QUANTIFICATION OF STEADY-STATE EXPRESSION OF MESSENGER-RNA FOR ALPHA-1-ADRENERGIC RECEPTOR SUBTYPES USING REVERSE TRANSCRIPTION AND A COMPETITIVE POLYMERASE CHAIN-REACTION

Three distinct alpha-1 adrenergic receptor subtypes (alpha-1 alpha, al pha-1b and alpha-1d) have been identified through molecular cloning. E xpression of alpha-1 adrenergic receptor mRNA has been detected in sev eral tissues, but previous studies with Northern blot analysis or RNas e protection assays have provided only semiquantitative information. F urthermore, the mRNA distribution of these subtypes in many rat tissue s is unknown. In the present study, we quantified alpha-1a, alpha-1b a nd alpha-1d adrenergic receptor mRNA in 19 adult rat tissues through t he use of reverse transcription (RT) and a competitive polymerase chai n reaction (PCR). This procedure used internal cRNA standards as compe titive templates that contained sequences complementary to the primers for alpha-1 alpha, alpha-1b and alpha-1d adrenergic receptors and tha t differed in molecular size from the native sequences. Total RNA was harvested from 19 freshly dissected organs of the rat. The PCR product s were labeled by inclusion of alpha-P-32-dCTP in the PCR. After elect rophoresis, ratios of competing to native radiolabeled products were u sed to interpolate the amount of native RNA originally present in each sample. The results revealed that the relative abundance of alpha-1a adrenergic receptor mRNA among various tissues was as follows: vas def erens > colon greater than or equal to stomach greater than or equal t o cerebral cortex > heart greater than or equal to small intestine gre ater than or equal to testis > prostate. The relative rank order of al pha-1b adrenergic receptor mRNA expression was heart greater than or e qual to cerebral cortex > liver, whereas that of alpha-1d adrenergic r eceptor mRNA was vas deferens > cerebral cortex greater than or equal to aorta > adrenal gland. Competitive RT-PCR is an accurate and sensit ive method for the measurement of alpha-1 adrenergic receptor mRNA in small tissue samples. The results of the present study indicate that a lpha-1 adrenergic receptor subtypes are discretely distributed among r at tissues, suggesting independent functions.