Effect of cGMP analogues and protein kinase G blocker on secretory activity, apoptosis and the cAMP/protein kinase A system in porcine ovarian granulosa cells in vitro

Q2 · BIOLOGY

Article

Author: Jozef Bulla ; Hans-Gottfried Genieser ; Alexander V Makarevich ; Juray Pivko ; Jan Kotwica ; Alexander V Sirotkin

The aim of the present study was to examine the role of cGMP-dependent intracellular mechanisms in control of ovarian functions. In the first series of experiments we studied the effects of the cGMP analogues 8-pCPT-cGMP (0.001-100 nM), Rp-8-pCPT-cGMPS (0. 01-100 nM), Rp-8-Br-cGMPS (0.01-100 nM), and Rp-8-Br-PET-cGMPS (0.01-100 nM) on the release of progesterone, insulin-like growth factor I (IGF-I) and oxytocin by cultured porcine granulosa cells. In a second series of experiments, the effects of Rp-8-Br-PET-cGMPS (50 nM) and KT5822 (100 ng/ml), specific inhibitor of cGMP-dependent protein kinase (PKG), on cAMP, PKA, oxytocin and the occurrence of apoptosis in cultured cells were compared. The release of hormones and IGF-I into the culture medium was evaluated using a RIA, while the percentage of cells containing visible oxytocin, cAMP, as well as the regulatory and catalytic subunits of PKA was assessed using immunocytochemistry. Occurrence of apoptosis in these cells was detected using the TUNEL method. The stimulatory (8-pCPT-cGMP and Rp-8-pCPT-cGMPS), inhibitory (Rp-8-Br-cGMPS) and biphasic (Rp-8-Br-PET-cGMPS) effect of cGMP analogues on progesterone release was observed. All cGMP analogues used suppressed IGF-I release. All cGMP analogues decreased oxytocin release, but 8-pCPT-cGMP and Rp-8-Br-cGMPS, when given at low doses (0.01-0.1 and 1-10 nM, respectively) stimulated oxytocin output. Both, Rp-8-Br-PET-cGMPS and KT5822 increased the rate of incidence of apoptosis and percentage of cells containing immunoreactive cAMP. Both Rp-8-Br-PET-cGMPS and KT5822 decreased the proportion of cells containing immunoreactive oxytocin and regulatory subunit of PAK KT5822, but not Rp-8-Br-PET-cGMPS, increased the number of cells containing catalytic subunit of PKA. The present observations suggest the involvement of cGMP and PKG in control of the production of steroid, nonapeptide hormone, growth factor, cAMP and cAMP-dependent PKA, as well as the induction of apoptosis in porcine ovarian cells.

01 Jan 1996·Circulation ResearchQ1 · MEDICINE

Low Increase in cGMP Induced by Organic Nitrates and Nitrovasodilators Improves Contractile Response of Rat Ventricular Myocytes

Q1 · MEDICINE

Article

Author: Schlüter, Klaus Dieter ; Kottenberg, Karin ; Nix, Petra ; Piper, Hans Michael ; Kojda, Georg ; Noack, Eike

Abstract Whether organic nitrates are bioactivated to NO in cardiac muscle cells and may thus directly affect cardiac contractile function has remained an open question. Therefore, we determined the effects of the organic nitrates glyceryl trinitrate (100 μmol/L), pentaerythritol tetranitrate (10 μmol/L), and isosorbide-5-mononitrate on electrically stimulated contractile response (CR) and cAMP and cGMP content of isolated adult rat ventricular cardiomyocytes compared with different concentrations of the spontaneous NO donors S -nitroso- N -acetyl-d,l-penicillamine (SNAP) and 2,2-diethyl-1-hydroxy-1-nitroso-hydrazine (DEA/NO). A high concentration of spontaneous NO donors (100 μmol/L) caused a large increase in cGMP content that was accompanied by a decrease in CR to 73.8±6.7% (SNAP) and 80.9±6.1% (DEA/NO) of the control values. Inhibition of cGMP-dependent protein kinase by 10 μmol/L KT 5822 converted this effect into a pronounced improvement of CR (163.5±14.0%). By contrast, the organic nitrates caused a small but significant increase in cGMP, which was accompanied by an increase in cAMP and CR identical to that induced by 10 nmol/L isoprenaline (141.6±6.4%). A similar effect was observed with a low concentration (1 μmol/L) of SNAP and DEA/NO. All increases in CR induced by nitrates were abolished after inhibition of cAMP-dependent protein kinase by Rp-cAMPS (10 μmol/L). The positive contractile effect of isoprenaline was enhanced by 1 μmol/L SNAP. This effect was also demonstrated in isolated rat papillary muscles. These results indicate that in cardiac muscle (1) organic nitrates are bioactivated to NO; (2) this results in a moderate increase in cGMP, which causes an improved CR by increasing cAMP and activating cAMP-dependent protein kinase; and (3) a large increase in cGMP, produced by high doses of NO donors, reduces CR because of the activation of cGMP-dependent protein kinase.

01 Feb 1993·Experimental Cell ResearchQ3 · MEDICINE

Inhibition of Chemotactic Peptide-Induced Development of Cell Polarity and Locomotion by the Protein Kinase C Inhibitor CGP 41 251 in Human Neutrophils Correlates with Inhibition of Protein Phosphorylation

Q3 · MEDICINE

Article

Author: Hansuli Keller ; Verena Niggli

Several protein kinase inhibitors of the staurosporine type displaying different enzyme specificity were used to study the role of protein kinase C (PKC) in motile neutrophil functions. Effects on protein phosphorylation, F-actin localization, morphology, and locomotion were determined. Only staurosporine, but not another inhibitor more specific for PKC (CGP 41 251) or inhibitors of cyclic nucleotide-dependent kinases (KT 5720, KT 5822), induced formation of F-actin-enriched surface protrusions at nanomolar concentrations in initially spherical neutrophils. The inhibitor with the highest specificity for PKC, CGP 41 251, had no effect on morphology and F-actin localization of resting cells up to 1 microM. However, this inhibitor suppressed front-tail polarity and locomotion in chemotactic peptide-stimulated cells. The cells became nonpolar with surface projections instead. Inhibition of front-tail polarity induced by CGP 41 251 strongly correlated with inhibition of in situ phosphorylation of a 67-kDa protein (IC50 = 0.2 microM), whereas induction of polarity by chemotactic peptide was associated with increased phosphorylation of this protein. Kinase inhibitors inactive on PKC (KT 5720, KT 5822) did not affect development of front-tail polarity. We conclude that a kinase involved in the phosphorylation of a 67-kDa protein, very likely a PKC isoform, may be important for the development of cell polarity and locomotion. In contrast, chemotactic peptide-induced formation of actin-containing protrusions may not be mediated by this enzyme.

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Indication	Highest Phase	Country/Location	Organization	Date
Neoplasms	Discovery	JP	Kyowa Hakko Kogyo Co., Ltd.	-

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