MECHANISM OF EXTRACELLULAR CA2-STIMULATED HORMONE-RELEASE FROM SHEEP THYROID PARAFOLLICULAR CELLS( RECEPTOR)

1. Expression of receptors to extracellular calcium enables parafollic ular cells of the thyroid gland (PF cells) to release calcitonin (CT) and serotonin (5-HT) in response to increased external Ca2+. Recently, a calcium-sensing receptor (CaR), similar to the G protein-coupled re ceptor for external Ca2+ cloned from parathyroid gland, was shown to b e expressed in PF cells. Using a highly purified preparation of sheep PF cells, we have examined the electrical and biochemical processes co upling CaR activation to hormone release. 2. Whole-cell recordings in the permeabilized-patch configuration show that elevated extracellular Ca2+ concentration ([Ca2+](o)) depolarizes these cells and induces os cillations in membrane potential. In voltage clamp, high [Ca2+](o) act ivates a cation conductance that underlies the depolarization. This co nductance is cation selective, with a reversal potential near -25 mV i ndicating poor ion selectivity. 3. The CaR expressed in these cells is activated by other multivalent cations with a rank order potency of G d3+ > Ba2+ > Ca2+ >> Mg2+. The insensitivity of these cells to high ex ternal Mg2+ contrasts with the reported sensitivity of the cloned CaR from parathyroid. 4. Elevation of [Ca2+](o) also stimulates increases in intracellular Ca2+ concentration ([Ca2+](i)) and this effect is lar gely inhibited by the Ca2+ channel blocker nimodipine, indicating that L-type voltage gated. Ca2+ channels contribute to the response to ele vated [Ca2+](o). 5. Elevated [Ca2+](o) induces an inward current under conditions where the only permeant external cation is Ca2+, indicatin g that influx via the cation conductance is another source of the incr eases in [Ca2+](i). 6. Extracellular Ca2+ stimulates 5-HT release with an EC50 of 1.5 mM. Nimodipine blocks 90% of the Ca-o(2+)-induced 5-HT release, while other inhibitors of voltage-gated calcium channels had no effect. These data support an important role for L-type Ca2+ chann els in CaR-induced hormone secretion. Although earlier studies indicat e that high [Ca2+](o) induces release of Ca2+ from intracellular store s, thapsigargin-induced depletion of these stores did not affect secre tion from these cells, indicating that Ca2+ influx is necessary and su fficient for the Ca-o(2+)-induced 5-HT secretion. 7. Inhibition of pro tein kinase C (PKC) using chelerythrine, staurosporine, or calphostin C inhibited Ca-o(2+)-induced 5-HT release by 50% while phorbol ester-i nduced 5-HT secretion was completely inhibited. Thus, PKC is an import ant component of the pathway linking CaR activation to hormone release . However, another as yet unknown second messenger also contributes to this pathway 8. We tested the contribution of two different phospholi pases to the CaR responses to determine the source of the PKC activato r diacylglycerol (DAG). Selective inhibition of phosphatidylinositol-s pecific phospholipase C (PI-PLC) with U73122 had no effect on the resp onse to elevated [Ca2+](o). However, pretreatment with D609, a selecti ve inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), inhibited Ca2+-induced 5-HT release to 50% of control indicating that phosphatidylcholine is a likely source of DAG in the response of PF c ells to elevated [Ca2+](o).