Functional characteristics of K‐Cl cotransport (COT) in vascular smooth muscle cells (VSMCs)

In VSMCs, K‐Cl COT (KCC), the electroneutral coupled movement of K and Cl ions is mediated by the KCC1,3&4 isoforms, is activated by vasodilators, and regulated by the nitric oxide pathway at the mRNA level (Adragna et al., Acta Physiol Scand 187 : –39, 2006). To further define the functional characteristics of KCl COT, we studied factors affecting K‐Cl COT activity such as culture protocol, passage and days in culture, flux protocol and its response to the loop diuretic bumetanide, a selective inhibitor of Na‐K‐Cl COT known to inhibit K‐Cl COT at higher doses in several tissues. K‐Cl COT was measured as the Cl‐dependent Rb influx in Cl and Cl‐free media (Br, NO 3 , I, SCN and sulfamate (Sf) as Cl replacement) as a function of time and in the presence or absence of bumetanide (0–1 mM), and with or without serum starvation. Our main findings are: The anion selectivity for Rb influx decreased according to the sequence: Br=Cl>>NO 3 =I=SCN=Sf (p<0.001 with respect to Cl, n=15–18). K‐Cl COT in Sf increased as a function of protein (p<0.0001, n=26); and decreased as a function of passage number (p=0.02, n=22). The effect of bumetanide on K‐Cl COT was dependent on the flux protocol (presence or absence of serum up to two days prior to flux), anion replacement and presence or absence of Na (N‐methyl‐D‐glucamine replacement). Further complications arose when Sf was used as Cl replacement since Rb influx in Sf was highly dependent on the presence or absence of GdCl 3 used to inhibit stretch‐activated K channels. Hence, experimental conditions play a critical role in the determination of K‐Cl COT and its response to bumetanide in VSMCs.