Cellular Mechanism Involved in the Synthesis of Cyclic GMP in Nervous Tissues

Summary

Intracellular cyclic GMP content responds to the stimulation of muscarinic receptor in a variety of tissues. Several aspects of the cellular mechanism involved in the synthesis of cyclic GMP were investigated. 1. In cultured bovine chromaffin cells, acetylcholine as well as muscarine stimulated the ³²Pi incorporation into phosphatidic acid, induced Ca²⁺ mobilization across the cells, and, in parallel, elevated intracellular cyclic GMP content. Phosphatidic acid added to culture medium also stimulated the efflux and influx of Ca²⁺ and the synthesis of cyclic GMP in bovine chromaffin cells and in neuroblastoma cells in the same fashion as acetylcholine. 2. We have succeeded in a purification of an endogenous activator for guanylate cyclase from rat brain and identified it as L-arginine. L-Arginine, but not D-arginine, activated soluble guanylate cyclase 10- to 20-fold at a low concentration (1−2 × 10^2212;5 M). The activation of the enzyme by L-arginine seemed to require Ca²⁺. Calcium accumulated in cells in response to muscarinic stimulation would activate guanylate cyclase in collaboration with L-arginine. 3. Using a specific monoclonal antibody, we demonstrated the cellular and subcellular localizations of guanylate cyclase in rat brain. An intense reaction was observed in the brain regions which were rich in muscarinic receptor. Electron microscopic examination revealed that guanylate cyclase was concentrated in the postsynaptic perikaryon and dendrites of some type of neurons indicating its involvement in neural transmission.

Since the study of George et al. (1970), it has been repeatedly demonstrated that acetylcholine, a-adrenergic agents and other transmitters elevated the intracellular cyclic GMP content in a variety of tissues and cultured cells. Goldberg et al. (1973) have postulated that cyclic GMP could be a second messenger of the stimulation of muscarinic receptor on an analogy of catecholamine-cyclic AMP system. Although a number of reports have accumulated dealing with the mechanism of cyclic GMP synthesis and the activation of guanylate cyclase that is responsible for the synthesis of cyclic GMP, the following essential questions have remained to be elucidated. 1. Most of guanylate cyclase in mammalian tissues were detected in soluble fraction. How is the signal of the stimulation of membrane receptor transduced to the activation of soluble enzyme? 2. Guanylate cyclase required a high concentration (3–5 mM) of manganese for its full activity. What is the physiological metal cofactor for the enzyme? 3. Although extracellular Ca²⁺ was essentially required for the elevation of cyclic GMP in tissues (Schultz et al. 1973), Ca²⁺ did not activate guanylate cyclase in cell-free systems. What is the role of Ca²⁺ in this reaction? 4. Acetylcholine and other transmitters that stimulated the synthesis of cyclic GMP in intact tissues did not activate guanylate cyclase in tissue homogenate or in purified enzyme preparations. What is the physiological regulator for guanylate cyclase activity?

On the other hand, it has been also well documented that acetylcholine stimulated the turnover of phosphatidylinositol by acting on a muscarinic receptor and accumulated phosphatidic acid in cell membrane of a variety of tissues. Michell (1975) has postulated that the turnover of phosphatidylinositol is intimately involved in the mechanism by which certain transmitters or hormones activate the Ca²⁺ gate in membrane. However, experimental evidence for this speculation has been scanty. Since the same compounds such as muscarinic and α-adrenergic agents stimulated both the turnover of phosphatidylinositol and cyclic GMP synthesis in tissues, both reactions might be coupled each other.

We report the role of phosphatidic acid in the muscarinic action of acetylcholine, the detection and identification of an endogenous activator for soluble guanylate cyclase, and the immunohistochemical demonstration of guanylate cyclase in rat brain.