Abstract
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 Ca2+-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 μm. Cells bathed in K+ saline (150 μ m) were less sensitive to UTP (∼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 μm) 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. 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 inhibit either PKC or phosphatase activity did not prevent abolition of UTP responses by PMA, nor did they modify basal channel activity.
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References
Barros, F., Kaczorowski, G.J., Katz, G.M., Vandlen, R.L., Reuben, J.P. 1986. Application of whole-cell voltage clamp in the study of neuroendocrine cells. In: Electrophysiological Techniques in Pharmacology. H.M. Geller, editor. pp. 149–168, Alan R. Liss, New York
Bean, B.P. 1992. Pharmacology and electrophysiology of ATP-activated ion channels. Trends Pharmacol. Sci. 13:87–90
Benham, C.D. 1989. ATP-activated channels gate calcium entry in single smooth muscle cells dissociated from rabbit ear artery. J. Physiol. 419:689–701
Benham, C.D., Tsien, R.W. 1987. A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle. Nature 328:275–278
Boeynaems, J.-M., Pearson, J.D. 1990. P2 purinoceptors on vas-cular endothelial cells: physiological significance and transduction mechanisms. Trends Pharmacol. Sci. 11:34–37
Breitwieser, G.E., Szabo, G. 1985. Uncoupling of cardiac muscarinic and β-adrenergic receptors from ion channels by a guanine nucleotide analogue. Nature 317:538–540
Davidson, J.S., Wakefield, I.K., Sohnius, U., Van der Merwe, P.A., Millar, R.P. 1990. A novel extracellular nucleotide receptor coupled to phosphoinositidase-C in pituitary cells. Endocrinology 126:80–87
Demolle, D., Lagneau, C., Boeynaems, J.-M. 1988. Stimulation of prostacyclin release from aortic smooth muscle cells by purine and pyrimidine nucleotides. Eur. J. Pharmacol. 155:339–343
Drummond, A.H. 1986. Inositol lipid metabolism and signal transduction in clonal pituitary cells. J. Experimental. Biol. 124:337–358
Flavahan, N.A., Shimokawa, H., Vanhoutte, P.M. 1991. Inhibition of endothelium-dependent relaxations by phorbol myristate acetate in canine coronary arteries; role of a pertussis toxin-sensitive G-protein. J. Pharmacol. Exp. Ther. 256: 50–55
Friel, D.D. 1988. An ATP-sensitive conductance in single smooth muscle cells from the rat vas deferens. J. Physiol. 401:361–380
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch. 391:85–100
Horn, R., Marty, A. 1988. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92:145–159
Inoue, R., Brading, A.F. 1990. The properties of the ATP-induced depolarization and current in single cells isolated from the guinea-pig urinary bladder. Br. J. Pharmacol. 100:619–625
Llano, I., Marty, A. 1987. Protein kinase C activators inhibit the inositol trisphosphate-mediated muscarinic current responses in rat lacrimal cells. J. Physiol. 394:239–248
Martin, S.C. 1992. ATP activates a Ca2+-dependent Cl− current in the rat thyroid cell line, FRTL-5. J. Membrane Biol. 125:243–253
Murrin, R.J., Boarder, M.R. 1992. Neuronal “nucleotide” receptor linked to phospholipase C and phospholipase D? Stimulation of PC12 cells by ATP analogues and UTP. Mol. Pharmacol. 41:561–568
Niedergerke, R., Page, S. 1981. Two physiological agents that appear to facilitate calcium discharge from the sarcoplasmic reticulum in frog heart cells: adrenalin and ATP. Proc. R. Soc. Lond. 213:325–344
Petersen, O.H., Wakui, M. 1990. Oscillating intracellular Ca2+ signals evoked by activation of receptors linked to inositol lipid hydrolysis: mechanism of generation. J. Membrane Biol. 118:93–105
Pfaffinger, P.J., Martin, J.M., Hunter, D.D., Nathanson, N.M., Hille, B. 1985. GTP-binding proteins couple cardiac muscarinic receptors to a K channel. Nature 317:536–538
Sanchez, M., Katz, G.M., Bale, T., Reuben, J.P. 1991a. Exposure of bovine aortic smooth muscle cells to UTP causes transient activation of calcium-dependent potassium channels. Biophys. J. 59:78a (Abstr.)
Sanchez, M., Katz, G.M., Bale, T., Reuben, J.P. 1991b. A putative pyrimidinergic receptor in bovine aortic smooth muscle cells: Pertussis toxin and phorbol ester sensitivity. Biophys. J. 59:80a (Abstr.)
Seifert, R., Burde, R., Schultz, G. 1989. Activation of NADPH oxidase by purine and pyrimidine nucleotides involves G proteins and is potentiated by chemotactic peptides. Biochem. J. 259:813–819
Seifert, R., Schultz, G. 1989. Involvement of pyrimidinoceptors in the regulation of cell functions by uridine and by uracil nucleotides. Trends Pharmacol. Sci. 10:365–369
Seifert, R., Wenzel, K., Eckstein, F., Schultz, G. 1989. Purine and pyrimidine nucleotides potentiate activation of NADPH oxidase and degranulation by chemotactic peptides and induce aggregation of human neutrophils via G proteins. Eur. J. Biochem. 181:277–285
Smith, C.D., Uhing, R.J., Snyderman, R. 1987. Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters. J. Biol. Chem. 262:6121–6127
Suzuki, H. 1985. Electrical responses of smooth muscle cells of the rabbit ear artery to adenosine triphosphate. J. Physiol. 359:401–415
Urquilla, P.R. 1978. Prolonged contraction of isolated human and canine cerebral arteries induced by uridine 5′-triphosphate. Stroke 9:133–136
von Kugelgen, I.V., Haussinger, D., Starke, K. 1987. Evidence for a vasoconstriction-mediating receptor for UTP, distinct from the P2 purinoceptor, in rabbit ear artery. Naunyn-Schmiedeberg's Arch. Pharmacol. 336:556–560.
von Kugelgen, I.V., Starke, K. 1990. Evidence for two separate vasoconstriction-mediating nucleotide receptors, both distinct from the P2x-receptor, in rabbit basilar artery: a receptor for pyrimidine nucleotides and a receptor for purine nucleotides. Naunyn-Schmiedeberg's Arch. Pharmacol. 341:538–546
Wakui, M., Osipchuk-Y. V., Petersen, O.K. 1990. Receptor-activated cytoplasmic Ca2+ spiking mediated by inositol trisphosphate is due to Ca2+-induced Ca2+ release. Cell 63: 1025–1032
Williams, D.L., Katz, G.M., Roy-Contancin, L., Reuben, J.P. 1988. Guanosine 5′-monophosphate modulates gating of high-conductance Ca2+-activated K+ channels in vascular smooth muscle cells. Proc, Natl. Acad. Sci. USA 85:9360–9364
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Sanchez-Fernandez, M., Katz, G.M., Suarez-Kurtz, G. et al. Mobilization of intracellular calcium in cultured vascular smooth muscle cells by uridine triphosphate and the calcium ionophore A23187. J. Membarin Biol. 135, 273–287 (1993). https://doi.org/10.1007/BF00211099
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DOI: https://doi.org/10.1007/BF00211099