Natural and synthetic agonists of the melanocortin receptor type 3 possess anti-inflammatory properties.

The effects of the natural and synthetic ligands for the melanocortin receptor type 3 (MC3-R) have been evaluated in a murine model of experimental gout. Systemic treatment of mice with gamma2-melanocyte-stimulating hormone (gamma2-MSH) and the synthetic agonist MTII inhibited accumulation of KC, interleukin-1 beta (IL-1beta), and PMN elicited by urate crystals in the peritoneal cavity. In vitro, macrophage (Mø) activation, determined as release of KC and IL-1beta, was inhibited by gamma2-MSH and MTII. The mixed MC3/4-R antagonist SHU9119 prevented the inhibitory actions of gamma2-MSH and MTII in vitro and in vivo, whereas the selective MC4-R antagonist HS024 was without effect. Western blotting also showed the presence of MC3-R protein on murine peritoneal Mø. Furthermore, agonism at the MC3-R evoked accumulation of cAMP within the Mø, which was inhibited by SHU9119. Thus, naturally occurring melanocortins, as well as the synthetic long-acting compound MTII, activate MC3-R on peritoneal Mø to inhibit the experimental inflammatory response.

Roles of endothelin receptors in the regional and systemic vascular responses to ET-1 in the anaesthetized ganglion-blocked rat: use of selective antagonists.

1. Endothelin-1 (ET-1) produces vasoconstriction, via activation of ETA and ETB receptors on vascular smooth muscle, and vasodilatation via ETB receptors on endothelial cells. Here we have used the ETA receptor-selective antagonist, BQ-123, the ETB receptor-selective antagonist, BQ-788 and the ETA/ETB receptor non-selective antagonist, PD 145065, to study the role of these receptors in mediating the haemodynamic changes induced by an infusion of ET-1 to the anesthetized ganglion-blocked rat. 2. Infusion of ET-1 (10 pmol kg-1 min-1) increased the mean arterial pressure (MAP) by 57.5 +/- 5.1 mmHg over 70 min. This pressor response was reduced by about 50% by coinfusion of BQ-123 (10 mmol kg-1 min-1), but was unaffected by either BQ-788 (10 nmol kg-1 min-1) or PD 145065 (10 nmol kg-1 min-1). 3. After infusion of ET-1 for 70 min the cardiac output had fallen from 102.6 +/- 11.3 to 55.7 +/- 7.6 ml min-1 and the total peripheral resistance had increased from 3.24 +/- 0.6 to 10.0 +/- 0.8 mmHg ml-1 min-1 (per 100g body weight). BQ-123 decreased the magnitudes of these changes whereas BQ-788 potentiated them. PD 145065 was without effect. 4. ET-1 increased the vascular resistances of all the organs studied except the brain and stomach. These changes were attenuated by BQ-123 in the kidneys, skin, adrenal glands and caecum and potentiated by BQ-788 in the kidneys, small intestine, large intestine and mesentery. PD 145065 had little effect on the individual tissues. 5. Thus, BQ-123, a selective ETA receptor antagonist, inhibits the pressor and vascular constrictor effects of ET-1 more actively than PD 145065. As BQ-788 potentiates some of the vasoconstrictor effects of ET-1 and increases the effects of ET-1 on total peripheral resistance, the predominant role of ETB receptors in the rat circulation is to limit the pressor effects of ET-1.

Reversal of established responses to endothelin-1 in vivo and in vitro by the endothelin receptor antagonists, BQ-123 and PD 145065.

1. Endothelin-1 binds almost irreversibly to its receptors and causes prolonged vasoconstrictions. Here we have studied the reversal of established responses to ET-1 in vivo and in vitro by BQ-123, an ETA receptor-selective antagonist, and/or PD 145065, an ETA/ETB receptor non-selective antagonist. 2. In anaesthetized rats pretreated with hexamethonium, infusion of ET-1 (10(-11) mol kg-1 min-1) increased the mean arterial pressure (MAP) from 93 +/- 1.5 mmHg to 137 +/- 2.4 mmHg after 70 min (n = 29). While the ET-1 infusion was continued an additional infusion of BQ-123 caused a gradual dose-dependent reduction in the pressor effect of ET-1. For instance, after a 60 min infusion of BQ-123 (10(-8) mol kg-1 min-1) the MAP was decreased by 29.3 +/- 4.3 mmHg (n = 4). 3. PD 145065 was a much weaker antagonist of the established pressor effects of ET-1. At 10(-8) mol kg-1 min-1 it had no significant effect and even at 10(-7) mol kg-1 min-1 the elevated blood pressure was only reduced by 11.8 +/- 8.0 mmHg (n = 5) after 60 min. Co-infusion of BQ-123 and PD 145065 caused smaller reductions in the established response to ET-1 than infusion of BQ-123 alone. 4. Sustained contractions of rat aortic rings induced by ET-1 (3 x 10(-9) M) and mediated by ETA receptors were slowly reversed by addition of BQ-123 (10(-5) M) or PD 145065 (10(-5) M). For instance,after 40 min the elevated tone was reduced 85.8 +/- 5.6% (n = 6) by PD 145065, and 77.1 +/- 6.7% (n = 6)by BQ-123. Thus, on the rat aortic rings in vitro both antagonists were equally effective against established responses to ET-1.5. ET-1 increased the perfusion pressure of the rat isolated perfused kidney by 138.1 +/- 7.6 mmHg(n = 14). Subsequent co-infusion of BQ-123 or PD 145065 reversed this increase with PD 145065 being more active. For instance, PD 145065 (10-6 M) reversed the increase in perfusion pressure by 56.9 +/- 8.8% (n = 5) and BQ-123 (10-6 M) reversed it by 22.8 +/- 8.0% (n = 5). This fits well with the vasoconstriction induced by ET-1 in the rat kidney being mediated by ETA and ETB receptors.6. Thus, sustained vasoconstrictions to ET-1 in vitro, mediated by either ETA or ETB receptors, may be reversed slowly by the subsequent application of receptor antagonists. Similarly, endothelin antagonists reverse the pressor effects of ET-1 in vivo although co-antagonism of ETA and ETB receptors or the co-administration of an ETA receptor antagonist, BQ-123, and a mixed antagonist, PD 145065 produces less reversal than the application of an ETA receptor-selective antagonist. This may be because PD 145065 also reduces vasodilatations induced by ET-1 in vivo, or could suggest that because of its peptide structure PD 145065 affects the elimination of ET-1.

Comparison of effects of chronic and acute administration of NG-nitro-L-arginine methyl ester to the rat on inhibition of nitric oxide-mediated responses.

1. Vascular responses to acetylcholine and sodium nitroprusside in vivo and in vitro, in the isolated perfused kidney and in rings of rat thoracic aorta, were measured in rats treated chronically with NG-nitro-L-arginine methyl ester (L-NAME; approx, 70 mg kg-1) and compared to responses in age-matched control animals, and age-matched animals after the acute administration of L-NAME (3-100 mumol kg-1). Parallel experiments examined alterations in responsiveness in rings of trachea and anococcygeus muscles taken from the same animals. 2. Chronic oral administration of L-NAME elevated the blood pressure in anaesthetized animals from 114 +/- 5 mmHg to 153 +/- 11 mmHg (n = 5). The hypotensive responses to both acetylcholine (1 nmol kg-1) and sodium nitroprusside (10 nmol kg-1) were enhanced by chronic L-NAME treatment (n = 5-7) whereas acute L-NAME administration enhanced only the response to sodium nitroprusside (n = 5). 3. After chronic treatment with L-NAME, the basal perfusion pressure in the isolated perfused kidney was elevated. However, vasodilator responses to either acetylcholine (1 nmol) or sodium nitroprusside (3 nmol) were unaltered (n = 5-7). The vasodilatation induced by acetylcholine was inhibited in a concentration-dependent manner by the administration of acute L-NAME (0.1 - 100 microM; n = 5), such that significant inhibition was seen at 10 microM L-NAME. The response to sodium nitroprusside was unaffected by L-NAME. 4. The relaxations of isolated rings of rat thoracic aorta induced by acetylcholine were inhibited in tissues prepared from rats treated chronically with L-NAME (n = 5-7). Acute administration of L-NAME (0.1-100 microM) concentration-dependently inhibited the relaxations induced by acetylcholine in this preparation, with significant inhibition occurring at 1 microM L-NAME (n = 5). Responses to sodium nitroprusside were unaffected by either chronic or acute exposure to L-NAME (n = 5-7).5. Relaxations of precontracted anococcygeus muscles induced by electrical field stimulation, or contractions of rings of trachea induced by carbachol or endothelin-1, were unaffected by chronic oral administration of L-NAME (n = 4-6). Acute addition of L-NAME (0.1-100 microM) to the organ baths inhibited in a concentration-dependent manner the relaxations of anococcygeus muscles taken from control animals, with a significant effect being seen at a concentration of 10 micro.M (n = 4-6).6. Our cardiovascular data are consistent with chronic oral administration of L-NAME inhibiting the production of nitric oxide (NO) within the vasculature, although the pattern of inhibition is not uniform between different tissues. Despite the inhibition of endothelial NO production, chronic L-NAME does not alter the vasodepressor activity of acetylcholine in vivo or in the isolated perfused kidney. This maybe explained by an enhanced responsiveness of guanylyl cyclase pathways, the increased release of vasodilators other than nitric oxide or a decreased importance of nitric oxide in resistance vessels compared with conductance vessels. The resistance of peripheral neuronal NO responses to chronic treatment with L-NAME indicates that selective inhibition of different isoforms of NOS may be achieved in vivo.

Use of the endothelin antagonists BQ-123 and PD 142893 to reveal three endothelin receptors mediating smooth muscle contraction and the release of EDRF.

1. We have compared the receptors mediating the contractions of rings of rat thoracic aorta or rabbit pulmonary artery and rat stomach strips in response to the endothelin/sarafotoxin (ET/SX) family of peptides and to those mediating endothelium-dependent vasodilations within the isolated perfused mesentery of the rat. To discriminate ETA receptors from ETB receptors we have used the criteria that ET-1 is more active than SX6c on ETA receptors, and that the ET/SX peptides are equiactive on ETB receptors. We have also assessed the effects of the ETA receptor-selective antagonist BQ-123, and the non-selective ET receptor antagonist PD 142893 on the responses of each preparation to the ET/SX peptides. 2. ET-1-induced constrictions of the rat thoracic aorta (EC50 3 x 10(-10) M), a prototypic ETA receptor-mediated response, or isolated perfused mesentery of the rat were antagonized by BQ-123 (10(-5) M) or PD 142893 (10(-5) M). SX6c did not constrict either the rat isolated perfused mesentery or the rat thoracic aorta. Thus, ETA receptors mediate these constrictions. 3. ET-1 and SX6c were approximately equipotent in constricting rabbit pulmonary artery rings (EC50S 3-6 x 10(-10) M). Neither BQ-123 (10(-5) M) nor PD 142893 antagonized the contractions induced by ET-1. These effects suggest mediation by ETB receptors but PD 142893 (10(-5) M) did give a 3 fold antagonism of constrictions induced by SX6c. 4. SX6c was more potent than ET-1 in contracting the rat stomach strip (threshold concentrations 10(-10) and 3 x 10(-10) M). Contractions to ET-1 or SX6c were unaffected by BQ-123 (10-5 M), again indicative of ETB receptor-mediated events. PD 142893 (10-5 M) was ineffective against ET-1 but produced a 3 fold antagonism of SX6c.5. In the rat isolated perfused mesentery ET-1 or SX6c (0.3-300pmol) were equipotent in producing dose-related vasodilatations that were unaffected by BQ-123 (10-6 M), indicative of an ETB receptor mediated response. In contrast to the other ETB-mediated responses, PD 142893 (10-6 M) strongly antagonized these vasodilatations.6. Thus, ETA receptors mediate constrictions of the rat thoracic aorta and rat isolated perfused mesentery whereas ETB receptors mediate constrictions of the rabbit pulmonary artery and rat stomach strip and endothelium-dependent dilatations within the mesentery. However, within the group of ETB receptor-mediated responses, endothelium-dependent vasodilatations are sensitive to PD 142893, whereas contractions of the isolated smooth muscle preparations are not. Thus, the receptor present on the endothelium responsible for the release of nitric oxide in response to the ET/SX peptides is most probably different from that present on smooth muscle that mediates BQ-123-insensitive contractions.

Characterization of endothelin receptors mediating the effects of the endothelin/sarafotoxin peptides on autonomic neurotransmission in the rat vas deferens and guinea-pig ileum.

1. To characterize the receptors mediating the effects of the endothelin/sarafotoxin family of peptides on the responses to electrical stimulation of the rat vas deferens (RVD) and guinea-pig ileum (GPI) we have used endothelin-1 (ET-1), ET-3, sarafotoxin 6b (SX6b) and SX6c as agonists and the endothelin-receptor antagonists BQ-123 (ETA receptor selective) and PD 142893 (non-selective). 2. In the RVD, ET-1 and SX6b increased the twitches induced by field stimulation starting at a threshold concentration of 10(-10) M while the threshold concentration for ET-3 was 3 x 10(-9) M. SX6c (up to 3 x 10(-8) M) did not potentiate the twitches. SX6b produced significantly (P < 0.05) greater potentiations than ET-1 at concentrations of 3 x 10(-9) M and higher, and 10(-7) M ET-3 also produced a significantly greater effect than ET-1 at the same concentration. Thus, at threshold the rank order of peptides was ET-1 = SX6b > ET-3 >>> SX6c, and at concentrations of 3 x 10(-8) M and higher, SX6b > ET-3 > ET-1 >>> SX6c. 3. In the presence of BQ-123 or PD 142893 (10(-5) M) the threshold concentrations for ET-1 to augment the twitches were increased 30 fold. In the same conditions neither SX6b nor ET-3 potentiated the responses. The relative activities of the endothelin/sarafotoxin peptides and the effectiveness of the endothelin receptor antagonists are consistent with postjunctional ETA receptors mediating these effects. 4. In the transmurally stimulated GPI the endothelin/sarafotoxin peptides produced two effects; an increase in the basal tension of the tissues and an inhibition of the twitch responses. To increase the basal tension the peptides had the order of potency ET-1 > SX6b>> ET-3 = SX6c. These direct effects of ET-1 or SX6b were strongly antagonized (100 fold) by either BQ-123 (10-5M) or PD 142893(10-5 M). Thus, ETA receptors mediate contractions of the GPI induced by these peptides.5. The endothelin/sarafotoxin peptides were approximately equipotent at depressing twitches of the GPI in response to transmural stimulation (EC50s, 4 x 10-11 to 1.5 x 10-10 M). The depressions induced byET-1 were unaffected by either BQ-123 (10-5 M) or PD 142893 (10-5 M). BQ-123 produced a small(three fold) antagonism of the inhibitory effects of ET-3 or SX6c. These results indicate that a receptor of the ETB type mediates the inhibitory effects of the endothelin/sarafotoxin peptides on neurotransmission in the GPI.6. Thus, both ETA receptors and ETB receptors mediate the effects of the endothelin/sarafotoxinpeptides on neurotransmission.

Endothelin ETA and ETB receptors mediate vasoconstriction and prostanoid release in the isolated kidney of the rat.

Endothelin-1 or sarafotoxin 6c (10(-12) to 10(-9) M) induced concentration-dependent increases in perfusion pressure in the isolated perfused kidney of the rat, and increased the release of 6-keto-prostaglandin F1 alpha, prostaglandin E2 and prostaglandin F2 alpha. The pressor effects of endothelin-1 were partially antagonised by BQ-123 (10(-6) M) and more strongly so by the endothelin ETA/B receptor non-selective antagonist PD 145065 (10(-6) M). PD 145065, but not BQ-123, also completely blocked the pressor effects of sarafotoxin 6c. The releases of prostanoids induced by endothelin-1 were greatly reduced by infusion of either BQ-123 or PD 145065, whereas PD 145065, but not BQ-123, strongly antagonised the releases induced by sarafotoxin 6c. These results indicate that the vasoconstrictions and the prostanoid releases induced by the endothelin/sarafotoxin peptides are mediated by the activation of both endothelin ETA and endothelin ETB receptors in the isolated perfused kidney of the rat.

Activation of ET(A) receptors is partially responsible for the rapid increase in haematocrit induced by bacterial lipopolysaccharide in the rat.

It is believed that changes in the production and release of endothelin-1 (ET-1) are mediated over prolonged periods, and, therefore, that it is unlikely that ET-1 mediates rapid responses within the circulation. Here we show that ET-1 is involved in the rapid changes produced by injection of LPS in vivo. In anaesthetised rats, a bolus of LPS induced an increase in haematocrit and a fall in blood pressure within 10 min. The increase in haematocrit was reduced by administration of antagonists selective for endothelin ET(A) receptors, while the accompanying decrease in MAP was potentiated. Thus, activation of ET(A) receptors is partially responsible for the rapid increase in haematocrit seen in this model. This clearly demonstrates that ET-1 can act as a rapid responder to acute cardiovascular challenges.

Inhibition of ETB receptors limits the efficacy of nonselective endothelin antagonists in vivo.

Endothelin-1 (ET-1) produces vasoconstriction via activation of ETA and ETB receptors on vascular smooth muscle, and vasodilatation via ETB receptors on endothelial cells. Here we have used the selective ETA receptor antagonist BQ-123, the selective ETB receptor antagonist BQ-788 and the nonselective ETA/ETB receptor antagonist PD 145065 to study the role of these receptors in mediating the hemodynamic changes induced by an infusion of ET-1 to the anesthetised, ganglion-blocked rat. ET-1 (10 pmol/kg/min) infused for 70 min induced an increase in the mean arterial pressure (MAP) and total peripheral resistance (TPR), as well as a fall in cardiac output (CO). ET-1 also induced a fall in the blood flow to all of the tissues studied except the heart and stomach. BQ-123 (10 nmol/kg/min) attenuated, whereas BQ-788 (10 nmol/kg/min) potentiated, most of these effects of ET-1. PD 145065 (10 nmol/kg/min), a potent antagonist in vitro, was largely without effect in vivo, even though the dose infused was 1,000 times that of ET-1. Therefore, BQ-123 inhibits the vasoconstrictor effects of ET-1 more actively than PD 145065. Because BQ-788 potentiates the effects of ET-1 on TPR and generally increases ET-1-induced regional vasoconstriction, this weaker effect of PD 145065 compared to BQ-123 is most likely due to its additional effect on ETB receptors. Therefore, nonselective receptor antagonists may well prove less efficacious in vivo than predicted from in vitro assays because they will reduce the ET-1-limiting processes on vasoconstriction mediated by ETB receptors.

ETA receptor blockade attenuates the hypertension but not renal dysfunction in DOCA-salt rats.

Endothelin (ET)-1 has potent renal and systemic vasoconstrictor properties, and thus we investigated whether ET-1 plays a role in increasing blood pressure and decreasing renal function in DOCA-salt hypertension. After a right nephrectomy, rats had DOCA or placebo pellets implanted subcutaneously and were given saline or tap water to drink, respectively. Additional groups of rats were given the ETA receptor antagonist A-127722 in their water. Rats were maintained in metabolic cages for monitoring excretory function and food and water intake. Three weeks after surgery, mean arterial pressure (MAP) was recorded in the conscious rats via a carotid artery catheter. As expected, DOCA-salt rats had significantly higher MAP compared with uninephrectomized controls (197 +/- 6 vs. 133 +/- 3 mmHg). Creatinine clearance, used as an estimate of glomerular filtration rate, was significantly reduced in DOCA-salt rats (2.9 +/- 0.4 vs. 6. 8 +/- 0.3 dl . day-1 . 100 g-1 body wt in controls). ETA receptor blockade with A-127722 significantly reduced MAP (156 +/- 8 mmHg) but had no effect on creatinine clearance of DOCA-salt-treated rats (2.8 +/- 0.3 dl . day-1 . 100 g-1 body wt). Plasma ET-1 levels were significantly raised after DOCA-salt treatment (1.4 +/- 0.5 pg/ml vs. 0.4 +/- 0.1 pg/ml in controls). A-127722 treatment increased circulating ET-1 levels in both placebo (2.3 +/- 0.5 pg/ml) and DOCA-salt (5.6 +/- 0.7 pg/ml) rats. However, ET-1 mRNA expression in renal cortical and medullary tissue was not affected by either A-127722 or DOCA-salt treatments. Thus ETA receptors appear to play a role in the maintenance and development of DOCA-salt hypertension but not in the accompanying reduction of renal function.

Upregulation of endothelin B receptors in kidneys of DOCA-salt hypertensive rats.

Experiments were designed to elucidate the role of endothelin B receptors (ET(B)) on arterial pressure and renal function in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Male Sprague-Dawley rats underwent uninephrectomy and were treated with either DOCA and salt (0.9% NaCl to drink) or placebo. DOCA-salt rats given the ET(B)-selective antagonist, A-192621, for 1 wk (10 mg. kg(-1). day(-1) in the food) had significantly greater systolic arterial pressure compared with untreated DOCA-salt rats (208 +/- 7 vs. 182 +/- 4 mmHg) whereas pressure in placebo rats was unchanged. In DOCA-salt, but not placebo rats, A-192621 significantly decreased sodium and water excretion along with parallel decreases in food and water intake. To determine whether the response in DOCA-salt rats was due to increased expression of ET(B) receptors, endothelin receptor binding was performed by using membranes from renal medulla. Maximum binding (B(max)) of [(125)I]ET-1, [(125)I]ET-3, and [(125)I]IRL-1620 increased from 227 +/- 42, 146 +/- 28, and 21 +/- 1 fmol/mg protein, respectively, in placebo rats to 335 +/- 27, 300 +/- 38, and 61 +/- 6 fmol/mg protein, respectively, in DOCA-salt hypertensive rats. The fraction of receptors that are the ET(B) subtype was significantly increased in DOCA-salt (0.88 +/- 0.07) compared with placebo (0.64 +/- 0.01). The difference between [(125)I]ET-3 and [(125)I]IRL-1620 binding is consistent with possible ET(B) receptor subtypes in the kidney. These results indicate that ET(B) receptors in the renal medulla are up-regulated in the DOCA-salt hypertensive rat and may serve to maintain a lower arterial pressure by promoting salt and water excretion.

Characterization of endothelin receptors mediating mechanical responses to the endothelins in the isolated stomach strip of the rat.

We have characterized the receptors mediating the mechanical responses of the isolated stomach strip to endothelin-1 (ET-1), endothelin-3 (ET-3) and the ETB-selective receptor agonists sarafotoxin 6c (SX6c) and IRL 1620. As antagonists we used BQ-123 (ETA receptor selective), BQ-788 (ETB receptor selective) and PD 145065 (ETA/ETB receptor nonselective). We have also compared the responses of the mature peptides to their precursors human big ET-1(1-38), porcine big ET-1(1-39) and big ET-3(1-41) amide. ET-1, ET-3, SX6c and IRL 1620 produced equipotent concentration-dependent contractions of the rat stomach strips that were antagonized by PD 145065 (10(-5) M) or BQ-788 (10(-5) M) but not by BQ-123 (10-5 M). This indicates that the ETB receptor mediates contractions to the endothelins in this preparation. In preparations precontracted with PGE2 (3 x 10(-8) M), ET-1, but not SX6c (both 3 x 10(-9) M), caused a transient (< 2 min) relaxation (approx. 40% of the induced tone). This relaxation was antagonized by BQ-123 (10(-5) M) but prolonged by BQ-788, and therefore mediated by ETA receptors. A single administration of 3 x 10(-7) M ET-1, ET-3, SX6c or IRL 1620 produced contractions that reached a maximal response after 1 to 3 min. The contractions were not maintained, although responses to ET-1 or ET-3 lost their tone less rapidly than those to SX6c or IRL 1620.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutrophil accumulation induced by bacterial lipopolysaccharide: effects of dexamethasone and annexin 1.

Annexin 1 (ANX-1) can reduce leucocyte migration in response to cytokines and chemokines in some rodent models of inflammation. However, its effectiveness against an inflammatory stimulus as strong as bacterial lipopolysaccharide (LPS) is unknown. Thus, we have examined whether ANX-1 can modulate LPS-induced neutrophil accumulation in the rat, as assessed by intravital microscopy and by myeloperoxidase (MPO) assay. The anti-inflammatory glucocorticoid, dexamethasone (DEX) was also studied for comparison. LPS superfusion induced adhesion of leucocytes to the endothelium and a subsequent increase in emigration from rat post-capillary venules over 2 h as assessed by intravital microscopy. Either ANX-1 or DEX was able to attenuate this adhesion and emigration of leucocytes. MPO activity in the lung, kidney and ileum was elevated after a 6-h exposure to LPS (intraperitoneal), indicating accumulation of neutrophils in these tissues. DEX attenuated the LPS-induced increase in MPO in the ileum but had no effect on MPO in the lungs or kidneys. This would suggest that the underlying mechanism by which neutrophils accumulate in the ileum, and more generally in the gastrointestinal compartment, is different from other vascular beds. ANX-1 had no effect on the LPS-induced increase in MPO activity in any of the tissues studied. Thus, from these data, ANX-1 appears to reduce leucocyte adhesion and emigration induced by a short-term (2 h), but not a longer (6 h) exposure to LPS.

Comparative studies with the endothelin receptor antagonists BQ-123 and PD 142893 indicate at least three endothelin receptors.

Endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6b (SX6b), and sarafotoxin 6C (SX6c) were used as agonists, and BQ-123 (ETA-selective) and PD 142893 (ET receptor-nonselective) were used as antagonists to characterize the receptors mediating the effects of the ET/SX peptides on a variety of isolated smooth-muscle preparations. Contractions of the rat thoracic aorta, rat isolated perfused mesentery, and guinea pig ileum and potentiation of twitch of the rat vas deferens were mediated by ETA receptors in that they showed the order of potency ET-1 = SX6b > ET-3 >> SX6C. These effects were antagonized by BQ-123 or PD 142893. Contractions of the rabbit pulmonary artery and rat stomach strip, inhibition of twitches in the guinea pig ileum, and vasodilatations of the rat isolated perfused mesentery showed the order of potency ET-1 = SX6b = ET-3 = SX6c and were unaffected by BQ-123, suggesting involvement of ETB receptors. However, in these tissues, PD 142893 antagonized only dilatations of the rat mesentery to ET-1 but not any of the other effects of ET-1. Thus, we suggest that there are three types of endothelin receptors: ETA and two subtypes of ETB.

Increased nitric oxide synthase-3 expression in kidneys of deoxycorticosterone acetate-salt hypertensive rats.

In addition to its hemodynamic effects, nitric oxide (NO) may play a role in the renal tubular handling of sodium. Experiments were conducted to determine possible changes in renal nitric oxide synthase-3 (NOS3) expression in rats treated with deoxycorticosterone acetate (DOCA) and high salt. All rats were uninephrectomized, and either a placebo or DOCA pellet was implanted subcutaneously. Placebo-treated rats were then given tap water to drink ad libitum, and DOCA-treated rats received a 0.9% NaCl solution to drink. Once a week, rats were placed in metabolic cages so that a 24-h urine sample could be collected. After 3 wk, the animals were sacrificed and the kidneys removed and prepared for subsequent immunohistochemical or Western blot analysis. Urinary excretion of nitrate and nitrite (NOx) was measured to provide an indication of the intrarenal production of NO. DOCA-salt hypertensive rats exhibited increased urinary NOx excretion (2.43 +/- 0.48 micromol NOx/mg creatinine) compared with the placebo control animals (1.17 +/- 0.06 micromol NOx/mg creatinine). Western blot analysis revealed that NOS3 protein levels in both the cortex and medulla were greater in DOCA-salt rats compared with placebo-treated animals. Immunohistochemical analysis of kidneys revealed that NOS3 expression in placebo rats was localized in vascular endothelial cells with slight, but detectable, immunoreactivity in medullary collecting ducts. In DOCA-salt rats, a very large increase in the intensity of immunostaining was detected in tubular epithelia of the proximal tubule, thick ascending limb of Henle's loop, and cortical and medullary collecting duct; immunoreactivity in endothelial cells appeared unchanged. These data suggest that increased tubular expression of NOS3 is responsible, at least in part, for the increased renal production of NO in DOCA-salt hypertension, and are consistent with a role for NO in the renal tubular response to salt loading.