Stability of lyophilized S-9 preparations

Lyophilized S-9 preparations were stored and desiccated at 4 °C and ?20 °C for up to 30 weeks. The Ames' Salmonella test and enzyme assays of oxidative-N-demethylation, C-hydroxylation and N-hydroxylation were run on the lyophilized preparation, and the specific activities were compared to S-9 stored at ?100 °C. Decreases in enzyme activity did occur in all preparations; however, lyophilized S-9 overall maintained higher activity than S-9 preparations stored at ?100 °C.     

Effect of apomorphine on rat brain opiate receptors

Stereospecific binding of apomorphine to rat brain opiate receptors was shown by assaying the competition of 7,8(n)--3H--naloxone and D-ala2-tyrosyl-3,5-3H--enkephalin (5-D-leucine) for opiate receptor binding. EC-NaCl50, the concentration of apomorphine which inhibited 50% binding of the radioactive naloxone and D-ala2, D-leu5-enkephalin in the absence of NaCl were 20 and 42 microM, respectively. EC+NaCl 50, the concentration of apomorphine which inhibited 50% binding of the radioactive naloxone in the presence of 100 mM NaCl was 17 microM. From the ratio of EC+NaCl 50 to EC-NaCl the value of "sodium shift" of effective concentration can be calculated as 0.85. From the data obtained it is concluded that apomorphine, like naloxone, is a "pure" antagonist but it has much less affinity for enkephalin and opiate binding sites. The probable mechanisms of the pharmacological action of apomorphine are discussed.     

Effect ot thyroliberin on rat brain opiate receptors

The ability of thyroliberin to interact with opiate receptors of the rat midbrain and hypothalamus has been studied. It was shown by competitive displacement analysis that thyroliberin did not replace labeled opioid peptides in opiate receptor binding sites when added in vitro at concentrations of up to 10(-5) M. The specific binding of opioid peptides was increased by 10-20% in the presence of 10(-7)-10(-6) M thyroliberin. This effect was, probably, due to the rise in the affinity of high-affinity opiate receptors. At the same time the affinity of low-affinity binding sites was decreased. It is suggested that the antagonistic properties of thyroliberin are mediated by the modulation of the binding characteristics of enkephalin-low-affinity opiate receptors.     

Alpha interferon interaction with opiate receptors in the rat brain

The preferential interactions of alpha-interferon (alpha-IFN) with delta and mu opiate receptors were studied. alpha-IFN (specific antiviral activity 2 X 10(3) U/mg protein) was shown to inhibit in the competitive manner 3H-naloxone and 3H-D-ala2, D-leu5-enkephalin (3H-DADL) specific binding to opiate receptor subpopulations. alpha-IFN was much more effective in decreasing 3H-DADL than 3H-naloxone binding in opiate receptors: K1 values averaged 160 +/- 30 and 1150 +/- 80 U/ml, respectively. IFN effective concentrations inhibiting 50% of 3H-naloxone opiate receptor binding in the absence or presence of 100 mmol/l NaCl were similar, and the "sodium shift" value was equal to 1. The independence of alpha-IFN activity of the presence of NA+ cations suggests the antagonist character of alpha-IFN interaction with opiate receptors. Thus, alpha-IFN employed appears to be an alpha-selective ligand displaying the in vitro properties of "pure" morphine antagonists.     

Development of opiate receptors and GTP-binding regulatory proteins in neonatal rat brain

The mu and delta opiate receptors present in rat brain were measured independently during postnatal development. The numbers of delta receptors were almost undetectable at birth and increased substantially during the first few weeks, whereas the numbers of mu receptors remained relatively constant. Activation of either of these receptors caused inhibition of adenylate cyclase, but inhibition coupled to mu receptors was much smaller than that associated with delta receptors at all ages. Attempts to use pertussis toxin-catalyzed ADP-ribosylation as an assay for the GTP-binding proteins Ni and No were hampered by the development of an NADase with age. However, specific antibodies directed against the alpha subunits of Ni or No allowed separate quantitation of these transducer proteins. Both increased with age. No is present at levels at least 5-fold higher than Ni in the adult rat brain. The N proteins are in vast excess over receptors and as such are unlikely to be limiting factors in receptor function. The data further suggest that the number of opiate receptors present throughout neonatal development is in excess over that required for optimal function.     

Cholecystokinin-8 suppressed 3H-etorphine binding to rat brain opiate receptors

Radio receptor assay (RRA) was adopted to analyse the influence of CCK-8 on 3H-etorphine binding to opiate receptors in rat brain synaptosomal membranes (P2). In the competition experiment CCK-8 (1pM to 1 microM) suppressed the binding of 3H-etorphine. This effect was completely reversed by proglumide at 1 microM. Rosenthal analysis for saturation revealed two populations of 3H-etorphine binding sites. CCK-8 (1pM to 1 microM) inhibited 3H-etorphine binding to the high affinity sites by an increase in Kd (up to +235%) and decrease in Bmax (up to -80%) without significant changes in the Kd and Bmax of the low affinity sites. This effect of CCK-8 (10nM) was also completely reversed by proglumide at 1 microM. Unsulfated CCK-8 (100pM to 1 microM) produced only a slight increase in Kd of the high affinity sites (+64%) without affecting Bmax. The results suggest that CCK-8 might be capable of suppressing the high affinity opioid binding sites via the activation of CCK receptor.     

Effects of paradoxical sleep deprivation on the activity of opiate receptors isolated from synaptic membranes of the rat brain]

The specific binding of 3H-naloxone with opiate receptors isolated from brain synaptic membranes of control and paradoxical sleep deprived rats were studied. This extreme state was shown to reduce the naloxone-binding activity of synaptic membranes by 35% and isolated receptors by 25-28%. The values of Kd and Bmax were calculated for isolated opiate receptors at different stages of purification. Considerable decrease of 3H-naloxone binding sites density (2 times) in the isolated opiate receptors with simultaneous increase of their affinity (3-4,5 times) was found following 24 hours paradoxical sleep deprivation. These findings suggest development of fixed alterations in the structure and functions of integral receptor proteins under extreme influences.     

Alcohol interactions with brain opiate receptors

Ethanol, added $\text{in vitro}$ to mouse caudate membranes, inhibited high-affinity binding of 0.2 nM ³H-dihydromorphine (³H-DHM) over an ethanol concentration range of 250–1,000 mM. At lower, physiologically-attainable ethanol concentrations (e.g.: 50 mM), ³H-DHM binding was significantly increased. Over the concentration range of 50–1,000 mM, ethanol inhibited 0.5 nM ³H-[D-Ala², D-Leu⁵] enkepha l in (³H-ENK) binding to mouse caudate tissue, and no stimulation of Ethanol inhibits opiate binding in a pseudo-competitive manner and, therefore, the concentration of ligand used to assess the effects of ethanol is of major importance. Results obtained with other alcohols which differ in their membrane: water partition coefficients suggest that alcohol effects on opiate binding are not solely dependent on the membrane-disordering properties of the alcohols.     

Differential distribution of opiate and neuroleptic receptors and dopamine-sensitive adenylate cyclase in rat brain.

In rat brain, the regional distribution of the neuroleptic receptor and of dopamine-sensitive adenylate cyclase was found to be very similar, but it differed markedly from the distribution of the opiate receptor. Neuroleptic receptor sites were detectable in the cortex and the hypophysis. After differential centrifugation of rat striatum homogenate, opiate and neuroleptic receptors were enriched in the microsomal fraction while dopamine-sensitive adenylate cyclase revealed a mitochondrial distribution pattern. This different subcellular localization of the neuroleptic receptor and the dopamine-sensitive adenylate cyclase suggests a different function for both receptors.     

Pregnancy related changes of opiate receptors identified in rat uterine membranes by 3H-naloxone binding

3H-Naloxone was used to demonstrate the presence of specific opiate binding sites in uterine membrane preparations of rats. 3H-Naloxone binding (0.41-27 nM) was found to be rapid, saturable and reversible showing two populations of binding sites with the characteristic of high (KD 2.2 nM; Bmax 46.6 fmol/mg prot.) and low (KD 18.1 nM; Bmax 143.7 fmol/mg prot.) affinity. The number and affinity of the binding sites labelled by 3H-naloxone in the uterus were measured in the rat at mid (14 days), late (21 days) pregnancy and at parturition. The high and low affinity recognition sites labelled by 3H-naloxone showed a consistent reduction during pregnancy and at parturition without changes in the affinity constant. We concluded that pregnancy and parturition are associated with significant changes in the number of the opiate receptors bound in the uterus by 3H-naloxone. This phenomenon which seems to be linked with the several pregnancy-related changes in the levels of endogenous peptides and hormones could be relevant to further explain the pregnancy related changes in pain perception and maternal behavior.     

Stereospecific interaction of opiate narcotics in binding of 3H-dihydromorphine to membranes of rat brain

Membranes from homogenates of corpus striatum bound ³H-dihydromorphine in a saturable fashion with a Km value of 1 × 10^?9M. The binding of ³H-dihydromorphine to the membranes was reduced to about 10% by 10^?7M levorphanol but not by 10^?7M dextrorphan. The binding of ³H-dihydromorphine became less sensitive to 10^?7M levorphanol when the concentration of ³H-dihydromorphine was greater than 2 × 10^?9M. Other opiate narcotics, e.g. morphine and $\text{l}$-methadone, were as effective as levorphanol in competition for the binding ³H-dihydromorphine with ED₅₀ values of 2–4 × 10^?9M. $\text{d}$-Methadone and dextrorphan were about 1/50 and 1/2000 as effective as their respective levo-isomers. The opiate antagonist, naloxone, also competed effectively for the binding sites with an ED₅₀ value of 3.3 × 10^?9M. Substances like acetylcholine, choline, serotonin, norepinephrine and dopamine were ineffective. Only ionophores specific for divalent cations stimulated the binding of ³H-dihydromorphine suggesting that some endogenous divalent cations may be inhibitory to the binding of the opiate narcotic. The receptors of ³H-dihydromorphine probably exist in the membranes of nerve endings and have a density of 6 × 10¹² sites per g in corpus striatum. We conclude that the described technique can successfully detect the opiate narcotic receptors in the central nervous system without the usual method of displacement.     

The binding of fluorocatecholamines to adrenergic and dopaminergic receptors in rat brain membranes

2-Fluoronorepinephrine (IC₅₀ ≈0.7 μM) is a relatively selective ligand for displacement of radioactive dihydroalprenolol from β₁-adrenergic receptors in membrane preparations from rat cerebral cortex. It is less potent (IC₅₀ ≈10 μM) in displacing dihydroalprenolol from β₂-adrenergic receptors in rat cerebellar membranes and in displacing clonidine from α₂-adrenergic receptors in rat cerebral cortical membranes. It is much less potent (IC₅₀ > 100 μM) in displacing WB-4101 from α₁-adrenergic receptors in rat cerebral cortical membranes. In contrast, 6-fluoronorepinephrine is relatively selective for α-adrenergic receptors, being at least 50–200 times more potent at such receptors than at β-adrenergic receptors. 5-Fluoronorepinephrine like norepinephrine does not exhibit remarkable selectivity towards α- and β-adrenergic receptors. The 2-, 5- and 6-fluorodopamines are more potent ligands at α₁-adrenergic receptors than at α₂- and β-adrenergic receptors but the specificity is not markedly affected by the position of the fluorine substituent. The results suggest that the specificity exhibited by the 2- and 6-fluoronorepinephrine at adrenergic receptors is not primarily due to fluorine-induced changes in the physicochemical properties of the aromatic ring, but instead to stereoselective interactions of the ethanolamine side chain of norepinephrine with fluorine at either the 2- or 6-ring positron. The fluorodopamines like dopamine itself are more potent at dopaminergic than at α- or β-adrenergic receptors. The 2-, 5- and 6-fluorodopamines are all nearly equipotent with dopamine in the displacement of radioactive spiroperidol from dopaminergic receptors in membrane preparations from rat striatum, while the 2- and 6-fluorodopamine are somewhat less potent than dopamine or 5-fluorodopamine in displacement of radioactive apomorphine in striatal membranes.     

Visualization of opiate receptors and opioid peptides in sequential brain sections

Autoradiographic and immunocytochemical studies were carried out on adjacent sections from formaldehyde-perfused rat brains in order to directly correlate the distribution of opiate receptors and opioid peptides. Perfusion fixation of the brains resulted in a partial loss of specific [3H]naloxone binding with essentially no change in the pharmacological properties of the remaining sites. When the distribution of sites was compared to that of enkephalin immunoreactivity in adjacent sections, striking correlations were observed in a number of areas throughout the neuraxis. Adjacent section autoradiography-immunocytochemistry should provide a useful tool for relating the anatomical distribution of opiate receptor subtypes to different opioid peptide neuronal systems.     

Disulfide bonds within the alpha-subunit of insulin receptors in rat liver and brain membranes

We have characterized inter- and intrasubunit disulfide bonds of insulin receptors using reductant-treated rat liver and brain membranes. In autoradiograms of 125I-insulin cross-linked to both membranes pretreated with dithiothreitol, the intensity of affinity-labeled bands of the alpha beta-heterodimer and alpha-subunit was increased. Interestingly, labeled 120 and 110 kDa bands considered to be the alpha-subunit in partially reduced liver and brain membranes moved to 130 and 120 kDa bands under further reduced conditions, respectively. Double electrophoresis of each partially reduced band in the presence of reductants clearly demonstrates that the alpha-subunit of insulin receptors contains intrasubunit disulfide bonds.     

Specific binding of mu- and delta-ligands by opiate receptors in the rat brain in the presence of reaferon

The ability of recombinant alpha 2-interferon (reaferon) to compete for opiate binding sites with mu- and delta-selective compounds was determined. Reaferon was found to inhibit the binding of 3H-D-ala2, D-leu5-enkephalin, and Ki value calculated was equal to 8.5 +/- 2.6 U.10(-3)/ml. The mu-agonists reception levels were decreased in the presence of reaferon at concentrations above 500 U/ml; the Ki values for 3H-morphine, 3H-dihydromorphine, 3H-RX 783006 were found to be 3.25 +/- 0.35, 4.28 +/- 0.81 and 6.51 +/- 1.27 U.10(-4)/ml, respectively. When reaferon was added into reaction medium at concentrations more than 5.10(3) U/ml the specific receptor binding of opiate antagonist 3H-naloxone was demonstrated to be increased and this effect was reversed with 100 mM NaCl. The existence of allosteric reaferon binding site which coupled with naloxone sensitive receptor was suggested to explain the results obtained.     

Monoclonal antibodies against the S2-serotonin receptor from rat brain that cross-react with dopamine and opiate receptors

A method for the production of plasmids giving different levels of expression of ovine growth hormone (oGH) variants in E. coli is described. The cDNA sequence coding for mature oGH was inserted into the multiple cloning site of plasmid pUC8 and random deletions were then introduced 3′ to the initiation codon. Clones producing GH (with varying N-terminal extensions) were identified by immunological screening. Levels of expression of GH-related protein, measured by immunoassay or on SDS-polyacrylamide gels, varied from over 20% to less than 0.05% of total cell protein. The coding sequence of plasmid pOGHe101, giving very high expression of variant oGH1, was determined.