Spontaneous cytosolic calcium pulses in Xenopus melanotrophs are due to calcium influx during phasic increases in the calcium permeability of the cell membrane.

Abstract

We recently discovered that melanotrophs of Xenopus laevis exhibit spontaneous pulse-like rises in cytosolic free calcium ([Ca2+]i) and that this cytosolic Ca pulsing is inhibited by the secreto-inhibitory transmitters dopamine, gamma-aminobutyric acid, and neuropeptide-Y and stimulated by the secretagogues CRF and TRH. Here we provide evidence for the factors responsible for the individual cytosolic Ca pulse and the repetitive behavior. Isolated melanotrophs of Xenopus were loaded with fura-2, and fluorescence was recorded from perifused single cells to measure [Ca2+]i and assess the patency of divalent cation channels by Mn quenching of fluorescence. Cytosolic Ca pulsing was arrested by omission of Ca and by the Ca channel blockers Ni and Co, but was unaffected by tetrodotoxin. Mn (0.3 mM) caused phasic quenching, each "quench" being synchronous with the rising phase of a cytosolic Ca pulse. Quenching was blocked by Ni, but was unaffected by tetrodotoxin. When introduced during the course of an individual Ca pulse, Ni aborted the pulse and [Ca2+]i collapsed. Extracellular K+ (10 mM) or K channel block with tetraethylammonium stimulated Ca pulsing. Threshold concentrations of Ni slowed Ca pulsing without reducing the amplitude of the individual pulses. The overshoots in quenching and Ca pulsing observed after exposure to Ni, dopamine, or baclofen are interpreted as off responses. It is concluded that each cytosolic Ca pulse is attributable, probably quantitatively, to Ca influx during a spontaneous increase in Ca permeability independent of Na-mediated action potentials, and that the pacemaker for the repetitive phenomenon is voltage sensitive and may involve Ca currents active around basal membrane potential.