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Involvement of Tetrodotoxin-Resistant Na⁺ Current and Protein Kinase C in the Action of Growth Hormone (GH)-Releasing Hormone on Primary Cultured Somatotropes from GH-Green Fluorescent Protein Transgenic Mice

Prince Henry’s Institute of Medical Research (S.-K.Y., K.W., C.C.), Melbourne 3168, Victoria, Australia; and Department of Physiology (S.-K.Y., H.P.), Monash University, Melbourne 3004, Victoria, Australia

Address all correspondence and requests for reprints to: Chen Chen, M.D., Ph.D., School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia. E-mail: chen.chen{at}uq.edu.au.

GHRH depolarizes the membrane of somatotropes, leading to an increase in intracellular free Ca²⁺ concentration and GH secretion. Na⁺ channels mediate the rapid depolarization during the initial phase of the action potential, and this regulates Ca²⁺ influx and GH secretion. GHRH increases a tetrodotoxin-sensitive somatotrope Na⁺ current that is mediated by cAMP. TTX-resistant (TTX-R) Na⁺ channels are abundant in sensory neurons and cardiac myocytes, but their occurrence and/or function in somatotropes has not been investigated. Here we demonstrate expression of TTX-R Na⁺ channels and a TTX-R Na⁺ current, using patch-clamp method, in green fluorescent protein-GH transgenic mouse somatotropes. GHRH (100nM) increased the TTX-R Na⁺ current in a reversible manner. The GHRH-induced increase in TTX-R Na⁺ current was not affected by the cAMP antagonist Rp-cAMP or protein kinase A inhibitors KT5720 or H89. The TTX-R current was increased by 8-bromoadenosine-cAMP (cAMP analog), forskolin (adenylyl-cyclase activator), and 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), but the additional, GHRH-induced increase in TTX-R Na⁺ currents was not affected. U-73122 (phospholipase C inhibitor) and protein kinase C (PKC) inhibitors, Gö-6983 and chelerythrine, blocked the effect of GHRH. PKC activators, phorbol dibutyrate and phorbol myristate acetate, increased the TTX-R Na⁺ current, but GHRH had no further effect on the current. Na⁺-free extracellular medium significantly reduced GHRH-stimulated GH secretion. We conclude that GHRH-induced increase in the TTX-R Na⁺ current in mouse somatotropes is mediated by the PKC system. An increase in the TTX-R Na⁺ current may contribute to the GHRH-induced exocytosis of GH granules from mouse somatotropes.