A hormonal role for endogenous opiate alkaloids: vascular tissues

The distribution of morphine-containing cells in the central nervous system, adrenal gland, and its presence in blood may serve to demonstrate that this signal molecule can act as a hormone besides its role in cell-to-cell signaling within the brain. This speculative review is the result of a literature evaluation with an emphasis on studies from our laboratory. Opioid peptides and opiate alkaloids have been found to influence cardiac and vascular function. They have also been reported to promote ischemic preconditioning protection in the heart. Given the presence of morphine and the novel mu(3) opiate receptor on vascular endothelial cells, including cardiac and vascular endothelial cells in the median eminence, it would appear that endogenous opiate alkaloids are involved in modulating cardiac function, possible at the hormonal level. This peripheral target tissue, via nitric oxide coupling to mu opiate receptors, may serve to down regulate the excitability of this tissue given the heart's high performance state as compared to that of the saphenous vein, a passive resistance conduit. With this in mind, morphine and other endogenous opiate alkaloids may function as a hormone.     

Proof of stimulating effect of dalargin of the process of cell division through opiate receptors

The role of opiate receptors on cell division in corneal epithelium during administration of dalargin was analysed. Naloxone injection/200 micrograms/kg/decreased MI two times, DNA-synthesis 1.4 times over 24 hours. Naloxone prevented dalargin effect on cell proliferation. Another testimonies of dalargin opiate-binding mitogenic effect were the results of the study with dalargin analogues. They are agonists of opiate receptors too. These drugs, as well as dalargin, in a dose 10 micrograms/kg increased DNA-synthesis 1.5 times, MI and MIK 2.2 times. It turned out, that the administration of another two analogues of dalargin, which are not ligands of opiate receptors, probably do not cause an adequate increase of DNA-synthesis and mitotic index.     

Opiate receptors in mice: genetic differences.

The concentration of opiate receptors in the brains of mice was determined by means of a naloxone-binding assay. The strains of mice used in these experiments were C57BL/6By, BALB/cBy, their reciprocal F₁ hybrids, and 7 recombinant-inbred strains derived by inbreeding from the F₂ generation. These strains could be divided into 3 groups on the basis of the number of opiate receptors: high (CXBH); low (CXBK); and intermediate (all the other strains). The difference in stereospecific binding of naloxone reflects a difference in the total number of receptor sites rather than in the affinity for the drug. The recombinantinbred strains also differ in their analgesic response to morphine, as previously determined by the tail-flick assay. The differences in the number of opiate receptors are not enough to account for the genetic difference in analgesic responsiveness. Both these parameters appear to be under different genetic control, and at least 2 genetic determinants may be involved in regulating the level of opiate receptors.     

The interference of naloxone hydrochloride in the teratogenic activity of opiates

Diamorphine hydrochloride, methadone hydrochloride, and the synthetic enkephalin analogue FK 33-824 are potent teratogens for the central nervous system in mouse embryos. They induce the "neurotropic syndrome of malformations," which is restricted to the central nervous system if administered during the critical period of neural tube closure. Pretreatment with corresponding equimolecular doses of the antagonist naloxone hydrochloride applied 30 minutes before treatment with the opiate agonists abolishes the major severe malformations, i.e., exencephaly, craniorachischisis, and brachyury, and reduces the number of cases of kinking of the spinal cord. Dilation of the fourth brain ventricle remains unaffected. It is suggested that the mechanism of interference in the teratogenicity of the opiates by naloxone hydrochloride reported here is based on competition for opiate receptors. In general, these observations are regarded as evidence that the pharmacological affinity of opiate agonists to receptors in the central nervous system is responsible for the malformations caused by them in this system.     

Opioids, feeding, and anorexias

This review summarizes recent work that focuses on the role of endogenous opioids (EOs) and opiate receptors in the control of food intake. Although the anorexic effect of opiate antagonists are now well accepted, the exact EO, site(s), and mechanism(s) of action remain to be established. However, accumulating evidence suggests that dynorphin, an endogenous ligand for kappa-type opiate receptors, is an important regulator (stimulant) of appetite. The roles of other EOs, such as beta-endorphin, are less clear. EOs appear to be involved in maintaining normal feeding behavior and are likely responsible for the overconsumption of fat in genetically obese and stressed subjects. Opiate antagonists block overconsumption of palatable foods, thus offering a promising approach to weight reduction for some overweight individuals. Anorexias may follow from a deficiency of kappa-type opioid activity, and surprisingly, can also result from excess opioid activity. Indeed, opiate antagonists of the mu type (naloxone) can enhance eating and weight gain in certain anorexic conditions. Therefore, it appears that excess opioid agonist activity may result in hyperphagia or anorexia (depending on the opiate receptor type). Finally, opiate antagonists may help normalize both types of pathological feeding states.     

Opioid peptides as intercellular messengers

Opioid peptides are endogenous substances present in central nervous system and various tissues whose actions are mediated by opiate receptors. They belong to two different classes: short peptides like the two pentapeptides enkephalin and substances of higher molecular weight like beta-endorphin. It appears that these various peptides play a messenger role between cells, either as neurotransmitters in the case of enkephalins or as hormones in the case of beta-endorphin.     

Electroacupuncture analgesia is mediated by stereospecific opiate receptors and is reversed by antagonists of type I receptors

Dextronaloxone, a recently synthesized stereoisomer, which was shown to possess much less opiate receptor affinity than levonaloxone, produces no reversal of electroacupuncture analgesia (EAA) in mice. Since levonaloxone completely reverses EAA, this proves that stereospecific opiate receptors are involved. It has been reported that there are two classes of opiate receptors: Type I and Type II. Type I opiate receptors may be responsible for opiate analgesia. Antagonists of Type I receptors, levonaloxone, naltrexone, cyclazocine and diprenorphine, all block electroacupuncture analgesia at low doses. All together, these results strongly support the hypothesis that electroacupuncture analgesia is mediated by opiate receptors. Possibly Type I receptors are the major component of this system.     

Characterization of type 2 opiate receptors

Recent evidence suggests that the Type 1 opiate receptor (in rat striatal patches) is a mobile receptor which is able to adopt a mu, delta, or kappa opiate receptor ligand selectivity pattern under appropriate conditions. In this paper, we have investigated such a possibility for Type 2 opiate receptors which are visualized diffusely over rat striatum. Ligand selectivity analysis suggested that the Type 2 opiate binding site is equivalent to a delta opiate receptor. The auto-radiographic distribution of Type 2 opiate binding sites is diffuse over most areas of rat brain. Thus, Type 2 opiate binding sites are different from Type 1 opiate receptors which are very discretely distributed in rat brain. Our results suggest that Type 2 opiate receptors, unlike Type 1 opiate receptors, are receptors locked in a delta-like ligand selectivity conformation.     

The significance of the mu- and delta-opiate receptors in realizing enkephalin action on the course of hypoxic hypoxia]

New enkephalins analogues have been synthesized. They are characterized by linear, cyclic and branched peptide chain. A relationship has been established between antihypoxic activity of opioid peptides an their interaction with opiate receptors. Compounds efficiently interacting with mu-receptors irrespective of delta-receptors affinity, promote longer survival of mice in hypoxia. The antihypoxic effect of opioids is proportional to their specificity to mu-receptors.     

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.     

Lack of antagonism by naloxone of the analgesic and locomotor stimulant actions of ketamine

Ketamine hydrochloride caused dose-related analgesia and ataxia in rats. In mice, ketamine caused dose-related analgesia and stimulation of locomotor activity. None of these actions of ketamine were appreciably altered by the narcotic antagonist naloxone. Thus, these actions of ketamine do not appear to be mediated by opiate receptors.     

Effects of exogenous and endogenous opiates on the hypothalamic--pituitary--gonadal axis in the male

Narcotics acutely depress serum testosterone levels in the male. Three mechanisms could be involved: an enhancement of the degradation of testosterone; a direct inhibition of testicular steroidogenesis; or, finally, an inhibition of the hypothalamic-pituitary-luteinizing hormone (LH) axis resulting in a reduction in LH-dependent testicular steroidogenesis. The currently available evidence indicates that narcotics do not affect the catabolism of testosterone by the liver or testicular steroidogenesis. Rather, the data favor a direct action on the hypothalamic--pituitary--LH axis, probably by inhibiting the secretion of LH-releasing hormone (LH-RH) from the hypothalamus. The effects of narcotics on serum LH appear to be mediated via specific opioid receptors, suggesting that a naturally occurring opioid-like substance exists that normally inhibits LH. In support of this conclusion, opiate receptor blockers markedly increase serum LH levels shortly after their subcutaneous administration. In addition, endogenous opioids also seem to participate in testosterone's negative feedback control of the hypothalamic--pituitary--LH axis. Thus, it appears that opiate drugs inhibit the function of the hypothalamic-pituitary-gonadal axis by occupying opiate receptors in the hypothalamus and, moreover, that endogenous opioids exist that normally bind to these receptors and regulate activity in this axis.     

Microsomal Opiate Receptors: Characterization of Smooth Microsomal and Synaptic Membrane Opiate Receptors

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.     

Naltrexone prevents footshock-induced performance deficit in rats

Three experiments demonstrate that inescapable footshock delivered to unrestrained rats produces analgesia as well as performance deficits in subsequent one-way shuttle acquisition. Both the performance and the antinociceptive effects are prevented by pretreatment with as little as 0.1 mg/kg i.p. of the opiate antagonist, naltrexone. These studies suggest that both effects are mediated through opiate receptors with similar underlying naltrexone pharmacodynamics.     

Interaction of [3H](–)-SKF-10,047 with Brain σ Receptors: Characterization and Autoradiographic Visualization

The sigma opiates differ from other opiates in their stimulatory and psychotomimetic actions. The sigma opiate [3H](-)-SKF-10,047 has been used to characterize sigma receptors in rat nervous tissue. Binding of [3H](-)-SKF-10,047 to rat brain membranes was of high affinity, saturable, and reversible. Scatchard analysis revealed the apparent interaction of this drug with two distinct binding sites characterized by affinities of 0.03 and 75 nM (5 mM Tris-HCl buffer, pH 7.4, at 4 degrees C). Competition analyses involving rank order determinations for a series of opiates and other drugs indicate that the high-affinity binding site is the mu opiate receptor. The lower-affinity site (revealed after suppression of mu and delta receptor binding) has been identified as the sigma opiate/phencyclidine receptor. In vitro autoradiography has been used to visualize neuroanatomical patterns of receptors labeled using [3H](-)-SKF-10,047 in the presence of normorphine and [D-Ala2,D-Leu5]enkephalin to block mu and delta interactions, respectively. Labeling patterns differ markedly from those for mu, delta, or kappa receptors. The highest densities (determined by quantitative autoradiography) are found in the medial portion of the nucleus accumbens, amygdaloid nucleus, hippocampal formation, central gray, locus coeruleus, and the parabrachial nuclei. Receptors in these structures could account for the stimulatory, mood-altering, and analgesic properties of the sigma opiates. Although not the most selective sigma opiate ligand, [3H](-)-SKF-10,047 binds to sigma opiate receptors in brain, and this interaction can be readily distinguished from its interactions with other classes of brain opiate receptors.     

Evidence for a central but not adrenal, opioid mediation in hypertension induced by brief isolation in the rat

Naloxone was found to provoke a hypotensive effect related to the dose on high blood pressure (BP) induced by short-term isolation in young rats. Another opiate antagonist, nalorphine, also reduced the arterial pressure of socially deprived rats. In contrast, naltrexone methylbromide that selectively blocked peripheral opiate receptors did not alter the elevated BP. To investigate whether adrenomedullary opioids were somehow implicated in the development of isolation-induced hypertension, bilaterally adrenalectomized rats were kept under social deprivation for 7 consecutive days. The data obtained indicated that high systolic BP developed in the same manner as in intact rats run in parallel. In conclusion, central opioids appear to be involved in BP elevation due to the stress generated by brief social deprivation in young rats.     

Multiple opiate receptors: emerging concepts

Increasing biochemical evidence indicates that the wide spectrum of opiate pharmacological actions are mediated via heterogeneous classes of receptors. μ receptors have been identified as the high affinity sites where morphine-like opiates exert their analgesic effects. δ receptors have a somewhat different CNS distribution and have been identified as sites relatively selective for the naturally occuring enkephalins. Recent biochemical studies provide evidence for two additional classes of opiate receptor sites which were originally proposed on the basis of physiological studies. Ketocyclazocine-like opiates produce their unique ataxic and sedative effects via interaction with K receptors, and SKF-10,047 (N-allylnorcyclazocine) and related opiates produce stimulant and psychotomimetic effects via interactions with σ receptors.Many opiate drugs interact at multiple receptor sites. Thus, the constellation of neuropharmacological actions of a particular opioid ligand may reflect its various potencies at a combination of μ, δ, K, and σ receptors.     

Opioid receptor agonists and Ca2+ modulation in human B cell lines.

Opiates and opioid peptides have been shown to modulate lymphocyte functions; however, little attention has been given to the type of receptors or receptor signaling mechanisms that are involved. Receptor-mediated signaling via ionized free Ca2+ is an event thought to be important in the triggering of lymphocyte activities. We report use of the calcium indicator dye, indo-1, and flow cytometry to identify B lymphocyte calcium responses to physiologic concentrations of opioid peptides. The human B cell lines Nalm 6 and JY responded to the naturally occurring opioid pentapeptide methionine-enkephalin or other opiate receptor agonists with a rapid, dose-dependent rise in free cytoplasmic Ca2+. This opioid peptide effect on Ca2+ modulation was inhibited by the opiate receptor antagonist naloxone. The synthetic enkephalin analogue DAMGO with specificity for mu-type opiate receptors and the synthetic opiate receptor agonists U50,488H and U69,593 with selectivity for kappa-type sites also stimulated calcium responses when applied to the B cell lines. These studies provide evidence that human B cell lines express functional opiate receptors of the mu- and kappa-types and suggest that such receptors, coupled with Ca2+ modulation, are instrumental in the B cell response to opiates and endogenous opioid neuropeptides.     

beta-Endorphin: radioreceptor binding assay. Relative potency of synthetic analogs with various chain lengths

An assay system is described to measure the specific binding of beta-endorphin to opiate sites (receptors) in rat brain membrane preparations using the tritiated hormone as the primary ligand. By this assay procedure, the radioreceptor activity of beta-endorphin and synthetic analogs with various chain lengths has been determined. The results suggest that both NH2- and COOH-terminal sequences of the molecule are involved in the interaction of beta-endorphin with opiate receptors.     

A Subclassification of Multiple Opiate Receptors by Means of Selective Tolerance Development

Abstract

The chronic activation of opiate receptors results in the development of tolerance. On theoretical grounds, the appearance of cross-tolerance between different opioids should imply that these compounds exhibit an identical pattern on receptor activation. Tests with isolated tissue preparations, e.g. the guinea-pig ileum, made highly tolerant in vivo to the μ-agonist morphine proved that these were of almost unchanged sensitivity to different types of receptor agonists such as K-ago-nists. Thus, the ability to selectively induce toleraance on particular types of opiate receptors represents a reliable tool for the differentiation and characterization of multiple opiate receptors. Moreover, this technique facilitated the demonstration of subtypes of the already known opiate receptors.