Effects of a kappa-opioid agonist on adrenocorticotropic and diuretic function in man

Although kappa-opiate receptors represent an important fraction of the total opiate receptor capacity in human brain their endocrine function is unknown. We determined the effects of a kappa-opiate receptor agonist on the secretion of vasopressin, ACTH and cortisol and on diuresis. The racemic benzomorphan kappa agonist MR 2033 or its opiate active (-)-isomer, MR 2034, inhibited the release of cortisol and ACTH in 12 trials in a naloxone reversible manner; plasma levels of vasopressin were not altered. The (+)-isomer, MR 2035, did not affect the secretion of cortisol or ACTH. Surprisingly, in five other subjects large increases were observed in vasopressin, ACTH and cortisol following the kappa-agonist, which were probably elicited indirectly by aversive effects of the opioid. The subjects in whom vasopressin release was not altered by MR 2033 and MR 2034 displayed large decreases in urine osmolality which were not antagonized by naloxone. The opiate inactive (+)-isomer, MR 2035, caused no diuretic response. Subjects in whom vasopressin release was stimulated did not show decreases in urine osmolality indicating that vasopressin is capable of antagonizing the diuretic action of the kappa-agonist. Our data show that a kappa-agonist inhibits secretion of cortisol and ACTH by acting at stereospecific opiate receptors and elicits diuresis by acting at stereospecific, but naloxone-insensitive non-classical opioid receptors. These data support the concept that different types of kappa-receptors can be distinguished in man.     

Morphine and Enkephalins Potently Inhibit [3H]Noradrenaline Release from Rat Brain Cortex Synaptosomes: Further Evidence for a Presynaptic Localization of μ-Opioid Receptors

Synaptosomes prepared from rat cerebral cortex and labeled with [3H]noradrenaline (NA) were superfused with calcium-free Krebs-Ringer-bicarbonate medium and exposed to 10 mM K+ plus 0.1 mM Ca2+ so that [3H]NA release was induced. 6,7-Dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99) strongly inhibited synaptosomal K+-induced [3H]NA release (EC50 = 5-10 nM) by activating alpha 2-adrenoceptors. Release was also inhibited (maximally by 40-50%) by morphine (EC50 = 5-10 nM), [Leu5]enkephalin (EC50 = approximately 300 nM), [D-Ala2,D-Leu5]enkephalin (DADLE), and Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAGO) (EC50 values = approximately 30 nM). In contrast to the mu-selective opioid receptor agonists morphine and DAGO, the highly delta-selective agonist [D-Pen2,D-Pen5]enkephalin (1 microM) did not affect [3H]-NA release. Furthermore, the inhibitory effect of DADLE, an agonist with affinity for both delta- and mu-opioid receptors, was antagonized by low concentrations of naloxone. The findings strongly support the view that, like alpha 2-adrenoceptors, mu-opioid receptors mediating inhibition of NA release in the rat cerebral cortex are localized on noradrenergic nerve terminals.     

MOR Is Not Enough: Identification of Novel mu-Opioid Receptor Interacting Proteins Using Traditional and Modified Membrane Yeast Two-Hybrid Screens

The mu-opioid receptor (MOR) is the G-protein coupled receptor primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. We have utilized a combination of traditional and modified membrane yeast two-hybrid screening methods to identify a cohort of novel MOR interacting proteins (MORIPs). The interaction between the MOR and a subset of MORIPs was validated in pulldown, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta and kappa opioid receptors, suggesting that they may represent general opioid receptor interacting proteins (ORIPS). Expression of several MORIPs was altered in specific mouse brain regions after chronic treatment with morphine, suggesting that these proteins may play a role in response to opioid agonist drugs. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction.     

Partial agonistic effect of 9-hydroxycorynantheidine on mu-opioid receptor in the guinea-pig ileum

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa that is reported to have opioid agonistic properties. The 9-demethyl analogue of mitragynine, 9-hydroxycorynantheidine, is synthesized from mitragynine. 9-Hydroxycorynantheidine inhibited electrically stimulated guinea-pig ileum contraction, but its maximum inhibition was weaker than that of mitragynine and its effect was antagonized by naloxone, suggesting that 9-hydroxycorynantheidine possesses partial agonist properties on opioid receptors. Receptor binding assays revealed that 9-hydroxycorynantheidine has high affinity for mu-opioid receptors. In an assay of the guinea-pig ileum, naloxone shifted the concentration-response curves for [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), (5alpha,7alpha,8beta)-(+)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69593) and 9-hydroxycorynantheidine to the right in a competitive manner. The pA(2) values of naloxone against 9-hydroxycorynantheidine and DAMGO were very similar, but not that against U69593. As indicated by the two assay systems, the opioid effect of 9-hydroxycorynantheidine is selective for the mu-opioid receptor. 9-Hydroxycorynantheidine shifted the concentration-response curve for DAMGO slightly to the right. Pretreatment with the mu-opioid selective and irreversible antagonist beta-funaltorexamine hydrochloride (beta-FNA) shifted the concentration-response curve for DAMGO to the right without affecting the maximum response. On the other hand, beta-FNA did not affect the curve for 9-hydroxycorynantheidine, but decreased the maximum response because of the lack of spare receptors. These studies suggest that 9-hydroxycorynantheidine has partial agonist properties on mu-opioid receptors in the guinea-pig ileum.     

Guanine nucleotides inhibit binding of agonists and antagonists to soluble opiate receptors

The guanine nucleotides GDP, GTP, and guanosine-5'-(beta, gamma-imido)triphosphate inhibit binding of opiates and opioid peptides to receptors solubilized from membranes of neuroblastoma X glioma NG108-15 hybrid cells. The inhibition reflects decreased affinity of receptors for opioid ligands. Whereas in membranes, only opioid agonist binding is sensitive to guanine nucleotide inhibition, both agonist and antagonist binding is reduced in the case of soluble receptors. Furthermore, soluble receptors are more sensitive to the effects of guanine nucleotides than are membrane-bound receptors. These observations are consistent with the suggestion that solubilized receptors may be complexes of an opiate binding protein and a guanine nucleotide-sensitive regulatory component.     

Opiate-Inhibited Adenylate Cyclase in Rat Brain Membranes Depleted of Gs-Stimulated Adenylate Cyclase

Opiate agonists inhibit adenylate cyclase in brain membranes, but under normal conditions the maximal inhibition is small (10-15%). When rat brain membranes were preincubated at pH 4.5, washed, and then assayed for adenylate cyclase at pH 7.4, stimulation of activity by agents (fluoride, guanylyl-5'-imidodiphosphate, cholera toxin) that act through the stimulatory GTP-binding coupling protein (Gs) protein was lost. At the same time, inhibition of basal adenylate cyclase by opiate agonists was increased to a maximum of 30-40%. Opiate inhibition was maximal at low magnesium concentrations (less than 5 mM), required guanine nucleotides, and decreased the Vmax, not Km, of the enzyme. Incubation of membranes with pertussis toxin lowered the apparent affinity for agonists in inhibiting activity. The delta opioid agonists were more potent than mu agonists, and the Ke values for naloxone in blocking agonist inhibition were similar for both mu and delta agonists (50-90 nM). These results suggest that inhibition of adenylate cyclase in brain is not mediated by mu opiate receptors, but whether classic high-affinity delta and kappa receptors are involved with this enzyme cannot be confirmed by these experiments.     

Changes in rat brain opiate receptor content upon castration and testosterone replacement.

The saturable binding of naltrexone-³H in the brains of castrated male rats exceeds that found in intact animals by a factor of two. This increase is androgen dependent since testosterone replacement reduced the binding to control levels. Scatchard analysis of the saturation curves revealed that the change in binding reflects increased available binding sites and is not due to increased binding affinity. This relationship between testosterone and brain opiate receptors provides for the participation of endorphins in the regulation of pituitary gonadotropins by gonadal hormones. The increased content of opiate receptors in the brains of castrated rats correlates with the greater brain N-demethylation of morphine establishing a further link between this biotransformation and agonist action.     

Regional cerebral glucose utilization in withdrawal following systemic and intracerebroventricular sufentanil administration

Regional cerebral glucose utilization (RCGU) and behavior were studied during naloxone-precipitated withdrawal in rats after chronic intravenous (IV) or intracerebroventricular (ICV) administration of sufentanil citrate, a potent, highly selective mu opiate agonist. Changes in RCGU were indistinguishable between the two groups (p<0.05) in 21 of 24 anatomically related limbic and brainstem structures known to be activated during withdrawal. Rats made dependent by ICV infusions of sufentanil had smaller RCGU changes in the lateral septal areas, lateral habenular nuclei and paratenial nuclei than rats made dependent by IV infusions of sufentanil. These observations are consistent with infusion artifact, given the proximity of these structures to the site of IVC infusion. All 24 structures had increased RCGU in experimental groups compared with controls (p<0.05). Although linear regression analysis suggests slightly greater RCGU changes in rats after IV sufentanil than in rats after ICV sufentanil (m=0.81), the changes in corresponding structures are highly correlated (r=0.96) indicating qualitatively almost identical RCGU changes. Behavioral changes paralleled RCGU changes and revealed slightly greater withdrawal in rats after IV sufentanil but no clear qualitative differences. Taken together, these results suggest that cerebral metabolic changes in withdrawal following chronic sufentanil administration result exclusively from effects at CNS opiate receptors and not from peripheral receptors. Additionally, the current study provides a model for the production of opiate dependence, by the ICV administration of a specific mu opiate receptor agonist that is relatively free of infusion artifact.     

Agonist-specific down regulation of mu-opioid receptors: Different cellular pathways are activated by different opioid agonists

Opioid agonists are known to induce down regulation of opioid receptors through the classical pathway that involves phosphorylation, clathrin-dependent endocytosis and lysosomal/endosomal degradation of the internalized receptors. As expected, exposure of mu-opioid receptor (MOR)-transfected HEK-293 cells to either DAMGO (a specific mu-opioid agonist) or etorphine (a wide spectrum opioid agonist) resulted in down regulation of the receptors that was blocked by the kinase inhibitor staurosporine, by hypertonic sucrose and by the lysosomal and proteasomal inhibitors chloroquine and lactacystin. High concentration of etorphine, but not of DAMGO, induced an additional process of down regulation that was resistant to staurosporine, to hypertonic sucrose and to chloroquine-lactacystin. Etorphine, but not DAMGO, also induced down regulation of mu-opioid receptors in isolated membranes of HEK cells. This membrane-delimited down regulation was blocked by selective inhibitors of protease enzymes, suggesting the involvement of membranous serine- and amino-peptidases. This membranous down regulation of opioid receptors was dependent on the concentration of etorphine and was blocked by the opioid antagonist naloxone. Etorphine induced similar down regulation in membranes of HEK-293 cells transfected with delta-opioid receptors (DOR) as well in membranes of cells that endogenously express opioid receptors. This agonist-specific membrane-delimited regulatory process appears to be physiologically relevant and should be taken into account when studying long term effects of opioid drugs.     

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.     

5'-Guanylylimidodiphosphate decreases affinity for agonists and apparent molecular size of a frog brain opioid receptor in digitonin solution

When assayed for specific opiate binding in the presence of 120 mM NaCl, digitonin extracts from frog (Rana ridibunda) brain membranes were found to contain about the same quantity (0.5 pmol/mg of protein) of high (Kdh = 0.4 nM) and of lower (Kdl = 15-20 nM) affinity sites for the opiate agonist [3H]etorphine. The two classes of [3H]etorphine binding sites displayed equally high (Kd = 0.3 nM) affinity for the opiate antagonist [3H]diprenorphine. 5'-Guanylylimidodiphosphate (GppNHp) selectively and potently (IC50 = 0.1 microM) inhibited high affinity binding of the tritiated agonist, and this inhibition resulted from the GppNHp-induced conversion of the high into the lower affinity sites for [3H]etorphine. Following centrifugation of the digitonin extract in sucrose gradients, opioid binding activity was found to be associated with two clearly separated macromolecular components of apparent sedimentation coefficients 11.5 and 9.7 S, respectively. The two components bound [3H]diprenorphine equally well, whereas the fast sedimented component bound [3H]etorphine better than did the slower sedimented one. In addition, labeling of the component of bigger apparent size with [3H]etorphine was considerably reduced in the presence of 50 microM GppNHp. Finally, in soluble extracts which had been (i) preincubated with and (ii) centrifuged in the presence of GppNHp, the fast sedimented component was no longer observed while there was about twice as much of the component of smaller apparent size as in control (no GppNHp) extracts. Together, these results demonstrated the existence of an opioid receptor-G protein complex which, in digitonin solution, was still amenable to regulation (dissociation) by guanine nucleotides.     

Purification of opioid receptor in the presence of sodium ions.

Opioid receptor was solubilized from rat brain membranes with a mixture of the detergents CHAPS and digitonin in the presence of protease inhibitors and 1 M NaCl. The solubilized receptor bound mu-opioid agonists and antagonists with affinities similar to those of native membrane receptor. The affinity of solubilized receptor for the agonist PL017 was greatly reduced by GTP gamma S, suggesting the receptor is still associated with G-protein. The solubilized material was passed through an opioid antagonist (10cd) affinity column and a wheat germ agglutinin column, set up in series, to obtain a partially purified receptor preparation. This partially purified material bound mu-agonist with low affinity and the binding affinity was no longer affected by GTP gamma S. The partially purified receptor was further purified by repeating the affinity and lectin chromatography with smaller size column. Binding of opioid antagonist [3H]diprenorphine to the partially or purified receptors was dependent upon the presence of sodium ions. The purified receptor showed saturable and stereospecific binding for opioid ligands, was predominantly of the mu-type, and exhibited as a diffuse band with a medium molecular mass of 62 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The average specific binding activity of the purified receptor was 18.8 +/- 2.3 pmol/micrograms protein, a value close to the theoretical estimation.     

Luteinizing hormone secretion following intracerebroventricular administration of morphine in the prepuberal gilt

Antagonism of endogenous opioids with naloxone stimulates luteinizing hormone (LH) release in mature but not prepuberal gilts. The present report demonstrates that the opiate agonist morphine (500 micrograms), administered intracerebroventricularly (ICV), reduced LH secretion in both ovariectomized mature and prepuberal gilts. We suggest that opioid receptors are functionally coupled to the GnRH secretory system in prepuberal gilts even though endogenous opioid peptide modulation of LH secretion was not demonstrable in our previous studies.     

Bimodal Opioid Regulation of Cyclic AMP Formation: Implications for Positive and Negative Coupling of Opiate Receptors to Adenylyl Cyclase

Abstract: A μ-selective opiate receptor agonist, sufentanil, can either increase or decrease the stimulated formation of cyclic AMP (cAMP) in the myenteric plexus. The direction of the opioid modulation of this second messenger depends on the concentration of opioid used. Low doses of opioid enhance, whereas higher concentrations inhibit, the magnitude of cAMP that is formed in response to electrical stimulation. Opioids exert this dual regulation on only stimulated cAMP formation. Basal levels are not affected. Opioid facilitation and inhibition of stimulated cAMP formation are blocked by naloxone, indicating mediation by opiate receptors. Because all experiments were conducted in the presence of a phosphodiesterase inhibitor, it is highly unlikely that opioid regulation of stimulated cAMP formation is due to changes in the rate of its degradation. Positive and negative coupling of μ-opiate receptors to adenylyl cyclase is the most plausible explanation for the bimodal opioid effects on cAMP content. The marked parallel between the current observations and the previously reported bimodal opioid regulation of evoked enkephalin release is consistent with the hypothesis that adenylyl cyclase is one biochemical substrate for the bimodal opiate receptor-coupled regulatory mechanism governing the stimulated release of this opioid peptide.     

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.     

Involvement of protein kinase C in the adaptive changes of cholinergic neurons to sympathetic denervation in the guinea pig myenteric plexus

Supersensitivity to muscarinic, kappa- and mu-opioid agents modulating cholinergic neurons in the guinea pig colon develops after chronic sympathetic denervation. A possible role for protein kinase C (PKC) in contributing to development of these sensitivity changes was investigated. The PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced acetylcholine (ACh) overflow in preparations obtained from normal animals. The facilitatory effect of PMA was significantly reduced after prolonged exposure to the phorbol ester and by the PKC inhibitors, chelerythrine and calphostin C. Subsensitivity to the facilitatory effect of PMA developed after chronic sympathetic denervation. In this experimental condition, immunoblot analysis revealed reduced levels of PKC in myenteric plexus synaptosomes. The facilitatory effect of the muscarininc antagonist, scopolamine, on ACh overflow was significantly reduced by the phospolipase C (PLC) inhibitor, U73122, chelerythrine and calphostin C, both in normal and denervated animals. However, in both experimental groups, PLC antagonists and PKC antagonists did not affect the inhibitory effect of the muscarinic agonist, oxotremorine-M on ACh overflow. The inhibitory effects of U69593 (kappa-opioid receptor agonist) and DAMGO (mu-opioid receptor agonist) on ACh overflow significantly increased in the presence of U73122, chelerythrine and calphostin C in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals.These results indicate that activation of PKC enhances ACh release in the myenteric plexus of the guinea pig colon. At this level, chronic sympathetic denervation entails a reduced efficiency of the enzyme. In addition, PKC is involved in the inhibitory modulation of ACh release mediated by muscarinic-, kappa- and mu-opioid receptors, although with different modalities. Muscarinic receptors inhibit PKC activity, whereas kappa- and mu-opioid receptors increase PKC activity. Both the inhibitory and the facilitatory effect on PKC involve modulation of PLC activity. The possibility that the change in PKC activity represents one of the biochemical mechanisms at the basis of development of sensitivity changes to opioid and muscarinic agents after chronic sympathetic denervation is discussed.     

Changes in the expression of G protein-coupled receptor kinases and beta-arrestin 2 in rat brain during opioid tolerance and supersensitivity

We previously demonstrated that chronic treatment of rats with the mu-opioid receptor agonist sufentanil induced pharmacological tolerance associated with mu-opioid receptor desensitization and down-regulation. Administration of the calcium channel blocker nimodipine during chronic treatment with sufentanil prevented mu-opioid receptor down-regulation, induced down-stream supersensitization, and produced supersensitivity to the opioid effects. The focus of the present study was to determine a role for G protein-coupled receptor kinases (GRKs) and beta-arrestin 2 in agonist-induced mu-opioid receptor signalling modulation during chronic opioid tolerance and supersensitivity. Tolerance was induced by 7-day chronic infusion of sufentanil (2 microgram/h). Supersensitivity was induced by concurrent infusion of sufentanil (2 microgram/h) and nimodipine (1 microgram/h) for 7 days. Antinociception was evaluated by the tail-flick test. GRK2, GRK3, GRK6 and beta-arrestin 2 immunoreactivity levels were determined by western blot in brain cortices. Acute and chronic treatment with sufentanil induced analgesic tolerance, associated with up-regulation of GRK2, GRK6, and beta-arrestin 2. GRK3 expression only was increased in the acutely treated group. When nimodipine was associated to the chronic opioid treatment, tolerance expression was prevented, and immunoreactivity levels of GRK2, GRK6 and beta-arrestin 2 recovered the control values. These data indicate that GRK2, GRK3, GRK6 and beta-arrestin 2 are involved in the short- and long-term adaptive changes in mu-opioid receptor activity, contributing to tolerance development in living animals. These observations also suggest that GRKs and beta-arrestin 2 could constitute pharmacological targets to prevent opioid tolerance development, and to improve the analgesic efficacy of opioid drugs.     

Differential Effect of Intrastriatal Kainic Acid on the Modulation of Dopamine Release by μ- and δ-Opioid Peptides: A Microdialysis Study

The present study investigated the effects of a striatal lesion induced by kainic acid on the striatal modulation of dopamine (DA) release by mu- and delta-opioid peptides. The effects of [D-Pen2,D-Pen5]-enkephalin (DPDPE) and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO), two highly selective delta- and mu-opioid agonists, respectively, were studied by microdialysis in anesthetized rats. In control animals both opioid peptides, administered locally, significantly increased extracellular DA levels. The effects of DPDPE were also observed in animals whose striatum had been previously lesioned with kainic acid. In contrast to the effects of the delta agonist, the significant increase induced by DAGO was no longer observed in lesioned animals. These results suggest that delta-opioid receptors modulating the striatal DA release, in contrast to mu receptors, are not located on neurons that may be lesioned by kainic acid.     

Differences of mu-opioid receptors between Lewis and F344 rats

F344 and Lewis rats show different responses to opioids in several experimental paradigms. In this study we have used the specific mu-opioid agonist DAMGO to find out if these differences could be attributed to heterogeneity of mu-opioid receptors. The density of [H3]DAMGO binding sites was similar in the brain cortex and spinal cord of both strains, but DAMGO affinity for mu-opioid receptors was higher in F344 tissues. Moreover, a parallel study of the effects of DAMGO on electrically-evoked twitches of isolated vasa deferentia revealed that this drug was also more effective in F344 preparations. These results suggest that mu-opioid receptors of F344 rats are more sensitive to pharmacological stimulation in vitro, which could be related to a higher drug affinity.