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.     

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.     

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.     

Does interferon exert its actions through opiate receptors

It had been reported that alpha interferon (alpha-IFN) induces endorphin-like effects such as analgesia and catatonia. These effects, reversed and prevented by naloxone, suggest that the alpha-IFN effect is mediated via opiate receptors. In order to examine this hypothesis, the present study was initiated. Extracellular cortical cell recording and microiontophoretic application of alpha-IFN, morphine, and naloxone, as well as the application of these three drugs on the coaxially stimulated guinea-pig ileum preparation were used. alpha-IFN application induced excitation in cortical cells and on the guinea-pig ileum. In contrast, the main effect elicited by morphine was a decrease in both preparations. Naloxone was able to reverse and/or prevent the morphine effects in both preparations, but failed to alter the effects induced by alpha-IFN. The present observations using the guinea-pig ileum preparation and cortical neurons recording failed to support the hypothesis that alpha-IFN effects are mediated via opiate receptors.     

In vitro interaction of ACTH with rat brain muscarinic receptors

ACTH-(1-24) inhibits the in vitro binding of the muscarinic antagonist [3H]QNB to membranes from rat brain. The magnitude of inhibition is dependent on the concentration of ACTH-(1-24). Kinetic analysis indicates a pure competitive inhibition which is suggestive of a reversible interaction of ACTH with muscarinic receptors. A mechanism involving an interaction of ACTH-(1-24) with the phospholipid core of the receptors is suggested. Structure activity studies point to a relation with reported effects of intracerebroventricularly administered ACTH on the turnover rate of acetylcholine and the ACTH-induced stretching and yawning syndrome.     

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.     

In vitro interaction of premazepam with benzodiazepine receptors in rat brain regions

Premazepam (PRZ) in vitro competitively displaced 3H-diazepam (DIA), 3H-flunitrazepam (FLU) and 3H-RO 15-1788 from their binding sites on rat brain synaptosomes, with a potency intermediate to other benzodiazepines (BDZs), and Hill coefficients near 1 in different brain regions. Incubation at 37 degrees C reduced premazepam's affinity for BDZ receptors to a lower extent than other benzodiazepines and had no effect on the Hill coefficient. The IC50 of PRZ on 3H-RO 15-1788 and 3H-FLU binding was markedly reduced by GABA in rat cortex, like those of reference classical BDZs, but was GABA-independent in the cerebellum. The IC50 of the BDZ antagonist, RO 15-1788 was unaffected by GABA in both brain areas. The possibility that PRZ behaves as a partial agonist in the cortex and as an antagonist in the cerebellum is discussed.     

Stability of opiate receptors of wet and lyophilized preparations of rat brain membranes

The effects of incubation of rat brain membranes at 0 degrees C on the specific binding of mu-ligands (naloxone, morphine) and the delta-ligand (D-Ala2, D-Leu5-enkephalin) to opiate receptors were studied. The effects of lyophilization of rat brain membranes on the properties of the opiate receptors were determined. The lyophilized brain membrane preparations revealed an extraordinarily high stability as compared to "wet" membranes. The experimental results suggest that morphine and D-Ala2, D-Leu5-enkephalin binding both to the high affinity and low affinity sites has different nature and point to the utility of stable and standard preparations of lyophilized membranes for the use in the receptor analysis of opiate and opioid peptides.     

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.     

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.     

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.     

Neurotoxin of the black widow spider and its interaction with receptors from the rat brain

A presynaptic neurotoxin isolated from the venom of the Central Asia spider karakurt (Black Widow Spider, Latrodectus mactans tredecimguttatus) is shown to consist of two identical subunits of mol. weight about 118 kDa. The iodinated neurotoxin binds to the rat brain synaptosomal plasma membranes with Kd 0.1 nM (Bmax 0.1 pmol/mg of protein) at 37 degrees C, and with Kd 0.35 nM (Bmax 0.2 pmol/mg of protein) at 5 degrees C. At intermediate temperatures both types of receptors are detectable. It is supposed that the dimeric form of the toxin interacts with a single class of receptors possessing lateral mobility in the membrane. By the use of different bifunctional reagents it is revealed that the neurotoxin interacts with a presynaptic membrane protein of mol. weight 95 kDa. A protein of the same size accompanied by a 71 kDa protein was isolated by the affinity chromatography of solubilized synaptosomal membranes on the absorbent, containing immobilized neurotoxin.     

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.     

A differential interaction of putative μ-selective agonists with opiate binding sites in rat brain

The availability of tritium-labelled sufentanil ([³H]SUF) allowed for a further radioligand analysis of opiate binding sites in rat brain. A comparison of the binding characteristics of [³H]SUF and [³H]dihydromorphine ([³H]DHM) revealed a very similar potency in their mutual displacement by unlabelled analogues. Furthermore, a series of putative μ-opiate agonists displayed equal potencies in displacing either [³H]SUF and [³H]DHM, the only striking exception being the highly μ-selective opioid peptide morphiceptin which was 33 times less potent in inhibiting [³H]SUF as compared to [³H]DHM binding. Additional experiments revealed further pronounced differences in [³H]SUF and [³H]DHM binding characteristics: the total amount of binding sites for [³H]SUF was 4 times higher than that for [³H]DHM and the regional distribution within particular brain areas displayed considerable differences. Furthermore, the binding of [³H]SUF was differentially modulated by sodium and GTP as compared to [³H]DHM binding. These data suggest that in rat brain, [³H]SUF interacts both with μ-opiate sites recognizing [³H]DHM and another type of opiate site, which cannot be equated with any of the, as yet, described δ- or κ-binding sites, and rather, represents a subclass of μ-opiate receptor sites. These experiments, thus, support the notion of subclasses (isoreceptors) for different types of opiate receptors.     

The interaction of N alpha-alkylenkephalins with opiate receptors. Tissue-dependent shifts in the opiate activity of methionine-enkephalin following N alpha-alkylation

Experimental conditions for the small scale alkylation of the alpha-amino group of the opioid peptide methionine-enkephalin using the method of reductive alkylation are described. The generality of the method is established by describing in detail the synthesis, purification, and structure elucidation of the N alpha-ethyl, isopropyl, phenethyl, and cyclopropylmethyl derivatives of methionine-enkephalin. By a combination of amino acid and mass spectral analysis it is possible to carry out the structure analysis of nanomole quantities of these modified peptides, making direct chemical modification and structure elucidation of carrier-free tritiated and radioiodinated enkephalins feasible. Using these chemical procedures, a homologous series of N alpha-n-alkyl derivatives of methionine-enkephalin has has been synthesized. It is shown that the relative binding and opiate activity of these N alpha-alkyl derivatives, using several standard in vitro assay systems, depends on the tissue source used. The results of this study are analyzed by considering recent reports indicating the existence of multiple opiate receptors.