Contribution of endothelin to coronary vasomotor tone is abolished after myocardial infarction

Left ventricular dysfunction in swine with a recent myocardial infarction (MI) is associated with neurohumoral activation, including increased catecholamines and endothelin (ET). Although the increase in ET may serve to maintain blood pressure and, hence, perfusion of essential organs such as the heart and brain, it could also compromise myocardial perfusion by evoking coronary vasoconstriction. In the present study, we tested the hypothesis that endogenous ET contributes to perturbations in myocardial O2 balance during exercise in remodeled myocardium of swine with a recent MI. For this purpose, 26 chronically instrumented swine (10 with and 16 without MI) were studied at rest and while running on a treadmill at 1-4 km/h. After MI, plasma ET increased from 3.2 +/- 0.4 to 4.9 +/- 0.3 pM (P < 0.05). In normal swine, blockade of ETA (by EMD-122946) or ETA-ETB (by tezosentan) receptors resulted in an increase in coronary venous PO2, i.e., coronary vasodilation at rest, which decreased during exercise. In contrast, neither ETA nor ETA-ETB receptor blockade resulted in coronary vasodilation in swine with MI. Coronary vasoconstriction to intravenous ET-1 infusion in awake resting swine was blunted after MI. To investigate whether factors released by cardiac myocytes contributed to decreased vascular responsiveness to ET, we performed ET-1 dose-response curves in isolated coronary arterioles (70-200 microm). Vasoconstriction to ET-1 in isolated arterioles from MI swine was enhanced. In conclusion, the vasoconstrictor influence of endogenous as well as exogenous ET on coronary circulation in vivo is reduced. Because the response of isolated coronary arterioles to ET is increased after MI, the reduced vasoconstrictor influence in vivo suggests modulation of ET receptor sensitivity by cardiac myocytes, which may serve to maintain adequate myocardial perfusion.     

Prostanoids suppress the coronary vasoconstrictor influence of endothelin after myocardial infarction

Myocardial infarction (MI) is associated with endothelial dysfunction resulting in an imbalance in endothelium-derived vasodilators and vasoconstrictors. We have previously shown that despite increased endothelin (ET) plasma levels, the coronary vasoconstrictor effect of endogenous ET is abolished after MI. In normal swine, nitric oxide (NO) and prostanoids modulate the vasoconstrictor effect of ET. In light of the interaction among NO, prostanoids, and ET combined with endothelial dysfunction present after MI, we investigated this interaction in control of coronary vasomotor tone in the remote noninfarcted myocardium after MI. Studies were performed in chronically instrumented swine (18 normal swine; 13 swine with MI) at rest and during treadmill exercise. Furthermore, endothelial nitric oxide synthase (eNOS) and cyclooxygenase protein levels were measured in the anterior (noninfarcted) wall of six normal and six swine with MI. eNOS inhibition with N(ω)-nitro-L-arginine (L-NNA) and cyclooxygenase inhibition with indomethacin each resulted in coronary vasoconstriction at rest and during exercise, as evidenced by a decrease in coronary venous oxygen levels. The effect of l-NNA was slightly decreased in swine with MI, although eNOS expression was not altered. Conversely, in accordance with the unaltered expression of cyclooxygenase-1 after MI, the effect of indomethacin was similar in normal and MI swine. L-NNA enhanced the vasodilator effect of the ET(A/B) receptor blocker tezosentan but exclusively during exercise in both normal and MI swine. Interestingly, this effect of L-NNA was blunted in MI compared with normal swine. In contrast, whereas indomethacin increased the vasodilator effect of tezosentan only during exercise in normal swine, indomethacin unmasked a coronary vasodilator effect of tezosentan in MI swine both at rest and during exercise. In conclusion, the present study shows that endothelial control of the coronary vasculature is altered in post-MI remodeled myocardium. Thus the overall vasodilator influences of NO as well as its inhibition of the vasoconstrictor influence of ET on the coronary resistance vessels were reduced after MI. In contrast, while the overall prostanoid vasodilator influence was maintained, its inhibition of ET vasoconstrictor influences was enhanced in post-MI remote myocardium.     

Coronary vasoconstrictor influence of angiotensin II is reduced in remodeled myocardium after myocardial infarction

The renin-angiotensin system plays an important role in cardiovascular homeostasis by contributing to the regulation of blood volume, blood pressure, and vascular tone. Because AT(1) receptors have been described in the coronary microcirculation, we investigated whether ANG II contributes to the regulation of coronary vascular tone and whether its contribution is altered during exercise. Since the renin-angiotensin system is activated after myocardial infarction, resulting in an increase in circulating ANG II, we also investigated whether the contribution of ANG II to the regulation of vasomotor tone is altered after infarction. Twenty-six chronically instrumented swine were studied at rest and while running on a treadmill at 1-4 km/h. In 13 swine, myocardial infarction was induced by ligation of the left circumflex coronary artery. Blockade of AT(1) receptors (irbesartan, 1 mg/kg iv) had no effect on myocardial O(2) consumption but resulted in an increase in coronary venous O(2) tension and saturation both at rest and during exercise, reflecting coronary vasodilation. Despite increased plasma levels of ANG II after infarction and maintained coronary arteriolar AT(1) receptor levels, the vasodilation evoked by irbesartan was significantly reduced both at rest and during exercise. In conclusion, despite elevated plasma levels, the vasoconstrictor influence of ANG II on the coronary circulation in vivo is reduced after myocardial infarction. This reduction in ANG II-induced coronary vasoconstriction may serve to maintain perfusion of the remodeled myocardium.     

20-Hydroxyeicosatetraenoic acid synthesis is increased in human neutrophils and platelets by angiotensin II and endothelin-1

The cytochrome P-450 arachidonic acid metabolite 20-HETE is central to the regulation of vascular tone, renal function, and blood pressure and is synthesized in the rat kidney in response to angiotensin II (ANG II) and endothelin-1 (ET-1). There are very few studies examining the cellular synthesis of 20-HETE in humans. We aimed to measure human neutrophil and platelet 20-HETE levels under basal conditions and after ANG II, ET-1, and calcium ionophore (CaI). 20-HETE was measured in human platelets and neutrophils after saline (control), CaI (2.5 μg/ml), and ANG II or ET-1 (10 nmol/l-1 μmol/l) incubations. The effect of cells, which were preincubated with the ω-hydroxylase inhibitor N-hydroxy-N'-(4-butyl-2-methylphenyl) (HET0016, 10 nM), ANG II types 1 or 2 (AT(1) or AT(2)) receptor inhibition with irbesartan (1 μmol/l) or PD-123319 (1 μmol/l), or endothelin receptor subtypes A or B (ET(A) or ET(B)) receptor inhibition with BQ-123 or BQ-778 (100 nmol/l), was studied. Neutrophil and platelet content and release of 20-HETE was significantly increased by CaI and blocked by the ω-hydroxylase inhibitor HET0016. ANG II and ET-1 significantly increased neutrophil and platelet content and release of 20-HETE. ANG II increased 20-HETE via the AT(2) receptor. ET-1 increased 20-HETE through the ET(B) receptor in platelets and both the ET(A) and ET(B) receptors in neutrophils. These studies show that human platelets and neutrophils synthesize 20-HETE in response to ANG II and ET-1. 20-HETE synthesis in both cell types was predominantly mediated via the AT(2) and ET(B) receptors. Stimulation via these receptor pathways has generally been thought to be cardioprotective and requires further studies in clinical situations associated with low-grade inflammation or where ANG II and ET-1 are elevated to clarify the role of 20-HETE.     

Functional role of angiotensin II type 1 and 2 receptors in regulation of uterine blood flow in nonpregnant sheep

The objective was to determine the receptor subtype of angiotensin II (ANG II) that is responsible for vasoconstriction in the nonpregnant ovine uterine and systemic vasculatures. Seven nonpregnant estrogenized ewes with indwelling uterine artery catheters and flow probes received bolus injections (0.1, 0.3 and 1 microg) of ANG II locally into the uterine artery followed by a systemic infusion of ANG II at 100 ng x kg(-1) x min(-1) for 10 min to determine uterine vasoconstrictor responses. Uterine ANG II dose-response curves were repeated following administration of the ANG II type 2 receptor (AT(2)) antagonist PD-123319 and then repeated again in the presence of an ANG II type 1 receptor (AT(1)) antagonist L-158809. In a second experiment, designed to investigate the mechanism of ANG II potentiation that occurred in the presence of AT(2) blockade, nonestrogenized sheep received a uterine artery infusion of L-158809 (3 mg/min for 5 min) prior to the infusion of 0.03 microg/min of ANG II for 10 min. ANG II produced dose-dependent decreases in uterine blood flow (P < 0.03), which were potentiated in the presence of the AT(2) antagonist (P < 0.02). Addition of the AT(1) antagonist abolished the uterine vascular responses and blocked ANG II-induced increases in systemic arterial pressure (P < 0.01). Significant uterine vasodilation (P < 0.01) was noted with AT(1) blockade in the second experiment, which was reversed by administration of the AT(2) antagonist or by the nitric oxide synthetase inhibitor N(omega)-nitro-L-arginine methyl ester. We conclude that the AT(1)-receptors mediate the systemic and uterine vasoconstrictor responses to ANG II in the nonpregnant ewe. AT(2)-receptor blockade resulted in a potentiation of the uterine vasoconstrictor response to ANG II, suggesting that the AT(2)-receptor subtype may modulate uterine vascular responses to ANG II potentially by release of nitric oxide.     

Coronary arteriolar vasoconstriction to angiotensin II is augmented in prediabetic metabolic syndrome via activation of AT1 receptors

The metabolic syndrome is associated with activation of the renin-angiotensin system. However, whether the coronary vascular response to ANG II is altered under this condition is unknown. Experiments were conducted in control and chronically high-fat-fed dogs with the prediabetic metabolic syndrome both in vitro (isolated coronary arterioles, 60-110 microm) and in vivo (anesthetized and conscious). We found that plasma renin activity and ANG II levels are elevated in high-fat-fed dogs and that this increase in ANG II is associated with a significant increase in ANG II-mediated coronary vasoconstriction in isolated coronary arterioles and in anesthetized open-chest dogs. The vasoconstriction to ANG II is abolished by ANG II type 1 (AT1) receptor blockade. In conscious chronically instrumented dogs, AT1 receptor blockade with telmisartan improved the balance between coronary blood flow and myocardial oxygen consumption in the high-fat-fed dogs but not in normal control dogs, i.e., the relationship between coronary venous Po2 and myocardial oxygen consumption was shifted upward, toward normal control values. Quantitative assessment of coronary arteriolar AT1 and ANG II type 2 (AT2) receptor mRNA levels by real-time PCR revealed no significant difference between normal control and high-fat-fed dogs; however, Western blot analysis showed a significant increase in AT1 receptor protein level with no change in AT2 receptor protein density. These findings indicate that AT1 receptor-mediated coronary constriction is augmented in the prediabetic metabolic syndrome and contributes to impaired control of coronary blood flow via increases in circulating ANG II and/or coronary arteriolar AT1 receptor density.     

Angiotensin II mediates uterine vasoconstriction through alpha-stimulation

Intravenous angiotensin II (ANG II) increases uterine vascular resistance (UVR), whereas uterine intra-arterial infusions do not. Type 2 ANG II (AT(2)) receptors predominate in uterine vascular smooth muscle; this may reflect involvement of systemic type 1 ANG II (AT(1)) receptor-mediated alpha-adrenergic activation. To examine this, we compared systemic pressor and UVR responses to intravenous phenylephrine and ANG II without and with systemic or uterine alpha-receptor blockade and in the absence or presence of AT(1) receptor blockade in pregnant and nonpregnant ewes. Systemic alpha-receptor blockade inhibited phenylephrine-mediated increases in mean arterial pressure (MAP) and UVR, whereas uterine alpha-receptor blockade alone did not alter pressor responses and resulted in proportionate increases in UVR and MAP. Although neither systemic nor uterine alpha-receptor blockade affected ANG II-mediated pressor responses, UVR responses decreased >65% and also were proportionate to increases in MAP. Systemic AT(1) receptor blockade inhibited all responses to intravenous ANG II. In contrast, uterine AT(1) receptor blockade + systemic alpha-receptor blockade resulted in persistent proportionate increases in MAP and UVR. Uterine AT(2) receptor blockade had no effects. We have shown that ANG II-mediated pressor responses reflect activation of systemic vascular AT(1) receptors, whereas increases in UVR reflect AT(1) receptor-mediated release of an alpha-agonist and uterine autoregulatory responses.     

Angiotensin receptors contribute to blood pressure homeostasis in salt-depleted SHR

This study evaluated the contribution of angiotensin peptides acting at various receptor subtypes to the arterial pressure and heart rate of adult 9-wk-old male conscious salt-depleted spontaneously hypertensive rats (SHR). Plasma ANG II and ANG I in salt-depleted SHR were elevated sevenfold compared with peptide levels measured in sodium-replete SHR, whereas plasma ANG-(1-7) was twofold greater in salt-depleted SHR compared with salt-replete SHR. Losartan (32.5 micromol/kg), PD-123319 (0.12 micromol. kg(-1). min(-1)), [d-Ala(7)]ANG-(1-7) (10 and 100 pmol/min), and a polyclonal ANG II antibody (0.08 mg/min) were infused intravenously alone or in combination. Combined blockade of AT(2) and AT((1-7)) receptors significantly increased the blood pressure of losartan-treated SHR (+15 +/- 1 mmHg; P < 0.01); this change did not differ from the blood pressure elevation produced by the sole blockade of AT((1-7)) receptors (15 +/- 4 mmHg). On the other hand, sole blockade of AT(2) receptors in losartan-treated SHR increased mean arterial pressure by 8 +/- 1 mmHg (P < 0.05 vs. 5% dextrose in water as vehicle), and this increase was less than the pressor response produced by blockade of AT((1-7)) receptors alone or combined blockade of AT((1-7)) and AT(2) receptors. The ANG II antibody increased blood pressure to the greatest extent in salt-depleted SHR pretreated with only losartan (+11 +/- 2 mmHg) and to the least extent in salt-depleted SHR previously treated with the combination of losartan, PD-123319, and [d-Ala(7)]ANG-(1-7) (+7 +/- 1 mmHg; P < 0.01). Losartan significantly increased heart rate, whereas other combinations of receptor antagonists or the ANG II antibody did not alter heart rate. Our results demonstrate that ANG II and ANG-(1-7) act through non-AT(1) receptors to oppose the vasoconstrictor actions of ANG II in salt-depleted SHR. Combined blockade of AT(2) and AT((1-7)) receptors and ANG II neutralization by the ANG II antibody reversed as much as 67% of the blood pressure-lowering effect of losartan.     

ETA and ETB receptors are expressed in vascular adventitial fibroblasts

The adventitia has been recognized to play important roles in vascular oxidative stress, remodeling, and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin (ET)-1 in response to ANG II. However, it is unclear whether ET-1 receptors are expressed in the adventitia. We therefore investigated the expression and roles of both ET(A) and ET(B) receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Adventitial fibroblasts were isolated and cultured from the mouse thoracic aorta by the explant method. Cultured cells were treated with ANG II (100 nmol/l) or ET-1 (10 pM) in the presence or absence of the ANG II type 1 receptor antagonist losartan (100 μM), the ET-1 receptor antagonists BQ-123 (ET(A) receptor, 1 μM) and BQ-788 (ET(B) receptor, 1 μM), and the ET(B) receptor agonist sarafotoxin 6C (100 nM). ET-1 peptide levels were determined by ELISA, whereas ET(A), ET(B), and collagen levels were determined by Western blot analysis. ANG II increased ET-1 peptide levels in a time-dependent manner. ANG II increased ET(A) and ET(B) receptor protein levels as well as collagen in a similar fashion. ANG II-induced collagen was reduced while in the presence of BQ-123, suggesting a role for the ET(A) receptor in the regulation of the extracellular matrix. ANG II treatment in the presence of BQ-788 significantly increased ET-1 peptide levels. Conversely, the ET(B) receptor agonist sarafotoxin 6C significantly decreased ET-1 peptide levels. These data implicate a role for the ET(B) receptor in the clearance of the ET-1 peptide. In conclusion, both ET(A) and ET(B) receptors are expressed in adventitial fibroblasts, which paves the ground for the biological significance of adventitial ET-1. The ET(A) receptor subtype mediates collagen I expression, whereas the ET(B) receptor subtype may play a protective role through increasing the clearance of the ET-1 peptide.     

Cerebrovascular ETB, 5-HT1B, and AT1 receptor upregulation correlates with reduction in regional CBF after subarachnoid hemorrhage

We hypothesize that cerebral ischemia leads to enhanced expression of endothelin (ET), 5-hydroxytryptamine (5-HT), and angiotensin II (ANG II) receptors in the vascular smooth muscle cells. Our aim is to correlate the upregulation of cerebrovascular receptors and the underlying molecular mechanisms with the reduction in regional and global cerebral blood flow (CBF) after subarachnoid hemorrhage (SAH). SAH was induced by injecting 250 microl blood into the prechiasmatic cistern in rats. The cerebral arteries were removed 0, 1, 3, 6, 12, 24, and 48 h after the SAH for functional and molecular studies. The contractile responses to ET-1, 5-carboxamidotryptamine (5-CT), and ANG II were investigated with myograph. The receptor mRNA and protein levels were analyzed by quantitative real-time PCR and immunohistochemistry, respectively. In addition, regional and global CBFs were measured by an autoradiographic method. As a result, SAH resulted in enhanced contractions to ET-1 and 5-CT. ANG II [via ANG II type 1 (AT(1)) receptors] induced increased contractile responses [in the presence of the ANG II type 2 (AT(2)) receptor antagonist PD-123319]. In parallel the ET(B), 5-HT(1B), and AT(1) receptor, mRNA and protein levels were elevated by time. The regional and global CBF showed a successive reduction with time after SAH. In conclusion, the results demonstrate for the first time that SAH induces the upregulation of ET(B), 5-HT(1B), and AT(1) receptors in a time-dependent manner both at functional, mRNA, and protein levels. These changes occur in parallel with a successive decrease in CBF. Thus there is a temporal correlation between the changes in receptor expression and CBF reduction, suggesting a linkage.     

Role of beta 2-adrenergic receptors on coronary resistance during exercise

The effects of regional alpha- and specific beta 2-adrenergic receptor blockade on measurements of late diastolic coronary resistance (LDCR) and mean coronary blood flow velocity (CBFV) during exercise were examined in 14 conscious adult mongrel dogs. Specific beta 2-adrenergic receptor blockade (ICI 118.551) significantly decreased CBFV and increased LDCR by blockade of beta 2-vasodilator tone independent of alpha-adrenergic receptor-mediated tone and independent of altering myocardial metabolism. alpha-Adrenergic receptor blockade (phentolamine, 1 mg) significantly increased CBFV and decreased LDCR by blocking sympathetically mediated vasoconstrictor tone. There was no significant difference in the magnitude of response between alpha- and beta 2-adrenergic receptor blockade. These results demonstrate that alpha- and beta 2-adrenergic receptors have a significant and evidently equal influence on CBFV and LDCR during exercise. Four weeks of daily exercise and left stellate ganglionectomy (LSGx) prevented phentolamine-induced vasodilation but not ICI 118.551-induced vasoconstriction. This suggests that daily exercise and LSGx significantly decreased the alpha-adrenergic receptor-mediated vasoconstrictor tone on the coronary circulation, resulting in an apparently greater role for the coronary vascular beta 2-adrenergic receptor on the control of CBFV and LDCR during exercise.     

Contribution of KATP+ channels to coronary vasomotor tone regulation is enhanced in exercising swine with a recent myocardial infarction

Previous studies demonstrated a decreased flow reserve in the hypertrophied myocardium early after myocardial infarction (MI). Previously, we reported that exacerbation of hemodynamic abnormalities and neurohumoral activation during exercise caused slight impairment of myocardial O(2) supply in swine with a recent MI. We hypothesized that increased metabolic coronary vasodilation [via ATP-sensitive K(+) (K(ATP)(+)) channels and adenosine] may have partially compensated for the increased extravascular compressive forces and increased vasoconstrictor neurohormones, thereby preventing a more severe impairment of myocardial O(2) balance. Chronically instrumented swine were exercised on a treadmill up to 85% of maximum heart rate. Under resting conditions, adenosine receptor blockade [8-phenyltheophylline (8-PT), 5 mg/kg i.v.] and K(ATP)(+) channel blockade (glibenclamide, 3 mg/kg i.v.) produced similar decreases in myocardial O(2) supply in normal and MI swine. However, while glibenclamide's effect waned in normal swine during exercise (P < 0.05), it was maintained in MI swine. 8-PT's effect was maintained during exercise and was not different between normal and MI swine. Finally, in normal swine combined treatment with 8-PT and glibenclamide produced a vasoconstrictor response that equaled the sum of the responses to blockade of the individual pathways. In contrast, in MI swine the vasoconstrictor response to 8-PT and glibenclamide was similar to that produced by glibenclamide alone. In conclusion, despite significant hemodynamic abnormalities in swine with a recent MI, myocardial O(2) supply and O(2) consumption in remodeled myocardium are still closely matched during exercise. This close matching is supported by increased K(ATP)(+) channel-mediated coronary vasodilation. Although the net vasodilator influence of adenosine was unchanged in remodeled myocardium, it became exclusively dependent on K(ATP)(+) channel opening.     

Central angiotensin II AT1 receptors mediate fetal swallowing and pressor responses in the near-term ovine fetus

Swallowed volumes in the fetus are greater than adult values (per body weight) and serve to regulate amniotic fluid volume. Central ANG II stimulates swallowing, and nonspecific ANG II receptor antagonists inhibit both spontaneous and ANG II-stimulated swallowing. In the adult rat, AT1 receptors mediate both stimulated drinking and pressor activities, while the role of AT2 receptors is controversial. As fetal brain contains increased ANG II receptors compared with the adult brain, we sought to investigate the role of both AT1 and AT2 receptors in mediating fetal swallowing and pressor activities. Five pregnant ewes with singleton fetuses (130 +/- 1 days) were prepared with fetal vascular and lateral ventricle (LV) catheters and electrocorticogram and esophageal electromyogram electrodes and received three studies over 5 days. On day 1 (ANG II), following a 2-h basal period, 1 ml artificial cerebrospinal fluid (aCSF) was injected in the LV. At time 4 h, ANG II (6.4 microg) was injected in the LV, and the fetus was monitored for a final 2 h. On day 3, AT1 receptor blocker (losartan 0.5 mg) was administered at 2 h, and ANG II plus losartan was administered at 4 h. On day 5, AT2 receptor blocker (PD-123319; 0.8 mg was administered at 2 h and ANG II plus PD-123319 at 4 h. In the ANG II study, LV injection of ANG II significantly increased fetal swallowing (0.9 +/- 0.1 to 1.4 +/- 0.1 swallows/min; P < 0.05). In the losartan study, basal fetal swallowing significantly decreased in response to blockade of AT1 receptors (0.9 +/- 0.1 to 0.4 +/- 0.1 swallows/min; P < 0.05), while central injection of ANG II in the presence of AT1 receptor antagonism did not increase fetal swallowing (0.6 +/- 0.1 swallows/min). In the PD-123319 study, basal fetal swallowing did not change in response to blockade of AT2 receptor (0.9 +/- 0.1 swallows/min), while central injection of ANG II in the presence of AT2 blockade significantly increased fetal swallowing (1.5 +/- 0.1 swallows/min; P < 0.05). ANG II caused significant pressor responses in the control and PD-123319 studies but no pressor response in the presence of AT1 blockade. These data demonstrate that in the near-term ovine fetus, AT1 receptor but not AT2 receptors accessible via CSF contribute to dipsogenic and pressor responses.     

NO and prostanoids blunt endothelin-mediated coronary vasoconstrictor influence in exercising swine

Withdrawal of the endothelin (ET)-mediated vasoconstrictor influence contributes to metabolic coronary vasodilation during exercise. Because production of nitric oxide (NO) and prostanoids increases with increasing shear stress and because NO and prostanoids are able to modify the release of ET, we hypothesized that the withdrawal of ET-mediated coronary vasoconstriction during exercise is mediated through NO and/or prostanoids. To test this hypothesis, 19 chronically instrumented swine were studied at rest and while running on a treadmill up to 85-90% of maximal heart rate. Blockade of ET(A)/ET(B) receptors with tezosentan resulted in an increase in coronary venous O(2) levels (i.e., in coronary vasodilation) at rest, which waned at increasing levels of exercise intensity. Inhibition of either NO synthase [N(omega)-nitro-l-arginine (l-NNA)] or cyclooxygenase (indomethacin) did not affect the response to tezosentan under resting conditions but unmasked a vasodilator response to tezosentan during exercise. The vasodilator response to tezosentan during exercise increased progressively after combined administration of l-NNA and indomethacin. These findings suggest that NO and prostanoids act synergistically to inhibit the vasoconstrictor influence of ET on the coronary circulation during exercise, thereby facilitating the exercise-induced vasodilation of coronary resistance vessels.     

Metabolic regulation of coronary vascular tone: role of endothelin-1

Coronary tone is determined by a balance between endogenously produced endothelin and metabolic dilators. We hypothesized that coronary vasodilation during augmented metabolism is the net result of decreased endothelin production and increased production of vasodilators. Isolated rat myocytes were stimulated at 0, 200, and 400 beats/min to modify metabolism. Supernatant from these preparations was added to isolated coronary arterioles with and without blocking vasoactive pathways (adenosine, bradykinin, and endothelin). Chronically instrumented swine were studied while resting and running on a treadmill before and after endothelin type A (ET(A)) receptor blockade. The vasodilatory properties of the supernatant increased with increased stimulation frequencies. Combined blockade of adenosine and bradykinin receptors abolished vasodilation in response to supernatant of stimulated myocytes. ET(A) blockade increased vasodilation to supernatant of unstimulated myocytes but did not affect dilation to supernatant of myocytes stimulated at 400 beats/min. In vivo, ET(A) blockade resulted in coronary vasodilation at rest, which waned during exercise. Thus endothelin has a tonic constrictor influence through the ET(A) receptor at low myocardial metabolic demand but its influence decreased during increased metabolism.     

Long-term AT1 receptor blockade improves metabolic function and provides renoprotection in Fischer-344 rats

Fischer-344 (F344) rats exhibit proteinuria and insulin resistance in the absence of hypertension as they age. We determined the effects of long-term (1 yr) treatment with the angiotensin (ANG) II type 1 (AT(1)) receptor blocker L-158,809 on plasma and urinary ANG peptide levels, systolic blood pressure (SBP), and indexes of glucose metabolism in 15-mo-old male F344 rats. Young rats at 3 mo of age (n = 8) were compared with two separate groups of older rats: one control group (n = 7) and one group treated with L-158,809 (n = 6) orally (20 mg/l) for 1 yr. SBP was not different between control and treated rats but was higher in young rats. Serum leptin, insulin, and glucose levels were comparable between treated and young rats, whereas controls had higher glucose and leptin with a similar trend for insulin. Plasma ANG I and ANG II were higher in treated than untreated young or older rats, as evidence of effective AT(1) receptor blockade. Urinary ANG II and ANG-(1-7) were higher in controls compared with young animals, and treated rats failed to show age-related increases. Protein excretion was markedly lower in treated and young rats compared with control rats (young: 8 +/- 2 mg/day vs. control: 129 +/- 51 mg/day vs. treated: 9 +/- 3 mg/day, P < 0.05). Long-term AT(1) receptor blockade improves metabolic parameters and provides renoprotection. Differential regulation of systemic and intrarenal (urinary) ANG systems occurs during blockade, and suppression of the intrarenal system may contribute to reduced proteinuria. Thus, insulin resistance, renal injury, and activation of the intrarenal ANG system during early aging in normotensive animals can be averted by renin-ANG system blockade.     

Prevention of pulmonary vascular remodeling and of decreased BMPR-2 expression by losartan therapy in shunt-induced pulmonary hypertension

The renin-ANG system has been reported to be overexpressed in pulmonary arterial hypertension (PAH). We investigated the effects of ANG receptor-1 blockade by losartan on hemodynamics and signaling molecules in a piglet overflow model of early PAH. Twenty-six 3-wk-old piglets were randomized to placebo or losartan therapy (1 mg.kg(-1).day(-1)) after anastomosis of the inominate to the main pulmonary artery or after a sham operation. Three months later, the animals underwent a hemodynamic evaluation, followed by pulmonary tissue sampling for morphometry, immunohistochemistry, and real-time quantitative-PCR. Chronic systemic-to-pulmonary shunting increased the pulmonary vascular resistance from 2.5 +/- 0.2 to 6.2 +/- 0.3 mmHg.l(-1).min.m(-2) and arteriolar medial thickness from 13.6 to 25.4%. These changes were associated with increased expressions of ANG II and its type 1 (AT1) and type 2 (AT2) receptors, endothelin-1 (ET-1) and its type B receptor (ETB), and angiopoietin-1, together with decreased expressions of bone morphogeneic protein receptor-1A and -2 (BMPR-1A and BMPR-2, respectively) and unchanged expression of the receptor tyrosine kinase with immunoglobulin and EGF homology domains-2 (Tie 2). Pretreatment with losartan decreased shunt-induced pulmonary vascular resistance and medial thickness by 51% and 35%, respectively. Losartan therapy was associated with persistent overexpressions of ANG II, AT2, ET-1, ETB, and angiopoietin-1 and with a return to normal of the BMPR-2 expression. These results suggest that ANG II contributes to left-to-right, shunt-induced PAH.     

Endothelin-1 expression in vascular adventitial fibroblasts

Endothelial cells are a major source of endothelin (ET)-1, but the possibility that vascular adventitial fibroblasts generate ET-1 has not been explored. We hypothesized that aortic adventitial fibroblasts have the ability to produce ET-1, which may contribute to extracellular matrix synthesis. Vascular adventitial fibroblasts were isolated from mouse aorta and incubated with various concentrations of angiotensin II (ANG II). mRNA levels of preproET-1 and type I procollagen were detected with relative RT-PCR. ET-1 levels in culture medium were measured with ELISA. Protein levels of procollagen were detected with Western blotting. ANG II (10 and 100 nM, 1 microM) induced a time- and concentration-dependent increase in preproET-1 mRNA levels (P < 0.05). Induction of preproET-1 mRNA was accompanied by release of immunoreactive peptide ET-1 (P < 0.05). ANG II-evoked increases in preproET-1 mRNA expression and ET-1 release were blocked by losartan (100 microM), an AT1 receptor antagonist, but not PD-123319 (100 microM), an AT2 receptor antagonist. To further confirm our findings, we cloned and then sequenced vascular fibroblast preproET-1 bidirectionally with T7 and M13 reverse sequencing primers. Their nucleotide sequences were identical to preproET-1 cDNA from mouse vascular endothelial cells (accession no. AB081657). Moreover, ANG II-induced type I procollagen mRNA and protein expression were inhibited by BQ-123 (10 microM), an ET(A) receptor inhibitor, but not BQ-788 (10 microM), an ET(B) receptor inhibitor, suggesting a significant role of adventitial ET-1 in regulation of extracellular matrix synthesis. The results demonstrate that vascular adventitial fibroblasts are able to synthesize and release ET-1 in response to ANG II.     

Altered role of smooth muscle endothelin receptors in coronary endothelin-1 and alpha1-adrenoceptor-mediated vasoconstriction in Type 2 diabetes

Regulation of vascular tone and blood flow involves interactions between numerous local and systemic vascular control signals, many of which are altered by Type 2 diabetes (T2D). Vascular responses to endothelin-1 (ET-1) are mediated by endothelin type A (ET(A)) and type B (ET(B)) receptors that have been implicated in cross talk with alpha(1)-adrenoceptors (alpha(1)-AR). ET(A) and ET(B) receptor expression and plasma ET-1 levels are elevated in T2D; however, whether this influences coronary alpha(1)-AR function has not been examined. Therefore, we examined the effect of ET(A) and ET(B) receptor inhibition on coronary vasoconstriction to ET-1 and alpha(1)-AR activation in a mouse model of T2D. Coronary vascular responses were examined in isolated mouse hearts from control and diet-induced T2D C57BL/6J mice. Responses to ET-1 and the selective alpha(1)-AR agonist phenylephrine (PE) were examined alone and in the presence of the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) alone or in combination with selective ET(A) or ET(B) receptor inhibitors BQ-123 and BQ-788, respectively. Vasoconstriction to ET-1 was enhanced, whereas ET(B), but not ET(A), receptor blockade reduced basal coronary tone in T2D hearts. In the presence of l-NAME, ET(A) receptor inhibition attenuated ET-1 vasoconstriction in both groups, whereas ET(B) inhibition abolished this response only in control hearts. In addition, ET(A) inhibition enhanced alpha(1)-AR-mediated vasoconstriction in T2D, but not control, hearts following l-NAME treatment. Therefore, in this model, enhanced coronary ET-1 responsiveness is mediated primarily through smooth muscle ET(B) receptors, whereas the interaction with alpha(1)-ARs is mediated solely through the ET(A) receptor subtype.