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a Laboratoire de Recherche en Pharmacologie Périnatale, Hôpital Ste-Justine and Departments of Obstetrics and Gynecology and of Pharmacology, University of Montréal, Montréal, Québec, Canada H3T 1C5
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-adrenergic agonists and other vasoconstrictors on aortic vessels during pregnancy in the rat has been reported [2, 3]. In mesenteric resistance arteries, it was observed that reduced sensitivity to phenylephrine, an 1-adrenergic vasoconstrictor, was not mediated by endothelium-derived relaxing factor (EDRF) or prostaglandins [4]. This indicates that endothelium, or at least EDRF, might not be the principal mechanisms involved in the blunted vasopressor responses in the vasculature during pregnancy. Some mechanisms have been proposed to explain these changes during pregnancy [5, 6]. For instance, down-regulation of membrane receptors for vasopressor ligands (vasopressin, norepinephrine, etc.) in vascular tissues, increased liberation of an endogenous vasodilator acting as physiological antagonist (prostacyclin, nitric oxide, etc.) to vasopressor, modifications of mechanical properties and/or tissue composition (ratio of smooth muscle to connective tissue, or of elastin to collagen, etc.) leading to increased compliance of vessels, and alterations (or uncoupling) of receptor-response coupling have been documented; but this work has not provided conclusive results so far.
Recently, we reported that functional involvement of voltage-operated calcium channels in myotropic responses to vasoconstrictors is impaired in aortic rings and mesenteric resistance arteries of pregnant compared to virgin rats [7, 8]. Calcium is a major intermediate in the mechanisms that trigger contraction in vascular smooth muscle. An elevation in intracellular calcium allows the interaction of contractile myofilaments, thus causing contraction [9]. Calcium required for this interaction comes from both intracellular stores and the extracellular space, the latter through calcium channels. In the present study, we tested the hypothesis that blunted responses to vasoconstrictor agents during normal pregnancy in the rat are linked to an altered mobilization of extracellular calcium in vascular smooth muscle through voltage-operated calcium channels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Sprague-Dawley rats (Charles River Canada, St-Constant, PQ, Canada) aged 10–11 wk were mated with age-matched males. The morning on which vaginal smears were found to contain spermatozoa was labeled Day 1 of pregnancy. The pregnant females were then placed in individual cages until used on the 22nd day of gestation. Virgin rats of the same age served as controls without regard to the stage of the estrous cycle. The animals were housed in facilities of the Research Centre at Hôpital Ste-Justine, which is accredited by the Canadian Council on Animal Care. The protocol was approved by the local animal care committee.
Chemicals
All salts employed in these experiments were of analytical grade and were obtained from Fisher Scientific (Montréal, PQ, Canada). Acetylcholine, Hepes, EGTA, and LaCl3 were purchased from Sigma Chemical Co. (St. Louis, MO). Nifedipine hydrochloride, Bay K 8644 (methyl ester), phenylephrine hydrochloride, verapamil, D-600, and diltiazem were obtained from Research Biochemical International (Natick, MA). 45Ca2+ (~10 Ci/g) and Ecolite(+) were obtained from ICN. [3H]Isradipine (specific activity 73 Ci/mmol) was obtained from DuPont Canada (Mississauga, ON, Canada). The dihydropyridines were prepared as stock solution in 95% ethanol in vials protected from light. Ethanol concentrations in the tissue baths did not exceed 0.1% and were verified by adding the same concentrations of vehicle in control tissues.
Organ Bath Assay
After decapitation, the thoracic aorta was rapidly removed and placed in cold Krebs bicarbonate solution (KBS). It was cleaned of fat and extraneous tissues and cut into four consecutive rings (2–3 mm), which were mounted on stainless steel hooks and placed in individual jacketed tissue baths (10 ml; Radnotti Glass, Monrovia, CA) maintained at 37°C. In order to exclusively study the regulation of Ca2+ mobilization in smooth muscle of the vessels, the lumen of each ring was rubbed with a wooden stick to remove the endothelium. In each experiment, four rings each for both virgin and pregnant rats were used. They were equilibrated for 60 min under 2.0 g passive tension, the optimal tension for both groups of tissues [2], with frequent washing and tension adjustment. The tissues were bathed in KBS of the following composition: 118 mM NaCl, 4.65 mM KCl, 25 mM NaHCO3, 2.5 mM CaCl2, 1.18 mM MgSO4, 1.18 mM KH2PO4, 5.5 mM dextrose. The solution was bubbled with a mixture of 95% O2:5% CO2; pH was 7.4. After equilibration, the tissues were challenged with 1.0 µM phenylephrine. At plateau response, acetylcholine (0.1 mM) was added to verify removal of the endothelium. Tension was measured by force-displacement transducers (FT-03; Grass Instruments, Quincy, MA) and recorded on a Grass polygraph (Model 7E) or computerized data acquisition system using Work Bench software (Kent Scientific, Litchfield, CT). The experiments with calcium channel blockers were performed under a sodium lamp to prevent photodegradation of these substances.
Protocol
Responses to phenylephrine in Ca2+-free KBS In order to verify the involvement of extra- and intracellular Ca2+ in the myotropic response to phenylephrine, aortic rings from virgin and pregnant rats were challenged with phenylephrine (20 nM). After the response was obtained and tissues were reequilibrated (~45 min), the rings were bathed in Ca2+-free KBS (CaCl2 omitted) for 15 min; then the effect of the same concentration of phenylephrine was measured. When this response had plateaued, CaCl2 (2.5 mM) was reintroduced in the bathing solution and the response recorded. Aortic rings were allowed to reequilibrate for 90 min, and the same protocol was again applied using phenylephrine at 1.0 µM as stimulant.
Concentration-response curves to calcium To evaluate the mobilization of extracellular Ca2+ in myotropic responses and the involvement of voltage-operated calcium channels, concentration-response curves to CaCl2 were measured after application of phenylephrine or KCl in the absence of CaCl2 in the bathing solution. Four aortic rings from each virgin and pregnant rat were used; two rings of each group were challenged with phenylephrine and the other two with potassium chloride (KCl). Phenylephrine (20 nM or 1.0 µM) and KCl (20 or 80 mM) was used in normal KBS to obtain a control contraction. After 60 min of equilibration, the aortic rings were bathed in calcium-free KBS supplemented with 0.5 mM EGTA. After 10 min, tissues were rinsed in the same solution for another 10 min; the solution was then changed for only calcium-free Krebs (EGTA omitted), and the rings were challenged with the same concentration of phenylephrine or KCl. Tissues were washed with calcium-free solution and restimulated (after 15 min relaxation) with the same concentration of vasoconstrictor. When this response had stabilized, a concentration-response curve was constructed with CaCl2 (10 µM–2.5 mM). At the end of this protocol, tissues were washed with normal KBS and allowed to rest for 90 min. The same protocol was repeated except that the second application of the stimulant in calcium-free KBS was immediately followed by the addition of D-600, a phenylalkylamine calcium channel blocker. The concentrations of the blocker used were 30 nM and 1.0 µM for KCl and phenylephrine stimulation, respectively.
Effects of D-600 on concentration-response curves to phenylephrine and KCl In order to precisely evaluate the differential effects of D-600 on myotropic responses to receptor-stimulated and depolarization-induced myotropic responses, cumulative concentration-response curves to both phenylephrine (10-9 to 10-4 M) and KCl (2–100 mM) were consequently measured in 4 adjacent aortic rings of both virgin and pregnant rats in the absence and the presence of different concentrations of D-600. The order of the stimulants alternated in each experiment. One tissue from each group was used as control while the other three were bathed in increasing concentrations of D-600 (10-8, 10-7, and 10-6 M) applied 10 min before the first concentration of either phenylephrine or KCl. An equilibration period of 90 min was allowed between the measurement of the two concentration-response curves.
Calcium Uptake and Influx Experiments
Measurement of 45Ca2+ uptake was used to evaluate the contribution of extracellular Ca2+ in myotropic responses. Aortic rings prepared as described above were used in duplicate under a passive tension of 2.0 g. A modified version of the protocol of Meisheri et al. [10] was used. Briefly, the rings were suspended in tissue baths bubbled with 100% O2 in a physiological salt solution (PSS) of the following composition: 118 mM NaCl, 4.65 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, 11 mM dextrose, 5 mM Hepes; pH was 7.40 at 37°C. After 1 h of equilibration, the protocol was started.
In the first experiments, basal and stimulated 45Ca2+ uptake was measured by simultaneous exposure of aortic rings from virgin rats to 0.5 µCi 45Ca2+ and KCl (100 mM). Stimulated 45Ca2+ uptake was measured in the absence and presence of calcium channel blockers: nifedipine (1 µM), diltiazem (10 µM), and verapamil (10 µM). 45Ca2+ uptake was also measured in aortic rings of pregnant and virgin rats stimulated with phenylephrine (1 µM), KCl (100 mM), and Bay K 8644 (1 µM). Each experiment included a basal uptake and a nonspecific uptake (in the presence of 50 mM LaCl3).
The time course for 1–30 min of basal and stimulated (100 mM KCl) calcium influx was assessed by exposing tissues to 45Ca2+. It was also measured after phenylephrine-sensitive calcium pools were emptied by stimulating the aortic rings with 1 µM phenylephrine in a calcium-free PSS containing 2 mM EGTA for 10 min. Two wash periods of 20 min each in calcium-free PSS followed. The stimulation with phenylephrine was repeated in the absence of EGTA. At the end of the last wash period, tissues were put into normal PSS containing 45Ca2+ (0.5 µCi) for various periods of time (2–45 min).
After the protocol was completed, tissues were placed in ice-cold calcium-free PSS containing 50 mM LaCl3 for 20 min. Rings were then blotted, weighted, and dissolved in H2O2:perchloric acid (1:1) for 2 h at 37°C. Scintillation liquid (5 ml; Ecolite (+); ICN, Montréal, PQ, Canada) was added, and radioactivity was counted in a beta counter (LS 500; Beckman, Orange, CA) with a counting efficacy of 50%. Specific uptake was calculated by subtracting nonspecific (in the presence of LaCl3) uptake from total uptake.
Binding of [3H]Isradipine to Aortic Rings
Studies of [3H]isradipine binding to intact aortic rings of rats were conducted according to the method of Morel and Godfraind and Godfraind et al. [11, 12]. These experiments were undertaken to verify whether, in polarized preparations, binding of dihydropyridine was affected by polarization status of the cells. Aortic rings (used as triplicates) of each group were attached to a glass rod to facilitate handling and then submerged, without application of passive tension, in PSS, bubbled with 95% O2:5% CO2, of the following composition: 122 mM NaCl, 15 mM NaHCO3, 5.9 mM KCl, 1.25 mM CaCl2, 1.2 mM MgCl2, 11 mM glucose. Tissues were equilibrated for 60 min in PSS and then placed in normal PSS containing 40 pM [3H]isradipine for 45 min. Aortic rings were then transferred in baths containing 5.9–100 mM KCl for 45 min with 40 pM [3H]isradipine. After this incubation period, tissues were blotted dry, weighted, dissolved, and counted as described above. All experiments had a nonspecific binding group, e.g., in the presence of 1.0 µM nifedipine for the two final incubations. Specific binding was obtained by subtracting the nonspecific from the total binding for each group.
[3H]Isradipine total and nonspecific binding was measured for various times. After the equilibration period, tissues were placed in normal PSS containing 40 pM [3H]isradipine for times between 15 and 90 min in baths containing 5.9 mM and 100 mM KCl. Nonspecific binding kinetics was evaluated by the presence of 1.0 µM nifedipine in separated tubes.
Data Analysis
Each concentration curve was analyzed by computer fitting to a 4-parameter logistic equation with the program Prism (GraphPad Software, San Diego, CA) to evaluate the concentration producing 50% of the maximal response (EC50) and the maximum asymptote of the curve (Emax, maximum response). When phenylephrine was used to prestimulate tissues in the measurement of concentration-response curves to CaCl2 (see Fig. 2), the lower asymptote used was the stabilized residual response to phenylephrine. Different curves in the same protocol were compared by a two-way ANOVA on mean pD2 (negative logarithm of the EC50), on mean Emax, and on the lower asymptote of the curve (i.e., when required). Data are expressed as mean experimental points with their standard error (SEM), together with the best-fitted curve to these points. 45Ca2+ uptake and [3H]isradipine binding data were compared using a two-way ANOVA. Data were considered significant when a probability smaller than 5% (p < 0.05) was reached.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To measure the contribution of extracellular calcium in the myotropic responses of aortic rings, concentration-response curves to CaCl2 were constructed in both groups of aortic rings in the absence and presence of D-600, a phenylalkylamine calcium channel blocker. Phenylephrine and KCl were both used at low (~EC50) and high (near maximal) concentrations to trigger contraction. These were applied in calcium-free KBS, after the KBS was changed from a calcium-free solution containing 0.5 mM EGTA, according to the protocol described above. Figure 2 describes the results obtained with phenylephrine (upper panels) and with KCl (lower panels). With the use of phenylephrine in the absence of calcium, there was a residual contraction that was significantly greater in aorta of pregnant than of virgin rats as indicated by the lower asymptote of the curves, which is described by the first response to 10-5 M calcium. In both groups of aorta, this residual response to phenylephrine was markedly blocked by D-600 (1.0 µM, open symbols), to a similar magnitude, in the two groups of tissues. Nevertheless, the residual responses remained higher in aortic rings of pregnant than of virgin rats. These residual contractions were not observed with KCl (Fig. 2, C and D).
When this residual contraction to phenylephrine had stabilized, a concentration-response curve to CaCl2 was measured. Each concentration of CaCl2 induced increases in tension that reached higher levels in tissues of pregnant than of virgin rats. Maximal contraction on these tissues was 1.00 ± 0.12 vs. 1.32 ± 0.11 g (p < 0.05) for virgin and pregnant rats with 20 nM phenylephrine but were not different upon stimulation with 1.0 µM phenylephrine (2.66 ± 0.11 and 2.41 ± 0.10 g, respectively; not significant). Considering the difference in the starting level of each curve from aortas of virgin and pregnant rats, the spread between lower asymptote and maximal response was not statistically different. D-600 (1.0 µM) markedly reduced the effects of the concentration-dependent addition of CaCl2 to a similar level in both groups of aortic rings in both prestimulatory conditions. The larger responses to CaCl2 in aortic rings of pregnant than of virgin rats also appear to be dependent on a higher level of residual contraction in the absence of calcium. Sensitivity to CaCl2 (pD2, i.e., negative logarithm of EC50) did not significantly vary in the two groups of aorta with the two concentrations of phenylephrine. These results suggest that once -adrenergic receptors are activated, sensitivity for calcium of the postreceptor coupling mechanisms is not modified in blood vessels by pregnancy. These results also support the observation in Figure 1 that intracellularly stored calcium released by -adrenergic stimulation is increased in vascular smooth muscle of pregnant rats.
Similar experiments were performed with KCl (20 and 80 mM) as the stimulating agent (Fig. 2, C and D). It should be noted that in these experiments, D-600 was used at a much lower concentration, e.g., 30 nM, since it is much more potent when KCl is used as a stimulant (see Fig. 3). KCl, in the absence of extracellular calcium, did not elicit contraction, as shown by the lower asymptote of the concentration-response curves. With the use of 20 mM KCl to prestimulate the tissues, contractions to CaCl2 were similar, except at the highest concentration of CaCl2, in both groups of tissues. When D-600 (30 nM) was added to the bathing solution, concentration-response curves to CaCl2 were similarly decreased on both tissues (Fig. 2C). With 80 mM KCl, maximal contraction to CaCl2 addition was similar in the two groups of tissues (virgin, 2.43 ± 0.11 g and pregnant, 2.59 ± 0.06 g; not significant). D-600 markedly reduced maximal contraction and sensitivity to CaCl2 in both groups of tissues. D-600 seems to be similarly effective in the two groups of tissues when KCl is used to trigger contractile mechanisms.
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Concentration-response curves to phenylephrine and KCl were measured in the absence or presence of D-600 at various concentrations (Fig. 3). On aortic rings from virgin animals (Fig. 3A), D-600 significantly reduced, in a concentration-dependent manner, both maximal contraction and sensitivity to phenylephrine. Similar effects were observed on aortic rings from pregnant rats (Fig. 3B). The decreased sensitivity to phenylephrine in the presence of D-600 was similar in aortic rings of pregnant and virgin rats, but a more important effect of the calcium channel blocker (at 0.1 and 1.0 µM) was observed on maximum responses to phenylephrine in virgin compared to pregnant rats.
The control (absence of D-600) concentration curve to KCl on tissues from virgin rats showed a higher maximal contraction than the curve from pregnant rats (2.54 ± 0.08 vs. 2.25 ± 0.11 g, respectively, p < 0.05). Each concentration of D-600 produced a significant progressive decrease in maximal response to KCl on tissues from both groups of rats (Fig. 3, C and D). Sensitivity to KCl was not modified in the presence of D-600. The blocking effect of D-600 on maximum responses to KCl was slightly larger in aortic rings of virgin than of pregnant rats.
The specificity of the 45Ca2+ uptake through voltage-operated calcium channels was assessed by stimulating the rings with 100 mM KCl and blocking the uptake with 3 classes of calcium channel blockers: nifedipine (1 µM), a dihydropyridine; verapamil (10 µM), a phenylalkylamine; and diltiazem (10 µM), a benzothiazepine (Fig. 4A). 45Ca2+ uptake increased more than 2-fold over basal uptake (180 ± 26 vs. 86 ± 9 µmol 45Ca2+/kg tissue [w/w], respectively, p < 0.05). The increase in 45Ca2+ uptake was completely blocked by the various voltage-gated calcium channel blockers used, i.e., nifedipine, verapamil, and diltiazem, indicating the very high specificity for these drugs to block voltage-gated calcium channels.
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Basal 45Ca2+ uptake was significantly lower in aortic rings of pregnant than of virgin rats (Fig. 4B). Maximal stimulation with 100 mM KCl or 1.0 µM phenylephrine increased 45Ca2+ uptake that reached, for each stimulant, similar levels in tissues of virgin and pregnant rats. In contrast, Bay K 8644, a dihydropyridine calcium channel activator, increased 45Ca2+ uptake in both groups of rings, but the difference between aorta of virgin and pregnant rats that was seen under basal conditions was maintained (p < 0.05).
The time course of basal 45Ca2+ uptake was also measured in aortic rings of virgin and pregnant rats (Fig. 5A). From 2 min up to 30 min, basal (not stimulated) uptake was significantly lower in tissues of pregnant than of virgin rats. Upon stimulation with 100 mM KCl (Fig. 5B), 45Ca2+ uptake increased markedly in comparison to that in the absence of stimulation (note the difference in the scale of the ordinate in both panels of Fig. 5). Stimulated 45Ca2+ uptake was significantly decreased in tissues of pregnant compared to virgin rats. The results show that 45Ca2+ uptake was delayed, an effect that was observed on both basal and stimulated (100 mM KCl) uptake.
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45Ca2+ uptake was also measured after emptying of phenylephrine-sensitive calcium pools in calcium-free Krebs supplemented with 2 mM EGTA. Upon simultaneous addition of 1.5 mM CaCl2 and 45Ca2+, basal 45Ca2+ uptake was statistically lower in aortic rings of pregnant compared to virgin rats (Fig. 6). Again, these results suggest that basal 45Ca2+ uptake is somewhat impaired in aortic rings of pregnant compared to virgin rats.
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Such a finding could be explained by a difference in the equilibrium of activated to inactivated state of voltage-operated calcium channels in the basal condition that will affect the basal activity of the channel as well as binding of dihydropyridines to channel molecules [12]. To test such a possibility, we measured binding of a tritiated voltage-gated calcium channel blocker, [3H]isradipine, in whole aortic rings of virgin and pregnant rats. The bathing solution had increasing concentrations of KCl to depolarize the tissues and thereby modify calcium channel affinity for the tritiated ligand. We observed similar elevation in [3H]isradipine binding, directly proportional to the increase in concentration of KCl in the bathing solution, for both groups of aorta (data not shown). The time course of [3H]isradipine binding was time-dependent (Fig. 7), as shown by an increase in binding with time that was significant in the presence of 100 mM KCl at all times. We also saw that the increase in binding in the presence of 100 mM KCl was higher on tissues of virgin (0.98 ± 0.15 vs. 2.20 ± 0.16 fm/mg tissue, at 5.9 mM and 100 mM KCl, respectively, p < 0.05) compared to pregnant rats (1.27 ± 0.10 vs. 1.78 ± 0.15 fm/mg tissue, at 5.9 mM and 100 mM KCl, respectively, p < 0.05). Nonspecific binding kinetics was similar in the two groups of tissues and did not vary significantly over time (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pregnancy-associated diminished responses to vasopressors have been consistently reported [3, 5, 13, 14], but the underlying mechanisms have not been elucidated. The contraction of vascular smooth muscle induced by 1-adrenoceptor activation is known to be caused by both calcium release from intracellular stores and calcium influx through calcium channels [15]. This increase in calcium acts on a complex cascade of enzymes that phosphorylate several elements of the contractile apparatus to cause contraction. In the present experiment we observed, in the absence of extracellular calcium, increased responses to phenylephrine, an 1-adrenoceptor agonist, in aortic rings of pregnant compared to virgin rats (Figs. 1 and 2). This residual contraction is apparently triggered by the release of intracellularly stored calcium, since it was also observed in the presence of EGTA in the bathing solution (Fig. 2). The increased residual response in aorta of pregnant rats could be caused by a higher quantity of Ca2+ in intracellular stores, a facilitated Ca2+ release from these stores, or a higher inhibition of the reuptake mechanisms by the stores. However, Ezimokhai et al. [13] reported that the effects of phenylephrine, in the absence of extracellular calcium (+ EGTA), were similar in aortic rings of virgin and pregnant rats. It appears that their protocol did not allow for complete refilling of the intracellular pools before testing phenylephrine.
We have previously reported that extracellular calcium mobilization could be impaired during pregnancy [7, 8]. To evaluate the influence of extracellular calcium in the decreased vascular reactivity of gestation, we measured concentration-response curves to CaCl2 on aortic rings prestimulated, in the absence of CaCl2, by receptor-dependent (phenylephrine) and voltage-dependent (KCl) stimuli (Fig. 2). Our results did not provide evidence for altered extracellular Ca2+ mobilization during pregnancy. However, they indicate that perhaps the initial activation and/or setting of voltage-operated calcium channels is altered in blood vessels of pregnant rats. Ezimokhai et al. [13] also reported concentration-response curves to CaCl2 after prestimulation (in the absence of Ca2+) with phenylephrine and KCl. They observed significantly greater responses for each CaCl2 addition in aortic rings from nonpregnant as compared to pregnant rats. Their experiments were carried out in intact aortic rings (endothelium present), while ours were performed on denuded preparations. It is suspected that this situation had an influence on the reactivity of the underlying smooth muscle, particularly upon reintroduction of calcium—an interpretation that was shared by these authors [13]. However, in the experiments reported here, endothelium-derived interacting influences are unlikely to have interfered.
Calcium channel blockers bind to the voltage-operated calcium channels and block voltage-dependent calcium influx. In so doing, they interfere with induced contractions [7, 16, 17]. The efficacy of D-600 was lower on tissues of pregnant rats under both phenylephrine and KCl (Fig. 3). The concentration-response curves to KCl in the presence of D-600 were similar to the ones we published earlier with nifedipine [7]. D-600, used in the present study, is a phenylalkylamine that binds to the 1 subunit of the calcium channel but at a site different from that for nifedipine [18]. These results show that the two types of calcium channel blockers act in similar way and with similar potency to block KCl responses. On the other hand, D-600 was clearly more efficient in the present study than nifedipine [7] in blocking responses to phenylephrine. Indeed, as shown in Figure 2, a concentration of D-600 that was 33 times larger (1 µM vs. 30 nM) was required to block the CaCl2 responses after stimulation with phenylephrine than with KCl. This confirms the previously reported observation [7] that voltage-operated calcium channels are implicated at a given level of the myotropic response of the aorta to phenylephrine. This raises at least a couple of question. Why would different calcium blockers have different efficacy against phenylephrine responses? Why are these blockers more efficient on CaCl2 responses after prestimulation with phenylephrine (Fig. 2, A and B) than on direct responses to the agonist (Fig. 3, A and B)? These questions remain to be fully investigated.
Calcium influx is needed for the tonic component of vascular smooth muscle contraction [13, 15]. It was reported that when the concentration of CaCl2 in the bathing solution was reduced to 0.8 from 1.6 mM, responses of aortic rings of pregnant rats to phenylephrine were less affected than those of their nonpregnant counterparts [13]. These authors conclude that the dependence of rat aorta on extracellular calcium for phenylephrine-induced contraction is diminished during pregnancy. Such decreased dependence on extracellular Ca2+ seen in pregnancy strongly suggests an impairment in the major calcium influx pathway, the voltage-operated calcium channels. Previous studies in our laboratory [7, 8] showed a decreased effect of calcium channel blockers of the dihydropyridine type on aorta and mesenteric resistance arteries during pregnancy. This could not be the result of regulation of voltage-operated calcium channels, since density and affinity of binding sites for [3H]isradipine were not changed in membrane preparations of aorta [7] or mesenteric vasculature [8] of pregnant rats. Similar results were reported by Ikeda et al. [19] in tissues from spontaneously hypertensive and normotensive rats. The results of that study show that physiological or pathological alterations of blood pressure do not involve a prior alteration of the density of voltage-gated calcium channels on the vascular smooth muscle. These results reinforce our belief that voltage-operated calcium channels are functionally altered during pregnancy and that this mechanism could be responsible for blunted responses to vasoconstrictor associated with this condition.
Specific binding of [3H]isradipine was examined over a wide range of KCl concentrations in the intact aorta from spontaneously hypertensive and Wistar-Kyoto rats [12]. These results indicated that proportions of 30% and 5% of calcium channels were in a high-affinity state in aorta of Wistar-Kyoto and spontaneously hypertensive rats, respectively, under physiological conditions. We have performed similar experiments in aorta of pregnant and virgin Sprague-Dawley rats and did not observe any difference between the two groups of rats. On the other hand, the time-course experiments, either in physiological or depolarizing (100 mM KCl) PSS, showed that binding of [3H]isradipine increased with time and was always significantly higher on aortic rings of virgin compared to pregnant rats (Fig. 7). This supports the concept of functional reduction of voltage-operated calcium channels in blood vessels of pregnant rats that is manifested by slower activation process.
When the cells are depolarized, the voltage-gated calcium channels open and extracellular calcium enters the cell [15, 19, 20]. To verify this aspect, we showed a total inhibition of 45Ca2+ uptake induced by KCl with a variety of calcium blockers, indicating that this influx was indeed mediated through such channels. Results shown in Figure 5A support the interpretation that function of these channels is impaired during pregnancy. Indeed, 45Ca2+ uptake was stimulated by a dihydropyridine calcium channel activator, Bay K 8644. Both basal and Bay K 8644-stimulated 45Ca2+ uptake were significantly higher in aorta of virgin than of pregnant animals. We have previously reported that the contractile effect of Bay K 8644 is markedly reduced during pregnancy [7]; the present results suggest that this observation may be the consequence of reduced voltage-operated calcium channel function, since a reduced 45Ca2+ uptake with Bay K 8644 was observed. Increase of the contractile effect of Bay K 8644 was reported in aorta of rats with coarctation-induced hypertension, indicating that a specific altered (increased) function of voltage-operated channel is linked to the development of hypertension in this model [21]. A similar mechanism, but of opposite direction, is proposed to occur during gestation on the basis of the present and previous [7] findings. The mechanism of this decreased calcium channel function suspected to be involved can be a delayed activation of Ca2+ influx from extracellular milieu. Indeed, both basal and KCl-stimulated (Fig. 5) 45Ca2+ uptake were delayed in aorta of pregnant compared to nonpregnant rats. Moreover, when phenylephrine-sensitive intracellular calcium pools had been previously emptied (Fig. 6), delayed uptake of 45Ca2+ was still observed in tissues of pregnant rats compared to nonpregnant.
We have previously shown that the decreased effect of Bay K 8644 on aortic rings of pregnant rats can be modulated with KCl [7]. In the present study, we have observed a reduced Bay K 8644-stimulated 45Ca2+ uptake on aortic rings from pregnant rats. Calcium channel modulators act by changing the opening probability of channels—activators (Bay K 8644) increasing and blockers (nifedipine) decreasing the probability [22]. In electrophysiological studies, dihydropyridine blockade of L-type calcium channels in vascular cells was enhanced by decreasing the holding potential [23]. These channels exist in three states: closed, activated, and inactivated [24]. The time a calcium channel spends in a given state is regulated by membrane potential, and the open configuration increases exponentially with membrane depolarization [25]. KCl acts by modifying the membrane potential and hence the conformation of the voltage-operated calcium channels. This is probably the mechanism by which KCl reestablishes the effects of Bay K 8644 on aortic rings of pregnant rats [7], indicating that the absence of direct effect of Bay K 8644 in aortic rings of pregnant rats is caused by an increased membrane potential. It is considered that a hyperpolarization of 2 mV could decrease the probability of there being open channels by around 30% [25], meaning that a greater proportion of the channels are in a closed state—the conformation of the channels to which blockers have the lower affinity [18]. It should be noted that Meyer et al. [26] have shown that smooth muscle of the rat mesenteric vascular bed is hyperpolarized by around 7 mV in pregnancy, an observation that supports our interpretation.
Our study clearly demonstrates a decrease of reactivity to vasoconstrictor agents on isolated aorta from pregnant rats compared to virgin ones. The blunted responses to vasoconstrictors could be attributed to a decreased function of calcium channels through a delayed extracellular calcium uptake by vascular smooth muscle. It is suggested that this results from a slower activation of calcium channels in vascular smooth muscle during pregnancy. Action of the calcium channel activator, Bay K 8644, could be modulated by KCl, suggesting a change in membrane potential during pregnancy. The mechanism of these modifications remains to be investigated.
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FOOTNOTES |
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Accepted: November 25, 1998.
Received: June 23, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 M. Simaan, S. Picard, J. St-Louis, and M. Brochu Functional alteration of dihydropyridine-sensitive Ca2+ channels in the adrenal glomerulosa of pregnant rats Am J Physiol Endocrinol Metab, May 1, 2000; 278(5): E925 - E932. [Abstract] [Full Text] [PDF]
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R. Venuto, G. Brown, M. Schoenl, and G. Losonczy Enhanced vascular effects of the Ca2+ channel agonist Bay K 8644 in pregnant rabbits Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2002; 282(4): R952 - R959. [Abstract] [Full Text] [PDF]
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