Chronic atropine administration up-regulates rat cortical muscarinic m1 receptor mRNA molecules: assessment with the RT/PCR

Abstract

The modulation of the rat cortical m1 muscarinic receptor mRNA was studied with a method of quantitation using the polymerase chain reaction after conversion to complementary DNA (cDNA) with AMV reverse transcriptase (RT/PCR). Primers specific to the C3 region of the m1 mRNA were employed. The y-intercepts from the linear regions of semilogarithmic plots of PCR product versus cycle number were used as measures of the levels of m1 muscarinic mRNA-measured relative to that of glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA (the ‘ratio’ method). Alternatively, m1 mRNA in total cortical RNA samples was quantitated from the increase in product elicited by adding a known amount of exogenous m1 muscarinic cDNA sequence (the ‘spiking’ method). This allowed calculation of absolute level of m1 mRNA, which was 4.1 pg/μg total RNA. We measured the level of the rat cortical m1 mRNA after 1 week of chronic receptor blockade with atropine, showing upregulation of 154% by the GAPDH/m1 ratio method and 145% by the spiking method. That this transcriptional alteration was specific was indicated by the finding that the level of GAP-43 mRNA was not affected by atropine treatment.