AJP - Cell Physiology, Vol 263, Issue 6 C1266-C1273, Copyright © 1992 by American Physiological Society
Regulation of pHi in Saos-2 cells by thrombin: roles of proteolytic activity and cytosolic calcium transients
E. Ofori-Darko and A. H. Tashjian Jr
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
Some, if not all, of the cellular actions of alpha-thrombin are now
believed to be mediated by proteolytic cleavage of the cell surface
thrombin receptor to yield a tethered ligand that initiates signal
transduction via the receptor. We have investigated the actions of
alpha-thrombin on the regulation of cytosolic free Ca2+ concentration
([Ca2+]i) and intracellular pH (pHi) in human osteoblast-like Saos-2 cells.
After acidification with nigericin, thrombin induced an acute increase of
[Ca2+]i and a rise in pHi. The action of thrombin on pHi was dependent on
activation of the Na(+)-H+ antiporter. Thrombin elicited parallel
concentration-dependent increases in both [Ca2+]i and pHi, and the rise in
[Ca2+]i was a prerequisite for the increase in pHi. Preincubation of
thrombin with the active site proteolytic inhibitor,
BOC-D-Phe-L-Pro-D,L-Lys-CF3, prevented the alkalinization response to
thrombin but had little or no effect on the thrombin-induced rise in
[Ca2+]i. Hirudin, a natural inhibitor of thrombin, acts by tight binding to
several discrete regions on the thrombin molecule. Preincubation of
thrombin with hirudin completely blocked the rise in both [Ca2+]i and pHi.
These results demonstrate that the thrombin-induced rise in [Ca2+]i alone
is not sufficient to cause alkalinization in Saos-2 cells. More
importantly, our findings reveal that not all of the cellular actions of
thrombin can be explained by proteolytic cleavage of the thrombin receptor
and suggest that different domains on the thrombin molecule may be required
for eliciting signals that raise [Ca2+]i and pHi in Saos-2 cells.
