Cyclic-AMP-Dependent Protein Kinase Activity Is Not Required by Parathyroid Hormone to Stimulate Phosphoinositide Signaling in Chondrocytes but Is Required to Transduce the Hormone′s Proliferative Effect

Abstract

Parathyroid hormone (PTH), an activator of both cAMP and phosphoinositide (PI) signaling in growth plate chondrocytes (GPCs), is generally believed to trigger each of these pathways through interactions with separate G proteins. Recently, however, activation of cAMP-dependent protein kinase (pkA) has been found to cause a stimulation of the PI cascade in hepatocytes. This finding raises the possibility that PTH stimulation of PI metabolism in GPCs may really be a secondary event, mediated through a primary stimulation of pkA. Experiments discussed in the present report indicate that the PTH stimulation of PI metabolism in GPCs is independent of pkA activity. The data show that (1) unlike the Ca²⁺ response evoked by PTH, the responses evoked by dibutyryl-cAMP or S_p diastereomer of cyclic adenosine-3′,5′-monophosphothioate, two activators of pkA, require an extracellular Ca²⁺ source; (2) also unlike PTH, activation of pkA by these same cAMP analogs does not cause an increase in cellular inositol-1,4,5-trisphosphate; and (3) specific inhibition of pkA with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinsulfanomide (H-89) or R_p diastereomer of cylic adenosine-3′,5′-monophosphothioate (R_p cAMPS) has no effect on the ability of PTH to evoke its normal Ca²⁺ response. Furthermore, data presented indicate that the PTH stimulation of GPC proliferation does not require Ca²⁺ signals, but rather is at least partially dependent on pkA. The data show that either loading the cells with the Ca²⁺ buffer bis-(o-ami-nophenoxy)ethane-N,N,N′,N′-tetracetic acid or depleting the cells of intracellularly stored Ca²⁺ is without effect on the stimulation of DNA synthesis by the hormone. Inhibition of pkA activity with H-89 or R_p-cAMPS, in contrast, leads to a significant reduction in the ability of PTH to stimulate its proliferative effect.