Quantitative measurement of mRNA coding for the receptors controlling acid secretion in the ovine fundus and antrum by using RT-PCR.

Abstract

Gastric acid secretion is stimulated by the action of gastrin, histamine and acetylcholine on their respective receptors. To determine the regulation of synthesis of these receptors during different gastric secretory states a competitive RT-PCR method for quantitating the mRNA for these receptors was developed.Partial cDNA clones (400-500 base pairs (bp)) for the ovine gastrin, histamine (H2) and acetylcholine (M3) receptors were isolated and sequenced. These cDNA constructs were modified by the inclusion of approximately 100 bp of unrelated sequence within the plasmids. cDNA was synthesized from a mixture of known amounts of RNA transcribed from the modified plasmids and from total RNA extracted from sheep stomach. Proportional coamplification of mixed cDNA was demonstrated using common primer sets.All three receptors were more highly expressed in the fundus than the antrum. The concentration of cholecystokinin-B/gastrin receptor mRNA was 75-fold higher in the fundus than in the antrum, and the concentration of both histamine and acetylcholine receptor mRNA were fivefold higher in the fundus than in the antrum. Infusion of gastrin caused a significant increase in fundic histamine mRNA receptor expression, but not in the expression of the gastrin or muscarinic receptors.No significant differences were observed in the levels of receptor mRNA between normal adult and fetal animals despite markedly reduced gastric secretion in the fetus, suggesting that gastric receptor gene expression is not the rate-limiting factor in determining gastric acid secretion in the neonatal animal.