A method for direct patch-clamp recording from smooth muscle cells embedded in functional brain microvessels.

Abstract

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. Small blood vessels were isolated from rabbit brain using an enzymatic and mechanical procedure. 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. Arterioles excluded trypan blue, constricted in response to 60 mM K+ and dilated in response to levcromakalim. Patch-clamp gigaOhm seals were made regularly on smooth muscle cells embedded in arterioles. The membrane potential recorded using amphotericin-B-containing patch pipettes averaged -72 mV. Short arteriolar segments could be voltage-clamped. Injection of depolarising current or bath application of 10 mM Ba2+ induced constriction of the entire arteriolar segment. Cell-attached patch, inside-out patch and outside-out patch recordings were made readily and K+ channel unitary currents were studied. The method is readily applied and has several advantages over previous methods for the study of ion channels in smooth muscle cells. 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.