Involvement of endothelins in deoxycorticosterone acetate-salt hypertension through the modulation of noradrenergic transmission in the rat posterior hypothalamus.

NEW FINDINGS: What is the central question of this study? Does ex vivo administration of endothelin-1 and endothelin-3 regulate noradrenergic transmission in the posterior hypothalamus of deoxycorticosterone acetate-salt hypertensive rats compared with normotensive rats? What is the main finding and its importance? Endothelin-1 and endothelin-3 enhanced diverse mechanisms leading to increased noradrenergic transmission in the posterior hypothalamus of deoxycorticosterone acetate-salt hypertensive rats. Unveiling the role of brain endothelins in hypertension would probably favour the development of new therapeutic targets for the treatment of essential hypertension, which still represents a challenging disease with high mortality. Brain catecholamines participate in diverse biological functions regulated by the hypothalamus. We have previously reported that endothelin-1 and endothelin-3 (ET-1 and ET-3) modulate catecholaminergic activity in the anterior and posterior hypothalamus of normotensive rats. The aim of the present study was to evaluate the interaction between endothelins and noradrenergic transmission in the posterior hypothalamus of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We assessed the effects of ET-1 and ET-3 on tyrosine hydroxylase activity and expression, neuronal noradrenaline (NA) release, neuronal NA transporter (NAT) activity and expression, monoamine oxidase activity and NA endogenous content and utilization (as a marker of turnover) in the posterior hypothalamus of DOCA-salt hypertensive rats. In addition, levels of ETA and ETB receptors were assayed in normotensive and hypertensive rats. Results showed that tyrosine hydroxylase activity and total and phosphorylated levels, NAT activity and content, NA release, monoamine oxidase activity and NA utilization were increased in DOCA-salt rats. Both ET-1 and ET-3 further enhanced all noradrenergic parameters except for total tyrosine hydroxylase level and NA endogenous content and utilization. The expression of ETA receptors was increased in the posterior hypothalamus of DOCA-salt rats, but ETB receptors showed no changes. These results show that ET-1 and ET-3 upregulate noradrenergic activity in the posterior hypothalamus of DOCA-salt hypertensive rats. Our findings suggest that the interaction between noradrenergic transmission and the endothelinergic system in the posterior hypothalamus may be involved in the development and/or maintenance of hypertension in this animal model.

The contractile effects of endothelins on isolated isthmic segment of human oviduct at the luteal phase of the menstrual cycle.

The purpose of this study was to investigate the effects of endothelins (ET) 1, 2 and 3 on isolated isthmic segments of the human oviduct at the luteal phase of menstrual cycle. Fallopian tubes were taken from 21 patients and the isthmic segments were mounted in an organ bath longitudinally. Tension of the isolated preparations was recorded with an isometric transducer. ET-1 and ET-2 triggered concentration-dependent tonic contractions of the isolated isthmic segment and inhibited rhythmic activity, while ET-3 caused no effect. Furthermore, the selective ET(A) antagonist BQ-123 and the selective ET(B) antagonist BQ-788 inhibited the ET-1 effects on both tone and spontaneous rhythmic contractions. These results suggested that during the luteal phase of the menstrual cycle, both ET(A) and ET(B) receptors participate in contractile effects of endothelins on isthmic segment of fallopian tubes, probably regulating the length of time the oocyte remains in the oviduct ampulla.

Endothelin-3 applied to the brain evokes opposite effects on bile secretion mediated by a central nitric oxide pathway.

We sought to establish Endothelin (ET-3) role in the central regulation of bile secretion in the rat. The intracerebroventricular (icv) injection of ET-3 evoked a cholestatic or a choleretic effect depending on the administered dose. Lower doses increased bile flow and bicarbonate excretion, whereas higher doses decreased bile flow and bile acid output. ET-3 effects were dependent on brain nitric oxide and independent of the autonomic nervous system or hemodynamic variations. A selective ETB antagonist abolished the cholestatic effect, whereas the choleretic effect was totally inhibited by either ETA or ETB selective blockade. These results show that ET-3 applied to the brain modified through a nitric oxide pathway distinct bile flow fractions depending on the administered dose and give further insights into the complexity of brain-liver interaction.

Endothelin-1 and endothelin-3 stimulate insulin release by isolated rat pancreatic islets.

Endothelins (ETs) are potent vasoconstrictive peptides released from the endothelium and other tissues, which act on target cells by receptorial calcium-mediated mechanisms. ET-1 levels are increased in diabetes, and observations suggest the involvement of ETs in the pathogenesis of diabetic angiopathy. However, it is not possible to exclude that ETs might also influence insulin secretion or function. In vivo infusion of ET-1 in rats induces hypoglycaemia and hyperinsulinemia and in vitro incubation with ET-1 stimulates insulin release by mouse islets. Therefore, ETs might be involved in a circulus vitiosus, resulting in hyperinsulinemia and diabetic angiopathy. The purpose of our study was to verify the effect of ET-1 on rat islets, in both the presence and absence of physiological glucose concentration. Moreover, we tested the effect of another isoform of endothelins, ET-3, and verified the involvement of extracellular calcium in such events. Islets were incubated with increasing ET-1 or ET-3, with or without glucose 5.6 mM. Other samples were prepared using calcium-free medium. Incubation in medium containing ET-1 and ET-3, in the presence of glucose and calcium, induced an increase in insulin release. When ET-1 and ET-3 were incubated without glucose and calcium, insulin release was not modified. Our studies demonstrate that: 1) ET-3, like ET-1, stimulates insulin release by rat isolated islets; 2) direct insulin stimulating effect on islets of both ET-1 and ET-3 is evident with physiological glucose concentrations and is calcium mediated. These results support the hypothesis of ET involvement in the regulation of insulin secretion.

A rat model of endothelin-3-induced middle cerebral artery occlusion with controlled reperfusion.

Surge hyperemia and mechanical damage to the cerebrovascular endothelium may serve to exacerbate the neuropathological outcome in animal models of focal cerebral ischemia. We have modified an existing model of endothelin-1-induced middle cerebral artery (MCA) occlusion to enable controlled reperfusion without damage to the cerebral vasculature. Endothelin-1 (ET-1) and endothelin-3 (ET-3) were injected via a double-injection cannula into brain parenchyma adjacent to the MCA of anesthetized rats to produce focal cerebral ischemia. ET-1 and ET-3 produced large ischemic lesions that were restricted to those cortical and subcortical structures supplied by the MCA. The volume of ischemic damage produced by 100 pmol of ET-1 and ET-3 was similar. The endothelin-A (ET(A)) receptor antagonist FR139317 (3 or 30 nmol) injected 10 min after ET-1 did not significantly alter the volume of damage. By contrast, the lesion produced by ET-3 was completely inhibited by FR139317 at the 10 min time-point. FR139317 partially attenuated the ET-3-induced lesion when administered 30 min post-occlusion, but injection 90 min following ET-3 produced a lesion not different to that produced by ET-3 alone. These findings were supported by laser Doppler flowmetry which determined FR139317 induces reperfusion when injected 10 or 90 min following ET-3. ET-3-induced MCA occlusion is therefore amenable to reversal by the ET(A) receptor antagonist FR139317, and this model may offer a means to investigate the neuropathology of reperfusion without the procedure-related artifacts associated with some reperfusion models.

Effect of coenzyme Q10 (CoQ10) on superoxide dismutase activity in ET-1 and ET-3 experimental models of cerebral ischemia in the rat.

The aim of the work was to evaluate the influence of CoQ10 on superoxide dismutase (SOD) activity levels in the rat model of cerebral ischemia induced by endothelins (ET-1 or ET-3). ETs (20 pmol) were injected into the right lateral cerebral ventricle and immediately CoQ10 was given intraperitoneally (10 mg/kg b.w.). In the brains of experimental animals subjected both to ET-1 and ET-2 administration there was observed a decrease of SOD activity in the brain stem, in the cerebrallum and in the cerebral cortex at all time intervals. ET-1, as compared to ET-3 evoked longer lasting disturbances in SOD activity. In the cerebellum and in the cerebral cortex positive effect of CoQ10 and recovery to the control values was noted after 4 hours in the group subjected to ET-3 injection and after 24 hours in the ET-1 treated animal. Investigated brain areas showed different sensitivity to ETs. Above data may indicate on beneficial effect CoQ10 in the cerebral ischemia via decrease of free radicals concentration.

Excitatory gastric motor and cardiovascular effects of endothelins in the dorsal vagal complex are mediated through ET(A) receptors.

We have shown recently that intracisternal administration of endothelin-(ET)1 and ET-3 evokes increases in gastric motor function and arterial blood pressure. The aim of our study was to investigate whether the dorsal vagal complex (DVC) is a medullary site of action for the gastric motor and cardiovascular effects of ET-1 and to identify the ET receptor subtype through which these effects are mediated. ET-1 (0.1-40 pmol/site) and ET-3 (1 and 100 pmol/site) were microinjected into the DVC of alpha-chloralose anesthetized rats, while monitoring intragastric pressure, contractile activity of greater curvature longitudinal and pyloric circular smooth muscle, arterial blood pressure and heart rate. ET-1, at doses of 0.1 to 40 pmol, increased intragastric pressure and, at doses of 10 and 40 pmol, increased pyloric contractile activity and arterial blood pressure. The increases in gastric motor function, but not the hypertension, induced by ET-1 (10 pmol) in the DVC were completely abolished by bilateral vagotomy. Spinal cord transection prevented increases in arterial blood pressure evoked by ET-1 (40 pmol). Because only the highest dose of ET-3 (100 pmol), microinjected into the DVC, increased intragastric pressure and pyloric contractile activity and no consistent changes in cardiovascular functions were noted, we hypothesized that the gastric motor and hypertensive responses to endothelins in the DVC are mediated via ET(A) receptors. This was supported by the observation that a selective ET(A) receptor antagonist, cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu) (BQ-123; 400 pmol), microinjected into the DVC 15 min before ET-1 (10 pmol) or ET-3 (100 pmol), completely blocked the gastric motor and cardiovascular responses to endothelins. We conclude that endothelins act in the brainstem at the level of the DVC to increase intragastric pressure and gastric contractile activity via a vagally mediated pathway and that both the gastric motor and hypertensive effects of endothelins in the DVC are mediated through ET(A) 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.

Effects of submucosal administration of endothelin-3 on rat gastric mucosa.

The present study was carried out to examine the effects of submucosal administration of endothelin on gastric mucosal integrity in rats. Injection of endothelin-3 into the submucosal space of the stomach induced gastric mucosal damage dose-dependently and site-specifically. The gastric injury was localized only at the injected site and the mucosal damage was associated with hemorrhage. Macroscopic and microscopic examinations revealed that mucosal injury had developed 15 min after endothelin application. Submucosal injection of either adrenalin or noradrenalin also induced gastric mucosal damage, but produced multiple gastric mucosal lesions; i.e., the macroscopic appearance of endothelin-induced gastric lesions differed from those produced by catecholamines. The endothelin-induced mucosal lesions were significantly inhibited by pretreatment with either atropine, pirenzepine, or ranitidine; or by vagotomy. In addition, NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, significantly enhanced the formation of gastric mucosal lesions. Thus, it appears that nitric oxide synthesis, possibly induced by endothelin, may play a role as an antiulcer mechanism in endothelin-induced gastric mucosal damage. Vagotomy and anti-cholinergic or anti-secretory treatment significantly attenuated the severity of the mucosal lesions, suggesting that vagal cholinergic pathways and acid secretion may influence the development of the gastric mucosal damage induced by endothelin-3. These results suggest that endothelin-3 may play an important role in the development of gastric ulceration; the submucosal application of endothelin-3 in the gastric mucosa may be a useful experimental model for investigating acute gastric mucosal ulceration.

Role of endothelin receptor subtypes in the systemic and renal responses to endothelin-1 in humans.

The authors recently reported that infusion of endothelin-1 in humans to obtain pathophysiological plasma levels causes profound renal vasoconstriction and sodium retention. The relative roles of the ETA- and ETB-receptor subtypes in these effects in humans is unknown. Such information is essential in view of the recent introduction of endothelin-receptor blockers in clinical medicine. The study presented here was designed to define the role of the ETA- and ETB-receptor subtypes in the renal actions of endothelin-1 in humans. Systemic infusion of endothelin-1, a nonselective receptor agonist, was compared with infusion of equimolar dosages of the ETB-selective agonist endothelin-3 in healthy volunteers. Endothelin-1 infusion was associated with an approximate 2.5-fold increase in plasma levels of endothelin-1. This was accompanied by an increase in blood pressure by approximately 6 mm Hg (P < 0.05). During endothelin-1 infusion, RPF decreased from 642 +/- 42 to 480 +/- 36 mL/min (P < 0.05) and GFR from 121 +/- 4 to 109 +/- 7 mL/min (P < 0.05). Sodium excretion rate decreased during endothelin-1 infusion, from a baseline value of 182 +/- 33 to 84 +/- 28 mumol/min at the end of the endothelin-1 infusion. Endothelin-3 infusion also resulted in a approximate 2.5-fold increase of plasma levels of endothelin-3. However, in contrast to the endothelin-1 infusion, endothelin-3 had no effect on blood pressure, renal hemodynamics, and electrolyte excretion. These results suggest that the systemic and renal vasoconstrictor effects of endothelin-1 in humans are predominantly mediated by the ETA receptors.

Effects of endothelins 1, 2 and 3 on mucosal blood flow in the upper airways.

The three endothlins 1, 2 and 3 (ET-1, ET-2 and ET-3) are 21-amino-acid peptides which in a previous study in the rabbit have been shown to increase both mucociliary activity in the maxillary sinus in vivo and ciliary beat frequency (CBF) in sinus and tracheal samples in vitro. We have also demonstrated positive immunohistochemical staining for endothelin in the epithelium of samples from both maxillary sinus and trachea. The aims of the present investigation were to study the effects of the three endothlins on mucosal blood flow in the maxillary sinus of rabbits and the nasal mucosa of humans. ET-1, ET-2 and ET-3 decreased mucosal blood flow in the maxillary sinus of rabbits measured in vivo with laser Doppler flowmetry (LDF). Significant dose-response relationships were established for all endothelins. A comparison of the regression lines of the dose-response relationships for ET-1 and ET-2 did not show any difference, whereas the regression line of the dose-response relationships for ET-3 differed significantly. Maximum effect was seen at 50.0 pmol/kg ET-1, which reduced blood flow by -74.4 +/- 7.0%. Vasoconstriction became evident 30 s after administration of the endothelins, reached its maximum after 1 min and lasted 10 to 20 min. ET-1 induced stronger vasconstriction than ET-2 and ET-3 at equal dosage, except at 100.0 pmol/kg, where ET-2 had the same effect as ET-1. No tachyphylaxis was observed after repeated 10.0 pmol/kg-dose of ET-1. Similarly, there was no tachyphylactic effect on mucociliary activity in vivo in the maxillary sinus of rabbits after repeated challenges with ET-1 at 10.0 pmol/kg. Pretreatment with the cyclooxygenase inhibitors diclofenac had no inhibitory effect on the vasoconstriction induced by ET-1 at 10.0 and 50.0 pmol/kg. In 6 human volunteers the peak blood flow in the nasal septum decreased by -41.8 +/- 11.6% (p < 0.05) and AUC (area under curve) values decreased by -612.0 +/- 322.4% (p < 0.05) after challenge with 0.1 nmol ET-1, delivered in aerosol form into the nose. The effects of the endothelins on the blood flow in the upper airways of rabbit and man indicate that they have a functional role in the regulation of the mucosal vasculature of the upper airways.