Adenylate Cyclase In Neuroblastoma Research Articles

Cholera toxin impairment of opioid-mediated inhibition of adenylate cyclase in neuroblastoma x glioma hybrid cells is due to a toxin-induced decrease in opioid receptor levels.

Cholera toxin treatment (up to 1 microgram/ml, 16 h) of neuroblastoma x glioma hybrid NG108-15 cells produced a decrease of some 35% in both delta opioid receptor-mediated stimulation of high-affinity GTPase activity and inhibition of forskolin-amplified adenylate cyclase. Coincident with these decreases was a down-regulation of some 35% in the delta opioid receptor population. A similar pattern of a decrease in signalling capacity was noted for the alpha 2B-adrenergic receptor in these cells after cholera toxin treatment. Half-maximal effects of cholera toxin on all of the parameters assayed were noted at concentrations between 2 and 5 ng/ml. Neither levels of Gi2, as assessed by immunoblotting with specific antisera, nor the intrinsic activity of the alpha subunit of the guanine-nucleotide-binding protein which acts as the inhibitory G-protein of the adenylate cyclase in these cells, as assessed by guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p)-mediated inhibition of adenylate cyclase, was lowered by cholera toxin treatment. Furthermore, levels of another pertussis toxin-sensitive G-protein (Go) expressed by these cells was also not lowered by cholera toxin treatment. However, as previously noted in other cells [Milligan, Unson & Wakelam (1989) Biochem. J. 262, 643-649], marked down-regulation of the alpha subunit of the stimulatory G-protein (Gs) of the adenylate cyclase cascade was observed in response to cholera toxin treatment. Previous studies [Klee, Milligan, Simonds & Tocque (1985) Mol. Aspects Cell Regul. 4, 117-129] have shown that cholera toxin treatment can result in a decrease in the maximal effectiveness of agonists which function to inhibit adenylate cyclase. These data have been used as evidence to suggest a functional interaction between Gs and 'Gi'. The results provided herein demonstrate that such effects of the toxin can be explained adequately by a decrease in the number of receptors that function to produce inhibition of adenylate cyclase.

5′-Deoxy-5′-methylthioadenosine: A nucleoside which differentiates between adenosine receptor types

The activities of an endogenous nucleoside, 5′-deoxy-5′-methylthioadenosine (MTA), on adenosine sensitive sites such as adenosine a 1 and A 2 receptors and the P-site, as well as on purine nucleoside transport, have been studied. This nucleoside competitively antagonized the A 2 receptor-mediated stimulation of neuroblastoma adenylate cyclase, produced a GTP-dependent and 8- p-sulfophenyltheophylline-sensitive inhibition of adenylate cyclase activity in rat cerebellar membranes, and decreased the spontaneous contractile activity of isolated segments of rabbit jejunum. MTA was neither active at the P-site nor did it diminish the binding of [ 3Hnitrobenzylthioinosine, a nucleoside transport inhibitor. We conclude that (a) MTA is an agonist at the adenosine A 1 receptor but an antagonist at the A 2 receptor, and (b) the adenosine receptor which causes relaxation of rabbit jejunum is not a neuroblastoma-type A 2 receptor which activates adenylate cyclase.

Cell-free desensitization of opioid inhibition of adenylate cyclase in neuroblastoma X glioma NG108-15 hybrid cell membranes.

When membranes from neuroblastoma X glioma NG108-15 hybrid cells were incubated in a cell-free system with opioid agonists, a time-, temperature-, and dose-dependent desensitization to opioid inhibition of adenylate cyclase activity was observed. The composition of the system during the incubation was manipulated to elucidate the biochemical mechanisms of desensitization. Receptor coupling appeared to be a prerequisite for desensitization, because both magnesium and sodium, which are necessary for coupling, were required for desensitization. Removal of ATP and addition of cyclic AMP or cyclic GMP had no effect on desensitization.

Dual pathways of receptor-mediated cyclic GMP generation in NG108-15 cells as differentiated by susceptibility to islet-activating protein, pertussis toxin

The cellular cGMP content increased in response to a variety of receptor agonists, which activate [e.g., prostaglandin (PG) E 1, E 2, and F 2α or inhibit (e.g., α-adrenergic, muscarinic, and opiate agonists) adenylate cyclase in neuroblastoma X glioma hybrid NG108-15 cells. The responses were additive when PGF 2α and enkephalin were mixed. The inhibitory guanine nucleotide regulatory protein (N i) is involved in adenylate cyclase inhibition; this function of N i is lost when it is ADP-ribosylated by islet-activating protein (IAP), pertussis toxin [ H. Kurose, T. Katada, T. Amano, and M. Ui (1983) J. Biol. Chem. 258, 4870–4875 ]. The cGMP rise induced by stimulation of the receptors linked to adenylate cyclase inhibition was also diminished by IAP; the time course and dose response for the IAP-induced diminution were the same between adenylate cyclase inhibition and cGMP generation. N i thus appears to mediate guanylate cyclase activation as well as adenylate cyclase inhibition initiated via the same receptors. Melittin also increased cGMP. No additivity was shown when enkephalin and melittin were combined, suggesting that phospholipase A 2 might play a role in N i-mediated guanylate cyclase activation. On the other hand, the PGF 2α-induced cGMP rise was associated with increased incorporation of 32 P i into phosphatidylinositol; was not affected by cholera toxin, IAP or forskolin; and showed no additivity when combined with A23187, which increased cGMP by itself. PGs would occupy receptors linked to phosphatidylinositol breakdown, thereby increasing the availability of intracellular Ca 2+, which is responsible for guanylate cyclase activation. Thus, dual pathways are proposed for a receptor-mediated cGMP rise in NG108-15 cells.

Effects of amino and ammonio derivatives of adenosine on smooth muscle preparations and mouse neuroblastoma adenylate cyclase.

Several amino- and ammonio-substituted derivatives of adenosine were tested as effectors of adenosine receptors in different smooth muscle preparations and mouse neuroblastoma adenylate cyclase. The compounds did not affect adenosine receptors in smooth muscles. N6-[3-(trimethylammonio)propyl]adenosine was a weak stimulator of adenylate cyclase, and 3'-amino-3'-deoxyadenosine and 3'-monomethylamino-3'-deoxyadenosine antagonized the stimulation of adenylate cyclase by 2-chloroadenosine.

Inhibition of neuroblastoma adenylate cyclase by cannabinoid and nantradol compounds

This study was undertaken to ascertain the effects of cannabinoid drugs on prostanoid-stimulated adenylate cyclase in neuroblastoma cells. This report demonstrates that Δ 9-tetrahydrocannabinol (THC) and levonantradol could decrease initial rate cyclic AMP accumulation in response to prostacyclin in intact cells. Basal accumulation was also diminished. Prostanoid-stimulated adenylate cyclase in a membrane preparation from these cells was inhibited by cannabinoid and nantradol compounds. However, this inhibition was not competitive with prostaglandin E 1 or prostacyclin. Further, inhibition was also observed when the enzyme was stimulated by peptide hormones at the secretin receptor. In contrast, enzyme activated by NaF was not inhibited by cannabinoid compounds. Cyclic AMP phosphodiesterase activity in subcellular fractions was unaltered by these agents. These data demonstrate that cannabinoid and nantradol compounds decrease cyclic AMP accumulation in neuronally derived cells, and that this results from an inhibition of basal and hormone-stimulated adenylate cyclase activity.

Interaction of manganese with NB41A neuroblastoma adenylate cyclase.

In vitro assay of the adenylate cyclase of NB41A neuroblastoma cells in the presence of increasing concentrations of MnCl2 suggested that the enzyme is modulated by both high- and low-affinity sites for manganese. MnCl2 in a concentration of 1 microM significantly stimulated adenylate cyclase activity, but increasing the concentration of manganese to 3 microM or 10 microM had no further effect. Raising MnCl2 to 0.1 or 1 mM, however, further stimulated enzyme activity. In addition to differences in affinity for manganese, the two classes of binding sites may be distinguished by differences in their interaction with other agents that affect adenylate cyclase activity. Millimolar manganese and magnesium appeared to compete for a common site on the enzyme and the effect of manganese in this range and the effect of guanyl nucleotide were synergistic. In contrast, the stimulation of activity by micromolar manganese appeared to be additive to the effects of either increasing magnesium or the addition of guanyl nucleotide to the assay media. Comparison of the substrate dependency of the reaction measured in the presence and absence of manganese suggests that the stimulation of adenylate cyclase activity involves increases in both the apparent Vmax of the reaction and the affinity for ATP. The results raise the possibility that the interaction of Mn2+ may play a role in the modulation of adenylate cyclase in vivo.

Inhibition by lithium of PGE1-sensitive adenylate cyclase in neuroblastoma X glioma hybrid cells: approach to the attenuation of the opiate withdrawal syndrome.

Exposure of neuroblastoma X glioma hybrid cells to LiCl (Li+) for 48 h significantly reduces prostaglandin E1 (PGE1)-dependent cyclic adenosine monophosphate (AMP) accumulation. When applied simultaneously with morphine for the same period, Li+ inhibits the excessive accumulation of cyclic AMP in response to PGE1 characteristically exhibited by these cells upon opiate withdrawal. Likewise, Li+ inhibits, in vitro, PGE1-dependent cyclic AMP accumulation in tissue slices from rat corpus striatum. Prophylactic administration of Li+ in the diet attenuates the withdrawal symptoms in morphine-addicted rats. These findings support clinical reports that suggest that prophylactic treatment with Li+ attenuates the severity of the opiate withdrawal syndrome.

Lipid requirement for long-lived morphine-dependent activations of adenylate cyclase of neuroblastoma X glioma hybrid cells.

Abstract:Morphine or norepinephrine at 10 μM inhibit adenylate cyclase of NG108‐15 neuroblastoma × glioma hybrid cells and gradually, over a period of 12 h or more, elicit a compensatory, persistent increase in adenylate cyclase activity. The cells are then dependent upon the receptor ligand for maintenance of normal cyclic AMP levels. Serum lipids or linoleic acid are required for the full expression of the long‐lived morphine‐dependent increase in adenylate cyclase activity, but serum lipids are not required for inhibition of the enzyme by morphine. Serum lipids are also not required for the full expression of the norepinephrine‐dependent increase in adenylate cyclase activity. In the NG108‐15 model system, therefore, the acquisition of dependence upon morphine can be dissociated from the inhibition of adenylate cyclase by morphine as well as from the acquisition of dependence upon another receptor ligand, norepinephrine.

Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha-adrenergic receptors. II. Long lived increase of adenylate cyclase activity mediated by alpha receptors.

Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha-adrenergic receptors. II. Long lived increase of adenylate cyclase activity mediated by alpha receptors.

Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha-adrenergic receptors. I. Inhibition of adenylate cyclase mediated by alpha receptors.

(-)-Norepinephrine and other catecholamines inhibit basal and prostaglandin E1-stimulated adenylate cyclase activities by 35 to 60% in homogenates of NG108-15 neuroblastoma x gloma hybrid cells and markedly reduce adenosine 3'35:'-monophosphate levels of intact cells, but do not affect guanosine 3':5'-monophosphate levels. The specificity of the NG108-15 receptor for ligands is that of an alpha receptor, possibly a presynaptic alpha 2 receptor. The inhibition of adenylate cyclase by norepinephrine is reversed by alpha receptor antagonists such as dihydroergotamine or phentolamine, but not by the beta receptor antagonist propranolol. The effect of norepinephrine on adenylate cyclase activity initially is dependent on GTP; half-maximal inhibition of enzyme activity by norepinephrine is obtained with 0.2 micron GTP. The inhibition of adenylate cyclase activity by norepinephrine is reduced by 10 mM NaF and is abolished by 0.05 mM guanyl-5'-yl imidodiphosphate. Inhibitions of NG108-15 adenylate cyclase mediated by alpha receptors, opiate receptors, and muscarinic acetylcholine receptors are not additive; this suggests that the three species of receptors can be functionally coupled to the same adenylate cyclase molecules or molecules regulating the enzyme.

Stereospecific and Nonstereospecific Effects of (+)- and (—)-Morphine: Evidence for a New Class of Receptors?

The unnatural (+) enantiomer of morphine had minimal activity in three opiate assays in vitro: the rat brain homogenate binding assay, the electrically stimulated guinea pig ileum assay, and the inhibition of adenylate cyclase in neuroblastoma X glioma hybrid cell homogenates. When (+)-morphine was microinfected into the periaqueductal gray (a site known to mediate morphine analgesia) of drug-naive rats, there was only minimal analgesia, but the hyperresponsivity usually observed after microinfection of (-)-morphine occurred. Also, when (+)-morphine was microinfected into the midbrain reticular formation of drug-naive rats, rotation similar to that following microinjection of (-)-morphine occurred. These behaviors were not blocked by naloxone. Significantly, they typically occur in precipitated abstinence in morphine-dependent rats. These observations suggest that there are at least two classes of receptors, one stereospecific and blocked by naloxone and the other only weakly stereospecific and not blocked by naloxone, and that precipitated abstinence may be due, in part, to a selective blockade of receptors of the former class but not of the latter.

Mode of action of endogenous opiate peptides

AS THE culmination of a search for endogenous ligands of the opiate receptor1–4, the structures of two closely related pentapeptides, isolated from porcine (brain, with morphine-like properties in pharmacological tests, have been reported by Hughes et al.5 The amino acid sequences of the pentapeptides, termed methionine enkephalin and leucine enkephalin, are Tyr-Gly-Gly-Phe-Met (Met5-enkephalin) and Tyr-Gly-Gly-Phe-Leu (Leu5-enkephalin), respectively. They behave as highly potent opiates in the mouse vas deferens and guinea pig ileum assays5. In addition, animals tolerant to morphine are also tolerant to enkephalin6. The action of the pentapeptides in the guinea pig ileum5 and when injected intracerebrally or intracerebroventricularly7–9 is very short lived, perhaps because of proteolytic degradation. A related peptide, with longer chain length has been shown to have a long duration of action10. Morphine and other narcotics have been shown to bind to the opiate receptor11–13 and inhibit adenylate cyclase activity in homogenates of neuroblastoma × glioma hybrid cells14,15, to inhibit cyclic AMP accumulation in intact hybrid cells15,16 and in rat brain homogenates17. We have tested the effects of Met5- and Leu5-enkephalin on NG108-15 adenylate cyclase activity, and show here that endogenous opiate peptides are potent, receptor-mediated, inhibitors of adenylate cyclase of neuroblastoma × glioma hybrid cells.

Neuroblastoma adenylate cyclase. Role of 2-chloroadenosine, prostaglandin E1, and guanine nucleotides in regulation of activity.

Neuroblastoma adenylate cyclase is activated by 2-chloroadenosine, prostaglandin E1, and 5'-guanylylimidodiphosphate [GMP-P(NH)P]. However, the process of activation by the first two compounds is different from that induced by the third. Prostaglandin E1 and 2-chloroadenosine activation is rapid, producing elevated activities which are constant throughout a 20-min assay. In contrast, GMP-P(NH)P activation is slow and although the activity is elevated within 1 min, it continues to increase for up to 12 min before attaining a maximal constant value. Activation is more rapid when either prostaglandin E1 or 2-chloroadenosine is present with GMP-P(NH)P. Activation of the enzyme by GMP-P(NH)P appears to be retarded by endogenous nucleotides as suggested by the following observations: (a) if the enzyme is incubated at 30 degrees with 5 mM MgCl2 for 5 to 7 min, GMP-P(NH)P then produces maximal activation without a detect able lag; (b) if, during this incubation, nucleotides, a nucleotide regenerating system, or EDTA (instead of MgCl2) are present, subsequent GMP-P(NH)P activation is slow; and (c) in the assays which contain a nucleotide regenerating systm and MgATP as substrate, the Km for GMP-P(NH)P is 6 +/- 2 muM. However, in the assays using MgAMP-P(NH)P as substrate but no nucleotide regenerating system, the Km is 0.5 +/- 0.2 muM. GPD and GTP do not replace GMP-P(NH)P as an enzyme activator in any of our assays systems, and in fact, are potent inhibitors of GMP-P(NH)P enzyme activation. Prostaglandin E1 and 2-chloradensine do not alter significantly the Km for GMP-P(NH)P but do decrease the ensyme's sensitivity of GDP. Proposed is a hysteretic model of neuroblastoma adenylate cyclase, which shows the enzyme responding slowly to rapid changes in GMP-P(NH)P concentration due to the slow displacement of the tightly bound endogenous guanine nucleotides by GMP-P(NH)P. Additionally, prostaglandin E1 and 2-chloroadenosine increase the rate of GMP-P(NH)P activation by decreasing the enzyme's affinity for these endogenous guanine nucleotides.

Mouse neuroblastoma cell adenylate cyclase: regulation by 2-chloroadenosine, prostaglandin E1 and the cations Mg2+, Ca2+ and Mn2+.

Abstract—Some basic kinetic properties of adenylate cyclase in cell free preparations of mouse neuroblastoma were investigated. Production of cAMP from ATP by the enzyme requires the presence of either Mg2+ or Mn2+ in addition to ATP. In the presence of Mg2+, the Km for ATP is 120 ± 15 μM and the interaction of ATP and adenylate cyclase appears to be non‐cooperative (Hill coefficient of 1). Magnesium ion concentrations in excess of the ATP concentration cause stimulation although similar excess concentrations of Mn2+ cause inhibition.Prostaglandin E1 and 2‐chloroadenosine activate the enzyme. The Km of the cyclase for 2‐chloroadenosine is 6 μm. Activation by 2‐chloroadenosine leads to an increase in Vmax but does not effect the Km for ATP. At a fixed ATP concentration, the extent of activation caused by prostaglandin E1 and 2‐chloroadenosine is inversely related to the Mg2+ concentration.Calcium ion causes inhibition of adenylate cyclase from 0.1 to 4mM with a Ki of 5 ± 10−4m. Ca2+ interaction with the enzyme in the absence or presence of either 2‐chloroadenosine or prostaglandin E1 appears cooperative (i.e. Hill coefficients of ˜2). Ca2+ inhibition is non‐competitive with respect to either ATP or 2‐chloroadenosine but is progressively diminished by increasing Mn2+ concentrations.Divalent cation effects and activation by 2‐chloroadenosine and prostaglandin E1 of the neuroblastoma adenylate cyclase are compared with ion effects and hormone activation of the enzyme obtained from non‐neuronal tissue.

Mouse neuroblastoma adenylate cyclase. Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity.

1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.

Effect of adrenergic agents on toad bladder response to ADH, 3',5'-AMP, and theophylline.

Effect of adrenergic agents on toad bladder response to ADH, 3',5'-AMP, and theophylline.