Effect of an Endothelin B Receptor Agonist on the Tumor Accumulation of Nanocarriers.

Enhancing blood flow to tumors is a prominent strategy for improving the tumor accumulation of macromolecular drugs through the enhanced permeability and retention (EPR) effect. IRL-1620 is an agonist of the endothelin B receptor, and is a promising molecule to enhance tumor blood flow by activating endothelial nitric oxide synthase. However, contradictory effects on tumor blood flow modulation have been reported because the effects of IRL-1620 may differ in different animal models. Here, we examined for the first time the effect of IRL-1620 on the EPR effect for PEGylated liposomes in a CT-26 murine colon cancer model. Co-injection of IRL-1620 at an optimum dose (3 nmol/kg) nearly doubled the tumor accumulation of liposomes compared with controls, indicating that IRL-1620 enhanced the EPR effect in the present colon cancer model. Co-injection of IRL-1620 is a promising strategy to improve the therapeutic effects of macromolecular drugs while reducing their side effects.

Sovateltide (IRL-1620) affects neuronal progenitors and prevents cerebral tissue damage after ischemic stroke.

Stimulation of endothelin B receptors by its agonist IRL-1620 (INN, sovateltide) provides neuroprotection and neurological and motor function improvement following cerebral ischemia. We investigated the effect of sovateltide on stem and progenitor cells mediated neural regeneration and its effect on the cerebral tissue repair and restoration of neurological and motor function. Sovateltide (5 µg/kg) was injected intravenously in permanent middle cerebral artery occluded (MCAO) rats at 4, 6, and 8 h at days 0, 3, and 6. Neurological and motor function tests were carried out pre-MCAO and at day 7 post-MCAO. At day 7, significantly reduced expression of neuronal differentiation markers HuC/HuD and NeuroD1 was seen in MCAO + vehicle than sham rats. Sovateltide treatment upregulated HuC/HuD and NeuroD1 compared to MCAO + vehicle and their expression was similar to sham. Expression of stem cell markers Oct 4 and Sox 2 was similar in rats of all of the groups. Significantly reduced infarct volume and DNA damage with recovery of neurological and motor function was observed in sovateltide-treated MCAO rats. These results indicate that sovateltide initiates a regenerative response by promoting differentiation of neuronal progenitors and maintaining stem cells in an equilibrium following cerebral ischemic stroke.

Phase 2 study of combination SPI-1620 with docetaxel as second-line advanced biliary tract cancer treatment.

BACKGROUND: This multicentre, open-label study evaluated the efficacy and safety of SPI-1620, an analogue of endothelin-1, administered in combination with docetaxel as second-line treatment for patients with advanced biliary tract cancer (ABTC). METHODS: Eligible patients received continuous cycles of combination therapy with SPI-1620 (11 µg m-2) and docetaxel (75 mg m-2) intravenously every 3 weeks until disease progression (PD) or intolerable toxicity. Tumour response was evaluated using computed tomography or magnetic resonance imaging every 2 cycles (6 weeks). The primary efficacy end point was progression-free survival (PFS); secondary end points included overall response rate (ORR), duration of response, and overall survival (OS) that were estimated using the Kaplan-Meier method. RESULTS: Of the 30 enrolled patients, 25 patients had qualifying events (PD or death), 1 patient was nonevaluable, and 4 patients were censored at the time of their last tumour assessment. Our primary end point of PFS ⩾5 months was not reached. Median PFS was 2.6 months (95% confidence interval (CI): 1.4-2.8), ranging from 0.7 to 8.4 months. The ORR was 10.3% (95% CI: 0.02-0.27). Eleven additional patients achieved stable disease. The OS was 4.87 months. The most common grade 3-4 toxicities were febrile neutropenia and neutropenia. CONCLUSIONS: The addition of docetaxel to SPI-1620 in second-line ABTC did not meet the pre-specified primary end point of PFS ⩾5 months in unselected patient population.

Safety and Efficacy of Sovateltide (IRL-1620) in a Multicenter Randomized Controlled Clinical Trial in Patients with Acute Cerebral Ischemic Stroke.

BACKGROUND: Sovateltide (IRL-1620, PMZ-1620), an endothelin-B receptor agonist, has been previously shown to increase cerebral blood flow, have anti-apoptotic activity and produce neurovascular remodeling when administered intravenously following acute cerebral ischemic stroke in rats. Its safety and tolerability were confirmed in healthy human volunteers (CTRI/2016/11/007509). OBJECTIVE: Our objective was to determine the safety, tolerability and efficacy of sovateltide as an addition to standard of care (SOC) in patients with acute cerebral ischemic stroke. METHODS: A prospective, multicentric, randomized, double-blind, placebo-controlled study was conducted to compare the safety (primary objective) and efficacy (secondary objective) of sovateltide in patients with acute cerebral ischemic stroke. Adult males or females aged 18-70 years who had experienced a radiologically confirmed ischemic stroke within the last 24 h were included in the study. Patients with intracranial hemorrhage and those receiving endovascular therapy were excluded. Patients randomized to the sovateltide group received three doses of sovateltide (each dose 0.3 µg/kg) administered as an intravenous bolus over 1 min at an interval of 3 ± 1 h on day 1, day 3 and day 6 (total dose of 0.9 µg/kg/day). Patients randomized to the placebo group received an equal volume of saline. Every patient in both groups received SOC for stroke. Efficacy was evaluated using neurological outcomes based on National Institute of Health Stroke Scale (NIHSS), modified Rankin Scale (mRS) and Barthel Index (BI) scores from day 1 through day 90. Quality of life was measured using the EuroQoL-5 Dimensions (EQ-5D) and Stroke-Specific Quality of Life (SSQoL) at 60 and 90 days of follow-up. RESULTS: A total of 40 patients with acute cerebral ischemic stroke were enrolled in this study, of whom 36 completed the 90-day follow-up. Patients received saline (n = 18; 11 male and 7 female) or sovateltide (n = 18; 15 male and 3 female) within 24 h of onset of stroke. The number of patients receiving investigational drug within 20 h of onset of stroke was 14/18 in the saline group and 10/18 in the sovateltide group. The baseline characteristics and SOC in both cohorts was similar. Sovateltide was well-tolerated, and all patients received complete treatment with no incidence of drug-related adverse events. Hemodynamic, biochemical or hematological parameters were not affected by sovateltide. Sovateltide treatment resulted in improved mRS and BI scores on day 6 compared with day 1 (p < 0.0001), an effect not seen in the saline group. Sovateltide increased the frequency of favorable outcomes at 3 months. An improvement of ≥ 2 points on the mRS was observed in 60 and 40% of patients in the sovateltide and saline groups, respectively (p = 0.0519; odds ratio [OR] 5.25). An improvement on the BI of ≥ 40 points was seen in 64 and 36% of the sovateltide and saline groups, respectively (p = 0.0112; OR 12.44). An improvement of ≥6 points on the NIHSS was seen in 56% of patients in the sovateltide group versus 43% in the saline group (p = 0.2714; OR 2.275). The number of patients with complete recovery (defined as an NIHSS score of 0 and a BI of 100) was significantly greater (p < 0.05) in the sovateltide group than in the saline group. An assessment of complete recovery using an mRS score of 0 did not show a statistically significant difference between the treatment groups. Sovateltide treatment resulted in improved quality of life as measured by the EQ-5D and SSQoL on day 90. CONCLUSION: Sovateltide was safe and well-tolerated and resulted in improved neurological outcomes in patients with acute cerebral ischemic stroke 90 days post-treatment. TRIAL REGISTRATION: The study is registered at CTRI/2017/11/010654 and NCT04046484.

Pharmacologic and pharmacokinetic rationale for combination therapy in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary vasculature characterized by elevated pulmonary artery pressure. Currently, there is no cure for this disease, and treatment is palliative. PAH therapies target 3 main pathways: prostacyclin, endothelin, and nitric oxide. The 3 distinct therapeutic pathways targeted have provided the opportunity to explore the efficacy of combination therapy. This article reviews the pharmacokinetic profiles of available therapies and existing clinical trial results that support combination therapy, as well as provides rationale and clinical guidance for combination therapy. Combination therapy may offer an additive and potentially synergistic benefit. The initiation and titration of combination therapy has several pharmacokinetic considerations, such as the onset of drugs' action and time to steady state, as well as potential drug interactions. Although great strides have been made in the treatment of PAH, unanswered questions still remain in regard to combination therapy: (1) Which combination is best? and (2) Is it most appropriate to maximize one drug and then initiate a second drug or to start both drugs simultaneously? Answers to these questions will help identify a safe and effective combination therapy and thus optimize treatment for patients with PAH.

Sovateltide (IRL-1620) activates neuronal differentiation and prevents mitochondrial dysfunction in adult mammalian brains following stroke.

The development of effective drugs for stroke is urgently required as it is the 2nd largest killer in the world and its incidence is likely to increase in the future. We have demonstrated cerebral endothelin B receptors (ETBR) as a potential target to treat acute cerebral ischemic stroke. However, the mechanism of ETBR mediated neural regeneration and repair remains elusive. In this study, a permanent middle cerebral artery occluded (MCAO) rat model was used. Sovateltide (an ETBR agonist) injected intravenously showed better survival and neurological and motor function improvement than control. Higher neuronal progenitor cells (NPCs) differentiation along with better mitochondrial morphology and biogenesis in the brain of sovateltide rats were noted. Exposure of cultured NPCs to hypoxia and sovateltide also showed higher NPC differentiation and maturation. This study shows a novel role of ETBR in NPCs and mitochondrial fate determination in cerebral ischemia, and in improving neurological deficit after stroke.

Leukemia inhibitory factor extends the lifespan of injured photoreceptors in vivo.

Survival and death of photoreceptors in degenerative diseases of the retina is controlled by a multitude of genes and endogenous factors. Some genes may be involved in the degenerative process itself whereas others may be part of an endogenous defense system. We show in two models of retinal degeneration that photoreceptor death strongly induces expression of leukemia inhibitory factor (LIF) in a subset of Muller glia cells in the inner nuclear layer of the retina. LIF expression is essential to induce an extensive intraretinal signaling system which includes Muller cells and photoreceptors and is characterized by an upregulation of Edn2, STAT3, FGF2 and GFAP. In the absence of LIF, Muller cells remain quiescent, the signaling system is not activated and retinal degeneration is strongly accelerated. Intravitreal application of recombinant LIF induces the full molecular pathway including the activation of Muller cells in wild-type and Lif(-/-) mice. Interruption of the signaling cascade by an Edn2 receptor antagonist increases whereas activation of the receptor decreases photoreceptor cell death. Thus, LIF is essential and sufficient to activate an extensive molecular defense response to photoreceptor injury. Our data establish LIF as a Muller cell derived neuronal survival factor which controls an intrinsic protective mechanism that includes Edn2 signaling to support photoreceptor cell survival and to preserve vision in the injured retina.

Repeated administration of ET(B) receptor agonist, IRL-1620, produces tachyphylaxis only to its hypotensive effect.

IRL-1620, a highly selective ET(B) receptor agonist, is presently in a phase I clinical trial (NCT00613691) in the United States for patients with recurrent or progressive carcinoma. The effect of acute repeated administration of IRL-1620 on the development of tachyphylaxis to changes in blood pressure, heart rate and blood flow (renal and cerebral) has not been studied. The present studies were conducted in urethane anesthetized rats to determine the cardiovascular effects of acute repeated intravenous administration of IRL-1620. In order to determine the tachyphylactic effect, each dose of IRL-1620 was administered at 0, 60, and 120min. It was found that IRL-1620 did not significantly affect heart rate. IRL-1620 produced a transient fall in blood pressure. A fall in mean arterial pressure (MAP) of 35.47% with 1.6microg/kg, 38.87% with 5.0microg/kg and 28.04% with 15.0microg/kg dose of IRL-1620 was observed. Repeated administration of a low dose (1.6microg/kg, i.v.) of IRL-1620 produced a fall in MAP but no tachyphylaxis was observed. However, repeated administration of IRL-1620 (5.0microg/kg, i.v.) produced a fall in MAP of 40.12%, 29.15%, and 21.61% with the first, second and third injections, respectively. IRL-1620 produced a consistent decrease in renal blood flow and increase in cerebral blood flow without any evidence of tachyphylaxis. Pretreatment with ET(A) antagonist BMS187824 (5mg/kg, i.v.), followed by three doses of 5microg/kg IRL-1620 at 60min intervals eliminated the development of tachyphylaxis to the transient hypotension, confirming the involvement of the ET(A) receptor in tachyphylactic development. The findings indicate development of tachyphylaxis to IRL-1620 only to the fall in blood pressure when given repeatedly at mid-high doses, while the decrease in renal and increase in cerebral blood flow were not affected with regards to tachyphylactic development.

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.

Apoptotic cardiomyocyte hypertrophy during sepsis and septic shock results from prolonged exposure to endothelin precursor.

Septic shock is a complex cardiovascular dysfunction which leads to regional circulatory alterations and multi-organ dysfunction in humans and animal models. To elucidate the role of stress-activated signaling molecules in the regulation of myocardial dysfunction, we have developed and standardized isolated ventricular myocyte techniques. These techniques allow the assessment of cardiodynamics at cellular (ventricular myocyte) level. These studies are carried out in a well defined model of systemic inflammatory response syndrome following polymicrobial sepsis in the rat. Evidence is provided that sepsis-induced myocardial dysfunction produces indications (signs) of early stages of heart failure. This evidence correlates with upregulation of stress-activated protein kinase cascade. These findings suggest that prolonged exposure to endothelin precursor causes decompensatory hypertrophy in adult rat ventricular myocytes (ARVMs) during sepsis. The decompensatory hypertrophy could, in turn, results in increased cytosolic caspases-3 activity in ARVMs.

Endothelin-1 activates ETA receptors to cause reflex scratching in BALB/c mice.

BACKGROUND AND PURPOSE: Endothelin-1 (ET-1) is present in murine and human skin and causes itch (pruritus) when injected in humans. This behavioural study examined the scratch reflex evoked by ET-1 in mice. EXPERIMENTAL APPROACH: An automated detector was used to determine whether ET-1 causes reflex scratching, the behavioural correlate of itching, in BALB/c mice. Selective agonists and antagonists were used to probe the ET receptor(s) involved. KEY RESULTS: ET-1 evoked dose-related reflex scratching lasting up to 20 min following intradermal injection (0.1-100 ng; 0.04-40 pmol). The ED(50) for ET-1 induced scratching was 2.1 ng and desensitization occurred with cumulative dosing. High doses of the ET(B) receptor agonist IRL1620 (10 microg; 5.5 nmol), also caused scratching (ED(50) 1.3 microg, 0.7 nmol). The ET(A) receptor antagonist BQ123 significantly reduced scratching evoked by ET-1 and IRL 1620, suggesting that both agonists caused scratching via an ET(A) receptor-dependent mechanism. The ET(B) receptor antagonist BQ788 significantly reduced scratching evoked by IRL1620 but had no effect on scratching evoked by ET-1. This indicated that activation of ET(B) receptors by high doses of ET(B) agonist, but not ET-1, can trigger scratching. CONCLUSION AND IMPLICATIONS: ET-1 is a potent endogenous activator of reflex scratching (itch). Mechanisms for ET-induced scratching are considered, including direct action of ET-1 on pruriceptive nerve endings and indirect actions via release of endogenous mediators such as histamine from mast cells. ET-1 and ET(A) receptors, possibly also ET(B) receptors, are potential targets for developing specific anti-pruritic drugs to treat pruritic skin disorders such as atopic dermatitis.

Pulmonary hypertension: evaluation and management.

Pulmonary hypertension (PH) is a hemodynamic state characterized by elevation in the mean pulmonary arterial pressure and pulmonary vascular resistance leading to right ventricular failure and premature death. PH can be the result of a variety of diseases of different etiologies. Pulmonary arterial hypertension (PAH) should be distinctly differentiated from pulmonary venous hypertension (PVH) as a result of left heart disease. PAH is commonly caused by or associated with an underlying pulmonary, cardiac, or systemic disease (APAH). In the absence of an identifiable etiology or associated underlying disease, PAH is referred to as idiopathic (IPAH). IPAH, formerly known as primary pulmonary hypertension (PPH), is a rare disease most commonly seen in women of childbearing age. Presenting symptoms and signs are nonspecific and include dyspnea on exertion, fatigue, and a loud pulmonary component of the second heart sound. Transthoracic Doppler echocardiography is an excellent noninvasive test to detect the presence of pulmonary hypertension, although every patient should receive a right heart catheterization to confirm the diagnosis. A detailed work up, including laboratory tests and imaging studies, is also indicated to rule out known causes of pulmonary hypertension. Several targeted treatment options have become available in recent years and include parenteral and inhaled prostanoids, oral endothelin receptor antagonists, and oral phosphodiesterase type-5 inhibitors. As a result of their complex care, patients should be referred to centers with expertise in pulmonary hypertension.

Effects of endothelin-induced prefrontal cortical lesions on delay discounting in the rat.

Stroke is one of the most prominent causes of neurological disability, and the number of stroke cases worldwide is expected to grow due to increases in both average life span and population. As such, new methods for both acute treatment and poststroke rehabilitation will be increasingly necessary. Although a number of approaches to restoring motor function poststroke are in development, there are few methods to alleviate the cognitive deficits caused by this disease. As well, there are very few preclinical models of stroke with a specific focus on higher-order cognitive functions. The goal of the current experiments was to examine the effects of bilateral ischemic lesions, produced by targeted microinjections of endothelin-1 (ET-1) in the medial (mPFC) and orbital (oPFC) prefrontal cortices of adult male Sprague-Dawley rats (n = 39) on inhibitory control as measured through a delay discounting paradigm. The ET-1 injections to the mPFC and oPFC resulted in average lesion volumes of 17.98 mm3 ± 2.841 mm3 (Mean ± SE) and 26.05 mm3 ± 4.052 mm3 (Mean ± SE), respectively. During delay discounting testing, wherein animals were offered a small, immediately available food reward versus a large, but delayed reward, it was found that animals with lesions to the oPFC were more likely to choose the immediately available reward as compared to their mPFC or control counterparts. We conclude that using ET-1 in the oPFC may be a new and viable method to study the effects of ischemic lesions on higher-order cognitive dysfunction poststroke. (PsycINFO Database Record

Evaluation of liposomal nanocarriers loaded with ETB receptor agonist, IRL-1620, using cell-based assays.

One common feature of most neurodegenerative diseases, including Alzheimer's disease (AD) and stroke, is the death of neuronal cells. Neuronal cell death is associated with apoptosis, generation of reactive oxygen species and oxidative stress. Neuronal cell death pathways can be reversed by endothelin B receptor agonist, IRL-1620, which was found to enhance neuroprotection by promoting vascular and neuronal growth in a rodent stroke model. Previous studies conducted at our institution indicated that the treatment with IRL-1620 significantly improved neurological and motor function while reducing oxidative stress and overall infarct area. IRL-1620 is a hydrophilic, 15 amino acid peptide and has a molecular weight of 1820Da. In this study, we have encapsulated IRL-1620 in PEGylated liposomes in order to enhance its efficacy. Each batch of liposomes encapsulating IRL-1620 was evaluated for particle size, polydispersity index, and charge (zeta potential) over a period of time to determine their stability. A dose-response bar graph was plotted based on the effect of neuroprotection by free IRL-1620 on differentiated neuronal PC-12 cells. The 1nM concentration was found to have the highest cell viability. The liposomes loaded with IRL-1620 were tested on differentiated neuronal PC-12 cells for their neuroprotective ability against apoptosis caused by removal of nerve growth factor (NGF) against free (non-encapsulated) IRL-1620. The liposomal IRL-1620 was found to proliferate the growth of serum-deprived differentiated PC-12 cells significantly (p<0.0001). In the western blot analysis, the expression of the anti-apoptotic marker, BCL-2 was found to be increased, and that of pro-apoptotic marker, BAX was found to be decreased with liposomal IRL-1620. The effects were found to be independent of the NGF levels. Finally the free IRL-1620 was found to cause neuronal outgrowth equivalent to the 75ng/ml NGF treatment.

Interaction between a rat model of cerebral ischemia and beta-amyloid toxicity: inflammatory responses.

BACKGROUND AND PURPOSE: Clinical data suggest that Alzheimer disease (AD) and stroke together potentiate cognitive impairment. Inflammatory mechanisms are involved in AD pathology and stroke and may be the mediator between AD and stroke toxicity. METHODS: AD was modeled by cerebroventricular injections of beta-amyloid (Abeta[25-35]) and subcortical lacunar infarcts by striatal endothelin injections. Inflammatory mechanisms were examined using immunohistochemical analysis. Memory and motor tasks were assessed using the Montoya staircase test. RESULTS: Abeta injections elicited increases in pathological and inflammatory correlates of AD in multiple forebrain sites. Increases in astrocytosis and reactive microglia in the hippocampus were enhanced with the combination of endothelin and Abeta(25-35). Abeta(25-35) treatment decreased performance in the Montoya staircase behavioral test. CONCLUSIONS: The enhanced inflammatory response with Abeta toxicity and ischemia may mediate the inability to improve behavioral performance caused by the stroke. Anti-inflammatory treatment may ameliorate the pathological and behavioral deficits associated with the combination of AD and stroke.

Effects of calcium channel blockers on coronary vasoconstriction induced by endothelin-1 in closed chest pigs.

The purpose of this study was to determine the effects of endothelin-1 on the coronary vascular bed of closed chest pigs. Endothelin-1 (3 to 30 pmol/kg body weight) was selectively administered into the left anterior descending coronary artery. Coronary blood flow and epicardial vessel diameter were measured by quantitative arteriography. Arterial pressure increased after a 30 pmol/kg dose and heart rate was not changed. Coronary blood flow and vessel diameter of the left anterior descending artery significantly decreased by 74% and 32%, respectively (p less than 0.01 versus control) after the 30 pmol/kg dose, whereas these variables modestly decreased in the left circumflex artery. Endothelin-1 in doses of 10 to 30 pmol/kg produced electrocardiographic ST segment elevation associated with decreased oxygen saturation of coronary sinus venous blood. Endothelin-induced coronary vasoconstriction was significantly inhibited after treatment with intravenous diltiazem (0.2 mg/kg, n = 6) or nifedipine (0.1 mg/kg, n = 5), but not after vehicle administration (n = 4). This study demonstrates that intracoronary administration of endothelin-1 causes significant myocardial ischemia through coronary vasoconstriction, which is inhibited by a calcium channel blocker. The data suggest that calcium influx into the smooth muscle cells appears to be involved at least in part in the mechanism of endothelin-induced coronary vasoconstriction in vivo.

Stimulation of endothelin B receptors by IRL-1620 decreases the progression of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by severe cognitive impairment that ultimately leads to death. Endothelin (ET) and its receptors have been considered as therapeutic targets for AD. Recent studies in our lab have shown that stimulation of ETB receptors provide significant neuroprotection following Aß1-40 administration. It is possible that IRL-1620 may be neuroprotective due to angiogenesis. However, the effect of IRL-1620 on neurovascular remodeling following Aß1-40 administration has not been established. The purpose of this study was to determine the effect of stimulation of ETB receptors by IRL-1620 on vascular and neuronal growth factors after Aß1-40 administration. Rats were treated with Aß1-40 (day 1, 7 and 14) in the lateral cerebral ventricles using stereotaxically implanted cannula and received three intravenous injections of IRL-1620 (an ETB agonist), and/or BQ788 (an ETB antagonist) at 2-h interval on day 8; experiments were performed on day 15. Rats were sacrificed for estimation of brain ETB receptors, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) expression using immunofluorescence and Western blot. In the Morris swim task, amyloid-ß (Aß)-treated rats showed a significant (p<0.0001) impairment in spatial memory. Rats treated with IRL-1620 significantly (p<0.001) reduced the cognitive impairment induced by Aß. BQ788 treatment completely blocked IRL-1620-induced improvement in cognitive impairment. IRL-1620 treatment enhanced the number of blood vessels labeled with VEGF compared to vehicle treatment. Additionally, cells showed increased (p<0.001) positive staining for NGF in IRL-1620-treated animals. ETB, VEGF and NGF protein expression significantly (p<0.001) increased in the brain of IRL-1620-treated rats as compared to vehicle. Pretreatment with BQ788 blocked the effects of IRL-1620, thus confirming the role of ETB receptors in the neurovascular remodeling actions of IRL-1620. Results of the present study demonstrate that IRL-1620 improves both acquisition (learning) and retention (memory) on the water maze task and enhances angiogenic and neurogenic remodeling. These findings indicate that the ETB receptor may be a novel therapeutic target for AD and other neurovascular degenerative disorders.

Endothelin inhibits basal and stimulated release of prolactin by human decidual cells.

Recent studies have shown that the basal release of PRL from anterior pituitary cells is inhibited by endothelin-1 (ET-1) and ET-3. To determine whether ET also regulates the synthesis and release of PRL by decidual cells, we examined the effects of ET on the synthesis and release of PRL from an enriched fraction of human decidual cells prepared by isopycnic centrifugation of enzymatically dispersed term decidual tissue. Exposure of decidual cells to ET-1 (10(-7) M) for 96 h caused a progressive decrease in basal PRL synthesis and release beginning 24 h after exposure with half-maximal inhibition occurring at an ET-1 concentration of 5 x 10(-9) M. Between 72-96 h of culture, ET-1-exposed cells synthesized 37.2 +/- 2.7% (SEM) and released 32.3 +/- 1.3% less PRL than control cells (P < 0.01). ET-1-exposed cells incubated with [35S]methionine between 72-96 h also released less [35S] PRL than control cells. Sarafotoxin S6C, an ETB receptor agonist, also inhibited basal PRL release, whereas BQ-123, an ETA receptor antagonist, had no effect on basal or on ET-1-mediated inhibition of PRL release. ET-1 also markedly inhibited the stimulation of PRL synthesis and release in response to insulin and insulin-like growth factor-1 (IGF-1). Cells exposed to insulin (100 ng/ml) and IGF-1 (50 ng/ml) alone released 61.2 +/- 3.6% and 40.0 +/- 3.8% more PRL, respectively, than control cells between 72-96 h of exposure. However, cells exposed simultaneously to insulin and ET-1 (10(-7) M) released only 17.1 +/- 3.5% more PRL than control cells during the same time period, and cells exposed simultaneously to IGF-1 and ET-1 released only 4.1 +/- 1.8% more PRL than controls during the same time interval (P vs. insulin or IGF-1 alone < 0.001 in each instance). Both basal and insulin- and IGF-1-stimulated PRL release were also inhibited by the ET isotypes ET-2 and ET-3, and by the ET-1 precursor, big ET. Since macrophages and decidual cells synthesize ET, and decidual tissue contains specific receptors for ET, the inhibitory action of ET on basal and stimulated PRL release may result from an autocrine and/or paracrine effect and appears to be mediated through the ETB receptor.

Differentiated metabolic stimulation of rat pituitary lobes by peripheral and central endothelin-1.

Intravenous infusion (14 nmol/min) and lateral cerebral ventricular injection (9 pmol) of endothelin-1 increased rates of glucose utilization (quantitative autoradiographic [14C]deoxyglucose method) by 75-219% in pars intermedia and distalis of the rat pituitary gland. In rats given intraventricular endothelin, glucose metabolism was also increased significantly in pars nervosa (+92%). Metabolic activation by central endothelin in pars intermedia and distalis was inhibited by intraventricular pretreatment with the dihydropyridine, nimodipine, indicating that endothelin stimulates energy metabolism and probably hormone secretion in melanotrophs and pars distalis cells via L-type calcium channels.

Low doses of endothelin-1 inhibit atrial natriuretic peptide secretion.

To clarify the interaction between endothelin-1 (ET-1) and atrial natriuretic peptide (ANP), the effects of ET-1 on ANP secretion were investigated in isolated perfused rat hearts and in conscious unrestrained rats. Perfusion with 10(-9) M ET-1 stimulated ANP secretion from the isolated perfused rat heart. However, 10(-10) M ET-1 significantly decreased ANP secretion for the initial 15 min of the perfusion period. The perfusion with 10(-11) M ET-1, which is near the plasma level of ET-1 (2 x 10(-12) M), inhibited ANP secretion throughout the perfusion period. The perfusion of 10(-12) M ET-1 slightly decreased ANP secretion. After the ET-1 perfusion period, ANP secretion increased in proportion to ET-1 dose. The inhibitory action of ET-1 on ANP secretion was almost abolished by simultaneous perfusion of indomethacin, a cyclooxygenase inhibitor, but not by methylene blue, an inhibitor of soluble guanylate cyclase. In conscious unrestrained rats the iv infusion of 1 pmol/kg.min ET-1, which doubled the plasma ET-1 level, slightly but significantly decreased the plasma ANP level 5 and 10 min after the initiation of the infusion. The infusion of 10 and 30 pmol/kg.min ET-1 increased the plasma ANP level. These results demonstrate that low doses of ET-1 exert an inhibitory and short-acting action on ANP secretion from the heart, although high doses of ET-1 exert stimulating and long-lasting action, and suggest that prostanoids are involved in this inhibitory action.