Modulation by extracellular pH of bradykinin-evoked activation of Ca(2+)-activated K+ channels in endothelial cells

Abstract

We used the patch-clamp technique to investigate, via the activation of Ca(2+)-activated potassium channels [K(Ca2+)channels], the effects of extracellular pH (pHo) on the bradykinin (BK)-stimulated rise in cytosolic Ca2+ concentration in bovine aortic endothelial cells (BAE). In cell-attached experiments, the external application of BK caused a transient activation of the K(Ca2+) channels. Increasing pHo from 7.3 to 9 maintained the channel activity, which was not inhibited by withdrawing the agonist. The channel-activation process could be blocked either by removing external Ca2+ or by depolarizing the cells with a high-K+ external solution. These results indicate that the Ca2+ influx triggered by BK contributes in maintaining the agonist-evoked response in high pHo. Changes in pHo produced a slight increase in the intracellular pH (pHi) measured fluorimetrically with the H+ indicator dye 2',7-bis(carboxyethyl)5(6')-carboxyfluorescein. However, increasing pHi by the external application of NH4Cl at physiological pHo caused a rapid decline and not an increase in the K(Ca2+) channel activity triggered by BK. In fura-2-loaded cells, alkaline pHo had no effect on the time course of the Ca2+ response to BK in external Ca(2+)-free conditions, suggesting that the Ca2+ extrusion process is not affected by pHo. Our results suggest that the BK-evoked Ca2+ influx, which is required to reload internal Ca2+ stores, is controlled by a mechanism depending on extracellular H+.

Ion Channels in Vascular Endothelium

“…They show Ca 2+ -and voltage-dependent activation and are blocked by TEA, charybdotoxin, d-tubocurarine (8,30,147), and by extracellular alkalinization (170). The NO-releaser LP-805 (72) potentiates outward Ca 2+ -dependent K + currents in bovine pulmonary artery ECs, but it is not clear whether this effect is direct or mediated by changes in [Ca 2+ ] i .…”

Molecular Responses of Endothelial Tissue to Kinins

“…Intracellular alkalinization, however, is strictly dependent on the presence of extracellular sodium and sensitive to inhibition by amiloride analogs (46,47); as such, it can be attributed to activation of the Na + -H + exchanger. Experimental evidence has suggested that exchanger activation is linked to both the release of Ca 2+ from intracellular stores and the agonist-induced transmembranous Ca 2+ influx (48). Indeed, a number of studies have linked changes in pH t with alterations in [Ca 2+ ]i.…”

“…For example, maintained intracellular alkalinization induced by either NH 4 + or methylamine has been reported to empty intracellular Ca 2+ stores without stimulating IP 3 formation (42). Although, in the absence of an agonist, intracellular alkalinization has not been associated with a significant increase in K + channel activity, an increased endothelial pHj has been shown to enhance bradykinin-induced activation of Kc a 2+ channels and prolong the transmembranous Ca 2+ influx (42,48). These data indicate that the agonist-evoked Ca 2+ entry pathway responsible for the refilling of intracellular Ca 2+ stores may be modulated by changes in pHj.…”

Divergent effects of extracellular and intracellular alkalosis on Ca2+ entry pathways in vascular endothelial cells

“…Up to now, only a few studies have addressed the question of how Ca# + entry into vascular endothelial cells is promoted during extracellular alkalosis. Alkalosis (pH o 8.5) was reported to augment Ca# + influx into store-depleted endothelial cells [19,20]. Since Ca# + influx into activated cells generally comprises the background leak Ca# + influx and the Ca# + influx occurring via activated Ca# + channels, the relative contribution of pH modulation of either component to the overall effect is still unclear.…”