MOBILIZATION OF INTRACELLULAR CALCIUM IN CULTURED VASCULAR SMOOTH-MUSCLE CELLS BY URIDINE TRIPHOSPHATE AND THE CALCIUM IONOPHORE A23187

The known action of uridine triphosphate (UTP) to contract some types of vascular smooth muscle, and the present finding that it is more pot ent than adenosine triphosphate in eliciting an increase in cytosolic Ca2+ concentration in aortic smooth muscle, led us to investigate the mode of action of this nucleotide. With this aim, cultured bovine aort a cells were subjected to patch-clamp methodologies under various cond itions. Nucleotide-induced variations in cytosolic Ca2+ were monitored by using single channel recordings of the high conductance Ca2+-activ ated K+ (Maxi-K) channel within on-cell patches as a reporter, and who le-cell currents were measured following perforation of the patch. In cells bathed in Na+-saline, UTP (>30 nM) induced an inward current, an d both Maxi-K channel activity and unitary current amplitude of the Ma xi-K channel transiently increased. Repetitive exposures elicited simi lar responses when 5 to 10 min wash intervals were allowed between cha llenges of nucleotide. Oscillations in channel activity, but not oscil lation in current amplitude were frequently observed with UTP levels > 0.1 muM. Cells bathed in K+ saline (150 mM) were less sensitive to UT P (approximately 5-fold), and did not show an increase in unitary Maxi -K current amplitude. Since the increase in amplitude occurs due to de polarization of the cell membrane, a change in amplitude was not obser ved in cells previously depolarized with K+ saline. The enhancement of Maxi-K channel activity in the presence of UTP was not diminished by Ca2+ entry blockers or by removal of extracellular Ca2+. However, in t he latter case, repetitive responses progressively declined. These obs ervations, as well as data comparing the action of low concentrations of Ca2+ ionophores (<5 muM) to that of UTP indicate that both agents e levate cytosolic Ca2+ by mobilization of this ion from intracellular p ools. However, the Ca2+ ionophore did not cause membrane depolarizatio n, and thus did not change unitary current amplitude. The effect of UT P on Maxi-K channel activity and current amplitude was blocked by pert ussis toxin and by phorbol 12-myristate 13-acetate (PMA), but was not modified by okadaic acid, or by inhibitors of protein kinase C (PKC). Our data support a model in which a pyrimidinergic receptor is coupled to a G protein, and this interaction mediates release of Ca2+ from in tracellular pools, presumably via the phosphatidyl inositol pathway. T his also results in activation of membrane channels that give rise to an inward current and depolarization. Ultimately, smooth muscle contra ction ensues. PKC does not appear to be directly involved, even though the UTP response is blocked by low nm levels of PMA. While the latter data implicate PKC in diminishing the UTP response, agents that inhib it either PKC or phosphatase activity did not prevent abolition of UTP responses by PMA, nor did they modify basal channel activity.