AUTORADIOGRAPHIC DEMONSTRATION OF THE DISTRIBUTION OF DIACYLGLYCEROL IN THE GASTRIC MUSCULARIS MUSCLE OF BUFO-MARINUS - IS DIACYLGLYCEROL ONE OF THE 2ND 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% pa raformaldehyde/2.5% glutaraldehyde, postfixed, washed and longitudinal sections (15 microns) were cut on a cryotome and placed on gelatinize d 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 a gonist-stimulated formation of cytidine diphosphate-DAG (CDP-DAG). The sections were processed through an autoradiographic technique using [ H-3]-cytidine, which binds to 1,2-diacylglycerol within the tissue to form CDP-DAG. Analysis of the developed tissue slides indicated that t he dose of ACh (10(-5) M) which had been predetermined to elicit autom aticity of the preparation in vitro increased the density of CDP-DAG g rains. 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 throu ghout the muscle fibers. The increase in the density of the grains whi ch was induced by the conditions eliciting automaticity suggest that D AG is one of the second messengers in the manifestation of automaticit y of the muscularis muscle of Bufo marinus.