Notably, avoidance of single-cell isolation means that enzymatic treatment is minimised and cells can be studied within their normal environment of the blood vessel wall. The method is readily applied and has several advantages over previous methods for the study of ion channels in smooth muscle cells. Cell-attached patch, inside-out patch and outside-out patch recordings were made readily and K+ channel unitary currents were studied. To achieve this, small glass capillaries are heated and pulled to fabricate glass micropipettes with an aperture size of 12 m. Injection of depolarising current or bath application of 10 mM Ba2+ induced constriction of the entire arteriolar segment. 2.1 Conventional Patch Clamping Technique In patch clamping a patch of membrane is isolated from the external solution to record the currents flowing into the patch. Short arteriolar segments could be voltage-clamped. The membrane potential recorded using amphotericin-B-containing patch pipettes averaged -72 mV. Patch-clamp gigaOhm seals were made regularly on smooth muscle cells embedded in arterioles. Arterioles excluded trypan blue, constricted in response to 60 mM K+ and dilated in response to levcromakalim. Vessels were identified under a microscope and the majority were small arterioles with a mean external diameter, in Ca2+-containing (1.5 mM) solution, of 29 microm and variable lengths of 100 microm or more. Small blood vessels were isolated from rabbit brain using an enzymatic and mechanical procedure. The aim of this project was to develop a method to enable routine application of all patch-clamp configurations to smooth muscle cells while they remain embedded in blood vessels.
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