Pioglitazone protects the myocardium against ischemia-reperfusion injury in eNOS and iNOS knockout mice

Endothelial nitric oxide synthase (eNOS) activation with subsequent inducible NOS (iNOS), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase-2 (COX2) activation is essential to statin inhibition of myocardial infarct size (IS). In the rat, the peroxisome proliferator-activated receptor-gamma agonist pioglitazone (Pio) limits IS, upregulates and activates cPLA2 and COX2, and increases myocardial 6-keto-PGF1alpha levels without activating eNOS and iNOS. We asked whether Pio also limits IS in eNOS-/- and iNOS-/- mice. Male C57BL/6 wild-type (WT), eNOS-/-, and iNOS-/- mice received 10 mg.kg(-1).day(-1) Pio (Pio+) or water alone (Pio-) for 3 days. Mice underwent 30 min coronary artery occlusion and 4 h reperfusion, or hearts were harvested and subjected to ELISA and immunoblotting. As a result, Pio reduced IS in the WT (15.4+/-1.4% vs. 39.0+/-1.1%; P<0.001), as well as in the eNOS-/- (32.0+/-1.6% vs. 44.2+/-1.9%; P<0.001) and iNOS-/- (18.0+/-1.2% vs. 45.5+/-2.3%; P<0.001) mice. The protective effect of Pio in eNOS-/- mice was smaller than in the WT (P<0.001) and iNOS-/- (P<0.001) mice. Pio increased myocardial Ser633 and Ser1177 phosphorylated eNOS levels in the WT and iNOS-/- mice. iNOS was undetectable in all six groups. Pio increased cPLA2, COX2, and PGI2 synthase levels in the WT, as well as in the eNOS-/- and iNOS-/-, mice. Pio increased the myocardial 6-keto-PGF1alpha levels and cPLA2 and COX2 activity in the WT, eNOS-/-, and iNOS-/- mice. In conclusion, the myocardial protective effect of Pio is iNOS independent and may be only partially dependent on eNOS. Because eNOS activity decreases with age, diabetes, and advanced atherosclerosis, this effect may be relevant in a clinical setting and should be further characterized.     

Collecting duct-specific endothelin B receptor knockout increases ENaC activity

Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na(+) excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na(+) regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na(+) intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na(+) regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na(+) feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na(+) retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.     

Deletion of the eNOS gene has a greater impact on the pulmonary circulation of male than female mice

Nitric oxide is involved in development and postnatal adaptation of the pulmonary circulation. This study aimed to determine whether genetic deletion of nitric oxide synthase (NOS) would lead to maldevelopment of the pulmonary arteries in fetal life, compromise adaptation to extrauterine life, and be associated with a pulmonary hypertensive phenotype in adult life and if any abnormalities were detected, were they sex dependent. Morphometric analyses were made on lung tissue from male and female fetal, newborn, 14-day-old, and adult endothelial NOS-deficient (eNOS-/-) or inducible NOS-deficient (iNOS-/-) and wild-type mice. Hemodynamic studies were carried out on adult mice with deletion of either eNOS or iNOS genes. We found that in eNOS-/- mice, lung development was normal in fetal, newborn, and adult lungs. Pulmonary arterial muscularity was greater than normal in both male and female eNOS-/- during fetal life and at birth, but the abnormality persisted only in male mice. Right ventricular hypertrophy was present in 14-day-old and adult male eNOS-/- but not in female mice. Adult male eNOS-/- mice had higher mean right ventricular and systemic pressures than female eNOS-/- mice (P < 0.05). Thus deletion of the eNOS gene was associated with structural evidence of pulmonary hypertension in both sexes during fetal life, but pulmonary hypertension persisted only in the male. In neither sex did iNOS or neuronal NOS appear to compensate for the eNOS deletion. Adult iNOS-/- mice did not have structural or hemodynamic evidence of pulmonary hypertension. Possible compensatory mechanisms are discussed.     

A potent and specific agonist, Suc-[Glu9,Ala11,15]-endothelin-1(8-21), IRL 1620, for the ETB receptor.

A series of C-terminal linear peptides of endothelin (ET)-1 and their N alpha-succinyl (Suc) analogs were synthesized and their binding affinities for the two subtypes of ET receptor, ETA and ETB, in porcine lung membranes were examined. Among the synthetic analogs, Suc-[Glu9,Ala11,15]-ET-1(8-21), IRL 1620, was the most potent and specific ligand for the ETB receptor (KiETA/KiETB approximately equal to 120,000) as judged by the Ki values for ETA (1.9 microM) and ETB (16 pM) receptors. IRL 1620 was 60 times more selective for the ETB receptor than ET-3 (KiETA/KiETB approximately equal to 1,900). IRL 1620 (10(-9)-10(-7) M) induced contractions of the guinea pig trachea with a comparable potency to those of ET-1 or ET-3, suggesting that IRL 1620 is a potent ETB receptor agonist.     

Mice with targeted deletion of eNOS develop hyperdynamic circulation associated with portal hypertension

Systemic vasodilation is the initiating event of the hyperdynamic circulatory state, being most likely triggered by increased levels of vasodilators, primarily nitric oxide (NO). Endothelial NO synthase (eNOS) is responsible for this event. We tested the hypothesis that gene deletion of eNOS and inducible NOS (iNOS) may inhibit the development of the hyperdynamic circulatory state in portal hypertensive animals. To test this hypothesis, we used mice lacking eNOS (eNOS-/-) or eNOS/iNOS (eNOS/iNOS-/-) genes. A partial portal vein ligation (PVL) was used to induce portal hypertension. Sham-operated animals were used as a control. Hemodynamic characteristics were tested 2 wk after surgery. As opposed to our hypothesis, PVL also caused significant reduction in peripheral resistance in eNOS-/- compared with sham animals (0.33 +/- 0.02 vs. 0.41 +/- 0.03 mmHg. min x kg body wt x ml(-1); P = 0.04) and in eNOS/iNOS-/- animals with PVL compared with that of the sham-operated group (0.44 +/- 0.02 vs. 0.54 +/- 0.04; P = 0.03). This demonstrates that, despite gene deletion of eNOS, the knockout mice developed hyperdynamic circulation. Compensatory vasodilator molecule(s) are upregulated in place of NO in the systemic and splanchnic circulation in portal hypertensive animals.     

Endothelin 1 and 3 enhance neuronal nitric oxide synthase activity through ETB receptors involving multiple signaling pathways in the rat anterior hypothalamus

We have previously reported that endothelin 1 and 3 (ET-1, ET-3) through the ETB receptor decrease norepinephrine release in the anterior hypothalamus and activate the nitric oxide (NO) pathway. In the present work we sought to establish the receptors and intracellular mechanisms underlying the increase in nitric oxide synthase (NOS) activity stimulated by ET-1 and ET-3 in the rat anterior hypothalamus. Results showed that ETs-stimulated NOS activity was inhibited by a selective ETB antagonist (BQ-788), but not by a selective ETA antagonist (BQ-610). In addition, NOS activity was not altered in the presence of an ETA agonist (sarafotoxin 6b), but it was enhanced in the presence of a ETB agonist (IRL-1620). Both Nomega-nitro-L-arginine methyl ester (NOS inhibitor), and 7-nitroindazole (neuronal NOS inhibitor) diminished ETs-stimulated NOS activity. The stimulatory effect of ETs on NOS activity was inhibited in the presence of PLC, PKC, PKA and CaMK-II inhibitors (U-73122, GF-109203X, H-89 and KN-62, respectively), and the IP3 receptor selective antagonist, 2-APB. Our results showed that both ET-1 and ET-3 modulate neuronal NOS activity through the ETB receptor in the rat anterior hypothalamus involving the participation of the PLC-PKC/IP3 pathway as well as PKA and CaMK-II.     

Nitric oxide mediates protective effect of endothelin receptor antagonism during myocardial ischemia and reperfusion

Endothelin (ET) receptor antagonism protects from ischemia-reperfusion injury. We hypothesized that the cardioprotective effect is related to nitric oxide (NO) bioavailability. Buffer-perfused rat and mouse hearts were subjected to ischemia and reperfusion. At the onset of ischemia, the rat hearts received vehicle, the dual endothelin type A/type B (ETA/ETB) receptor antagonist bosentan (10 microM), the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 100 microM), the combination of bosentan and L-NMMA or the combination of bosentan, L-NMMA, and the NO substrate L-arginine (1 mM). Hearts from wild-type and endothelial NO synthase (eNOS)-deficient mice received either vehicle or bosentan. Myocardial performance, endothelial function, NO outflow, and eNOS expression were monitored. Bosentan significantly improved myocardial function during reperfusion in rats and in wild-type mice, but not in eNOS-deficient mice. The functional protection afforded by bosentan was inhibited by L-NMMA, whereas it was restored by L-arginine. Myocardial expression of eNOS (immunoblotting) increased significantly in bosentan-treated rat hearts compared with vehicle hearts. Recovery of NO outflow during reperfusion was enhanced in the bosentan-treated rat heart. The endothelium-dependent vasodilator adenosine diphosphate increased coronary flow by 18 +/- 9% at the end of reperfusion in the bosentan group, whereas it reduced coronary flow by 7 +/- 5% in the vehicle group (P < 0.001). The response to the endothelium-independent dilator sodium nitroprusside was not different between the two groups. In conclusion, the dual ETA/ETB receptor antagonist bosentan preserved endothelial and cardiac contractile function during ischemia and reperfusion via a mechanism dependent on endothelial NO production.     

Cloning and functional expression of human cDNA for the ETB endothelin receptor.

We report the cloning of human cDNA encoding an ETB (non-isopeptide-selective) subtype of the endothelin receptor. The predicted amino acid sequence of the human ETB endothelin receptor was 87.8% and 62.9% identical with the previously cloned rat ETB and ETA receptors, respectively. COS cells transiently transfected with the cloned cDNA expressed specific, high-affinity binding sites for endothelin isopeptides and responded to the peptides with a transient increase of [Ca2+]i; endothelin-1 and endothelin-3 exhibited approximately equal potencies both in displacing 125I-labeled endothelin-1 binding and in eliciting [Ca2+]i transients. The ETB receptor mRNAs were expressed in various human tissues and also in the intact porcine aortic intimal cells ex vivo.     

Hypoxia-induced reactive oxygen species downregulate ETB receptor-mediated contraction of rat pulmonary arteries

Production of reactive oxygen species (ROS) may be increased during hypoxia in pulmonary arteries. In this study, the role of ROS in the effect of hypoxia on endothelin (ET) type B (ETB) receptor-mediated vasocontraction in lungs was determined. In rat intrapulmonary (approximately 0.63 mm ID) arteries, contraction induced by IRL-1620 (a selective ETB receptor agonist) was significantly attenuated after 4 h of hypoxia (30 mmHg Po2) compared with normoxic control (140 mmHg Po2). The effect was abolished by tiron, a scavenger of superoxide anions, but not by polyethylene glycol (PEG)-conjugated catalase, which scavenges H2O2. The hypoxic effect on ETB receptor-mediated vasoconstriction was also abolished by endothelium denudation but not by nitro-L-arginine and indomethacin. Exposure for 4 h to exogenous superoxide anions, but not H2O2, attenuated the vasoconstriction induced by IRL-1620. Confocal study showed that hypoxia increased ROS production in pulmonary arteries that were scavenged by PEG-conjugated SOD. In endothelium-intact pulmonary arteries, the ETB receptor protein was reduced after 4 h of exposure to hypoxia, exogenous superoxide anions, or ET-1. BQ-788, a selective ETB receptor antagonist, prevented these effects. ET-1 production was stimulated in endothelium-intact arteries after 4 h of exposure to hypoxia or exogenous superoxide anions. This effect was blunted by PEG-conjugated SOD. These results demonstrate that exposure to hypoxia attenuates ETB receptor-mediated contraction of rat pulmonary arteries. A hypoxia-induced production of superoxide anions may increase ET-1 release from the endothelium and result in downregulation of ETB receptors on smooth muscle.     

DOCA-salt treatment enhances responses to endothelin-1 in murine corpus cavernosum

The penis is kept in the flaccid state mainly via a tonic activity of norepinephrine and endothelins (ETs). ET-1 is important in salt-sensitive forms of hypertension. We hypothesized that cavernosal responses to ET-1 are enhanced in deoxycorticosterone acetate (DOCA)-salt mice and that blockade of ETA receptors prevents abnormal responses of the corpus cavernosum in DOCA-salt hypertension. Male C57BL/6 mice were unilaterally nephrectomized and treated for 5 weeks with both DOCA and water containing 1% NaCl and 0.2% KCl. Control mice were uninephrectomized and received tap water with no added salt. Animals received either the ETA antagonist atrasentan (5 mg x day(-1) x kg(-1) body weight) or vehicle. DOCA-salt mice displayed increased systolic blood pressure (SBP), and treatment with atrasentan decreased SBP in DOCA-salt mice. Contractile responses in cavernosal strips from DOCA-salt mice were enhanced by ET-1, phenylephrine, and electrical field stimulation (EFS) of adrenergic nerves, whereas relaxations were not altered by IRL-1620 (an ETB agonist), acetylcholine, sodium nitroprusside, and EFS of nonadrenergic noncholinergic nerves. PD59089 (an ERK1/2 inhibitor), but not Y-27632 (a Rho-kinase inhibitor), abolished enhanced contractions to ET-1 in cavernosum from DOCA-salt mice. Treatment of DOCA-salt mice with atrasentan did not normalize cavernosal responses. In summary, DOCA-salt treatment in mice enhances cavernosal reactivity to contractile, but not to relaxant, stimuli, via ET-1/ETA receptor-independent mechanisms.     

Inotropic effects of ETB receptor stimulation and their modulation by endocardial endothelium, NO, and prostaglandins

Endothelin (ET)-1 acts on ETA and ETB receptors. The latter include ETB1 (endothelial) and ETB2 (muscular) subtypes, which mediate opposite effects on vascular tone. This study investigated, in rabbit papillary muscles (n = 84), the myocardial effects of ETB stimulation. ET-1 (10(-9) M) was given in the absence or presence of BQ-123 (ETA antagonist). The effects of IRL-1620 (ETB1 agonist, 10(-10)-10(-6) M) or sarafotoxin S6c (ETB agonist, 10(-10)-10(-6) M) were evaluated in muscles with intact or damaged endocardial endothelium (EE); intact EE, in the presence of NG-nitro-L-arginine (L-NNA); and intact EE, in the presence of indomethacin (Indo). Sarafotoxin S6c effects were also studied in the presence of BQ-788 (ETB2 antagonist). ET-1 alone increased 64 +/- 18% active tension (AT) but decreased it by 4 +/- 2% in the presence of BQ-123. In muscles with intact EE, sarafotoxin S6c alone did not significantly alter myocardial performance. Sarafotoxin S6c (10(-6) M) increased, however, AT by 120 +/- 27% when EE was damaged and by 39 +/- 8% or 23 +/- 6% in the presence of l-NNA or Indo, respectively. In the presence of BQ-788, sarafotoxin S6c decreased AT (21 +/- 3% at 10(-6) M) in muscles with intact EE, an effect that was abolished when EE was damaged. IRL-1620 also decreased AT (22 +/- 3% at 10(-6) M) in muscles with intact EE, an effect that was abolished when EE was damaged or in the presence of L-NNA or Indo. In conclusion, the ETB-mediated negative inotropic effect is presumably due to ETB1 stimulation, requires an intact EE, and is mediated by NO and prostaglandins, whereas the ETB-mediated positive inotropic effect, observed when EE was damaged or NO and prostaglandins synthesis inhibited, is presumably due to ETB2 stimulation.     

Endothelial E- and P-selectin expression in iNOS- deficient mice exposed to polymicrobial sepsis

In vitro, nitric oxide (NO) decreases leukocyte adhesion to endothelium by attenuating endothelial adhesion molecule expression. In vivo, lipopolysaccharide-induced leukocyte rolling and adhesion was greater in inducible NO synthase (iNOS)-/- mice than in wild-type mice. The objective of this study was to assess E- and P-selectin expression in the microvasculature of iNOS-/- and wild-type mice subjected to acute peritonitis by cecal ligation and perforation (CLP). E- and P-selectin expression were increased in various organs within the peritoneum of wild-type animals after CLP. This CLP-induced upregulation of E- and P-selectin was substantially reduced in iNOS-/- mice. Tissue myeloperoxidase (MPO) activity was increased to a greater extent in the gut of wild-type than in iNOS-/- mice subjected to CLP. In the lung, the reduced expression of E-selectin in iNOS-/- mice was not associated with a decrease in MPO. Our findings indicate that NO derived from iNOS plays an important role in sepsis-induced increase in selectin expression in the systemic and pulmonary circulation. However, in iNOS-/- mice, sepsis-induced leukocyte accumulation is affected in the gut but not in the lungs.     

PTHrP fragments 1-16 and 1-23 do not bind to either the ETA or the ETB endothelin receptors

Because of some isofunctional similarities with endothelin-1 (ET-1), it has been suggested that PTHrP(1-16) and PTHrP(1-23) could interact with osteoblast cells via ETA receptors. To document this interaction, we used the thoracic rat aorta and the guinea-pig lung parenchyma paradigms as ETA and ETB models, respectively. In addition, we also performed a series of competition experiments against [125I]ET-1, using transfected cells expressing the ETA or ETB receptor. So far, no agonistic nor antagonistic activities were observed in the ETA and ETB bioassays with the PTHrP fragments. Furthermore, both fragments were unable to displace [125I]ET-1 bound to cells expressing the ETA or ETB receptor.     

Simultaneous changes in intracellular calcium and tension induced by endothelin-1 and sarafotoxin S6c in guinea pig isolated gallbladder: influence of indomethacin

The relationships between changes in intracellular Ca2+ and smooth muscle tension triggered by endothelin-1 and the selective endothelin ETB receptor agonist sarafotoxin S6c, as well as their susceptibility to modification by the nonselective cyclooxygenase blocker indomethacin, were assessed in guinea pig isolated gallbladder strips. Cumulative additions of either agonist (1, 10, and 100 nM) induced simultaneous graded, strongly correlated, slowly developing, and sustained changes in tension and intracellular Ca+2 (Fura-2 technique). Sarafotoxin S6c was more effective than endothelin-1 in raising intracellular Ca2+ at 1 or 10 nM, but their abilities to cause contractions were similar at all concentrations. Indomethacin (5.6 microM) markedly inhibited the changes in both intracellular Ca2+ and tension caused by all concentrations of sarafotoxin S6c (in response to 100 nM, increases in Ca2+ fluorescence intensity and tension were inhib ited from 7.7 +/- 0.7 to 4.0 +/- 0.4% and from 460 +/- 100 to 160 +/- 40 mg, respectively) but only reduced the contraction triggered by 100 nM endothelin-1 (from 560 +/- 100 to 230 +/- 70 mg). Endothelin-1 caused greater prostacyclin release from gallbladder than sarafotoxin S6c (at 100 nM, 6-keto-PGF1alpha levels in the medium rose 4.8- and 2.8-fold, respectively; P < 0.05) and slightly increased thromboxane A2 release (1.6-fold; P < 0.05). Thus, gallbladder contractions triggered by combined ETA/ETB or selective ETB receptor stimulation (with endothelin-1 or sarafotoxin S6c, respectively) are strongly correlated with increases in intracellular Ca2+ but differentially affected by indomethacin. It remains to be assessed if this difference is because endothelin-1 triggers greater prostacyclin release than sarafotoxin S6c and (or) is due to the coupling of ETA and ETB receptors to distinct patterns of generation of cyclooxygenase-derived eicosanoids.     

Endothelin-1 activates endothelial cell nitric-oxide synthase via heterotrimeric G-protein betagamma subunit signaling to protein jinase B/Akt

Endothelin-1 has dual vasoactive effects, mediating vasoconstriction via ETA receptor activation of vascular smooth muscle cells and vasorelaxation via ETB receptor activation of endothelial cells. Although it is commonly accepted that endothelin-1 binding to endothelial cell ETB receptors stimulates nitric oxide (NO) synthesis and subsequent smooth muscle relaxation, the signaling pathways downstream of ETB receptor activation are unknown. Here, using a model in which we have utilized isolated primary endothelial cells, we demonstrate that ET-1 binding to sinusoidal endothelial cell ETB receptors led to increased protein kinase B/Akt phosphorylation, endothelial cell nitric-oxide synthase (eNOS) phosphorylation, and NO synthesis. Furthermore, eNOS activation was not dependent on tyrosine phosphorylation, and pretreatment of endothelial cells with pertussis toxin as well as overexpression of a dominant negative G-protein-coupled receptor kinase construct that sequesters betagamma subunits inhibited Akt phosphorylation and NO synthesis. Taken together, the data elucidate a G-protein-coupled receptor signaling pathway for ETB receptor-mediated NO production and call attention to the absolute requirement for heterotrimeric G-protein betagamma subunits in this cascade.     

Distinct roles of nitric oxide synthases and interstitial cells of Cajal in rectoanal relaxation

Nitric oxide (NO) relaxes the internal anal sphincter (IAS), but its enzymatic source(s) remains unknown; neuronal (nNOS) and endothelial (eNOS) NO synthase (NOS) isoforms could be involved. Also, interstitial cells of Cajal (ICC) may be involved in IAS relaxation. We studied the relative roles of nNOS, eNOS, and c-Kit-expressing ICC for IAS relaxation using genetic murine models. The basal IAS tone and the rectoanal inhibitory reflex (RAIR) were assessed in vivo by a purpose-built solid-state manometric probe and by using wild-type, nNOS-deficient (nNOS-/-), eNOS-deficient (eNOS-/-), and W/W(v) mice (lacking certain c-Kit-expressing ICC) with or without L-arginine or N(omega)-nitro-L-arginine methyl ester (L-NAME) treatment. Moreover, the basal tone and response to electrical field stimulation (EFS) were studied in organ bath using wild-type and mutant IAS. In vivo, the basal tone of eNOS-/- was higher and W/W(v) was lower than wild-type and nNOS-/- mice. L-arginine administered rectally, but not intravenously, decreased the basal tone in wild-type, nNOS-/-, and W/W(v) mice. However, neither L-arginine nor L-NAME affected basal tone in eNOS-/- mice. In vitro, L-arginine decreased basal tone in wild-type and nNOS-/- IAS but not in eNOS-/- or wild-type IAS without mucosa. The in vivo RAIR was intact in wild-type, eNOS-/-, and W/W(v) mice but absent in all nNOS-/- mice. EFS-induced IAS relaxation was also reduced in nNOS-/- IAS. Thus the basal IAS tone is largely controlled by eNOS in the mucosa, whereas the RAIR is controlled by nNOS. c-Kit-expressing ICC may not be essential for the RAIR.     

Modulation of mouse cardiac function in vivo by eNOS and ANP

To study the role of endothelial nitric oxide synthase (eNOS) in cardiac function, we compared eNOS expression, contractility, and relaxation in the left ventricles of wild-type and eNOS-deficient mice. eNOS immunostaining is localized to the macro- and microvascular endothelium throughout the myocardium in wild-type mice and is absent in eNOS-/- mice. Whereas blood pressure is elevated in eNOS-/- mice, baseline cardiac contractility (dP/dt(max)) is similar in wild-type and eNOS-/- mice (9,673 +/- 2, 447 and 9,928 +/- 1,566 mmHg/s, respectively). The beta-adrenergic agonist isoproterenol (Iso) at doses of >/=1 ng causes enhanced increases in dP/dt(max) in eNOS-/- mice compared with wild-type controls in vivo (P < 0.01) as well as in Langendorff isolated heart preparations (P < 0.02). beta-Adrenergic receptor binding (B(max)) is not significantly different in the two groups of animals (B(max) = 41.4 +/- 9.4 and 36.1 +/- 5.1 fmol/mg for wild-type and eNOS-/-). Iso-stimulated ventricular relaxation is also enhanced in the eNOS-/- mice, as measured by dP/dt(min) in the isolated heart. However, baseline ventricular relaxation is normal in eNOS-/- mice (tau = 5.2 +/- 1.0 and 5.6 +/- 1.5 ms for wild-type and eNOS-/-, respectively), whereas it is impaired in wild-type mice after NOS inhibition (tau = 8.3 +/- 2.4 ms). cGMP levels in the left ventricle are unaffected by eNOS gene deletion (wild-type: 3.1 +/- 0.8 pmol/mg, eNOS-/-: 3.1 +/- 0.6 pmol/mg), leading us to examine the level of another physiological regulator of cGMP. Atrial natriuretic peptide (ANP) expression is markedly upregulated in the eNOS-/- mice, and exogenous ANP restores ventricular relaxation in wild-type mice treated with NOS inhibitors. These results suggest that eNOS attenuates both inotropic and lusitropic responses to beta-adrenergic stimulation, and it also appears to regulate baseline ventricular relaxation in conjunction with ANP.     

Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor.

We describe novel potent endothelin (ET) antagonists that are highly potent and selective for the ETA receptor (selective to ET-1). Of the synthetic analogs based on ETA antagonist BE-18257A isolated from Streptomyces misakiensis (IC50 value for ETA receptor on porcine aortic smooth muscle cells (VSMCs); 1.4 microM), the compounds BQ-123 and BQ-153 greatly improved the binding affinity of [125I]ET-1 for ETA receptors on VSMCs (IC50; 7.3 and 8.6 nM, respectively), whereas they barely inhibited [125I]ET-1 binding to ETB receptors (nonselective with respect to isopeptides of ET family) in the cerebellar membranes (IC50; 18 and 54 microM, respectively). Associated with the increased affinity for ETA receptors, these peptides antagonized ET-1-induced constriction of isolated porcine coronary artery. However, there was a small amount of ET-1-induced vasoconstriction resistant to these antagonists, which paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the aortic smooth muscle layer. These data suggest that the artery has both ETA and ETB receptors responsible for ET-1-induced vasoconstriction. The antagonists shifted the concentration-response curve to the right for ET-1 in the coronary artery, and increased the apparent dissociation constant in the Scatchard analysis of [125I]ET-1 binding on the VSMCs without affecting the binding capacity, indicative of the competitive antagonism for ETA receptor. In conscious rats, pretreatment with the antagonists markedly antagonized ET-1-induced sustained pressor responses in dose-dependent fashion without affecting ET-1-induced transient depressor action, suggesting that the pressor action is mediated by ETA receptors, while the depressor action is mediated by ETB receptors. In addition, pretreatment with the potent antagonists prevented ET-1-induced sudden death in mice. Thus, these potent ETA antagonists should provide a powerful tool for exploring the therapeutic uses of ETA antagonists in putative ET-1-related disorders.     

Reperfusion injury in skeletal muscle is reduced in inducible nitric oxide synthase knockout mice.

Inducible nitric oxide synthase (iNOS) participates in many pathological events, and selective inhibition of iNOS has been shown to reduce ischemia-reperfusion (I/R) injury in different tissues. To further confirm its role in this injury process, I/R injury was observed in denervated cremaster muscles of iNOS-deficient (iNOS-/-) and wild-type mice. After 3-h ischemia and 90-min reperfusion, blood flow in reperfused muscle was 80 +/- 8.5% (mean +/- SE) of baseline at 10-min reperfusion and completely returned to the preischemia baseline after 20 min in iNOS-/- mice. In contrast, blood flow was 32 +/- 7.4% at 10 min and increased to 60 +/- 20% of the baseline level at 90 min in wild-type mice (P < 0.001 vs. iNOS-/- mice at all time points). The increased muscle blood flow in iNOS-/- mice was associated with significantly less vasospasm in all three sizes of arterial vessel size categories. The weight ratio to the contralateral muscle not subjected to I/R was greater in wild-type mice (173 +/- 11%) than in iNOS-/- mice (117 +/- 3%; P < 0.01). Inflammation and neutrophil extravasation were also more severe in wild-type mice. Western blot analysis demonstrated an absence of iNOS protein band in iNOS-/- mice and upregulation of iNOS protein expression in wild-type mice. Our results confirm the importance of iNOS in I/R injury. Upregulated iNOS exacerbates I/R injury and appears to be a therapeutic target in protection of tissues against this type of injury.