In this study high concentrations

In this study, high concentrations (0.5–4 mM) of thuringiensin were required to stimulate the basal adenylate cyclase activity Fig. 2, Fig. 3, Fig. 4, but low concentrations of thuringiensin (1–100 μM) were sufficient to inhibit the forskolin-stimulated adenylate cyclase activity in rat cerebral cortical membranes Fig. 5, Fig. 6. Additionally, an adenylate cyclase irreversible inhibitor MDL-12330A Gadea et al., 1999, Guellaen et al., 1977 also inhibited the basal adenylate cyclase activity (data not shown) and forskolin-stimulated adenylate cyclase activity (Fig. 6). Forskolin classically was considered to be an activator that directly interacts with the catalytic subunit of adenylate cyclase in astrocytoma cell, Cathepsin Inhibitor 1 or cerebral cortical cultures of rats Barovsky and Brooker, 1985, Emanuelli et al., 2001, Rosenberg and Li, 1996, Sano et al., 1983. Moreover, the stimulation effect of 1 mM thuringiensin roughly equaled to that of 10 μM forskolin on basal adenylate cyclase activity in rat cerebral cortical membranes Fig. 2, Fig. 5. Because of the log-level difference in concentrations, thuringiensin acts as a weak activator of adenylate cyclase or an inhibitor of forskolin-stimulated adenylate cyclase in rat brain. Considering, the stimulation of adenylate cyclase was shown at high concentration (mM level) of thuringiensin alone; in contrast, the forskolin-stimulated adenylate cyclase activity was inhibited with low level of thuringiensin. From another point of view, thuringiensin could be regarded as a partial agonist which competed with the full agonist causing the decrease in the cAMP production. The elevation of cAMP can be attributed to direct increase in the rate of enzyme reaction (adenylate cyclase activity) or an activation of Gs protein. For the forskolin-stimulated enzyme, thuringiensin can be regarded to inhibit adenylate cyclase itself Fig. 5, Fig. 6. The increase in cAMP production by adenylate cyclase of E. coli required a high concentration of thuringiensin (Fig. 4). Moreover, the commercial adenylate cyclase from E. coli is a partially purified protein free of membranes (Yang and Epstein, 1983), in which thuringiensin may have directly interacted. In fact, reports have demonstrated that mammalian adenylate cyclase, except ADCY9, is directly activated by forskolin Hanoune et al., 1997, Jackman and Bobik, 1986, Ruiz et al., 1986, Seamon et al., 1981. These findings confirm the effect of thuringiensin directly acting on adenylate cyclase. Furthermore, the inhibitory effects of thuringiensin on forskolin-stimulated adenylate cyclase may be due to the structure analogues as other adenoside analogues (Tesmer et al., 2000). Analogues of adenosides known as P (purine)-site inhibitors inhibit all the isoforms of adenylate cyclase Johnson and Shoshani, 1990, Tesmer et al., 2000. Some inhibitors may play a role as physiological regulators of adenylate cyclase. The most potent inhibitor of adenylate cyclase may ultimately serve as a useful therapeutic agent for cholera and other serious diarrheal diseases that activated intestinal enzyme activity inhibited on pharmacodynamic properties Desaubry et al., 1996, Dessauer et al., 1999. Thus, enzyme inhibitors can counter reverse the pathogenic effects by acting locally and being cleared rapidly from the systemic circulation. In summary, our data demonstrated that thuringiensin can activate the basal adenylate cyclase activity in cerebral cortical membrane preparation of rats, and thuringiensin cannot compete with ATP as a substrate for adenylate cyclase activity. Moreover, comparing the thuringiensin concentrations that activated the basal adenylate cyclase activity and inhibited the forskolin-stimulated adenylate cyclase activity in rat brain, thuringiensin can be regarded as a weak activator of adenylate cyclase or an inhibitor of forskolin-stimulated adenylate cyclase agent. Literatures have demonstrated that cAMP is an important effector involved in the learning and memory process of the brain Ando et al., 1987, Levitzki, 1987, Telegdy and Kokavszky, 2000. Meanwhile, thuringiensin could be detected in brain tissue by intraperitoneal administration (Sebesta and Horska, 1973). Because thuringiensin acted on adenylate cyclase, it may disturb cAMP-mediated signal transduction pathways in neuroendocrine systems. However, the effect of thuringiensin on the physiological significance of brain requires further investigation. Moreover, it is necessary to clarify the effect of thuringiensin on various isoforms of adenylate cyclase in the cerebral cortex as well as those of species differences in the future.