RESUMO
Epoxyeicosatrienoic acids (EETs) are also known as epoxyeicosanoids that have renal and cardiovascular actions. These renal and cardiovascular actions can be regulated by soluble epoxide hydrolase (sEH) that degrades and inactivates EETs. Extensive animal hypertension studies have determined that vascular, epithelial transport, and anti-inflammatory actions of EETs lower blood pressure and decrease renal and cardiovascular disease progression. Human studies have also supported the notion that increasing EET levels in hypertension could be beneficial. Pharmacological and genetic approaches to increase epoxyeicosanoids in several animal models and humans have found improved endothelial vascular function, increased sodium excretion, and decreased inflammation to oppose hypertension and associated renal and cardiovascular complications. These compelling outcomes support the concept that increasing epoxyeicosanoids via sEH inhibitors or EET analogs could be a valuable hypertension treatment.
Assuntos
Anti-Hipertensivos/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Eicosanoides/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Epóxido Hidrolases/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Animais , Eicosanoides/metabolismo , Epóxido Hidrolases/metabolismo , Humanos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Transdução de SinaisRESUMO
We showed recently that increasing kidney epoxyeicosatrienoic acids (EETs) by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for EETs degradation, retarded the development of renal dysfunction and progression of aorto-caval fistula(ACF)-induced congestive heart failure (CHF) in Ren-2 transgenic hypertensive rats (TGR). In that study the final survival rate of untreated ACF TGR was only 14 % but increased to 41 % after sEH blockade. Here we examined if sEH inhibition added to renin-angiotensin system (RAS) blockade would further enhance protection against ACF-induced CHF in TGR. The treatment regimens were started one week after ACF creation and the follow-up period was 50 weeks. RAS was blocked using angiotensin-converting enzyme inhibitor (ACEi, trandolapril, 6 mg/l) and sEH with an sEH inhibitor (sEHi, c-AUCB, 3 mg/l). Renal hemodynamics and excretory function were determined two weeks post-ACF, just before the onset of decompensated phase of CHF. 29 weeks post-ACF no untreated animal survived. ACEi treatment greatly improved the survival rate, to 84 % at the end of study. Surprisingly, combined treatment with ACEi and sEHi worsened the rate (53 %). Untreated ACF TGR exhibited marked impairment of renal function and the treatment with ACEi alone or combined with sEH inhibition did not prevent it. In conclusion, addition of sEHi to ACEi treatment does not provide better protection against CHF progression and does not increase the survival rate in ACF TGR: indeed, the rate decreases significantly. Thus, combined treatment with sEHi and ACEi is not a promising approach to further attenuate renal dysfunction and retard progression of CHF.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Benzoatos/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Indóis/uso terapêutico , Insuficiência Renal/prevenção & controle , Ureia/análogos & derivados , Animais , Fístula Arteriovenosa , Benzoatos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Epóxido Hidrolases/antagonistas & inibidores , Feminino , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/mortalidade , Masculino , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Insuficiência Renal/etiologia , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
Endothelial and vascular smooth cells generate cytochrome P450 (CYP) arachidonic acid metabolites that can impact endothelial cell function and vascular homeostasis. The objective of this review is to focus on the physiology and pharmacology of endothelial CYP metabolites. The CYP pathway produces two types of eicosanoid products: epoxyeicosatrienoic acids (EETs), formed by CYP epoxygenases, and hydroxyeicosatetraenoic acids (HETEs), formed by CYP hydroxylases. Advances in CYP enzymes, EETs, and 20-HETE by pharmacological and genetic means have led to a more complete understanding of how these eicosanoids impact on endothelial cell function. Endothelial-derived EETs were initially described as endothelial-derived hyperpolarizing factors. It is now well recognized that EETs importantly contribute to numerous endothelial cell functions. On the other hand, 20-HETE is the predominant CYP hydroxylase synthesized by vascular smooth muscle cells. Like EETs, 20-HETE acts on endothelial cells and impacts importantly on endothelial and vascular function. An important aspect for EETs and 20-HETE endothelial actions is their interactions with hormonal and paracrine factors. These include interactions with the renin-angiotensin system, adrenergic system, puringeric system, and endothelin. Alterations in CYP enzymes, 20-HETE, or EETs contribute to endothelial dysfunction and cardiovascular diseases such as ischemic injury, hypertension, and atherosclerosis. Recent advances have led to the development of potential therapeutics that target CYP enzymes, 20-HETE, or EETs. Thus, future investigation is required to obtain a more complete understanding of how CYP enzymes, 20-HETE, and EETs regulate endothelial cell function.
Assuntos
Doenças Cardiovasculares/fisiopatologia , Eicosanoides/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Animais , Ácido Araquidônico/metabolismo , Citocromo P-450 CYP2J2 , Sistema Enzimático do Citocromo P-450/metabolismo , Células Endoteliais/metabolismo , HumanosRESUMO
The detailed mechanisms determining the course of congestive heart failure (CHF) and associated renal dysfunction remain unclear. In a volume overload model of CHF induced by creation of aorto-caval fistula (ACF) in Hannover Sprague-Dawley (HanSD) rats we explored the putative pathogenetic contribution of epoxyeicosatrienoic acids (EETs), active products of CYP-450 dependent epoxygenase pathway of arachidonic acid metabolism, and compared it with the role of the renin-angiotensin system (RAS). Chronic treatment with cis-4-[4-(3-adamantan-1-yl-ureido) cyclohexyloxy]benzoic acid (c-AUCB, 3 mg/l in drinking water), an inhibitor of soluble epoxide hydrolase (sEH) which normally degrades EETs, increased intrarenal and myocardial EETs to levels observed in sham-operated HanSD rats, but did not improve the survival or renal function impairment. In contrast, chronic angiotensin-converting enzyme inhibition (ACEi, trandolapril, 6 mg/l in drinking water) increased renal blood flow, fractional sodium excretion and markedly improved survival, without affecting left ventricular structure and performance. Hence, renal dysfunction rather than cardiac remodeling determines long-term mortality in advanced stage of CHF due to volume overload. Strong protective actions of ACEi were associated with suppression of the vasoconstrictor/sodium retaining axis and activation of vasodilatory/natriuretic axis of the renin-angiotensin system in the circulating blood and kidney tissue.
Assuntos
Benzoatos/uso terapêutico , Epóxido Hidrolases/antagonistas & inibidores , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Renal/prevenção & controle , Ureia/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/sangue , Ácido 8,11,14-Eicosatrienoico/metabolismo , Angiotensina I/sangue , Angiotensina II/sangue , Inibidores da Enzima Conversora de Angiotensina , Animais , Benzoatos/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Compostos de Epóxi/metabolismo , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/diagnóstico por imagem , Rim/metabolismo , Masculino , Miocárdio/metabolismo , Fragmentos de Peptídeos/sangue , Distribuição Aleatória , Ratos , Insuficiência Renal/sangue , Insuficiência Renal/etiologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Ultrassonografia , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
AIM: It has been demonstrated that tumour necrosis factor-alpha (TNF-α) via its receptor 2 (TNFR2) plays a role in the cardioprotective effects of preconditioning. It is also well known that chronic hypoxia is associated with activation of inflammatory response. With this background, we hypothesized that TNF-α signalling may contribute to the improved ischaemic tolerance of chronically hypoxic hearts. METHODS: Adult male Wistar rats were kept either at room air (normoxic controls) or at continuous normobaric hypoxia (CNH; inspired O2 fraction 0.1) for 3 weeks; subgroups of animals were treated with infliximab (monoclonal antibody against TNF-α; 5 mg kg(-1), i.p., once a week). Myocardial levels of oxidative stress markers and the expression of selected signalling molecules were analysed. Infarct size (tetrazolium staining) was assessed in open-chest rats subjected to acute coronary artery occlusion/reperfusion. RESULTS: CNH increased myocardial TNF-α level and expression of TNFR2; this response was abolished by infliximab treatment. CNH reduced myocardial infarct size from 50.8 ± 4.3% of the area at risk in normoxic animals to 35.5 ± 2.4%. Infliximab abolished the protective effect of CNH (44.9 ± 2.0%). CNH increased the levels of oxidative stress markers (3-nitrotyrosine and malondialdehyde), the expression of nuclear factor κB and manganese superoxide dismutase, while these effects were absent in infliximab-treated animals. CNH-elevated levels of inducible nitric oxide synthase and cyclooxygenase 2 were not affected by infliximab. CONCLUSION: TNF-α plays a role in the induction of ischaemia-resistant cardiac phenotype of CNH rats, possibly via the activation of protective redox signalling.
Assuntos
Adaptação Fisiológica/fisiologia , Hipóxia/metabolismo , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Coração/efeitos dos fármacos , Infliximab/farmacologia , Masculino , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Arachidonic acid metabolites, eicosanoids, are key contributors to vascular function and improper eicosanoid regulation contributes to the progression of cardiovascular diseases. Epoxyeicosatrienoic acids (EETs) are synthesized from arachidonic acid by epoxygenase enzymes to four regioisomers, 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET. These EETs have interesting beneficial effects like vasodilation, anti-inflammation, and anti-platelet aggregation that could combat cardiovascular diseases. There is mounting evidence that each regioisomeric EET may have unique vascular effects and that the contribution of individual EETs to vascular function differs from organ to organ. Over the past decade EET analogs and antagonists have been synthesized to determine EET structure function relationships and define the contribution of each regioisomeric EET. A number of studies have demonstrated that EET analogs induce vasodilation, lower blood pressure and decrease inflammation. EET antagonists have also been used to demonstrate that endogenous EETs contribute importantly to cardiovascular function. This review will discuss EET synthesis, regulation and physiological roles in the cardiovascular system. Next we will focus on the development of EET analogs and what has been learned about their contribution to vascular function. Finally, the development of EET antagonists and how these have been utilized to determine the cardiovascular actions of endogenous epoxides will be discussed. Overall, this review will highlight the important knowledge garnered by the development of EET analogs and their possible value in the treatment of cardiovascular diseases.
Assuntos
Fenômenos Fisiológicos Cardiovasculares , Eicosanoides/fisiologia , Animais , Doenças Cardiovasculares/tratamento farmacológico , Eicosanoides/antagonistas & inibidores , Eicosanoides/metabolismo , HumanosRESUMO
The soluble epoxide hydrolase enzyme (SEH) and vascular remodeling are associated with cardiovascular disease. Although inhibition of SEH prevents smooth muscle cell proliferation in vitro, the effects of SEH inhibition on vascular remodeling in vivo and mechanisms of these effects remain unclear. Herein we determined the effects of SEH antagonism in an endothelium intact model of vascular remodeling induced by flow reduction and an endothelium denuded model of vascular injury. We demonstrated that chronic treatment of spontaneously hypertensive stroke-prone rats with 12-(3-adamantan-1-yl-ureido) dodecanoic acid, an inhibitor of SEH, improved the increment of inward remodeling induced by common carotid ligation to a level that was comparable with normotensive Wistar Kyoto rats. Similarly, mice with deletion of the gene responsible for the production of the SEH enzyme (Ephx2(-/-)) demonstrated enhanced inward vascular remodeling induced by carotid ligation. However, the hyperplastic response induced by vascular injury that denudes the endothelium was unabated by SEH inhibition or Ephx2 gene deletion. These results suggest that SEH inhibition or Ephx2 gene deletion antagonizes neointimal formation in vivo by mechanisms that are endothelium dependent. Thus SEH inhibition may have therapeutic potential for flow-induced remodeling and neointimal formation.
Assuntos
Artérias Carótidas/fisiopatologia , Lesões das Artérias Carótidas/fisiopatologia , Epóxido Hidrolases/antagonistas & inibidores , Epóxido Hidrolases/fisiologia , Animais , Pressão Sanguínea/fisiologia , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/etiologia , Proliferação de Células , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Epóxido Hidrolases/genética , Hiperplasia/patologia , Hiperplasia/fisiopatologia , Ácidos Láuricos/farmacologia , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Túnica Íntima/patologia , Túnica Íntima/fisiopatologiaRESUMO
Studies of the cytochrome P450 arachidonic acid (AA) monooxygenase, now established as a major pathway for the bioactivation of this physiological important fatty acid, have uncovered new and important roles for this enzyme system in the regulation of kidney function, including renal hemodynamics and tubular ion transport. Associations between genetically controlled alterations in blood pressure and the activity and/or transcriptional regulation of the kidney Cyp2c AA epoxygenases and Cyp4a omega-hydroxylases revealed a role for these enzymes in the pathophysiology of hypertension, a leading cause of cardiovascular, cerebral, and renal morbidity and mortality. Furthermore, analysis of associations between genetic variants of human CYP4A11 and hypertension suggest a potential role for this gene as a determinant of polygenic blood pressure control in humans. These results are providing conceptually novel approaches for studies of the molecular basis of human hypertension that could lead to new strategies for the early diagnosis and clinical management of this devastating disease.
Assuntos
Ácido Araquidônico/metabolismo , Pressão Sanguínea/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos MutantesRESUMO
Impaired cytochrome P450 epoxygenase enzyme (Cyp2c) regulation contributes to renal damage in angiotensin salt-sensitive hypertension (ANG/HS). We hypothesized that interleukin-6 null mice (IL6-/-) would improve Cyp2c regulation and reduce renal damage in hypertensive mice fed a high salt diet. Systolic blood pressure increased to a greater extent in ANG/HS hypertension as compared to angiotensin (ANG) hypertension but blood pressure did not differ between WT and IL6-/- hypertensive groups. Albuminuria, a marker for renal injury, increased significantly in ANG/HS hypertension in WT mice (5,113 +/- 1,050 mug/day) and was attenuated in the ANG/HS IL6-/- group (1,306 +/- 385 mug/day). Renal Cyp2c protein expression significantly decreased with ANG/HS hypertension in WT mice as compared to high salt alone. However, the ability to upregulate Cyp2c expression in response to a high salt diet was restored in the ANG/HS IL6 deficient hypertensive mice. Renal expression of soluble epoxide hydrolase, which inactivates protective epoxygenase metabolites, was significantly reduced in ANG/HS IL6-/- hypertensive mice compared to the ANG/HS WT group. These data suggest that IL6, while having no effect on blood pressure, impairs regulation of epoxygenase producing Cyp2c, which could contribute to the development of renal injury in angiotensin salt-sensitive hypertension.
Assuntos
Pressão Sanguínea/fisiologia , Modelos Animais de Doenças , Hipertensão/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-6/fisiologia , Cloreto de Sódio na Dieta/administração & dosagem , Animais , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/enzimologia , Hipertensão/prevenção & controle , Interleucina-6/biossíntese , Camundongos , Camundongos Knockout , Cloreto de Sódio na Dieta/efeitos adversosRESUMO
This study examined the role of intrarenal ANG II in the renal vascular reactivity changes occurring in the remaining kidney undergoing adaptation following contralateral nephrectomy. Renal blood flow responses to intrarenal injections of ANG II (0.25 to 5 ng) were measured in anesthetized euvolemic male Wistar rats 1, 4, 12, and 24 wk after uninephrectomy (UNX) or sham procedure (SHAM). At week 4, renal vasoconstriction induced by 2 ng ANG II was greater in UNX (69 +/- 5%) than in SHAM rats (50 +/- 3%; P < 0.01). This response was inhibited, by 50 and 66%, and by 20 and 25%, in SHAM and UNX rats, after combined injections of ANG II and losartan, or PD-123319 (P < 0.05), respectively. Characteristics of ANG II receptor binding in isolated preglomerular resistance vessels were similar in the two groups. After prostanoid inhibition with indomethacin, renal vasoconstriction was enhanced by 42 +/- 8% (P < 0.05), only in SHAM rats, whereas after 20-HETE inhibition with HET0016, it was reduced by 53 +/- 16% (P < 0.05), only in UNX rats. These differences vanished after concomitant prostanoid and 20-HETE inhibition in the two groups. After UNX, renal cortical protein expression of cytochrome P-450 2c23 isoform (CYP2c23) and cyclooxygenase-1 (COX-1) was unaltered, but it was decreased for CYP4a and increased for COX-2. In conclusion, renal vascular reactivity to ANG II was significantly increased in the postuninephrectomy adapted kidney, independently of protein expression, but presumably involving interactions between 20-HETE and COX in the renal microvasculature and changes in the paracrine activity of ANG II and 20-HETE.
Assuntos
Angiotensina II/farmacologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Circulação Renal/efeitos dos fármacos , Circulação Renal/fisiologia , Vasoconstritores/farmacologia , Adaptação Fisiológica/fisiologia , Amidinas/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Ciclo-Oxigenase 1/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Citocromo P-450 CYP2J2 , Sistema Enzimático do Citocromo P-450/metabolismo , Dinoprostona/urina , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Imidazóis/farmacologia , Indometacina/farmacologia , Losartan/farmacologia , Masculino , Microcirculação/efeitos dos fármacos , Microcirculação/fisiologia , Nefrectomia , Norepinefrina/farmacologia , Piridinas/farmacologia , Ratos , Ratos Wistar , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
Autoregulation of renal blood flow is an established physiological phenomenon, however the signalling mechanisms involved remain elusive. Autoregulatory adjustments in preglomerular resistance involve myogenic and tubuloglomerular feedback (TGF) influences. While there is general agreement on the participation of these two regulatory pathways, the signalling molecules and effector mechanisms have not been identified. Currently, there are two major hypotheses being considered to explain the mechanism by which TGF signals are transmitted from the macula densa to the afferent arteriole. The adenosine hypothesis proposes that the released adenosine triphosphate (ATP) is hydrolysed to adenosine and this product stimulates preglomerular vasoconstriction by activation of A(1) receptors on the afferent arteriole. Alternatively, the P2 receptor hypothesis postulates that ATP released from the macula densa directly stimulates afferent arteriolar vasoconstriction by activation of ATP-sensitive P2X(1) receptors. This hypothesis has emerged from the realization that P2X(1) receptors are heavily expressed along the preglomerular vasculature. Inactivation of P2X(1) receptors impairs autoregulatory responses while afferent arteriolar responses to A(1) adenosine receptor activation are retained. Autoregulatory behaviour is markedly attenuated in mice lacking P2X(1) receptors but responses to adenosine A(1) receptor activation remain intact. More recent experiments suggest that P2X(1) receptors play an essential role in TGF-dependent vasoconstriction of the afferent arteriole. Interruption of TGF-dependent influences on afferent arteriolar diameter, by papillectomy or furosemide treatment, significantly attenuated pressure-mediated afferent arteriolar vasoconstriction in wild-type mice but had no effect on the response in P2X(1) knockout mice. Collectively, these observations support an essential role for P2X(1) receptors in TGF-mediated afferent arteriolar vasoconstriction.
Assuntos
Receptores Purinérgicos P2/fisiologia , Circulação Renal/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Arteríolas/fisiologia , Homeostase/fisiologia , Camundongos , Camundongos Knockout , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X , Fatores de Crescimento Transformadores/fisiologiaRESUMO
Arachidonic acid can be metabolized by cytochrome p450 (CYP450) enzymes to 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosa-trienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE). These arachidonic acid metabolites are involved in the regulation of renal epithelial transport and vascular function. 20-HETE and EETs are produced in the renal microvascular smooth muscle cells and endothelial cells, respectively. 20-HETE constricts the preglomerular arterioles by inhibiting K(+) channels, and contributes importantly to renal blood flow autoregulatory responsiveness of the afferent arterioles. EETs dilate the preglomerular arterioles by activating the renal smooth muscle cell Ca(2+)-activated K(+) channels and hyperpolarizing smooth muscle cells. These EET actions are consistent with their identification as endothelium-derived hyperpolarizing factors (EDHFs). In the kidney, EETs and 20-HETE are also produced in the proximal tubule and the thick ascending loop of Henle, and these metabolites modulate ion transport in the proximal tubules and the thick ascending limb by inhibiting Na(+)-K(+)-ATPase and the Na(+)-K(+)-2Cl(-) cotransporter. CYP450 metabolites also act as second messengers for many paracrine and hormonal agents, including endothelin, nitric oxide, and angiotensin II. The production of kidney CYP450 arachidonic acid metabolites is altered in diabetes, pregnancy, hepatorenal syndrome, and in various models of hypertension, and it is likely that changes in this system contribute to the abnormalities in renal function that are associated with many of these conditions.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Rim/enzimologia , Preparações Farmacêuticas/metabolismo , Animais , Ácido Araquidônico/metabolismo , Humanos , Rim/fisiologia , Transdução de Sinais/fisiologiaRESUMO
This study was conducted to test the hypothesis that the cytochrome P-450 (CYP450) metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the afferent arteriolar response to P2 receptor activation. Afferent arteriolar responses to ATP, the P2X agonist, alpha,beta-methylene ATP and the P2Y agonist UTP were determined before and after treatment with the selective CYP450 hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE). Stimulation with 1.0 and 10 microM ATP elicited an initial preglomerular vasoconstriction of 12 +/- 1% and 45 +/- 4% and a sustained vasoconstriction of 11 +/- 1% and 11 +/- 2%, respectively. DDMS or 20-HEDE significantly attenuated the sustained afferent arteriolar constrictor response to ATP. alpha,beta-Methylene ATP (1 microM) induced a rapid initial afferent vasoconstriction of 64 +/- 3%, which partially recovered to a stable diameter 10 +/- 1% smaller than control. Both DDMS and 20-HEDE significantly attenuated the initial vasoconstriction and abolished the sustained vasoconstrictor response to alpha,beta-methylene ATP. UTP decreased afferent diameter by 50 +/- 5% and 20-HEDE did not change this response. In addition, the ATP-induced increase in the intracellular Ca2+ concentration in preglomerular microvascular smooth muscle cells was significantly attenuated by 20-HEDE. Taken together, these results are consistent with the hypothesis that the CYP450 metabolite 20-HETE participates in the afferent arteriolar response to activation of P2X receptors.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Receptores Purinérgicos P2/metabolismo , Vasoconstrição/fisiologia , Trifosfato de Adenosina/farmacologia , Amidas/farmacologia , Animais , Arteríolas/fisiologia , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Masculino , Músculo Liso Vascular/fisiologia , Cloreto de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X , Receptores Purinérgicos P2Y1 , Artéria Renal/fisiologia , Sulfonas/farmacologia , Uridina Trifosfato/metabolismo , Vasoconstrição/efeitos dos fármacosRESUMO
In the kidney, epoxyeicosatrienoic acids (EETs) have been suggested to be endothelium-derived hyperpolarizing factors (EDHFs). The aim of the present study was to determine the contribution of EETs to the preglomerular vasodilation elicited by bradykinin. Sprague-Dawley rats were studied utilizing an in vitro perfused juxtamedullary nephron preparation. The afferent arteriolar diameter was determined and the diameter averaged 19 +/- 1 microm (n = 26) at a renal perfusion pressure of 100 mm Hg. Addition of 1, 10 and 100 nM bradykinin to the perfusate dose-dependently increased afferent arteriolar diameter by 5 +/- 1, 12 +/- 2 and 17 +/- 2%, respectively. The nitric oxide inhibitor N(omega)-nitro-L-arginine reduced bradykinin-induced afferent arteriolar vasodilation by 50%, and the diameter increased by 9 +/- 2% in response to 100 nM bradykinin. Epoxygenase inhibitors N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide or miconazole greatly attenuated the nitric oxide-independent component of the vasodilation elicited by bradykinin. Cyclooxygenase (COX) inhibition attenuated the nitric oxide-independent vasodilation elicited by 1 nM bradykinin but did not significantly affect the vascular response to 100 nM bradykinin. Combined inhibition of nitric oxide, COX and epoxygenase pathways completely abolished bradykinin-mediated afferent arteriolar vasodilation. In additional studies, renal microvessels were isolated and incubated with bradykinin and samples were analyzed by NICI/GC/MS. Under control conditions, renal microvascular EET levels averaged 49 +/- 9 pg/mg/20 min (n = 7). In the presence of bradykinin, EET levels were significantly higher and averaged 81 +/- 11 pg/mg/20 min (n = 7). These data support the concept that EETs are EDHFs and contribute to the nitric oxide-independent afferent arteriolar vasodilation elicited by bradykinin.
Assuntos
Arteríolas/fisiologia , Bradicinina/farmacologia , Sistema Enzimático do Citocromo P-450/fisiologia , Rim/irrigação sanguínea , Óxido Nítrico/fisiologia , Oxigenases/fisiologia , Vasodilatação/efeitos dos fármacos , Ácido 8,11,14-Eicosatrienoico/sangue , Amidas/farmacologia , Animais , Arteríolas/efeitos dos fármacos , Técnicas de Cultura , Inibidores de Ciclo-Oxigenase/farmacologia , Citocromo P-450 CYP2J2 , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Indometacina/farmacologia , Rim/enzimologia , Cinética , Masculino , Microcirculação/efeitos dos fármacos , Nitroarginina/farmacologia , Oxigenases/antagonistas & inibidores , Prostaglandina-Endoperóxido Sintases/fisiologia , Ratos , Ratos Sprague-Dawley , Circulação Renal/efeitos dos fármacosRESUMO
OBJECTIVES: Epoxygenase metabolites produced by the kidney affect renal blood flow and tubular transport function and 11,12-epoxyeicosatrienoic acid (11,12-EET) has been putatively identified as an endothelium-derived hyperpolarizing factor. The current studies were performed to determine the influence of 11,12-EET on the regulation of afferent arteriolar diameter in angiotensin II-infused hypertensive rats. MATERIALS AND METHODS: Male Sprague-Dawley rats received angiotensin II (60 ng/min) or vehicle via an osmotic minipump. Angiotensin II-infused hypertensive and vehicle-infused normotensive rats were studied for 2 weeks following implantation of the minipump. Renal microvascular responses to the sulfonimide analog of 11,12-EET (11,12-EET-SI) and angiotensin II were observed utilizing the in-vitro juxtamedullary nephron preparation. Renal cortical epoxygenase enzyme protein levels were quantified by Western blot analysis. Renal microvessels were also isolated and epoxygenase metabolite levels measured by negative ion chemical ionization (NICI)/gas chromatography-mass spectroscopy. RESULTS: Systolic blood pressure averaged 118 +/- 2 mmHg prior to pump implantation and increased to 185 +/- 7 mmHg in rats infused with angiotensin II for 2 weeks. Afferent arteriolar diameters of 2-week normotensive animals averaged 22 +/- 1 microm. Diameters of the afferent arterioles were 17% smaller in hypertensive rats (P< 0.05); however, arterioles from both groups responded to 11,12-EET-SI (100 nmol) with similar 15-17% increases in diameter. As we previously demonstrated, the afferent arteriolar reactivity to angiotensin II was enhanced in angiotensin II-infused animals. Interestingly, elevation of 11,12-EET-SI levels to 100 nmol reversed the enhanced vascular reactivity to angiotensin II associated with angiotensin II hypertension. Renal microvascular EET levels were not different between groups and averaged 81 +/- 9 and 87 +/- 13 pg/mg per 30 min in normotensive and hypertensive animals, respectively. Renal cortical microsomal levels of the epoxygenase CYP2C23 and CYP2C11 proteins were also similar in normotensive and angiotensin II hypertensive rats. CONCLUSIONS: Taken together, these data support the concept that renal microvascular 11,12-EET activity and levels may not properly offset the enhanced angiotensin II renal vasoconstriction during angiotensin II hypertension.
Assuntos
Angiotensina II/farmacologia , Circulação Renal/efeitos dos fármacos , Sulfonamidas/farmacologia , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/sangue , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animais , Arteríolas/fisiopatologia , Citocromo P-450 CYP2J2 , Sistema Enzimático do Citocromo P-450/metabolismo , Sinergismo Farmacológico , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Córtex Renal/metabolismo , Masculino , Microcirculação/efeitos dos fármacos , Microssomos/metabolismo , Ratos , Ratos Sprague-Dawley , Sulfonamidas/sangue , VasoconstriçãoRESUMO
This study tested the hypothesis that P2X receptor activation increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in preglomerular microvascular smooth muscle cells (MVSMC) by evoking voltage-dependent calcium influx. MVSMC were obtained and loaded with the calcium-sensitive dye fura 2 and studied by using single-cell fluorescence microscopy. The effect of P2X receptor activation on [Ca(2+)](i) was assessed by using the P2X receptor-selective agonist alpha,beta-methylene-ATP and was compared with responses elicited by the endogenous P2 receptor agonist ATP. alpha,beta-Methylene-ATP increased [Ca(2+)](i) dose dependently. Peak increases in [Ca(2+)](i) averaged 37 +/- 11, 73 +/- 15, and 103 +/- 21 nM at agonist concentrations of 0.1, 1, and 10 microM, respectively. The average peak response elicited by 10 microM alpha,beta-methylene-ATP was approximately 34% of the response obtained with 10 microM ATP. alpha,beta-Methylene-ATP induced a transient increase in [Ca(2+)](i) before [Ca(2+)](i) returned to baseline, whereas ATP induced a biphasic response including a peak response followed by a sustained plateau. In Ca(2+)-free medium, ATP induced a sharp transient increase in [Ca(2+)](i), whereas the response to alpha,beta-methylene-ATP was abolished. Ca(2+) channel blockade with 10 microM diltiazem or nifedipine attenuated the response to alpha,beta-methylene-ATP, whereas nonspecific blockade of Ca(2+) influx pathways with 5 mM Ni(2+) abolished the response. Blockade of P2X receptors with the novel P2X receptor antagonist NF-279 completely but reversibly abolished the response to alpha,beta-methylene-ATP. These results indicate that P2X receptor activation by alpha,beta-methylene-ATP increases [Ca(2+)](i) in preglomerular MVSMC, in part, by stimulating voltage-dependent Ca(2+) influx through L-type Ca(2+) channels.
Assuntos
Sinalização do Cálcio/fisiologia , Glomérulos Renais/irrigação sanguínea , Músculo Liso Vascular/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Diltiazem/farmacologia , Técnicas In Vitro , Masculino , Níquel/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (-/-) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (-/-) mice and minimizes Cyp 4a12 expression and arachidonate omega-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (-/-) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 omega-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.
Assuntos
Androgênios/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Hipertensão/enzimologia , Oxigenases de Função Mista/metabolismo , Androgênios/sangue , Animais , Ácido Araquidônico/metabolismo , Pressão Sanguínea , Castração , Citocromo P-450 CYP4A , Sistema Enzimático do Citocromo P-450/genética , Di-Hidrotestosterona/farmacologia , Indução Enzimática/efeitos dos fármacos , Feminino , Deleção de Genes , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/genética , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Oxigenases de Função Mista/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Circulação Renal/fisiologia , Caracteres Sexuais , Testosterona/farmacologia , Resistência VascularRESUMO
The proximal tubules play a critical role in providing the kidneys with their ability to regulate body fluid volume and electrolyte composition. One major function is the reabsorption of sodium and other electrolytes which is caused, in part, by angiotensin II (Ang II), a peptide that is made up of eight amino acids. By activating Ang II receptors on both the basolateral and luminal membranes, Ang II serves a major function in determining how the kidney processes.
RESUMO
BACKGROUND: Enhanced intrarenal angiotensin II (Ang II) activity may contribute to diabetic nephropathy. The proximal tubule is a proposed site of significant intrarenal Ang II production. We determined the effect of early diabetes on mRNA expression of components of the proximal tubule renin-angiotensin system. METHODS: Three groups of male Sprague-Dawley rats were studied after two weeks: (1) control (C), (2) streptozotocin-induced diabetes (STZ), and (3) STZ-induced diabetes, with normoglycemia maintained by insulin implants (STZ-I). Competitive reverse transcription-polymerase chain reaction was used to assay mRNA for renin, angiotensinogen, and angiotensin-converting enzyme in suspensions of proximal tubules; plasma and kidney levels of Ang II were measured by radioimmunoassay, and Western analysis of Ang II subtype 1 (AT1) receptors was performed. RESULTS: STZ rats tended to have increased plasma and intrarenal levels of Ang II compared with C and STZ-I rats. In proximal tubules, mRNA for renin was significantly increased in STZ rats, with reversal to control values in STZ-I rats (C, 2432 +/- 437 vs. STZ, 5688 +/- 890 fg/0.25 microg RNA, P < 0.05 vs. C, N = 9, vs. STZ-I, 1676 +/- 376 fg/0.25 microg RNA, P = NS vs. C). In STZ rats, the AT1 receptor antagonist losartan caused a further fivefold increase in proximal tubule renin mRNA, associated with proximal tubular renin immunostaining. STZ had no significant effect on mRNA expression for angiotensinogen or angiotensin-converting enzyme in proximal tubules. By Western blot analysis, cortical and proximal tubule AT1 receptor protein expression was significantly decreased in STZ rats. CONCLUSIONS: These data suggest activation of the proximal tubule renin-angiotensin system in early STZ diabetes, mediated at least partly by enhanced expression of renin mRNA. Increased local production of Ang II could contribute to tubulointerstitial injury in this model.