Distribution of angiotensin II receptor expression in the microcirculationof striated muscle

Objective: The current study was undertaken to localize and identify angiot ensin II (Ana II) receptor subtypes in the microcirculation of striated mus cle. Methods: Cremaster muscles from 7- to 8-week-old Sprague-Dawley rats were e xcised, placed in a dissection solution maintained at 4 degreesC. and 3 bra nch orders (if arterioles and venules. as well as capillaries and a muscle specimen, were microdissected under a stereomicroscope. Reverse transcripti on polymerase chain reaction (RT-PCR) methods were developed for purificati on and amplification of extremely small amounts of RNA (<5 ng/<mu>L) from w hole tissue samples. RNA was isolated from each sample, reverse transcribed . and the cDNA products were amplified by polymerase chain reactions (PCR) specifically primed with either AT(1a). At-1b, or AT(2) receptor primers. T he products were electrophoretically size-fractionated on an a-arose gel, s tained with ethidium bromide to visualize DNA bands, and analyzed to determ ine the presence or absence of AT receptor subtypes, Protein expression was confirmed by Western blotting pooled samples with specific antiserum, Results: AT(1a) and AT(2) receptors were found in nearly all orders of both arterioles and venules, as well as the skeletal muscle biopsies. AT(1b) re ceptors. if present, were only observed on a few instances in the arteriole s. Furthermore. PCR reaction, specifically primed for skeletal muscle cell (MHC2B) and endothelial cell (eNOS) specific proteins demonstrated that the re was no cross-contamination between the vessels and the skeletal muscle b iopsies. Conclusions: This study describes a unique method for the isolation and pre paration of microvessels and provides, the first data directly demonstratin g the presence of AT(1) and AT(2) receptors in microvessels as well as in s keletal muscle fibers.