Functional regulation of K‐Cl cotransport (COT) by the nitric oxide (NO) pathway, vasoconstrictors (VCs) and inhibitors of the contractile apparatus (ICA) in vascular smooth muscle cells (VSMCs)

VSMC K‐Cl COT (KCC), the electroneutral coupled movement of K and Cl ions is mediated by the KCC1,3&4 isoforms and regulated by the NO pathway at the mRNA level. Here, we tested whether the NO pathway, VCs and ICA regulate K‐Cl COT activity. K‐Cl COT was measured as the Cl‐dependent Rb influx in Cl and sulfamate (Cl replacement) in the presence or absence of 0.1&1.0 mM 8‐Br‐cGMP and 1 mM sodium nitroprusside (SNP), and with the inhibitors LY83583 (20 μM) and KT5823 (1 μM) and the NO donors NOC‐18, NOC‐5 and NOR‐3 (0.1&1.0 mM). The effect of VCs and ICA was tested with SNP (0.05–1.0 mM), 10 μM prostaglandine (PG) F 2α ,, 50 μM trifluoperazine (TFP), an inhibitor of calmodulin, 100 μM ML‐9, a selective inhibitor of myosin light chain (MLC) kinase 10 μM norepinephrine (NE) and 75 μM U46619, (increases MLC phosphorylation in VSMCs). Our main findings are: 8‐Br‐cGMP and SNP increased K‐Cl COT by more than twofold, whereas LY83583 and KT5823 reversed the SNP‐activation to control levels or below. All NO donors significantly activated K‐Cl COT. SNP activated in a dose‐dependent manner and PGF 2α, completely inhibited K‐Cl COT, whereas in cells pre‐treated withPGF 2α, 1 mM SNP activated even further. In addition, TFP and ML‐9 significantly stimulated whereas NE and U46610 inhibited K‐Cl COT. In addition, U46619 + 0.5 mM SNP reduced by 50% the inhibition observed upon treatment with U46619 alone. Results indicate that in addition to regulating mRNA expression, the NO pathway; VCs and ICA regulate the activity of K‐Cl COT in VSMCs (Supported by NAHA).