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Am J Physiol Heart Circ Physiol 291: H2282-H2289, 2006. First published May 12, 2006; doi:10.1152/ajpheart.00321.2006
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Regulation of {alpha}1-adrenoceptor-mediated contractions of uterine arteries by PKC: effect of pregnancy

Hongying Zhang, DaLiao Xiao, Lawrence D. Longo, and Lubo Zhang

Center for Perinatal Biology, Department of Physiology and Pharmacology, Loma Linda University School of Medicine, Loma Linda, California

Submitted 28 March 2006 ; accepted in final form 11 May 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Protein kinase C (PKC) plays an important role in the regulation of uterine artery contractility and its adaptation to pregnancy. The present study tested the hypothesis that PKC differentially regulates {alpha}1-adrenoceptor-mediated contractions of uterine arteries isolated from nonpregnant (NPUA) and near-term pregnant (PUA) sheep. Phenylephrine-induced contractions of NPUA and PUA sheep were determined in the absence or presence of the PKC activator phorbol 12,13-dibutyrate (PDBu). In NPUA sheep, PDBu produced a concentration-dependent potentiation of phenylephrine-induced contractions and shifted the dose-response curve to the left. In contrast, in PUA sheep, PDBu significantly inhibited phenylephrine-induced contractions and decreased their maximum response. Simultaneous measurement of contractions and intracellular free Ca2+ concentrations ([Ca2+]i) in the same tissues revealed that PDBu inhibited phenylephrine-induced [Ca2+]i and contractions in PUA sheep. In NPUA sheep, PDBu increased phenylephrine-induced contractions without changing [Ca2+]i. Western blot analysis showed six PKC isozymes, {alpha}, betaI, betaII, {delta}, {epsilon}, and {zeta}, in uterine arteries, among which betaI, betaII, and {zeta} isozymes were significantly increased in PUA sheep. In contrast, PKC-{alpha} was decreased in PUA sheep. In addition, analysis of subcellular distribution revealed a significant decrease in the particulate-to-cytosolic ratio of PKC-{epsilon} in PUA compared with that in NPUA sheep. The results suggest that pregnancy induces a reversal of PKC regulatory role on {alpha}1-adrenoceptor-mediated contractions from a potentiation in NPUA sheep to an inhibition in PUA sheep. The differential expression of PKC isozymes and their subcellular distribution in uterine arteries appears to play an important role in the regulation of Ca2+ mobilization and Ca2+ sensitivity in {alpha}1-adrenoceptor-mediated contractions and their adaptation to pregnancy.

calcium mobilization; calcium sensitivity; adaptation; sheep

PREGNANCY IS ASSOCIATED WITH a significant decrease in uterine vascular tone and a striking increase in uterine blood flow, which ensures normal fetal development. Multiple mechanisms are involved in the adaptation of uterine artery contractility during pregnancy (3, 12, 38, 48, 50). Zhang's laboratory (45, 48, 50) has recently demonstrated in ovine uterine arteries that protein kinase C (PKC) plays an important role in the regulation of uterine artery contractility, and the pregnancy-associated decrease in myogenic tone of the uterine artery is primarily regulated through the PKC signaling pathway. In addition to its regulatory effect of myogenic tone (10, 45), PKC interacts with {alpha}1-adrenoceptors and modulates {alpha}1-adrenoceptor-mediated contractions of vascular smooth muscle (12, 35, 37). Activation of {alpha}1-adrenoceptors plays a key role in the regulation of uterine artery smooth muscle contractions (7, 12, 28, 41, 51). Yet, whether or to what extent PKC regulates {alpha}1-adrenoceptor-mediated contractions of the uterine artery and their adaptation to pregnancy remains unclear.

Activation of {alpha}1-adrenoceptors leads to hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG). Ins(1,4,5)P3 binds to Ins(1,4,5)P3 receptors and stimulates Ca2+ release from intracellular Ca2+ stores. On the other hand, DAG activates PKC that has been suggested to play a key role in {alpha}1-adrenoceptor-mediated Ca2+ sensitization (4, 13, 24, 31, 36, 40). In addition to its coupling to {alpha}1-adrenoceptors, activation of PKC by phorbol esters, synthetic analogs of DAG, has been shown to regulate {alpha}1-adrenoceptor-mediated contractions in vascular smooth muscle. Both PKC-mediated inhibition and potentiation of {alpha}1-adrenoceptor-mediated contractions have been reported. Thus activation of PKC by phorbol esters inhibited norepinephrine-induced contractions in rat aorta, and cat and sheep cerebral arteries (2, 6, 9, 25, 27, 34, 39, 53). On the other hand, activation of PKC potentiated {alpha}1-adrenoceptor-mediated contractions in rabbit aorta, rat mesenteric arteries, femoral arteries, corpora cavernosa, and vas deferens (1, 14, 18, 32, 33). The effect of potentiation was abolished by depleting PKC after prolonged treatment with phorbol esters (14). The controversial effects of PKC on {alpha}1-adrenoceptor-mediated contractions in different arteries from different species may be due in part to the diversity of PKC isozymes, which have different enzymatic properties, substrates, functions, and different subcellular distributions in different blood vessels and species (15, 20, 21, 23, 26, 37).

The present study tested the hypothesis that activation of PKC differentially regulates {alpha}1-adrenoceptor-mediated contractions of uterine arteries from nonpregnant and pregnant sheep. Concentration-response curves of phenylephrine-induced contractions of the uterine arteries were conducted in the absence or presence of the PKC activator phorbol 12,13-dibutyrate (PDBu). To evaluate the role of Ca2+ in the PKC-mediated effects, phenylephrine-induced contractions and intracellular free Ca2+ concentrations ([Ca2+]i) were measured simultaneously in the same tissues of uterine arteries. In addition, the differential expression of PKC isozymes and their subcellular distributions in uterine arteries from nonpregnant and pregnant sheep were determined.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue preparation. Nonpregnant and near-term pregnant (~140 day gestation) ewes were anesthetized with thiamylal (10 mg/kg), administered via the external left jugular vein. The ewes were then intubated, and anesthesia was maintained with 1.5–2.0% halothane in O2 throughout the surgery. An incision was made in the abdomen to expose the uterus. The uterine arteries were isolated and removed without stretching and were placed in a modified Krebs solution (pH 7.4) of the following composition: (in mM) 115.2 NaCl, 4.7 KCl, 1.80 CaCl2, 1.16 MgSO4, 1.18 KH2PO4, 22.14 NaHCO3, 0.03 EDTA, and 7.88 dextrose. The Krebs solution was oxygenated with a mixture of 95% O2-5% CO2. After the tissues were removed, animals were euthanized 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 in the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.

Contraction studies. The third (in nonpregnant ewes) and fourth (in pregnant ewes) branches of the main uterine arteries with similar external diameter were dissected and cut into 2-mm ring segments. Isometric tension was measured in the Krebs solution in a tissue bath at 37°C as described previously (50). Briefly, each ring was equilibrated for 60 min and then gradually stretched to the optimal resting tension as determined by the tension that developed in response to 120 mM KCl added at each stretch level. Tissues were then stimulated with cumulative additions of phenylephrine in approximate one-half log increments to generate a concentration-response curve, and contractile tensions were recorded with an online computer. After phenylephrine was washed away, tissues were relaxed to the baseline and were recovered at the resting tension for 30 min. The second concentration-response curves of phenylephrine-induced contractions were then repeated in the absence or presence of PDBu (10, 30, and 100 nM for uterine arteries from nonpregnant ewes, and 0.1, 0.3, and 1 µM for uterine arteries during pregnancy, for 10 min). The different concentration ranges of PDBu were chosen in uterine arteries from nonpregnant and pregnant ewes, based on our previous findings that PDBu was 10 times more potent in contracting uterine arteries from nonpregnant than pregnant ewes [pD2 (–log EC50), 6.64 ± 0.07 vs. 5.62 ± 0.17] (48). To determine the effects of PDBu on phenylephrine-induced contractions, concentrations of PDBu less than EC50 values in uterine arteries from nonpregnant and pregnant ewes, respectively, were utilized in the present study. The concentrations of phenylephrine were chosen to produce full concentration-response curves in uterine arteries from both nonpregnant and pregnant ewes.

Simultaneous measurement of [Ca2+]i and tension. Smooth muscle [Ca2+]i was measured simultaneously with muscle contractions in the same tissues as described previously (52). 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 HEPES buffer containing (in mM) 115.2 NaCl, 4.7 KCl, 1.80 CaCl2, 1.16 MgSO4, 1.18 KH2PO4, 10.0 HEPES, 0.03 EDTA, and 7.88 dextrose (pH 7.4) under a resting tension of 0.5 g for 40 min, followed by stimulation with 120 mM KCl once and recovery to the resting tension for 30 min. The tissue was then loaded with 5 µM fura-2 AM for 3 h in the presence of 0.02% cremophor EL and 0.25% DMSO at 25°C. After being loaded, the tissue was washed with HEPES buffer at 37°C for 45 min to allow for hydrolysis of fura-2 ester groups by endogenous esterase. The tissue was then stimulated twice with 120 mM KCl. After recovery for 30 min, the tissue was stimulated with phenylephrine (3 µM for uterine arteries from both nonpregnant and pregnant ewes to produce submaximal contractions, and ~30–200 nM for uterine arteries from nonpregnant ewes to produce <50% of KCl maximum) in the absence or presence of 0.1 µM PDBu. The experimental protocol is shown on the diagram in Fig. 1. Agonist-induced changes in contractile force 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 (Rf340/380) were recorded with a time constant of 250 ms and stored with the force signal on a computer.


Figure 1
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  		Fig. 1. Experimental protocol. PDBu, phorbol 12,13-dibutyrate.

 
Measurement of PKC isozymes. To determine the protein expression of PKC isozymes in uterine arteries, tissues (third or fourth branches of uterine arteries from nonpregnant and pregnant ewes, respectively) were homogenized in the lysis buffer containing 20 mM Tris·HCl, 250 mM sucrose, 5 mM EDTA, 5 mM EGTA, 0.2% Triton X-100, 10 mM beta-mercaptoethanol, 1 mM benzamidine, 1 mM PMSF, 50 µM leupetin, 1 mM dithiothreitol, and 2 µg/ml aprotinin (pH 7.5). Homogenates were centrifuged at 6,000 g for 5 min at 4°C, and the supernatants were collected. Protein was quantified with a protein assay kit (Bio-Rad). Western blotting was performed as described in Western blot analysis.

Measurement of PKC isozyme distribution and relative activity. To determine the distribution of PKC isozymes in cytosolic and particulate fractions of uterine arterial smooth muscle, tissues (third or fourth branches of uterine arteries from nonpregnant and pregnant ewes, respectively) were homogenized in ice-cold homogenization buffer A containing (in mM) 20 Tris·HCl, 250 sucrose, 5 EDTA, 5 EGTA, 10 beta-mercaptoethanol, 1 benzamidine, 1 PMSF, 50 leupetin, and 1 dithiothreitol and 2 µg/ml aprotinin (pH 7.5). The homogenates were centrifuged at 100,000 g for 20 min at 4°C, and the supernatants were collected and used as the cytosolic fraction (S). The pellets were resuspended in homogenization buffer A containing 1% Triton X-100 by stirring for overnight at 4°C, diluted with the buffer A to a final concentration of 0.2% Triton X-100, and then centrifuged at 100,000 g for 20 min at 4°C. The supernatants were collected and referred to as the particulate fraction (P). Protein concentrations were determined with a protein assay kit (Bio-Rad). Immunoreactive bands for PKC isozymes in cytosolic and particulate fractions were determined by Western blotting using specific PKC isozyme antibodies as described in Western blot analysis. The ratio of P to S was used to determine the relative activity of PKC isozymes.

Western blot analysis. Protein-matched samples obtained from the tissues or cytosolic and particulate fractions were subjected to electrophoresis on 7.5% sodium dodecylsulfate-polyacrylamide gel and then transferred electrophoretically to nitrocellulose membranes. The membranes were incubated at room temperature for 1 h in Tris-buffered saline solution containing 5% dried milk and 0.5% Tween 20, followed by incubation with primary anti-PKC isozyme antibodies overnight at 4°C and secondary antibody for 1 h at room temperature. Polyclonal antibodies to PKC-{alpha}, -betaI, -betaII, -{delta}, -{epsilon}, -{zeta}, and -{gamma} were used. Bands were detected with enhanced chemiluminsecence, visualized on Hyperfilm, and analyzed with the Kodak 1D image analysis software. To determine the total PKC levels, the same amount of protein (5 µg) from each sample was loaded to the gel. To normalize the loading variation of each sample, the corresponding actin level presented in each sample was determined by using monoclonal anti-actin as primary antibody. To determine the ratio of P to S, equal volume (10 µl) of cytosolic and particulate fractions, respectively, from each sample was loaded to the gel.

Materials. Phenylephrine, PDBu, and anti-actin antibody were obtained from Sigma (St. Louis, MO). Anti-PKC isozyme antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Fura-2 AM was from Molecular Probes (Eugene, OR). All electrophoretic and immunoblot reagents were from Bio-Rad. General laboratory reagents were of analytical grade or better and were purchased from Sigma and Fisher Scientific. All drugs were prepared freshly each day and kept on ice throughout the experiment.

Data analysis. Data 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. Differences were evaluated for statistical significance (P < 0.05) by one-way ANOVA and Student's t-test.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of PDBu on phenylephrine-induced contractions. Figure 2 shows that phenylephrine produced concentration-dependent contractions of uterine arteries from both nonpregnant and pregnant ewes. In uterine arteries from nonpregnant ewes, PDBu (10, 30, and 100 nM) induced concentration-dependent contractions and produced a concentration-dependent potentiation of phenylephrine-induced contractions. In the presence of 100 nM PDBu, the concentration-response curve of phenylephrine-induced contractions was markedly shifted to the left with a significant increase in the pD2 value from 6.27 ± 0.10 to 8.86 ± 0.28 (P < 0.001), representing an over 300-fold increase in the potency of phenylephrine-induced contractions in uterine arteries from nonpregnant ewes. The maximal responses of phenylephrine-induced contractions were not affected with PDBu (Figs. 2 and 3). In uterine arteries during pregnancy, PDBu failed to induce contractions up to 1 µM, which produced a comparable contraction (1.30 ± 0.34 g) as that induced by 100 nM PDBu in uterine arteries from nonpregnant ewes (1.38 ± 0.74 g). As shown in Fig. 2, in contrast to its effect in uterine arteries from nonpregnant ewes, PDBu produced a concentration-dependent inhibition of phenylephrine-induced contractions in uterine arteries during pregnancy. Whereas the pD2 values were not affected, the maximal responses of phenylephrine-induced contractions were significantly decreased by 27%, 37%, and 68% in the presence of 0.1, 0.3, and 1 µM PDBu, respectively (Figs. 2 and 3). In uterine arteries from both nonpregnant and pregnant ewes, KCl-induced contractions were not affected with PDBu (data not shown).


Figure 2
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  		Fig. 2. Effect of PDBu on concentration-response curves of phenylephrine-induced contractions in uterine arteries from nonpregnant and pregnant ewes. Data are means ± SE of tissues from 4 animals.

 

Figure 3
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  		Fig. 3. Effect of PDBu on pD2 (–log EC50) value and maximal response (Tmax) of phenylephrine-induced contractions in uterine arteries from nonpregnant and pregnant ewes. Data are means ± SE of tissues from 4 animals. *P < 0.05, control vs. PDBu treatment.

 
Effect of PDBu on phenylephrine-induced changes of [Ca2+]i. Figure 4 shows the effects of PDBu on phenylephrine-induced increases in [Ca2+]i and contractile tensions, measured simultaneously in the same tissues, in uterine arteries from both nonpregnant and pregnant ewes. In uterine arteries during pregnancy, the submaximal concentration (3 µM) of phenylephrine induced significant increases in [Ca2+]i and contractile tension. PDBu (100 nM) produced a minimal contraction in the absence of changes in [Ca2+]i. In the presence of PDBu, phenylephrine-induced increases in [Ca2+]i and contractions were significantly inhibited (Fig. 4, top). In uterine arteries from nonpregnant ewes, 100 nM PDBu did not affect contractions and [Ca2+]i induced by the submaximal concentration (3 µM) of phenylephrine (Fig. 4, middle). However, PDBu significantly increased contractions produced by lower concentrations (~30–200 nM) of phenylephrine, in the absence of significant changes in phenylephrine-mediated [Ca2+]i (Fig. 4, bottom). After PDBu was washed away from the tissues, phenylephrine produced similar levels of increases in tension and [Ca2+]i as those before the PDBu treatment in uterine arteries from both nonpregnant and pregnant ewes (Fig. 4), eliminating the potential time factor that could affect the responses of uterine arteries to phenylephrine during the experimental procedure.


Figure 4
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  		Fig. 4. Effect of PDBu on phenylephrine-induced intracellular free Ca2+ concentrations ([Ca2+]i) and contractions in uterine arteries from nonpregnant and pregnant ewes. Changes of tension ({Delta}Tension) and [Ca2+]i ({Delta}Rf340/380) induced by PDBu and phenylephrine before (PE1) and after (PE2) PDBu treatment were measured simultaneously in the same tissues in experimental protocol as described in METHODS. PE3 depicts recovery of phenylephrine-induced [Ca2+]i and contractions after washing away PDBu from tissues. Top: uterine arteries during pregnancy were treated with 3 µM phenylephrine and 0.1 µM PDBu (n = 3). Middle: uterine arteries from nonpregnant ewes were treated with 3 µM phenylephrine and 0.1 µM PDBu (n = 4). Bottom: uterine arteries from nonpregnant ewes were treated with ~30–200 nM phenylephrine and 0.1 µM PDBu (n = 6). Data are means ± SE. *P < 0.05, PE1 vs. PE2.

 
Expression of PKC isozymes in uterine arteries. Figure 5 shows the expression of at least 6 PKC isozymes, {alpha}, betaI, betaII, {delta}, {epsilon}, and {zeta}, in ovine uterine arteries. PKC-{gamma} was not detected in uterine arteries from either nonpregnant or pregnant ewes. When compared with uterine arteries from nonpregnant ewes, vessels during pregnancy showed significantly increased expression levels of PKC-betaI, PKC-betaII, and PKC-{zeta} but significantly decreased levels of PKC-{alpha} (Fig. 5). There were no significant differences in the expression levels of PKC-{delta} and PKC-{epsilon} between uterine arteries from nonpregnant and pregnant ewes (Fig. 5). The distribution of PKC isozymes in cytosolic and particulate fractions in the uterine arteries is shown in Figure 6. When compared with that of uterine arteries from nonpregnant ewes, the ratio of P to S distribution for PKC isozymes in uterine arteries during pregnancy was significantly decreased for PKC-{epsilon} but not significantly different for PKC-{alpha}, -betaI, -betaII, -{delta}, and -{zeta} (Fig. 6).


Figure 5
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  		Fig. 5. PKC isozymes in uterine arteries from nonpregnant and pregnant ewes. PKC isozymes were detected by Western blot analysis and expressed as percentage of standard of each isozyme blotted in same membrane. Data are means ± SE of tissues from 4 animals. *P < 0.05, pregnant vs. nonpregnant.

 

Figure 6
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  		Fig. 6. Subcellular distribution of PKC isozymes in uterine arteries from nonpregnant and pregnant ewes. Cytosolic (S) and particulate (P) fractions were prepared from uterine arteries as described in METHODS. PKC isozymes were detected by Western blot analysis and expressed as ratio of P to S fractions. Data are means ± SE of tissues from 4 animals. *P < 0.05, pregnant vs. nonpregnant.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study offers the following new findings: 1) activation of PKC potentiated {alpha}1-adrenoceptor-induced contractions in uterine arteries from nonpregnant ewes but inhibited the contractions in uterine arteries during pregnancy; 2) PKC activation blocked {alpha}1-adrenoceptor-mediated increases of [Ca2+]i in uterine arteries during pregnancy but had no significant effect on the Ca2+ mobilization in uterine arteries from nonpregnant ewes; and 3) PKC isozyme expression and subcellular distribution were differentially regulated in uterine arteries from nonpregnant and pregnant ewes, with significantly increased expression levels of PKC-beta and PKC-{zeta} but decreased PKC-{alpha} and the basal activity of PKC-{epsilon} in uterine arteries from pregnant, compared with nonpregnant, ewes.

It has been suggested that PKC plays an important role in regulating vascular myogenic response (10). A recent study (45) from our laboratory has demonstrated that myogenic tone of the uterine artery is reduced during pregnancy, which is primarily regulated through the PKC signal pathway. In addition to its role in the regulation of myogenic tone, the present study demonstrated that activation of PKC interacted with {alpha}1-adrenoceptors and modulated {alpha}1-adrenoceptor-mediated contractions of the uterine artery. To maintain the similar vessel diameters for uterine arteries from nonpregnant and pregnant ewes in the study, the third and fourth branches of the main uterine arteries from nonpregnant and pregnant ewes, respectively, were used in the present study. These vessels have been extensively studied in our previous studies (46, 48–50), and for a comparative purpose, they were used in the present study. Similarly, the third and fourth generation uterine arteries from nonpregnant and pregnant ewes were utilized in determining the role of Ca2+ and Ca2+ channels in regulating basal and angiotensin II-induced prostacyclin production in the uterine artery (29). Although few studies examined the potential cellular and subcellular differences between branch generations of small uterine arteries, numerous studies examined structural and signal proteins in combination of all branches of the uterine artery and showed differences between uterine arteries from nonpregnant and pregnant ewes, including the studies of PKC (11, 30). In the present study, we have shown that the smaller branches (beyond the fourth generation) of uterine arteries from pregnant and nonpregnant ewes, utilized in the studies of simultaneous measurement of [Ca2+]i and contractions, demonstrate the same characteristics of tissue response to PDBu and phenylephrine stimulations observed in the third and fourth branches, i.e., PDBu-mediated potentiation of phenylephrine-induced contraction in uterine arteries from nonpregnant ewes but inhibition of that in uterine arteries during pregnancy. These findings suggested that the characteristics of PKC-mediated effects were not altered between branches of small uterine arteries. The finding that PKC differentially regulated {alpha}1-adrenoceptor-mediated contractions in uterine arteries from nonpregnant and pregnant ewes is intriguing and suggests another important mechanism through which PKC regulates the adaptation of uterine artery contractility to pregnancy. Previous studies (1, 2, 6, 9, 14, 18, 25, 27, 32–34, 39) have demonstrated that PKC can either inhibit or potentiate {alpha}1-adrenoceptor-mediated contractions in different arteries from different species. However, to our knowledge, it has not been previously demonstrated that activation of PKC can inhibit and potentiate {alpha}1-adrenoceptor-mediated contractions in the same artery of the same species at different physiological states, i.e., pregnancy and nonpregnancy. Thus the present finding of a potentiation of {alpha}1-adrenoceptor-mediated contractions in uterine arteries from nonpregnant ewes, but an inhibition in uterine arteries during pregnancy, provides a physiological importance of PKC in regulating the adaptation of {alpha}1-adrenoceptor-mediated contractions of the uterine artery during pregnancy. The finding that activation of PKC had no effect on KCl-induced contractions suggests that the effect of PKC is selective to {alpha}1-adrenoceptor-mediated signal pathways in the uterine artery. Similar findings were obtained in cat cerebral arteries, in which phorbol esters potentiated phenylephrine-induced, but not KCl-induced, contractions (39).

In the present study, activation of PKC significantly enhanced the sensitivity of {alpha}1-adrenoceptor-mediated contractions by increasing the pD2 value in uterine arteries from nonpregnant ewes. Yet the maximal response was not affected. In contrast, in vessels during pregnancy, PKC significantly decreased the maximal {alpha}1-adrenoceptor-mediated contractions without affecting the sensitivity. These findings suggest that PKC modulates smooth muscle contractile apparatus at different steps of signal transduction pathways responding to {alpha}1-adrenoceptor stimulation in uterine arteries from nonpregnant and pregnant ewes. In vascular smooth muscle cells, activation of {alpha}1-adrenoceptors leads to an increase in free intracellular Ca2+ concentrations from intracellular Ca2+ stores via Ins(1,4,5)P3 stimulation, resulting in myosin light chain phosphorylation and contractions (35). In the present study, we found that activation of PKC abolished {alpha}1-adrenoceptor-induced increase in [Ca2+]i in uterine arteries during pregnancy but had no significant effect on [Ca2+]i in uterine arteries from nonpregnant ewes. The inhibition of [Ca2+]i was accompanied by decreased contractions, measured simultaneously in the same tissues. This suggests a predominant mechanism of decreased Ca2+ mobilization in the PKC-mediated attenuation of {alpha}1-adrenoceptor-mediated contractions in uterine arteries during pregnancy. Consistent with the present finding, previous studies in ovine cerebral arteries demonstrated that PDBu inhibited norepinephrine-induced increases in intracellular Ca2+ concentrations and contractions (27). Whereas the mechanisms of PKC-mediated inhibition of Ca2+ mobilization in uterine arteries during pregnancy are not clear at present, it has been demonstrated that activation of PKC decreases agonist binding affinity of {alpha}1-adrenoceptors, increases phosphorylation of {alpha}1-adrenoceptors, destabilizes {alpha}1-adrenoceptor mRNA, downregulates {alpha}1-adrenoceptors, and promotes uncoupling of {alpha}1-adrenoceptors from inositol phospholipid metabolism (2, 5, 6, 19, 25, 27, 34).

In contrast to uterine arteries during pregnancy, the finding that PDBu had no effect on {alpha}1-adrenoceptor-mediated Ca2+ mobilization but potentiated the contractions in vessels from nonpregnant ewes is intriguing and provides strong evidence of increased Ca2+ sensitivity in the PKC-mediated potentiation of {alpha}1-adrenoceptor-induced contractions in uterine arteries from nonpregnant ewes. In the present studies of simultaneous measurement of [Ca2+]i and contractions, the finding that PDBu had no effect on phenylephrine-induced contractions at a high concentration (3 µM) but potentiated it at low concentrations (Fig. 4, middle and bottom) was consistent with the studies of concentration-dependent contractions that showed the PKC-mediated potentiation occurring in the lower range concentrations of phenylephrine (Fig. 2). These results validated that loading of tissues with fura-2 did not alter characteristics of tissue response to PDBu and phenylephrine stimulation, albeit the tissue size, and hence the contractions were much smaller in the studies of simultaneous measurement of [Ca2+]i and contractions. Consistent with the present findings, PKC has been implicated in the regulation of myofilament Ca2+ sensitivity in vascular smooth muscle (10, 15, 43). We have demonstrated that PKC modulates Ca2+ sensitivity in the uterine artery predominantly through the thin-filament regulatory pathway, i.e., independent of changes in phosphorylation of myosin light chain (50). Furthermore, the thin-filament pathway is an important component in {alpha}1-adrenoceptor-mediated contractions, particularly in uterine arteries from nonpregnant ewes (47, 50). Taken together, we speculate that activation of PKC in uterine arteries from nonpregnant ewes produces a permissive and priming effect on the thin-filament pathway, resulting in enhanced {alpha}1-adrenoceptor-induced contractions.

The apparent opposite effects of PKC on {alpha}1-adrenoceptor-mediated contractions in uterine arteries from nonpregnant and pregnant ewes may be due in part to the differential expression of PKC isozymes that show different enzymatic properties, substrates, and functions (15, 23, 26). The {alpha}, beta, {delta}, {epsilon}, and {zeta} isozymes of PKC have been detected in vascular smooth muscle (44). Although not all of these isozymes appear to be in all vascular smooth muscle tissues, the present study demonstrated their presence in ovine uterine arteries. The finding of significantly increased expression levels of PKC-beta and PKC-{zeta} in uterine arteries from pregnant, compared with nonpregnant, ewes suggests a potential mechanism for these isozymes in inhibiting {alpha}1-adrenoceptor-mediated Ca2+ mobilization and contractions in vessels during pregnancy. PKC-beta is a conventional PKC isozyme that is activated by DAG and phorbol esters in the presence of Ca2+ (15). Previous studies have shown that overexpression of PKC-beta inhibits agonist-induced Ca2+ mobilization, and inhibition of PKC-beta results in a dramatic increase in agonist-mediated Ca2+ mobilization (17, 22, 42). In contrast to PKC-beta, inhibition of PKC-{zeta} by a specific inhibitory peptide did not alter agonist-induced Ca2+ mobilization (17). In addition, PKC-{zeta} is an atypical PKC isozyme that is not activated by Ca2+, DAG, or phorbol esters (15). These results suggest that the increased PKC-beta in uterine arteries during pregnancy is likely to play an important role in the PKC-mediated inhibition of {alpha}1-adrenoceptor-mediated Ca2+ mobilization and contractions in vessels during pregnancy. Although PKC-{zeta} may be less likely to be involved in the PDBu-mediated inhibition of intracellular Ca2+ mobilization, the increased PKC-{zeta} in uterine arteries during pregnancy may be important in proliferation and remodeling of the uterine artery during pregnancy, because it translocates from perinuclear localization into the nucleus on activation and functions in control of gene expression (8, 23, 44). In contrast to increased PKC-beta and PKC-{zeta}, the expression levels of PKC-{alpha} and PKC-{epsilon} in the particulate fraction were significantly decreased in uterine arteries during pregnancy. Both PKC-{alpha} and PKC-{epsilon} have been implicated in contractions of vascular smooth muscle through increasing Ca2+ sensitivity (16, 43, 44). Taken together, these findings suggest that the adaptation of the uterine artery to pregnancy is associated with the upregulation of the PKC isozyme(s) that inhibit intracellular Ca2+ mobilization and with the downregulation of the PKC isozymes that increase Ca2+ sensitivity.

In summary, we have shown the opposite effects of PKC on {alpha}1-adrenoceptor-induced contractions in uterine arteries from nonpregnant and pregnant ewes. Activation of PKC enhances {alpha}1-adrenoceptor-induced contractions in uterine arteries from nonpregnant ewes by increasing Ca2+ sensitivity but inhibits the contractions in uterine arteries during pregnancy by decreasing intracellular Ca2+ mobilization. The differential regulation of PKC isozyme expression, with the upregulation of PKC-beta and PKC-{zeta} and with the downregulation of PKC-{alpha} and PKC-{epsilon}, is likely to play an important role in the adaptation of uterine artery Ca2+ homeostasis from pro-Ca2+ sensitivity in vessels from nonpregnant ewes to inhibition of Ca2+ mobilization in vessels during pregnancy. From the physiological perspective, the uterine circulation during pregnancy functions as a low-resistance shunt to accommodate the large increase of uteroplacental blood flow, required for normal fetal development. In addition to growth and remodeling of vessels, the decreased uterine artery resistance is accomplished by increased endothelial nitric oxide release, decreased myogenic response, and a reversible sympathetic denervation of the uterine artery. Although the decreased sympathetic innervation may sensitize postsynaptic {alpha}1-adrenoceptor signal pathways, the present finding of the increased inhibitory effect of PKC on {alpha}1-adrenoceptor-mediated contractions in the uterine artery during pregnancy reveals another important mechanism in maintaining the low uterine vascular tone during pregnancy. The potential effect of steroid hormones in the regulation of differential expression of PKC isozymes in the uterine artery during pregnancy presents an intriguing area for future investigation.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by National Institutes of Health Grants HL-57787, HL-67745, and HD-31226 and by the Loma Linda University School of Medicine. D. Xiao is a recipient of a Postdoctoral Fellowship Award from The Regents of the University of California Tobacco-Related Disease Research Program (Award No. 14FT-0075).


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Zhang, Center for Perinatal Biology, Dept. of Physiology and Pharmacology, Loma Linda Univ. School of Medicine, Loma Linda, CA 92350 (e-mail: lzhang{at}llu.edu)

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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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