Ping-Yee Law

Demonstration and Characterization of Opiate Inhibition of the Striatal Adenylate Cyclase

Abstract: The conditions in which Leu5‐enkephalin inhibition of striatal adenylate cyclase was observed were defined. It was determined that enkephalin inhibition was dependent on GTP. The apparent Km for GTP in opiate inhibition was determined to be 0.5 and 2 μM when 0.1 mM‐ and 0.5 mM‐ATP were used as substrate. ITP, but not CTP or UTP, could substitute for GTP in the reaction. Though the addition of monovalent cations—Na+,K+, Li+, Cs+, and choline+—stimulated striatal adenylate cyclase activity, enkephalin inhibition of striatal adenylate cyclase did not require Na+ when theophylline was used as the phosphodiesterase inhibitor. Under optimal conditions, i.e., 20 μM‐GTP and 100 mM‐Na+, Leu5‐enkephalin inhibited the striatal adenylate cyclase activity by 23–27%. When the enkephalin regulation of the cyclase activity was further characterized, it was observed that Leu5‐enkephalin inhibited the rate of the enzymatic reaction. Kinetic analysis revealed that the opioid peptide decreases Vmax values but not the Km values for the substrates Mg2+ and Mg‐ATP. Agents such as MnCl2, NaF, and guanyl‐5′‐ylimido‐diphosphate, which directly activated the adenylate cyclase, antagonized the opiate inhibition. Levorphanol and (–)naloxone were more potent than dextrorphan and (+)naloxone in inhibiting adenylate cyclase and in reversing the enkephalin inhibition, respectively. There were differences in the potencies of various opiate peptides in their inhibition of striatal adenylate cyclase activity, with Met5‐ > Leu5‐enkephalin > β‐endorphin. The opiate receptor through which the enkephalin inhibition was observed is most likely δ in nature, since in the presence of either Na+ or K+, the magnitude of the alkaloid inhibition was reduced, whereas the peptide inhibition was either potentiated or not affected.

Decrease in δ-opioid receptor density in rat brain after chronic [d-Ala2,d-Leu5]enkephalin treatment

Chronic treatment of Sprague-Dawley rats with [D-Ala2,D-Leu5]enkephalin (DADLE) resulted in the development of tolerance to the antinociceptive effect of this opioid peptide. When opioid receptor binding was measured, time-dependent decreases in [3H]diprenorphine binding to the P2 membranes prepared from the cortex, midbrain and striatum were observed. Scatchard analysis of the saturation binding data revealed a decrease in Bmax values and no change in the Kd values of [3H]diprenorphine binding to these brain regions, indicative of down-regulation of the receptor. This reduction in the opioid receptor binding activities could be demonstrated to be due to the DADLE effect on the delta-opioid receptors in these brain regions. When [3H]DADLE binding was carried out in the presence of morphiceptin, a significant reduction in the delta-opioid receptor binding was observed in all brain areas tested. mu-Opioid receptor binding decrease was observed only in the striatum after 5 days of DADLE treatment. Additionally, the onset of delta-opioid receptor decrease in the midbrain area was rapid, within 6 h of the initiation of the chronic DADLE treatment. Thus, analogous to previous observations in which chronic etorphine treatment preferentially reduced mu-opioid receptor binding, chronic DADLE treatment preferentially reduced delta-opioid receptor binding activity.

Pertussis toxin treatment modifies opiate action in the rat brain striatum

In this report we present evidence that a guanine nucleotide regulatory protein, Gi, mediates opiate action in the rat brain striatum. Opiates inhibit basal adenylate cyclase activity in rat brain striatum. This effect on adenylate cyclase is dose-dependently attenuated by pretreatment of membranes with pertussis toxin, which ADP-ribosylates a protein with a molecular mass of 41,000 daltons. This protein co-migrates with the GTP-binding subunit of Gi, which mediates inhibition of adenylate cyclase. Several brain regions were compared for the extent of radiolabeling and effects on adenylate cyclase activity. Although Gi was found in each region examined, opiate inhibition of adenylate cyclase is clearly seen only in the striatum.

β-Endorphin: Structure-activity relationships in the guinea pig ileum and opiate receptor binding assays

Abstract The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of βh-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of βh-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.

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 (Ni) and stimulatory (Ns) components. Inactivation of Ni 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 Ns, 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 Ni resulting in the unimpeded expression of Ns activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which Ni regulates adenylate cyclase in this cell line.

Role of Opioid Receptors in Narcotic Tolerance/ Dependence

The signal feature of opioid drugs is their ability to induce tolerance and dependence when given chronically to humans or experimental animals. Tolerance may be defined as a state in which the dose of drug required to achieve a given effect is larger than normal. Dependence is a state in which regular doses of the drug are required to prevent withdrawal symptoms.

Cell-Free Desensitization of Opioid Inhibition of Adenylate Cyclase in Neuroblastoma × Glioma NG108–15 Hybrid Cell Membranes

Abstract: When membranes from neuroblastoma |MX 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.

Effects of cycloheximide and tunicamycin on opiate receptor activities in neuroblastoma X glioma NG108-15 hybrid cells.

The molecular mechanism of opiate receptor down-regulation and desensitization was investigated by studying the effects of cycloheximide and tunicamycin on opiate receptor activities in neuroblastoma X glioma NG108-15 hybrid cells. Cycloheximide inhibited [35S]methionine and [3H]-glucosamine incorporation by hybrid cells, while tunicamycin inhibited [3H]glucosamine incorporation only. Exposing hybrid cells to these two agents did not alter the viability of the cell. Treatment of NG108-15 cells with cycloheximide or tunicamycin produced a decrease in [3H]diprenorphine binding dependent on both time and concentrations of inhibitors, with no measurable modification in the ability of etorphine to regulate intracellular cyclic AMP production. Cycloheximide attenuated [3H]-diprenorphine binding by decreasing both the number of sites, Bmax, and the affinity of the receptor, Kd. Tunicamycin treatment produced a decrease in Bmax with no apparent alteration in Kd values. Cycloheximide and tunicamycin did not potentiate the rate or magnitude of etorphine-induced down-regulation or desensitization of opiate receptor in NG108-15 cells. Furthermore, there was an apparent antagonism in cycloheximide action on receptor down-regulation. The reappearance of opiate binding sites after agonist removal was affected by these two inhibitors. Cycloheximide and tunicamycin eliminated the increase in [3H]diprenorphine binding in the chronic etorphine-treated cells after agonist removal. These two inhibitors did not alter the resensitization of hybrid cells to etorphine. Thus, the site of opiate agonist action to induce receptor down-regulation and desensitization is not at the site of protein synthesis or protein glycosylation. These data substantiate previously reported observations that receptor down-regulation and receptor desensitization are two different cellular adaptation processes.

Attenuation of Enkephalin Activity in Neuroblastoma × Glioma NG108—15 Hybrid Cells by Phospholipases

Abstract: The role of membrane phospholipids in enkephalin receptor‐mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity in neuroblastoma × glioma NG108‐15 hybrids was studied by selective hydrolysis of lipids with phospholipases. When NG108‐15 cells were treated with phospholipase C from Clostridium welchii at 37°C, an enzyme concentration‐dependent decrease in adenylate cyclase activity was observed. The basal and prostaglandin E1 (PGE1)‐stimulated adenylate cyclase activities were more sensitive to phospholipase C (EC 3.1.4.3) treatment than were the NaF‐5′‐guanylyl‐imidodiphosphate (Gpp(NH)p)‐sensitive adenylate cyclase activities. Further, Leu5‐enkephalin inhibition of basal or PGE1‐stimulated adenylate cyclase activity was attenuated by phospholipase C treatment, characterized by a decrease of enkephalin potency and of maximal inhibitory level. [3H]d‐Ala2‐Met5‐enkephalinamide binding revealed a decrease in receptor affinity with no measurable reduction in number of binding sites after phospholipase C treatment. Although opiate receptor was still under the regulation of guanine nucleotide after phospholipase C treatment, adenylate cyclase activity was more sensitive to the stimulation of Gpp(NH)p. Thus, the reduction of opiate agonist affinity was not due to the uncoupling of opiate receptor from N‐component. Further, treatment of NG108‐15 hybrid cell membrane with phospholipase C at 24°C produced analogous attenuation of enkephalin potency and efficacy without alteration in receptor binding. The reduction in enkephalin potency could be reversed by treating NG108‐15 membrane with phosphatidylcholine, but not with phosphatidylserine, phosphatidylinositol, or cerebroside sulfate. The enkephalin activity in NG108‐15 cells was not altered by treating the cells with phospholipase A2 or phospholipase C from Bacillus cereus. Hence, apparently, there was a specific lipid dependency in enkephalin inhibition of adenylate cyclase activity.

DEMONSTRATION AND CHARACTERIZATION OF A STEREOSPECIFIC OPIATE RECEPTOR IN THE NEUROBLASTOMA N18TG2 CELLS

Abstract— Morphine has been observed to have only a minor effect on the prostaglandin E1 (PGE1) stimulated adenylate cyclase or the basal cyclase activity in the neuroblastoma N18TG2 calls. However, this ineffectiveness of the opiates was not due to the absence of opiate receptor in this cell line. Contrary to previous observations, neuroblastoma N18TG2 cells possessed a high affinity, stereospecific opiate receptor. When [3H]dihydromorphine and [3H]naloxone binding were determined, a single component receptor with Kdiss= 25‐31 nm and with a capacity of 165 fmol/mg protein could be observed. This receptor has similar properties to those observed in the brain homogenates. The naloxone specific binding was dependent on the pH of the incubation medium and maximal binding occurred at pH 7.6. The agonist binding was inhibited by the alkali metal cations and divalent cations, while the antagonist binding was not affected by the cations significantly. There was no observable reversal of the Na+ inhibitory effect on agonist binding by the addition of Mn2+ to the incubation mixtures. Opiate binding to the neuroblastoma N18TG2 cells could be attenuated by pretreating the cells with N‐ethylmaleimide or proteolytic enzymes. Of the lipases tested, only phospholipase A2 has an inhibitory effect on the naloxone binding. Fractionation of the cell homogenates with differential centrifugation and purification of the membrane fractions by sucrose gradients suggested the localization of the receptor at the plasma membranes. Thus, the receptor in the neuroblastoma N18TG2 cells closely resembles those observed in the brain homogenates