Isolation and Pharmacological Characterization of Six Opioidergic Picralima nitida Alkaloids.

The seeds of the akuamma tree (Picralima nitida) have been used as a traditional treatment for pain and fever. Previous studies have attributed these effects to a series of indole alkaloids found within the seed extracts; however, these pharmacological studies were significantly limited in scope. Herein, an isolation protocol employing pH-zone-refining countercurrent chromatography was developed to provide six of the akuamma alkaloids in high purity and quantities sufficient for more extensive biological evaluation. Five of these alkaloids, akuammine (1), pseudo-akuammigine (3), akuammicine (4), akuammiline (5), and picraline (6), were evaluated against a panel of >40 central nervous system receptors to identify that their primary targets are the opioid receptors. Detailed in vitro investigations revealed 4 to be a potent kappa opioid receptor agonist, and three alkaloids (1-3) were shown to have micromolar activity at the mu opioid receptor. The mu opioid receptor agonists were further evaluated for analgesic properties but demonstrated limited efficacy in assays of thermal nociception. These findings contradict previous reports of the antinociceptive properties of the P. nitida alkaloids and the traditional use of akuamma seeds as analgesics. Nevertheless, their opioid-preferring activity does suggest the akuamma alkaloids provide distinct scaffolds from which novel opioids with unique pharmacologic properties and therapeutic utility can be developed.

Development of a cyclic adenosine monophosphate assay for Gi-coupled G protein-coupled receptors by utilizing the endogenous calcitonin activity in Chinese hamster ovary cells.

Activation of G(i)-coupled G protein-coupled receptor (GPCRs) by their ligands leads to inhibition of adenylyl cyclase (AC) and reduction of cyclic adenosine monophosphate (cAMP) levels in cells. The traditional cAMP assay for G(i)-coupled GPCRs commonly uses forskolin, a nonspecific AC activator, to increase the basal cAMP level in cells to create an assay window for ligand detection. However, there is still a need to develop a nonforskolin-based cAMP assay because of the challenges inherent in titrating the concentration of forskolin to achieve a reliable assay window, along with issues related to the cAMP-independent effects of forskolin. Herein, we describe such an assay by utilizing the endogenous activity of the calcitonin receptor in Chinese hamster ovary (CHO) cells. The calcitonin receptor is a G(s)-coupled GPCR that, when activated by calcitonin, leads to the stimulation of AC and increases cAMP in cells. Thus, we use calcitonin, instead of forskolin, to increase the basal cAMP level in CHO cells to achieve an assay window. We demonstrated that calcitonin peptides robustly increased cAMP accumulation in several CHO cell lines stably expressing well-known G(i)-coupled GPCRs, such as the Dopamine D2 receptor, the Opioid µ receptor, or the Cannabinoid receptor-1. Agonists of these G(i)-coupled GPCRs attenuated calcitonin-induced cAMP production in their receptor stable cell lines. On the other hand, antagonists and/or inverse agonists blocked the effects of their agonists on calcitonin-induced cAMP production. This calcitonin-based cAMP assay has been demonstrated to be sensitive and robust and exhibited acceptable assay windows (signal/noise ratio) and, thus, can be applied to screen for agonists and antagonists/inverse agonists of G(i)-coupled GPCRs in high-throughput screening formats.

The mechanism involved in the repression of the mu opioid receptor gene expression in CEM x174 cells infected by simian immunodeficiency virus.

Morphine can promote the pathogenesis of human acquired immunodeficiency syndrome through binding to the mu opioid receptor (MOR) in immune cells. Previous investigation has suggested that expression of the MOR gene in lymphocytes is triggered by cooperative interaction between transcription factors, specificity protein 1 (Sp1) and Ying Yang 1 (YY1), in the promoter region. However, the specific molecular mechanism by which immunodeficiency virus infection impacts regulation of the MOR gene expression in lymphocytes is still unclear. In this study, it was demonstrated that SIV (SIVmac239) infection may result in gradual reduction of the MOR gene expression and Sp1 during a period of 48 h postinfection by analysis of quantitative real-time RT-PCR and Western blotting. The results of methylation-specific PCR showed that two of 14 CpG islands adjacent to the Sp1 and YY1 elements in the promoter region were methylated, which together with reduced Sp1, contributed to the failure of interaction of Sp1 with YY1 and their binding to the elements, as determined by coimmunoprecipitation, chromatin immunoprecipitation-real-time PCR, and EMSAs. The repression of the MOR gene secondary to SIVmac239 infection could be abolished by the demethylating agent 5-aza-2'-deoxycytidine. Transfection with Sp1-expressing vector (PN3-Sp1) was also able to enhance the activity of the promoter in SIVmac239-infected cells. We therefore concluded that aberrant methylation of the promoter and reduction of Sp1 resulting from SIVmac239 infection led to the silencing of the MOR gene. This finding will be helpful in understanding the synergistic mechanism of HIV infection and morphine addiction in the pathogenesis of AIDS.

Beta-endorphin, Met-enkephalin and corresponding opioid receptors within synovium of patients with joint trauma, osteoarthritis and rheumatoid arthritis.

OBJECTIVE: Intra-articularly applied opioid agonists or antagonists modulate pain after knee surgery and in chronic arthritis. Therefore, the expression of beta-endorphin (END), Met-enkephalin (ENK), and mu and delta opioid receptors (ORs) within synovium of patients with joint trauma (JT), osteoarthritis (OA) and rheumatoid arthritis (RA) were examined. METHODS: Synovial samples were subjected to double immunohistochemical analysis of opioid peptides with immune cell markers, and of ORs with the neuronal markers calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH). RESULTS: END and ENK were expressed by macrophage-like (CD68(+)) and fibroblast-like (CD68(-)) cells within synovial lining layers of all disorders. In the sublining layers, END and ENK were mostly expressed by granulocytes in patients with JT, and by macrophages/monocytes, lymphocytes and plasma cells in those with OA and RA. Overall, END- and ENK-immunoreactive (IR) cells were more abundant in patients with RA than in those with OA and JT. ORs were found on nerve fibres and immune cells in all patients. OR-IR nerve fibres were significantly more abundant in patients with RA than in those with OA and JT. muORs and deltaORs were coexpressed with CGRP but not with TH. CONCLUSIONS: Parallel to the severity of inflammation, END and ENK in immune cells and their receptors on sensory nerve terminals are more abundant in patients with RA than in those with JT and OA. These findings are consistent with the notion that, with prolonged and enhanced inflammation, the immune and peripheral nervous systems upregulate sensory nerves expressing ORs and their ligands to counterbalance pain and inflammation.

Modulation of cardiovascular excitatory responses in rats by transcutaneous magnetic stimulation: role of the spinal cord.

This study investigated the efficacy of magnetic stimulation on the reflex cardiovascular responses induced by gastric distension in anesthetized rats and compared these responses to those influenced by electroacupuncture (EA). Unilateral magnetic stimulation (30% intensity, 2 Hz) at the Jianshi-Neiguan acupoints (pericardial meridian, P 5-6) overlying the median nerve on the forelimb for 24 min significantly decreased the reflex pressor response by 32%. This effect was noticeable by 20 min of magnetic stimulation and continued for 24 min. Median nerve denervation abolished the inhibitory effect of magnetic stimulation, indicating the importance of somatic afferent input. Unilateral EA (0.3-0.5 mA, 2 Hz) at P 5-6 using similar durations of stimulation similarly inhibited the response (35%). The inhibitory effects of EA occurred earlier and were marginally longer (20 min) than magnetic stimulation. Magnetic stimulation at Guangming-Xuanzhong acupoints (gallbladder meridian, GB 37-39) overlying the superficial peroneal nerve on the hindlimb did not attenuate the reflex. Intravenous naloxone immediately after termination of magnetic stimulation reversed inhibition of the cardiovascular reflex, suggesting involvement of the opioid system. Also, intrathecal injection of delta- and kappa-opioid receptors antagonists, ICI174,864 (n=7) and nor-binaltorphimine (n=6) immediately after termination of magnetic stimulation reversed inhibition of the cardiovascular reflex. In contrast, the mu-opioid antagonist CTOP (n=7) failed to alter the cardiovascular reflex. The endogenous neurotransmitters for delta- and kappa-opioid receptors, enkephalins and dynorphin but not beta-endorphin, therefore appear to play significant roles in the spinal cord in mediating magnetic stimulation-induced modulation of cardiovascular reflex responses.

Gender and age influences on human brain mu-opioid receptor binding measured by PET.

OBJECTIVE: Both age and gender are being increasingly recognized as important factors influencing CNS structure and function. However, there are relatively few data on actual neurochemical differences between the sexes in human subjects or on their interaction with age. One of the central neurotransmitter systems for which sex differences have been suggested by animal models and clinical human data is the opioid. In this study the authors examined age- and gender-associated variations in mu-opioid receptor binding with positron emission tomography (PET). METHOD: Healthy human subjects were studied with PET and the radiotracer [11C]carfentanil, a selective mu-opioid agonist. Two separate subject groups were examined: one group of 24 men and 12 women was studied in a retrospective analysis of data, and a second group of 12 men and 18 women was recruited prospectively and studied with a higher-resolution scanner. RESULTS: Mu-opioid receptor binding potential (Bmax/Kd) was found to increase with age in neocortical areas and the putamen. Sex differences, with higher mu-opioid binding in women, were observed in a number of cortical and subcortical areas. Gender-by-age interactions were observed in the thalamus and the amygdala; in vivo mu-opioid binding declined in postmenopausal women to levels below those of men. CONCLUSIONS: These data imply that both age and gender are important variables to consider in the interpretation of investigations of human function in which the opioid system plays a role. Also, women's reproductive status (reproductive age versus postmenopausal) may influence the function of CNS opioid systems.

The organization of the thalamostriatal projection from the lateral posterior thalamic nuclear complex (LP) in the pigmented rat.

We investigated the thalamostriatal projection of the rat using biotinylated dextran amine (BDA) and wheat-germ agglutinin horseradish peroxidase (WGA-HRP). To obtain the patch/matrix compartments of the striatum (ST), we used mu-opioid receptor (MOR) immunoreaction labeling. Thus, an MOR-positive 'patch' was indicated by a darkly stained spot, while the MOR-negative 'matrix' was displayed as a non-immunoreactive region. A small injection of BDA was made in a subregion of the lateral posterior thalamic nucleus (LP). The LP-ST fibers originated in all subregions of LP and terminated in the dorsocaudal portion of ST, where the corticostriatal fibers from the visual cortex terminate (Serizawa et al. 1994). These LP-ST fibers and terminals were concentrated in the MOR-negative matrix compartment. Electron microscopic observations showed that the LP-ST terminals made asymmetrical synaptic contacts mainly (70%, n = 30) with the dendritic spines of the presumptive ST-output neurons, and fewer (30%) contacted dendritic shafts. The present results provide anatomical support for the contention that ST-output spiny neurons of the matrix that project to the pars reticulata of the substantia nigra or globus pallidus, may be influenced directly by the LP-ST projection.

Regulation of cannabinoid and mu opioid receptors in rat lumbar spinal cord following neonatal capsaicin treatment.

In vitro receptor binding and quantitative autoradiography were used to determine whether cannabinoid receptors in rat lumbar spinal cord are localized to the central terminals of nociceptive primary afferents. Rats were treated as neonates with capsaicin to destroy sensory C-fibers. The densities of cannabinoid and mu opioid receptors in the spinal cord of the adult rats were compared with age-matched vehicle controls. Neonatal capsaicin produced a moderate but reliable suppression (16%) of [3H]CP55,940 binding to cannabinoid receptors. By contrast, the binding of [3H][D-Ala2-MePhe4,Gly-ol5]enkephalin (DAMGO) to mu receptors was depleted by approximately 60% in near adjacent sections. These data suggest that only a subpopulation of cannabinoid receptors is situated on the central terminals of primary afferent C-fibers. The present data provide anatomical evidence for a dissociation between cannabinoid and mu opioid modulation of sensory transmission at the level of the primary afferent inputs to the spinal cord.

Immunohistochemical localization of mu-opioid receptors in the central nervous system of the rat.

Of the three major types of opioid receptors ( mu, delta, kappa) in the nervous system, mu-opioid receptor shows the highest affinity for morphine that exerts powerful effects on nociceptive, autonomic, and psychological functions. So far, at least two isoforms of mu-opioid receptors have been cloned from rat brain. The present study attempted to examine immunohistochemically the distribution of mu-opioid receptors in the rat central nervous system with two kinds of antibodies to recently cloned mu-opioid receptors (MOR1 and MOR1B). One antibody recognized a specific site for MOR1, and the other bound to a common site for MOR1 and MOR1B. Intense MOR1-like immunoreactivity (LI) was seen in the 'patch' areas and subcallosal streak in the striatum, medial habenular nucleus, medial terminal nucleus of the accessory optic tract, interpeduncular nucleus, median raphe nucleus, parabrachial nuclei, locus coeruleus, ambiguous nucleus, nucleus of the solitary tract, and laminae I and II of the medullary and spinal dorsal horns. Many other regions, including the cerebral cortex, amygdala, thalamus, and hypothalamus, also contained many neuronal elements with MOR1-LI. The distribution pattern of the immunoreactivity revealed with the antibody to the common site for MOR1 and MOR1B (MOR1/1B-LI) was almost the same as that of MOR1-LI. Both MOR1-LI and MOR1/1B-LI were primarily located in neuronal cell bodies and dendrites. However, the immunoreactivities were observed in the accessory optic tract, fasciculus retroflexus, solitary tract, and primary afferent fibers in the superficial layers of the medullary and spinal dorsal horns. The presynaptic location of MOR1-LI and MOR1/1B-LI was confirmed by lesion experiments: Enucleation, placing a lesion in the medial habenular nucleus, removal of the nodose ganglion, or dorsal rhizotomy resulted in a clear reduction of the immunoreactivities, respectively, in the nuclei of the accessory optic tract, some subnuclei of the interpeduncular nucleus, nucleus of the solitary tract, or laminae I and II of the spinal dorsal horn. The results indicate that the mu-opioid receptors are widely distributed in the brain and spinal cord, mainly postsynaptically and occasionally presynaptically. Opioids, including morphine, may inhibit the excitation of neurons via the postsynaptic mu-opioid receptors, and also suppress the release of neurotransmitters and/or neuromodulators from axon terminals through the presynaptic mu-opioid receptors.

Tolerance of hypothalamic beta-endorphin neurons to mu-opioid receptor activation after chronic morphine.

The mu-opioid receptor is an autoreceptor on hypothalamic beta-endorphin neurons that when activated inhibits cell firing via increasing an inwardly rectifying potassium conductance. The membrane hyperpolarization to DAMGO ([D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin) in beta-endorphin and other arcuate (ARC) neurons was investigated in hypothalamic slices from control and morphine-treated, ovariectomized guinea pigs. Chronic morphine treatment caused both a decreased potency (EC50 220 +/- 10 nM vs. 64 +/- 3 nM in controls) and a decreased efficacy (Vmax: -7.1 +/- 1.1 mV vs. -10.7 +/- 0.6 mV in controls) of DAMGO in a population of ARC neurons including beta-endorphin neurons. In another population of ARC neurons from morphine-treated animals, DAMGO was less potent (EC50: 110 +/- 4 nm) than in controls (EC50: 64 +/- 3nM), but there was not a significant change in the efficacy of DAMGO. Twenty percent of ARC neurons did not exhibit any signs of tolerance. The density of mu-opioid receptors labeled with the antagonist radioligand [3H]diprenorphine was found to be significantly decreased in the ARC and surrounding mediobasal hypothalamus after morphine treatment (Bmax: 217 +/- 9 vs. 276 +/- 16 fmol/mg protein in controls), which is consistent with the altered response in beta-endorphin neurons. In summary, chronic morphine treatment decreases mu-opioid receptor density and the functional coupling of mu-opioid receptors to K+ channels in ARC neurons. This expression of morphine tolerance by beta-endorphin (ARC) neurons may serve as a homeostatic mechanism to maintain opioid control of a variety of systems ranging from reproduction to motivation and reward.

Effect of ovariectomy on the binding characteristics of hypothalamic mu opioid receptors in the rat.

The present experiments have been performed to analyze whether ovariectomy, performed in adult female rats, might influence the binding characteristics of hypothalamic mu binding sites. To this purpose, different groups of ovariectomized females have been killed by decapitation 1, 3, 7, 15 and 21 days after surgery, and the binding characteristics (Bmax and Kd) of the mu receptors have been determined in their hypothalami. Dihydromorphine was used as a specific mu ligand. The data obtained have been compared with those derived from the hypothalami of adult female rats in the morning of proestrus, when the density of hypothalamic mu opioid receptors reaches its highest concentration. Blood from trunk vessels was also collected for the evaluation of serum LH levels by radioimmunoassay. The results obtained have shown that one day after ovariectomy, the number of hypothalamic mu opioid binding sites is as high as that found in proestrus animals. The number of these binding sites tends to decrease on days 3, 7 and 15 after surgery. On day 21 after gonadectomy, the number of mu binding sites results to be significantly lower than that observed in the hypothalami of intact female rats in the morning of proestrus and in the hypothalami of female animals castrated since one day. The value of the Kd did not show any variation on days 1, 3, 15 and 21; however, a significant increase of this parameter (which indicates a decrease of the affinity of the ligand for its receptors) was observed on day 7 after ovariectomy.(ABSTRACT TRUNCATED AT 250 WORDS)