Guanine nucleotides inhibit binding of agonists and antagonists to soluble opiate receptors

The guanine nucleotides GDP, GTP, and guanosine-5'-(beta, gamma-imido)triphosphate inhibit binding of opiates and opioid peptides to receptors solubilized from membranes of neuroblastoma X glioma NG108-15 hybrid cells. The inhibition reflects decreased affinity of receptors for opioid ligands. Whereas in membranes, only opioid agonist binding is sensitive to guanine nucleotide inhibition, both agonist and antagonist binding is reduced in the case of soluble receptors. Furthermore, soluble receptors are more sensitive to the effects of guanine nucleotides than are membrane-bound receptors. These observations are consistent with the suggestion that solubilized receptors may be complexes of an opiate binding protein and a guanine nucleotide-sensitive regulatory component.     

Chronic Activation of Inhibitory δ-Opioid Receptors Cross-Regulates the Stimulatory Adenylate Cyclase-Coupled Prostaglandin E1 Receptor System in Neuroblastoma × Glioma (NG108-15) Hybrid Cells

Abstract: The present article investigates chronic opioid regulation of the stimulatory adenylate cyclase-coupled prostaglandin E₁ (PGE₁) receptor system in neuroblastoma × glioma (NG108-15) hybrid cells. Persistent activation of δ-opioid receptors by morphine (10 µmol/L; 3 days) substantially down-regulates the number of PGE₁ binding sites by ～30%, without affecting their affinity. Radioligand binding studies performed in the presence of GTPγS (100 µmol/L) further revealed that the remaining PGE₁ binding sites are still capable of interacting functionally with their associated stimulatory G proteins, G_s. On the postreceptor level, neither changes in the abundance nor in the intrinsic activity of the α subunit of G_s (G_sα) were found during the state of opioid dependence, as has been verified by western blot and S49 cyc^? reconstitution experiments, respectively. Evaluation of the functional interaction between PGE₁ receptors and G_s by means of receptor-stimulated, cholera toxin-catalyzed ADP-ribosylation of G_sα revealed a significant increase in the ability of PGE₁ receptors to activate G_sα (3.3-fold increase in EC₅₀; p < 0.05) in cells chronically exposed to morphine. This effect was completely blocked by coincubation of the cells together with the opiate antagonist naloxone (100 µmol/L; 3 days), whereas precipitation of morphine withdrawal by naloxone (100 µmol/L) had no further effect on sensitization in PGE₁ receptor/G_s coupling. These findings provide evidence that the stimulatory adenylate cyclase-coupled PGE₁ receptor system represents a potential target of chronic δ-opioid receptor activation in NG108-15 hybrid cells. They further suggest that sensitization in stimulatory signal transduction plays a critical role in the generation of opioid dependence.     

Opiate binding to membrane preparations of neuroblastoma x glioma hybrid cells NG108-15: effects of ions and nucleotides.

Interaction of a number of opiate agonists with the opiate receptors in NG108-15 cell membranes is influenced by ions, as well as certain nucleotides. Steady state binding of [³H]leu-enkephalin is increased by Mg⁺⁺ and decreased by Na⁺, GMP-P(NH)P, GTP, GDP, ITP and IMP-P(NH)P. Half-maximal inhibition produced by GMP-P(NH)P occurred at 4.6 μM. The dissociation of [³H]leu- and [³H]met-enkephalin, as well as [³H]etorphine, from these opiate receptors was also shown to be altered by both ions and nucleotides.     

Desensitization of PGE1 receptors in neuroblastoma-glioma hybrid cells

Prostaglandin E₁ receptor sites were measured in homogenates of NG108-15 neuroblastoma-glioma hybrid cells after exposure of intact cells to PGE₁. Scatchard analysis of competitive binding studies showed that incubation of NG108-15 cells in the presence of 2.5 μM PGE₁ for 16 h resulted in a loss of PGE₁ receptors and an increase in the dissociation constant of the remaining receptors. Thus, cells challenged with PGE₁ not only lose adenylate cyclase activity, but also lose PGE₁ receptors and decreased the affinity of the remaining receptors for PGE₁.     

A novel analogue of clonidine with opiate-receptor agonist activity

A new analogue of the α₂-adrenergic receptor ligand clonidine, N-(4-hydroxphenacetyl)-4-aminoclonidine, was synthesized. The analogue possesses opiate-receptor agonist activity in addition to α-adrenergic partial agonist activity. The analogue elicits inhibition of adenylate cyclase of NG108-15 neuroblastoma × glioma hybrid cells; most of the inhibition is reversed by the opiate-receptor antagonist naloxone. The analogue also inhibits the binding of [³H]D-Ala²-Met⁵-enkephalinamide and [³H]dihydromorphine to rat brain opiate receptors. The structure of the analogue suggests common elements in the ligand binding sites of α- and opiate receptors and may lead to a new class of opiate analgesics.     

Azidomorphine is an agonist of high-affinity opioid receptor binding sites

Azidomorphine at low concentration (10^–9 M) inhibits the high-affinity binding site of labeled naloxone in rat brain membrane preparations. In the presence of Na⁺ and guanine nucleotides the displacement curves of azidomorphine are increased toward high concentrations, whereas Mg²⁺ ions decrease the IC₅₀ values; This demonstrates the agonist behavior of azidomorphine in binding experiments. When compared with morphine, azidomorphine displayed five-fold lower IC₅₀ values. Based on the presented results, azidomorphine appears to be a good candidate for photoaffinity labeling of opiate receptors.     

Interrelationships among prostaglandins, vasopressin and cAMP in renal papillary collecting tubile cells in culture

To determine the influence of prostaglandins on cAMP metabolism in renal papillary collecting tubule (RPCT) cells, intracellular cAMP levels were measured after incubating cells with prostaglandins (PGs) alone or in combination with arginine vasopressin (AVP). PGE₁, PGE₂ and PGI₂, but not PGD₂ or PGF_2α, increased intracellular cAMP concentrations. At maximal concentrations (10⁻⁵ tthe effects of PGE₂ plus PGI₂ (or PGE₁), but not of PGI₂ plus PGE₁, were additive suggesting that at least two different PG receptors may be present in RPCT cell populations. Bradykinin treatment of RPCT cells caused an accumulation of intracellular cAMP which was blocked by aspirin and was quantitatively similar to that observed with 10⁻⁵ PGE₂. PGs, when tested at concentrations (e.g. 10⁻⁹ ) which had no independent effect on intracellular cAMP levels, did not inhibit the AVP-induced accumulation of intracellular cAMP in RPCT cells. These results indicate that PGs do not block AVP-induced accumulation of intracellular cAMP in RPCT cells at concentrations of PGs which have been shown to inhibit the hydroosmatic effect of AVP on perfused collecting tubule segments. However, at higher concentrations of PGs (e.g. 10⁻⁵ ), the effects of AVP plus PGE₁, PGE₂, PGI₂ or bradykinin on intracellular cAMP levels were not additive. Thus, under certain conditions, there is an interaction between PGs and AVP at the level of cAMP metabolism in RPCT cells.     

Characteristics of45Ca uptake stimulated by high KCl of differentiated and undifferentiated NG108-15 and PC12h cells

The characteristics of KCl-stimulated⁴⁵Ca uptake by neuroblastoma x glioma hybrid NG108-15 cells induced to differentiate with dibutyryl cAMP (Bt₂cAMP) and of PC12h pheochromocytoma cells induced to differentiate with nerve growth factor (NGF) were studied. The extent and rate of KCl-stimulated⁴⁵Ca uptake by differentiated NG108-15 cells induced with Bt₂cAMP were significantly higher than those of the undifferentiated cells. However, differentiation of PC12h cells induced with NGF did not enhance their extent or rate of KCl-stimulated⁴⁵Ca uptake. The effects of Ca agonist and antagonists indicated that the characteristics of KCl-stimulated⁴⁵Ca uptake by Bt₂cAMP-treated NG108-15 cells and NGF-treated PC12h cells mainly reflected those of peripheral L-type voltage-sensitive calcium channels activated by high KCl. These results suggest that differentiated neural cells did not all show an enhanced capacity for KCl-stimulated⁴⁵Ca uptake, although the characteristic patterns of differentiation (extension of neurite-like processes, etc.) and that of effect by Ca agonist or antagonists on NG108-15 cells and PC12h cells were similar.     

Rabies virus decreases agonist binding to opiate receptors of mouse neuroblastoma-rat glioma hybrid cells 108-CC-15

An acute and a persistent infection with Rabies virus (HEP Flury) was established in the CNS-derived cell line 108-CC-15 (NG 108-15). These cells possess specific membrane receptors to many hormones and neurotransmitters including opiate receptors. In both cases we found an increase in the dissociation constant (K_D) for the agonist ³H -etorphine as estimated by Scatchard plot analysis. However, in both cases there was no change in the number of opiate receptors per cell compared to uninfected cells. These studies complete our previous published observations of the impairment of receptor functions in rabies virus infected cells (1).     

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.     

Guanine Nucleotides Modulate Cortical S2 Serotonin Receptors

Abstract

Many radiolabelled receptors coupled to intracellular adenylate cyclase activity have been found to be modulated by physiological modulators such as GTP (guanosine triphosphate) and Gpp(NH)p (guanosine-imido-diphosphate). In particular, the apparent affinity of agonists competing for the binding of ³H-antagonist-labelled receptors is reduced in the presence of GTP and Gpp(NH)p. We report herein the agonist-specific effects of GTP and Gpp(NH)p on rat brain cortical S² serotonin receptors. The agonists serotonin, 5-methoxytryptamine, bufotenine, and tryptamine display threefold lower affinities for S₂ serotonin receptors in the presence of 10^-4M GTP or Gpp(NH)p than in the absence of the nucleotides. The antagonists spiperone, cinanserin, cyproheptadine and methysergide are unaffected by the guanine nucleotides. The Hill coefficients of the agonists increase from between 0.70–0.80 to 0.90–1.00 due to guanine nucleotides. ATP, ADP, and GDP have little or no effect. This pattern of guanine nucleotide effects has been found with receptors which are modulated by a guanine nucleotide regulatory protein and may indicate that the S₂ serotonin receptor may be coupled to intracellular adenylate cyclase activity.     

Binding of Aminoalkylindoles to Noncannabinoid Binding Sites in NG108-15 Cells

1. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma × glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharamcological characteristics of [³H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes.2. Although the K _D of specifid [³H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the B _max was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [³H]WIN 55212-2 binding.However, IC₅₀ values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the K _i values for CP55,940 were1.2nM in brain and >5000nM in NG108-15 membranes. Moreover, sodium and GTP--S decreased [³H]WIN 55212-2 binding in brain but not in NG108-15membranes.3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindolebinding sites are not coupled to G proteins.     

Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via alpha-adrenergic, cholinergic, and opiate receptors in neuroblastoma x glioma hybrid cells

Exposure of NG108-15 hybrid cells to islet-activating protein (IAP), pertussis toxin, caused strong ADP-ribosylation of one of the membrane proteins with a molecular weight of 41,000. This ADP-ribosylation was paralleled by decreases in the inhibition of cAMP accumulation in intact cells or associated with reversal of the inhibition of GTP-dependent membrane adenylate cyclase, via alpha-adrenergic, cholinergic muscarinic, or opiate receptors. The affinity of these receptors for agonists was lowered by guanyl-5'-yl beta-gamma-imidodiphosphate (Gpp(NH)p) reflecting their coupling to the guanine nucleotide regulatory protein in this cell line. This effect of Gpp(NH)p was lost in membranes of IAP-treated cells; in the absence of Gpp(NH)p, the affinity for agonist was lower in treated than in nontreated cells. In contrast, the function of these receptors to bind antagonists remained unaltered in IAP-treated cells. Thus, IAP treatment of NG108-15 cells caused specific uncoupling of negative signal transduction from inhibitory receptors to the adenylate cyclase catalytic unit via the guanine nucleotide regulatory protein, as a result of ADP-ribosylation of one of the subunits of the regulatory protein.     

Involvement of Both Inhibitory and Stimulatory Guanine Nucleotide Binding Proteins in the Expression of Chronic Opiate Regulation of Adenylate Cyclase Activity in NG108-15 Cells

Abstract: Chronic etorphine treatment of neuroblastoma × glioma NG108-15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108-15 cells is under tonic regulation by both guanine nucleotide regulatory (N_i) and stimulatory (N_s) components. Inactivation of N_i by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor-mediated activation of N_s, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E₁, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through N_i resulting in the unimpeded expression of N_s activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which N_i regulates adenylate cyclase in this cell line.     

Modification of neuroblastoma X glioma hybrid NG108-15 adenylate cyclase by vanadium ions

Vanadium ions activate as well as inhibit the activity of the NG108-15 adenylate cyclase $\text{in vitro}$ in the absence of any hormone. Below 5mM ion, ortho- and metavanadate activate; the maximal increase in activity is 2-fold. Vanadyl sulfate, at 0.1–0.1mM, activates to a similar magnitude as does vanadate over these concentrations; above 0.1mM, it inhibits. Activation of the enzyme by vanadate is not additive to that induced by PGE₁ or NaF. Vanadium ions do not alter the Ka for PGE₁-activation, nor the Ki for Dala met amide-inhibition, nor diminish the efficacy of opiate, muscarinic and alpha adrenergic regulation of the enzyme. However, the mechanisms by which NaF and vanadium ions activate must differ. Vanadium, unlike NaF, does not attenuate the ability of hormone receptors to direct inhibition of adenylate cyclase.     

Heterogeneity of Nucleotide Receptors in NG108-15 Neuroblastoma and C6 Glioma Cells for Mediating Phosphoinositide Turnover

Abstract: We have compared the characteristics of receptors for nucleotide analogues and the involvement of phospholipase C (PLC) in the effector mechanism in NG108-15 neuroblastoma and C6 glioma cells. The relative potency of these analogues to stimulate inositol phosphate (IP) formation is UTP > UDP ? 2-methylthio-ATP (2-MeSATP), GTP > ATP, CTP > ADP > UMP in NG108-15 cells and ATP > UTP > ADP > GTP > UDP ? 2Me-SATP, CTP, UMP in C6 glioma cells. α,β-Methylene-ATP, β,γ-methylene-ATP, AMP, and adenosine had little or no effect in both types of cells. The EC₅₀ values were 3 and 106 µM for UTP in NG108-15 and C6 glioma cells, respectively. The EC₅₀ value for ATP in C6 glioma cells was 43 µM. 2-MeSATP was threefold more potent than ATP in NG108-15 cells but had little effect in C6 glioma cells at 1 mM. In NCB-20 cells, a similar rank order of potency to that found in NG108-15 cells, i.e., UTP ? GTP > ATP > CTP, was observed. In both NG108-15 and C6 glioma cells, preincubation with ATP or UTP caused a pronounced cross-desensitization of subsequent nucleotide-stimulated IP production. ATP and UTP displayed no additivity in terms of IP formation at maximally effective concentrations. In contrast, endothelin-1, bradykinin, and NaF interacted in an additive manner with either nucleotide in stimulating PI hydrolysis. Pretreatment with pertussis toxin did not affect ATP-, UTP-, and GTP-stimulated IP generation in these cells, indicating that nucleotide receptors coupled to PLC by a pertussis toxin-resistant G protein in both cell types. Short-term treatment of the cells with protein kinase C (PKC) activators [phorbol 12-myristate 13-acetate (PMA) and octylindolactam V] produced a dose-dependent inhibition of ATP- and UTP-induced IP formation with a greater extent and higher susceptibility in C6 glioma cells than in NG108-15 cells. Furthermore, a 24-h exposure of the cells to PMA resulted in an obvious attenuation of nucleotide-induced IP formation in C6 glioma cells but failed to change the response in NG108-15 cells. These results suggest that distinct nucleotide receptors that respond to ATP and UTP with different selectivity exist in NG108-15 and C6 glioma cells. These heterogeneous nucleotide receptors coupled to PLC undergo discriminative modulation by PKC. NG108-15 and NCB-20 neuroblastoma are two cell lines that showed the highest specificity to extracellular UTP rather than ATP among the nucleotide receptors so far studied in various cells, suggesting the presence of a pyrimidine receptor in these cells.     

Modulation of sodium-sensitive GTPase by partial opiate agonists. An explanation for the dual requirement for Na+ and GTP in inhibitory regulation of adenylate cyclase

Opiates and opioid peptides inhibit adenylate cyclase and stimulate specific low Km GTPase activity in membranes from neuroblastoma x glioma NG108-15 hybrid cells. The effects of opiate agonists on both enzymes are mediated by high affinity stereospecific receptors and require Mg2+, GTP, and Na+. In the presence of Mg2+, Na+ inhibits basal GTPase activity; opiates stimulate GTP hydrolysis by antagonizing the Na+-induced inhibition. Activation of GTPase leads, in turn, to inactivation of GTP-stimulated adenylate cyclase activity. The intrinsic activities (or efficacies) of a series of opiates are identical for stimulation of GTPase and inhibition of adenylate cyclase. These results provide a mechanism for the dual requirement for Na+ and GTP in the inhibitory coupling of opiate receptors to the adenylate cyclase system in these cells and may be of general significance to the action of other inhibitory hormones.     

Modulation of human myometrial PGE2 receptor by GTP characterization of receptor subtype

We studied PGE₂ specific binding sites in human myometrial microsomes prepared from uterine specimens obtained by hysterectomy (women between 38 and 55 years of age). Competition experiments showed that the potency order for various prostaglandins (PGs) was : PGE₂ ≥ PGE₁ PGF_2α > Iloprost ≥ Carbacyclin ZK 110841 (PGD₂ analogue). These relative affinities indicated that the receptor was of the EP type.In kinetic experiments GTP, GppNHp and GTPγS increased the rate of PGE₂ binding (steady state was reached more rapidly in the presence of nucleotides) but maximal specific binding was not significantly different. Complete dissociation could not be obtained, even in the presence of GTP. Only 50% of maximal binding was readily dissociable. The dissociation rate was 4.56.10⁻⁴ sec⁻¹ (half time of about 660 sec) and in the presence of GTP analogues it was slightly increased (k−1 = 7.16 10⁻⁴ sec⁻¹ half time 420 sec.). Scatchard analysis of saturation curves showed an increase in ligand receptor affinity in the presence of GTP or nucleotide analogues: the Kd shifted from 9.66 ± 2.8.10⁻⁹ M to 4.96 ± 1.25.10⁻⁹M, but the number of binding sites did not change significantly (310 ± 37 to 350 ± 17 fmol/mgP). The effect of GTP was observed at a concentration of 5.10⁻⁴M. GppNHp and GTPγS were effective at 1.10⁻⁵M. Pretreatment of myometrial membranes with pertussis or cholera toxins had no effect on PGE₂ binding to membrane sites. Our conclusion is that GTP induced conversion of a population of low affinity sites into a population of higher affinity sites. This effect of guanine nucleotides was described in adipocytes and kidney medulla.Competition studies with PGE₂ analogues (sulprostone, 17-phenyl-ω-trinor PGE₂, M&B 28,767, misoprostol, butaprost) showed that this receptor mediates a contractile response and is probably an EP₃ subtype.     

Down regulation of enkephalin (δ) receptors Demonstration in membrane-bound and solubilized receptors

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue, d-Ala²-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to δ-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the α₂-adrenergic ligand, [³H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of δ-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.