Abstract
Objective:The current study was undertaken to localize and identify angiotensin II (Ang II) receptor subtypes in the microcirculation of striated muscle. Methods:Cremaster muscles from 7- to 8-week-old Sprague-Dawley rats were excised, placed in a dissection solution maintained at 4 °C, and 3 branch orders of arterioles and venules, as well as capillaries and a muscle specimen, were microdissected under a stereomicroscope. Reverse transcription polymerase chain reaction (RT-PCR) methods were developed for purification and amplification of extremely small amounts of RNA (<5 ng/µL) from whole 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 AT1a, AT1b, or AT2receptor primers. The products were electrophoretically size-fractionated on an agarose gel, stained with ethidium bromide to visualize DNA bands, and analyzed to determine the presence or absence of AT receptor subtypes. Protein expression was confirmed by Western blotting pooled samples with specific antiserum. Results:AT1aand AT2receptors were found in nearly all orders of both arterioles and venules, as well as the skeletal muscle biopsies. AT1breceptors, if present, were only observed on a few instances in the arterioles. Furthermore, PCR reactions specifically primed for skeletal muscle cell (MHC2B) and endothelial cell (eNOS) specific proteins demonstrated that there was no crosscontamination between the vessels and the skeletal muscle biopsies. Conclusions:This study describes a unique method for the isolation and preparation of microvessels and provides the first data directly demonstrating the presence of AT1and AT2receptors in microvessels as well as in skeletal muscle fibers. Microcirculation (2001) 8, 275–281.