Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca2+ concentration ([Ca2+]i) remain unknown. We analyzed mechanisms regulating resting [Ca2+]i in dissociated rat melanotrophs by Ca2+-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca2+ channels (VGCCs) as well as removal of extracellular Ca2+ resulted in a rapid and reversible decrease in [Ca2+]i, indicating constitutive Ca2+ influx through L-type VGCCs. Reduction of extracellular Na+ concentration (replacement with NMDG+) similarly decreased resting [Ca2+]i. When cells were champed at -80 mV, decrease in the extracellular Na+ resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na+ caused hyperpolarisation. The holding current shifted in negative direction when extracellular K+ concentration was increased from 5 mM to 50 mM in the presence of K+ channel blockers, Ba2+ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca2+]i. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca2+]i in rat melanotrophs.

Faculty of Biology Medicine and Health University of Manchester M13 9PT Manchester UK; Achucarro Centre for Neuroscience IKERBASQUE Basque Foundation for Science 48011 Bilbao Spain

Institut des Sciences Biologiques Neurosciences cognition Centre National de la Recherche Scientifique 3 rue Michel Ange Paris France; MMDN Institut National de la Santé et de la Recherche Médicale U1198 Université Montpellier Montpellier France; Ecole Pratique des Hautes Etudes Sorbonne Paris France; Department of Pharmacology and Toxicology Faculty ofMedicine Charles University at Plzen Plzen Czech Republic

Intensive Care Medicine University Hospital University of Occupational and Environmental Health Kitakyushu Japan

Laboratory of Veterinary Physiology Joint Department of Veterinary Medicine Faculty of Agriculture Tottori University Tottori Japan

Laboratory of Veterinary Physiology Joint Department of Veterinary Medicine Faculty of Agriculture Tottori University Tottori Japan; The United Graduate School of Veterinary Science Yamaguchi University Yamaguchi Japan

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