INTRODUCTION

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EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) has been proposed to regulate smooth muscle contraction. Activation of ERK is dependent on a dual phosphorylation on Tyr¹⁸⁵ and Thr¹⁸⁷ by mitogen-activated/extracellular-regulated kinase kinase or MEK (5, 6, 34). It has been demonstrated in many studies that different agents that produce contractions of the smooth muscle, activate ERK at the same time (2, 9, 10, 13, 14, 23, 39). However, conflicting results were obtained regarding a role for ERK in smooth muscle contractile regulation. A cause-and-effect relationship between ERK activation and ₁-adrenoceptor-mediated contraction was demonstrated in ferret aorta (10). In addition, the addition of activated ERK to permeabilized airway smooth muscle strips resulted in a contraction by increasing Ca²⁺ sensitivity of contractile proteins (13). In contrast, studies with permeabilized rabbit vascular smooth muscle preparations showed no effect of ERK on Ca²⁺ sensitivity (32). Furthermore, in the swine carotid artery, inhibition of MEK with PD-98059 had no effect on histamine and phorbol 12,13-dibutyrate (PDBu)-mediated contractions (15).

It is unknown whether ERK plays a role in the regulation of uterine artery contractility, and more importantly, whether pregnancy effects the potential role of ERK in the uterine artery. Pregnancy is associated with a growth of uterine vasculature and a dramatic increase in uterine artery blood flow. It has been shown recently that the pregnancy-induced increase in uterine artery endothelial vasodilator production is mediated in part by a marked alteration in the signaling pathway, including activation of the ERK pathway (7). In the present study, we tested the hypothesis that the ERK pathway played an important role in the regulation of uterine artery contractility in an agonist-dependent manner. Furthermore, we tested the hypothesis that pregnancy selectively enhanced the role of ERK in ₁-adrenoceptor-induced contractions and its effect in suppressing protein kinase C (PKC)-mediated contraction in the uterine artery. Specifically, we examined the effect of PD-98059 (a selective MEK inhibitor) on phenylephrine-, serotonin (5-HT)-, PDBu-, and KCl-induced contractions in uterine arteries of both pregnant and nonpregnant sheep. ERK activity was measured using a phosphospecific ERK1/2 antibody. We also examined the role of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺ sensitivity in the ERK-mediated response, and we tested the hypothesis that both the Ca²⁺-dependent and independent components were involved in the ERK pathway 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% to 2.0% halothane in oxygen throughout the surgery. An incision in the abdomen was made and the uterus exposed. The uterine arteries were isolated and removed without stretching and 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, animals were killed with euthanasia solution (T-61, Hoechst-Roussel; Somervile, 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.

Contraction studies. The third (nonpregnant) and fourth (pregnant) branches of the main uterine arteries 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. In some rings the endothelium was removed by gentle rotation of the arterial rings on an approximately sized, rough-surfaced blunt hypodermic needle as described previously (20). Contractile responses were quantified in Krebs solution in tissue baths at 37°C as described previously (20). Isometric tensions were measured. After 60 min of equilibration in the tissue bath, each ring was stretched to the optimal resting tension (1 g) as determined by the tension developed in response to potassium chloride (120 mM) added at each stretch level. Concentration-response curves were obtained by cumulative addition 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 (log EC₅₀) values. All responses are normalized to the maximal high KCl (120 mM) contraction.

Immunoblotting. Arterial rings were equilibrated in the tissue bath, and the optimal tension was obtained as described above. The tissues were then incubated for 30 min with PD-98059 (30 µM) or vehicle alone in the organ bath (37°C). After incubation, they were stimulated with phenylephrine (3 µM). The reaction was stopped by snap-freezing the tissues in liquid nitrogen and stored at 80°C until used. Frozen samples were homogenized in a lysis buffer containing 150 mM NaCl, 50 mM Tris · HCl, 10 mM EDTA, 0.1% Tween 20, 0.1% -mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml leupeptin, and 5 µg/ml aprotinin, pH 7.4. Sample homogenates were then centrifuged at 4°C for 5 min at 6,000 g, and the supernatants were collected. Protein was quantified in the supernatant using protein assay kit from Bio-Rad. Samples with equal protein were loaded on 10% polyacrylamide gel with 0.1% sodium dodecyl sulfate (SDS) and were separated by electrophoresis at 100 V for 2 h. Proteins were then transferred onto immobilon P membrane at 30 V for 50 min at room temperature using a semidry blotter (Bio-Rad; Richmond, CA). Nonspecific binding sites in the membranes were blocked by an overnight incubation at 4°C in a Tris-buffered saline solution (TBS) containing 5% dry milk. The membranes were washed in TBS (3 × 15 min) and then incubated with rabbit phospho-p44/42 MAP kinase (Tyr²⁰²/Tyr²⁰⁴) antibody (at 1:1,000 dilution; New England Biolabs; Beverly, MA). Total p44/42 MAP kinase (ERK1/2) was determined using anti-ERK1/2 antibody (New England Biolabs). Membranes were washed using TBS and then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2,000) obtained from Amersham (Arlington Heights, IL). Immunoreactive bands were visualized by enhanced chemiluminescence. The blots were exposed to hyperfilm. Results were quantified by scanning densitometer (model 670, Bio-Rad). The data were normalized by actin and presented as the percentage of the control protein levels within each group.

Measurement of PKC activity. PKC activity was determined as previously described (8, 37). Briefly, after treatment, tissues were homogenized in buffer A containing (in mM) 20 Tris · HCl, 250 sucrose, 5 EDTA, 5 EGTA, 1 PMSF, 10 -mercaptoethanol, and 1 benzamide. The homogenate was centrifuged at 100,000 g for 60 min at 4°C, and the supernatant was used as the cytosolic fraction. The pellet corresponding to the membrane particulate fraction was solubilized in buffer A containing Triton X-100 at a final concentration of 0.1% by stirring on ice for 45 min at 4°C, followed by centrifugation at 100,000 g for 60 min at 4°C to remove insoluble membrane particles. Cytosolic and solubilized membrane fractions were applied to DEAE-cellulose columns that had been preequilibrated with buffer A including 0.1% Triton X-100. The DEAE columns were washed with 5 ml of buffer A and 5 ml of buffer B containing 20 mM Tris · HCl, 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 mM -mercaptoethanol, and 1 mM benzamide, and 0.1% Triton X-100. PKC was eluted with 2 ml of buffer B including 400 mM NaCl. Protein concentrations were determined with a protein assay kit (Bio-Rad). PKC activity was determined in the cytosol and solubilized membrane particulate fractions using a PKC ELISA assay that utilizes a synthetic peptide and a monoclonal antibody that recognizes the phosphorylated form of the peptide (Upstate Biotech).

Simultaneous measurement of [Ca²+]_i and tension. Simultaneous recordings of contraction and free [Ca²⁺]_i in the same tissue were conducted as described previously (36, 44). 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 (fura 2-AM) for 3 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. 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 by a photomultiplier tube. 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. Phenylephrine, PDBu, 5-HT, PD-98059, staurosporine, and calphostin C were obtained from Sigma (St. Louis, MO). All electrophoretic and immunoblot reagents were from Bio-Rad. Fura 2-AM was obtained from Molecular Probes (Eugene, OR). PKC activity ELISA assay kit was obtained from Upstate Biotech (Lake Placid, NY). All drugs were prepared fresh each day and kept on ice throughout the experiment.

Data analysis. Concentration-response curves were analyzed by computer-assisted nonlinear regression to fit the data using GraphPad Prism (GraphPad software; San Diego, CA). Results were expressed as means ± SE obtained from the number (n) of experimental animals given. Differences were evaluated for statistical significance (P < 0.05) by one-way ANOVA and paired Student t-test.

	RESULTS

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Effect of PD-98059 on agonist-mediated contractions. Figure 1 shows the effect of PD-98059 on the phenylephrine-induced contraction of the uterine artery. PD-98059 showed no effect on basal tension in the uterine arteries of both nonpregnant and pregnant sheep. As depicted in Fig. 1, PD-98059 did not effect phenylephrine-induced contractions in nonpregnant uterine arteries (pD₂: 5.76 ± 0.12 vs. 5.50 ± 0.14, P > 0.05) but significantly shifted the phenylephrine concentration-response curve to the right in uterine arteries from pregnant sheep (pD₂: 6.25 ± 0.02 vs. 5.44 ± 0.24, P < 0.05). The maximal response was not effected (Fig. 1). Removal of the endothelium did not change the inhibition of PD-98059 on phenylephrine-induced contractions (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of PD-98059 (PD) on phenylephrine (PE)-induced contraction in ovine uterine arteries. Arterial rings were pretreated with 30 µM PD or with the vehicle DMSO (control) for 30 min and then subjected to the cumulative addition of PE in Krebs solution. Data are expressed as percentages of contraction produced by 120 mM KCl, and each point represents mean ± SE of 4 animals. The pD2 (log EC50) values were presented in the text. A: pregnant uterine artery. B: nonpregnant uterine artery.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effect of PD on serotonin (5-HT)-induced contraction in ovine uterine arteries. Arterial rings were pretreated with 30 µM PD or with the vehicle DMSO (control) for 30 min and then subjected to the cumulative addition of 5-HT in Krebs solution. Data are expressed as percentages of contraction produced by 120 mM KCl, and each point represents mean ± SE of 4 animals. The pD2 (log EC50) values were presented in the text. A: pregnant uterine artery. B: nonpregnant uterine artery.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of PD on phorbol 12,13-dibutyrate (PDBu)-induced contraction in ovine uterine arteries. Arterial rings were pretreated with 30 µM PD-98059 or with the vehicle DMSO (control) for 30 min and then subjected to the cumulative addition of PDBu in Krebs solution. Data are expressed as percentages of contraction produced by 120 mM KCl, and each point represents mean ± SE of 4 animals. The pD2 (log EC50) values were presented in the text. A: pregnant uterine artery. B: nonpregnant uterine artery.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of PD on PDBu-stimulated protein kinase C (PKC) activity in ovine uterine artery. Pregnant uterine arteries were pretreated with 30 µM PD, 0.1 µM staurosporine (Stau), and 0.1 µM calphostin C (Calp) for 30 min before the addition of PDBu (3 µM, 30 min). PKC activity in the cytosolic and particulate fractions was determined as described in METHODS. Data are means ± SE of 4 animals. aP < 0.05, vs. rest; bP < 0.05, vs. PDBu alone.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effect of PD on KCl-induced contraction in ovine uterine arteries. Arterial rings were pretreated with 30 µM PD or with the vehicle DMSO (control) for 30 min and then subjected to the cumulative addition of KCl in Krebs solution. Data are means ± SE of 4-6 animals. The pD2 (log EC50) values were presented in the text. A: pregnant uterine artery. B: nonpregnant uterine artery.

Effect of ₁-adrenergic agonist on the ERK activation. Figure 6 shows total ERK1/2 protein levels in uterine arteries from nonpregnant and pregnant sheep and demonstrates that pregnancy is associated with an increase in ERK2 protein levels in the uterine artery. To demonstrate that the effect of PD-98059 observed in the contraction was associated with inhibition of ERK activation, we measured the phenylephrine-induced phosphorylation of ERK using an ERK1/2 phospho-MAP kinase antibody. Figure 7 depicts data from experiments in which pregnant uterine artery rings in tissue baths were exposed to phenylephrine (3 µM) following PD-98059 pretreatment (30 min) or the vehicle (DMSO) alone. The tissues were treated with phenylephrine for 5 min followed by immediately being frozen in liquid nitrogen to stop the reaction. As shown in Fig. 7, phenylephrine significantly increased tyrosyl-phosphorylation of two proteins with molecular masses of 44 kDa (207% above control) and 42 kDa (236% above control). PD-98059 alone did not effect basal p44/p42 MAP kinase phosphorylation, but significantly inhibited phenylephrine-induced tyrosyl-phosphorylation of both ERK1 and ERK2 by 50% and 53%, respectively (Fig. 7).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of pregnancy on total extracellular signal-regulated kinase (ERK)1/2 protein levels in uterine arteries. A: scanned images of representative anti-p44/42-mitogen-activated protein kinase (MAPK) immunoblot from pregnant (P) and nonpregnant (NP) uterine arteries. Two bands detected correspond to the 44-kDa isoform (ERK1) and 42-kDa isoform (ERK2). B: ERK2 (42 kDa) determined by densitometry. *P < 0.05, vs. nonpregnant. C: ERK1 (44 kDa) determined by densitometry. Data are means ± SE of 10 animals.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Effect of PE on ERK activation in uterine arteries. Pregnant uterine arteries were pretreated with 30 µM PD or with the vehicle DMSO (control, C) for 30 min before the addition of PE. When the contraction reached its steady state (PE alone or PE + PD group) (5 min), the rings were snap-frozen in liquid N2. A: scanned images of representative anti-phospho-MAP kinase immunoblot from uterine artery treated as above. Two bands detected correspond to the 44-kDa isoform (ERK1) and 42-kDa isoform (ERK2). B: phosphorylation of ERK2 (42 kDa) determined by densitometry and expressed relative to -actin density. C: phosphorylation of ERK1 (44 kDa) determined by densitometry and expressed relative to -actin density. Data are means ± SE of 4 animals. aP < 0.05, control vs. PE; bP < 0.05, PE vs. PE + PD.

Effect of PD-98059 on phenylephrine-induced changes in [Ca²+]_i and Ca²⁺ sensitivity. To examine the potential effect of ERK on agonist-mediated Ca²⁺ concentration and Ca²⁺ sensitivity, contractile tension and [Ca²⁺]_i were measured simultaneously in the same tissue as described in METHODS. 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 fluorescence ratio (R_340/380). Typical traces of simultaneous measurement of phenylephrine-stimulated increase in [Ca²⁺]_i and muscle tension development in the pregnant uterine artery are shown in Fig. 8. Because preliminary studies demonstrated that 10 and 30 µM PD-98059 completely blocked phenylephrine-induced contractions in this preparation, subsequent studies were performed using 3 µM PD-98059. PD-98059 significantly decreased basal [Ca²⁺]_i as evidenced of reducing the fura 2 R_340/380 from 0.198 ± 0.012 to 0.146 ± 0.008 (P < 0.05) in pregnant but not nonpregnant (0.099 ± 0.01 vs. 0.092 ± 0.009, P > 0.05) uterine arteries. Figure 8 shows the real time effect of PD-98059 on phenylephrine-induced [Ca²⁺]_i response and contractile tension in intact pregnant uterine arteries. As shown in Fig. 8, 10 µM phenylephrine caused an increase in [Ca²⁺]_i and contractile tension simultaneously. After wash and recovery, the same tissue was pretreated with 3 µM PD-98059 for 30 min and then challenged again with 10 µM phenylephrine. Both the phenylephrine-induced [Ca²⁺]_i and contractile tension were significantly reduced in the presence of PD-98059 (Fig. 8). The responses to phenylephrine were completely recovered after the removal of PD-98059 (Fig. 8). Quantitative analysis of the data revealed that PD-98059 decreased phenylephrine-induced contractile tension and [Ca²⁺]_i by 71% and 53%, respectively (P < 0.05, paired t-test) (Fig. 9). In addition, the simultaneous measurement of [Ca²⁺]_i with tension in the same intact tissue allowed us to determine directly the precise relationship between fura 2 R_340/380 and tension in the uterine artery and thus to estimate Ca²⁺ sensitivity of myofilaments. As shown in Fig. 9, the contraction of the uterine artery from a pregnant sheep at a given amount of increase in [Ca²⁺]_i mediated by phenylephrine was significantly decreased by PD-98059. In contrast, PD-98059 had no effect on [Ca²⁺]_i and Ca²⁺ sensitivity induced by phenylephrine in uterine arteries from nonpregnant sheep (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Effect of PD on PE-induced contraction and intracellular Ca2+ concentration ([Ca2+]i) in the uterine artery. Representative traces show the effect of PD on PE-induced [Ca2+]i (fura 2 signal Rf340/f380, A) and contraction (B) recorded simultaneously in the same tissue of pregnant uterine artery loaded with fura 2. Tissue was stimulated first with 10 µM PE. After washout, it was treated with 3 µM PD for 30 min, followed by the same concentration of PE. After washout, the same tissue was stimulated with PE again.

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Effect of PD on PE-induced [Ca2+]i and Ca2+ sensitivity in the uterine artery. PE (10 µM)-induced [Ca2+]i (fura 2 signal Rf340/f380) and contraction were recorded simultaneously in the absence (control) or presence of PD (3 µM for 30 min) in the same tissue of pregnant uterine artery loaded with fura 2 as shown in Fig. 7. Data are means ± SE of 4 animals. *P < 0.05.

	DISCUSSION

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The present study has demonstrated that the ERK pathway plays a key role in the regulation of uterine artery contractility. More importantly, the effect of ERK on the uterine artery is regulated by pregnancy. There are several important observations in the present study. First, among the several agonists tested, PD-98059 selectively inhibited 5-HT-induced contractions in the nonpregnant uterine artery. Second, pregnancy selectively augmented the inhibition of PD-98059 on ₁-adrenoceptor-induced, but not 5-HT-induced, contractions in uterine arteries. Third, in agreement with the previous finding in the rat thoracic aorta (22), the PDBu-induced contraction was significantly attenuated in the pregnant uterine artery. PD-98059 did not effect the PDBu-mediated contraction in nonpregnant uterine arteries, but significantly increased it in pregnant uterine arteries. In accordance, PD-98059 significantly enhanced PDBu-stimulated PKC activity in pregnant uterine artery. Fourth, PD-98059 had no effect on KCl-mediated contractions in both nonpregnant and pregnant uterine arteries. Fifth, activation of ₁-adrenoceptors increased tyrosyl-phosphorylation of ERK1/2, which was blocked by PD-98059. Sixth, PD-98059-mediated inhibition of the phenylephrine-induced contraction was associated with a decrease in both [Ca²⁺]_i concentration and Ca²⁺ sensitivity of contractile proteins in the uterine artery.

The agonist-stimulated activation of ERK has been well documented in cultured smooth muscle cells (16, 21, 28, 29) and intact smooth muscle (3, 10, 23). The lack of effect of PD-98059 on KCl-induced contractions in both nonpregnant and pregnant uterine arteries suggests that the function of Ca²⁺ channels may not be regulated by the ERK pathway in uterine arteries. This is in agreement with several previous findings (10, 12, 30, 39). On the other hand, it was also reported that tyrosine kinase inhibitors inhibited L-type, voltage-gated Ca²⁺ channels (2, 23, 40). Similarly, several previous studies have explored the role of ERK in the regulation of agonist-mediated arterial contractions in different animal models and arterial types, but the results are controversial (10, 13, 15, 32). The present finding that PD-98059 inhibited 5-HT-induced contractions of the uterine artery is in agreement with the previous study in which 5-HT-mediated contractions were inhibited by PD-98059 in rat aorta, mesenteric artery, and tail artery (39). We have previously demonstrated (42, 44) that 5-HT-elicited contractions of the uterine artery are mediated by the increase of inositol (1,4,5)-trisphosphate, leading to release of Ca²⁺ from intracellular stores. The present results suggest the involvement of the ERK pathway in 5-HT-induced contractions of the uterine artery. It has been demonstrated that 5-HT stimulates the activation of ERK1/2 in arterial smooth muscle (12, 39). The finding that the inhibitions of PD-98059 on 5-HT-induced contractions were not different in pregnant and nonpregnant uterine arteries suggests that the effect of ERK on the 5-HT-mediated contraction is not regulated by pregnancy.

Unlike its effect on the 5-HT-mediated contraction, PD-98059 showed no effect on the phenylephrine-induced contractions in the nonpregnant uterine artery. This is contrary to the previous finding in the ferret aorta in which PD-98059 inhibited phenylephrine-induced contractions (10). However, PD-98059 did not effect phenylephrine-induced contractions in rat mesenteric resistance arteries (30). These results suggest that the role of ERK in the ₁-adrenoceptor-mediated contraction show a considerable heterogeneity in vessel types. Nevertheless, PD-98059 did inhibit phenylephrine-mediated contractions in the pregnant uterine artery. In consistent with the contraction results, Western analysis indicated that phenylephrine increased protein tyrosyl-phosphorylation of both p42 and p44 MAP kinase in pregnant uterine artery, which was blocked by PD-98059. These results suggest that pregnancy upregulates the coupling of the ERK pathway to ₁-adrenoceptor-mediated contractions in the uterine artery. Given that both 5-HT and ₁-adrenoceptor-mediated contractions share a common downstream signal, i.e., inositol (1,4,5)-trisphosphate, in the uterine artery (19, 42-44), it is intriguing that PD-98059 differentially regulated 5-HT and phenylephrine-induced contractions in uterine arteries. This would suggest a specific coupling of the ERK pathway to individual receptor signaling pathways. Although the cellular mechanisms for this selectivity are not presently clear, it is postulated that scaffolding proteins may play an important role.

It is generally believed that during pregnancy the uterine vasculature acts as a low-resistance shunt to accommodate the large increase in uteroplacental blood flow required for normal fetal development. The mechanisms for the attenuated vascular tone may involve a decreased role of endogenous vasoconstrictors and/or an increased role of both endogenous vasodilator and placental angiogenic factors (31, 35, 41). Given that ₁-adrenoceptors play a key role in moment-to-moment regulation of uterine vascular tone, the finding that pregnancy selectively upregulated the role of ERK in ₁-adrenoceptor (but not 5-HT)-mediated contractions in the uterine artery warrants a physiological significance of the ERK pathway in the regulation of uterine artery contractility during pregnancy. Nonetheless, it is not clear at present whether and to what extent the enhanced ERK pathway in ₁-adrenoceptor-mediated signaling effects the vascular tone of pregnant uterine artery. It has been shown recently that the pregnancy-induced increase in uterine artery endothelial vasodilator production is mediated in part by a marked alteration in the signaling pathway including activation of the ERK pathway (7). The present finding that the inhibition of PD-98059 on the phenylephrine-induced contraction was not changed with and without the endothelium in both pregnant and nonpregnant uterine arteries suggests a less important role of ERK on the uterine artery endothelium.

The finding that the PDBu-induced contraction was significantly attenuated in the pregnant uterine artery is in agreement with previous results in rat thoracic aorta (22), suggesting that the role of PKC in the regulation of uterine vascular tone is downegulated during pregnancy. It has been well documented that PKC plays an important role in the regulation of the sustained phase of contraction in vascular smooth muscle (18). In consistent with the previous findings (1, 15, 30, 39), PD-98059 had no effect on the PDBu-induced contraction in nonpregnant uterine arteries. To our surprise, PD-98059 significantly increased PDBu-induced contractions in pregnant uterine arteries. To our knowledge, it has not been reported previously that PD-98059 increases contractions to any agonists examined. It has been proposed in several studies that PKC is an upstream signal of the activation of the ERK pathway in the vascular smooth muscle (24-27, 29, 30). In contrast, the present result that the PBDu-induced contraction was increased after the ERK inhibition by PD-98059 would suggest a role for ERK in the regulation of PKC as a downstream signal in the uterine artery of pregnant animals. This is supported by the finding that PD-98059 significantly enhanced PDBu-stimulated PKC activity in pregnant uterine arteries. The finding that PD-98059 increased PDBu-induced contractions of pregnant uterine arteries and eliminated its difference between nonpregnant and pregnant uterine arteries is likely to have physiological significance and suggests that ERK may play a very important role in increased uterine blood flow by suppressing the PKC-mediated contraction during pregnancy. Consistent with this notion, the present study demonstrated a significant increase in ERK-2 protein levels in the uterine arteries of pregnant sheep, compared with the uterine arteries of nonpregnant sheep. This is in agreement with our recent findings in uterine artery endothelial cells in which pregnancy is associated with an enhancement in ERK-2 signaling pathway (7, 11).

Whereas the mechanisms underlying ERK-mediated inhibition of PKC-induced contractions are not clear at present, our study demonstrated that PD-98059 inhibited the phenylephrine-mediated contraction by decreasing both phenylephrine-induced intracellular Ca²⁺ concentration and Ca²⁺ sensitivity. To our knowledge, our results are the first to show a direct relation between PD-98059-mediated inhibitions of agonist-induced contraction and Ca²⁺ concentration in intact muscle. Previous studies have suggested that PD-98059 inhibits agonist-induced contraction by decreasing Ca²⁺ sensitivity of contractile proteins through the inhibition of ERK-mediated phosphorylation of caldesmon (4, 10, 12, 17, 18). Studies in isolated smooth muscle cells indicated that PD-98059 did not effect agonist-induced [Ca²⁺]_i (33, 38). However, the effect of PD-98059 on Ca²⁺ concentration in intact smooth muscle was not examined. We have developed a method to measure contractile tension and [Ca²⁺]_i simultaneously in the same intact arterial ring (44). This allowed us to directly determine the precise relationship between fura 2 R_340/380 and tension in the uterine artery and thus to estimate not only Ca²⁺ concentration but also Ca²⁺ sensitivity of myofilaments with unimpaired excitation-contraction coupling processes and retained regulatory targets for second messenger pathways. The present study clearly demonstrated that the PD-98059-mediated inhibition of phenylephrine-induced contraction was associated with a decrease in both Ca²⁺ concentration and Ca²⁺ sensitivity in the uterine artery. Our findings suggest, therefore, that, in addition to the Ca²⁺-independent pathway as proposed previously, the ERK signaling pathway also involves the Ca²⁺-dependent component of vascular contractions. It is noteworthy that PD-98059 decreased not only the agonist-induced Ca²⁺ concentration, but also the basal Ca²⁺ concentration. This raises the possibility that ERK may play a role in the regulation of basal tone of the uterine artery.

In summary, the results indicate that ERK plays an important role in the regulation of uterine artery contractility, and its effect is agonist dependent. More importantly, pregnancy selectively enhances the role of ERK in ₁-adrenoceptor-mediated contractions and its effect in suppressing the protein kinase C-mediated contraction in the uterine artery. In addition, both the Ca²⁺-dependent and independent components are involved in the ERK pathway in the uterine artery. The physiological role of the ERK pathway and its mechanisms in the adaptation of uterine vascular reactivity to pregnancy are important avenues for future studies.