We evaluated the acute effect of
homocysteine on the
iberiotoxin-sensitive, Ca(2+)-activated K(+) (BK(Ca)) channels of the porcine coronary artery smooth muscle cells.
NS 1619 (1 to 30 microM) caused a concentration-dependent enhancement of the BK(Ca) amplitude (recorded using the whole-cell, membrane-
rupture configuration) only with an elevated [Ca(2+)](i) of approximately 444 nM, but not with [Ca(2+)](i) of approximately 100 nM.
Homocysteine (30 microM) caused a small inhibition ( approximately 16%) of the BK(Ca) amplitude ([Ca(2+)](i)= approximately 444 nM), and a greater inhibition ( approximately 77%) was observed with 100 microM
NADH present in the pipette
solution. The inhibition persisted after washing. With
NADPH (100 microM), a smaller magnitude of inhibition ( approximately 34%) of the BK(Ca) amplitude was recorded. The NS 1619-mediated enhancement of the BK(Ca) amplitude (with elevated [Ca(2+)](i) plus
NADH in the pipette) was attenuated by
homocysteine. The
homocysteine-mediated inhibition of the BK(Ca) amplitude was suppressed by
Tiron (10 mM) or
diphenylene iodonium (30 nM), applied alone, but not by
superoxide dismutase (500 U/ml) and
catalase (500 U/ml). Generation of
superoxide (O(2)(-)) of the smooth muscle cells (with
NADH presence), measured using the
lucigenin-enhanced chemiluminescence, was markedly increased by
angiotensin II (100 nM) and
homocysteine (30 microM). The chemiluminescence signal was sensitive to
apocynin (300 microM) or
Tiron, applied alone, but not to
superoxide dismutase and
catalase. In conclusion, our results demonstrate that acute
homocysteine application inhibits the
iberiotoxin-sensitive BK(Ca) channels (with elevated [Ca(2+)](i) and
NADH present) which is probably caused by the
NADH oxidase activation and the concomitant generation of intracellular
superoxide.