Heparin-insensitive calcium release from intracellular stores triggered by the recombinant human parathyroid hormone receptor.

1. In the present study we have characterized the parathyroid hormone (PTH)-induced calcium signalling in 293 cells stably transfected with the human PTH receptor cDNA. In these cells, human PTH-1(1-38) strongly stimulates adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation (EC50 = 0.39 nM) but fails to activate phosphoinositide (PI) turnover. The latter pathway is strongly activated, however, by carbachol (CCh) acting through endogenous M3-muscarinic receptors. 2. Despite the lack of detectable inositol phosphate (IP) formation, hPTH-(1-38) elicited calcium transients (EC50 = 11.2 nM) which were comparable to the signals evoked by CCh. These signals are independent of cyclic AMP generation as cyclic AMP elevating agents did not mimic or modify the PTH response. 3. The PTH-stimulated calcium signal still occurred in calcium-free medium but was absent in cells pretreated with thapsigargin, an inhibitor of the calcium pump of the endoplasmic reticulum (ER). hPTH-(1-38) did not accelerate Mn(2+)-influx through the plasma membrane. These data indicate that PTH releases calcium from intracellular stores. 4. Using heparin, an inhibitor of the IP3-activated calcium release channel of the ER, we tested whether the formation of a low amount of IP3, escaping detection by our biochemical assay, might be the origin of the PTH-induced calcium response. However, intracellular infusion of heparin through patch pipettes in voltage clamp experiments failed to block hPTH-(1-38)-induced calcium signals, whereas it abolished the CCh response. 5. The PTH response, like the CCh response, was insensitive to micromolar concentrations of ryanodine and ruthenium red, eliminating the possibility that hPTH-(1-38) stimulates calcium-induced calcium release through ryanodine receptors.6. We conclude that the recombinant human PTH receptor stimulates calcium release from intracellular stores through a novel pathway not involving IP3- or ryanodine receptors.