New drugs and emerging therapeutic targets in the endothelin signaling pathway and prospects for personalized precision medicine.

During the last thirty years since the discovery of endothelin-1, the therapeutic strategy that has evolved in the clinic, mainly in the treatment of pulmonary arterial hypertension, is to block the action of the peptide either at the ET(A) subtype or both receptors using orally active small molecule antagonists. Recently, there has been a rapid expansion in research targeting ET receptors using chemical entities other than small molecules, particularly monoclonal antibody antagonists and selective peptide agonists and antagonists. While usually sacrificing oral bio-availability, these compounds have other therapeutic advantages with the potential to considerably expand drug targets in the endothelin pathway and extend treatment to other pathophysiological conditions. Where the small molecule approach has been retained, a novel strategy to combine two vasoconstrictor targets, the angiotensin AT(1) receptor as well as the ET(A) receptor in the dual antagonist sparsentan has been developed. A second emerging strategy is to combine drugs that have two different targets, the ET(A) antagonist ambrisentan with the phosphodiesterase inhibitor tadalafil, to improve the treatment of pulmonary arterial hypertension. The solving of the crystal structure of the ET(B) receptor has the potential to identify allosteric binding sites for novel ligands. A further key advance is the experimental validation of a single nucleotide polymorphism that has genome wide significance in five vascular diseases and that significantly increases the amount of big endothelin-1 precursor in the plasma. This observation provides a rationale for testing this single nucleotide polymorphism to stratify patients for allocation to treatment with endothelin agents and highlights the potential to use personalized precision medicine in the endothelin field.

Endothelin and diabetic complications: a brain-centric view.

The global epidemic of diabetes is of significant concern. Diabetes associated vascular disease signifies the principal cause of morbidity and mortality in diabetic patients. It is also the most rapidly increasing risk factor for cognitive impairment, a silent disease that causes loss of creativity, productivity, and quality of life. Small vessel disease in the cerebral vasculature plays a major role in the pathogenesis of cognitive impairment in diabetes. Endothelin system, including endothelin-1 (ET-1) and the receptors (ET(A) and ET(B)), is a likely candidate that may be involved in many aspects of the diabetes cerebrovascular disease. In this review, we took a brain-centric approach and discussed the role of the ET system in cerebrovascular and cognitive dysfunction in diabetes.

Regulations of astrocytic functions by endothelins: roles in the pathophysiological responses of damaged brains.

The receptors for endothelins (ETs) are classified into the ET(A) and ET(B) types. ET(B) receptors are highly expressed in astrocytes, but pharmacological usages of this receptor are not clarified. In this article, recent studies on the pathophysiological roles of astrocytic ET(B) receptors in the brain are reviewed. The administration of ET(B) agonists and antagonists in nerve injury models showed that several astrocytic functions are regulated by ET(B) receptors. The activation of ET(B) receptors causes morphological alterations and proliferation of cultured astrocytes. Astrocytes produce various bio-active substances that can affect damage to nerve tissues. ETs stimulate the production of neurotrophic factors by astrocytes. This action improves impaired brain functions. On the other hand, the production of matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF), which induce brain edema, also are stimulated by ETs. These findings indicate that astrocytic functions are effectively regulated by modulations of ET(B) receptors. In brain insults and neurodegenerative diseases, these functions of astrocytes affect the protection and repair of damaged nerve tissues. Thus, astrocytic ET(B) receptors could be a target for novel types of neuroprotective drugs.

Endothelin modulates the cardiovascular effects of clonidine in the rat.

Clonidine decreases mean arterial pressure (MAP) by acting as an α(2)-adrenergic receptor (AR) agonist in the central nervous system; it also acts on peripheral α-ARs to produce vasoconstriction. Endothelin (ET) has been shown to modulate the action of ARs. The present study was conducted to determine the involvement of ET in cardiovascular effects of clonidine. Intravenous administration of clonidine (10, 30 and 90µgkg(-1)) produced a dose-dependent decrease in MAP and heart rate (HR). Treatment with ET-1 (100, 300 and 900ngkg(-1)) significantly attenuated clonidine (10µgkg(-1)) induced fall in MAP and HR. Rats treated with ET-1 (900ngkg(-1)) showed an increase in MAP and HR after clonidine administration compared to untreated rats, while ET(A/B) antagonist, TAK-044 (1mgkg(-1)) and ET(A) antagonist, BMS-182874 (9mgkg(-1)) potentiated the hypotensive effect of clonidine. ET(B) receptor agonist, IRL-1620 (5µgkg(-1)) produced significant attenuation of clonidine induced fall in MAP and HR, while ET(B) receptor antagonist, BQ-788 (0.3mgkg(-1)), potentiated the hypotensive effect of clonidine. Prazosin (0.1mgkg(-1)) completely blocked ET-1 induced changes in cardiovascular effects of clonidine. Clonidine-induced contraction of rat abdominal aortic ring was potentiated by ET-1, which was completely blocked by prazosin. Clonidine produced an increase in ET(A) receptor expression in the brain and abdominal aorta while ET(B) receptors were not affected. It is concluded that ET enhances the responsiveness of vascular ARs to the constrictor effect of clonidine and ET antagonists potentiate the hypotensive effect of clonidine suggesting that a combination of ET antagonist with clonidine may be a useful option to treat hypertension.

Endothelin signaling pathways in rat adrenal medulla.

1. We further characterized the effect of endothelins (ETs) on receptor-mediated phosphoinositide (PI) turnover, nitric oxide synthase (NOS) activation, and cGMP formation in whole rat adrenal medulla. 2. The PI hydrolysis was assessed as accumulation of inositol monophosphates (InsP(1)) in the presence of 10 mM LiCl in whole tissue and the analysis of inositol-1-phosphate by Dowex anion exchange chromatography. NOS activity was assayed by monitoring the conversion of radiolabeled L-arginine to L-citrulline. Cyclic GMP formation was assessed as accumulation of cGMP in whole tissue in the presence of phosphodiesterase inhibition, and the amount of cGMP formed was determined by radioimmuno-antibody procedure. 3. ET-1 and ET-3 increased PI turnover by 30% in whole adrenal medulla prelabeled with [(3)H] myoinositol. Both ETs isoforms, at equimolar doses, increased NOS activity and cGMP levels in similar degree. The selective ET(B) receptor agonist, IRL-1620, also increased cGMP formation, mimicking the effects of ETs, while IRL-1620 did not alter the PI metabolism. ETs-induced InsP(1) accumulation and cGMP was dependent on extracellular calcium. The effect of ETs on PI turnover was inhibited by neomycin. The L-arginine analogue, N-nitro-L-arginine (L-NAME), and two inhibitors of soluble guanylyl cyclase, methylene blue and ODQ, significantly inhibited the increase in cGMP production induced by ETs or IRL-1620. The selective ET(A) receptor antagonist, BQ 123, inhibited the ETs-induced increase in PI turnover, while the selective ET(B) receptor antagonist, BQ 788, was ineffective. Likewise, BQ 788, significantly inhibited ET-1- or ET-3-induced NOS activation and cGMP generation but not ETs-induced InsP(1) accumulation. 4. Our data indicate that stimulation of PI turnover and NO-induced cGMP generation constitutes ETs signaling pathways in rat adrenal medulla. The former action is mediated through activation of ET(A) receptor, while the latter through the activation of ET(B) receptor. These results support the role of endothelins in the regulation of adrenal medulla function.

Pharmacology and physiopathology of the brain endothelin system: an overview.

The endothelin system, consisting of three peptides, two peptidases and two G-protein coupled receptors, is widely expressed in the brain cell types and brain-derived tumor cell lines. The stimulation of endothelin receptors elicits a variety of short- and long-term changes at cellular level but the effects of the pharmacological modulation of the endothelin system in brain physiology and pathophysiology are, at the present time, poorly understood. Altered expression of endothelins (ETs) in reactive astrocytes has been observed in many pathological conditions of the human brain, such as infarcts, lacunae, traumatic conditions, Alzheimer's disease and inflammatory diseases of the brain. In addition, recent studies have shown that endothelin antagonists might inhibit growth and induce cell death in human melanoma cells in vitro and in vivo, and have emphasized a possible role of endothelin peptides as autocrine or paracrine factor in the proliferation and dissemination of tumor cell lines. Given the fact that brain cell and a variety of brain tumor cell lines express functional endothelin receptors, further studies are warranted to demonstrate a possible therapeutic role of endothelin agonists and antagonist in the pharmacological treatment of brain-related diseases and brain tumors.

Endothelins negatively regulate glial glutamate transporter expression.

Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system which at high extracellular levels leads to neuronal over-stimulation and subsequent excitotoxic neuronal cell death. Both the termination of glutamatergic neurotransmission and the prevention of neurotoxic extracellular glutamate concentrations are predominantly achieved by the uptake of extracellular glutamate into astroglia through the high-affinity glutamate transporters, excitatory amino acid transporter-2/glutamate transporter-1 (EAAT-2/GLT-1) and EAAT-1/glutamate aspartate transporter (GLAST). Although several injury-induced growth factors such as epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) potently stimulate the expression of glutamate transporters in cultured astroglia, GLT-1 and/or GLAST expression temporarily decreases during acute brain injuries eventually contributing to secondary neuronal cell death. We now demonstrate that the stimulatory influences of these injury-regulated growth factors are overridden by endothelins (ETs), a family of peptides also upregulated in the injured brain. Exposure of cultured cortical astroglia to ET-1, ET-2, and ET-3 resulted in a major loss of basal glutamate transporter expression after 72 hours and the complete prevention of the known stimulatory influences of dibutyryl cyclic (dbc)AMP, pituitary adenylate cyclase-activating polypeptide (PACAP), EGF, and TGFalpha on both GLT-1 and GLAST expression. With all ET isoforms, the inhibitory effects were detectable with similar low nanomolar concentrations and persisted in endothelin B-receptor deficient astroglia, suggesting that the inhibitory action is equally induced by endothelin A and B receptors. In astroglial cultures maintained with endothelins alone or in combination with PACAP, the inhibitory action was remarkably long-lasting and was still detectable after 7 days. In apparent contrast, glutamate transporter expression partially recovered between days 5 and 7 in cultures maintained with a combination of ETs and the injury-regulated growth factors EGF or TGFalpha. These findings point to ETs as major mediators of injury-dependent down-regulation of glial glutamate transporters and subsequent glutamate-induced brain damage.

Role of endothelin in regulation of resistance, fluid-exchange, and capacitance functions of the systemic circulation.

This article reviews data at the in vivo whole animal and human level. The importance of both flow and pressure recordings and of the methods used to record these variables is emphasized. Exogenous administration of endothelin-1 evokes a transient depressor response mediated by endothelial endothelinB receptors, but the predominate effect of endothelin-1 is a sustained increase in blood pressure resulting from increases in total peripheral resistance. Resistance in the superior mesenteric, renal, and hindquarter vascular beds of animals and forearm resistance in humans is increased. Both endothelinA and, to a lesser extent, endothelinB receptors on vascular smooth muscle mediate the increases in resistance. Endothelin-1 evokes decreases in the precapillary/postcapillary resistance ratio, resulting in increased capillary pressure and net transcapillary filtration. Endothelin-1 evokes increases in mean circulatory filling pressure in animals and in constriction of the human dorsal hand vein. This venoconstrictor activity is mediated primarily through endothelinA and to a lesser extent endothelinB receptors. Endogenously generated endothelin contributes to the hemodynamic effects of angiotensin and vasopressin in certain animal models of hypertension. Antagonists of endothelin evoke modest hemodynamic changes in healthy humans and in some healthy animals, and they decrease vascular resistance dramatically in several salt-sensitive rat models of hypertension and also in some hypertensive human subjects. Thus, endogenously generated ET appears to play a modest role in the healthy organism, but it likely plays a major role in many pathophysiological states as described in companion articles in this issue.

The brain endothelin system as potential target for brain-related pathologies.

The endothelin system, consisting of three peptides, two peptidases and two G-protein coupled receptors, is widely expressed in the brain cell types and brain-derived tumor cell lines. The stimulation of endothelin receptors elicits a variety of short- and long-term changes at cellular level but the effects of the modulation of the endothelin system in brain physiology and pathophysiology are, at the present time, poorly understood. Altered expression of endothelins (ETs) in reactive astrocytes has been observed in many pathological conditions of the human brain, such as infarcts, lacunae, traumatic conditions, Alzheimer's disease and inflammatory diseases of the brain. In addition, recent studies have shown that endothelin antagonists might inhibit growth and induce cell death in human melanoma cells in vitro and in vivo, and have emphasized a possible role of endothelin peptides as autocrine or paracrine factor in the proliferation and dissemination of tumor cell lines. Given the fact that brain cell and a variety of brain tumor cell lines express all the components of the endothelin system, further studies are warranted to demonstrate a possible therapeutic role of endothelin antagonist in the pharmacological treatment of brain diseases and brain tumors.

The role of endothelin-B receptors in myocardial and endothelial reperfusion injury after heart transplantation.

BACKGROUND: This study was designed to investigate the effects of the selective endothelin-B (ET-B) receptor agonist IRL1620 and the selective ET-B receptor antagonist BQ788 on myocardial and endothelial function after reversible deep hypothermic ischemia and reperfusion. METHODS: Isogenic intraabdominal heterotopic heart transplantation was performed on Lewis rats. After one hour of cold ischemic preservation, reperfusion was started either after application of saline vehicle or IRL1620 or BQ788 or both. Left ventricular pressure-volume relations and myocardial blood flow were assessed after one and 24 hours of reperfusion. Endothelium-dependent vasodilatation to acetylcholine (ACH) and endothelium-independent vasodilatation to sodium nitroprusside (SNP) were also determined. RESULTS: IRL1620 attenuated and BQ788 improved myocardial contractility significantly as indicated by the left or upward shift of the systolic pressure-volume relation, respectively, and significantly changed myocardial blood flow during early reperfusion (p<0.05). Although myocardial function and baseline myocardial blood flow were similar in both groups after 24 hours of reperfusion, endothelium-dependent vasodilatation was still significantly lower in the IRL1620 and higher in the BQ788 group (p<0.05). CONCLUSIONS: These results suggest that activation of the ET-B receptors contributes to reperfusion injury after cardiac preservation in a rat heart transplant model.

Cellular mechanisms involved in rabbit carotid body excitation elicited by endothelin peptides.

The present study evaluated the effects of endothelin (ET) peptides on carotid sinus nerve (CSN) activity, catecholamine (CA) release, and second messenger signaling pathways in rabbit carotid bodies superfused in vitro, and in dissociated chemosensory type I cells. ET-1 (1.0 microM) and ET-3 (1.0 microM) did not alter basal CSN activity and CA release, but they potentiated nerve activity (P<0. 05) and CA release (P<0.05) evoked by hypoxia. Under basal conditions, ET-1 and ET-3 (1.0 microM each) elevated tissue cyclic AMP (cAMP) levels nearly 3-fold (P<0.001, ET-1; P<0.05, ET-3) and inositol phosphate (IP(n)) levels nearly 4-fold (P<0.01, ET-1). Hypoxia evoked an increase in carotid body cAMP, and this response was also potentiated in the presence of 1.0 microM ET-1 (P<0.01) or 1.0 microM ET-3 (P<0.001). Patch-clamp studies of isolated type I cells showed that 100 nM ET-1 elevated the peak amplitude of voltage-sensitive (L-type) Ca(2+)-currents by an average of 37.6% (P<0.001). Fluorescent Ca(2+)-imaging revealed that 100 nM ET-1 did not alter [Ca(2+)](i) under basal conditions, but that [Ca(2+)](i)-responses evoked by hypoxia were potentiated by 87% (P<0. 01). Our data indicate that ET augments chemoreceptor responses by activating second messenger signaling pathways which promote the phosphorylation of Ca(2+)-channel protein, thereby enhancing stimulus-evoked intracellular Ca(2+) levels.

Developmental aspects of endothelin receptors in rabbit lower urinary tract.

Since evidence of development/age-related alterations of endothelin receptors in circulation and respiration systems has been increasing, we attempted to investigate the pharmacological characterization of endothelin receptors in neonatal, premature, and mature male rabbit lower urinary tract. The biochemical properties of ET receptors were examined in the lower urinary tracts of 1-day (neonatal)-, 6-week (premature)-, and 1-year(mature)-old male rabbits with binding technique utilizing [(125)I]ET-1. The rank orders of the densities (B(max) values) of endothelin receptors in the bladder dome, bladder base, and urethra of different aged rabbits were bladder dome, 1 day > 6 week &vbar;Ls 1 year, bladder base, 1 day > 6 week &vbar;Ls 1 year, and urethra, 1 day > 6 week > 1 year. The pharmacological profiles of these binding sites inhibited by various kinds of endothelin receptor compounds showed similar K(i) values and similar proportions of endothelin receptor subtypes in the same regions of 1-day-, 6-week-, and 1-year-old animals. Our data clearly demonstrated the presence of regional difference and development-related changes in the density and subtype specificity of endothelin receptors in the lower urinary tract of the male rabbit. Neurourol. Urodynam. 19:71-85, 2000.

Pharmacological and molecular biological evidence for ETA endothelin receptor subtype mediating mechanical responses in the detrusor smooth muscle of the human urinary bladder.

The aim of this study was to characterize endothelin receptor subtypes of the detrusor muscle of the human urinary bladder. The receptor subtypes mediating endothelin (ET)-1-induced activity in the human detrusor smooth muscles have been characterized using isometric contraction and reverse transcription-polymerase chain reaction (RT-PCR). ET-1 (a non-selective ET receptor agonist; 10(-10) M to 10(-6) M) exhibited concentration-dependent contractions in human urinary bladder with a plateau at concentrations above 3x10(-7) M. Neither IRL1620 nor sarafotoxin S6c (both ETB-selective agonists; 10(-10) M to 10(-6) M) elicited contractile activity in the human urinary bladder detrusor smooth muscle. FR139317 (an ETA-selective antagonist; 10(-7) M to 10(-5) M) produced a marked shift to the right of the ET-1 concentration-response curve in human urinary bladder detrusor smooth muscle (from the Schild plot TpA2=7.96; slope=0.95). In contrast, RES701-1 (an ETB-selective antagonist; 10(-7) M to 10(-5) M) had no effect on the ET-1 concentration-response curve. RT-PCR revealed positive amplification of ETA receptor mRNA fragment, but not ETB. These results indicate that the ET-1-induced contractile effects of urinary bladder detrusor smooth muscle seem to be mediated mainly by the ETA receptor, not by the ETB receptor.

Signaling mechanisms induced by endothelin agonists in human adrenal glomerulosa cells.

Endothelin-1 (ET-1) exhibits secretagogue and trophic actions on the adrenal zona glomerulosa (ZG). Little information is available on the intracellular signaling events that follow stimulation of ET receptors on ZG cells. This study examined the expression of ET receptor subtypes and their involvement in transduction mechanisms induced by ET agonists on human ZG cells in primary culture. RT-PCR allowed the detection of both ETA and ETB receptor mRNAs in these cells. ET-1 induced a concentration-dependent increase in inositol phosphate (IP) accumulation in the presence of LiCl, whereas ETB agonists were inactive. The ET-1-induced increase in IP accumulation was prevented by BQ-123. ET-1 evoked an increase in [Ca2+]i, which was partially prevented by BQ-788. IRL 1620 also delayed the rise in [Ca2+]i. These results show that in human adrenal ZG cells, ET-1 induces an increase in IP accumulation through ETA receptor activation and evokes a rise in [Ca2+]i via stimulation of both ETA and ETB receptors.

Effect of centrally administered endothelin agonists on systemic and regional blood circulation in the rat: role of sympathetic nervous system.

The aims of the present study were to determine (1) the hypotensive and regional circulatory effects of centrally administered endothelin (ET) ETA and ETB agonists, and (2) the role of the sympathetic nervous system in the mediation of hypotensive effects due to centrally administered ET-1. The systemic haemodynamics and regional blood circulation in urethane anaesthetized rats following intracerebroventricular (i.c.v.) administration of ET-1, ET-2, SRT6b, ET-3 and SRT6c (10, 30 and 90 ng) were determined by a radioactive microsphere technique. The effect of centrally administered ET-1 on sympathetic nerve activity was also analysed. Systemic haemodynamics and regional blood circulation were determined before (baseline) and 30 min after administration of ET agonists. Cumulative administration of three doses of saline (5 microliters, i.c.v. at 30 min intervals) did not produce any significant cardiovascular effects. ET-1, ET-2 and SRT6b produced a decrease in blood pressure (51%, 47% and 41%, respectively) along with a decrease in cardiac output (58%, 60% and 45%, respectively) and stroke volume. Heart rate and total peripheral resistance were not affected. ET-1, ET-2 and SRT6b also produced a significant reduction in blood flow to the brain, kidneys, heart, portal, mesentery and pancreas, gastrointestinal tract (GIT) and musculoskeletal system. The effect of ET-2 on the cardiovascular system was less intense in comparison with ET-1 and SRT6b. Centrally administered specific ETB receptor agonists ET-3 and SRT6c did not produce any change in systemic haemodynamics and regional blood flow. Centrally administered ET-1 (90 ng) produced a significant decrease (61%) in sympathetic nerve activity 30 min after drug administration, along with a fall in blood pressure. It is concluded that centrally administered ETA agonists produce significant cardiovascular effects mediating through the sympathetic nervous system.

Characterization of endothelin receptors in human varicose veins.

PURPOSE: Experiments were designed to characterize endothelin receptors in human varicose veins. Three groups of veins were studied: (1) varicose vein (VV) tributaries of the greater saphenous vein from patients who were undergoing vein stripping for primary varicosity; (2) greater saphenous veins (SVs) from the same patients; and (3) greater saphenous veins from patients without varicosity who were undergoing arterial reconstruction (control). METHODS: Veins were either cut into rings and suspended in organ chambers for measurement of isometric force, prepared for receptor binding of membrane proteins, or were prepared for measurement of preproendothelin mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Endothelin-1 (10(-11) to 10(-7) mol/L) produced similar concentration-dependent contractions in rings with or without endothelium. Maximal tensions were significantly greater in control veins compared with either SVs or VVs. Sarafotoxin S6c (10(-11) to 3 x 10(-7) mol/L), which is selective for the endothelin-B receptor, also produced concentration-dependent increases in tension in all veins. Sarafotoxin S6c responses in VVs were shifted significantly rightward compared with either SVs or control. Maximal tensions to sarafotoxin S6c also were significantly greater in control veins compared with either SVs or VVs. In receptor binding studies, the number of binding sites as defined by competitive inhibition of 125I-endothelin-1 by endothelin-1 was less in VVs than control veins. Competitive inhibition of 125I-endothelin-1 with endothelin-3 (both A and B receptors) or sarafotoxin S6c (B receptors only) suggests that the difference in receptor number between varicose and nonvaricose veins is attributable to differences in the endothelin-B receptor subtype. Binding affinities were not significantly different for either of the receptor subtypes in all veins studied. Preproendothelin mRNA as quantitated by RT-PCR tended to be higher in VVs compared with either SVs or control veins. CONCLUSIONS: Decreased contractions to endothelin-1 in both varicose and saphenous veins of patients with primary varicosity may be associated with a decrease in the number of receptors. These receptors may be downregulated in response to increased production of endothelin-1, which is regulated at the transcriptional level.

Las endotelinas estimulan la formación de GMPc en la eminencia media y en la médula suprarrenal de la rata / The endothelins stimu the formation of GMPc at the median eminence and at the adrenal medulla of the rat

En el presente trabajo investigamos el efecto de las endotelinas (ETs) sobre la formación de GMPC, mediada por receptores, en la eminencia media (EM) y en la médula suprarrenal (MSR) de la rata. Las isoformas de ETs producen un incremento de la producción de GMPc, siendo este de magnitud similar y dependiente de la concentración. El IRL 1620, un agonista selectivo del receptor ETB, también produce incremento de la formación en la producción de GMPc en ambas estructuras. El incremento en la producción de GMPc inducido por las ETs o por el IRL fue inhibido por el BQ788, un antagonista del receptor ETB. Nuestros resultados demuestran que la estimulación del receptor ETb en estructuras nerviosas como la EM y la MSR produce un incremento de la formación de GMPc. Estos hallazgos sugieren un papel funcional de las ETs mediado por el GMPc en estas estructuras

Endothelin subtypes: effect on isolated rhesus monkey ciliary muscle.

The effects of endothelin (ET)-1, ET-2, ET-3 and sarafotoxin S6C on the contractile response in the longitudinal and coronal vectors of the isolated rhesus monkey ciliary muscle were studied. Fresh ciliary muscle strips from young and middleaged rhesus monkeys were mounted in an apparatus capable of monitoring contractile force simultaneously in the two vectors. The responses to the ET compounds were measured and compared to those produced with 1 microM carbachol. ET-1 produced contractions of up to 15% of the near-maximum response to carbachol in both vectors of 66% of ciliary muscle strips studied. In responsive strips, the maximal ET-1 induced contraction was approximately equal in both vectors, but the longitudinal vector was approximately 5-fold more sensitive than the circular. All ciliary muscle strips responded reproducibly to carbachol. None of the other ET compounds tested had any effect, suggesting that the ETA receptor may predominate in rhesus monkey ciliary muscle. Because ET-1 induces only weak and interindividually variable contraction in isolated rhesus monkey ciliary muscle strips, ET-1's enhancement of outflow facility with only minimal induction of accommodation in the living monkey may be due to effects directly on the trabecular meshwork. However, the 5-fold greater potency of ET-1 in the longitudinal compared to the circular contractile vector may indicate that selective contraction of the longitudinal portion of the ciliary muscle with consequent deformation of the trabecular meshwork but not the lens is also involved.

Tissue endothelin-1 levels in perfused rat heart following stimulation with agonists and in ischaemia and reperfusion.

The purpose of this study was to compare coronary and interstitial endothelin-1 (ET-1) levels in perfused rat hearts under several experimental conditions, because the cardiac tissue concentration of ET-1 is not clear. Hearts were perfused in an upside-down position with a colloid-free buffer at a constant flow rate of 9 ml/min/g heart wet weight, and immunoreactive ET-1 was determined in timed collections of coronary effluent and interstitial transudate produced by the ventricles and appearing on their surface. Basal ET-1 release into effluent was 0.26 +/- 0.007 pg/min/g, and 0.005 +/- 0.0012 pg/min/g in transudate. Basal ET-1 concentration was 0.11 +/- 0.005 pg/ml (transudate) and 0.03 +/- 0.002 pg/ml (effluent), indicating four-fold higher transudate than effluent levels (P < 0.05). Following perfusion of hearts with collagenase to remove endothelial cells, ET-1 release into effluent was reduced to one-third and completely abolished in transudate, indicating that the peptide originated from the vascular endothelium. Perfusion of hearts with angiotensin II (0.1 mumol/l) or thrombin (5 U/ml) increased coronary perfusion pressure and ET-1 secretion, but little affected the transudate/effluent ET-1 concentration ratio (5.5 and 3.2, respectively). When coronary flow was reduced to ischaemic level (1 ml/min/g over several hours), ET-1 secretion rates into effluent were decreased by 55-65%, but increased three- to four-fold on reperfusion at normal flow (P < 0.05). The ET-1 concentrations in both fluids were still always below 1 pg/ml. No change in coronary perfusion pressure compared to time-matched normoxic controls was observed. In the presence of the ET-1 converting enzyme inhibitor, phosphoramidon (1.7 mumol/l), ischaemia-induced increases of ET-1 secretion were attenuated, and this was accompanied by a time-dependent rise in coronary perfusion pressure up to 60% (P < 0.05). These are the first measurements of endogenous cardiac tissue ET-1 levels; they do not support a vasoconstrictor (pro-ischaemic) action of endogenous ET-1 in rat hearts following ischaemia/reperfusion, but rather point to a possible vasodilator role of the peptide under these conditions.

Endothelin-1 and its receptors A and B in human aldosterone-producing adenomas.

Endothelin-1 stimulates aldosterone secretion by interacting with specific receptors. Accordingly, we wished to investigate endothelin-1, endothelin-A (ETA) receptor, and endothelin-B (ETB) receptor gene expression, localization, and properties in aldosterone-producing adenomas and in the normal human adrenal cortex. We carried out 125I-endothelin-1 displacement studies with cold endothelin-1, endothelin-3, the specific ETA antagonist BQ-123, and the specific ETB weak agonist sarafotoxin 6 C and coanalyzed data with the nonlinear iterative curve-fitting program LIGAND. We also studied gene expression with reverse transcription-polymerase chain reaction with specific primers for endothelin-1, ETA, and ETB complementary DNA. Normal adrenal cortices from consenting kidney cancer patients (n = 2) and aldosterone-producing adenomas (n = 4) were studied; for the latter, surrounding normal cortex and kidney biopsy tissue served as controls. To further localize the receptor subtypes, tissue sections were studied by autoradiography in the presence and absence of 500 nmol/L BQ-123, 100 nmol/L sarafotoxin 6 C, and 1 mumol/L cold endothelin-1. In all tissues examined, endothelin-1, ETA, and ETB messenger RNAs were easily detected. However, in aldosterone-producing adenomas, both receptors' genes were expressed at a higher level than in the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)