RESUMO
CGS 35601 is a triple vasopeptidase inhibitor (VPI) of angiotensin converting enzyme (ACE), neutral endopeptidase (NEP), and endothelin (ET) converting enzyme-1 (ECE-1), with respective IC(50) values of 22, 2, and 55 nM. The aim of the present study was to establish the hemodynamic profile of Zucker diabetic fatty (Zdf)-Fatty rats, a high-fat diet gene-prone model developing spontaneous Type 2 diabetes (T2D) and the effects of CGS 35601. Male Zdf-Fatty (14 weeks, n = 17-23), Zdf-Lean (14 weeks, n = 8-10), and Wistar (14 weeks, n = 9-10) rats on distinct diets were implanted with a catheter in the left carotid and placed individually in a metabolic cage for 30 days. The hemodynamic profile and some metabolic biomarkers were assessed daily. After a 7-day stabilization period, the Zdf-Fatty rats were divided into two groups: Group 1, controls (n = 7-10) receiving vehicle-saline (250 microl/hr) and Group 2, (n = 10-13) receiving increasing doses of CGS 35601 (0.1, 1, and 5 mg/kg/day x 6 days each, intra-arterially) followed by a 5-day washout period. Mean arterial blood pressure (MABP) of young Zdf-Fatty rats was compared with age-matched Zdf-Lean and Wistar rats, which were found similar. MABP decreased by 5.9% (from baseline at 102 +/- 5 to 96 +/- 4 mmHg), 12.7% (to 89 +/- 6 mmHg) and 21.6% (to 80 +/- 4 mmHg), at 0.1, 1, and 5 mg/kg/day, respectively, in CGS 35601-treated Zdf-Fatty rats. Systolic and diastolic blood pressures were similarly reduced. The heart rate was not affected. Hyperglycemic status and insulin-resistance were not modulated by short-term treatment. CGS 35601 presented an excellent short-term safety profile. This novel molecule and class of VPI may be of interest for lowering vascular tone. Further long-term studies, once cardiovascular and renal complications have developed in this T2D rat model are warranted to define the efficacy of this class of VPI.
Assuntos
Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/metabolismo , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Animais , Ácido Aspártico Endopeptidases/administração & dosagem , Ácido Aspártico Endopeptidases/farmacologia , Combinação de Medicamentos , Enzimas Conversoras de Endotelina , Indóis/administração & dosagem , Indóis/agonistas , Masculino , Metaloendopeptidases/administração & dosagem , Metaloendopeptidases/farmacologia , Neprilisina/administração & dosagem , Neprilisina/farmacologia , Peptidil Dipeptidase A/administração & dosagem , Peptidil Dipeptidase A/farmacologia , Ratos , Ratos Wistar , Ratos ZuckerRESUMO
We previously reported that CGS 35601, a potent triple inhibitor of angiotensin-converting enzyme, neutral endopeptidase, and endothelin-converting enzyme 1, completely normalized mean arterial blood pressure (MABP) in 36-week-old spontaneously hypertensive rats, a normal renin model. The aim of the present study was to determine the effects of this triple vasopeptidase inhibitor (VPI) on the hemodynamic profile of instrumented, conscious, and unrestrained Dahl salt-sensitive (DSS) rats, a gene-prone, high-salt diet-induced low-renin hypertension model. Male DSS rats (mean weight [+/-SEM], 385 +/- 10 g) were fed a normal diet (Group 1) or a high-salt diet (Groups 2 and 3; 8% NaCl in food) for 6 weeks and then instrumented with a carotid catheter and placed individually in metabolic cages for 30 days. The hemodynamic, hematological, and biochemical profiles were assessed daily. Dose-dependent treatment started after a 7-day stabilization period in Groups 1 and 2 (vehicle dosage, 250 microl/hr) and Group 3 (CGS 35601 dosages of 0.1, 1, and 5 mg/kg/day for 6 days per dose by means of constant intra-arterial infusion), followed by a 5-day washout period. Two additional groups included normotensive Wistar rats (Group 4) and DSS rats that received a double high-salt solid (8% NaCl) and liquid (1% NaCl) diet (Group 5). The MABP in rats receiving CGS 35601 decreased in a dose-dependent fashion toward the baseline level observed in DSS rats receiving a normal diet. The heart rate was unaffected. The hemodynamic profile returned to normal during the washout period. This novel triple VPI is a potent and effective antihypertensive agent with a safe short-term profile that may be of interest for treating hypertension and other cardiovascular diseases. Other hypertensive rat models are being tested.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Renina/sangue , Sódio na Dieta/farmacologia , Animais , Ácido Aspártico Endopeptidases/administração & dosagem , Ácido Aspártico Endopeptidases/farmacologia , Combinação de Medicamentos , Enzimas Conversoras de Endotelina , Masculino , Metaloendopeptidases/administração & dosagem , Metaloendopeptidases/farmacologia , Neprilisina/administração & dosagem , Neprilisina/farmacologia , Peptidil Dipeptidase A/administração & dosagem , Peptidil Dipeptidase A/farmacologia , Ratos , Ratos Endogâmicos DahlRESUMO
CGS 35601 is a triple vasopeptidase inhibitor (VPI) of angiotensin-converting enzyme, neutral endopeptidase, and endothelin-converting enzyme-1 with respective IC50 values of 22, 2, and 55 nM. We characterized the safety profile and toxicity of escalating doses of CGS 35601 over a 20-day period in chronically instrumented, unrestrained, conscious, male, spontaneously hypertensive rats (SHR). Once instrumented with an arterial catheter, the SHR were placed in metabolic cages allowing daily assessment of hemodynamics and blood sampling for biochemical and hematological measurements. After a 7-day stabilization period, the SHR were divided into 2 groups: Gr. 1, (n = 13 to 18) receiving CGS 35601 at 0.01, 0.1, 1 and 5 mg kg(-1) day(-1) (continuous i.a. infusion) for 5 consecutive days/dose, followed by a 5-day washout; and Gr. 2, (n = 10) receiving vehicle (saline). The highest dose of CGS 35601 dose-dependently reduced MABP from 156 +/- 4 up to 94 +/- 5 mm Hg, whereas heart rate, metabolic, electrolytic, and hematological profiles, growth, diuresis, and renal activity were unaffected, and no hepatic or liver toxicities were observed. These results suggest that this novel triple VPI presents no safety concerns at this stage and may become of interest for the treatment of hypertension and other cardiovascular disorders. Long-term chronic experiments are needed to assess possible angioedema and increases in vascular permeability.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Hipertensão/tratamento farmacológico , Indóis/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Neprilisina/antagonistas & inibidores , Inibidores da Enzima Conversora de Angiotensina/toxicidade , Animais , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Enzimas Conversoras de Endotelina , Frequência Cardíaca/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Indóis/toxicidade , Masculino , Estrutura Molecular , Ratos , Ratos Endogâmicos SHRRESUMO
The aim of the present study was to assess several biochemical and physiological endpoint parameters alongside controlled hemorrhagic and recovery phases of chronically instrumented, conscious and unrestrained healthy rats. Male Sprague-Dawley rats (12-14 weeks; 430+/-20 g; n=22-18) were instrumented with a saline-perfused femoral arterial catheter and placed individually in a metabolic cage for up to 20 days, allowing instant assessments of the hemodynamic profile and blood and urine sampling for hematological profile and biochemical measurements to assess hepatic, renal and metabolic functions. In addition, body weight, food and water intake, and diuresis were monitored daily. After a 7-day stabilization period, the rats underwent severe and acute hemorrhagic shock (HS) (removal of 50% of total circulating blood volume), kept in hypovolemic shock for an ischemic period of 50 min and then resuscitated over 10 min. Gr. 1 was re-infused with autologous shed blood (AB; n=10) whereas Gr. 2 was infused 1:1 with a solution of sterile saline-albumin (SA; 7% w/v) (n=8-12). Ischemic rats recovered much more rapidly following AB re-infusion than those receiving SA. Normal hemodynamic and biochemical profiles were re-established after 24 h. Depressed blood pressure lasted 4-5 days in SA rats. The hematological profile in the SA resuscitated rats was even more drastically affected. Circulating plasma concentrations of hemoglobin (-40%), hematocrit (-50%), RBC (-40%) and platelets (-41%) counts were still severely decreased 24 h after the acute ischemic event whereas WBC counts increased 2.2-fold by day 4. It took 5-9 days for these profiles to normalize after ischemia-reperfusion with SA. Diuresis increased in both groups (by 45+/-7% on day 1) but presented distinct electrolytic profiles. Hepatic and renal functions were normal in AB rats whereas altered in SA rats. The present set of experiments enabled us to validate a model of HS in conscious rats and the use of an integrated in vivo platform as a valuable tool to characterize HS-induced stress and to test new classes of blood substitutes in real time, post-event, over days.
Assuntos
Substitutos Sanguíneos/uso terapêutico , Hemodinâmica/efeitos dos fármacos , Choque/tratamento farmacológico , Choque/fisiopatologia , Doença Aguda , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/fisiopatologia , Animais , Biomarcadores , Proteínas Sanguíneas/análise , Modelos Animais de Doenças , Eletrólitos/sangue , Eletrólitos/urina , Testes de Função Renal , Lipídeos/sangue , Falência Hepática Aguda/metabolismo , Falência Hepática Aguda/fisiopatologia , Testes de Função Hepática , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Choque/metabolismo , TelemetriaRESUMO
BACKGROUND: PGD(2) is the major prostanoid released by mast cells during an allergic response. Its role in the allergic response, however, remains unclear. OBJECTIVE: Because the accumulation of eosinophils is a feature of allergic reactions, we investigated the role of PGD(2) in the modulation of eosinophil function. METHODS: Circulating human eosinophils were isolated and challenged with PGD(2). The effects of PGD(2) on various eosinophil functions were then analyzed. RESULTS: PGD(2) binds with high affinity preferentially to 2 receptors, DP and chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH2). We show that both DP and CRTH2 are detectable on circulating eosinophils. We demonstrate that PGD(2) (1-10 nmol/L) induces a rapid change in human eosinophil morphology and an increase in chemokinesis and promotes eosinophil degranulation. These effects are induced by the CRTH2-selective agonist 13-14-dihydro-15-keto-PGD(2) (DK-PGD(2)) but not by the DP-selective agonist BW245C. These results suggest a role for CRTH2 in the modulation of eosinophil movement and in triggering the release of cytotoxic proteins. Finally, we demonstrate that BW245C, but not DK-PGD(2), can delay the onset of apoptosis in cultured eosinophils, presumably through interaction with DP. CONCLUSION: These data support the hypothesis that PGD(2) controls eosinophil functions through 2 pharmacologically distinct receptors with independent functions. Blockade of PGD(2)-mediated effects on human eosinophils may reduce the damage caused by these cells during an allergic response, but inhibition of both receptors may be required.
Assuntos
Apoptose , Eosinófilos/fisiologia , Receptores Imunológicos/fisiologia , Receptores de Prostaglandina/fisiologia , Degranulação Celular/efeitos dos fármacos , Movimento Celular , Células Cultivadas , Eosinófilos/efeitos dos fármacos , Eosinófilos/patologia , Humanos , Prostaglandina D2/farmacologia , RNA Mensageiro/análise , Receptores Imunológicos/genética , Receptores de Prostaglandina/genéticaRESUMO
Pre-clinical and clinical studies are currently underway to evaluate the potential of phosphodiesterase-4 (PDE4) inhibitors for the treatment of chronic obstructive pulmonary disease and other inflammatory conditions of the airways. The most common side effect associated with this class of compounds is emesis. The squirrel monkey provides a model for evaluating the efficacy of PDE4 inhibitors and their emetic potential. The distribution of three PDE4 isoforms (A, C and D) has been investigated in the squirrel monkey medulla and nodose ganglion to determine which isoform(s) could be responsible for the emetic adverse effects. The distribution of PDE4 isoforms was delineated using immunohistochemistry with antibodies specific for PDE4A, PDE4C and PDE4D and by in situ hybridization with isoform-selective riboprobes. PDE4A was present in the medulla where expression was mostly restricted to glial cells and the vasculature. PDE4C was not detected in either the medulla or nodose ganglion. Finally, the PDE4D isoform was localized to neurons in the nodose ganglion and found through many structures of medulla including the area postrema, neurons of the nucleus tractus solitarius and locus coeruleus. These data are consistent with a role for PDE4D in the emetic response.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Bulbo/enzimologia , Gânglio Nodoso/enzimologia , Animais , Sequência de Bases , Western Blotting , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Feminino , Imuno-Histoquímica , Hibridização In Situ , Isoenzimas/metabolismo , Masculino , Microscopia de Fluorescência , Dados de Sequência Molecular , Sondas RNA , Reflexo/fisiologia , Saimiri , Substância P/metabolismo , Vômito/fisiopatologiaRESUMO
The action of prostaglandin E(2) (PGE(2)) in the preoptic area is thought to play an important role in producing fever. Pharmacologic evidence suggests that, among the four subtypes of E-series prostaglandin (EP) receptors, i.e., EP(1), EP(2), EP(3), and EP(4), the EP(1) receptor mediates fever responses. In contrast, evidence from mice with EP receptor gene deletions indicates that the EP(3) receptor is required for the initial (<1 hour) fever after intravenous (i.v.) lipopolysaccharide (LPS). To investigate which subtypes of EP receptors mediate systemic infection-induced fever, we assessed the coexpression of Fos-like immunoreactivity (Fos-IR) and EP(1-4) receptor mRNA in nuclei in the rat hypothalamus that have been shown to be involved in fever responses. Two hours after the administration of i.v. LPS (5 microg/kg), Fos-IR was observed in the ventromedial preoptic nucleus, the median preoptic nucleus, and the paraventricular hypothalamic nucleus. In these nuclei, EP(4) receptor mRNA was strongly expressed and the Fos-IR intensely colocalized with EP(4) receptor mRNA. Strong EP(3) receptor mRNA expression was only seen within the median preoptic nucleus but Fos-IR showed little coexpression with EP(3) receptor mRNA. EP(2) receptor mRNA was not seen in the PGE(2) sensitive parts of the preoptic area. Although approximately half of the Fos-immunoreactive neurons also expressed EP(1) receptor mRNA, EP(1) mRNA expression was weak and its distribution was so diffuse in the preoptic area that it did not represent a specific relationship. In the paraventricular nucleus, EP(4) mRNA was found in most Fos-immunoreactive neurons and levels of EP(4) receptor expression increased after i.v. LPS. Our findings indicate that neurons expressing EP(4) receptor are activated during LPS-induced fever and suggest the involvement of EP(4) receptors in the production of fever.
Assuntos
Febre/fisiopatologia , Hipotálamo/metabolismo , Lipopolissacarídeos/metabolismo , Neurônios/metabolismo , Receptores de Prostaglandina E/genética , Animais , Contagem de Células , Dinoprostona/metabolismo , Febre/patologia , Hipotálamo/patologia , Lipopolissacarídeos/farmacologia , Masculino , Neurônios/patologia , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Área Pré-Óptica/citologia , Área Pré-Óptica/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Prostaglandina E Subtipo EP1 , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP3 , Receptores de Prostaglandina E Subtipo EP4RESUMO
The inhibition of PGE(2) synthesis resulting from sustained NSAIDs therapy has been linked to gastrointestinal irritations and ulceration. The multiple physiological effects of PGE(2) in the gut are mediated through the activation of four receptors termed EP(1-4). The aim of the study was to determine the precise distribution of the four prostaglandin E(2) receptors in the rat stomach, small intestine, and colon. We used non-radioactive in situ hybridization techniques on paraffin-embedded tissue. Mucous cells of the stomach and goblet cells of the small intestine and colon were found to express mRNA for all four EP subtypes. A positive hybridization signal for EP(1), EP(3), and EP(4) was detected in the parietal cells of the stomach whereas the chief cells expressed low levels of EP(1) and EP(3). The EP(1) and EP(3) receptor mRNA could also be detected in the muscularis mucosa, longitudinal muscle and enteric ganglias of the stomach and small intestine. However, close examination of the enteric ganglias indicated that most of the positive labeling was localized to the glial cells, although some neurons did express EP(3). In conclusion, we have detailed the distribution of prostanoid EP receptors in the gut at the cellular level, giving new insights to the role of prostaglandins in gastrointestinal functions.
Assuntos
Intestino Grosso/química , Intestino Delgado/química , Receptores de Prostaglandina E/análise , Estômago/química , Animais , Hibridização In Situ , Mucosa Intestinal/química , Mucosa Intestinal/citologia , Intestino Grosso/citologia , Intestino Grosso/inervação , Intestino Delgado/citologia , Intestino Delgado/inervação , Masculino , Músculo Liso/química , Músculo Liso/citologia , RNA Complementar , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores de Prostaglandina E/genética , Estômago/citologia , Estômago/inervaçãoRESUMO
1 We characterized the regulation of cyclooxygenase-2 (COX-2) at the mRNA, protein and mediator level in two rat models of acute inflammation, carrageenan-induced paw oedema and mechanical hyperalgesia. 2 Carrageenan was injected in the hind paw of rat at low (paw oedema) and high doses (hyperalgesia). COX-2 and prostaglandin E2 (PGE2) levels were measured by RT-PCR and immunological assays. We also determined the distribution of COX-2 by immunohistochemistry. 3 The injection of carrageenan produced a significant and parallel induction of both COX-2 and PGE2. This induction was significantly higher in hyperalgesia than in paw oedema. This was probably due to the 9 fold higher concentration of carrageenan used to provoke hyperalgesia. 4 Immunohistochemical examination showed COX-2 immunoreactivity in the epidermis, skeletal muscle and inflammatory cells of rats experiencing hyperalgesia. In paw oedema however, only the epidermis showed positive COX-2 immunoreactivity. 5 Pretreatment with indomethacin completely abolished the induction of COX-2 in paw oedema but not in hyperalgesia. 6 These results suggest that multiple mechanisms regulate COX-2 induction especially in the more severe model. In carrageenan-induced paw oedema, prostanoid production have been linked through the expression of the COX-2 gene which suggest the presence of a positive feedback loop mechanism.
Assuntos
Carragenina/toxicidade , Inflamação/enzimologia , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Sequência de Bases , Ciclo-Oxigenase 2 , Primers do DNA , Dinoprostona/biossíntese , Dinoprostona/metabolismo , Indução Enzimática , Imuno-Histoquímica , Indometacina/farmacologia , Inflamação/induzido quimicamente , Isoenzimas/biossíntese , Masculino , Prostaglandina-Endoperóxido Sintases/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
To gain insight into the role of prostanoids in human kidney function, we examined the distribution of cyclooxygenase (COX) 1 and COX-2 by immunofluorescence and immunohistochemistry in human kidneys from adults of various age groups. COX-1 was detected in the collecting ducts, thin loops of Henle and portions of the renal vasculature. COX-2 was detected in the renal vasculature, medullary interstitial cells, and the macula densa. In addition, COX-2 immunoreactivity was noted in afferent arteries and the macula densa of the renal cortex and was more evident in the kidneys of older adults.
Assuntos
Idoso/fisiologia , Isoenzimas/metabolismo , Túbulos Renais Distais/citologia , Túbulos Renais Distais/enzimologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Adolescente , Adulto , Idoso de 80 Anos ou mais , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Feminino , Humanos , Isoenzimas/imunologia , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , Prostaglandina-Endoperóxido Sintases/imunologia , Valores de Referência , Coloração e Rotulagem/métodosRESUMO
We report the cloning, functional expression and cell-specific localization of the rat homologue of the prostaglandin D2 receptor (DP). In situ hybridization, utilizing multiple digoxigenin-labelled riboprobes and their complementary sense controls, was performed to determine the detailed distribution of DP receptor mRNA in the central nervous system and the gastrointestinal tract. Within the brain, the leptomeninges and choroid plexus expressed DP receptor mRNA. Transcripts detected in the spinal cord were localized to the sensory and motor neurons of the dorsal and ventral horns, respectively, suggesting a role for the DP receptor in the modulation of central nervous system processes, including pain transmission. Within the gastrointestinal tract (stomach, duodenum, ileum and colon) signals were highly localized to the mucous-secreting goblet cells and the columnar epithelium. These findings suggest a novel biological role for prostaglandin D2-mediated activity at the DP receptor, namely mucous secretion. In addition, radioligand binding assays (saturation analyses and equilibrium competition assays) and functional assays (measuring cAMP accumulation) were performed to characterize the recombinant rat DP receptor expressed in human embryonic kidney (HEK) 293(EBNA) cells. A single site of binding (K(D) = 14 nM, Bmax = 115 fmol/mg protein) was measured for prostaglandin D2-specific binding to the rat DP receptor. Prostaglandin D2 proved to be a potent agonist at the rat DP receptor (EC50 = 5 nM). The rank order of efficacy for DP receptor specific agonists [prostaglandin D2 = prostaglandin J2 = BW 245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropylhydantoin)) > L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)] reflected the affinity with which the ligands bound to the receptor.
Assuntos
Prostaglandina D2/fisiologia , Receptores Imunológicos , Receptores de Prostaglandina/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Linhagem Celular , Membrana Celular/metabolismo , Clonagem Molecular , AMP Cíclico/biossíntese , Epitopos , Expressão Gênica , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Oligopeptídeos , Peptídeos/genética , Sondas RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ensaio Radioligante , Ratos , Receptores de Prostaglandina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Several endocrine and neuronal functions are governed by the cAMP-dependent signalling pathway. In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signalling pathway is mediated by a family of cAMP-responsive nuclear factors. This family consists of a large number of members that may act as activators or repressors. These factors contain the basic domain/ leucine zipper motifs and bind as dimers to cAMP-response elements (CRE). The function of CRE-binding proteins (CREBs) is modulated by phosphorylation by several kinases. Direct activation of gene expression by CREB requires phosphorylation by the cAMP-dependent protein kinase A to the serine-133 residue. Among the repressors, ICER (Inducible cAMP Early Repressor) deserves special mention. ICER is generated from an alternative CREM promoter and constitutes the only inducible cAMP-responsive element binding protein. Furthermore, ICER negatively autoregulates the alternative promoter, thus generating a feedback loop. In contrast to the other members of the CRE-binding protein family, ICER expression is tissue specific and developmentally regulated. The kinetics of ICER expression are characteristic of an early response gene. Our results indicate that CREM plays a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. We have previously shown that the transcriptional activator CREM is highly expressed in postmeiotic cells. Spermiogenesis is a complex process by which postmeiotic male germ cells differentiate into mature spermatozoa. This process involves remarkable structural and biochemical changes that are under the hormonal control of the hypothalamic-pituitary axis. We have addressed the specific role of CREM in spermiogenesis using CREM-mutant mice generated by homologous recombination. Analysis of the seminiferous epithelium from mutant male mice reveals that spermatogenesis stops at the first step of spermiogenesis. Late spermatids are completely absent, while there is a significant increase in apoptotic germ cells. A series of postmeiotic germ cell-specific genes are not expressed. Mutant male mice completely lack spermatozoa. This phenotype is reminiscent of cases of human infertility. We have shown that ICER is regulated in a circadian manner in the pineal gland, the site of the hormone melatonin production. This night-day oscillation is driven by the endogenous clock (located in the suprachiasmatic nucleus, SCN). The synthesis of melatonin is regulated by a rate-limiting enzyme, the serotonin N-acetyltransferase (NAT). By using the CREM-deficient mice and by analysis of the regulatory region of the gene encoding the serotonin NAT, we have established that ICER is responsible for the amplitude and rhythmicity of NAT and thus for the oscillation in the hormonal synthesis of melatonin.
Assuntos
AMP Cíclico/farmacologia , Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , Proteínas Repressoras , Transdução de Sinais/fisiologia , Adenilil Ciclases/metabolismo , Animais , Modulador de Elemento de Resposta do AMP Cíclico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/fisiologia , Humanos , MasculinoAssuntos
AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Repressoras , Animais , Sequência de Bases , Modulador de Elemento de Resposta do AMP Cíclico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA/genética , Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Fosforilação , Transdução de Sinais , Espermatogênese , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Spermatogenesis is a complex differentiation process under the hormonal control of the hypothalamic-pituitary axis. The CREM gene encodes activators and repressors of cAMP-mediated gene transcription. The transcript corresponding to the activator isoform CREMtau is found at high levels in pachytene spermatocytes onwards. The CREMtau protein, however, is present only in post-meiotic spermatids where it activates the transcription of testis-specific genes, such as the protamines and transition proteins. Mice in which the CREM gene has been inactivated by homologous recombination have been generated. Homozygous male mutant mice are sterile and produce no spermatozoa. Histological analysis of the seminiferous tubules reveal a complete arrest of spermatogenesis at the first step of spermiogenesis. CREM deficiency results in the lack of post-meiotic gene expression and a ten-fold increase in apoptotic germ cells. These results demonstrate the essential role of CREM in spermatogenesis and are reminiscent of some cases of male infertility.
Assuntos
Modulador de Elemento de Resposta do AMP Cíclico/genética , Espermatogênese/fisiologia , Espermatozoides/citologia , Animais , Diferenciação Celular , AMP Cíclico/fisiologia , Modulador de Elemento de Resposta do AMP Cíclico/deficiência , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Infertilidade Masculina , Masculino , Camundongos , Camundongos Knockout , Espermatozoides/fisiologia , Testículo/fisiologiaRESUMO
The CREM gene encodes both repressors and activators of cAMP-dependent transcription in a tissue and developmentally regulated manner. In addition, multiple and cooperative phosphorylation events regulate the function of the CREM proteins. CREM plays a key physiological and developmental role within the hypothalamic-pituitary axis. There is a functional switch in CREM expression during the development of male germ cells which is directed by the pituitary hormone FSH. The CREM protein in germ cells is a powerful activator which appears to function as a master-switch in the regulation of postmeiotic genes. CREM is inducible by activation of the cAMP signalling pathway with the kinetics of an early response gene. The induction is transient, cell-specific, does not involve increased transcript stability and does not require protein synthesis. The subsequent decline in CREM expression requires de novo protein synthesis. The induced transcript encodes ICER and is generated from an alternative, intronic promoter. ICER functions as a powerful repressor of cAMP-induced transcription, and represses the activity of its own promoter, thus constituting a negative autoregulatory loop.
Assuntos
AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sequência Consenso , Sequência Conservada , Modulador de Elemento de Resposta do AMP Cíclico , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Hormônio Foliculoestimulante/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Modelos Biológicos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Repressoras/biossíntese , Proteínas Repressoras/metabolismo , Células de Sertoli/fisiologia , Transdução de Sinais , Espermatogênese , Testículo/fisiologiaRESUMO
Human beta1- and beta2-adrenergic receptor (beta1 AR and beta2 AR) coupling and desensitization characteristics were compared in defined heterologous expression systems. Significant differences in the coupling efficacies of the two subtypes were found in both Chinese hamster fibroblasts and murine Ltk- fibroblasts, which were used as surrogate cell lines. At the maximal level of stimulation with the nonselective beta-adrenergic agonist isoproterenol, beta1 AR-mediated adenylyl cyclase activation represented 70% and 20% of that mediated by beta2 AR in Chinese hamster and murine Ltk- fibroblasts, respectively. Sustained (15 min) stimulation with subsaturating concentration of isoproterenol (< 10% of receptor occupancy) led to identical desensitization of the beta-adrenergic-stimulated adenylyl cyclase activity for both beta1 AR and beta2AR-expressing cells. In contrast, when a nearly saturating concentration of isoproterenol (> 90% of receptor occupancy) was used to promote desensitization, the extent of desensitization observed for beta2 AR-expressing cells was 1.7-2-fold higher than that of the beta1 AR. The carboxyl domain of several G protein-coupled receptors has been shown to play important roles in both coupling efficacy and agonist-promoted desensitization. Therefore, we examined the contribution of this receptor domain in the subtype-selective phenotypes described above. A chimeric receptor composed of the first six transmembrane domains of the beta1 AR and of the seventh transmembrane domain and carboxyl tail of the beta2 AR maintained a coupling efficacy characteristic of the beta1 AR, whereas the extent of desensitization resulting from high receptor occupancy was identical to that of the beta2 AR. These results therefore suggest that the carboxyl portion of the beta1 AR and beta2 AR determines their subtype-selective desensitization patterns but not their respective coupling efficacies.
Assuntos
Adenilil Ciclases/metabolismo , Receptores Adrenérgicos beta 1/fisiologia , Receptores Adrenérgicos beta 2/fisiologia , Animais , Cricetinae , Cricetulus , Humanos , Isoproterenol/farmacologia , Camundongos , Fenótipo , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/efeitos dos fármacosRESUMO
Spermiogenesis is a complex process by which postmeiotic male germ cells differentiate into mature spermatozoa. This process involves remarkable structural and biochemical changes including nuclear DNA compaction and acrosome formation. Transcription activator CREM (cyclic AMP-responsive element modulator) is highly expressed in postmeiotic cells, and CREM may be responsible for the activation of several haploid germ cell-specific genes involved in the structuring of the spermatozoon. The specific role of CREM in spermiogenesis was addressed using CREM-mutant mice generated by homologous recombination. Analysis of the seminiferous epithelium in mutant male mice reveals postmeiotic arrest at the first step of spermiogenesis. Late spermatids are completely absent, and there is a significant increase in apoptotic germ cells. We show that CREM deficiency results in the lack of postmeiotic cell-specific gene expression. The complete lack of spermatozoa in the mutant mice is reminiscent of cases of human infertility.
Assuntos
Apoptose , Proteínas de Ligação a DNA/fisiologia , Proteínas Repressoras , Espermatogênese , Espermatozoides/citologia , Animais , Modulador de Elemento de Resposta do AMP Cíclico , Proteínas de Ligação a DNA/genética , Feminino , Fertilidade/genética , Fertilidade/fisiologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Proteínas/genética , Contagem de Espermatozoides , Espermatozoides/metabolismoRESUMO
In eukaryotes, transcriptional regulation upon stimulation of the adenylate cyclase signalling pathway is mediated by a family of cAMP-responsive nuclear factors. This family consists of a large number of members which may act as activators or repressors. These factors contain the basic domain/leucine zipper motifs and bind as dimers to cAMP-response elements (CRE). The function of CRE-binding proteins is modulated by phosphorylation by several kinases. The ICER (inducible cAMP early repressor) protein is the only inducible member of this family. The induction of this powerful repressor is likely to be important for the transient nature of cAMP-induced gene expression. CRE-binding proteins have been found to play an important role in the physiology of the pituitary gland, in regulating spermatogenesis, in the response to circadian rhythms and in the molecular basis of memory.
Assuntos
Núcleo Celular/genética , AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Modulador de Elemento de Resposta do AMP Cíclico , Regulação da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Proteínas Repressoras/genética , Fatores de Transcrição/fisiologiaRESUMO
We recently reported that tyramine caused concentration-dependent relaxation of rat aorta, which was endothelium independent and was not exerted via alpha 1-adrenoceptors (AR), alpha 2AR, beta 1AR, beta 2AR, or receptors for 5-hydroxytryptamine, histamine, and adenosine. The present studies were done on endothelium-denuded strips to determine structure-vasorelaxant activity after blockade of beta AR by propranolol plus irreversible blockade of alpha 1AR with benextramine. Vasorelaxation under these conditions was limited to noncatecholamines, and their vasorelaxant potencies were methoxyphenamine > tyramine > p-hydroxyephedrine > L-amphetamine > L-ephedrine > phenylethylamine > synephrine > methoxamine > octopamine. beta 3AR agonists (BRL 37344 and CGP 12177A) did not produce vasorelaxation, although tyramine could compete for cyanopindolol binding to murine L cells expressing human beta 2AR or beta 3AR. There was no significant specific binding of [3H]tyramine to aortic membrane preparations after the inhibition of monamine oxidase. Yohimbine, which has a high affinity for Drosophila tyramine receptors, also caused dose-dependent vasorelaxation like tyramine. It is concluded that tyramine and several other phenylethylamines produce relaxation of rat aorta, which does not involve any of the known adrenoceptors but may be exerted via novel tyramine receptors.
Assuntos
Aorta/efeitos dos fármacos , Fenetilaminas/farmacologia , Tiramina/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Masculino , Ratos , Ratos Sprague-Dawley , Vasodilatação , Ioimbina/farmacologiaRESUMO
The functional effects of long-term activation of beta-adrenoceptors were investigated by measuring adenylyl cyclase activity, cyclic AMP accumulation and cyclic AMP-dependent protein kinase activity in CHW and L cells expressing either human beta 2- or beta 3-adrenoceptors. Pre-incubation of CHW and L cells expressing beta 2-adrenoceptors with 10 microM isoprenaline for 24 h produced a marked reduction in the total receptor number and dramatically reduced the capacity of the receptor to stimulate adenylyl cyclase maximally. In contrast, the ability of beta 3-adrenoceptors number was observed in L but not in CHW cells. Maximal levels of intracellular cyclic AMP concentrations were reached during the first hour of receptor activation with isoprenaline in all four cell lines. In the absence of phosphodiesterase inhibitors, cyclic AMP decreased to basal levels within 24 h of continuous stimulation. This phenomenon occurred more rapidly in cells expressing the beta 2- than the beta 3-adrenoceptors. These results confirm that, at the level of adenylyl cyclase stimulation and cyclic AMP accumulation, the beta 3-adrenoceptor is more resistant than the beta 2-adrenoceptor to long-term desensitization. However, when cyclic AMP-dependent protein kinase activity was considered, a 24 h stimulation of beta 2- and when cyclic AMP-dependent protein kinase activity was considered, a 24 h stimulation of beta 2- and beta 3-adrenoceptor expressing cells led to the desensitization of the kinase in L but not in CHW cells. In conclusion, long-term desensitization may have distinct functional effects on cell signalling depending on the receptor subtype and the cell type considered. These findings might have practical implications for future strategies involving long-term therapies with receptor agonists.