RESUMO
Regulation of the kallikrein-kinin system in cerebral inflammation is still unclear. Here, we used reverse-transcription polymerase chain reaction (RT-PCR) techniques to show that lipopolysaccharide (LPS) activates the kallikrein-kinin system by enhancing liberation of bradykinin (BK), and alters mRNA levels of kallikrein-kinin system components, including high molecular weight (H-) and low molecular weight (L-) kininogens, in ECPC4 cells, a cell line of mouse choroid plexus epithelium. LPS treatment increased liberation of immunoreactive bradykinin in the supernatant of ECPC4 cells, and addition of LPS (500 ng/ml) to cultures resulted in elevation of H- and L-kininogen mRNA levels in ECPC4 cells within 24-48 h. Furthermore, LPS treatment elevated bradykinin type 2 and type 1 receptor mRNA levels within 4h, but did not change tissue kallikrein or plasma kallikrein mRNA levels. On the other hand, expression of pro-inflammatory mediators interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and cyclooxygenase-2 mRNA increased within 4-8h after addition of LPS to ECPC4 cells. The addition of IL-1beta and TNF-alpha to investigate the major mediator for kininogen expression in ECPC4 cells remarkably induced expression of H- and L-kininogen mRNAs in ECPC4 cells. These results suggest that LPS activates the kallikrein-kinin system in the choroid plexus via autocrine induction of IL-1beta and TNF-alpha.
Assuntos
Plexo Corióideo/metabolismo , Citocinas/genética , Encefalite/líquido cefalorraquidiano , Mediadores da Inflamação/metabolismo , Calicreínas/líquido cefalorraquidiano , Cininas/líquido cefalorraquidiano , Animais , Bradicinina/líquido cefalorraquidiano , Bradicinina/efeitos dos fármacos , Bradicinina/genética , Linhagem Celular , Líquido Cefalorraquidiano/metabolismo , Plexo Corióideo/efeitos dos fármacos , Plexo Corióideo/fisiopatologia , Ciclo-Oxigenase 2/genética , Encefalite/induzido quimicamente , Encefalite/fisiopatologia , Interleucina-1beta/genética , Interleucina-1beta/farmacologia , Calicreínas/efeitos dos fármacos , Calicreínas/genética , Cininas/efeitos dos fármacos , Cininas/genética , Lipopolissacarídeos , Camundongos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores da Bradicinina/efeitos dos fármacos , Receptores da Bradicinina/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
The generation of kinins on the surface of vascular endothelium has been postulated in two pathways involving plasma kallikrein and tissue kallikrein; the former pathway has been well documented, but the latter is controversial. To clarify the presence of a kinin-generating system on endothelium, we examined whether human umbilical vein endothelial cells (HUVEC) synthesize and release tissue kallikrein in vitro. Kallikrein-like activity hydrolyzing a peptide Pro-Phe-Arg-4-methyl-coumaryl-7-amide was detected in the culture medium of HUVEC and was inhibited by aprotinin but not by soybean trypsin inhibitor. Western blotting of HUVEC medium using anti-human tissue kallikrein antibodies demonstrated the release of tissue kallikrein from HUVEC, and the reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blotting revealed the expression of tissue kallikrein mRNA in HUVEC. HUVEC metabolically labeled with [35S]methionine released radioactive proteins corresponding to tissue kallikrein. RT-PCR also showed the expression of low-molecular-weight kininogen (L-kininogen) mRNA in HUVEC. The cGMP levels in HUVEC were significantly elevated by the incubation with angiotensin converting enzyme inhibitor, lisinopril, and the elevation was completely inhibited by aprotinin or bradykinin B2-receptor antagonist, FR172357. These results suggest that the endothelial cells continuously release an active form of tissue kallikrein which enables generation of kinins on the vascular endothelium.