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Am J Physiol Regul Integr Comp Physiol 278: R295-R303, 2000;
0363-6119/00 $5.00
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Vol. 278, Issue 2, R295-R303, February 2000

Pregnancy-associated reduction in vascular protein kinase C activity rebounds during inhibition of NO synthesis

Celia A. Kanashiro, Kathy L. Cockrell, Barbara T. Alexander, Joey P. Granger, and Raouf A. Khalil

Department of Physiology and Biophysics and Center for Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Vascular reactivity has been shown to be reduced during pregnancy and to be enhanced during chronic inhibition of nitric oxide (NO) synthesis in pregnant rats; however, the cellular mechanisms involved are unclear. The purpose of this study was to investigate whether the pregnancy-induced changes in vascular reactivity are associated with changes in the amount and/or activity of vascular protein kinase C (PKC). Active stress as well as the amount and activity of PKC was measured in deendothelialized thoracic aortic strips from virgin and pregnant rats untreated or treated with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). In virgin rats, the PKC activator phorbol 12,13-dibutyrate (PDBu, 10-6 M) and the alpha -adrenergic agonist phenylephrine (Phe, 10-5 M) caused significant increases in active stress and PKC activity that were inhibited by the PKC inhibitors staurosporine and calphostin C. Western blot analysis in aortic strips of virgin rats showed significant amount of the alpha -PKC isoform. Both PDBu and Phe caused significant translocation of alpha -PKC from the cytosolic to the particulate fraction. Compared with virgin rats, the PDBu- and Phe-stimulated active stress and PKC activity as well as the amount and the PDBu- and Phe-induced translocation of alpha -PKC were significantly reduced in late pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. The PDBu- and Phe-induced changes in active stress and the amount, distribution, and activity of alpha -PKC in virgin rats treated with L-NAME were not significantly different from that in virgin rats, whereas the changes in pregnant rats treated with L-NAME + the NO synthase substrate L-arginine were not significantly different from that in pregnant rats. These results provide evidence that a PKC-mediated contractile pathway in vascular smooth muscle is reduced during pregnancy and significantly enhanced during chronic inhibition of NO synthesis. The results suggest that one possible mechanism of the pregnancy-associated changes in vascular reactivity may involve changes in the amount and activity of the alpha -PKC isoform.

nitric oxide; vascular smooth muscle; hypertension


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NORMAL PREGNANCY IS CHARACTERIZED by increased plasma volume, increased renal blood flow, decreased total peripheral resistance, and reduced sensitivity to circulating pressor agents (9, 10, 13, 15). The hemodynamic changes associated with normal pregnancy have been attributed, in part, to an increase in nitric oxide (NO) synthesis (3, 5, 6, 29, 38). An increase in the production of NO by many cell types, including vascular endothelial cells, during pregnancy has been suggested to control the total peripheral vascular resistance and blood pressure by direct vasodilatory actions and by blunting the responsiveness to circulating vasoconstrictors (31, 38). This is supported by reports that the expression and specific actions of NO synthase are elevated during late gestation in rats (1, 5) and that the plasma level, metabolic production, and urinary excretion of cGMP, a second messenger of NO and a cellular mediator of vascular smooth muscle relaxation, are increased during pregnancy (4).

We have previously reported that the vascular reactivity to the alpha -adrenergic agonist phenylephrine (Phe) is decreased in pregnant rats and increased in late pregnant rats chronically treated with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) compared with virgin rats and suggested alteration of the signaling pathways downstream from receptor activation as one possible cellular mechanism of the pregnancy-associated changes in vascular reactivity (7, 20).

In several cell types, agonist-receptor interaction is coupled to increased breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) and production of diacylglycerol (DAG), which activates protein kinase C (PKC), an enzyme that enhances the cellular responses to Ca2+ (19, 34). Biochemical studies in several cell types including vascular smooth muscle have shown that PKC is mainly cytosolic under resting conditions and undergoes translocation to the particulate fraction when it is activated by DAG or phorbol esters (19, 34). Also, direct activation of PKC by phorbol esters causes sustained contraction of vascular smooth muscle (8, 24, 35) with no significant change in intracellular Ca2+ concentration ([Ca2+]i) (17, 32). These reports have suggested a role for PKC in regulating the contractile responses of vascular smooth muscle, at least in part, by increasing the Ca2+ sensitivity of the contractile proteins. However, PKC is not a simple enzyme but rather a family of several isoforms (19, 34). These PKC isoforms appear to have different enzyme properties, substrates, and functions and to exhibit different subcellular distributions in the same blood vessel from different species and in different vessels from the same species (22, 27).

Although the changes in PKC isoforms have been well characterized in systemic blood vessels of normal male rats and ferrets (21, 22, 27), it is not clear whether the decreased vascular reactivity observed during late pregnancy and the enhanced vascular reactivity observed during inhibition of NO synthesis in late-pregnant rats are associated with changes in PKC isoforms of vascular smooth muscle. The present study was designed to investigate whether the pregnancy-induced changes in vascular reactivity are associated with changes in the amount and/or activity of specific PKC isoforms in vascular smooth muscle. Active stress as well as the amount, distribution, and activity of specific PKC isoforms were measured in rat thoracic aortic strips isolated from virgin and pregnant rats untreated or treated with L-NAME. The effects of the alpha -adrenergic agonist phenylephrine (Phe) were compared with direct activation of PKC by phorbol esters, and the reversibility of these effects by PKC inhibitors was also investigated.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Mature female Sprague-Dawley rats (10-12 wk of age) were purchased from Harlan Sprague Dawley (Indianapolis, IN). Virgin rats were either untreated (n = 24) or treated with L-NAME (n = 24). Pregnant rats were studied at day 6 or early pregnancy (n = 8), day 13 or midpregnancy (n = 8), and days 19-21 or late pregnancy (n = 36) and 3 days postpartum (n = 8). Other late-pregnant rats were treated with L-NAME (n = 36) or with L-NAME and L-arginine (n = 18). The first day of pregnancy was verified by the presence of sperm in vaginal smears (full term is 21 days). The average weight of virgin rats was 240 ± 5.8 g compared with 349 ± 2.9 g in late-pregnant rats. All procedures were performed in accordance with the guidelines of the Animal Care and Use Committee at the University of Mississippi Medical Center and the American Physiological Society.

Protocol for L-NAME treatment. Pregnant and virgin rats in the untreated groups received drinking water. Pregnant and virgin rats in the treated groups received L-NAME (Sigma, St. Louis, MO) at a dose of ~4 mg · kg-1 · day-1. This dose of L-NAME has been shown to cause significant elevation of blood pressure in pregnant rats while having minimal effect in virgin rats (7, 20, 30). L-NAME treatment of the pregnant rats began at day 15 of gestation and continued for 4-6 days before killing the rats and harvesting the tissues at days 19-21 of gestation. Because water intake in pregnant rats was approximately two times that in virgin rats, the amount of L-NAME in the drinking water was adjusted to maintain a daily dose of ~4 mg · kg-1 · day-1 in both the pregnant and virgin rats. Some of the L-NAME-treated pregnant rats simultaneously received L-arginine (Sigma) in the drinking water at a dose of ~80 mg · kg-1 · day-1 for the same period of time (4-6 days). L-Arginine did not significantly affect the amount of drinking water in pregnant rats. Therefore, the amount of L-NAME the animals ingested was similar between pregnant rats treated with L-NAME and pregnant rats treated with L-NAME + L-arginine. After this protocol, the recorded systolic blood pressure in a subset of rats on the day of the experiment, using an automated sphygmomanometer with a tail cuff device, was 118 ± 3 mmHg (n = 8) in virgin rats, 125 ± 6 mmHg (n = 10) in virgin rats treated with L-NAME, 113 ± 5 mmHg (n = 8) in late-pregnant rats, 172 ± 6 mmHg (n = 10) in late-pregnant rats treated with L-NAME, and 124 ± 3 mmHg (n = 12) in late-pregnant rats simultaneously treated with L-NAME and L-arginine as previously described (7, 20).

Tissue preparation. Rats were terminally anesthetized by inhalation of chloroform. The thoracic aorta was removed, placed in oxygenated Krebs solution, and cleaned of connective tissue. The aorta was cut transversely into 3-mm wide rings. The endothelium was removed by rubbing the vessel interior with forceps. Rings were cut open into strips. One end of the strip was attached to a glass hook using a thread loop, and the other end was connected to a Grass force transducer (FT03, Astro-Med, West Warwick, RI).

Isometric tension. Thoracic aortic strips were stretched to Lmax [1.5× their initial unloaded length (L)] and allowed to equilibrate for 1 h in an organ bath filled with 50 ml Krebs solution continuously bubbled with 95% O2 and 5% CO2 at 37°C. The changes in isometric tension were recorded on a Grass polygraph (model 7D, Astro-Med). Removal of the endothelium was routinely verified by the absence of ACh (10-6 M)-induced vasorelaxation in tissue strips precontracted with Phe (3 × 10-7 M).

Tissue fractions. Thoracic aortic strips (~50 mg) were homogenized in a homogenizing buffer containing 20 mM MOPS, 4% SDS, 10% glycerol, 2.3 mg dithiothreitol (DTT), 1.2 mM EDTA, 0.02% BSA, 5.5 µM leupeptin, 5.5 µM pepstatin, 2.15 µM aprotinin, and 20 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride, using a 2 ml tight-fitting glass homogenizer (Kontes Glass, Vineland, NJ) at 4°C. The homogenate was centrifuged at 10,000 g for 2 min. The supernatant was used as the whole tissue fraction. Other tissue samples were stimulated with phorbol 12,13-dibutyrate (PDBu; 10-6 M) or Phe (10-5 M) in the presence or absence of the PKC inhibitors staurosporine (10-6 M) or calphostin C (10-6 M) for 30 min. Control tissues were incubated with the vehicle DMSO or the inactive 4-alpha PDBu. The strips were rapidly transferred to ice-cold equilibrating buffer A then homogenized in a homogenizing buffer B at 4°C. The homogenate was centrifuged at 100,000 rpm for 20 min at 4°C (Ultra-Centrifuge TL-100, Beckman, Houston, TX). The supernatant was used as the cytosolic fraction. The pellet was resuspended in a homogenizing buffer containing 1% Triton X-100 for 20 min. The homogenate was diluted with homogenizing buffer to a final concentration of 0.2% Triton X-100 and centrifuged at 100,000 rpm for 20 min at 4°C. The supernatant was used as the particulate fraction. Protein concentrations in tissue fractions were determined using a protein assay kit (Bio-Rad, Hercules, CA).

PKC activity. The cytosolic and particulate fractions were applied to DEAE-cellulose columns (0.8 × 4.0 cm; Bio-Rad) preequilibrated in buffer A. The column was washed with 10 ml of buffer A, and the protein was eluted with 1 ml of 0.1 M NaCl in buffer A. We selected 0.1 M NaCl to elute the fractions, because using 0.0-0.3 M NaCl linear gradient showed that the peak PKC activity was consistently detected in the 0.1 M NaCl eluting fraction (18). The PKC activity was determined in aliquots of the cytosolic and particulate fractions by measuring the incorporation of 32P from [gamma 32P]ATP (ICN Radiochemicals, Irvine, CA) into histone IIIS (18). One unit of PKC activity is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of 32P into histone IIIS. The final assay mixture contained 25 mM Tris · HCl (pH 7.5), 10 mM MgCl2, 200 µg/ml histone IIIS, 80 µg/ml phosphatidylserine, 30 µg/ml diolein, [gamma 32P]ATP (1-3 × 105 counts · min-1 · nmol-1), 0.5-3.0 µg protein, and 1 mM CaCl2. After 5 min of incubation at 30°C, the reaction was stopped by removing 25 µl from the assay tubes and spotting onto phosphocellulose discs. The discs were washed 3 × 5 min with 5% TCA, placed in 4 ml Ecolite scintillation cocktail, and the radioactivity was measured in a liquid scintillation counter (Beckman LS 6500).

Immunoblotting. Protein-matched samples of the whole tissue, cytosolic, and particulate fractions were subjected to electrophoresis on 8% SDS polyacrylamide gels, then transferred electrophoretically to nitrocellulose membranes. The membranes were incubated in 5% nonfat dry milk in PBS-Tween buffer at 22°C for 1 h, washed with PBS-Tween 3 × 5 min, then incubated in the primary anti-PKC antibody solution at 4°C overnight. Polyclonal antibodies to alpha -, beta -, and gamma -PKC isoforms were obtained primarily from GIBCO (Grand Island, NY). The specificity of the antibodies was confirmed by the observation that the peptide controls were successful only with the peptide to which the antibody was raised. To maintain the labeling conditions in the immunoblotting assays constant, we used the same titer of the polyclonal anti-PKC antibodies (1:500) and the same concentration of protein (10 µg) in all tissue samples. These concentrations gave significant immunoreactive signals while remaining on the linear portion of the titration curve. To confirm the results with the GIBCO polyclonal antibodies, we also used polyclonal anti-PKC antibodies (1:500) from Sigma, polyclonal anti-PKC antibodies (1:100) from Chemicon International (Temecula, CA), and monoclonal anti-PKC antibodies (1:100) from Seikagaku America (Ijamsville, MD) and obtained similar results. Actin was used as an internal control protein and was detected using a monoclonal anti-actin antibody (1:500) from Sigma. The nitrocellulose membranes were washed 5 × 15 min in PBS-Tween then incubated in horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody for 1.5 h. The blots were washed with PBS-Tween 5 × 15 min and visualized with enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL). PBS-Tween contained (in mM) 80 Na2HPO4, 20 NaH2PO4, and 100 NaCl and 0.05% Tween. The reactive bands corresponding to PKC isoforms were analyzed quantitatively by optical densitometry using a high-resolution imaging densitometer and Molecular Analyst software (Bio-Rad).

Solutions. Normal Krebs contained (in mM) 120 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 11.5 dextrose at pH 7.4 when bubbled with 95% O2 and 5% CO2. Equilibrating buffer A contained (in mM) 25 Tris · HCl (pH 7.5), 5 EGTA, 0.02 leupeptin, 0.2 phenylmethylsulfonylfluoride (PMSF), and 1 DTT. Homogenizing buffer B had the same composition as buffer A plus sucrose 250 mM.

Drugs and chemicals. Stock solution of L-Phe HCl (Sigma) was prepared as 10-2 M in distilled water. PDBu and 4-alpha PDBu (Alexis Laboratory, San Diego, CA) and staurosporine and calphostin C (Kamiya Laboratory, Seattle, WA) were dissolved in DMSO to form a stock solution of 10-3 M. The final concentration of DMSO in solution was 0.1%. All other chemicals were of reagent grade or better.

Statistical analysis. The developed force was corrected for the cross-sectional area of each individual aortic strip and expressed as active stress (N/m2) using the equation stress = force/cross-sectional area; where cross sectional area = wet weight/(tissue density × length of the strip), and tissue density = 1.055 g/cm3. Data are presented as the means ± SE. Data were compared using one-way ANOVA with Scheffe's F test and Student's t-test for unpaired data with P < 0.05 considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of PDBu and Phe on active stress. In aortic strips of virgin rats incubated in normal Krebs solution, PDBu (10-6 M) and Phe (10-5 M) caused significant increase in active stress to a maximum of 10.2 ± 1.3 × 103 (n = 8) and 9.7 ± 1.2 × 103 N/m2 (n = 8), respectively (Fig. 1). In late-pregnant rats, the PDBu- and Phe-induced stress was significantly reduced to 6.3 ± 1.3 × 103 (n = 8) and 5.8 ± 1.2 × 103 N/m2 (n = 8), respectively, compared with that in virgin rats (Fig. 1). In contrast, the PDBu- and Phe-induced stress in late-pregnant rats treated with L-NAME was significantly increased to 15.8 ± 1.1 × 103 N/m2 (n = 8) and 15.3 ± 1.0 × 103 N/m2 (n = 8), respectively. The PDBu- and Phe-induced stress was not significantly different between virgin rats treated with L-NAME and virgin rats or between pregnant rats simultaneously treated with L-NAME and L-arginine and untreated pregnant rats. No significant changes in active stress were observed in tissue samples treated with the vehicle DMSO or the inactive 4-alpha PDBu (data not shown).


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  		Fig. 1.   Phorbol 12,13-dibutyrate (PDBu)- and phenylephrine (Phe)-induced contractions in rat thoracic aorta. Thoracic aortic strips from virgin and pregnant (Preg) rats untreated or treated with NG-nitro-L-arginine methyl ester (L-NAME) and pregnant rats treated with L-NAME + L-arginine (L-Arg) were incubated in normal Krebs (2.5 mM Ca2+), then stimulated with 10-6 M PDBu (A) or 10-5 M Phe (B), and maximal steady-state active stress was measured. Data bars represent means ± SE of measurements in individual thoracic aortic strips of 8 rats from each group. * Significantly different (P < 0.05) from respective measurements in virgin rats. dagger  Not significantly different from respective measurements in untreated pregnant rats. Stauro, staurosporine; Calph C, calphostin C.

We tested the effect of two chemically unrelated PKC inhibitors on PDBu- and Phe-induced contraction. In aortic strips of all groups of rats, staurosporine (10-6 M) caused significant inhibition of the contractile responses-induced by PDBu or Phe that reached a steady state in ~30 min. Calphostin C (10-6 M) also caused significant inhibition of PDBu- and Phe-induced contraction (Fig. 1). However, the inhibitory effects of calphostin C were slower in onset than staurosporine and reached steady state in ~1 h.

PKC activity at different stages of gestation. Activated PKC has been shown to undergo translocation from the cytosolic to the particulate fraction of many cell types including vascular smooth muscle (19, 34). In unstimulated aortic strips of virgin rats, the basal PKC activity was greater in the cytosolic fraction than the particulate fraction (Fig. 2A). Both PDBu (10-6 M) and Phe (10-5 M) caused significant increase in PKC activity in the particulate fraction and a concomitant decrease in the cytosolic fraction (Fig. 2, B and C).


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  		Fig. 2.   PKC activity at different stages of pregnancy. Protein kinase C (PKC) activity was measured in cytosolic and particulate fractions of tissue samples isolated from rats during early (day 6), mid- (day 13), and late pregnancy (days 19-21) as well as 3 days postpartum as described in METHODS. Figure indicates distribution of PKC activity in cytosolic and particulate fractions of unstimulated tissue samples (A) or tissue samples stimulated with 10-6 M PDBu (B) or 10-5 M Phe (C) for 30 min. Data bars represent means ± SE of measurements in individual aortic strips of 8 rats from each group. * Significantly different (P < 0.05) from respective measurements in virgin rats.

The basal PDBu- and Phe-induced changes in PKC activity were measured during early, mid-, and late pregnancy as well as 3 days postpartum. During all stages of pregnancy the basal PKC activity in the cytosolic fraction was significantly greater than that in the particulate fraction (Fig. 2A). However, during late pregnancy, the basal PKC activity in both the cytosolic and particulate fractions was significantly reduced compared with that in virgin rats. Also, during early and midpregnancy, the basal PKC activity in the cytosolic fraction was ~1.5-fold of that in the particulate fraction. On the other hand, during late pregnancy, the basal PKC activity in the cytosolic fraction was greater than twofold of that in the particulate fraction (Fig. 2A).

At different stages of pregnancy, PDBu (10-6 M; Fig. 2B) and Phe (10-5 M; Fig. 2C) did not significantly change the total (cytosolic + particulate) PKC activity compared with that in unstimulated tissues (Fig. 2A) but caused significant increase in PKC activity in the particulate fraction and a concomitant decrease in the cytosolic fraction (Fig. 2, B and C). During late pregnancy, the PDBu- and Phe-stimulated PKC activity was significantly reduced in both the cytosolic and particulate fractions compared with that in virgin rats (Fig. 2, B and C). Also, the PDBu-and Phe-stimulated PKC activity in the particulate fraction was approximately twofold of that in the cytosolic fraction during early and midpregnancy but only slightly higher than that in the cytosolic fraction in late pregnancy (Fig. 2, B and C). In the postpartum rats, the total PKC activity and the relative PKC activity in the particulate and cytosolic fractions were not significantly different from those in virgin rats (Fig. 2, A-C).

Effect of L-NAME treatment on PKC activity. In virgin rats, the basal particulate to cytosolic (P/C) PKC activity ratio was 1.02 ± 0.10 (n = 9; Fig. 3A). PDBu (10-6 M; Fig. 3B) and Phe (10-5 M; Fig. 3C) caused significant increases in the P/C PKC activity compared with that in unstimulated tissues (Fig. 3A). The basal PDBu- and Phe-stimulated P/C PKC activity was significantly reduced in late-pregnant rats but significantly increased in late-pregnant rats treated with L-NAME compared with virgin rats. The basal PDBu- and Phe-stimulated PKC activity was not significantly different between virgin rats treated with L-NAME and virgin rats or between pregnant rats simultaneously treated with L-NAME and L-arginine and untreated pregnant rats. The PKC inhibitors staurosporine and calphostin C inhibited the PDBu- (Fig. 3B) and Phe-induced (Fig. 3C) increases in PKC activity to values not significantly different from those observed in unstimulated tissue samples (Fig. 3A).


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  		Fig. 3.   Effect of L-NAME treatment on vascular PKC activity. PKC activity was measured in cytosolic and particulate fraction of thoracic aortic strips from virgin and Preg rats untreated or treated with L-NAME and pregnant rats treated with L-NAME + L-arginine. Basal particulate to cytosolic (P/C) PKC activity ratio was measured in tissue samples untreated or treated with 10-6 M Stauro or Calph C (A). Other tissue samples were stimulated with 10-6 M PDBu (B) or 10-5 M Phe (C), and P/C PKC activity was measured in presence or absence of 10-6 M Stauro or Calph C. Data bars represent means ± SE of measurements in individual aortic strips of 9 rats from each group. * Significantly different (P < 0.05) from respective measurements in virgin rats. dagger  Not significantly different from respective measurements in untreated Preg rats.

PKC isoforms in virgin and pregnant rats. Immunoblots were performed in the tissue samples using primary antibodies specific to the Ca2+-dependent alpha -, beta -, and gamma -PKC isoforms. A significant immunoreactive band at ~80 kDa was observed with specific antiserum to alpha -PKC isoenzyme (Fig. 4). The specificity of the alpha -PKC reactive band was confirmed by the loss of immunoreactive signal in the presence of specific synthetic peptide to which the antibody was raised. No significant immunoreactive bands were detected with antibodies to beta - or gamma -PKC isoform. In virgin rats, the optical density (OD) per microgram of protein for alpha -PKC was 0.12 ± 0.01 (n = 6; Fig. 4). The OD per microgram of protein for alpha -PKC was significantly reduced in late-pregnant rats but significantly increased in late-pregnant rats treated with L-NAME. The OD per microgram of protein for alpha -PKC was not significantly different between virgin rats treated with L-NAME and virgin rats or between pregnant rats simultaneously treated with L-NAME and L-arginine and untreated pregnant rats (Fig. 4). Immunoblots for the internal control protein actin did not show any significant difference in tissue samples from the different groups of rats.


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  		Fig. 4.   Expression of alpha -PKC isoform in virgin and Preg rats. Whole tissue fractions of thoracic aortic strips isolated from virgin and Preg rats untreated or treated with L-NAME and pregnant rats treated with L-NAME + L-arginine were prepared for Western blot analysis using alpha -PKC antibody (GIBCO, 1:500). Position of molecular mass marker is shown on right. Figure is representative of results obtained from at least 4 experiments. Autoradiographs were scanned by optical densitometry, and amounts of alpha -PKC are expressed as optical density (OD) per microgram of protein. Data bars represent means ± SE of measurements in 4-6 experiments. * Significantly different (P < 0.05) from respective measurements in virgin rats. dagger  Not significantly different from respective measurements in untreated Preg rats.

Distribution of alpha -PKC in thoracic aorta of pregnant rats. In unstimulated tissue samples from late-pregnant rats, the OD per microgram of protein for alpha -PKC was greater in the cytosolic fraction than the particulate fraction (Fig. 5A). PDBu (10-6 M; Fig. 5B) and Phe (10-5 M; Fig. 5C) caused significant redistribution of alpha -PKC from the cytosolic to the particulate fraction. In late-pregnant rats treated with L-NAME, the basal PDBu- and Phe-induced distribution of alpha -PKC in the particulate fraction was greater than that in the cytosolic fraction. Treatment of the tissue samples with staurosporine did not significantly change the basal PDBu- or Phe-induced distribution of alpha -PKC observed in tissue samples of pregnant rats or pregnant rats treated with L-NAME. In contrast, treatment of the tissue samples with calphostin C significantly inhibited the PDBu- and Phe-induced translocation of alpha -PKC in pregnant rats and pregnant rats treated with L-NAME (Fig. 5, B and C) to levels not significantly different from those observed in the unstimulated tissue samples (Fig. 5A).


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  		Fig. 5.   Distribution of alpha -PKC isoform in Preg rats. Cytosolic and particulate fractions of thoracic aortic strips from Preg rats untreated or treated with L-NAME were prepared for Western blot analysis using alpha -PKC antibody (GIBCO, 1:500). Autoradiographs were scanned by optical densitometry, and amounts of alpha -PKC were expressed as OD per microgram of protein. Figure shows basal distribution of alpha -PKC in cytosolic and particulate fractions of tissue samples untreated or treated with 10-6 M Calph C or Stauro (A). Other tissue samples were stimulated with 10-6 M PDBu (B) or 10-5 M Phe (C) in presence or absence of 10-6 M Calph C or Stauro. Data bars represent means ± SE of measurements in 4-6 experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study showed that in thoracic aortic smooth muscle of virgin rats, the PKC activator PDBu caused significant increases in contraction and PKC activity that were completely inhibited by two chemically unrelated PKC inhibitors with two different sites of action on the PKC molecule (16, 19, 25, 34). The alpha -adrenergic agonist Phe also caused significant contraction and increase in PKC activity that were significantly inhibited by the PKC inhibitors staurosporine and calphostin C at the same concentrations that completely inhibited the phorbol ester-induced responses. These results are consistent with other reports (21, 23, 27) and suggest that PKC is involved in the Phe-induced contraction of rat thoracic aortic smooth muscle.

Although changes in vascular PKC activity have been described during the ovarian cycle of nonpregnant ewes (28), the nonpregnant rats used in the present study were selected at random, regardless of the stage of the ovarian cycle. Because the ovarian cycle in rats, unlike larger mammals such as the ewes, is more frequent (every 4-5 days) and the estrous stage is shorter (~12 h), the present results should roughly represent the average changes in vascular PKC activity during all stages of the ovarian cycle.

We previously reported that the vascular reactivity of systemic vessels to alpha -adrenergic agonists is reduced in late-pregnant rats (7, 20). Although a decrease in alpha -adrenergic sensitivity has been reported in the pulmonary artery and aorta of pregnant ewes (37), we have previously shown that the sensitivity to the alpha -adrenergic agonist Phe was not significantly affected in late pregnant rats, suggesting that the decrease in vascular reactivity to Phe in rats may involve other signaling events downstream from receptor activation (20). In the present study, the contractile responses induced by PDBu and Phe were smaller in thoracic aortic strips from late-pregnant rats compared with virgin rats. Also, the basal PDBu- and Phe-stimulated PKC activity was significantly reduced during late pregnancy compared with virgin rats but returned to the levels observed in virgin rats after a 3-day postpartum period. These results suggest that the decreased reactivity of aortic smooth muscle to PDBu and Phe during late pregnancy in rats is associated with a decrease in the amount and/or the activity of PKC and are in agreement with reports that PKC activity is reduced during late gestation in the ewes and gilts (12, 28).

Although a clear relationship between the hemodynamic changes observed during late pregnancy and the vascular PKC activity is difficult to discern at the present time, such a relationship is conceivable. It has been suggested that an increase in endogenous NO production during late pregnancy causes a decrease in vascular reactivity (29, 38), perhaps through increased formation of cGMP in vascular smooth muscle (4, 5). NO per se could cause reversible inactivation of PKC either directly through the formation of disulfide bridges with the PKC molecule (14) or indirectly through the inhibition of phosphatidylinositol breakdown and consequently decreased DAG production (33, 39). Also, increased cGMP production has been shown to inhibit PKC and to cause relaxation of PKC-mediated contractions in rat aorta (26, 36) by mechanisms involving inhibition of phospholipid metabolism and decreased DAG formation (2). Thus the pregnancy-associated decreases in PKC activity could be related, at least in part, to the increased production of NO and cGMP that we and others reported to occur during late pregnancy (1, 4, 5). On the basis of these premises, one would predict that blocking NO production during late pregnancy would bring the vascular reactivity and PKC activity back to the level observed in virgin rats. However, we observed that the PDBu- and Phe-induced contraction and PKC activity in pregnant rats treated with L-NAME were significantly greater than those in virgin rats. These results suggest that treatment of pregnant rats with L-NAME not only inhibits NO synthesis, but may also increase the synthesis of or sensitivity to other vasoactive compounds that would increase the phorbol ester- and Phe-induced PKC activity and vascular reactivity. This is consistent with a recent study showing that long-term inhibition of NO synthesis during mid- to late gestation in rats is associated with elevated plasma levels of endothelin-1 (11).

The immunoblot analysis in the present study showed significant amounts of the alpha -PKC isoform in aortic smooth muscle of virgin rats. These results are consistent with other reports that have shown significant amounts of alpha -PKC in the aorta of male ferrets and rats (21, 27). We also found that both phorbol esters and Phe caused significant translocation of alpha -PKC from the cytosolic to the particulate fraction, suggesting that this PKC isoform may be involved in the phorbol ester- and Phe-induced contraction. Interestingly, the amount of alpha -PKC was significantly reduced in late-pregnant rats but significantly increased in pregnant rats treated with L-NAME. Also, the phorbol ester- and Phe-induced translocations of alpha -PKC were reduced in pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. These results suggest that the observed reduction in vascular reactivity in pregnant rats and its enhancement during inhibition of NO synthesis are related, in part, to underlying changes in the amount and activity of the alpha -PKC isoform in vascular smooth muscle. The causes of the pregnancy-associated changes in alpha -PKC are not clear at the present time but could be related, among other factors, to changes in the rate of phospholipids turnover and DAG production in vascular smooth muscle and should represent important areas for future investigations.

The present study showed that compared with the L-NAME-treated pregnant rats, in pregnant rats simultaneously treated with L-NAME and L-arginine, the PDBu- and Phe-induced contraction and the activation and translocation of alpha -PKC were significantly reduced to levels not significantly different from those observed in the untreated pregnant rats, lending support to the contention that the enhanced responses may be due to inhibition of the L-arginine-NO pathway.

Finally, we observed that the inhibitory effects of the PKC inhibitors calphostin C and staurosporine on PDBu- and Phe-induced PKC activity and alpha -PKC translocation were different, which could be due to the differences in their site of action. Calphostin C interacts with the regulatory domain of PKC at the DAG/phorbol ester binding site (25), which may explain why it inhibited both alpha -PKC translocation and PKC activity. On the other hand, staurosporine interacts with the catalytic domain of PKC at the ATP binding site (16), which may explain why it inhibited PKC activity but did not prevent alpha -PKC translocation.

In conclusion, a PKC-mediated contractile pathway in vascular smooth muscle is reduced during pregnancy and significantly enhanced in pregnant rats pretreated with the NO synthase inhibitor L-NAME. The results suggest that the pregnancy-associated changes in vascular reactivity may reflect changes in the amount and activity of the alpha -PKC isoform.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-51971 and HL-33849 (J. P. Granger) and grants from American Heart Association (grant-in-aid, Mississippi affiliate) and the National Heart, Lung, and Blood Institute (HL-52696 to R. A. Khalil).


    FOOTNOTES

C. A. Kanashiro is a recipient of postdoctoral fellowship from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. A. Khalil, Dept. of Physiology and Biophysics, Univ. of Mississippi Medical Center, 2500 N. State Street, Jackson, MS 39216-4505 (E-mail: rkhalil{at}physiology.umsmed.edu).

Received 26 January 1999; accepted in final form 3 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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