James E. Zadina

Opiate tolerance and dependence: receptors, G-proteins, and antiopiates

Despite the existence of a large body of information on the subject, the mechanisms of opiate tolerance and dependence are not yet fully understood. Although the traditional mechanisms of receptor down-regulation and desensitization seem to play a role, they cannot entirely explain the phenomena of tolerance and dependence. Therefore, other mechanisms, such as the presence of antiopiate systems and the coupling of opiate receptors to alternative G-proteins, should be considered. A further complication of studies of opiate tolerance and dependence is the multiplicity of endogenous opiate receptors and peptides. This review will focus on the endogenous opioid system--peptides, receptors, and coupling of receptors to intracellular signaling via G-proteins--in the context of their roles in tolerance and dependence. Opioid peptides include the recently discovered endomorphins and those encoded by three known genes--pro-opiomelanocortin, pro-enkephalin, and pro-dynorphin. They bind to three types of receptors--mu, delta, and kappa. Each of the receptor types is further divided into multiple subtypes. These receptors are widely known to be coupled to G-proteins of the Gi and Go subtypes, but an increasing body of results suggests coupling to other G-proteins, such as Gs. The coupling of opiate receptors to Gs, in particular, has implications for tolerance and dependence. Alterations at the receptor and transduction level have been the focus of many studies of opiate tolerance and dependence. In these studies, both receptor down-regulation and desensitization have been demonstrated in vivo and in vitro. Receptor down-regulation has been more easily observed in vitro, especially in response to morphine, a phenomenon which suggests that some factor which is missing in vitro prevents receptors from down-regulating in vivo and may play a critical role in tolerance and dependence. We suggest that antiopiate peptides may operate in vivo in this capacity, and we outline the evidence for the antiopiate properties of three peptides: neuropeptide FF, orphanin FQ/nociceptin, and Tyr-W-MIF-1. In addition, we provide new results suggesting that Tyr-W-MIF-1 may act as an antiopiate at the cellular level by inhibiting basal G-protein activation, in contrast to the activation of G-proteins by opiate agonists.

Tumor Necrosis Factor-α: a Neuromodulator in the CNS

PAN, W., ZADINA, J.E., HARLAN, R.E., WEBER, J.T., BANKS, W.A. AND KASTIN, A.J. Tumor necrosis factor-α: a neuromodulator in the CNS. NEUROSCI BIOBEHAV REV 21(5) 603–613, 1997.—In the central nervous system (CNS), the cytokine tumor necrosis factor-α (TNFα) is produced by both neurons and glial cells, participates in developmental modeling, and is involved in many pathophysiological conditions. There are activity-dependent expressions of TNFα as well as low levels of secretion in the resting state. In contrast to the conventional view of a cytotoxic effect of TNFα, accumulating evidence suggests a beneficial effect when TNFα is applied at optimal doses and at specific periods of time. The bimodal effect is related to subtypes of receptors, activation of different signal transduction pathways, and the presence of other molecules that alter the intracellular response elements such as immediate-early genes. TNFα may be an important neuromodulator in development of the CNS, diseases of demyelination and degeneration, and in the process of regeneration. It could induce growth-promoting cytokines and neurotrophins, or it could increase the production of antiproliferative cytokines, nitric oxide, and free radicals, thereby contributing to apoptosis.

ISOLATION OF RELATIVELY LARGE AMOUNTS OF ENDOMORPHIN-1 AND ENDOMORPHIN-2 FROM HUMAN BRAIN CORTEX

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) were previously isolated from bovine brain. Both peptides showed the greatest selectivity and affinity for the mu opiate receptor of any endogenous substance found to date and may serve as natural ligands for the mu-opiate receptor. We have purified them from the fronto-parietal cortex of human brain tissue by solid phase extraction and high performance liquid chromatography. Peptide content was followed by a specific and sensitive radioimmunoassay with an antibody that was generated against endomorphin-1. The isolated endomorphins showed full biological activity. The tetrapeptides were found in human brain in much higher amounts than in bovine frontal cortex.

Localization of endomorphin-2-like immunoreactivity in the rat medulla and spinal cord.

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are endogenous ligands that have greater affinity and selectivity for the mu-opiate receptor than any other known mammalian peptide. A polyclonal antiserum, screened for specificity to endomorphin-2 by immunodot-blot assay and preabsorption controls, was used for localization of this peptide. Immunocytochemistry performed on the brainstem, spinal cord, and sensory ganglia of rats by the avidin-biotin-peroxidase method revealed a continuous dense aggregation of endomorphin-2-like immunoreactive varicose fibers in the superficial laminae of the dorsal horn of the medulla and spinal cord. Immunoreactive fibers were detected in the dorsal root as well as within the dorsal root ganglia. The results suggest that endomorphin-2 is synthesized in primary sensory neurons in ganglia, transported to the superficial dorsal horn, and released near neurons expressing mu receptors. Its distribution appears to represent a functional unit likely to be associated with modulation of nociceptive stimuli.

Endomorphins: Novel Endogenous μ‐Opiate Receptor Agonists in Regions of High μ‐Opiate Receptor Density

Abstract: Endomorphin‐1 (Tyr‐Pro‐Trp‐Phe‐NH2, EM‐1) and endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH2, EM‐2) are peptides recently isolated from brain that show the highest affinity and selectivity for the m (morphine) opiate receptor of all the known endogenous opioids. The endomorphins have potent analgesic and gastrointestinal effects. At the cellular level, they activate G‐proteins (35S‐GTP γ‐S binding) and inhibit calcium currents. Support for their role as endogenous ligands for the μ‐opiate receptor includes their localization by radioimmunoassay and immunocytochemistry in central nervous system regions of high μ receptor density. Intense EM‐2 immunoreactivity is present in the terminal regions of primary afferent neurons in the dorsal horn of the spinal cord and in the medulla near high densities of μ receptors. Chemical (capsaicin) and surgical (rhizotomy) disruption of nociceptive primary afferent neurons depletes the immunoreactivity, implicating the primary afferents as the source of EM‐2. Thus, EM‐2 is well‐positioned to serve as an endogenous modulator of pain in its earliest stages of perception. In contrast to EM‐2, which is more prevalent in the spinal cord and lower brainstem, EM‐1 is more widely and densely distributed throughout the brain than EM‐2. The distribution is consistent with a role for the peptides in the modulation of diverse functions, including autonomic, neuroendocrine, and reward functions as well as modulation of responses to pain and stress.

Differential involvement of μ-opioid receptor subtypes in endomorphin-1- and -2-induced antinociception

We investigated the role of mu-opioid receptor subtypes in both endomorphin-1 and endomorphin-2 induced antinociception in mice using supraspinally mediated behavior. With tail pressure as a mechanical noxious stimulus, both intracerebroventricularly (i.c.v.) and intrathecally (i.t.) injected-endomorphins produced potent and significant antinociceptive activity. Antinociception induced by i.t. and i.c.v. injection of endomorphin-1 was not reversed by pretreatment with a selective mu1-opioid receptor antagonist, naloxonazine (35 mg/kg, s.c.). By contrast, antinociception induced by i.t. and i.c.v. endomorphin-2 was significantly decreased by mu1-opioid receptor antagonist. Antinociception of both i.t. and i.c.v. endomorphin-1 and -2 was completely reversed by pretreatment with beta-funaltrexamine (40 mg/kg, s.c.). The results indicate that endomorphins may produce antinociception through the distinct mu1 and mu2 subtypes of mu-opioid receptor.

Chronic corticosterone impairs memory performance in the Barnes maze.

Chronic stress has been reported to impair spatial memory and cause hippocampal impairment in rodents. Glucocorticoids are believed to be the active agent in this impairment. Studies have demonstrated that chronic glucocorticoid administration results in animals being impaired in the Morris water maze (MWM) or eight-arm radial maze. Although both of these methods are well established means of testing spatial memory, neither might be considered optimal for studying the behavioral effects of stress. The Morris maze is itself highly stressful to the animals. The eight-arm maze relies on a food reward to motivate the animals, and glucocorticoids have profound effects on hunger and satiety. We therefore investigated behavioral deficits of corticosterone-treated animals in the two previously used mazes and the Barnes circular platform maze (BCM), a test similar in design to the Morris maze, but one that does not require the animal to perform a highly stressful swim. Consistent with results in other tests, we found that animals that had been treated for 3 months with stress-equivalent concentrations of glucocorticoids showed significantly impaired behavior in the Barnes maze.

The neurotrophins and their receptors: Structure, function, and neuropathology

The neurotrophins are a family of polypeptides that promote differentiation and survival of select peripheral and central neurons. Nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and neurotrophin-5 are included in this group. In recent years, tremendous advances have been made in the study of these factors. This has stimulated our review of the field, characterizing the neurotrophins from initial isolation to molecular analysis. The review also discusses their synthesis, localization, and responsive tissues, in both the periphery and CNS. The complex receptor interactions of the neurotrophins are also analyzed, as are putative signal transduction mechanisms. Discussion of the observed and postulated involvement in neuropathological disorders leads to the conclusion that the neurotrophins are involved in the function and dysfunction of the nervous system.

Endomorphin-2 is an endogenous opioid in primary sensory afferent fibers

Evidence is presented that the recently discovered endogenous mu-selective agonist, endomorphin-2, is localized in primary sensory afferents. Endomorphin-2-like immunoreactivity was found to be colocalized in a subset of substance P- and mu opiate receptor-containing fibers in the superficial laminae of the spinal cord and spinal trigeminal nucleus. Disruption of primary sensory afferents by mechanical (deafferentation by dorsal rhizotomy) or chemical (exposure to the primary afferent neurotoxin, capsaicin) methods virtually abolished endomorphin-2-like immunoreactivity in the dorsal horn. These results indicate that endomorphin-2 is present in primary afferent fibers where it can serve as the endogenous ligand for pre- and postsynaptic mu receptors and as a major modulator of pain perception.

Pharmacological modulation of anxiety-like phenotypes in adult zebrafish behavioral models.

Zebrafish (Danio rerio) are becoming increasingly popular in neurobehavioral research. Here, we summarize recent data on behavioral responses of adult zebrafish to a wide spectrum of putative anxiolytic and anxiogenic agents. Using the novel tank test as a sensitive and efficient behavioral assay, zebrafish anxiety-like behavior can be bi-directionally modulated by drugs affecting the gamma-aminobutyric acid, monoaminergic, cholinergic, glutamatergic and opioidergic systems. Complementing human and rodent data, zebrafish drug-evoked phenotypes obtained in this test support this species as a useful model for neurobehavioral and psychopharmacological research.