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EXTRACELLULAR MATRIX, CELL INTERACTIONS

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

Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Cientificas y Tecnicas, Buenos Aires, Argentina

Submitted 10 August 2006 ; accepted in final form 11 May 2007

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.

phosphatases; protein kinase A; protein kinase C; epidermal growth factor