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

Law, Peter K.; Griffin, Michael T.; Koehler, Jane E.; Loh, Horace H.

doi:10.1111/j.1471-4159.1983.tb12681.x

Cited by 13 publications

(5 citation statements)

References 27 publications

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“…We also demonstrated that the isolated opiate-binding material contained lipids as well as proteins, and these were apparently critical to its activity. This is consistent with a large and diverse body of evidence indicating that both components are necessary for binding to membrane-bound opiate receptors (18)(19)(20). We now report purification of this material to apparent homogeneity.…”

supporting

confidence: 89%

“…As previously reported (17), chromatography of the solubilized brain membranes on Affi-Gel 102 with a linear gradient of NaCl yields two protein peaks. Peak A was eluted at 0.35 M NaCl (fractions [10][11][12][13][14][15][16][17][18][19][20], and peak B at 0.6 M NaCl (fractions [22][23][24][25][26][27][28][29][30][31][32][33][34]. Moreover, the peak A proteins, but not the peak B material, exhibited significant opiate-binding activity.…”

Section: Resultsmentioning

confidence: 99%

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

Cho¹,

Hasegawa²,

Ge³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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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.

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supporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

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

Cho¹,

Hasegawa²,

Ge³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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“…The sensitivity of opioid receptors to lipids has been well documented. Ligand binding (Lin and Simon, 1978;Abood et al, 1980) and receptor coupling to adenylate cyclase (Law et al, 1983) and low-K, GTPase (Lazar and Medzihradsky, 1990) were inhibited by phospholipase treatment, and unsaturated fatty acids decreased opioid receptor binding in cultured NG 108-15 cells (Ho and Cox, 1982;McGee and Kenimer, 1982). Acidic phospholipids containing polyunsaturated fatty acids enhanced ligand binding to the partially purified p-opioid receptor (Hasegawa et al, 1987), whereas phospholipids inhibited opioid receptor binding in rat brain membranes (Remmers and Medzihradsky, 1987).…”

mentioning

confidence: 99%

Modulation of Opioid Receptor Binding by Cis and Trans Fatty Acids

Remmers

Nordby

Medzihradsky

1990

Journal of Neurochemistry

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In synaptosomal brain membranes, the addition of oleic acid (cis), elaidic acid (trans), and the cis and trans isomers of vaccenic acid, at a concentration of 0.87 mumol of lipid/mg of protein, strongly reduced the Bmax and, to a lesser degree, the binding affinity of the mu-selective opioid [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAMGO). At comparable membrane content, the cis isomers of the fatty acids were more potent than their trans counterparts in inhibiting ligand binding and in decreasing membrane microviscosity, both at the membrane surface and in the core. However, trans-vacenic acid affected opioid receptor binding in spite of just marginally altering membrane microviscosity. If the receptors were uncoupled from guanine nucleotide regulatory protein, an altered inhibition profile was obtained: the impairment of KD by the fatty acids was enhanced and that of Bmax reduced. Receptor interaction of the delta-opioid [3H](D-Pen2,D-Pen5)enkephalin was modulated by lipids to a greater extent than that of [3H]DAMGO: saturable binding was abolished by both oleic and elaidic acids. The binding of [3H]naltrexone was less susceptible to inhibition by the fatty acids, particularly in the presence of sodium. In the absence of this cation, however, cis-vaccenic acid abolished the low-affinity binding component of [3H]naltrexone. These findings support the membrane model of opioid receptor sequestration depicting different ionic environments for the mu- and delta-binding sites. The results of this work show distinct modulation of different types and molecular states of opioid receptor by fatty acids through mechanisms involving membrane fluidity and specific interactions with membrane constituents.

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“…The cellular response to opiate agonist was determined by measuring either the intracellular cAMP level or the adenylate cyclase activity in membrane preparations as described previously (Law et al, 19833). Intracellular cAMP levels were determined by the method of Schutz and Daly (1973) using [3H]adenine to label the ATP pools.…”

Section: Intracellular Camp and Adenylate Cyclase Quantitationmentioning

confidence: 99%

Effect of Phospholipases on Chronic Opiate Action in Neuroblastoma × Glioma NG108‐15 Hybrid Cells

Griffin

Law²,

Loh³

1986

Journal of Neurochemistry

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Chronic treatment of neuroblastoma × 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 phospho‐lipids 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 μM for 4 h) hybrid cells. In addition, incubation of hybrid cells with phospholipase C concentrations of >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.

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

Cited by 13 publications

References 27 publications

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

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

Modulation of Opioid Receptor Binding by Cis and Trans Fatty Acids

Effect of Phospholipases on Chronic Opiate Action in Neuroblastoma × Glioma NG108‐15 Hybrid Cells

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