Post-translational control of endothelial nitric oxide synthase: why isn't calcium/calmodulin enough?

Endothelial nitric oxide synthase (eNOS) is important for cardiovascular homeostasis, vessel remodeling, and angiogenesis. Given the impact of endothelium- derived nitric oxide (NO) in vascular biology, much work in the past several years has focused on the control of NO synthesis by regulatory proteins that influence its function. Indeed calcium-activated calmodulin is important for regulation of NOS activity. Herein we discuss why other proteins, in addition to calmodulin, are necessary for eNOS regulation and summarize the biology of negative and positive regulators of eNOS function in vitro, in cells, and in blood vessels.

Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells.

Vascular endothelial growth factor (VEGF) utilizes a phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling pathway to protect endothelial cells from apoptotic death. Here we show that PI 3-kinase/Akt signaling promotes endothelial cell survival by inhibiting p38 mitogen-activated protein kinase (MAPK)-dependent apoptosis. Blockade of the PI 3-kinase or Akt pathways in conjunction with serum withdrawal stimulates p38-dependent apoptosis. Blockade of PI 3-kinase/Akt also led to enhanced VEGF activation of p38 and apoptosis. In this context, the pro-apoptotic effect of VEGF is attenuated by the p38 MAPK inhibitor SB203580. VEGF stimulation of endothelial cells or infection with an adenovirus expressing constitutively active Akt causes MEKK3 phosphorylation, which is associated with decreased MEKK3 kinase activity and down-regulation of MKK3/6 and p38 MAPK activation. Conversely, activation-deficient Akt decreases VEGF-stimulated MEKK3 phosphorylation and increases MKK/p38 activation. Activation of MKK3/6 is not dependent on Rac activation since dominant negative Rac does not decrease p38 activation triggered by inhibition of PI 3-kinase. Thus, cross-talk between the Akt and p38 MAPK pathways may regulate the level of cytoprotection versus apoptosis and is a new mechanism to explain the cytoprotective actions of Akt.

Acidic hydrolysis as a mechanism for the cleavage of the Glu(298)-->Asp variant of human endothelial nitric-oxide synthase.

The 894G-->T polymorphism within exon 7 of the human endothelial nitric-oxide synthase (eNOS) gene codes for glutamate or aspartate, respectively, at residue 298 and has been associated with several diseases of cardiovascular origin. A recent report indicates that Asp(298)-eNOS (E298D) is cleaved intracellularly to 100- and 35-kDa fragments, suggesting a mechanism for reduced endothelial function. Here we have documented the precise cleavage site of the E298D variant as a unique aspartyl-prolyl (Asp(298)--Pro(299)) bond not seen in wild-type eNOS (Glu(298)). We show that E298D-eNOS, as isolated from cells and in vitro, is susceptible to acidic hydrolysis, and the 100-kDa fragment can be generated ex vivo by increasing temperature at low pH. Importantly, cleavage of E298D was eliminated using a sample buffer system designed to limit acidic hydrolysis of Asp--Pro bonds. These results argue against intracellular processing of E298D-eNOS and suggest that previously described fragmentation of E298D could be a product of sample preparation. We also found that eNOS turnover, NO production, and the susceptibility to cellular stress were not different in cells expressing WT versus E298D-eNOS. Finally, enzyme activities were identical for the respective recombinant enzymes. Thus, intracellular cleavage mechanisms are unlikely to account for associations between the exon 7 polymorphism and cardiovascular diseases.

Sphingosine 1-phosphate activates Akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells.

Sphingosine 1-phosphate (SPP) binds to members of the endothelial differentiation gene family (EDG) of receptors and leads to diverse signaling events including cell survival, growth, migration and differentiation. However, the mechanisms of how SPP activates these proangiogenic pathways are poorly understood. Here we show that SPP signals through the EDG-1 receptor to the heterotrimeric G protein G(i), leading to activation of the serine/threonine kinase Akt and phosphorylation of the Akt substrate, endothelial nitric-oxide synthase (eNOS). Inhibition of G(i) signaling, and phosphoinositide 3-kinase (PI 3-kinase) activity resulted in a decrease in SPP-induced endothelial cell chemotaxis. SPP also stimulates eNOS phosphorylation and NO release and these effects are also attenuated by inhibition of G(i) signaling, PI 3-kinase, and Akt. However, inhibition of NO production did not influence SPP-induced chemotaxis but effectively blocked the chemotactic actions of vascular endothelial growth factor. Thus, SPP signals through G(i) and PI 3-kinase leading to Akt activation and eNOS phosphorylation.

The sonic hedgehog receptor patched associates with caveolin-1 in cholesterol-rich microdomains of the plasma membrane.

The Hedgehog signaling pathway is involved in early embryonic patterning as well as in cancer; however, little is known about the subcellular localization of the Hedgehog receptor complex of Patched and Smoothened. Since Hh has been found in lipid rafts in Drosophila, we hypothesized that Patched and Smoothened might also be found in these cholesterol-rich microdomains. In this study, we demonstrate that both Smoothened and Patched are in caveolin-1-enriched/raft microdomains. Immunoprecipitation studies show that Patched specifically interacts with caveolin-1, whereas Smoothened does not. Fractionation studies show that Patched and caveolin-1 can be co-isolated from buoyant density fractions that represent caveolae/raft microdomains and that Patched and caveolin-1 co-localize by confocal microscopy. Glutathione S-transferase fusion protein experiments show that the interaction between Patched and caveolin-1 involves the caveolin-1 scaffolding domain and a Patched consensus binding site. Immunocytochemistry data and fractionation studies also show that Patched seems to be required for transport of Smoothened to the membrane. Depletion of plasmalemmal cholesterol influences the distribution of the Hh receptor complex in the caveolin-enriched/raft microdomains. These data suggest that caveolin-1 may be integral for sequestering the Hh receptor complex in these caveolin-enriched microdomains, which act as a scaffold for the interactions with the Hh protein.

In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation.

Caveolin-1, the primary coat protein of caveolae, has been implicated as a regulator of signal transduction through binding of its "scaffolding domain" to key signaling molecules. However, the physiological importance of caveolin-1 in regulating signaling has been difficult to distinguish from its traditional functions in caveolae assembly, transcytosis, and cholesterol transport. To directly address the importance of the caveolin scaffolding domain in vivo, we generated a chimeric peptide with a cellular internalization sequence fused to the caveolin-1 scaffolding domain (amino acids 82-101). The chimeric peptide was efficiently taken up into blood vessels and endothelial cells, resulting in selective inhibition of acetylcholine (Ach)-induced vasodilation and nitric oxide (NO) production, respectively. More importantly, systemic administration of the peptide to mice suppressed acute inflammation and vascular leak to the same extent as a glucocorticoid or an endothelial nitric oxide synthase (eNOS) inhibitor. These data imply that the caveolin-1 scaffolding domain can selectively regulate signal transduction to eNOS in endothelial cells and that small-molecule mimicry of this domain may provide a new therapeutic approach.

Reconstitution of an endothelial nitric-oxide synthase (eNOS), hsp90, and caveolin-1 complex in vitro. Evidence that hsp90 facilitates calmodulin stimulated displacement of eNOS from caveolin-1.

The activity of endothelial nitric-oxide synthase (eNOS) is regulated by its subcellular localization, phosphorylation and through its interaction with different proteins. The association of eNOS with caveolin-1 (Cav) is believed to maintain eNOS in an inactive state; however, increased association of eNOS to heat shock protein 90 (hsp90) is observed following activation. In this study, we investigate the relationship between caveolin and hsp90 as opposing regulatory proteins on eNOS function. Immunoprecipitation of Cav-1 from bovine lung microvascular endothelial cells shows that eNOS and hsp90 are present in the Cav-1 complex. eNOS and hsp90 from the lysate also interact with exogenous glutathione S-transferase-linked caveolin-1 (GST-Cav), and the addition of calcium-activated calmodulin (CaM) to the GST-Cav complex partially inhibited the association of eNOS and hsp90. Purified eNOS associates with GST-Cav specifically through the caveolin-scaffolding domain (residues 82-101); however, the addition of CaM slightly, but nonstatistically, reduces eNOS binding to GST-Cav. When hsp90 is present in the binding reaction, the addition of increasing concentrations of CaM significantly displaces eNOS and hsp90 from GST-Cav. eNOS enzymatic activity is also less sensitive to inhibition by the caveolin scaffolding peptide (residues 82-101) when eNOS is prebound to hsp90. Collectively, our results show that the actions of CaM on eNOS dissociation from caveolin are facilitated in the presence of hsp90.

ET(B) receptor blockade potentiates the pressor response to big endothelin-1 but not big endothelin-2 in the anesthetized rabbit.

The precursor of endothelin-1, big endothelin-1, is considered to be a more reliable marker of systemic production of vasoactive peptide. However, it is largely unclear whether ET(B) receptor-dependent clearance and endothelium-derived relaxing factors affect the precursor in a similar manner to mature ET-1. These ET(B)-dependent modulations of big ET-1 and big ET-2 pressor properties were therefore studied in the anesthetized rabbit. When injected into the left cardiac ventricle, ET-1 and ET-2 (0.01 to 1 nmol/kg) each induced biphasic responses (a depressor followed by a pressor response), whereas big ET-1 and big ET-2 (0.1 to 3 nmol/kg) caused only protracted pressor responses. The highest dose of big ET-1 caused significantly greater responses than ET-1, ET-2, or big ET-2. A selective ET(A) receptor antagonist, BQ-123 (1 mg/kg), markedly reduced pressor responses to all 4 peptides, whereas blockade of ET(B) receptors with BQ-788 (0.25 mg/kg) sharply potentiated the responses to ET-1, ET-2, and big ET-1, but not to big ET-2. Indomethacin (10 mg/kg) sharply potentiated the pressor response to ET-1 (1 nmol/kg), but not big ET-1, at all time points. In control animals, ET-1, but not big ET-1, also triggered an indomethacin-sensitive increase in circulating prostacyclin. Finally, systemically administered big ET-1, but not big ET-2, induced a phosphoramidon-sensitive increase in plasma IR-ET. Our results suggest a significant limiting role of ET(B) receptors on pressor responses to big ET-1. In contrast, the same receptor entities do not modulate the hemodynamic properties of the ET-2 precursor, given that, unlike big ET-1, it is poorly converted in the pulmonary or systemic circulation in anesthetized rabbits.

Regulation of endothelium-derived nitric oxide production by the protein kinase Akt.

Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation, but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.

Nitric oxide limits the eicosanoid-dependent bronchoconstriction and hypotension induced by endothelin-1 in the guinea-pig.

1. This study attempts to investigate if endogenous nitric oxide (NO) can modulate the eicosanoid-releasing properties of intravenously administered endothelin-1 (ET-1) in the pulmonary and circulatory systems in the guinea-pig. 2. The nitric oxide synthase blocker N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 microM; 30 min infusion) potentiated, in an L-arginine sensitive fashion, the release of thromboxane A2 (TxA2) stimulated by ET-1, the selective ET(B) receptor agonist IRL 1620 (Suc-[Glu9,Ala11,15]-ET-1(8-21)) or bradykinin (BK) (5, 50 and 50 nM, respectively, 3 min infusion) in guinea-pig isolated and perfused lungs. 3. In anaesthetized and ventilated guinea-pigs intravenous injection of ET-1 (0.1-1.0 nmol kg(-1)), IRL 1620 (0.2-1.6 nmol kg(-1)), BK (1.0-10.0 nmol kg(-1)) or U 46619 (0.2-5.7 nmol kg(-1)) each induced dose-dependent increases in pulmonary insufflation pressure (PIP). Pretreatment with L-NAME (5 mg kg(-1)) did not change basal PIP, but increased, in L-arginine sensitive manner, the magnitude of the PIP increases (in both amplitude and duration) triggered by each of the peptides (at 0.25, 0.4 and 1.0 nmol kg(-1), respectively), without modifying bronchoconstriction caused by U 46619 (0.57 nmol kg(-1)). 4. The increases in PIP induced by ET-1, IRL 1620 (0.25 and 0.4 nmol kg(-1), respectively) or U 46619 (0.57 nmol kg(-1)) were accompanied by rapid and transient increases of mean arterial blood pressure (MAP). Pretreatment with L-NAME (5 mg kg(-1); i.v. raised basal MAP persistently and, under this condition, subsequent administration of ET-1 or IRL 1620, but not of U-46619, induced hypotensive responses which were prevented by pretreatment with the cyclo-oxygenase inhibitor indomethacin. 5. Thus, endogenous NO appears to modulate ET-1-induced bronchoconstriction and pressor effects in the guinea-pig by limiting the peptide's ability to induce, possibly via ET(B) receptors, the release of TxA2 in the lungs and of vasodilatory prostanoids in the systemic circulation. Furthermore, it would seem that these eicosanoid-dependent actions of ET-1 in the pulmonary system and on systemic arterial resistance in this species are physiologically dissociated.

Endothelin-B receptor-dependent modulation of the pressor and prostacyclin-releasing properties of dynamically converted big endothelin-1 in the anesthetized rabbit.

Big endothelin-1 (ET-1) injected systemically in vivo induces a long-lasting pressor response, in contrast to its active metabolite ET-1, which alters in a biphasic fashion the mean arterial pressure (MAP) of the anesthetized rabbit. In this study we investigated the effect of selective ETA or ETB receptor blockade on the pressor response and increase in plasma prostacyclin (PGI2) levels (determined by RIA) induced by big ET-1 in the anesthetized rabbit. Pretreatment (5 min) of the rabbit with the ETB receptor antagonist BQ-788 (0.25 mg/kg) potentiated the ET-1 (1 nmol/kg) and, interestingly, big ET-1 (0.5 nmol/kg) induced pressor responses. The selective ETA receptor antagonist BQ-123 (1 mg/kg) significantly reduced the big ET-1 (0.5 and 3 nmol/kg) pressor responses. Big ET-1 (3 nmol/kg) injected i.v. induced an increase in plasma PGI2 levels in contrast to intra-arterial (i.a.) administration. This increase was prevented by BQ-123 (1 mg/kg) but not by BQ-788 (0.25 nmol/kg). Furthermore, in the presence of BQ-788, i.a. administration of big ET-1 (3 nmol/kg) induced a significant release of PGI2. These results show that vasodilator ETB receptors may be activated after conversion of big ET-1 to its active metabolite. Furthermore, after pulmonary conversion of big ET-1, ETA receptors may be responsible for the release of vasodilator and anti-aggregatory prostacyclin, which modulates the big ET-1-induced responses in the rabbit.

ET(B) receptor and nitric oxide synthase blockade induce BQ-123-sensitive pressor effects in the rabbit.

Endothelin-1 (0.25 nmol/kg, injected into the left cardiac ventricle) induces a protracted increase of mean arterial pressure that is significantly reduced by the selective ET(A) receptor antagonist BQ-123 (1 and 10 mg/kg) in the anesthetized rabbit. The sole administration of the selective ET(B) antagonist BQ-788 (0.25 mg/kg) induces a pressor response abolished by BQ-123 (1 mg/kg). Concomitant to the increase in mean arterial pressure, BQ-788 induces a significant increase in plasma levels of endothelin-1 and its precursor big endothelin-1. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg) also increases arterial blood pressure, and the response is reduced dose-dependently by BQ-123 (1 and 10 mg/kg). In addition, the administration of BQ-788 in the presence of L-NAME induced a further increase in arterial blood pressure. The duration of the pressor response to L-NAME is also significantly reduced by an endothelin-converting enzyme inhibitor, phosphoramidon (10 mg/kg). Finally, L-NAME induces an increase in plasma levels of big endothelin-1 but not endothelin-1. Our results illustrate that blockade of either nitric oxide synthase or ET(B) receptors triggers a raise in plasma levels of endothelin-1 or its precursor. These later moieties are suggested to be significantly involved, through the activation of ET(A) receptors, in the pressor effects of L-NAME and BQ-788 in the anesthetized rabbit.

Pharmacology of two novel mixed ETA/ETB receptor antagonists, BQ-928 and 238, in the carotid and pulmonary arteries and the perfused kidney of the rabbit.

1. In the present study, we have pharmacologically characterized two novel mixed endothelin ETA/ETB receptor antagonists, namely BQ-928 and BQ-238, in ETA and ETB preparations, the rabbit carotid artery (RbCA) and the rabbit pulmonary artery (RbPA), respectively. These two antagonists were compared to established ETA (BQ-123 and BMS 182874), ETB (BQ-788) and mixed ETA/ETB (SB 209670) receptor antagonists. 2. In the RbCA, the ETA monoreceptor preparation, BQ-238 and BQ-928 had apparent affinities (pA2) of 7.42 +/- 0.22 and 7.22 +/- 0.18, respectively, BQ-788 being inactive in this preparation. In the ETB monoreceptor preparation, the RbPA (when IRL-1620 was used as an ETB receptor agonist), the pA2 for BQ-238 was 7.05 +/- 0.14 and for BQ-928 was 8.43 +/- 0.04. BQ-123 and BMS 182874 were inactive in this preparation. Similar to SB 209670, BQ-238 but not BQ-928 had a higher affinity for the ETA than the ETB receptor. 3. All of the antagonists were tested for their ability to block and reverse endothelin-l-induced vasoconstrictions in the rabbit perfused kidney. In this preparation endothelin-1-induced increases in vascular resistance have been shown to be mediated solely by ETA receptors. All compounds (except BQ-788) blocked the pressor effects of endothelin within the kidney; the calculated IC50 values for BQ-123, BMS 182874, SB 209670, BQ-928 and BQ-238 were 0.4 microM, 2 microM, 0.01 microM, 0.4 microM and 0.09 microM, respectively. 4. In all experiments in the rabbit perfused kidney, endothelin-1 was readministered for a third time, 60 min following cessation of infusion of the above-mentioned antagonists. The response to the third infusion of endothelin-1 following cessation of infusion of BQ-123, BMS 182874 and SB 209670 was not significantly different from that to the third infusion of endothelin in control conditions. However, the response to endothelin-1 was significantly higher than control in tissues pre-infused with BQ-788 or BQ-928 (56 +/- 9 and 41.6 +/- 15%, respectively, n = 8 each, P < 0.05). 5. Our results suggest that in a system where ETA receptor activation is responsible for vasoconstriction and ETB-receptor activation for vasodilatation. ETA receptor selective antagonists or mixed ETA/ETB receptor antagonists which possess high affinity for ETA receptors do not induce hyperresponsiveness to endothelin-1. In contrast, ETB selective antagonists or mixed antagonists possessing a high affinity for ETB receptors (such as BQ-928) interfere with the ETB-receptor-dependent physiological antagonism of endothelin-1-induced pressor responses in these same tissues.

Pharmacology of endothelins: vascular preparations for studying ETA and ETB receptors.

Three rabbit vessels, the carotid and pulmonary arteries and the jugular vein were investigated to identify vascular monoreceptor systems (either ETA or ETB) to be used in structure-activity studies on endothelins and their antagonists. The RbCA has been found to behave as a monoreceptor ETA preparation, since it shows much greater sensitivity to ET-1 than to ET-3 and is insensitive to IRL 1620. The contractile response of the RbCA to ET-1 is reduced in the presence of BQ-123 but is not influenced by BQ-788. The RbPA behaves as a pure ETB system when stimulated with the ETB selective agonist IRL 1620. The contractile effect of IRL 1620 is reduced in the presence of BQ-788 but is not influenced by BQ-123. The RbJV responds to ET-1 and to IRL 1620 with contractions that are reduced by both BQ-123 and BQ-788, respectively. THe RbJV appears to be a mixed ETA and ETB system in which the two functional sites play an equivalent role in the stimulatory contractile response. Thus, contractile ETA and ETB receptors have been found in arterial and venous vessels of the rabbit and some of these vessels provide sensitive and selective (either ETA or ETB) preparations that appear to be adequate for pharmacological studies on ET receptor agonists or antagonists.

Pharmacological properties of endothelins and big endothelins in ketamine/xylazine or urethane anesthetized rats.

Endothelin-1 (ET-1) is a potent endogenous vasoconstrictor peptide formed through a specific conversion of its intermediate precursor, big ET-1, by an endothelin-converting enzyme (ECE). The present study evaluates the capacity of the ECE to convert the three big endothelins (big ET-1, big ET-2, and big ET-3), by comparing the pressor responses to these peptides with those induced by their respective metabolites (ET-1, -2, and -3) in the rat in vivo, anesthetized either with a mixture of ketamine/xylazine or with urethane. The mean basal arterial pressure under urethane anesthesia was not significantly different from that of ketamine/xylazine-treated animals (90/15 mg/kg; intramuscularly), although the basal heart rate was significantly higher in the former animals (urethane: 407 +/- 10 beats/min, ketamine/xylazine: 276 +/- 4 beats/min, P < .01; n = 8 to 17). In ketamine/xylazine and hexamethonium-treated rats (5-min infusion, 10 mg/kg intravenously), intravenous injection of ET-1 (1 nmol/kg) and big ET-1 (1 nmol/kg) induced potent vasopressor effects which lasted for more than 20 min. ET-2 (1 nmol/kg) produced similar pressor responses while big ET-2 (1-37) and big ET-2 (1-38) were twofold less potent than ET-2 (P < .05; n = 3 to 4). Big ET-3 induced a pressor effect only at 4 nmol/kg and was found to be at least 10 times less potent than ET-3. In animals anesthetized with urethane (1.5 g/kg intraperitoneally), the pressor responses induced by the endothelins and their intermediate precursors, as well as the pressor responses to angiotensin II and norepinephrine, were reduced by more than 60% (P < .01) when compared to ketamine/xylazine-treated animals. Big ET-3 was found inactive under urethane anesthesia. Ganglion blockade by hexamethonium did not affect the response to ET-1, big ET-1, ET-3, or big ET-3 in rats anesthetized with either ketamine/xylazine or urethane. On the other hand, big ET-2 (1-38), in contrast to ET-2 or big ET-1, did not release prostacyclin from the rat perfused lung, thus indicating that big ET-2 (1-38) is poorly converted in the pulmonary vasculature, and that the phosphoramidon-sensitive ECE responsible for the pressor effects of big ET-2 is localized elsewhere in the systemic circulation. Our results also show that the choice of anesthetics is crucial for the proper monitoring of the pressor responses to endothelins as well as other pressor agents. Nonetheless, even in what we consider as optimal conditions of anesthesia (threshold dose for the pressor response to ET-1 in ketamine/xylazine-treated rats: 0.01 nmol/kg), big ET-3 remains far less active than big ET-1 as a pressor peptide in the rat, suggesting a preferential processing of the latter by the ECE.

Different pressor and bronchoconstrictor properties of human big-endothelin-1, 2 (1-38) and 3 in ketamine/xylazine-anaesthetized guinea-pigs.

1. In the present study, the precursors of endothelin-1, endothelin-2 and endothelin-3 were tested for their pressor and bronchoconstrictor properties in the anaesthetized guinea-pig. In addition, the effects of big-endothelin-1 and endothelin-1 were assessed under urethane or ketamine/xylazine anaesthesia. 2. When compared to ketamine/xylazine, urethane markedly depressed the pressor and bronchoconstrictor properties of endothelin-1 and big-endothelin-1. 3. Under ketamine/xylazine anaesthesia, the three endothelins induced a biphasic increase of mean arterial blood pressure. In contrast, big-endothelin-1, as well as big-endothelin-2 (1-38), induced only sustained increase in blood pressure whereas big-endothelin-3 was inactive at doses up to 25 nmol kg-1. 4. Big-endothelin-1, but not big-endothelin-2, induced a significant increase in airway resistance. Yet, endothelin-1, endothelin-2 and endothelin-3 were equipotent as bronchoconstrictor agents. 5. Big-endothelin-1, endothelin-1 and endothelin-2, but not big-endothelin-2, triggered a marked release of prostacyclin and thromboxane A2 from the guinea-pig perfused lung. 6. Our results suggest the presence of a phosphoramidon-sensitive endothelin-converting enzyme (ECE) which is responsible for the conversion of big-endothelin-1 and big-endothelin-2 to their active moieties, endothelin-1 and 2. However, the lack of bronchoconstrictor and eicosanoid-releasing properties of big-endothelin-2, as opposed to endothelin-2 or big-endothelin-1, suggests the presence of two distinct phosphoramidon-sensitive ECEs in the guinea-pig. The ECE responsible for the systemic conversion of big-endothelins possesses the same affinity for big-endothelin-l and 2 but not big-endothelin-3. In contrast, in the pulmonary vasculature is localized in the vicinity of the sites responsible for eicosanoid release, an ECE which converts more readily big-endothelin-1 than big-endothelin-2.

Hyperresponsiveness to ET-1 after treatment with a mixture of ETA and ETB receptor antagonists in the rabbit in vivo and in vitro.

We tested the reversibility of the response to endothelin-1 (ET-1) in the rabbit renal vasculature after termination of an infusion of either a selective ETA receptor antagonist, BQ-123, a selective ETB receptor antagonist, BQ-788, or a mixture of both antagonists. Previous studies from our laboratory have shown that, as in humans, vasoconstriction of the rabbit renal vasculature induced by ET-1 is mediated solely via the activation of ETA receptors. Therefore, a 15-min infusion of BQ-123 (1 microM) prevented the vasoconstrictor response to ET-1 (10 nM). Sixty minutes after the end of the infusion, the vasoconstrictor response to ET-1 was fully restored to control levels. Conversely, when the selective ETB receptor antagonist was administered, the vasoconstrictor response to ET-1 was markedly potentiated. After the infusion of the antagonist was ended there was no further potentiation of the response to ET-1. Co-infusion of supramaximal concentrations of BQ-123 (1 microM) and BQ-788 (10 nM) still reduced by about 90% the vasoconstrictor response to ET-1. However, after removal of the antagonists, the vasoconstriction to ET-1 was potentiated by more than 75% compared to control (n = 8; p < 0.05). Similarly, a mixture of both antagonists was less effective than BQ-123 alone in reducing the pressor effect of ET-1 in anesthetized rabbits. These results show that blockade of ETB receptors produces marked potentiation in the vasoconstrictor responses to ET-1. Furthermore, a rebound hyperresponsiveness is found after treatment with a mixture of BQ-123 and BQ-788. These results suggest that the systemic and renal vasculatures would respond to ET-1 with enhanced vasoconstriction after treatment with mixed ETA/ETB receptor antagonists owing to the slow reversibility of the antagonism of the ETB receptor-mediated release of nitric oxide.

Characterization of endothelin receptors and endothelin-converting enzyme activity in the rabbit lung.

We had earlier shown that endothelin-1 (ET-1)-induced vasoconstriction and prostacyclin (PGI2) release in the rabbit kidney are exclusively linked to ETA receptor activation. In contrast, another study showed that ET-1 inhibited platelet aggregation ex vivo through the release of PGI2 solely via the activation of ETB receptors. In an attempt to identify the organs involved in the ET-1-induced release of PGI2 in vivo, we characterized the receptors responsible for the release of this eicosanoid and also monitored the activity of the endothelin-converting enzyme (ECE) in rabbit pulmonary lobe. In this perfused organ, a 5-min infusion of ET-1 (50 nM) triggered a marked release of PGI2 and an increase in perfusion pressure, both of which were mimicked by the selective ETB agonist IRL 1620 (0.5 microM). Furthermore, a selective ETB antagonist, BQ-788 (10 nM), markedly blunted the release of PGI2 induced by ET-1 (50 nM). On the other hand, ET-2 was also able to trigger the release of PGI2 and to increase the perfusion pressure in this organ. The immediate precursor of ET-1, big ET-1 (0.5 microM), also induced a protracted increase in perfusion pressure. In contrast, big ET-2 (0.5 microM) was significantly weaker than big ET-1 in increasing perfusion pressure. Our results suggest that the receptors responsible for the release of PGI2 in the lung are (in contrast to the kidney) predominantly of the ETB type. Furthermore, the ECE localized in the rabbit pulmonary vasculature is relatively selective for big ET-1. On the basis of these results, we suggest that ETB-mediated inhibition of platelet aggregation ex vivo is due to a marked release of PGI2 generated from the pulmonary vasculature in the rabbit.

Block of endothelin-1-induced release of thromboxane A2 from the guinea pig lung and nitric oxide from the rabbit kidney by a selective ETB receptor antagonist, BQ-788.

1. The present study characterizes the receptors responsible for endothelin-1-induced release of thromboxane A2 from the guinea pig lung and of endothelium-derived nitric oxide from the rabbit perfused kidney, by the use of the selective ETA receptor antagonist, BQ-123, and a novel selective ETB receptor antagonist, BQ-788. 2. In the guinea pig perfused lung, endothelin-1 (ET-1) (5 nM) induced a marked increase of thromboxane A2 which was reduced by 17 +/- 5.0, 70 +/- 1.0 and 93 +/- 1.2% by BQ-788 infused at concentrations of 1, 5 and 10 nM respectively. In contrast, BQ-123 (0.1 and 1.0 microM) had little or no effect on the ET-1-induced release of thromboxane A2. 3. In the same perfused model, the selective ETB agonist, IRL 1620 (50 nM), stimulated the release of thromboxane A2, but not prostacyclin. The eicosanoid-releasing properties of IRL 1620 were abolished by BQ-788 at 10 nM, yet were unaffected by BQ-123 (1 microM). 4. In the rabbit perfused kidney, BQ-788 (10 nM) potentiated the increase of perfusion pressure induced by endothelin-1 (1, 5 and 10 nM) by approximately 90%, but not that induced by angiotensin II (1 microM). Furthermore, the selective ETB receptor antagonist did not reduce the release of prostacyclin triggered by either peptide. 5. In another series of experiments, pretreatment of the perfused kidney with a nitric oxide synthase inhibitor, L-NAME (100 microM), potentiated the pressor responses to both endothelin-1 and angiotensin II. Under L-NAME treatment, BQ-788 did not further potentiate the pressor response to endothelin-1. 6 Our results illustrate the predominant role of ETB receptor activation in the release of thromboxane A2 and nitric oxide triggered by endothelin-l in the guinea pig perfused lung and rabbit kidney respectively.

Endothelin-1 induces vasoconstriction and prostacyclin release via the activation of endothelin ETA receptors in the perfused rabbit kidney.

Endothelin-1 (0.005 and 0.01 nmol) induced a dose-dependent increase in perfusion pressure in the perfused rabbit kidney. These pressor effects were markedly reduced by an endothelin ETA receptor antagonist, BQ-123 (0.1 microM). Similarly, the release of prostacyclin triggered by intra-arterial infusion of endothelin-1 (10 nM) was significantly reduced in a concentration-dependent manner when the kidney was pretreated with BQ-123 (0.5-1 microM). In contrast, two selective ETB receptor agonists, BQ-3020 and IRL 1620, were found to be inactive, both as pressor agents and releasers of prostacyclin at doses (for the pressor effects) and concentrations (for the prostacyclin generation) 50-100 times higher than those of endothelin-1. BQ-123 (1 microM) did not modify the pressor or prostanoid-releasing properties of angiotensin II. These results confirm our previous observations suggesting that pressor responses and prostanoid release induced by endothelin-1 are mediated via the selective activation of ETA receptors in the perfused rabbit kidney.