Whole-cell voltage clamp was used in conjunction with the fluorescent Ca2+ indicator indo-1 to measure extracellular Ca2+ entry and intracellular Ca2+ concentrations ([Ca2+]i) in rat gonadotrophs identified with the reverse haemolytic plaque assay. 2. Depolarizations to potentials more positive than -40 mV elicited inward Ca2+ current (ICa) and transient elevations of [Ca2+]i. 3. The relationship between [Ca2+]i elevations and Ca2+ entry with different Ca2+ buffer concentrations in the pipette showed that endogenous Ca2+ buffers normally bind approximately 99% of the Ca2+ entering the cell. 4. With [Ca2+]i elevations less than 500 nM, decay of [Ca2+]i could be approximated by an exponential whose time constant increased with the concentration of exogenous Ca2+ buffers. 5. Inhibitors of intracellular Ca(2+)-ATPases, thapsigargin, cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ), caused [Ca2+]i to rise. Application of BHQ during [Ca2+]i oscillations induced by gonadotrophin-releasing hormone (GnRH) terminated the oscillation in a slowly decaying elevation. BHQ slowed the decay of depolarization-induced [Ca2+]i elevations about 3-fold. 6. Taking into account the Ca2+ buffering properties of the cytoplasm permitted estimation of the fluxes and rate constants for Ca2+ movements in gonadotrophs. The intracellular store is a major determinant of Ca2+ homeostasis in gonadotrophs.
