We cloned the complete coding sequences of porcine Gpr3, Gpr6, and Gpr12 genes. Further, on the basis of their high levels of sequence similarity, these genes are identified as a subfamily of G protein-coupled recepto...We cloned the complete coding sequences of porcine Gpr3, Gpr6, and Gpr12 genes. Further, on the basis of their high levels of sequence similarity, these genes are identified as a subfamily of G protein-coupled receptors. These putative protein sequences also showed high sequence identity with other mammalian orthologs, including several highly conserved motifs. A wide expression of the Gpr3 gene in pigs was observed through tissue distribution analysis by reverse transcriptase-polymerase chain reaction （RT-PCR） and real-time PCR, specially in the brain, pituitary, fat, liver and oocyte, where its strong expression was observed. The Gpr3 gene was found to be located on chromosome 6 and a single exon coded for the entire open reading frame. Expression of porcine Gpr3 in HEK293 cells resulted in constitutive activation of adenylate cyclase （AC） similar in amplitude to that produced by fully stimulated Gs coupled receptors. Moreover, sphingosine 1-phosphate （S1P） could increase AC activation via the constitutively active Gpr3 receptor. When a Gpr3-green fluorescent protein （GFP） construct was expressed in HEK293 cells, GFP-labeled Gpr3 protein was shown to be localized in the plasmalemma and subcellular membranes. After S1P treatment, agonist-mediated internalization could be visualized by confocal microscopy. In short, our findings suggest the porcine Gpr3, Gpr6, and Gpr12 genes as a subfamily of G protein-coupled receptors, and porcine Gpr3 was a constitutively active G protein-coupled receptor. Constitutive activation of AG and agonist-mediated internalization of Gpr3 receptor could be modulated by the S1 P, suggesting that S1P might act as an activator for porcine Gpr3 receptor.展开更多
Neuropathic pain is of serious clinical concern and only about half of patients achieve partial relief with currently-available treatments,so it is critical to find new drugs for this condition.Recently,the cellsurfac...Neuropathic pain is of serious clinical concern and only about half of patients achieve partial relief with currently-available treatments,so it is critical to find new drugs for this condition.Recently,the cellsurface trafficking of pain-related receptors has been suggested as an important mechanism underlying persistent neuropathic pain.Here,we used the short peptide GluA_（2-3y）,which specifically inhibits the GluA2-dependent endocytosis of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors,and tested its anti-nociceptive effect in the periaqueductal grey（PAG） of intact rats and rats with neuropathic pain.Intra-PAG injection of 0.15,1.5,7.5,and 15 pmol of GluA_（2-3y） induced dose-dependent increases in hindpaw withdrawal latencies to noxious thermal and mechanical stimuli in intact rats,suggesting that GluA2 cell-surface trafficking in the PAG is involved in pain modulation.Furthermore,GluA_（2-3y） had much stronger anti-nociceptive effects in rats with neuropathic pain induced by sciatic nerve ligation.Interestingly,the intra-PAG injection of 15 pmol GluA_（2-3y） had an analgesic effect similar to 10 ug（35nmol） morphine in rats with neuropathic pain.Taken together,our results suggested that GluA2 trafficking in the PAG plays a critical role in pain modulation,and inhibiting GluA2 endocytosis with GluA_（2-3y） has potent analgesic effects in rats with neuropathic pain.These findings strongly support the recent hypothesis that targeting receptor trafficking could be a new strategy for the treatment of neuropathic pain.展开更多
Objective To study the redistribution of ET1 receptors in two subcellular organelles, the sarcolemmal membrane and the light vesicle, of rat heart during the progress of septic shock Methods Male Sprague Dawley ...Objective To study the redistribution of ET1 receptors in two subcellular organelles, the sarcolemmal membrane and the light vesicle, of rat heart during the progress of septic shock Methods Male Sprague Dawley rats weighing from 270 to 320?g were randomly divided into three groups: control, early sepsis, and late sepsis Each group included six rats Sepsis was induced by cecal ligation and puncture (CLP) Control rats were sham operated After operation for 9 hours or 18 hours, animals of the three groups were anesthetized with sodium pentobarbital (60?mg/kg IP) and the hearts were removed for preparation of sarcolemma and light vesicle Hemodynamic parameters were determined with polygraph via femoral artery and intraventricular cannula ET1 receptor was assayed by [ 125 I] ET1 binding Results Heart rate, cardiac output and left ventricular +dp/dt max undergo biphasic changes: an increase in early phase of sepsis (9?h after CLP) followed by a decrease in late phase of sepsis (18?h after CLP) Mean arterial blood pressure and left ventricular dp/dt max remained relatively unaltered during early phase of sepsis but was decreased during late phase of sepsis Although septic rat heart exhibited biphasic cardiodynamic changes, myocardial function showed signs of progressive deterioration during the development of sepsis, as indicated by a progressive elevation of LVEDP [ 125 I] ET1 bindings to cardiac membranes exhibited a saturable process with a single component binding characteristic for all three experimental groups In sarcolemmal membrane fraction, the maximum binding capacity (B max ) calculated from scatchard plot was increased 30% ( P <0 01) during early phase of sepsis but decreased 24% ( P <0 01) during late phase of sepsis The affinity [the reciprocal of the dissociation contant (Kd)] for [ 125 I] ET1 binding in sarcolemmal membranes remained unaffected during early and late phases of sepsis In light vesicle fraction, the B max for [ 125 I] ET1 binding was decreased by 19% ( P <0 05) during early phase of sepsis but increased by 38% ( P <0 01) during late phase of sepsis The affinity for [ 125 I] ET1 binding in light vesicles was unaltered in early and late phases of sepsis It should be mentioned that the sum of Bmax of sarcolemmal and light vesicle fractions was increased by 25% ( P <0 01) during early phase of sepsis but was decreased by 17% ( P <0 01) during late phase of sepsis Conclusions These data indicated that a biphasic intracellular redistribution of ET1 receptor in the heart might contribute to the development of the initial hyperdynamic and subsequent hypodynamic state during sepsis展开更多
基金Project supported by the National High-Tech R&D Program(863)of China(No.2006AA10Z136)a Grant-in-Aid for Innovative Training of Doctoral Students in Jiangsu Province of China(No.CXLX11-0701)
文摘We cloned the complete coding sequences of porcine Gpr3, Gpr6, and Gpr12 genes. Further, on the basis of their high levels of sequence similarity, these genes are identified as a subfamily of G protein-coupled receptors. These putative protein sequences also showed high sequence identity with other mammalian orthologs, including several highly conserved motifs. A wide expression of the Gpr3 gene in pigs was observed through tissue distribution analysis by reverse transcriptase-polymerase chain reaction （RT-PCR） and real-time PCR, specially in the brain, pituitary, fat, liver and oocyte, where its strong expression was observed. The Gpr3 gene was found to be located on chromosome 6 and a single exon coded for the entire open reading frame. Expression of porcine Gpr3 in HEK293 cells resulted in constitutive activation of adenylate cyclase （AC） similar in amplitude to that produced by fully stimulated Gs coupled receptors. Moreover, sphingosine 1-phosphate （S1P） could increase AC activation via the constitutively active Gpr3 receptor. When a Gpr3-green fluorescent protein （GFP） construct was expressed in HEK293 cells, GFP-labeled Gpr3 protein was shown to be localized in the plasmalemma and subcellular membranes. After S1P treatment, agonist-mediated internalization could be visualized by confocal microscopy. In short, our findings suggest the porcine Gpr3, Gpr6, and Gpr12 genes as a subfamily of G protein-coupled receptors, and porcine Gpr3 was a constitutively active G protein-coupled receptor. Constitutive activation of AG and agonist-mediated internalization of Gpr3 receptor could be modulated by the S1 P, suggesting that S1P might act as an activator for porcine Gpr3 receptor.
基金supported by the National Natural Science Foundation of China (30670658)support from the Minzu University 985 Academic Team-building Fund (YLDX01013, 2015MDTD13C and 25C)the 111 Project of China (B08044)
文摘Neuropathic pain is of serious clinical concern and only about half of patients achieve partial relief with currently-available treatments,so it is critical to find new drugs for this condition.Recently,the cellsurface trafficking of pain-related receptors has been suggested as an important mechanism underlying persistent neuropathic pain.Here,we used the short peptide GluA_（2-3y）,which specifically inhibits the GluA2-dependent endocytosis of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors,and tested its anti-nociceptive effect in the periaqueductal grey（PAG） of intact rats and rats with neuropathic pain.Intra-PAG injection of 0.15,1.5,7.5,and 15 pmol of GluA_（2-3y） induced dose-dependent increases in hindpaw withdrawal latencies to noxious thermal and mechanical stimuli in intact rats,suggesting that GluA2 cell-surface trafficking in the PAG is involved in pain modulation.Furthermore,GluA_（2-3y） had much stronger anti-nociceptive effects in rats with neuropathic pain induced by sciatic nerve ligation.Interestingly,the intra-PAG injection of 15 pmol GluA_（2-3y） had an analgesic effect similar to 10 ug（35nmol） morphine in rats with neuropathic pain.Taken together,our results suggested that GluA2 trafficking in the PAG plays a critical role in pain modulation,and inhibiting GluA2 endocytosis with GluA_（2-3y） has potent analgesic effects in rats with neuropathic pain.These findings strongly support the recent hypothesis that targeting receptor trafficking could be a new strategy for the treatment of neuropathic pain.
文摘Objective To study the redistribution of ET1 receptors in two subcellular organelles, the sarcolemmal membrane and the light vesicle, of rat heart during the progress of septic shock Methods Male Sprague Dawley rats weighing from 270 to 320?g were randomly divided into three groups: control, early sepsis, and late sepsis Each group included six rats Sepsis was induced by cecal ligation and puncture (CLP) Control rats were sham operated After operation for 9 hours or 18 hours, animals of the three groups were anesthetized with sodium pentobarbital (60?mg/kg IP) and the hearts were removed for preparation of sarcolemma and light vesicle Hemodynamic parameters were determined with polygraph via femoral artery and intraventricular cannula ET1 receptor was assayed by [ 125 I] ET1 binding Results Heart rate, cardiac output and left ventricular +dp/dt max undergo biphasic changes: an increase in early phase of sepsis (9?h after CLP) followed by a decrease in late phase of sepsis (18?h after CLP) Mean arterial blood pressure and left ventricular dp/dt max remained relatively unaltered during early phase of sepsis but was decreased during late phase of sepsis Although septic rat heart exhibited biphasic cardiodynamic changes, myocardial function showed signs of progressive deterioration during the development of sepsis, as indicated by a progressive elevation of LVEDP [ 125 I] ET1 bindings to cardiac membranes exhibited a saturable process with a single component binding characteristic for all three experimental groups In sarcolemmal membrane fraction, the maximum binding capacity (B max ) calculated from scatchard plot was increased 30% ( P <0 01) during early phase of sepsis but decreased 24% ( P <0 01) during late phase of sepsis The affinity [the reciprocal of the dissociation contant (Kd)] for [ 125 I] ET1 binding in sarcolemmal membranes remained unaffected during early and late phases of sepsis In light vesicle fraction, the B max for [ 125 I] ET1 binding was decreased by 19% ( P <0 05) during early phase of sepsis but increased by 38% ( P <0 01) during late phase of sepsis The affinity for [ 125 I] ET1 binding in light vesicles was unaltered in early and late phases of sepsis It should be mentioned that the sum of Bmax of sarcolemmal and light vesicle fractions was increased by 25% ( P <0 01) during early phase of sepsis but was decreased by 17% ( P <0 01) during late phase of sepsis Conclusions These data indicated that a biphasic intracellular redistribution of ET1 receptor in the heart might contribute to the development of the initial hyperdynamic and subsequent hypodynamic state during sepsis
