CA2-DEPENDENT NONSELECTIVE CATION AND POTASSIUM CHANNELS ACTIVATED BYBRADYKININ IN PIG CORONARY-ARTERY ENDOTHELIAL-CELLS()

1. Using the cell-attached and inside-out modes of the patch-clamp tec hnique, we studied the Ca2+-dependent ionic channels activated by brad ykinin in cultured pig coronary artery endothelial cells to further un derstand electrophysiological events underlying cellular activation. 2 . In the cell-attached mode, bradykinin (94 nM) activated two types of Ca2+-dependent channels: a high conductance K+ channel (285 pS in hig h symmetrical K+), whose open state probability was increased by depol arization, and a lower conductance inwardly rectifying non-selective c ation 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 mu M at +10 and -30 mV, respectively. Such local concentrations should be reached in the presence of bradykinin, which induces a mean maxima l cytosolic Ca2+ rise of 1.3 mu M. 4. The 285 pS K+ channel was inhibi ted by d-tubocurarine, which acted by reducing the mean open time dura tion (flickering pattern), finally reducing the channel conductance. 5 . Divalent cations such as Ca2+ could flow through the 44 pS non-selec tive cation channel, with nearly the same permeability (P) as monovale nt cations (P-K:P-Na:P-Ca = 1:1:0.7). 6. The cation channel appeared t o be more sensitive to Ca2+ than the K+ channel, with a half-maximal o pen probability induced by 0.7 mu M Ca2+ on the intracellular side of tile membrane. 7. In contrast to the K+ channel, the cation channel me an open time was clearly increased by bradykinin. This effect was dela yed compared with the increase in the channel open state probability a nd was rapidly lost in the inside-out configuration. Caffeine also act ivated the cation channel but more transiently than bradykinin and wit hout any effect on the open duration. 8. In the absence of extracellul ar Ca2+, the bradykinin-induced increase in cytosolic free Ca2+ was sh ortened 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 con clude that bradykinin activates two types of Ca2+-dependent channels i n coronary endothelial cells: a high conductance K+ channel regulated by membrane potential, and an inwardly rectifying cation channel allow ing Ca2+ entry the cation channel being about 6 times more sensitive t o 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 cati on channels could contribute to increase the cytoplasmic Ca2+ level, a ctivate Ca2+-dependent K+ channels, thus triggering membrane hyperpola rization when the endothelial cell is stimulated by a vasoactive agoni st such as bradykinin.