ALPHA-ADRENOCEPTOR REGULATION OF INOSITOL PHOSPHATES, INTERNAL CALCIUM AND MEMBRANE CURRENT IN DDT1 MF-2 SMOOTH-MUSCLE CELLS

The effect of alpha-1-adrenoceptor stimulation on inositol phosphates (InsPs), intracellular Ca2+ and membrane current was measured in vas deferens DDT1 MF-2 cells. The InsPs were analyzed after labelling the cells with [H-3]myo-inositol using high performance liquid chromatography and the internal Ca2+ concentration was determined by measuring fluorescence using Indo-1 as probe. Noradrenaline stimulated the formation of inositol mono-, bis-, tris- and tetrakisphosphate (InsP, InsP2, InsP3 and InsP1) concentration-dependently in the presence of LiCl, but no changes occurred in the formation of inositol pentakis- and hexakisphosphate (InsP5 and InsP6). Various isomers of InsP3 and InsP4 were detected after noradrenaline (10(-5) M) stimulation (without LiCl). Only the formation of the putative second messenger Ins(1,4,5)P3 formation was increased in the presence of noradrenaline. The internal Ca2+ concentration was enhanced both in the presence and absence of external Ca2+ upon addition of noradrenaline. The response in the presence of extracellular Ca2+ was not affected by diltiazem (10(-5) M). The increase in cytoplasmic Ca2+ could be repeatedly evoked, but could be elicited only once under Ca2+-free conditions. The relation between the noradrenaline concentration and the rise in internal Ca2+ was similar to that obtained for the InsP formation. Half-maximal effects were obtained at about 1-mu-M. The membrane current measured in these cells by using the whole-cell patch-clamp method was not changed by the agonist (10(-5) M). These results suggest that noradrenaline acts on DDT1 MF-2 smooth muscle cells, represented by the formation of InsP3 and enhancement of internal Ca2+ originating from internal structures, via the alpha-1B-adrenoceptor subtype.