1 The effects of 12-O-tetradecanoyl 4p-phorbol 13-acetate (P-TPA) on the inositol 1,4,5-trisphosphate (IP3) production, Ca2+ release from the intracellular Ca2+ stores and sensitization of contractile apparatus, induced by prostaglandin Fu (PGF2.) and U46619, a thromboxane A2-mimetic, were studied, using fura-2-loaded and -unloaded rat thoracic aortic strips. 2 Both eicosanoids had characteristic patterns of responses in Ca2+-free, 2 mM EGTA-containing solution (Ca2'-free solution). They induced transient increases in intracellular Ca2+ concentration ([Ca2+],) without corresponding transient contraction, but produced delayed, sustained contraction, where [Ca2+], was returned to the basal level.3 Treatment with ,-TPA for 60 min reduced the eicosanoids-induced IP3 production, suggesting that the treatment inhibits PIP2 breakdown. 4 The treatment also attenuated [Ca2+]1 transient induced by the eicosanoids, but not by caffeine (an IP3-independent releaser of stored Ca2+), in fura-2-loaded preparations incubated in Ca2+-free solution.5 In contrast in the presence of P-TPA, the sustained contractions evoked by the eicosanoids in Ca2'-free solution were potentiated, suggesting that the sites of actions of P-TPA and the eicosanoids may differ from each other. 6 PGF,A and U46619 utilize different and parallel signal transduction pathways to release Ca2+ by IP3 produced by PIP2 breakdown (,-TPA-sensitive), and to increase the sensitivity of contractile apparatus, in which protein kinase C may not be involved (P-TPA-insensitive).Keywords: Vascular smooth muscle; prostaglandin F2CC; U46619; calcium; fura-2; Ca stores; protein kinase C; phorbol ester; inositol trisphosphate (Litten et al., 1987) and sensitizing contractile apparatus by activating protein kinase C (PKC; Rasmussen et al., 1987;Ruzycky & Morgan, 1989;Karaki, 1989 (Orellana et al., 1985;Leeb-Lundberg et al., 1985;Roth et al., 1986;Litten et al., 1987;Slivka & Insel, 1988;Kaya et al., 1989;Araki et al., 1989). This providues us with a possible method of addressing this point, i.e., to learn which of the functional response(s) could be induced by the PIP2 breakdown reaction. Thus, the effects of 1 2-O-tetradecanoyl 4P-phorbol 13-acetate (P-TPA) on IP3 production,
Measurement of [Ca2+]i in fura-2-loaded preparationThe experiment was carried out as described previously (Sato et al., 1988;Hisayama et al., 1990). The strips were incubated with 5 1M fura-2/AM in normal PSS for 3-4 h at room temperature in the presence of 0.2% Cremophor EL, then rinsed with the solution for 15min. Thereafter, experiments were performed with a double wavelength excitation fluorimeter (CAF 100, Japan spectroscopic Co., Tokyo, Japan) where the fura-2-loaded strips was fixed horizontally in a bath that was bubbled with 100% 02 at 37°C. The mechanical activity was monitored isometrically. Simultaneously, 500nm fluorescence emitted by 340nm and 380nm excitation (F34o and F380, respectively) were measured by successive alternating illuminations (48 Hz), and the ratio (R340/380) of F340 to ...
