RESUMEN
Radix puerariae, a traditional Chinese herbal medication, has been used to treat patients with diabetic kidney disease (DKD). Our previous studies demonstrated that puerarin, the active compound of radix puerariae, improves podocyte injury in type 1 DKD mice. However, the direct molecular target of puerarin and its underlying mechanisms in DKD remain unknown. In this study, we confirmed that puerarin also improved DKD in type 2 diabetic db/db mice. Through RNA-sequencing odf isolated glomeruli, we found that differentially expressed genes (DEGs) that were altered in the glomeruli of these diabetic mice but reversed by puerarin treatment were involved mostly in oxidative stress, inflammatory and fibrosis. Further analysis of these reversed DEGs revealed protein kinase A (PKA) was among the top pathways. By utilizing the drug affinity responsive target stability method combined with mass spectrometry analysis, we identified guanine nucleotide-binding protein Gi alpha-1 (Gnai1) as the direct binding partner of puerarin. Gnai1 is an inhibitor of cAMP production which is known to have protection against podocyte injury. In vitro, we showed that puerarin not only interacted with Gnai1 but also increased cAMP production in human podocytes and mouse diabetic kidney in vivo. Puerarin also enhanced CREB phosphorylation, a downstream transcription factor of cAMP/PKA. Overexpression of CREB reduced high glucose-induced podocyte apoptosis. Inhibition of PKA by Rp-cAMP also diminished the effects of puerarin on high glucose-induced podocyte apoptosis. We conclude that the renal protective effects of puerarin are likely through inhibiting Gnai1 to activate cAMP/PKA/CREB pathway in podocytes.
Asunto(s)
Diabetes Mellitus Experimental , Nefropatías Diabéticas , Podocitos , Animales , Apoptosis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Glucosa/metabolismo , Guanidina/metabolismo , Guanidina/farmacología , Guanidina/uso terapéutico , Humanos , Isoflavonas , Ratones , Nucleótidos/metabolismo , Podocitos/metabolismoRESUMEN
Dominant GNAO1 mutations cause an emerging group of childhood-onset neurological disorders characterized by developmental delay, intellectual disability, movement disorders, drug-resistant seizures and neurological deterioration. GNAO1 encodes the α-subunit of an inhibitory GTP/GDP-binding protein regulating ion channel activity and neurotransmitter release. The pathogenic mechanisms underlying GNAO1-related disorders remain largely elusive and there are no effective therapies. Here, we assessed the functional impact of two disease-causing variants associated with distinct clinical features, c.139A > G (p.S47G) and c.662C > A (p.A221D), using Caenorhabditis elegans as a model organism. The c.139A > G change was introduced into the orthologous position of the C. elegans gene via CRISPR/Cas9, whereas a knock-in strain carrying the p.A221D variant was already available. Like null mutants, homozygous knock-in animals showed increased egg laying and were hypersensitive to aldicarb, an inhibitor of acetylcholinesterase, suggesting excessive neurotransmitter release by different classes of motor neurons. Automated analysis of C. elegans locomotion indicated that goa-1 mutants move faster than control animals, with more frequent body bends and a higher reversal rate and display uncoordinated locomotion. Phenotypic profiling of heterozygous animals revealed a strong hypomorphic effect of both variants, with a partial dominant-negative activity for the p.A221D allele. Finally, caffeine was shown to rescue aberrant motor function in C. elegans harboring the goa-1 variants; this effect is mainly exerted through adenosine receptor antagonism. Overall, our findings establish a suitable platform for drug discovery, which may assist in accelerating the development of new therapies for this devastating condition, and highlight the potential role of caffeine in controlling GNAO1-related dyskinesia.
Asunto(s)
Proteínas de Caenorhabditis elegans , Discinesias , Acetilcolinesterasa/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cafeína/farmacología , Evaluación Preclínica de Medicamentos , Discinesias/tratamiento farmacológico , Discinesias/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Proteínas de Unión al GTP/genética , Mutación , Neurotransmisores/metabolismoRESUMEN
Transient receptor potential melastatin 3 (TRPM3) channels are activated by heat, and chemical ligands such as pregnenolone sulphate (PregS) and CIM0216. Here, we show that activation of receptors coupled to heterotrimeric Gi/o proteins inhibits TRPM3 channels. This inhibition was alleviated by co-expression of proteins that bind the ßγ subunits of heterotrimeric G-proteins (Gßγ). Co-expression of Gßγ, but not constitutively active Gαi or Gαo, inhibited TRPM3 currents. TRPM3 co-immunoprecipitated with Gß, and purified Gßγ proteins applied to excised inside-out patches inhibited TRPM3 currents, indicating a direct effect. Baclofen and somatostatin, agonists of Gi-coupled receptors, inhibited Ca2+ signals induced by PregS and CIM0216 in mouse dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited inward currents induced by CIM0216 in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in mice. Our data uncover a novel signaling mechanism regulating TRPM3 channels.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/farmacología , Subunidades gamma de la Proteína de Unión al GTP/farmacología , Canales Catiónicos TRPM/efectos de los fármacos , Animales , Baclofeno/antagonistas & inhibidores , Escala de Evaluación de la Conducta , Calcio/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Células HEK293 , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Pregnenolona/farmacología , Somatostatina/antagonistas & inhibidoresRESUMEN
From the brain tissue of Wistar rats,we purified a bioregulator, which is active at ultralow doses. Using reversed-phase HPLC, we prepared a homogenous polypeptide with a molecular weight of 4749 +/- 2 Da, which is responsible for the biological activity of the bioregulator. Using the CD spectroscopy method, we calculated the percentage of canonical elements of the secondary polypeptide structure in a solution. Using the methods of proteomics, we revealed that the structure of the investigated polypeptide was similar to the N-terminal sequence of a fragment of guanine-nucleotide binding G0-protein subunit alpha-1.
Asunto(s)
Encéfalo/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Hígado/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Química Encefálica , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/aislamiento & purificación , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Peso Molecular , Estructura Secundaria de Proteína , Ratas , Ratas Wistar , Técnicas de Cultivo de TejidosRESUMEN
BACKGROUND: Basic secretagogues of connective tissue mast cells act as receptor mimetic agents that trigger mast cells by activating G proteins. This leads to simultaneous propagation of two signaling pathways: one that culminates in exocytosis, while the other involves protein tyrosine phosphorylation and leads to release of arachidonic acid metabolites. We have previously shown that introduction of a peptide that comprises the C-terminal end of G alpha i3 into permeabilized mast cells inhibits basic secretagogue-induced exocytosis [Aridor et al., Science 1993;262:1569-1572]. We investigated whether cell-permeable peptides, composed of the C-terminus of G alpha i3 fused with importation sequences, affect mast cell function. METHODS: Following preincubation with the fused peptides, rat peritoneal mast cells were activated by compound 48/80 and analyzed for histamine and prostaglandin D2 release and protein tyrosine phosphorylations. RESULTS: We demonstrate that out of three importation sequences tested only G alpha i3 peptide fused with the Kaposi fibroblast growth factor importation sequence (ALL1) inhibited release of histamine. ALL1 as well as a cell-permeable peptide that corresponds to G alpha i2 also blocked compound 48/80-stimulated protein tyrosine phosphorylation, though the latter did not block histamine release. ALL1 effect was G protein-specific, as it was incapable of blocking protein tyrosine phosphorylation stimulated by pervanadate. CONCLUSION: ALL1, a transducible G alpha i3-corresponding peptide, blocks the two signaling pathways in mast cells: histamine release and protein tyrosine phosphorylation. Cell permeable peptides that block these two signaling cascades may constitute a novel approach for preventing the onset of the allergic reaction.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Mediadores de Inflamación/fisiología , Mastocitos/efectos de los fármacos , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Permeabilidad de la Membrana Celular , Evaluación Preclínica de Medicamentos , Subunidad alfa de la Proteína de Unión al GTP Gi2/farmacología , Liberación de Histamina/efectos de los fármacos , Integrina beta3/química , Mastocitos/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/farmacología , Péptidos/síntesis química , Fosforilación/efectos de los fármacos , Prostaglandina D2/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducina/farmacología , p-Metoxi-N-metilfenetilamina/antagonistas & inhibidoresRESUMEN
Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca(2+)-sensing receptor (CaR) was visualized in single CaR-expressing human embryonic kidney (HEK) 293 cells using ratiometric fluorescence resonance energy transfer-dependent cAMP sensors based on protein kinase A and Epac. Stimulation of CaR rapidly reversed or prevented agonist-stimulated elevation of cAMP through a dual mechanism involving pertussis toxin-sensitive Galpha(i) and the CaR-stimulated increase in intracellular [Ca2+]. In parallel measurements with fura-2, CaR activation elicited robust Ca2+ oscillations that increased in frequency in the presence of cAMP, eventually fusing into a sustained plateau. Considering the Ca2+ sensitivity of cAMP accumulation in these cells, lack of oscillations in [cAMP] during the initial phases of CaR stimulation was puzzling. Additional experiments showed that low-frequency, long-duration Ca2+ oscillations generated a dynamic staircase pattern in [cAMP], whereas higher frequency spiking had no effect. Our data suggest that the cAMP machinery in HEK cells acts as a low-pass filter disregarding the relatively rapid Ca2+ spiking stimulated by Ca(2+)-mobilizing agonists under physiological conditions.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , AMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Receptores Sensibles al Calcio/fisiología , Acetilcisteína/análogos & derivados , Acetilcisteína/metabolismo , Técnicas Biosensibles/métodos , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Eritromicina/análogos & derivados , Eritromicina/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Toxina del Pertussis/farmacología , Receptores Sensibles al Calcio/efectos de los fármacos , Sensibilidad y Especificidad , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Chemokines are important mediators in immune responses and inflammatory processes. Calcitonin gene-related peptide (CGRP) is produced in dorsal root ganglion (DRG) neurons. In this study, CGRP radioimmunoassay was used to investigate whether the chemokines CCL2 and CXCL1 could trigger CGRP release from cultured DRG neurons of neonatal rats and, if so, which cellular signaling pathway was involved. The results showed that CCL2 and CXCL1 ( approximately 5-100 ng/ml) evoked CGRP release and intracellular calcium elevation in a pertussis toxin (PTX)-sensitive manner. The CGRP release by CCL2 and CXCL1 was significantly inhibited by EGTA, omega-conotoxin GVIA (an N-type calcium channel blocker), thapsigargin, and ryanodine. Pretreatment of DRG neurons for 30 min with the inhibitors of phospholipase C (PLC) and protein kinase C (PKC) but not mitogen-activated protein kinases (MAPKs) significantly reduced CCL2- or CXCL1-induced CGRP release and intracellular calcium elevation. Intraplantar injection of CCL2 or CXCL1 produced hyperalgesia to thermal and mechanical stimulation in rats. These data suggest that CCL2 and CXCL1 can stimulate CGRP release and intracellular calcium elevation in DRG neurons. PLC-, PKC-, and calcium-induced calcium release from ryanodine-sensitive calcium stores signaling pathways are involved in CCL2- and CXCL1-induced CGRP release from primary nociceptive neurons, in which chemokines produce painful effects via direct actions on chemokine receptors expressed by nociceptive neurons.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/metabolismo , Quimiocina CCL2/farmacología , Quimiocinas CXC/farmacología , Ganglios Espinales/citología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Neuronas Aferentes/efectos de los fármacos , Nociceptores/fisiología , Análisis de Varianza , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Quelantes/farmacología , Quimiocina CCL2/inmunología , Quimiocina CXCL1 , Quimiocinas CXC/inmunología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/inmunología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Neuronas Aferentes/metabolismo , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Toxina del Pertussis/farmacología , Radioinmunoensayo/métodos , Ratas , Rianodina/farmacología , Tapsigargina/farmacología , Factores de Tiempo , omega-Conotoxina GVIA/farmacologíaRESUMEN
The P2Y13 receptor has recently been identified as a new P2Y receptor sharing a high sequence homology with the P2Y12 receptor as well as similar functional properties: coupling to Gi and responsiveness to ADP (Communi et al., 2001). In the present study, the pharmacology of the P2Y13 receptor and its differences with that of the P2Y12 receptor have been further characterized in 1321N1 cells (binding of [33P]2-methylthio-ADP (2MeSADP) and of GTPgamma[35S]), 1321N1 cells coexpressing Galpha16 [AG32 cells: inositol trisphosphate (IP3) measurement, binding of GTPgamma[35S]) and Chinese hamster ovary (CHO)-K1 cells (cAMP assay)]. 2MeSADP was more potent than ADP in displacing [33P]2MeSADP bound to 1321N1 cells and increasing GTPgamma[35S] binding to membranes prepared from the same cells. Similarly, 2MeSADP was more potent than ADP in stimulating IP3 accumulation after 10 min in AG32 cells and increasing cAMP in pertussis toxin-treated CHO-K1 cells stimulated by forskolin. On the other hand, ADP and 2MeSADP were equipotent at stimulating IP3 formation in AG32 cells after 30 s and inhibiting forskolininduced cAMP accumulation in CHO-K1 cells. These differences in potency cannot be explained by differences in degradation rate, which in AG32 cells was similar for the two nucleotides. When contaminating diphosphates were enzymatically removed and assay of IP3 was performed after 30 s, ATP and 2MeSATP seemed to be weak partial agonists of the P2Y13 receptor expressed in AG32 cells. The stimulatory effect of ADP on the P2Y13 receptor in AG32 cells was antagonized by reactive blue 2, suramin, pyridoxal-phosphate-6-azophenyl-2',4'disulfonic acid, diadenosine tetraphosphate, and 2-(propylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (AR-C67085MX), but not by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS-2179) (up to 100 microM). The most potent antagonist was N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (ARC69931MX) (IC50 = 4 nM), which behaved in a noncompetitive way. The active metabolite of clopidogrel was unable to displace bound 2MeSADP at concentrations up to 2 microM.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Fosfatos de Inositol/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Difosfato/farmacología , Animales , Células CHO , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , AMP Cíclico/metabolismo , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Cinética , Receptores Purinérgicos P2/efectos de los fármacos , Tionucleótidos/farmacologíaRESUMEN
Substance P (SP) excites large neurons of the nucleus basalis (NB) by inhibiting an inward rectifier K(+) channel (Kir). The properties of the Kir in NB (KirNB) in comparison with the G protein-coupled Kir (GIRK) were investigated. Single-channel recordings with the cell-attached mode showed constitutively active KirNB channels, which were inhibited by SP. When the recording method was changed from the on-cell to the inside-out mode, the channel activity of KirNB remained intact with its constitutive activity unaltered. Application of Gbeta(1gamma2) to inside-out patches induced activity of a second type of Kir (GIRK). Application of Gbeta(1gamma2), however, did not change the KirNB activity. Sequestering Gbeta(1gamma2) with Galpha(i2) abolished the GIRK activity, whereas the KirNB activity was not affected. The mean open time of KirNB channels (1.1 ms) was almost the same as that of GIRKs. The unitary conductance of KirNB was 23 pS (155 mM [K(+)](o)), whereas that of the GIRK was larger (32-39 pS). The results indicate that KirNB is different from GIRKs and from any of the classical Kirs (IRKs). Whole-cell current recordings revealed that application of muscarine to NB neurons induced a GIRK current, and this GIRK current was also inhibited by SP. Thus, SP inhibits both KirNB and GIRKs. We conclude that the excitatory transmitter SP has two types of Kirs as its effectors: the constitutively active, Gbetagamma-independent KirNB channel and the Gbetagamma-dependent GIRK.
Asunto(s)
Encéfalo/metabolismo , Neuronas/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio/metabolismo , Animales , Encéfalo/citología , Encéfalo/fisiología , Células Cultivadas , Electrofisiología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Proteínas de Unión al GTP/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Neurotransmisores/metabolismo , Fosforilación , Canales de Potasio/fisiología , Canales de Potasio de Rectificación Interna/fisiología , Proteínas Proto-Oncogénicas/farmacología , Ratas , Ratas Long-Evans , Sustancia P/farmacologíaRESUMEN
Previous studies have suggested that heterotrimeric G proteins and tyrosine kinases may be involved in lipopolysacchaide (LPS) signaling events. Signal transduction pathways activated by LPS we examined in human pomonocytic THP-l cells. We hypothesized that Gi proteins and Src tyrosine kinase differentially affect mitogen-activated protein (MAP) kinases (MAPK) and nuclear factor kappa(NF-kappaB) activation. Post-receptor coupling to Ga, proteins were examined using pertussis toxin (PTx),which inhibits Galpha i receptor-coupling. The involvement of the Src family of tyrosine kinases was examined using the selective Src tyrosine kinase inhibitor pyrazolopyrimidine-2 (PP2). Pretreatment of THP-1 cells with PTx attenuated LPS-induced activation of c-Jun-N-terminal kinase (JNK) and p38 kinase, and production of tumor necrosis factor-alpha (TN-alpha) and thromboxane B2 (TXB2). Pretreatment with PP2 inhibited TNF-alpha and TxB2 production, but had no effect on p38 kinase or JNK signaling. Therefore, the Ga i-coupled signaling pathways and Src tyrosine kinase-coupled signaling pathways are necessary for LPS-induced TNF-alpha and TxB2 production, but differ in their effects on MAPK activation. Neither PTx nor PP2 inhibited LPS-induced activation of interleukin receptor activated kinase (IRAK) or inhibited translocation of NF-kappaB. However, PP2 inhibited LPS-induced NF-kappaB transactivation of a luciferase reporter gene construct in a concentration-dependent manner. Thus, LPS induction of Src tyrosine kinases may be essential in downstream NF-kappaB tansactivation of genes following DNA binding. PTx had no effect on NF-kaapaB activation of the reporter construct. These data suggest upstream divergence in signaling through Galpha i,pathways leading to MAPK activation and other signaling events leading to IkappaBalpha degradation and NF-kaapaB DNA binding.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Lipopolisacáridos/farmacología , Familia-src Quinasas/farmacología , Técnicas de Cultivo de Célula , ADN/metabolismo , Humanos , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/farmacología , Monocitos , FN-kappa B/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Transducción de Señal , Tromboxano B2/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
It was shown previously that platelet-activating factor receptors (PAF-Rs) inhibit invasiveness of colonic and kidney epithelial cells induced by the src and Met oncogenes via a pertussis toxin-sensitive mechanism. Therefore, Madin-Darby canine kidney (MDCKts.src) cells were stably transfected with constitutively activated forms of Galphao, Galphai1, Galphai2, Galphai3 (AGalphao/i), two Gbetagamma sequestering proteins [C-terminal end of beta-adrenergic receptor kinase (ct-betaARK) and the Galphat subunit of retinal G-protein transducin], and Gbeta1-Ggamma2 subunits alone or in combination. Cellular invasion induced by src, Met, and leptin was abrogated by the AGalphao/i, ct-betaARK, and Galphat-positive clones, but was induced by coexpression of Gbeta1gamma2. In contrast, invasion stimulated by the trefoil factors (TFFs) pS2 and intestinal trefoil factor in MDCKts.src cells or human colonic epithelial cells PCmsrc and HCT8/S11 was insensitive to PAF, AGalphao, AGalphai1, and AGalphai2, but was abolished by AGalphai3 and the protease-activated receptor-1 (PAR-1) agonist thrombin receptor-activating peptide. Depletion of free Gbetagamma heterodimers by ct-betaARK resulted in a remarkable decrease of cellular adhesion and spreading on collagen matrix. Our data demonstrate the following: 1) PAF-Rs impair cellular invasion induced by src, Met, and leptin via the activation of Galphao and Galphai1 to -3; 2) invasion induced by TFFs is selectively inhibited by PAR-1 receptors and Galphai3 activation; and 3) Gbetagamma dimers are required as positive effectors of invasion pathways induced by oncogenes and epigenetic factors. Thus, redistribution of Galphao/Galphai and Gbeta/gamma heterotrimeric G-proteins by PAF-R and PAR-1 exert differential functions on positive and negative signaling pathways involved in cellular invasion and may serve as potential targets for anticancer therapy.
Asunto(s)
Proteínas de Caenorhabditis elegans , Movimiento Celular/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Proteínas de Unión al GTP Heterotriméricas/farmacología , Proteínas Proto-Oncogénicas/farmacología , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular Transformada , Células Cultivadas , Perros , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Proteínas de Unión al GTP Heterotriméricas/biosíntesis , Ratones , Ratones Desnudos , Invasividad Neoplásica , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Toxina del Pertussis , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transfección , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The administration of efficacious doses of morphine or beta-endorphin causes acute tolerance (tachyphylaxis) to the effects of additional administrations of these opioids. Mice intracerebroventricularly (icv)-injected with biologically active myristoylated (myr(+))-G(i2)alpha subunits developed no tachyphylaxis to morphine antinociception in the tail-flick test. This treatment increased the potency of opioid-induced analgesia during the declining phase. Moreover, animals showing tachyphylaxis to opioid effects exhibited normal responses to the agonists after icv-administration of myr(+)-G(i2)alpha subunits. In morphine tolerant/dependent mice, an icv dose of 12 pmol/mouse myr(+)-G(i2)alpha subunits facilitated complete restoration of morphine antinociception in only 4 or 5 days instead of the 10 to 11 days required for post-dependent mice. This was observed when myr(+)-G alpha subunits were injected within the first 24 h of chronic morphine administration -- but not later when long-term tolerance takes place. These results suggest that during the course of an opioid effect a progressive reduction of receptor-regulated G-proteins occurs, and hence tachyphylaxis develops. Exogenous administration of myr(+)-G alpha subunits may be of therapeutic potential in improving agonist activity and accelerating the recovery of post-dependent receptors.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Receptores Opioides mu/fisiología , Taquifilaxis/fisiología , Analgésicos Opioides/farmacología , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Inyecciones Intraventriculares , Masculino , Ratones , Morfina/sangre , Morfina/metabolismo , Morfina/farmacología , Dependencia de Morfina/fisiopatología , Miristatos/metabolismo , Naloxona/farmacología , Nociceptores/efectos de los fármacos , Nociceptores/fisiología , Síndrome de Abstinencia a Sustancias/fisiopatología , Factores de Tiempo , betaendorfina/farmacologíaRESUMEN
Platelet activation results in shape change, aggregation, generation of thromboxane A2, and release of granule contents. We have recently demonstrated that secreted ADP is essential for thromboxane A2-induced platelet aggregation (J. Biol. Chem. 274: 29108-29114, 1999). The aim of this study was to investigate the role of secreted ADP interacting at P2 receptor subtypes in platelet secretion. Platelet secretion induced by the thromboxane A2 mimetic U46619 was unaffected by adenosine-3'phosphate-5'-phosphate, a P2Y1 receptor selective antagonist. However, AR-C66096, a selective antagonist of the P2T(AC) receptor, inhibited U46619-induced platelet secretion, indicating an important role for Gi signaling in platelet secretion. Selective activation of either the P2T(AC) receptor or the alpha2A adrenergic receptor did not cause platelet secretion, but potentiated U46619-induced platelet secretion. SC57101, a fibrinogen receptor antagonist, failed to inhibit platelet secretion, demonstrating that outside-in signaling was not required for platelet secretion. Since Gi signaling results in reduction of basal cAMP levels through inhibition of adenylyl cyclase, we investigated whether this is the signaling event that potentiates platelet secretion. SQ22536 or dideoxyadenosine, inhibitors of adenylyl cyclase, failed to potentiate U46619-induced primary platelet secretion, indicating that reduction in cAMP levels does not directly contribute to platelet secretion. Wortmannin, a selective inhibitor of PI-3 kinase, minimally inhibited U46619-induced platelet secretion when it was solely mediated by Gq, but dramatically ablated the potentiation of Gi signaling. We conclude that signaling through the P2T(AC) receptor by secreted ADP causes positive feedback on platelet secretion through a PI-3 kinase pathway.
Asunto(s)
Plaquetas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Tromboxano A2/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Adenosina Difosfato/farmacología , Adenosina Difosfato/fisiología , Adenosina Trifosfato/metabolismo , Plaquetas/efectos de los fármacos , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/fisiología , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Humanos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Antagonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2/efectos de los fármacos , Serotonina/metabolismo , Transducción de Señal , Tromboxano A2/metabolismoRESUMEN
Studies were conducted to characterize receptors for prostaglandin (PG) F(2alpha) (PGF(2alpha)) and PGE(2), and the signalling pathways regulating total nitric oxide synthase activity and progesterone production in rabbit corpora lutea (CL) of different luteal stages. CL were obtained at days 4, 9 and 13 of pseudopregnancy and cultured in vitro for 2 h with PGF(2alpha) or PGE(2) and with activators and inhibitors of G protein (Gp), phospholipase C (PLC), protein kinase C (PKC), adenylate cyclase (AC) and protein kinase A (PKA). High affinity PGF(2alpha) receptor (K(d)=1.9+/-0.6 nM mean+/-s.e.m. ) concentrations increased (P< or =0.01) four- to five-fold from early to mid- and late-luteal phases (50.6+/-8.5, 188.3+/-36.1 and 231.4+/-38.8 fmol/mg protein respectively). By contrast, PGE(2) receptor (K(d)=1.6+/-0.5 nM) concentrations decreased (P< or =0.01) from day 4 to day 9 and 13 (27.5+/-7.7, 12.4+/-2.4 and 16.5+/-3.0 fmol/mg protein respectively). The Gp-dependent AC/PKA pathway was triggered only on day 4 CL, mimicking the PGE(2) treatment and increasing progesterone production. In both day 9 and day 13 CL, the Gp-activated PLC/PKC pathway evoked a luteolytic effect similar to that induced by PGF(2alpha). The time-dependent selective resistance to PGF(2alpha) and PGE(2) by rabbit CL is mediated by factors other than a lack of luteal receptor-ligand interactions.
Asunto(s)
Cuerpo Lúteo/metabolismo , Dinoprost/farmacología , Proteínas de Unión al GTP/farmacología , Fase Luteínica/metabolismo , Receptores de Prostaglandina/metabolismo , Transducción de Señal/fisiología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenilil Ciclasas/farmacología , Análisis de Varianza , Animales , Colforsina/farmacología , Técnicas de Cultivo , Dinoprost/metabolismo , Dinoprostona/metabolismo , Dinoprostona/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/farmacología , Imipramina/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Progesterona/análisis , Progesterona/biosíntesis , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Seudoembarazo , Conejos , Receptores de Prostaglandina E/metabolismo , Estadísticas no Paramétricas , Estaurosporina/farmacología , Fosfolipasas de Tipo C/farmacologíaRESUMEN
Besides having a role in signal transduction some trimeric G-proteins may be involved in a late stage of exocytosis. Using immunocytochemistry and confocal microscopy we found that Gi(3)-protein resides mainly in the plasma membrane, whereas Gi(1/2-)protein is preferentially associated with secretory granules. To study the function of trimeric Gi(3)- and Gi(1/2)-proteins, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. We report here that mastoparan, an activator of trimeric G-proteins, enhances calcium-induced secretory activity in rat melanotrophs. The introduction of synthetic peptides corresponding to the C-terminal domain of the (&agr;)-subunit of Gi(3)- and Gi(1/2)-proteins indicated that Gi(3 )peptide specifically blocked the mastoparan-stimulated secretory activity, which indicates an involvement of a trimeric Gi(3)-protein in mastoparan-stimulated secretory activity. Flash photolysis of caged Ca(2+)-elicited biphasic capacitance increases consisting of a fast and a slower component. Injection of anti-Gi(3) antibodies selectively inhibited the slow but not the fast component of secretory activity in rat melanotrophs. We propose that the plasma membrane-bound Gi(3)-protein may be involved in regulated secretion by specifically controlling the slower kinetic component of exocytosis.