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Autoradiographic demonstration of the distribution of diacylglycerol in the gastric muscularis muscle of Bufo marinus: is diacylglycerol one of the second messengers in the muscularis muscle?

The distribution of 1,2-diacylglycerol (DAG) in the muscularis muscle of Bufo marinus has been determined using autoradiographic techniques. Strips of the muscle from the body of the stomach were fixed in 4% paraformaldehyde/2.5% glutaraldehyde, postfixed, washed and longitudinal sections (15 microns) were cut on a cryotome and placed on gelatinized slides. The sections were then incubated in the presence and absence of agonist, acetylcholine (ACh) 10(-5) M or carbachol (CCh) 10(-5) M plus Li+ (10 mM) in the medium which causes a marked potentiation of agonist-stimulated formation of cytidine diphosphate-DAG (CDP-DAG). The sections were processed through an autoradiographic technique using [3H]-cytidine, which binds to 1,2-diacylglycerol within the tissue to form CDP-DAG. Analysis of the developed tissue slides indicated that the dose of ACh (10(-5) M) which had been predetermined to elicit automaticity of the preparation in vitro increased the density of CDP-DAG grains. When the muscle strips were pretreated with ACh (10(-5) M) and CCh (10(-5) M) in the presence of LiCl (10 mM), the density of the CDP-DAG grains were further enhanced. CDP-DAG grains were localized throughout the muscle fibers. The increase in the density of the grains which was induced by the conditions eliciting automaticity suggest that DAG is one of the second messengers in the manifestation of automaticity of the muscularis muscle of Bufo marinus.