Differences in intracellular calcium signaling after activation of the thrombin receptor by thrombin and agonist peptide in osteoblast-like cells.

Abstract

Thrombin and the thrombin receptor agonist peptide (TRAP) caused a rise in intracellular calcium concentration ([Ca2+]i) in the human osteoblast-like cell line Saos-2. Striking differences in the [Ca2+]i signals elicited by these agonists were revealed. In cell populations, thrombin induced a transient increase in [Ca2+]i while TRAP caused a biphasic [Ca2+]i response consisting of an initial peak and a sustained plateau phase. In individual cells, thrombin mainly caused a single [Ca2+]i transient while TRAP induced repetitive [Ca2+]i spikes. Neither tyrosine phosphorylation, cAMP-dependent phosphorylation, nor pertussis toxin-sensitive G proteins appeared to be involved in thrombin receptor [Ca2+]i signaling in this cell line. However, the sustained [Ca2+]i response caused by TRAP was converted into a transient, thrombin-like response by pretreatment with serine/threonine phosphatase inhibitors. Pretreatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) abrogated thrombin receptor [Ca2+]i signaling, and TRAP-induced Ca2+ entry was inhibited by the acute treatment with PMA. In contrast, Ca2+ entry stimulated by thapsigargin was not sensitive to agents affecting serine/threonine phosphorylation. The observation that thrombin and TRAP, despite being agonists for a common receptor, induce dissimilar [Ca2+]i responses indicates that binding of TRAP alone is insufficient to fully regulate the thrombin receptor in Saos-2 cells.