RESUMO
Loss of function of the peritoneal membrane is associated with peritonitis. Adenosine levels in sites of inflammation were shown to increase and exhibit immunoregulatory effects. Our aim was to elucidate the regulatory role of adenosine during peritonitis and to test the involvement of peritoneal mesothelial cells (PMC) in adenosine regulation. In a mice model of Escherichia coli peritonitis, the adenosine A(2A) receptor (A(2A)R) agonist (CGS21680) prevented leukocyte recruitment and reduced tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) levels. Peritonitis induced the elevation of adenosine with a peak at 24 h. Analysis of adenosine receptor levels on peritoneum showed that A(1) receptor (A(1)R) protein levels peak at 12 h after inoculation and then return to baseline at 24 h, whereas high affinity A(2A)R protein levels peak at 24 h concomitantly with the peak of adenosine concentration. Low affinity A(2B) receptor (A(2B)R) levels elevated slowly, remaining elevated up to 48 h. In human PMC (HPMC), the early cytokines, IL-1-alpha, and TNF-alpha upregulated the A(2B) and A(2A) receptors. However, interferon-gamma (IFN-gamma) upregulated the A(2B)R and decreased A(2A)R levels. Treatment with the A(2A)R agonist reduced IL-1-dependent IL-6 secretion from HPMC. In conclusion, the kinetics of adenosine receptors suggest that at early stage of peritonitis, the A(1)R dominates, and later its dominance is replaced by the G stimulatory (Gs) protein-coupled A(2A)R that suppresses inflammation. Early proinflammatory cytokines are an inducer of the A(2A)R and this receptor reduces their production and leukocyte recruitment. Future treatment with adenosine agonists should be considered for attenuating the damage to mesothelium during the course of acute peritonitis.
Assuntos
Adenosina/metabolismo , Inflamação/genética , Peritonite/imunologia , Peritonite/metabolismo , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A1 de Adenosina , Agonistas do Receptor A2 de Adenosina , Antagonistas do Receptor A2 de Adenosina , Animais , Anti-Hipertensivos/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Escherichia coli , Feminino , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos , Peritonite/microbiologia , Fenetilaminas/farmacologia , Agonistas do Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismo , Teobromina/análogos & derivados , Teobromina/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Xantinas/farmacologiaRESUMO
Single chain variants of the heterodimeric gonadotropins were engineered by tethering the genes of the individual subunits into one polypeptide. In tethered human (h) gonadotropins, the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) beta subunit serves as an effective linker to enhance the secretion of the analogs compared to variants lacking the CTP. The gonadotropin subunits of non-primate, non-equid species lack a CTP domain that precludes the use of a homologous CTP in tethered analogs in many species. Here we used the bovine LH as a model to examine the impact of the CTP domain of the hCGbeta subunit (denoted as huCTP) and of a previously untranslated CTP-like sequence decoded from the bovine LHbeta gene on the secretion and bioactivity of tethered analogs. This cryptic CTP (designated boCTP) was incorporated into the bovine LHbeta reading frame by deletion frame-shift mutations analogous to these that presumably occurred in primates and equids. We genetically engineered single chain variants in which the beta and alpha subunit domains were linked directly or via the heterologous huCTP or the homologous boCTP sequences and expressed them in CHO cells. The data suggest that the tethered analogs were expressed and N-glycosylated, but unlike the huCTP, the boCTP appears as devoid of mucin O-glycans. The incorporation of the boCTP or huCTP linkers enhanced by about 3fold the rate and efficiency of secretion from the transfected cells. The tether variants were bioactive, as estimated by induction of steroid production in immortalized granulosa cells expressing the rat LH receptor. Furthermore, the variants were about equally potent, as judged by their EC50s (0.7-0.9 ng/ml). Thus, the hCGbeta CTP maintains pro-secretory determinants without inhibiting receptor activation when applied as a linker in tethered bovine LH, implying that these CTP features are preserved when the domain is incorporated into non-primate single chain analogs. The study suggests that the boCTP and huCTP domains are advantageous for the secretion of tethered bovine gonadotropins, and also demonstrates strategies for the design of bioactive LH analogs in ruminant species.