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K Influx Parameters in Rat Aortic Vascular Smooth Muscle Cells (RASMCs)
Author(s) -
Sharma Neelima,
Lauf Peter K,
Adragorma C
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.1115.4
Subject(s) - cotransporter , bumetanide , ouabain , potassium , chemistry , flux (metallurgy) , microbiology and biotechnology , sodium , biophysics , biology , organic chemistry
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, IC 50 = 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 IC 50 = 0.1 μM. OBIS KCC activity (53%) saturated with increasing Cl mol fractions, remainder in 100% sulfamate due to channel Rb influx. KCC V max = ~ 1 and 2 nmol/(mg protein × min), and K m = 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.