Role of Protein Kinase C Isozymes in the Regulation of alpha₁-Adrenergic Receptor-Mediated Contractions in Ovine Uterine Arteries

Abstract

Previously, we demonstrated that activation of protein kinase C (PRKC) enhanced alpha₁-adrenergic receptor-induced contractions in nonpregnant ovine uterine arteries but inhibited the contractions in pregnant ovine uterine arteries. The present study tested the hypothesis that differential regulation of PRKC isozyme activities contributes to the different effects of phorbol 12, 13-dibutyrate (PDBu) on alpha₁-adrenergic receptor-mediated contractions between the pregnant and nonpregnant ovine uterine arteries. Phenylephrine-induced contractions of ovine nonpregnant and pregnant uterine arteries were determined in the absence or presence of the PRKC activator PDBu and/or conventional and novel PRKC isozyme inhibitor GF109203X, PRKC isozyme-selective inhibitory peptides for conventional PRKC, PRKCB1, PRKCB2, and PRKCE, respectively. GF109203X produced a concentration-dependent inhibition of phenylephrine-induced contractions in both nonpregnant and pregnant uterine arteries, and it reversed the PDBu-mediated potentiation and inhibition of phenylephrine-induced contractions in non-pregnant and pregnant uterine artieries, respectively. In addition, PRKCB1, PRKCB2, and PRKCE inhibitory peptides blocked the PDBu-mediated responses in both nonpregnant and pregnant uterine arteries. Western blot analysis showed that PDBu induced a membrane translocation of PRKCA, PRKCB1, PRKCB2, and PRKCE in pregnant uterine arteries, and PRKCB1, PRKCB2, and PRKCE in nonpregnant uterine arteries. The results disprove the hypothesis that the dichotomy of PRKC mechanisms in the regulation of alpha₁-adrenergic receptor-induced contractions in nonpregnant and pregnant uterine arteries is caused by the activation of different PRKC isozymes, and suggest downstream mechanisms of differential subcellular distributions for the distinct functional effects of PRKC isozymes in the adaptation of uterine arteries to pregnancy.

INTRODUCTION

During pregnancy, the uterine artery maintains low resistance, resulting from growth and vascular remodeling and relaxation to accommodate a large increase in uteroplacental blood flow to ensure normal fetal development. Multiple mechanisms are involved in the adaptation of uterine artery contractility during pregnancy [1–5]. Activation of α₁ adrenergic receptors is an important mechanism in the regulation of uterine artery smooth muscle contractions [1, 6–9]. Protein kinase C (PRKC) plays a key role in α₁-adrenergic receptor-mediated contractions of vascular smooth muscle [1, 10, 11] and pressure-dependent myogenic tone of the uterine artery [3, 5, 12]. Recently, we have demonstrated that activation of PRKC by phorbol-12, 13-dibutyrate (PDBu) potentiates α₁-adrenergic receptor-induced contractions in uterine arteries from nonpregnant sheep (nonpregnant artery), but inhibits the contractions in uterine arteries from pregnant animals (pregnant artery) [13]. In addition, we have shown that differential regulations of the thick and thin filament pathways play an important role in the dichotomy of PRKC mechanisms in the regulation of α₁-adrenergic receptor-induced contractions in nonpregnant and pregnant uterine arteries [14]. However, it remains unknown whether and to what extent to which activation of differential PRKC isozymes contributes to these pregnancy-specific effects of PDBu observed in the uterine arteries.

PRKC, a serine/threonine kinase family, consists of at least 11 isozymes that are further classified into three subfamilies: the conventional isozymes (PRKCA, PRKCB1, PRKCB2, and PRKCC), the novel isozymes (PRKCD, PRKCE, PRKCH, and PRKCQ), and the atypical isozymes (PRKCZ, PRKCI, and PRKC lambda). We have recently demonstrated that PRKCA, PRKCB, PRKCD, PRKCE, and PRKCZ isozymes are expressed in ovine uterine arteries [13]. If arteries from pregnant sheep are compared with those from nonpregnant animals, the basal activity of PRKCE is decreased, as is the expression level of PRKCA. In contrast, expression levels of PRKCB and PRKCZ are increased in arteries from pregnant sheep. Each PRKC isozyme has unique enzymatic properties, substrates, functions, and subcellular distributions in different blood vessels and species [11, 15–19]. Despite extensive studies, the physiologic role of each individual PRKC isozyme in the regulation of vascular contractility, including the uterine artery, remains unclear. This is likely due to the lack of highly selective pharmacologic agents to either inhibit or activate the isozymes. The aminoalkyl bisindolylmaleimide, GF109203X, has been demonstrated to be a potent and selective inhibitor of PRKC and to inhibit activity of conventional and novel PRKC isozymes [20–26]. More recent discovery of PRKC isozyme-selective translocation inhibitory peptides [27] allows determination of individual PRKC isozyme functions. Following activation, each PRKC isozyme translocates to its unique subcellular sites and binds to isozyme-specific anchoring proteins, receptors for activated C-kinase (RACKs). Each isozyme has a specific RACK-selective binding site that is exposed only after the activation of PRKC. PRKC isozyme-selective inhibitory peptides, containing isozyme-specific RACK-binding sites, have been demonstrated to inhibit translocation of the corresponding PRKC isozymes and, consequently, inhibit their isozyme-unique function [27–29].

The function of PRKC isozymes in the adaptation of uterine artery contractility during pregnancy is unknown. The present study tests the hypothesis that differential regulation of PRKC isozyme activities contributes to the different effects of PDBu on α₁ adrenergic receptor-mediated contractions between the pregnant and nonpregnant uterine arteries. Concentration-response curves of phenylephrine-induced contractions of the uterine arteries were conducted in the absence or presence of PRKC activator PDBu and/or PRKC inhibitor GF109203X, PRKC isozyme-selective translocation inhibitory peptides. In addition, PDBu-mediated PRKC isozyme translocations were determined in the uterine arteries.

MATERIALS AND METHODS

Tissue Preparation

Tissues were prepared as previously described [13]. A total of 4–10 animals were used in each group. Nonpregnant and near-term pregnant (139.8 ± 0.2 days’ gestation; range, 138–141 days) ewes were anesthetized with thiamylal (10 mg/kg) administered via the external left jugular vein. The ewes then were 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 at room temperature. The Krebs solution was oxygenated with a mixture of 95%O₂/5% CO₂. After the tissues were removed, animals were killed with T-61 euthanasia solution (Hoechst-Roussel, Somerville, NJ). All procedures and protocols used in the present study were approved by the Animal Research Committee of Loma Linda University and followed the guidelines in the National Institutes of Health’s “Guide for the Care and Use of Laboratory Animals.”

Contraction Studies

The third (nonpregnant) and fourth (pregnant) branches of the main uterine arteries with similar external diameters were used in the present studies, as previously described [13]. Uterine arteries were dissected and cut into 2-mm ring segments, as previously described [3, 5, 13, 14, 30]. Isometric tension was measured in the Krebs solution in a tissue bath at 37°C as previously described [13]. Briefly, each ring was equilibrated for 60 min and then gradually stretched to the optimal resting tension as determined by the tension developed in response to 120 mM KCl added at each stretch level. Previous studies demonstrated that 120 mM KCl produced maximal contractions in the uterine arteries [3, 30]. Tissues then were 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 washing away phenylephrine, 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 then were repeated in the same tissue in the absence or presence of PDBu (0.03 and 0.1 μM for nonpregnant uterine arteries; 0.3 and 1 μM for pregnant uterine arteries; 10 min) alone and/or in combination with GF109203X (0.1, 0.3, and 1 μM for 20 min), PRKC isozyme-selective inhibitory peptides for conventional PRKC, PRKCB1, PRKCB2, or PRKCE (3 μM for 20 min), respectively. The effects of PDBu and PRKC inhibitors on phenylephrine-induced contractions were determined by comparing the two phenylephrine-induced concentration-response curves in the same tissues before and after the treatment with PDBu and the inhibitors. Time control studies showed no significant time-related shift of phenylephrine-response curves. Contractions were expressed as percentage of the KCl response.

Measurement of PRKC Isozyme Translocation

PDBu-stimulated PRKC isozyme translocation and contractions were measured simultaneously in the same uterine arteries. Pregnant and nonpregnant uterine artery rings were equilibrated in the tissue bath, and the optimal tensions were obtained. Tissues then were subjected to stimulation with PDBu (0.2 μM for nonpregnant; 1 μM for pregnant) for 10 min. At the end of treatment, the tissues were quickly frozen in liquid N₂, and cytosolic and particulate fractions were prepared as previously described [13]. Briefly, the tissues were homogenized in ice-cold homogenization buffer A containing Tris-HCl 20 mM, sucrose 250 mM, EDTA 5 mM, EGTA 5 mM, β-mercaptoethanol 10 mM, benzamidine 1 mM, phenylmethylsulfonyl fluoride 1 mM, leupetin 50 μM, dithiothreitol 1 mM, and aprotinin 2 μg/ml, 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. The pellets were resuspended in homogenization buffer A containing 1% Triton X-100 by stirring 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. Protein concentrations were determined with a protein assay kit (Bio-Rad). Protein samples (5 μg) of 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-PRKC isozyme antibodies overnight at 4°C and secondary antibody for 1 h at room temperature. Polyclonal antibodies to PRKCA, PRKCB1, PRKCB2, and PRKCE were used. Bands were detected with enhanced chemiluminsecence (ECL), visualized on Hyperfilm, and analyzed with the Kodak 1D image analysis software. To normalize the loading variation of each sample, the corresponding actin level presented in each sample was determined using monoclonal antiactin as primary antibody.

Materials

Phenylephrine, PDBu, GF109203X, and antiactin antibody were obtained from Sigma (St. Louis, MO). Anti-PRKC isozyme antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The PRKC isozyme-selective inhibitory peptides for conventional PRKC, PRKCB1, PRKCB2, and PRKCE were from KAI Pharmaceuticals (San Francisco, CA). These peptides were modified with conjugation of peptide carriers via Cys-Cys bonds to facilitate their transportation through biologic membranes into cells. Once in the cells, the Cys-Cys bonds were reduced to enable the exit of the carriers while trapping the peptides inside the cells [28]. In both nonpregnant and pregnant uterine arteries, the peptide carrier alone had no significant effects on PDBu-mediated responses on phenylephrine-induced contractions (data not shown). 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

Concentration-response curves were analyzed by computer-assisted nonlinear regression to fit the data using GraphPad Prism (GraphPad Software, San Diego, CA) to obtain the values of pD₂ (−log EC₅₀) and the maximum response (E_max). Results were expressed as means ± SEM, and the differences were evaluated for statistical significance (P < 0.05) by one-way ANOVA followed by Neuman-Keuls post-hoc tests.

RESULTS

Effect of GF109203X on Phenylephrine-Induced Contractions

Figure 1 shows that phenylephrine produced concentration-dependent contractions of uterine arteries from both nonpregnant and pregnant ewes. In agreement with the previous findings [5], the pD₂ values were significantly increased in uterine arteries from pregnant (6.2 ± 0.1) compared with nonpregnant (5.5 ± 0.1) animals. GF109203X (0.1, 0.3, and 1 μM), a selective inhibitor for conventional and/or novel PRKC isozymes, produced a concentration-dependent inhibition of phenylephrine-induced contractions and shifted the concentration-response curves to the right. As shown in Table 1, in both arteries, 0.3 and 1 μM GF109203X significantly decreased the pD₂ values, with no significant effects on the maximal responses.

FIG. 1.

Effect of GF109203X (GF) on concentration-response curves of phenylephrine-induced contractions in uterine arteries. Phenylephrine-induced, concentration-dependent contractions were obtained in the absence or presence of GF109203X (100 nM, 300 nM, and 1 μM). Data are expressed as a percentage of 120 mM KCl-induced response (%KCl) and are means ± SEM of tissues from 4 to 10 animals.

TABLE 1.

Effect of GF109203X (GF) on concentration-response curves of phenylephrine-induced contractions in uterine arteries.

	pD2b			Emaxc
GF concentrationa	0.1 μM	0.3 μM	1 μM	0.1 μM	0.3 μM	1 μM
NPUA (n = 4)
−GF	5.5 ± 0.1	5.5 ± 0.1	5.5 ± 0.2	307.6 ± 20.0	261.2 ± 12.9	304.9 ± 22.8
+GF	5.5 ± 0.1	5.1 ± 0.1*	4.8 ± 0.1d	321.7 ± 19.0	261.1 ± 12.2	261.7 ± 19.5
PUA (n = 10)
−GF	6.2 ± 0.1	6.3 ± 0.1	6.2 ± 0.1	199.0 ± 8.6	188.9 ± 5.2	185.2 ± 5.7
+GF	6.0 ± 0.0	5.8 ± 0.0d	5.4 ± 0.0d	210.0 ± 2.0	188.6 ± 1.4	172.3 ± 1.7

^aNPUA, Nonpregnant uterine arteries; PUA, pregnant uterine arteries; n = the number of ewes.

^bNegative log EC₅₀.

^cMaximal contraction expressed as a percentage of KCl.

^dValues are significantly different vs. control; P < 0.05.

Effect of GF109203X on PDBu-Affected Phenylephrine-Induced Contractions

Our previous study demonstrated that PDBu suppressed phenylephrine-induced contractions in pregnant uterine arteries but potentiated the contractions in nonpregnant uterine arteries [13]. Consistent with the previous findings, PDBu (0.3 and 1 μM) dose-dependently inhibited phenylephrine-induced contractions in pregnant uterine arteries, with marked decreases in the maximal response (Fig. 2). GF109203X blocked PDBu-mediated responses and reversed its effect on phenylephrine-induced maximal responses in the pregnant uterine arteries (Table 2). In the presence of 1 μM GF109203X and 0.3 μM or 1 μM PDBu, respectively, the pD₂ values of phenylephrine-induced contractions were not significantly different from each other (5.1 ± 0.3 vs. 5.0 ± 0.3; P > 0.05; Table 2), and they were not significantly different from the pD₂value of phenylephrine-induced contractions in the presence of 1 μM GF109203X alone (5.4 ± 0.0; P > 0.05), as shown in Table 1. In nonpregnant uterine arteries, also consistent with the previous findings [13], PDBu (0.03 and 0.1 μM) produced a concentration-dependent potentiation of phenylephrine-induced contractions, with significant increases in the pD₂ values but not the maximal responses. GF109203X blocked PDBu-mediated responses (Fig. 3 and Table 3). In the presence of 1 μM GF109203X and 0.03 μM or 0.1 μM PDBu, respectively, the pD₂ values of phenylephrine-induced contractions were not significantly different from each other (4.8 ± 0.2 vs. 5.0 ± 0.2; P > 0.05; Table 3), and they were not significantly different from the pD₂ value of phenylephrine-induced contractions in the presence of 1 μM GF109203X alone (4.8 ± 0.1; P > 0.05), as shown in Table 1.

FIG. 2.

Effect of GF109203X (GF) on PDBu-inhibted, phenylephrine-induced contractions in pregnant uterine arteries. Phenylephrine-induced, concentration-dependent contractions were obtained in the absence or presence of PDBu (0.3 μM, upper panel; 1 μM, lower panel) and 1 μM GF109203X. Data are expressed as a percentage of 120 mM KCl-induced response (%KCl), and are means ± SEM of tissues from four animals.

TABLE 2.

Effect of GF109203X (GF) on PDBu-inhibited phenylephrine-induced contractions in pregnant uterine arteries.

	PDBu 0.3 μMa (n = 4)		PDBu 1 μMa (n = 4)
Treatment	pD2	Emax	pD2	Emax
Control	6.6 ± 0.0	209.9 ± 5.3	6.6 ± 0.0	180.3 ± 5.3
PDBu	5.3 ± 0.7b	30.4 ± 5.0b	6.5 ± 0.3	14.0 ± 5.9b
PDBu + GF	5.1 ± 0.3b	213.3 ± 19.1c	5.0 ± 0.3b	145.6 ± 19.0c

^apD₂, Negative log EC₅₀; E_max, maximal contraction expressed as a percentage of KCl; n = the number of ewes.

^bValues are significantly different vs. control; P < 0.05.

^cValues are significantly different vs. PDBu; P < 0.05.

FIG. 3.

Effect of GF109203X (GF) on PDBu-potentiated, phenylephrine-induced contractions in nonpregnant uterine arteries. Phenylephrine-induced, concentration-dependent contractions were obtained in the absence or presence of PDBu (0.03 μM, upper panel; 0.1 μM, lower panel) and 1 μM GF109203X. Data are expressed as a percentage of 120 mM KCl-induced response (%KCl) and are means ± SEM of tissues from four animals.

TABLE 3.

Effect of GF109203X (GF) on PDBu-potentiated phenylephrine-induced contractions in nonpregnant uterine arteries.

	PDBu 0.03 μMa (n = 4)		PDBu 0.1 μMa (n = 4)
Treatment	pD2	Emax	pD2	Emax
Control	5.2 ± 0.1	328.4 ± 11.5	5.3 ± 0.1	287.7 ± 12.7
PDBu	5.5 ± 0.1b	343.1 ± 13.5	8.4 ± 0.3b	279.5 ± 17.3
PDBu + GF	4.8 ± 0.2c	319.2 ± 40.3	5.0 ± 0.2c	231.9 ± 29.6

^apD₂, Negative log EC₅₀; E_max, maximal contraction expressed as a percentage of KCl; n = the number of ewes.

^bValues are significantly different vs. control; P < 0.05.

^cValues are significantly different vs. PDBu; P < 0.05.

Effect of PRKC Isozyme-Selective Inhibitory Peptides on PDBu-Affected Phenylephrine-Induced Contractions

To determine the cause and effect relations between individual PRKC isozymes and PDBu-mediated responses, we determined the effects of PRKC isozyme-selective inhibitory peptides for conventional PRKC, PRKCB1, PRKCB2, and PRKCE on PDBu-mediated effects on phenyl-ephrine-induced contractions in pregnant and nonpregnant uterine arteries. In pregnant uterine arteries, there were no significant differences in the pD₂ values of phenylephrine-induced contractions among the treatment groups (Table 4). PDBu-mediated inhibition of the maximal response of phenylephrine-induced contractions was partially blocked by specific conventional PRKC, PRKCB1, PRKCB2, and PRKCE inhibitory peptides (Fig. 4 and Table 4). In nonpregnant uterine arteries, PDBu-mediated increases in the pD₂ of phenylephrine-induced contractions were inhibited with PRKC isozyme-selective inhibitory peptides for conventional PRKC, PRKCB1, PRKCB2, and PRKCE (Fig. 5 and Table 4). The maximal responses were not significantly different among the treatment groups in nonpregnant uterine arteries (Table 4).

TABLE 4.

Effect of PRKC isozyme-selective translocation inhibitory peptides (TIP) on PDBu-affected phenylephrine-induced contractions in uterine arteries.

	Nonpregnant uterine arteries (n = 4)b		Pregnant uterine arteries (n = 6)b
Treatmenta	pD2	Emax	pD2	Emax
Control	5.2 ± 0.1	299.9 ± 12.0	6.1 ± 0.0	198.6 ± 3.0
PDBu	7.7 ± 0.2c	235.8 ± 12.6	5.4 ± 0.4	37.7 ± 5.8c
PDBu + cPRKC TIP	6.0 ± 0.2d	248.0 ± 15.3	6.1 ± 0.4	65.3 ± 10.9cd
PDBu + PRKCB1 TIP	5.5 ± 0.2d	249.1 ± 21.0	5.6 ± 0.2	96.9 ± 10.6cd
PDBu + PRKCB2 TIP	5.3 ± 0.3d	304.7 ± 39.0	5.4 ± 0.3	74.4 ± 10.1cd
PDBu + PRKCE TIP	5.4 ± 0.4d	237.2 ± 32.8	5.3 ± 0.3	85.8 ± 11.1cd

^acPRKC, Conventional PRKC.

^bpD₂, Negative log EC₅₀; E_max, maximal contraction expressed as a percentage of KCl; n = number of ewes.

^cValues are significantly different vs. control; P < 0.05.

^dValues are significantly different vs. PDBu; P < 0.05.

FIG. 4.

Effect of PRKC isozyme-selective translocation inhibitory peptides (TIP) on PDBu-inhibited, phenylephrine-induced contractions in pregnant uterine arteries. Phenylephrine-induced, concentration-dependent contractions were obtained in the absence or presence of PDBu (1 μM) and conventional PRKC (cPRKC) TIP (3 μM), PRKCB1 TIP (3 μM), PRKCB2 TIP (3 μM), and PRKCE TIP (3 μM), respectively. Data are expressed as a percentage of 120 mM KCl-induced response (%KCl), and are means ± SEM of tissues from six animals.

FIG. 5.

Effect of PRKC isozyme-selective translocation inhibitory peptides (TIP) on PDBu-potentiated, phenylephrine-induced contractions in nonpregnant uterine arteries. Phenylephrine-induced, concentration-dependent contractions were obtained in the absence or presence of PDBu (0.1 μM) and conventional PRKC (cPRKC) TIP (3 μM), PRKCB1 TIP (3 μM), PRKCB2 TIP (3 μM), and PRKCE TIP (3 μM), respectively. Data are expressed as a percentage of 120 mM KCl-induced response (%KCl), and are means ± SEM of tissues from four animals.

PDBu-Induced Translocation of PRKC Isozymes in Uterine Arteries

In pregnant uterine arteries, PDBu significantly increased the levels of PRKCA (137%), PRKCB1 (229%), PRKCB2 (22%), and PRKCE (566%) in membrane particulate fractions, suggesting that PDBu induced translocation and activation of all four PRKC isozymes in pregnant vessels (Fig. 6). In nonpregnant uterine arteries, PDBu significantly increased the levels of PRKCB1 (74%), PRKCB2 (258%), and PRKCE (153%) in membrane particulate fractions without affecting PRKCA levels, suggesting that PDBu induced translocation and activation of PRKCB1, PRKCB2, and PRKCE, but not PRKCA, in nonpregnant vessels (Fig. 6).

FIG. 6.

PDBu-induced membrane translocation of PRKC isozymes in uterine arteries. Membrane translocation of PRKC isozymes was determined with Western blotting, as described in Materials and Methods. The results are expressed as a percentage of control of each isozyme blotted in the same membrane. Data are means ± SEM of tissues from four animals. *P < 0.05 versus control.

DISCUSSION

The present study has demonstrated that PRKC activation is involved in α₁-adrenergic receptor-mediated contractions in ovine uterine arteries and is responsible for the effects of PDBu on α₁-adrenergic receptor-mediated contractions of nonpregnant and pregnant uterine arteries. More importantly, individual isozymes of conventional PRKC, PRKCB, and PRKCE were identified to be involved in the PDBu-mediated responses.

It has been suggested that PRKC plays a key role in α₁-adrenergic receptor-mediated smooth muscle contractions [21, 31–35]. In agreement with the previous findings, the present study showed that GF109203X, an inhibitor for conventional and novel PRKC isozymes, concentration-dependently inhibited phenylephrine-induced contractions in both nonpregnant and pregnant uterine arteries. The relative inhibition produced by GF109203X was similar in uterine arteries from nonpregnant and pregnant animals. This suggests a role of conventional and novel PRKC isozymes in α₁-adrenergic receptor-mediated contractions in the uterine artery. The concentrations of GF109203X used in the present study were within the range shown to inhibit PRKC in previous studies [20, 22, 26].

Our previous study demonstrated that PDBu inhibited α₁-adrenergic receptor-mediated contractions in pregnant uterine arteries [13]. In the present study, we found that PDBu-mediated responses were blocked by GF109203X, suggesting a cause-effect relation between PRKC activation and PDBu-mediated inhibitory effects in pregnant uterine arteries. The finding that GF109203X has a dual effect on α₁-adrenergic receptor-mediated contractions (i.e., inhibition of α₁-adrenergic receptor-mediated contractions and blockade of PDBu-mediated inhibitory effects on α₁-adrenergic receptor-mediated contractions) is intriguing and suggests a two-compartment model of PRKC in the regulation of α₁-adrenergic receptor-mediated contractions in pregnant uterine arteries. One compartment of PRKC may be tightly associated with α₁-adrenergic receptors and forms a “signalsome,” which is activated by phenylephrine and participates in α₁-drenergic receptor-mediated contractions. The other, more diffusely distributed compartment of PRKC locates distal to α₁-adrenergic receptors, which is activated nonselectively by phorbol esters and mediates an inhibitory effect on α₁-adrenergic receptor-mediated contractions. It has been well demonstrated that the function of each PRKC isozyme depends on the subcellular location and the availability of protein substrates that can be phosphorylated by the isozyme at the site of anchoring [28]. Whereas PDBu was used as an experimental tool in the present study, physiologic activation of the diffuse and non-receptor-coupled PRKC compartment has been well demonstrated in response to pressure and stretch of vascular smooth muscle, which plays an important role in the regulation of myogenic tone [36].

The other possibility is that different PRKC isozymes may be involved in the regulation of α₁-adrenergic receptor-mediated responses. GF109203X is a nonselective PRKC inhibitor that blocks both conventional and novel PRKC isozymes. We have demonstrated the presence of PRKCA, PRKCB1, PRKCB2, and PRKCE, but not PRKCC, isozymes in uterine arteries [13]. In the present study, we have used the selective inhibitory peptides for PRKCA, PRKCB1, PRKCB2, and PRKCE isozymes to determine the role of PRKC isozymes in PDBu-mediated responses. The inhibitory activities of these peptides are obtained at an intracellular concentration of 5–50 nM [28]. In pregnant uterine arteries, we found that the inhibitory effect of PDBu on α₁-adrenergic receptor-induced contractions was partially, but significantly, reversed by inhibition of conventional PRKC, PRKCB1, PRKCB2, and PRKCE, respectively, suggesting an involvement of these isozymes in the PDBu-mediated inhibitory effect. Previous studies have shown that overexpression of PRKCB inhibits agonist-induced Ca²⁺ mobilization, and inhibition of PRKCB results in a dramatic increase in agonist-mediated Ca²⁺ release [37–39]. In addition, it has been demonstrated that PRKCE inhibits L-type Ca²⁺ current, and PRKCE inhibition increases phenylephrine-induced maximal contractions [40, 41]. Given our previous finding that PDBu significantly inhibited α₁-adrenergic receptor-mediated increases in intracellular Ca²⁺ concentrations in pregnant uterine arteries [13], the present results suggest that the inhibitory effect of PDBu on α₁-adrenergic receptor-mediated contractions in pregnant uterine arteries is caused by activation of PRKCB and PRKCE isozymes, resulting in a decrease in α₁-adrenergic receptor-mediated Ca²⁺ mobilization. This is supported with the finding that PDBu induced a membrane translocation of PRKCB and PRKCE in pregnant uterine arteries. It should be noted that none of these PRKC-isozyme selective peptide inhibitors fully reversed the PDBu-mediated inhibition of phenylephrine-induced contractions in pregnant uterine arteries. This is possibly due to either insufficient inhibitor concentrations or the presence of an additive/synergistic effect of PRKC isozymes in PDBu-mediated responses.

In nonpregnant uterine arteries, it appears that PRKC activation has a consistent and positive regulatory role in α₁-adrenergic receptor-mediated contractions. GF109203X inhibited phenylephrine-stimulated contractions and blocked PDBu-mediated potentiation of phenylephrine-induced contractions. This suggests a single functional compartment of PRKC isozyme(s) in the regulation of α₁-adrenergic receptor-mediated contractions in nonpregnant uterine arteries. Unlike pregnant uterine arteries, PRKCB and PRKCE inhibitor peptides produced a complete inhibition of PDBu-mediated potentiation of α₁-adrenergic receptor-induced contractions in nonpregnant uterine arteries, suggesting that activation of PRKCB and PRKCE isozymes contributes in parallel to the PDBu-mediated effect. Consistent with this finding, PDBu increased the membrane translocation of PRKCB and PRKCE in nonpregnant uterine arteries. The role of PRKCA is not clear at the present, given that PDBu did not increase its membrane translocation. We have recently demonstrated that PDBu potentiates α₁-adrenergic receptor-induced contractions in nonpregnant uterine arteries by increasing the Ca²⁺ sensitivity [13]. Given the previous findings that PRKCB and PRKCE isozymes regulated vascular smooth muscle contractions by increasing the Ca²⁺ sensitivity [42–47], the present study suggests that PDBu activates PRKCB and PRKCE and increases the Ca²⁺ sensitivity, resulting in the potentiation of α₁-adrenergic receptor-induced contractions in nonpregnant uterine arteries.

The finding that activation of PRKCB and PRKCE participates in apparent opposite effects of PDBu on α₁-adrenergic receptor-induced contractions in nonpregnant and pregnant uterine arteries is intriguing and suggests that the dichotomy of PRKC mechanisms in the regulation of α₁-adrenergic receptor-induced contractions in nonpregnant and pregnant uterine arteries is not caused by the activation of different PRKC isozymes. Although previous studies have shown that different PRKC isozymes have unique enzymatic properties, substrates, functions in different blood vessels, and species [11, 15–19], the present study is the first one to show that activation of the same PRKC isozymes (i.e., PRKCB and PRKCE) exhibits opposite effects in the same vessel in different physiologic states (i.e., pregnancy and nonpregnancy). This finding suggests that the downstream mechanisms of PRKC isozymes are involved.

Because the function of each PRKC isozyme requires localization to the specific subcellular sites such as plasma membrane, cytoskeletal filaments, or myofilaments, and ability of the isozyme to phosphorylate substrates co-localized to the sites, the distinct functional effects of the same PRKC isozymes with essentially identical catalytic activities can be achieved by their binding and proximity to a particular set of substrates [28]. It is possible that pregnancy alters the subcellular distribution of PRKC isozymes, resulting in regulation of different sets of substrates in the uterine arteries. We have demonstrated previously that activation of PRKC enhances the contractions in nonpregnant uterine arteries through its effects on thin filament regulatory pathway and activation of extracellular signal-regulated kinase/caldesmon and actin polymerization, but inhibits α₁-adrenergic receptor-mediated contractions in pregnant uterine arteries through downregulation of Ca²⁺-depedent thick filament pathway and decreased myosin light chain phosphorylation [14].

Taken together, our results suggest a transition of subcellular localization of PRKCB and PRKCE from thin filaments to thick filaments in uterine arteries during pregnancy, resulting in an upregulation in coupling of the PRKC isozymes that inhibit α₁-adrenergic receptor-mediated contractions and a downregulation in coupling of the PRKC isozymes that increase α₁-adrenergic receptor-induced contractions. 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 vascular 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 α₁-adrenergic receptor signal pathways, as demonstrated in the present study as well as previous studies [5, 48, 49], the present finding of the increased inhibitory effect of PRKC on α₁-adrenergic receptor-mediated contractions in the pregnant uterine artery reveals another important mechanism in maintaining the low uterine vascular tone in pregnancy. Given the finding that steroid hormones induce modulation of cytosolic and membrane-bound regulatory proteins, including PRKC, and regulate their functions [50–54], future studies are needed to investigate the mechanisms of steroid hormones in pregnancy adaptation of PRKC isozyme subcellular distribution and its role in the regulation of α₁-adrenergic receptor signaling pathways in the uterine arteries