P2 purinoceptor-mediated inositol phosphate formation in relation to cytoplasmic calcium in DDT1 MF-2 smooth muscle cells.

The effect of P2 purinoceptor stimulation on inositol phosphate (InsP) formation in relation to the intracellular Ca2+ concentration was measured in vas deferens DDT1 MF-2 smooth muscle cells. The different [3H]myo-inositol-labelled InsP fractions were analyzed by high performance liquid chromatography and intracellular Ca2+ was determined by measuring fluorescence using Indo-1 as indicator. Stimulation with ATP (10(-4) M) resulted in an enhanced formation of inositol mono-, bis-, tris- and tetrakisphosphate (InsP1, InsP2, InsP3 and InsP4), but no changes occurred in the formation of inositol pentakis- and hexakisphosphate (InsP5 and InsP6). The putative second messenger Ins(1,3,4,5)P4 rapidly increased after addition of the agonist, reaching a maximum after about 2 min. The isomer Ins(1,4,5)P3 showed a delayed rise starting after about 2 min. The formation of Ins(1,3,4,5)P4 in the presence of ATP (2 min) was concentration-dependent, reaching a half maximal value at about 50 microM of the agonist. The intracellular Ca2+ concentration showed an initial increase after P2 purinoceptor stimulation, reaching a plateau after 2 min. Both the top of the initial phase and the plateau value of the response reached a half maximal value at an ATP concentration of about 7 microM. This Ca2+ response could be evoked repeatedly by ATP and was not affected by diltiazem (10(-5) M). In the absence of external Ca2+, the internal Ca2+ concentration increased transiently in the presence of ATP without showing the plateau phase. This response could be evoked only once under Ca2(+)-free conditions.(ABSTRACT TRUNCATED AT 250 WORDS)