Laura J. Sim

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.

Anatomical distribution of mu, delta, and kappa opioid- and nociceptin/orphanin FQ-stimulated [35S]Guanylyl-5?-O-(?-Thio)-triphosphate binding in guinea pig brain

An in vitro autoradiographic technique has recently been developed to visualize receptor‐activated G‐proteins by using agonist‐stimulated [35S]guanylyl‐5′‐O‐(γ‐thio)‐triphosphate ([35S]GTPγS) binding in the presence of excess guanosine 5′‐diphosphate. This technique was used to localize opioid‐activated G‐proteins in guinea pig brain, a species that contains the three major types of opioid receptors. This study used selective μ, δ, and κ opioid agonists as well as nociceptin or orphanin FQ (N/OFQ) peptide, an endogenous ligand for an orphan opioid receptor‐like (ORL1) receptor, to stimulate [35S]GTPγS binding in guinea pig brain sections. Opioid receptor specificity was confirmed by blocking agonist‐stimulated [35S]GTPγS binding with the appropriate antagonists. In general, the distribution of agonist‐stimulated [35S]GTPγS binding correlated with previous reports of receptor binding autoradiography, although quantitative differences suggest regional variations in receptor coupling efficiency. Mu, δ, and κ opioid‐stimulated [35S]GTPγS binding was found in the caudate‐putamen, nucleus accumbens, amygdala, and hypothalamus. Mu‐stimulated [35S]GTPγS binding predominated in the hypothalamus, amygdala, and brainstem, whereas κ‐stimulated [35S]GTPγS binding was particularly high in the substantia nigra and cortex and was moderate in the cerebellum. N/OFQ‐stimulated [35S]GTPγS binding was highest in the cortex, hippocampus, and hypothalamus and exhibited a unique anatomical distribution compared with opioid‐stimulated [35S]GTPγS binding. The present study extends previous reports on opioid and ORL1 receptor localization by anatomically demonstrating functional activity produced by μ, δ, and κ opioid and ORL1 receptor activation of G‐proteins. J. Comp. Neurol. 386:562–572, 1997.

Effects of long-term exposure to Δ9-THC on expression of cannabinoid receptor (CB1) mRNA in different rat brain regions

The time course of changes across 21 days of continuous exposure to Δ9-tetrahydrocannabinol (Δ9-THC) was assessed for the level of cannabinoid receptor (CB1) mRNA expression in three different rat brain regions: cerebellum, hippocampus and corpus striatum. Expression levels of CB1 mRNA were determined using semi-quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) following a protocol which included a gene standard, 28S ribonucleic acid protein (rRNA), for normalization of levels of RNA in the three different brain regions. The levels of CB1 mRNA were assessed in four different rats at each of seven time points (6 h, and 1, 2, 3, 7, 14 and 21 days) during a 21-day Δ9-THC one dose day−1 (10 mg kg−1) treatment regimen. In the cerebellum and hippocampus, CB1 mRNA levels were increased above vehicle control animals at 7 and 14 days of treatment. In the striatum the levels of CB1 transcripts were severely reduced from days 2–14. CB1 message expression in all three brain areas returned to vehicle control levels by day 21 of Δ9-THC treatment, a time at which behavioral tolerance has been previously reported. An additional measure, receptor stimulated GTPγS binding, performed over the same time period revealed differential desensitization within the 3 brain areas as a function of chronic exposure to Δ9-THC. Hippocampus was the earliest to desensitize decreasing to 35% of control by treatment day 7, followed by a decrease in the cerebellum to that same level on day 14 of treatment. The striatum showed only half that degree of desensitization (65%) over the entire 21-day treatment period. Comparisons suggests that CB1 message may be regulated by different effector systems in each of the three areas during chronic Δ9-THC exposure.

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.

Endomorphin-Stimulated [35S]GTPγS Binding in Rat Brain: Evidence for Partial Agonist Activity at μ-Opioid Receptors

Abstract: Endomorphin‐1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at μ‐opioid receptors. In the present study, the effect of endomorphin‐1 on μ receptor‐coupled G proteins was compared with that of the μ agonist DAMGO by using agonist‐stimulated [35S]GTPγS binding in rat brain. [35S]GTPγS autoradiography revealed a similar localization of endomorphin‐1 and DAMGO‐stimulated [35S]GTPγS binding in areas including thalamus, caudate‐putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin‐1‐stimulated labeling in all regions examined. Although the distribution of endomorphin‐1‐stimulated [35S]GTPγS binding resembled that of DAMGO, the magnitude of endomorphin‐1‐stimulated binding was significantly lower than that produced by DAMGO. Concentration‐effect curves of endomorphin‐1 and DAMGO in thalamic membranes confirmed that endomorphin‐1 produced only 70% of DAMGO‐stimulated [35S]GTPγS binding. Differences in maximal stimulation of [35S]GTPγS binding between DAMGO and endomorphin‐1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin‐1‐stimulated [35S]GTPγS binding revealed a lower apparent Bmax value than that obtained with DAMGO. Endomorphin‐1 also partially antagonized DAMGO stimulation of [35S]GTPγS binding. These results demonstrate that endomorphin‐1 is a partial agonist for G protein activation at the μ‐opioid receptor in brain.

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.

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.

Baroreceptor input regulates osmotic control of central vasopressin secretion.

Sinoaortic baroreceptor denervation (SAD) results in increased osmotically induced secretion of vasopressin (VP) and oxytocin (OT) and increased cardiovascular responses to many centrally acting pressor agents. Studies were conducted to determine whether SAD increases the cardiovascular and endocrine responses to direct and peripheral osmotic stimulation of the supraoptic nucleus (SON). SON microdialysis was performed in urethane-anesthetized male rats with measurement of dialysate peptides, mean arterial pressure (MAP) and heart rate. Experiment 1 tested the effect of direct stimulation of the SON with hypertonic NaCl in SAD, sham-operated (control) and intake-matched (matched) rats. Osmotically induced VP release into the SON was significantly greater in SAD than in control or matched groups. VP release peaked at 36 +/- 13 and 15 +/- 7 pg in SAD and controls, respectively, with no increase observed in the matched group. Plasma VP was significantly elevated after SON osmotic stimulation with no differences observed among the groups. The pressor response to osmotic stimulation was greater in SAD (29 +/- 4 mm Hg) than in control (20 +/- 3 mm Hg) and matched animals (15 +/- 3 mm Hg). Experiment 2 tested the effect of intraperitoneal injection of hypertonic NaCl on SON VP and OT release. SAD rats showed an increased central VP response to peripheral osmotic stimulation, a 64-fold increase in SAD as compared to a 4-fold one in controls. Central OT release was not significantly altered (peak of 22 +/- 6 vs. 11 +/- 4 pg, SAD vs. control). A direct SON osmotic challenge given 3.5 h after the intraperitoneal test confirmed an increased VP responsiveness in the SAD group. Plasma VP and OT were significantly increased after intraperitoneal hypertonic saline with no difference observed between groups. The MAP response to intraperitoneal hypertonic saline was greater in the SAD group with an elevation of 37 +/- 4 versus 18 +/- 3 mm Hg observed in SAD versus control subjects. These results demonstrate that baroreceptor denervation produces a state of heightened osmotic sensitivity for VP neurons, with evidence for increased central VP release to both direct and peripheral hypertonic NaCl stimulation.

Effects of intracerebroventricular administration of ?-funaltrexamine on DAMGO-stimulated [35S]GTP-?-S binding in rat brain sections

Intracerebroventricular administration of β‐funaltrexamine (β‐FNA) reduces the density of μ opioid receptors as measured by in situ autoradiography by 40–50% throughout the brain, with little regional variation [Martin et al. (1993) J. Pharmacol. Exp. Ther. 267:506–514] Recently an assay has been developed to study opioid stimulation of [35S]GTP‐γ‐S binding autoradiographically in situ using slide‐mounted brain sections [Sim et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92:7242–7246]. The present study was undertaken to determine the effect of μ opioid receptor alkylation on G protein activation by the μ opioid agonist DAMGO. Animals were injected intracerebroventricularly with 40 nmol of β‐FNA or saline and sacrificed 24 hours later. DAMGO stimulated [35S]GTP‐γ‐S binding with an anatomical specificity consistent with the localization of μ opioid receptors. The percent stimulation by DAMGO ranged from ∼50 to 100% in the regions studied. β‐FNA significantly decreased G protein activation by DAMGO in regions that are consistent with its reported long‐lasting and insurmountable antagonism of the antinociceptive (medial thalamus, central gray) and reinforcing (nucleus accumbens) effects of μ opioid agonists [Adams et al. (1990) J. Pharmacol. Exp. Ther. 255:1027–1032; Martin et al. (1995) J. Pharmacol. Exp. Ther. 272:1135–1140]. However, the effects of β‐FNA were not equal in all brain regions. This may indicate regional differences in the coupling efficiency of μ opioid receptors with G proteins, or in the effects of β‐FNA on μ opioid receptor binding or on μ opioid receptor‐stimulated G protein activity. Synapse 27:177–182, 1997.

Calcium and cAMP mediated stimulation of Fos in cultured hypothalamic tyrosine hydroxylase-immunoreactive neurons

Immediate-early genes, such as c-fos, couple extracellular signals to genetic changes in the cell. We have previously demonstrated that depolarization with 50 mM KCl increases Fos immunoreactivity in hypothalamic tyrosine hydroxylase (TH) and oxytocin immunoreactive (-ir) neurons in primary culture. This Fos activation occurs within 1.5-2 h in TH-ir cells. In the present study, we examined the effects of depolarization, glutamate receptor activation and adenylyl cyclase stimulation on Fos-ir to determine the possible mechanism(s) of Fos activation in TH-ir neurons. Hypothalamic cultures were treated with KCl, glutamate or forskolin, and Fos and TH were visualized immunocytochemically. Forskolin increased the percentage of Fos/TH-ir neurons in a dose-dependent manner, with a maximal stimulation of 53.4 +/- 4.5% Fos/TH-ir neurons at 30 microM forskolin. The dose-response curve for glutamate was steep, with a maximal stimulation of 24.8 +/- 2.1% Fos-ir neurons at 100 microM. 50 mM KCl resulted in 50.0 +/- 0.8% Fos/TH-ir neurons. Pretreatment with verapamil decreased KCl induced Fos-ir by 57%, glutamate by 65% and forskolin by 39%. Combined drug administration demonstrated significant additivity between forskolin and glutamate, and forskolin and KCl, however, no significant additivity was found with KCl and glutamate. The results are discussed in terms of cAMP and calcium mediation of the Fos response to these stimuli.