Discrimination by temperature of opiate agonist and antagonist receptor binding.

Variations in incubation temperature can markedly differentiate opiate receptor binding of agonists and antagonists. In the presence of sodium increasing incubation temperatures from 0° to 30° reduces receptor binding of ³H-naloxone by 50% while tripling the binding of the agonist ³H-dihydromorphine. Lowering incubation temperature from 25° to 0° reduces the potency of morphine in inhibiting ³H-naloxone binding by 9-fold while not affecting the potency of the antagonist nalorphine. At temperatures of 25° and higher the number of binding sites for opiate antagonists is increased by sodium and the number of sites for agonists is decreased by sodium with no changes in affinity. By contrast, in the presence of sodium lowering of incubation temperature to 0° increases opiate receptor binding of the antagonist naloxone by enhancing its affinity for binding sites even though the total number of binding sites are not changed.     

Electron spin resonance study of the synaptosome opiate receptor: kinetics of stereospecific binding of spin labeled morphine.

Morphine, spin labeled on the 3- or 6-position has been used as the opiate ligand in a study of the time course of stereospecific opiate binding to intact synaptosomes isolated from non-cerebellar rat brain. The broadening of electron spin resonance lines induced by immobilization of the ligand on binding has been used to determine the concentration of bound opiate. The stereospecificity of the reaction was measured by comparing ligand binding in the presence of thousand-fold molar excesses of dextrorphan or levorphanol. Using both static and flow techniques, the binding process has been continuously monitored at times greater than 4.8 s after mixing spin labeled morphine with synaptosomes. It is shown that for this ligand and receptor preparation, binding takes place primarily during a delayed, abrupt process whose rate and time of onset are temperature dependent and reflect the presence of added opiate agonist or antagonist.     

Evidence for nuclear sites of stereospecific opiate binding in neuroblastoma cells in continuous culture.

A class of stereospecific, saturable receptors for narcotic drugs has been found in a clone of mouse neuroblastoma cells in continuous culture. Cells grown in the presence of 10(-8) M etorphine, a synthetic opiate, had an increased doubling time. The neuroblastoma cell nucleus was found to be the sole site for the high affinity binding of etorphine. It was found that these nuclear receptors (1) became saturated at a concentration of 2 X 10(-9) M etorphine, (2) had a dissociation constant of 5 X 10(-11) M etorphine, (3) were capable of binding etorphine stereospecifically at 37 degrees C but not at 4 degree C, and t4) were protein in nature as evidenced by treatment with proteolytic enzymes. More importantly, the receptor activity appeared to be chromatin-associated.     

Effect of the opiate antagonist naloxone on body temperature in rats.

The specific opiate antagonist naloxone was used to assess the hypothesis that an endogenous opioid plays a significant role in temperature regulation in the rat. A very slight hypothermic effect was observed at a naloxone dose sufficient to block the opiate receptors. Even under conditions of cold stress, the magnitude of the effect was so small as to lend little support to the hypothesis.     

On the specificity of naloxone as an opiate antagonist.

Since the discovery of endogenous opioid peptides in brain (68,69,97,113, 128) and the pituitary gland (26,81,105,125) there has been considerable interest in their possible roles in a variety of physiological and pharmacological processes. Many studies have used antagonism by naloxone as a criterion for implicating endogenous opiates in a process, assuming that naloxene has no pharmacological actions other than those related to blockade of opiate receptors. The doses of naloxene used are often higher than those required to antagonize the analgesic and other effects of morphine. However, multiple forms of opiate receptors are present in nervous tissue and higher concentrations of naloxene are required to antagonize effects mediated by some of these receptors (83). Although the earlier literature supports the assumption that the effects of naloxene are due to the blockade of opiate receptors (87), there are an increasing number of reports which indicate that naloxene may have pharmacological actions unrelated to opiate receptor blockade. The subsequent review serves to emphasize that antagonism by naloxene is a necessary but not sufficient criterion for invoking the mediation of a response by an endogenous opiate (61). Additional lines of evidence which serve to strengthen the conclusion that endogenous opiates mediate a process will be considered.     

Impact of aromatic residues within transmembrane helix 6 of the human gonadotropin-releasing hormone receptor upon agonist and antagonist binding.

To investigate the impact of aromatic residues within transmembrane helix 6 (TMH6) of the human gonadotropin-releasing hormone receptor (GnRH-R) on agonist and antagonist binding, residues Y(283), Y(284), W(289), Y(290), W(291), and F(292) were exchanged to alanine and analyzed comprehensively in functional reporter gene and ligand binding assays. Whereas receptor mutants Y(283)A, Y(284)A, and W(291)A were capable of neither ligand binding nor signal transduction, mutants W(289)A, Y(290)A, and F(292)A were functional: the F(292)A mutant behaved like wild-type receptor, while mutants W(289)A and Y(290)A differentiated between agonistic and antagonistic ligands. On the basis of the high-resolution X-ray structure of bovine rhodopsin as well as available data on GnRH-R mutants, models for ligand-receptor interactions are proposed. The model for D-Trp(6)-GnRH (Triptorelin) binding, representing a superagonistic ligand, is in full accordance to available data. Furthermore, new interactions are proposed: pGlu(1) interacts with N(212) in transmembrane helix 5, Tyr(5) with Y(290), and D-Trp(6) with W(289). The binding behavior of mutants W(289)A and Y(290)A corresponds to the proposed binding model for the antagonist Cetrorelix. In summary, our data as presented indicate that Y(290) plays a key function in agonist but not antagonist binding.     

Pupillary effects of leucine and methionine enkephalin in rats after intraperitoneal administration

Changes in pupil size after peripheral administration of met-enkephalin, leu-enkephalin, or morphine were studied in the rat. With a simple pupillographic technique, the pupil diameter of male, S.D. rats (250–300 g) was measured by a series of photographs taken every 60 sec for at least 45 min after the last drug injection. Morphine (8 mg/kg, SC) caused mydriasis characterized by rapid and marked fluctuations of pupil size. Mydriasis also occurred after leu-enkephalin (5 and 10 mg/kg, IP) and met-enkephalin (20 mg/kg, IP). Both peptides induced morphine-like fluctuations. When given 15 min after morphine, leu-enkephalin (5 and 10 mg/kg) increased the mydriatic effect of morphine from 172 percent of control to 224 and 272 percent, respectively. Met-enkephalin (20 mg/kg, but not 10 mg/kg) also enhanced the mydriatic response of morphine, to 244 percent of control. These interactions appear to represent simple addition rather than potentiation. The effects of both peptides were reversed by naloxone (1 mg/kg, SC), suggesting an opiate receptor interaction for the pupillary effects of the enkephalins. The rat pupil thus provides one of the few in vivo models permitting quantification of enkephalin action after parenteral administration.     

The effect of ethanol upon early development in mice and rats. VI. In vivo effect of acetaldehyde upon preimplantation stages in rats

In completion of the previously outlined "experimental alcohol blastopathy", the role of acetaldehyde in the induction of preimplantation pathological changes in rat embryos has been controlled. Two experimental models were used: the direct administration of acetaldehyde by gavage and the blockage of acetaldehyde metabolization by ANTALCOL (an aldehyde-dehydrogenase blocking compound). The main results were as follows: The exogenous acetaldehyde in the blood of pregnant animals has an obvious effect upon the developmental rate during the late preimplantation period (retarding segmentation, blastulation), and in one of the experimental models upon the oviductal-uterine migration rate. The increase of the blood acetaldehyde level by blockage of its further metabolization has a more marked effect as compared with the direct intravenous administration of the substance. According to our previous observations the intravenous application of ethanol on the same day (day 4) has no such effect. The direct noxious influence upon the developing preimplantation embryos (fragmentation) of the increased level of acetaldehyde obtained by ANTALCOL treatment is similar but more marked than this effect obtained previously by ethanol administration. The same effect observed after the direct administration of the substance is less marked than the effect of ANTALCOL treatment but more marked than the effect of intravenous ethanol administration. These results attest that acetaldehyde may contribute (alone or together with the effect of ethanol) to the induction of "experimental alcohol blastopathy". The less marked action of the substance proper introduced into the blood stream may be due--in our opinion--to its possible alteration during the period between distillation and application.     

Radiosensitivity during the irradiation of animals in an altered gaseous environment. A comparative study on mice and rats of the radiation-protective effect of oxygen-deficient gas mixtures

The radioprotective effect of gas hypoxic mixtures containing 5, 7, 8, 10 and 15% of oxygen on mice and rats was comparatively studied. The dependence of DMF upon oxygen concentration in the mixture was approximated by a hyperbolic function similar to the dependence of the radiomodifying effect of circulatory hypoxia caused by radioprotective agents of the indolylalkylamine series.     

Differential effect of pH on thromboxane A2/prostaglandin H2 receptor agonist and antagonist binding in human platelets.

The effects of changes in pH on the binding of agonists and antagonists to the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were determined. Competition binding studies were performed with the TXA2/PGH2 mimetic [1S-1 alpha,2 beta (5Z), 3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4'-iodophenoxy)-1-buteny) 7-oxabicyclo-[2.2.1]-heptan-2-yl]-5-heptenoic acid ([125I]BOP). The pH optimum for binding of [125I] BOP to washed human platelets was broad with a range of pH 4-6 in contrast to that of the TXA2/PGH2 receptor antagonist 9,11-dimethyl-methano-11,12-methano-16-(3-iodo-4-hydroxyl)-13-aza-15 alpha,beta-omega-tetranorthromboxane A2 ([125I]PTA-OH) which was 7.4. Scatchard analysis of [125I]BOP binding in washed platelets at pH 7.4, 6.0, and 5.0 revealed an increase in affinity (Kd = 1.16 +/- 0.06, 0.64 +/- 0.09, and 0.48 +/- 0.05 nM, respectively) and an increase in the number of receptors (Bmax = 2807 +/- 415, 5397 +/- 636, and 7265 +/- 753 sites/platelet, respectively). The potency of I-BOP to induce shape change in washed platelets at pH 6.0 was also significantly increased from an EC50 value of 0.34 +/- 0.016 nM at pH 7.4 to 0.174 +/- 0.014 nM at pH 6.0 (n = 6, p less than 0.05). In contrast, the EC50 value for thrombin was unaffected by the change in pH. In competition binding studies with [125I]BOP, the affinity of the agonists U46619 and ONO11113 were increased at pH 6.0 compared to 7.4. In contrast, the affinity of the TXA2/PGH2 receptor antagonists I-PTA-OH, SQ29548, and L657925 were either decreased or unchanged at pH 6.0 compared to 7.4. Diethyl pyrocarbonate and N-bromosuccinimide, reagents used to modify histidine residues, reversed the increase in affinity of [125I]BOP at pH 6.0 to values equivalent to those at pH 7.4. In solubilized platelet membranes, the effects of NBS were blocked by coincubation with the TXA2/PGH2 mimetic U46619. The results suggest that agonist and antagonist binding characteristics are different for the TXA2/PGH2 receptor and that histidine residue(s) may play an important role in the binding of TXA2/PGH2 ligands to the receptor.     

Localization of agonist and antagonist binding domains of the human neurokinin-1 receptor.

To identify the molecular determinants of ligand-receptor interactions, the extracellular domain of the human neurokinin-1 receptor was systematically substituted with the corresponding sequences from the other two neurokinin receptor subtypes. Three residues within the first extracellular segment and 2 residues of the second segment are required for the optimal binding of all three natural peptide agonists. The divergent nature of 4 of the 5 residues supports the hypothesis that the peptide binding site on the neurokinin-1 receptor is not highly conserved in the other two receptor subtypes. In contrast, substitution of part of the third extracellular segment and the fourth extracellular segment with the corresponding amino acids of the human neurokinin-3 receptor results in an increase in neurokinin B affinity without affecting substance P binding, suggesting that the two peptides do not interact with the same set of functional groups on the receptor. Among the four extracellular regions, only parts of the third and fourth segments affect the binding of the quinuclidine antagonist L-703,606, and these two regions may partially account for the neurokinin-1 receptor subtype specificity of this non-peptide antagonist. These studies demonstrate that both the extracellular and transmembrane domains of the neurokinin-1 receptor are involved in the binding of substance P and related peptides.     

Hypotensive effects of hemopressin and bradykinin in rabbits, rats and mice. A comparative study

Hemopressin is a novel vasoactive nonapeptide derived from hemoglobin's alpha-chain as recently reported by Rioli et al. [Rioli V, Gozzo FC, Heimann AS, Linardi A, Krieger JE, Shida CS, et al. Novel natural peptide substrates for endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme. J Biol Chem 2003;278(10):8547-55]. In anesthetized male Wistar rats, this peptide exhibited hypotensive actions similar to those of bradykinin (BK) when administered intravenously (i.v.), and was found to be metabolized both in vitro and in vivo by several peptidases, including the angiotensin-converting enzyme (ACE). In this study, these findings were expanded upon by examining: (i) the degradation kinetics following incubation with ACE purified from rabbit lung and (ii) the blood pressure lowering effects of HP and BK injected i.v. or intra-arterially (i.a.) in male rabbits, rats, and mice. Our findings demonstrate that, in vitro, HP and BK are both degraded by ACE, but at different velocity rates. Furthermore, both HP and BK induced transient hypotension in all animals tested, although the responses to HP relative to the administration sites were significantly lower (by 10-100-fold) on an equimolar basis compared to those of BK. In rabbits, the decrease of blood pressure induced by HP (10-100 nmol/kg) did not differ whether it was administered i.v. or i.a., suggesting an absence of pulmonary/cardiac inactivation in contrast to BK (0.1-1 nmol/kg). The in vivo effect of HP was significantly potentiated in rabbits immunostimulated with bacterial lipopolysaccharide (LPS), but was unaffected by both the B2 receptor antagonist HOE 140 (0.1 micromol/kg) and captopril (100 microg/kg), contrary to BK. Therefore, HP acts as a weak hypotensive mediator, which does not activate kinin B2 receptors, but uses a functional site and/or signaling paths appearing to be up-regulated by LPS.     

Recognition of opioid agonist and antagonist in the opioid receptor binding site

By treating the rat crude synaptosomal fraction with 5,5'-dithio-bis-(2-nitrobenzoic acid), DTNB, a marked decrease of stereo-specific binding of opioid agonist (dihydromorphine or D-Ala-D-Leu-enkephalin) was observed, but there was no effect in the case of the binding of opioid antagonist (naloxone or diprenorphine). The decrease of the agonist binding in the presence of 500 microM of DTNB was nearly equal to that of 100 mM of NaCl. The ability of opioids to inhibit 3H-naloxone binding in the absence of DTNB was compared to their inhibitory potency in the presence of 500 microM of DTNB to obtain DTNB response ratio. This ratio closely correlated with sodium index of each opioid. Potency of the inactivation of the agonist binding by congeners of DTNB changed with net charge of the reagents, and 2,2'-dithiobis-(5-nitropyridine), bearing a positive charge, was most effective. These results suggest that an aliphatic sulfhydryl group, being sensitive to DTNB is located to the active center of an anionic binding site for the agonist, and controls opioid agonist binding through a proton transfer mechanism.     

Antibody binding to the beta-subunit of deglycosylated chorionic gonadotropin converts the antagonist to an agonist

The murine Leydig tumor cell line, MLTC-1, has a gonadotropin-responsive adenylate cyclase system. Binding of human chorionic gonadotropin (hCG) stimulates the accumulation of cyclic AMP in these cells. Chemically deglycosylated hCG (DG-hCG) is an antagonist that binds with high affinity to the gonadotropin receptor, but fails to stimulate adenylate cyclase. This antagonism can be reversed if the binding of DG-hCG is followed by treatment of the DG-hCG-receptor complex with antibodies against hCG. Polyclonal antibodies against DG-hCG were raised in rabbits. These antibodies were strongly cross-reactive with hCG, bound to both the alpha- and beta-subunits of hCG and DG-hCG, and reversed the antagonism of DG-hCG. The antiserum was divided into two fractions by affinity chromatography on hCG-Sepharose. The fraction that was not retained reacted only with DG-hCG (DG-hCG antibodies) and, on Western blots, bound to both the alpha- and beta-subunits of DG-hCG. DG-hCG antibodies did not reverse the antagonism of DG-hCG. However, using 125I-protein A, we were able to detect binding of these antibodies to the cell surface DG-hCG-receptor complex. The fraction of antibodies retained by the affinity column reacted with both DG-hCG and hCG (DG-hCG/hCG antibodies). On Western blots, DG-hCG/hCG antibodies bound to the beta-subunit, but only weakly to the alpha-subunit of both hCG and DG-hCG. These antibodies also bound to the cell surface DG-hCG-receptor complex. In addition, DG-hCG/hCG antibodies were able reverse the antagonism of DG-hCG. Reversal of DG-hCG antagonism by the whole antiserum was blocked by the beta- but not the alpha-subunit of hCG. Polyclonal antiserum against the beta- but not the alpha-subunit of hCG reversed the antagonism of DG-hCG. From these results, we conclude that antibody binding to specific determinants common to both native and deglycosylated beta-subunit reverses the antagonism of DG-hCG. In addition, antibodies directed against unique determinants on the deglycosylated beta-subunit are not capable of reversing the antagonism of DG-hCG.     

The effect of ethanol upon early development in mice and rats. VIII. The effect of chronic consumption of some beverages upon preimplantation development in rats

The effects of chronic consumption of some beverages (plum-brandy 24% and cognac 20%) upon preimplantation development in rats were studied. The control of possible effects was performed on day 5 by usual flushing, examination and photographying of oviductal and uterine embryos. In order to evaluate the effect of the beverage applied, the following criteria were used: mean litter size, migration of the embryos from the oviduct to the uterus, the developmental stage attained by the pre-implantation embryos and the appearance of pathological embryos. The main results were the following: both beverages applied influenced the preimplantation development; with respect to the developmental rate and to the induction of pathological changes, the effect of both beverages was similar (retardation and an increased, number of pathological morulae and blastocysts); a different action could be detected as to the mean litter size and to the migration of preimplantation embryos: plum-brandy reduced more substantially the mean litter size, whereas cognac had a more marked retarding effect upon the migration of embryos from the oviduct to the uterus: all the changes detected show a more or less marked "litter-effect". The present data were compared with the corresponding effects of chronic ethanol administration observed previously in our laboratory. No obvious potentiating effect of beverage congeners could be established. The findings are discussed in connection with other experimental models of alcohol embryo and fetopathy.     

The effect of ethanol upon early development in mice and rats. II. In vitro effect upon early postimplantation rat embryos

The direct effect of ethanol upon in vitro cultured 9.5 day rat embryos was investigated (2, 4, 8 and 10% ethanol added to the culture medium). The main effects recorded were as follows: 1. Significant increase of the number of "dying" embryos (beating heart without yolk sac circulation); 2. No significant increase of mortality; 3. Significant increase of the number of living embryos with deficient blood circulation; 4. Significant retardation of coiling in living embryos with a significant dose-effect relation, when the effects of 20/00 and 80/00 ethanol were compared; 5. Lowering of the mean somite number in living embryos; 6. Various macro- and macroscopical pathological changes (mainly necrotic areas in the central nervous system).     

Centrally administered GABA and GABA-selective agonist and antagonist in rats: histoenzymological cytophotometric study

In the present study, the effect of intracerebroventricular (icv) injection of GABA, its agonist--muscimol, and antagonist--picrotoxin, has been studied on histoenzymological alterations of acetylcholinesterase (AChE). butyrylcholinesterase (BuChE), monoamine oxidase (MAO), and succinic dehydrogenase (SDH) by cytophotometric technique. This study was conducted on medial preoptic area (mPOA), nucleus paraventricularis hypothalami (PVH), area lateralis hypothalami (LHA), nucleus dorsomedialis hypothalami (DMH), and nucleus ventromedialis hypothalami (VMH). Results showed that GABA and muscimol inhibited AChE, BuChE, MAO, and SDH in all the areas while picrotoxin stimulated these enzymes. These changes in enzyme activity by GABA, muscimol, and picrotoxin and their possible mode of action are discussed.