INTRODUCTION

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PREGNANCY IS ASSOCIATED WITH a striking increase in uterine blood flow. It has been proposed that endothelial nitric oxide (NO) plays an important role in the pregnancy-associated increase in vasodilation of the uterine artery (31). Previous studies (5, 40, 44) demonstrated that pregnancy was associated with elevated plasma and urinary levels of nitrate, suggesting an increase in NO synthesis. Accordingly, plasma and urinary cGMP levels were increased in pregnant versus nonpregnant animals and humans (6, 16, 21, 31). It has been shown that basal activity of NO synthase (NOS) in the uterine artery endothelium is elevated in pregnant versus nonpregnant guinea pigs (36), sheep (18, 20, 21), and humans (26). In sheep, this increase was specific to the uterine artery because no differences were noted in NOS activity among omental arteries due to pregnancy (20). It was also suggested that the endothelium contained virtually all of the NOS activity in the sheep uterine vascular wall (20). More recently (2, 21, 39), it has been demonstrated that the expression of endothelial NOS protein and mRNA is increased by pregnancy in ovine uterine, but not systemic, artery endothelium. Our recent study (39) in the isolated perfused ovine uterine artery supported the interpretation that increased endothelial NOS (eNOS) protein expression in the uterine artery during pregnancy was associated with elevations of NO release.

To our knowledge, there is no study yet to correlate directly NO release and endothelium-dependent relaxation, and to determine the potential differences between nonpregnant and pregnant uterine arteries. Furthermore, it is unknown whether NO induces relaxation of the uterine artery by decreasing smooth muscle intracellular Ca²⁺ concentration ([Ca²⁺]_i) or by attenuating Ca²⁺ sensitivity of contractile proteins, and whether pregnancy affects Ca²⁺ handling in response to NO in the uterine artery. In the present study, we tested the hypothesis that the pregnancy-associated increase in endothelium-dependent relaxation of the uterine artery was mediated predominantly by an increase in NO release, and signal transduction pathways distal to NO production were not changed by pregnancy. We also tested the hypothesis that the NO-induced relaxation of the uterine artery was mediated primarily by a decrease in smooth muscle free [Ca²⁺]_i. By measuring [Ca²⁺]_i and tension simultaneously in the same tissue, the present study further examined the role of Ca²⁺ sensitivity in NO-mediated relaxation of the uterine artery, and tested the hypothesis that pregnancy had no effect on NO-mediated Ca²⁺ sensitivity in the uterine artery.

	METHODS

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Tissue preparation. Nonpregnant and pregnant (~140 days gestation) sheep were anesthetized with thiamylal (10 mg/kg) administered via the external left jugular vein. The ewes were then intubated and anesthesia was maintained on 1.5-2.0% halothane in oxygen throughout surgery. An incision in the abdomen was made and the uterus was exposed. The uterine arteries were isolated and removed without stretching and were placed into a modified Krebs solution (pH 7.4) of the following composition (in mM): 115.21 NaCl, 4.7 KCl, 1.80 CaCl_2, 1.16 MgSO₄, 1.18 KH₂PO₄, 22.14 NaHCO₃, and 7.88 dextrose. EDTA (0.03 mM) was added to suppress oxidation of amines. The Krebs solution was oxygenated with a mixture of 95% oxygen-5% carbon dioxide. After the tissues were removed, the animals were killed with T-61 (euthanasia solution, Hoechst-Roussel; Somerville, NJ). All procedures and protocols used in the present study were approved by the Animal Research Committee of Loma Linda University and followed the guidelines put forward in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Measurement of arterial tension. Third (nonpregnant) and fourth (pregnant) branches of the main uterine arteries with a similar diameter (~0.8 mm) were separated from the surrounding tissue, and special care was taken to avoid touching the luminal surface. The arteries were cut into 2-mm ring segments with ~7 mg/wet wt for both nonpregnant and pregnant tissues. To determine the role of basal NO release in contractions of the uterine artery, we first examined the effect of endothelial removal on norepinephrine-induced contractions. The endothelium was removed by gentle rotation of the rings on an appropriately sized, rough-surfaced blunt hypodermic needle, and validation of endothelium removal was demonstrated by the elimination of the endothelium-dependent relaxation induced by ATP (11). Contractile responses of uterine artery rings were quantified in 10 ml of Krebs solution in tissue baths at 37°C as described previously (11). Isometric tensions were measured. After 60 min of equilibration in the tissue bath, each ring was stretched to the optimal resting tension as determined by the tension developed in response to three exposures of potassium chloride (120 mM) added at different stretch levels. Concentration-response curves were obtained by cumulative additions of the agonist in approximate one-half log increments. EC₅₀ values for the agonist in each experiment were taken as the molar concentration at which the contraction-response curve intersected 50% of the maximum response, and were expressed as pD₂ (logEC₅₀) values. To determine the endothelium-dependent relaxation, the endothelium-intact uterine arteries were precontracted with a submaximal concentration (1 µM) of phenylephrine, followed by a cumulative addition of the Ca²⁺ ionophore A23187 and sodium nitroprusside (SNP), respectively.

Measurement of NO. From the relaxation study, 0.1 ml of the bath solution was collected before and after each dose of the Ca²⁺ ionophore A23187. Samples were flash-frozen in liquid N₂ and stored at 80°C. NO was measured by chemiluminescence method, as described previously (39, 44). Because of the instability of NO in oxygenated physiological solution, it is rapidly converted to nitrite and further to nitrate. Nitrite and nitrate are relatively stable in the solution, and are readily reduced back to NO in vanadium(III)/HCl solution. The samples (100 µl) were injected into the gas purge vessel containing 5 ml vanadium(III)/HCl to react for 1 min and reduce nitrite/nitrate in the sample back to NO. To achieve high reducing efficiency, the reduction was performed at 90°C. NO in the sample was then "stripped" into the head space of the gas purge vessel by bubbling it with helium (12 ml/min) for 60 s. NO in the head space was drawn into the NO analyzer (model 270B, Sievers Instruments; Boulder, CO) and mixed with O₃ in the front of a cooled Hamamatsu, red-sensitive photomutiplier tube. Signals from the detector were analyzed with the use of an on-line computer as area under the peak. The measurement reflected the combined concentrations of nitrite, nitrate, and NO of each sample.

Simultaneous measurement of [Ca²+]_i and tension. Simultaneous recordings of tension and free [Ca²⁺]_i in uterine artery smooth muscle were obtained as described previously by us (43). Briefly, the arterial ring was attached to an isometric force transducer in a 5-ml tissue bath mounted on a CAF-110 intracellular Ca²⁺ analyzer (Jasco; Tokyo, Japan). The tissue was equilibrated in Krebs buffer under a resting tension of 0.5 g for 40 min. The ring was then loaded with 5 µM fura 2-acetoxymethyl ester (AM) for 2 h in the presence of 0.02% Cremophor EL at room temperature (25°C). After the ring was loaded, the tissue was washed with Krebs solution at 37°C for 30 min to allow for hydrolysis of fura 2 ester groups by endogenous esterase. Contractile tension and fura 2 fluorescence were measured simultaneously at 37°C in the same tissue. The tissue was illuminated alternatively (125 Hz) at excitation wavelengths of 340 and 380 nm, respectively, by means of two monochromators in the light path of a 75-W xenon lamp. Fluorescence emission from the tissue was measured at 510 nm with the use of a photomultiplier. The fluorescence intensity at each excitation wavelength (F₃₄₀ and F₃₈₀, respectively) and the ratio of these two fluorescence values (R_340/380) were recorded with a time constant of 250 ms and stored with the force signal on a computer.

Materials. Norepinephrine, N^G-nitro-L-arginine (L-NNA), phenylephrine, and SNP were obtained from Research Biomedicals (Natick, MA). The Ca²⁺ ionophore A23187 was purchased from Sigma (St. Louis, MO). Sodium nitrate and vanadium(III) chloride were from Aldrich (Milwaukee, WI). Fura 2-AM was obtained from Molecular Probes (Eugene, OR).

Data analysis. Concentration-response curves were analyzed by computer-assisted nonlinear regression to fit the data by using Prism software (GraphPad Software; San Diego, CA). For NO data analysis, the area under the peak was continuously integrated during sample measurement using the data-acquisition software WorkBench (Kent Scientific; Litchfield, CT). Linear regression of the standard curve and comparison of regression lines were analyzed with the use of Prism software (GraphPad). Results were expressed as means ± SE, and the differences were evaluated for statistical significance (P < 0.05) by analysis of variance.

	RESULTS

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The effect of endothelium removal on norepinephrine-induced contractions of the uterine arteries is shown in Fig. 1. As shown in Fig. 1A, removal of the endothelium shifted the norepinephrine concentration-response curve to the left in the pregnant uterine arteries, and increased the pD₂ value from 6.0 ± 0.16 in the intact arteries to 6.54 ± 0.14 in the denuded arteries (P < 0.05). In contrast, endothelium removal had no effect on norepinephrine-induced contractions in nonpregnant uterine arteries (Fig. 1B).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of endothelium removal on norepinephrine-induced contractions of the uterine artery. Cumulative concentration-response curves to norepinephrine were obtained with endothelium-intact and denuded arterial ring segments from pregnant (A) and nonpregnant (B) uterine arteries. Data are means ± SE of the tissues from 4 to 5 animals. Inset: mean values of molar contraction response curve intersected at 50% of the maximum response [pD2 (logEC50)]. *P < 0.05.

To confirm the role of NO in the above experiments, norepinephrine-induced contractions of the uterine arteries were performed in the absence and presence of NOS inhibitor L-NNA (100 µM, 20 min). As shown in Fig. 2A, L-NNA shifted the norepinephrine concentration-response curve to the left in the pregnant uterine arteries, and increased the pD₂ value from 6.36 ± 0.06 to 6.91 ± 0.18 (P < 0.05). In contrast, L-NNA did not significantly change the pD₂ values of norepinephrine-induced contractions of nonpregnant uterine arteries (Fig. 2B).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effects of NG-nitro-L-arginine (L-NNA) on norepinephrine-induced contractions of the uterine artery. Cumulative concentration-response curves to norepinephrine were obtained with the arterial ring segments, in the absence or presence of L-NNA (100 µM, 20 min), from pregnant (A) and nonpregnant (B) uterine arteries. Data are means ± SE of the tissues from 3 to 6 animals. Inset: mean values of pD2 (logEC50). *P < 0.05.

The endothelium-dependent relaxation induced by the Ca²⁺ ionophore A23187 was examined in the uterine arteries precontracted with the ₁-adrenoceptor agonist phenylephrine (1 µM). As shown in Fig. 3, the Ca²⁺ ionophore A23187 produced concentration-dependent relaxation in both pregnant and nonpregnant uterine arteries with the maximum relaxation of 47% and 88% in nonpregnant and pregnant uterine arteries, respectively (Fig. 3B). Removal of the endothelium abolished the A23187-induced relaxation. Unlike A23187, the endothelium-independent relaxation mediated by SNP was not different between nonpregnant and pregnant uterine arteries (Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effects of pregnancy on endothelium-dependent relaxation of the uterine artery. Arterial ring segments of endothelium-intact uterine arteries were precontracted with 1 µM phenylephrine, followed by a cumulative addition of the Ca2+ ionophore A23187. A: typical experiment. B: dose-response curves obtained with nonpregnant and pregnant uterine arteries. Data are means ± SE of the tissues from 6 to 12 animals. *P < 0.05, pregnant vs. nonpregnant.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effects of pregnancy on endothelium-independent relaxation of the uterine artery. Uterine arteries were precontracted with 1 µM of phenylephrine, followed by a cumulative addition of sodium nitroprusside (SNP). Data are means ± SE of the tissues from 18 to 28 animals.

Figure 5 illustrates the Ca²⁺ ionophore A23187-stimulated NO release from the uterine arteries. Basal levels of NO release in phenylephrine precontracted uterine arteries were significantly different between pregnant (20.4 ± 2.5 pmol/100 µl) and nonpregnant (9.5 ± 2.8 pmol/100 µl) uterine arteries (P < 0.05). Phenylephrine had no effect on basal NO release in both nonpregnant and pregnant uterine arteries. The A23187-stimulated NO release was significantly higher in pregnant than in nonpregnant uterine arteries at all but one (0.01 µM) doses used (Fig. 5). Figure 6 shows a significant correlation between the A23187-mediated NO release and relaxation of pregnant uterine arteries (r² = 0.95, P < 0.05). The slope of 0.96 was not significantly different from unity. In contrast to pregnant uterine arteries, there was no linear correlation of the A23187-mediated NO release and relaxation in nonpregnant uterine arteries (Fig. 6).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effects of pregnancy on nitric oxide (NO) release in the uterine artery. Ca2+ ionophore A23187-stimulated NO release was determined in the arterial ring segments of nonpregnant and pregnant uterine arteries. NO was measured as NOX (NO, nitrite, and nitrate) as described in METHODS. Data are means ± SE of the tissues from 5 to 9 animals. *P < 0.05, pregnant vs. nonpregnant.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Correlation of the Ca2+ ionophore A23187-induced NO release and relaxation of uterine arteries. A linear correlation is shown between A23187 (0.01-10 µM)-stimulated increases in NO release and relaxation of phenylephrine precontracted uterine arteries from pregnant (r2 = 0.95) but not nonpregnant animals. Data are means ± SE of the tissues from 5 to 12 animals. The slope of 0.96 ± 0.13 obtained in pregnant arteries was not significantly different from one.

To examine the role of Ca²⁺ in the A23187-mediated relaxation of the uterine arteries, smooth muscle tension and [Ca²⁺]_i were measured simultaneously in the same tissue of endothelium-intact arterial rings as described in METHODS. As shown in Fig. 7, the Ca²⁺ ionophore A23187 evoked concentration-dependent reductions of [Ca²⁺]_i in both pregnant and nonpregnant uterine arteries precontracted with 1 µM of phenylephrine. The A23187-induced reduction of [Ca²⁺]_i was significantly increased in pregnant, compared with nonpregnant, uterine arteries (Fig. 7A). The [Ca²⁺]_i-tension relationships depicted from the data of simultaneous measurements of [Ca²⁺]_i and tension in the same tissue indicated that there was a positive correlation between these two parameters in the presence of cumulative doses of A23187 in both pregnant and nonpregnant uterine arteries (Fig. 7B). The slopes (tension/[Ca²⁺]_i) of the lines were the same in pregnant (0.87 ± 0.08) and nonpregnant (0.89 ± 0.07) uterine arteries, and were not significantly different from unity.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Effects of pregnancy on the A23187-induced reduction of intracellular Ca2+ concentration ([Ca2+]i) and tension in the uterine artery. Smooth muscle [Ca2+]i and tension were measured simultaneously in the same tissues of the endothelium-intact arterial rings loaded with fura 2. A: dose-response curves of the A23187-mediated reduction of [Ca2+]i in the arterial rings precontracted with 1 µM of phenylephrine. B: A23187-induced [Ca2+]i-tension relationships obtained with phenylephrine-precontracted arterial rings. Data are means ± SE of tissues from 5 animals. *P < 0.05, pregnant vs. nonpregnant. Slope values (tension/[Ca2+]i) are presented in the text.

Unlike the A23187, the SNP-induced reduction of [Ca²⁺]_i was the same in pregnant and nonpregnant uterine arteries (Fig. 8A). Similar to the A23187, there were significant linear correlations between SNP-induced relaxation and reduction of [Ca²⁺]_i in pregnant and nonpregnant uterine arteries precontracted with phenylephrine (Fig. 8B). Although there was no difference between the slopes (tension/[Ca²⁺]_i) obtained in nonpregnant (2.16 ± 0.14) and pregnant (2.01 ± 0.34) uterine arteries (P > 0.05), they were significantly higher than unity (P < 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Effects of pregnancy on the SNP-induced reduction of [Ca2+]i and tension in the uterine artery. Smooth muscle [Ca2+]i and tension were measured simultaneously in the same tissues of the endothelium-intact arterial rings loaded with fura 2. A: dose-response curves of the SNP-mediated reduction of [Ca2+]i in the arterial rings precontracted with 1 µM of phenylephrine. B: SNP-induced [Ca2+]i-tension relationships obtained with phenylephrine-precontracted arterial rings. Data are means ± SE of tissues from 5 animals. Slope values (tension/[Ca2+]i) are presented in the text.

	DISCUSSION

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The present study has demonstrated that pregnancy upregulates endothelial NO release and the endothelium-dependent relaxation of the uterine artery. There are several unique findings in the present study. First, by simultaneous measurement of the A23187-induced NO release and relaxation of uterine arteries, we were able to correlate directly the NO release and relaxation induced by the A23187 and to evaluate potential changes in tissue sensitivity to NO mediated by pregnancy. The finding that the slope of the NO-tension relationship was not different from one in pregnant uterine artery indicates that the pregnancy-associated increase in relaxation is primarily due to increases in NO release, and signal transduction pathways downstream of NO production may not be changed by pregnancy. Furthermore, this is supported by the discovery that SNP-induced relaxation was not changed by pregnancy. The effect of pregnancy on SNP-mediated responses has been studied previously, but the results were controversial (12, 20, 27, 28, 35, 39). In the present study, consistent with the lack of effect of pregnancy on the SNP-induced relaxation, pregnancy had no effect on the SNP-mediated reductions in smooth muscle [Ca²⁺]_i.

The finding of increased NO release in uterine artery rings by pregnancy is consistent with our previous results obtained in the perfused uterine artery preparation (39). The increase in basal NO release in pregnant uterine arteries is likely to play an important role in refractoriness to -adrenoceptor agonists occurring during pregnancy in uterine arteries (1, 19, 24, 35). In the present study, endothelium removal significantly increased norepinephrine-induced contractions of pregnant uterine arteries and shifted its dose-response curve to the left, suggesting an important role of endothelium-mediated substances in the regulation of contractile sensitivity of norepinephrine in pregnant uterine arteries. The involvement of NO in this process was confirmed by the results of L-NNA, a potent NOS inhibitor (25), in which the norepinephrine dose response curve was shifted to the left in a similar manner. Similar findings were obtained in guinea pig uterine arteries in which the removal of the endothelium and/or treatment with NOS inhibitor increased the activity of the pregnant vessel so that it became like a nonpregnant vessel (34, 35). Many factors could be involved in the pregnancy-induced increase in NO release in the uterine artery. Among them, the upregulation of eNOS expression in the uterine artery endothelium is likely to play a key role (2, 21, 39). Alternatively, because eNOS is regulated by phosphorylation (7, 9), changes in cell signaling, e.g., activation of the extracellular signal-regulated protein kinase-1/2 signaling pathway, in response to pregnancy, may also play a key role in the increased eNOS activity in uterine artery endothelial cells (2).

In accordance with the increases in NO release, the A23187-mediated endothelium-dependent relaxation of the uterine artery was significantly increased by pregnancy. In contrast to our findings, Weiner et al. (34, 35) demonstrated that pregnancy enhanced the ACh-mediated relaxation, but had no effect on the A23187-stimulated relaxation in guinea pig uterine arteries. Although the relaxation response of nonpregnant tissues to the A23187 was weak in the present study compared with that in the guinea pig study (35), it is comparable to the finding in the rabbit carotid artery, where A23187 produced 46% relaxation of phenylephrine-precontracted tissues (8). In our study, we failed to see the ACh-mediated relaxation in either nonpregnant or pregnant sheep uterine arteries (data not shown). This suggests either a lack of muscarinic receptors on the sheep uterine artery endothelium or an uncoupling of the receptors to NO signaling pathways. Because the A23187-induced relaxation is receptor independent, our results do not examine the potential changes in endothelial receptors and coupling signals by pregnancy, but rather focus on changes in NOS. Hence, the increase in A23187-mediated endothelium-dependent relaxation of the uterine artery by pregnancy is likely due to an increase in NOS levels and/or activity, leading to an enhanced NO release. This is confirmed by the excellent correlation between the A23187-induced relaxation and NO release in pregnant uterine arteries.

The second finding of the present study is that, by simultaneous measurement of smooth muscle tension and [Ca²⁺]_i in the same intact arterial ring, we were able to examine two key parameters, [Ca²⁺]_i and Ca²⁺ sensitivity of contractile proteins, in the A23187-induced, endogenous NO-mediated relaxation of the uterine arteries. The excellent correlation of the A23187-mediated reduction in [Ca²⁺]_i and tension in phenylephrine-precontracted uterine arteries confirms the obligatory role of Ca²⁺ reduction in the A23187-mediated relaxation of both nonpregnant and pregnant uterine arteries (38). Although it is likely that fura 2 was loaded into both endothelial and smooth muscle cells of the uterine arteries, the setting of the preparation with only abluminal smooth muscle side exposed to the light path in the present study allowed us to measure predominantly, if not exclusively, Ca²⁺ changes in smooth muscle cells, and the endothelial interference of the smooth muscle cell [Ca²⁺]_i signal was minimal. Similar findings were obtained in the previous study (33). The present finding that endogenous NO decreased [Ca²⁺]_i in uterine artery smooth muscle cells is consistent with previous studies (3, 4, 13, 15, 37, 41, 42). Multiple mechanisms have been proposed to explain NO-mediated reduction of Ca²⁺ in vascular smooth muscle cells, including inhibition of intracellular Ca²⁺ release by D-myo-inositol 1,4,5-trisphosphate (13, 29, 30, 32), activation of voltage-gated (42) and Ca²⁺-dependent (3) potassium channels, and increases of Ca²⁺ effluxes via ATP-dependent Ca²⁺ pump (10). A recent study (4) suggested that NO decreased intracellular Ca²⁺ by refilling of intracellular stores by sarcoplasmic reticulum Ca²⁺-ATPase and inhibition of store-operated Ca²⁺ influx in vascular smooth muscle.

In accordance with the increase in A23187-induced relaxation in pregnant uterine arteries, the A23187-mediated reduction in smooth muscle cell [Ca²⁺]_i was significantly enhanced by pregnancy. This is likely due to the increased NO release in pregnant uterine arteries. The simultaneous measurement of [Ca²⁺]_i with tension allowed us to estimate Ca²⁺ sensitivity of myofilaments with unimpaired signaling pathways in the uterine artery. With this method, there are two interesting findings in the present study. The first is that the slopes of the [Ca²⁺]_i-tension relationship of A23187-induced reductions in smooth muscle [Ca²⁺]_i and tension were not significantly different from one in both nonpregnant and pregnant uterine arteries. This suggests that the A23187-induced, endogenous NO-mediated relaxation of the uterine artery is mediated primarily by a decrease in intracellular Ca²⁺, but not by changes in Ca²⁺ sensitivity of contractile proteins. Although numerous studies have demonstrated that NO-mediated reduction in intracellular Ca²⁺ plays an important role in vasorelaxation of a variety of vessels, the present study is the first to clearly demonstrate that endogenous NO-mediated relaxation is independent of changes in myofilament Ca²⁺ sensitivity. This is in contrast with the finding in the rat tail artery, in which both NO and nitroglycerine decreased vasoconstrictor responses to norepinephrine but had no effect on intracellular Ca²⁺ (33). The reason for this difference is not clear at present but may be due in part to the differences in species and/or artery types used. The second interesting finding is that the slopes of the [Ca²⁺]_i-tension relationship of A23187-induced reductions of Ca²⁺ and tension are the same in nonpregnant and pregnant uterine arteries, suggesting that pregnancy does not change Ca²⁺ sensitivity in endogenous NO-mediated relaxation in the uterine artery. This reinforces the notion that the pregnancy-associated increase in endothelium-dependent relaxation of the uterine artery is mediated primarily by an increase in NO production, leading to an increased reduction in smooth muscle cell [Ca²⁺]_i.

The quite different results obtained with the NO donor SNP represent the third unique finding of the present study. Unlike A23187, the slopes of the [Ca²⁺]_i-tension relationship mediated by SNP were significantly greater than unity in both nonpregnant and pregnant uterine arteries. SNP caused a dose-dependent and near-complete relaxation of phenylephrine-precontracted uterine arteries. This was accompanied by decreases in [Ca²⁺]_i, but it was not of a comparable magnitude to the decrease in force. With the maximum relaxation, there was ~50% reduction in [Ca²⁺]_i. This suggests that the SNP-induced relaxation of the uterine artery is mediated by reductions of both [Ca²⁺] and Ca²⁺ sensitivity in the smooth muscle. Although it is not entirely clear at present why the NO donor and the endogenous NO have different effects on Ca²⁺ sensitivity in vascular smooth muscle, it has been reported (14, 23) that SNP-induced relaxation of vascular smooth muscle is not associated with the proportional decrease in [Ca²⁺]_i, suggesting a decrease in Ca²⁺ sensitivity. It has not been determined whether the SNP-induced decrease in Ca²⁺ sensitivity is mediated by the NO/cGMP-dependent pathway, although it has been demonstrated that 8-bromo-cGMP causes Ca²⁺ desensitization in vascular smooth muscle (17). It is not clear whether differences in a global increase in NO by a NO donor or a focal increase in NO by endogenous NO affects downstream signal pathways in the smooth muscle. Nonetheless, in agreement with the present finding that endogenous NO-induced relaxation of the uterine artery depended completely on the reduction in [Ca²⁺]_i, it has been demonstrated that in pressurized/perfused cerebral arteries loaded with fura 2, luminal application of 2-methylthioadenosine triphosphate and release of endogenous NO induce nearly one-to-one ratio in reductions of vascular tone and [Ca²⁺]_i (22). In the present study, pregnancy did not affect SNP-mediated responses either in the reduction of [Ca²⁺]_i or in the [Ca²⁺]_i-tension relationship in the uterine arteries, further confirming that signal transduction pathways distal to the NO production in the uterine artery may not be changed by pregnancy.

In summary, the major findings of this study are 1) pregnancy increases uterine artery endothelial NO release and the endothelium-dependent relaxation of the uterine artery, 2) the reduction of smooth muscle [Ca²⁺]_i fully accounts for endogenous NO-mediated relaxation induced by A23187, which is not changed by pregnancy, and 3) the endothelium-independent relaxation of the uterine artery induced by the NO donor SNP is mediated by decrease in both [Ca²⁺]_i and Ca²⁺ sensitivity and is not affected by pregnancy. From these results, we conclude that the pregnancy-associated increase in the endothelium-dependent relaxation of the uterine artery is mediated by the upregulation of NO release resulting in a decrease in smooth muscle [Ca²⁺]_i, and signal transduction pathways downstream of the NO production play minimum role. The increased endothelium-dependent relaxation is likely to play a key role in the adjustment of uterine vascular tone with a dramatic increase in uterine blood flow during pregnancy.