INTRODUCTION

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CHRONIC HYPOXIA has been known for years to have profound effects on vascular contractility in a variety of vascular beds and to produce a sustained attenuation of uterine, cerebral, pulmonary, and systemic vasoreactivity in response to a variety of agonists such as serotonin [5-hydroxytryptamine (5-HT)], norepinephrine, phenylephrine, angiotensin II, and arginine vasopressin (7, 12, 13, 29, 30). Yet the cellular and biochemical mechanisms underlying chronic hypoxia-mediated vascular responses are not well understood. Previous studies from this laboratory have suggested that chronic hypoxia suppresses vascular smooth muscle pharmacomechanical coupling by altering the function of cell membrane receptors as well as the postreceptor mechanisms (12, 13, 29). The finding that chronic hypoxia significantly decreased 5-HT-induced D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] synthesis (13) suggests that chronic hypoxia may suppress agonist-mediated Ca²⁺ mobilization in vascular smooth muscle.

Not only does free intracellular Ca²⁺ concentration ([Ca²⁺]_i) play an important role in determining contractions of vascular smooth muscle, but also Ca²⁺ sensitivity of smooth muscle myofilaments provides a key determinant of contractile force (15, 16, 28). It has been well documented that, compared with K⁺ stimulation, agonists produce greater contractions at a given Ca²⁺ concentration in smooth muscle (6, 16, 27). This agonist-enhanced Ca²⁺ sensitivity of contractile proteins can be subject to physiological and/or pathophysiological modulations. For example, in sheep cerebral arteries, 5-HT-enhanced Ca²⁺ sensitivity was GTP dependent and was physiologically modulated (2). Our recent finding that for a given amount of Ins(1,4,5)P₃ synthesis induced by 5-HT less contraction was produced in the uterine artery from chronically hypoxic animals than that from control ones (13) lets us hypothesize that chronic hypoxia inhibits Ca²⁺ sensitivity of myofilaments in the uterine artery. To our knowledge, no studies have yet been performed to directly examine the effect of chronic hypoxia on the regulation of Ca²⁺ homeostasis and Ca²⁺ sensitivity in vascular smooth muscle. Thus the object of this investigation was to study further the effect of chronic hypoxia on intracellular Ca²⁺ mobilization and Ca²⁺ sensitivity of myofilaments in the uterine artery. To avoid permeabilization of smooth muscle cells, we have adopted a method to measure [Ca²⁺]_i and muscle contraction simultaneously in intact vascular smooth muscle.

	MATERIALS AND METHODS

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Tissue preparation. Uterine arteries were obtained from normoxic and chronically hypoxic pregnant sheep as reported previously (12, 13). Briefly, time-dated pregnant sheep were obtained from Nebeker Ranch (Lancaster, CA; altitude ~300 m) with arterial PO₂ (Pa_O2) of 102 ± 2 mmHg. Uterine arteries were obtained from near-term pregnant sheep (~140 days gestation). To induce chronic hypoxia, we transported the animals at 30 days gestation to Barcroft Laboratory, White Mountain Research Station (Bishop, CA; altitude 3,820 m; Pa_O2 60 ± 2 mmHg), and kept them there for ~110 days, whereas the control animals were maintained near sea level (~300 m) throughout the gestation period. The animals were transported to our laboratory immediately before the studies. The maternal arterial blood gas values were measured immediately before and after transportation of sheep to the laboratory, and blood samples (0.5 ml) taken from the femoral artery with a chronic catheter were measured with a blood gas analyzer (ABL300 Radiometer, Copenhagen, Denmark, at 39°C) (13). Anesthesia of the animals was rapidly induced with intravenous injection of thiamylal (10 mg/kg). The ewes were then intubated, and anesthesia was maintained on 1.5-2.0% halothane in oxygen throughout surgery. An incision was made in the abdomen and the uterus was exposed. The fourth branches of the main uterine arteries were isolated without stretching and were placed into a modified Krebs solution (pH 7.4) of the following composition (in mM): 115.21 NaCl, 4.70 KCl, 1.80 CaCl₂, 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 removal of the tissues, animals were killed with T-61 (euthanasia solution, Hoechst-Roussel, Somerville, NJ).

Simultaneous measurement of [Ca²⁺]_i and contraction. Smooth muscle [Ca²⁺]_i was measured simultaneously with muscle contraction as reported by Sato et al. (27). 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. After two stimulations of the tissue with 120 mM KCl, tissue autofluorescence intensity was measured at 510 nm induced by 340- and 380-nm excitations, respectively. The ring was then loaded with 5 µM fura 2-acetoxymethyl ester (fura 2-AM) for 4 h in the presence of 0.02% Cremophor EL at room temperature (25°C). After loading, 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.

Materials. Fura 2-AM was obtained from Molecular Probes (Eugene, OR). 5-HT was purchased from Research Biomedicals (Natick, MA). Ionomycin, EGTA, Cremophor EL, KCl, and other chemicals were from Sigma (St. Louis, MO).

Data analysis. Concentration-response curves were analyzed by computer-assisted nonlinear regression to fit the data using GraphPad Prism (GraphPad Software, San Diego, CA). Half-maximal effective concentration (EC₅₀) values for an 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 (log EC₅₀) values. Comparison of regression lines was performed using GraphPad Prism (GraphPad Software). 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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In the arterial rings loaded with fura 2, an increase in [Ca²⁺]_i resulted in an increase in F₃₄₀, a decrease in F₃₈₀, and an increase in R_340/380. Typical traces of simultaneous measurement of 5-HT-stimulated increases in [Ca²⁺]_i and muscle tension development in the uterine artery smooth muscle are shown in Fig. 1. The time delay between the increase in the fluorescence ratio and the contractile force development due to 5-HT stimulation was 2.7 ± 0.33 s (n = 10), which was longer than that due to KCl stimulation (1.1 ± 0.01 s, n = 10).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Typical simultaneous recordings of tension development and fura 2 fluorescence in the uterine artery. The arterial ring was loaded with 5 µM fura 2. Fura 2 fluorescence was measured, using a Jasco CAF-110 fluorometer, simultaneously with tension development (see MATERIALS AND METHODS). 5-Hydroxytryptamine (5-HT; 10 µM) was added as indicated by arrowhead. F340 and F380, fluorescence intensities at excitation wavelengths of 340 and 380 nm, respectively; Ratio(f340/f380), ratio of F340 to F380.

As shown in Fig. 2, addition of 120 mM KCl or 10 µM 5-HT increased both R_340/380 and muscle tension in the uterine artery. The rate of increase in the fluorescence due to 5-HT was slightly slower than that due to high KCl. For both KCl- and 5-HT-induced responses, washing of the tissues with normal Krebs solution decreased R_340/380, which was followed by muscle relaxation. Figure 2 also depicts that, in the same tissue, although the 10 µM 5-HT-induced increment in [Ca²⁺]_i was similar to that induced by 120 mM KCl, the contractile tension mediated by 5-HT was significantly greater than that by KCl. As shown in Fig. 3, contractile tension of the uterine artery at a given amount of increase in [Ca²⁺]_i mediated by KCl and 5-HT was 16.9 ± 4.0 and 29.8 ± 6.9 (g/R_340/380), respectively (P < 0.05, n = 4).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Typical recordings of effect of 5-HT and KCl on intracellular free Ca2+ concentration ([Ca2+])i and tension in the uterine artery. 5-HT- and KCl-mediated muscle contraction and increment in [Ca2+]i were measured simultaneously in the same arterial ring (see MATERIALS AND METHODS). R340/380, ratio of F340 to F380.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   5-HT- and KCl-mediated muscle contraction at a given increment of [Ca2+]i in the uterine artery. Contraction and increase of [Ca2+]i induced by 10 µM 5-HT (filled bars) or 120 mM KCl (open bars) were measured simultaneously in the same arterial ring. Data are from 4 individual experiments (ex1-ex4). Mean values of 5-HT- and KCl-mediated tension at a given increment of [Ca2+]i are presented in the text.

Cumulative additions of KCl induced concentration-dependent increases in contraction of the uterine arteries from both normoxic and chronically hypoxic animals with pD₂ values of 1.73 ± 0.05 and 1.68 ± 0.07, respectively (P > 0.05, n = 5) and maximum contractions of 3.19 ± 0.35 and 2.86 ± 0.37 g, respectively (P > 0.05) (Fig. 4). In accordance with the contractile responses, chronic hypoxia showed no effect on KCl-mediated concentration-dependent increases in [Ca²⁺]_i in uterine arteries (Fig. 5). The pD₂ values were 1.74 ± 0.03 for normoxic arteries and 1.71 ± 0.16 for chronically hypoxic ones (P > 0.05, n = 5).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of chronic hypoxia on KCl-induced contractions of the uterine artery. Cumulative concentration-response curves for KCl were obtained with arterial ring segments from normoxic () and chronically hypoxic () animals. Data are means ± SE of tissues from 5 animals. Mean values of pD2 (log EC50, where EC50 is half-maximal effective concentration) and the maximal responses to KCl are presented in text.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effect of chronic hypoxia on KCl-induced increase in [Ca2+]i of the uterine artery. Cumulative concentration-response curves for KCl were obtained with fura 2-loaded arterial ring segments from normoxic () and chronically hypoxic () animals. Data are means ± SE of tissues from 5 animals. Mean values of the pD2 are presented in text.

The [Ca²⁺]_i-tension relationships depicted from the data of simultaneous measurement of [Ca²⁺]_i and tension in the same tissue (see MATERIALS AND METHODS) indicated that there was a positive correlation between these two parameters in the presence of cumulative doses of KCl in the uterine arteries from both normoxic and chronically hypoxic animals (Fig. 6). The slopes (g tension/nM [Ca²⁺]_i) of the lines were the same in the normoxic arteries (0.015 ± 0.003) and chronically hypoxic ones (0.015 ± 0.002) (P > 0.05, n = 5).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effect of chronic hypoxia on KCl-mediated [Ca2+]i-tension relationship in the uterine artery. [Ca2+]i-tension relationship was obtained by simultaneous measurement of [Ca2+]i and tension induced by cumulative addition of KCl in arterial ring segments loaded with fura 2 from normoxic () and chronically hypoxic () animals. Data are means ± SE of tissues from 5 animals. Mean values of slope (g tension/nM [Ca2+]i) are presented in text.

In contrast to KCl-induced responses, chronic hypoxia significantly decreased contractile sensitivity of the uterine arteries in response to 5-HT (Fig. 7). The pD₂ values of 5-HT-mediated concentration-dependent contractions were 7.20 ± 0.10 and 6.63 ± 0.13 for normoxic and chronically hypoxic arteries, respectively (P < 0.05, n = 6). The maximum contractile responses were 5.03 ± 0.19 g for normoxic arteries and 3.89 ± 0.14 g for chronically hypoxic arteries (P < 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effect of chronic hypoxia on 5-HT-induced contractions of the uterine artery. Cumulative concentration-response curves for 5-HT were obtained with arterial ring segments from normoxic () and chronically hypoxic () animals. Data are means ± SE of tissues from 6 animals. Mean values of pD2 and maximal responses to 5-HT are presented in text.

In agreement with contractile studies, chronic hypoxia also inhibited the potency of 5-HT in stimulating increases in [Ca²⁺]_i in the uterine arteries (Fig. 8). The pD₂ value of 5-HT-induced increases in [Ca²⁺]_i was decreased from 7.46 ± 0.18 in normoxic arteries to 6.86 ± 0.11 in chronically hypoxic arteries (P < 0.05, n = 5), although the maximum responses were slightly higher in chronically hypoxic arteries (416.1 ± 11.8 nM) than that in normoxic ones (320.9 ± 12.7 nM) (P > 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Effect of chronic hypoxia on 5-HT-induced increase in [Ca2+]i of the uterine artery. Cumulative concentration-response curves for 5-HT were obtained with fura 2-loaded arterial ring segments from normoxic () and chronically hypoxic () animals. Data are means ± SE of tissues from 5 animals. Mean values of pD2 and the maximal responses to 5-HT are presented in text.

Figure 9 shows a positive correlation between muscle tension and [Ca²⁺]_i increased by the cumulative additions of 5-HT in both normoxic and chronically hypoxic arteries. Whereas the slope (g tension/nM [Ca²⁺]_i) of the [Ca²⁺]_i-tension relationship mediated by 5-HT in normoxic arteries (0.024 ± 0.002) was significantly greater than that mediated by KCl (0.015 ± 0.003) (P < 0.05), it significantly decreased in chronically hypoxic arteries (0.013 ± 0.001) (P < 0.01, n = 6). There was no significant difference between the slopes of the [Ca²⁺]_i-tension relationship determined from cumulative additions of KCl (0.015 ± 0.002) and 5-HT (0.013 ± 0.001) in chronically hypoxic arteries (P > 0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Effect of chronic hypoxia on 5-HT-mediated [Ca2+]i-tension relationship in the uterine artery. [Ca2+]i-tension relationship was obtained by simultaneous measurement of [Ca2+]i and tension induced by cumulative addition of 5-HT in arterial ring segments loaded with fura 2 from normoxic () and chronically hypoxic () animals. Data are means ± SE of tissues from 6 animals. Mean values of the slope (g tension/nM [Ca2+]i) are presented in text.

	DISCUSSION

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The major finding of this study is that chronic hypoxia does not affect the electromechanical coupling elicited by KCl but significantly suppresses pharmacomechanical coupling mediated by the activation of GTP-binding protein-coupled receptors in smooth muscle of the uterine artery. The two major components of pharmacomechanical coupling of vascular smooth muscle, Ca²⁺ mobilization and Ca²⁺ sensitivity of myofilaments, were significantly suppressed in response to chronic hypoxia.

The relationship of Ca²⁺ and tension development in vascular smooth muscle can be studied in intact or permeabilized preparations, where [Ca²⁺]_i is either measured or controlled, respectively. Although permeabilization of cell membrane has been commonly used in a variety of smooth muscle preparations (1-3, 18, 19), potential regulatory elements in the cell may be lost during permeabilization (15). In the present study, we were able to measure changes in [Ca²⁺]_i simultaneously with tension in isolated intact uterine artery rings. The correlation of [Ca²⁺]_i and tension obtained for both KCl and 5-HT stimulation is a predictable confirmation of the obligatory role that Ca²⁺ plays in vascular smooth muscle contraction and is in good agreement with earlier findings (16, 27). The simultaneous measurement of [Ca²⁺]_i with tension in the same intact tissue allowed us to determine directly the precise relationship between [Ca²⁺]_i and tension in the uterine artery and thus to estimate Ca²⁺ sensitivity of myofilaments with unimpaired excitation-contraction coupling processes and retained regulatory targets for second messenger pathways. The finding of the delay between the agonist-mediated increase in [Ca²⁺]_i and tension development indicates one major component of the common long delay between agonist-receptor interaction and contraction in the smooth muscle and is likely due to prephosphorylation reactions, such as Ca²⁺-calmodulin diffusion and/or activation of myosin light-chain kinase (MLCK) and, to a lesser extent, the time course of myosin phosphorylation (28).

In most smooth muscles, high-KCl-mediated contractions are completely dependent on extracellular Ca²⁺, and L-type Ca²⁺ channel blockers inhibit contraction. In the present study, high KCl induced a sustained increase in [Ca²⁺]_i followed by a sustained contraction in the uterine artery. Simultaneous measurement of [Ca²⁺]_i and tension induced by cumulative addition of KCl indicated a one-to-one ratio in the increments of [Ca²⁺]_i and tension. Furthermore, addition of the L-type Ca²⁺ channel blocker verapamil (10 µM) completely inhibited the high-KCl-stimulated increases in [Ca²⁺]_i and muscle tension (data not shown). Similar results were obtained in other smooth muscle preparations (16, 27). These data suggest that high KCl contracts smooth muscle of the uterine artery, like most other smooth muscles, through electromechanical coupling and primarily by increasing Ca²⁺ influx through L-type Ca²⁺ channels. The finding that the KCl-induced Ca²⁺ mobilization and [Ca²⁺]_i-tension relationship were the same in the normoxic and chronically hypoxic arteries in the present study suggests that mild chronic hypoxia does not affect the L-type Ca²⁺ channel in the uterine artery. Studies in rabbit systemic arteries also indicated that KCl-evoked contractions were not affected by moderate hypoxia and that receptor-operated rather than voltage-operated processes were particularly vulnerable to hypoxia (22). However, the finding that KCl-mediated electromechanical coupling was not changed by chronic hypoxia in the present study does not necessarily preclude the possible changes in membrane potential of vascular smooth muscle in response to chronic hypoxia. In fact, many studies suggest that acute hypoxia activates ATP-dependent K⁺ (K⁺_ATP) channels and relaxes smooth muscle (14, 23, 25). Although studies of the effect of chronic hypoxia on K⁺ channels and smooth muscle membrane potential are extremely limited, one recent study in conscious instrumented rats suggests that opening of K⁺_ATP channels is not involved in the attenuated vasoreactivity associated with chronic hypoxia (9).

In contrast to KCl-mediated responses, the 5-HT-induced Ca²⁺ mobilization and [Ca²⁺]_i-tension relationship in the uterine artery were significantly altered by chronic hypoxia. Chronic hypoxia also significantly suppressed the norepinephrine-mediated Ca²⁺ mobilization and [Ca²⁺]_i-tension relationship in the uterine artery (31). It is unclear whether chronic hypocapnia may also contribute to the changes in responses of the uterine artery observed in the present study. Although arterial PCO₂ moderately decreased from 35 to 29 mmHg, the blood pH was not altered (from 7.44 to 7.46).

There are two major components in G protein-coupled receptor-mediated pharmacomechanical coupling: 1) the agonist-induced increase in [Ca²⁺]_i by either release of Ca²⁺ from intracellular stores or by Ca²⁺ influx, and 2) the agonist-induced increase in Ca²⁺ sensitivity of myofilaments. In light of the findings that 5-HT stimulated a rapid increase in Ins(1,4,5)P₃ (34, 36) as well as Ca²⁺ influx through a voltage-independent Ca²⁺ channel (35) in the uterine artery, the 5-HT-induced increase in [Ca²⁺]_i in the present study is likely due to the combined Ca²⁺ release from internal stores and the influx of extracellular Ca²⁺. It has been firmly established that Ins(1,4,5)P₃ releases Ca²⁺ from intracellular stores in a wide variety of cells and tissues, including smooth muscle (4, 8, 28). Although it has been suggested that Ins(1,4,5)P₃ plays an important role in agonist-mediated, voltage-insensitive entry of external Ca²⁺ (5, 32), it is not clear at the present time that such a mechanism operates during 5-HT-induced contraction in the uterine artery. In our recent studies, we have found that chronic hypoxia significantly suppresses the coupling efficiency between activation of 5-HT₂ receptors and Ins(1,4,5)P₃ synthesis, leading to a decrease in intracellular Ins(1,4,5)P₃ levels in the uterine artery (13). Although the decreased Ins(1,4,5)P₃ synthesis will certainly lead to a decrease in [Ca²⁺]_i, mechanisms downstream, such as Ins(1,4,5)P₃-receptor affinity and density, may also contribute to chronic hypoxia-mediated suppression in 5-HT-induced Ca²⁺ mobilization in the uterine artery. On its formation, Ins(1,4,5)P₃ diffuses into cytosol, where it binds to the Ins(1,4,5)P₃ receptors located on the sarcoplasmic reticulum and/or sarcolemma membrane and mediates Ca²⁺ release (5, 32, 33). The finding that chronic hypoxia inhibited contractile sensitivity of the uterine artery to Ins(1,4,5)P₃, i.e., for a given amount of Ins(1,4,5)P₃ synthesized less contraction was produced in the chronically hypoxic arteries (13), suggests that the chronic hypoxia-mediated decrease in 5-HT-induced Ca²⁺ mobilization in the uterine artery may be due in part to changes in the Ins(1,4,5)P₃ receptor. Indeed, depending on vascular beds, either Ins(1,4,5)P₃ binding affinity to the Ins(1,4,5)P₃ receptor or the density of the receptor could be altered by chronic hypoxia. We have found that chronic hypoxia significantly decreases Ins(1,4,5)P₃ binding affinity to the Ins(1,4,5)P₃ receptor without affecting Ins(1,4,5)P₃ receptor numbers in the uterine artery (31). In contrast, chronic hypoxia significantly decreased Ins(1,4,5)P₃-receptor density without changing Ins(1,4,5)P₃ binding affinity in cerebral arteries (38). Although it was not examined in the present study, the potential effect of chronic hypoxia on the Ca²⁺ channels responsible for agonist-stimulated influx of Ca²⁺ into cells is a promising area for future investigation.

Modulation of myosin phosphorylation independent of changes in [Ca²⁺]_i is another major component of pharmacomechanical coupling. In the present study, when [Ca²⁺]_i and tension were measured simultaneously in the same arterial ring, 5-HT always produced greater contraction than KCl for a given increment in [Ca²⁺]_i. The result suggests that 5-HT-mediated modulation of the Ca²⁺ sensitivity of the contractile apparatus is also a key mechanism governing contractility of the uterine artery. A similar finding of agonist-induced increase in Ca²⁺ sensitivity of myofilaments has also been demonstrated in other smooth muscle preparations (6, 16, 27). Although it is clear that Ca²⁺ sensitivity of myofilaments of vascular smooth muscle is primarily determined by the relative activities of MLCK and myosin light-chain phosphatase (MLCP) (15), many studies suggested that agonist-mediated sensitization of myofilaments to Ca²⁺ was due to inhibition of MLCP, most likely through a GTP-binding protein-dependent pathway (10, 19). We have recently demonstrated that in rabbit and sheep cerebral arteries 5-HT enhances Ca²⁺ sensitivity of myofilaments through GTP-dependent mechanisms (1-3). Both trimeric (28) and monomeric (17) GTP-binding proteins are proposed to be involved in the enhancement of Ca²⁺ sensitivity of myofilaments.

In the present study, chronic hypoxia significantly inhibited 5-HT-enhanced Ca²⁺ sensitivity in the uterine artery. The finding that chronic hypoxia did not affect the KCl-mediated [Ca²⁺]_i-tension relationship but suppressed the slope of the 5-HT-mediated [Ca²⁺]_i-tension relationship suggests that chronic hypoxia specifically inhibits signal transduction processes of GTP-binding protein-coupled receptors. However, the precise cellular and molecular mechanisms underlying the chronic hypoxia-mediated decrease in Ca²⁺ sensitivity of smooth muscle remain uncertain. One of the second messengers that is thought to play an important role in the inhibition of MLCP is protein kinase C (15, 28). Activation of plasma membrane-associated phosphoinositide-specific phospholipase C stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate and generates Ins(1,4,5)P₃ and diacylglycerol, which, in turn, activates protein kinase C. Because chronic hypoxia suppresses this pathway (13), it is likely to decrease protein kinase C activity, leading to an increase in MLCP activity.

Alternatively, the decrease in the agonist-enhanced Ca²⁺ sensitivity in response to chronic hypoxia may be due to GTP-binding protein-independent mechanisms. We have found that chronic hypoxia significantly increases intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels in the uterine artery (37), which is likely due to the chronic hypoxia-induced increase in endothelial nitric oxide release, as shown in other studies (20, 26). In light of the finding that cGMP has a profound effect on Ca²⁺ mobilization in vascular smooth muscle (21), we have shown that 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), a membrane-permeable analog of cGMP, decreases agonist-induced Ins(1,4,5)P₃ synthesis and Ins(1,4,5)P₃ binding affinity to the Ins(1,4,5)P₃ receptor in the uterine artery (37). Not only does cGMP regulate Ca²⁺ mobilization, but also it inhibits Ca²⁺ sensitivity of myofilaments in vascular smooth muscle. In permeabilized vascular smooth muscle, addition of cGMP-preactivated, cGMP-dependent protein kinase reduced myosin phosphorylation and contractile tension proportionally despite a constant [Ca²⁺]_i, suggesting that cGMP decreased the Ca²⁺ sensitivity of contractile proteins (24). We also found that in the uterine artery, when [Ca²⁺]_i and contractile tension were measured simultaneously, addition of 8-BrcGMP completely relaxed 5-HT-induced contraction but inhibited only 50% of increased [Ca²⁺]_i (unpublished observation). Although it may be unlikely that elevated cGMP was responsible for the decreases in 5-HT-induced Ca²⁺ mobilization and Ca²⁺ myofilament sensitivity in uterine arteries in the present study because the study has been done on endothelium-denuded arteries, potential sustained effects of cGMP that were not immediately reversible on removal of the endothelium should not be precluded.

In conclusion, the present study provides, for the first time, the evidence that mild chronic hypoxia produces, among other effects, a direct effect on vascular smooth muscle of the uterine artery and impairs pharmacomechanical coupling of agonist-mediated contractions by inhibiting Ca²⁺ mobilization and Ca²⁺ sensitivity of myofilaments. Whereas KCl-induced contraction was mediated predominantly by an increase in [Ca²⁺]_i, activation of the GTP-binding protein-coupled receptor modulates uterine artery contractions by changing both cytosolic Ca²⁺ concentration and the sensitivity of contractile elements to Ca²⁺. The finding that KCl-mediated contraction and Ca²⁺ responses were not altered suggests that electromechanical coupling processes may not be affected by chronic hypoxia. Whereas protein kinase C and cGMP and/or cGMP-dependent protein kinase may play a role in chronic hypoxia-mediated attenuation of the agonist-enhanced Ca²⁺ sensitivity, many other mechanisms remain to be explored. For example, small monomeric GTP-binding proteins, such as Rho A p21 or Ras p21, mediate the agonist-activated pathway for the inhibition of MLCP and enhancement of Ca²⁺ sensitivity of smooth muscle contractions (10, 17). Modulation of the function of these small GTP-binding proteins could provide a potential target for the effect of chronic hypoxia. It should be borne in mind that the effect of chronic hypoxia on vascular smooth muscle is mediated by multiple modulators, and each modulator may act on more than one site.