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1.
J Recept Signal Transduct Res ; 39(1): 9-17, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31223051

RESUMO

Although multiple roles of dopamine through D1-like (D1 and D5) and D2-like (D2, D3, and D4) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via Gs/olf or Gi/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gαq activation in rat brain membranes was investigated. Agonist-induced Gαq activation was assessed by increase in guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding to Gαq determined by [35S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gαq functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D1-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D2-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D1-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT2A receptor, but not by dopamine D1-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D1-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT2A receptor has been mentioned.


Assuntos
Membrana Celular/metabolismo , Córtex Cerebral/metabolismo , Dopamina/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Dopamina D1/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Dopaminérgicos/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley
2.
J Biol Chem ; 294(21): 8351-8360, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-30962282

RESUMO

Phosphodiesterase-6 (PDE6) plays a central role in both rod and cone phototransduction pathways. In the dark, PDE6 activity is suppressed by its inhibitory γ-subunit (Pγ). Rhodopsin-catalyzed activation of the G protein transducin relieves this inhibition and enhances PDE6 catalysis. We hypothesized that amino acid sequence differences between rod- and cone-specific Pγs underlie transducin's ability to more effectively activate cone-specific PDE6 than rod PDE6. To test this, we analyzed rod and cone Pγ sequences from all major vertebrate and cyclostome lineages and found that rod Pγ loci are far more conserved than cone Pγ sequences and that most of the sequence differences are located in the N-terminal region. Next we reconstituted rod PDE6 catalytic dimer (Pαß) with various rod or cone Pγ variants and analyzed PDE6 activation upon addition of the activated transducin α-subunit (Gtα*-GTPγS). This analysis revealed a rod-specific Pγ motif (amino acids 9-18) that reduces the ability of Gtα*-GTPγS to activate the reconstituted PDE6. In cone Pγ, Asn-13 and Gln-14 significantly enhanced Gtα*-GTPγS activation of cone Pγ truncation variants. Moreover, we observed that the first four amino acids of either rod or cone Pγ contribute to Gtα*-GTPγS-mediated activation of PDE6. We conclude that physiological differences between rod and cone photoreceptor light responsiveness can be partially ascribed to ancient, highly conserved amino acid differences in the N-terminal regions of Pγ isoforms, demonstrating for the first time a functional role for this region of Pγ in the differential activation of rod and cone PDE6 by transducin.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/química , Guanosina 5'-O-(3-Tiotrifosfato)/química , Células Fotorreceptoras Retinianas Cones/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Animais , Catálise , Bovinos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo
3.
Eur J Pharmacol ; 854: 1-8, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-30951717

RESUMO

Cannabinoid CB1 and CB2 receptors are activated by Δ9-tetrahydrocannabinol, a psychoactive component of marijuana. The cannabinoid CB1 receptor is primarily located in the brain and is responsible for the psychoactive side effects, whereas the cannabinoid CB2 receptor is located in immune cells and is an attractive target for immune-related maladies. We identify small molecules that selectively bind to the cannabinoid CB2 receptor and can be further developed into therapeutics. The affinity of three molecules, ABK5, ABK6, and ABK7, to the cannabinoid CB2 receptor was determined with radioligand competition binding. The potency of G-protein coupling was determined with GTPγS binding. The three compounds bound selectively to the cannabinoid CB2 receptor, and no binding to the cannabinoid CB1 receptor was detected up to 10 µM. Immunoblotting studies show that the amount of ERK1/2 and MEK phosphorylation increased in a Gi/o-dependent manner. Furthermore, an immune cell line (Jurkat cells) was treated with ABK5, and as a result, inhibited cell proliferation. These three compounds are novel cannabinoid CB2 receptor agonists and hold promise to be further developed to treat inflammation and the often-associated pain.


Assuntos
Receptor CB2 de Canabinoide/agonistas , Ligação Competitiva , Avaliação Pré-Clínica de Medicamentos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Humanos , Células Jurkat , Ligantes , Receptor CB2 de Canabinoide/metabolismo
4.
Neuroscience ; 396: 66-72, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30458219

RESUMO

Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.


Assuntos
Ativação do Canal Iônico/efeitos da radiação , Luz , Microvilosidades/metabolismo , Microvilosidades/efeitos da radiação , Células Fotorreceptoras de Invertebrados/citologia , Canais de Receptores Transientes de Potencial/metabolismo , Canais de Receptores Transientes de Potencial/efeitos da radiação , Animais , Diacilglicerol Quinase/biossíntese , Diglicerídeos/farmacologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/efeitos da radiação , Drosophila melanogaster , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Proteína Quinase C/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , Canais de Receptores Transientes de Potencial/isolamento & purificação , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/genética
5.
Behav Pharmacol ; 30(4): 358-362, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30212383

RESUMO

Sex differences in µ-opioid receptor (MOR) agonist-induced antinociception have been reported in nonhuman primates. The degree to which µ-opioid receptor agonist sex differences in nonhuman primates extend to other behavioral endpoints remains unknown. The present study compared the behavioral effects of three MOR ligands (fentanyl, buprenorphine, and naltrexone) that varied in efficacy to stimulate [S]-GTPγS binding (from highest to lowest: fentanyl, buprenorphine, and naltrexone) in male and female rhesus monkeys. Male (n=3) and female (n=3) monkeys were trained to respond under a fixed-ratio 10 schedule of food presentation during daily sessions consisting of multiple components. Once rates of responding were stable, cumulative dose-effect functions were determined for intramuscular fentanyl (0.00032-0.032 mg/kg), buprenorphine (0.001-1 mg/kg), and naltrexone (0.01-0.1 mg/kg). Fentanyl dose-dependently decreased rates of responding in both sexes and the corresponding ED50 values were not significantly different. Buprenorphine dose-dependently decreased rates of responding in females, but not males. Naltrexone did not significantly alter behavior in either females or males. Overall, these results suggest that the expression of sex differences in MOR pharmacology depends upon both the efficacy of the MOR ligand and the behavioral endpoint.


Assuntos
Comportamento Animal/efeitos dos fármacos , Receptores Opioides mu/metabolismo , Fatores Sexuais , Analgésicos/farmacologia , Animais , Buprenorfina/farmacologia , Condicionamento Operante/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Fentanila/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Ligantes , Macaca mulatta , Masculino , Naltrexona/farmacologia , Receptores Opioides mu/agonistas
6.
Basic Clin Pharmacol Toxicol ; 124(6): 649-659, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30507034

RESUMO

The biochemical abnormalities in transmembrane signal transduction mediated through G protein-coupled receptors (GPCRs) have been postulated as underlying pathophysiology of psychiatric diseases such as schizophrenia and mood disorders. In the present study, the experimental conditions of agonist-induced [35 S]GTPγS binding in postmortem human brain membranes were optimized, and the responses induced by a series of agonists were pharmacologically characterized. The [35 S]GTPγS binding assay was performed in postmortem human prefrontal cortical membranes by means of filtration techniques, and standardized as to GDP concentration, membrane protein content, MgCl2 and NaCl concentrations in assay buffer, incubation period and effect of white matter contamination. Under the standard assay conditions, the specific [35 S]GTPγS binding was stimulated by the addition of 15 compounds in a concentration-dependent manner. Of these agonists, R(+)-8-OH-DPAT, UK-14,304, DAMGO and DPDPE showed apparently biphasic concentration-response curves. As for these four responses, only higher-potency site was pharmacologically characterized. The receptors involved in the responses investigated were 5-HT1A receptor (probed with R(+)-8-OH-DPAT or 5-HT), α2A -adrenoceptor (UK-14,304 or (-)-epinephrine), M2 /M4 mAChRs (carbachol), adenosine A1 receptor (adenosine), histamine H3 receptor (histamine), group II mGlu (l-glutamate), GABAB receptor (baclofen), µ-opioid receptor (DAMGO or endomophin-1), δ-opioid receptor (DPDPE or SNC-80) and NOP (nociceptin). Although dopamine also activated specific [35 S]GTPγS binding, this response was likely mediated via α2A -adrenoceptor, but not dopamine receptor subtypes. The present study provides us with fundamental aspects of the strategy for elucidation of probable abnormalities of neural signalling mediated by G proteins activated through multiple GPCRs in the brain of psychiatric patients.


Assuntos
Proteínas de Ligação ao GTP/agonistas , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Córtex Pré-Frontal/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ligação Competitiva , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Receptor A1 de Adenosina/metabolismo , Receptor 5-HT2C de Serotonina/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Receptores de GABA-B/metabolismo , Receptores Histamínicos H3/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Receptores Opioides mu/metabolismo , Receptores sigma/metabolismo , Adulto Jovem
7.
Mol Cancer Res ; 17(4): 963-973, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30567972

RESUMO

Uveal melanoma is the most common intraocular tumor in adults and often metastasizes to the liver, leaving patients with few options. Recurrent activating mutations in the G proteins, Gαq and Gα11, are observed in approximately 93% of all uveal melanomas. Although therapeutic intervention of downstream Gαq/11 targets has been unsuccessful in treating uveal melanoma, we have found that the Gαq/11 inhibitor, FR900359 (FR), effectively inhibits oncogenic Gαq/11 signaling in uveal melanoma cells expressing either mutant Gαq or Gα11. Inhibition of oncogenic Gαq/11 by FR results in cell-cycle arrest and induction of apoptosis. Furthermore, colony formation is prevented by FR treatment of uveal melanoma cells in 3D-cell culture, providing promise for future in vivo studies. This suggests direct inhibition of activating Gαq/11 mutants may be a potential means of treating uveal melanoma. IMPLICATIONS: Oncogenic Gαq/11 inhibition by FR900359 may be a potential treatment option for those with uveal melanoma.


Assuntos
Depsipeptídeos/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa de Proteínas de Ligação ao GTP/antagonistas & inibidores , Melanoma/tratamento farmacológico , Neoplasias Uveais/tratamento farmacológico , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/isolamento & purificação , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Insetos/citologia , Sistema de Sinalização das MAP Quinases , Melanoma/metabolismo , Melanoma/patologia , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia
8.
Biomol NMR Assign ; 13(1): 131-137, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30539422

RESUMO

G-proteins are essential switch points at the cell membrane that control downstream signaling by their ability to adopt an inactive, GDP-bound or an active, GTP-bound state. Among other exchange factors, G-protein coupled receptors (GPCRs) induce exchange of GDP to GTP and thus promote the active state of the G-protein. The nucleotide-binding α subunit of the G-protein undergoes major conformational changes upon nucleotide binding. Thus, an NMR analysis of the two distinct nucleotide-bound states is essential for a more detailed understanding of associated structural changes. Here, we provide an NMR backbone as well as methyl group resonance assignment of an inhibitory G-alpha subunit subtype 1 (Gαi,1) in the GDP-bound form and show that, in contrast to the GTP-bound form, large parts of the protein are mobile, presumably caused by a loose arrangement of the two subdomains in Gα that tightly interact with each other only in the GTP-bound state. As the GDP-bound form represents the GPCR-binding-competent state, the presented NMR data will be essential for further studies on G-protein-GPCR interactions and dynamics in solution for receptor systems that couple to G-proteins containing an inhibitory Gα,1 subunit.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Guanosina Difosfato/química , Ressonância Magnética Nuclear Biomolecular , Guanosina 5'-O-(3-Tiotrifosfato)/química , Humanos , Proteínas Mutantes/química , Estrutura Secundária de Proteína
9.
ACS Chem Neurosci ; 10(1): 518-527, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30188693

RESUMO

Allosteric modulators have attracted significant interest as an alternate strategy to modulate CB1 receptor signaling for therapeutic benefits that may avoid the adverse effects associated with orthosteric ligands. Here we extended our previous structure-activity relationship studies on the diarylurea-based CB1 negative allosteric modulators (NAMs) by introducing five-membered heterocycles to replace the 5-pyrrolidinylpyridinyl group in PSNCBAM-1 (1), one of the first generation CB1 allosteric modulators. Many of these compounds had comparable potency to 1 in blocking the CB1 agonist CP55,940 stimulated calcium mobilization and [35S]GTP-γ-S binding. Similar to 1, most compounds showed positive cooperativity by increasing [3H]CP55,940 binding, consistent with the positive allosteric modulator (PAM)-antagonist mechanism. Interestingly, these compounds exhibited differences in ability to increase specific binding of [3H]CP55,940 and decrease binding of the antagonist [3H]SR141716. In saturation binding studies, only increases in [3H]CP55,940 Bmax, but not Kd, were observed, suggesting that these compounds stabilize low affinity receptors into a high affinity state. Among the series, the 2-pyrrolyl analogue (13) exhibited greater potency than 1 in the [35S]GTP-γ-S binding assay and significantly enhanced the maximum binding level in the [3H]CP5,5940 binding assay, indicating greater CB1 receptor affinity and/or cooperativity.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Piridinas/farmacologia , Receptor CB1 de Canabinoide/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Humanos , Compostos de Fenilureia/química , Piridinas/química , Relação Estrutura-Atividade
10.
Neuroscience ; 390: 293-302, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30176322

RESUMO

Clinical studies have reported lower effectivity of opioid drugs in therapy of neuropathic pain. Therefore, to determine the changes in endogenous opioid systems in this pain more precisely, we have studied the changes in the pain-related behavior on days 1, 14, and 28 following a chronic constriction injury (CCI) to the sciatic nerve in mice. In parallel, we have studied the changes of µ-(MOP), δ-(DOP) and κ-(KOP) receptors, proenkephalin (PENK) and prodynorphin (PDYN) mRNA levels, as well as GTPγS binding of opioid receptors on the ipsi- and contralateral parts of the spinal cord and thalamus on the 14th day following CCI, as on this day the greatest manifestation of pain-related behavior was observed. On ipsilateral spinal cord, the decrease in MOP/DOP/KOP receptor and increase in PDYN/PENK mRNA expression was observed. In thalamus, MOP/DOP/KOP receptor expression decreased contralaterally. On ipsilateral side, there were no changes in PDYN/PENK or DOP/KOP receptor expression, but MOP mRNA decreased. The spinal GTPγS binding of MOP/DOP/KOP receptor ligands decreased on the ipsilateral side, yet the effect was less pronounced for DOP receptor ligands. In thalamus, a decrease was observed on the contralateral side for all opioid receptor ligands, especially for DOP ligand. A less pronounced decrease in GTPγS binding of spinal DOP ligands may indicate a weaker stimulation of ascending nociceptive pathways, which could explain the absence of decreased activity of DOP receptor ligands in neuropathy. These findings may suggest that drugs with a higher affinity for the DOP receptor will perform better in neuropathic pain.


Assuntos
Encefalinas/metabolismo , Neuralgia/metabolismo , Precursores de Proteínas/metabolismo , Receptores Opioides/metabolismo , Medula Espinal/metabolismo , Tálamo/metabolismo , Animais , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Masculino , Camundongos , Limiar da Dor , RNA Mensageiro/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo
11.
J Med Chem ; 61(17): 7525-7545, 2018 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-30117738

RESUMO

Past studies have shown that it has been difficult to discover and develop potent and selective κ opioid receptor antagonists, particularly compounds having potential for clinical development. In this study, we present a structure-activity relationship (SAR) study of a recently discovered new class of tetrahydroisoquinoline κ opioid receptor antagonists which led to (3 R)-7-hydroxy- N-{(1 S)-2-methyl-1-[(-4-methylpiperidine-1-yl)methyl]propyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (12) (4-Me-PDTic). Compound 12 had a Ke = 0.37 nM in a [35S]GTPγS binding assay and was 645- and >8100-fold selective for the κ relative to the µ and δ opioid receptors, respectively. Calculated log BB and CNS (central nervous system) multiparameter optimization (MPO) and low molecular weight values all predict that 12 will penetrate the brain, and pharmacokinetic studies in rats show that 12 does indeed penetrate the brain.


Assuntos
Encéfalo/efeitos dos fármacos , Antagonistas de Entorpecentes/química , Antagonistas de Entorpecentes/farmacologia , Receptores Opioides kappa/antagonistas & inibidores , Tetra-Hidroisoquinolinas/química , Animais , Células CHO , Cricetulus , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Masculino , Antagonistas de Entorpecentes/metabolismo , Ensaio Radioligante , Ratos Sprague-Dawley , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/genética , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Relação Estrutura-Atividade
12.
Biochem Pharmacol ; 157: 258-265, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30099006

RESUMO

Brain endocannabinoid system is proposed to play a role in the pathogenesis of affective disorders. In the present study, we analyzed the functionality of the cannabinoid receptor type 1 (CB1 receptor) at different transduction levels in prefrontal cortex (PFC) of depressed suicide victims. We examined stimulation of [35S]GTPγS binding, activation of Gα protein subunits and inhibition of adenylyl cyclase by the cannabinoid agonist WIN55,212-2, as well as [3H]CP55,940 binding, in PFC homogenates from suicide victims with major depression (MD) and matched control subjects. CB1 receptor-stimulated [35S]GTPγS binding was significantly greater in the PFC of MD compared with matched controls (23%, p < 0.05). This increase was most evident in the PFC from MD subgroup with negative blood test for antidepressants (AD) at the time of death (AD-free) (38%, p < 0.05), being absent when comparing the AD-treated MD cases with their controls. The density of CB1 receptors and their coupling to adenylyl cyclase were similar between MD and control cases, regardless of the existence of AD intake. Analysis of [35S]GTPγS-labelled Gα subunits allowed for the detection of upregulated CB1 receptor coupling to Gαo, but not to Gαi1, Gαi2, Gαi3, Gαz subunits, in the PFC from AD-free MD suicides. These results suggest that increased CB1 receptor functionality at the Gαi/o protein level in the PFC of MD subjects is due to enhanced coupling to Gαo proteins and might be modulated by AD intake. These data provide new insights into the role of endocannabinoid neurotransmission in the pathobiology of MD and suggest its regulation by ADs.


Assuntos
Transtorno Depressivo Maior/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Suicídio , Adenilil Ciclases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Transtorno Depressivo Maior/enzimologia , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Córtex Pré-Frontal/metabolismo , Regulação para Cima
13.
PLoS One ; 13(7): e0201184, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30044876

RESUMO

Our lab has previously shown that nitric oxide (NO) can alter the synaptic response properties of amacrine cells by releasing Cl- from internal acidic compartments. This alteration in the Cl- gradient brings about a positive shift in the reversal potential of the GABA-gated current, which can convert inhibitory synapses into excitatory synapses. Recently, we have shown that the cystic fibrosis transmembrane regulator (CFTR) Cl- channel is involved in the Cl- release. Here, we test the hypothesis that (acidic) synaptic vesicles are a source of NO-releasable Cl- in chick retinal amacrine cells. If SVs are a source of Cl-, then depleting synaptic vesicles should decrease the nitric oxide-dependent shift in the reversal potential of the GABA-gated current. The efficacy of four inhibitors of dynamin (dynasore, Dyngo 4a, Dynole 34-2, and MiTMAB) were evaluated. In order to deplete synaptic vesicles, voltage-steps were used to activate V-gated Ca2+ channels and stimulate the synaptic vesicle cycle either under control conditions or after treatment with the dynamin inhibitors. Voltage-ramps were used to measure the NO-dependent shift in the reversal potential of the GABA-gated currents under both conditions. Our results reveal that activating the synaptic vesicle cycle in the presence of dynasore or Dyngo 4a blocked the NO-dependent shift in EGABA. However, we also discovered that some dynamin inhibitors reduced Ca2+ signaling and L-type Ca2+ currents. Conversely, dynasore also increased neurotransmitter release at autaptic sites. To further resolve the mechanism underlying the inhibition of the NO-dependent shift in the reversal potential for the GABA-gated currents, we also tested the effects of the clathrin assembly inhibitor Pitstop 2 and found that this compound also inhibited the shift. These data provide evidence that dynamin inhibitors have multiple effects on amacrine cell synaptic transmission. These data also suggest that inhibition of endocytosis disrupts the ability of NO to elicit Cl- release from internal stores which may in part be due to depletion of synaptic vesicles.


Assuntos
Células Amácrinas/metabolismo , Cetilpiridínio/metabolismo , Dinaminas/metabolismo , Endocitose , Óxido Nítrico/metabolismo , Vesículas Sinápticas/metabolismo , Actinas/metabolismo , Células Amácrinas/efeitos dos fármacos , Animais , Ânions/metabolismo , Proteínas Aviárias/metabolismo , Canais de Cálcio/metabolismo , Técnicas de Cultura de Células , Embrião de Galinha , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dinaminas/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Vesículas Sinápticas/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo
14.
J Med Chem ; 61(17): 7546-7559, 2018 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-30032602

RESUMO

Animal pharmacological studies suggest that potent and selective κ opioid receptor antagonists have potential as pharmacotherapies targeting depression, anxiety, and substance abuse (opiates, alcohol, nicotine, cocaine). We recently reported lead compound 1 as a new class of κ opioid receptor antagonists with only one basic amine group. Analogues were synthesized and evaluated for their in vitro opioid receptor antagonist properties using a [35S]GTPγS binding assay. All analogues were pure opioid receptor antagonists with no agonist activity. Compounds 1, 8, 9, 13, and 14 ( Ke values 0.058-0.64 nM) are highly potent and highly selective for the κ relative to the µ and δ opioid receptors. Favorable calculated physiochemical properties were confirmed in rat PK studies, demonstrating brain penetration for selected compounds 1, 9, and 13. High κ opioid receptor potency and selectivity and highly favorable calculated physiochemical and PK properties for brain penetration suggest these compounds should be considered for further development.


Assuntos
Antagonistas de Entorpecentes/química , Antagonistas de Entorpecentes/farmacologia , Receptores Opioides kappa/antagonistas & inibidores , Tetra-Hidroisoquinolinas/química , Animais , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Masculino , Antagonistas de Entorpecentes/farmacocinética , Ensaio Radioligante , Ratos Sprague-Dawley , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/genética , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Relação Estrutura-Atividade
15.
J Alzheimers Dis ; 64(1): 117-136, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29865071

RESUMO

The endocannabinoid system, which modulates emotional learning and memory through CB1 receptors, has been found to be deregulated in Alzheimer's disease (AD). AD is characterized by a progressive decline in memory associated with selective impairment of cholinergic neurotransmission. The functional interplay of endocannabinoid and muscarinic signaling was analyzed in seven-month-old 3xTg-AD mice following the evaluation of learning and memory of an aversive stimulus. Neurochemical correlates were simultaneously studied with both receptor and functional autoradiography for CB1 and muscarinic receptors, and regulations at the cellular level were depicted by immunofluorescence. 3xTg-AD mice exhibited increased acquisition latencies and impaired memory retention compared to age-matched non-transgenic mice. Neurochemical analyses showed changes in CB1 receptor density and functional coupling of CB1 and muscarinic receptors to Gi/o proteins in several brain areas, highlighting that observed in the basolateral amygdala. The subchronic (seven days) stimulation of the endocannabinoid system following repeated WIN55,212-2 (1 mg/kg) or JZL184 (8 mg/kg) administration induced a CB1 receptor downregulation and CB1-mediated signaling desensitization, normalizing acquisition latencies to control levels. However, the observed modulation of cholinergic neurotransmission in limbic areas did not modify learning and memory outcomes. A CB1 receptor-mediated decrease of GABAergic tone in the basolateral amygdala may be controlling the limbic component of learning and memory in 3xTg-AD mice. CB1 receptor desensitization may be a plausible strategy to improve behavior alterations associated with genetic risk factors for developing AD.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Endocanabinoides/metabolismo , Oxazinas/metabolismo , Transdução de Sinais/genética , Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/genética , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Canabinoides/farmacologia , Colinérgicos/farmacologia , Cicloexanóis/farmacocinética , Modelos Animais de Doenças , Endocanabinoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Presenilina-1/genética , Radioisótopos/farmacocinética , Transdução de Sinais/efeitos dos fármacos , Proteínas tau/genética
16.
Sci Signal ; 11(532)2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29844055

RESUMO

Resistance to inhibitors of cholinesterase-8A (Ric-8A) and Ric-8B are essential biosynthetic chaperones for heterotrimeric G protein α subunits. We provide evidence for the direct regulation of Ric-8A cellular activity by dual phosphorylation. Using proteomics, Western blotting, and mutational analyses, we determined that Ric-8A was constitutively phosphorylated at five serines and threonines by the protein kinase CK2. Phosphorylation of Ser435 and Thr440 in rat Ric-8A (corresponding to Ser436 and Thr441 in human Ric-8A) was required for high-affinity binding to Gα subunits, efficient stimulation of Gα subunit guanine nucleotide exchange, and mediation of Gα subunit folding. The CK2 consensus sites that contain Ser435 and Thr440 are conserved in Ric-8 homologs from worms to mammals. We found that the homologous residues in mouse Ric-8B, Ser468 and Ser473, were also phosphorylated. Mutation of the genomic copy of ric-8 in Caenorhabditis elegans to encode alanine in the homologous sites resulted in characteristic ric-8 reduction-of-function phenotypes that are associated with defective Gq and Gs signaling, including reduced locomotion and defective egg laying. The C. elegans ric-8 phosphorylation site mutant phenotypes were partially rescued by chemical stimulation of Gq signaling. These results indicate that dual phosphorylation represents a critical form of conserved Ric-8 regulation and demonstrate that Ric-8 proteins are needed for effective Gα signaling. The position of the CK2-phosphorylated sites within a structural model of Ric-8A reveals that these sites contribute to a key acidic and negatively charged surface that may be important for its interactions with Gα subunits.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Conformação Proteica , Ratos , Serina/química , Serina/genética , Serina/metabolismo , Transdução de Sinais , Treonina/química , Treonina/genética , Treonina/metabolismo
17.
Br J Pharmacol ; 175(14): 3050-3059, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29722902

RESUMO

BACKGROUND AND PURPOSE: Opioid δ receptor agonists are potent antihyperalgesics in chronic pain models, but tolerance develops after prolonged use. Previous evidence indicates that distinct forms of tolerance occur depending on the internalization properties of δ receptor agonists. As arrestins are important in receptor internalization, we investigated the role of arrestin 2 (ß-arrestin 1) in mediating the development of tolerance induced by high- and low-internalizing δ receptor agonists. EXPERIMENTAL APPROACH: We evaluated the effect of two δ receptor agonists with similar analgesic potencies, but either high-(SNC80) or low-(ARM390) internalization properties in wild-type (WT) and arrestin 2 knockout (KO) mice. We compared tolerance to the antihyperalgesic effects of these compounds in a model of inflammatory pain. We also examined tolerance to the convulsant effect of SNC80. Furthermore, effect of chronic treatment with SNC80 on δ agonist-stimulated [35 S]-GTPγS binding was determined in WT and KO mice. KEY RESULTS: Arrestin 2 KO resulted in increased drug potency, duration of action and decreased acute tolerance to the antihyperalgesic effects of SNC80. In contrast, ARM390 produced similar effects in both WT and KO animals. Following chronic treatment, we found a marked decrease in the extent of tolerance to SNC80-induced antihyperalgesia and convulsions in arrestin 2 KO mice. Accordingly, δ receptors remained functionally coupled to G proteins in arrestin 2 KO mice chronically treated with SNC80. CONCLUSIONS AND IMPLICATIONS: Overall, these results suggest that δ receptor agonists interact with arrestins in a ligand-specific manner, and tolerance to high- but not low-internalizing agonists are preferentially regulated by arrestin 2.


Assuntos
Analgésicos/farmacologia , Benzamidas/farmacologia , Tolerância a Medicamentos/fisiologia , Piperazinas/farmacologia , Receptores Opioides delta/agonistas , beta-Arrestina 1/metabolismo , Analgésicos/uso terapêutico , Animais , Benzamidas/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Masculino , Camundongos Knockout , Dor/tratamento farmacológico , Dor/metabolismo , Piperazinas/uso terapêutico , Receptores Opioides delta/metabolismo , Convulsões/induzido quimicamente , beta-Arrestina 1/genética
18.
Environ Toxicol Pharmacol ; 60: 58-65, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29660611

RESUMO

Male Sprague-Dawley rats (8-9 weeks-old) were exposed for three days (acute exposure) or eight weeks (subchronic exposure) to purified air or concentrated ambient fine particles, PM2.5 (≤2.5 µm; 15 to 18-fold of ambient air; 370-445 µg/m3). In membranes from rat prefrontal cortex (PFC) or striatum, the density and function of dopamine D2-like receptors (D2Rs) were assessed by [3H]-spiperone binding and dopamine-stimulated [35S]-GTPγS binding, respectively. Glial activation was evaluated by immunoperoxidase labeling of the glial fibrillary acidic protein (GFAP). In the PFC, no significant changes in D2R density or signaling were observed after the acute and subchronic exposure to PM2.5. In the striatum, acute exposure to PM2.5 decreased D2R density, with no effect on signaling efficacy, whereas subchronic exposure did not affect D2R density but reduced signaling efficacy. Both acute and subchronic exposure to PM2.5 induced reactive gliosis in the striatum but not in the PFC. These results indicate that exposure to PM2.5 induces astrocyte activation and alters striatal dopaminergic transmission.


Assuntos
Corpo Estriado/metabolismo , Gliose/induzido quimicamente , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Material Particulado/toxicidade , Receptores de Dopamina D2/metabolismo , Animais , Membrana Celular , Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/metabolismo , Masculino , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Sprague-Dawley , Testes de Toxicidade
19.
Methods ; 147: 213-220, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29510249

RESUMO

Characterisation of receptors can involve either assessment of their ability to bind ligands or measure receptor activation as a result of agonist or inverse agonist interactions. This review focuses on G protein-coupled receptors (GPCRs), examining techniques that can be applied to both receptors in membranes and after solubilisation. Radioligand binding remains a widely used technique, although there is increasing use of fluorescent ligands. These can be used in a variety of experimental designs, either directly monitoring ligand itself with techniques such as fluorescence polarisation or indirectly via resonance energy transfer (fluorescence/Forster resonance energy transfer, FRET and bioluminescence resonance energy transfer, BRET). Label free techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are also increasingly being used. For GPCRs, the main measure of receptor activation is to investigate the association of the G protein with the receptor. The chief assay measures the receptor-stimulated binding of GTP or a suitable analogue to the receptor. The direct association of the G protein with the receptor has been investigated via resonance energy techniques. These have also been used to measure ligand-induced conformational changes within the receptor; a variety of experimental techniques are available to incorporate suitable donors and acceptors within the receptor.


Assuntos
Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/fisiologia , Regulação Alostérica , Transferência Ressonante de Energia de Fluorescência , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Conformação Proteica
20.
Purinergic Signal ; 14(2): 177-190, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29492786

RESUMO

Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A1 receptor and G-protein was assessed by means of two [35S]GTPγS binding assays, i.e., conventional filtration method and [35S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A1 receptor-mediated Gαi-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [35S]GTPγS binding determined with conventional assay derives from functional activation of Gαi/o proteins (not restricted only to Gαi-3) coupled to adenosine A1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [35S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %Emax values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A1 receptor/G-protein interaction in postmortem human brain tissue.


Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Imunoprecipitação/métodos , Receptor A1 de Adenosina/metabolismo , Animais , Ligação Competitiva , Feminino , Humanos , Antagonistas de Receptores Purinérgicos P1 , Ratos , Radioisótopos de Enxofre/metabolismo
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