Buffering action of endogenous nitric oxide on the adrenocortical secretagogue effect of endothelins in the rat

The secretagogue effect of endothelins (ETs) on the rat adrenal cortex is mediated by the ETB receptor. ETB receptors are coupled with nitric oxide (NO) synthase (NOS), and NO is known to inhibit steroid-hormone secretion from adrenal cortex. We investigated whether ETB-mediated NO production interferes with the stimulatory action of ETs on rat adrenal cortex. The selective agonist of ETB receptor BQ-3020 concentration-dependently increased aldosterone secretion from dispersed zona glomerulosa (ZG) cells and corticosterone secretion from dispersed zona fasciculata-reticularis (ZF/R) cells, and the NOS inhibitor NG-nitro-L-arginine methylester (L-NAME) potentiated the effect of BQ-3020 in a concentration-dependent manner. The guanylate cyclase inhibitor Ly-83583, at a concentration suppressing guanylin- and L-arginine-induced cyclic-GMP release from dispersed adrenocortical cells, did not affect the secretory response of ZG and ZF/R cells to BQ-3020. ET-1, an agonist of both ETA and ETB receptors, stimulated the release of both aldosterone and corticosterone by in situ perfused rat adrenal gland. This effect was potentiated by L-NAME and unaffected by Ly-83583. Collectively, our findings allow us to suggest that endogenous NO exerts in vivo and in vitro a cyclic-GMP-independent buffering action on the ETB receptor-mediated adrenocortical secretagogue action of ETs.     

Mechanisms and receptor subtypes involved in the stimulatory action of endothelin-1 on rat adrenal zona glomerulosa

Endothelin (ET)-1 is the prototype of a family of 21-amino acid residue hypertensive peptides, acting through two subtypes of receptors, named ETA and ETB. ETs and their receptors are expressed in the adrenal cortex and medulla, and ET-1 enhances both corticosteroid and catecholamine release. ET-1 concentration-dependently (from 10(-11) to 10(-8) M) increased aldosterone secretion of both dispersed rat zona glomerulosa (ZG) cells and adrenal slices containing a core of medullary chromaffin tissue, but the response of the latter preparations was significantly more intense than that of the formers. The stimulatory effect of 10(-8) M ET-1 on dispersed ZG cells was blocked by the ETB-receptor antagonist BQ-788 (10(-7) M), but not by the ETA-receptor antagonist BQ-123 (10(-7) M); conversely, both ET-receptors antagonists counteracted aldosterone response of adrenal slices to ET-1. The -adrenoceptor antagonist l-alprenolol (10(-6) M) did not affect aldosterone response of dispersed ZG cells to ET-1 (10(-8) M), but it significantly lowered that of adrenal slices. l-Alprenolol also counteracted the aldosterone response of adrenal slices to the pure activation of ETB or ETA receptors, as obtained by using the selective ETB-receptor agonist BQ-3020 (10(-8) M) or ET-1 (10(-8) M) plus BQ-788 (10(-7) M). ET-1 concentration-dependently (from 10(-9) to 10(-8)/10(-7) M) stimulated catecholamine release by adrenal slices, and the effect was counteracted by both BQ-123 and BQ-788 (10(-7) M). Collectively, our findings suggest that, when the integrity of adrenal tissue is preserved, a two-fold mechanism underlies the aldosterone secretagogue action of ET-1 in the rat: i) a direct mechanism mediated by ETB receptors located on ZG cells; and ii) an indirect mechanism involving the ETA and ETB receptor-mediated local release of catecholamines, which in turn stimulate ZG cells in a paracrine manner.     

IGF-I enhances cortisol secretion from guinea-pig adrenal gland: in vivo and in vitro study

Insulin-like growth factor (IGF)-I is a ubiquitously synthesized peptide that, along with IGF-II, acts via the IGF-R type I receptor. IGF-I and its receptor are expressed in the adrenal gland of humans and bovines, the secretion of which they seem to stimulate. As in humans and cows, the main glucocorticoid hormone secreted by guinea-pig adrenals is cortisol, and hence we have studied the adrenocortical effects of IGF-I in this species. In vivo experiments showed that prolonged IGF-I administration raised the plasma concentration of cortisol in both normal and dexamethasone/captopril-treated guinea pigs, thereby ruling out the possibility that IGF-I may act by activating the hypothalamic-pituitary-adrenal axis and the renin-angiotensin system. In vitro experiments demonstrated that IGF-I enhanced basal, but not maximally agonist [ACTH and angiotensin-II (Ang-II)]-stimulated, cortisol secretion from freshly dispersed guinea-pig inner adrenocortical cells. The IGF-I immuno-neutralization suppressed the IGF-I secretagogue effect, without altering the cortisol response to both ACTH and Ang-II. IGF-I raised cyclic-AMP and inositol triphosphate release from dispersed guinea-pig cells, and the effect was reversed by the adenylate cyclase inhibitor SQ-22536 and the phospholipase-C (PLC) inhibitor U-73122. SQ-22536, U-73122, the protein kinase (PK) A inhibitor H-89 and the PKC inhibitor calphostin-C decreased by approximately 50% the cortisol response of dispersed cells to IGF-I, and the combined exposure to SQ-22536 and U-73122 abolished it. We conclude that IGF-I stimulates glucocorticoid secretion from guinea-pig adrenocortical cells, acting via selective receptors coupled to both the adenylate cyclase/PKA- and PLC/PKC-dependent signaling cascades.     

Endothelin-3 at low concentrations attenuates inflammatory responses via the endothelin B2 receptor

Objective

The aim of this study was to clarify a role of endothelins (ETs: ET-1, -2, and -3) and their receptors (ETA, ETB1, and ETB2) in inflammatory responses.

Methods

Male Wistar rats (180–220 g) were used. The effects of ETs in the absence or presence of the ETA antagonist BQ-123/the selective ETB2 antagonist BQ-788, and the effect of the selective ETB1 agonist IRL-1620 and the nonselective ETB agonist BQ-3020, on rat hind paw oedema induced by several proinflammatory substances were examined. The involvement of nitric oxide (NO) in the effects of ETs on the paw oedema was investigated using the NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME).

Results

ET-3, which acts mainly on ETB, at low concentrations specifically inhibited platelet-activating factor (PAF)-induced paw oedema, whereas neither ET-1 nor ET-2, both of which act on ETA and ETB, showed inhibitory activity. The inhibition by ET-1 and ET-3 (each 0.5 pmol/paw) in the presence of BQ-123 (66.4 ± 6.7 % and 65.4 ± 22.6 %, respectively), was comparable to that by ET-3 (0.5 pmol/paw) alone (65.4 ± 10.9 %), whereas neither ET-1 nor ET-3 in the presence of BQ-788 showed inhibitory activity. BQ-3020 (0.5 pmol/paw) inhibited the oedema by 50.9 ± 6.0 %, whereas IRL-1620 showed almost no activity. Additionally, L-NAME markedly attenuated the inhibitory effects of ET-3 on PAF-induced paw oedema. These results indicate that ETB2 may mediate NO production and attenuation of PAF-induced inflammatory responses. Moreover, ET-3 (0.5 pmol/paw) inhibited the oedema induced by ET-1 at higher dose and zymosan by 76.6 ± 11.0 and 85.4 ± 13.6 %, respectively, indicating that ET-3 at lower concentrations inhibits the paw oedema induced by various inflammatory substances.

Conclusions

ET-3 at low concentrations may attenuate inflammatory responses via ETB2 activation and NO production.     

Effects of endothelin-1 on epithelial ion transport in human airways

Endothelin-1 (ET-1) exerts many biological effects in airways, including bronchoconstriction, airway mucus secretion, cell proliferation, and inflammation. We investigated the effect of ET-1 on Na absorption and Cl secretion in human bronchial epithelial cells. Addition of 10(-7) M ET-1 had no effect on the inhibition of the short circuit current (Isc) induced by amiloride, a Na channel blocker. Addition of 10(-7) M ET-1 to the apical bath in the presence of amiloride increased Isc in cultured human bronchial epithelial cells studied in Ussing chambers. No effect was observed when ET-1 was added to basolateral bath, indicating that the involved ET-1 receptors are likely present only in the apical membrane of the cells. Use of Cl-free solutions and bumetanide reduced the ET-1-induced increases in Isc, indicating that ET-1 stimulates Cl secretion. The ET-1-induced increase in Isc was prevented by exposure to the ETB receptor antagonist BQ-788 but not to the ETA receptor antagonist BQ-123. ET-1 did not raise intracellular Ca levels, but increased the intracellular concentration of cAMP. These findings indicate that ET-1 is a Cl secretagogue in human airways and acts presumably through apically located ETB receptors and activation of the cAMP pathway.     

Effects of selective and non-selective endothelin antagonists on ischemia-reperfusion damage in the isolated perfused murine liver.

The objective of the present study was to clarify the differential effects of endothelinA (ETA) and ETB antagonism in the early phase of ischemia-reperfusion damage. Male Sprague Dawley rats were randomly divided into 4 groups: control (n = 10), bosentan (40 nM; n = 10), BQ-485 (20 nM; n = 10), and BQ-788 (50 nM; n = 10) to compare the effects of ETA or ETB or both ETA and ETB antagonists against the warm ischemia-reperfusion damage of murine livers. Isolated livers were perfused with oxygenated Krebs-Henseleit bicarbonate buffer solution and ET antagonists for 30 min before inducing warm ischemia (non-recirculating system). After 40 min without perfusate, measurements (portal pressure, O2 tensions of influent and effluent perfusate, liver enzymes, etc.) were taken up to 60 min after reperfusion. The BQ-788 group had significantly more liver damage than did the other groups, and more O2 consumption than did the bosentan group. BQ-485 and bosentan were more protective at some points after reperfusion. Antagonism of only the ETB receptors is detrimental, but antagonism of the ETA receptors appears to have a role in protecting the liver from warm ischemia-reperfusion damage in the early phase.     

Comparison of the signaling mechanisms involved in the ETB receptor-mediated secretagogue action of endothelin-1 on dispersed zona glomerulosa cells and capsule-zona glomerulosa preparations of the rat adrenal gland

Endothelin-1 (ET-1) is a hypertensive peptide, which is expressed in the rat adrenal gland, where it stimulates aldosterone secretion from zona glomerulosa (ZG) by activating the ETb receptor subtype. A higher effectiveness of ET-1 has been frequently observed when the integrity of adrenal tissue is preserved. Hence, we compared the aldosterone secretagogue action of ET-1 on dispersed rat ZG cells and capsule-ZG strips. ET-1 concentration-dependently raised aldosterone output by both preparations with similar potency. However, the efficacy of the maximal effective concentration of ET-1 (10-8 M) was about 2.7-fold higher in capsule-ZG strips. The ETb-receptor antagonist BQ-788 (10-7 M) abolished aldosterone response to 10-8 M ET-1 in both ZG preparations, while the ETa receptor antagonist BQ-123 was ineffective. The aldosterone secretagogue action of 10-8 M ET-1 on dispersed ZG cells was concentration-dependently suppressed by the protein kinase (PK) inhibitor calphostin-C. Conversely, both calphostin-C and the nitric oxide (NO) synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) evoked a concentration-dependent partial reversal of the aldosterone response to 10-8 M ET-1 of capsule-ZG strips. The NO donor L-arginine enhanced basal aldosterone yield of capsular strips, but not dispersed ZG cells. The PKA, cyclooxygenase and lipoxygenase inhibitors H-89, indomethacin and phenidone, as well as the beta-adrenoceptor antagonist l-alprenolol, were ineffective. Collectively, these findings allow us to conclude that in the rat i) the ETb receptor-mediated PKC activation is the main signaling mechanism involved in the direct stimulatory effect of ET-1 on ZG cells; and ii) the higher responsiveness of capsular strips to ET-1 may be accounted for by the ETb receptor-mediated release by stromal elements of NO, which in turn increases aldosterone secretion from ZG cells in a paracrine manner.     

Acute effects of endothelins on endogenous adrenomedullin system in the rat heart: Immunocytochemical and autoradiographic studies

Many lines of evidence indicate that adrenomedullin (AM) through its coronary vasodilatory and inotropic effects, exerts a major cardiac protective action. Conversely, endothelins (ETs) exert a cardiac detrimental action, which seems to be mainly mediated by the ETA receptor, whose activation promotes coronary constriction and decreases cardiac blood flow. Hence, we have investigated by immunocytochemistry (ICC) and autoradiography the acute effects of ET-1 on endogenous AM system of adult rat heart. Isolated hearts were perfused for 20 min, according to the Langendorff technique, with 2x10(-9) M ET-1[1-21] or ET-1[1-31], which are mixed ETA/ETB and selective ETA receptor agonists, respectively. ICC demonstrated AM-immunoreactivity (IR) in cardiomyocytes, endocardium and especially in the wall of coronary vessels. Quantitative densitometry showed that ET-1[1-31], but not ET1[1-21], significantly decreased AM-IR in coronary vessels, thereby suggesting that ET-1 elicits AM release in the heart through the activation of ETA receptors. Autoradiography demonstrated [125I]AM-binding sites in cardiomyocytes and especially in the wall of coronary venules, and treatment with both ET-1s did not apparently affect them. This location af AM receptors suggests that AM raises cardiac blood flow by evoking coronary artery dilation indirectly, probably via its stimulating effect on NO production, and by decreasing postcapillary resistance via cardiac vein dilation. Collectively, our findings indicate that important functional interrelationships occur between ET and AM systems in the rat heart, where ETs may at least partly hamper their own ETA receptor-mediated decrease in blood flow by increasing the local release of endogenous AM.     

Localization and distribution of endothelin receptor subtypes in pulmonary vasculature of normal and hypoxia-exposed rats

To clarify the roles of two different endothelin (ET) receptors in the pulmonary vasculature, the localization and distribution of endothelin-A (ETA) and ETB receptors were investigated in rat lung under normal and hypoxic conditions by an immunohistochemical method. We also carried out in situ hybridization for ETB receptor. In normal rats, ETA receptor is localized in the media of the pulmonary artery and vein with predominant distribution in such proximal segments as elastic arteries and large muscular arteries. ETB receptor is expressed in the intima and media of pulmonary vessels. The distribution of ETB receptor in the media predominates in the distal segments of the pulmonary artery, whereas its distribution in the intima is greater in the proximal segments. Immunoreactivity for ETA receptor increases in the media of the distal segments of the pulmonary artery after exposure to hypobaric hypoxia. Semiquantitative evaluation showed immunoreactivity for ETA receptor in the pulmonary arteries accompanying the terminal bronchioles, respiratory bronchioles, and alveolar ducts to be increased by 2.5-, 5-, and 20-fold after 14 d exposure to hypoxia, respectively. The messenger RNA and immunoreactivity for ETB receptor increased significantly in the intima of the distal segments of pulmonary artery after 7 and 14 d exposure to hypoxia. These results suggest that the vasoconstrictive effects of ET-1 are exerted mainly through ETA receptor in the proximal segments of the pulmonary artery and vein, whereas its effects in the distal segments are mediated by ETA and ETB receptors in normal rats. ETA receptors that increase in resistance arteries after exposure to hypoxia appear to play an important role in the vascular remodeling associated with hypoxic pulmonary hypertension. Because ETB receptors in the endothelium mediate ET-1-induced vasodilatory effects, the increase in endothelial ETB receptors may counteract the development of hypoxic pulmonary hypertension.     

Endothelin receptor expression in human lungs of newborns with congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a major cause of refractory respiratory failure in the neonatal period and is characterized by persistent pulmonary hypertension of the newborn (PPHN) and pulmonary hypoplasia. Endothelin-1 (ET-1) dysregulation may play a significant role in the pathophysiology of PPHN and ET-1 acts through binding to type A (ETA) and type B (ETB) receptors. Therefore, ETA and ETB receptor protein expression was studied using immunohistochemistry in 10 lung specimens obtained from newborns with CDH, and 4 normal lung specimens, in order to explore whether dysregulation of ETA and ETB expression contributes to PPHN. ETA and ETB mRNAs were then quantified using real-time RT-PCR in laser-microdissected pulmonary resistive arteries. In the lungs of newborns with CDH, immunohistochemistry of both ETA and ETB receptors demonstrated over-expression in the thickened media of pulmonary arteries. Using laser microdissection and real-time RT-PCR, higher levels of ETA and ETB mRNA were found in CDH pulmonary arteries than in controls: this increase was more pronounced for ETA mRNA. This study provides the first demonstration of ET-1 receptor dysregulation in association with structural alteration of pulmonary arteries in newborns with CDH and PPHN. This dysregulation preferentially affects the ETA receptor. These results suggest that dysregulation of ET-1 receptors may contribute to PPHN associated with CDH.     

Endothelin synthesis and receptors in porcine kidney

As insufficient information on the endothelin (ET) system in the porcine kidney is available at present, we investigated renal ET-1 synthesis and ET receptors in this species. Because ET specifically affects renal and glomerular haemodynamics and distal tubular reabsorption, we studied ET-1 synthesis in isolated glomeruli and in inner medullary collecting duct (IMCD) cells and preproET-1 mRNA in renal cortex, isolated glomeruli and papillary tissue. In addition, we characterized density and properties of ET receptors in membranes from isolated glomeruli and papillary tissue. In contrast to isolated IMCD cells, which synthesized 120 +/- 11 fmol h(-1) mg-1 protein of ET-1, no such synthesis was found with isolated glomeruli in our assay system. Nevertheless, with RT-PCR preproET(-1) mRNA was clearly present in renal cortex and glomeruli as well as in papillary tissue. Glomerular membranes were found to have ET receptors with Bmax of 1.6 +/- 0.2 pmol mg-1 protein and Kd of 311 +/- 33 pmol L(-1). Using BQ-123 (10-5 M), a specific blocker of ETA receptors, we found that 58% of total receptors are ETA receptors. Thus, presumably 42% are ETB receptors (Bmax 0.7 +/- 0.1 pmol mg-1 protein; Kd 429 +/- 110 pmol L(-1)). Bosentan (10-5 M), an ETA- and ETB-receptor antagonist, blocked all ET receptors in glomerular membranes. Papillary membranes showed ET receptors with Bmax of 2.1 +/- 0.2 pmol mg-1 protein and Kd of 137 +/- 11 pmol L(-1). In the presence of BQ-123 (10-5 M) we found that all receptors are ETB receptors (Bmax 2.3 +/- 0.4 pmol mg-1 protein; Kd 162 +/- 25 pmol L(-1)). Bosentan (10-5 M) again blocked all ET receptors in papillary membranes, thus confirming our previous finding that IMCD cells possess high-affinity ETB receptors mediating the diuretic effects of ET. Thus, in the porcine kidney the ET system may act in an autocrine/paracrine manner at the glomerular as well as at the IMCD level.     

Effect of endothelin on DNA synthesis in human bronchial epithelium: role of MAP kinase cascade]

To elucidate the effect of endothelin (ET) on airway epithelial cell proliferation, we measured intracellular DNA levels and assessed a possible contribution of mitogen activated protein kinase (MAPK) cascade to the ET action. Incubation of transformed human bronchial epithelial (16 HBE) cells with ET in the serum-free medium caused time-dependent increases in DNA synthesis and MTT reduction, an effect that was attenuated by the MAPK kinase inhibitor PD 98059 in a concentration-dependent manner. Western blot analysis showed that ET induced the expression of phosphorylated MAPK protein, indicating an activation of MAPK, and that this effect was inhibited in the presence of PD 98059 or the ETA receptor antagonist BQ-123. These results suggest that ET may stimulate the proliferation of human airway epithelium via ETA receptors and the concomitant activation of MAPK cascade.     

Staphylococcal exfoliative toxins cleave alpha- and beta-melanocyte-stimulating hormones

The staphylococcal exfoliative toxins (ETs) A and B (ETA and ETB) are 27-kDa exotoxins produced by certain strains of Staphylococcus aureus and are the causative agents of staphylococcal scalded-skin syndrome. The crystal structures of the ETs strongly indicate that the proteins are members of the serine protease family of enzymes, although protease activity until now has not yet been conclusively demonstrated. Here, we show that the peptide beta-melanocyte-stimulating hormone (beta-MSH) is cleaved by ETA and that both ETA and ETB are capable of cleaving alpha-MSH. Both toxins exhibit cleavage at specific glutamic acid residues in MSH peptides. Moreover, biologically inactive mutants of ETA were incapable of cleaving beta-MSH.     

Scarlet fever and types of erythrogenic toxins produced by the infecting streptococcal strains.

Group A streptococcal strains were isolated from the throats of 46 children suffering from scarlet fever. For detection of erythrogenic toxins (ETs), the culture supernatants were concentrated 100 times by ethanol precipitation and solubilisation in acetate buffer. ELISA was used to identify ETA and double immunodiffusion to identify ETB and ETC. The presence of the ETA gene was detected by a specific DNA probe. ETA (alone or in combination with ETB and/or ETC) was found in 51.9% of the strains, ETB (alone or in combination with ETA and/or ETC) in 76.9% and ETC (in combination with ETA and ETB) in 28.9%. Only 5.8% of strains did not produce any detectable ET. In SDS-PAGE, supernatants of ETB-producing strains showed a pronounced band in either the region of the proteinase zymogen or the active proteinase. There was no correlation between the type of erythrogenic toxin and the serological M or T type of the producing strain. The mitogenic potency of culture supernatants did not differ significantly irrespective of the toxin type(s) present. Culture supernatants of strains without a detectable amount of the known ETs were highly mitogenic, indicating the production of other streptococcal mitogens. A correlation with clinical symptoms was determined with regard to exanthema and fever. Strains producing two or three toxins caused a more intense exanthema. Patient temperature was higher (greater than or equal to 38 degrees C) when the infecting strain produced ETB. The toxin-producing patterns of the strains of this study were compared with those isolated during the last epidemic outbreak of scarlet fever in East Germany.     

Selective blockade of the endothelin subtype A receptor decreases early atherosclerosis in hamsters fed cholesterol.

Recent studies suggest that endothelin and its receptors may be involved in atherogenesis. To test this hypothesis, cholesterol-fed hamsters were treated with a selective endothelin subtype A (ETA) receptor antagonist BMS-182874. Characterization of hamster atherosclerotic plaques indicated that they contained a fibrous cap of smooth muscle cells, large macrophage-foam cells, and epitopes of oxidized low density lipoprotein. Messenger RNA for both ETA and ETB receptors was detected in aortic endothelial cells, in medial smooth muscle cells, and in macrophage-foam cells and smooth muscle cells of the fibro-fatty plaques. BMS-182874 inhibited the endothelin-1-induced pressor response whereas the depressor effect was unaltered, suggesting that vascular ETA receptors were selectively blocked in vivo. In hyperlipidemic hamsters, BMS-182874 decreased the area of the fatty streak by reducing the number and size of macrophage-foam cells. The results indicated that ETA receptors and thus endothelin promoted the early inflammatory phase of atherosclerosis.