Phorbol ester-induced expression of the common, low-affinity binding site for primary prostanoids in vascular smooth muscle cells

We showed in an earlier study (Hanasaki, K., and Arita, H. (1989) Biochim. Biophys. Acta 1013, 28-35) that there is a common, low-affinity binding site for primary prostanoids in cultured vascular smooth muscle cells (VSMC). This site, called the "primary prostaglandin (PG) site," can be evaluated by radioreceptor assay using 3H]PGF2 alpha and 3H]PGE1. Comparison of the capacity of several PGF2 alpha analogs to displace both radioligand bindings indicated strict requirements of the 15-hydroxy group as well as the 13,14-double bond in the omega-side chain of prostaglandins for recognition of this site. Treatment of VSMC with phorbol 12-myristate 13-acetate (PMA), a known protein kinase C activator, led to concentration- and time-dependent increases in the binding activities of 3H] PGF2 alpha as well as 3H]PGE1, which could be completely suppressed by the addition of protein kinase C inhibitor, H-7. The PMA effects could be mimicked by phorbol 12,13-dibutylate, but not by inactive phorbol ester. Scatchard analyses revealed an approximately 8-fold increase in the binding density with unaltered binding affinity after PMA treatment. This expression of the primary PG site was blocked by the addition of cycloheximide and actinomycin D. In contrast, PMA did not affect the binding activity for the thromboxane A2/prostaglandin H2 receptor in VSMC. These results suggest that the expression of the primary PG site is regulated by a protein kinase C-dependent mechanism in VSMC.