Cytosolic Ca2+ transients are not required for platelet-derived growth factor to induce cell cycle progression of vascular smooth muscle cells in primary culture. Actions of tyrosine kinase.
We investigated interrelations among changes in cytosolic Ca2+ concentrations ([Ca2+]i), tyrosine phosphorylation, and the cell cycles of rat aorta smooth muscle cells in primary culture, as stimulated with platelet-derived growth factor (PDGF). Changes in [Ca2+]i were monitored using the microfluorometry of Fura-2. The phase of the cell cycle and the extent of tyrosine phosphorylation were examined by immunocytochemical analysis of monoclonal antibodies against cell cycle-specific nuclear antigens and against phosphotyrosine, respectively. Prior to the application of PDGF, the cell cycle was synchronized in the G0 phase by serum deprivation for 24 h. In the presence of extracellular Ca2+, PDGF induced an initial transient (first component) and a subsequent lower steady-state (second component) elevation of [Ca2+]i. NiCl2 and the removal of extracellular Ca2+ inhibited the second, but not the first, component. The first component was inhibited by pretreatment with ryanodine. These results are compatible with the notion that the first and second components may be mediated mainly through the release of intracellular Ca2+ and the influx of extracellular Ca2+, respectively. After pretreatment with ryanodine and in the presence of NiCl2, PDGF also stimulated the entry of G0 cells into G1 phase, but there were no [Ca2+]i transients. Genistein, a tyrosine kinase blocker, inhibited tyrosine phosphorylation induced by PDGF and blocked the first, but not the second, component of [Ca2+]i elevation induced by PDGF. However, genistein did not inhibit the release of intracellular Ca2+ induced by angiotensin II or by caffeine. Genistein prevented G0 cells from entering the G1 phase, as induced by PDGF, but this was not the case when serum was reapplied to the growth medium. Similar results were obtained with another tyrosine kinase blocker, tyrphostin. These data suggest that in vascular smooth muscle cells: 1) an increase in [Ca2+]i is not required for competent (G0 to G1) cell proliferation induced by PDGF; and 2) tyrosine kinase plays an important role in the release of intracellular Ca2+ and in cell proliferation, as induced by PDGF.
Kobayashi
S
S
Division of Molecular Cardiology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Nishimura
J
J
Kanaide
H
H
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D009570:Nitriles / Q000494:pharmacology; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012433:Ryanodine / Q000494:pharmacology; D015290:Second Messenger Systems; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D007529:Isoflavones; D009570:Nitriles; D010982:Platelet-Derived Growth Factor; D000804:Angiotensin II; D012433:Ryanodine; D019000:Phosphotyrosine; D002110:Caffeine; D014443:Tyrosine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D002118:Calcium