence of NONO2P at 3 nM for 5 min (Fluorescence intensity (FI), AU: 13557.75 ± 300.19, n = 4), 1 μM for 5 min (FI, AU:14963.50 ± 176.95, n = 4), 3 nM for 30 min (FI, AU: 14096.75 ± 365.91, n = 4), and 1 μM for 30 min (FI, AU: 13869.75 ± 434.07, n = 4) compared to vehicle levels (FI, AU: 12149.00 ± 338.58, n = 4). The ROS levels were higher in cells treated with higher concentration of NONO2P (1 μM vs. 3 nM for 5 min), when compared to the basal levels (FI, AU: 12323.00 ± 1054.47, n = 3). Interestingly, NONO2P at 1 μM for 30 min did not exacerbate the generation of ROS levels when compared to a 5 min treatment with NONO2P (Fig. 6).

Fig. 6.

NONO2P increases ROS production in endothelial cells. Fluorescent images of concentration of ROS in representative slices of mouse endothelial cells preloaded with ROS-sensitive probe DHE (10 μM) and stimulated with NONO2P (3 nM or 1 μM) at different times (5 or 30 min). Fluorescence of DHE was recorded in the absence or presence of NONO2P (A). Average of DHE fluorescence intensity in mouse endothelial cells loaded with DHE after the addition of NONO2P (3 nM or 1 μM for 5 or 30 min) (B). *p < 0.05; **p < 0.01 NONO2P versus vehicle, ^$$p < 0.01 NONO2P 1 μM versus basal, ^# p < 0.05 NONO2P 3 nM for 5 min versus NONO2P 1 μM for 5 min. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni post-test.

4. Discussion

The main findings of this study were that NONO2P had hypotensive effects in non-anesthetized normotensive and hypertensive rats. Further, this NO donor can promote negative inotropic action in isolated atria and induce a pronounced endothelium-dependent and –independent vasorelaxation in isolated rat coronary artery rings. We also observed that exposure to NONO2P seems to induce vascular tolerance in mesenteric rings, and stimulate ROS production in cultured endothelial cells, which was not enough to counterbalance the relaxation induced by this compound.

Ruthenium-derived NO donors may provide a promising method for treating hypertension in situations of emergency compared to SNP, which can be toxic due to cyanide production [21]. Some investigations have been conducted to assess the hypotensive effect of nitrosyl ruthenium complexes in conscious animals. For instance, Gaitani and colleagues reported that the hypotensive effect caused by trans-[RuCl([15] aneN₄)NO]²⁺ was considerably greater in severe hypertensive 2 K-1C rats than in moderate hypertensive 2 K-1C rats [22]. Similarly, the hypotensive effect to trans-[Ru(NO)(NH₃)₄(POEt)₃](PF₆)₃ was more pronounced in both secondary (angiotensin II- and phenylephrine-induced hypertension) and primary (SHR) hypertension when compared to normotensive rats [23]. It has been shown that Ru(terpy)(bdq)NO⁺]³⁺ (TERPY), another NO donor, is less potent when compared to SNP to cause a hypotensive response. However, the hypotensive effect induced by TERPY was more pronounced in SHR than in Wistar rats [12]. In the present study, the effects of NONO2P on cardiovascular parameters in Wistar rats and SHR in non-anesthetized conditions were studied in vivo for the first time. We found that intravenous administration of NONO2P caused a hypotensive response in both Wistar rats and SHR, accompanied by tachycardia as a probable reflex response, similar to SNP-induced effects, as observed in BP recording (unpublished data). However, the hypotensive effect of NONO2P was reduced in hypertensive rats, when compared to the effect produced in normotensive rats. Interestingly, we observed that after the infusion of NONO2P (10 mg/kg), the MAP in SHR returned to levels similar to those observed in Wistar rats. Before the infusion, SHR exhibited significantly higher MAP compared to Wistar rats, but following NONO2P administration, MAP normalized to values comparable to those in normotensive control mice. It is important to emphasize that NONO2P does not release cyanide, unlike SNP. Therefore, this compound could also be used in clinical applications, as a pharmacological tool, without producing harmful metabolites. In addition, NONO2P may offer a safer alternative for clinical applications, particularly in conditions where NO-mediated vasodilation is desirable. Few investigations have shown that exogenous NO effects present with a biphasic [24,25] or do not affect cardiac contractility [26]. For instance, in the administration of S-nitroso-N-acetyl-dl-penicillamine (SNAP), another NO donor, depending on the concentration utilized, either raises (low NO concentrations) or lowers (high NO concentrations) the index of cardiac contractility [25,27]. Moreover, SNP exhibited a negative inotropic effect in isolated papillary muscles and enhanced the economy of isometrically contracting papillary muscles from rabbits, which may contribute to the improvement reported in nitrate-treated patients with coronary heart disease [28]. Based on these premises, we also investigated whether NONO2P plays a role on the cardiac function. Indeed, we observed that NONO2P caused a negative inotropic effect without altering cardiac rhythmicity. Unfortunately, the exact mechanism(s) by which NONO2P induced these responses are unclear. Overall, based on our findings in vascular and cardiac function, we suggest that the hypotensive effect induced by NONO2P is a result of a sum of a decrease in peripheral vascular resistance and cardiac contractility.

Although most of NO donors release NO or another molecule that will be converted to NO, it is well known that distinct vascular beds (e.g., large arteries vs. resistance arteries) may respond differently to vasodilators. Additionally, NO donors may trigger distinct pathways in different vascular beds. In this way, investigating the mechanisms associated with the relaxation induced by NO donors in distinct vascular beds is necessary [29]. For instance, it has been demonstrated that the effect of SNP can differ among rabbit femoral, mesenteric, and aorta arteries [30,31]. In the same way, cis-[Ru(bpy)₂(py)NO₂](PF₆) (RuBPY)-induced relaxation exhibited different potencies and maximum effects in distinct vascular beds, including the aorta, mesenteric, coronary, and basilar arteries [29]. Furthermore, it has been shown that the sGC enzyme appears to play a major role in TERPY-induced relaxation in mesenteric resistance arteries than the aorta [2,32]. In the present study, NONO2P-induced relaxation produced distinct relaxation responses in the superior mesenteric and coronary arteries, suggesting tissue-specific responses. NONO2P generated monophasic relaxation in the coronary artery, in contrast to the biphasic concentration-effect curve induced by NONO2P in the superior mesenteric artery. The contrast between these two types of blood vessels highlights the heterogeneity of the vascular system. Blood vessels in different parts of the body may exhibit diverse responses to vasodilatory agents, like NO donors. These variations could be due to differences in the size of the vessels (large vs. resistance arteries), where large arteries use NO as the main mechanism for vasodilation whereas resistance arteries present with other mechanisms such as endothelium-dependent hyperpolarization (EDH), as an alternative pathway to relax the arteries [33]. Further, the expression of various receptors, enzymes, or signaling pathways is different among vascular beds.

Regarding the relaxation induced by NONO2P in coronary arteries, it is important to highlight that this donor could potentially alleviate the symptoms of angina. NONO2P promoted vasorelaxation in a concentration-dependent manner in both endothelium-intact and –denuded coronary artery, indicating that the vasorelaxant effect caused by NONO2P is most likely by direct action of this donor on vascular smooth muscle. Nonetheless, the presence of endothelium impaired NONO2P-induced relaxation in endothelium-intact coronary rings compared to the endothelium-denuded rings. Our results are consistent with previous findings involving other NO donor compounds, such as TERPY [34,35]. Accordingly, endothelium negatively modulated the vasorelaxation caused by TERPY in aorta [34] and mesenteric resistance arteries [35] from Wistar rats. The authors suggested that TERPY’s metabolite(s) can induce endothelium nitric oxide synthase (eNOS) uncoupling, elevating superoxide production in the tissue. Hydrogen peroxide or peroxynitrite, which are both quickly produced from superoxide, can activate the cyclooxygenase (COX) enzyme to produce thromboxane A2 and decreasing TERPY-induced relaxation [34].

In mesenteric preparations where the contractile factor used was phenylephrine, NONO2P exhibited a similar maximum response when compared to NTG. However, NONO2P appears to be more potent than NTG to produce vasorelaxation in superior mesenteric artery with functional endothelium, while in the coronary artery, NONO2P and NTG share the same potency and maximum response. Our data indicates that both donors, NONO2P and NTG, have a lower vasorelaxant effect on the coronary artery than the mesenteric artery.

Although important as a pharmacological tool, it is well known that available NO donors still present with some limitations, including the development of vascular tolerance [6,20,36,37]. In this way, we investigated the possibility of NONO2P to induce vascular tolerance. Indeed, the concentration-relaxation curve for NONO2P was shifted to the right in isolated mesenteric arteries that were pre-exposed to NONO2P at 3 nM. These results suggest NONO2P induces tolerance in ex vivo tissue. This tolerance may be a result of cellular adaptation to the continuous presence of NO, a phenomenon commonly observed in prolonged exposure to NO-releasing compounds. Further research is needed to elucidate the underlying mechanisms of this tolerance phenomenon induced by NONO2P, which may involve alterations in endothelial or VSM cells signaling pathways. Investigating the temporal aspects of NO exposure and its effects on vascular function can provide valuable insights into optimizing the use of NO donors in clinical settings and developing more effective treatments for cardiovascular diseases. Further, specific experiments are also required to compare the development of tolerance between different NO donors, including NTG and isosorbide mononitrate, and the potential advantages or limitations of NONO2P when compared to these donors. The concept of oxidative stress and vascular tolerance are closely related [6]. Paulo and colleagues indicated that ROS generation was increased in human umbilical vein endothelial cells (HUVECs) after stimulation with NTG [20]. Here, we also measured the level of ROS in cultured endothelial cells treated with NONO2P in a time and concentration-dependent manner. Accordingly, NONO2P enhanced the levels of ROS in endothelial cells. The exposure to NONO2P at 1 μM for 5 min resulted in higher ROS levels when compared to NONO2P at 3 nM for the same duration. Given that, it has been shown that antioxidants maintain vascular sensitivity [20] and enhanced hypotensive effect [38] to NO donors, it would be interesting to investigate whether the combination of NONO2P and antioxidants could prevent the development of vascular tolerance. Further, the use of antioxidants and NONO2P could improve the hypotensive effect induced by this donor, especially in SHR animals where oxidative stress is present in the cardiovascular system [39].

4.1. Conclusion

In conclusion, we have shown that the NONO2P administration caused hypotension, lowered atrium contractility and induced mesenteric and coronary artery relaxation. These results may shed light on the potential therapeutic use of NONO2P, particularly in treating cardiovascular diseases such as angina and hypertensive crises. On the other hand, this compound still has some limitations including the development of vascular tolerance. Therefore, further studies are required to investigate if NONO2P can be advantageous over other NO donors such as isosorbide mononitrate and SNP.

Abbreviations:

Ach

acetylcholine chloride

BP

blood pressure

CEUA

animal use ethics Committee

cGMP

cyclic guanosine monophosphate

CN⁻

cyanide

COX

cyclooxygenase

CONCEA

National Council for Animal Experiments Control

DBP

diastolic blood pressure

DHE

dihydroethidium

EC₅₀

concentration of a drug that produces 50% of maximal response

EDH

endothelium-dependent hyperpolarization

Emax

maximal effect

eNOS

endothelium nitric oxide synthase

FI

fluorescence intensity

HR

heart rate

HUVECs

human umbilical vein endothelial cells

K⁺

potassium

LAD

left anterior descending coronary artery

MAP

mean arterial pressure

NO

nitric oxide

NONO2P

cis-[Ru(NO)(NO₂)(phen)₂](PF₆)₂

NTG

nitroglycerin

PAP

pulsatile arterial pressure

PE

(−)-phenylephrine hydrochloride

pEC50

negative logarithm of the EC50

ROS

reactive oxygen species

RuBPY

cis-[Ru(bpy)₂(py)NO₂](PF₆)

SNAP

S-nitroso-N-acetyl-dl-penicillamine

SBP

systolic blood pressure

SEM

standard error of the mean

sGC

soluble guanylate cyclase

SHR

spontaneously hypertensive rats

SNP

sodium nitroprusside salt

TERPY

Ru(terpy)(bdq)NO⁺]³⁺

VSM

vascular smooth muscle

5-HT

serotonin

Data availability

Data will be made available on request.