Extracellular ATP and bradykinin increase cGMP in vascular endothelial cells via activation of PKC

A. F. Castro1, C. Amorena1,3, A. Müller1, G. Ottaviano1, M. T. Tellez-Iñon2, and A. C. Taquini1

1 Instituto de Investigaciones Cardiológicas, Facultad de Medicina, Universidad de Buenos Aires, 1122 Buenos Aires; 2 Instituto de Ingeniería Genética y Biología Molecular (Consejo Nacional de Investigaciones Científicas y Técnicas), 1428 Buenos Aires; and 3 Escuela de Ciencia y Tecnologia, Universidad Nacional de General San Martin, Buenos Aires, Argentina

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Vasodilation by agents such as bradykinin and ATP is dependent on nitric oxide, the endothelium-dependent relaxing factor (EDRF). The release of EDRF results in elevation of cGMP in endothelial and smooth muscle cells (9). The signaling pathway that leads to increases in cGMP is not completely understood. The role of protein kinase C (PKC) in the elevation of cGMP induced by ATP and bradykinin was studied in cultured porcine aortic endothelial cells, by measuring PKC phosphorylation of a substrate and by measuring cGMP levels by radioimmunoassay. Extracellular ATP and bradykinin simultaneously elevated cGMP levels and PKC activity. The PKC inhibitors staurosporine, calphostin C, and Cremophor EL (T. Tamaoki and H. Nakano. Bio/Technology 8: 732-735, 1990; F. K. Zhao, L. F. Chuang, M. Israel, and R. Y. Chuang. Biochem. Biophys. Res. Commun. 159: 1359-1367, 1989) prevented the elevation of cGMP elicited by ATP and reduced that produced by bradykinin. Cremophor did not affect the elevation of cGMP by nitroprusside, an agent that directly increases guanylate cyclase activity (9). The PKC activator phorbol 12-myristate 13-acetate, but not a phorbol ester analog inactive on PKC, also elevated cGMP levels. These results suggest that EDRF agonists elevate cGMP in endothelial cells via PKC stimulation.

endothelium-dependent relaxing factor; phorbol ester; protein kinase C inhibitors

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

VASODILATION BY AGENTS SUCH as bradykinin and ATP is dependent on endothelial cells' release of nitric oxide, the endothelium-dependent relaxing factor (EDRF). EDRF, produced from the enzymatic degradation of L-arginine, stimulates guanylate cyclase from endothelial and smooth muscle cells, which results in elevation of cGMP levels (9). The signaling pathway that leads to increases in cGMP by EDRF-releasing agents is not completely understood. A possible role of protein kinase C (PKC) has been proposed but not definitively established. It has been suggested that PKC activation could participate in a negative feedback regulating the receptor-activated synthesis and release of EDRF (8, 25). In addition, it has been shown that stimulation or inhibition of PKC reduces and increases, respectively, nitric oxide synthase activity of endothelial cells (6, 22). In contrast to the above observations, PKC-activating phorbol esters induce relaxation of norepinephrine-contracted aortic rings, and it was suggested that this effect is due to release of EDRF (24). In support of this idea, an increase in the activity of nitric oxide synthase from macrophages and brain has been shown after phosphorylation by PKC (14, 21). Furthermore, PKC inhibition reduces the endothelial nitric oxide synthase activity stimulated by agonists (3). As outlined above, the role of PKC in the regulation of EDRF release in endothelial cells is controversial. The controversies could arise from the use of different preparations and/or the indirect nature of the experiments, i.e., conclusions were based only on the use of pharmacological agents without direct measurements of PKC activity.

The aim of this work was to assess the contribution of PKC to the agonist-induced elevation of endothelial cGMP levels by obtaining simultaneous measurements of PKC activity and cGMP in cultured endothelial cells stimulated by agonists that increase cGMP through stimulation of EDRF release. This study suggests that EDRF agonists elevate cGMP in endothelial cells via PKC stimulation.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. Endothelial cells were isolated from thoracic aortas of porcine fetuses by a modification of the method of Jaffe et al. (16). Each aorta was cut longitudinally, and the endothelial surface was exposed to 1 mg/ml collagenase in Hanks' solution for 10 min at 37°C. The endothelial cells were scraped off with a Teflon spatula and harvested in M-199 medium containing 20% FCS, 5 IU/ml heparin (to inhibit the proliferation of smooth muscle cells, see Ref. 11), and 2 mM L-glutamine. The cells were then placed in 35-mm plastic petri dishes and incubated at 37°C in a 5% CO2-95% O2 atmosphere. Confluence was reached in 7-10 days. The identity of the endothelial cells was verified by detection of factor VIII antigen by immunofluorescence (27).

Three days after the monolayers reached confluence, the culture medium was removed and the cells were washed three times with a physiological salt solution (PSS) containing (in mmol/l) 10 HEPES, 138 NaCl, 5 KCl, 1.25 CaCl2, 1.25 MgSO4, 1 NaH2PO4, and 10 glucose. The pH was titrated to 7.4 with NaOH. After cells were washed, they were preincubated in PSS for 60 min at 37°C. For PKC activity assays, the following experimental groups were studied: 1) control, in absence of agonists, 2) 10 µM ATP, 3 min, 3) 1 µM bradykinin, 3 min, 4) 1 µM phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, 15 min, 5) 1 µM 4alpha -phorbol 12,13-didecanoate (PDD), a phorbol ester analog inactive on PKC, 15 min. The incubation solution was removed at the times indicated, and the monolayers still attached to the culture vessels were rapidly frozen in liquid nitrogen and kept at -70°C until further analysis. For cGMP measurements, after the medium was removed and cells were washed with PSS, the endothelial cells were incubated for 30 min in PSS containing 1 mM 3-isobutyl-1-methylxanthine, an inhibitor of cyclic 3',5'-nucleotide phosphodiesterases. Experimental groups were as follows: 1) control, in absence of drugs, 2) agonist of EDRF (in which either 10 µM ATP or 1 µM bradykinin was added to the incubation solution), 3) antagonist of EDRF [100 µM NG-nitro-L-arginine methyl ester (L-NAME) alone or combined with either 10 µM ATP or 1 µM bradykinin], 4) PKC inhibitors (160 µg/ml Cremophor, 10 nM staurosporine, or 2.5 µM calphostin C alone or combined with either 10 µM ATP or 1 µM bradykinin), and 5) phorbol esters (1 µM PMA or 1 µM PDD as control). The cells were incubated with ATP or bradykinin for 3 min or with phorbol esters for 15 min. The cells were preincubated with the inhibitors for at least 30 min. The incubation solution was removed at the times indicated, and the monolayers still attached to the culture vessels were rapidly frozen in liquid nitrogen and kept at -70°C until further analysis.

Endothelial PKC activity. For PKC assays, endothelial cells were harvested in buffer A, which contained 20 mM Tris · HCl, 2 mM EDTA, 0.5 mM EGTA, 5 mM beta -mercaptoethanol, 250 mM sucrose, 0.1 mM phenylmethylsulfonyl fluoride, 0.02 mM leupeptin, 1 mM benzamidine, 2 µg/ml soybean (trypsin inhibitor), and 10 µg/ml aprotinin, pH 7.4. Cells were lysed by sonication at 4°C, and the lysate was centrifuged at 100,000 g for 60 min at 4°C. The resulting supernatant was considered the cytosolic (soluble) fraction. The pellet was resuspended in buffer A containing 1% Triton X-100, incubated for 60 min at 4°C with continuous mixing, and then centrifuged at 100,000 g for 60 min at 4°C. The supernatant was considered the membrane (particulate) fraction. Crude preparations were used except when indicated. Partial purification of soluble and particulate fractions was carried out by DEAE-cellulose chromatography (DE52, Whatman) essentially as described (10). The fractions (1 mg protein) were applied to DEAE-cellulose columns (0.5 × 1.5 cm) previously equilibrated with buffer A. The columns were washed with 2 volumes of buffer A and eluted with solutions containing 150 or 400 mM NaCl. PKC activity was determined by measuring 32P incorporation from [gamma -32P]ATP into synthetic PKC substrate peptide analog to a fragment of glycogen synthase (GS). The reaction mixture consisted of 25 µM GS in 20 mM Tris · HCl, 10 mM MgCl2, and 50 µM [gamma -32P]ATP [specific activity 500-1,000 counts · min-1 (cpm) · pmol-1] plus 0.5 mM EGTA or 0.5 mM CaCl2, 60 µg/ml phosphatidylserine, and 3 µg/ml diolein in a total volume of 60 µl. The pH of the reaction mixture was 7.4, and incubations were carried out at 30°C for 10 min. Reactions were started by addition of [gamma -32P]ATP and stopped by spotting a 40-µl aliquot of the reaction mixture onto Whatman P-81 phosphocellulose filters (2 × 2 cm), which were immediately soaked in 75 mM H3PO4. The filters were washed three times in the same solution, for 20 min each, dried, and assayed for radioactivity in scintillation mixture. PKC activity was calculated as the difference between 32P incorporated into the GS substrate peptide in presence of CaCl2-phosphatidylserine-diolein vs. EGTA. Results were expressed as picomoles of 32P incorporated per milligram of protein per minute (pmol · mg-1 · min-1). Protein concentrations were measured by the method of Lowry et al. (19).

Immunoblotting. For immunoblots, samples were subjected to 10% SDS-PAGE according to Laemmli (18) and transferred to nitrocellulose membranes. The membranes were blocked for 30 min at room temperature with 5 g/100 ml milk in Tris-buffered saline (TBS; 25 mM Tris · HCl and 154 mM NaCl, pH 8.0) containing 2 g/100 ml glycine and incubated overnight at 4°C with a polyclonal antibody against a consensus sequence present in three PKC isoforms (alpha , beta , and gamma ; anti-PAN PKC). Antibody concentration was 2 µg/ml in TBS. The blots were washed with TBS and incubated for 60 min at room temperature with horseradish peroxidase-conjugated secondary antibody for enhanced chemiluminescence (ECL) detection or with goat anti-rabbit biotinylated antibody for alkaline phosphatase detection.

Endothelial cGMP levels. Measurements of cGMP were performed in the acetylated samples by radioimmunoassay (NEN, Boston, MA). 125I-cGMP activity was measured in a gamma well counter (LKB Wallac Clinigamma 1272). Results are expressed as picomoles of cGMP per milligram of protein (pmol/mg).

Materials. Collagenase was from Worthington (Freehall, NJ), and tissue culture medium was from GIBCO (Grand Island, NY). The PKC substrate GS was purchased from the Saint Vincent's Institute for Medical Research (Fitzroy, Australia). The anti-PAN PKC antibody was purchased from Upstate Biotechnology (Lake Placid, NY), and the horseradish peroxidase-conjugated secondary antibody was from Amersham (Arlington Heights, IL). The ECL Western blotting detection kit, cGMP radioimmunoassay kit, and [gamma -32P]ATP were from DuPont NEN (Boston, MA). The secondary antibody and the chemicals for alkaline phosphatase detection were purchased from Vector (Burlingame, CA). Other chemicals were from Sigma (St. Louis, MO).

Statistics. Results are expressed as means ± SE and were evaluated using the Student's t-test for nonpaired data.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The redistribution of PKC from the cytosol to the membrane is a good index of PKC activation by agonists (7). Because PKC subcellular distribution depends on the state of growth of pig aortic endothelial cells (31), it was critical to validate that confluent cells under our growing conditions have PKC activity mostly associated with the cytosolic fraction. Several PKC isoforms have been found in cultured porcine aortic endothelial cells (12), and, based on the information available, we selected an antibody that recognizes alpha -, beta -, and gamma -PKC. Figure 1 illustrates the presence of an immunoreactive band consistent with PKC in Western blots of soluble and particulate fractions of cultured endothelial cells. The signal was stronger in the soluble fraction. Figure 1 also shows that partially purified PKC is eluted from a DEAE column in the 150 mM NaCl fraction, consistent with previous observations (10). The antibody used reacts only with members of the classic PKC family, and, to determine whether the results above correlate with a global distribution of PKC activity in the cytosol, we performed a GS phosphorylation assay. The results of this assay were similar to those obtained in the immunologic PKC distribution assay, confirming that the PKC activity is predominantly located in the cytosol (Fig. 2).


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Fig. 1.   Immunoblots of soluble and particulate fractions of cultured endothelial cells. Nonspecific: lysate of endothelial cells incubated with secondary antibody (no primary antibody present). Brain PKC: partially purified bovine brain protein kinase C (PKC). Cytosol and membrane: crude soluble and particulate fractions (50 µg each), respectively. Elution 1: first 150 mM NaCl elution fraction. Elution 2: second 150 mM NaCl elution fraction. Elution 3: 400 mM NaCl elution fraction. Molecular mass markers are indicated at left. See MATERIALS AND METHODS for additional details. Similar results were obtained in 2 additional experiments.


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Fig. 2.   Effect of ATP and bradykinin (BK) on PKC activity of cultured endothelial cells. Cells were incubated with ATP (10 µM, 3 min) or BK (1 µM, 3 min). Percent of the total PKC activity in soluble (open columns) and particulate fractions (solid columns) in the absence and presence of agonists is illustrated. No. of experiments = 3-6.

To investigate the effects of ATP and bradykinin on the PKC activity of endothelial cells, we measured the percentage of the total PKC activity in soluble and particulate fractions of endothelial cells. We selected a 3-min incubation period to test the effect of ATP on the PKC activity on the basis of the time course of ATP-induced elevation in cGMP (see below and Fig. 3). ATP increased the percentage of the total PKC activity of the particulate fraction (Fig. 2). This activation of PKC by ATP accounted for an elevation in the PKC activity of the particulate fraction, without a change in the activity of the soluble fraction, i.e., there was elevation of total PKC activity by ~55% [86.40 ± 4.91 pmol · mg-1 · min-1 in the absence of agonist (n = 4) vs. 132.77 ± 9.64 pmol · mg-1 · min-1 in the presence of ATP (n = 4), P < 0.05]. Although PKC activity per milligram protein was higher in partially purified soluble and particulate fractions (data not shown), the distribution was similar to that of the crude preparations shown in Fig. 2. ATP increased the percentage of total activity in the purified-particulate fractions from 16 to 54%.


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Fig. 3.   Time course of the effect of ATP on cGMP in cultured endothelial cells. ATP (10 µM), with or without NG-nitro-L-arginine methyl ester (L-NAME; 100 µM), was added at time 0 (control). No. of experiments = 4.

Bradykinin, 3-min incubation, also increased the percentage of the total PKC activity of the particulate fraction (Fig. 2). The activation of PKC by bradykinin was comparable to that of PMA (i.e., increase in the PKC activity of the particulate fraction without change in the total PKC activity, described below). In fact, bradykinin reduced the PKC activity of the soluble fraction [191.5 ± 8.5 pmol · mg-1 · min-1 in the absence of agonist (n = 3) vs. 33.5 ± 8.5 pmol · mg-1 · min-1 in the presence of bradykinin (n = 3), P < 0.05] and increased the PKC activity of the particulate fraction [29.5 ± 17.1 pmol · mg-1 · min-1 in the absence of agonist (n = 3) vs. 237.0 ± 44.5 pmol · mg-1 · min-1 in the presence of bradykinin (n = 3), P < 0.05].

PMA, 15-min incubation, reduced the PKC activity of the soluble fraction [84.2 ± 2.4 pmol · mg-1 · min-1 in the presence of the inactive phorbol ester analog, PDD (n = 3), vs. 16.9 ± 1.4 pmol · mg-1 · min-1 in the presence of PMA (n = 3), P < 0.05] and increased the PKC activity of the particulate fraction [5.7 ± 0.5 pmol · mg-1 · min-1 in the presence of PDD (n = 3) vs. 64.1 ± 4.7 pmol · mg-1 · min-1 in the presence of PMA (n = 3), P < 0.05]. The total PKC activity in the presence of PMA was not significantly different from that in the presence of the phorbol ester analog inactive on PKC, PDD. Note that incubation with PDD had no effect on the distribution of PKC activity between soluble and particulate fractions; the percentage of the total PKC activity in particulate fractions of cells treated with PDD did not increase (6%, compared with control in Fig. 2).

To evaluate the ability of our cultured endothelial cells to increase cGMP in response to agonists, we tested the effect of ATP and bradykinin, agents known to induce EDRF synthesis and cGMP levels in endothelial cells (9, 20). ATP produced a largely transient elevation in cGMP levels, which was inhibited by incubation of cultured endothelial cells with L-NAME, which inhibits EDRF synthesis (Fig. 3; Ref. 9). A 3-min exposure to bradykinin (1 µM) also increased cGMP levels, which was inhibited 75% by L-NAME (Fig. 4). Note that, at 3 min of exposure to ATP and bradykinin, both cGMP levels and PKC activity increased (see Fig. 2). These data indicate that the agonists increased cGMP levels predominantly by stimulation of EDRF release.


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Fig. 4.   Effect of BK on cGMP in cultured endothelial cells. Content of cGMP was measured in cells exposed to BK (1 µM, 3 min) preincubated with or without L-NAME (100 µM). No. of experiments = 4.

To demonstrate the role of PKC stimulation in the elevation of cGMP by EDRF-releasing agents, we employed PKC inhibitors [both staurosporine and the more specific blockers calphostin C and Cremophor (29, 32)]. Cremophor is a polyoxyethylated derivative of castor oil that is used as solvent for the immunosuppressant cyclosporine A. It is a highly specific inhibitor of PKC that blocks PMA-induced phosphorylation in human leukemia cells (5, 32). To validate the effect of this novel PKC inhibitor in endothelial cells, we performed a cell-free assay in which the PKC activity was measured in the presence of increasing concentrations of Cremophor. For these experiments, PKC was partially purified by DEAE-cellulose chromatography. Figure 5 shows that Cremophor inhibited the PKC activity of endothelial cells in a dose-dependent manner. On the other hand, 160 µg/ml Cremophor had no effect on the elevation of cGMP induced by 1 mM nitroprusside [61.10 ± 3.55 pmol/mg in the absence of Cremophor (n = 3) vs. 58.60 ± 2.01 pmol/mg in the presence of Cremophor (n = 3)], an agent known to directly increase the guanylate cyclase activity (9). These results suggest that Cremophor inhibits the PKC activity of endothelial cells and that its effect is exerted before the guanylate cyclase activation step. As shown in Fig. 6, the PKC inhibitors staurosporine, calphostin C, and Cremophor prevented the elevation in cGMP elicited by ATP and reduced that produced by bradykinin. Moreover, the activation of PKC by PMA increased the levels of cGMP 2.5-fold, whereas the phorbol ester inactive on PKC, PDD, did not affect the basal levels of cGMP (Fig. 7).


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Fig. 5.   Inhibition of endothelial PKC activity by Cremophor. PKC activity is expressed as percent of control. No. of experiments = 3.


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Fig. 6.   Effects of PKC inhibition on agonist-induced cGMP levels of cultured endothelial cells. cGMP levels were normalized to those under control condition in the absence of drugs. Content of cGMP was measured in cells exposed to ATP (10 µM, 3 min) or BK (1 µM, 3 min) with or without preincubation with the PKC inhibitors Cremophor (160 µg/ml), staurosporine (10 nM), or calphostin C (2.5 µM). No. of experiments = 3-5. * P < 0.05 compared with their corresponding control (i.e., ATP or BK alone). # P < 0.05 compared with control in the absence of drugs.


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Fig. 7.   Effects of PKC activation on cGMP levels of cultured endothelial cells. Content of cGMP was measured in cells without or with 4alpha -phorbol 12,13-didecanoate (PDD; 1 µM, 15 min) or phorbol 12-myristate 13-acetate (PMA; 1 µM, 15 min). No. of experiments = 3. * P < 0.05 compared with control.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The aim of this work was to determine the role of PKC in the increase of cGMP induced by EDRF-releasing agents in cultured endothelial cells. Our results support the notion that PKC stimulation is necessary for the elevation of cGMP by ATP, a well-known EDRF-releasing agent. In addition, they show that PKC stimulation is partially responsible for the elevation of cGMP by another EDRF-releasing agent, bradykinin. The main results of our studies show that 1) ATP and bradykinin stimulate PKC activity, as determined by the increase in the percentage of the total PKC activity in the particulate fraction, and 2) elevation of cGMP levels produced by ATP and bradykinin is abolished and reduced, respectively, by PKC inhibitors.

The redistribution of PKC between cytosol and membranes is a good index of changes in PKC activity for most PKC isoenzymes (7). On the other hand, increases in total PKC activity as a result of increases in the PKC activities of cytosolic and/or membrane fractions have also been reported (10, 30). In the present experiments, an increase in total PKC activity as a result of elevation in the PKC activity of the membrane fraction indicates that ATP stimulated PKC in cultured endothelial cells. The activation of PKC by bradykinin and PMA was similar. In fact, there was a redistribution of PKC activity from cytosol to membranes without a change in the total PKC activity. These results suggest that the mechanism of PKC activation by both agonists is different. For bradykinin, it is clear that the activation involved the translocation of PKC to the membrane where it can be activated by endogenous activators, like 1,2-diacylglycerol. The mechanism of PKC activation by ATP seems to be more complicated. The effect of ATP may be due an elevation in cellular PKC activity independent of translocation to the membrane (true activation of PKC), which results in an activated state of PKC. This would explain the increase of total PKC activity by ATP, but we cannot rule out parallel PKC activation through translocation from cytosol to the membrane. A possible explanation for a different activity state of the enzyme is a change of PKC activity by phosphorylation, since it has been reported that phosphorylation of PKC alters the kinetic properties of the enzyme (15).

The fact that PKC inhibition prevented the elevation of cGMP elicited by ATP, and reduced that induced by bradykinin, stresses the role of PKC stimulation in the response of cGMP to the agonists. This interpretation is further supported by the observation that three chemically unrelated PKC inhibitors, Cremophor, calphostin C, and staurosporine, have similar effects on the agonist-stimulated increase of cGMP levels in endothelial cells. Indeed, although none of the PKC inhibitors is entirely specific, it is unlikely that three chemically unrelated blockers affect the cGMP increase by mechanisms independent of PKC inhibition. In contrast with our results, it has been shown that UCN-01, another PKC inhibitor, increased the bradykinin-stimulated release of EDRF (12). However, the authors failed to detect PKC activation by bradykinin in their system, suggesting that UCN-01 did not affect EDRF release by PKC inhibition.

It has been previously shown that Cremophor, at a concentration shown here to inhibit PKC, reverses the relaxation of vascular rings elicited by ATP, acetylcholine, and carbamylcholine and that its effect is reversed by L-arginine (1). The observation that Cremophor affects the cGMP elevation produced by both ATP and bradykinin indicates that its effect is not at the level of the ATP or bradykinin receptors. Furthermore, because Cremophor had no effect on either the increase of cGMP or the relaxation of aortic rings by nitroprusside, as methylene blue does (1), we conclude that the PKC inhibition prevents or reduces the increase in EDRF brought about by ATP and bradykinin, respectively, and that the inhibitor does not act by blocking guanylate cyclase. The ATP and bradykinin-induced increase of cGMP blocked by L-NAME, a competitive blocker of EDRF synthesis (9), confirmed that the increase of cGMP is mediated by an increase in EDRF production. In this context, it is relevant to mention that there is a parallelism between endothelial EDRF production and cGMP levels, which is the result of the stimulation of guanylate cyclase by EDRF (20, 25).

The fact that PKC inhibition did not block completely cGMP elevation by bradykinin suggests PKC-sensitive and PKC-insensitive components of bradykinin-stimulated increase in cGMP. Evidences suggest a PKC-independent arachidonic acid release, the precursor of prostacyclin (which releases EDRF, Ref. 26), in bradykinin-stimulated endothelial cells (4). The mechanism of the prostacyclin-stimulated EDRF release remains to be examined; however, it could explain the lack of complete inhibition of bradykinin-stimulated increases in cGMP with the PKC inhibitors.

Finally, additional evidence for a relationship between PKC stimulation and cGMP levels comes from the results showing elevation of cGMP by PMA but not by the PKC-inactive phorbol, PDD. PMA produced the well-known stimulation of PKC (17) and also elevated cGMP. The latter effect is consistent with the vascular relaxation produced by PKC-activating phorbol esters (Ref. 24 and data not shown). The effect of the phorbol esters on vascular relaxation seems to be due to an increase in EDRF production and an elevation in cGMP because relaxation is blocked by methylene blue (24) and is not present in endothelium-denuded arterial rings (Ref. 24 and data not shown).

From these results, we conclude that PKC stimulation elevates cGMP levels probably through EDRF production. Previous studies have shown that PKC modulation affects EDRF release and nitric oxide synthase activity stimulated by agonists (6, 8, 25), but the results are controversial. On the basis of results in preparations pretreated with phorbol esters, it was suggested that PKC activation participates in a negative feedback, regulating the receptor-activated synthesis and release of EDRF (8, 25). Because phorbol esters uncouple G proteins from phospholipase C (28), they can reduce the response to EDRF agonists. In contrast, our results suggest that PKC exerts a positive regulation on the receptor-activated synthesis and release of EDRF. Together, the results suggest a dual regulation by PKC: 1) a negative feedback in the coupling between membrane receptors and phospholipase C, observed with preincubation with phorbol esters, and 2) a positive regulation, likely exerted at a step distal to phospholipase C activation and before guanylate cyclase activation. Two results support the latter interpretation. First, PKC activation is necessary for EDRF release by ATP, since inhibition of PKC abolishes the increase of cGMP, independent of the facilitation of the receptor response by inhibition of the PKC-dependent negative feedback. Second, the inhibition of PKC by Cremophor had no effect on either the increase of cGMP or the relaxation of aortic rings by nitroprusside, a direct activator of guanylate cyclase. Hence, it can be speculated that PKC regulates EDRF production through the endothelial nitric oxide synthase, probably by phosphorylation. In support of this idea, it was reported that phosphorylation of brain and macrophage nitric oxide synthases by PKC results in increased enzyme activity (14, 21). Furthermore, inhibition of PKC reduces the endothelial nitric oxide synthase activity stimulated through purinoceptors 2u and 2y in bovine aortic endothelial cells (3). The release of EDRF by ATP in endothelial cells is mediated by the P2 purinoceptors, and P2u and P2y receptor subtypes are present in porcine aortic endothelial cells (23). In contrast with the results above and our observations, reduced activity of the brain nitric oxide synthase after phosphorylation by PKC (2) and increased expression and activity of endothelial nitric oxide synthase by PKC inhibition have been reported (22). Moreover, inhibition of nitric oxide synthase activity by PMA has been observed in crude endothelial cell homogenates (6). These results are not consistent with the observation that the vascular relaxation produced by PKC-activating phorbol esters seems to be due to an increase in EDRF production (Ref. 24 and data not shown) and with the elevation in cGMP by phorbol esters reported here. The reason for these discrepancies is unknown but could reflect the use of preparations from different origins and/or differences in the experimental approaches. Differences in regulation of PKC isoforms in response to EDRF agonists, as demonstrated in other systems (7), could explain some of the discrepancies. The present results suggest that PKC stimulation exerts a positive regulation in the elevation of cGMP; however, whether or not this reflects a regulation of nitric oxide synthase activity remains unclear.

In summary, our results show that ATP and bradykinin, two EDRF-releasing agents, elevate cGMP by stimulation of PKC and suggest that this effect is due to stimulation of EDRF production.

    ACKNOWLEDGEMENTS

We are grateful to Drs. G. Altenberg and L. Reuss for their helpful criticism and suggestions in the preparation of this manuscript.

    FOOTNOTES

This work was supported by funds from Consejo Nacional de Investigaciones Científicas y Técnicas (Grant 337570092 to A. C. Taquini and C. Amorena) and Universidad Nacional de General San Martin (to C. Amorena), Argentina.

Address for reprint requests and present address of A. F. Castro: Dept. of Physiology and Biophysics, Univ. of Texas Medical Branch, Galveston, TX 77555-0641.

Received 1 February 1996; accepted in final form 20 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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6.   Davda, R. K., L. J. Chandler, and N. J. Guzman. Protein kinase C modulates receptor-independent activation of endothelial nitric oxide synthase. Eur. J. Pharmacol. 266: 237-244, 1994[Medline].

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Am J Physiol Cell Physiol 275(1):C113-C119
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