RESUMO
Cyclooxygenase-2 (COX-2) is constitutively expressed in a subset of thick ascending limb cells in the cortex and medulla and increases when the renin-angiotensin and kallikrein-kinin systems are activated. Although the contribution of angiotensin II to the regulation of COX-2 is known, the effects of bradykinin on COX-2 expression have not been determined in this nephron segment. We evaluated expression of B2 bradykinin receptors in thick ascending limb cells containing COX-2 and the effect of bradykinin on COX-2 expression in primary cultured medullary thick ascending cells. The presence of B2 receptors was studied in renal sections by immunohistochemistry with antibodies against B2, COX-2, and Tamm-Horsfall glycoprotein. B2 receptors were detected on the apical and basolateral portion of the thick ascending cells. These cells also contained COX-2, suggesting that COX-2 expression may be regulated via B2 receptor. Incubation of cultured medullary thick ascending cells with bradykinin (10(-7) to 10(-5) mol/L) induced a significant increase on COX-2 protein expression. Maximal expression of COX-2 was observed 4 hours after exposure to bradykinin (10(-7) mol/L), effect abolished by a B2 receptor antagonist (HOE-140; 10(-6) mol/L). Prostaglandin E2 production increased when these cells were challenged with bradykinin for 4 hours, indicating that COX-2 was enzymatically active. We have demonstrated (1) the presence of B2 receptors in thick ascending limb cells expressing COX-2 and (2) the stimulatory effect of bradykinin on COX-2 protein expression, via B2 receptors, in cultured medullary thick ascending cells. We suggest that bradykinin can affect ion transport in the thick ascending limb via a COX-2-mediated mechanism.
Assuntos
Bradicinina/metabolismo , Isoenzimas/metabolismo , Rim/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina , Ciclo-Oxigenase 2 , Dinoprostona/biossíntese , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Receptor B2 da Bradicinina/metabolismoRESUMO
We examined the rat proximal tubule (PT) response to endothelin-1 (ET-1) in terms of 20-hydroxyeicosatetraenoic acid (HETE) dependency. Arachidonic acid (AA) (1 microM) decreased ouabain-sensitive (86)Rb uptake from 2.1 +/- 0.1 to 0.3 +/- 0.08 ng Rb. 10 microg protein(-1). 2 min(-1) (P < 0.05); 20-HETE (1 microM) had similar effects. Dibromododecenoic acid (DBDD) (2 microM), an inhibitor of omega-hydroxylase, abolished the inhibitory action of AA on (86)Rb uptake whereas the PT response to 20-HETE was unaffected. ET-1 at 0.1, 1, 10, and 100 nM reduced (86)Rb uptake from 2.8 +/- 0.3 in control PTs to 2.4 +/- 0.2, 1.7 +/- 0.1, 0.67 +/- 0.08, and 0.1 +/- 0.03 ng Rb. 10 microg protein(-1). 2 min(-1), respectively. DBDD (2 microM) abolished the inhibitory effect of ET-1 on (86)Rb uptake as did BMS182874 (1 microM), an ET(A)-selective receptor antagonist. ET-1 (100 nM) significantly increased PT 20-HETE release by approximately 50%, an effect prevented by DBDD. N(omega)-nitro-L-arginine-methyl ester (L-NAME), given for 4 days to inhibit nitric oxide synthase (NOS), increased arterial pressure from 92 +/- 12 to 140 +/- 8 mmHg and increased endogenous release of 20-HETE from isolated PTs (measured by gas chromatography/mass spectrometry). In L-NAME-treated PTs, but not in control PTs, 0.1 microM AA inhibited ouabain-sensitive (86)Rb uptake by > 40%; the response to AA was attenuated by DBDD. We conclude that, in the PTs, 1) 20-HETE is a second messenger for ET-1 and 2) conversion of AA to 20-HETE is augmented when NOS is inhibited.
Assuntos
Ácido Araquidônico/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Endotelina-1/metabolismo , Túbulos Renais Proximais/metabolismo , Animais , Transporte Biológico/fisiologia , Antagonistas dos Receptores de Endotelina , Inibidores Enzimáticos/farmacologia , Ácidos Hidroxieicosatetraenoicos/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A , Receptores de Endotelina/metabolismo , Radioisótopos de Rubídio/farmacocinética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Adrenalectomized (ADX) and sham-operated rats received either dexamethasone (DEX) or vehicle. Renal tissue was used for morphologic analysis, assessment of cyclooxygenase-2 (COX-2) protein expression and mRNA accumulation, and quantitation of COX-2 activity. In untreated or shamoperated rats, COX-2 protein was observed in a subset of tubular epithelial cells (<2%), which were located mainly in the cortex. All COX-2-positive cells also expressed Tamm-Horsfall glycoprotein, a highly selective marker for thick ascending limb (TAL) cells. After ADX, >30% of TAL cells expressed COX-2 in a manner consistent with recruitment of COX-2-positive TAL cells toward the medulla. Treatment of ADX rats with DEX reduced the number of COX-2-positive cells to that observed in sham-operated or intact rats. COX-2 mRNA accumulation was increased by ADX and partially attenuated by treatment with DEX. Western blot analysis of cortical microsomes revealed a substantial increase in COX-2 expression in ADX rats, compared with ADX/DEX-treated, sham-operated, or intact rats. The increase in COX-2 protein expression was associated with a twofold increase in prostaglandin E(2) formation by cortical microsomes obtained from ADX rats, compared with sham-operated rats. It is concluded that ADX induces expression of enzymatically active COX-2, such that expression occurs in the cortical TAL and proceeds in a defined pattern toward the outer medullary TAL. It is suggested that ADX induces expression of TAL cells that, in the basal state, do not express COX-2 protein.