1 Instituto de Investigaciones
Cardiológicas, 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
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.
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).
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-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
-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
[
-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
[
-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
[
-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 (,
, and
; 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
[-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -,
-, and
-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).
|
|
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 · mg1 · 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%.
|
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 · mg1 · 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 · mg1 · 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.
|
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).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Amorena, C.,
A. Castro,
A. Müller,
and
M. F. Villamil.
Direct vascular effects in the rat of the vehicles used for the intravenous and oral administration of cyclosporin A.
Clin. Sci. (Colch.)
79:
149-154,
1990[Medline].
2.
Bredt, D. S.,
C. D. Ferris,
and
S. H. Snyder.
Nitric oxide synthase regulatory sites.
J. Biol. Chem.
267:
10976-10981,
1992
3.
Brown, C. A.,
V. Patel,
G. Wilkinson,
and
M. Boarder.
P2 purinoceptor-stimulated conversion of arginine to citrulline in bovine endothelial cells is reduced by inhibition of protein kinase C.
Biochem. Pharmacol.
52:
1849-1854,
1996[Medline].
4.
Buckley, B.,
A. Barchowsky,
R. Dolor,
and
R. Whorton.
Regulation of arachidonic acid release in vascular endothelium.
Biochem. J.
280:
281-287,
1991[Medline].
5.
Chuang, L. F.,
M. Israel,
and
R. Y. Chuang.
Cremophor EL inhibits 12-o-tetradecanoylphorbol-13-acetate (TPA)-induced protein phosphorylation in human myeloblastic leukemia ML-1 cells.
Anticancer Res.
11:
1517-1521,
1991[Medline].
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].
7.
Dekker, L. V.,
and
P. J. Parker.
Protein kinase Ca question of specificity.
Trends Biochem. Sci.
19:
73-77,
1994[Medline].
8.
De Nucci, G.,
R. J. Gryglewski,
T. D. Warner,
and
J. R. Vane.
Receptor-mediated release of endothelium-derived relaxing factor and prostacyclin from bovine aortic endothelial cells is coupled.
Proc. Natl. Acad. Sci. USA
85:
2334-2338,
1988[Abstract].
9.
Furchgott, R. F. Introduction to EDRF research.
J. Cardiovasc. Pharmacol. 22, Suppl. 7: S1-S2, 1993.
10.
Gomez, M. L.,
E. E. Medrano,
E. G. A. Cafferatta,
and
M. T. Tellez-Iñón.
Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells.
Exp. Cell Res.
175:
74-80,
1988[Medline].
11.
Guyton, J. R.,
R. D. Rosenberg,
A. W. Clowes,
and
M. J. Karnovsky.
Inhibition of rat arterial smooth muscle cell proliferation by heparin.
Circ. Res.
46:
625-634,
1980[Medline].
12.
Hecker, M.,
A. Luckhoff,
and
R. Busse.
Modulation of endothelial autacoid release by protein kinase C: feedback inhibition or non specific attenuation of receptor-dependent cell activation?
J. Cell. Physiol.
156:
571-578,
1993[Medline].
13.
Hirata, K.,
R. Kuroda,
T. Sakoda,
M. Katayama,
I. Nobutaka,
M. Suematsu,
S. Kawashima,
and
M. Yokoyama.
Inhibition of endothelial nitric oxide synthase activity by protein kinase C.
Hypertension
25:
180-185,
1995
14.
Hortelano, S.,
A. M. Genaro,
and
L. Bosca.
Phorbol esters induce nitric oxide synthase and increase arginine influx in cultured peritoneal macrophages.
FEBS Lett.
320:
135-139,
1993[Medline].
15.
Huang, K.-P.,
K.-F. J. Chan,
T. J. Singh,
H. Nakabayashi,
and
F. L. Huang.
Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase.
J. Biol. Chem.
261:
12134-12140,
1986
16.
Jaffe, E. A.,
R. N. Nachman,
C. G. Becker,
and
C. R. Minick.
Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.
J. Clin. Invest.
52:
2745-2756,
1973[Medline].
17.
Kraft, A. S.,
and
W. B. Anderson.
Phorbol esters increase the amount of Ca2+, phospholipid- dependent protein kinase associated with plasma membrane.
Nature
301:
621-623,
1983[Medline].
18.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head bacteriophage.
Nature
227:
680-685,
1970[Medline].
19.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
20.
Martin, W.,
D. G. White,
and
A. H. Henderson.
Endothelium-derived relaxing factor and atriopeptin II elevate cyclic GMP levels in pig aortic endothelial cells.
Br. J. Pharmacol.
93:
229-239,
1988[Abstract].
21.
Nakane, M.,
J. Mitchell,
U. Förstermann,
and
F. Murad.
Phosphorylation by calcium calmodulin-dependent protein kinase II and protein kinase C modulates the activity of nitric oxide synthase.
Biochem. Biophys. Res. Commun.
180:
1396-1402,
1991[Medline].
22.
Ohara, Y.,
H. S. Sayegh,
J. J. Yamin,
and
D. Harrison.
Regulation of endothelial constitutive nitric oxide synthase by protein kinase C.
Hypertension
25:
415-420,
1995
23.
Pirotton, S.,
D. Communi,
S. Motte,
R. Janssens,
and
J. M. Boeynaems.
Endothelial P2-purinoceptors: subtypes and signal transduction.
J. Auton. Pharmacol.
16:
353-356,
1996[Medline].
24.
Sakata, K.,
and
H. Karaki.
Phorbol ester-induced release of endothelium-derived relaxing factor.
Eur. J. Pharmacol.
179:
207-210,
1990[Medline].
25.
Smith, J. A.,
and
D. Lang.
Release of endothelium-derived relaxing factor from pig cultured aortic endothelial cells, as assessed by changes in endothelial cell cyclic GMP content, is inhibited by a phorbol ester.
Br. J. Pharmacol.
99:
565-571,
1990[Abstract].
26.
Shimokawa, H.,
N. A. Flavahan,
R. R. Lorenz,
and
P. M. Vanhoutte.
Prostacyclin releases endothelium-derived relaxing factor and potentiates its action in coronary arteries of the pig.
Br. J. Pharmacol.
95:
1197-1203,
1995[Abstract].
27.
Schwartz, S. M.
Selection and characterization of bovine aortic endothelial cells.
In Vitro (Rockville)
14:
966-980,
1978[Medline].
28.
Smith, C. D.,
R. J. Uhing,
and
R. Snyderman.
Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters.
J. Biol. Chem.
262:
6121-6127,
1987
29.
Tamaoki, T.,
and
H. Nakano.
Potent and specific inhibitors of protein kinase C of microbial origin.
Bio/Technology
8:
732-735,
1990[Medline].
30.
Tang, E. K. Y.,
and
M. D. Houslay.
Glucagon, vasopressin and angiotensin all elicit a rapid, transient increase in hepatocyte protein kinase C activity.
Biochem. J.
283:
341-346,
1992[Medline].
31.
Uratsuji, Y.,
and
P. E. DiCorleto.
Growth-dependent subcellular redistribution of protein kinase C in cultured porcine aortic endothelial cells.
J. Cell. Physiol.
136:
431-438,
1988[Medline].
32.
Zhao, F. K.,
L. F. Chuang,
M. Israel,
and
R. Y. Chuang.
Cremophor EL, a widely used parenteral vehicle, is a potent inhibitor of protein kinase C.
Biochem. Biophys. Res. Commun.
159:
1359-1367,
1989[Medline].