PTH secretion is controlled by changes in the extracellular (EC) [Ca2+]. High EC Ca2+ inhibits, and low EC Ca2+ maximally stimulates PTH release. The EC [Ca2+] also modulates PTH biosynthesis through effects on pre- proPTH mRNA levels and PTH gene transcription. These latter effects are likely to be crucial in the chronic adaptation to changes in serum Ca2+ in vivo. Ca2+ is thought to interact with a recently identified membrane Ca2+ sensor which couples to phospholipase C activation, 1,4,5-InsP3 formation, sustained increases in [Ca2+]i, and eventually, to the inhibition of PTH secretion. Sustained intracellular Ca2+ responses in parathyroid cells require EC Ca2+ and, we hypothesize, result from the opening of membrane Ca2+ Channels. Little information is available on the pharmacologic, biochemical, or molecular properties of Ca2+ influx pathways in parathyroid cells. By whole-cell patch-clamping, we have recorded Ca2+ currents which are voltage-insensitive, cation-selective and blocked by La3+ and Gd3+. In microflurimetry studies, Gd3+ markedly reduces intracellular Ca2+ responses to high EC [Ca2+], underscoring the potential importance of Gd3+-blockable Currents in mediating Ca2+ influx. Upon further analysis, the Ca2+ currents are comprised of 2 components. One component is a voltage-insensitive current whose conductance is dependent on changes in the EC [Ca2+]. This current is blocked by dihydropyridine Ca2+ channel antagonists and negatively modulated by protein kinase A. The other current component is voltage-dependent and regulated by protein kinase C. The studies proposed have 4 aims: (1) to investigate the role of sustained increases in [Ca2+]i in mediating chronic suppression of PTH secretion and biosynthesis, by measuring PTH release and pre-proPTH mRNA levels in cells incubated with agents which selectively induce transient or transient plus sustained increases in [Ca2+]i; (2) to define the properties of Ca2+ channels in parathyroid cells and assess their regulation by phosphorylation and guanyl nucleotides; (3) to assess the contribution of Ca2+ currents to sustained increases in [Ca2+]i and their role in high EC Ca2+-induced suppression of PTH secretion/biosynthesis, using channel agonists and antagonists; and (4) to isolate a cDNA from a parathyroid cDNA library, which encodes a dihydropyridine-sensitive Ca2+ channel, as expressed in Xenopus oocytes, and to determine whether this channel can couple to the Ca2+ sensor. Ca2+ channels in parathyroid cells may serve as a key mechanism for transducing signals initiated by the interaction of EC Ca2+ with the Ca2+ sensor. These channels are likely to contribute to the longterm adaptation to Ca2+ deficiency states or chronic hypercalcemic conditions in vivo.
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