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

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 m M sodium, [ 3 H]Tyr-d-Ala-Gly-(Me)Phe-Gly-ol ([ 3 H]-DAMGO) and [ 3 H]naltrexone bound to Μ receptor with K D values of 3.1 and 0.32 n M 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 [ 3 H]DAMGO, without significantly affecting the other corresponding binding parameters. Depolarizing concentrations of KCl (34 m M ) in physiological buffer decreased the intracellular sodium levels by 67%, but this did not decrease the [ 3 H]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 [ 3 H]DAMGO and [ 3 H]naloxone binding to intact cells with EC 50 values of 24 and 3,600 n M , 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 m M sodium, and typical sigmoidal dose-response curves were obtained only in the presence of 118 m M 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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65917/1/j.1471-4159.1997.68031053.x.pd

Altered Microviscosity at Brain Membrane Surface Induces Distinct and Reversible Inhibition of Opioid Receptor Binding

In synaptosomal membranes from rat and monkey brain cortex, the addition of petroselenic (18:1, cis -Δ 6 ) acid, oleic (18:1, m-Δ 9 ) acid, and vaccenic (18:1, cis -Δ 11 ) acid or their corresponding methyl esters at 0.5 Μmol/mg of membrane protein caused a similar 7–10% decrease in the microviscosity of the membrane core, whereas at the membrane surface the microviscosity was reduced 5–7% by the fatty acids but only 1% by their methyl esters. Concomi-tantly, the fatty acids, but not the methyl esters, inhibited the specific binding of the tritiated Μ-, Δ-, and K-opioids Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO), [D-Pen 2 ,D-Pen 5 ]-enkephalin (DPDPE), and U69,593, respectively. As shown with oleic acid, the sensitivity of opioid receptor binding toward inhibition by fatty acids was in the order Δ > ΜΚ k , whereby the binding of [ 3 H]DPDPE was abolished, but significant inhibition of [ 3 H]U69,593 binding, determined in membranes from monkey brain, required membrane modification with a twofold higher fatty acid concentration. Except for the unchanged K D of [ 3 H]U69,593, the inhibition by oleic acid involved both the B max and affinity of opioid binding. Cholesteryl hemisuccinate (0.5–3 Μmol/mg of protein), added to membranes previously modified by fatty acids, reversed the fluidization caused by the latter compounds and restored inhibited Μ-, Δ-, and k -opioid binding toward control values. In particular, the B max of [ 3 H]-DPDPE binding completely recovered after being undetectable. The results implicate membrane surface fluidity in the modulation of opioid receptor binding, reveal distinct sensitivity of Δ, Μ, and K receptors toward that modulation, and identify unsaturated fatty acids and cholesterol as possible endogenous regulators of opioid receptor function.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66380/1/j.1471-4159.1992.tb08432.x.pd

Altered Transition Between Agonist-and Antagonist-Favoring States of Μ-Opioid Receptor in Brain Membranes with Modified Microviscosity

In unmodified synaptosomal brain membranes the presence of NaCl inhibited the binding to Μ receptors of the tritiated opioid agonists etorphine, Tyr-D-Ala-Gly-(Me)Phe-Gly-ol, and sufentanil by 53, 43, and 37%, respectively, and increased that of the antagonist [ 3 H]naltrexone by 54%. On the other hand, in membranes whose microviscosity was increased by incorporation of cholesteryl hemi-succinate (CHS) the effects of sodium on opioid agonist and antagonist binding were abolished and strongly reduced, respectively. Furthermore, in the modified membranes the ability of sodium to protect the opioid receptor from inactivation by the sulfhydryl-reactive agent N -ethyl-maleimide (NEM) was diminished. In CHS-treated membranes whose elevated microviscosity was reduced by the incorporation of oleic acid, the effectiveness of sodium in modulating opioid binding and attenuating receptor inactivation by NEM was restored. The results implicate membrane microviscosity in the mechanism by which sodium modulates the conversion between agonist-and antagonist-favoring states of Μ opioid receptor.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65931/1/j.1471-4159.1993.tb03630.x.pd

Opioid Signal Transduction in Intact and Fragmented SH-SY5Y Neural Cells

Parameters of ligand binding, stimulation of low- K m GTPase, and inhibition of adenylate cyclase were determined in intact human neuroblastoma SH-SY5Y cells and in their isolated membranes, both suspended in identical physiological buffer medium. In cells, the Μ-selective opioid agonist [ 3 H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol ([ 3 H]DAMGO) bound to two populations of sites with K D values of 3.9 and 160 n M , with <10% of the sites in the high-affinity state. Both sites were also detected at 4°C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [ 3 H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [ 3 H]naltrexone by DAMGO became monophasic with only low-affinity binding present. The toxin specifically reduced high-affinity agonist binding but had no effect on the binding of [ 3 H]naltrexone. In isolated membranes, both agonist and antagonist bound to a single population of receptor sites with affinities similar to that of the high-affinity binding component in cells. Addition of GTP to membranes reduced the B max for [ 3 H]DAMGO by 87% and induced a linear ligand binding component; a low-affinity binding site, however, could not be saturated. Compared with results obtained with membranes suspended in Tris buffer, agonist binding, including both receptor density and affinity, in the physiological medium was attenuated. The results suggest that high-affinity opioid agonist binding represents the ligand-receptor-guanine nucleotide binding protein (G protein) complex present in cells at low density due to modulation by endogenous GTP. Opioid receptor coupling to adenylate cyclase in intact and fragmented cells occurred with similar efficiency: DAMGO inhibited adenylate cyclase with K i , values of 11 n M in cells and 26 n M in lysates, with 30% maximal inhibition in both preparations. Receptor coupling to G protein in membranes occurred with similar parameters: DAMGO stimulated low- K m GTPase with a K s of 31 n M and an S max of 48%. Both effector responses were blocked by naloxone and were strongly impaired by rigorous cell homogenization. These results indicate that opioid signal transduction in intact SH-SY5Y cells and their appropriately isolated membranes functions with similar efficiencies involving a large reserve of uncoupled receptors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66450/1/j.1471-4159.1992.tb10032.x.pd

Assessment of membrane permeability in primary cultures of neurons and glia in response to osmotic perturbation

In primary cultures of neurons and astrocytes from rat brain, the cellular contents of K + and Na + were used to assess plasma membrane permeability in response to osmotic shock. In contrast to trypan blue exclusion, the measure of ionic content reflected both transient and permanent membrane damage, and was also applicable to aggregates of cells. In steady state, the neurons and glia exhibited a K + to Na + ratio of 3–5, and the mean cellular contents (ΜEq/mg protein) of K + and Na + were 0.72 and 0.17 for astrocytes and 0.78 and 0.23 for neurons. Both hypo- and hypertonicity resulted in marked efflux of cell K + , but elevation of cellular Na + occurred only under severe hypertonic conditions. Relative to neurons, astrocytes displayed considerably higher resistence to osmotic shock. During subsequent isotonic incubation, these cells were able to completely recover from transient membrane damage caused by a 10-min exposure to fourfold hypertonicity. Permanent changes in glial permeability were obtained only after a 20-min hypertonic shock. In contrast, 5 min hypertonic treatment of neurons decreased the ratio of cellular K + to Na + from 4.5 to 1. This ratio was restored twofold by isotonic incubation, but decreased permanently to below 1 after 10 min of hypertonic shock. The results describe marked differences in the osmotic fragility of neurons and glia and demonstrate that the determination of cellular K + and Na + provides a sensitive and accurate indicator of membrane permeability in neural cells propagated as surface-growing cultures. The approach has wide-ranging applicability.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50216/1/490130304_ftp.pd

Heterogeneity of opioid receptor binding in brain slices

A methodological approach was established for the study of ligand binding to multiple opioid receptors in slices from rat brain striatum. Specific binding of radiolabeled opiates was resolved from total binding with enantiomers or excess unlabeled ligand. Equilibrium binding of triated etorphine, dihydromorphine, and ethylketocyclazocine, and competitive displacement of [ 3 H]dihydromorphine by the unlabeled opiates were used to assess both high and low affinity receptor sites. The high-affenity binding components of the radiolabeled opiates were characterized by linear Scatchard plots, K d values of 2.8–3.7 nM, and binding site densities of 180-297 fmol/mg protein. The displacement of [ 3 H]etorphine by morphine and ethylketocyclazocine displayed Hill coefficients of 0.62 and 0.47, respectively, and revealed receptor sites with much lower affinities than those described by the direct binding of these opiates. On the other hand, both morphine and ethylketocyclazocine displaced [ 3 H]dihydromorphine with similar high potencies (apparent K d s, 3-4 nM). The results support the feasibility of using brain slices as a cellular preparation to study opioid receptor mechanisms.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50218/1/490180214_ftp.pd

Glucaric acid as an indicator of use of enzyme‐inducing drugs

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/116979/1/cpt1974154417.pd

The uptake of pentazocine into brain

A simple procedure for the extraction from tissues and determination by gas chromatography of pentazocine was developed. The mean recovery from brain was 96%.The time- and dose-dependent uptake of pentazocine into the brain was investigated and correlated with the plasma levels of the drug. The results showed a rapid entry of pentazocine into the drug. The results showed a rapid entry of pentazocine into at 2 min and 10 min, respectively, after the i.p. administration. After the injection of a single dose, the ratio, concentration of pentazocine in plasma/concentration of pentazocine in brain was constant from 10-90 min. This ratio remained unchanged over the range of 25-100 mg/kg of administered drug.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33633/1/0000141.pd

The determination of benzomorphan derivatives in plasma by gas chromatography

A specific, sensitive and simple procedure for the extraction from plasma and determination by gas chromatography of benzomorphan derivatives is presented.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33632/1/0000140.pd

Relationship Between Opioid-Receptor Occupancy and Stimulation of Low- K m GTPase in Brain Membranes

Treatment of rat brain membranes with the irreversible opioid ligand cis -3-methylfentanylisothiocyanate (Superfit) was used to reduce gradually the number of available binding sites for the Δ-selective agonist [ 3 H][d-Ser 2 , Leu 5 lenkephalin-Thr 6 ([ 3 H]DSLET). Subsequently, the correlation between ligand binding and low- K m GTPase was investigated. Alkylation with 10 ΜM and 25 ΜM Superfit inactivated 66% and 71% of high-affinity ( K D , 1 n M ) binding sites without decreasing the affinity of the remaining sites and the stimulation of low-. K m GTPase by DSLET. Following exposure of the membranes to 50 ΜM and 75 ΜM Superfit, ligand binding was confined to the low-affinity ( K D , 20 n M ) sites. In these membranes, the Δ-agonists DSLET and [d-Pen 2 ,D-Pen 5 ]enkephalin still stimulated low- K m GTPase, and these effects were blocked by ICI 174864 ( N,N- diallyl-Tyr-AIB-AIB-Phe-Leu-OH; AIB, Α-aminoisobutyric acid), a Δ-selective antagonist. A similar relationship between low-affinity ligand binding and GTPase stimulation was observed following alkylation of the Δ-opioid receptor with the nonselective irreversible antagonist Β-chlomaltrexamine in the presence of protective concentrations of DSLET. The results reveal spare receptor sites in the coupling of the Δ-opioid receptor to low- K m GTPase in brain and identify low-affinity ligand binding as a functional component in the process.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65675/1/j.1471-4159.1989.tb01862.x.pd