Thaw C N, Raaka E G, Gershengorn M C
Am J Physiol. 1984 Sep;247(3 Pt 1):C150-5. doi: 10.1152/ajpcell.1984.247.3.C150.
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.
钴对促甲状腺激素释放激素（TRH）刺激的催乳素分泌的抑制作用，已被解释为表明钙内流增强在GH3细胞中TRH作用过程中起着至关重要的作用。然而，这种解释是基于这样一种假设，即钴离子（Co2+）仅通过阻断细胞外钙离子（Ca2+）的内流，与细胞外表面结合来拮抗钙介导的过程。在本报告中，我们提供证据表明Co2+作用于细胞内位点来抑制催乳素分泌。当GH3细胞在含有1.5 mM Ca2+的培养基中孵育时，Co2+抑制基础分泌以及50 mM K+和TRH诱导的分泌；半数最大效应发生在0.1至0.3 mM Co2+之间。当细胞在含有0.05和0.003 mM Ca2+（这两种浓度可消除50 mM K+诱导的催乳素分泌）的培养基中孵育时，Co2+仍然抑制基础分泌和TRH刺激的催乳素分泌。Co2+还抑制由1-甲基-3-异丁基黄嘌呤、二丁酰腺苷3',5'-环磷酸（cAMP）和血管活性肠肽刺激的催乳素分泌，这三种促分泌剂的作用是升高细胞内cAMP，这一机制似乎不涉及增强的Ca2+内流。最后，通过细胞内捕获的二价阳离子螯合剂喹啉2的荧光猝灭，表明细胞内存在Co2+。这些数据表明Co2+进入GH3细胞，并且Co2+对催乳素分泌的抑制不涉及细胞外Ca2+。我们认为Co2+不仅阻断GH3细胞中的Ca2+通道，而且在细胞内位点抑制催乳素分泌。因此，Co2+的抑制作用不应被解释为表明刺激分泌过程中需要Ca2+内流。
