Functional and molecular expression of volume‐regulated chloride channels in canine vascular smooth muscle cells

Membrane stretch or increases in transmural pressure cause contraction of vascular smooth muscle cells, a response referred to as myogenic tone (Meininger & Davis, 1992). This response to mechanical force is accompanied by changes in the membrane conductance, the cytoskeleton and the activation or inactivation of a variety of second messenger systems (Osol, 1995). Early studies (Sparks, 1964; Uchida & Bohr, 1969; Harder, 1984) revealed that the myogenic response is associated with membrane depolarization and exhibits some dependence on extracellular Ca2+. Although modulation of large-conductance Ca2+-activated K+ channels (Brayden & Nelson, 1992) and stretch-activated non-selective cation channels (Davis, Donovitz & Hood, 1992) has been implicated in the myogenic response, the exact mechanisms responsible for the initial myogenic membrane depolarization are unknown. It has recently been shown that pressure-induced depolarization and contraction of cerebral artery smooth muscle is inhibited by various Cl− channel blockers, suggesting that activation of Cl− channels may be responsible for myogenic membrane depolarization of these arteries (Nelson, Conway, Knot & Brayden, 1997). Although Ca2+-activated Cl− channels are known to be ubiquitous in smooth muscle (Large & Wang, 1996), the Cl− channels involved in the myogenic response were insensitive to niflumic acid, an effective blocker of Ca2+-activated Cl− channels, leading to speculation that these Cl− channels may be activated by membrane stretch, like those involved in cell volume regulation in many other tissues (Strange, Emma & Jackson, 1996). However, there is presently no direct evidence showing that volume-regulated Cl− channels are functionally or molecularly expressed in vascular smooth muscle. In other tissues, these channels are known to possess many common biophysical and pharmacological properties, including activation by hypotonic solutions, outward rectification in symmetrical Cl− solutions, anion selectivity sequence of SCN− > I− > Br− > Cl− > F−, and sensitivity to block by hypertonic solutions, stilbene compounds, external nucleotides and the antioestrogen compound tamoxifen (Strange et al. 1996; Okada, 1997). Although three different proteins, P-glycoprotein (P-Gp; Valverde, Diaz, Sepulveda, Gill, Hyde & Higgins, 1992), pIcln (Paulmichl, Li, Wickman, Ackerman, Peralta & Clapham, 1992) and ClC-2 (Grunder, Thiemann, Pusch & Jentsch, 1992), have been proposed as molecular candidates for volume-sensitive Cl− channels, recent data suggest that P-Gp and pIcln may instead be regulators of endogenous Cl− channels, and the properties of expressed ClC-2 channels fail to exhibit many of the characteristic properties of volume-sensitive Cl− currents (ICl,vol) found in native cells (Okada, 1997). We have recently identified another member of the ClC Cl− channel family, ClC-3, as the gene encoding ICl,vol in heart and many other mammalian cells (Duan, Winter, Cowley, Hume & Horowitz, 1997). We therefore tested for functional expression of ICl,vol in smooth muscle cells isolated from canine pulmonary and renal arteries using conventional patch-clamp techniques, and performed quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to examine molecular expression of ClC-3.