REGULATION OF GLIBENCLAMIDE-SENSITIVE K+ CURRENT BY NUCLEOTIDE PHOSPHATES IN ISOLATED RABBIT PULMONARY MYOCYTES

Objective: Although the existence of ATP-sensitive K+ (K-ATP) channels in vascular muscle is widely accepted, there appears to be little con sensus as to what the primary regulator of these channels is under phy siological or pathophysiological conditions. Recent evidence has sugge sted that nucleotide diphosphates (NDPs) may play a more important rol e than ATP. However, since the properties of vascular K-ATP channels a re quite diverse, and the effects of these nucleotides are poorly unde rstood, the aim of this study was to test the hypothesis that intracel lular ATP can regulate whole-cell K-ATP current (I-K,I-ATP) in the abs ence of NDPs. Methods: Single cells were isolated from rabbit main pul monary artery by enzymatic treatment with papain. Whole-cell patch cla mp experiments were performed in cells dialysed with different nucleot ides. The effect of the K-ATP channel activator, levcromakalim (10 mu M), was investigated at a holding potential of -60 mV. The contributio n of I-K,I-ATP to the holding current was defined as the current which was blocked by glibenclamide following washout of levcromakalim. Resu lts: Lowering the intracellular ATP concentration ([ATP](i)) from 1 to 0.1 mM, in the presence or absence of GTP, enhanced the levcromakalim -induced current (I-kev) by similar to 2.5 fold and increased a gliben clamide-sensitive background K+ current (I-glib). However, I-glib, was larger with GTP and the total glibenclamide-sensitive current (I-lev + I-glib) increased with time. Significant activation of I-glib failed to occur when the pipette contained no nucleotides and the responses to levcromakalim were generally much smaller than seen with ATP. GDP ( 0.5 mM), in the absence of pipette ATP, activated a large background K + current which had similar properties to I-lev. Consistent with this was the observation that I-lev became substantially reduced in the pre sence of GDP, presumably because a significant amount of I-K,I-ATP was already activated. Conclusions: The response to levcromakalim in isol ated cells from pulmonary artery was, as expected for an agent activat ing K-ATP channels, modulated by changes in the pipette [ATP]. This ef fect was not dependent on the presence of other pipette nucleotides, a lthough the possibility cannot be excluded that metabolites from the c ellular breakdown of ATP are essential for normal channel regulation. GDP could also activate I-K,I-ATP under conditions where the channel i s probably in a low phosphorylation state. The time-dependent effects of GTP require further work to determine the precise mechanism, but ma y suggest that GTP and/or G-proteins are involved in the regulation of K-ATP channels.