Endothelin-1 (ET1) is a peptide that was initially identified as a strong inductor of vascular contraction. In the last 25 years, there have been several biological processes identified in which ET1 seems to play a critical role. In particular, genetic studies have unveiled that ET1 is important for neuronal development, growth and function. Experimental studies identified ET1 as a regulator of the interaction between sympathetic neurons and cardiac myocytes. This might be of clinical importance since patients suffering from heart failure are characterized by disrupted norepinephrine homeostasis in the heart. This review summarizes the important findings on the role of ET1 for sympathetic neurons and norepinephrine homeostasis in the heart.

In preclinical studies, endothelin receptor A (ETA) antagonists (ETAi) attenuated the progression of heart failure (HF). However, clinical HF trials failed to demonstrate beneficial effects of ETAi. These conflicting data may be explained by the possibility that established HF drugs such as adrenergic receptor blockers interfered with the mechanism of ETAi action in clinical trials. Here we report that mice lacking ETA only in sympathetic neurons (SN-KO) showed less adverse structural remodeling and cardiac dysfunction in response to pathological pressure overload induced by transverse aortic constriction (TAC). In contrast, mice lacking ETA only in cardiomyocytes (CM-KO) were not protected. TAC led to a disturbed sympathetic nerve function as measured by cardiac norepinephrine (NE) tissue levels and [(124)I]-metaiodobenzylguanidine-PET, which was prevented in SN-KO. In a rat model of HF, ETAi improved cardiac and sympathetic nerve function. In cocultures of cardiomyocytes (CMs) and sympathetic neurons (SNs), endothelin-1 (ET1) led to a massive NE release and exaggerated CM hypertrophy compared with CM monocultures. ETA-deficient CMs gained a hypertrophic response through wild-type SNs, but ETA-deficient SNs failed to mediate exaggerated CM hypertrophy. Furthermore, ET1 mediated its effects indirectly via NE in CM-SN cocultures through adrenergic receptors and histone deacetylases, resulting in activation of the prohypertrophic transcription factor myocyte enhancer factor 2. In conclusion, sympathetic ETA amplifies ET1 effects on CMs through adrenergic signaling pathways. Thus, antiadrenergic therapies may blunt potentially beneficial effects of ETAi. Taken together, this may indicate that patients with β blocker intolerance or disturbed sympathetic nerve function could be evaluated for a potential benefit from ETAi.

The aim of this study was to establish the cellular source of ET-like peptides affecting PRL secretion. Fluorescence double label immunocytochemistry and confocal laser scanning microscopy were used to demonstrate cellular colocalization for PRL and endothelin-1 (ET1)-like immunoreactivities in the anterior lobe of the pituitary gland of rats. An ET-specific reverse hemolytic plaque assay was applied to demonstrate that lactotrophs are capable of releasing ET-like peptides. A PRL-specific reverse hemolytic plaque assay was used to assess the influence of the released endogenous ETs on PRL secretion. ET(A)-specific receptor antagonists BQ123 and BQ610, and endothelin convertase enzyme inhibitory peptide, [22Val]big ET1-(16-38), increased PRL secretion, whereas the ET(B) receptor-specific antagonist BQ788 was ineffective. The ET(A) antagonist BQ123-induced increase in PRL secretion followed a bell-shaped dose-response curve in cells obtained from female rats, whereas it followed a sigmoid curve in males. Frequency distribution of PRL plaque sizes using logarithmically binned data revealed two subpopulations of lactotrophs with differential responsiveness to endogenous ETs. These data demonstrate that a large proportion of lactotrophs is capable of expressing and secreting ET-like peptides in biologically significant quantities. As low pituitary cell density in reverse hemolytic plaque assay minimizes cell to cell communications, these findings constitute direct proof of autocrine regulation of PRL secretion by ET-like peptides.

OBJECTIVE

In recent years, renal collecting duct-specific endothelin-1 (ET1), endothelin A (ETA) and endothelin B (ETB) receptors as well as nitric oxide synthase 1 (NOS1) knockout mice have been developed with subsequent identification for an integral role in regulation of sodium water homeostasis and ultimately blood pressure. The focus of this review is to integrate these models and to propose a scheme for the control of sodium excretion by the collecting duct and the endothelin/ETB/NOS system.

RESULTS

NOS1 splice variants are expressed in the kidney, especially in the collecting duct. Mice express predominantly NOS1β in the medulla, with NOS1α and NOS1β in the cortex, whereas rats express NOS1α and NOS1β in both the cortex and medulla. Novel transcription of collecting duct ET1 mediated by epithelial sodium channels, mitochondrial Na/Ca exchangers and glucocorticoids has been determined. ET1 via the ETB receptor increases nitric oxide production in both rat and mouse collecting ducts, suggesting that NOS1β is linked to ET1-dependent NOS activation in the kidney. As well, genetic deletion of NOS1 splice variants in the collecting duct results in a salt-sensitive hypertensive phenotype in mice, much like the collecting duct ET1 and collecting duct ETB knockout mice.

CONCLUSIONS

In the collecting duct, the ET1/nitric oxide pathways are intimately linked, and deletion of collecting duct ET1, ETB receptor or NOS1β results in a salt-sensitive phenotype, which is at least partially dependent on dysregulation of sodium and water reabsorption.

Overstimulation of endothelin type A (ET(A)) and nucleotide (P2Y) Gα(q)-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gα(q)-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca(2+) levels were assessed in isolated ASMCs loaded with Ca(2+)-sensitive dyes, P2Y(2) and ET(A) receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y(2) receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET(A) receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET(A) and P2Y(2) receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease.

We performed a proteomic investigation on primary cultures of neonatal rat cardiomyocytes after treatment with 10 nM endothelin-1 (ET1) for 48 h, an in vitro model for cardiac hypertrophy. Two-dimensional gel electrophoresis profiles of cell lysates were compared after colloidal Coomassie Blue staining. 12 protein spots that significantly changed in density due to ET1 stimulation were selected for in-gel digestion and identified through mass spectrometry. Of these, 8 spots were increased and 4 were decreased. Four of the increased proteins were identified as desmin, the cardiac component of intermediate filaments and one as alpha-B-crystallin, a molecular chaperone that binds desmin. All the desmins increased 2- to 5-fold, and alpha-B-crystallin increased 2-fold after ET1 treatment. Desmin cytoskeleton has been implicated in the regulation of mitochondrial activity and distribution, as well as in the formation of amyloid bodies. Mitochondria-specific fluorescent probe MitoTracker indicated mitochondrial redistribution in hypertrophic cells. An increase of amyloid aggregates containing desmin upon treatment with ET1 was detected by filter assay. Of the four proteins that showed decreased abundance after ET1 treatment, the chaperones hsp60 and grp75 were decreased 13- and 9-fold, respectively. In conclusion, proteomic profiling of ET1-stimulated rat neonatal cardiomyocytes reveals specific changes in cardiac molecular phenotype mainly involving intermediate filament and molecular chaperone proteins.

The pathogenesis of arterial hypertension in autosomal dominant polycystic kidney disease (ADPKD) is complex and likely dependent on interaction of hemodynamic, endocrine and neurogenic factors. We decided to evaluate the role of endothelin (ET1) and nitric oxide (NO) in the regulation of arterial blood pressure (BP) and to determine plasma levels of ET1 and NO in the group of patients with ADPKD. The ADPKD group (18 patients, 6 men + 12 women, mean age 44.6+/-11.7 years, with creatinine clearancecorrig>> 1.1 ml/s) was compared with a control group of 27 healthy volunteers of comparable age. Plasma levels of ET1 assessed by direct RIA determination in the group of ADPKD patients (11.03+/-1.8 fmol/ml) were significantly increased (p<0.001) in comparison with the control group (2.66+/-0.58 fmol/ml), while no significant differences were observed between normotensive and hypertensive patients in the ADPKD group. Serum levels of NO were evaluated according to the determination of serum levels of their metabolites - nitrites/nitrates. Serum levels of NO in the group of ADPKD patients (39.85+/-.38 micro mol/l) were significantly higher (p<0.05) in comparison with the control group (22.7+/-1.20 micro mol/l), whereas in the ADPKD group no significant differences were observed between normotensive and hypertensive patients. Thus, our study supports the concept of complex alteration of both vasoconstrictor and vasodilator systems in the pathogenesis of arterial hypertension in ADPKD.

Endothelin (ET) is a potent vasoconstrictive peptide that may play a role in vascular pathology in general and diabetic nephropathy in particular. The aim of this study was to investigate (1) alterations of urinary ET1 (UET1) in adolescents and young adults with insulin-dependent diabetes mellitus (IDDM) and (2) the relation of UET1 to other indices of diabetic nephropathy and to risk factors of diabetic angiopathy in general. In 130 IDDM subjects aged 15.2+/-4.9 years with a diabetes duration of 7.3+/-5.1 years, UET1 by radioimmunoassay, urinary albumin by nephelometry, plasma renin by immunoradiometric assay, hemoglobin A1c (HbA1c) by high-performance liquid chromatography, and routine biochemistry analyses were determined. Forty-eight controls, healthy siblings of the diabetics of comparable age, were similarly studied. Total 24-hour UET1 excretion was higher in diabetics than in controls (10,866+/-7,270 and 6,598+/-3,294 pg/24 h, respectively, P=.000). This difference was also noted if male and female diabetics were separately compared with controls. In diabetics with normoalbuminuria (<20 microg/min), total 24-hour UET1 excretion was also higher than in controls (P=.002). In diabetics but not in controls, 24-hour UET1 values were higher in males than in females (P=.018). In IDDM subjects, UET1 showed a linear relationship with age (P=.002), urinary albumin (P=.000), serum creatinine (P=.001), systolic blood pressure (P=.038), triglycerides (P=.003), and HbA1c (P=.041). Multiple regression analysis demonstrated that the variables interacting independently with UET1 were urinary albumin (P=.003) and serum creatinine (P=.038). UET1 is elevated early (in adolescence) in IDDM subjects, and it is positively correlated with the degree of albuminuria. These data suggest that the amount of UET1 possibly reflects the severity of diabetic renovascular damage. It may thus be speculated that UET1 could be used as another index of diabetic nephropathy or its progress.

We have investigated the effects of endothelin-1 (ET1) on phospholipid hydrolysis and 3H-arachidonic acid (AA) release and prostaglandin synthesis in the rabbit iris sphincter smooth muscle. ET1 actions are concentration- and time dependent with an EC50 for AA release of 1 nM and t1/2 value of 1.5 min. We have identified the AA metabolites released by ET1, employing HPLC, as both cyclooxygenase and lipoxygenase products. The AA released by ET1 appears to derive mainly from the phosphoinositides through phospholipase A2, rather than phospholipase C activation. A key role for phospholipase A2 in AA release in the sphincter muscle is supported by the following observations. (1) Pretreatment of the labeled sphincter with the phorbol ester, PDBu (100 nM) inhibited ET1-stimulated IP3 formation, but it potentiated ET1-stimulated AA release. (2) Pretreatment of the labeled tissue with isoproterenol (5 M) inhibited ET1-stimulated IP3 production without altering AA release. (3) The potency for ET1-stimulated AA release (EC50 = 1 nM) was much higher than that for IP3 formation (EC50 = 45 nM). (4) There were considerable increases, rather than decreases, in 1, 2-diacyl-glycerol formation (1.2-folds) and its phosphorylated product, phosphatidic acid (2.6-folds) by ET1. It is concluded that in the rabbit iris sphincter ET1 is a potent agonist for AA release and eicosanoid synthesis and that AA is released from phosphoinositides mainly through activation of phospholipase A2.

A modified method of injection into the cerebellomedullary (CM) cistern of mice was developed based on fixation of the mouse with a special mask under inhalation anesthesia, and exposure of the sagittal suture of the cranium and midline of the nape to allow us to visualize injection point directly. The accuracy of the modified method was evaluated using the temporal and spatial intracranial distribution of dye by intracisternal injection of 6- microliter methylene blue aqueous solution. A high concentration of dye was found in the CM cistern, the ventral cisterns, and intracranial proximal portion of the main cranial nerves at 1 hour after injection. The color of the dye in the CM cistern and the ventral cisterns was lighter, and the dye had reached the intracranial distal portion of the main cranial nerves at 6 hours after injection. The dye was completely eliminated by cerebrospinal fluid (CSF) circulation at 24 hours after injection. No severe brain injury was found in any of the 20 mice. Intracisternal injection was successful in all 14 mice sacrificed 1 hour or 6 hours after injection according to the confirmation of dye distribution. The effects of central administration of endothelin-1 (ET1) were evaluated on cerebral blood supply, constriction of cerebral arteries, and change of respiration in mice. Three doses of ET1 were studied: 2 micrograms (0.8 nmol), 4 micrograms (1.6 nmol), and 6 micrograms (2.4 nmol). Cerebral blood flow (CBF) was monitored for 60 minutes following injection using a laser Doppler probe. Intracisternal ET1 injection induced dose-dependent reduction of CBF, constriction of cerebral arteries, and respiratory depression in mice. This modified method of injection into the CM cistern under direct visualization provides accurate and reproducible injection into the CSF, and can be used to investigate the effects of various chemical substances on the central nervous system in mice.

The purpose of this study was to determine the effects of staining solutions on the different shades of a dental composite, and to compare these effects with that of distilled water. Two shades of effect (ET1 and ET2), two shades of enamel (EL and EM), and two shades of dentin (DA1 and DA2) were used. Specimens were immersed in three staining solutions (chlorhexidine gluconate, filtered coffee, and orange juice) and distilled water. Color changes were characterized using the CIELAB color space. Since significant interactions occurred, the staining effect of each solution on each shade was determined using one-way ANOVA and the levels of factor in one-way ANOVA were compared using Tukey's method (p < or = 0.05). The staining solutions were found to be more chromogenic than distilled water, and EM and DA2 shades were found to be more color-stable. Based on the color change results in this study, chlorhexidine gluconate (1.45), filtered coffee (1.43), and orange juice (1.02) were determined as having "slight" staining effects on the tested shades. As for the tested shades in this study, they revealed acceptable color change results.

Nitroxyl (HNO) is a redox congener of NO. We now directly compare the antihypertrophic efficacy of HNO and NO donors in neonatal rat cardiomyocytes and compare their contributing mechanisms of actions in this setting. Isopropylamine-NONOate (IPA-NO) elicited concentration-dependent inhibition of endothelin-1 (ET1)-induced increases in cardiomyocyte size, with similar suppression of hypertrophic genes. Antihypertrophic IPA-NO actions were significantly attenuated by l-cysteine (HNO scavenger), Rp-8-pCTP-cGMPS (cGMP-dependent protein kinase inhibitor), and 1-H-(1,2,4)-oxodiazolo-quinxaline-1-one [ODQ; to target soluble guanylyl cyclase (sGC)] but were unaffected by carboxy-PTIO (NO scavenger) or CGRP8-37 (calcitonin gene-related peptide antagonist). Furthermore, IPA-NO significantly increased cardiomyocyte cGMP 3.5-fold (an l-cysteine-sensitive effect) and stimulated sGC activity threefold, without detectable NO release. IPA-NO also suppressed ET1-induced cardiomyocyte superoxide generation. The pure NO donor diethylamine-NONOate (DEA-NO) reproduced these IPA-NO actions but was sensitive to carboxy-PTIO rather than l-cysteine. Although IPA-NO stimulation of purified sGC was preserved under pyrogallol oxidant stress (in direct contrast to DEA-NO), cardiomyocyte sGC activity after either donor was attenuated by this stress. Excitingly IPA-NO also exhibited acute antihypertrophic actions in response to pressure overload in the intact heart. Together these data strongly suggest that IPA-NO protection against cardiomyocyte hypertrophy is independent of both NO and CGRP but rather utilizes novel HNO activation of cGMP signaling. Thus HNO acutely limits hypertrophy independently of NO, even under conditions of elevated superoxide. Development of longer-acting HNO donors may thus represent an attractive new strategy for the treatment of cardiac hypertrophy, as stand-alone and/or add-on therapy to standard care.

BACKGROUND

Atrial fibrillation (AF) is characterized by electrical and structural remodeling of the atria with atrial fibrosis being one hallmark. Angiotensin II (AngII) is a major contributing factor and blockage of its type I receptor (AT1R) prevents remodeling to some extent. Here we explored the effects of the AT1R antagonist irbesartan on global gene expression and profibrotic signaling pathways after induction of rapid atrial pacing (RAP) in vivo in pigs.

RESULTS

Microarray-based RNA profiling was used to screen left atrial (LA) tissue specimens for differences in atrial gene expression in a model of acute RAP. RAP caused an overall expression profile that reflected AngII-induced ROS production, tissue remodeling, and energy depletion. Of special note, the mRNA levels of EDN1, SGK1, and CTGF encoding pro-endothelin, stress- and glucocorticoid activated kinase-1, and of connective tissue growth factor were identified to be significantly increased after 7h of rapid pacing. These specific expression changes were additionally validated by RT-qPCR or immunoblot analyses in LA, RA, and partly in LV samples. All RAP-induced differential gene expression patterns were partially attenuated in the presence of irbesartan. Similar results were obtained after RAP of HL-1 cardiomyocytes in vitro. Furthermore, exogenously added endothelin-1 (ET1) induced CTGF expression concomitant to the transcriptional activation of SGK1 in HL-1 cells.

CONCLUSIONS

RAP provokes substantial changes in atrial and ventricular myocardial gene expression that could be partly reversed by irbesartan. ET1 contributes to AF-dependent atrial fibrosis by synergistic activity with AngII to stimulate SGK1 expression and enhance phosphorylation of the SGK1 protein which, in turn, induces CTGF. The latter has been consistently associated with tissue fibrosis. These findings suggest ETR antagonists as being beneficial in AF treatment.

BACKGROUND

Despite the availability of numerous transgenic mouse lines to study the role of individual genes in promoting neural repair following stroke, few studies have availed of this technology, primarily due to the lack of a reproducible ischemic injury model in the mouse. Intracortical injections of Endothelin-1 (ET1) a potent vasoconstrictive agent, reliably produces focal infarcts with concomitant behavioral deficits in rats. In contrast, ET1 infarcts in mice are significantly smaller and do not generate consistent behavioral deficits.

METHODS

We have modified the ET1 ischemia model to target the anterior forelimb motor cortex (aFMC) and show that this generates a reproducible focal ischemic injury in mice with consistent behavioral deficits. Furthermore, we have developed a novel analysis of the cylinder test by quantifying paw-dragging behavior.

RESULTS

ET1 injections which damage deep layer neurons in the aFMC generate reproducible deficits on the staircase test. Cylinder test analysis showed no forelimb asymmetry post-injection; however, we observed a novel paw-dragging behavior in mice which is a positive sign of damage to the FMC.

METHODS

Previous ET1 studies have demonstrated inconsistent behavioral deficits; however, targeting ET1 injections to the aFMC reliably results in staircase deficits. We show that analysis of paw-dragging behavior in the cylinder test is a more sensitive measure of damage to the FMC than the classical forelimb asymmetry analysis.

CONCLUSIONS

We have developed a focal ischemic injury model in the mouse that results in reproducible behavioral deficits and can be used to test future regenerative therapies.

Vitamin A and its derivatives, so-called retinoids, can prevent squamous metaplasia induced not only by vitamin A deficiency but also by carcinogenic hydrocarbons. An aromatic retinoid, such as ET1, has been shown to prevent chemically induced papillomas in mice and to amplify certain immunologic reactions. Heavy smokers, 106 volunteers, were submitted to fibrobronchoscopy with bronchial biopsies. An index of metaplasia (IM) was calculated on the basis of microscopical examination of a total of 9,633 sections of 1,010 biopsies. Despite the subjectivity of the estimates of cigarette consumption, this was significantly (P less than 0.02) and positively correlated to the IM. Eighty-five percent of the women had a low IM as compared to only 42% of the men (P less than 0.01), although there was no significant difference in the reported cigarette consumption. Fifty-two subjects had an IM greater than 15% and were given 25 mg ET1 orally daily for 6 months. The bronchoscopy was repeated in 30 patients following completion of the 6-month treatment. The IM was significantly (P less than 0.01) reduced after treatment.

Based on the previous finding that locally produced nitric oxide (NO) and endothelin (ET) exert competing effects on choroidal resistance vessels, the present study sought to further characterize the pharmacology of ET in the choroid. The specific goal was to quantify the choroidal blood flow responses to acute changes in perfusion pressure before and after administering endothelin 1 (ET1), a non-selective ET antagonist, and selective antagonists for the endothelin A (ETA) and endothelin B (ETB) receptor subtypes. Anesthetized rabbits were instrumented with an ear artery cannula to measure mean arterial pressure (MAP), occluders on the aorta and vena cava to control MAP, and a vitreous cannula to measure intraocular pressure (IOP). Choroidal blood flow was measured by laser Doppler flowmetry with a vitreous fiber optic probe. The protocol entailed changing the ocular perfusion pressure by varying MAP before and after ET1 (0.9 microg kg(-1), i.v., n = 14), non-selective ET blockade (A-182086, 3 mg kg(-1), i.v., n = 10), selective ETA blockade (FR-139317, 3 mg kg(-1), i.v., n = 12), and selective ETB blockade (A-192621, 3 mg kg(-1), i.v., n = 14). ET1 and ETB blockade shifted the choroidal pressure-flow relation downward, while the non-selective antagonist and the selective ETA antagonist had no effect. The choroid had a biphasic response to exogenous ET1 as seen in other tissues (i.e. initial brief dilation followed by prolonged constriction) that was blocked by the non-selective antagonist whereas the ETA antagonist enhanced the dilation and blocked the constriction, and the ETB antagonist blocked the dilation and enhanced the constriction. These results indicate that ETA and ETB receptors are present and mediate opposing effects on choroidal vascular resistance. The results also suggest that endogenous ET preferentially elicits ETB vasodilation, most likely by stimulating endothelial nitric oxide release.

We propose a novel generalized ensemble method, a virtual-system coupled multicanonical molecular dynamics (V-McMD), to enhance conformational sampling of biomolecules expressed by an all-atom model in an explicit solvent. In this method, a virtual system, of which physical quantities can be set arbitrarily, is coupled with the biomolecular system, which is the target to be studied. This method was applied to a system of an Endothelin-1 derivative, KR-CSH-ET1, known to form an antisymmetric homodimer at room temperature. V-McMD was performed starting from a configuration in which two KR-CSH-ET1 molecules were mutually distant in an explicit solvent. The lowest free-energy state (the most thermally stable state) at room temperature coincides with the experimentally determined native complex structure. This state was separated to other non-native minor clusters by a free-energy barrier, although the barrier disappeared with elevated temperature. V-McMD produced a canonical ensemble faster than a conventional McMD method.

OBJECTIVE

To investigate the role of feedback by Ca²⁺-sensitive plasma-membrane ion channels in endothelin 1 (Et1) signaling in vitro and in vivo. Methods. Et1 responses were imaged from Fluo-4-loaded smooth muscle in isolated segments of rat retinal arteriole using two-dimensional (2-D) confocal laser microscopy. Vasoconstrictor responses to intravitreal injections of Et1 were recorded in the absence and presence of appropriate ion channel blockers using fluorescein angiograms imaged using a confocal scanning laser ophthalmoscope. Results. Et1 (10 nM) increased both basal [Ca²⁺](i) and the amplitude and frequency of Ca²⁺-waves in retinal arterioles. The Ca²⁺-activated Cl⁻-channel blockers DIDS and 9-anthracene carboxylic acid (9AC) blocked Et1-induced increases in wave frequency, and 9AC also inhibited the increase in amplitude. Iberiotoxin, an inhibitor of large conductance (BK) Ca²⁺-activated K⁺-channels, increased wave amplitude in the presence of Et1 but had no effect on frequency. None of these drugs affected basal [Ca²⁺](i). The voltage-operated Ca²⁺-channel inhibitor nimodipine inhibited wave frequency and amplitude and also lowered basal [Ca²⁺](i) in the presence of Et1. Intravitreal injection of Et1 caused retinal arteriolar vasoconstriction. This was inhibited by DIDS but not by iberiotoxin or penitrem A, another BK-channel inhibitor. Conclusions. Et1 evokes increases in the frequency of arteriolar Ca²⁺-waves in vitro, resulting in vasoconstriction in vivo. These responses, initiated by release of stored Ca²⁺, also require positive feedback via Ca²⁺-activated Cl⁻-channels and L-type Ca²⁺-channels.

BACKGROUND

The alteration of endothelin (ET) levels in diabetic patients with cardiac autonomic neuropathy (CAN) has not been studied extensively and its correlation with cardiac function parameters has not been discussed.

OBJECTIVE

The aim of the present study was to discuss the correlation between the degree of cardiac autonomic neuropathy, plasma big-ET levels, and cardiac functions in diabetic patients who were clinically free of cardiovascular disease.

METHODS

Twenty subjects (32.1 +/- 7.8 years, 11 men, 9 women) with insulin-dependent diabetes mellitus (IDDM) were studied to evaluate the relationship between circulating big-endothelin (big-ET1) levels, CAN, and cardiac functions. The severity of CAN was scored according to Ewing's criteria. Cardiac functions were assessed using Doppler echocardiography.

RESULTS

Left ventricular systolic function in the patient group was within normal limits and comparable with the values of the control group (n = 10). The mean E/A values of diabetics with CAN (1.15 +/- 0.33, p = 0.004) and without CAN (1.34 +/- 0.17) were significantly lower than those of controls (1.57 +/- 0.27). Diabetics with CAN had significantly higher big-ET1 values (81.1 +/- 94 pg/ml) compared with others (12.4 +/- 5.9 and 21.1 +/- 17.7 pg/ml, p = 0.04). Circulating big-ET1 levels showed a significant correlation with E/A values in the control group (p = 0.01, r = -0.7) and with peak A values (p = 0.003, r = 0.64) in diabetics. The CAN score correlated negatively with E/A values (p = 0.01, r = 0.54).

CONCLUSIONS

High big-ET levels might have an important role in the pathogenesis or consequences of diastolic dysfunction in diabetics with CAN. Their role in cardiac autonomic neuropathy and diastolic dysfunction should be investigated further.

Endothelin (ET) may play a role in vasospasm after subarachnoid hemorrhage (SAH). The aim of our study was to test whether the systemic administration of bosentan, a nonpeptidic ET(A) and ETB receptor antagonist, could reverse vasospasm without inducing hypotension. In rabbits (single-hemorrhage model) and in dogs (double-hemorrhage model), SAH was induced; after vasospasm was established, the animals received intravenously either saline or a 30 mg/kg bolus of bosentan. The cross-sectional area of the basilar artery was analyzed by quantitative angiography. In rabbits (n = 13), bosentan reversed basilar vasospasm to the same extent as did an intravertebral injection of sodium nitroprusside. In dogs (n = 10), bosentan reversed only 52 +/- 10% of the vasospasm reversible by papaverine. Bosentan did not alter the heart rate or the arterial blood pressure in either the rabbits or the dogs. In the cerebrospinal fluid, SAH increased endothelin-1 (ET1) and big ET1 by 6 and 3.8 times, respectively; in the basilar artery, SAH increased ET1 concentration, big ET1 concentration, and ET-converting enzyme activity by 1.3, 2, and 2.7 times, respectively. In addition, a local involvement of ET was also suggested by the relaxing effect of bosentan on basilar artery rings from rabbits with SAH and not from control rabbits. Receptor binding studies performed on dog basilar arteries revealed a shift in the phenotype expression of ET receptors from the A to the B type after SAH. We conclude that ET plays a major role in SAH and that systemic ET blockade might selectively dilate spastic arteries.

The present study employed enzyme-immunoassay to examine the effect of ethanol on endothelin-1 and/or -2(ET1 + 2) release from human umbilical vein endothelial cells. Thirty minutes of exposure to ethanol increased the release of immunoreactive ET1 + 2 from cultured endothelial cells in a dose-dependent manner. However, ethanol at concentrations of less than 400 mM did not induce any LDH release from the endothelial cells. Trypan blue exclusion test revealed that 400 mM solution of ethanol decreased the cell viability to 7.7%. Thus, ethanol was found to directly stimulate ET1 + 2 release from cultured human umbilical vein endothelial cells. This reaction of vascular endothelial cells against ethanol may be related to ethanol-induced cardiovascular diseases such as hypertension, myocardial infarction and stroke, as well as fatal alcohol syndrome.

Confluent cultures of two renal collecting duct cell lines (M-1 and mIMCD-K2 cells derived from cortical and inner medullary collecting ducts, respectively) express endothelin1 (ET1), transforming growth factor-beta (TGF beta; both TGF beta 1 and TGF beta 2), and both types of the TGF beta receptor. Experiments were performed to test whether endogenous TGF beta may be a paracrine modulator of ET1 expression in these cells. Treatment of M-1 and mIMCD-K2 cells with TGF beta 2 antisense oligodeoxynucleotides (ODN) significantly reduced ET1 messenger RNA (mRNA) and ET secretion (as well as TGF beta 2 mRNA) in a concentration-dependent manner, whereas control ODN were without significant effects. To produce ET inhibition, antisense ODN had to be present in the basolateral medium, whereas its sole presence in the apical medium was without effect. In addition, a pan-specific TGF beta antibody caused a significant reduction of ET1 mRNA expression and ET1 secretion. M-1 cells were found to express high levels of the mRNA for plasminogen activator of both tissue and urokinase types. Addition of the nonspecific serine protease inhibitor aprotinin (50 micrograms/ml) to the medium for 24 h significantly reduced the secretion of ET1. These results suggest that secretion of endogenous TGF beta, at least in part activated by the plasminogen/plasmin system, participates in the regulation of ET1 synthesis and secretion by collecting duct cell lines.

OBJECTIVE

Patients with hypertensive left-ventricular hypertrophy (LVH) have lower coronary flow reserve (CFR). Whether carvedilol can improve CFR of patients with hypertensive LVH is unknown. We aimed to investigate the effects of carvedilol on CFR in patients with hypertensive LVH.

METHODS

Sixty-three patients were randomly divided into two groups for treatment with carvedilol or metoprolol. The peak diastolic coronary flow velocity in the left anterior descending coronary artery at rest and at maximal vasodilation with dipyridamole infusion was recorded by transesophageal echocardiography (TEE), then CFR was calculated at baseline and at the end of 6 months of therapy. Left-ventricular mass index (LVMI) was calculated by 2-D echocardiography. Endothelium-dependent and -independent reactivity of the brachial artery was measured. Levels of plasma endothelin-1 (ET1), nitric oxide (NO) and other metabolites were monitored and analyzed before and after 6-month therapy.

RESULTS

Both blood pressure and heart rate decreased significantly in the two treatment groups after therapy (p<0.05). With carvedilol treatment, LVMI was lower (p<0.05), endothelium function of the brachial artery was higher (p<0.05), and peak diastolic coronary flow velocity at rest and at maximal vasodilation after dipyridamole infusion was significantly higher (p<0.05) than with metoprolol treatment, which led to a significantly higher CFR (p<0.05). Changes in CFR and LVMI with carvedilol treatment were inversely correlated (R(2)=0.474, p=0.036). With carvedilol treatment, plasma level of ET-1 was lower, but that of NO was significantly higher than with metoprolol treatment (both p<0.05).

CONCLUSIONS

The CFR of patients with hypertensive LVH but not coronary artery disease could increase with 6-month carvedilol therapy.

OBJECTIVE

To investigate endothelin 1 (Et1)-dependent Ca(2+)-signaling at the cellular and subcellular levels in retinal arteriolar myocytes.

METHODS

Et1 responses were imaged from Fluo-4-loaded smooth muscle in isolated segments of rat retinal arteriole using confocal laser microscopy.

RESULTS

Basal [Ca(2+)](i), subcellular Ca(2+)-sparks, and cellular Ca(2+)-oscillations were all increased during exposure to Et1 (10 nM). Ca(2+)-spark frequency was also increased by 90% by 10 nM Et1. The increase in oscillation frequency was concentration dependent and was inhibited by the EtA receptor (Et(A)R) blocker BQ123 but not by the EtB receptor antagonist BQ788. Stimulation of Ca(2+)-oscillations by Et1 was inhibited by a phospholipase C blocker (U73122; 10 μM), two inhibitors of inositol 1,4,5-trisphosphate receptors (IP(3)Rs), xestospongin C (10 μM), 2-aminoethoxydiphenyl borate (100 μM), and tetracaine (100 μM), a blocker of ryanodine receptors (RyRs).

CONCLUSIONS

Et1 stimulates Ca(2+)-sparks and oscillations through Et(A)Rs. The underlying mechanism involves the activation of phospholipase C and both IP(3)Rs and RyRs, suggesting crosstalk between these Ca(2+)-release channels. These findings suggest that phasic Ca(2+)-oscillations play an important role in the smooth muscle response to Et1 within the retinal microvasculature and support an excitatory, proconstrictor role for Ca(2+)-sparks in these vessels.