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Department of Obstetrics and Gynecology, University of Vermont, College of Medicine, Burlington, Vermont
Submitted 1 August 2005 ; accepted in final form 16 November 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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acetylcholine; intraluminal pressure; fura-2; manganese quenching
Extensive studies have shown that late pregnancy is a state of increased basal and stimulated production of endothelium-derived vasodilators in the maternal uterine circulation (2, 3, 12, 17, 29, 43, 49, 52). Uterine vasodilation, induced both by chemical (ACh) or mechanical stimulation (shear stress), was significantly enhanced during gestation, suggesting that some common mechanism(s) might underlie the pregnancy-induced adaptive changes in endothelial cell function (1, 9, 10, 30, 36, 38, 49, 54). Enhanced release of nitric oxide (NO) and prostacyclin in uterine arteries in response to agonist stimulation has been documented in both animal and human pregnancy and is associated with elevated endothelial NO synthase (eNOS) and cyclooxygenase activity and expression (2, 3, 29, 37, 49, 53, 54, 56).
An increase in the concentration of intracellular Ca2+ ([Ca2+]i) in the cytoplasm of endothelial cells is a specific mechanism that translates the effects of mechanical or chemical stimulation into cellular responses (16, 40). The Ca2+-calmodulin complex of endothelial cells is known to play an essential role in controlling the activity of eNOS, an enzyme involved in the generation of the powerful vasodilator NO in a variety of vascular beds (16, 27). Elevation of endothelial cell [Ca2+]i is also importantly involved in the production of two other endothelium-derived vasodilators, prostacyclin and endothelium-derived hyperpolarizing factor (EDHF) (6, 7, 20). At present, the role of endothelial Ca2+ signaling in the gestational enhancement of endothelium-dependent vasodilation in the maternal uterine circulation remains uncertain. Some important information on the role of Ca2+ signaling in NO production has been obtained from studies (2, 56) using cultured endothelial cells or, more recently, freshly isolated endothelial cells from sheep uterine arteries. Mechanisms controlling intracellular levels of Ca2+ in native endothelial cells of intact uterine arteries are largely unknown. Endothelial cells in the walls of small resistance arteries are subjected to physical forces (intraluminal pressure and shear stress) that can significantly affect their function (11). Recent studies (13, 14, 48, 55) indicate that endothelial and smooth muscle cells can be electrically and chemically coupled through myoendothelial gap junctions, adding more complexity to the potential mechanisms that control endothelial cell [Ca2+]i in uterine arteries and their modulation by pregnancy.
In the present study, we hypothesized that pregnancy upregulates Ca2+ signaling in endothelial cells of intact pressurized uterine arteries and that this results in augmented endothelial vasodilator influence. The purpose of this study was to 1) compare the basal levels of [Ca2+]i in endothelial cells of pressurized uterine arteries from late-pregnant (LP) rats and age-matched nonpregnant (NP) controls, 2) explore the effects of intraluminal pressure and associated vasoconstriction on basal levels of endothelial cell [Ca2+]i, and 3) study endothelial cell [Ca2+]i and diameter responses to ACh in vessels from NP versus LP animals.
The data from the present study indicate that late pregnancy is a state of enhanced basal and stimulated Ca2+ signaling in endothelial cells of intact pressurized uterine vessels that may represent an important underlying mechanism for the gestational increase in endothelium-mediated vasodilation in the maternal uterine circulation.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Adult (14–16 wk old) NP or LP (19–20 days) female Sprague-Dawley rats (n = 69) were anesthetized by an intraperitoneal injection of methohexital sodium (Brevital, 50 mg/kg) or Nembutal (50 mg/kg) and euthanized by decapitation. The abdominal wall was transected, and the entire uterus and uterine vasculature were rapidly removed and pinned in a dissecting dish filled with aerated cold physiological salt solution (PSS; see Solutions and drugs for composition). First or second order uterine radial arteries were identified within the mesometrial arcade and dissected free of connective tissue. Only uteroplacental arteries (radial arteries feeding the placenta) were dissected from the vasculature of pregnant uteri and used for the present study. Arterial segments were cannulated within an arteriograph, placed on the stage of an inverted microscope, and continuously superfused with aerated (10% O2-5% CO2-85% N2) PSS at 37°C. Initial intraluminal pressure was set at 10 mmHg using the servo pressure system (Living System Instrumentations, Burlington, VT). All experiments were performed under no intraluminal flow conditions.
Endothelial cell loading with fura-2. After a 20-min equilibration period at 10 mmHg and 37°C, endothelial cells were loaded with a Ca2+-sensitive dye fura-2 (5 µM). Background fluorescence and arterial autofluorescence were measured before starting the loading procedure (51). Loading of endothelial cells with AM form of fura-2 (fura-2 AM) was performed by intraluminal perfusion of fura-2 AM-containing solution at room temperature for 5 min, followed by 10 min of washout with regular PSS. Similar experimental approaches have been successfully used in numerous studies aimed to selectively load the endothelial cells of pressurized vessels with fura-2 (24, 34, 48). In a separate set of experiments, arteries were loaded intraluminally with BAPTA AM (50 µM) for 15–20 min and then with a combination of fura-2 AM and BAPTA AM for an additional 10 min.
In some experiments, after the experimental protocol was completed, the endothelium was removed by passing air bubbles through the lumen of the arterial segment for 5–6 min, followed by 1–2 min of perfusion with regular PSS. This procedure resulted in a nearly complete disappearance of fura-2 signal, confirming the selective loading of endothelial cells. The reduction in fura-2 fluorescence after excitation with 340 nm and 380 nm wavelengths was 91 ± 2% and 89 ± 3%, respectively (n = 8). Residual fluorescence was occasionally generated by a small number of endothelial cells located around the end of each cannula, which were not accessible to perfusion with air bubbles.
Smooth muscle cell loading with fura-2. After an equilibration period of 20 min at 37°C at 10 mmHg and measurement of background fluorescence and arterial autofluorescence, smooth muscle cells within the wall of pressurized arteries were loaded with 5 µM fura-2. An arterial segment was incubated extraluminally in fura-2 AM loading solution at room temperature in the dark for 45–60 min under no perfusion conditions. Fura-2-loaded arteries were washed 2–3 times and then continuously superfused at 3 ml/min with aerated PSS at 37°C.
Measurement of endothelial or smooth muscle cell [Ca2+]i in pressurized arteries. Ratiometric measurements of fura-2 fluorescence from endothelial or smooth muscle cells were performed by using a photomultiplier system (IonOptix, Milton, MA). Experimental ratios were corrected for background fluorescence and autofluorescence taken from each artery before loading with fura-2. Corrected ratios of 510-nm emission were obtained at a sampling rate of 5 Hz from arteries alternately excited at 340 and 380 nm (or 360 nm in Mn2+ quenching experiments). The arterial lumen diameter was simultaneously monitored by using the SoftEdge Acquisition Subsystem (IonOptix). All experimental protocols were started after an additional 15- to 20-min equilibration period at 10 mmHg to allow intracellular deesterification of fura-2 AM (BAPTA AM).
ACh-induced endothelial cell [Ca2+]i responses and dilatation of pressurized uterine arteries. To minimize mechanical stimulation of endothelial and smooth muscle cells within the arterial wall during the equilibration period, cannulated arteries were initially pressurized to 10 mmHg. The diameter and levels of endothelial [Ca2+]i were recorded during 5–10 min, followed by an elevation of intraluminal pressure to 50 mmHg in the majority of tested arteries. In contrast to uterine radial arteries of NP rats, vessels of LP animals can develop vasoconstriction (myogenic tone) in response to elevations of pressure that exceed 50 mmHg. Therefore, to avoid the development of myogenic tone and to equalize experimental conditions for arteries of NP and LP rats, they were pressurized to a similar level (50 mmHg).
Ten to fifteen minutes after the elevation of intraluminal pressure, phenylephrine was added in increasing concentrations (1–3 doses) to produce a constriction of 40–60% of maximally dilated arteries (Dmax). Higher levels of preconstriction (70–80%) occasionally resulted in a significant elevation in endothelial cell [Ca2+]i, complicating the calculations of [Ca2+]i responses to ACh, and were not used in the present study. The concentrations of phenylephrine applied to produce desirable levels of preconstriction were typically 0.1–0.5 and 0.05–0.1 µM for arteries of NP and LP rats, respectively. After the stabilization of vasoconstriction, one or two concentrations (threshold and maximal) of ACh were tested during 3 min for each artery. Initial transient (maximal) and sustained (in the end of 3-min period of ACh application) components of the endothelial [Ca2+]i response, and corresponding initial and sustained levels of vasodilation, were measured in each artery (shown in Fig. 4A). A combination of papaverine (100 µM, a phosphodiesterase inhibitor) and diltiazem (10 µM, a calcium channel blocker) was applied at the end of each experiment to obtain the Dmax. ACh-induced vasodilation was expressed as the percentage of Dmax.
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Mn2+-quenching protocol. Arteries were loaded with fura-2 AM intraluminally, as described above, in Ca2+-free HEPES-PSS. After an equilibration period of 15 min, intraluminal pressure was elevated from 10 to 50 mmHg. After an additional 10 min, 200 µM MnCl2 was added to the perfusate for the next 20 min. At the end of each experiment, 10 µM ionomycin and 1 mM MnCl2 were applied to induce maximal fura-2 quenching. This procedure yielded a reduction in the fluorescence signal to the level comparable to that recorded before loading the vessels with fura-2. Fluorescence was measured while the arteries were excited with an isosbestic wavelength of 360 nm (F360). The rate of fura-2 quenching during the first 10 min of Mn2+ application (linear decline) was expressed as a percentage per minute, where 100% was the level before adding MnCl2 and 0% was F360 fluorescence obtained after exposing the arteries to a combination of ionomycin and MnCl2.
Solutions and drugs. The PSS contained (in mM) 119 NaCl, 4.7 KCl, 24.0 NaHCO3, 1.2 KH2PO4, 1.6 CaCl2, 1.2 MgSO4, 0.023 EDTA, and 11.0 glucose, pH = 7.4. Ca2+-free solution was made by omitting the CaCl2 from regular PSS and adding 5 mM EGTA. For the fura-2 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. Ca2+-free HEPES-PSS used in the experiments with Mn2+ quenching of fura-2 contained (in mM) 141.8 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.7 MgSO4, 0.5 EDTA, 10 HEPES, and 5.0 glucose. The pH of the solution was adjusted to 7.4 by using NaOH.
All chemicals were purchased from Sigma Chemical (St. Louis, MO) with the exception of ionomycin and nigericin, which were obtained from Calbiochem (La Jolla, CA). Fura-2 AM, BAPTA AM, and pluronic acid were purchased from Invitrogen (Carlsbad, CA). Fura-2 AM was dissolved in dehydrated DMSO as a 1 mM stock solution, frozen in small aliquots, and used within 1 wk of preparation. Phenylephrine, ACh, and papaverine were dissolved in deionized water on the experimental day and used on the same day only. Diltiazem was prepared as a 10 mM stock solution in deionized water and kept refrigerated until use (2–3 wk). Ionomycin and nigericin were prepared as 10 mM stock solutions in methanol and kept at –20°C until use.
Calculations and statistical analysis.
Smooth muscle cell or endothelial cell [Ca2+]i was calculated by using the following equation (21): [Ca2+]i = Kd
(R – Rmin)/(Rmax – R), where R is experimentally measured ratio (340/380 nm) of fluorescence intensities, Rmin is a ratio in the absence of [Ca2+]i, Rmax is a ratio at Ca2+-saturated fura-2 conditions, and is a ratio of the fluorescence intensities at 380 nm excitation wavelength at Rmin and Rmax. Rmin, Rmax, and were determined by an in situ calibration procedure from the arteries treated with nigericin (5 µM) and ionomycin (10 µM). Calibration was performed for two separate sets of vessels loaded intraluminally (endothelial cell loading, n = 9) or extraluminally (smooth muscle cell loading, n = 8) with fura-2. These values were then pooled and used to convert the ratio values into a [Ca2+]i. The Kd (dissociation constant for fura-2) was 282 nM, as determined by using in situ titration of Ca2+ in fura-2-loaded small arteries (25). Arterial diameter, pressure, and ratio values were simultaneously recorded by using an IonOptix data acquisition program and imported into Sigma Plot and Sigma Stat programs for graphical representation, calculations, and statistical analysis. Data are expressed as means ± SE, where n is the number of arterial segments studied. One to three arteries from the same animal were used on each experimental day. Only one vessel per animal was used for a particular protocol. A paired or unpaired Student's t-test or two-way repeated-measures ANOVA was used to determine the significance of differences between sets of data, with P < 0.05 considered as significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Pregnancy augments ACh-induced endothelial cell [Ca2+]i rise and dilatation of uterine arteries. We next studied and compared the effects of different concentrations of ACh on endothelial cell [Ca2+]i levels and diameters of arteries from NP and LP rats. Representative changes in endothelial cell [Ca2+]i and lumen diameter in response to phenylephrine and ACh application are shown in Fig. 4A. Under these experimental conditions, phenylephrine-induced constriction was associated with little (10–20 nM) or no increase in [Ca2+]i. The application of ACh resulted in a transient [Ca2+]i rise, followed by a slow [Ca2+]i decline to a relatively sustained level. The [Ca2+]i elevation was clearly associated with an arterial dilatation: the onset of [Ca2+]i elevation always preceded the onset of dilatation (at ACh 10 µM by 11 ± 2 s, n = 20). For comparison, smooth muscle cell [Ca2+]i and diameter responses with the use of an identical experimental protocol are shown in Fig. 4B. The application of phenylephrine was followed by a significant elevation of smooth muscle cell [Ca2+]i and vasoconstriction. ACh reduced smooth muscle cell [Ca2+]i to a nearly basal level that was associated with complete vasodilation.
To obtain precise correlations between endothelial cell [Ca2+]i levels and the degree of vasodilation, only one or two (threshold and maximal) concentrations of ACh were tested on each artery. The representative changes in [Ca2+]i and the diameters of arteries from LP and NP rats in response to application of 0.3 and 1 µM ACh are shown in Fig. 5, A and B. As evident from these records, ACh-induced [Ca2+]i elevation and associated dilatation were enhanced in uterine arteries of LP rats compared with NP controls. In some arteries, ACh application triggered an oscillatory pattern of [Ca2+]i response.
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The role of endothelial cell [Ca2+]i in ACh-induced vasodilation of uterine arteries.
To further explore the role of Ca2+ signaling in endothelium-dependent vasodilation of uterine arteries, we studied the effect of cytosolic Ca2+ chelation with BAPTA on ACh-stimulated [Ca2+]i and diameter responses. Loading arteries of both LP and NP rats with BAPTA resulted in almost complete inhibition of both transient and sustained endothelial cell [Ca2+]i elevations and associated dilatations in response to 10 µM ACh (Fig. 7A). Figure 7, B and C, summarizes the transient [Ca2+]i changes and initial vasodilations obtained from BAPTA-loaded and control vessels. These data indicate a critical role of endothelial cell [Ca2+]i elevation in ACh-induced dilatation of uterine arteries. We next analyzed the correlation between [Ca2+]i levels in endothelial cells and the arterial dilatations elicited by ACh in arteries from NP and LP animals. There is evidence that the initial [Ca2+]i elevation in response to stimulation of endothelial cells with VEGF is associated with Ca2+-dependent NO production that is later switched to phosphorylation-dependent activation of eNOS, which is less dependent on sustained [Ca2+]i levels (5). Initial agonist-induced [Ca2+]i rise can also trigger the hyperpolarization of endothelial cells that is associated with EDHF-mediated vasodilation (40). Therefore, we plotted initial transient [Ca2+]i elevations against initial dilatations of the arteries by using data shown in Fig. 6, A and E. There was a significant positive correlation between endothelial cell [Ca2+]i elevation and dilatation with correlation coefficients (r2) of 0.89 and 0.75 for NP and LP rats, respectively. Threshold concentrations of cytosolic Ca2+ required to elicit 10–15% of dilatation were not different between LP and NP groups (
150 nM). The concentrations of endothelial cytosolic Ca2+ associated with a maximal dilatation were 400 nM for both NP and LP animals (Fig. 8).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Increased basal Ca2+ influx in intact endothelium of uterine arteries in late pregnancy.
Our results indicate that late pregnancy is a state of elevated basal [Ca2+]i in endothelial cells of intact pressurized uterine arteries. There is evidence that endothelial and smooth muscle cells are electrically and chemically coupled through gap junctions, raising the possibility for smooth muscle being a source of elevated [Ca2+]i in endothelial cells (13, 14, 48, 55). However, in arteries of LP rats pressurized at 10 mmHg, resting levels of [Ca2+]i were significantly lower in smooth muscle cells (75 ± 2 nM) compared with endothelial cells (126 ± 8 nM). In addition, sustained levels of endothelial cell [Ca2+]i were not affected by pressure elevation from 10 to 50 mmHg. Similar to observations reported for intact pressurized coronary arteries (24), our results show no significant changes in the sustained levels of endothelial cell [Ca2+]i in the arteries of LP rats that developed myogenic tone in response to pressure elevation, despite significant elevation of [Ca2+]i in smooth muscle cells (200 nM). In some of our experiments during strong phenylephrine-induced vasoconstriction, in which arterial diameters were reduced by 70–80%, a significant elevation in endothelial cell [Ca2+]i was occasionally observed. These [Ca2+]i responses were delayed from the beginning of the constriction by 1–2 min and likely resulted from Ca2+ diffusion from smooth muscle cells. It seems that in uterine vessels the Ca2+ diffusion is negligible at moderate levels of smooth muscle cell [Ca2+]i (200 nM, myogenic vasoconstriction) but becomes more significant when [Ca2+]i reaches higher levels. Mechanical deformation of endothelial cells due to severe narrowing of the arteries might be an additional or alternative mechanism for endothelial [Ca2+]i elevation during strong uterine vasoconstriction (50). Collectively, our data do not favor Ca2+ diffusion from smooth muscle cells into endothelial cells through myoendothelial gap junctions as a major cause for elevated basal endothelial cell [Ca2+]i during pregnancy; the latter is rather mediated by specific changes in Ca2+ signaling in endothelial cells of uterine arteries.
Basal levels of endothelial cell [Ca2+]i can be reduced on withdrawal of Ca2+ from the extracellular solution; this effect was more pronounced in uterine arteries from LP rats compared with NP controls. We also observed a severalfold increase in the rate of Mn2+ quenching in fura-2 fluorescence, providing direct evidence for enhanced basal endothelial Ca2+ influx in late pregnancy. Endothelial cells are typically lacking L-type-voltage-gated Ca2+ channels, and capacitative and noncapacitative Ca2+-permeable channels are the major pathway for Ca2+ entry in endothelial cells of the majority of blood vessels (40). Upregulation of cellular events controlling the active state of Ca2+-permeable channels and/or their overexpression in the membrane of endothelial cells of uteroplacental arteries may be responsible for increased Ca2+ entry during pregnancy. Influx of Ca2+ into endothelial cells is also regulated by the electrochemical gradient for Ca2+ ions (22, 24, 40). Whether the membrane of endothelial cells is more hyperpolarized in pregnancy compared with the NP state and whether this mechanism contributes to pregnancy-enhanced basal Ca2+ influx remain to be determined.
Increased basal Ca2+ influx shown in the present study might be one of the underlying mechanisms for augmented basal production of NO as well as other endothelial autacoids in late pregnancy. The basal activity of Ca2+-dependent eNOS of guinea-pig uterine arteries was upregulated fourfold in the course of pregnancy (53). Basal generation of NO was increased in uterine arteries of pregnant animals and attenuated the phenylephrine-induced vasoconstriction (17, 29, 49, 52–54). In a previous study of Gokina et al. (18), inhibition of basal NO production with N
-nitro-l-arginine, a potent inhibitor of NO synthase, significantly enhanced pressure-induced constriction of rat uteroplacental arteries. Reduced basal endothelial Ca2+ influx might be one of the causes of enhanced vasoconstriction of uteroplacental arteries in compromised pregnancies that are characterized by endothelial dysfunction. Pregnancy-augmented permeability of endothelial cells to Ca2+ found in the present study can explain the beneficial effects of calcium supplementation in preventing a pregnancy-induced hypertension (28).
Pregnancy potentiates transient increase in endothelial [Ca2+]i in response to elevation in intraluminal pressure.
One novel observation of the present study is that an elevation of intraluminal pressure resulted in a transient [Ca2+]i increase in endothelial cells of rat uterine arteries. This initial increase in [Ca2+]i occurs 2 s after the arterial distension secondary to the pressure elevation and was significantly potentiated in late pregnancy (Figs. 1 and 2). It is unlikely that smooth muscle cells serve as a source of transient pressure-induced [Ca2+]i elevation in endothelial cells for the following reasons. Most uterine arteries of NP and LP rats did not develop myogenic tone and did not show a significant increase in smooth muscle [Ca2+]i in response to pressure elevation from 10 to 50 mmHg. The majority of LP arteries pressurized at 60 mmHg does develop myogenic tone associated with initial followed by sustained increase in smooth muscle cell [Ca2+]i of 150–200 nM (Fig. 2). However, pressure-induced initial [Ca2+]i elevations in smooth muscle cells were significantly smaller compared with those in endothelial cells (300–400 nM). These data are not consistent with Ca2+ diffusion as a cause for the transient [Ca2+]i endothelial response to pressure elevation.
In our system, intraluminal pressure elevation was achieved by a short-term flow of PSS inside the vessel with a subsequent increase in vessel volume. A transient shear stress created by the flow might be a stimulus for [Ca2+]i elevation in endothelial cells of uterine arteries observed in the present study. Flow-induced shear stress has been demonstrated to be associated with a moderate elevation of [Ca2+]i in the cytoplasm of endothelial cells of intact pressurized microvessels (11, 15, 34). Another plausible mechanism of transient [Ca2+]i increase might be Ca2+ influx through stretch-sensitive channels in the membrane of endothelial cells and/or Ca2+ release from internal stores in response to the distension of uterine arteries by pressure elevation. Ca2+-permeable, stretch-activated ion channels were described in both cultured endothelial cells and in the intact endothelium, and they might play a significant role in the regulation of endothelial function during vasomotor activity (26, 35, 39).
In vivo, uterine arteries are continually exposed to systemic pulsatile pressure. Therefore, transient [Ca2+]i increases in response to rhythmic changes in shear stress or circumferential stretch imposed on endothelial cells might be efficient in elevating [Ca2+]i and triggering Ca2+-dependent production and release of vasodilators under in vivo conditions. Recent studies (11, 41, 42, 47) demonstrated that both pulsatile pressure and pulsatile shear stress were associated with endothelial [Ca2+]i elevation and generation of endothelium-derived autacoids. Although the nature of the pressure-induced transient [Ca2+]i increase in endothelium of uterine arteries remains to be determined, the marked enhancement of this response in late pregnancy might be an important adaptive mechanism that contributes to augmented vasodilation in the maternal uterine circulation.
ACh-stimulated elevation in endothelial cell [Ca2+]i is augmented in late pregnancy. The results of the present study demonstrate that, in intact pressurized uterine arteries, ACh elicited endothelial Ca2+ mobilization in a dose-dependent manner that always preceded the development of vasodilation. The degree of ACh-induced vasodilation closely correlated with the level of [Ca2+]i rise, and the chelation of intracellular Ca2+ with BAPTA abolished [Ca2+]i elevation as well as vasodilation in response to the application of ACh. Collectively, these data indicate that an increase in endothelial [Ca2+]i is a crucial step in ACh-induced vasodilation and implicate Ca2+ as a mediator of the production and/or release of endothelium-derived vasodilators in uterine resistance arteries.
Both transient and sustained levels of [Ca2+]i achieved in endothelial cells as a result of stimulation with ACh were significantly higher in arteries of LP rats compared with their NP counterparts (Fig. 7). An analysis of our data has shown that the enhanced basal level of [Ca2+]i during pregnancy contributes to overall levels of [Ca2+]i rise during stimulation with ACh. A subtraction of basal levels from the total [Ca2+]i elevation eliminates the significance of differences in the responses of arteries from LP and NP rats induced by low doses of ACh. However, at high concentrations of ACh, the net increase in [Ca2+]i for both transient and sustained components was enhanced in pregnancy, indicating a fundamental change in the mechanisms of endothelial Ca2+ signaling during gestation. ACh-induced stimulation of muscarinic receptors of endothelial cells results in G protein-mediated activation of PLC- with a subsequent formation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol. The Ins(1,4,5)P3-dependent Ca2+ release from the endoplasmic reticulum, involved in the initial ACh-induced [Ca2+]i rise, is followed by a sustained [Ca2+]i elevation due to the activation of capacitative Ca2+ influx (40). An understanding of the intracellular mechanisms responsible for the regulation of receptor-stimulated Ca2+ signaling in endothelial cells of uterine arteries and the modulation of these mechanisms in normal and abnormal pregnancies remains an important area for future research.
Analysis of the endothelial cell [Ca2+]i-dilatation relationships of ACh-stimulated uterine arteries has shown that complete vasodilation can be achieved as a result of relatively minor changes in [Ca2+]i: from 100 to 400 nM ( Fig. 8). In rat coronary artery, the [Ca2+]i required for half-maximal activation of eNOS was 160 nM, and maximal activation of eNOS was achieved at 300 nM (24). In another study (33) utilizing ex vivo aortic valves, the NO production in response to stimulation of endothelial cells with agonists quantitatively correlated with [Ca2+]i transients in the range of 100–400 nM. Therefore, ACh-induced [Ca2+]i elevations found in the present study very likely result in a significant eNOS activation and NO production. The endothelial cell [Ca2+]i rise in response to ACh might also significantly activate EDHF, another important mediator of endothelium-dependent vasodilation in uterine arteries of animals and humans (9, 23). It is currently unknown whether similar [Ca2+]i-diameter relationships will occur in response to the stimulation of endothelial cells of uterine arteries with other Ca2+-mobilizing agonists. In cultured endothelial cells from sheep uterine arteries, angiotensin II and growth factors have been shown to stimulate the production of NO without Ca2+ mobilization (2). Recent studies (33) demonstrate that the Ca2+ requirement for NO production depends on the type of agonist used, suggesting that some other mechanisms may be involved in eNOS activation.
The precise causes of pregnancy-increased Ca2+ signaling in endothelial cells of uteroplacental arteries, shown in the present study, remain unknown. A dependence of basal levels of endothelial cell [Ca2+]i on sex has been reported, implicating sex hormones in the regulation of Ca2+ signaling in vascular endothelium. Basal levels of [Ca2+]i were significantly higher in endothelial cells of aortic valves and coronary arteries of females compared with males (24, 44). These data provide indirect evidence that suggests that estrogen and progesterone, the most important hormones of pregnancy, can potentially play a critical role in the regulation of Ca2+ signaling in endothelial cells of uterine arteries during gestation.
In conclusion, basal levels of endothelial cell [Ca2+]i and pressure-induced and ACh-stimulated [Ca2+]i responses are significantly potentiated during gestation. These data implicate augmented Ca2+ signaling as one of the mechanisms for enhanced endothelium-mediated vasodilation of uteroplacental arteries in late pregnancy. Exploring the causal factors responsible for gestational enhancement of endothelial cell Ca2+ signaling will be important for understanding the mechanisms underlying the abnormal behavior of uterine resistance arteries in pregnancies complicated with hypertension, gestational diabetes, or preeclampsia.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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) versus LP (
) rats. One or two doses (threshold and maximal) of ACh were tested for each artery. C and D: net increase in T (C) and S (D) components of ACh-induced [Ca2+]i responses as function of ACh concentration, obtained after subtraction of basal levels from total levels of [Ca2+]i in arteries of NP versus LP rats. E and F: degree of I (E) and S (F) vasodilation as function of ACh concentrations in arteries from NP and LP rats. ACh-induced vasodilation is expressed as percentage of maximal dilatation of arteries in response to 100 µM papaverine and 10 µM diltiazem (Dmax). *P < 0.05, significantly different using unpaired Student's t-test. On all graphs, averaged number for a given concentration of ACh was obtained from 6–8 NP or 5–8 LP vessels.
