Na⁺/Ca²⁺ (NCX) EXCHANGE IN MYOGENICALLY ACTIVE ARTERIOLES

Hema Raina, Kerryn Popa, Simon Potocnik, Neela Kotecha, Alex Wiedenmann, Clemens Schneeweiss, Michael A. Hill, Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora 3083.

Arteriolar myogenic vasoconstriction is associated with smooth muscle membrane depolarisation and opening of L-type voltage-gated Ca²⁺ channels. These events may be linked by the action of non-selective cation channels which would likely passage Na⁺ under physiological conditions. In addition to modulating Ca²⁺ levels, this may activate Na⁺ extrusion mechanisms. To determine a possible role for NCX in myogenically active isolated (rat) skeletal muscle arterioles , studies were conducted at several levels. Functional significance was determined by manipulation of extracellular Na⁺ levels and inhibition of the exchanger using the relatively selective inhibitor KB-R7943. Changes in diameter, intracellular Ca²⁺ and membrane potential were measured in response to these manipulations. Identification of the protein was accomplished by western blotting using antibodies raised against the NCX1 isoform. Total RNA was extracted from arterioles and real time PCR, using appropriate primers and sybr-green fluorescence, was performed to demonstrate levels of expression of mRNA for the NCX 1, 2 and 3 isoforms relative to the expression of GAPDH. mRNA samples extracted from brain, liver and smooth muscle cells were used for comparison. Reduction of extracellular Na⁺ (137, 100 and 25 mM) caused significant steady-state vasoconstriction and an increase in Ca²⁺_i. Inhibition of NCX (30µM KB-R7943) in arterioles at physiological levels of extracellular Na⁺ (137 mM) caused membrane hyperpolarization and vasodilatation. Western blotting showed the presence of a protein of approximately 120 kD consistent with studies in other tissues. PCR showed that arterioles expressed mRNA for all three isoforms with relative expression being NCX1 > NCX2 > NCX3. The data are consistent with a functional role for the NCX in arterioles, however, complexity may exist due to the expression of multiple isoforms.