Parathyroid hormone (PTH) release is regulated by changes in the extracellular (EC) [Ca2+]. High Ca2+ inhibits, and low Ca2+ stimulates PTH secretion. Raising EC Ca2+ produces prompt, sustained increases in intracellular free Ca2+ ([Ca 2+]i) and the Ca2+- mobilizing second-messenger inositol trisphosphate (Insp3)- 1,4,5-Insp3 releases Ca 2+ from intracellular stores in a variety of cells and, in T-cells, can also stimulate Ca2+ influx by opening membrane Ca2+ channels. In sea urchin eggs and lacrimal cells, 1,3,4,5 InsP4, generated from 1,4,5-Insp3 by 1,4,5-InsP3 3-kinase and ATP, acts together with 1,4,5-Insp3 to mediate membrane Ca2+ influx. The role of 1,4,5-Insp3 and 1,3,4,5-Insp4 in the regulation of EC Ca2+- induced increases in [Ca 2+]i has not been addressed nor have the Ca2+ influx mechanisms in parathyroid cells been studied directly by patch-clamp techniques. Our hypothesis is that parathyroid cells express receptors or sensors for Ca 2+ which when activated induce rapid and sustained increases in [Ca2+], due to intracellular Ca2+ mobilization and membrane Ca2+ influx. We propose that 1,4,5-Insp3 mediates the initial release of intracellular Ca2+ and that membrane Ca2+ channels-- gated either by 1,4,5-Insp3 and/or 1,3,4,5-InsP4, by a GTP-binding protein, or by the Ca2+ receptor molecule itself -- are responsible for sustained increases in [Ca2+]i . The goal of the current proposal is to identify and characterize the mechanisms for Ca2+ mobilization in parathyroid cells using electrophysiologic and biochemical approaches. Specifically, we will determine whether high EC Ca2+ induces 1 4,5-InsP3 and 1,3,4,5Insp4 accumulation; whether 1,3,4,5-Insp4 regulates the reuptake of Ca2+ released by 1,4,5-Insp3; and whether 1,4,5-Insp3 production is to essential to Ca2+ mobilization and uptake. We will accomplish the latter by blocking phosphatidyl inositol 4,5-bis P04 (PIP2) hydrolysis and 1,4,5-lnsp3 generation with a phospholipase C inhibitor and a monoclonal antibody which binds to PIP2 and by the 1,4,5-Insp3 analogue 1,4,5-Insp3S, which mobilizes 1,4,5-Insp3-sensitive Ca2+ pools and resists phosphorylation to 1,3,4,5Insp4- By patch-clamping, we will assess whether these cells express voltage-insensitive Ca2+ channels which are regulated by second-messengers or guanyl nucleotides or directly by the Ca2+ receptor molecule. These approaches should provide important insights into the regulation of [Ca2+]i in parathyroid cells and identify Ca2+ influx mechanisms that may be involved in the pathogenesis of PTH hypersecretory states.
Showing the most recent 10 out of 24 publications
