Fedor Medzihradsky

Stereospecific binding of 3H-phencyclidine in brain membranes.

Phencyclidine (PCP) displaceable binding of 3H-PCP to glass-fiber filters was eliminated and total binding markedly reduced by initial treatment of the discs with 0.05% polyethyleneimine. Assessed with treated filters, unlabeled PCP displaced 3H-PCP in both rat and pigeon brain membranes with an EC50 of 1 microM. Of similar high inhibitory potency were dextrorphan, levorphanol, SKF 10047 and ketamine, while morphine, naloxone and etorphine had EC50 values higher then 1 mM. Using the dissociative anesthetic dexoxadrol and its inactive isomer levoxadrol as displacing agents, stereospecific binding of 3H-PCP was obtained in rat and pigeon brain membranes. The markedly higher potency of dexoxadrol, relative to levoxadrol, in displacing bound 3H-PCP is compatible with behavioral data for these enantiomers. However, they were equipotent in displacing 3H-PCP bound to glass-fiber filters in the absence of tissue. Heat denaturation, but not freezing, abolished stereospecific binding of 3H-PCP, which was also absent in rat liver membranes. The stereospecific binding component in brain displayed biphasic saturability at 60-70 nM and 300-400 nM, respectively.

A comparison of the ATPase activity of the glial cell fraction and the neuronal perikaryal fraction isolated in bulk from rat cerebral cortex1

SINCE SKOU (1957) first described a Na-K-activated, Mgdependent, and ouabain-sensitive adenosinetriphosphatase in crab nerve, considerable evidence has accumulated to implicate this enzyme in the transport of Na and K (see reviews by SKOU, 1965; and ALBERS, 1967). The activity of this ATPase is high in membrane preparations from various organs, the highest activity being that of membranes from nervous tissues (BONTING. SWON and HAWKINS, 1961). The enzyme is intimately associated with the membranes, but its solubilization and stabilization in solution have recently been accomplished (MEDZIHRADSKY, UINE and H o r n , 1967). Also, the regional distribution of Na-K ATPase has been mapped in the brain of the rabbit (HARMONY, URBL-HOLMGREN and URBAY, 1967) and the rhesus monkey (FAHN and G3d, 1968). In view of the cellular heterogeneity of brain and of unsettled questions concerning the role of glial cells and of their interrelations with neurons, we have compared the activities of Na-K ATPase and total ATPase of the neuronal perikaryal fraction and glial cell fraction obtained from cerebral cortex as two separate populations by a bulk-isolation procedure. In addition, we determined the intracellular distribution of these two enzymic activities in neuronal perikarya by the use of standard differential centrifugation techniques.

Receptor binding, antagonist, and withdrawal precipitating properties of opiate antagonists☆

A number of opiate antagonists and the dextro isomers of some of these drugs were studied for antagonism of acute opiate effects on ilea isolated from opiate-naive guinea pigs, precipitation of a withdrawal contraction of ilea isolated from morphine-dependent guinea pigs, precipitation of withdrawal in morphine-dependent rhesus monkeys and stereospecific displacement of 3H-etorphine binding to rat-brain membranes. With the exception of d-naloxone, all of the compounds displaced 3H-etorphine. With the exception of d-naloxone, nalorphine, and quaternary nalorphine, all of the antagonists caused a contraction of ilea isolated from morphine-dependent guinea pigs. Moreover, the IC 50 values of the compounds for displacing 3H-etorphine binding were well correlated with both their Ke values for antagonism in the ileum (r = 0.95) and with their EC 50 values for precipitating a contraction in this preparation (r = 0.92). Generally, the concentration of antagonist necessary to precipitate half maximal contracture was 30-fold greater than the Ke value of the antagonist. Most of the opiate antagonists also precipitated withdrawal when administered to morphine-dependent rhesus monkeys and their in vivo potencies were well correlated with their in vitro potencies in ileum (with Ke: r = 0.95; with EC 50: r = 0.99) and in displacing 3H-etorphine (r = 0.95). The quaternary derivative of naltrexone, however, was an effective opiate antagonist only in vitro, and was ineffective in precipitating withdrawal in morphine-dependent rhesus monkeys. These results suggest that the receptor sites labeled by 3H-etorphine are the same as those involved in antagonism of acute opiate actions and in precipitation of withdrawal.

Cyclic, disulfide- and dithioether-containing opioid tetrapeptides: Development of a ligard with high delta opioid receptor selectivity and affinity

Tetrapeptides of primary sequence Tyr-X-Phe-YNH2, where X is D-Cys or D-Pen (penicillamine) and where Y is D-Pen or L-Pen, were prepared and were cyclized via the side chain sulfurs of residues 2 and 4 to disulfide or dithioether-containing analogs. These peptides are related to previously reported penicillamine-containing pentapeptide enkephalin analogs but lack the central glycine residue of the latter and were designed to assess the effect of decreased ring size on opioid activity. Binding affinities of the tetrapeptides were determined to both mu and delta opioid receptors. Binding affinity and selectivity in the tetrapeptide series were observed to be highly dependent on primary sequence. For example, L-Pen4 analogs displayed low affinity and were nonselective, while the corresponding D-Pen4 diastereomers were of variable affinity and higher selectivity. Among the latter compounds were examples of potent analogs in which selectivity shifted from delta selective to mu selective as the ring size was increased. The relatively high binding affinity and delta receptor selectivity observed with one of the carboxamide terminal disulfide analogs led to the synthesis of the corresponding carboxylic acid terminal, Tyr-D-Cys-Phe-D-PenOH. This analog displayed delta receptor binding selectivity similar to that of the standard delta ligand, [D-Pen2,D-Pen5]enkephalin (DPDPE), and was found to have a 3.5-fold higher binding affinity than DPDPE. All the tetrapeptides were further evaluated in the isolated mouse vas deferens (mvd) assay and all displayed opioid agonist activity. In general, tetrapeptide potencies in the mouse vas deferens correlated well with binding affinities but were somewhat lower. Receptor selectivity in the mvd, assessed by examining the effect of opioid antagonists on the tetrapeptide concentration-effect curves, was similar to that determined in the binding studies.

Selectivity of ligand binding to opioid receptors in brain membranes from the rat, monkey and guinea pig

Conditions for the equilibrium binding to opioid receptor of [3H]sufentanil (mu selective), [3H][D-Pen2,D-Pen5]enkephalin (delta selective), and [3H]U69,593 (kappa selective) were established in membranes from rat brain cerebrum, monkey cortex, or guinea pig cerebellum. The selectivity index of various opioid alkaloids and peptides in binding to the mu, delta, or kappa opioid receptors was expressed as the ratio of their EC50 values in displacing two selective radiolabeled ligands: [3H]sufentanil/[3H](D-Pen2,D-Pen5)enkephalin (selectivity: mu/delta), [3H]sufentanil/[3H]U69,593 (selectivity: mu/kappa), or [3H][D-Pen2,D-Pen5]enkephalin/[3H]U69,593 (selectivity: delta/kappa). High resolution in binding selectivity was observed: in rat brain the mu/delta selectivity for Tyr-D-Ala-Gly-(Me)Phe-Gly-ol and sufentanil were 0.02 and 0.03, whereas for [D-Pen2,D-Pen5]enkephalin and ICI 174,864 they were 1,200 and 998. Compared to mu opiates, the specific binding of delta and kappa agonists was less sensitive to sodium. The results describe a routinely applicable methodological approach for the assessment of selective ligand binding to the mu, delta and kappa opioid receptors in rodent and monkey brain membranes.

Measures of Viability in Isolated Cells

Abstract Various methods were compared for estimation of cell viability. Cell count, uptake of trypan blue, release of cellular LDH, as well as cell contents of K+ and Na+ were determined in leukocytes, spleen cells, and Ehrlich cells exposed to adverse conditions. After incubation of these cells with NaF, the cell count remained essentially constant; the extent of cell staining and release of LDH increased to a limited level in suspensions of leukocytes and spleen cells and was virtually unchanged in experiments with Ehrlich cells. On the other hand, pretreatment with NaF induced rapid and marked changes in contents of K+ and Na+ in all three cell types. Exposure of the cells to hypertonic conditions caused more pronounced, but similar overall effects. As a consequence of increased tonicity, the cell count decreased and considerable leakage of LDH occurred in all the cells investigated. Whereas the contents of K+ and Na+ in all pretreated cells changed markedly, the cellular uptake of trypan blue was again the least responsive index of cell permeability. The results showed cell staining by trypan blue to be a relatively poor measure of cell viability and suggest use of the ratio cellular K + Na + or, even more efficiently, the cell content of K+ as sensitive indicators of plasma membrane integrity.

Scatchard Analysis of Opiate Receptor Binding

The study of [3H]naltrexone binding in a membrane preparation from rat brain revealed that experimental conditions of the opiate receptor assay markedly influenced the outcome of the corresponding Scatchard analysis. The use of inappropriate concentrations of the unlabeled displacing drugs to assess stereospecific [3H]naltrexone interaction resulted in Scatchard plots which mimicked cooperativity in binding. These monophasic plots also indicated that sodium affects antagonist binding by changing receptor affinity ( KD ) but not the density of sites. When assessed under correct experimental conditions the Scatchard plots for specific [3H]naltrexone binding were biphasic. Sodium, without affecting the KD , increased and decreased the number of high- and low-affinity sites, respectively. Thus, at 25° the total number of opiate receptor sites for [3H]naltrexone binding in the absence and presence of sodium was statistically indistinguishable. Initial membrane incubation competed with sodium in increasing the density of high-affinity [3H]naltrexone-binding sites. After kinetic resolution and computer analysis considering several binding models, the data for specific [3H]naltrexone binding provided best fit for two saturable sites. At 25° and in the absence of sodium the respective approximate KD values were 0.4 and 30 nM, with densities of 200 and 350 fmoles/mg of protein. Under a variety of experimental conditions, including different temperatures and exposure of the membranes to trypsin or freezing, some of these binding parameters differed, but the biphasic nature of specific [3H]naltrexone binding was unaltered. Identical biphasic Scatchard plots were obtained if specific binding of [3H]naltrexone was determined with excess unlabeled naltrexone, morphine, ethylketocyclazocine, or SKF 10047; with dextrorphan and levorphanol; or with enantiomers of naloxone as displacing ligands. Terminating the binding assay by rapid centrifugation yielded results identical with those obtained when quick filtration was used. The dissociation of bound [3H]naltrexone was resolved into a rapid component and a slow component. In the presence of high concentrations of the drug an additional rapid component of dissociation became apparent. The KD values calculated from the two rapid components of dissociation and the corresponding rate constants of association agreed well with those determined for the high- and low-affinity [3H]naltrexone-binding sites by Scatchard analysis. The nature of the slow dissociation component with markedly high affinity has to await further clarification. The results of this study characterize the interaction of naltrexone with opiate receptor, contribute to the understanding of the mechanism of the sodium effect, and describe the role of methodology in evaluating ligand binding.

Receptor-mediated stimulation of brain GTPase by opiates in normal and dependent rats

In membranes from rat brain striatum, opiate agonists stimulated low-K GTPase. Half-maximal enhancement of enzyme activity was obtained with 0. 09m microM morphine and 3.8 microM levorphanol. This order of potency corresponded to that of the affinities of these compounds in binding to opiate receptor. The effect was inhibited by the antagonist naloxone. As shown by the use of the enantiomers levorphanol and dextrorphan, only the pharmacologically active stereoisomer stimulated GTPase. In membranes isolated from morphine-dependent rats, the activity of GTPase was reduced 20-40% relative to that in control rats. After the precipitation of morphine abstinence by naloxone, brain GTPase activity was intermediate between the respective values for naive and dependent animals.

Glucaric acid as an indicator of use of enzyme-inducing drugs.

The usefulness of urinary glucaric acid determinations to measure enzyme induction by drugs was investigated by correlating its excretion rate with the daily drug regimen of 100 hospitalized patients. The urinary excretion of glucaric acid was higher in patients than in normal controls. Patients treated with enzyme inducers excreted more glucaric acid than patients not getting them, as well as more than normal controls. In patients on multiple‐drug therapy, the effect of small doses of known enzyme inducers was masked, but even here relatively high doses of potent inducers significantly increased glucaric acid excretion. The results also suggest that urinary excretion of glucaric acid may have value as an indicator of the use of certain drugs by patients when such information is not revealed by the history.

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

Abstract: In synaptosomal membranes from rat and monkey brain cortex, the addition of petroselenic (18:1, cis‐Δ6) acid, oleic (18:1, m‐Δ9) acid, and vaccenic (18:1, cis‐Δ11) 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‐Pen2,D‐Pen5]‐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 > μκk, whereby the binding of [3H]DPDPE was abolished, but significant inhibition of [3H]U69,593 binding, determined in membranes from monkey brain, required membrane modification with a twofold higher fatty acid concentration. Except for the unchanged KD of [3H]U69,593, the inhibition by oleic acid involved both the Bmax 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 Bmax of [3H]‐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.