CALCIUM REGULATION OF INTRACELLULAR PH IN PITUITARY INTERMEDIATE LOBE MELANOTROPES

The regulatory activities of both intracellular calcium ([Ca2+]i) and intracellular pH (pH(i)) have greatly increased interest in the study of their interdependence. We have designed an epifluorescence video microscope that will image the fluorescence from two ratio dyes, indo-1 (for [Ca2+]i) and SNARF-1 (for pH(i)), at video rates. We examined primary cultures of pituitary intermediate lobe melanotropes loaded with both dyes. After experimentation, cells were positively identified by fluorescence immunohistochemistry. K+-induced depolarization of melanotropes produced increases in [Ca2+]i due to activation of L-type Ca channels. A secondary Ca2+ peak or oscillations were often seen. After treatment with carbonyl cyanide m-chlorophenylhydrozone, depolarization produced a rise in intracellular [Ca2+]i as well as oscillations. After thapsigargin or cyclopiazonic acid treatment, depolarization produced a primary Ca2+ elevation, but the secondary Ca2+ changes disappeared. This suggests that the oscillations were due to Ca2+ release from an endoplasmic reticulum type of intracellular store. All of these increases in [Ca2+]i were also directly coupled to a rise in intracellular H+. The close association between intracellular Ca2+ and H+ suggests that the observed pH(i) changes were due to the release of H+ upon binding of Ca2+ to intracellular buffers. This direct obligate coupling of intracellular Ca2+ and H+ suggests the possibility that pH-dependent cellular processes are directly activated by sudden increases in intracellular Ca2+ levels. This second messenger type of signaling system would be activated whether the Ca2+ was released from intracellular stores or entered the cell via plasma membrane Ca2+ channels.