It is well known that GH-PRL secreting GH 3 cells express constitutive neuronal nitric oxide synthase (nNOS) and produce nitric oxide (NO •). In addition, these cells possess plasma membrane prolactin (PRL) receptors which can be responsible for an autocrine 'short-loop' feedback. The aim of the present study was to investigate whether the activation of PRL receptors modulates the expression of the different spliced forms of nNOS gene, and the transductional mechanisms involved in this action. In GH 3 cells, both exon 2-containing nNOSa and exon 2-lacking nNOSb were timedependently expressed, whereas the other two isoforms eNOS and iNOS were not. The antibodies directed against the residues 53-68 of the external domain common to both the long and short form of rat PRL receptors, and the selective D 2 agonist cabergoline (1 nM) reduced both basal and exogenous PRL-induced expressions of nNOSa and nNOSb, but to a greater extent for the b splicing form. In line with these results, oPRL (1 and 10 lM) added to the incubation medium increased to a greater extent the expression of nNOSb form than of the nNOSa. The receptor and non-receptor protein tyrosine kinase (PTK) inhibitors, genistein (10 lM), the Srcspecific tyrosine kinase inhibitor PP2 (100 lM), the MAPK inhibitor PD 098059 (50 nM) and the two PI3¢-K inhibitors, wortmannin (300 nM) and LY-294002 (25 lM) prevented both basal and exogenous PRL-induced expression of nNOSa and nNOSb isoforms. In addition, exogenous PRL induced a phosphorylation of protein kinase B (PKB) (Akt) that was prevented both by the two MAPK inhibitors PD 098059 and U 0126, and by the PI3¢-K inhibitors wortmannin and LY-294002. Up-regulation of the expression of the two splicing forms of nNOS elicited by PRL-receptor activation was mirrored by the increased synthesis of NO• . In conclusion, PRL receptor activation up-regulated the expression of both nNOSa and nNOSb proteins via a PTK, PI3¢-K, MAPK and PKB signalling transduction components. This action may represent the molecular mechanism by which PRL exerts the 'short-loop' feedback on its own secretion. Keywords: nitric oxide, nNOS, MAPK, P13¢-K, PKB, prolactin. The free radical gaseous mediator nitric oxide (NO • ) has been reported to be involved in a wide array of physiological functions (Beckman and Koppenol 1996;Ignarro 1996;Moncada et al. 1997). Among these multiple functions NO Address correspondence and reprint requests to Dr Lucio Annunziato, MD, Division of Pharmacology, Department of Neurosciences, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, Italy. E-mail: lannunzi@unina.it Abbreviations used: FBS, fetal bovine serum; MAPK, mitogenactivated protein kinase; nNOS, neuronal nitric oxide synthase; NO • , nitric oxide; oPRL, ovine prolactin; PI3¢-K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PKG, cGMP-dependent protein tyrosine kinase; PRL, prolactin; PTK, protein tyrosine kinase.
The present study investigates the potential role of the Ca2+-calmodulin-dependent type I phosphodiesterase (PDE)-cGMP-protein kinase G (PKG) pathway in spontaneous [Ca2+]i oscillations in GH3 cells using fura-2 single cell videoimaging. Vinpocetine (2.5-50 microM), a selective inhibitor of type I PDE, induced a concentration-dependent inhibition of spontaneous [Ca2+]i oscillations in these pituitary cells, and at the same time produced an increase of the intracellular cGMP content. The cell permeable cGMP analog N2,2'-O-dibutyryl-cGMP (dB-cGMP) (1 mM) caused a progressive reduction of the frequency and the amplitude of spontaneous [Ca2+]i oscillations when added to the medium. KT5823 (400 nM), a selective inhibitor of cGMP-dependent protein kinase (PKG), produced an increase of baseline [Ca2+]i and the disappearance of spontaneous [Ca2+]i oscillations. When KT5823 was added before vinpocetine, the PKG inhibitor counteracted the [Ca2+]i lowering effect of the cGMP catabolism inhibitor. Finally, the removal of extracellular Ca2+ or the blockade of L-type voltage-sensitive calcium channels (VSCC) by nimodipine produced a decrease of cytosolic cGMP levels. Collectively, the results of the present study suggest that spontaneous [Ca2+]i oscillations in GH3 cells may be regulated by the activity of type I PDE-cGMP-PKG pathway.
Continuous and long-lasting exposure to tert-butylhydroperoxide (t-BOOH) increased the number of apoptotic SH-SY5Y human neuroblastoma cells both in the presence and in the absence of the intracellular Ca(2+) ion chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). In addition, t-BOOH exposure induced activation of CPP32, as demonstrated by poly-(ADP-ribose) polymerase (PARP) cleavage, and of ICH-1L caspases. Exposure to t-BOOH also induced a time-dependent release of cytochrome c. Interestingly, in the presence of BAPTA, CPP32 activation still occurred, whereas ICH-1L activation was blocked. Ac-DEVD-CHO, an inhibitor of CPP32 activity, prevented the appearance of apoptotic cells, whereas the inhibitor of ICH-1L activity Z-VDVAD-FMK did not. Collectively, these findings demonstrate that in SH-SY5Y neuroblastoma cells exposure to continuous and long-lasting oxidative stress induced activation of caspase-3 that was independent of intracellular Ca(2+) ion concentration ([Ca(2+)](i)) elevation but led to cell apoptosis. In contrast, caspase-2 activation was dependent on [Ca(2+)](i) increase but did not result in apoptosis.
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