RESUMO
Mice deficient in the anti-oxidant enzyme glutathione peroxidase-1 (Gpx1) have a greater susceptibility to cerebral injury following a localized ischemic event. Much of the response to ischemia-reperfusion is caused by aberrant responses within the microvasculature, including inflammation, diminished endothelial barrier function (increased vascular permeability), endothelial activation, and reduced microvascular perfusion. However, the role of Gpx1 in regulating these responses has not been investigated. Wild-type and Gpx1-/- mice underwent focal cerebral ischemia via mid-cerebral artery occlusion followed by measurement of cerebral perfusion via laser Doppler and intravital microscopy. Post-ischemic brains in wild-type mice displayed significant deficit in microvascular perfusion. However, in Gpx1-/- mice, the deficit in cerebral blood flow was significantly greater than that in wild-type mice, and this was associated with significant increase in infarct size and increased vascular permeability. Ischemia-reperfusion also resulted in expression of matrix metalloproteinase-9 (MMP-9) in endothelial cells. The absence of Gpx1 was associated with marked increase in pro-MMP-9 expression as well as potentiated MMP-9 activity. Pre-treatment of Gpx1-/- mice with the anti-oxidant ebselen restored microvascular perfusion, limited the induction and activation of MMP-9, and attenuated the increases in infarct size and vascular permeability. These findings demonstrate that the anti-oxidant function of Gpx1 plays a critical role in protecting the cerebral microvasculature against ischemia-reperfusion injury by preserving microvascular perfusion and inhibiting MMP-9 expression.
Assuntos
Isquemia Encefálica/enzimologia , Artérias Cerebrais/enzimologia , Circulação Cerebrovascular/genética , Glutationa Peroxidase/genética , Microcirculação/enzimologia , Traumatismo por Reperfusão/enzimologia , Animais , Antioxidantes/farmacologia , Azóis/farmacologia , Isquemia Encefálica/genética , Isquemia Encefálica/fisiopatologia , Artérias Cerebrais/diagnóstico por imagem , Artérias Cerebrais/fisiopatologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/fisiopatologia , Isoindóis , Fluxometria por Laser-Doppler , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcirculação/diagnóstico por imagem , Microcirculação/fisiopatologia , Compostos Organosselênicos/farmacologia , Estresse Oxidativo/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/fisiopatologia , Ultrassonografia , Vasculite do Sistema Nervoso Central/enzimologia , Vasculite do Sistema Nervoso Central/genética , Glutationa Peroxidase GPX1RESUMO
The role of nitric oxide (NO)- and prostacyclin (PGI(2))-independent mechanism, potentially attributable to endothelium-derived hyperpolarizing factor (EDHF), has not been extensively studied in human skin microcirculation. The aim of our study was to elucidate the contribution of the NO- and PGI(2)-independent mechanism to microvascular reactivity of cutaneous microcirculation. Skin perfusion was measured on the volar aspect of the forearm in 12 healthy male subjects (mean age 25.0 +/- 1.5), using laser Doppler (LD) fluxmetry. Combined endothelial nitric oxide synthase (eNOS) and cyclooxygenase (COX) inhibition was achieved by an intradermal injection (10 microl) of the eNOS inhibitor, L(omega)-monomethyl L-arginine (L-NMMA, 10 mM) and the COX inhibitor, diclofenac (10 mM); saline was injected as a control. LD flux was assessed at rest and after an iontophoretical application of acetylcholine (ACh, 1%), an endothelial agonist and sodium nitroprusside (SNP, 1%), an endothelium-independent agonist, respectively. Combined eNOS and COX inhibition had no effect on the baseline LDF (12.5 +/- 2.3 PU (perfusion units) in control vs. 10.9 +/- 1.8 PU in the treated site). On the other hand, the ACh-stimulated increase in LDF was significantly attenuated after eNOS and COX inhibition (390.5 +/- 43.5%), compared to the control (643.7 +/- 80.3% increase, t-test, P < 0.05). Nevertheless, at least 60% of ACh-mediated vasodilatation was preserved after combined eNOS and COX inhibition. eNOS and COX inhibition had no impact on the SNP-stimulated increase in LDF (768.8 +/- 70.5% in control vs. 733.5 +/- 54.6% in the treated site). These findings indicate that NO- and PGI(2)-independent mechanism plays an important role in the regulation of blood flow in the human skin microcirculation.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Diclofenaco/farmacologia , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Pele/irrigação sanguínea , Vasodilatação/efeitos dos fármacos , ômega-N-Metilarginina/farmacologia , Acetilcolina/administração & dosagem , Adulto , Fatores Biológicos/metabolismo , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/administração & dosagem , Diclofenaco/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Epoprostenol/metabolismo , Antebraço , Humanos , Injeções Intradérmicas , Iontoforese , Fluxometria por Laser-Doppler , Masculino , Microcirculação/efeitos dos fármacos , Microcirculação/enzimologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Nitroprussiato/administração & dosagem , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fatores de Tempo , Vasodilatadores/administração & dosagem , ômega-N-Metilarginina/administração & dosagemRESUMO
The distribution of NADPH-diaphorase-reactive (NADPH-dr) neurons and neuronal processes in the cerebral cortex and basal forebrain and their association with parenchymal vessels were studied in normal adult rats using NADPH-d histochemical protocol. The intensely stained cortical interneurons and reactive subcortically originating afferents, and stained microvessels were examined through a light microscope at law (x250) and high (x630) magnifications. NADPH-dr interneurons were concentrated in layers 2-6 of the M1 and M2 areas. However, clear predominance in their concentration (14 +/- 0.8 P < 0.05 per section) was found in layer 6. A mean number of labeled neurons in auditory (AuV), granular and agranular (GI, AIP) areas of the insular cortex was calculated to reach 12.3 +/- 0.7, 18.5 +/- 1.0 and 23.3 +/- 1.7 units per section, respectively (P < 0.05). The distinct apposition of labelled neurons to intracortical vessels was found in the M1, M2. The order of frequency of neurovascular coupling in different zones of the cerebral cortex was as following sequence: AuV (31.2%, n = 1040) > GI (18.0%, n = 640) > S1 (13.3%, n = 720) > M1 (6.3%, n = 1360). A large number of structural associations between labeled cells and vessels in the temporal and insular cortex indicate that NADPH-d-reactive interneurons can contribute to regulation of the cerebral regional blood flow in these areas.
Assuntos
Córtex Cerebral , NADPH Desidrogenase/metabolismo , Neurônios Aferentes , Animais , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/enzimologia , Córtex Cerebral/fisiologia , Córtex Cerebral/ultraestrutura , Fibras Colinérgicas/enzimologia , Fibras Colinérgicas/fisiologia , Fibras Colinérgicas/ultraestrutura , Histocitoquímica , Técnicas In Vitro , Interneurônios/enzimologia , Interneurônios/fisiologia , Interneurônios/ultraestrutura , Masculino , Microcirculação/enzimologia , Microcirculação/fisiologia , Microcirculação/ultraestrutura , Neurônios Aferentes/enzimologia , Neurônios Aferentes/fisiologia , Neurônios Aferentes/ultraestrutura , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos WistarRESUMO
Aminopeptidase A (APA) generated brain angiotensin III, one of the main effector peptides of the brain renin angiotensin system, exerting a tonic stimulatory effect on the control of blood pressure in hypertensive rats. The distribution of APA in human brain has not been yet studied. We first biochemically characterized human brain APA (apparent molecular mass of 165 and 130 kDa) and we showed that the human enzyme exhibited similar enzymatic characteristics to recombinant mouse APA. Both enzymes had similar sensitivity to Ca(2+). Kinetic studies showed that the K(m) (190 mumol/L) of the human enzyme for the synthetic substrate-l-glutamyl-beta-naphthylamide was close from that of the mouse enzyme (256 mumol/L). Moreover, various classes of inhibitors including the specific and selective APA inhibitor, (S)-3-amino-4-mercapto-butyl sulfonic acid, had similar inhibitory potencies toward both enzymes. Using (S)-3-amino-4-mercapto-butyl sulfonic acid, we then specifically measured the activity of APA in 40 microdissected areas of the adult human brain. Significant heterogeneity was found in the activity of APA in the various analyzed regions. The highest activity was measured in the choroids plexus and the pineal gland. High activity was also detected in the dorsomedial medulla oblongata, in the septum, the prefrontal cortex, the olfactory bulb, the nucleus accumbens, and the hypothalamus, especially in the paraventricular and supraoptic nuclei. Immunostaining of human brain sections at the level of the medulla oblongata strengthened these data, showing for the first time a high density of immunoreactive neuronal cell bodies and fibers in the motor hypoglossal nucleus, the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the Roller nucleus, the ambiguus nucleus, the inferior olivary complex, and in the external cuneate nucleus. APA immunoreactivity was also visualized in vessels and capillaries in the dorsal motor nucleus of the vagus and the inferior olivary complex. The presence of APA in several human brain nuclei sensitive to angiotensins and involved in blood pressure regulation suggests that APA in humans is an integral component of the brain renin angiotensin system and strengthens the idea that APA inhibitors could be clinically tested as an additional therapy for the treatment of certain forms of hypertension.
Assuntos
Angiotensinas/metabolismo , Vias Autônomas/enzimologia , Pressão Sanguínea/fisiologia , Encéfalo/enzimologia , Glutamil Aminopeptidase/metabolismo , Neurônios/enzimologia , Adulto , Idoso , Animais , Vias Autônomas/anatomia & histologia , Encéfalo/anatomia & histologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Evolução Molecular , Feminino , Glutamil Aminopeptidase/química , Glutamil Aminopeptidase/isolamento & purificação , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Masculino , Camundongos , Microcirculação/enzimologia , Pessoa de Meia-Idade , Neuroquímica/métodos , Especificidade da EspécieRESUMO
Matrix metalloproteinase-9 (MMP-9) activation plays an important role in blood-brain barrier (BBB) dysfunction after central nervous system injury. Oxidative stress is also implicated in the pathogenesis after cerebral ischemia and spinal cord injury (SCI), but the relationship between MMP-9 activation and oxidative stress after SCI has not yet been clarified. We examined MMP-9 expression after SCI using copper/zinc-superoxide dismutase (SOD1) transgenic (Tg) rats. Our results show that MMP-9 activity significantly increased after SCI in both SOD1 Tg rats and their wild-type (Wt) littermates, although the increase was less in the SOD1 Tg rats. This pattern of MMP-9 expression was further confirmed by immunostaining and Western blot analysis. In situ zymography showed that gelatinolytic activity increased after SCI in the Wt rats, while the increase was less in the Tg rats. Evans blue extravasation increased in both the Wt and Tg rats, but was less in the SOD1 Tg rats. Inhibitor studies showed that, with an intrathecal injection of SB-3CT (a selective MMP-2/MMP-9 inhibitor), the MMP activity, Evans blue extravasation, and apoptotic cell death decreased after SCI. We conclude that increased oxidative stress after SCI leads to MMP-9 upregulation, BBB disruption, and apoptosis, and that overexpression of SOD1 in Tg rats decreases oxidative stress and further attenuates MMP-9 mediated BBB disruption.
Assuntos
Barreira Hematoencefálica/enzimologia , Células Endoteliais/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Degeneração Neural/enzimologia , Estresse Oxidativo/genética , Traumatismos da Medula Espinal/enzimologia , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Barreira Hematoencefálica/fisiopatologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Inibidores Enzimáticos/farmacologia , Azul Evans/farmacocinética , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Metaloproteinase 9 da Matriz/genética , Microcirculação/enzimologia , Microcirculação/patologia , Microcirculação/fisiopatologia , Degeneração Neural/genética , Degeneração Neural/fisiopatologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/irrigação sanguínea , Medula Espinal/enzimologia , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/fisiopatologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Regulação para Cima/genéticaRESUMO
Rho kinase (ROCK) and nitric oxide (NO) are important targets in cardiovascular diseases. Therefore, we investigated the possible influence of NO on Rho kinase (ROCK-2 isoform) expressions in cultured rat coronary microvascular endothelial cells. The cells were isolated from Wistar rats on a Langendorff system, and were incubated overnight (approximately 16 h) with an NO generator, A-23187 (10 to 10 M), NO donors, such as sodium nitroprusside (10 to 10 M), glyceryl trinitrate (10 to 10 M), 2,2'-(hydroxynitrosohydrazono)bis-ethanimine (10 to 10 M), and NaNO2 (10 to 10 M) or a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methylester (2 x 10 M), or two ROCK inhibitors, (+)-(R)-trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10 M) and fasudil (10 M) in the absence or presence of thrombin (4 U/mL). ROCK-2 and endothelial NOS (eNOS) expressions were detected by Western blotting. Moreover, nitrite/nitrate levels were detected by Griess method in the presence of the ROCK inhibitors. The NO donors and the NO generator had no significant effects on ROCK-2 expression. Y-27632 and fasudil did not alter eNOS expression and NO production. Nitrite/nitrate levels were 4.4 +/- 0.32 microM in control and 4.0 +/- 0.93 microM and in Y-27632 group. These results demonstrate that prolong NO donation could not suppress the expression of ROCK-2 protein, and the ROCK inhibitor did not change e-NOS expression and NO production in the cultured rat coronary microvascular endothelial cells.
Assuntos
Vasos Coronários/enzimologia , Células Endoteliais/enzimologia , Óxido Nítrico/fisiologia , Quinases Associadas a rho/biossíntese , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Animais , Calcimicina/farmacologia , Células Cultivadas , Vasos Coronários/citologia , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Técnicas In Vitro , Masculino , Microcirculação/citologia , Microcirculação/enzimologia , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroglicerina/farmacologia , Nitroprussiato/farmacologia , Piridinas/farmacologia , Ratos , Ratos Wistar , Nitrito de Sódio/farmacologia , Triazenos/farmacologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
1. Endothelial nitric oxide synthase (NOS3) is important for vascular homeostasis. The role of protein kinase G (PKG) in regulation of NOS3 activity was studied in primary cultures of newborn lamb lung microvascular endothelial cells (LMVEC). 2. We determined the presence of PKG in fetal and neonatal LMVEC as well as subcellular localization of PKG isoforms in the neonatal cells by fluorescence immunohistochemistry. We used diaminofluorescein (DAF) fluorophore to measure nitric oxide (NO) production from neonatal LMVEC. We confirmed that NO measured was from constitutive NOS3 by inhibiting it with NOS inhibitors. 3. To identify a role for PKG in basal NO production, we measured NO release from LMVEC cells using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM; 0.5-0.8 micromol/L) with and without prior stimulation with the PKG activator 8-bromo-cGMP (8-Br-cGMP; 0.3 and 3 micromol/L) or prior PKG inhibition with beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (BPC; 0.3 and 3 micromol/L). With the same drugs, we determined the role of PKG on cellular expression of NOS3 and serine 116 phosphorylated NOS (pSer116-NOS) by qualitative and quantitative immunofluorescence assays, as well as western blotting. 4. Because PKG 1 beta was distributed throughout the cytosol in a punctate expression, we used 2 mmol/L cyclodextrin, a cholesterol extractor, to determine a role for lipid vesicles in PKG regulation of NO production. 5. Protein kinase G 1 beta gave a punctate appearance, indicating its presence in intracellular vesicles. Nitric oxide production decreased by approximately 20% with 300 nmol/L and 3 micromol/L 8-Br cGMP (P < 0.05) and increased by 20.8 +/- 3.7% with 3 micromol/L BPC (P < 0.001), indicating that both stimulated and basal PKG activity has inhibitory effects on basal NOS3 function. Nitric oxide synthase immunofluorescence and immunoblot expression were decreased and pSer116-NOS immunofluorescence was increased by 800 nmol/L 8-Br-cGMP and 170 micromol/L (Z)-1-[2-(2-aminoethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1, 2-diolate (DETANONOate). The effect of cyclodextrin indicated that cholesterol extraction interfered with PKG inhibition of NOS. Further examination of pSer116-NOS by immunohistochemistry showed it abundant in the endoplasmic reticulum and colocalized with PKG 1 beta, especially in nuclear vesicles. 6. We conclude that endothelial PKG is involved in endogenous regulation of basal NOS3 activity with the involvement of lipid structures, the endoplasmic reticulum and the nucleus. Protein kinase G 1 beta is colocalized with pSer116-NOS, indicating that PKG action may involve serine 116 phosphorylation on NOS.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Células Endoteliais/enzimologia , Pulmão/irrigação sanguínea , Lipídeos de Membrana/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Organelas/enzimologia , Animais , Animais Recém-Nascidos , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Ciclodextrinas/farmacologia , Citosol/enzimologia , Relação Dose-Resposta a Droga , Retículo Endoplasmático/enzimologia , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Fluoresceínas , Corantes Fluorescentes , Pulmão/embriologia , Microcirculação/enzimologia , Microscopia de Fluorescência/métodos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Compostos Nitrosos/farmacologia , Organelas/efeitos dos fármacos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Subunidades Proteicas/metabolismo , Transporte Proteico , Serina/metabolismo , Ovinos , Fatores de TempoRESUMO
NADPH-diaphorase histochemistry was used to study the distribution and density of labeled neurons in the limbic structures and hypothalamus in intact rat. NADPH-diaphorase positive neurons were registered in the basal forebrain-medial septal nucleus (MS), the nuclei of the diagonal band of Broca (VDB, HDB), substancia innominata (SI) and the nucleus basalis of Meynert (B). These areas largely overlap with the cholinergic CH1-CH4 forebrain system of the rodent brain. The order of density of labeled neurons in different regions of the basal forebrain was as following sequence: HDB > VDB > SI > B. The highest densities of the reactive neurons (> 1000 labeled neurons per section 200x200 microm2) was found in the islands of Calleja (ICjs). In the supraoptic (SO) and paraventricular (Pa) nuclei of hypothalamus were recorded > 130 and > 100 labeled units, respectively. The lowest density of labeled neurons was recorded within the SI-B complex: < 10 reactive units. Reactive neurons, their dendrites and axon-like processes within the ICjs, SO, Pa, the lateral nucleus hypothalamus (LH) often surround arterioles which traverse the structures. We suggest that NADPH-diaphorase-reactive (NO-generating) neurons within the ICjs and hypothalamus are involved in regulation of the regional, blood flow (RBF) that is important to adapt the blood flow to changes in neuronal activity of the basal forebrain structures.
Assuntos
Hipotálamo/enzimologia , NADPH Desidrogenase/metabolismo , Neurônios/enzimologia , Animais , Hipotálamo/irrigação sanguínea , Hipotálamo/citologia , Imuno-Histoquímica , Sistema Límbico/irrigação sanguínea , Sistema Límbico/citologia , Sistema Límbico/enzimologia , Masculino , Microcirculação/enzimologia , Microcirculação/metabolismo , Óxido Nítrico/biossíntese , Prosencéfalo/irrigação sanguínea , Prosencéfalo/citologia , Prosencéfalo/enzimologia , Ratos , Ratos WistarRESUMO
Data providing direct evidence for a causative link between endothelial dysfunction, microvascular disease and diabetic end-organ damage are scarce. Here we show that activated protein C (APC) formation, which is regulated by endothelial thrombomodulin, is reduced in diabetic mice and causally linked to nephropathy. Thrombomodulin-dependent APC formation mediates cytoprotection in diabetic nephropathy by inhibiting glomerular apoptosis. APC prevents glucose-induced apoptosis in endothelial cells and podocytes, the cellular components of the glomerular filtration barrier. APC modulates the mitochondrial apoptosis pathway via the protease-activated receptor PAR-1 and the endothelial protein C receptor EPCR in glucose-stressed cells. These experiments establish a new pathway, in which hyperglycemia impairs endothelial thrombomodulin-dependent APC formation. Loss of thrombomodulin-dependent APC formation interrupts cross-talk between the vascular compartment and podocytes, causing glomerular apoptosis and diabetic nephropathy. Conversely, maintaining high APC levels during long-term diabetes protects against diabetic nephropathy.
Assuntos
Apoptose , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/prevenção & controle , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/prevenção & controle , Endotélio Vascular/patologia , Podócitos/patologia , Proteína C/fisiologia , Substituição de Aminoácidos/genética , Animais , Apoptose/genética , Linhagem Celular Transformada , Células Cultivadas , Citoproteção/genética , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/genética , Endotélio Vascular/enzimologia , Ativação Enzimática/genética , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/enzimologia , Glomérulos Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Microcirculação/enzimologia , Microcirculação/patologia , Podócitos/enzimologia , Proteína C/biossíntese , Proteína C/genética , Transdução de Sinais/genética , Trombomodulina/fisiologiaRESUMO
Transient reduction in coronary perfusion pressure in the isolated mouse heart increases microvascular resistance (paradoxical vasoconstriction) by an endothelium-mediated mechanism. To assess the presence and extent of paradoxical vasoconstriction in hearts from normal and diabetic rats and to determine whether increased heme oxygenase (HO)-1 expression and HO activity, using cobalt protoporphyrin (CoPP), attenuates coronary microvascular response, male Wistar rats were rendered diabetic with nicotinamide/streptozotocin for 2 wk and either CoPP or vehicle was administered by intraperitoneal injection weekly for 3 wk (0.5 mg/100 g body wt). The isolated beating nonworking heart was submitted to transient low perfusion pressure (20 mmHg), and coronary resistance (CR) was measured. During low perfusion pressure, CR increased and was associated with increased lactate release. In diabetic rats, CR was higher, HO-1 expression and endothelial nitric oxide synthase were downregulated, and inducible nitric oxide synthase and O(2)(-) were upregulated. After 3 wk of CoPP treatment, HO activity was significantly increased in the heart. Upregulation of HO-1 expression and HO activity by CoPP resulted in the abolition of paradoxical vasoconstriction and a reduction in oxidative ischemic damage. In addition, there was a marked increase in serum adiponectin. Elevated HO-1 expression was associated with increased expression of cardiac endothelial nitric oxide synthase, B-cell leukemia/lymphoma extra long, and phospho activator protein kinase levels and decreased levels of inducible nitric oxide synthase and malondialdehyde. These results suggest a critical role for HO-1 in microvascular tone control and myocardial protection during ischemia in both normal and mildly diabetic rats through the modulation of constitutive and inducible nitric oxide synthase expression and activity, and an increase in serum adiponectin.
Assuntos
Adiponectina/sangue , Fármacos Cardiovasculares/farmacologia , Vasos Coronários/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Heme Oxigenase (Desciclizante)/biossíntese , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/enzimologia , Protoporfirinas/farmacologia , Animais , Fármacos Cardiovasculares/uso terapêutico , Vasos Coronários/enzimologia , Vasos Coronários/fisiopatologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/fisiopatologia , Indução Enzimática , Ácido Láctico/metabolismo , Masculino , Malondialdeído/metabolismo , Microcirculação/efeitos dos fármacos , Microcirculação/enzimologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Niacinamida , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo III , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Protoporfirinas/uso terapêutico , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Estreptozocina , Superóxidos/metabolismo , Fatores de Tempo , Regulação para Cima , Resistência Vascular/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Proteína bcl-X/metabolismoRESUMO
In the present study, we assessed the effects of chemical inhibitors shown to be selective for protein kinase C (PKC) isoforms on lung barrier function both in vitro and in vivo. Rottlerin, a purported inhibitor of PKCdelta, but not other chemical inhibitors, dose dependently promoted barrier dysfunction in lung endothelial cells in vitro. This barrier dysfunction correlated with structural changes in focal adhesions and stress fibers, which were consistent with functional changes in cell stiffness. To determine whether the effects noted in vitro correlated with changes in intact lungs, we tested the effects of rottlerin in the formation of pulmonary edema in rats using both ex vivo and in vivo models. Isolated, perfused lungs demonstrated a significant increase in filtration coefficients on exposure to rottlerin, compared with vehicle-treated lungs, an effect that correlated with increased extravasation of Evan's blue dye (EBD)-conjugated albumin. Additionally, compared with vehicle, the ratio of the wet lung weights to dry lung weights was significantly greater on exposure of animals to rottlerin; rottlerin also produced a dose-dependent increase in EBD extravasation into the lungs. These effects on lung edema occurred without any increase in right ventricular pressures. Microscopic assessment of edema in the ex vivo lungs demonstrated perivascular cuffing, with no evidence of septal capillary leak, in rottlerin-exposed lungs. Taken together, rottlerin increases barrier dysfunction in pulmonary endothelial cell monolayers and causes pulmonary edema in rats; results suggestive of an important role for PKCdelta in maintaining lung endothelial barrier function.
Assuntos
Acetofenonas/toxicidade , Benzopiranos/toxicidade , Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Pulmão/irrigação sanguínea , Proteína Quinase C-delta/antagonistas & inibidores , Inibidores de Proteínas Quinases/toxicidade , Edema Pulmonar/induzido quimicamente , Actomiosina/metabolismo , Animais , Carbazóis/toxicidade , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/enzimologia , Células Endoteliais/ultraestrutura , Azul Evans , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Indóis/toxicidade , Masculino , Maleimidas/toxicidade , Microcirculação/efeitos dos fármacos , Microcirculação/enzimologia , Proteína Quinase C-delta/metabolismo , Edema Pulmonar/enzimologia , Edema Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
BACKGROUND: Abnormalities of endogenous fibrinolysis are linked to diabetic macrovascular disease; whether key vascular endothelial regulatory proteins, such as tissue plasminogen activator (tPA), are altered in diabetic neuropathy microvasculature is unknown. This neuropathologic case: control study investigates the hypothesis that tPA expression is regionally deficient in microvessels in human diabetic neuropathy. METHODS: tPA and von Willebrand factor (vWF), a vascular endothelial cell marker, are detected on vascular endothelium by immunoperoxidase methods with specific antibodies on formalin fixed paraffin embedded sural nerve biopsies from six diabetic and six axonal neuropathy control nerves without vasculopathy. The proportion of microvessels in each nerve region expressing tPA is determined by the ratio of tPA positive vessels/total vWF positive vessels on serial sections. RESULTS: tPA expression is lower in diabetic neuropathy cases compared to controls in all regions, including epineurial (62.4 +/- 8.6% vs 91.0 +/- 1.6%, p < 0.02) and endoneurial microvessels (51.7 +/- 7.1% vs 91.5 +/- 2.9%, p < 0.001). CONCLUSIONS: These results demonstrate a four- to sixfold increase in the number of peripheral nerve microvessels lacking immunodetectable tissue plasminogen activator in the epineurial and endoneurial vessels in diabetes, suggesting that impaired endogenous fibrinolysis might contribute to microvascular ischemia in human diabetic neuropathy.
Assuntos
Neuropatias Diabéticas/enzimologia , Nervo Sural/irrigação sanguínea , Nervo Sural/enzimologia , Ativador de Plasminogênio Tecidual/metabolismo , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/enzimologia , Neuropatias Diabéticas/fisiopatologia , Endotélio Vascular/enzimologia , Humanos , Microcirculação/enzimologia , Microcirculação/fisiopatologia , Nervo Sural/fisiopatologia , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tecidual/deficiênciaRESUMO
Elevated oxidative stress plays a key role in diabetes-associated vascular disease. In this study, we tested the hypothesis that high glucose-induced oxidative stress was associated with changes in the expression of NADPH oxidase, superoxide dismutase (SOD) and endothelial nitric oxide synthase (eNOS). Oxidative stress was assessed in cell cultures of mouse microvessel endothelial cells (MMECs) by fluorescence labelling with dihydroethidium, lucigenin-enhanced chemiluminescence and determining NADPH oxidase subunit and eNOS expression with real-time polymerase chain reaction protocol and Western blotting. Oxidative stress and expression of the NADPH oxidase subunit, p22phox, were both increased, SOD1 and 3 expression lowered and eNOS significantly elevated in MMECs treated with 40 mM glucose for 72 h compared to low glucose medium. Oxidative stress, p22phox mRNA, eNOS mRNA, and protein were lowered by concurrent incubation with sepiapterin. When eNOS protein expression in endothelial cells was significantly decreased by eNOS siRNA treatment, superoxide generation was significantly higher in the MMECs grown in low glucose, but reduced in those grown in high glucose for 72 h. Thus, exposure of MMECs to high glucose results in increased oxidative stress that is associated with increased eNOS and NADPH oxidase subunit expression, notably p22phox, and decreased expression of SOD1 and 3.
Assuntos
Grupo dos Citocromos b/metabolismo , Células Endoteliais/metabolismo , Glucose/metabolismo , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo , Acetofenonas/farmacologia , Alcaloides/farmacologia , Animais , Benzofenantridinas/farmacologia , Células Cultivadas , Grupo dos Citocromos b/antagonistas & inibidores , Grupo dos Citocromos b/genética , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Glucose/farmacologia , Camundongos , Microcirculação/citologia , Microcirculação/enzimologia , Microcirculação/metabolismo , NADPH Oxidase 4 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III , Estresse Oxidativo/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Pterinas/farmacologia , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Superóxidos/metabolismo , TransfecçãoRESUMO
Sepsis-induced acute lung injury (ALI) is characterized by injury of the pulmonary microvascular endothelial cells (PMVEC) leading to high-protein pulmonary edema. Inducible NO synthase (iNOS) mediates trans-PMVEC protein leak in septic mice in vivo and in murine PMVEC under septic conditions in vitro, but the role of iNOS in human PMVEC protein leak has not been addressed. We hypothesized that iNOS in human neutrophils, but not human PMVEC, mediates septic trans-PMVEC protein leak in vitro. We isolated human PMVEC from lung tissue using magnetic bead-bound anti-PECAM antibody and assessed Evans blue albumin leak across human PMVEC monolayers under septic conditions in the presence/absence of human neutrophils. PMVEC were used at passages 3-4, seeded on 3 mum Transwell inserts and grown to confluence. Cytomix-stimulated trans-PMVEC albumin leak was not attenuated by pre-treatment with 1400 W, a selective iNOS inhibitor, or l-NAME, a non-selective NOS inhibitor. In neutrophil-PMVEC co-culture, basal unstimulated trans-EB-albumin leak was 0.6+/-0.3%, which was increased by cytomix stimulation to 11.5+/-4.4%, p<0.01. Cytomix-stimulated EB-albumin leak in neutrophil-PMVEC co-cultures was inhibited by pre-treatment with 1400 W (3.8+/-1.0%, p<0.05) or l-NAME (4.0+/-1.1%, p<0.05). Pre-treatment of neutrophil-PMVEC co-cultures with PEG-SOD (superoxide scavenger) and FeTPPS (peroxynitrite scavenger) also significantly attenuated neutrophil-dependent cytomix-stimulated leak (4.7+/-3.0%, p<0.05; 0.5+/-1.0%, p<0.01, respectively). In conclusion, trans-human PMVEC albumin leak under septic conditions is dependent on iNOS activity specifically in neutrophils, but not in PMVEC themselves. Septic neutrophil-dependent trans-PMVEC albumin leak may be mediated by peroxynitrite.
Assuntos
Albuminas/metabolismo , Endotélio Vascular/enzimologia , Pulmão/irrigação sanguínea , Neutrófilos/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Sepse/enzimologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Humanos , Microcirculação/efeitos dos fármacos , Microcirculação/enzimologia , NG-Nitroarginina Metil Éster/farmacologia , Neutrófilos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Espermina/análogos & derivados , Espermina/farmacologiaRESUMO
The aim of this study was to examine the effects of posthemorrhagic hypovolemia and hypotension upon the microvascular endothelial cells and on activity of antioxidant enzymes in blood, and to investigate the influence of intravenously injected endothelin-1 in rats. The experiment was conducted on 48 rats anesthetized with ethylurethane, subjected to controlled hypotension (under 35-40 mmHg) for 60 min. Endothelin-1 was administered intravenously once at a dose of 50 pmol/kg in the 5th min of hemorrhagic shock. The control group had blood volume restored after 5 min of hypovolemia with hypotension. The arterial blood pressure, systolic and diastolic, and heart rate were monitored. After 60 min, morphological changes in the capillary endothelium of the small intestine were assessed, using electron microscope, and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) in blood were measured. Animals with hypovolemia and hypotension, had edematous endothelial cells with injured cell-membrane and mitochondria, alongside of the enhancement in SOD activity (p < 0.05) and drop in the activity of CAT and GSH-PX. No signs of vascular endothelium injuries and no reduced enzymatic activities, except for GSH-PX, were observed after restoring the normal blood pressure by means of endothelin-1 in animals with hypovolemia. Hemorrhagic shock caused injuries in intestinal microcascular endothelium. Intravenously administered endothelin-1 quickly restored normal blood pressure, maintained it over a long time, and prevented the consequences of ischemia in microcirculation, thereby prolonging the survival for animals in hemorrhagic shock.
Assuntos
Endotelina-1/uso terapêutico , Endotélio Vascular/efeitos dos fármacos , Choque Hemorrágico/tratamento farmacológico , Animais , Antioxidantes/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Endotelina-1/administração & dosagem , Endotélio Vascular/enzimologia , Endotélio Vascular/ultraestrutura , Frequência Cardíaca/efeitos dos fármacos , Injeções Intravenosas , Intestino Delgado/irrigação sanguínea , Intestino Delgado/efeitos dos fármacos , Masculino , Microcirculação/efeitos dos fármacos , Microcirculação/enzimologia , Microcirculação/ultraestrutura , Microscopia Eletrônica de Transmissão , Ratos , Ratos Wistar , Choque Hemorrágico/patologia , Choque Hemorrágico/fisiopatologiaRESUMO
The mortality rate for septic patients with acute renal failure is approximately doubled compared with patients with sepsis alone. Unfortunately, the treatment for sepsis-induced renal failure has advanced little during the last several decades. Because sepsis is often caused by lipopolysaccharide (LPS), a mouse model of LPS challenge was used to study the development of kidney injury. We hypothesized that inducible nitric-oxide synthase (iNOS)-catalyzed nitric oxide production and that generation of reactive nitrogen species (RNS) might play a role in the microcirculatory defect and resulting tubular injury associated with LPS administration. Fluorescent intravital videomicroscopy was used to assess renal peritubular capillary perfusion and document RNS generation by renal tubules in real time. As early as 6 h after LPS administration (10 mg/kg i.p.), RNS generation (rhodamine fluorescence), redox stress [NAD(P)H autofluorescence], and the percentage of capillaries without flow were each significantly increased compared with saline-treated mice (p < 0.05). The generation of RNS was supported by the detection of nitrotyrosine-protein adducts in the kidney using immunohistochemistry. The iNOS inhibitor l-N(6)-(1-iminoethyl)-lysine (l-NIL; 3 mg/kg i.p.) completely blocked the increase in rhodamine fluorescence and NAD(P)H autofluorescence and prevented the capillary defects at 6 h after LPS administration. These results suggest that iNOS-derived RNS is an important contributor to the peritubular capillary perfusion defects and RNS generation that occur during sepsis and emphasize that pharmacological inhibition of iNOS may provide beneficial effects during sepsis by improving renal capillary perfusion and reducing RNS generation in the kidney.
Assuntos
Inibidores Enzimáticos/farmacologia , Nefropatias , Rim/irrigação sanguínea , Lisina/análogos & derivados , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Espécies Reativas de Nitrogênio/metabolismo , Animais , Imuno-Histoquímica , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/metabolismo , Nefropatias/enzimologia , Nefropatias/metabolismo , Nefropatias/fisiopatologia , Lipopolissacarídeos , Lisina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação/efeitos dos fármacos , Microcirculação/enzimologia , Microcirculação/metabolismo , Microscopia de Vídeo , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
We determined the effect of oxygen [approximately 100 Torr (normoxia) and approximately 30-40 Torr (hypoxia)] on functions of endothelial nitric oxide (NO) synthase (NOS-3) and its negative regulator caveolin-1 in ovine fetal and neonatal lung microvascular endothelial cells (MVECs). Fetal NOS-3 activity, measured as NO production with 0.5-0.9 microM 4-amino-5-methylamino-2,7-difluorofluorescein, was decreased in hypoxia by 14.4% (P < 0.01), inhibitable by the NOS inhibitor N-nitro-L-arginine, and dependent on extracellular arginine. Caveolar function, assessed as FITC-BSA (160 microg/ml) endocytosis, was decreased in hypoxia by 13.5% in fetal and 22.8% in neonatal MVECs (P < 0.01). NOS-3 and caveolin-1 were physically associated, as demonstrated by coimmunoprecipitation and colocalization, and functionally associated, as shown by cross-activation of endocytosis, by their specific antibodies and activation of NOS by albumin. Caveolin peptide, containing the sequence for the PKC phosphorylation site of caveolin, and caveolin antiserum against the site increased NO production and endocytosis by 12.3% (P < 0.05) and 16% (P < 0.05), respectively, in normoxia and increased endocytosis by 25% (P < 0.001) in hypoxia. PMA decreased NO production in normoxia and hypoxia by 19.32% (P < 0.001) and 11.8% (P < 0.001) and decreased endocytosis in normoxia by 20.35% (P < 0.001). PKC kinase activity was oxygen sensitive, and threonine phosphorylation was enhanced in hypoxia. Pertussis toxin increased caveolar and NOS functions. These data support our hypothesis that increased Po(2) at birth promotes dissociation of caveolin-1 and NOS-3, with an increase in their activities, and that PKC and an oxygen-sensitive cell surface G protein-coupled receptor regulate caveolin-1 and NOS-3 interactions in fetal and neonatal lung MVECs.
Assuntos
Caveolina 1/metabolismo , Células Endoteliais/enzimologia , Hipóxia/metabolismo , Pulmão/irrigação sanguínea , Óxido Nítrico Sintase Tipo III/metabolismo , Oxigênio/farmacologia , Animais , Especificidade de Anticorpos , Caveolina 1/imunologia , Células Cultivadas , Regulação para Baixo/fisiologia , Endocitose/fisiologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Hipóxia/tratamento farmacológico , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Microcirculação/efeitos dos fármacos , Microcirculação/enzimologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/imunologia , Toxina Pertussis/farmacologia , Fosforilação , Gravidez , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Serina/metabolismo , Ovinos , Treonina/metabolismoAssuntos
Antioxidantes/fisiologia , Endotélio Vascular/enzimologia , Heme Oxigenase-1/fisiologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Óxido Nítrico/fisiologia , Ativação Enzimática/fisiologia , Heme Oxigenase-1/biossíntese , Humanos , Microcirculação/enzimologia , NADPH Oxidase 1RESUMO
NO is known to induce expression of heme oxygenase-1, an antioxidant enzyme in blood vessels. We tested whether NO might modulate the endothelial NADPH oxidase function via heme oxygenase-1. In human microvascular endothelial cells, the NO donor DETA-NONOate (0.1 to 1 mmol/L) strongly induced expression of heme oxygenase-1 but not Cu/Zn superoxide dismutase. This was associated with a reduction of the superoxide-generating capacity of NADPH oxidase, an effect that depended on de novo gene transcription and heme oxygenase-1 activity. Activation of NADPH oxidase by tumor necrosis factor (TNF) alpha increased generation of reactive oxygen species. DETA-NONOate alone had little effect on TNF-stimulated reactive oxygen species, but it enhanced the TNF response when: (1) heme oxygenase-1 expression was blocked with specific small-interfering RNA; (2) heme oxygenase-1 activity was blocked by zinc-protoporphyrin; or (3) NADPH oxidase activity was blocked by diphenyleneiodonium. Moreover, the heme oxygenase-1 end product bilirubin directly inhibited fully functional NADPH oxidase and seemed to interrupt the assembly and activation of the oxidase. In conclusion, NO may modulate superoxide production by NADPH oxidase in human vascular endothelial cells, at least partly by inducing heme oxygenase-1. Our results indicate that suppression of NADPH oxidase-dependent reactive oxygen species formation may represent a novel mechanism underlying the cardiovascular protective actions of heme oxygenase-1 and bilirubin.
Assuntos
Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Heme Oxigenase-1/fisiologia , NADPH Oxidases/metabolismo , Óxido Nítrico/fisiologia , Células Cultivadas , Endotélio Vascular/metabolismo , Indução Enzimática/fisiologia , Heme Oxigenase-1/biossíntese , Humanos , Líquido Intracelular/metabolismo , Microcirculação/citologia , Microcirculação/enzimologia , Microcirculação/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/fisiologia , Doadores de Óxido Nítrico/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/antagonistas & inibidoresRESUMO
OBJECTIVE: Oxygen free radical production in hypertension may be associated with elevated arteriolar tone and organ injury. Previous results suggest an enhanced level of oxygen free radical formation in microvascular endothelium and in circulating neutrophils associated with xanthine oxidase activity in the spontaneously hypertensive rats (SHR) compared with their normotensive controls, the Wistar Kyoto rats (WKY). The aim of this study was to gain more detailed understanding of where oxidative enzymes are located in the microcirculation. METHODS: An approach was developed to delineate the cellular distribution of two selected oxidative enzymes, xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH) dependent oxidase (protein 67-kDa fraction). Immunolabeling with peroxidase substrate was utilized, which permits full delineation of the primary antibody in all microvascular structures of the mesentery. RESULTS: Xanthine oxidase is present in the endothelium of all segments of the microcirculation, in mast cells, and in parenchymal cells of the mesentery. NADPH oxidase can be detected in the endothelium, leukocytes, and mast cells and with lower levels in parenchymal cells. The mesentery of WKY and SHR has similar enzyme distributions with enhancements on the arteriolar and venular side of the microcirculation that coincide with the sites of enhanced free radical production recently reported. Immune label measurements under standardized conditions indicate that both enzymes are significantly enhanced in the SHR. Adrenalectomy, which serves to reduce the blood pressure and free radical production of the SHR to normotensive levels, leads to a reduction of NADPH and xanthine oxidase to normotensive levels, while supplementation of adrenalectomized SHR with dexamethasone significantly increases the oxidase expression in several parts of the microcirculation to levels above the WKY rats. CONCLUSION: The results indicate that enhanced expression of NADPH and xanthine oxidase in the SHR depends on an adrenal pathway that is detectable in the arteriolar and venular network at high and low pressure regions of the circulation.