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 potent 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 aorta cells were subjected to patch-clamp methodologies under various conditions. Nucleotide-induced variations in cytosolic Ca2+ were monitored by using single channel recordings of the high conductance Ca(2+)-activated K+ (Maxi-K) channel within on-cell patches as a reporter, and whole-cell currents were measured following perforation of the patch. In cells bathed in Na(+)-saline, UTP (> 30 nM) induced an inward current, and both Maxi-K channel activity and unitary current amplitude of the Maxi-K channel transiently increased. Repetitive exposures elicited similar responses when 5 to 10 min wash intervals were allowed between challenges of nucleotide. Oscillations in channel activity, but not oscillation in current amplitude were frequently observed with UTP levels > 0.1 microM. Cells bathed in K+ saline (150 mM) were less sensitive to UTP (approximately 5-fold), and did not show an increase in unitary Maxi-K current amplitude. Since the increase in amplitude occurs due to depolarization of the cell membrane, a change in amplitude was not observed 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 the latter case, repetitive responses progressively declined. These observations, as well as data comparing the action of low concentrations of Ca2+ ionophores (< 5 microM) to that of UTP indicate that both agents elevate cytosolic Ca2+ by mobilization of this ion from intracellular pools. However, the Ca2+ ionophore did not cause membrane depolarization, and thus did not change unitary current amplitude. The effect of UTP on Maxi-K channel activity and current amplitude was blocked by pertussis 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 intracellular pools, presumably via the phosphatidyl inositol pathway. This also results in activation of membrane channels that give rise to an inward current and depolarization. Ultimately, smooth muscle contraction ensues.(ABSTRACT TRUNCATED AT 400 WORDS)
Sanchez-Fernandez
M
M
Department of Membrane Biochemistry and Biophysics, Merck Research Laboratories, Rahway, New Jersey 07065.
Katz
G M
GM
Suarez-Kurtz
G
G
Kaczorowski
G J
GJ
Reuben
J P
JP
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D003570:Cytidine Triphosphate / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism*; D037342:Pertussis Toxin; D015221:Potassium Channels / Q000032:analysis / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D015221:Potassium Channels; D010566:Virulence Factors, Bordetella; D000001:Calcimycin; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D014544:Uridine Triphosphate