K channel activation by nucleotide diphosphates and its inhibition by glibenclamide in vascular smooth muscle cells
Department of Pharmacology and Clinical Pharmacology, St. George's Hospital Medical School, London SW17 0RE
- 1Whole-cell and inside-out patch recordings were made from single smooth muscle cells that had been isolated enzymatically and mechanically from the rabbit portal vein.
- 2In whole-cells the inclusion in the recording pipette solution of nucleotide diphosphates (NDPs), but not tri- or monophosphates, induced a K-current that developed gradually over 5 to 15 min. Intracellular 1 mm guanosine 5′-diphosphate (GDP) induced a slowly developing outward K-current at − 37 mV that reached a maximum on average of 72 ± 4 pA (n = 40). Half maximal effect was estimated to occur with about 0.2 mm GDP. Except for ADP, other NDPs had comparable effects. At 0.1 mm, ADP was equivalent to GDP but at higher concentration ADP was less effective. ADP induced its maximum effect at 1 mm but had almost no effect at 10 mm.
- 3In 14% of inside-out patches exposed to 1 mm GDP at the intracellular surface, characteristic K channel activity was observed which showed long (> 1 s) bursts of openings separated by longer closed periods. The current-voltage relationship for the channel was linear in a 60 mm:130 mm K-gradient and the unitary conductance was 24 pS.
- 4Glibenclamide applied via the extracellular solution was found to be a potent inhibitor of GDP-induced K-current (IK(GDP)) in the whole-cell. The Kd was 25 nm and the inhibition was fully reversible on wash-out.
- 5(IK(GDP)) was not evoked if Mg ions were absent from the pipette solution. In contrast the omission of extracellular Mg ions had no effect on outward or inward (IK(GDP)).
- 6Inclusion of 1 mm ATP in the recording pipette solution reduced (IK(GDP)) and also attenuated its decline during long (25 min) recordings.
- 7When perforated-patch whole-cell recording was used, metabolic poisoning with cyanide and 2-deoxy-d-glucose induced a glibenclamide-sensitive K-current. This current was not observed when conventional whole-cell recording was used. Possible reasons for this difference are discussed.
- 8These K channels appear similar to ATP-sensitive K channels but we refer to them as nucleotide diphosphate-dependent K channels (KNDP) to emphasise what seems to be a primary role for nucleotide diphosphates in their regulation.