The goal of the present study was to determine if voltage-sensitive calcium channels are present in bovine aortic endothelial cell plasmalemma and if they contribute to the rise in cytosolic calcium produced by bradykinin. After bradykinin (100 nM) exposure, endothelial cell associated fura-2 fluorescence peaked within 10-20 seconds and then declined to a steady level 2- to 3-fold above resting values. Pretreatment with lanthanum (20 microM) abolished the steady level produced by bradykinin but had little effect on the initial, transient rise in cytosolic calcium. Chelation of extracellular calcium with EGTA before addition of bradykinin resulted in a substantial decrease in the fura-2 transient and elimination of the long-lasting component. Nimodipine (3 microM) and nitrendipine (1 microM) were without effect on either phase of the bradykinin-induced response. Moreover, elevation of extracellular potassium failed to produce a rise in intracellular calcium. With the use of the tight seal technique to voltage clamp the cells, inwardly rectifying and calcium-activated potassium currents were found to exist in the endothelial cells. Addition of bradykinin (100 nM) elicited a calcium-activated potassium current that was eliminated in the absence of intracellular potassium. No voltage-sensitive calcium currents were activated when the cells were exposed to 10 mM or 110 mM calcium chloride in the presence or absence of bradykinin. The binding of [3H](+)PN200-110 to endothelial cell membrane preparations was 1-3 orders of magnitude lower than that observed in PC-12, GH3, or BC3H1 cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARY1. The role of action potentials in adrenaline secretion was investigated in the rat adrenal medulla. The effects of various treatments on adrenaline secretion from the perfused adrenal medulla were compared with the effects of similar treatments on spike frequency in dissociated adrenal chromaffin cells.2. KCl concentrations between 10 and 20 mm increased the extracellularly recorded spike frequency of dissociated adrenal chromaffin cells. Upon perfusion by a KCl concentration of 30 mM there was an initial brief burst of spikes followed by a period of inactivity in the continued presence of 30 mM-KCl. Tetrodotoxin (TTX, 6/M) decreased the amplitude and frequency of the KCl evoked spikes.3. The rate of adrenaline secretion from the isolated perfused rat adrenal gland increased as the KCl concentration was raised to 10 and up to 120 mm. Secretion which was evoked by KCl concentrations between 10 and 20 mm was partially inhibited by TTX. At KCl concentrations of 30 mm or greater evoked secretion was no longer affected by TTX.4. CoCl2 (5 mM) blocked KCl increase of spike frequency and also blocked stimulation of adrenaline secretion by all concentrations of KCl tested.5. Tetraethylammonium chloride (10 mM), which decreased spike frequency but greatly prolonged the spike duration, enhanced secretion induced by 15 mM-KCl.6. The results are consistent with the following interpretation. The TTX insensitive portion of the KCl stimulated adrenaline secretion is due to Ca influx through voltage dependent Ca channels which are open as a consequence of the steady-state level of KCl depolarization. The TTX sensitive portion of secretion is indicative of an extra increment of Ca influx during spike activity enhanced by KCl. This increment of Ca influx may occur through voltage dependent Ca channels whose activation is facilitated by the voltage changes caused during the TTX sensitive Na component of the spike and possibly through the Na channel itself.7. Stimulation of secretion by acetylcholine (ACh) in the perfused adrenal medulla was half maximal at 15 /LM and began to saturate around

SUMMARY1. The pharmacological sensitivities and physiological contributions of two types of Ca2+-activated K+ channels (BK and SK) in GH3 cells were examined by the outside-out, whole-cell and cell-attached modes of the patch-clamp technique.2. BK channels (250-300 pS in symmetrical 150 mM-K') in outside-out patches were blocked by external tetraethylammonium (TEA) and by 50 nm-charybdotoxin (CTX), but were not blocked by apamin.3. SK channels (9-14 pS in symmetrical 150 mM-K') in outside-out patches were blocked by external TEA and by apamin, but were not blocked by 50 nM-CTX.4. The dissociation constant (Kd) for TEA block of SK channels (31 +0-37 mM)was 12-fold greater than the Kd for the BK channels (260 + 21 gM). The TEA blockade of both channels was not strongly voltage dependent; for both channels the TEA binding site sensed less than 20 % of the membrane electric field. 5. Application of blockers of the BK channels (1 mM-TEA and 50 nM-CTX) to whole cells under current clamp prolonged action potential duration; whereas application of apamin, a selective blocker of the SK channel, inhibited a slowly decaying after-hyperpolarization and had little effect on action potential duration. Apamin also increased the firing rate in 30 % of the spontaneously pacing cells.6. It is suggested that BK channels contribute to action potential repolarization; whereas SK channels contribute to the regulation of action potential firing rate.

Bradykinin (BK)-stimulated release of endothelium-derived relaxing factor has been linked to a rise in cytosolic Ca2+ concentration and a change of K+ permeability of the endothelial cell. In the present study, measurement of BK-induced changes in fura-2 fluorescence and 86Rb+ efflux were used to monitor changes in cytosolic Ca2+ and K+ permeability in cultured bovine aortic endothelial cells. In the presence of normal extracellular Ca2+, BK induced a fourfold increase in cytosolic Ca2+, which peaked at 20 s and declined within 1 min to a value that was 50% of the peak level. Subsequently, cytosolic Ca2+ decreased and approached basal levels within 8 min. In the absence of Ca2+, BK produced a 1.5- to 2-fold increase in cytosolic Ca2+ that peaked within 20 s and declined to basal levels within 2 min. Addition of Ca2+ to the Ca-free reaction buffer 3-5 min after addition of BK resulted in a two-to threefold increase in cytosolic Ca2+ that declined slowly back to basal levels. Thus Ca2+ influx can occur in response to BK at a time when there is minimal elevation of cytosolic Ca2+ above the resting level. Under all conditions tested, 86Rb+ efflux paralleled changes in the cytosolic Ca2+, suggesting that efflux occurred through Ca2+-activated K+ channels. Isosmotic substitution of Na+ with N-methyl-D-glucamine did not affect the BK-stimulated changes in cytosolic Ca2+ or 86Rb+ efflux, suggesting that Na+-Ca2+ exchange plays little role in the BK response. These results suggest that BK stimulates Ca2+ influx via a BK receptor-operated channel or a channel activated by some internal messenger other than Ca2+.

Single Ca2+-activated K+ channels were studied in membrane patches from the GH3 anterior pituitary cell line. In excised inside-out patches exposed to symmetrical 150 mM KCl, two channel types with conductances in the ranges of 250-300 pS and 9-14 pS were routinely observed. The activity of the large conductance channel is enhanced by internal Ca2+ and by depolarization of the patch membrane. This channel contributes to the repolarization of Ca2+ action potentials but has a Ca2+ sensitivity at-50 mV that is too low for it to contribute to the resting membrane conductance. The small conductance channel is activated by much lower concentrations of Ca2+ at -50 mV, and its open probability is not strongly voltage sensitive. In cell-attached patches from voltage-clamped cells, the small conductance channels were found to be active during slowly decaying Ca2+-activated K+ tails currents and during Ca2+-activated K+ currents stimulated by thyrotropin-releasing hormone induced elevations of cytosolic calcium. In cell-attached patches on unclamped cells, the small conductance channels were also active at negative membrane potentials when the frequency of spontaneously firing action potentials was high or during the slow afterhyperpolarization following single spontaneous action potentials of slightly prolonged duration. The small conductance channel may thus contribute to the regulation of membrane excitability.