Biochemical Evidence of Functional Interaction Between μ- and δ-Opioid Receptors in SK-N-BE Neuroblastoma Cell Line

Abstract: Radioligand binding assays and functional experiments revealed that the SK-N-BE neuroblastoma cell line expresses a similar ratio of μ- and δ-opioid receptors, both negatively coupled to adenylyl cyclase through pertussis toxin-sensitive G proteins. Our findings also indicate that some functional interaction occurred between the two opioid subtypes; in fact, long-term exposure to [ d -Ala²- N -methyl-Phe⁴-Gly-ol⁵]enkephalin (DAMGO), a μ-selective agonist, sensitized the functional response of the δ-selective agonist but not vice versa. It is interesting that in acute interaction experiments, we observed a shift to the right of the concentration-effect curve of either DAMGO or [ d -Pen^2,5]enkephalin (DPDPE), a δ-selective agonist, as a result of DPDPE or DAMGO administration, respectively. In addition, low doses of naloxone, an antagonist selective for μ receptors, increased the inhibitory effect of [ d -Ala², d -Met⁵]enkephalinamide (DAME), a mixed μ/δ agonist, on adenylyl cyclase activity. Taken overall, these data support the hypothesis of the existence of a cross talk between μ and δ receptors in the SK-N-BE cell line.     

Calcium Independence of Phosphoinositide Hydrolysis-Induced Increase in Cyclic AMP Accumulation in SK-N-SH Human Neuroblastoma Cells

Previous work has shown that stimulation of muscarinic receptors in various cell lines increases intracellular cyclic AMP (cAMP) levels. This unusual response has been hypothesized to be mediated by stimulation of calcium/calmodulin-sensitive adenylate cyclase, secondary to inositol trisphosphate (IP3)-mediated calcium mobilization. To test this hypothesis, we stimulated muscarinic receptors in SK-N-SH human neuroblastoma cells while blocking the IP3-mediated rise in intracellular calcium concentration using two different methods. Loading cells with the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the carbachol-mediated intracellular calcium release without abolishing the carbachol-mediated increase in cAMP level. Similarly, in cells preexposed to carbachol, the agonist-induced change in intracellular calcium level was blocked, but the cAMP response was not. Thus, both of these methods failed to block the muscarinic receptor-mediated increase in cAMP level, thereby demonstrating that this cAMP level increase is not mediated by a detectable rise in intracellular calcium concentration.     

The δ-Opioid Receptor Regulates Activity of Ryanodine Receptors in the Human Neuroblastoma Cell Line SK-N-BE

Abstract: Recent studies have demonstrated that opioid agonists affect the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) either by regulating plasma membrane Ca²⁺-channel activity or by mobilizing intracellular Ca²⁺ stores. The present report documents the [Ca²⁺]_i increase induced by opioid agonists in a human neuroblastoma cell line, SK-N-BE, expressing δ-opioid receptors. In the presence, as well as in the absence, of extracellular Ca²⁺, opioid agonists enhanced significantly [Ca²⁺]_i, whereas carbachol, known to mobilize specifically inositol 1,4,5-trisphosphate-sensitive intracellular Ca²⁺ stores, acted only in the presence of extracellular Ca²⁺. The opioid-induced increase in [Ca²⁺]_i was not affected by treatments modifying the trimeric G_i, G_o, and G_s protein transduction mechanisms or the activity of adenylyl cyclase. The Ca²⁺-ATPase pump-inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid-induced [Ca²⁺]_i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK-N-BE cells, δ-opioid receptors mobilize Ca²⁺ from intracellular ryanodine-sensitive stores and the mechanism involved is independent of G_i/G_o and G_s proteins and protein kinase A activation.     

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.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

Differentiation of Human Neuroblastoma Cells: Marked Potentiation of Prostaglandin E-Stimulated Accumulation of Cyclic AMP by Retinoic Acid

Neuroblastoma cells in culture contain low levels of cyclic AMP, a second messenger which plays a major role in neuronal maturation. In this study, human neuroblastoma cells, SK-N-SH-SY5Y, were induced to differentiate by treatment with either nerve growth factor (50 ng/ml), retinoic acid (10 microM), dibutyryl cyclic AMP (1 mM), or 12-O-tetradecanoylphorbol-13-acetate (0.1 microM), and the ability of several neurotransmitters or hormones to stimulate adenylyl cyclase was tested. Although all four differentiation factors caused morphological changes towards a neuronal phenotype, only retinoic acid dramatically enhanced cyclic AMP accumulation, specifically upon stimulation with prostaglandin E1 (PGE1). PGE2 was also active, but less potent, than PGE1, whereas the other cyclic AMP-stimulating agents tested were largely unaffected. Further, the rapid desensitization of the PGE1-cyclic AMP response observed in control cells after 20 min of PGE1 exposure did not occur in retinoic acid-treated cells, and the EC50 values for PGE1 were reduced from approximately 240 to 14 nM after retinoic acid treatment. The increased sensitivity to PGE was associated with an increase of high-affinity PGE1 binding sites, whereas the Gs coupling proteins and adenylyl cyclase were not measurably affected. A similar enhancement of the PGE1-cyclic AMP response by retinoic acid was also observed in two additional human neuroblastoma cell lines tested, Kelly and IMR-32, suggesting that up-regulation of the prostaglandin response by retinoic acid is common among neuroblastoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of μ-Opioid Receptor in Neural Cells by Extracellular Sodium

Abstract: SH-SY5Y neural cells expressing μ- and δ-opioid receptors were maintained viable in isotonic, sodium-free buffer in vitro. Intracellular sodium levels were manipulated by various methods, and ligand binding to intact cells was studied. In physiological buffer containing 118 mM sodium, [³H]Tyr-d -Ala-Gly-(Me)Phe-Gly-ol ([³H]-DAMGO) and [³H]naltrexone bound to μ receptor with K_D values of 3.1 and 0.32 nM and B_max values of 94 and 264 fmol/mg of protein, respectively. Replacement of sodium by choline decreased the affinity of the antagonist and increased B_max for [³H]DAMGO, without significantly affecting the other corresponding binding parameters. Depolarizing concentrations of KCl (34 mM) in physiological buffer decreased the intracellular sodium levels by 67%, but this did not decrease the [³H]DAMGO binding to the cells. Incubation of cells with monensin and ouabain increased the intracellular sodium levels dramatically (from 78 to 250 and 300 nmol/mg, respectively), with no changes in agonist binding parameters. Ethylisopropylamiloride inhibited [³H]DAMGO and [³H]naloxone binding to intact cells with EC₅₀ values of 24 and 3,600 nM, respectively. Adenylyl cyclase activities measured in intact cells, at different concentrations of sodium, showed the physiological significance of this ion in signal transduction. Potency of DAMGO in inhibiting the forskolin-stimulated adenylyl cyclase activity was significantly higher at lower concentrations of sodium. However, inhibition reached the maximal level only at 50 mM sodium, and typical sigmoidal dose-response curves were obtained only in the presence of 118 mM sodium. Furthermore, even at low or high intracellular sodium levels, DAMGO inhibition of cyclic AMP levels was normal. These results support a role for extracellular sodium in regulating not only the ligand interactions with the receptor, but also the signal transduction through the μ receptor.     

Activation of Type II Adenylyl Cyclase by the Cloned μ-Opioid Receptor: Coupling to Multiple G Proteins

Abstract: Opioid receptors are multifunctional receptors that utilize G proteins for signal transduction. The cloned δ-opioid receptor has been shown recently to stimulate phospholipase C, as well as to inhibit or stimulate different isoforms of adenylyl cyclase. By using transient transfection studies, the ability of the cloned μ-opioid receptor to stimulate type II adenylyl cyclase was examined. Coexpression of the μ-opioid receptor with type II adenylyl cyclase in human embryonic kidney 293 cells allowed the μ-selective agonist, [d -Ala², N-Me-Phe⁴,Gly⁵-ol]enkephalin, to stimulate cyclic AMP accumulation in a dose-dependent manner. The opioid-induced stimulation of type II adenylyl cyclase was mediated via pertussis toxin-sensitive G_i proteins, because it was abolished completely by the toxin. Possible coupling between the μ-opioid receptor and various G protein α subunits was examined in the type II adenylyl cyclase system. The opioid-induced response became pertussis toxin-insensitive and was enhanced significantly upon co-expression with the α subunit of G_z, whereas those of G_q, G₁₂, or G₁₃ inhibited the opioid response. When pertussis toxin-sensitive G protein α subunits were tested under similar conditions, all three forms of α_i and both forms of α_o were able to enhance the opioid response to various extents. Enhancement of type II adenylyl cyclase responses by the co-expression of α subunits reflects a functional coupling between α subunits and the μ-opioid receptor, because such potentiations were not observed with the constitutively activated α subunit mutants. These results indicate that the μ-opioid receptor can couple to G_i1–3, G_o1–2, and G_z, but not to G_s, G_q, G₁₂, G₁₃, or G_t.     

α2-Adrenergic and Muscarinic Cholinergic Receptors Have Oposing Actions on Cyclic AMP Levels in SK-N-SH Human Neuroblastoma Cells

Forskolin and vasoactive intestinal polypeptide (VIP) were shown to increase cyclic AMP accumulation in a human neuroblastoma cell line, SK-N-SH cells. The alpha 2-adrenergic agonist UK 14304 decreased forskolin-stimulated cyclic AMP levels by 40 +/- 2%, with an EC50 of 83 +/- 20 nM. This response was blocked by pretreatment with pertussis toxin (PT) (EC50 = 1 ng/ml) or by the alpha 2-antagonists yohimbine, idazoxan, and phentolamine. Antagonist IC50 values were 0.3 +/- 0.1, 2.2 +/- 0.3, and 1.4 +/- 0.1 microM, respectively. This finding suggests the presence of normal inhibitory coupling of SK-N-SH cell alpha 2-adrenergic receptors to adenylate cyclase via the inhibitory GTP-binding protein species, Gi. Muscarinic receptors in many target cell types are coupled to inhibition of adenylate cyclase. However, in SK-N-SH cells, muscarinic agonists synergistically increased (67-95%) the level of cyclic AMP accumulation elicited by forskolin or VIP. EC50 values for carbamylcholine (CCh) and oxotremorine facilitation of the forskolin response were 1.2 +/- 0.2 and 0.3 +/- 0.1 microM, respectively. Pharmacological studies using the muscarinic receptor subtype-preferring antagonists 4-diphenylacetoxy-N-methylpiperidine, pirenzepine, and AF-DX 116 indicated mediation of this response by the M3 subtype. IC50 values were 14 +/- 1, 16,857 +/- 757, and 148,043 +/- 16,209 nM, respectively. CCh-elicited responses were unaffected by PT pretreatment. Muscarinic agonist binding affinity was indirectly measured by the ability of CCh to compete for [3H]quinuclidinyl benzilate binding sites on SK-N-SH cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic Receptor-Stimulated Phosphoinositide Turnover in Human SK-N-SH Neuroblastoma Cells: Differential Inhibition by Agents that Elevate Cyclic AMP

The possibility that an increased intracellular concentration of cyclic AMP (cAMP) can regulate the extent of muscarinic receptor-stimulated phosphoinositide (PPI) turnover in the human neuroblastoma cell line SK-N-SH was examined. Addition of either forskolin (or its water-soluble analog, L-85,8051), theophylline, isobutylmethylxanthine, or cholera toxin, agents that interact with either the catalytic unit of adenylate cyclase, cAMP phosphodiesterase, or the guanine nucleotide binding protein linked to adenylate cyclase activation, resulted in a 45-181% increase in cAMP concentration and a 27-70% inhibition of carbachol-stimulated inositol phosphate release. Through the use of digitonin-permeabilized cells, the site of inhibition was localized to a step at, or distal to, the guanine nucleotide binding protein that regulates phospholipase C activity. In contrast, when intact SK-N-SH cells were exposed to prostaglandin E1, the ensuing increases in cAMP were not accompanied by an inhibition of stimulated PPI turnover. These differential effects of increased cAMP concentrations on stimulated PPI turnover may reflect the compartmentation of cAMP within SK-N-SH cells.     

Effects of Vasoactive Intestinal Peptide and Other Peptides on Cyclic AMP Accumulation in Intact Pieces and Isolated Horizontal Cells of the Teleost Retina

The effects of vasoactive intestinal peptide (VIP) and several other peptides have been examined on cyclic AMP accumulation in intact pieces and isolated horizontal cells of the teleost (carp) retina. VIP was the most effective peptide examined, inducing a dose-related response, and an approximately fivefold increase in cyclic AMP production when used at a concentration of 10 microM. Porcine histidine isoleucine-containing peptide and secretin, peptides structurally related to VIP, also stimulated cyclic AMP accumulation, but at concentrations of 10 microM induced responses which were only approximately 40% and 10%, respectively, of the response observed with 10 microM VIP. In contrast, several other peptides, including glucagon, neurotensin, somatostatin, luteinizing hormone-releasing hormone, alpha-melanocyte-stimulating hormone, cholecystokinin octapeptide26-33, gastrin-releasing peptide, thyrotropin-releasing hormone, and VIP10-28 were totally inactive. The response to 10 microM VIP was not antagonized by several dopamine antagonists, indicating the presence of a population of specific VIP receptors coupled to adenylate cyclase, distinct from the population of dopamine receptors coupled to adenylate cyclase also known to be present in this tissue. Finally, experiments involving the use of fractions of isolated horizontal cells indicate that these neurons possess a population of VIP receptors coupled to cyclic AMP production which would appear to share a common pool of adenylate cyclase with a population of similarly coupled dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased Cyclic AMP Degradation in NG 108-15 Neuroblastoma × Glioma Hybrid Cells and S49 Lymphoma Cells Chronically Treated with Drugs that Inhibit Adenylate Cyclase

The increase in hormone-stimulated cyclic AMP accumulation observed in a variety of intact cells after chronic pretreatment with drugs that inhibit adenylate cyclase activity has been attributed to an increase in adenylate cyclase activity following withdrawal of the inhibitory drug. In NG 108-15 mouse neuroblastoma X rat glioma hybrid cells (NG cells) chronically treated with the muscarinic cholinergic agonist carbachol, we have found a significant decrease in the apparent degradation rate constant for cyclic AMP, in addition to an increase in the prostaglandin E1 (PGE1)-stimulated cyclic AMP synthesis rate in intact cells. In carbachol-pretreated NG cells that were stimulated with a maximally effective dose of PGE1, and that accumulated steady-state cyclic AMP concentrations fourfold or more higher than in control cells, the apparent rate constant for degradation was about 53% lower than the value for control cells. In carbachol-pretreated cells stimulated with a submaximal dose of PGE1 to yield a steady-state cyclic AMP concentration comparable to control cells, the apparent rate constant was 31% lower than the value for control cells. In S49 mouse lymphoma cells (S49 cells) chronically treated with an analog of the inhibitory agonist somatostatin, the first-order rate constant for cyclic AMP degradation in intact cells following isoproterenol stimulation was 29% lower than the value for control cells. Despite these changes in the kinetics of cyclic AMP degradation in intact NG cells and S49 cells, there was either no change or a minimal change (less than 10%) in phosphodiesterase activities assayed in extracts of cells chronically exposed to inhibitory drugs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Multiple Effectors by the Cloned δ-Opioid Receptor: Stimulation of Phospholipase C and Type II Adenylyl Cyclase

Abstract: The δ-opioid receptor is known to regulate multiple effectors in various tissues. When expressed in human embryonic kidney 293 cells, the cloned δ-opioid receptor inhibited cyclic AMP (cAMP) accumulation in response to the δ-selective agonist [ d -Pen², d -Pen⁵]enkephalin. The inhibitory response of [ d -Pen², d -Pen⁵]enkephalin was dependent on the expression of the δ-opioid receptor and exhibited an EC₅₀ of 1 n M . The receptor showed ligand selectivity and a pharmacological profile that is appropriate for the δ-opioid subtype. The inhibition was blocked by the opiate antagonist naloxone or by pretreatment of the cells with pertussis toxin. Cotransfection of the δ-opioid receptor with type II adenylyl cyclase and an activated mutant of α_s converted the δ-opioid signal from inhibition to stimulation of cAMP accumulation. It is interesting that when transfected into Ltk⁻ fibroblasts, the cloned δ-opioid receptor was able to stimulate the formation of inositol phosphates (EC₅₀ = 8 n M ). This response was sensitive to pertussis toxin. The opioid-mediated formation of inositol phosphates exhibited the same ligand selectivity as seen with the inhibition of cAMP accumulation. The ability of the δ-opioid receptor to couple to G proteins other than G_i was also examined. Cotransfection studies revealed that the δ-opioid receptor can utilize G_z to regulate cAMP accumulation and to stimulate the formation of inositol phosphates.     

Effects of C-Terminal Truncation of the Recombinant δ-Opioid Receptor on Phospholipase C and Adenylyl Cyclase Coupling

Abstract: Opioid receptors belong to the superfamily of guanine nucleotide binding (G) protein-coupled receptors. There is now growing evidence in support of a stimulatory coupling of opioid receptors to phospholipase C (PLC), via a pertussis toxin-sensitive G protein, leading to the generation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]. We have generated two C-terminal truncation mutants of the δ-opioid receptor lacking the final 15 or 37 amino acids and examined their coupling to PLC and adenylyl cyclase. d -[Pen^2,5]-enkephalin (DPDPE) mediated Ins(1,4,5)P₃ formation and cyclic AMP inhibition was measured in whole cells and assayed using radioreceptor mass assays. DPDPE produced a time- and dose-dependent increase in Ins(1,4,5)P₃ mass formation in Chinese hamster ovary (CHO) cells expressing the δ_wt, δ₁₅, and δ₃₇ receptors. As the C terminus was truncated, the time to maximum stimulation (15 s in CHOδ_wt, 60 s in CHOδ₁₅, and 120 s in CHOδ₃₇) increased and removal of the C terminus resulted in a prompt return to basal Ins(1,4,5)P₃ levels. Whereas the dose-response curves to Ins(1,4,5)P₃ formation and cyclic AMP inhibition remained largely unaffected by C-terminal truncation, there were large differences in the pEC/IC₅₀ values, with cyclic AMP inhibition being the more potent, perhaps indicating G_iα coupling to adenylyl cyclase and G_iβ/γ coupling to PLC. Collectively, these data indicate that the C terminus of the δ-opioid receptor is unimportant in the acute coupling to adenylyl cyclase but may have a role to play in PLC coupling. We hypothesize that an intact C terminus is required to allow normal “strong” coupling of receptor to G_i and that truncation weakens this link as reflected in an increased time to peak. In addition, if the coupling is weak, the acute response to agonist stimulation rapidly uncouples.     

Differential Effect of Intrastriatal Kainic Acid on the Modulation of Dopamine Release by μ- and δ-Opioid Peptides: A Microdialysis Study

The present study investigated the effects of a striatal lesion induced by kainic acid on the striatal modulation of dopamine (DA) release by mu- and delta-opioid peptides. The effects of [D-Pen2,D-Pen5]-enkephalin (DPDPE) and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO), two highly selective delta- and mu-opioid agonists, respectively, were studied by microdialysis in anesthetized rats. In control animals both opioid peptides, administered locally, significantly increased extracellular DA levels. The effects of DPDPE were also observed in animals whose striatum had been previously lesioned with kainic acid. In contrast to the effects of the delta agonist, the significant increase induced by DAGO was no longer observed in lesioned animals. These results suggest that delta-opioid receptors modulating the striatal DA release, in contrast to mu receptors, are not located on neurons that may be lesioned by kainic acid.     

Regulation of Spontaneous Activity of the δ-Opioid Receptor: Studies of Inverse Agonism in Intact Cells

Abstract: Adenylyl cyclase activity was measured following labelling of the cellular ATP pool with [³H]adenine in intact Rat-1 fibroblasts that had been stably transfected to express the murine δ-opioid receptor (clone D2). Basal [³H]cyclic AMP accumulation was low and was increased substantially by the addition of the diterpene forskolin. The synthetic enkephalin d -Ala², d -Leu⁵ enkephalin (DADLE) produced strong inhibition of forskolin-amplified [³H]cyclic AMP production, whereas the δ-opioid ligand ICI174864 augmented forskolin-amplified adenylyl cyclase activity. Naloxone was unable to mimic the effects of ICI174864, and coincubation of the cells with these two ligands attenuated the effect of ICI174864. The EC₅₀ (9.4 ± 0.6 × 10⁻⁸ M ) for ICI174864 augmentation of forskolin-stimulated adenylyl cyclase was equal to its estimated K _i. Pertussis toxin pretreatment of clone D2 cells prevented both this effect of ICI174864 and the inhibition produced by DADLE. Use of a Cytosensor microphysiometer demonstrated that treatment of clone D2 cells with DADLE increased and that with ICI174864 decreased the basal rate of cellular proton extrusion. By using these two distinct experimental strategies, ICI174864 was shown to function in a manner anticipated for an inverse agonist, demonstrating that such effects can be observed in intact cells and are not restricted to assays performed on membrane preparations.     

Regulation of Cyclic AMP Accumulation by Peptide Hormone Receptors in Immunocytochemically: Defined Astroglial Cells

Primary cultures of neonatal murine brain have been reported to express multiple receptors that regulate adenylate cyclase activity. Since for the most part these results were obtained with mixed cell cultures, it has been difficult to define receptor profiles for specific cell types. With this concern in mind a series of studies has been initiated designed to identify specific receptors present on highly purified, immunocytochemically defined astroglia derived from the cerebral cortices of neonatal rats. In this study the capacity of a variety of peptide hormones to regulate cyclic AMP metabolism in these cells was examined. Fibroblasts derived from the meninges represent a predictable source of contamination in primary CNS culture. Thus, to assign more clearly specific receptors to the astroglial cell population, receptor-mediated regulation of cyclic AMP accumulation was also examined in fibroblasts. Cyclic AMP accumulation in astroglia was stimulated by catecholamines (acting at beta 1-adrenergic receptors), prostaglandin E1, vasoactive intestinal polypeptide, alpha-melanocyte-stimulating hormone, and adrenocorticotropin. Bombesin, luteinizing hormone-releasing hormone, neurotensin, thyrotropin-releasing hormone, somatostatin, secretin, and vasopressin did not significantly increase cyclic AMP levels in these cultures. Catecholamines, acting at alpha 2-adrenergic receptors, and somatostatin inhibited agonist-stimulated cyclic AMP accumulation. In meningeal cell cultures catecholamines (acting at beta 2- and alpha 2-adrenergic receptors) and prostaglandin E1 regulated cyclic AMP levels. However, vasoactive intestinal peptide did not stimulate and somatostatin did not inhibit cyclic AMP accumulation in these cells.     

GABAB Receptor-Mediated Enhancement of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Production in Slices of Rat Cerebral Cortex

Basal and vasoactive intestinal peptide (VIP)-stimulated accumulations of cyclic AMP were measured in slices of rat cerebral cortex. Neither gamma-aminobutyric acid (GABA) nor the selective GABAB receptor agonist (-)-baclofen stimulated basal cyclic AMP accumulation, whereas VIP caused a large dose-dependent increase in cyclic AMP levels. However, in the presence of 100 microM (-)-baclofen, the effects of VIP on cyclic AMP accumulation were significantly enhanced, with the responses to 1 microM and 10 microM VIP being approximately doubled. The enhancing effects of (-)-baclofen was dose related (1-1,000 microM), but an enhancing effect was not observed with 100 microM (+)-baclofen. In the presence of the GABA uptake inhibitor nipecotic acid (1 mM), GABA caused a similar dose-related enhancement of the VIP response. The ability of either GABA or (-)-baclofen to augment VIP-stimulated production of cyclic AMP was not mimicked by the GABAA, agonists isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and was not antagonized by the GABAA antagonist bicuculline. The putative GABAB antagonist 5-aminovaleric acid (1 mM) significantly reduced the effect of (-)-baclofen. The ability of (-)-baclofen to enhance VIP-stimulated accumulation of cyclic AMP was observed in slices of rat cerebral cortex, hippocampus, and hypothalamus. These results indicate that GABA and (-)-baclofen can enhance VIP-stimulated accumulation of cyclic AMP in rat brain slices via an interaction with specific GABAB receptors.