Biomedical subjects

H H Loh

Publications and source records attributed to H H Loh.

At least 145 records · Page 8Linked to original sources

PubMed1987

Biological activity of mu opioid receptor.

The purification of the mu-opioid receptor has proved to be more difficult than most other receptors for at least two reasons. First, they are easily inactivated by most detergents and the second problem is the heterogeneous existence of the opioid receptor. Despite these problems, this laboratory, and several others, have recently reported purification of the mu-opioid receptor. The 58,000 molecular weight of the purified mu-opioid receptor agrees closely with the value reported by other investigators. In addition, it was found that this purified mu-opioid receptor was associated with a phosphatase activity. While the relevance of this phosphatase activity to opioid receptor action remains to be determined, it is interesting to note that this activity was stimulated by Mn++ and Mg+ ions, both of which also increase opioid binding.

Animals↗

PubMed1987

Lipid requirement for mu opioid receptor binding.

We have previously shown that a partially purified mu opioid receptor from bovine brain requires lipids to exhibit full binding activity. In the present report, we have determined the specificity of this lipid requirement. Lipids active in this regard were found always to contain an acidic head group and a fatty acid with two or more double bonds. Free, polyunsaturated fatty acids were also able to confer high binding activity on the partially purified opioid receptor. The possible roles lipids play in opioid binding are discussed in light of these data.

Animals↗

PubMed1987

Decrease in delta and mu opioid receptor binding capacity in rat brain after chronic etorphine treatment.

The modulatory effect of continued activation of opiate receptors with agonist on the receptor level was examined in current studies. Rats were rendered tolerant to etorphine by s.c. implantation of osmotic minipumps containing 3 mg/ml of etorphine for up to 7 days. During this period, there were a time-dependent increase in the AD50 values of etorphine to inhibit the tail-flick response and an increase in naloxone-precipitated withdrawal signs. When these animals and others were sacrificed and the opiate receptor binding properties were examined, it could be demonstrated that there was also a time-dependent decrease in the amount of [3H]diprenorphine specifically bound, with maximal attenuation reached 3 days after implantation. There was no alteration in alpha 2 adrenergic receptor binding. This observed decrease in [3H]diprenorphine binding was not due to the presence of nonwashed etorphine in the membrane, for acute administration of the same dose of etorphine before sacrificing did not produce an attenuation of the opiate receptor binding. Further examination of opiate receptor binding in the brain regions of cortex, midbrain and striatum revealed the greatest decrease in the amount of [3H]diprenorphine bound in striatal region after chronic etorphine treatment, a 68% decrease. When the relative decrease in mu and delta opioid receptor binding was determined by carrying out [3H]-D-Ala2, DLeu5-enkephalin binding in the presence of 1 microM morphiceptin, it was observed that, 3 days after etorphine treatment, there was a decrease in mu opioid receptor binding, with minimal change in delta opioid receptor binding in both brain regions of striatum and midbrain. Only in cortex was a decrease in binding of both receptor subtypes observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Mu-opioid receptor is associated with phosphatase activity.

A preparation of purified mu opioid receptor from bovine brain hydrolyzes p-nitrophenylphosphate. This phosphatase activity has a pH optimum of 9.0, a Km of 9.0 microM, and is stimulated by Mn++ and Mg++ ions. Evidence that the observed activity is not due to a contaminant in the opioid receptor preparation includes 1) the activity is associated primarily with 60,000 molecular weight material which is much smaller than bovine brain alkaline phosphatase; and 2) the activity could not be absorbed by antibodies specific for bovine alkaline phosphatase. Thus this appears to be the first demonstration of enzymatic activity associated with an opioid receptor.

4-Nitrophenylphosphatase↗

PubMed1986

Effects of dynorphin1-13 on opiate binding and dopamine and GABA uptake in stroked cat brain.

We previously reported that the opioid peptide dynorphin1-13 improves survival chances in stroked cats. Some evidence also suggests that changes in dopamine and gamma-aminobutyric acid (GABA) uptake may be associated with stroke. In the present study, therefore, we determined binding of the opiate [3H]ethylketocyclazocine (EKC), as well as dopamine and GABA uptake in various brain regions of control, stroked and dynorphin1-13-treated stroked cats. Cats were stroked by middle cerebral artery occlusion. In the EKC binding study, the Kd of the high-affinity site of the occluded cortex was significantly increased, relative to that of both the unoccluded side and control cortex. Dynorphin1-13 treatment reversed this effect, lowering the Kd to control level. In the dopamine uptake study, the Km was decreased and Vmax was increased significantly in unoccluded cortex, compared with that in the occluded cortex or in control cortex. Again, dynorphin1-13 reversed these effects, raising the Km and lowering the Vmax. However, the Km of occluded cortex was also increased so that it became significantly higher than that of control cortex. The Km of unoccluded subcortex in stroked cats treated with dynorphin1-13 was significantly reduced compared with control. In the GABA uptake study, there was no significant change in any parameter. The change in opioid binding observed here and its reversal by dynorphin1-13 are consistent with the notion that the peptide's beneficial effect on stroke is mediated through opiate receptors. Since opioid systems in the brain are known to have association with dopaminergic ones, the change in dopamine uptake could also be the result of an opioid effect.

Animals↗

PubMed1986

Competitive binding of dynorphin-(1-13) and beta-endorphin to cerebroside sulfate in solution.

Circular dichroism was used as a probe for competitive binding of two opioid peptides, dynorphin-(1-13) and beta-endorphin, with cerebroside sulfate, a membrane lipid thought to be part of the morphine receptor complex. The rationale was that bound beta-endorphin is partially helical but bound dynorphin-(1-13) remains unordered, thus making it possible to detect the degree of binding of beta-endorphin. The addition of dynorphin-(1-13) to a cerebroside sulfate solution of beta-endorphin invariably displaced beta-endorphin from the peptide-lipid complex, but the addition of beta-endorphin had little effect on dynorphin-(1-13) bound to the lipid. Similar results were obtained for competitive binding of the two peptides with two other amphiphiles, sodium dodecyl and decyl sulfate. The maximum number of binding sites on dynorphin-(1-13) and beta-endorphin was between five and six, which coincides with the five positively charged side chains plus an alpha NH+3 group at the NH2 terminus on both peptide molecules. The results support our working hypothesis that dynorphin-(1-13) may displace beta-endorphin bound to the receptor, which in turn can account for the inhibition of beta-endorphin-induced analgesia by dynorphin-(1-13).

Binding, Competitive↗

PubMed1986

Purification to apparent homogeneity of a mu-type opioid receptor from rat brain.

A mu-opioid-specific receptor was purified to apparent homogeneity from rat brain membranes by 6-succinylmorphine affinity chromatography, gel filtration, wheat germ agglutinin affinity chromatography, and isoelectric focusing. The purified receptor had a molecular weight of 58,000 as determined by NaDodSO4/polyacrylamide gel electrophoresis and was judged to be homogeneous by the following criteria: (i) a single band was detected by autoradiography after NaDodSO4/polyacrylamide gel electrophoresis of 125I-labeled receptor and (ii) the purified preparation had a specific opioid-binding activity of 17,720 pmol/mg of protein, close to the theoretical value. In addition, the Mr 58,000 value agrees closely with that determined by covalently labeling purified receptor with bromoacetyl-[3H]dihydromorphine or with 125I-labeled beta-endorphin and dimethyl suberimidate.

Animals↗

PubMed1986

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

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

Adenosine Triphosphate↗

PubMed1986

Effect of phospholipases on chronic opiate action in neuroblastoma X glioma NG108-15 hybrid cells.

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with opiate agonist resulted in loss of the acute opiate inhibition of adenylate cyclase activity with a concomitant increase in the enzymatic activity observable on addition of the antagonist naloxone. The role of membrane lipids in the cellular expression of these chronic opiate effects was investigated by the hydrolysis of phospholipids with various lipases. Treatment with phospholipase C from Clostridium welchii produced an enzyme concentration-dependent decrease of prostaglandin E1-stimulated adenylate cyclase activity in control or etorphine-treated (1 microM for 4 h) hybrid cells. In addition, incubation of hybrid cells with phospholipase C concentrations of greater than or equal to 0.5 U/ml completely abolished the compensatory increase in adenylate cyclase activity after chronic opiate treatment. This attenuation of the increase in adenylate cyclase activity by phospholipase C could be prevented by inclusion of phosphatidylcholine but not of phosphatidic acid during the enzymatic incubations. The specificity of the phospholipids involved in expression of the chronic opiate effect could be demonstrated further by the absence of effect exhibited by phospholipase C from Bacillus cereus and phospholipase D. Hydrolysis of the acyl side chains of phospholipids with phospholipase A2 did not alter the chronic opiate effect after removal of lysophosphatides with bovine serum albumin. Because the guanylylimidodiphosphate- and NaF-sensitive adenylate cyclase activities were not affected by these phospholipase treatments, the expression of the compensatory increase in adenylate cyclase activity is mediated via an increase in the coupling between hormonal receptor and adenylate cyclase with the participation of the polar head groups of the phospholipids and not the hydrophobic side chains.

Adenylyl Cyclase Inhibitors↗

PubMed1986

Enzymatic reconstitution of brain membrane and membrane opiate receptors.

A new method using lysophosphatide and acyl-CoA as detergents has been used to solubilize the rat brain opiate receptor. After solubilization, lysophosphatide and acyl-CoA can be almost completely removed by an enzymatic reaction that uses an acyltransferase from rat liver microsomes and reconstitutes the solubilized receptor in membranous vesicles. Morphological studies performed with negative staining and freeze-fracture electron microscopy revealed that the general appearance and intramembrane particle distribution of fracture faces in the reconstituted membrane are similar to those of the native membrane; this indicates that hydrophobic protein components of the original membrane were incorporated during reconstitution. Reconstituted membrane, however, contained higher levels of phosphatidylcholine and lower levels of cholesterol. The activities of the membrane-bound enzymes Na+, K+-ATPase and Ca2+, Mg2+-ATPase in the reconstituted system were 24 and 3%, respectively, those of the native membrane. Although binding of opiate ligands to the reconstituted membrane was stereospecific and saturable, higher concentrations of some of the unlabeled ligands were required to inhibit binding of the radiolabeled ligands. These changes in receptor characteristics are likely due to changes in lipid composition, physical state, and/or distribution of the lipids in the reconstituted membrane bilayer. This conclusion is supported by an increase in the affinity of opiate ligands for reconstituted membrane after adjustment of the latter's lipid composition to match more closely that of the original membrane. This was accomplished by treatment with phospholipid exchange protein to remove the excess phosphatidylcholine and by incorporation of cholesterol into the reconstituted membrane.

Acyltransferases↗

PubMed1986

Determination of the sizes of the opioid receptors in their membrane environment by radiation inactivation.

Opioids, like other drugs, are thought to initiate their effects by association with their specific receptors. However, very little is known about the opioid receptor as a molecular entity. The binding components have been solubilized in detergent and purified by different approaches, but the molecular size of soluble opioid receptor complexes reported by different groups varied from 23,000 to 750,000. In this study, the technique of radiation inactivation by gamma rays was used to investigate the apparent size of the opioid receptor in rat brain membranes under different conditions. The molecular sizes of opioid receptor complexes were estimated as 313,000 +/- 13,500 in the presence of [D-Ala2, D-Leu5] enkephalin, NaCl and Gpp (NH)p; as 165,000 +/- 8,500 in the presence of NaCl only, or of both NaCl and Gpp (NH)p; as 217,000 +/- 6,600 in the presence of Gpp (NH)p only; and as 286,000 +/- 60,900 in the presence of MgCl2 only. A simple model has been proposed to explain these different apparent target sizes of opioid receptors obtained under different conditions.

Animals↗

PubMed1985

Neuroblastoma X glioma NG108-15 hybrid cells cultured in a serum-free chemically defined medium: effects on acute and chronic opiate regulation of adenylate cyclase activity.

Neuroblastoma X glioma NG108-15 hybrid cells cultured in a chemically defined medium within 3 cell passages, exhibited viability, growth rate and morphology similar to those of cells grown in medium supplemented with 5% fetal calf serum. Hybrid cells cultured in the chemically defined medium within these periods of time also did not exhibit a difference in basal adenylate cyclase activity nor in the enzymatic activities stimulated by adenosine, forskolin, NaF, GppNHp or Mn2+. Furthermore, opiate receptor density in chemically defined medium cultured cells remained identical to that in cells cultured in 5% fetal calf serum. The acute and chronic effects of opiates on adenylate cyclase were similar for cells grown under either set of conditions.

Adenylyl Cyclases↗

PubMed1985

Effect of pertussis toxin treatment on the down-regulation of opiate receptors in neuroblastoma X glioma NG108-15 hybrid cells.

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with 10 nM [D-Ala2,D-Leu5] enkephalin (DADLE) results in a reduction of cell-surface opiate delta receptors. Whether opiate receptor internalization requires the activation of the guanine nucleotide-binding protein (Ni) is unclear. Hence, activation of Ni was attenuated by treating hybrid cells with 100 ng/ml pertussis toxin (PT) for 3 h, which resulted in a decrease in DADLE's ability to inhibit adenylate cyclase activity. Despite this prior treatment with PT, chronic exposure of these cells to 10 nM DADLE resulted in a time-dependent decrease in both [3H]diprenorphine and [3H]DADLE binding. This reduction in 3H-ligand binding in cells previously treated with PT represented internalization of the receptors because translocation was observed of bound [3H]DADLE from plasma membrane fractions to the lysosomal fractions in the Percoll gradients. Thus, opiate receptors internalize without activation of Ni. The internalization of opiate receptors was not accompanied by Ni. By measuring the amount of the 41-kDa alpha subunit being labeled by PT with [32P]NAD+, it was determined that plasma membrane preparations, of both the control cells and cells treated with 10 nM of DADLE for 4 h, contained equal concentrations of Ni, 2 pmol of Ni/mg of protein. Additionally, there was no measurable alteration in the amount of PT substrate in the lysosomal fractions of the DADLE-treated cells as compared to that of control cells. Chronic DADLE treatment resulted in a decrease in Km value of NAD+ in the ADP-ribosylation of 41-kDa subunit of Ni. In summary, opiate receptors internalize as agonist-receptor complexes without the guanine nucleotide-binding component.

Adenylate Cyclase Toxin↗

PubMed1985

Multiple affinity states of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Opiate agonist association rate is a function of receptor occupancy.

The existence of multiple affinity states for the opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells has been demonstrated by competition binding studies with tritiated diprenorphine and [D-Ala2, D-Leu5]enkephalin (DADLE). In the presence of 10 mM Mg2+, all receptors exist in a high affinity state with Kd = 1.88 +/- 0.16 nM. Addition of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) decreased the affinity of DADLE to Kd = 8.08 +/- 0.93 nM. However, in the presence of 100 mM Na+, which is required for opiate inhibition of adenylate cyclase activity, analysis of competition binding data revealed three sites: the first, consisting of 17.5% of total receptor population has a Kd = 0.38 +/- 0.18 nM; the second, 50.6% of the population, has a Kd = 6.8 +/- 2.2 nM; and the third, 31.9% of the population, has a Kd of 410 +/- 110 nM. Thus, in the presence of sodium, a high affinity complex between receptor (R), GTP binding component (Ni), and ligand (L) was formed which was different from that formed in the absence of sodium. These multiple affinity states of receptor in the hybrid cells are agonist-specific, and the percentage of total opiate receptor in high affinity state is relatively constant in various concentrations of Na+. Multiple affinity states of opiate receptor can be demonstrated further by Scatchard analysis of saturation binding studies with [3H]DADLE. In the presence of Mg2+, or Gpp(NH)p, analysis of [3H]DADLE binding demonstrates that opiate receptor can exist in a single affinity state, with apparent Kd values of [3H]DADLE in 10 mM Mg2+ = 1.75 +/- 0.28 nM and in 10 microM Gpp(NH)p = 0.85 +/- 0.12 nM. There is a reduction of Bmax value from 0.19 +/- 0.02 nM in the presence of Mg2+ to 0.14 +/- 0.03 nM in the presence of Gpp(NH)p. In the presence of 100 mM Na+, Scatchard analysis of saturation binding of [3H]DADLE reveals nonlinear plots; two-site analysis of the curves yields Kd = 0.43 +/- 0.09 and 7.9 +/- 3.2 nM. These Kd values are analogous to that obtained with competition binding studies. Again, this conversion of single site binding Scatchard plots to multiple sites binding plots in the presence of Na+ is restricted to 3H-agonist binding only.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1985

Isolation and purification of morphine receptor by affinity chromatography.

Brain membranes were solubilized by sonication and Triton X-100 extraction and applied to an affinity column consisting of a 6-succinyl morphine derivative of Affi Gel-102. A fraction exhibiting high opiate binding was eluted by tris-buffer containing naloxone, CHAPS and NaCl. This fraction consisted of both proteins and acidic lipids. The opiate binding properties of this purified material exhibited many properties similar to those of membrane bound receptors of the u-type, including high affinity, stereospecificity, Na-effect and rank order in affinity for opiates. This opiate binding material was highly sensitive to both trypsin and N-ethylmaleimide. Based on the protein content of the isolated membrane receptor, a 3200-fold purification over the original brain P2 fraction was achieved.

Animals↗

PubMed1985

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.

Adenosine Diphosphate Ribose↗

PubMed1985

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.

Adenylyl Cyclase Inhibitors↗

PubMed1985

Determination of chlorpromazine and its major metabolites by gas chromatography/mass spectrometry: application to biological fluids.

A method for the quantitative determination of chlorpromazine and five of its major metabolites in a single sample of biological fluid in the ng/ml range has been developed utilizing gas chromatography/mass spectrometry with selected ion recording. The assay is highly specific and quantification is accomplished by an inverse stable isotope dilution technique, using deuterium-labeled variants of the compounds as internal standards. In this way the concentrations of chlorpromazine and five of its major metabolites (the sulfoxide, the N-oxide, the monodemethylated, the didemethylated, and the 7-hydroxylated compounds) can be determined in biological fluids. Levels in humans have been measured both in plasma and in red blood cells and are compared to those found in related in vitro studies.

Biotransformation↗