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Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy
VPH-06232; No of Pages 7 Vascular Pharmacology xxx (2015) xxx–xxx
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Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy Hydralazine induces vasodilation via a prostacyclin pathway Nicole Maille a, Natalia Gokina a, Maurizio Mandalà a,b, Ilsley Colton a, George Osol a,⁎ a b
Department of Obstetrics and Gynecology and Reproductive Sciences, University of Vermont College of Medicine, USA Department of Biology, Ecology and Earth Science, University of Calabria, Italy
a r t i c l e
i n f o
Article history: Received 14 May 2015 Received in revised form 23 June 2015 Accepted 17 July 2015 Available online xxxx Keywords: Hypertension Endothelium Vascular smooth muscle Prostacyclin Pregnancy
a b s t r a c t The cellular mechanisms of hydralazine-induced relaxation were investigated in isolated mesenteric resistance arteries from pregnant rats. Administration of hydralazine relaxed phenylephrine-constricted mesenteric arteries with an EC50 of 3.6 ± 0.3 μM and an efficacy of 75 ± 6.2%. These vasodilatory effects were abolished by: (1) preconstriction with a potassium depolarizing solution, (2) endothelial denudation (for concentrations of hydralazine b 10 μM), (3) addition of non-selective cyclooxygenase-1 and cyclooxygenase-2 inhibitors, and (4) pretreatment with a prostacyclin receptor antagonist (R01138452). Nitric oxide synthase (NOS) inhibition did not significantly alter the sensitivity or magnitude of the vasodilatory response; surprisingly, exposure to hydralazine also did not elevate endothelial cell Ca2+, suggesting a novel mechanism of activation. In summary, hydralazine is a potent resistance artery vasodilator that affects both endothelial and vascular smooth muscle (VSM) cells in a concentration-dependent manner. At clinically relevant concentrations (b10 μM), its effects in the splanchnic resistance vasculature are: (1) primarily endothelial in origin, require (2) hyperpolarization and (3) activation of COX, and (4) are mediated by the PGI2 (IP) receptor. © 2015 Published by Elsevier Inc.
1. Introduction Hydralazine is an antihypertensive drug that is used clinically to treat gestational hypertension resulting from conditions such as preeclampsia. Little, however, is known about its cellular mechanism of action on the vascular wall during pregnancy. Previous studies in male and nonpregnant female animal models have attributed its blood pressure-lowering effects to decreased total peripheral resistance rather than changes in cardiac output [1]. Several earlier studies have examined arterial responses to the administration of hydralazine, and have documented vasodilation in a number of in vitro arterial preparations including the rabbit aorta [2–4], rabbit renal artery [5], rat caudal artery [6], porcine coronary artery [7], and human digital artery [8]. The specific mechanisms, however, are poorly defined. For example, there is conflicting evidence regarding the role of the endothelium in hydralazine-induced relaxation. In vessels from nonpregnant animals, dilation still occurred in the absence of the endothelium in rat aortic rings [9–11], but not in rabbit aorta [4,12,13], rat caudal artery [14], and porcine coronary strips [7]. To date, several different mechanisms ⁎ Corresponding author at: Department of Obstetrics and Gynecology, The University of Vermont, College of Medicine, Burlington, VT 05405, USA. E-mail address: [email protected] (G. Osol).
have been proposed, including altered prostaglandin production, Ca2 + handling, membrane hyperpolarization, and cGMP production [7,15,16]. Interpretation has been further complicated by variable responses due to vessel type [5,17]. Some studies used conduit vessels, such as the aorta, which do not contribute to peripheral resistance, and/or concentrations of hydralazine far above those present in human plasma under clinical conditions (N100 μM) [18]. It is well known that pregnancy causes changes in systemic and uterine vessel reactivity and function through changes in both the endothelium and vascular smooth muscle cells [19–22]. It is therefore important to understand the mechanism by which hydralazine induces vasodilation during pregnancy. Since the splanchnic vasculature holds approximately one fourth of the total arterial blood volume, and given that it is a major contributor to overall peripheral resistance and blood pressure regulation, we used mesenteric resistance arteries from pregnant rats to evaluate the vasodilator effects of hydralazine during gestation. We hypothesized that hydralazine relaxation involves mechanisms that involve a combination of endothelial and vascular smooth muscle actions. The results indicate that, at concentrations that are comparable to those used clinically in humans (b10 μM) [18,23,24], hydralazine action on the vascular wall is predominantly endothelial, requires membrane hyperpolarization, and is mediated by cyclooxygenase-induced prostacyclin receptor (IP) activation.
http://dx.doi.org/10.1016/j.vph.2015.07.009 1537-1891/© 2015 Published by Elsevier Inc.
Please cite this article as: N. Maille, et al., Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.07.009
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2. Materials and methods
relaxed diameter determined in relaxing solution containing diltiazem and papaverine.
2.1. Animals Timed late pregnant (day 20/22 of gestation; n = 30) thirteen week old female Sprague–Dawley rats were purchased from Charles River Canada (Saint Constant QC, Canada) and housed at the University of Vermont Small Animal Facility under a 12 L:12D photoperiod; food and water were provided ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Vermont. Animals were euthanized with an intraperitoneal injection of pentobarbital sodium (50 mg/mL), followed by decapitation with a small animal guillotine. The abdominal cavity was then opened and a section of the mesentery 5 cm distal to the pylorus was excised and pinned in a Sylgard-lined Petri dish containing cold (4 °C) HEPES-physiological saline solution (HEPES-PSS, pH = 7.4). 2.2. Isolated vessel preparation Third-order mesenteric arteries with diameters between 200 and 300 μm at 50 mm Hg were dissected free of surrounding adipose and connective tissue and cannulated in the chamber of an arteriograph (Instrumentation and Model Facility, University of Vermont, Burlington, VT). For some experiments, vessels were mechanically denuded via hair abrasion [25] followed by air bolus perfusion [26] for 10 min at a low perfusion pressure (10 mm Hg). Deionized water was then gently passed through the lumen of the artery for 30–60 s to remove any endothelial debris and subsequently replaced with HEPES solution. Following cannulation of the distal end of each vessel, mesenteric arteries were pressurized to 50 mm Hg using a pressure-servo system (Living Systems Instrumentation, Burlington, VT) and positioned on the stage of an inverted microscope (Zeiss, West Germany). A pressure of 50 mm Hg was chosen for these experiments as it approximates in vivo conditions and prevents possible confounding effects by myogenic tone that appears at higher transmural pressures HEPES-PSS. In denuded vessels, successful removal of the endothelium was verified by the complete loss of acetylcholine (ACh)-induced relaxation at 1 μM in vessels preconstricted with phenylephrine. Each vessel was examined for leakage by turning off the servo null pressure feedback system in the absence of flow. If no decrease in the transmural pressure occurred within 2 min, the vessel was considered suitable; otherwise, it was removed and a fresh artery was cannulated, pressurized, and re-tested for leaks. 2.3. Concentration–response curves Vessels were equilibrated for 40 min in HEPES-PSS (pH = 7.4) at 37 °C. All experiments were performed at an intraluminal pressure of 50 mm Hg without intraluminal flow. Arteries were preconstricted using a superfusate containing either phenylephrine or a high potassium (35 mM) depolarizing solution to produce a 40–60% reduction in lumen diameter. Changes in intraluminal diameter were measured using a video dimension analyzer (Living Systems Instrumentation, Burlington, VT), as previously described [27,28], and recorded on LabView software. Hydralazine was administered in increasing concentrations (0.1–100 μM), and the resulting changes in diameter recorded once dilation stabilized at each concentration. At the end of each experiment, vessels were treated with relaxing solution containing a mixture of the L-type Ca2+ channel blocker, diltiazem (10 μM) and the phosphodiesterase inhibitor, papaverine (100 μM) to assure maximal vasodilation. Pharmacologic sensitivity was calculated using a standard curve analysis (SigmaPlot version 11.0; Systat Software Inc., San Jose CA) which determined the concentration of hydralazine necessary to produce 50% of the maximal vasodilatory effect (EC50). Efficacy was defined as the maximal relaxation to hydralazine as a percentage of the fully
2.4. Role of nitric oxide and prostaglandin production in hydralazineinduced vasodilation Vessels were pre-treated for 20 min with a combination of NωNitro-L-arginine (L-NNA, 100 μM) and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 μM) to inhibit nitric oxide (NO) production prior to exposure to phenylephrine. Other studies have shown this combination to be more effective in inhibiting nitric oxide synthase (NOS) than either drug alone [29]. To investigate the role of prostaglandins in hydralazine-induced vasodilation, vessels preconstricted with phenylephrine were treated with non-specific cyclooxygenase-1 and cyclooxygenase-2 (COX-1/2) inhibitors (indomethacin or ibuprofen, each at 10 μM) for 20 min prior to hydralazine administration. In some experiments, the PGI2 (IP) receptor antagonist, R01138452 (10 μM), was administered in the same manner as the COX-1/2 inhibitors to block the downstream effects of PGI2 production. 2.5. Effects of KCl depolarization on hydralazine-induced vasodilation Vessels were depolarized with a high potassium (35 mM) depolarizing solution prior to exposure to a single high concentration of hydralazine (100 μM). The relative (%) inhibition of hydralazineinduced vasodilation under depolarized conditions was calculated by the following equation: 1 − (% dilation of KCl-constricted vessel / % dilation of phenylephrine-constricted vessel) × 100. 2.6. Measurement of endothelial cell [Ca2+]i Endothelial cells (ECs) were loaded with Ca2 + sensitive fura-2 by perfusing the lumen of cannulated arteries with fura-2 AM-containing solution (5 μM) for 5 min at room temperature, as previously described [30]. To remove excess fura-2, the lumen was perfused with regular PSS for 10 min by using a 20 mm Hg intraluminal pressure gradient. Ratiometric measurements of fura‐2 fluorescence from ECs were performed using a photomultiplier system (IonOptix Inc. Milton, MA). Experimental ratios were corrected for background fluorescence taken from each artery before loading with fura 2-AM. Background-corrected ratios of 510 nm emission were obtained at a sampling rate of 5 Hz from arteries alternately excited at 340 and 380 nm. The experimental protocol was started following an additional 15 min equilibration period at 10 mm Hg to allow intracellular deesterification of fura-2 AM. In these experiments, hydralazine was tested at an intraluminal pressure of 50 mm Hg without phenylephrine to eliminate movement artifacts secondary to constriction. Following application of hydralazine, vessels were given ACh (10 μM) as a positive control for EC calcium elevation. EC intracellular calcium concentration ([Ca2+]i) was calculated using the following equation [31]: [Ca2+]i = Kdβ(R − Rmin) / (Rmax − R), where R is an experimentally measured ratio (340/380 nm) of fluorescence intensities, Rmin is a ratio in the absence of [Ca2+]i and Rmax is a ratio at Ca2+-saturated fura-2 conditions, β is a ratio of the fluorescence intensities at 380 nm excitation wavelength at Rmin and Rmax. Rmin, Rmax and β were determined by an in situ calibration procedure from the arteries treated with ionophores ionomycin (10 μM) and nigericin (5 μM). These values were then pooled and used to convert the ratio values into a [Ca2+]i. The Kd (the dissociation constant for fura-2) was 282 nM, as determined by in situ titration of Ca2+ in fura-2 loaded small arteries [32]. 2.7. Drugs and solutions HEPES-PSS contained the following (in mM): 141.8 sodium chloride, 4.7 potassium chloride, 1.7 magnesium sulfate, 2.8 calcium chloride, 1.2
Please cite this article as: N. Maille, et al., Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.07.009
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potassium phosphate, 10.0 HEPES, 0.5 EDTA, and 5.0 dextrose. High potassium (35 mM) HEPES solution contained the following (in mM): 106.8 sodium chloride, 35 potassium chloride, 1.7 magnesium sulfate, 2.8 calcium chloride, 1.2 potassium phosphate, 10.0 HEPES, 0.5 EDTA, and 5.0 dextrose. Both solutions were prepared with deionized water and titrated with sodium hydroxide to a physiologic pH of 7.4. For the endothelial calcium experiments, oxygenated (aerated with a mixture of 5% CO2, 10% O2, and 85% N2) PSS was used. PSS was composed of the following (in mM): 119 sodium chloride, 4.7 potassium chloride, 24.0 sodium bicarbonate, 1.2 magnesium sulfate, 0.023 EDTA, 1.6 calcium chloride, 1.2 potassium phosphate, and 11.0 glucose (pH 7.4). For the calibration procedure, we used a solution of the following composition: (in mM): 140 KCl, 20 NaCl, 5 HEPES, 5 EGTA, and 1 MgCl2 and 5 μM nigericin and 10 μM ionomycin, pH = 7.1. All chemicals for HEPES-PSS and solutions for reactivity experiments were purchased from Fisher Scientific (Fair Lawn, NJ). Pentobarbital sodium was purchased from Ovation Pharmaceuticals (Deerfield, IL). Phenylephrine, hydralazine, indomethacin, ibuprofen, L-NNA, L-NAME, papaverine, diltiazem, ionomycin and nigericin were purchased from Sigma Chemical Co. (St. Louis, MO). R01138452 was purchased from Cayman Chemical Co. (Ann Arbor, MI) and fura-2AM from Invitrogen (Carlsbad, CA). Stock solutions for indomethacin, ibuprofen, R01138452, and fura-2AM were prepared in dehydrated dimethyl sulfoxide (DMSO, Acros Organics, Fair Lawn, NJ). Other drug stock solutions were prepared with HEPES solution, all on a daily basis. 2.8. Statistical analysis All data are presented as mean ± SEM. The % dilation at each dose of hydralazine was averaged for all experiments within the same treatment. Differences in response to hydralazine for the phe-constricted vs. high potassium constricted vessels were determined by an unpaired ttest. A two way repeated measures analysis of variance with the Holm– Sidak multiple comparisons test was used to compare the control group to the treatment groups. P-values b 0.05 were considered significant. 3. Results 3.1. Relaxation of intact vessels in phenylephrine vs. KCl The lumen diameter of third order mesenteric vessels used in this study averaged 257 ± 5.2 μm at 50 mm Hg under fully relaxed conditions (n = 46 total vessels used for experimentation). Phenylephrine (0.5–5 μM) resulted in progressive constriction that averaged 45 ± 1.3%. The dilatory response to hydralazine was detected within 10 min of administration, and the maximum effect was obtained within 20 min. As shown in the experimental tracing in Fig. 1a, increasing concentrations of hydralazine induced relaxation in all vessels. The concentration required for half-maximal relaxation (EC50) was 3.6 ± 0.3 μM. Maximum dilation was observed at 100 μM (Fig. 1b) with an efficacy of 75 ± 6.2%. 3.2. Effects of endothelial denudation At concentrations below 30 μM, the vasodilatory effects of hydralazine were completely abolished in endothelium-denuded vessels. However, at 30 and 100 μM, hydralazine relaxed the denuded vessels 15 ± 11.5% and 72 ± 13.1%, respectively (n = 3; Fig. 1b), showing that it also had some direct action on VSM. 3.3. Mechanism of action High K+ depolarizing solution greatly attenuated vasodilatory responses to hydralazine such that exposure to 100 μM hydralazine produced weak vasodilation (6 ± 1.1%; n = 3; Fig. 2). This result
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suggests that hyperpolarization is an important component of the underlying mechanism. Vessels were pre-treated with NOS inhibitors L-NNA and L-NAME (n = 7) to evaluate the role of NO. A second group of vessels were treated with the COX-1/2 inhibitor indomethacin (10 μM; n = 5) to inhibit the production of prostaglandins. Responses to hydralazine were not significantly affected by inhibiting NO production, as the EC50 was calculated to be 3.9 ± 2.3 μM (Fig. 3). Efficacy was also unchanged (61 ± 10.1%). On the other hand, preventing prostaglandin production using indomethacin completely abolished the vasodilatory-response to hydralazine (Fig. 4a) and unmasked a slight vasoconstrictor effect at concentrations N 3 μM. To minimize the likelihood of non-specific effects, additional experiments were performed using a second COX-1/2 inhibitor ibuprofen (10 μM; n = 5), which yielded similar results. The role of PGI2, a known vasodilator prostanoid, was evaluated by pretreating vessels with 10 μM of the specific IP receptor antagonist RO1138452 [33,34]. In its presence, vasodilatory responses to hydralazine were completely abolished (Fig. 4b). Endothelial cells were loaded with fura-2 via intraluminal perfusion to examine the effect of hydralazine on endothelial [Ca2+]I, as this is a common observation in response to endothelium-dependent vasodilators such as ACh [35]. Following dye-loading, vessels were superfused with a solution containing 10 μM hydralazine for 20 min to elicit a maximum dilator effect. Surprisingly, there were no detectable changes in endothelial cell [Ca2+]i in response to hydralazine (n = 3), although a significant rise in endothelial [Ca2 +]i was observed in response to subsequent application of 10 μM ACh, which was used as a positive control. A representative tracing is shown in Fig. 5. 4. Discussion The main findings of this study (summarized in Fig. 6) are that: (1) hydralazine is a potent vasodilator of mesenteric resistance vessels from pregnant rats with direct actions on both endothelium and vascular smooth muscle. At clinically-relevant concentrations (1–10 μM), however, its primary actions are (2) endothelial, and involve a mechanism that requires (3) hyperpolarization and a (4) cyclooxygenase/prostacyclin/IP receptor signaling pathway. An earlier in vivo study in dogs noted that the splanchnic vascular bed is particularly highly sensitive to hydralazine [36] further highlighting the importance of understanding its mechanism in resistance vessels from this circulation. Although this drug has been used to treat hypertension in pregnant women for N60 years, and is still commonly used, to our knowledge, this is the first study to specifically examine its effects on resistance arteries from pregnant animals. Moreover, unlike several other studies have used mesenteric artery rings under isometric conditions, this study used pressurized vessels that allow direct measurement of lumen diameter, a principal determinant of blood flow resistance under physiological conditions. During treatment for hypertension and gestational hypertension, human plasma levels of hydralazine are on the order of 0.1–5 μM [18, 23,24]. In studies on conduit vessels such as the rabbit aorta and renal artery [3,4], endothelial denudation showed mixed results, with effects at ≤ 10 μM hydralazine being reduced or completely eliminated, but observable at concentrations N 10 μM. Our results indicate that its effects are completely endothelium-dependent, which is consistent with earlier findings in nonpregnant animal models [12–14]. Although we only tested one relatively high concentration of hydralazine (100 μM) in the KCl experiments, it is unlikely that any effects would have been seen at lower concentrations. The observation that hyperpolarization is requisite for dilation is consistent with one earlier report in rat caudal arteries [6]. In this regard, it was interesting to note that there were no measurable increases in endothelial cell calcium in response to hydralazine, since an increase in endothelial [Ca2+]i is typically associated with endothelial production of NO, EDHF and PGI2 [37–39]. Clearly, this was not a limitation of methodology, as ACh
Please cite this article as: N. Maille, et al., Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.07.009
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Fig. 1. The vasodilatory effects of hydralazine on preconstricted mesenteric arteries from late pregnant rats. (A) Representative experimental tracing showing the relationship between hydralazine concentration and arterial lumen diameter. Arrowheads indicate the application of phenylephrine to induce preconstriction. Increasing concentrations (0.5, 0.75, 1.0 μM) of phenylephrine were used to constrict the vessel to 40% before adding cumulative concentrations of hydralazine. To test the health of the endothelium, 1 μM ACh was added to fully dilate the vessel. Here, and elsewhere, % dilation was calculated relative to the response evoked with a maximum relaxing solution of 100 μM papaverine and 10 μM diltiazem (fully relaxed diameter is shown by the dotted line). (B) Hydralazine produced a concentration-dependent vasodilation of mesenteric arteries preconstricted with phenylephrine. The concentration of hydralazine needed to produce a half maximal response (EC50) was calculated as 3.6 μM using a best-fit curve analysis. Removal of the endothelium abolished vasodilation to lower concentrations of hydralazine (0.1, 1.0, 3.0 and 10 μM), while some endothelium-independent dilation was noted at higher concentrations (30, 100 μM). Values are means ± SEM; n = number of experiments. *: P b 0.05 compared to control.
induced a substantial calcium increase when given to the same vessels following hydralazine. Vasodilatory regulation by the endothelium is primarily attributable to three main signaling molecules; NO, endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin (PGI2) [40–42]. In this study,
Fig. 2. The vasodilatory effects of hydralazine require membrane hyperpolarization. Preconstriction with a 35 mM potassium depolarizing solution virtually eliminated arterial vasodilation in response to a high concentration (100 μM) of hydralazine. Values are means ± SEM; Phe = phenylephrine. HDZ = hydralazine. Phe = phenylephrine. n = 17 for Phe-constricted vessels and n = 3 for KCl-constricted vessels. *: P b 0.05 compared to control vessels constricted with Phe.
inhibition of NOS did not significantly alter hydralazine-induced relaxation, suggesting that hydralazine does not elicit the production of NO in this vascular bed. Wei et al. [7] also predicted an NO-independent mechanism in porcine coronary strips after finding that the addition of hemoglobin (a scavenger of NO) and L-NOARG (an inhibitor of NO production from L-Arg) did not prevent hydralazine-induced relaxation.
Fig. 3. Hydralazine-induced vasodilation is not mediated by NO production. Inhibition of NOS on vessels preconstricted with phenylephrine does not affect dilation to hydralazine. L-NNA = Nω-Nitro-L-arginine. L-NAME = Nω-Nitro-L-arginine methyl ester hydrochloride. Values are means ± SEM; n = number of experiments.
Please cite this article as: N. Maille, et al., Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.07.009
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Fig. 5. Hydralazine treatment failed to elevate intracellular endothelial calcium in mesenteric arteries.(A) Representative experimental tracing of endothelial intracellular calcium in response to a single 10 μM concentration of hydralazine, followed by treatment with ACh. Note the absence of calcium elevation to hydralazine, and robust elevation in response to ACh. [Ca 2 + ] i = Intracellular Ca 2 + concentration. (B) Summary of endothelial cell [Ca2+]i. Only treatment with ACh produced a response significantly different from the basal level. Values shown are means ± SEM; n = number of experiments. *: P b 0.05 compared to control and hydralazine.
Fig. 4. Hydralazine vasodilation is abolished by COX-1/2 inhibition, or by treatment with the prostacyclin receptor antagonist, RO1138452. (A) COX-1/2 inhibited vessels by indomethacin and ibuprofen. Values shown are means ± SEM; n = number of experiments. *: P b 0.05 compared to control. (B) The IP receptor antagonist RO1138452 was administered to vessels prior to hydralazine exposure. Values shown are means ± SEM; n = number of experiments. *: P b 0.05 compared to control.
On the other hand, inhibition of COX-1/2 with indomethacin and ibuprofen completely prevented hydralazine-induced vasodilation, indicating this to be a primary signaling pathway in mesenteric resistance vessels from pregnant animals. The latter distinction is important, as other studies have shown that pregnancy alters the balance of vasodilator release in resistance vessels, e.g. is associated with an enhanced EDHF component, at least in small uterine vessels from pregnant rats [43]. Because its identity is not known, and specific inhibitors are not available, we cannot eliminate a contribution of EDHF in response to hydralazine. However, the fact that pretreatment with RO1138452 – an antagonist with a high selectivity for IP receptors over other prostanoid receptors [33,34], – completely abolished the vasodilatory effects of hydralazine, suggests PGI2 to be the primary signaling pathway, at least under these in vitro conditions. Attenuation of the vasodilatory response with COX-1/2 inhibition further supports this notion, as EDHF effects are typically unaffected by cyclooxygenase blockade. Further support for this pathway comes from other studies that have shown that hydralazine increases vasodilator prostaglandins production in dogs [16], and that pretreating men with indomethacin prior to hydralazine treatment attenuated its hypotensive effects [44]. Additional evidence comes from a study that showed that the hemodynamic effects of hydralazine and PGI2 were strikingly similar, and suggested that PGI2 responsiveness (e.g. to iloprost, a synthetic prostacyclin analog) could be used to predict a patient's response to hydralazine [45].
Hydralazine also reduces the synthesis of the platelet-derived vasoconstrictor thromboxane A2 in vitro [46] and in hypertensive rabbits [47], supporting a cyclooxygenase mechanism in non-vascular tissues. Paradoxically, vessels pretreated with indomethacin and ibuprofen constricted to increasing doses of hydralazine, most prominently at concentrations ≥30 μM. This would suggest that hydralazine may also elicit minor vasoconstrictor responses that are normally overshadowed by the drug's more prominent vasodilatory effects. A similar constrictor effect was noted in the splanchnic vasculature by Maekawa et al. [48] when awake dogs were treated with indomethacin prior to hydralazine administration. They were able to abolish the response to indomethacin by co-treating with phentolamine, suggesting that hydralazine may also activate α-adrenoceptors. It is also interesting to note
Fig. 6. Proposed vasodilatory mechanism of hydralazine during late pregnancy at concentrations below 30 μM. Hydralazine appears to act via the prostacyclin (PGI2) pathway, most likely in an endothelium-dependent manner upstream of cyclooxygenase (COX). After being stimulated by hydralazine, the endothelium produces PGI2 which can then bind to PGI2 receptors on adjacent vascular smooth muscle (VSM) cells. AA = arachidonic acid, HDZ = hydralazine, IP = prostacyclin receptor, PGH2 = prostaglandin H2, PGI2 = prostacyclin, and PGIS = PGI2 synthase.
Please cite this article as: N. Maille, et al., Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.07.009
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that, pre-treatment with indomethacin reversed the hypotensive effect of hydralazine (average reduction of 23 mm Hg in mean aortic pressure), as the authors noted a “paradoxic” increase of 11 mm Hg. We could not find any studies that address this question in a clinical setting, but this would be a study of interest in view of the widespread use of over-thecounter NSAIDS having COX antagonistic properties (e.g. aspirin, ibuprofen, naproxen). It has been previously published that rabbit aorta strips [47] and cultured smooth muscle cells [49,50] treated with hydralazine release two vasodilator prostaglandins, PGI2 and prostaglandin E2. This may explain the apparent paradox that vasoconstriction was evident in the presence of COX inhibition but not IP blockade since RO1148452 may have blocked the vasodilatory effects PGI2, but not of PGE2, and the latter would still have been able to bind to its receptor and thus counteract the vasoconstriction secondary to α-adrenergic receptor activation. At concentrations N 30 μM, hydralazine had additional, endotheliumindependent actions on the resistance artery wall. These effects were also eliminated by COX-1/2 inhibition, or by treatment with RO1148452, suggesting that PGI2 may be produced by VSM in response to higher (pharmacological) concentrations of hydralazine. Additional experiments are required to substantiate this speculation. In a series of preliminary studies (data not shown), we observed regional differences in resistance artery sensitivity to hydralazine in both pregnant and nonpregnant states. In both cases, the EC50 in mesenteric arteries was significantly lower than that of similarly- sized uterine arteries (P N 0.05), although there were no differences in pregnant vs. nonpregnant animals. Regional differences in response to hydralazine have also been noted by other researchers in the nonpregnant rabbit [5,17] and dog [48]. In summary, this is the first study to characterize the vasoactive effects of hydralazine in resistance arteries during pregnancy. Our results indicate that its actions are complex, with direct endothelial and vascular smooth muscle effects that depend on concentration. At clinically-relevant concentrations, however, the vasodilatory action of hydralazine is primarily mediated by it activation of the cyclooxygenasePGI2 pathway in endothelium, and by IP receptor activation on vascular smooth muscle.
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Contents lists available at ScienceDirect
Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph
Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy Hydralazine induces vasodilation via a prostacyclin pathway Nicole Maille a, Natalia Gokina a, Maurizio Mandalà a,b, Ilsley Colton a, George Osol a,⁎ a b
Department of Obstetrics and Gynecology and Reproductive Sciences, University of Vermont College of Medicine, USA Department of Biology, Ecology and Earth Science, University of Calabria, Italy
a r t i c l e
i n f o
Article history: Received 14 May 2015 Received in revised form 23 June 2015 Accepted 17 July 2015 Available online xxxx Keywords: Hypertension Endothelium Vascular smooth muscle Prostacyclin Pregnancy
a b s t r a c t The cellular mechanisms of hydralazine-induced relaxation were investigated in isolated mesenteric resistance arteries from pregnant rats. Administration of hydralazine relaxed phenylephrine-constricted mesenteric arteries with an EC50 of 3.6 ± 0.3 μM and an efficacy of 75 ± 6.2%. These vasodilatory effects were abolished by: (1) preconstriction with a potassium depolarizing solution, (2) endothelial denudation (for concentrations of hydralazine b 10 μM), (3) addition of non-selective cyclooxygenase-1 and cyclooxygenase-2 inhibitors, and (4) pretreatment with a prostacyclin receptor antagonist (R01138452). Nitric oxide synthase (NOS) inhibition did not significantly alter the sensitivity or magnitude of the vasodilatory response; surprisingly, exposure to hydralazine also did not elevate endothelial cell Ca2+, suggesting a novel mechanism of activation. In summary, hydralazine is a potent resistance artery vasodilator that affects both endothelial and vascular smooth muscle (VSM) cells in a concentration-dependent manner. At clinically relevant concentrations (b10 μM), its effects in the splanchnic resistance vasculature are: (1) primarily endothelial in origin, require (2) hyperpolarization and (3) activation of COX, and (4) are mediated by the PGI2 (IP) receptor. © 2015 Published by Elsevier Inc.
1. Introduction Hydralazine is an antihypertensive drug that is used clinically to treat gestational hypertension resulting from conditions such as preeclampsia. Little, however, is known about its cellular mechanism of action on the vascular wall during pregnancy. Previous studies in male and nonpregnant female animal models have attributed its blood pressure-lowering effects to decreased total peripheral resistance rather than changes in cardiac output [1]. Several earlier studies have examined arterial responses to the administration of hydralazine, and have documented vasodilation in a number of in vitro arterial preparations including the rabbit aorta [2–4], rabbit renal artery [5], rat caudal artery [6], porcine coronary artery [7], and human digital artery [8]. The specific mechanisms, however, are poorly defined. For example, there is conflicting evidence regarding the role of the endothelium in hydralazine-induced relaxation. In vessels from nonpregnant animals, dilation still occurred in the absence of the endothelium in rat aortic rings [9–11], but not in rabbit aorta [4,12,13], rat caudal artery [14], and porcine coronary strips [7]. To date, several different mechanisms ⁎ Corresponding author at: Department of Obstetrics and Gynecology, The University of Vermont, College of Medicine, Burlington, VT 05405, USA. E-mail address: [email protected] (G. Osol).
have been proposed, including altered prostaglandin production, Ca2 + handling, membrane hyperpolarization, and cGMP production [7,15,16]. Interpretation has been further complicated by variable responses due to vessel type [5,17]. Some studies used conduit vessels, such as the aorta, which do not contribute to peripheral resistance, and/or concentrations of hydralazine far above those present in human plasma under clinical conditions (N100 μM) [18]. It is well known that pregnancy causes changes in systemic and uterine vessel reactivity and function through changes in both the endothelium and vascular smooth muscle cells [19–22]. It is therefore important to understand the mechanism by which hydralazine induces vasodilation during pregnancy. Since the splanchnic vasculature holds approximately one fourth of the total arterial blood volume, and given that it is a major contributor to overall peripheral resistance and blood pressure regulation, we used mesenteric resistance arteries from pregnant rats to evaluate the vasodilator effects of hydralazine during gestation. We hypothesized that hydralazine relaxation involves mechanisms that involve a combination of endothelial and vascular smooth muscle actions. The results indicate that, at concentrations that are comparable to those used clinically in humans (b10 μM) [18,23,24], hydralazine action on the vascular wall is predominantly endothelial, requires membrane hyperpolarization, and is mediated by cyclooxygenase-induced prostacyclin receptor (IP) activation.
http://dx.doi.org/10.1016/j.vph.2015.07.009 1537-1891/© 2015 Published by Elsevier Inc.
Please cite this article as: N. Maille, et al., Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.07.009
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N. Maille et al. / Vascular Pharmacology xxx (2015) xxx–xxx
2. Materials and methods
relaxed diameter determined in relaxing solution containing diltiazem and papaverine.
2.1. Animals Timed late pregnant (day 20/22 of gestation; n = 30) thirteen week old female Sprague–Dawley rats were purchased from Charles River Canada (Saint Constant QC, Canada) and housed at the University of Vermont Small Animal Facility under a 12 L:12D photoperiod; food and water were provided ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Vermont. Animals were euthanized with an intraperitoneal injection of pentobarbital sodium (50 mg/mL), followed by decapitation with a small animal guillotine. The abdominal cavity was then opened and a section of the mesentery 5 cm distal to the pylorus was excised and pinned in a Sylgard-lined Petri dish containing cold (4 °C) HEPES-physiological saline solution (HEPES-PSS, pH = 7.4). 2.2. Isolated vessel preparation Third-order mesenteric arteries with diameters between 200 and 300 μm at 50 mm Hg were dissected free of surrounding adipose and connective tissue and cannulated in the chamber of an arteriograph (Instrumentation and Model Facility, University of Vermont, Burlington, VT). For some experiments, vessels were mechanically denuded via hair abrasion [25] followed by air bolus perfusion [26] for 10 min at a low perfusion pressure (10 mm Hg). Deionized water was then gently passed through the lumen of the artery for 30–60 s to remove any endothelial debris and subsequently replaced with HEPES solution. Following cannulation of the distal end of each vessel, mesenteric arteries were pressurized to 50 mm Hg using a pressure-servo system (Living Systems Instrumentation, Burlington, VT) and positioned on the stage of an inverted microscope (Zeiss, West Germany). A pressure of 50 mm Hg was chosen for these experiments as it approximates in vivo conditions and prevents possible confounding effects by myogenic tone that appears at higher transmural pressures HEPES-PSS. In denuded vessels, successful removal of the endothelium was verified by the complete loss of acetylcholine (ACh)-induced relaxation at 1 μM in vessels preconstricted with phenylephrine. Each vessel was examined for leakage by turning off the servo null pressure feedback system in the absence of flow. If no decrease in the transmural pressure occurred within 2 min, the vessel was considered suitable; otherwise, it was removed and a fresh artery was cannulated, pressurized, and re-tested for leaks. 2.3. Concentration–response curves Vessels were equilibrated for 40 min in HEPES-PSS (pH = 7.4) at 37 °C. All experiments were performed at an intraluminal pressure of 50 mm Hg without intraluminal flow. Arteries were preconstricted using a superfusate containing either phenylephrine or a high potassium (35 mM) depolarizing solution to produce a 40–60% reduction in lumen diameter. Changes in intraluminal diameter were measured using a video dimension analyzer (Living Systems Instrumentation, Burlington, VT), as previously described [27,28], and recorded on LabView software. Hydralazine was administered in increasing concentrations (0.1–100 μM), and the resulting changes in diameter recorded once dilation stabilized at each concentration. At the end of each experiment, vessels were treated with relaxing solution containing a mixture of the L-type Ca2+ channel blocker, diltiazem (10 μM) and the phosphodiesterase inhibitor, papaverine (100 μM) to assure maximal vasodilation. Pharmacologic sensitivity was calculated using a standard curve analysis (SigmaPlot version 11.0; Systat Software Inc., San Jose CA) which determined the concentration of hydralazine necessary to produce 50% of the maximal vasodilatory effect (EC50). Efficacy was defined as the maximal relaxation to hydralazine as a percentage of the fully
2.4. Role of nitric oxide and prostaglandin production in hydralazineinduced vasodilation Vessels were pre-treated for 20 min with a combination of NωNitro-L-arginine (L-NNA, 100 μM) and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 μM) to inhibit nitric oxide (NO) production prior to exposure to phenylephrine. Other studies have shown this combination to be more effective in inhibiting nitric oxide synthase (NOS) than either drug alone [29]. To investigate the role of prostaglandins in hydralazine-induced vasodilation, vessels preconstricted with phenylephrine were treated with non-specific cyclooxygenase-1 and cyclooxygenase-2 (COX-1/2) inhibitors (indomethacin or ibuprofen, each at 10 μM) for 20 min prior to hydralazine administration. In some experiments, the PGI2 (IP) receptor antagonist, R01138452 (10 μM), was administered in the same manner as the COX-1/2 inhibitors to block the downstream effects of PGI2 production. 2.5. Effects of KCl depolarization on hydralazine-induced vasodilation Vessels were depolarized with a high potassium (35 mM) depolarizing solution prior to exposure to a single high concentration of hydralazine (100 μM). The relative (%) inhibition of hydralazineinduced vasodilation under depolarized conditions was calculated by the following equation: 1 − (% dilation of KCl-constricted vessel / % dilation of phenylephrine-constricted vessel) × 100. 2.6. Measurement of endothelial cell [Ca2+]i Endothelial cells (ECs) were loaded with Ca2 + sensitive fura-2 by perfusing the lumen of cannulated arteries with fura-2 AM-containing solution (5 μM) for 5 min at room temperature, as previously described [30]. To remove excess fura-2, the lumen was perfused with regular PSS for 10 min by using a 20 mm Hg intraluminal pressure gradient. Ratiometric measurements of fura‐2 fluorescence from ECs were performed using a photomultiplier system (IonOptix Inc. Milton, MA). Experimental ratios were corrected for background fluorescence taken from each artery before loading with fura 2-AM. Background-corrected ratios of 510 nm emission were obtained at a sampling rate of 5 Hz from arteries alternately excited at 340 and 380 nm. The experimental protocol was started following an additional 15 min equilibration period at 10 mm Hg to allow intracellular deesterification of fura-2 AM. In these experiments, hydralazine was tested at an intraluminal pressure of 50 mm Hg without phenylephrine to eliminate movement artifacts secondary to constriction. Following application of hydralazine, vessels were given ACh (10 μM) as a positive control for EC calcium elevation. EC intracellular calcium concentration ([Ca2+]i) was calculated using the following equation [31]: [Ca2+]i = Kdβ(R − Rmin) / (Rmax − R), where R is an experimentally measured ratio (340/380 nm) of fluorescence intensities, Rmin is a ratio in the absence of [Ca2+]i and Rmax is a ratio at Ca2+-saturated fura-2 conditions, β is a ratio of the fluorescence intensities at 380 nm excitation wavelength at Rmin and Rmax. Rmin, Rmax and β were determined by an in situ calibration procedure from the arteries treated with ionophores ionomycin (10 μM) and nigericin (5 μM). These values were then pooled and used to convert the ratio values into a [Ca2+]i. The Kd (the dissociation constant for fura-2) was 282 nM, as determined by in situ titration of Ca2+ in fura-2 loaded small arteries [32]. 2.7. Drugs and solutions HEPES-PSS contained the following (in mM): 141.8 sodium chloride, 4.7 potassium chloride, 1.7 magnesium sulfate, 2.8 calcium chloride, 1.2
Please cite this article as: N. Maille, et al., Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.07.009
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potassium phosphate, 10.0 HEPES, 0.5 EDTA, and 5.0 dextrose. High potassium (35 mM) HEPES solution contained the following (in mM): 106.8 sodium chloride, 35 potassium chloride, 1.7 magnesium sulfate, 2.8 calcium chloride, 1.2 potassium phosphate, 10.0 HEPES, 0.5 EDTA, and 5.0 dextrose. Both solutions were prepared with deionized water and titrated with sodium hydroxide to a physiologic pH of 7.4. For the endothelial calcium experiments, oxygenated (aerated with a mixture of 5% CO2, 10% O2, and 85% N2) PSS was used. PSS was composed of the following (in mM): 119 sodium chloride, 4.7 potassium chloride, 24.0 sodium bicarbonate, 1.2 magnesium sulfate, 0.023 EDTA, 1.6 calcium chloride, 1.2 potassium phosphate, and 11.0 glucose (pH 7.4). For the calibration procedure, we used a solution of the following composition: (in mM): 140 KCl, 20 NaCl, 5 HEPES, 5 EGTA, and 1 MgCl2 and 5 μM nigericin and 10 μM ionomycin, pH = 7.1. All chemicals for HEPES-PSS and solutions for reactivity experiments were purchased from Fisher Scientific (Fair Lawn, NJ). Pentobarbital sodium was purchased from Ovation Pharmaceuticals (Deerfield, IL). Phenylephrine, hydralazine, indomethacin, ibuprofen, L-NNA, L-NAME, papaverine, diltiazem, ionomycin and nigericin were purchased from Sigma Chemical Co. (St. Louis, MO). R01138452 was purchased from Cayman Chemical Co. (Ann Arbor, MI) and fura-2AM from Invitrogen (Carlsbad, CA). Stock solutions for indomethacin, ibuprofen, R01138452, and fura-2AM were prepared in dehydrated dimethyl sulfoxide (DMSO, Acros Organics, Fair Lawn, NJ). Other drug stock solutions were prepared with HEPES solution, all on a daily basis. 2.8. Statistical analysis All data are presented as mean ± SEM. The % dilation at each dose of hydralazine was averaged for all experiments within the same treatment. Differences in response to hydralazine for the phe-constricted vs. high potassium constricted vessels were determined by an unpaired ttest. A two way repeated measures analysis of variance with the Holm– Sidak multiple comparisons test was used to compare the control group to the treatment groups. P-values b 0.05 were considered significant. 3. Results 3.1. Relaxation of intact vessels in phenylephrine vs. KCl The lumen diameter of third order mesenteric vessels used in this study averaged 257 ± 5.2 μm at 50 mm Hg under fully relaxed conditions (n = 46 total vessels used for experimentation). Phenylephrine (0.5–5 μM) resulted in progressive constriction that averaged 45 ± 1.3%. The dilatory response to hydralazine was detected within 10 min of administration, and the maximum effect was obtained within 20 min. As shown in the experimental tracing in Fig. 1a, increasing concentrations of hydralazine induced relaxation in all vessels. The concentration required for half-maximal relaxation (EC50) was 3.6 ± 0.3 μM. Maximum dilation was observed at 100 μM (Fig. 1b) with an efficacy of 75 ± 6.2%. 3.2. Effects of endothelial denudation At concentrations below 30 μM, the vasodilatory effects of hydralazine were completely abolished in endothelium-denuded vessels. However, at 30 and 100 μM, hydralazine relaxed the denuded vessels 15 ± 11.5% and 72 ± 13.1%, respectively (n = 3; Fig. 1b), showing that it also had some direct action on VSM. 3.3. Mechanism of action High K+ depolarizing solution greatly attenuated vasodilatory responses to hydralazine such that exposure to 100 μM hydralazine produced weak vasodilation (6 ± 1.1%; n = 3; Fig. 2). This result
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suggests that hyperpolarization is an important component of the underlying mechanism. Vessels were pre-treated with NOS inhibitors L-NNA and L-NAME (n = 7) to evaluate the role of NO. A second group of vessels were treated with the COX-1/2 inhibitor indomethacin (10 μM; n = 5) to inhibit the production of prostaglandins. Responses to hydralazine were not significantly affected by inhibiting NO production, as the EC50 was calculated to be 3.9 ± 2.3 μM (Fig. 3). Efficacy was also unchanged (61 ± 10.1%). On the other hand, preventing prostaglandin production using indomethacin completely abolished the vasodilatory-response to hydralazine (Fig. 4a) and unmasked a slight vasoconstrictor effect at concentrations N 3 μM. To minimize the likelihood of non-specific effects, additional experiments were performed using a second COX-1/2 inhibitor ibuprofen (10 μM; n = 5), which yielded similar results. The role of PGI2, a known vasodilator prostanoid, was evaluated by pretreating vessels with 10 μM of the specific IP receptor antagonist RO1138452 [33,34]. In its presence, vasodilatory responses to hydralazine were completely abolished (Fig. 4b). Endothelial cells were loaded with fura-2 via intraluminal perfusion to examine the effect of hydralazine on endothelial [Ca2+]I, as this is a common observation in response to endothelium-dependent vasodilators such as ACh [35]. Following dye-loading, vessels were superfused with a solution containing 10 μM hydralazine for 20 min to elicit a maximum dilator effect. Surprisingly, there were no detectable changes in endothelial cell [Ca2+]i in response to hydralazine (n = 3), although a significant rise in endothelial [Ca2 +]i was observed in response to subsequent application of 10 μM ACh, which was used as a positive control. A representative tracing is shown in Fig. 5. 4. Discussion The main findings of this study (summarized in Fig. 6) are that: (1) hydralazine is a potent vasodilator of mesenteric resistance vessels from pregnant rats with direct actions on both endothelium and vascular smooth muscle. At clinically-relevant concentrations (1–10 μM), however, its primary actions are (2) endothelial, and involve a mechanism that requires (3) hyperpolarization and a (4) cyclooxygenase/prostacyclin/IP receptor signaling pathway. An earlier in vivo study in dogs noted that the splanchnic vascular bed is particularly highly sensitive to hydralazine [36] further highlighting the importance of understanding its mechanism in resistance vessels from this circulation. Although this drug has been used to treat hypertension in pregnant women for N60 years, and is still commonly used, to our knowledge, this is the first study to specifically examine its effects on resistance arteries from pregnant animals. Moreover, unlike several other studies have used mesenteric artery rings under isometric conditions, this study used pressurized vessels that allow direct measurement of lumen diameter, a principal determinant of blood flow resistance under physiological conditions. During treatment for hypertension and gestational hypertension, human plasma levels of hydralazine are on the order of 0.1–5 μM [18, 23,24]. In studies on conduit vessels such as the rabbit aorta and renal artery [3,4], endothelial denudation showed mixed results, with effects at ≤ 10 μM hydralazine being reduced or completely eliminated, but observable at concentrations N 10 μM. Our results indicate that its effects are completely endothelium-dependent, which is consistent with earlier findings in nonpregnant animal models [12–14]. Although we only tested one relatively high concentration of hydralazine (100 μM) in the KCl experiments, it is unlikely that any effects would have been seen at lower concentrations. The observation that hyperpolarization is requisite for dilation is consistent with one earlier report in rat caudal arteries [6]. In this regard, it was interesting to note that there were no measurable increases in endothelial cell calcium in response to hydralazine, since an increase in endothelial [Ca2+]i is typically associated with endothelial production of NO, EDHF and PGI2 [37–39]. Clearly, this was not a limitation of methodology, as ACh
Please cite this article as: N. Maille, et al., Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.07.009
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Fig. 1. The vasodilatory effects of hydralazine on preconstricted mesenteric arteries from late pregnant rats. (A) Representative experimental tracing showing the relationship between hydralazine concentration and arterial lumen diameter. Arrowheads indicate the application of phenylephrine to induce preconstriction. Increasing concentrations (0.5, 0.75, 1.0 μM) of phenylephrine were used to constrict the vessel to 40% before adding cumulative concentrations of hydralazine. To test the health of the endothelium, 1 μM ACh was added to fully dilate the vessel. Here, and elsewhere, % dilation was calculated relative to the response evoked with a maximum relaxing solution of 100 μM papaverine and 10 μM diltiazem (fully relaxed diameter is shown by the dotted line). (B) Hydralazine produced a concentration-dependent vasodilation of mesenteric arteries preconstricted with phenylephrine. The concentration of hydralazine needed to produce a half maximal response (EC50) was calculated as 3.6 μM using a best-fit curve analysis. Removal of the endothelium abolished vasodilation to lower concentrations of hydralazine (0.1, 1.0, 3.0 and 10 μM), while some endothelium-independent dilation was noted at higher concentrations (30, 100 μM). Values are means ± SEM; n = number of experiments. *: P b 0.05 compared to control.
induced a substantial calcium increase when given to the same vessels following hydralazine. Vasodilatory regulation by the endothelium is primarily attributable to three main signaling molecules; NO, endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin (PGI2) [40–42]. In this study,
Fig. 2. The vasodilatory effects of hydralazine require membrane hyperpolarization. Preconstriction with a 35 mM potassium depolarizing solution virtually eliminated arterial vasodilation in response to a high concentration (100 μM) of hydralazine. Values are means ± SEM; Phe = phenylephrine. HDZ = hydralazine. Phe = phenylephrine. n = 17 for Phe-constricted vessels and n = 3 for KCl-constricted vessels. *: P b 0.05 compared to control vessels constricted with Phe.
inhibition of NOS did not significantly alter hydralazine-induced relaxation, suggesting that hydralazine does not elicit the production of NO in this vascular bed. Wei et al. [7] also predicted an NO-independent mechanism in porcine coronary strips after finding that the addition of hemoglobin (a scavenger of NO) and L-NOARG (an inhibitor of NO production from L-Arg) did not prevent hydralazine-induced relaxation.
Fig. 3. Hydralazine-induced vasodilation is not mediated by NO production. Inhibition of NOS on vessels preconstricted with phenylephrine does not affect dilation to hydralazine. L-NNA = Nω-Nitro-L-arginine. L-NAME = Nω-Nitro-L-arginine methyl ester hydrochloride. Values are means ± SEM; n = number of experiments.
Please cite this article as: N. Maille, et al., Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.07.009
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Fig. 5. Hydralazine treatment failed to elevate intracellular endothelial calcium in mesenteric arteries.(A) Representative experimental tracing of endothelial intracellular calcium in response to a single 10 μM concentration of hydralazine, followed by treatment with ACh. Note the absence of calcium elevation to hydralazine, and robust elevation in response to ACh. [Ca 2 + ] i = Intracellular Ca 2 + concentration. (B) Summary of endothelial cell [Ca2+]i. Only treatment with ACh produced a response significantly different from the basal level. Values shown are means ± SEM; n = number of experiments. *: P b 0.05 compared to control and hydralazine.
Fig. 4. Hydralazine vasodilation is abolished by COX-1/2 inhibition, or by treatment with the prostacyclin receptor antagonist, RO1138452. (A) COX-1/2 inhibited vessels by indomethacin and ibuprofen. Values shown are means ± SEM; n = number of experiments. *: P b 0.05 compared to control. (B) The IP receptor antagonist RO1138452 was administered to vessels prior to hydralazine exposure. Values shown are means ± SEM; n = number of experiments. *: P b 0.05 compared to control.
On the other hand, inhibition of COX-1/2 with indomethacin and ibuprofen completely prevented hydralazine-induced vasodilation, indicating this to be a primary signaling pathway in mesenteric resistance vessels from pregnant animals. The latter distinction is important, as other studies have shown that pregnancy alters the balance of vasodilator release in resistance vessels, e.g. is associated with an enhanced EDHF component, at least in small uterine vessels from pregnant rats [43]. Because its identity is not known, and specific inhibitors are not available, we cannot eliminate a contribution of EDHF in response to hydralazine. However, the fact that pretreatment with RO1138452 – an antagonist with a high selectivity for IP receptors over other prostanoid receptors [33,34], – completely abolished the vasodilatory effects of hydralazine, suggests PGI2 to be the primary signaling pathway, at least under these in vitro conditions. Attenuation of the vasodilatory response with COX-1/2 inhibition further supports this notion, as EDHF effects are typically unaffected by cyclooxygenase blockade. Further support for this pathway comes from other studies that have shown that hydralazine increases vasodilator prostaglandins production in dogs [16], and that pretreating men with indomethacin prior to hydralazine treatment attenuated its hypotensive effects [44]. Additional evidence comes from a study that showed that the hemodynamic effects of hydralazine and PGI2 were strikingly similar, and suggested that PGI2 responsiveness (e.g. to iloprost, a synthetic prostacyclin analog) could be used to predict a patient's response to hydralazine [45].
Hydralazine also reduces the synthesis of the platelet-derived vasoconstrictor thromboxane A2 in vitro [46] and in hypertensive rabbits [47], supporting a cyclooxygenase mechanism in non-vascular tissues. Paradoxically, vessels pretreated with indomethacin and ibuprofen constricted to increasing doses of hydralazine, most prominently at concentrations ≥30 μM. This would suggest that hydralazine may also elicit minor vasoconstrictor responses that are normally overshadowed by the drug's more prominent vasodilatory effects. A similar constrictor effect was noted in the splanchnic vasculature by Maekawa et al. [48] when awake dogs were treated with indomethacin prior to hydralazine administration. They were able to abolish the response to indomethacin by co-treating with phentolamine, suggesting that hydralazine may also activate α-adrenoceptors. It is also interesting to note
Fig. 6. Proposed vasodilatory mechanism of hydralazine during late pregnancy at concentrations below 30 μM. Hydralazine appears to act via the prostacyclin (PGI2) pathway, most likely in an endothelium-dependent manner upstream of cyclooxygenase (COX). After being stimulated by hydralazine, the endothelium produces PGI2 which can then bind to PGI2 receptors on adjacent vascular smooth muscle (VSM) cells. AA = arachidonic acid, HDZ = hydralazine, IP = prostacyclin receptor, PGH2 = prostaglandin H2, PGI2 = prostacyclin, and PGIS = PGI2 synthase.
Please cite this article as: N. Maille, et al., Mechanism of hydralazine-induced relaxation in resistance arteries during pregnancy, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.07.009
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that, pre-treatment with indomethacin reversed the hypotensive effect of hydralazine (average reduction of 23 mm Hg in mean aortic pressure), as the authors noted a “paradoxic” increase of 11 mm Hg. We could not find any studies that address this question in a clinical setting, but this would be a study of interest in view of the widespread use of over-thecounter NSAIDS having COX antagonistic properties (e.g. aspirin, ibuprofen, naproxen). It has been previously published that rabbit aorta strips [47] and cultured smooth muscle cells [49,50] treated with hydralazine release two vasodilator prostaglandins, PGI2 and prostaglandin E2. This may explain the apparent paradox that vasoconstriction was evident in the presence of COX inhibition but not IP blockade since RO1148452 may have blocked the vasodilatory effects PGI2, but not of PGE2, and the latter would still have been able to bind to its receptor and thus counteract the vasoconstriction secondary to α-adrenergic receptor activation. At concentrations N 30 μM, hydralazine had additional, endotheliumindependent actions on the resistance artery wall. These effects were also eliminated by COX-1/2 inhibition, or by treatment with RO1148452, suggesting that PGI2 may be produced by VSM in response to higher (pharmacological) concentrations of hydralazine. Additional experiments are required to substantiate this speculation. In a series of preliminary studies (data not shown), we observed regional differences in resistance artery sensitivity to hydralazine in both pregnant and nonpregnant states. In both cases, the EC50 in mesenteric arteries was significantly lower than that of similarly- sized uterine arteries (P N 0.05), although there were no differences in pregnant vs. nonpregnant animals. Regional differences in response to hydralazine have also been noted by other researchers in the nonpregnant rabbit [5,17] and dog [48]. In summary, this is the first study to characterize the vasoactive effects of hydralazine in resistance arteries during pregnancy. Our results indicate that its actions are complex, with direct endothelial and vascular smooth muscle effects that depend on concentration. At clinically-relevant concentrations, however, the vasodilatory action of hydralazine is primarily mediated by it activation of the cyclooxygenasePGI2 pathway in endothelium, and by IP receptor activation on vascular smooth muscle.
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