title: Ca(2+)-dependent non-selective cation and potassium channels activated by bradykinin in pig coronary artery endothelial cells.
abstract: 1. Using the cell-attached and inside-out modes of the patch-clamp technique, we studied the Ca(2+)-dependent ionic channels activated by bradykinin in cultured pig coronary artery endothelial cells to further understand electrophysiological events underlying cellular activation. 2. In the cell-attached mode, bradykinin (94 nM) activated two types of Ca(2+)-dependent channels: a high conductance K+ channel (285 pS in high symmetrical K+), whose open state probability was increased by depolarization, and a lower conductance inwardly rectifying non-selective cation channel (44 pS in high symmetrical K+). 3. The 285 pS K+ channel was half-maximally activated by cytosolic Ca2+ levels of 1.6 and 4.5 microM at +10 and -30 mV, respectively. Such local concentrations should be reached in the presence of bradykinin, which induces a mean maximal cytosolic Ca2+ rise of 1.3 microM. 4. The 285 pS K+ channel was inhibited by d-tubocurarine, which acted by reducing the mean open time duration (flickering pattern), finally reducing the channel conductance. 5. Divalent cations such as Ca2+ could flow through the 44 pS non-selective cation channel, with nearly the same permeability (P) as monovalent cations (PK: PNa: PCa = 1:1:0.7). 6. The cation channel appeared to be more sensitive to Ca2+ than the K+ channel, with a half-maximal open probability induced by 0.7 microM Ca2+ on the intracellular side of the membrane. 7. In contrast to the K+ channel, the cation channel mean open time was clearly increased by bradykinin. This effect was delayed compared with the increase in the channel open state probability and was rapidly lost in the inside-out configuration. Caffeine also activated the cation channel but more transiently than bradykinin and without any effect on the open duration. 8. In the absence of extracellular Ca2+, the bradykinin-induced increase in cytosolic free Ca2+ was shortened temporally by 52% and reduced in amplitude by 88%, whereas the bradykinin-induced hyperpolarization was not significantly reduced in amplitude but was shortened by 70%, thus illustrating the major role of Ca2+ influx in endothelial cell activation by bradykinin. 9. We conclude that bradykinin activates two types of Ca(2+)-dependent channels in coronary endothelial cells: a high conductance K+ channel regulated by membrane potential, and an inwardly rectifying cation channel allowing Ca2+ entry, the cation channel being about 6 times more sensitive to Ca2+ than the K+ channel. The increase in cation channel open state probability involves an increase in open number, like the K+ channel, but also involves a rise in channel open duration. Ca2+ entry via cation channels could contribute to increase the cytoplasmic Ca2+ level, activate Ca(2+)-dependent K+ channels, thus triggering membrane hyperpolarization when the endothelial cell is stimulated by a vasoactive agonist such as bradykinin.
authors.affiliation: Department of Zoology and Animal Biology, Geneva 4, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Baron
authors.forename: M
authors.initials: M
authors.lastname: Frieden
authors.forename: F
authors.initials: F
authors.lastname: Chabaud
authors.forename: J L
authors.initials: JL
authors.lastname: Bény
mesh_terms: D000818:Animals; D001711:Biotransformation / Q000187:drug effects / Q000502:physiology; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology*; D002412:Cations / Q000378:metabolism; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D007473:Ion Channels / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D010539:Permeability / Q000187:drug effects; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D013552:Swine
chemical_list: D002412:Cations; D005456:Fluorescent Dyes; D007473:Ion Channels; D015221:Potassium Channels; D001920:Bradykinin; D002118:Calcium; D016257:Fura-2