[Ca 2+ ] i Inhibition of K + Channels in Canine Pulmonary Artery

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Abstract
Experiments were performed on smooth muscle cells isolated from canine pulmonary artery to identify the type of K+ channel modulated by hypoxia and examine the possible role of [Ca2+]i in hypoxic K+ channel inhibition. Whole-cell patch-clamp experiments revealed that hypoxia (induced by the O2 scavenger, sodium dithionite) reduced macroscopic K+ currents, an effect that could be prevented by strong intracellular buffering of [Ca2+]i. The inhibitory effects of hypoxia were mimicked by acute exposure of cells to caffeine and could be prevented by caffeine pretreatment, suggesting an important obligatory role of [Ca2+]i in hypoxic inhibition of K+ currents. Exposure of cells to low concentrations of 4-aminopyridine (4-AP, 1 mmol/L) prevented hypoxic inhibition of macroscopic K+ currents, whereas low concentrations of tetraethylammonium were without effect, suggesting that the target K+ channel inhibited by hypoxia is a voltage-dependent delayed rectifier K+ channel, which is inhibited by [Ca2+]i. Hypoxia failed to consistently modify the activity of large-conductance (118 picosiemens [pS] in physiological K+) Ca2+-activated K+ channels in inside-out membrane patches but reduced open probability of smaller-conductance (25-pS) delayed rectifier K+ channels in cell-attached membrane patches. In inside-out membrane patches, 1 μmol/L Ca2+ added to the cytoplasmic surface significantly reduced open probability of small-conductance (25-pS) 4-AP–sensitive delayed rectifier K+ channels. Whole-cell current measurements using symmetrical K+ to increase driving force for small currents active near the cell’s resting membrane potential revealed the presence of a 4-AP–sensitive K+ current that activated near −65 mV and was inhibited by hypoxia. Simultaneous measurements of changes in [Ca2+]i, using the Ca2+ indicator indo 1, and membrane potential revealed that hypoxia causes an initial rise of [Ca2+]i, which precedes hypoxia-induced membrane depolarization. It is concluded that in canine pulmonary arterial cells an early key event in hypoxic pulmonary vasoconstriction is release of Ca2+ from caffeine-sensitive intracellular Ca2+ stores, which causes inhibition of delayed rectifier K+ channels and membrane depolarization, possibly leading to subsequent activation of Ca2+ entry through voltage-dependent Ca2+ channels.