1 Section of Nephrology, Department of Medicine, and 2 Department of Urology, Case Western Reserve University, Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Changes in
activity or expression of protein kinase C (PKC), reactive oxygen
products, and nitric oxide (NO) may account for the alteration in cell
behavior seen in diabetes. These changes have been proposed to be part
of the pathophysiology of erectile dysfunction. We sought to ascertain
if corpus cavernosal vascular smooth muscle cells (CCSMC) grown in a
high glucose milieu exhibit changes in the activity and expression of
PKC isoforms, NO, and reactive oxygen products and to find out if these
changes are prevented by -tocopherol. Rat CCSMC were
grown in 5, 15, and 30 mM glucose concentrations for 3, 7, and 14 days.
PKC isoform expression was assayed with isoform-specific antibodies. In
CCSMCs grown in 30 mM glucose for 2-wk, PKC-
2-isoform
was upregulated (n = 4; P < 0.01), whereas the
expression of
-,
-,
-, and
1-isoforms was
unchanged. NO as measured by nitrate-to-nitrite ratio was greatly
diminished at 14 days in 30 mM (n = 4; P < 0.002)
compared with 5 mM glucose. Reactive oxygen products were upregulated
at 14 days when they were assayed by the fluorescent probe
dichlorofluorescein diacetate bis(acetoxy-methyl) (DCFH-DA) (n = 5; P < 0.01). When these same cells were
exposed to
-tocopherol for 14 days, there was a reduction of
PKC-
2 (57.8%; P < 0.01; n = 4) and a reduction in reactive oxygen product formation (71.1%;
P < 0.001; n = 4), along with an increase in
nitrate-to-nitrite ratio (43.9%; P < 0.01, n = 4). These results suggest that there may be an
interrelationship between PKC, NO, and reactive oxygen product
formation in CCSMC exposed to a high glucose environment.
diabetes mellitus; nitric oxide synthase; protein kinase
C-2
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ERECTILE DYSFUNCTION (ED) is a cause of major morbidity in the United States (27). It is estimated that 50-70% of all diabetic men will suffer with ED during their lifetime, and the disease is related to pathology in the vascular tree (36, 38). ED appears to strike diabetic men at an earlier age than their nondiabetic cohorts. Whether or not ED is related to glycemic control in non-insulin-dependent diabetes mellitus has not been established.
Although several neurotransmitters have been postulated to play a role in the vasodilatory process, endothelium-derived relaxation factor(s), now established to be nitric oxide (NO) and synthesized by NO synthase (NOS), appears to be the main mediator of penile erection (8, 21, 34). Physiologically, NO release with concomitant penile vascular vasodilation permits blood under systemic pressure to enter into the corpus cavernosal sinusoidal spaces, leading to erection (4-7, 11, 27). It has been proposed that disorders that impair NO synthesis and/or release and changes in protein kinase C (PKC) expression are responsible, in part, for ED (4, 6). However, the responsible subcellular mechanisms remain ill defined.
Several lines of evidence suggest that the enzyme PKC specifically
mediates the activation of many cellular events. It has been
demonstrated that in diabetes (hyperglycemia) PKC mass is increased
(retina, glomeruli, and heart) and NO bioactivity is subsequently
altered (20, 22, 23, 24). Recent work (23) has shown that PKC activity
and expression may be modulated by the generation of reactive oxygen
product formation, and therefore subsequent cellular events regulated
by PKC activity are altered. We demonstrate that not only is a specific
PKC isoform (2) upregulated in CCSMC grown under high
glucose conditions but that this change can be prevented with exposure
to
-tocopherol (vitamin E).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture
Rat penile tissue was surgically removed. The corpus tissue was isolated and placed in media containing FBS, ITS (insulin, transferrin, and selenium), and HCODetermination of PKC Isoform Expression
PKC isoforms in CCSMC were detected with PKC isoform-specific antibodies. Polyclonal antibodies againstDetermination of PKC-2 Activity
NO Analysis
We have sought to determine whether there is a change in NO release as measured by standard nitrate-to-nitrite ratio (nitrite/nitrate) measurements in these same cells grown in 5, 15, and 30 mM glucose. With identical conditions to the experiments above, we incubated cultured CCSMC and then measured for nitrate/nitrite as has been performed previously at time 0, 3, 7, and 14 days (3).Reactive Oxygen Product Formation
Intracellular reactive oxygen species in cultured cells may be detected by using the fluorescent probe dichlorofluorescein diacetate bis(acetoxy-methyl) (9). Third-to-fifth passages CCSMC were grown on glass coverslips (9 × 50 mm) in 5, 15, and 30 mM glucose. Twenty-four hours before all studies, the medium was changed from 10% FBS to 0.5% FBS to halt cell growth. We examined cells subcultured in the varying glucose concentrations for 3, 7, and 14 days. Fluorescent measurements are made with an LS-5B spectrofluorometer (Norwalk, CT) with the coverslip mounted in a temperature-controlled flow through cuvette at an angle of 60° to the incident beam and calculations made every 5 s. Intracellular dye was alternately excited at wavelengths of 492 and 440 nm and emission at 525 nm. By using conventional fluorescent ratiometric analysis, one is able to calculate the precise change in reactive oxygen product formation.Vitamin E Experiments
Solutions
The standard HCOMaterials
Dichlorofluorescein diacetate bis(acetoxy-methyl) was obtained from Molecular Probes (Eugene, OR). DMEM, FBS, penicillin, streptomycin, and phosphate buffered saline (PBS) solution were purchased from GIBCO Laboratories (Grand Island, NY). Fibroblast growth factor, insulin, transferrin, and selenium were obtained from Collaborative Research (Bedford, MA). All PKC antibodies were purchased from Santa Cruz. Plastic cuvettes, phorbol 12-myristate, and other laboratory chemicals (phorbol esters) were purchased from Sigma (St. Louis, MO). Tissue culture flasks and petri dishes were obtained from Falcon (Lincoln Park, NJ).Statistics
Data are reported as means ± SE in all the tables. Statistical significance was judged by the unpaired Student's t-test. In experiments in which glucose concentrations were used (see Tables 1-4), the data were compared with control (normal glucose). ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of PKC Isoforms in Culture
Cells were grown under 5, 15, and 30 mM glucose. The cells were then harvested, and with specific PKC isoform antibodies, we performed Western blot analysis (Fig. 1; n = 4 for each glucose concentration). Changes in isoform expression are summarized in Table 1. There was no statistical difference in the expression for
|
|
To determine if PKC-2 is activated (translocated), we
isolated membrane and cytosolic fractions. Activity is determined by how much isoform in bound to the membrane. As shown in Table
2, when CCSMCs grown in 30 mM glucose for
14 days are acutely exposed to 100 nM phorbol 12-myristate, there is a
statistical increase in the membrane-bound fraction that is maximal
within 60 min. Interestingly, there is a significant amount of PKC
2 bound to the membrane after the cells have been in 30 mM glucose for 14 days. This suggests that the isoform is active in the
basal state (before phorbol exposure).
|
Production of Nitrate/Nitrite
Using the standard assay as a surrogate for NO production, we measured nitrate/nitrite and examined whether there is a change under hyperglycemic conditions. As is shown (Table 3), it appears that cells grown in the highest glucose concentration readily express the least amount of nitrate/nitrite. This decrease, a reflection of NO release, occurs after prolonged exposure to hyperglycemia and also is concentration dependent. This is again further evidence supporting the existence of changes induced in high glucose media that may be responsible for the pathobiology seen in ED.
|
Reactive Oxygen Products
We then sought to assess whether we can affect the production of reactive oxygen products in 5, 15, and 30 mM glucose. Reactive oxygen products increased when the cells were grown in a high glucose (30 mM) vs. a low glucose (5 mM) concentration. Reactive oxygen product formation increase was not evident at 24, or at 48 h (data not shown), but was statistically evident at 72 h and persisted for 2 wk (Table 4). There is an increase in reactive oxygen products with prolonged incubation in high glucose conditions.
|
Response to -Tocopherol
Expression of PKC isoforms in culture.
We sought to ascertain if, by blocking reactive oxygen production with
-tocopherol, we could alter PKC
2 production. Cells were grown as described above, but 50 µg of
-tocopherol were added
daily (every day) for 14 days. We used 50 µg of
-tocopherol because maximal effect was evident at this dose (75 and 100 µg did
not inhibit cell PKC expression any further; data not shown). The cells
were then harvested and, using PKC isoform antibodies to
2, we performed Western blot analysis. Cells grown in 30 mM glucose were exposed to 50 µg of daily
-tocopherol for 7 and 14 days. As shown in Fig. 2, PKC
2, while still present, was greatly reduced after
prolong exposure to
-tocopherol.
|
Production of nitrate/nitrite.
We then measured the effect of the daily administration of 50 µg of
-tocopherol on the production of nitrate/nitrite in cells exposed to
30 mM glucose. As is shown in Fig. 3, when
cells incubated in 30 mM were exposed to daily
-tocopherol, the
cells had a significant increase in nitrate/nitrite compared with those
cells not exposed to this reactive oxygen scavenger. This effect was
initially seen but was not statistically evident at 7 days. Therefore,
cells continuously exposed to
-tocopherol even in the presence of 30 mM glucose are able to restore, in part, NO bioactivity.
|
Reactive oxygen product.
We measured changes in reactive oxygen product when the cells were
exposed to daily 50 µg of -tocopherol. Reactive oxygen product was
decreased after prolonged exposure to
-tocopherol even in the face
of 30 mM glucose. This effect was seen at 3 days and maintained in the
continued presence of
-tocopherol after 2 wk (Fig.
4).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There is a growing body of evidence that suggests that
hyperglycemic-induced diabetic injury modulates cell biological
behavior and that this is reflected in changes in interrelated changes in PKC, NO, and reactive oxygen species (18, 28). We have demonstrated that in these cells subcultured in hyperglycemia there is
an increase in PKC-2 and reactive oxygen species with concomitant reduced NO production. These effects are prevented, in
part, by exposure to
-tocopherol.
PKC has demonstrated participation in the cellular response to injury,
in the inflammatory process, and in the immune response, and that
signaling through PKC is essential for the activation of neutrophils,
exocytosis, cytokine release, and cellular proliferation (1, 16, 41).
The role of glucose and alterations in PKC- has been the focus of
intensive investigations. Elevation of blood hexose level in diabetes
of experimental galactosemia has been found to induce abnormalities of
metabolism, which may play a role in the development of diabetic
complications (25, 40). Activity of PKC has been found to be elevated
in retina, glomeruli, and heart of diabetic animals in purified
microvessels from diabetic rats and in endothelial and mesangial cells
cultured in high glucose or galactose (12, 24). Moreover, recent work
(M. B. Ganz and T. Kern, unpublished observations) has
established that the rat non-insulin-dependent diabetes mellitus heart
is more specifically associated with a PKC-
distribution
defect. The elevated enzyme activity has been attributed,
in part, to excessive production of diacylglycerol from glucose or
galactose (19, 20, 39). Normalization of the diabetes-induced
abnormality of PKC activity (
-isoform and other PKC isoforms)
corrects diabetes-induced abnormalities in metabolism and physiology
(blood flow in retina and kidney and peripheral nerve conduction).
Finally, diabetes-induced metabolic abnormalities in some tissues are
normalization of PKC activity with specific inhibitors (18, 24).
It has been proposed that oxidative stress may play a role in ED
through the generation of free radicals that, in turn, lead to
modulation of PKC activity with eventual disordered NO production. Many
tissues in the diabetic show significant evidence of oxidative stress,
including supranormal levels of reactive oxygen species (26). It has
now been demonstrated that reactive oxygen species can directly
increase the activity of PKC independent of diacylglycerol production
and that inhibition of PKC activity can inhibit the hyperglycemia-induced increase in free radical production (26). Moreover, phosphorylation of phospholipase D and PKC- in fibroblasts is induced by reactive oxygen species along with other changes in PKC
(31, 32). These results demonstrate that the expression of reactive
oxygen species occurs before PKC isoform expression changes are
evident. In addition, we have demonstrated the presence of the specific
reactive oxygen species marker, nitrotyrosine, in the diabetic human
penile corpus cavernosum (Seftel, personal communication).
Hyperglycemia-induced decreased NO content has been demonstrated in a number of cells (10, 33). The direction of change (increase or decrease in NO) is dependent on the cell type (14, 15). We have demonstrated the constitutive expression of eNOS in primary human CCSMC in culture and have demonstrated that when these cells are exposed to 15 and 30 mM glucose, they show a significant decrease in eNOS expression (Seftel, unpublished observations). Thus the CCSMC appear to be a source of NO production, which may be negatively affected by hyperglycemia at the level of eNOS.
Investigators have demonstrated that the activation of PKC increases venular permeability that requires the production of NO. These data indicate that NO may be a target protein of a specific PKC isoform (29, 32, 35). Pharmacological inhibition studies of total PKC activity have demonstrated an alteration of NO mRNA and a change in the release of NO. In addition, the hyperpermeability of effect of PKC-activating agents could be blocked by NO inhibitors, supporting the concept that PKC displays its signaling effect by modulating the activity of NOs in at least the endothelium (30, 35, 37). Coincubation of endothelial cells with inhibitors of PKC increased the accumulation of nitrite but did not restore it to the levels obtained when cells were cultured in 5 mmol/l glucose. It is conceivable that reactive oxygen species formation may lead to a direct decrease in eNOS expression as an early event in hyperglycemia-mediated cavernosal disease as some have reported (2, 10).
Reactive oxygen species and peroxides can cause protein cross-linking
or fragmentation, DNA breaks, lipid peroxidation, and membrane damage
and might be responsible for some of the sequelae of hyperglycemia
(20). Administration of antioxidants including -tocopherol has been
found to prevent oxidative stress in the retina and heart of the
diabetic rats and to partially inhibit the development of retinopathy
in those rats (28). The link to PKC has not been definitively
demonstrated. Information on the interrelationship of
hyperglycemia-induced abnormalities of penile metabolism provides new
insight on the role that reactive oxygen species coupled with NO has in
the pathogenesis of ED in diabetes.
![]() |
ACKNOWLEDGEMENTS |
---|
M. B. Ganz was supported by a Dept. of Veterans Affairs Merit Review. He is an established investigator (9600485) of the American Heart Association. A. D. Seftel was supported by a Veterans Affairs Merit Award.
![]() |
FOOTNOTES |
---|
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: M. B. Ganz, Section of Nephrology, 111K (W), Cleveland VA Medical Center, 10701 East Blvd., Cleveland, OH 44106 (E-mail: mbg4{at}po.cwru.edu).
Received 12 April 1999; accepted in final form 2 September 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alcantara, O.,
L. Obeid,
Y. Hannun,
P. Ponka,
and
D. H. Boldt.
Regulation of protein kinase C (PKC) expression by iron: effect of different iron compounds on PKC- and PKC-
gene expression and role of the 5'-flanking region of the PKC-
gene in the response to ferric transferrin.
Blood
84:
3510-3517,
1994
2.
Aliev, G. A. U.,
P. A. U. Bodin,
and
G. Burnstock.
Free radical generators cause changes in endothelial and inducible nitric oxide synthases and endothelin-1 immunoreactivity in endothelial cells from hyperlipidemic rabbits.
Mol. Gen. Metab.
63:
191-197,
1998[ISI][Medline].
3.
Archer, S. L.,
K. A. Freude,
and
P. J. Shultz.
Effect of graded hypoxia on the induction and function of inducible nitric oxide synthase in rat mesangial cells.
Circ. Res.
77:
21-28,
1995
4.
Bredt, D. S.,
C. D. Ferris,
and
S. H. Snyder.
Nitric oxide synthase regulatory sites.
J. Biol. Chem.
267:
10976-10981,
1992
5.
Bredt, D. S.,
and
S. H. Snyder.
Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme.
Proc. Natl. Acad. Sci. USA
87:
682-685,
1990[Abstract].
6.
Bredt, D. S.,
and
S. H. Snyder.
Nitric oxide: a physiologic messenger molecule.
Annu. Rev. Biochem.
63:
175-195,
1994[ISI][Medline].
7.
Burnett, A. L.,
C. J. Lowenstein,
D. S. Bredt,
T. S. K. Chang,
and
S. H. Snyder.
Nitric oxide: a physiologic mediator of penile erection.
Science
257:
401-403,
1992[ISI][Medline].
8.
Burnett, A. L.,
S. L. Tillman,
T. S. K. Chang,
J. I. Epstein,
C. J. Lowenstein,
D. S. Bredt,
S. H. Snyder,
and
P. C. Walsh.
Immunohistochemical localization of nitric oxide synthase in the autonomic innervation of the human penis.
J. Urol.
150:
73-76,
1993[ISI][Medline].
9.
Cathcart, R.,
E. Schwiers,
and
B. N. Ames.
Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay.
Anal. Biochem.
134:
111-116,
1983[ISI][Medline].
10.
Chakravarthy, U.,
R. G. Hayes,
A. W. Stitt,
E. McAuley,
and
D. B. Archer.
Constitutive nitric oxide synthase expression in retinal vascular endothelial cells is suppressed by high glucose and advanced glycation end products.
Diabetes
47:
945-952,
1998[Abstract].
11.
Forstermann, U.,
and
H. Kleinert.
Nitric oxide synthase: expression and expressional control of the three isoforms.
Naunyn Schmiedeberg Arch. Pharmacol.
352:
351-364,
1995[ISI][Medline].
12.
Ganz, M. B.,
T. S. Kern,
and
A. Kumar.
Apoptosis is decreased and PKC II is increased in diabetic rat glomeruli.
J. Am. Soc. Nephrol.
9:
558,
1998.
13.
Ganz, M. B.,
B. Saksa,
R. Saxena,
K. Hawkins,
and
J. R. Sedor.
PDGF and IL-1 induce and activate specific protein kinase C isoforms in mesangial cells.
Am. J. Physiol. Renal Fluid Electrolyte Physiol.
271:
F108-F113,
1996
14.
Geng, Y.,
Q. Wu,
and
G. K. Hansson.
Protein kinase C activation inhibits cytokine-induced nitric oxide synthesis in vascular smooth muscle cells.
Biochim. Biophys. Acta.
1223:
125-132,
1994[ISI][Medline].
15.
Hirata, K.,
R. Kuroda,
T. Sakoda,
M. Katayama,
N. Inoue,
M. Suematsu,
S. Kawashima,
and
M. Yokoyama.
Inhibition of endothelial nitric oxide synthase activity by protein kinase C.
Hypertension
25:
180-185,
1995
16.
Hsieh, L. L.,
S. Hoshina,
and
I. B. Weinstein.
Phenotypic effects of overexpression of PKC 1 in rat liver epithelial cells.
J. Cell. Biochem.
41:
179-188,
1989[ISI][Medline].
18.
Ishii, H.,
M. R. Jirousek,
D. Koya,
C. Takagi,
P. Xia,
A. Clermont,
S.-E. Bursell,
T. S. Kern,
L. M. Ballas,
W. F. Heath,
L. E. Stramm,
E. P. Feener,
and
G. L. King.
Amelioration of vascular dysfunctions in diabetic rats by an oral PKC inhibitor.
Science
272:
728-731,
1996b[Abstract].
19.
Kern, T. S.,
and
R. L. Engerman.
Comparison of retinal lesions in alloxan-diabetic rats and galactose-fed rats.
Curr. Eye Res.
13:
863-867,
1994[ISI][Medline].
20.
Kern, T. S.,
R. A. Kowluru,
and
R. L. Engerman.
Effect of antioxidants on the development of retinopathy in diabetes and galactosemia.
Invest. Opthalmol. Visual Sci.
37:
970-976,
1996[ISI].
21.
Kim, N.,
K. M. Azadzoi,
I. Goldstein,
and
I. Saenz de Tejada.
A nitric oxide-like factor mediates nonadrenergic-noncholinergic neurogenic relaxation of penile corpus cavernosum smooth muscle.
J. Clin. Invest.
88:
112-118,
1991[ISI][Medline].??
22.
King, G. L.,
H. Ishii,
and
D. Koya.
Diabetic vascular dysfunctions: a model of excessive activation of protein kinase C.
Kidney Int. Suppl.
51:
S77-S85,
1997.
23.
King, G. L.,
M. Kunisaki,
Y. Nishio,
T. Inoguchi,
T. Shiba,
and
P. Xia.
Biochemical and molecular mechanisms in the development of diabetic vascular complications.
Diabetes
45, Suppl.3:
S105-S108,
1996[ISI][Medline].
24.
Kowluru, R.,
M. R. Jirousek,
L. Stramm,
R. L. Engerman,
and
T. S. Kern.
Diabetes-induced disorders of retinal protein kinase C and Na, K-ATPase are inhibited by LY333531 (Abstract).
Diabetes
45:
16A,
1996.
25.
Kowluru, R.,
T. S. Kern,
and
R. L. Engerman.
Abnormalities of retinal metabolism in diabetes or galactosemia. II. Comparison of gamma-glutamyl transpeptidase in retina and cerebral cortex, and effects of antioxidant therapy.
Curr. Eye Res.
13:
891-896,
1994[ISI][Medline].
26.
Kowluru, R.,
T. S. Kern,
and
R. L. Engerman.
Antioxidants prevent retinal glutathione dysmetabolism in diabetes (Abstract).
Invest. Ophthalmol. Vis. Sci.
36:
S1066,
1995[ISI].
27.
Krane, R. J.,
I. Goldstein,
and
I. Saenz de Tejada.
Impotence.
N. Engl. J. Med.
321:
1648-1659,
1989[ISI][Medline].
28.
Kunisaki, M.,
U. Fumio,
H. Nawata,
and
G. L. King.
Vitamin E normalizes diacylglycerol-protein kinase C activation induced by hyperglycemia in rat vascular tissues.
Diabetes
45, Suppl.3:
S117-S119,
1996[ISI][Medline].
29.
Mattar, A. L.,
C. K. Fujihara,
M. O. Ribeiro,
G. de Nucci,
and
R. Zatz.
Renal effects of acute and chronic nitric oxide inhibition in experimental diabetes.
Nephron
74:
136-143,
1996[ISI][Medline].
30.
Miller, B. W.,
L. D. Baier,
and
A. R. Morrison.
Overexpression of protein kinase C-zeta isoform increases cyclooxygenase-2 and inducible nitric oxide synthase.
Am. J. Physiol. Cell Physiol.
273:
C130-C136,
1997
31.
Min, D. S.,
E. C. Kim,
and
J. H. Exton.
Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H2O2 in Swiss 3T3 fibroblasts.
J. Biol. Chem.
273:
29986-29994,
1998
32.
Minamino, T.,
M. Kitakaze,
K. Node,
H. Funaya,
and
M. Hori.
Inhibition of nitric oxide synthesis increases adenosine production via an extracellular pathway through activation of protein kinase C.
Circulation
96:
1586-1592,
1997
33.
Muniyappa, R.,
P. R. Srinivas,
J. L. Ram,
M. F. Walsh,
and
J. R. Sowers.
Calcium and protein kinase C mediate high-glucose-induced inhibition of inducible nitric oxide synthase in vascular smooth muscle cells.
Hypertension
31:
289-295,
1998
34.
Rajfer, J.,
W. J. Aronson,
P. A. Bush,
F. J. Dorey,
and
L. J. Ignarro.
Nitric oxide as mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neuorotransmission.
N, Engl. J. Med.
326:
90-94,
1992[Abstract].
35.
Ramírez, M. M.,
D. D. Kim,
and
W. N. Durán.
Protein kinase C modulates microvascular permeability through nitric oxide synthase.
Am. J. Physiol. Heart Circ. Physiol.
271:
H1702-H1705,
1996
36.
Saenz de Tejada, I.,
I. Goldstein,
K. Azadzoi,
R. Krane,
and
R. Cohen.
Impaired neurogenic and endothlium-mediated relaxation of penile smooth muscle from diabetic men with impotence.
N. Engl. J. Med.
320:
1025-1030,
1989[Abstract].
37.
Studer, R. K.,
F. R. DeRubertis,
and
P. A. Craven.
Nitric oxide suppresses increases in mesangial cell protein kinase C, transforming growth factor , and fibronectin synthesis induced by thromboxane.
J. Am. Soc. Nephrol.
7:
999-1005,
1996[Abstract].
38.
Virag, R.,
D. Frydman,
and
P. Bouilly.
Is impotence an arterial disorder?
Lancet
1:
181-184,
1985[ISI][Medline].
39.
Wakasaki, H.,
D. Koya,
F. J. Schoen,
M. R. Jirousek,
D. K. Ways,
B. D. Hoit,
R. A. Walsh,
and
G. L. King.
Targeted overexpression of protein kinase C 2 isoform in myocardium causes cardiomyopathy.
Proc. Natl. Acad. Sci. USA
94:
9320-9325,
1997
40.
Xia, P.,
T. Inoguchi,
T. S. Kern,
R. L. Engerman,
P. J. Oates,
and
G. L. King.
Characterization of the mechanism for the chronic activation of DAG-PKC pathway in diabetes and hypergalactosemia.
Diabetes
43:
1122-1129,
1994[Abstract].
41.
Zauli, G.,
G. Visani,
A. Bassini,
E. Caramelli,
E. Ottaviani,
L. Bertolaso,
V. Bertagnolo,
P. Borgatti,
and
S. Capitani.
Nuclear translocation of protein kinase C- and -zeta isoforms in HL-60 cells induced to differentiate along the granulocytic lineage by all-trans retinoic acid.
Br. J. Haematol.
93:
542-550,
1996[ISI][Medline].