K Influx Parameters in Rat Aortic Vascular Smooth Muscle Cells (RASMCs)

Abstract

Background RASMCs serve as major experimental tool in cardiovascular research. Here we studied further the effect of cell culture passage on K influxes with Rb as congener. Methods Rb uptake was measured by atomic absorption spectrophotometry in RASMCs ± inhibitors of the Na/K pump (ouabain, O), Na-K-2Cl cotransport (NKCC, bumetanide, B), and K-Cl cotransport (KCC, Cl replacement by sulfamate). Rb influx was calculated in nmol/mg protein × time, with O inhibition maximized for NKCC and KCC measurements. RASMC identity was verified immunologically. Results Positive IF anti-αactin staining. Rb uptake nonlinear with time in the absence (total, T) and presence of 1 mM O, and both O and 10 μM B. Rb flux by NKCC {O-insensitive (OIS) – [O+B-insensitive, OBIS]) = > 30% than that by the Na/K pump (T- OIS). Presence of O during the flux alone or flux + preincubation inhibited the Na/K pump, IC50 = 0.14 or 0.17 mM and 45% and 60% of T at 2 mM O, respectively. At 2 mM O, B inhibited 80% of OIS Rb influx with an IC50 = 0.1 μM. OBIS KCC activity (53%) saturated with increasing Cl mol fractions, remainder in 100% sulfamate due to channel Rb influx. KCC Vmax = ~ 1 and 2 nmol/(mg protein × min), and Km = 89 and 59 mM [Cl]o, with Hill n=1, in old vs young RASMCs passages, respectively. Conclusions Na/K pump, NKCC and KCC measurement required inhibitor presence in both preincubation + flux. Apparent Na/K pump O affinity was consistent with the low rodent α1Na/K ATPase isoform O affinity and NKCC B affinity s close to that for renal NKCC2. KCC parameters, except Hill n, were age-dependent, consistent with KCC1, 3 & 4 isoforms present in RASMCs.