Insulin augments vasodilatory response elicited by amlodipine via nitric oxide-dependent vasodilation in isolated rat aortas

Abstract

Background

High-dose insulin and euglycemic therapy are widely used to treat calcium channel blocker toxicity. However, the effect of insulin on vasodilation evoked by the dihydropyridine calcium channel blocker amlodipine remains unknown. This study examined the effect of insulin on amlodipine-induced vasodilation in isolated rat aortas with specific emphasis on mechanisms associated with nitric oxide (NO).

Methods

The study assessed the roles of N^W-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor; methylene blue, a general guanylate cyclase suppressor; 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of NO-sensitive guanylate cyclase; and endothelial removal in modulating the NO-dependent signaling cascade underlying amlodipine-induced vasodilation. This study explored how insulin and various pharmacological inhibitors influenced the vasodilatory effects of amlodipine and verapamil in rat aortic tissues with or without an intact endothelium.

Results

In aortas with intact endothelium, amlodipine-induced relaxation was significantly suppressed by L-NAME, methylene blue, and ODQ. Insulin enhanced amlodipine-induced vasodilation in endothelium-intact aortas, whereas it had no effect on the vasodilatory response to amlodipine in endothelium-denuded aortas. Moreover, L-NAME, methylene blue, and ODQ eliminated insulin-mediated augmentation of amlodipine-induced vasodilation in endothelium-intact aortas. However, in endothelium-intact aortas, insulin exhibited no impact on the vasodilatory effects triggered by verapamil. Amlodipine increased endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein endothelial cells (HUVECs). Additionally, combined treatment with insulin and amlodipine further increased amlodipine-induced eNOS phosphorylation in HUVECs.

Conclusions

These findings suggest that insulin contributes to the amplification of amlodipine’s NO-dependent vasodilatory response in aortas, which appears to be mediated by increased NO production.

Introduction

Calcium channel blockers are frequently used to treat hypertension, specific arrhythmias, and coronary artery disease [1]. When used in therapeutic doses, dihydropyridine calcium channel blockers, such as amlodipine, primarily target L-type calcium channels in the smooth muscle cells of blood vessels, resulting in vasodilation, which helps in treating high blood pressure [1]. In contrast, non-dihydropyridine calcium channel antagonists, such as verapamil and diltiazem, predominantly affect L-type calcium channels in heart muscle cells, rendering them effective in treating cardiac arrhythmias and angina [1]. However, toxic doses of amlodipine or verapamil affect L-type calcium channels in both vascular smooth and cardiac muscle cells, causing severe low blood pressure and bradycardia [1]. The treatment of calcium channel blocker toxicity involves the use of vasopressors, intravenous calcium, high-dose insulin combined with euglycemic therapy (HIET), and lipid emulsions [1,2]. The mechanism of HIET in the treatment of calcium channel blocker toxicity involves a positive inotropic effect, delivering adenosine triphosphate to the heart muscle cells, and addressing hyperglycemia [3–5].

Additionally, insulin reduces afterload via endothelial nitric oxide (NO)-dependent vasodilation caused by increased NO production, contributing to HIET-mediated recovery from calcium channel blocker toxicity [6–8]. Amlodipine produces NO in coronary microvessels, whereas diltiazem (a non-dihydropyridine calcium channel blocker) does not significantly enhance NO production [9]. Amlodipine and the R-isomer of amlodipine produce NO, whereas the S-isomer does not affect NO production [10]. Moreover, intra-arterial amlodipine reduces perfusion pressure in the femoral artery in vivo [11]. The nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester attenuates amlodipine-induced perfusion pressure reduction [11]. Thus, the racemic antihypertensive drug amlodipine induces vasodilation primarily through the blockade of L-type calcium channels by its S-isomer and, to some extent, through the release of endothelial NO by its R-isomer [9–12]. A recent retrospective clinical study demonstrated that HIET for treating dihydropyridine calcium channel blocker (amlodipine) toxicity requires higher doses of vasopressors used for the treatment of refractory vasoplegia, such as epinephrine (median maximum dose: amlodipine group, 0.31 μg/kg/min vs. non-dihydropyridine group, 0.09 μg/kg/min) and increased use of non-specific guanylate cyclase (GC) inhibitors, such as methylene blue (used in 7 of 18 cases in the amlodipine group vs. 0 of 15 in the non-dihydropyridine group), than that required for treating toxicity caused by non-dihydropyridine calcium channel blockers (verapamil or diltiazem) [13]. Considering previous reports [6–12], this retrospective study suggests the novel hypothesis that a synergistic interaction between amlodipine and insulin may enhance NO-mediated vasodilation through augmented NO production [13]. Hence, the increased requirement for vasopressors observed during HIET in amlodipine toxicity may be attributed to severely reduced afterload caused by augmented NO release induced by insulin and amlodipine [6–13].

However, despite previous reports suggesting a possible synergistic interaction between amlodipine and insulin in terms of NO release, the effect of their interaction on vasodilation in isolated vessels has not yet been investigated [6–13]. Moreover, the underlying mechanism associated with the increased vasopressor requirement in amlodipine toxicity following HIET, compared with non-dihydropyridine calcium channel blocker toxicity, remains unknown. Thus, to examine the possible mechanism underlying the high-dose vasopressor requirement during HIET for amlodipine toxicity, this in vitro investigation aimed to explore the influence of insulin on the vasodilatory response induced by amlodipine in isolated rat aortas, with specific emphasis on elucidating the mechanisms involving endothelial NO. We hypothesize that insulin enhances the NO-mediated vasodilatory response to amlodipine in endothelium-intact vessels, potentially explaining the increased vasopressor requirement during clinical HIET therapy.

Materials and Methods

The experimental procedures received official approval from the Institutional Animal Care and Use Committee at Gyeongsang National University (GNU-230102-R0002; 2, January 2023). All animal handling and research activities adhered strictly to the ethical standards outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This is not a blinded study.

Isolation of rat aortic tissue and measurement of isometric tension

Adult male Sprague–Dawley rats, sourced from Koatech (Pyeongtaek, Gyeonggi-do, Republic of Korea) and weighing 240–280 g, were euthanized by introducing 100% carbon dioxide gas through a narrow inlet into their enclosure, inducing anesthesia. Rat aortas were isolated, and their isometric tension was measured following established methods [14]. The thoracic cavity was accessed, allowing the removal of the descending thoracic aorta, which was subsequently immersed in Krebs solution containing sodium bicarbonate (25 mM), glucose (11 mM), sodium chloride (118 mM), calcium chloride (2.4 mM), potassium chloride (4.7 mM), monopotassium phosphate (1.2 mM), and magnesium sulfate (1.2 mM). Using microscopic guidance, adjacent adipose and connective tissues were meticulously dissected away. The thoracic aortas were then cut into uniform rings approximately 2.5 mm in length. In selected preparations, the endothelial lining was carefully removed by inserting two 25-gauge needles into the vessel lumen and gently rolling them to mechanically denude the inner surface. The prepared aortic segments were affixed to a Grass FT-03 isometric force transducer (Grass Instrument) and immersed in an organ chamber kept at a stable temperature of 37°C. As described by Klöss et al. [15], to attain equilibrium, a baseline resting tension of 24.5 mN was maintained for 90 min, with the Krebs solution replaced every 30 min. The Krebs solution, aerated with 95% oxygen and 5% carbon dioxide, ensured a pH of 7.4. The endothelial integrity of the aorta was evaluated by inducing sustained contractions with phenylephrine (10⁻⁷ M), followed by the introduction of acetylcholine (10⁻⁵ M). An aortic segment was considered to have an intact endothelium if it exhibited more than 80% relaxation in response to acetylcholine. Successful removal of the endothelial layer was verified when acetylcholine (10⁻⁵ M) elicited less than 15% relaxation in vessels precontracted with phenylephrine (10⁻⁸ M).

Experimental protocols

First, we examined the effect of endothelial denudation and various inhibitors that block the endothelial NO-mediated vasodilation pathway on amlodipine-induced vasodilation in isolated rat aortas (Table 1). After phenylephrine (10⁻⁶ M) induced a prolonged and stable contraction of the aorta, regardless of endothelium presence, amlodipine (3 × 10⁻⁷ M), which slightly exceeds the toxic plasma concentration (approximately 1.55 × 10⁻⁷ M compared with the therapeutic plasma amlodipine concentration of 0.8 × 10⁻⁸ to 0.2 × 10⁻⁷ M) [16], was added to the organ bath to induce vasodilation. Subsequently, vasodilatory effects were monitored over a 1 h period following the administration of amlodipine. To investigate the underlying mechanisms, endothelium-intact aortic rings were incubated for 20 min with various inhibitors (Table 1): N^W-nitro-L-arginine methyl ester (L-NAME, 10⁻⁴ M), a NOS blocker; methylene blue (10⁻⁶ M), a non-selective GC inhibitor; and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10⁻⁵ M), a selective inhibitor of NO-sensitive GC [14]. After establishing a sustained contraction in the aortic tissue using phenylephrine (10⁻⁶ M) independent of the presence of these inhibitors, amlodipine (3 × 10⁻⁷ M) was added to the bath, and its vasorelaxant effects were continuously recorded for 1 h.

Table 1.

Summary of Experimental Protocols for Isometric Tension Measurement

Experimental protocols	Drug incubation time	Pre-contraction for vasodilation study	Response
Effect of endothelial denudation on the amlodipine-induced vasodilation		PE (10−6 M)	Vasodilation monitoring for 60 min induced by the addition of amlodipine (3 × 10−7 M) after plateau of PE-induced contraction in endothelium-intact or -denuded aorta.
Effect of various inhibitors on the amlodipine-induced vasodilation in endothelium-intact aorta	Pretreatment with L-NAME (10−4 M), MB (10−6 M), and ODQ (10−5 M) for 20 min	PE (10−6 M)	Vasodilation monitoring for 60 min induced by the addition of amlodipine (3 × 10−7 M) after plateau of PE-induced contraction in the presence or absence of various inhibitors.
Effect of insulin on the amlodipine-induced vasodilation in endothelium-intact or -denuded aorta		PE (10−6 M)	Vasodilation monitoring for 60 min induced by the addition of amlodipine (3 × 10−7 M) after plateau of PE-induced contraction. Insulin (10−7 or 5 × 10−7 M) was added into some organ bath 10 min after amlodipine-induced vasodilation.
Effect of insulin on the amlodipine-induced vasodilation in endothelium-intact aorta pretreated with various inhibitors	Pretreatment with L-NAME (10−4 M), MB (10−6 M), and ODQ (10−5 M) for 20 min	PE (10−6 M)	Vasodilation monitoring for 60 min induced by the addition of amlodipine (3 × 10−7 M) after plateau of PE-induced contraction in the presence of inhibitor. Insulin (10−7 M) was added into some organ bath 10 min after amlodipine-induced vasodilation.
Effect of insulin on the SNP-induced vasodilation in endothelium-denuded aorta	Pretreatment with insulin (10−7 or 5 × 10−7 M) for 20 min	PE (10−6 M)	SNP dose (10-10 to 10−6 M)-response curve produced by cumulative addition of SNP after plateau of PE-induced contraction in presence or absence of insulin.
Effect of insulin on the verapamil-induced vasodilation in endothelium-intact aorta		PE (10−6 M)	Vasodilation monitoring for 60 min induced by the addition of verapamil (10−7 M) after plateau of PE-induced contraction. Insulin (10−7 or 5 × 10−7 M) was added into some organ bath 10 min after verapamil-induced vasodilation.
Effect of amlodipine or amlodipine plus insulin on the PE-induced contraction in endothelium-intact aorta	Amlodipine (3 × 10−7 M) for 40 min or amlodipine (3 × 10−7 M) for 20 min followed by insulin (10−7 or 5 × 10−7 M) for 20 min		PE dose (10−9 to 10−5 M)-response curve induced by cumulative addition of PE in the presence or absence of amlodipine alone or amlodipine plus insulin.

PE: phenylephrine, L-NAME: N^W-nitro-L-arginine methyl ester, MB: methylene blue, ODQ: 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, SNP: sodium nitroprusside.

Second, we investigated the influence of insulin on the vasodilatory response induced by amlodipine in both endothelium-intact and endothelium-denuded aortas (Table 1). After consistent and stable contractions elicited by phenylephrine (10⁻⁶ M) were observed in both endothelium-intact and endothelium-denuded aortas, amlodipine (3 × 10⁻⁷ M) was added to the organ bath to induce vasodilation. Insulin (10⁻⁷ or 5 × 10⁻⁷ M), which falls within the range of serum insulin concentrations (approximately 10⁻⁴ to 10⁻⁷ M) observed during high-dose insulin therapy for amlodipine toxicity [17], was added to the organ bath 10 min after amlodipine administration. Vasodilation was monitored for 1 h after amlodipine administration.

Third, to explore the contribution of NO to the insulin-enhanced vasodilatory response elicited by amlodipine in aortic segments with an intact endothelium, tissues were pre-incubated for 20 min with different pharmacological inhibitors (Table 1): L-NAME (10⁻⁴ M), a NOS inhibitor; methylene blue (10⁻⁶ M), a broad-spectrum GC blocker; and ODQ (10⁻⁵ M), a selective inhibitor of NO-sensitive GC. After phenylephrine (10⁻⁶ M) induced a consistent and sustained contraction in the aorta with intact endothelium in the presence of the inhibitors, amlodipine (3 × 10⁻⁷ M) was added to the organ bath. Insulin (10⁻⁷ M) was added to the organ bath 10 min after amlodipine administration to examine its effect on amlodipine-induced vasodilation in endothelium-intact aortas pretreated with the various inhibitors. Vasodilation was assessed over a 1 h period after amlodipine administration. In addition, the effect of insulin on the vasodilatory response elicited by the NO donor sodium nitroprusside in endothelium-denuded aortas was investigated (Table 1). Following a 20 min pre-incubation of endothelium-denuded aortic rings with insulin (10⁻⁷ or 5 × 10⁻⁷ M), phenylephrine (10⁻⁶ M) was applied, resulting in a sustained contractile response regardless of insulin presence. Sodium nitroprusside (10⁻¹⁰–10⁻⁶ M) was then added to the organ bath to induce vasodilation.

Fourth, the influence of insulin on vasodilation triggered by verapamil was investigated using isolated rat aortic segments with intact endothelium (Table 1). Once phenylephrine (10⁻⁶ M) elicited a consistent and prolonged contraction in these vessels, verapamil (10⁻⁷ M) was added to the organ chamber to induce and assess the resulting vasorelaxation. Subsequently, insulin (10⁻⁷ and 5 × 10⁻⁷ M) was added to the organ bath 10 min after verapamil administration to examine verapamil-induced vasodilation in endothelium-intact aortas in the presence or absence of insulin. Vasodilation was assessed for 1 h after verapamil administration.

Fifth, the effect of amlodipine alone or in combination with insulin on phenylephrine-induced contraction was examined in endothelium-intact aortas (Table 1). This experiment aimed to confirm, from the opposite perspective, whether insulin enhances amlodipine-induced vasodilation. Aortic rings were exposed to amlodipine (3 × 10⁻⁷ M) either as a sole treatment for a continuous 40 min period or were first incubated with amlodipine for 20 min, followed by insulin (10⁻⁷ or 5 × 10⁻⁷ M) for an additional 20 min. Phenylephrine (10⁻⁹ to 10⁻⁵ M) was then cumulatively added to the organ bath.

Cell culture

Human umbilical vein endothelial cells (HUVECs; C-0003-5C, American Type Culture Collection) were maintained in endothelial cell medium (ECM; ScienCell) supplemented with 15% fetal bovine serum, 1% endothelial cell growth supplement, 100 U/mL penicillin, and 100 μg/mL streptomycin, all from ScienCell, according to previously described methods [14]. Cells in passages 3–5 were subjected to a 4-h serum deprivation period in ECM before drug pretreatment.

Western blot

The phosphorylation of endothelial NOS (eNOS) at Ser1177 in HUVECs was measured through western blotting, following the protocol outlined in previous studies [14]. HUVECs were exposed to amlodipine (3 × 10⁻⁷ M) for 10 min, a combination of insulin (5 × 10⁻⁷ M) and amlodipine (3 × 10⁻⁷ M) for 10 min, or insulin (5 × 10⁻⁷ M) alone for 10 min to assess the phosphorylation status of eNOS. The cells were collected in radioimmunoprecipitation assay buffer supplemented with phosphatase and protease inhibitor cocktails. The protein content of the supernatant was assessed following centrifugation at 20 000 × g for 15 min at 4°C, employing the bicinchoninic acid protein assay kit (Thermo Fisher Scientific). A total of 20 µg of protein from each sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.5% Tween-20 (TBST) for 1 h at room temperature (25°C), followed by overnight incubation at 4°C with primary antibodies: anti-phospho-eNOS (Ser1177, 1:1,000), anti-eNOS (1:1,000), and anti-β-actin (1:10 000). After three washes of 10 min each with TBST, membranes were treated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:5,000 dilution) for 1 h at room temperature. Protein bands were visualized using the WesternBright^TM ECL detection kit (Advansta, Menlo, CA, USA), and images were captured using the ChemiDoc^TM Touch Imaging System (Bio-Rad Laboratories, Inc.). Relative protein levels were quantified using ImageJ software (version 1.45; National Institutes of Health). The relative density of eNOS phosphorylation was normalized against the total protein levels, utilizing β-actin as the loading control.

Materials

All reagents employed in this study were of the highest available purity. Amlodipine, verapamil, insulin, L-NAME, methylene blue, ODQ, sodium nitroprusside, and phenylephrine were obtained from Sigma-Aldrich (St. Louis). Anti-phospho-eNOS and anti-eNOS antibodies were acquired from Cell Signaling Technology and BD Biosciences, respectively. Amlodipine and ODQ were dissolved in dimethyl sulfoxide (DMSO) in the organ bath to a final concentration of 0.1%. All remaining chemicals were dissolved in distilled water unless stated otherwise.

Statistical analysis

The main objective of this study was to assess how endothelial denudation, pharmacologic inhibitors, and insulin modulate vasodilatory responses to amlodipine. The Kolmogorov-Smirnov test was employed to evaluate the normal distribution of the data. Data on vasodilation induced by amlodipine, verapamil or sodium nitroprusside and phenylephrine-induced contraction were expressed as a percentage of the contraction induced by phenylephrine (10⁻⁶ M) and 60 mM KCl, respectively, to account for variations in aortic size, such as length and thickness, which can affect absolute contractile responses. This normalization facilitated comparison of vasodilatory and vasoconstrictive responses across experimental groups. The magnitude of maximal vasodilation induced by amlodipine or verapamil, and maximal vasoconstriction induced by phenylephrine, was quantified as a percentage of vasodilation or vasoconstriction relative to the contraction induced by phenylephrine (10⁻⁶ M) and 60 mM KCl, respectively. Within each treatment group, maximal vasodilation induced by amlodipine or verapamil, or maximal contraction induced by phenylephrine, was summarized as the mean ± standard deviation (SD) from the raw data. The longitudinal changes in vasodilation induced by amlodipine, and verapamil, according to treatment and time, were visualized as estimated marginal means with SD. The longitudinal changes in vasodilation induced by sodium nitroprusside, or in vasoconstriction induced by phenylephrine, according to treatment and concentration were presented as estimated marginal means with SD.

To evaluate between-treatment differences at each time point and the temporal trends in vascular responses across treatment conditions, we performed linear mixed-effects modeling (LMM) with a treatment × time or treatment × concentration interaction term specified as the primary fixed effect, testing whether the rate of change (slope) in vascular response differed significantly among treatment groups [18]. The model included random effects for slope only, to account for inter-individual variation in the rate of response change over time, with individual aortic ring preparations as the grouping variable (random intercepts were not included). Fixed effects included time (or concentration as a continuous variable), treatment group (categorical), and their interaction (treatment × time or treatment × concentration). P values for fixed effects in the LMM were calculated using Wald tests applied to restricted maximum likelihood, assuming large-sample asymptotics. Post-hoc pairwise comparisons of estimated marginal means were conducted using Bonferroni correction to control for Type I error inflation. Degrees of freedom were calculated assuming large-sample asymptotics.

Sample sizes were determined using the resource equation approach, as outlined in reference [19].

Maximal vasodilation induced by amlodipine or verapamil, and phenylephrine-induced maximal contraction, were analyzed using unpaired Student’s t-test or one-way analysis of variance (ANOVA), followed by Bonferroni’s multiple comparison test. Additionally, the effects of amlodipine and insulin, administered alone or in combination, on eNOS phosphorylation were evaluated using one-way ANOVA, followed by Bonferroni-adjusted post-hoc comparisons. P values were derived from F-tests or t-tests, and a two-sided P < 0.05 was considered statistically significant. All analyses were performed using Stata version 14.2 (Stata Corp LLC) or Prism 5.0 (GraphPad Software Inc.).

Results

Influence of endothelial removal, distinct pharmacological inhibitors, and insulin—individually or in combination—on vasodilation elicited by amlodipine

The vasodilatory response to amlodipine in isolated aortas, with or without endothelium, showed a significant treatment–time interaction effect (Fig. 1A; P <0.001). Vasodilatory responses to amlodipine (3 × 10⁻⁷ M) were significantly greater in aortic rings with intact endothelium compared to those lacking the endothelial lining (Fig. 1A; P <0.001 vs. endothelium-denuded at 15–60 min; Figs. 2A and C) (amlodipine-induced maximal vasodilation [P <0.001 vs. endothelium-denuded]: endothelium-intact aorta, 48.2 ± 11.0% vs. endothelium-denuded aorta, 21.8 ± 4.7%).

Fig. 1.

(A) Influence of endothelial denudation (n = 6) on vasodilation induced by amlodipine (3 × 10⁻⁷ M) in isolated rat aortic segments. (B) Impact of N^W-nitro-L-arginine methyl ester (L-NAME, 10⁻⁴ M, n = 5), methylene blue (MB, 10⁻⁶ M, n = 6), and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10⁻⁵ M, n = 6) on amlodipine (3 × 10⁻⁷ M)-induced vasodilation in aortas with intact endothelium. The data are presented as estimated marginal mean ± SD and expressed as the percentage of contraction produced by phenylephrine (10⁻⁶ M). n represents the number of independent rats from which the aortas were obtained. A: Treatment : P < 0.001, time: P < 0.001, and treatment × time: P < 0.001. ^aP = 0.003 and ^bP < 0.001 versus endothelium-denuded. 95% CI at endothelium-intact versus endothelium-denuded: −10.57 to −3.23 , −13.41 to −6.01, −16.51 to −9.17, −19.20 to −11.80, −21.96 to −14.62, −24.04 to −16.65, −26.04 to −18.70, −27.86 to −20.46, −28.97 to −21.63, −30.21 to −22.81, and −30.05 to −22.71 at 10 to 60 min, respectively. B: Treatment: P < 0.001, time: P < 0.001, and treatment × time: P < 0.001. ^aP = 0.014: MB versus control at 10 min, ^aP = 0.013: ODQ versus control at 10 min. ^bP < 0.001: L-NAME, MB, and ODQ versus control at 15 to 60 min. 95% CI at L-NAME versus control: 6.09 to 12.98, 9.80 to 16.70, 13.12 to 19.91, 16.56 to 23.45, 19.10 to 26.00, 20.82 to 27.71, 22.47 to 29.36, 23.34 to 30.34, 23.59 to 30.49, and 23.77 to 30.67 at 15 to 60 min, respectively; 95% CI at methylene blue versus control: 2.64 to 9.20, 6.70 to 13.25, 10.36 to 16.91, 13.68 to 20.23, 17.68 to 24.23, 20.88 to 27.43, 23.05 to 29.61, 24.65 to 31.20, 25.70 to 32.25, 26.45 to 33.00, and 26.63 to 33.18 at 10 to 60 min, respectively; 95% CI at ODQ versus control: 2.76 to 9.53, 5.71 to 12.48, 8.44 to 15.12, 11.03 to 17.80, 14.18 to 20.95, 16.50 to 23.12, 18.35 to 25.12, 19.49 to 26.26, 19.92 to 26.69, 19.92 to 26.69, and 19.81 to 26.58 at 10 to 60 min, respectively.

Fig. 2.

The original trace illustrates the impact of insulin (10⁻⁷ M) on amlodipine (3 × 10⁻⁷ M)-induced vasodilation in rat aortas with intact (A and B) and denuded (C and D) endothelium, pre-constricted with phenylephrine (10⁻⁶ M).

The vasodilatory response to amlodipine in isolated endothelium-intact aortas, with or without inhibitors (L-NAME, methylene blue, or ODQ), showed a significant treatment–time interaction effect (Fig. 1B; P < 0.001). The vasodilatory effect of amlodipine (3 × 10⁻⁷ M) in the aorta with an intact endothelium was significantly suppressed by the application of L-NAME (10⁻⁴ M), a NOS inhibitor; methylene blue (10⁻⁶ M), a non-specific GC inhibitor; and ODQ (10⁻⁵ M), an NO-sensitive GC inhibitor (Fig. 1B; P < 0.001: L-NAME, methylene blue or ODQ vs. the control at 15–60 min; P = 0.014, and 0.013 in methylene blue, and ODQ vs. control at 10 min, respectively) (amlodipine-induced maximal vasodilation [P <0.001 vs. control]: L-NAME, 27.6 ± 4.2%; methylene blue, 24.9 ± 6.9%; and ODQ, 31.6 ± 5.9% vs. control, 54.8 ± 7.4%). In addition, as DMSO (0.1%), which corresponds to the DMSO concentration used for dissolving ODQ (10⁻⁵ M), did not significantly alter amlodipine (3 × 10⁻⁷ M)-induced vasodilation in endothelium-intact aortas (0.1% DMSO vs. control), this result suggests that ODQ-mediated inhibition of vasodilation evoked by amlodipine (3 × 10⁻⁷ M) is attributable to ODQ itself.

The vasodilatory response to amlodipine in isolated endothelium-intact aortas, with or without insulin, showed a significant treatment–time interaction effect (Fig. 3A; P <0.001). In aortic segments with preserved endothelium, insulin at concentrations of 10⁻⁷ and 5 × 10⁻⁷ M enhanced the vasodilatory effect of amlodipine in a dose-dependent manner (Figs. 2A, B, and 3A; 10⁻⁷ M insulin: P = 0.006 vs. control at 15 min; 5 × 10⁻⁷ M insulin: P = 0.004 vs. control at 15 min; 10⁻⁷ and 5 × 10⁻⁷ M insulin: P < 0.001 vs. control at 20–60 min; 5 × 10⁻⁷ M insulin: P = 0.021, and 0.005 vs. 10⁻⁷ M insulin at 55 and 60 min, respectively) (amlodipine-induced maximal vasodilation [P < 0.001 vs. control]: 10⁻⁷ M insulin; 59.5 ± 8.2%, and 5 × 10⁻⁷ M insulin; 68.4 ± 10.6% vs. control; 48.5 ± 7.1%). However, the vasodilatory response to amlodipine in isolated endothelium-denuded aortas, with or without insulin, showed no significant treatment–time interaction effect (Fig. 3B; P = 0.620). Insulin (10⁻⁷ M) did not affect amlodipine (3 × 10⁻⁷ M)-induced vasodilation in endothelium-denuded aortas (Figs. 2C, D, and 3B) (amlodipine-induced maximal vasodilation [P = 0.384 vs. control]: 10⁻⁷ M insulin; 24.1 ± 5.4% vs. control; 22.6 ± 4.3%).

Fig. 3.

The impact of insulin on amlodipine (3 × 10⁻⁷ M)-induced vasodilation in rat aortas with intact (A, n = 8) and denuded (B, n = 6) endothelium. Results are presented as estimated marginal mean ± SD and expressed as the percentage of phenylephrine (10⁻⁶ M)-induced contraction. n represents the number of independent rats from which the aortas were collected. A: Treatment: P < 0.001, time: P < 0.001, and treatment × time: P < 0.001. ^aP = 0.006: 10⁻⁷ M insulin versus control at 15 min. ^aP = 0.004: 5 ×10⁻⁷ M insulin versus control at 15 min. ^bP < 0.001: 10⁻⁷ M or 5 × 10⁻⁷ M insulin versus control at 20 to 60 min. ^cP = 0.021 and ^dP = 0.005 at 5 × 10⁻⁷ versus 10⁻⁷ M insulin at 55 and 60 min, respectively. 95% CI at 10⁻⁷ M insulin versus control: −12.75 to −4.05, −14.34 to −5.62, −15.20 to −6.48, −16.32 to −7.61, −17.24 to −8.52, −17.51 to −8.79, −17.43 to −8.72, −16.98 to −8.26, −16.03 to −7.31, and −15.29 to −6.57 at 15 to 60 min, respectively; 95% CI at 5 × 10⁻⁷ M insulin versus control: −14.07 to −4.61, −16.77 to −7.31, −19.29 to −9.83, −21.01 to −11.55, −22.04 to −12.94, −23.45 to −13.99, −24.23 to −14.77, −24.03 to −14.57, −24.52 to −15.06, and −24.61 to −15.15 at 15 to 60 min, respectively; 95% CI at 5 × 10⁻⁷ M insulin versus 10⁻⁷ M insulin: −12.76 to −3.49, and −13.59 to −4.32 at 55 and 60 min, respectively. B: Treatment: P = 0.565, time: P < 0.001, and treatment × time: P = 0.620.

The vasodilatory response to amlodipine in isolated endothelium-intact aortas pretreated with inhibitor (methylene blue, ODQ), with or without insulin (methylene blue: Fig. 4B, P = 0.973; ODQ: Fig. 4C, P = 0.983), showed no significant treatment–time interaction effect. In endothelium-intact aortic tissues pretreated with L-NAME (10⁻⁴ M), methylene blue (10⁻⁶ M), or ODQ (10⁻⁵ M), the addition of insulin (10⁻⁷ M) had no significant effect on the vasodilatory response elicited by amlodipine (3 × 10⁻⁷ M) (Table 2), as illustrated in Figs. 4A, B, and C. These results indicate that insulin-mediated potentiation of amlodipine-induced vasodilation is likely driven by a pathway involving NOS, NO, and GC.

Fig. 4.

Insulin (10⁻⁷ M) modulation of amlodipine (3 × 10⁻⁷ M)-elicited vasorelaxation was evaluated in endothelium-intact rat aortic rings pre-incubated with one of the following: N^W nitro L‑arginine methyl ester (L‑NAME; 10⁻⁴M; A; n  =  6), methylene blue (MB; 10⁻⁶ M; B; n  =  6), or 1H [1,2,4]oxadiazolo[4,3 a]quinoxalin 1 one (ODQ; 10⁻⁵ M; C; n  =  6). Data are shown as estimated marginal mean ± SD and expressed as the percentage of contraction induced by phenylephrine (10⁻⁶ M). n indicates the number of independent rats from which aortas were isolated. A: Treatment : P = 0.292, time: P < 0.001, and treatment × time: P = 0.046. B: Treatment: P = 0.445, time: P < 0.001, and treatment × time: P = 0.973. C: Treatment: P = 0.439, time: P < 0.001, and treatment × time: P = 0.983.

Table 2.

Effect of Insulin on Amlodipine-induced Maximal Vasodilation in Isolated Endothelium-intact Rat Aorta Pretreated with Various Inhibitors

Group	n	Amlodipine (3 × 10−7 M)-induced maximal vasodilation (%)	P value	MD (95% CI)
L-NAME (10−4 M)	6	27.6 ± 4.2	0.165	2.2 (−1.03
L-NAME (10−4 M) plus insulin (10−7 M)	6	29.9 ± 4.5		to 5.69)
MB (10−6 M)	6	24.9 ± 6.9	0.745	0.8 (−4.16
MB (10-6 M) plus insulin (10−7 M)	6	25.7 ± 7.2		to 5.75)
ODQ (10−5 M)	6	31.6 ± 5.9	0.384	1.8 (−2.44 to 6.15)
ODQ (10−5 M) plus insulin (10−7 M)	6	33.4 ± 5.6

L-NAME: N^W-nitro-L-arginine methyl ester, MB: methylene blue, ODQ: 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. n indicates the number of independent rats from which the aortas were obtained. Data regarding amlodipine (3 × 10⁻⁷ M)-induced maximal vasodilation are shown as mean ± SD and expressed as the percentage of relaxation from phenylephrine (10⁻⁶ M)-induced contraction. MD: mean difference.

Effect of insulin on the vasodilatory response elicited by sodium nitroprusside in endothelium removed aorta

The vasodilatory response to sodium nitroprusside in isolated endothelium-denuded aortas, with or without insulin, showed no significant treatment–concentration interaction effect (Fig. 5A; P > 0.999). Insulin (10⁻⁷ and 5 × 10⁻⁷ M) did not affect vasodilation induced by sodium nitroprusside in the aorta after endothelium removal (Fig. 5A).

Fig. 5.

(A) The impact of insulin on vasorelaxation induced by sodium nitroprusside (SNP) was assessed in aortas lacking endothelium. Results (n = 5) are presented as estimated marginal mean ± standard deviation and quantified as a percentage relative to contractions induced by phenylephrine (10⁻⁶ M). The value of N denotes the number of independent rats from which the vascular tissues were obtained. Treatment : P = 0.999, concentration: P < 0.001, and treatment × concentration: P > 0.999. (B) The influence of insulin on verapamil (10⁻⁷ M)-mediated vasodilation was evaluated in aortas with intact endothelium. Measurements (n = 5) are expressed as estimated marginal mean ± SD and calculated as the proportion of the contractile response to phenylephrine (10⁻⁶ M). n corresponds to the number of independent rats used to obtain the isolated aortic segments. Treatment: P = 0.766, time: P < 0.001, and treatment × time: P = 0.772.

Influence of insulin on verapamil-evoked vasodilatory responses in isolated rat aortic segments

The vasodilatory response to verapamil in isolated endothelium-intact aortas, with or without insulin, showed no significant treatment–time interaction effect (Fig. 5B; P = 0.772). Insulin (10⁻⁷ and 5 × 10⁻⁷ M) did not affect verapamil (10⁻⁷ M)-induced vasodilation in endothelium-intact aortas (Fig. 5B) (verapamil-induced maximal vasodilation at 60 min [P = 0.892]: 10⁻⁷ M insulin; 46.1 ± 6.7%, and 5 × 10⁻⁷ M insulin; 44.8 ± 6.7% vs. control; 45.2 ± 4.9%).

Impact of amlodipine, either independently or in conjunction with insulin, on phenylephrine-induced vasoconstriction in isolated rat aortas with intact endothelium

The vasoconstrictive response to phenylephrine in isolated endothelium-intact aortas pretreated with amlodipine, with or without insulin, showed a significant treatment–concentration interaction effect (Fig. 6; P < 0.001). The combination of amlodipine and insulin inhibited phenylephrine-induced contraction more effectively than amlodipine alone (Fig. 6; P = 0.007: 3 × 10⁻⁷ M amlodipine plus 10⁻⁷ M insulin vs. 3 × 10⁻⁷ M amlodipine at 3 × 10⁻⁷ M phenylephrine; P < 0.001: 3 × 10⁻⁷ M amlodipine plus 5 × 10⁻⁷ M insulin vs. 3 × 10⁻⁷ M amlodipine at 3 × 10⁻⁷ M phenylephrine; P < 0.001: 3 × 10⁻⁷ M amlodipine plus 10⁻⁷ M insulin or 3 × 10⁻⁷ M amlodipine plus 5 × 10⁻⁷ M insulin vs. 3 × 10⁻⁷ M amlodipine at 10⁻⁶ to 10⁻⁵ M phenylephrine) (10⁻⁵ M phenylephrine-induced maximal contraction [P < 0.001: 3 × 10⁻⁷ M amlodipine plus 5 × 10⁻⁷ M insulin vs. 3 × 10⁻⁷ M amlodipine alone; P = 0.001: 3 × 10⁻⁷ M amlodipine plus 10⁻⁷ M insulin vs. 3 × 10⁻⁷ M amlodipine alone]: 3 × 10⁻⁷ M amlodipine plus 10⁻⁷ M insulin; 46.0 ± 10.4%, and 3 × 10⁻⁷ M amlodipine plus 5 × 10⁻⁷ M insulin; 41.8 ± 10.8% vs. 3 × 10⁻⁷ M amlodipine alone; 61.7 ± 10.1%).

Fig. 6.

Impact of amlodipine alone or in combination with insulin on phenylephrine-induced contractile responses in rat aortic rings with intact endothelium. Results (n = 7) are presented as estimated marginal mean ± SD and expressed relative to the contractile force generated by 60 mM potassium chloride. The value of n represents the number of independent rats from which the aortic tissues were harvested. Treatment: P < 0.001, concentration: P < 0.001, and treatment × concentration: P < 0.001. ^aP = 0.007: 3 × 10⁻⁷ M amlodipine plus 10⁻⁷ M insulin versus 3 × 10⁻⁷ M amlodipine at 3 × 10⁻⁷ M phenylephrine; ^bP < 0.001: 3 × 10⁻⁷ M amlodipine plus 10⁻⁷ M insulin versus 3 × 10⁻⁷ M amlodipine at 10⁻⁶ to 10⁻⁵ M phenylephrine. ^cP < 0.001: 3 × 10⁻⁷ M amlodipine plus 5 × 10⁻⁷ M insulin versus 3 × 10⁻⁷ M amlodipine at 3 × 10⁻⁷ to 10⁻⁵ M phenylephrine. 95% CI at 3 × 10⁻⁷ M amlodipine plus 10⁻⁷ M insulin versus 3 × 10⁻⁷ M amlodipine: −17.32 to −5.18, −20.18 to −8.05, −21.01 to −8.87, and −21.79 to −9.66 at 3 × 10⁻⁷ to 10⁻⁵ M phenylephrine, respectively; 95% CI at 3 × 10⁻⁷ M amlodipine plus 5 × 10⁻⁷ M insulin versus 3 × 10⁻⁷ M amlodipine: −19.91 to −8.07, −23.15 to −11.30, −24.85 to −13.00, and −25.81 to −13.96 at 3 × 10⁻⁷ to 10⁻⁵ M phenylephrine, respectively.

Effect of amlodipine and insulin, alone or combined, on eNOS (Ser1177) phosphorylation in HUVECs

Although HUVECs, which do not directly correspond to the endothelial cells of rat aortas, were used to evaluate eNOS phosphorylation, amlodipine at 3 × 10⁻⁷ M significantly promoted phosphorylation of eNOS at the Ser1177 site in HUVECs (Fig. 7; P = 0.005 compared to control [0.3% DMSO]). Moreover, co-treatment with insulin (5 × 10⁻⁷ M) and amlodipine resulted in an even greater increase in eNOS phosphorylation than amlodipine alone (Fig. 7; P = 0.004 vs. the amlodipine group). In addition, insulin (5 × 10⁻⁷ M) alone slightly increased eNOS phosphorylation (Fig. 7; P = 0.012 vs. control).

Fig. 7.

Influence of insulin (5 × 10⁻⁷ M) on amlodipine (3 × 10⁻⁷ M)-stimulated phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 in human umbilical vein endothelial cells. Results (n = 4) are presented as mean ± SD. n reflects the number of independently conducted experiments. ^aP = 0.012, and ^bP = 0.005 versus control (dimethyl sulfoxide [DMSO]: 0.3%). ^cP = 0.004 versus amlodipine alone. p-eNOS: phosphorylated eNOS. t-eNOS: total eNOS. 95% CI at 3 × 10⁻⁷ M amlodipine versus control: 0.12 to 0.67; 95% CI at 3 × 10⁻⁷ M amlodipine plus 5 × 10⁻⁷ M insulin versus 3 × 10⁻⁷ M amlodipine: 0.13 to 0.68; 95% CI at 5 × 10⁻⁷ M insulin alone versus control: 0.06 to 0.61.

Discussion

This study demonstrates that insulin enhances amlodipine-induced vasodilation in aortas with an intact endothelium by augmenting the production of endogenous NO through NO-dependent mechanisms. The key findings of this study were as follows: 1) Insulin markedly enhanced the vasodilatory effect of amlodipine in aortas with intact endothelium, whereas it had no impact on amlodipine-induced vasodilation in aortas lacking the endothelial layer. 2) L-NAME, methylene blue, and ODQ abolished the insulin-mediated augmentation of amlodipine-induced vasodilation in the endothelium-intact aorta. 3) Insulin increased amlodipine-induced eNOS phosphorylation in HUVECs.

Endothelial NO is synthesized from L-arginine via eNOS activity [20]. After its production, NO moves from the endothelium to the surrounding vascular smooth muscle [20]. This NO then stimulates GC, leading to an increase in cyclic guanosine monophosphate (cGMP) [20]. As a result, cGMP activates cGMP-dependent protein kinases, which then initiate a cascade that results in vasodilation [20]. Racemic amlodipine and the R-isomer of amlodipine produce nitrite in isolated canine coronary vessels, and this effect is inhibited by L-NAME and the B2-kinin receptor antagonist HOE-140 [10]. In addition, amlodipine induces nitrite formation in the aorta, which is inhibited by L-NAME and HOE-140 [9]. These previous reports suggest that amlodipine-induced NO production contributes to endothelial NO-dependent vasodilation [9,10]. Supporting these previous reports [9,10], vasodilation caused by amlodipine was more pronounced in aortas with intact endothelium than in those without, indicating that the vasodilatory effects of amlodipine depend on the endothelium. Consistent with previous reports [9,10,12], pretreatment with L-NAME, methylene blue, and ODQ inhibited vasodilation caused by amlodipine. Additionally, amlodipine increases cGMP formation in endothelium-intact aortas, whereas L-NAME inhibits amlodipine-induced cGMP formation [12]. Moreover, L-NAME decreases amlodipine-induced perfusion pressure reduction in vivo [11]. Taken together with previous in vitro and in vivo studies [9–12], these results suggest that amlodipine-induced vasodilation is mediated in part by pathways involving endothelial NO, GC, and cGMP. Insulin augmented the vasodilatory response to amlodipine in the aorta with preserved endothelium in a concentration-dependent manner (Fig. 3A). However, this did not affect the vasodilation caused by amlodipine in the aorta that had been stripped of endothelium (Fig. 3B). Collectively, these results imply that insulin-mediated augmentation of amlodipine-induced vasodilation is endothelium-dependent. Prior treatment with L-NAME, ODQ, or methylene blue effectively eliminated the insulin-mediated increase in the vasodilatory response to amlodipine in the aorta with endothelium (Fig. 4). Therefore, the enhancement of amlodipine-induced vasodilation by insulin seems to be driven by an endogenous pathway involving NO and GC. Insulin increases skeletal muscle blood flow, which is inhibited by a NOS inhibitor [7]. Additionally, insulin decreases forearm vascular resistance, which is inhibited by a NOS inhibitor [8]. Moreover, insulin increases NO-mediated vasodilation induced by acetylcholine or methacholine [6,7]. However, it did not significantly increase vasodilation induced by the NO donor sodium nitroprusside, consistent with the results of the current study [6,7]. Overall, these studies suggest that the vasodilatory effect of insulin is mediated by endothelial NO [6–8]. Moreover, the non-specific GC inhibitor methylene blue, which is one of the frequently used vasopressors during HIET for amlodipine toxicity, is also used for the treatment of NO-induced vasoplegic shock [13,21]. According to previous reports [6–13], during HIET for the treatment of amlodipine toxicity, a potential synergistic interaction between amlodipine and insulin, particularly in relation to NO production, may contribute to augmented vasodilation. Nevertheless, the specific effects of insulin on vasodilation induced by toxic doses of amlodipine have yet to be elucidated. Considering previous reports and all the results from isometric tension studies [6–13,21], insulin-mediated augmentation of vasodilation induced by amlodipine may result from increased endogenous NO production by insulin and amlodipine via additive or synergistic interactions in the endothelium-intact aorta because insulin did not affect vasodilation produced by NO donor sodium nitroprusside. Conversely, amlodipine plus insulin inhibited phenylephrine-induced contraction more than amlodipine alone in endothelium-intact aortas (Fig. 6), suggesting that vasopressor support may be more necessary during HIET in the amlodipine toxicity [13]. Endothelial phosphoinositide-3 kinase-induced NO production attenuates phenylephrine-induced contraction, suggesting that this pathway also reduces contraction induced by G protein-coupled receptor agonists [22]. Activation of phosphoinositide-3 kinase triggers the downstream signaling molecule Akt, which subsequently promotes eNOS phosphorylation and induces vasodilation [23]. Considering previous reports [22,23], additional research is required to investigate the potential upstream signaling mechanisms, such as the activation of phosphoinositide-3 kinase and Akt, involved in insulin-enhanced vasodilation induced by amlodipine. Diltiazem does not enhance NO production [9]. In addition, L-NAME does not alter the perfusion pressure reduced by verapamil in vivo [11]. Similar to previous reports [9,11], and in contrast to the insulin-mediated augmentation of amlodipine-induced vasodilation, insulin did not affect the vasodilation induced by the non-dihydropyridine calcium channel blocker verapamil. Further studies examining the mechanisms underlying the differential effects of insulin on vasodilation induced by amlodipine and verapamil are needed.

Phosphorylation of eNOS at Ser1177 activates the enzyme, resulting in enhanced eNOS activity and increased NO production [24]. Consistent with the tension study results on amlodipine-induced vasodilation, amlodipine increased eNOS phosphorylation in HUVECs. Moreover, co-treatment with insulin and amlodipine further increased amlodipine-induced eNOS phosphorylation. Thus, these results suggest that insulin may enhance amlodipine-induced vasodilation through endothelial NO release, although direct evidence from rat aortic endothelial cells is lacking. However, additional studies are required to specifically investigate whether insulin influences amlodipine-induced eNOS phosphorylation in rat aortic endothelial cells.

This study has several limitations. First, the experiment used rat aorta to measure isometric tension but employed HUVECs to assess eNOS phosphorylation. In addition, amlodipine increases intracellular calcium levels in HUVECs, which is associated with increased eNOS activation [12]. Second, small resistance arterioles such as the rat mesenteric artery are mainly involved in vascular resistance, which is associated with blood pressure [25,26], whereas this investigation used rat aorta, which is a major conduit vessel. The extrapolation to systemic vascular resistance should be approached with caution. Thus, further studies involving resistance arterioles are needed to validate the translational relevance of these results. Third, the research was performed in vitro; however, in vivo studies are essential to more thoroughly evaluate the effects of insulin and L-NAME on the cardiovascular depression induced by a toxic dose of amlodipine. Fourth, further clinical studies to confirm these hypothesis-generating results are needed. In other words, because this study used conduit aortas rather than small resistance arterioles, and HUVECs to examine eNOS phosphorylation (introducing cross-species mismatching), and did not include an in vivo component, caution should be exercised when extrapolating these findings to clinical applications. Although this study has several limitations in terms of clinical extrapolation, these findings suggest a novel hypothesis: insulin (5 × 10⁻⁷ M)-mediated enhancement of vasodilation induced by a high, potentially toxic dose of amlodipine (3 × 10⁻⁷ M) may contribute to severe vasodilation during HIET used in the treatment of amlodipine-induced toxicity [13,16,17], which is speculative. Thus, further study is needed to confirm this hypothesis.

Collectively, these findings imply that insulin enhances amlodipine-induced vasodilation through a NO-dependent mechanism in rat aortas, likely driven by potentially elevated NO synthesis. These findings suggest a novel, hypothesis-generating observation that insulin-mediated enhancement of amlodipine-induced vasodilation may contribute to severe vasodilation during HIET in the management of amlodipine toxicity. However, as these findings are speculative, further in vivo and clinical studies to examine the effects of insulin on hemodynamic changes including vascular resistance induced by amlodipine toxicity, are needed to validate the results obtained from this in vitro study.