Evidence for cAMP-dependent protein kinase in mediating the parathyroid hormone-stimulated rise in cytosolic free calcium in rabbit connecting tubules

We investigated cellular mechanisms mediating the parathyroid hormone (PTH)-induced increase in cytosolic free Ca2+ concentration ([Ca2+]i) in isolated perfused rabbit connecting tubules. Prior and/or concomitant exposure to 0.5 mM of N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-8), a cyclic nucleotide-dependent protein kinase inhibitor, abolished the rise in [Ca2+]i produced by 0.1 nM PTH in five connecting tubules and suppressed it by approximately 50% in another five. In the latter, there was a delayed onset in the rise of [Ca2+]i. Such responses contrasted to the prompt increase in [Ca2+]i in PTH-stimulated control tubules. However, when H-8 was withdrawn, [Ca2+]i rose within minutes to reach a plateau value similar to the uninhibited response to PTH in controls, indicating rapidly reversible inhibition by H-8. In an otherwise identical protocol, 0.5 mM H-8 also reversibly suppressed the rise in [Ca2+]i induced by 0.175 mM 8-Br-cAMP. In contrast to the stimulatory effect of 8-Br-cAMP on [Ca2+]i, 1 mM 8-Br-cGMP caused no increase. At a concentration of 0.4 mM, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), a well-characterized cAMP-dependent protein kinase inhibitor, totally abolished the rise in [Ca2+]i caused by 0.1 nM PTH. We conclude that a cAMP-dependent protein kinase plays an important role in the PTH-stimulated rise in [Ca2+]i in the rabbit connecting tubule. Since the increase in [Ca2+]i was shown previously to depend on extracellular Ca2+, we propose that cAMP-dependent protein phosphorylation is important in mediating PTH-stimulated Ca2+ fluxes across plasma membranes of connecting tubule cells.