Mu-opioid ([3H]DAGO) binding in slices of rat brain: Inhibition by sodium ions,guanyl-5′-yl imidodiphosphate and by the adrenergic neurotoxins DSP4 and xylamine

We have characterized and quantified the specific binding of the μ-agonist [³H] DAGO to 300 μM slices of hypothalamus and cerebral cortex. The receptors have many of the opioid characteristics previously demonstrated in homogenate assays. Binding is reversible, saturable, stereospecific and of high affinity. The δ-opioids DTLET and DSLET are 36- and 30-fold respectively, less effective than DAGO in competing for the binding site. Assays can be routinely performed in the presence of physiological concentrations of sodium, though in TRIS buffer the affinity and the number of receptors is increased. Protection of the ligand against proteolytic degradation is unnecessary. Unexpectedly, we observed that GppNHp inhibits [³H] DAGO binding to brain slices. This suggests an allosteric modification of the μ-receptor in the membranes of intact cells. The μ-receptors are also blocked by the adrenergic neurotoxins DSP4 and xylamine. This re-emphasizes our contention that care should be exercised in the use of these drugs. The technique is simple, rapid and involves minimal disruption of tissue. It should provide new opportunities for the study of cell surface opioid receptor subtypes in intact tissue.     

Neuroleptics andβ-carbolines displace (3H)imipramine from its binding sites in human and rat tissues

Summary Most investigations dealing with the pharmacological characterization of (³H)imipramine binding sites focus on tricyclic antidepressants (TCA). This approach seemed to be justified since imipramine belongs to that chemical group.Langer and coworkers, however, introduced a tetrahydro--carboline (THC) as a possible endogenous ligand. Thus, the high affinity of imipramine towards the binding sites might not be due to its special chemical structure but due to its tricyclic nature. In the present paper the structure-activity-relationships of neuroleptics and-carbolines were investigated and compared with that of tricyclic antidepressants.Among the tricyclic neuroleptics those with an electron attracting substituent (-Cl) exerted highest affinity. The effect was attenuated by a long, cyclic side chain. The affinity of tricyclic neuroleptics was only slightly weaker than that of 6-Meo-THC the suggested endogenous ligand. The experiments with other THCs supported the observation that an electron attracting substituent increases the affinity of a compound to the (³H)imipramine binding sites.Comparison of the binding characteristics of (³H)imipramine to membranes of human brain and thrombocytes as well as those of rat brain and thrombocytes revealed no differences among both species. Furthermore, the displacing potencies of neuroleptics were very similar with only slightly more activity in human tissue.As a methodological aspect the applicability of the Lowry method to determine the protein concentration is discussed.     

Lack of G protein-coupled sigma receptors in rat brain membranes: receptor-mediated high-affinity GTPase activity and [35S]GTPγS binding studies

Summary. Although sigma () receptors have been identified as an independent receptor family distinct from opioid and phencyclidine receptors, the physiological roles of these receptors are largely unknown. It is controversial whether there exist metabotropic receptors that are coupled with heterotrimeric G proteins. In the present study, the stimulatory effects of ligands on high-affinity GTPase activity and [³⁵S]GTPS binding were determined in the membranes prepared from rat cerebral cortex, hippocampus, and striatum. In either G protein activation assay, none of the ligands examined had stimulatory effect in any brain regions, except for unambiguous concentration-dependent increase in [³⁵S]GTPS binding by (+)-3-(3-hydroxyphenyl)-N-(1-propyl) piperidine [(+)-3-PPP] in striatal membranes. However, the competition study clearly showed this response was mediated through dopamine D₂-like receptors, but not receptors. It is concluded that receptors are not coupled to heterotrimeric G proteins, at least those of G_i/o type.     

³H]Chiba-1001(methyl-SSR180711) has low in vitro binding affinity and poor in vivo selectivity to nicotinic alpha-7 receptor in rodent brain

Neuronal nicotinic acetylcholine receptor (nAChR) agonists active at the alpha-7 (α-7) receptor subtype are potential therapeutics for cognitive deficits in schizophrenia, Alzheimer's disease, and other mental disorders. SSR180711, an α-7 selective partial agonist, has been shown to improve preclinical cognition. A novel positron emission tomography (PET) radioligand, 11C-Chiba1001, is a close analog of SSR180711. We labeled Chiba-1001 with tritium in order to evaluate its utility as a preclinical radioligand tool. In vitro, the binding affinity of [3H]Chiba-1001 at the α-7 receptor was low (K(d) = 120-180 nM) in both HEK239 cell membranes expressing human α-7 receptor and in native rat hippocampus membranes. The α-7 selective ligands AZD0328, ARR17779, and MLA did not inhibit [3H]Chiba-1001 binding (K(i) > 10,000 nM). In rat hippocampal membranes, Chiba-1001 and SSR180711 inhibited [3H]Chiba-1001 binding (K(i) = 220 and 230 nM, respectively), consistent with the literature reports. The in vivo binding profile of the radioligand was examined in normal rat, wild type mouse, and α-7 knockout mouse brain. We found that [3H]Chiba-1001 lacks adequate and specific brain regional uptake in rat and mouse brain. No significant inhibition of the radioligand binding was obtained following pretreatment of the animal with AZ11637326, AZD0328, or MLA. Our results indicate that [3H]Chiba-1001 has low affinity for α-7 nAChRs in vitro and poor α-7 regional and pharmacological selectivity in the rodent brain.     

Muscarinic acetylcholine receptor-mediated activation of G(q) in rat brain membranes determined by guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding using an anti-G protein scintillation proximity assay

In the present study, we performed antibody-capture guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) scintillation proximity assay (SPA), in which immuno-capture of Gα subunits following [³⁵S]GTPγS binding was combined with SPA technology, in rat brain membranes. Preliminary experiments using a series of agonists and commercially available anti-Gα antibodies indicated the increase in specific [³⁵S]GTPγS binding to Gα_q determined with the anti-Gα antibody sc-393 and evoked by carbamylcholine chloride (CCh) was pharmacologically relevant. The experimental conditions were optimized as for the concentrations of GDP, MgCl₂, and NaCl, the dilution of the anti-Gα_q antibody, and membrane protein contents incubated. Under the optimized conditions, CCh-stimulated specific [³⁵S]GTPγS binding to Gα_q in a concentration-dependent and saturable manner with an EC₅₀ of around 10 μM in all of the membranes prepared from rat hippocampus, cerebral cortex, and striatum. The maximum responses were varied according to the brain regions, with the rank order in magnitude of hippocampus > cerebral cortex > striatum. The addition of MT-7, a snake toxin with high selectivity for M₁ over the other muscarinic acetylcholine receptors (mAChRs) (M₂–M₅), almost completely extinguished CCh-stimulated [³⁵S]GTPγS binding to Gα_q, even at a concentration as low as 1 nM. These results indicate that the functional coupling between M₁ mAChR and Gα_q can be investigated in rat native brain membranes by means of antibody-capture SPA/[³⁵S]GTPγS binding assay. The assay developed in the present study would provide a useful strategy for investigation of possible pathophysiological alterations in neuropsychiatric disorders such as Alzheimer’s disease and schizophrenia as well as for drug discovery.     

The sequestration of [3H]spiperone by lymphocytes in schizophrenics and their first-degree relatives: a limited vulnerability marker?

The sequestration of [3H]spiperone by lymphocytes was studied in preserved cells obtained from 22 schizophrenic subjects and 40 of their relatives, and the results were compared with those obtained from 25 healthy control subjects. Mean displaceable sequestration values, obtained from measurements made at a single radioligand concentration (1nM) which optimised the relative contribution of "high affinity" sequestration, were found to be similar for all groups of subjects. Furthermore, displaceable spiperone sequestration was abnormally high in only a small proportion of the schizophrenics (13.6%) and their relatives (5%). There was no evidence that either exposure to neuroleptic medication or duration of illness had an effect on sequestration values. The results suggest that, at least until the required experimental conditions are better established, [3H]spiperone sequestration by lymphocytes does not offer a useful vulnerability marker for schizophrenia.     

In vitro and in vivo comparison of [³H](+)-PHNO and [³H]raclopride binding to rat striatum and lobes 9 and 10 of the cerebellum: a method to distinguish dopamine D₃ from D₂ receptor sites

In vitro binding characteristics of the dopamine D₃/D₂ antagonist [³H]raclopride were compared to the D₃/D₂ agonist [³H](+)‐PHNO in membrane preparations from rat striatum, cerebellum Lobules 9 and 10 (CB L9,10), and other cerebellar regions. In striatum, both radioligands labeled a single binding site. [³H](+)‐PHNO showed higher affinity, though lower B_max, compared with [³H]raclopride and was sensitive to inhibition by Gpp(NH)p. [³H](+)‐PHNO showed significant specific binding to CB L9,10 membranes with higher affinity compared to striatal membranes. [³H](+)‐PHNO binds to a high‐ and a low‐affinity binding site in CB L9,10 membranes; the high‐affinity site was not Gpp(NH)p‐sensitive. [³H](+)‐PHNO did not significantly bind cerebellum left hemisphere membranes. Very low specific binding of [³H]raclopride was found in CB L9,10. The selective dopamine D₃ antagonist SB‐277011 did not displace the binding of either ligand to striatal membranes but potently inhibited the binding of [³H](+)‐PHNO in CB L9,10 membranes. The highly selective D₂ antagonist SV‐156 showed the opposite profile. In vivo experiments were consistent with and supported by in vitro results. In summary, [³H](+)‐PHNO and [³H]raclopride mainly label dopamine D₂ receptors in rat striatum, with [³H](+)‐PHNO labeling a D population. In vitro and in vivo, [³H](+)‐PHNO labels CB L9,10 dopamine D₃ receptors that are apparently in a high affinity state whereas [³H]raclopride gave only very low signal in this region. The present approaches appear useful for selectively labeling dopamine D₃ and D₂ receptors in different rat brain regions and offer the possibility to demonstrate D₃ versus D₂ receptor selectivity of compounds using native rat brain tissue. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Effects of GABAA-ergic ligands on Cl− transport induced by the Cl−, HCO 3 − -ATPase from carp (Cyprinus carpio L.) brain reconstituted in proteoliposomes

A Cl^?, HCO ₃ ^? -ATPase isolated from the plasma membranes of fish brain was reconstituted in artificial proteoliposomes. The original sensitivity to GABA_A-ergic ligands was preserved in the reconstituted enzyme. GABA was shown to activate the liposome-embedded enzyme in a concentration range from 10 to 100 μM while picrotoxin (100 μM) removed this activating affect. A Cl^?-sensitive fluorescent probe (MEQ) was used to study the ATP-dependent Cl^? transport through the membrane of the proteoliposomes. ATP (in concentrations of 1.5 mM and higher) was found to induce Cl^?-transport into the proteoliposomes. This ATP-dependent transport was increased in the presence of GABA (100 μM), while the latter effect could also be blocked by picrotoxin (100 μM). It was concluded that the Cl^?, HCO ₃ ^? -ATPase could be involved in ATP-dependent Cl^?-transport and regulated by activators and blockers of GABA_A receptors.     

Ligustrazine inhibits high voltage-gated Ca~(2+) and TTX-resistant Na~+ channels of primary sensory neuron and thermal nociception in the rat:a study on peripheral mechanism

Objective Ligustrazine, also named as tetramethylpyrazine, is a compound purified from Ligusticum chuanxiong hort and has ever been testified to be a calcium antagonist. The present investigation was to determine the antinoci-ceptive effect of ligustrazine and, if any, the peripheral ionic mechanism involved. Methods Paw withdrawal Latency ( PWL) to noxious heating was measured in vivo and whole-cell patch recording was performed on small dorsal root ganglion (DRG) neurons. Results Intraplantar injection of ligustrazine (0.5 mg in 25μl) significantly prolonged the withdrawal latency of ipsilateral hindpaw to noxious heating in the rat. Ligustrazine not only reversibly inhibited high-voltage gated calcium current of dorsal root ganglion (DRG) neuron in dose-dependent manner with IC50 of 1.89 mmol/L, but also decreased tetrodotoxin (TTX) -resistant sodium current in relatively selective and dose-dependent manner with IC50 of 2.49 mmol/L. Conclusion The results suggested that ligustrazine could elevate the threshold of thermal nociception through inhibiting the high-voltage gated calcium current and TTX-resistant sodium current of DRG neuron in the rat.     

Epigenetic regulation of cholinergic receptor M1 (CHRM1) by histone H3K9me3 impairs Ca2+ signaling in Huntington’s disease

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by an expanded trinucleotide CAG repeat in the gene coding for huntingtin. Deregulation of chromatin remodeling is linked to the pathogenesis of HD but the mechanism remains elusive. To identify what genes are deregulated by trimethylated histone H3K9 (H3K9me3)-dependent heterochromatin, we performed H3K9me3-ChIP genome-wide sequencing combined with RNA sequencing followed by platform integration analysis in stable striatal HD cell lines (STHdhQ7/7 and STHdhQ111/111) cells. We found that genes involving neuronal synaptic transmission including cholinergic receptor M1 (CHRM1), cell motility, and neuronal differentiation pathways are downregulated while their promoter regions are highly occupied with H3K9me3 in HD. Moreover, we found that repression of CHRM1 gene expression by H3K9me3 impairs Ca²⁺-dependent neuronal signal transduction in stable cell lines expressing mutant HD protein. Thus, our data indicate that the epigenetic modifications, such as aberrant H3K9me3-dependent heterochromatin plasticity, directly contribute to the pathogenesis of HD.     

Genetic deletion of the angiotensin-(1–7) receptor Mas leads to alterations in gut villi length modulating TLR4/PI3K/AKT and produces microbiome dysbiosis

Renin-Angiotensin System (RAS) is an important peptide cascade involved in physiological processes. RAS homeostasis disruption produces several cardiovascular and metabolic disorders, such as arterial hypertension, atherosclerosis, acute myocardial infarct, obesity, diabetes, metabolic syndrome and increases gastrointestinal tract (GIT) cell proliferation. Angiotensin (Ang)-(1–7) peptide is the main RAS counter-regulatory axis effector. It is formed from ACE2 enzyme and acts mainly through Mas receptor (MasR). In this context, the aim of the present study was to evaluate alterations in small intestine morphology and intestinal microbiota composition in MasR knockout C57BL/6 mice. We analyzed glucose tolerance; insulin sensitivity and blood collected for biochemical parameters as well as small intestine tissues samples for immunohistochemistry. mRNA and bacteria gDNA expression evaluation. mRNA expression was evaluated by qRT-PCR for TLR4, PI3K and AKT. The main results showed that Mas-R-knockout mice presented lower body weight. MasR-knockout mice also presented increased fasted blood glucose and total cholesterol with reduced HDL, lower glucose tolerance and impaired insulin sensitivity. Increased intestinal mucosa length, increased intestinal villi, reduced Lieberkühn crypt depth. The increased expression of cell proliferation markers Ki-67 and Cyclin D1 and increased TLR4, PI3K and AKT expressions were observed with augmented Bacteroidetes and decreased amount of Firmicutes. That results suggests that MasR deletion generated changes in intestinal microbiota, possibly due to a lower neutral amino acids absorption followed by a compensatory increase in intestinal villi length associated with disbiosis and LPS overproduction that ultimately lead to proliferation and cell inflammation.