Opioids, Noradrenaline and GTP Analogs Inhibit Cholera Toxin Activated Adenylate Cyclase in Neuroblastoma × Glioma Hybrid Cells

Abstract

D-Ala2-Met5-enkephalin, morphine, and noradrenaline inhibit the adenylate cyclase in homogenates of neuroblastoma x glioma hybrid cells in a dose-dependent manner even after the enzyme has been preactivated by cholera toxin. Half-maximal inhibition and extent of inhibition are the same with native or cholera toxin-activated enzyme. The inhibition caused by opioids or noradrenaline are antagonized by naloxone or phentolamine, respectively. The effect of D-Ala2-Met5-enkephalin on cholera toxin-activated enzyme is immediate in onset and rapidly reversed by the addition of naloxone. Guanyl-5'-yl-imidodiphosphate stimulates basal activity but inhibits the enzyme activated by cholera toxin or prostaglandin E1. Stimulation occurs at a concentration of 100 microM or above, inhibition even at 0.1 microM. The inhibitory effect of the non-hydrolysable GTP analog is antagonized by GTP. Guanyl-5'-yl-methylenediphosphonate, another nonhydrolysable GTP analog, inhibits basal as well as cholera toxin-stimulated or prostaglandin E1-stimulated adenylate cyclase. Other guanine derivatives such as GDP, GMP, cyclic GMP, guanyl-5'-yl-phosphoric acid amide and guanosine have no effect under the same conditions. The results may be taken as a piece of evidence for two separate guanyl nucleotide-binding sites accompanying the adenylate cyclase in the hybrid cells and mediating, respectively, stimulation and inhibition of the enzyme by hormones.

Studies of the Regulation of Basal Adenylate Cyclase Activity by Membrane Polyunsaturated Fatty Acids in Cultured Neuroblastoma

“…As discussed previously, this could be explained by an increase in the number of adenylate cyclase molecules in the linoleate-supplemented cells. Adrenergic (Propst and Hamprecht, 1981) and muscarinic (Bruni et al, 1985) agonists have been demonstrated previously to inhibit basal adenylate cyclase in cultured neural cells, and we have seen similar responses in this study. The reason for the altered responsiveness to these agonists in PUFA-enriched cells is not clear; however, it may be due to changes in ligand-receptor interactions (perhaps due to altered receptor conformation) and/or in receptor-effector coupling.…”

Cell Cycle‐Dependent Expression of Specific Opiate Binding with Variable Coupling to Adenylate Cyclase in a Neurotumor Hybrid Cell Line NG108–15

“…Observations that GTP inhibits enkephalin [~-Ala~D-Leu'] binding (Chang et al, 1981) and that both the nonhydrolyzable GTP analog Gpp(NH)p and opiates can inhibit adenylate cyclase activity in broken NG108-I5 cells (Propst and Hamprecht, 1981) have been taken as evidence that a CTPbinding protein is responsible for mediating the inhibitory coupling of the opiate receptor to adenylate cyclase. Further, recent evidence has been presented that suggests that opiates act by stimulating an enzyme responsible for GTP hydrolysis .…”

Bimodal regulation of adenylate cyclase

“…However, this effect of cholera toxin is not universally encountered in dually regulated systems. Inhibitory effects were retained following toxin treatment of Chinese hamster ovarian, neuroblastoma X glioma hybrid ceils and platelets [40][41][42].…”