| Abstrakt: |
Cobalt inhibition of stimulated prolactin secretion has been interpreted as demonstrating an essential role for enhanced calcium influx in the action of thyrotropin-releasing hormone (TRH) in GH3 cells. However, this interpretation is based on the assumption that cobalt ion (Co2+) binds to the external surface of cells to antagonize calcium-mediated processes only by blocking influx of extracellular calcium ion (Ca2+). In this report, we present evidence that Co2+ acts at an intracellular locus (or loci) to inhibit prolactin secretion. When GH3 cells were incubated in medium containing 1.5 mM Ca2+, Co2+ inhibited basal as well as 50 mM K+- and TRH-induced secretion; half-maximal effect occurred between 0.1 and 0.3 mM Co2+. When cells were incubated in medium containing 0.05 and 0.003 mM Ca2+, concentrations that abolish 50 mM K+-induced prolactin secretion, Co2+ still inhibited basal and TRH-stimulated prolactin secretion. Co2+ also inhibited prolactin secretion stimulated by 1-methyl-3-isobutylxanthine, dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), and vasoactive intestinal peptide, three secretagogues that act to elevate intracellular cAMP, a mechanism which appears not to involve enhanced Ca2+ influx. Last, the presence of Co2+ within the cell was shown by fluorescence quenching of intracellularly trapped Quin 2, a chelator of divalent cations. These data demonstrate that Co2+ enters GH3 cells and that Co2+ inhibition of prolactin secretion does not involve extracellular Ca2+. We suggest that Co2+ not only blocks Ca2+ channels in GH3 cells, but it inhibits prolactin secretion at an intracellular locus (loci). Hence, inhibition by Co2+ should not be interpreted as demonstrating a requirement for Ca2+ influx in stimulated secretion. |
