Ruoyu Xiao

Differences in G-protein activation by μ- and δ-opioid, and cannabinoid, receptors in rat striatum

Receptor activation of G-proteins can be measured by agonist-stimulated [35S]GTPγS binding in the presence of excess guanosine diphosphate (GDP). To determine whether opioid and cannabinoid receptor-mediated G-protein activation correlate with their receptor densities, this study compared opioid- and cannabinoid-stimulated [35S]guanylyl-5′-O-(γ-thio)-triphosphate (GTPγS) binding with the corresponding Bmax values of receptor binding in rat striatum. Scatchard analysis revealed that the Bmax of cannabinoid receptor binding was approximately ten times higher than that of μ- or δ-opioid receptor binding. However, comparable levels of cannabinoid- and μ- and δ-opioid-stimulated [35S]GTPγS binding were observed in the caudate-putamen by [35S]GTPγS autoradiography in brain sections. Scatchard analysis of net agonist-stimulated [35S]GTPγS binding in membranes showed that the Bmax of cannabinoid-stimulated binding was only twice that of μ- or δ-opioid-stimulated binding. Thus, the calculated amplification factors for μ- and δ-opioid receptors are seven times that of cannabinoid receptors.

Identification of opioid receptor-like (ORL1) peptide-stimulated [35S]GTPγS binding in rat brain

Recent reports have identified an endogenous peptide ligand for the opioid receptor-like (ORL1) receptor. In the present study, ORL1 peptide-stimulated [35S]GTPγS binding was assessed in rat cortical membranes and brain sections to localize ORL1 receptor-activated G-proteins. In membrane assays, with 20 γM GDP, ORL1 peptide stimulated [35S]GTPγS binding by approximately twofold with an ED50 value of 20 nM. ORL1 peptide- stimulated [35S]GTPγS binding was unaffected by opioid or other G-protein-coupled receptor antagonists. In brain sections, ORL1 peptide-stimulated [35S]GTPγS binding was identified in regions including cortex, amygdala, hypothalamus, thalamus and brain stem. The anatomical distribution of ORL1 peptide-stimulated [35S]GTPγS binding suggests its involvement in cognition, emotion and homeostasis.

In Vitro Autoradiographic Localization of 5-HT1A Receptor-Activated G-Proteins in the Rat Brain

Serotonin 5-HT1A receptors belong to the superfamily of G-protein-coupled receptors. Receptor activation of G-proteins can be determined by agonist-stimulated [35S]GTPgammaS binding in the presence of excess GDP, and in vitro autoradiographic adaptation of this technique allows visualization of receptor-activated G-proteins in tissue sections. The present study was performed to examine 5-HT1A receptor activation of G-proteins using 8-OH-DPAT-stimulated [35S]GTPgammaS binding in membranes and brain sections. In hippocampal membranes, 8-OH-DPAT stimulated [35S]GTPgammaS binding by twofold, with an ED50 value of 25 nM. 5-HT1 antagonists, but not 5-HT2 antagonists, increased the ED50 of 8-OH-DPAT in a manner consistent with competitive antagonists. Scatchard analysis of [35S]GTPgammaS binding showed that 8-OH-DPAT induced the formation of high affinity [35S]GTPgammaS binding sites with a KD for GTPgammaS of 3.2 nM. [35S]GTPgammaS autoradiography, performed in brain sections with the 5-HT1A agonist 8-OH-DPAT, revealed high levels of 5-HT1A-stimulated [35S]GTPgammaS binding in the hippocampus, lateral septum, prelimbic cortex, entorhinal cortex, and dorsal raphe nucleus. 5-HT1A-stimulated [35S]GTPgammaS binding in sections was blocked by the addition of the 5-HT1 antagonist methiothepin. These results show that the use of agonist-stimulated [35S]GTPgammaS autoradiography for the 5-HT1A receptor system should provide new information regarding signal transduction in specific brain regions.

Agonist-stimulated [35S]GTPγS binding in brain modulation by endogenous adenosine

Coupling of receptors to G-proteins can be assessed by the ability of specific agonists to stimulate [35S]GTPgammaS binding in both brain membranes and sections in the presence of excess GDP. In some brain regions, however, high basal activity makes it difficult to detect agonist-stimulated [35S]GTPgammaS binding. The present study suggests a modification of the assay to reduce basal [35S]GTPgammaS binding and thus increase the signal:noise ratio. Adenosine A1 receptors belong to the class of G-protein-coupled receptors that activate Gi/Go proteins in brain. In the present study, the A1 agonist R(-)N6-(2-phenylisopropyl)adenosine (R-PIA) stimulated [35S]GTPgammaS binding in brain regions known to contain A1 receptors, including cerebellum, hippocampus and dentate gyrus, medial geniculate body, superior colliculus, certain thalamic nuclei, cerebral cortex, piriform cortex, caudate-putamen, and nucleus accumbens. Treatment of sections and membranes with adenosine deaminase (ADase), which is typically used in adenosine assays to eliminate endogenous adenosine, reduced basal [35S]GTPgammaS binding. In addition, for cannabinoid and mu-opioid agonists, the percent stimulation of [35S]GTPgammaS binding was approximately doubled when ADase was included in the assay. These results suggest that endogenous adenosine contributes significantly to basal [35S]GTPgammaS binding in certain brain regions, and that this activity may be reduced by the addition of ADase, thus improving the signal:noise ratio of agonist-stimulated [35S]GTPgammaS binding.

Striatal CB1 and D2 receptors regulate expression of each other, CRIP1A and delta opioid systems

Although biochemical and physiological evidence suggests a strong interaction between striatal CB1 cannabinoid (CB1R) and D2 dopamine (D2R) receptors, the mechanisms are poorly understood. We targeted medium spiny neurons of the indirect pathway using shRNA to knockdown either CB1R or D2R. Chronic reduction in either receptor resulted in deficits in gene and protein expression for the alternative receptor and concomitantly increased expression of the cannabinoid receptor interacting protein 1a (CRIP1a), suggesting a novel role for CRIP1a in dopaminergic systems. Both CB1R and D2R knockdown reduced striatal dopaminergic‐stimulated [35S]GTPγS binding, and D2R knockdown reduced pallidal WIN55212‐2‐stimulated [35S]GTPγS binding. Decreased D2R and CB1R activity was associated with decreased striatal phosphoERK. A decrease in mRNA for opioid peptide precursors pDYN and pENK accompanied knockdown of CB1Rs or D2Rs, and over‐expression of CRIP1a. Down‐regulation in opioid peptide mRNAs was followed in time by increased DOR1 but not MOR1 expression, leading to increased [D‐Pen2, D‐Pen5]‐enkephalin‐stimulated [35S]GTPγS binding in the striatum. We conclude that mechanisms intrinsic to striatal medium spiny neurons or extrinsic via the indirect pathway adjust for changes in CB1R or D2R levels by modifying the expression and signaling capabilities of the alternative receptor as well as CRIP1a and the DELTA opioid system.

Allosteric modulation of adenosine A1 receptor coupling to G-proteins in brain

2‐Amino‐4,5,6,7‐tetrahydrobenzo(β)thiophen‐3‐yl 4‐chlorophenylmethanone (T62) is a member of a group of allosteric modulators of adenosine A1 receptors tested in animal models of neuropathic pain to increase the efficacy of adenosine. To determine its mechanisms at the level of receptor‐G‐protein activation, the present studies examined the effect of T62 on A1‐stimulated [35S]guanosine‐5′‐O‐(γ‐thio)‐triphosphate ([35S]GTPγS) binding in brain membranes, and by [35S]GTPγS autoradiography using the A1 agonist, phenylisopropyladenosine (PIA), to activate G‐proteins. In hippocampal membranes, T62 increased both basal and PIA‐stimulated [35S]GTPγS binding. The effect of T62 was non‐competitive in nature, since it increased the maximal effect of PIA, with no effect on agonist potency. GTPγS saturation analysis showed that T62 increased the number of G‐proteins activated by agonist but had no effect on the affinity of activated G‐proteins for GTPγS. [35S]GTPγS autoradiography showed that the neuroanatomical localization of T62‐stimulated [35S]GTPγS binding was identical to that of PIA‐stimulated activity. The increase in PIA‐stimulated activity by T62 varied between brain regions, with areas of lower A1 activation producing the largest percent modulation by T62. These results suggest a mechanism of allosteric modulators to increase the number of activated G‐proteins per receptor, and provide a neuroanatomical basis for understanding potential therapeutic effects of such drugs.

Chronic intrathecal morphine administration produces homologous mu receptor/G-protein desensitization specifically in spinal cord.

Previous studies have shown that chronic i.v. treatment with morphine or heroin decreased mu opioid receptor activation of G-proteins in specific brain regions. The present study examined the effect of intrathecal (i.t.) morphine administration on receptor/G-protein coupling in the spinal cord. In spinal cord membranes, [35S]GTP gamma S binding was stimulated by agonists of several G-protein-coupled receptors, including mu opioid (DAMGO), delta opioid (DPDPE), GABA(B) (baclofen), cannabinoid CB(1) (WIN 55,212-2), muscarinic cholinergic (carbachol) and adenosine A(1) (PIA). [35S]GTP gamma S autoradiography revealed that most of this agonist activation of G-proteins was localized to laminae I and II of dorsal horn. To determine the effects of chronic morphine on these receptor activities, rats were treated for 7 days with 0.11 mg/kg/day i.t. morphine, and receptor activation of G-proteins was determined by [35S]GTP gamma S autoradiography of brain and spinal cord. In spinal cord sections, chronic morphine treatment decreased DAMGO-stimulated [35S]GTP gamma S binding in laminae I and II at all levels of spinal cord examined. There were no effects of morphine treatment on [35S]GTP gamma S stimulation in spinal cord by other receptor systems examined (Adenosine A(1) and GABA(B)), and no significant effects of chronic i.t. morphine treatment were observed in brain sections. These data show that homologous desensitization of mu receptor/G-protein coupling occurs specifically in spinal cord following chronic morphine administration.

Oral Donepezil Reduces Hypersensitivity after Nerve Injury by a Spinal Muscarinic Receptor Mechanism

Background:Cholinesterase inhibitors which reach the central nervous system produce pain relief but are poorly tolerated because of gastrointestinal side effects. Here, the authors tested whether donepezil, a central nervous system penetrant cholinesterase inhibitor with a low incidence of gastrointestinal side effects, would relieve hypersensitivity in an animal model of neuropathic pain. Methods:Male rats were anesthetized, and the L5 and L6 spinal nerves were ligated unilaterally. Hypersensitivity was measured by withdrawal threshold to von Frey filament application to the hind paw after oral donepezil, and antagonists administered centrally and peripherally. Efficacy of chronic oral donepezil to relieve hypersensitivity was tested, and activation of G proteins by M2 muscarinic receptors was determined by carbachol-stimulated [35S]guanosine triphosphate γS autoradiography in brain and spinal cord. Results:Spinal nerve ligation resulted in hypersensitivity that was more severe ipsilateral than contralateral to surgery. Oral donepezil reduced hypersensitivity bilaterally in a dose-dependent manner for 2 h, and this effect was blocked by spinal but not supraspinal or peripheral muscarinic receptor antagonism. Oral donepezil maintained efficacy over 2 weeks of twice daily administration, and this treatment did not lead to desensitization of muscarinic receptor–coupled G proteins in brain or spinal cord. Conclusions:Donepezil, a well-tolerated cholinesterase inhibitor used in the treatment of Alzheimer dementia, reduces hypersensitivity in this rat model of neuropathic pain by actions on muscarinic receptors in the spinal cord. Lack of tolerance to this effect, in contrast to rapid tolerance to direct receptor agonists, suggests that cholinesterase inhibition may be useful in the treatment of neuropathic pain.

κ Opioid receptor stimulation of [35S] GTPγS binding in guinea pig brain

Abstract Although only one gene for κ opioid receptors has been cloned to date, κ1 and κ2 receptors have been defined pharmacologically, with drugs such as bremazocine binding to both putative κ receptor subtypes. To examine whether κ receptor subtypes can be distinguished at the level of the G-protein, the ability of the κ1 agonist (trans-(dl)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide) methane sulfonate (U-50488H) to stimulate [35S]guanosine-5′-O-(γ-thio)-triphosphate ([35S]GTPγS) binding in guinea pig brain was compared with that of bremazocine and dynorphin. In membranes prepared from guinea pig striatum, both bremazocine and U-50488H stimulated [35S]GTPγS binding with the same relative efficacy, while dynorphin produced at least two-fold greater efficacy than the other two agonists. In vitro autoradiography of agonist-stimulated [35S]GTPγS binding revealed similar regional distributions of bremazocine- and U-50488H-activated G-proteins. In striatal membranes, the κ antagonist nor-binaltorphimine (nor-BNI) blocked both bremazocine- and U-50488H-stimulated [35S]GTPγS binding with similar Ke values. In agonist additivity experiments, the stimulation of [35S]GTPγS binding by the δ agonist [ d -pen2,5, p-Cl-Phe4]enkephalin (p-Cl-DPDPE) was approximately additive with the two κ agonists. Stimulation of [35S]GTPγS binding by the μ agonist [ d -Ala2, N-Me4, Gly5-ol]-enkephalin (DAMGO) was additive with U-50488H, but not with bremazocine, reflecting the μ antagonist properties of this compound. The combination of bremazocine and U-50488H together produced no greater stimulation of binding than either agonist alone, indicating that they were binding to the same site. These results demonstrate that bremazocine and U-50488H activate G-proteins in guinea pig brain through the same receptor, and suggest that κ2 receptors are not coupled through the same signal transduction mechanisms as κ1 receptors.

Involvement of the Lateral Amygdala in the Antiallodynic and Reinforcing Effects of Heroin in Rats after Peripheral Nerve Injury

Background:Neuropathic pain alters opioid self-administration in rats. The brain regions altered in the presence of neuropathic pain mediating these differences have not been identified, but likely involve ascending pain pathways interacting with the limbic system. The amygdala is a brain region that integrates noxious stimulation with limbic activity. Methods:&mgr;-Opioid receptors were blocked in the amygdala using the irreversible antagonist, &bgr;-funaltrexamine, and the antiallodynic and reinforcing effects of heroin were determined in spinal nerve-ligated rats. In addition, the effect of &bgr;-funaltrexamine was determined on heroin self-administration in sham-operated rats. Results:&bgr;-Funaltrexamine decreased functional activity of &mgr;-opioid receptors by 60 ± 5% (mean ± SD). Irreversible inhibition of &mgr;-opioid receptors in the amygdala significantly attenuated the ability of doses of heroin up to 100 &mgr;g/kg to reverse hypersensitivity after spinal nerve ligation. Heroin intake by self-administration in spinal nerve-ligated rats was increased from 5.0 ± 0.3 to 9.9 ± 2.1 infusions/h after administration of 2.5 nmol of &bgr;-funaltrexamine in the lateral amygdala, while having no effect in sham-operated animals (5.8 ± 1.6 before, 6.7 ± 0.9 after). The antiallodynic effects of 60 &mgr;g/kg heroin were decreased up to 4 days, but self-administration was affected for up to 14 days. Conclusions:&mgr;-Opioid receptors in the lateral amygdala partially meditate heroins antiallodynic effects and self-administration after peripheral nerve injury. The lack of effect of &bgr;-funaltrexamine on heroin self-administration in sham-operated subjects suggests that opioids maintain self-administration through a distinct mechanism in the presence of pain.