Critical role for CuZn-superoxide dismutase in preventing angiotensin II-induced endothelial dysfunction

The goal of the present study was to test the hypothesis that the CuZn isoform of superoxide dismutase (CuZnSOD) protects against angiotensin II (Ang II)-induced endothelial dysfunction. Vascular responses of carotid arteries from control, CuZnSOD-deficient (CuZnSOD(+/-)), and CuZnSOD transgenic mice were examined in vitro after overnight incubation with either vehicle or Ang II (1 or 10 nmol/L). In control mice, acetylcholine produced concentration-dependent relaxation that was not affected by 1 nmol/L Ang II. In contrast, relaxation to acetylcholine in arteries from CuZnSOD+/- mice was markedly and selectively attenuated after incubation with 1 nmol/L Ang II (eg, 100 micromol/L acetylcholine produced 93+/-6% and 44+/-15% relaxation in vehicle- and Ang II-treated arteries, respectively). A higher concentration of Ang II (10 nmol/L) selectively impaired relaxation to acetylcholine in arteries from control mice (eg, 100 micromol/L acetylcholine produced 96+/-4% and 45+/-7% relaxation in vehicle- and Ang II-treated vessels, respectively). In contrast, 10 nmol/L Ang II had no effect on responses to acetylcholine in carotid arteries from CuZnSOD transgenic mice (or in control mice treated with the superoxide scavenger Tiron [1 mmol/L]). Superoxide levels in control mice were higher in aorta treated with Ang II than with vehicle and were markedly reduced in CuZnSOD transgenic mice. These findings provide the first direct evidence that CuZnSOD limits Ang II-mediated impairment of endothelial function and that loss of 1 copy of the CuZnSOD gene is sufficient to enhance Ang II-induced vascular dysfunction.     

Heme oxygenase-1 induction does not improve vascular relaxation in angiotensin II hypertensive mice

BACKGROUND: Induction of heme oxygenase-1 (HO-1) attenuates the development of angiotensin II (Ang II)-dependent hypertension in mice. However, the mechanism by which HO-1 lowers blood pressure in this model is not clear. This study was designed to determine whether induction of HO-1 results in an improvement in vascular relaxation in Ang II hypertensive mice. METHODS: Mice were treated with either of the vehicles (control), the HO-1 inducer cobalt protoporphyrin (CoPP;50 mg/kg), Ang II(1 microg/kg/min, 14 days), or Ang II + CoPP. CoPP was administered as a single bolus dose 2 days prior to subcutaneous implantation of the osmotic minipump containing Ang II. Vascular relaxation was examined in isolated carotid arteries precontracted with the thromboxane mimetic U46619 (0.4 microg/ml). RESULTS: Endothelial dependent relaxation to acetylcholine (ACh; 1 micromol/l) was significantly impaired in Ang II-treated mice compared to control mice (56 +/- 3% vs. 40 +/- 4%, P < 0.05, n > or = 6). Similarly, endothelial independent relaxation to sodium nitroprusside (SNP; 1 micromol/l) was significantly impaired in Ang II mice (56 +/- 6% vs. 28 +/- 6%, P < 0.05, n > or = 6). Relaxation in response to the carbon monoxide donor, CORM-A1 (100 micromol/l), was attenuated after Ang II treatment (75 +/- 7% vs. 59 +/- 7%,P < 0.05, n > or = 6). CoPP treatment induced HO-1 but not HO-2 protein in the aorta, as measured by western blot analysis. CoPP treatment had no effect on vascular responses in control mice and did not improve ACh (26 +/- 5%, n = 15), SNP (23 +/- 4%, n = 15), or CORM-A1 (46 +/- 7%, n = 10) dependent relaxation in Ang II treated mice. CONCLUSIONS: These results suggest that induction of HO-1 lowers Ang II-dependent hypertension through a mechanism independent of improved vascular relaxation.     

Angiotensin II regulates adrenal vascular tone through zona glomerulosa cell-derived EETs and DHETs

Elevated concentrations of aldosterone are associated with several cardiovascular diseases. Angiotensin II (Ang II) increases aldosterone secretion and adrenal blood flow. This concurrent increase in steroidogenesis and adrenal blood flow is not understood. We investigated the role of zona glomerulosa (ZG) cells in the regulation of vascular tone of bovine adrenal cortical arteries by Ang II. ZG cells enhanced endothelium-dependent relaxations to Ang II. The ZG cell-dependent relaxations to Ang II were unchanged by removing the endothelium-dependent response to Ang II. These ZG cell-mediated relaxations were ablated by cytochrome P450 inhibition, epoxyeicosatrienoic acid (EET) antagonism, and potassium channel blockade. Analysis of ZG cell EET production by liquid chromatography/mass spectrometry demonstrated an increase in EETs and dihydroxyeicosatrienoic acids with Ang II stimulation. These EETs and dihydroxyeicosatrienoic acids produced similar concentration-dependent relaxations of adrenal arteries, which were attenuated by EET antagonism. Whole-cell potassium currents of adrenal artery smooth muscle cells were increased by Ang II stimulation in the presence of ZG cells but decreased in the absence of ZG cells. This increase in potassium current was abolished by iberiotoxin. Similarly, 14,15-EET induced concentration-dependent increases in potassium current, which was abolished by iberiotoxin. ZG cell aldosterone release was not directly altered by EETs. These data suggest that Ang II stimulates ZG cells to release EETs and dihydroxyeicosatrienoic acids, resulting in potassium channel activation and relaxation of adrenal arteries. This provides a mechanism by which Ang II concurrently increases adrenal blood flow and steroidogenesis.     

Acetylcholine-induced relaxation and hyperpolarization in small bovine adrenal cortical arteries: role of cytochrome P450 metabolites

The present study characterizes the vascular responses of isolated small bovine adrenal cortical arteries to acetylcholine, an endogenous neurotransmitter in the adrenal gland. Acetylcholine (10(-10) to 10(-6) m) elicited a concentration-dependent relaxation, with a maximal relaxation of 96 +/- 1% and EC50 of 4.2 nm. The relaxation was abolished by endothelial removal and attenuated by the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NA, 30 microm) but not by the cyclooxygenase inhibitor indomethacin (10 microm). The maximal relaxation and EC50 of acetylcholine in the presence of L-NA were 87 +/- 4% and 22 nm, respectively. The acetylcholine-induced, indomethacin- and L-NA-resistant relaxation was eliminated by high K+ and markedly inhibited by the cytochrome P450 inhibitors SKF 525A (10 microm) and miconazole (10 microm). The maximal relaxations and EC50s with SKF 525A and miconazole were 56 +/- 8 and 72 +/- 2% and 0.8 and 0.5 microm, respectively. In indomethacin- and L-NA-treated arteries, acetylcholine induced a smooth muscle hyperpolarization, which was blocked by SKF 525A (3 +/- 1 mV vs. 15 +/- 2 mV of control). Arachidonic acid (10(-9) to 10(-5) m) and 14,15-epoxyeicosatrienoic acid (14,15-EET, 10(-9) to 10(-5) m), a cytochrome P450 metabolite of arachidonic acid, also evoked relaxations in small adrenal arteries, with maximal relaxations of 56 +/- 4 and 90 +/- 5%, respectively. The arachidonic acid-induced relaxation was blocked by SKF 525A. Using high-pressure liquid chromatography and gas chromatography/mass spectrometry analysis, EETs were identified in small adrenal arteries. These results demonstrate that acetylcholine is a potent vasodilator of small adrenal cortical arteries. The acetylcholine-induced relaxation is largely mediated by an endothelium-dependent hyperpolarization mechanism, presumably through cytochrome P450 metabolites of arachidonic acid.     

Investigation into the sources of superoxide in human blood vessels: angiotensin II increases superoxide production in human internal mammary arteries

BACKGROUND: Increased vascular superoxide anion (.O(2)(-)) production contributes to endothelial dysfunction and hypertension in animal models of cardiovascular disease. Observations in experimental animals suggest that angiotensin II (Ang II) increases.O(2)(-) production by activation of vascular NAD(P)H oxidase. We studied the sources of.O(2)(-) production in human blood vessels and investigated whether, and by what mechanism, Ang II might alter vascular.O(2)(-) production. METHODS AND RESULTS: Internal mammary arteries (IMAs) and saphenous veins (SVs) were collected at the time of cardiac surgery. Vessels were incubated in Krebs buffer at 37 degrees C.O(2)(-) was measured by lucigenin chemiluminescence. Basal. O(2)(-) concentrations were greater in IMAs than SVs. Inhibitors of NAD(P)H oxidase (10 micromol/L to 200 micromol/L diphenyleneiodonium) and xanthine oxidase (1 mmol/L allopurinol) caused reductions in.O(2)(-) concentrations in both IMAs and SVs. Western blotting of superoxide dismutase proteins demonstrated similar expression in IMAs and SVs. Vessels were also incubated in the presence or absence of Ang II (1 pmol/L to 1 micromol/L). Ang II increased.O(2)(-) production in IMAs at 4 hours of incubation (control, 978+/-117 pmol. min(-1). mg(-1); 1 micromol/L of Ang II, 1690+/-213 pmol. min(-1). mg(-1); n=27, P=0.0001, 95% CI 336, 925) but not in SVs. This effect was completely inhibited by coincubation of IMAs with DPI (100 micromol/L), a nonspecific Ang II antagonist ([sar(1), thre(8)]-Ang II, 1 micromol/L) and a specific Ang II type 1 (AT(1)) receptor antagonist (losartan, 1 micromol/L). Conclusions-. O(2)(-) production is greater in human IMAs than in SVs. NAD(P)H oxidase and xanthine oxidase are sources of.O(2)(-) production in these vessels. The vasoactive peptide Ang II increases.O(2)(-) production in human arteries by an AT(1) receptor-dependent mechanism.     

14,15-epoxyeicosatrienoic acid represents a transferable endothelium-dependent relaxing factor in bovine coronary arteries

Bradykinin causes arterial relaxation and hyperpolarization, which is mediated by a transferable endothelium-derived hyperpolarizing factor (EDHF). In coronary arteries, epoxyeicosatrienoic acids (EETs) are involved in the EDHF response. However, the role of EETs as transferable mediators of EDHF-dependent relaxation remains poorly defined. Two small bovine coronary arteries were cannulated and perfused in tandem in the presence of the nitric oxide synthase inhibitor, nitro-L-arginine (30 micromol/L), and the cyclooxygenase inhibitor, indomethacin (10 micromol/L). Luminal perfusate from donor arteries with intact endothelium perfused endothelium-denuded detector arteries. Detector arteries were constricted with U46619 and diameters were monitored. Bradykinin (10 nmol/L) added to detector arteries did not induce dilation (5+/-2%), whereas bradykinin addition to donor arteries dilated detector arteries by 26.5+/-7% (P<0.05). These dilations were blocked by donor artery endothelium removal and detector artery treatment with the EET-selective antagonist, 14,15-epoxyeicosa-5(Z)-monoenoic acid (14,15-EEZE; 10 micromol/L, -5+/-6%) but not 14,15-EEZE treatment of donor arteries (20+/-5%). 14,15-EET (0.1 to 10 micromol/L) added to detector arteries induced maximal dilations of 82+/-5% that were inhibited 50% by detector artery treatment with 14,15-EEZE (32+/-12%) but not donor artery treatment with 14,15-EEZE. Liquid chromatography-electrospray ionization mass spectrometry analysis verified the presence of 14,15-EET in the perfusate from an endothelium-intact but not denuded artery. These results show that bradykinin stimulates donor artery 14,15-EET release that dilates detector arteries. 14,15-EEZE blocked the donor artery, endothelium-dependent, bradykinin-induced relaxations, and attenuated relaxations to 14,15-EET. These results suggest that EETs are transferable EDHFs in coronary arteries.     

Steroid-producing cells regulate arterial tone of adrenal cortical arteries

Adrenal blood flow is coupled to adrenal hormone secretion. ACTH increases adrenal blood flow and stimulates the secretion of aldosterone and cortisol in vivo. However, ACTH does not alter vascular tone of isolated adrenal cortical arteries. Mechanisms underlying this discrepancy remain unsolved. The present study examined the effect of zona glomerulosa (ZG) cells on cortical arterial tone. ZG cells (10(5) to 10(7) cells) and ZG cell-conditioned medium relaxed preconstricted adrenal arteries (maximal relaxations = 79 +/- 4 and 66 +/- 4%, respectively). In adrenal arteries coincubated with a small number of ZG cells (0.5-1 x 10(6)), ACTH (10(-12) to 10(-8) m) induced concentration-dependent relaxations (maximal relaxation = 67 +/- 4%). Similarly, ACTH (10(-8) m) dilated (55 +/- 10%) perfused arteries embedded in adrenal cortical slices. ZG cell-dependent relaxations to ACTH were endothelium-independent and inhibited by high extracellular K(+) (60 mm); the K(+) channel blocker, iberiotoxin (100 nm); the cytochrome P450 inhibitors SKF 525A (10 microm) and miconazole (10 microm); and the epoxyeicosatrienoic acid (EET) antagonist 14,15-EEZE (2 microm). Four EET regioisomers were identified in ZG cell-conditioned media. EET production was stimulated by ACTH. We conclude that ZG cells release EETs and this release is stimulated by ACTH. Interaction of endocrine and vascular cells represents a mechanism for regulating adrenal blood flow and couples steroidogenesis to increased blood flow.     

Angiotensin-(1-7) downregulates the angiotensin II type 1 receptor in vascular smooth muscle cells

Angiotensin (Ang)-(1-7) is a biologically active peptide of the renin-angiotensin system that has both vasodilatory and antiproliferative activities that are opposite the constrictive and proliferative effects of angiotensin II (Ang II). We studied the actions of Ang-(1-7) on the Ang II type 1 (AT(1)) receptor in cultured rat aortic vascular smooth muscle cells to determine whether the effects of Ang-(1-7) are due to its regulation of the AT(1) receptor. Ang-(1-7) competed poorly for [(125)I]Ang II binding to the AT(1) receptor on vascular smooth muscle cells, with an IC(50) of 2.0 micromol/L compared with 1.9 nmol/L for Ang II. The pretreatment of vascular smooth muscle cells with Ang-(1-7) followed by treatment with acidic glycine to remove surface-bound peptide resulted in a significant decrease in [(125)I]Ang II binding; however, reduced Ang II binding was observed only at micromolar concentrations of Ang-(1-7). Scatchard analysis of vascular smooth muscle cells pretreated with 1 micromol/L Ang-(1-7) showed that the reduction in Ang II binding resulted from a loss of the total number of binding sites [B(max) 437.7+/-261.5 fmol/mg protein in Ang-(1-7)-pretreated cells compared with 607.5+/-301.2 fmol/mg protein in untreated cells, n=5, P<0.05] with no significant effect on the affinity of Ang II for the AT(1) receptor. Pretreatment with the AT(1) receptor antagonist L-158,809 blocked the reduction in [(125)I]Ang II binding by Ang-(1-7) or Ang II. Pretreatment of vascular smooth muscle cells with increasing concentrations of Ang-(1-7) reduced Ang II-stimulated phospholipase C activity; however, the decrease was significant (81.2+/-6.4%, P<0.01, n=5) only at 1 micromol/L Ang-(1-7). These results demonstrate that pharmacological concentrations of Ang-(1-7) in the micromolar range cause a modest downregulation of the AT(1) receptor on vascular cells and a reduction in Ang II-stimulated phospholipase C activity. Because the antiproliferative and vasodilatory effects of Ang-(1-7) are observed at nanomolar concentrations of the heptapeptide, these responses to Ang-(1-7) cannot be explained by competition of Ang-(1-7) at the AT(1) receptor or Ang-(1-7)-mediated downregulation of the vascular AT(1) receptor.     

Effects of urotensin II in human arteries and veins of varying caliber

BACKGROUND:-Urotensin II (UII) is the ligand for the GPR14 receptor and the most potent vasoconstrictor in the cynomolgus monkey. UII also contracts rat thoracic aorta. We studied the effect of human UII (hUII) in human blood vessels Methods and Results-Small subcutaneous resistance arteries, internal mammary arteries, saphenous veins, and small subcutaneous veins were studied using standard techniques. Subcutaneous resistance arteries constricted in response to norepinephrine (maximum tension, 2.84+/-0.38 mN/mm; the concentration required to produce 50% of the maximum response [EC(50)], 0.52+/-0.07 micromol/L) and endothelin-1 (maximum tension, 4.19+/-0.93 mN/mm; EC(50), 1.6+/-0.1 nmol/L). hUII did not contract these arteries, internal mammary arteries, or either type of vein, but it was a potent vasoconstrictor in rat thoracic aorta (maximum tension, 2.36+/-0.2 mN/mm; EC(50), 1.13+/-0.36 nmol/L). CONCLUSIONS:-hUII has no vasoconstrictor action in human arteries and veins of different sizes and vascular beds. Marked species differences in the actions of UII question its importance in human cardiovascular regulation.     

Angiotensin II stimulates DNA and protein synthesis in vascular smooth muscle cells from human arteries: role of extracellular signal-regulated kinases.

OBJECTIVE: This study investigates the growth effects and associated signaling pathways of angiotensin II (Ang II) in human vascular smooth muscle cells. METHODS: Cultured vascular smooth muscle cells derived from resistance arteries (< 300 microm diameter) from subcutaneous gluteal biopsies of healthy subjects (n = 6) and human aortic vascular smooth muscle cells were used. Cells were studied between passages 3 and 6. Both 3H-thymidine and 3H-leucine incorporation were measured as indices of vascular smooth muscle cell hyperplasia (DNA synthesis) and cell hypertrophy (protein synthesis), respectively. Growth effects of Ang II (10(-12) - 10(-6) mol/l), in the absence and presence of 10(-5) mol/l losartan (AT1 antagonist) and PD123319 (AT2 antagonist), were determined. Ang II-induced effects were compared to those of endothelin-1. To determine whether extracellular signal-regulated kinase (ERK)-dependent pathways play a role in Ang II-mediated growth, cells were pretreated with the selective ERK kinase (MEK) inhibitor, PD98059 (10(-5) mol/l). ERK activation was determined by Western blot in the absence and presence of PD98059. RESULTS: Ang II dose-dependently increased 3H-thymidine incorporation in cells from aorta (Emax = 276 +/- 10.4% of control) and resistance arteries (Emax = 284 +/- 5.1% of control). Ang II also stimulated 3H-leucine incorporation in cells from aorta (Emax = 162 +/- 11.6 of control) and resistance arteries (Emax 175 +/- 10% of control). Unlike Ang II, endothelin-1 failed to significantly alter cellular growth, except at high concentrations (> 10(-7) mol/l), where it had a weak stimulatory effect Losartan, but not PD123319, blocked Ang II-stimulated growth responses. Ang II significantly increased phosphorylation of ERK-1 and ERK-2, with maximum responses obtained at 5 min. PD98059 inhibited Ang II-stimulated ERK activity and abrogated agonist-induced DNA and protein synthesis. Losartan, but not PD123319 inhibited Ang II-induced phosphorylation of ERK-1 and ERK-2. CONCLUSIONS: Ang II stimulates both hyperplasia and hypertrophy in vascular smooth muscle cells from human arteries. These growth effects are mediated via Ang II receptors of the AT1 subtype that are linked to ERK-dependent signaling pathways.     

Stable 5,6-epoxyeicosatrienoic acid analog relaxes coronary arteries through potassium channel activation

5,6-epoxyeicosatrienoic acid (5,6-EET) is a cytochrome P450 epoxygenase metabolite of arachidonic acid that causes vasorelaxation. However, investigations of its role in biological systems have been limited by its chemical instability. We developed a stable agonist of 5,6-EET, 5-(pentadeca-3(Z),6(Z),9(Z)-trienyloxy)pentanoic acid (PTPA), in which the 5,6-epoxide was replaced with a 5-ether. PTPA obviates chemical and enzymatic hydrolysis. In bovine coronary artery rings precontracted with U46619, PTPA (1 nmol/L to 10 micromol/L) induced concentration-dependent relaxations, with maximal relaxation of 86+/-5% and EC50 of 1 micromol/L. The relaxations were inhibited by the cyclooxygenase inhibitor indomethacin (10 micromol/L; max relaxation 43+/-9%); the ATP-sensitive K+ channel inhibitor glybenclamide (10 micromol/L; max relaxation 49+/-6%); and the large conductance calcium-activated K+ channel inhibitor iberiotoxin (100 nmol/L; max relaxation 38+/-6%) and abolished by the combination of iberiotoxin with indomethacin or glybenclamide or increasing extracellular K+ to 20 mmol/L. Whole-cell outward K+ current was increased nearly 6-fold by PTPA (10 micromol/L), which was also blocked by iberiotoxin. Additionally, we synthesized 5-(pentadeca-6(Z),9(Z)-dienyloxy)pentanoic acid and 5-(pentadeca-3(Z),9(Z)-dienyloxy)pentanoic acid (PDPA), PTPA analogs that lack the 8,9 or 11,12 double bonds of arachidonic acid and therefore are not substrates for cyclooxygenase. The PDPAs caused concentration-dependent relaxations (max relaxations 46+/-13% and 52+/-7%, respectively; EC50 1micromol/L), which were not altered by glybenclamide but blocked by iberiotoxin. These studies suggested that PTPA induces relaxation through 2 mechanisms: (1) cyclooxygenase-dependent metabolism to 5-ether-containing prostaglandins that activate ATP-sensitive K+ channels and (2) activation of smooth muscle large conductance calcium-activated K+ channels. PDPAs only activate large conductance calcium-activated K+ channels.     

Impaired Ca2+ signaling attenuates P2X receptor-mediated vasoconstriction of afferent arterioles in angiotensin II hypertension

This study tested the hypothesis that afferent arteriolar responses to purinoceptor activation are attenuated, and Ca2+ signaling mechanisms are responsible for the blunted preglomerular vascular reactivity in angiotensin II (Ang II) hypertension. Experiments determined the effects of ATP, the P2X1 agonist beta,gamma-methylene ATP or the P2Y agonist UTP on arteriolar diameter using the juxtamedullary nephron technique and on renal myocyte intracellular Ca2+ concentration ([Ca2+]i) using single cell fluorescence microscopy. Six or 13 days of Ang II infusion significantly attenuated the vasoconstrictor responses to ATP and beta,gamma-methylene ATP (P<0.05). During exposure to ATP (1, 10, and 100 micromol/L), afferent diameter declined by 17+/-2%, 29+/-3%, and 30+/-2% in normal control rats and 8+/-3%, 7+/-3%, and 22+/-3% in kidneys of Ang II-infused rats (13 days). Renal myocyte intracellular calcium responses to ATP or beta,gamma-methylene ATP were also decreased in Ang II hypertensive rats. In myocytes of control rats, peak increases in [Ca2+]i averaged 107+/-21, 170+/-38, and 478+/-79 nmol/L at ATP concentrations of 1, 10, and 100 micromol/L, respectively. Ang II infusion for 13 days decreased the peak responses to ATP (1, 10, and 100 micromol/L) to 65+/-13, 102+/-20, and 367+/-73 nmol/L, respectively. The peak increases in [Ca2+]i in response to beta,gamma-methylene ATP were also reduced in Ang II hypertensive rats. However, angiotensin hypertension did not change the UTP-mediated vasoconstrictor responses or the myocyte calcium responses to UTP. These results indicate that the impaired autoregulatory response observed in Ang II-dependent hypertension can be attributed to impairment of P2X1 receptor-mediated signal transduction.     

Angiotensin II dilates bovine adrenal cortical arterioles: role of endothelial nitric oxide

Adrenal steroidogenesis is modulated by humoral and neuronal factors and blood flow. Angiotensin II (AII) stimulates adrenal cortical aldosterone and cortisol production and medullary catecholamine release. However, AII regulation of adrenal vascular tone has not been characterized. We examined the effect of AII on diameters of cannulated bovine adrenal cortical arteries. Cortical arteries (average internal diameter = 230 microm) were constricted with U46619 and concentration-diameter responses to AII (10(-13) to 10(-8) mol/liter) were measured. In endothelium-intact arteries, AII induced dilations at low concentrations (maximum dilation = 25 +/- 6% at 10(-10) mol/liter) and constrictions at high concentrations (maximum constriction = 25 +/- 18% at 10(-8) mol/liter). AII constrictions were blocked by the angiotensin type 1 (AT1) receptor antagonist, losartan (10(-6) mol/liter). AII dilations were enhanced by losartan (maximal dilation = 48 +/- 8%), abolished by endothelial cell removal or N-nitro-L-arginine (L-NA, 3 x 10(-5) mol/liter) and inhibited by the angiotensin type 2 (AT2) receptor antagonist, PD123319 (10(-6) mol/liter, maximal dilation = 18 +/- 4%). In a 4,5-diaminofluorescein diacetate nitric oxide (NO) assay of isolated cortical arteries, AII stimulated NO production, which was abolished by PD123319, L-NA, or endothelial cell removal. Western immunoblot of arterial homogenates and endothelial and zona glomerulosa cell lysates revealed 48-kD and 50-kD bands corresponding to AT1 and AT2 receptors, respectively, in all three and a 140-kD band corresponding to endothelial NO synthase in endothelial cells and arteries. Our results demonstrate that AII stimulates adrenal cortical arterial dilation through endothelial cell AT2 receptor activation and NO release and AT1 receptor-dependent constriction.     

Nongenomic effects of aldosterone in the human heart: interaction with angiotensin II

Aldosterone exerts rapid "nongenomic" effects in various nonrenal tissues. Here, we investigated whether such effects occur in the human heart. Trabeculae and coronary arteries obtained from 57 heart valve donors (25 males; 32 females; 17 to 66 years of age) were mounted in organ baths. Aldosterone decreased contractility in atrial and ventricular trabeculae by maximally 34+/-3% and 15+/-4%, respectively, within 5 to 15 minutes after its application. The protein kinase C (PKC) inhibitor chelerythrine chloride, but not the mineralocorticoid receptor antagonists spironolactone and eplerenone, blocked this effect. Aldosterone also relaxed trabeculae that were prestimulated with angiotensin II (Ang II), and its negative inotropic effects were mimicked by hydrocortisone (at 10-fold lower potency) but not 17beta-estradiol. Aldosterone concentrations required to reduce inotropy were present in failing but not in normal human hearts. Previous exposure of coronary arteries to 1 micromol/L aldosterone or 17beta-estradiol (but not hydrocortisone) doubled the maximum contractile response (Emax) to Ang II. DeltaEmax correlated with extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (P<0.01). Spironolactone and eplerenone did not block the potentiating effect of aldosterone. Studies in porcine renal arteries showed that potentiation also occurred at pmol/L aldosterone levels but not at 17beta-estradiol levels <1 micromol/L. Aldosterone did not potentiate the alpha1-adrenoceptor agonist phenylephrine. In conclusion, aldosterone induces a negative inotropic response in human trabeculae (thereby antagonizing the positive inotropic actions of Ang II) and potentiates the vasoconstrictor effect of Ang II in coronary arteries. These effects are specific and involve PKC and ERK 1/2, respectively. Furthermore, they occur in a nongenomic manner, and require pathological aldosterone concentrations.     

Torasemide inhibits angiotensin II-induced vasoconstriction and intracellular calcium increase in the aorta of spontaneously hypertensive rats.

Torasemide is a loop diuretic that is effective at low once-daily doses in the treatment of arterial hypertension. Because its antihypertensive mechanism of action may not be based entirely on the elimination of salt and water from the body, a vasodilator effect of this drug can be considered. In the present study, the ability of different concentrations of torasemide to modify angiotensin II (Ang II)-induced vascular responses was examined, with the use of an organ bath system, in endothelium-denuded aortic rings from spontaneously hypertensive rats. Ang II-induced increases of intracellular free calcium concentration ([Ca(2+)](i)) were also examined by image analysis in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. A dose-response curve to Ang II was plotted for cumulative concentrations (from 10(-9) to 10(-6) mol/L) in endothelium-denuded aortic rings (pD(2)=7.5+/-0.3). Isometric contraction induced by a submaximal concentration of Ang II (10(-7) mol/L) was reduced in a dose-dependent way by torasemide (IC(50)=0.5+/-0.04 micromol/L). Incubation of VSMCs with different concentrations of Ang II (from 10(-10) to 10(-6) mol/L) resulted in a dose-dependent rise of [Ca(2+)](i) (pD(2)=7.5+/-0.3). The stimulatory effect of [Ca(2+)](i) induced by a submaximal concentration of Ang II (10(-7) mol/L) was blocked by torasemide (IC(50)=0.5+/-0.3 nmol/L). Our findings suggest that torasemide blocks the vasoconstrictor action of Ang II in vitro. This action can be related to the ability of torasemide to block the increase of [Ca(2+)](i) induced by Ang II in VSMCs. It is proposed that these actions might be involved in the antihypertensive effect of torasemide observed in vivo.     

Tempol augments angiotensin II-induced AT2 receptor-mediated relaxation in diabetic rat thoracic aorta

OBJECTIVE: To assess angiotensin II type 2 receptor-mediated responses in thoracic aorta of streptozotocin-induced diabetic rats. METHODS: The concentration-dependent relaxation response (in the presence of an AT1 receptor blocker) to angiotensin II (Ang II) was studied in phenylephrine (PE) or potassium chloride (KCl) precontracted rat thoracic aortic rings isolated from male Sprague-Dawley rats pretreated with streptozotocin (65 mg/kg i.p.) or vehicle 8 weeks prior to the study. RESULTS: Ang II-induced relaxation response (% relaxation), evident only in the presence of an AT1 receptor blocker, was significantly enhanced in aortic rings isolated from diabetic (55%) compared to control (25%) rats. Tempol (100 micromol/l) augmented the relaxation response in aortic rings isolated from diabetic (80%) but not control (28%) rats. N-nitro-l-arginine methyl ester (L-NAME) (100-300 micromol/l) [a nitric oxide (NO) synthase inhibitor] partially inhibited the relaxation response in diabetic (25%) and control (15%) rats. However, l-NAME (100 micromol/l) and glipizide or butanedione monoxime (1 micromol/l) (ATP-sensitive K channel blockers) together completely blocked the relaxation response. [H]Ang II saturation binding at the AT2 receptor was enhanced in aortic membranes from diabetic [maximum binding capacity, (Bmax)=1.14 +/- 0.06 fmol/mg protein] compared to control rats (Bmax=0.75 +/- 0.03 fmol/mg protein), with no change in the dissociation equilibrium constant (Kd) value (2.55 +/- 0.12 versus 2.22 +/- 0.15 nmol/l). CONCLUSIONS: The results suggest enhanced AT2-receptor density and function [mediated by a nitric oxide and ATP-sensitive K channel-dependent relaxation response (in presence of an AT1 receptor blocker)] in thoracic aorta isolated from diabetic rats. This could be a compensatory mechanism, which may be therapeutically exploited.     

Characterization of vasoconstrictor responses in small bovine adrenal cortical arteries in vitro

The adrenal gland is highly vascularized with tightly regulated blood flow that is closely correlated with steroidogenesis. Mechanisms involved in the regulation of adrenal blood flow and vascular tone are largely unknown. The present study characterizes the contractile responses of isolated small cortical arteries from bovine adrenal glands. In endothelium-intact arteries, K(+), the thromboxane mimetic U46619, 5-hydroxytryptamine (5-HT), and endothelin-1 (ET-1) induced concentration-dependent contractions, whereas phenylephrine, norepinephrine, and ACTH were without effect. The EC(50)s for K(+), U46619, 5-HT, and ET-1 were 45 +/- 3 mm, 150 +/- 24 nm, 370 +/- 38 nm, and 2.8 +/- 0.8 nm, respectively. Contractions induced by U46619, 5-HT, and ET-1 were blocked by the thromboxane receptor antagonist SQ 29,548, the 5-HT(2A) receptor antagonist ketanserin, and the ET(A) receptor antagonist BQ 123, respectively. Removal of the endothelium caused a marked leftward shift of concentration responses to high K(+), U46619, 5-HT, and ET-1, and revealed contractile responses to phenylephrine and norepinephrine. In U46619-preconstricted arteries, BQ 123 converted ET-1-induced contractions to relaxations (maximal relaxation of 57 +/- 8%), which were subsequently blocked by the ET(B) receptor antagonist BQ 788. The ET(B)-mediated relaxations were endothelium dependent and inhibited by the nitric oxide synthase inhibitor N-nitro-l-arginine, the cytochrome P450 inhibitor SKF 525A, and high extracellular K(+), but not by the cyclooxygenase inhibitor indomethacin. These results demonstrate that small adrenal cortical arteries are highly responsive to various vasoconstrictor agents. The forceful contractile responses of these arterioles are consistent with their potential role in the regulation of adrenal blood flow.     

Involvement of Rho-kinase and the actin filament network in angiotensin II-induced contraction and extracellular signal-regulated kinase activity in intact rat mesenteric resistance arteries

We have previously shown that angiotensin II (Ang II) and pressure increase extracellular signal-regulated kinase (ERK) 1/2 activity synergistically in intact, pressurized resistance arteries in vitro. However, the mechanisms by which pressure and Ang II activate ERK1/2 in intact resistance arteries remain to be determined. The purpose of the present study was to investigate the involvement of Rho-kinase and the actin filament network in Ang II- and pressure-induced ERK1/2 activation, as well as in the contractile response induced by Ang II. Mesenteric resistance arteries (200 to 300 microm) were isolated, mounted in an arteriograph, and stimulated by pressure, Ang II, or both. Activation of ERK1/2 was then measured by an in-gel assay. In mesenteric resistance arteries maintained at 70 mm Hg, Ang II (0.1 micromol/L) induced contraction (29+/-1.4% of phenylephrine, 10 micromol/L-induced contraction) and significantly increased ERK1/2 activity. Selective inhibition of Rho-kinase by Y-27632 (10 micromol/L) and selective disruption of the actin filament network by cytochalasin B (10 micromol/L) both decreased the Ang II-induced contraction by 78+/-1.2% and 87+/-1.9%, respectively, and significantly diminished ERK1/2 activity. In the absence of Ang II, increasing intraluminal pressure from 0 to 70 or 120 mm Hg increased ERK1/2 activity. ERK1/2 activity at 120 mm Hg was similar to that observed at 70 mm Hg in the presence of Ang II. Pressure-induced ERK1/2 activation was markedly attenuated by cytochalasin B but not by Y-27632. These results indicate that whereas pressure-induced ERK1/2 activation requires an intact actin filament network, but not Rho-kinase, the activation of ERK1/2 and the contraction induced by Ang II require both Rho-kinase and an intact actin filament network in isolated, intact mesenteric resistance arteries.     

Angiotensin II stimulates extracellular signal-regulated kinase activity in intact pressurized rat mesenteric resistance arteries

The activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was assessed in isolated rat mesenteric resistance arteries (200-micrometer diameter) in a pressure myograph and stimulated for 5 minutes by angiotensin II (Ang II, 0.1 micromol/L) with a pressure of 70 mm Hg. ERK1/2 activity was measured by using an in-gel assay, and ERK1/2 phosphorylation was measured by Western blot analysis with use of a phospho-specific ERK1/2 antibody. Ang II (0.1 micromol/L) induced contraction (28% of phenylephrine contraction, 10 micromol/L). ERK kinase inhibitor PD98059 (10 micromol/L) attenuated this contraction by 36% but not that to phenylephrine or K(+) (60 mmol/L). In unpressurized arteries, Ang II increased ERK1/2 activity by 26%, and pressure (70 mm Hg) itself increased ERK1/2 activity by 72%. Ang II and pressure together acted synergistically, increasing ERK1/2 activity by 264%. Thus, in pressurized vessels, Ang II (0.1 micromol/L) increased ERK1/2 activity by 112%, calculated as [(364/172)-1]x100, which was confirmed by a measured 72% increase in ERK1/2 phosphorylation. Ang II type 1 receptor blockade by candesartan (10 micromol/L) abolished the Ang II-induced increase in ERK1/2 activity, but Ang II type 2 receptor blockade (PD123319, 10 micromol/L) did not. The Ang II-induced increase in ERK1/2 activity was inhibited by protein kinase C inhibitors Ro-31-8220 (1 micromol/L) and Go-6976 (300 nmol/L) and tyrosine kinase inhibitors genistein (1 micromol/L, general) and herbimycin A (1 micromol/L, c-Src family). The present findings show for the first time in intact resistance arteries that ERK1/2 activation is rapidly regulated by Ang II, is synergistic with pressure, and is involved in contraction. The ERK1/2 signaling pathway apparently includes upstream protein kinase C and c-Src.     

Interaction between AT1 and alpha1-adrenergic receptors in cardiomyopathic hamsters

BACKGROUND: Cross talk between angiotensin AT1 and alpha1-adrenergic receptors has been reported previously and points to the existence of physiologic regulation between the renin-angiotensin system and the sympathetic nervous system at the receptor level. This regulation may play an important role in the control of blood pressure and may be modified in different cardiovascular pathologies. Nevertheless, neither the physiologic actions nor the clinical relevance of the interaction between these 2 receptors has yet been established. To reveal these aspects in relation to heart failure, the interaction between vascular AT1 and alpha1-adrenergic receptors was evaluated in the Syrian cardiomyopathic hamster model. METHODS AND RESULTS: The vascular response of each individual receptor to vasoactive agonist was assessed in the presence and absence of antagonists of the other receptor using aortic rings from 11-month-old Syrian cardiomyopathic hamsters. Age-matched golden hamsters were used as controls. In control hamsters, concentration-response curves for the norepinephrine (NE)-induced contraction were significantly displaced to the left after 100 mmol/L losartan incubation. The maximal tension achieved (Emax) values increased by 26+/-4.3% after incubation (P < .05). Similar results were obtained when 20 micromol/L enalapril was used to block angiotensin II (Ang II) synthesis. NE concentration-response curves were also displaced to the left and Emax increased by 27%+/-8.0% (P < .05). The concentrations that induce 50% of the maximal contraction (EC50) were 22.2+/-0.2 nmol/L for untreated and 27.1+/-2.0 nmol/L for losartan-treated aortic rings (n = 8, P > .05). However, EC50 values were significantly reduced in aortic rings treated with enalapril (7.51+/-0.16 nmol/L, n = 8, P < .05). Blockade of alpha1 receptor with 10 micromol/L prazosin increased the response to Ang II by 32% (n = 6, P < .05). In contrast, when these experiments were repeated in aortic rings from cardiomyopathic animals, no interaction between the 2 receptors was observed. NE concentration-response curves, Emax (9.6%+/-2.8% increase after enalapril. and 5.8%+/-6.5% increase after losartan, P > .05) and EC50 values (14.7+/-0.7 nmol/L without treatment, 17.5+/-1.5 nmol/L with enalapril and 11.1+/-0.8 with losartan, n = 8, P > .05) were similar. Furthermore, in cardiomyopathic animals, prazosin did not modify the vascular response to Ang II. CONCLUSIONS: An interaction exists between vascular AT1 and alpha1-adrenergic receptors in control hamsters but not in cardiomyopathic animals. This interaction seems to be bidirectional and counterregulatory. The lack of this regulation may promote a state of enhanced vascular wall activity, which could contribute to the increased vasoconstriction and total peripheral resistance characteristic of heart failure.