Different kinases regulate activation of voltage-dependent calcium channels by depolarization in GH3 cells

Abstract

The L-type Ca2+ channel is the primary voltage-dependent Ca2+-influx pathway in many excitable and secretory cells, and direct phosphorylation by different kinases is one of the mechanisms involved in the regulation of its activity. The aim of this study was to evaluate the participation of Ser/Thr kinases and tyrosine kinases (TKs) in depolarization-induced Ca2+ influx in the endocrine somatomammotrope cell line GH3. Intracellular Ca2+ concentration ([Ca2+]i) was measured using a spectrofluorometric method with fura 2-AM, and 12.5 mM KCl (K+) was used as a depolarization stimulus. K+ induced an abrupt spike (peak) in [Ca2+]i that was abolished in the presence of nifedipine, showing that K+ enhances [Ca2+]i, preferably activating L-type Ca2+ channels. H89, a selective PKA inhibitor, significantly reduced depolarization-induced Ca2+ mobilization in a concentration-related manner when it was applied before or after K+, and okadaic acid, an inhibitor of Ser/Thr phosphatases, which has been shown to regulate PKA-stimulated L-type Ca2+ channels, increased K+-induced Ca2+ entry. When PKC was activated by PMA, the K+-evoked peak in [Ca2+]i, as well as the plateau phase, was significantly reduced, and chelerythrine (a PKC inhibitor) potentiated the K+-induced increase in [Ca2+]i, indicating an inhibitory role of PKC in voltage-dependent Ca2+ channel (VDCC) activity. Genistein, a TK inhibitor, reduced the K+-evoked increase in [Ca2+]i, but, unexpectedly, the tyrosine phosphatase inhibitor orthovanadate reduced not only basal Ca2+ levels but, also, Ca2+ influx during the plateau phase. Both results suggest that different TKs may act differentially on VDCC activation. Activation of receptor TKs with epidermal growth factor (EGF) or vascular endothelial growth factor potentiated K+-induced Ca2+ influx, and AG-1478 (an EGF receptor inhibitor) decreased it. However, inhibition of the non-receptor TK pp60 c-Src enhanced K+-induced Ca2+ influx. The present study strongly demonstrates that a complex equilibrium among different kinases and phosphatases regulates VDCC activity in the pituitary cell line GH3: PKA and receptor TKs, such as vascular endothelial growth factor receptor and EGF receptor, enhance depolarization-induced Ca2+ influx, whereas PKC and c-Src have an inhibitory effect. These kinases modulate membrane depolarization and may therefore participate in the regulation of a plethora of intracellular processes, such as hormone secretion, gene expression, protein synthesis, and cell proliferation, in pituitary cells.

Novel Pituitary Actions of TAC3 Gene Products in Fish Model: Receptor Specificity and Signal Transduction for Prolactin and Somatolactin α Regulation by Neurokinin B (NKB) and NKB-Related Peptide in Carp Pituitary Cells

“…[Ca 2ϩ ]i mobilization during the peak phase is consistent with the role of IP 3 receptors, because the [Ca 2ϩ ]i channels are responsible for [Ca 2ϩ ]i release from IP 3 -sensitive Ca 2ϩ stores (51). In pituitary cell lines (eg, GH 3 cells), PKA and PKC activation are also known to up-regulate VSCC activity (52), which may contribute to [Ca 2ϩ ]e entry during the shoulder phase. To our knowledge, the biphasic Ca 2ϩ response linked with NKB and the functional involvement of CaM and CaMK-II in the pituitary actions of tachykinins have not been reported in mammals.…”

Constitutive activity of the A2A adenosine receptor and compartmentalised cyclic AMP signalling fine-tune noradrenaline release

“…Activity of Src-family kinases is a possible cause of release inhibition [44][45][46]. We tested the involvement of Src kinases by including the specific inhibitor PP1 in the release experiment and probing the effect of 8-Br-cAMP.…”

“…Our data, however, point to a role of Src-family kinases. Previous reports have demonstrated Src-dependent release inhibition in neuronal cells [44][45][46]. A consequence of Src-kinase activity may be the restriction of VDCC with calcium channels and/or HCN representing potential phosphorylation substrates [57,58].…”

Expression, regulation and biological actions of growth hormone (GH) and ghrelin in the immune system

“…PKA also phosphorylates L-type Ca 2+ channel in the anterior pituitary cells, which facilitates Ca 2+ influx. Elevated intracellular Ca 2+ [Ca 2+ ]i stimulates exocytosis of GH from the secretory granules [38]. Increased cholinergic tone enhances the GH response to GHRH [39] (Fig.…”