RESUMO
G protein-coupled receptors (GPCRs) are the largest class of membrane proteins with around 800 members in the human genome/proteome. Extracellular signals such as hormones and neurotransmitters regulate various biological processes via GPCRs, with GPCRs being the bodily target of 30-40% of current drugs on the market. Complete identification and understanding of GPCR functionality will provide opportunities for novel drug discovery. Yeast expresses three different endogenous GPCRs regulating pheromone and sugar sensing, with the pheromone pathway offering perspectives for the characterization of heterologous GPCR signaling. Moreover, yeast offers a ''null" background for studies on mammalian GPCRs, including GPCR activation and signaling, ligand identification, and characterization of disease-related mutations. This review focuses on modifications of the yeast pheromone signaling pathway for functional GPCR studies, and on opportunities and usage of the yeast system as a platform for human GPCR studies. Finally, this review discusses in some further detail studies of adenosine receptors heterologously expressed in yeast, and what Geoff Burnstock thought of this approach.
Assuntos
Regulação Fúngica da Expressão Gênica , Receptores Acoplados a Proteínas G/biossíntese , Receptores Purinérgicos P1/biossíntese , Saccharomyces cerevisiae/metabolismo , Agonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Humanos , Engenharia de Proteínas/métodos , Agonistas do Receptor Purinérgico P1/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Receptores Acoplados a Proteínas G/genética , Receptores Purinérgicos P1/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The influence of adenosine receptors on blood pressure in salt-sensitive hypertension is unknown. Here, we examined the effects of salt diets on arterial blood pressures (radiotelemetry) in female and male Dahl salt-sensitive wild-type versus female and male Dahl salt-sensitive A1, A2A, or A2B receptor knockouts (A1KOs, A2AKOs, and A2BKOs, respectively). At baseline, all rats were on a 0.3% salt diet; then separate groups were switched to either 4% or 8% salt diet for 2 weeks. Compared with wild-types, baseline pressures were not affected by knockout of A1 or A2B receptors; yet, mean, systolic, and diastolic pressures were significantly (P<0.01) higher in A2AKOs versus wild-types, an effect independent of sex. During the second week on a 4% salt diet, mean, systolic, and diastolic blood pressures (mm Hg, mean±SEM) in female A1KOs (176±5, 209±5, and 147±4, respectively) and A2BKOs (166±8, 198±9, and 139±8, respectively) were significantly lower (P<0.001) than wild-type on a 4% salt diet (202±4, 240±5, and 172±3, respectively). Male A1KOs and A2BKOs were not protected against 4% salt diet-induced hypertension. This female advantage was overwhelmed by an 8% salt diet. Female and male A2AKOs were more salt sensitive, a phenotype that was apparent in male A2AKOs on 4% and 8% salt diets and in females on 8% salt diet. Female A1KOs and A2BKOs were less susceptible to salt-induced stroke and experienced improved survival. Adenosine receptors influence blood pressure and survival in salt-sensitive rats, and the impact of deleting adenosine receptors on blood pressure and survival depends on salt diet and sex.
Assuntos
Pressão Sanguínea/fisiologia , Regulação da Expressão Gênica , RNA/genética , Receptores Purinérgicos P1/genética , Cloreto de Sódio na Dieta/farmacologia , Animais , Dieta Hipossódica , Modelos Animais de Doenças , Feminino , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Ratos , Ratos Endogâmicos Dahl , Receptores Purinérgicos P1/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Angiogenesis, formation of new blood vessels is an important process involved in neovascular diseases and tumor progression. Understanding and defining novel therapeutic targets of neovascular diseases like retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration have been hindered by a lack of appropriate animal models. Zebrafish provides an excellent vertebrate model to study above disorders since its circulatory system and retinal layers are similar to mammals. Adenosine is a known mediator of angiogenesis in hypoxic condition and adenosine receptor antagonists such as theophylline, theobromine are known to exert antiangiogenic properties. We evaluated the anti-angiogenic potential of a methylxanthine pentoxifylline (PTX) with various concentrations (0.1-1mM) at 50% epiboly stage (5.2 hpf) of zebrafish embryos and studied the mRNA expression of major angiogenic factors like vegfaa and its receptors under normal conditions and when treated with an adenosine analog NECA (5'-N-ethylcarboxamidoadenosine). Upregulation of adenosine receptors, hif-1α and vegfaa by NECA could possibly mimic hypoxic condition, but PTX downregulated vegfaa and other growth factors at 1mM concentration. Vegfa protein expression was also downregulated by PTX in the retina and the compound did not damage the retinal cells. Embryos treated with PTX generated abnormal phenotypic variants with poor vasculature, tail bending and developmental delay at 1mM. Survival rates, heart rate and hatching rates were also significantly lower. Targeting the vegf signaling pathway with small molecules inhibiting adenosine receptors in addition to antagonizing vegf might be a promising approach to treat neovascular diseases of the retina and also tumors.
Assuntos
Inibidores da Angiogênese/farmacologia , Modelos Animais , Neovascularização Patológica/metabolismo , Pentoxifilina/farmacologia , Receptores Purinérgicos P1/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Retina/efeitos dos fármacos , Retina/metabolismo , Peixe-ZebraRESUMO
BACKGROUND: Adenosine, a purine nucleoside plays important roles in the pathogenesis of cancer initiation and promotion via interaction with four adenosine receptors. In the present study we examined the differential expression pattern of adenosine receptors in the malignant and adjacent normal human prostate tissues. METHODS: Prostate cancer tissue samples and adjacent normal tissues were obtained from 20 patients undergoing radical prostatectomy and histopathological diagnosis was confirmed for each sample. Total RNA was extracted and reverse transcribed into cDNA and the mRNA expression levels of adenosine receptors were investigated by Taq-man real-time RT-PCR experiment. Quantitative protein analysis was done by Western blotting experiment. Moreover, the mRNA and protein expression levels of adenosine receptors were measured after androgen treatment. RESULT: Taq-man real-time RT-PCR measurements show different expression levels of adenosine receptor transcripts. A2B adenosine receptor was predominantly expressed in tumor tissues (2.4-fold) followed by significantly expression of A3 (1.6-fold) and A2A adenosine receptors (1.5-fold) compared to adjacent normal tissues. The presence of adenosine receptors at protein levels in prostate cancer tissues compared with normal tissues was shown the following rank order: A2B > A3 > A2A > A1 . Androgen receptor regulates adenosine receptors mRNA and protein expression in AR-positive LNCaP cells, which was not seen in AR-negative PC-3 cells. CONCLUSION: These results indicated for the first time, the differential mRNA expression profile and protein levels of adenosine receptors in the human prostate cancer. Interestingly, the A2B adenosine receptor followed by A3 is highly expressed in prostate tumor samples in comparison with the adjacent normal tissues. The findings support the possible key role of A2B adenosine receptor in promoting cancer cell growth and suggest that A2B may be a novel target for prostate cancer treatment.
Assuntos
Neoplasias da Próstata/metabolismo , Receptores Purinérgicos P1/biossíntese , Idoso , Western Blotting , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Purinérgicos P1/classificação , Receptores Purinérgicos P1/genéticaRESUMO
Long-term renal allograft survival has not improved despite improvements in short term outcomes. Graft loss is characterized histologically by the development of interstitial fibrosis and tubular atrophy (IFTA). Mechanisms underlying the development of IFTA are multifactorial and include ischemia-reperfusion injury (IRI). Therapeutic options to reduce IFTA include management of immunologic causes, such as rejection, but despite these efforts IFTA can still occur and leads to the inexorable destruction of the transplanted kidney. The adenosine A2B receptor (A2BR) has recently been implicated in the development of renal fibrosis. We performed an observational study to examine the mRNA expression of the adenosine receptors after renal ischemia up to the development of renal fibrosis in a mouse model of unilateral IRI. A2BR was the only adenosine receptor that showed elevated expression following ischemia until the development of renal fibrosis 4 weeks after injury. At 2 weeks after ischemia, increased expression of the fibrotic markers transforming growth factor ß and Collagen-1α was observed. Expression of hypoxia inducible factor 1α and endothelin-1, which lie downstream of A2BR activation and have been recognized to promote renal fibrosis, were also significantly up-regulated at 2 weeks after ischemia. Expression of fibrotic markers returned to baseline by 4 weeks after ischemia, indicating resolution of injury with the concurrent development of renal fibrosis and reduced renal function. Our data suggest that A2BR may be a therapeutic target in reducing the development of renal fibrosis after ischemia.
Assuntos
Regulação da Expressão Gênica , Transplante de Rim , Rim/patologia , Receptores Purinérgicos P1/genética , Traumatismo por Reperfusão/genética , Animais , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores Purinérgicos P1/biossíntese , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HomólogoRESUMO
Extracellular adenosine is an essential negative regulator of immune reactions that acts by signaling via 4 distinct adenosine receptors. We evaluated adenosine receptor expression in Lewis rats presenting with experimental autoimmune myasthenia gravis (EAMG) to determine whether the expression of adenosine receptors are changed in the development and progression of EAMG. Lymphocyte A1AR and A2AAR mRNA and protein levels from lymphocytes harvested from the lymph nodes, spleen, and peripheral blood mononuclear cells (PBMCs) of EAMG rats were decreased. A modest but not significant increase in A2BAR levels was observed in EAMG lymphocytes harvested from lymph nodes and PBMCs. No changes in A3AR expression were observed in lymphocytes harvested from lymph nodes, spleen, or PBMCs following EAMG induction. Results presented in this report showed that the expression levels and the distribution pattern of adenosine receptors were altered in EAMG lymphocytes.
Assuntos
Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/metabolismo , Receptores Purinérgicos P1/biossíntese , Animais , Autoanticorpos/sangue , Autoanticorpos/imunologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Ratos , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase em Tempo Real , Receptores Purinérgicos P1/imunologiaRESUMO
BACKGROUND: Peripheral blood mononuclear cells and isolated polymorphonuclear neutrophilis were used to evaluate gene expression studies. Unfortunately, there are many methodological problems related to these cellular models, limiting their use. The aim was to evaluate a fast and easy procedure for the extraction of total RNA from leukocytes obtained from human whole blood (WB) < 10 mL; to determine adenosine receptor (AR) mRNA expression in WB samples of normal subjects and to establish the most stable reference genes for data normalization. METHODS: mRNA expression was performed by Real-Time PCR. RESULTS: The most stably expressed genes were TPT1, EEF1A, and RPL13A. Similar levels of mRNA expression were observed for A2aR, A2bR, and A3R while lower levels were measured for A1R (p = 0.02 A1R vs. A2aR; p = 0.04 A1R vs. A3R). CONCLUSIONS: Our study represents an important and useful starting point for future investigations devoted to evaluate the expression of ARs in human diseases.
Assuntos
Leucócitos/fisiologia , RNA Mensageiro/sangue , Receptores Purinérgicos P1/genética , Adulto , Análise de Variância , Animais , Eletroforese em Gel de Ágar , Marcadores Genéticos/genética , Humanos , Leucócitos/química , Leucócitos/metabolismo , Camundongos , RNA Mensageiro/biossíntese , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Purinérgicos P1/biossíntese , Receptores Purinérgicos P1/sangue , Valores de Referência , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
A(1) adenosine receptors (A1AR) are required for the modulation of afferent arteriolar tone by changes in luminal NaCl concentration implying that extracellular adenosine concentrations need to change in synchrony with NaCl. The present experiments were performed in mice with a null mutation in the gene for the major equilibrative nucleoside transporter ENT1 to test whether interference with adenosine disposition by cellular uptake of adenosine may modify TGF characteristics. Responses of stop flow pressure (P(SF)) to maximum flow stimulation were measured in mice with either C57Bl/6 or SWR/J genetic backgrounds. Maximum flow stimulation reduced P(SF) in ENT1(-/-) compared with wild-type (WT) mice by 1.6 ± 0.4 mmHg (n = 28) and 5.8 ± 1.1 mmHg (n = 17; P < 0.001) in C57Bl/6 and by 1.4 ± 0.4 mmHg (n = 15) and 9 ± 1.5 mmHg (n = 9; P < 0.001) in SWR/J. Plasma concentrations of adenosine and inosine were markedly higher in ENT1(-/-) than WT mice (ado: 1,179 ± 78 and 225 ± 48 pmol/ml; ino: 179 ± 24 and 47.5 ± 9 pmol/ml). Renal mRNA expressions of the four adenosine receptors, ENT2, and adenosine deaminase were not significantly different between WT and ENT1(-/-) mice. No significant differences of glomerular filtration rate or mean arterial blood pressure were found while plasma renin concentration, and heart rates were significantly lower in ENT1(-/-) animals. In conclusion, TGF responsiveness is significantly attenuated in the absence of ENT1, pointing to a role of nucleoside transport in the NaCl-synchronous changes of extracellular adenosine levels in the juxtaglomerular apparatus interstitium.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/fisiologia , Deleção de Genes , Túbulos Renais/fisiologia , Adenosina/sangue , Adenosina Desaminase/biossíntese , Animais , Pressão Arterial/genética , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/biossíntese , Feminino , Taxa de Filtração Glomerular/genética , Frequência Cardíaca/genética , Inosina/sangue , Glomérulos Renais/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Purinérgicos P1/biossíntese , Renina/sangueRESUMO
Ectonucleoside triphosphate diphosphohydrolase-1 hydrolyzes extracellular ATP and ADP to AMP. Previously, we showed that CD39 is expressed at several sites within the kidney and thus may impact the availability of type 2 purinergic receptor (P2-R) ligands. Because P2-Rs appear to regulate urinary concentrating ability, we have evaluated renal water handling in transgenic mice (TG) globally overexpressing hCD39. Under basal conditions, TG mice exhibited significantly impaired urinary concentration and decreased protein abundance of AQP2 in the kidney compared with wild-type (WT) mice. Urinary excretion of total nitrates/nitrites was significantly higher in TG mice, but the excretion of AVP or PGE(2) was equivalent to control WT mice. There were no significant differences in electrolyte-free water clearance or fractional excretion of sodium. Under stable hydrated conditions (gelled diet feeding), the differences between the WT and TG mice were negated, but the decrease in urine osmolality persisted. When water deprived, TG mice failed to adequately concentrate urine and exhibited impaired AVP responses. However, the increases in urinary osmolalities in response to subacute dDAVP or chronic AVP treatment were similar in TG and WT mice. These observations suggest that TG mice have impaired urinary concentrating ability despite normal AVP levels. We also note impaired AVP release in response to water deprivation but that TG kidneys are responsive to exogenous dDAVP or AVP. We infer that heightened nucleotide scavenging by increased levels of CD39 altered the release of endogenous AVP in response to dehydration. We propose that ectonucleotidases and modulated purinergic signaling impact urinary concentration and indicate potential utility of targeted therapy for the treatment of water balance disorders.
Assuntos
Antígenos CD/biossíntese , Apirase/biossíntese , Água/metabolismo , Animais , Antígenos CD/genética , Apirase/genética , Western Blotting , Primers do DNA , Desamino Arginina Vasopressina/farmacologia , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Capacidade de Concentração Renal/efeitos dos fármacos , Capacidade de Concentração Renal/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nucleotidases/metabolismo , Concentração Osmolar , Reação em Cadeia da Polimerase em Tempo Real , Receptores Purinérgicos P1/biossíntese , Receptores Purinérgicos P2Y/biossíntese , Fármacos Renais/farmacologia , Privação de ÁguaRESUMO
Chronic heart failure (HF) is associated with increased systemic (plasma) and reduced local (myocardial) adenosine levels. The final biological action of adenosine in a particular organ or cell population may depend on the relative degree of expression and signaling efficiency of individual adenosine receptor (AR) subtypes. The aim of this study was to determine the myocardial expression of ARs, in the different chambers of failing versus normal minipig hearts. Cardiac tissue was collected from minipigs without (n = 5) and with HF (n = 5). ARs, adenosine deaminase, and tumor necrosis factor-α (TNF-α) mRNA expression were evaluated by real time-polymerase chain reaction. ARs were expressed in all cardiac regions. After 3 weeks of pacing, the only significant change was observed in A2BR mRNA expression in the left ventricle (P = 0.02), with a similar trend for A3R, A2AR, and A1R. A trend toward higher expression of mRNA adenosine deaminase in the myocardium of pigs with HF was observed. TNF-α mRNA expression was higher after HF in all cardiac chambers (left ventricle: P = 0.009), and a significant correlation was observed between TNF-α and A2BR (r = 0.80, P < 0.0001). In this study, A2BR mRNA resulted in being overexpressed in the left ventricle of pigs with HF as well as TNF-α expression possibly testifying a link between AR expression, inflammation, and HF.
Assuntos
Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/biossíntese , Receptores Purinérgicos P1/biossíntese , Animais , Doença Crônica , Colágeno/metabolismo , Coração/diagnóstico por imagem , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/fisiopatologia , Hemodinâmica/fisiologia , Imagem Cinética por Ressonância Magnética , Masculino , Miocárdio/patologia , Tomografia por Emissão de Pósitrons , Reação em Cadeia da Polimerase em Tempo Real , Receptores Purinérgicos P1/genética , Suínos , Porco MiniaturaRESUMO
INTRODUCTION: Many aspects of skeletal muscle regeneration are now considered to be controlled by the innate immune system, specifically macrophages, but the mechanisms for activation and modulation of the innate immune system during injury are not well understood. METHODS: We analyzed the expression of toll-like receptors (TLRs) and adenosine receptors during traumatic skeletal muscle injury. mRNA expression and immunostaining of these receptors were evaluated in mouse skeletal muscle injured by freezing. RESULTS: Expression of nearly all mammalian TLRs was induced at 1 and/or 3 days postinjury with a common trend for higher expression at day 3. Injury also elicited a dramatic increase in the expression of adenosine receptors A(2B) and A(3) but not A(1) and A(2A) . CONCLUSIONS: Both receptor types may be potential targets for stimulation of skeletal muscle tissue regeneration and functional restoration after injury.
Assuntos
Regulação da Expressão Gênica , Músculo Esquelético/lesões , Músculo Esquelético/fisiologia , Receptores Purinérgicos P1/biossíntese , Regeneração/fisiologia , Receptores Toll-Like/biossíntese , Animais , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND AND PURPOSE: Adenosine is a major endogenous regulator of macrophage function, and activates four specific adenosine receptors (A(1), A(2A), A(2B) and A(3)). Here, we have assessed in human lung macrophages the modulation of the expression of adenosine receptor mRNA by lipopolysaccharide (LPS), and the relative contributions of the different adenosine receptors to LPS-induced production of tumour necrosis factor (TNF)-alpha and chemokines. EXPERIMENTAL APPROACH: Lung macrophages isolated from resected lungs were stimulated with LPS and treated with adenosine receptor agonists or/and antagonists. Adenosine receptor expression was assessed with qRT-PCR. Cytokines were measured in lung macrophage supernatants with elisa. KEY RESULTS: LPS increased (about 400-fold) mRNA for A(2A) adenosine receptors, decreased mRNA for A(1) and A(2B), but had no effect on A(3) adenosine receptor mRNA. The adenosine receptor agonist NECA inhibited TNF-alpha production concentration dependently, whereas the A(1) receptor agonist, CCPA, and the A(3) receptor agonist, AB-MECA, inhibited TNF-alpha production only at concentrations affecting A(2A) receptors. NECA also inhibited the production of CCL chemokines (CCL2, CCL3, CCL4, CCL5) and CXCL chemokines (CXCL9 and CXCL10), but not that of CXCL1, CXCL8 and CXCL5. Reversal of NECA-induced inhibition of TNF-alpha and chemokine production by the selective A(2A) adenosine receptor antagonist ZM 241385, but not the A(2B) receptor antagonist, MRS 1754, or the A(3) receptor antagonist, MRS 1220, indicated involvement of A(2A) receptors. CONCLUSIONS AND IMPLICATIONS: LPS up-regulated A(2A) adenosine receptor gene transcription, and this receptor subtype mediated inhibition of the LPS-induced production of TNF-alpha and of a subset of chemokines in human lung macrophages.
Assuntos
Quimiocinas/biossíntese , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Receptores Purinérgicos P1/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Acetamidas/farmacologia , Células Cultivadas , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Pulmão/citologia , Macrófagos Alveolares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Subunidades Proteicas , Agonistas do Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , Purinas/farmacologia , Quinazolinas/farmacologia , Receptores Purinérgicos P1/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triazinas/farmacologia , Triazóis/farmacologiaRESUMO
Hyperglycemia-induced alterations of adenosine receptors (ARs) expression are implicated in the pathomechanism leading to impaired function of the lymphocytes in diabetes. However, the signaling pathways utilized by glucose to regulate ARs expression are unknown. This work was undertaken to investigate the impact of high glucose level on the ARs expression in rat B lymphocytes. The results presented in this report demonstrate that rat B lymphocytes express all four types of ARs at the mRNA and protein level. Exposing B cells to high glucose (25 mM) suppressed the expression of A(1)-AR, A(2B)-AR, and A(3)-AR, but had no effect on the expression of A(2)A-AR. A selective inhibitor of Ca(2+)-dependent protein kinase C (PKC) isoforms suppressed the high glucose effect on A(1)-AR expression. Inhibition of PKC-delta with rottlerin blocked the high glucose effect on A(1)-AR mRNA level. An inhibitor of Raf-1 kinase completely blocked the high glucose effect on A(2B)-AR expression. The suppression of A(1)-AR and A(2B)-AR mRNA expression induced by high glucose was blocked by an inhibitor (PD98059) of MAPK kinase (MEK). In conclusion, high glucose utilizes a signaling pathway involving some elements of the MAPK pathway and different PKC isoforms to suppress the expression of A(1)-AR, A(2B)-AR, and A(3)-AR in rat B lymphocytes.
Assuntos
Linfócitos B/efeitos dos fármacos , Glucose/farmacologia , Proteína Quinase C/fisiologia , Receptores Purinérgicos P1/biossíntese , Animais , Linfócitos B/enzimologia , Linfócitos B/metabolismo , Western Blotting , Técnicas de Cultura de Células , Células Cultivadas , Relação Dose-Resposta a Droga , Masculino , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/imunologia , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos WistarRESUMO
Transgenic mice that express human equilibrative nucleoside transporter subtype 1 (hENT1) under the control of a neuron-specific enolase promoter have been generated. Southern blot and PCR revealed the presence of the transgene in five founder mice. Mice from each founder line were examined by reverse transcriptase (RT)-PCR and found to express hENT1 in RNA isolated from whole brain, cerebral cortex, striatum, hippocampus, and cerebellum but not liver, kidney, heart, lung or skeletal muscle. Cortical synaptosomes prepared from transgenic mice had significantly increased [(3)H]adenosine uptake and [(3)H]nitrobenzylthioinosine binding, relative to samples from wild-type mice. In behavioral tests, transgenic mice had altered responses to caffeine and ethanol, two drugs that inhibit and enhance, respectively, adenosine receptor activity. Caffeine-induced locomotor stimulation was attenuated whereas the hypnotic effect of ethanol was enhanced in transgenic mice. Caffeine was more potent in inhibiting ethanol-induced motor incoordination in wild-type than in transgenic mice. No differences in expression of mouse genes for adenosine receptors, nucleoside transporters, or purine metabolizing enzymes were detected by RT-PCR analyses. These data indicate that expression of hENT1 in neurons does not trigger adaptive changes in expression of adenosine-related genes. Instead, hENT1 expression affects dynamic changes in endogenous adenosine levels, as revealed by altered behavioral responses to drugs that affect adenosine receptor signalling.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/biossíntese , Transportador Equilibrativo 1 de Nucleosídeo/genética , Regulação da Expressão Gênica/fisiologia , Neurônios/metabolismo , Adenosina/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Cafeína/administração & dosagem , Cafeína/antagonistas & inibidores , Transportador Equilibrativo 1 de Nucleosídeo/fisiologia , Etanol/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Células PC12 , Ratos , Receptores Purinérgicos P1/biossíntese , Receptores Purinérgicos P1/genética , Regulação para Cima/genéticaRESUMO
Adenosine mediates Na+ reabsorption in the proximal tubule (PT) and other segments by activating adenosine type 1 receptors (A1-AR). We tested the hypothesis that A1-AR in the PT is regulated by salt intake and participates in the kidney adaptation to changes in salt intake. Absolute fluid reabsorption (Jv) was measured by direct in vivo microperfusion and recollection in rats maintained on low (LS; 0.03% Na, wt/wt)-, normal (NS; 0.3% Na)-, and high-salt (HS; 3.0% Na) diets for 1 wk. The effect of microperfusion of BG9719 a highly selective inhibitor of A1-ARs or adenosine deaminase (AD), which metabolizes adenosine, was measured in each group. Jv was higher in PT from LS rats (LA: 2.8 +/- 0.2 vs. NS: 2.1 +/- 0.2 nl.min(-1).mm(-1), P < 0.001). Jv in HS rats was not different from NS. BG9719 reduced Jv in LS rats by 66 +/- 6% (LS: 2.8 +/- 0.2 vs LS+CVT: 1.3 +/- 0.3 nl.min(-1).mm(-1), P < 0.001), which was greater than its effect in NS (45 +/- 4%) or HS (41 +/- 4%) rats. AD reduced Jv similarly, suggesting that A1-ARs are activated by local production of adenosine. Expression of A1-AR mRNA and protein was higher (P < 0.01) in microdissected PTs in LS rats compared with NS and HS. We conclude that A1-ARs in the PT are increased by low salt intake and that A1-AR participates in the increased PT reabsorption of solute and fluid in response to low salt intake.
Assuntos
Túbulos Renais Proximais/fisiologia , Receptores Purinérgicos P1/fisiologia , Cloreto de Sódio na Dieta/administração & dosagem , Animais , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P1/biossíntese , Regulação para Cima , Equilíbrio Hidroeletrolítico/fisiologia , Xantinas/farmacologiaRESUMO
L-Arginine increases myocardial nitric oxide production. Nitric oxide mediates many of the cardiovascular actions of adenosine and modulates adenosine metabolism. In this study, we examined the effect of chronic L-arginine (5%) intake on cardiac nitric oxide synthase (NOS) and adenosine receptor expression and cardiac function in rat Langendorff-isolated perfused hearts. Our results show that 4-week chronic l-arginine ingestion increases the weight of rat hearts by 17.6% (P < 0.05). L-Arginine treatment decreased the expression of all the cardiac adenosine receptors, with reductions in adenosine A(1) (20-fold), A(2A) (7.7-fold), A(2B) (76-fold) and A(3) (25.6-fold) mRNA (P < 0.05). NOS expression was variably affected with no change in the expression of NOS(1) and 4.2-fold down-regulation of NOS(3) expression with chronic L-arginine treatment (P < 0.05). NOS(2) was expressed in control tissues; however, in L-arginine-treated hearts the amount of NOS(2) mRNA was reduced to non-detectable levels. Following chronic L-arginine treatment, an increase in coronary perfusion pressure was observed (P < 0.05). Purine efflux was used as an indicator of metabolic efficiency. L-Arginine did not alter catecholamine-induced purine efflux (P > 0.05); however, noradrenaline-mediated increases in contractility and myocardial oxygen consumption were reduced. Vasodilator responses to 5'-N-ethylcarboxamidoadenosine (NECA) were reduced in hearts from l-arginine-treated rats and the NOS inhibitor N omega-nitro-L-arginine methyl ester (3 microM) did not inhibit responses to NECA. In conclusion, 4-week dietary supplementation of L-arginine reduced the expression of cardiac adenosine receptors and NOSs with a subsequent decrease in noradrenaline-stimulated cardiac function and adenosine receptor-mediated coronary vasodilation.
Assuntos
Arginina/farmacologia , Miocárdio/metabolismo , Óxido Nítrico Sintase/biossíntese , Receptores Purinérgicos P1/biossíntese , Animais , Arginina/administração & dosagem , Suplementos Nutricionais , Regulação para Baixo , Masculino , Ratos , Ratos WistarRESUMO
The in vitro differentiation of P19 murine embryonal carcinoma cells to neurons resembles developmental stages which are encountered during neuronal development. Three days following induction to neuronal differentiation by retinoic acid, most cells of the P19 population lost expression of the stage specific embryonic antigen (SSEA-1) and expressed the neural progenitor cell specific antigen nestin. Beginning from day 4 of differentiation nestin expression was down-regulated, and expression of neuron-specific enolase as marker of differentiated neurons increased. The molecular mechanisms underlying neuronal differentiation are poorly understood. We have characterized the participation of purinergic ionotropic (P2X) and metabotropic (P2Y) receptors at mRNA transcription and protein levels as well as ATP-induced Ca2+ transients during neuronal differentiation of P19 cells. Gene and protein expression of P2X2, P2X6, P2Y2, and P2Y6 receptors increased during the course of differentiation, whereas P2X3, P2X4, P2Y1 and P2Y4 receptor expression was high in embryonic P19 cells and then decreased following induction of P19 cells to differentiation. P2X1 receptor protein expression was only detected on days 2 and 4 of differentiation. Although P2X5 and P2X7 mRNA transcription was present, no protein expression for this receptor subunit could be detected throughout the differentiation process. In undifferentiated cells, mainly ionotropic P2X receptors contributed to the ATP-induced Ca2+-response. In neuronal-differentiated P19 cells, the ATP-induced Ca2+-response was increased and the metabotropic component predominated. Purinergic receptor function is implicated to participate in neuronal maturation, as cholinergic and glutamate-N-methyl-D-aspartate (NMDA) induced calcium responses were affected when cells were differentiated in the presence of purinergic receptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin or reactive blue-2. Our data suggest that inhibition of P2Y1 and possibly P2X2 receptors led to a loss of NMDA receptor activity whereas blockade of possibly P2X2 and P2Y2 purinergic receptors during neuronal differentiation of P19 mouse led to inhibition of cholinergic receptor responses.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Neoplásicas/metabolismo , Neurônios/metabolismo , Receptores Colinérgicos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Purinérgicos/biossíntese , Animais , Western Blotting , Linhagem Celular , Células-Tronco de Carcinoma Embrionário , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Antagonistas Purinérgicos , Antagonistas de Receptores Purinérgicos P1 , Antagonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Receptores Muscarínicos/biossíntese , Receptores Muscarínicos/genética , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Receptores Purinérgicos/genética , Receptores Purinérgicos P1/biossíntese , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P2/biossíntese , Receptores Purinérgicos P2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suramina/farmacologia , Triazinas/farmacologiaRESUMO
CD73 (ecto-5'-nucleotidase) on human gingival fibroblasts plays a role in the regulation of intracellular cAMP levels through the generation of adenosine, which subsequently activates adenosine receptors. In this study, we examined the involvement of ecto-adenosine deaminase, which can be anchored to CD26 on human gingival fibroblasts, in metabolizing adenosine generated by CD73, and thus attenuating adenosine receptor activation. Ecto-adenosine deaminase expression on fibroblasts could be increased by pre-treatment with a lysate of Jurkat cells, a cell line rich in cytoplasmic adenosine deaminase. Interestingly, the cAMP response to adenosine generated from 5'-AMP via CD73 and the ability of 5'-AMP to induce hyaluronan synthase 1 mRNA were significantly decreased by the pre-treatment of fibroblasts with Jurkat cell lysate. This inhibitory effect was reversed by the specific adenosine deaminase inhibitor. These results suggest that ecto-adenosine deaminase metabolizes CD73-generated adenosine and regulates adenosine receptor activation.
Assuntos
Adenosina Desaminase/metabolismo , Gengiva/enzimologia , Receptores Purinérgicos P1/biossíntese , 5'-Nucleotidase/metabolismo , Adenosina/biossíntese , Adenosina/metabolismo , Adenosina Desaminase/biossíntese , Adolescente , Células Cultivadas , Criança , AMP Cíclico/metabolismo , Dipeptidil Peptidase 4/biossíntese , Feminino , Fibroblastos/enzimologia , Fibroblastos/microbiologia , Gengiva/citologia , Glucuronosiltransferase/biossíntese , Humanos , Hialuronan Sintases , MasculinoRESUMO
The proliferative response of T lymphocytes is a crucial step in cell-mediated immunity. This study was undertaken to investigate the mechanisms leading to the impaired proliferative response of diabetic T lymphocytes. T cells that had been isolated from the spleen of normal rats and cultured in medium containing 20 mm glucose and no insulin displayed the same degree of proliferative impairment as cells isolated from diabetic rats. The rate of T-cell proliferation, when induced with concanavalin A or anti-CD3 and anti-CD28 antibodies, was not affected by the inhibition of nucleoside transporters. T cells cultured at high glucose concentrations in the absence of insulin displayed decreased expression of adenosine kinase, and released measurable extracellular quantities of adenosine. Under resting conditions, the level of cAMP was 5.9-fold higher in these cells compared to cells grown in low glucose and in the presence of insulin. Experiments with specific adenosine receptor agonists and antagonists showed that adenosine-induced suppression of diabetic T cell proliferation was mediated by the A2A adenosine receptor, but not by the A2B receptor. Treatment of diabetic T cells with 10 microm H-89, a specific protein kinase A inhibitor, restored T-cell proliferation. These results show that suppressed proliferation of diabetic T lymphocytes is evoked by the decreased expression of adenosine kinase, leading to the outflow of adenosine from the cell. Extracellular adenosine then stimulates the A2A receptor and induces cAMP production, leading to the activation of protein kinase A, and suppression of T-cell proliferation.