6ß-Hydroxytestosterone, a metabolite of testosterone generated by CYP1B1, contributes to vascular changes in angiotensin II-induced hypertension in male mice.

BACKGROUND: Previously, we showed that 6ß-hydroxytestosterone (6ß-OHT), a cytochrome P450 1B1 (CYP1B1)-derived metabolite of testosterone, contributes to angiotensin II (Ang II)-induced hypertension in male mice. This study was conducted to test the hypothesis that 6ß-OHT contributes to increased vascular reactivity, endothelial dysfunction, vascular hypertrophy, and reactive oxygen species production associated with Ang II-induced hypertension. METHODS: Eight- to 10-week-old intact or castrated C57BL/6 J (Cyp1b1+/+ and Cyp1b1-/-) mice were anesthetized for implantation of a micro-osmotic pump which delivered Ang II (700 ng/kg/day) or saline for 14 days. Mice were injected with 6ß-OHT (15 µg/g b.w every third day), flutamide (8 mg/kg every day), or its vehicle. Blood pressure was measured via tail-cuff. Vascular reactivity, endothelial-dependent and endothelial-independent vasodilation, media to lumen ratio, fibrosis by collagen deposition, and reactive oxygen species production by dihydroethidium staining were determined in the isolated thoracic aorta. RESULTS: The response of thoracic aorta to phenylephrine and endothelin-1 was increased in Ang II-infused Cyp1b1+/+ mice compared to intact Cyp1b1-/- or castrated Cyp1b1+/+ and Cyp1b1-/- mice; these effects of Ang II were restored by treatment with 6ß-OHT. Ang II infusion caused endothelial dysfunction, as indicated by decreased relaxation of the aorta to acetylcholine in Cyp1b1+/+ but not Cyp1b1-/- or castrated Cyp1b1+/+ and Cyp1b1-/- mice. 6ß-OHT did not alter Ang II-induced endothelial dysfunction in Cyp1b1+/+ mice but restored it in Cyp1b1-/- or castrated Cyp1b1+/+ and Cyp1b1-/- mice. Ang II infusion increased media to lumen ratio and caused fibrosis and reactive oxygen species production in the aorta of Cyp1b1+/+ mice. These effects were minimized in the aorta of Cyp1b1-/- or castrated Cyp1b1+/+ and Cyp1b1-/- mice and restored by treatment with 6ß-OHT. Treatment with the androgen receptor antagonist flutamide reduced blood pressure and vascular hypertrophy in castrated Ang II-infused mice injected with 6ß-OHT. CONCLUSIONS: 6ß-OHT is required for the action of Ang II to increase vascular reactivity and cause endothelial dysfunction, hypertrophy, and increase in oxygen radical production. The effect of 6ß-OHT in mediating Ang II-induced hypertension and associated hypertrophy is dependent on the androgen receptor. Therefore, CYP1B1 could serve as a novel target for the development of therapeutics to treat vascular changes in hypertensive males.

Deletion of DGCR8 in VSMCs of adult mice results in loss of vascular reactivity, reduced blood pressure and neointima formation.

DiGeorge syndrome chromosomal region 8 (DGCR8), a double-stranded-RNA-binding protein, participates in the miRNA biogenesis pathway and contributes to miRNA maturation by interacting with the RNAase III enzyme Drosha in cell nuclei. To investigate the role of DGCR8 in vascular smooth muscle cells (VSMCs) at the postnatal stages, we generated tamoxifen-inducible VSMC specific knockout (iKO) mice by crossing DGCR8loxp/loxp with VSMC specific tamoxifen-inducible Cre transgenic mice SMA-Cre-ERT2. DGCR8iKO mice display reduced body weight one month following tamoxifen treatment and died around 3 months. Blood pressure and vascular reactivity were significantly reduced in DGCR8iKO mice compared to control. Furthermore, loss of DGCR8 in VSMCs inhibited cell proliferation, migration and neointima formation. VSMC differentiation marker genes, including SMA and SM22, were downregulated in DGCR8 iKO mice. The majority of miRNAs were downregulated in DGCR8iKO mice. Disruption of the DGCR8-mediated miRNA biogenesis pathway attenuated multiple signaling pathways including ERK1/2 and AKT. Our results demonstrate that the DGCR8-mediated miRNA pathway is required for maintaining blood pressure, vascular reactivity and vascular wall remodeling at the postnatal stages.

Cytochrome P450 1B1 Contributes to the Development of Atherosclerosis and Hypertension in Apolipoprotein E-Deficient Mice.

Cytochrome P450 (CYP) 1B1 contributes to vascular smooth muscle cell growth and hypertension in male mice. This study was conducted to determine the contribution of CYP1B1 to the development of atherosclerosis and hypertension and associated pathogenesis in 8-week-old male apolipoprotein E-deficient (ApoE(-/-)/Cyp1b1(+/+)), and ApoE- and CYP1B1-deficient (ApoE(-/-)/Cyp1b1(-/-)) mice fed a normal or atherogenic diet for 12 weeks. A separate group of ApoE(-/-)/Cyp1b1(+/+) mice on an atherogenic diet was injected every third day with the CYP1B1 inhibitor, 2,3',4,5'-tetramethoxystilbene (300 µg/kg), or its vehicle, dimethyl sulfoxide (30 µL, IP); systolic blood pressure was measured by the tail cuff method. After 12 weeks, mice were euthanized, blood collected for lipid analysis, and aortas harvested for measuring lesions and remodeling, and for infiltration of inflammatory cells by histological and immunohistochemical analysis, respectively, and for reactive oxygen species production. Blood pressure, areas of lipids and collagen deposition, elastin breaks, infiltration of macrophages and T lymphocytes, reactive oxygen species generation in the aorta, and plasma lipid levels were increased in ApoE(-/-)/Cyp1b1(+/+) mice on an atherogenic diet; these changes were minimized in mice given 2,3',4,5'-tetramethoxystilbene, and in ApoE(-/-)/Cyp1b1(-/-) mice on an atherogenic diet; absorption/production of lipids remained unaltered in these mice. These data suggest that aortic lesions, hypertension, and associated pathogenesis in ApoE(-/-)/Cyp1b1(+/+) mice on an atherogenic diet are most likely dependent on CYP1B1-generated oxidative stress and increased plasma lipid levels independent of blood pressure and absorption of lipids. CYP1B1 could serve as a novel target for developing drugs to treat atherosclerosis and hypertension caused by hypercholesterolemia.

Cytosolic phospholipase A2α is critical for angiotensin II-induced hypertension and associated cardiovascular pathophysiology.

Angiotensin II activates cytosolic phospholipase A(2)α (cPLA2α) and releases arachidonic acid from tissue phospholipids, which mediate or modulate ≥1 cardiovascular effects of angiotensin II and has been implicated in hypertension. Because arachidonic acid release is the rate limiting step in eicosanoid production, cPLA2α might play a central role in the development of angiotensin II-induced hypertension. To test this hypothesis, we investigated the effect of angiotensin II infusion for 13 days by micro-osmotic pumps on systolic blood pressure and associated pathogenesis in wild type (cPLA2α(+/+)) and cPLA2α(-/-) mice. Angiotensin II-induced increase in systolic blood pressure in cPLA2α(+/+) mice was abolished in cPLA2α(-/-) mice; increased systolic blood pressure was also abolished by the arachidonic acid metabolism inhibitor, 5,8,11,14-eicosatetraynoic acid in cPLA2α(+/+) mice. Angiotensin II in cPLA2α(+/+) mice increased cardiac cPLA2 activity and urinary eicosanoid excretion, decreased cardiac output, caused cardiovascular remodeling with endothelial dysfunction, and increased vascular reactivity in cPLA2α(+/+) mice; these changes were diminished in cPLA2α(-/-) mice. Angiotensin II also increased cardiac infiltration of F4/80(+) macrophages and CD3(+) T lymphocytes, cardiovascular oxidative stress, expression of endoplasmic reticulum stress markers p58(IPK), and CHOP in cPLA2α(+/+) but not cPLA2α(-/-) mice. Angiotensin II increased cardiac activity of ERK1/2 and cSrc in cPLA2α(+/+) but not cPLA2α(-/-) mice. These data suggest that angiotensin II-induced hypertension and associated cardiovascular pathophysiological changes are mediated by cPLA2α activation, most likely through the release of arachidonic acid and generation of eicosanoids with predominant prohypertensive effects and activation of ≥1 signaling molecules, including ERK1/2 and cSrc.

Disruption of the cytochrome P-450 1B1 gene exacerbates renal dysfunction and damage associated with angiotensin II-induced hypertension in female mice.

Recently, we demonstrated in female mice that protection against ANG II-induced hypertension and associated cardiovascular changes depend on cytochrome P-450 (CYP)1B1. The present study was conducted to determine if Cyp1b1 gene disruption ameliorates renal dysfunction and organ damage associated with ANG II-induced hypertension in female mice. ANG II (700 ng·kg(-1)·min(-1)) infused by miniosmotic pumps for 2 wk in female Cyp1b1(+/+) mice did not alter water consumption, urine output, Na(+) excretion, osmolality, or protein excretion. However, in Cyp1b1(-/-) mice, ANG II infusion significantly increased (P < 0.05) water intake (5.50 ± 0.42 ml/24 h with vehicle vs. 8.80 ± 0.60 ml/24 h with ANG II), urine output (1.44 ± 0.37 ml/24 h with vehicle vs. 4.30 ± 0.37 ml/24 h with ANG II), and urinary Na(+) excretion (0.031 ± 0.016 mmol/24 h with vehicle vs. 0.099 ± 0.010 mmol/24 h with ANG II), decreased osmolality (2,630 ± 79 mosM/kg with vehicle vs. 1,280 ± 205 mosM/kg with ANG II), and caused proteinuria (2.60 ± 0.30 mg/24 h with vehicle vs. 6.96 ± 0.55 mg/24 h with ANG II). Infusion of ANG II caused renal fibrosis, as indicated by an accumulation of renal interstitial α-smooth muscle actin, collagen, and transforming growth factor-ß in Cyp1b1(-/-) but not Cyp1b1(+/+) mice. ANG II also increased renal production of ROS and urinary excretion of thiobarburic acid-reactive substances and reduced the activity of antioxidants and urinary excretion of nitrite/nitrate and the 17ß-estradiol metabolite 2-methoxyestradiol in Cyp1b1(-/-) but not Cyp1b1(+/+) mice. These data suggest that Cyp1b1 plays a critical role in female mice in protecting against renal dysfunction and end-organ damage associated with ANG II-induced hypertension, in preventing oxidative stress, and in increasing activity of antioxidant systems, most likely via generation of 2-methoxyestradiol from 17ß-estradiol.

Estrogen metabolism by cytochrome P450 1B1 modulates the hypertensive effect of angiotensin II in female mice.

To determine the role of cytochrome P450 (CYP) 1B1 in the sex difference in response to angiotensin II (Ang II)-induced hypertension, female Cyp1b1(+/+) and Cyp1b1(-/-) mice were infused with Ang II (700 ng/kg per minute) or vehicle with or without ovariectomy. In addition, mice were treated with the CYP1B1 inhibitor, 2,3',4,5'-tetramethoxystilbene (TMS; 300 µg/kg IP, every third day), and 17-ß estradiol metabolites, 2-hydroxyestradiol (2-OHE), 4-OHE, or 2-methoxyestradiol (1.5 mg/kg per day IP, for 2 weeks) and systolic blood pressure (SBP) measured. Ang II increased SBP more in Cyp1b1(-/-) than in Cyp1b1(+/+) mice (119±3-171±11 versus 120±4-149±4 mm Hg; P<0.05). Ang II caused cardiovascular remodeling and endothelial dysfunction and increased vascular reactivity and oxidative stress in Cyp1b1(-/-) but not in Cyp1b1(+/+)mice. The Ang II-induced increase in SBP was enhanced by ovariectomy and TMS in Cyp1b1(+/+) but not in Cyp1b1(-/-) mice. 2-OHE did not alter Ang II-induced increase in SBP in Cyp1b1(+/+) mice but minimized it in Cyp1b1(-/-) mice, whereas 4-OHE enhanced Ang II-induced increase in SBP in Cyp1b1(+/+) mice but did not alter the increased SBP in Cyp1b1(-/-) mice. 2-OHE-derived catechol-O-methyltransferase metabolite, 2-methoxyestradiol, inhibited Ang II-induced increase in SBP in Cyp1b1(-/-) mice. Ang II increased plasma levels of 2-methoxyestradiol in Cyp1b1(+/+) but not in Cyp1b1(-/-) mice and increased activity of cardiac extracellular signal-regulated kinase 1/2, p38 mitogen-activated kinase, c-Src, and Akt in Cyp1b1(-/-) but not in Cyp1b1(+/+) mice. These data suggest that CYP1B1 protects against Ang II-induced hypertension and associated cardiovascular changes in female mice, most likely mediated by 2-methoxyestradiol-inhibiting oxidative stress and the activity of these signaling molecules.

Cytochrome P450 1B1 contributes to increased blood pressure and cardiovascular and renal dysfunction in spontaneously hypertensive rats.

PURPOSE: We investigated the contribution of cytochrome P450 (CYP) 1B1 to hypertension and its pathogenesis by examining the effect of its selective inhibitor, 2,4,3',5'-tetramethoxystilbene (TMS), in spontaneously hypertensive rats (SHR). METHODS: Blood pressure (BP) was measured bi-weekly. Starting at 8 weeks, TMS (600 µg/kg, i.p.) or its vehicle was injected daily. At 14 weeks, samples were collected for measurement. RESULTS: TMS reversed increased BP in SHR (207 ± 7 vs. 129 ± 2 mmHg) without altering BP in Wistar-Kyoto rats. Increased CYP1B1 activity in SHR was inhibited by TMS (RLU: aorta, 5.4 ± 0.7 vs. 3.7 ± 0.7; heart, 6.0 ± 0.8 vs. 3.4 ± 0.4; kidney, 411 ± 45 vs. 246 ± 10). Increased vascular reactivity, cardiovascular hypertrophy, endothelial and renal dysfunction, cardiac and renal fibrosis in SHR were minimized by TMS. Increased production of reactive oxygen species and NADPH oxidase activity in SHR, were diminished by TMS. In SHR, TMS reduced increased plasma levels of nitrite/nitrate (46.4 ± 5.0 vs. 28.1 ± 4.1 µM), hydrogen-peroxide (36.0 ± 3.7 vs. 14.1 ± 3.8 µM), and thiobarbituric acid reactive substances (6.9 ± 1.0 vs. 3.4 ± 1.5 µM). Increased plasma levels of pro-inflammatory cytokines and catecholamines, and cardiac activity of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, c-Src tyrosine kinase, and protein kinase B in SHR were also inhibited by TMS. CONCLUSIONS: These data suggests that increased oxidative stress generated by CYP1B1 contributes to hypertension, increased cytokine production and sympathetic activity, and associated pathophysiological changes in SHR. CYP1B1 could be a novel target for developing drugs to treat hypertension and its pathogenesis.

Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase.

Lysophosphatidic acid (LPA) has been implicated as a mediator of several cardiovascular functions, but its potential involvement in the control of vascular tone is obscure. Here, we show that both LPA (18:1) and VPC31143 (a synthetic agonist of LPA1-3 receptors) relax intact mouse thoracic aorta with similar Emax values (53.9 and 51.9% of phenylephrine-induced precontraction), although the EC50 of LPA- and VPC31143-induced vasorelaxations were different (400 vs. 15 nM, respectively). Mechanical removal of the endothelium or genetic deletion of endothelial nitric oxide synthase (eNOS) not only diminished vasorelaxation by LPA or VPC31143 but converted it to vasoconstriction. Freshly isolated mouse aortic endothelial cells expressed LPA1, LPA2, LPA4 and LPA5 transcripts. The LPA1,3 antagonist Ki16425, the LPA1 antagonist AM095, and the genetic deletion of LPA1, but not that of LPA2, abolished LPA-induced vasorelaxation. Inhibition of the phosphoinositide 3 kinase-protein kinase B/Akt pathway by wortmannin or MK-2206 failed to influence the effect of LPA. However, pharmacological inhibition of phospholipase C (PLC) by U73122 or edelfosine, but not genetic deletion of PLCε, abolished LPA-induced vasorelaxation and indicated that a PLC enzyme, other than PLCε, mediates the response. In summary, the present study identifies LPA as an endothelium-dependent vasodilator substance acting via LPA1, PLC, and eNOS.

Cytochrome P450 1B1 gene disruption minimizes deoxycorticosterone acetate-salt-induced hypertension and associated cardiac dysfunction and renal damage in mice.

Previously, we showed that the cytochrome P450 1B1 inhibitor 2,3',4,5'-tetramethoxystilbene reversed deoxycorticosterone acetate (DOCA)-salt-induced hypertension and minimized endothelial and renal dysfunction in the rat. This study was conducted to test the hypothesis that cytochrome P450 1B1 contributes to cardiac dysfunction, and renal damage and inflammation associated with DOCA-salt-induced hypertension, via increased production of reactive oxygen species and modulation of neurohumoral factors and signaling molecules. DOCA-salt increased systolic blood pressure, cardiac and renal cytochrome P450 1B1 activity, and plasma levels of catecholamines, vasopressin, and endothelin-1 in wild-type (Cyp1b1(+/+)) mice that were minimized in Cyp1b1(-/-) mice. Cardiac function, assessed by echocardiography, showed that DOCA-salt increased the thickness of the left ventricular posterior and anterior walls during diastole, the left ventricular internal diameter, and end-diastolic and end-systolic volume in Cyp1b1(+/+) but not in Cyp1b1(-/-) mice; stroke volume was not altered in either genotype. DOCA-salt increased renal vascular resistance and caused vascular hypertrophy and renal fibrosis, increased renal infiltration of macrophages and T lymphocytes, caused proteinuria, increased cardiac and renal nicotinamide adenine dinucleotide phosphate-oxidase activity, caused production of reactive oxygen species, and increased activities of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and cellular-Src; these were all reduced in DOCA-salt-treated Cyp1b1(-/-) mice. Renal and cardiac levels of eicosanoids were not altered in either genotype of mice. These data suggest that, in DOCA-salt hypertension in mice, cytochrome P450 1B1 plays a pivotal role in cardiovascular dysfunction, renal damage, and inflammation, and increased levels of catecholamines, vasopressin, and endothelin-1, consequent to generation of reactive oxygen species and activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and cellular-Src independent of eicosanoids.

Contribution of cytochrome P450 1B1 to hypertension and associated pathophysiology: a novel target for antihypertensive agents.

The aim of this review is to discuss the contribution of cytochrome P450 (CYP) 1B1 in vascular smooth muscle cell growth, hypertension, and associated pathophysiology. CYP1B1 is expressed in cardiovascular and renal tissues, and mediates angiotensin II (Ang II)-induced activation of NADPH oxidase and generation of reactive oxygen species (ROS), and vascular smooth muscle cell migration, proliferation, and hypertrophy. Moreover, CYP1B1 contributes to the development and/or maintenance of hypertension produced by Ang II-, deoxycorticosterone (DOCA)-salt-, and N(ω)-nitro-L-arginine methyl ester-induced hypertension and in spontaneously hypertensive rats. The pathophysiological changes, including cardiovascular hypertrophy, increased vascular reactivity, endothelial and renal dysfunction, injury and inflammation associated with Ang II- and/or DOCA-salt induced hypertension in rats, and Ang II-induced hypertension in mice are minimized by inhibition of CYP1B1 activity with 2,4,3',5'-tetramethoxystilbene or by Cyp1b1 gene disruption in mice. These pathophysiological changes appear to be mediated by increased production of ROS via CYP1B1-dependent NADPH oxidase activity and extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src.

Cytochrome P450 1B1 contributes to renal dysfunction and damage caused by angiotensin II in mice.

Cytochrome P450 1B1 contributes to the development of angiotensin II-induced hypertension and associated cardiovascular pathophysiology. In view of the critical role of angiotensin II in the kidney, as well as in salt and water homeostasis, and blood pressure regulation, we determined the contribution of cytochrome P450 1B1 to renal dysfunction and injury associated with angiotensin II-induced hypertension in male Cyp1b1(+/+) and Cyp1b1(-/-) mice. Angiotensin II infusion (700 ng/kg per minute) given by miniosmotic pumps for 13 and 28 days increased systolic blood pressure in Cyp1b1(+/+) mice; this increase was significantly reduced in Cyp1b1(-/-) mice. Angiotensin II increased renal Cyp1b1 activity, vascular resistance, and reactivity to vasoconstrictor agents and caused endothelial dysfunction in Cyp1b1(+/+) but not Cyp1b1(-/-) mice. Angiotensin II increased water consumption and urine output, decreased urine osmolality, increased urinary Na(+) and K(+) excretion, and caused proteinuria and albuminuria in Cyp1b1(+/+) mice that was diminished in Cyp1b1(-/-) mice. Infusion of angiotensin II for 28 but not 13 days caused renal fibrosis, tubular damage, and inflammation in Cyp1b1(+/+) mice, which was minimized in Cyp1b1(-/-) mice. Angiotensin II increased levels of 12- and 20-hydroxyeicosatetraenoic acids; reactive oxygen species; and activity of NADPH oxidase, extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src in the kidneys of Cyp1b1(+/+) but not Cyp1b1(-/-) mice. These data suggest that increased thirst, renal dysfunction, and injury and inflammation associated with angiotensin II-induced hypertension in mice depend on cytochrome P450 1B1 activity, thus indicating that cytochrome P450 1B1 could serve as a novel target for treating renal disease and hypertension.

Involvement of cytochrome P-450 1B1 in renal dysfunction, injury, and inflammation associated with angiotensin II-induced hypertension in rats.

We investigated the contribution of cytochrome P-450 1B1 (CYP1B1) to renal dysfunction and organ damage associated with ANG II-induced hypertension in rats. ANG II (300 ng·kg(-1)·min(-1)) or vehicle were infused for 2 wk, with daily injections of a selective CYP1B1 inhibitor, 2,4,3',5'-tetramethoxystilbene (TMS; 300 µg/kg ip), or its vehicle. ANG II increased blood pressure and renal CYP1B1 activity that were prevented by TMS. ANG II also increased water intake and urine output, decreased glomerular filtration rate, increased urinary Na(+) and K(+) excretion, and caused proteinuria, all of which were prevented by TMS. ANG II infusion caused hypertrophy, endothelial dysfunction, and increased reactivity of renal and interlobar arteries to vasoconstrictor agents and renal vascular resistance and interstitial fibrosis as indicated by accumulation of α-smooth muscle actin, fibronectin, and collagen, and inflammation as indicated by increased infiltration of CD-3(+) cells; these effects were inhibited by TMS. ANG II infusion also increased production of reactive oxygen species (ROS) and activities of NADPH oxidase, ERK1/2, p38 MAPK, and c-Src that were prevented by TMS. TMS alone had no effect on any of the above parameters. These data suggest that CYP1B1 contributes to the renal pathophysiological changes associated with ANG II-induced hypertension, most likely via increased ROS production and activation of ERK1/2, p38 MAPK, and c-Src and that CYP1B1 could serve as a novel target for treating renal disease associated with hypertension.

Cytochrome P450 1B1 contributes to angiotensin II-induced hypertension and associated pathophysiology.

Hypertension is the leading cause of cardiovascular diseases, and angiotensin II is one of the major components of the mechanisms that contribute to the development of hypertension. However, the precise mechanisms for the development of hypertension are unknown. Our recent study showing that angiotensin II-induced vascular smooth muscle cell growth depends on cytochrome P450 1B1 led us to investigate its contribution to hypertension caused by this peptide. Angiotensin II was infused via miniosmotic pump into rats (150 ng/kg per minute) or mice (1000 µg/kg per day) for 13 days resulting in increased blood pressure, increased cardiac and vascular hypertrophy, increased vascular reactivity to vasoconstrictor agents, increased vascular reactive oxygen species production, and endothelial dysfunction in both species. The increase in blood pressure and associated pathophysiological changes were minimized by the cytochrome P450 1B1 inhibitor 2,3',4,5'-tetramethoxystilbene in both species and was markedly reduced in Cyp1b1(-/-) mice. These data suggest that cytochrome P450 1B1 contributes to angiotensin II-induced hypertension and associated pathophysiological changes. Moreover, 2,3',4,5'-tetramethoxystilbene, which prevents both cytochrome P450 1B1-dependent and -independent components of angiotensin II-induced hypertension and inhibits associated pathophysiological changes could be clinically useful in the treatment of hypertension and associated cardiovascular and inflammatory diseases.

2,3',4,5'-Tetramethoxystilbene prevents deoxycorticosterone-salt-induced hypertension: contribution of cytochrome P-450 1B1.

Reactive oxygen species (ROS) contribute to various models of hypertension, including deoxycorticosterone acetate (DOCA)-salt-induced hypertension. Recently, we have shown that ROS, generated by cytochrome P-450 1B1 (CYP1B1) from arachidonic acid, mediate vascular smooth muscle cell growth caused by angiotensin II. This study was conducted to determine the contribution of CYP1B1 to hypertension and associated pathophysiological changes produced by DOCA (30 mg/kg) given subcutaneously per week with 1% NaCl + 0.1% KCl in drinking water to uninephrectomized rats for 6 wk. DOCA-salt treatment increased systolic blood pressure (SBP). Injections of the selective inhibitor of CYP1B1, 2,3',4,5'-tetramethoxystilbene (TMS; 300 µg/kg ip every 3rd day) initiated at the 4th week of DOCA-salt treatment normalized SBP and decreased CYP1B1 activity but not its expression in the aorta, heart, and kidney. TMS also inhibited cardiovascular and kidney hypertrophy, prevented the increase in vascular reactivity and endothelial dysfunction, and minimized the increase in urinary protein and K(+) output and the decrease in urine osmolality, Na(+) output, and creatinine clearance associated with DOCA-salt treatment. These pathophysiological changes caused by DOCA-salt treatment and associated increase in vascular superoxide production, NADPH oxidase activity, and expression of NOX-1, and ERK1/2 and p38 MAPK activities in the aorta, heart, and kidney were inhibited by TMS. These data suggest that CYP1B1 contributes to DOCA-salt-induced hypertension and associated pathophysiological changes, most likely as a result of increased ROS production and ERK1/2 and p38 MAPK activity, and could serve as a novel target for the development of agents like TMS to treat hypertension.

Mechanisms of nitric oxide-mediated, neurogenic vasodilation in mesenteric resistance arteries of toad Bufo marinus.

This study determined the role of nitric oxide (NO) in neurogenic vasodilation in mesenteric resistance arteries of the toad Bufo marinus. NO synthase (NOS) was anatomically demonstrated in perivascular nerves, but not in the endothelium. ACh and nicotine caused TTX-sensitive neurogenic vasodilation of mesenteric arteries. The ACh-induced vasodilation was endothelium-independent and was mediated by the NO/soluble guanylyl cyclase signaling pathway, inasmuch as the vasodilation was blocked by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and the NOS inhibitors N(omega)-nitro-l-arginine methyl ester and N(omega)-nitro-l-arginine. Furthermore, the ACh-induced vasodilation was significantly decreased by the more selective neural NOS inhibitor N(5)-(1-imino-3-butenyl)-l-ornithine. The nicotine-induced vasodilation was endothelium-independent and mediated by NO and calcitonin gene-related peptide (CGRP), inasmuch as pretreatment of mesenteric arteries with a combination of N(omega)-nitro-l-arginine and the CGRP receptor antagonist CGRP-(8-37) blocked the vasodilation. Clotrimazole significantly decreased the ACh-induced response, providing evidence that a component of the NO vasodilation involved Ca(2+)-activated K(+) or voltage-gated K(+) channels. These data show that NO control of mesenteric resistance arteries of toad is provided by nitrergic nerves, rather than the endothelium, and implicate NO as a potentially important regulator of gut blood flow and peripheral blood pressure.

Neurally-derived nitric oxide regulates vascular tone in pulmonary and cutaneous arteries of the toad, Bufo marinus.

In this study, the role of nitric oxide (NO) in regulation of the pulmocutaneous vasculature of the toad, Bufo marinus was investigated. In vitro myography demonstrated the presence of a neural NO signaling mechanism in both arteries. Vasodilation induced by nicotine was inhibited by the soluble guanylyl cyclase (GC) inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, and the NO synthase (NOS) inhibitor, N(omega)-nitro-l-arginine (l-NNA). Removal of the endothelium had no significant effect on the vasodilation. Furthermore, pretreatment with N(5)-(1-imino-3-butenyl)-l-ornithine (vinyl-l-NIO), a more specific inhibitor of neural NOS, caused a significant decrease in the nicotine-induced dilation. In the pulmonary artery only, a combination of l-NNA and the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP((8-37)), completely blocked the nicotine-induced dilation. In both arteries, the vasodilation was also significantly decreased by glibenclamide, an ATP-sensitive K(+) (K(+)(ATP)) channel inhibitor. Levcromakalim, a K(+)(ATP) channel opener, caused a dilation that was blocked by glibenclamide in both arteries. In the pulmonary artery, NO donor-mediated dilation was significantly decreased by pretreatment with glibenclamide. The physiological data were supported by NADPH-diaphorase histochemistry and immunohistochemistry, which demonstrated NOS in perivascular nerve fibers but not the endothelium of the arteries. These results indicate that the pulmonary and cutaneous arteries of B. marinus are regulated by NO from nitrergic nerves rather than NO released from the endothelium. The nitrergic vasodilation in the arteries appears to be caused, in part, via activation of K(+)(ATP) channels. Thus, NO could play an important role in determining pulmocutaneous blood flow and the magnitude of cardiac shunting.

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri.

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.

Mechanisms of vasodilation in the dorsal aorta of the elephant fish, Callorhinchus milii (Chimaeriformes: Holocephali).

This study investigated vasodilator mechanisms in the dorsal aorta of the elephant fish, Callorhinchus milii, using anatomical and physiological approaches. Nitric oxide synthase could only be located in the perivascular nerve fibres and not the endothelium of the dorsal aorta, using NADPH histochemistry and immunohistochemistry. In vitro organ bath experiments demonstrated that a NO/soluble guanylyl cyclase (GC) system appeared to be absent in the vascular smooth muscle, since the NO donors SNP (10(-4) mol l(-1)) and SIN-1 (10(-5) mol l(-1)) were without effect. Nicotine (3 x 10(-4) mol l(-1)) mediated a vasodilation that was not affected by ODQ (10(-5) mol l(-1)), L-NNA (10(-4) mol l(-1)), indomethacin (10(-5) mol l(-1)), or removal of the endothelium. In contrast, the voltage-gated sodium channel inhibitor, tetrodotoxin (10(-5) mol l(-1)), significantly decreased the dilation induced by nicotine, suggesting that it contained a neural component. Pre-incubation of the dorsal aorta with the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(8-37) (10(-6) mol l(-1)) also caused a significant decrease in the nicotine-induced dilation. We propose that nicotine is mediating a neurally-derived vasodilation in the dorsal aorta that is independent of NO, prostaglandins and the endothelium, and partly mediated by CGRP.

Nitric oxide control of the dorsal aorta and the intestinal vein of the Australian short-finned eel Anguilla australis.

This study investigated the mechanisms by which nitric oxide (NO) regulates the dorsal aorta and the intestinal vein of the Australian short-finned eel Anguilla australis. NADPH diaphorase histochemistry and immunohistochemistry using a mammalian endothelial nitric oxide synthase (NOS) antibody could not demonstrate NOS in the endothelium of either blood vessel; however, NOS could be readily demonstrated in the endothelium of the rat aorta that was used as a control. Both blood vessels contained NADPH diaphorase positive nerve fibres and nerve bundles, and immunohistochemistry using a neural NOS antibody showed a similar distribution of neural NOS immunoreactivity in the perivascular nerves. In vitro organ bath physiology showed that a NO/soluble guanylyl cyclase (GC) system is present in the dorsal aorta and the intestinal vein, since the soluble GC inhibitor oxadiazole quinoxalin-1 (ODQ; 10(-5) mol l(-1)) completely abolished the vasodilatory effect of the NO donor, sodium nitroprusside (SNP; 10(-4) mol l(-1)). In addition, nicotine (3 x 10(-4) mol l(-1)) mediated a vasodilation that was not affected by removal of the endothelium. The nicotine-mediated dilation was blocked by the NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA; 10(-4) mol l(-1)), and ODQ (10(-5) mol l(-1)). More specifically, the neural NOS inhibitor, N(omega)-propyl-L-arginine (10(-5) mol l(-1)), significantly decreased the dilation induced by nicotine (3 x 10(-4) mol l(-1)). Furthermore, indomethacin (10(-5) mol l(-1)) did not affect the nicotine-mediated dilation, suggesting that prostaglandins are not involved in the response. Finally, the calcium ionophore A23187 (3 x 10(-6) mol l(-1)) caused an endothelium-dependent dilation that was abolished in the presence of indomethacin. We propose the absence of an endothelial NO system in eel vasculature and suggest that neurally derived NO contributes to the maintenance of vascular tone in this species. In addition, we suggest that prostaglandins may act as endothelially derived relaxing factors in A. australis.