Modulation of L-type Ca2+ channel currents by various protein kinase activators and inhibitors in rat clonal pituitary GH3 cell line

L-type Ca2+ channels play an important role in regulating cytosolic Ca2+ and thereby regulating hormone secretions in neuroendocrine cells. Since hormone secretions are also regulated by various kinds of protein kinases, we investigated the role of some kinase activators and inhibitors in the regulation of the L-type Ca2+ channel currents in rat pituitary GH3 cells using the patch-clamp technique. Phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, and vanadate, a protein tyrosine phosphatase (PTP) inhibitor, increased the Ba2+ current through the L-type Ca2+ channels. In contrast, bisindolylmaleimide I (BIM I), a PKC inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, suppressed the Ba2+ currents. Forskolin, an adenylate cyclase activator, and isobutyl methylxanthine (IBMX), a non-specific phosphodiesterase inhibitor, reduced Ba2+ currents. The above results show that the L-type Ca2+ channels are activated by PKC and PTK, and inhibited by elevation of cyclic nucleotides such as cAMP. From these results, it is suggested that the regulation of hormone secretion by various kinase activity in GH3 cells may be attributable, at least in part, to their effect on L-type Ca2+ channels.

The role of K+ channels on spontaneous action potential in rat clonal pituitary GH3 cell line

The types of K+ channel which determine the pattern of spontaneous action potential (SAP) were investigated using whole-cell variation of patch clamp techniques under current- and voltage-clamp recording conditions in rat clonal pituitary GH3 cells. Heterogeneous pattern of SAP activities was changed into more regular mode with elongation of activity duration and afterhyperpolarization by treatment of TEA (10 mM). Under this condition, exposure of the class III antiarrhythmic agent E-4031 (5 micrometer) to GH3 cells hardly affected SAP activities. On the other hand, the main GH3 stimulator thyrotropin-releasing hormone (TRH) still produced its dual effects (transient hyperpolarization and later increase in SAP frequency) in the presence of TEA. However, addition of BaCl2 (2 mM) in the presence of TEA completely blocked SAP repolarization process and produced membrane depolarization in all tested cells. This effect was observed even in TEA-untreated cells and was not mimicked by higher concentration of TEA (30 mM). Also this barium-induced membrane depolarization effect was still observed after L-type Ca2+ channel was blocked by nicardipine (10 micrometer). These results suggest that barium-sensitive current is important in SAP repolarization process and barium itself may have some depolarizing effect in GH3 cells.

Regulation of the contraction induced by emptying of intracellular Ca2+ stores in cat gastric smooth muscle

To investigate the mechanism of smooth muscle contraction induced by emptying of intracellular Ca2+ stores, we measured isometric contraction and 45Ca2+ influx. CaCl2 increased Ca2+ store emptying- induced contraction in dose-dependent manner, but phospholipase C activity was not affected by the Ca2+ store emptying-induced contraction. The contraction was inhibited by voltage-dependent Ca2+ channel antagonists dose dependently, but not by TMB-8 (intracellular Ca2+ release blocker). Both PKC inhibitors (H-7 and staurosporine) and tyrosine kinase inhibitors (genistein and methyl 2,5-dihydroxycinnamic acid) significantly inhibited the contraction, but calmodulin antagonists (W-7 and trifluoperazine) had no inhibitory effect on the contraction. The combined inhibitory effects of protein kinase inhibitors, H-7 and genistein, together with verapamil were greater than that of each one alone. In Ca2+ store-emptied condition, 45Ca2+ influx was significantly inhibited by verapamil, H-7 or genistein but not by trifluoperazine. However combined inhibitory effects of protein kinase inhibitors, H-7 and genistein, together with verapamil were not observed. Therefore, this kinase pathway may modulate the sensitivity of contractile protein. These results suggest that contraction induced by emptying of intracellular Ca2+ stores was mediated by influx of extracellular Ca2+ through voltage-dependent Ca2+ channel, also protein kinase C and/or tyrosine kinase pathway modulates the Ca2+ sensitivity of contractile protein.

Involvement of Spontaneously Formed Cyclic Nucleotides in Cat Gastric Muscle Relaxation

Muscle strips and muscle cells from cat stomach were used to investigate whether spontaneously formed cyclic nucleotides were involved in the inhibition of gastric smooth muscle contraction. A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), increased the levels of both cyclic GMP (cGMP) and cyclic AMP (cAMP) in resting state cells, while decreasing acetylcholine-induced muscle contraction. Under the influence of IBMX, SQ22536, an adenylyl cyclase inhibitor and methylene blue, a guanylyl cyclase inhibitor completely blocked increases in cAMP and cGMP respectively, without any effect on contraction. However, the combination of SQ22536 and methylene blue completely blocked increases in both cAMP and cGMP levels and stimulated contractions markedly even in the presence of IBMX. Muscle contraction inhibitors such as isoprenaline, vasoactive intestinal polypeptide and sodium nitroprusside also appeared to increase cyclic nucleotide levels which decreased contraction. Which nucleotide increased the most was dependent on the agonist used. Therefore, irrespective of the cyclic nucleotide class, the spontaneous formation of cyclic nucleotides should be considered in evaluating the mechanism of gastric smooth muscle relaxation.