Neuroblastoma × Glioma Hybrid Cells Synthesize Enkephalin-Like Opioid Peptides

Partially purified extracts from neuroblastoma X glioma hybrid cells 108CC15 inhibit, like opioids, the prostaglandin E1-evoked formation of cyclic AMP in a dose-dependent manner in the same hybrid cells. The inhibition is prevented by the opioid antagonist naloxone. In addition, the same extract competes with [3H]naloxone and [3H]Leu-enkephalin for binding to opioid receptors of hybrid cell membranes and to a specific antiserum, respectively. The opioid activity in the extracts is destroyed by carboxypeptidase A and leucine aminopeptidase, but not by trypsin. Further purification of the extracts by HPLC, TLC, or high-voltage paper electrophoresis reveals in each case two active fractions which behave like Met- and Leu-enkephalin. The Met-enkephalin-like, but not the Leu-enkephalin-like, fraction is inactivated by treatment with BrCN. Dimethylaminonaphtylsulfonyl (dansyl) derivatives of Met- and Leu-enkephalin correspond to [3H]dansyl derivatives of Met-like substances from hybrid cells. Three to four times as much Met-enkephalin-like as Leu-enkephalin-like material is present in the extract. The overall concentration of opioid peptides in the hybrid cells varies between 0.03 and 1.0 pmol Leu-enkephalin equivalents per mg protein. The amount of opioids in the hybrid cells is strongly dependent on the cell density. The findings suggest that neuroblastoma X glioma hybrid cells contain opioid peptides that are very similar, if not identical, to Met- and Leu-enkephalin. Opioid activity can also be detected in other neuronal cell lines and even in glioma cells.     

Opioid binding site in EL-4 thymoma cell line

Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of [3H] bremazocine indicated a single site with a KD = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10(6) cells (51 pmols/mg total cell proteins). To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of [3H] bremazocine with an IC50 value = 0.57 microM. The two stereoisomers levorphanol and dextrorphan showed the same affinity for this site (IC50 = 2.9 microM and 1.9 microM respectively). While morphine, [D-Pen2, D-Pen5] enkephalin and beta-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC50 = 60 microM, that was similar to naloxone (IC50 = 69 microM).     

Subcellular Compartmentation of Opioid Receptors: Modulation by Enkephalin and Alkaloids

Abstract: A subclone of NG108–15 neuroblastoma-glioma hybrid cells was used to study the intracellular distribution of opioid receptors. Subcellular organelles were separated on self-generating Percoll-sucrose gradients and the enzymes β-glucuronidase, galactosyltransferase, 5′-nucleotidase, and glucose-6-phosphatase were used as markers to localize the various structures. Analysis of the receptor distribution from untreated cells shows that the plasma membranes contained the highest receptor density, but a significant portion of the opioid binding sites was unevenly distributed between the lysosomes, microsomes, and Golgi elements. The enzyme markers indicated that appearance of opioid receptors in these intracellular structures does not result merely from contamination with plasma membranes. About 11% of the receptors appeared in a fraction lighter than plasma membranes. The antilysosomal agent chloroquine altered the intracellular compartmentation of the receptors, possibly by blocking their translocation in the cells. Leu-enkephalin induced time-dependent loss of receptors from all four intracellular compartments examined, but a kinetic analysis showed that the rate of receptor loss in these fractions was not identical. Thus, the percent of receptors appearing in the lysosomal fraction that could still bind [³H]-D-Ala²D-Leu⁵-enkephalin in vitro was increased on treatment with Leu-enkephalin. As an additional approach to follow the intracellular fate of the receptors, cells were labeled with [³H]diprenorphine, chased with various unlabeled opiates, and the distribution of ³H-ligand-receptors in the cells was monitored. Leu-enkephalin and etorphine altered the distribution of receptor-bound [³H]diprenorphine between the plasma membranes, lysosomes, and Golgi elements, whereas morphine had no such effect. The study sheds light on the role of intracellular structures in the metabolism of opioid receptors in untreated and opioid-treated cells.     

Differential Regulation by Butyrate and Dibutyryl Cyclic AMP of δ-Opioid, α2-Adrenergic, and Muscarinic Cholinergic Receptors in NCB-20 Cells

Long-term treatment of NCB-20 cells with sodium butyrate resulted in a marked increase in the specific binding of [3H]D-Ala2,D-Leu5 enkephalin. This increase was concentration and time dependent, with an EC50 of about 480 microM and a maximal effect detected after 3-day treatment. At saturating concentration of butyrate (1 mM) the increase was three- to fourfold of the untreated control. Scatchard analysis revealed that the butyrate effect was due to an increase in the density of the opioid receptor binding sites. Butyrate also induced a smaller (about twofold) increase in the density of muscarinic cholinergic receptor binding assessed by using [3H]quinuclidinyl benzilate, whereas alpha 2-adrenergic receptor binding assessed by using [3H]clonidine was not significantly affected. The butyrate-induced opioid receptor binding could be totally abolished by the presence of cycloheximide, suggesting that the butyrate effect involves synthesis of the receptor protein. Butyrate treatment did not affect basal and prostaglandin E1-stimulated cyclic AMP levels but caused a three- to fourfold decrease in the IC50 of D-Ala2,D-Leu5 enkephalin for attenuating these cyclic AMP levels and approximately 25% increase in the maximal extent of attenuation. In contrast to the butyrate effect, long-term treatment of NCB-20 cells with 1 mM dibutyryl cyclic AMP induced an 80% decrease in the opioid and alpha 2-adrenergic receptor bindings and a 57% loss of muscarinic cholinergic receptor binding. This down-regulation of muscarinic cholinergic receptor binding sites was associated with a 35% decrease of carbachol-induced phosphoinositide breakdown, whereas the receptor up-regulation induced by butyrate was found to increase the carbachol response by about threefold. The differential regulation by butyrate and dibutyryl cyclic AMP suggests that the butyrate effect is mediated by a mechanism independent of intracellular cyclic AMP. The induction by butyrate of opioid-receptors and muscarinic cholinergic receptors in NCB-20 cells may provide a useful system for studying the regulation of gene expression of these receptor proteins.     

Opioid peptides derived from food proteins. The exorphins.

Peptides with opioid activity are found in pepsin hydrolysates of wheat gluten and alpha-casein. The opioid activity of these peptides was demonstrated by use of the following bioassays: 1) naloxone-reversible inhibition of adenylate cyclase in homogenates of neuroblastoma X-glioma hybrid cells; 2) naloxone-reversible inhibition of electrically stimulated contractions of the mouse vas deferens; 3) displacement of [3H]dihydromorphine and [3H-Tyr, dAla2]met-enkephalin amide from rat brain membranes. Substances which stimulate adenylate cyclase and increase the contractions of the mouse vas deferens but do not bind to opiate receptors are also isolated from gluten hydrolysates. It is suggested that peptides derived from some food proteins may be of physiological importance.     

An Assessment of the Role of Opioid Receptors in the Response to Cannabimimetic Drugs

Cannabimimetic drugs have been shown to inhibit adenylate cyclase activity in N18TG2 neuroblastoma cells. This investigation examines the possible role of opioid receptors in the cannabimimetic response. Opioid receptors of the delta subtype were found on N18TG2 membranes using [3H]D-Ala2-D-Leu5-enkephalin. No mu or kappa receptors were detected using selective ligands for these sites. The delta binding affinity and capacity were unaltered by cannabimimetic drugs. To test if cannabimimetic drugs may modulate opioid effector mechanisms, cyclic AMP metabolism was determined in intact cells and in membranes. N18TG2 adenylate cyclase was inhibited by the cannabimimetic drugs delta 9-tetrahydrocannabinol and desacetyllevonantradol, and by the opioid agents morphine, etorphine, and D-Ala2-Met5-enkephalinamide. The opioid inhibition was reversed by naloxone and naltrexone; however, the cannabimimetic response was unaffected. Both cannabimimetic and opioid drugs decreased cyclic AMP accumulation in intact cells, but opioid antagonists blocked the response only to the latter. Thus, cannabimimetic effects are observed even though opioid receptors are blocked by antagonist drugs. The interaction between desacetyllevonantradol and etorphine was neither synergistic nor additive at maximal concentrations, suggesting that these two drugs operate via the same effector mechanism. Other neuronal cell lines having an opioid response were also examined. The cannabimimetic inhibition of cyclic AMP accumulation in NG108-15 neuroblastoma X glioma cells was not as great as the response in N18TG2. N4TG1 neuroblastoma cells did not respond to cannabimimetic drugs under any conditions tested. Thus, the cannabimimetic inhibition of adenylate cyclase is not universally observed, and the efficacy of the cannabimimetic response does not correlate with the efficacy of the opioid response.     

The effect of GABA on in vitro binding of two novel non-benzodiazepines, PK 8165 and CGS 8216, to benzodiazepine receptors in the rat brain

The effect of gamma-aminobutyric acid (GABA) on the binding of PK 8165, a quinoline derivative, and CGS 8216, a pyrazoloquinoline, was assessed in two different regions of the rat brain. PK 8165, a compound with reported anxiolytic properties, inhibited [3H]-propyl beta-carboline-3-carboxylate labeled receptors in the cerebellum with an IC50 of 844 nM and 370 nM in the absence and presence of micro M GABA, respectively. GABA (100 micro M) was less effective in the cerebral cortex, decreasing the IC50 value from 280 to 197 nM. In saturation isotherm studies with [3H]-CGS 8216, a benzodiazepine receptor antagonist, GABA (100 micro M) induced a small but significant reduction in the apparent affinity of [3H]-CGS 8216 for benzodiazepine receptors in the cerebral cortex but the Bmax was unchanged.     

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.     

Transferred nuclear Overhauser effect analyses of membrane-bound enkephalin analogues by 1H nuclear magnetic resonance: correlation between activities and membrane-bound conformations

Leu-enkephalin, [D-Ala2]Leu-enkephalin, and [D-Ala2]Leu-enkephalinamide (agonists) and [L-Ala2]Leu-enkephalin (inactive analogue) bind to lipid bilayer consisting of phosphatidylcholine and phosphatidylserine. The conformations that these compounds assume, once bound to perdeuterated phospholipid bilayer, have been shown to be unique, as shown by the transferred nuclear Overhauser effect (TRNOE) of 1H NMR spectroscopy. In addition, their location in the bilayer was analyzed by TRNOE in the presence of spin-labeled phospholipids. These analyses showed a clear relationship between the activity and the peptide-membrane interaction. The three active peptides, when bound to membranes, adopt the same conformation, characterized by a type II' beta-turn around Gly3-Phe4 and a gamma-turn around Gly2 (or D-Ala2). The inactive analogue, [L-Ala2]Leu-enkephalin, displayed a completely different TRNOE pattern corresponding to a different conformation in the membrane-bound state. The tyrosine residue of the active compounds is not inserted into the interior of membrane, but it is inserted into the bilayer for the L-Ala2 analogue. According to these results, [L-Ala2]Leu-enkephalin may be explained to be inactive because the mode of binding to the membranes is different from that of active compounds.     

Specific opioid binding sites for dermorphin in rat brain. A radioreceptor assay using the tritiated hormone as primary ligand

Dermorphin, a heptapeptide amide isolated from amphibian skin, is the most potent of the naturally occurring opioid peptides. (3H)-dermorphin (52 Ci/mmol, 1294 GBq/mmol) was prepared by catalytic tritiation of the synthetic (2,5-iodotyrosyl 1,5)-dermorphin precursor. High affinity specific binding sites for dermorphin were labeled in rat brain membranes using tritiated dermorphin as primary ligand. The binding was saturable and time-dependent. Scatchard analysis revealed a single population of non-interacting high affinity sites (Kd = 0.86 nM). Dermorphin and the specific opiate antagonist naloxone inhibited specific (3H)-dermorphin binding in a concentration dependent manner. The displacement curves could be fit to a simple competitive model assuming only one population of binding sites, with IC 50 of 1.6 nM and 3.4 nM for dermorphin and naloxone, respectively. The use of tritiated dermorphin will be helpful to ascertain unequivocally the selectivity of dermorphin for the different opioid receptor subtypes in the central nervous system.     

Dopamine-stimulated cyclic AMP and binding of [3H] dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [3H] dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1.10(-8) M and was half-maximal at 7.9 +/- 3.4 10(-7) M. The increase at 1.10(-5) M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1.10(-9) M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC50 value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1.10(-5) M dopamine was 2.3 +/- 0.9.10(-6) M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [3H] Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37 degrees C, it was rapid, reversible, saturable and stereospecific. The Kd value for high affinity binding sites was 0.43 +/- 0.1.10(-7) M and 4.7 +/- 1.6.10(-7) M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2 +/- 0.4.10(-7) M epinine, 4.1 +/- 1.8.10(-6) M fluphenazine, 8.0 +/- 1.6.10(-6) M haloperidol, 4.2 +/- 1.2.10(-6) M cis-flupenthixol, 2.7 +/- 4.0.10(-5) M trans-flupenthixol, less than 1.10(-5) M apomorphine, sulpiride, naloxone and isoproterenol.     

Cyclic somatostatin octapeptide analogues with high affinity and selectivity toward mu opioid receptors

A series of cyclic conformationally restricted penicillamine containing somatostatin octapeptide analogues have been prepared by standard solid phase synthetic techniques and tested for their ability to inhibit specific [125I]CGP 23,996 (des-Ala1-,Gly2-[desamino-Cys3Tyr11]-dicarba3, 14-somatostatin), [3H]naloxone or [3H]DPDPE ([D-Pen2-D-Pen5]enkephalin) binding in rat brain membrane preparations. We now report structure-activity relationship studies with the synthesis of our most potent and selective mu opioid receptor compound D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, which we refer to as Cys2Tyr3Orn5Pen7-amide. While this octapeptide exhibited high affinity (IC50 = 2.80 nM) for an apparently single population of binding sites (nH = 0.89 +/- 0.1) and exceptional selectivity for mu opioid receptors with an IC50(DPDPE)/IC50 (naloxone) ratio of 4,829, it also displayed very low affinity for somatostatin receptors (IC50 = 22,700 nM). Thus, Cys2Tyr3Orn5Pen7-amide may be the ligand of choice for further characterization of mu opioid receptors and for examining the physiological role of this class of receptors.     

Reduced prostaglandin E2 (PGE2) receptors on atopic T lymphocytes

We have recently demonstrated that atopic T lymphocytes have decreased sensitivity to prostaglandin E2 (PGE2). In order to determine whether this decreased sensitivity was reflected at the receptor level, we have employed a radioligand binding assay utilizing [3H]PGE2. We have demonstrated a single specific reversible binding site for [3H]PGE2 on normal T cells (N = 10) with a mean KD (+/-SD) of 32.2 (+/-25.0) nM, a binding capacity of 20.2 (+/-13.0) pM, and a mean of 1004 (+/-118) receptors per cell. Atopic T cells (N = 10) were also found to have a single specific binding site for [3H]PGE2 with a mean KD of 24.9 (+/-17.8) nM, a binding capacity of 7.1 (+/-10.1) pM, and a mean of 372 (+/-61) receptors per cell. These radioligand binding studies were correlated with functional studies in the same subjects. Phytohemagglutinin-stimulated protein synthesis ([3H]leucine uptake) was suppressed in a dose-dependent fashion by PGE2 (10(-6)-10(-12) M). The maximal effect of PGE2 on normal T cells was 10(-6) M PGE2 with an IC50 of 10(-12) M. Atopic T cells responded quantitatively less than normal T cells to PGE2. Further, the maximum suppression of protein synthesis by PGE2 occurred at 10(-6) M with an IC50 of 10(-10) to 10(-11) M. These studies suggest that part of the decreased sensitivity of atopic T cells to PGE2 may result from a reduction in PGE2 binding sites.     

An affinity label for delta-opioid receptors derived from [D-Ala2]deltorphin I.

A series of potential affinity label derivatives of the amphibian opioid peptide [D-Ala2]deltorphin I were prepared by incorporation at the para position of Phe3 (in the 'message' sequence) or Phe5 (in the 'address' sequence) of an electrophilic group (i.e. isothiocyanate or bromoacetamide). The introduction of the electrophile was accomplished by incorporating Fmoc-Phe(p-NHAlloc) into the peptide, followed later in the synthesis by selective deprotection of the Alloc group and modification of the resulting amine. While para substitution decreased the delta-opioid receptor affinity, selected analogs retained nanomolar affinity for delta receptors. [D-Ala2,Phe(p-NCS)3]deltorphin I exhibited moderate affinity (IC50=83 nM) and high selectivity for delta receptors, while the corresponding amine and bromoacetamide derivatives showed pronounced decreases in delta-receptor affinity (80- and >1200-fold, respectively, compared with [D-Ala2]deltorphin I). In the 'address' sequence, the Phe(p-NH2)5 derivative showed the highest delta-receptor affinity (IC50=32 nM), while the Phe(p-NHCOCH2Br)5 and Phe(p-NCS)5 peptides displayed four- and tenfold lower delta-receptor affinities, respectively. [D-Ala2,Phe(p-NCS)3]deltorphin I exhibited wash-resistant inhibition of [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) binding to delta receptors at a concentration of 80 nM. [D-Ala2, Phe(p-NCS)3]deltorphin I represents the first affinity label derivative of one of the potent and selective amphibian opioid peptides, and the first electrophilic affinity label derivative of an agonist containing the reactive functionality in the 'message' sequence of the peptide.     

Opioid activities and structures of alpha-casein-derived exorphins

Exorphins, peptides with opioid activity, have previously been isolated from pepsin hydrolysates of alpha-casein [Zioudrou, C., Streaty, R. A., & Klee, W. A. (1979) J. Biol. Chem. 254, 2446-2449]. Analysis of these peptides shows that they correspond to the sequences 90-96, Arg-Tyr-Leu-Gly-Tyr-Leu-Glu, and 90-95, Arg-Tyr-Leu-Gly-Tyr-Leu, of alpha-casein. These peptides, as well as two of their analogues Tyr-Leu-Gly-Tyr-Leu-Glu (91-96) and Tyr-Leu-Gly-Tyr-Leu (91-95), have now been synthesized and characterized. Their opioid activity was examined by three different bioassays: (a) displacement of D-2-alanyl[tyrosyl-3,5-3H]enkephalin-(5-L-methioninamide) and [3H]dihydromorphine from rat brain membranes; (b) naloxone-reversible inhibition of adenylate cyclase in homogenates of neuroblastoma x glioma hybrid cells; (c) naloxone-reversible inhibition of electrically stimulated contractions of the mouse vas deferens. The synthetic peptide of sequence 90-96 was the most potent opioid in all three bioassays and its potency was similar to that of the isolated alpha-casein exorphins. The synthetic peptides were totally resistant to hydrolysis by trypsin and homogenates of rat brain membranes, but were partially inactivated by chymotrypsin and subtilisin. The difference in opioid activity of alpha-casein exorphins may be related to differences in conformational flexibility observed by NMR spectroscopy.     

Identification and characterization of the beta-adrenergic receptor on neuroblastoma × glioma hybrid NG108-15 cells

1. Using [3H]DHA and unlabeled L-alprenolol, a substantial amount of over 64% specific binding of beta-adrenergic receptor has been identified on the neuroblastoma x glioma hybrid NG108-15 cell, which has been proven to display numerous functional characteristics of intact neurons. 2. Beta-adrenergic receptor binding on intact NG108-15 cells does not change significantly upon morphological differentiation, induced by 1 mM dibutyryl cyclic AMP (dBcAMP). 3. The [3H]DHA binding on intact NG108-15 cells is rapid, saturable, and reversible, having a t1/2 of 1.0 min for association and 3.5 min for dissociation. 4. The affinity constant (Kd) and maximum binding capacity (Bmax) for binding of [3H]DHA to beta-adrenergic receptors on NG108-15 cells have been estimated by Scatchard plot analysis to be 2.5 and 0.23 nM, respectively. Further analysis indicates a single class of receptors for [3HDHA binding on NG108-15 cells. 5. Studies on kinetic properties have revealed on-rate (K + 1) and off-rate (K - 1) constants of 0.7 X 10(-9) M min-1 and 0.19 min-1, respectively. Further, the IC50 value and inhibition constant (Ki) for unlabeled L-alprenolol to inhibit [3HDHA binding on NG108-15 cells have been estimated to be 10(-5) and 8.9 X 10(-6) M, respectively. 6. The rank-order potency of catecholamine agonists, (-)ISO greater than (+)ISO greater than EPI greater than NE, reveals the presence of type 2 receptor for the beta-adrenergic binding on both differentiated and undifferentiated NG108-15 cells. 7. The present study indicates that the clonal neuroblastoma x glioma hybrid NG108-15 cell line possesses substantial amounts of beta-adrenergic receptors with characteristics similar to those on neuronal cells.     

Effect of tunicamycin on functions of PGE1 receptors from mouse mastocytoma P-815 cells

Prostaglandin E1 (PGE1) receptors from mouse mastocytoma P-815 cells were found to bind to a wheat germ agglutinin (WGA)-Agarose column, suggesting that the receptors are glycoproteins. To further elucidate the role of carbohydrate moieties in the PGE1 receptors for their binding activity to ligand, the P-815 cells were treated with tunicamycin, swainsonine or monensin. Tunicamycin, an inhibitor of N-glycosylation, dose- and time-dependently inhibited the binding of PGE1 to mastocytoma P-815 cells. Neither swainsonine, an inhibitor of Golgi mannosidase II, nor monensin, an inhibitor of processing beyond the high mannose stage, altered PGE1 binding properties of the cells. The inhibition of PGE1 binding by tunicamycin was observed when incorporation of [3H]glucosamine into macromolecules was inhibited. The inhibitory effect was not on their affinity but on their number of binding sites. Subcellular distributions of [3H]PGE1-binding activity showed that decreases in the binding activity by tunicamycin were highest in plasma membrane fractions. Treatment of membranes with various endo- and exoglycosidases did not affect PGE1 binding. PGE1-stimulated cyclic AMP accumulation in the cells was also inhibited by tunicamycin. These results suggest that PGE1 receptors of mastocytoma P-815 cells are glycoproteins and that inhibition of N-glycosylation of PGE1 receptors by tunicamycin results in the arrest of the translocation of newly synthesized receptors to the surface of mastocytoma P-815 cells.     

In vitro effects of lead ions on peripheral benzodiazepine receptors and adenylyl cyclase activity in the mantle of Mytilus galloprovincialis

As an extension of our previous work, where the density of peripheral benzodiazepine receptors (PBR) increased in mantle mitochondria of the marine mollusk Mytilus galloprovincialis Lmk. under chronic exposure to lead, the present study investigates the in vitro effects of an exogenous source of lead ions on PBR and on adenylyl cyclase (AC) complex in mantle membranes of mussels collected from a non-polluted coastal area. PBR binding experiments used the specific isoquinoline carboxamide derivative [3H]PK 11195, and AC activity was measured using a modified procedure adapted to M. galloprovincialis. Lead ions (Pb2+) dose-dependently decreased either the [3H]PK 11195 specific binding in mitochondria or basal AC velocity in plasma membranes of mussel mantle. The IC50 values for lead ions were 10 microM with [3H]PK 11195 binding and 25 microM with AC activity, with maximal inhibition values of 60% and 70%, respectively. Moreover, lead behaved as a non-competitive inhibitor on [3H]PK 11195 binding and as a 'mixed' inhibitor on AC activity. The present results suggest that some of the early effects induced by lead in mussel cell metabolism consist in significant changes of the PBR density and cyclic AMP production in the mantle of M. galloprovincialis.     

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

Abstract: Monolayer cultures of neuroblastome × glioma hybrid (clonal) cell line NG108-15, synchronized by the isoleucine/glutamine deprivation method, showed maximal expression of opiate binding sities at the same point in the cell cycle at which prostaglandin E₁(PGE₁) had a maximum stimulatory effect of cyclinc AMP synthesis. However, the capacity of enkephalin (D-Ala²D-Leu⁵] to block the stimulation of cyclic AMP synthesis by PGE₁ was not related to the number of opiate receptors expressed. The K₁ for the inhibition of cyclic AMP synthesis by opioid peptides increased substanitilly during the period of the cell cycle at which maximal expression of opiate binding sites occurred, making the effectivel level of inhibition of adenylate cyclase activity by 0.1μM enkephalin [D-Al²D-Leu³] the same throght the cell cycle. Data are presented to suggest the enkephalin receptor coupling to adenylate cyclase, via a GTP-binding protein, is maximal during G₁ phase (which may approximate the state of the differentiated neuron) and minimal during S + G₂ phase, just prior to cell division, when many receptors are uncoupled.     

Analgesic, receptor binding, and peripheral opioid activities of synthetic dermorphin-dynorphin hybrid peptides

The effects of substituting the enkephalin moiety of dynorphin with the dermorphin sequence were studied on the receptor preference, analgesic, and peripheral opioid potencies by using synthetic dermorphin-dynorphin hybrid peptides as the probe. Replacement of the enkephalin moiety of dynorphin with the dermorphin or dermorphin1-5 sequences caused a remarkable increase in analgesic potency, and a 3-6 fold increase in potency of binding against [3H]-dihydromorphine. The potency of receptor binding against [3H]-EKC was also increased by incorporation of the whole dermorphin sequence into the dynorphin molecule. In the presence of NaCl (100 mM), the effect of enhancing binding against [3H]-EKC due to dermorphin substitution disappeared, suggesting the contribution of opioid mu-receptor. Peripheral opioid activities assayed by various smooth muscle preparations showed that dermorphin incorporation caused a decreased in the potency of inhibition of the contractions of the guinea pig ileum and the rabbit vas deferens, no change in potency on the mouse vas deferens, and a marked increase in the inhibition of the rat vas deferens. Among the peripheral opioid activities only that assayed with the rat vas deferens appears to correlate approximately with the analgesic and the receptor binding activities. Judging from the relative potencies obtained from all assays, it is evident that the N-terminal dermorphin moiety, but not the C-terminal dynorphin fragment, dominates the opioid activity and receptor preference of the hybrid peptide.