Interactions of gangliosides with adenylate cyclase and cyclic AMP phosphodiesterase in rat brain homogenates and synaptosomes [proceedings].

Gangliosides are a complex group of acid glycolipids characterized by the presence of sialic acid residues (Klenk, 1942). 'Ihey are present in high concentrations in neuronal tissue and appear to be concentrated in nerve endings (Pert et al., 1974). As such, they may be important in the process of neurotransmission (McIlwain, 1961) or may be involved in membrane receptors (Cuatrecasas, 1974) which mediate drug action. It is also now firmly established that cyclic AMP plays a pivotol role in regulating many metabolic processes (Robison et al., 1971), including its importance in the nervous system (Nathanson, 1977). The cellular concentration of cyclic AMP is modulated largely by the relative activities of adenylate cyclase (EC 4.6.1.1) and phosphodiesterase (EC 3.1.4.17). It has been suggested that various lipids may be involved in the regulation of these enzymes (De Robertis et al., 1967; Pichard & Cheung, 1977; Davis & Daly, 1978). The present report demonstrates that gangliosides (in vitro) are capable of inhibiting cyclic AMP phosphodiesterase and adenylate cyclase. Cyclic AMP phosphodiesterase was prepared and assayed as follows. Rats (Wistar, male, 120-15Og) were decapitated, and the brains rapidly excised, weighed, placed in 5vol of 5 0 m ~ Tris/HCI buffer, pH 7.5, at 4OC and homogenized. After centrifugation at 3000g for 30min at 4OC the supernatant was used as the enzyme source. Enzyme activity was estimated by the isotopic procedure described by Boudreau & Drummond (1975) with a substrate concentration of 1 p ~ . Adenylate cyclase activity was estimated by the method of White & Karr (1978) with one exception. The assay buffer contained, 50mM-glyCylglycine, 10 mM-theophylline, 1 mwdithiothreitol, 8 mhf-MgCI,, 60p~-CaCI , and 1 mM-ATP, pH 7.5. Synaptosomes were prepared as described by Whittaker & Barker (1972). Protein was determined by the method of Lowry et al. (195 I), with bovine serum albumin (Cohn fraction V) as standard. Gangliosides (type 11) purified from bovine brain by the method of Trans & Lauter (1962), and other lipids were obtained from Sigma (London). Pure gangliosides (GM,, GD,, and GT,) were obtained from Supelco Inc., Bellefonte, PA 16823, U.S.A. Table 1 reveals the concentration-related inhibitory response of gangliosides on low-K, cyclic AMP phosphodiesterase from rat brain homogenates and synaptosomes. Conversely, inhibition of basal adenylate cyclase activity was not concentration-dependent. However, the effects on prostaglandin E,stimulated adenylate cyclase activity are more pronounced. It is well known that gangliosides act as membrane receptors for various toxins (van Heyningen, 1974), and the addition of exogenous monosialoganglioside (GM ,) increases the response of adenylate cyclase to cholera toxin and also in the binding or function of many biologically important macromolecules, e.g. lutropin (luteinizing hormone) (Lee et al., 1977), 5-hydroxytryptamine (serotonin) (Ochoa & Bangham, 1976) and wheatgerm agglutinin (Redwood & Polefka, 1976). From our results it appears that gangliosides and prostaglandin El do interact in synaptosomes, although the exact mode of interaction remains unclear. In view of these paradoxical findings it is apparent that gangliosides may play a modulating role in controlling the activity of cyclic AMP phosphodiesterase in vivo. It should be noted, however, that DLsphingosine, N-acetylneuraminic acid, sphingosine, sphingomyelin, cerebrosides and ceramides had no effect on either enzyme. It is possible that gangliosides obtained by the method of Trans & Lauter (1962) may contain a contaminant that is in fact capable of inhibiting cyclic AMP phosphodiesterase, and basal and prostaglandin El-stimulated adenylate cyclase activity. Davis & Daly (1978) have reported that mixed brain gangliosides (lOpg/ml) activated cyclic AMP phosphodiesterase by about 2.2-fold. These results are difficult to reconcile with our findings; but their results were obtained against the Ca2+dependent phosphodiesterase. It appears likely that gangliosides do have, as yet unknown, roles in the function of these two enzymes.