Endothelin Receptor Expression Research Articles

High Fat Diet Induced Lipid Deposition and Perilipin 2 Expression in Mouse Liver Is Attenuated by Endothelin-1 Receptor A Blockade

Obesity is associated with dyslipidemia and lipid deposition into liver tissue. Circulating endothelin-1 (ET-1) is elevated in patients with obesity, and our lab has demonstrated that ET-1 is increased in liver of mice fed high fat (HFD). Interestingly, treatment with atrasentan attenuates reduces circulating triglycerides and free fatty acids and reduces lipid deposition into liver of mice fed HFD for 10 weeks; however, the mechanisms are not known. Perilipin 2 (Plin2) is a protein expressed in adipocytes and hepatocytes that is integral to lipid droplet formation by forming a coating around the droplet. We hypothesized that ET-1 receptor blockade reduces liver lipid deposition in obese mice by reducing Plin2 expression. To test this hypothesis, mice were fed normal (NMD) or HFD for 10 weeks and treated with the ET-1 receptor A (ETA) antagonist atrasentan for weeks 9-10. ET-1 receptor expression was significantly increased in HFD fed mice compared to NMD. As previously demonstrated, HFD fed mice had significantly higher liver triglyceride content compared to NMD controls, and this was attenuated in mice treated with atrasentan (ATR). As expected, Plin2 expression in liver tissue was significantly elevated in HFD mice compared to NMD (91.7±17.4 vs. 60.8±14.4 copies/pg RNA), an affect that was abolished in mice treated with atrasentan (NMD, 60.8±14.4; HFD, 91.7±17.4; NMD + ATR, 70.2±10.8; HFD + ATR, 26.3±5.8 copies/10pg RNA). These data suggest that ET-1 promotes lipid accumulation in liver of obese mice by reducing Plin2 expression. Funding: R01 DK124437, P30GM149404, and T32HL105324. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unaltered Long-Term Maternal Endothelin System in a Gestational Hypertensive Rat Model

INTRODUCTION: Cardiovascular diseases (CVDs) are two to four times more likely to affect women with a history of hypertensive disorders of pregnancy (HDPs). One of the etiologies of CVDs is endothelial dysfunction, which results from an imbalance in the production of endothelin-1 (ET-1) and nitric oxide (NO). Although blood pressure (BP) is normalized postpartum, we hypothesize that a transient increase in BP during HDPs may cause ongoing endothelial dysfunction. We aimed to discover the impact of HDPs in the development of cardiovascular diseases after long-term postpartum changes in endothelin-A receptor (ETAR) and endothelin-B receptor (ETBR) expression and the concentration of ET-1 and NO. MATERIALS AND METHOD: Twenty-four female Sprague-Dawley (SD) rats were assigned into four groups (n = 6), including two treatment and two control groups. All rats were sacrificed on Day 30 postpartum. The NO and ET-1 concentrations were determined by ELISA and the ETAR and ETBR expression of the mesenteric arteries were measured by immunohistochemistry studies. RESULTS: The mean concentrations of ET-1 and NO were not significantly different in all groups. There was no significant difference in the mean immunoreactivity of ETAR and ETBR percentages area in the tunica intima and media in all groups. CONCLUSION: No evidence demonstrates significant changes in the endothelin system of the resistance arteries at the proteomic level in the long-term duration following HDP. Further investigation of its potential chronic effect warrants a deeper analysis at the molecular and ultrastructural levels.

Histopathological Evaluation of Pulmonary Thromboendarterectomy Specimens of Chronic Thromboembolic Pulmonary Hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a condition with a poor prognosis in which the pulmonary arteries are occluded by organized thrombi. P ulmonary thromboendarterectomy (PEA) is an effective treatment for CTEPH; however, the literature on its histopathological examination is lacking. This study aimed to investigate the histopathological findings and protein and gene expression in PEA specimens, establish an optimal histopathological evaluation method, and clarify the mechanisms of thrombus organization and disease progression in CTEPH. In total, 50 patients with CTEPH who underwent PEA were analyzed. The patients were categorized according to their clinical data into two groups: good and poor postoperative courses. The relationship between their histopathological findings and the clinical course was examined. Immunohistochemical studies confirmed the expression of oxidants, antioxidants, and s mooth muscle cell (SMC) differentiation markers and their changes during the progression of thrombus organization. The mRNA expression analysis of 102 samples from 27 cases included oxidants, antioxidants, and vasoconstrictor endothelin-1. In the PEA specimens, colander-like lesions (aggregations of recanalized blood vessels with well-differentiated SMCs) were significantly more common in the good postoperative course group than in the poor postoperative course group; analysis of proteins and genes proposed that oxidative and antioxidant mechanisms were involved. In the colander-like lesions, there was an increase in endothelin-1 mRNA and protein expression of endothelin receptor A. Colander-like lesions in PEA specimens must be identified. Additionally, SMC differentiation in recanalized vessels and the expression of vasoconstrictors and their receptors may contribute to the progression of CTEPH.

Immunohistochemical Localization of Endothelin- 1 and Endothelin A Receptor in Human Primary Tooth Enamel Organ.

Enamel organ (EO) is an ectodermal derived structure, which is involved in the different aspects of tooth development. Tooth development shares the same regulatory molecules and genes expressed in other developing organs. Endothelin- 1 (ET-1) and Endothelin A receptor (ETAR), (ET-1/ETAR) axis, are involved in differentiation of embryonic stem cells and organ development. The present study aimed to investigate the ET-1 and ETAR expression profiles during the development of human primary tooth EO with the relatively large sample size. In this experimental study, 33 human fetuses aged from 13 to 23 weeks (3 samples from each fetal age) were collected. The sampleswere divided into three age groups (<16 weeks, <19 weeks, ≥19 weeks) and cut for hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining. A two-way ANOVA test was conducted to examine the expression levels of ET-1 and ETAR in different layers of human primary tooth EO. The statistical significance was assumed at p ≤ 0.05. There were statistically significant differences between the expression levels of ET-1/ETAR axis in the four-layered human primary tooth EO in different fetal ages (13-23 weeks). Besides, there were significant differences between the expression levels of ET-1/ETAR axis in all layers of human primary enamel organ and types of teeth. Due to the profile of expression of ET-1/ETAR axis, it can be concluded that this axis contributes to the differentiation of all human primary EO layers and secretion of enamel. ET-1/ETAR axis is one of the signaling molecules, which may have crucial roles in tooth development.

Development, synthesis and evaluation of novel fluorescent Endothelin-B receptor probes

The endothelin (ET) signaling system is comprised of three endothelin peptides (ET-1, -2 and -3) and two corresponding endothelin-A and -B receptors (ETAR and ETBR), which belong to the G-protein coupled receptor (GPCR) superfamily. The endothelin axis, as this system is also referred to, contributes to the maintenance of vascular tone, functions as regulator of inflammation and proliferation and helps in balancing water homeostasis. In pathological settings, the ET axis is known to contribute to endothelial activation in cardiovascular diseases, to cell proliferation, chemoresistance and metastasis in cancer and to inflammation and fibrosis in renal disease. Antagonists of ETAR and ETBR, either subtype-specific compounds or substances with high affinity to both receptors, have been developed for more than 30 years. In the preclinical context, in vivo imaging of endothelin receptor expression has been utilized to assess ET-axis contribution to e.g. cancer or myocardial infarction. In this work, we present the development and synthesis of two novel ETBR-specific fluorescent probes, based on the available antagonists BQ788 and IRL2500 and their preliminary evaluation in a breast cancer context.

Stretching Promotes Wound Contraction Through Enhanced Expression of Endothelin Receptor B and TRPC3 in Fibroblasts.

The conditioned medium from stretched keratinocyte was added to the fibroblast populated collagen lattice. Then, we analyzed the levels of endothelin receptor in the human hypertrophic scar tissue and stretched fibroblasts. To address the function of TRPC3, we have used an overexpression system with the collagen lattice. Finally, the TRPC3 overexpressing fibroblasts were transplanted to mouse dorsal skin, and the rate of skin wound contraction was assessed. Conditioned medium from stretched keratinocytes increased the rate of contraction of fibroblast populated collagen lattice. In human hypertrophic scar and stretched fibroblasts, endothelin receptor type B was increased. Cyclic stretching of TRPC3 overexpressing fibroblasts activated NFATc4, and stretched human fibroblasts showed more activation of NFATc4 in response to ET-1. The wound treated with TRPC3 overexpressing fibroblasts showed more contraction than control wound. These findings suggest that cyclical stretching of wounds have an effect on both keratinocytes and fibroblasts, where keratinocytes secret more ET-1, and fibroblasts develop more sensitivity to ET-1 by expressing more endothelin receptors and TRPC3.

Endothelin A receptors contribute to senescence of brain microvascular endothelial cells.

Cellular senescence plays a pivotal role in the aging and progression of neurodegenerative diseases, including vascular cognitive impairment and dementia (VCID). In postmortem brains from individuals with VCID, endothelin-1 (ET-1) levels closely correlate with blood barrier breakdown and cerebral hypoperfusion. Brain microvascular endothelial cells (BMVECs), previously thought to have exclusively endothelin B receptors, also possess endothelin A (ETA) receptors; however, the functional significance of this receptor in BMVECs is not known. We hypothesize that ETA receptors mediate BMVEC senescence. Serum-starved human BMVECs (HBEC5i) were incubated with ET-1 (1µmol/L) in the presence/absence of ETA receptor antagonist BQ-123 (20µmol/L). Cells were collected for Western blot and quantitative real-time PCR analyses. Treatment of ET-1 increased protein expression of ETA receptor, while it was prevented by the ETA receptor antagonist. ET-1 increased p21, p16, p53, LIF1 and cyclin D1 protein levels, and β-galactosidase accumulation, which were prevented in the presence of ETA blockade. While there was no change in tight junction proteins, ET-1 decreased adherent junction protein vascular endothelial cadherin (VE-cadherin) levels. In conclusion, ET-1 upregulates ETA receptors in BMVECs in an autocrine manner and triggers the activation of senescence. These in vitro findings need to be further studied in vivo to establish the role of ETA receptors in the progression of endothelial senescence in VCID.

DMSO-soluble smoking particles up-regulate the vascular endothelin receptors through AMPK-SIRT1 and MAPK pathways

Smoking is a well-known risk factor for cardiovascular diseases. However, the mechanisms behind how smoking leads to vasospasm are not fully elucidated. Endothelin receptors are involved in the pathogenesis of cardiovascular diseases. This study examined whether cigarette smoke could induce up-regulation of vascular endothelin receptors through AMPK-SIRT1 and MAPK pathways. The results show that DMSO-soluble smoking particles (DSP) up-regulated the protein expressions of endothelin receptors and the contractile responses. Furthermore, the inhibition of MAPK or activation of AMPK-SIRT1 markedly attenuated DSP-enhanced vasoconstriction and the protein expression of endothelin receptors. The in vivo study showed that cigarette smoke increased the blood pressure of the rats and down-regulated p-AMPKα, SIRT1, and up-regulated endothelin receptors, p-ERK1/2, and p-P38 protein expressions. However, treatment with resveratrol attenuated vasoconstriction, endothelin receptor proteins expression, and blood pressure in vivo. In conclusion, this suggests that cigarette smoke up-regulates the vascular endothelin receptors through AMPK-SIRT1 and MAPK pathways.

Celastrol: A new potential therapeutic option in pulmonary hypertension associated to bronchopulmonary dysplasia

Bronchopulmonary dysplasia is a common pathology of premature newborns caused by oxidative stress and mechanical ventilation, resulting in hypoalveolization and impaired angiogenesis. It can be complicated by pulmonary hypertension (HTP-DBP) with excess mortality. Celastrol has anti-inflammatory and antioxidant properties. So, we addressed its potential therapeutic interest in HTP-DBP. Different concentrations of celastrol were used in a murine model of hyperoxic HTP-DBP (14 days with 90% O 2 ) and we evaluated hemodynamic parameters with echocardiography and lung remodeling with histological methods. Pulmonary arterial (PA) reactivity was addressed with myograph. Cytoplasmic calcium (Ca2 + i) response to endothelin-1 (ET-1) was evaluated in human fetal PA smooth muscle cells (fPASMC) with spectrophotometry in normoxic (21% O 2 ) and hyperoxic conditions (48 h with 60% O 2 ). Inflammation was addressed with IL6 ELISA assay in fPASMC. Celastrol reduces mortality at doses of 1 and 0.5 mg/kg/d compared to the hyperoxic control group. At 1 mg/kg/d, it normalizes PAAT (PA acceleration time) ( P < 0.001) and reduces vascular hyperreactivity to phenylephrine and ET-1 ( P < 0.001) with no effect on the alteration of the response to acetylcholine induced by hyperoxia. At doses of 0.1, 0.5 and 1 mg/kg/d, it decreases right heart hypertrophy and vascular remodeling (wall thickness) but has no effect on alveolization and vascular density (vWF staining). Finally, although celastrol (0.1 and 0.3 μM) decreases the increased Ca2 + i response to ET-1 in hyperoxia in fPASMC, expression of ET-1 receptors (Western blotting and qPCR for ET-A and ET-B) was unchanged. However, celastrol 0.5 μM decreases ET-1 concentration measured with an ELISA assay in human umbilical arterial endothelial cells. An increased concentration of IL6 was also observed in fPASMC in hyperoxia and was decreased by celastrol 0.5 μM. Celastrol has a preventive dose-dependent effect on some hallmarks of HTP induced by hyperoxia and could thus be considered as an interesting new potential therapeutic option for HTP-DBP.

Effect and Mechanism of Endothelin Receptor A Inhibitor BQ-123 Combined with Electroacupuncture on Tibia Cancer Pain in Rats.

Objective To research the impact and mechanism of endothelin receptor A inhibitor BQ-123 combined with electroacupuncture on tibia cancer pain in rats. Methods Sprague-Dawley (SD) rats were randomly divided into sham group (SHAM group) and bone cancer pain model group (BCP group). The behavior of SD rats was measured. The histology of the right tibia was observed by hematoxylin-eosin (HE) staining. The remaining rats were randomly divided into model, BQ-123, electroacupuncture, and BQ-123+ electroacupuncture group. Behavioral tests were performed, and mechanical pain threshold (MWT) and thermal pain threshold (TWL) were measured. The expressions of α-smooth muscle actin (αSMA), ETAR (endothelin A receptor), ETB (End of Transmission Block), P-Phosphatidylinositol 3-kinase (PI3K), and P-Protein kinase B (Akt) were detected by real-time fluorescence quantitative PCR and western blot. Results In the BCP group, bone structure was severely damaged, local tissue swelling was obvious, bone trabecula was missing, and bone cortex was discontinuous. The optical density of Glial fibrillary acidic protein (GFAP) and CD11b immunoreactive signal in BCP group was significantly increased, and most of the ETAR of endothelin receptor was comapped with NeuN, and a small part of GFAP was comapped with CD11b, but no comapped with CD11b. The AS score of BQ-123+ electroacupuncture group was significantly lower than that of BQ-123 group and electroacupuncture group (P < 0.05), whereas the MWT and TWL values were significantly higher than that of the BQ-123 group and electroacupuncture group (P < 0.05). The mRNA expression of α-SMA and ETAR in BQ-123+ electroacupuncture group was lower than that in BQ-123 and electroacupuncture group, and the protein expression of P-PI3K and P-Akt in BQ-123+ electroacupuncture group was lower as well. Conclusion BQ-123 may inhibit the activation of PI3K/Akt signal path combined with electroacupuncture to alleviate the effects of tibia cancer pain in rats.

Effect of celastrol on pulmonary hypertension associated to bronchopulmonary dysplasia

Bronchopulmonary dysplasia is a common pathology of premature newborns caused by oxidative stress and mechanical ventilation, resulting in hypoalveolization and impaired angiogenesis. It can be complicated by pulmonary hypertension (HTP-DBP) with excess mortality. Celastrol has anti-inflammatory and antioxidant properties and we addressed its potential therapeutic interest in HTP-DBP. Different concentrations of celastrol were used in a murine model of hyperoxic HTP-DBP (14 days with 90% O2) and we evaluated hemodynamic parameters with echocardiography and lung remodeling with histological methods. Pulmonary arterial (PA) reactivity was addressed with myograph. Cytoplasmic calcium (Ca2 + i) response to endothelin-1 (ET-1) was evaluated in human fetal PA smooth muscle cells (fPASMC) with spectrophotometry in normoxic (21% O2) and hyperoxic conditions (48 h with 60% O2). Inflammation was addressed with IL6 ELISA assay in fPASMC. Celastrol reduces mortality at doses of 1 and 0.5 mg/kg/d compared to the hyperoxic control group. At 1 mg/kg/d, it normalizes PAAT (PA acceleration time) ( P < 0.001) and reduces vascular hyperreactivity to phenylephrine ( P < 0.001) with no effect on the alteration of the response to acetylcholine induced by hyperoxia. At doses of 0.1, 0.5 and 1 mg/kg/d, it decreases right heart hypertrophy and vascular remodeling (wall thickness) but has no effect on alveolization and vascular density (vWF staining). Finally, although celastrol (0.1 and 0.3 μM) decreases the increased Ca2 + i response to ET-1 in hyperoxia in fPASMC, expression of ET-1 receptors (Western blotting and qPCR for ET-A and ET-B) was unchanged. However, celastrol 0.5 μM decreases ET-1 concentration measured with an ELISA assay in human umbilical arterial endothelial cells. An increased concentration of IL6 was also observed in fPASMC in hyperoxia and was decreased by celastrol 0.5 μM. Celastrol has a preventive dose-dependent effect on some hallmarks of HTP induced by hyperoxia and could thus be considered as an interesting new potential therapeutic option for HTP-DBP.

Endosomal disentanglement of a transducible artificial transcription factor targeting endothelin receptor A

Cell-penetrating peptides (CPPs) hold great promise for intracellular delivery of therapeutic proteins. However, endosomal entrapment of transduced cargo is a major bottleneck hampering their successful application. While developing a transducible zinc finger protein-based artificial transcription factor targeting the expression of endothelin receptor A, we identified interaction between the CPP and the endosomal membrane or endosomal entanglement as a main culprit for endosomal entrapment. To achieve endosomal disentanglement, we utilized endosome-resident proteases to sever the artificial transcription factor from its CPP upon arrival inside the endosome. Using this approach, we greatly enhanced the correct subcellular localization of the disentangled artificial transcription factor, significantly increasing its biological activity and distribution invivo. With rational engineering of proteolytic sensitivity, we propose a new design principle for transducible therapeutic proteins, helping CPPs attain their full potential as delivery vectors for therapeutic proteins.

TLR4-Endothelin Axis Controls Syncytiotrophoblast Motility and Confers Fetal Protection in Placental Malaria.

Pregnancy-associated malaria is often associated with adverse pregnancy outcomes. Placental circulatory impairments are an intriguing and unsolved component of malaria pathophysiology. Here, we uncovered a Toll-like receptor 4 (TLR4)-TRIF-endothelin axis that controls trophoblast motility and is linked to fetal protection during Plasmodium infection. In a cohort of 401 pregnancies from northern Brazil, we found that infection during pregnancy reduced expression of endothelin receptor B in syncytiotrophoblasts, while endothelin expression was only affected during acute infection. We further show that quantitative expression of placental endothelin and endothelin receptor B proteins are differentially controlled by maternal and fetal TLR4 alleles. Using murine malaria models, we identified placental autonomous responses to malaria infection mediated by fetally encoded TLR4 that not only controlled placental endothelin gene expression but also correlated with fetal viability protection. In vitro assays showed that control of endothelin expression in fetal syncytiotrophoblasts exposed to Plasmodium-infected erythrocytes was dependent on TLR4 via the TRIF pathway but not MyD88 signaling. Time-lapse microscopy in syncytiotrophoblast primary cultures and cell invasion assays demonstrated that ablation of TLR4 or endothelin receptor blockade abrogates trophoblast collective motility and cell migration responses to infected erythrocytes. These results cohesively substantiate the hypothesis that fetal innate immune sensing, namely, the TRL4-TRIF pathway, exerts a fetal protective role during malaria infection by mediating syncytiotrophoblast vasoregulatory responses that counteract placental insufficiency.

Transcriptomic analysis and ednrb expression in cochlear intermediate cells reveal developmental differences between inner ear and skin melanocytes.

In the inner ear, the neural crest gives rise to the glia of the VIII ganglion and two types of melanocytic cells: The pigmented cells of the vestibular system and intermediate cells of the stria vascularis. We analyzed the transcriptome of neonatal intermediate cells in an effort to better understand the development of the stria vascularis. We found that the expression of endothelin receptor B, which is essential for melanocyte development, persists in intermediate cells long after birth. In contrast, skin melanocytes rapidly downregulate the expression of EdnrB. Our findings suggest that endothelins might have co‐opted new functions in the inner ear during evolution of the auditory organ.

Monitoring Endothelin-A Receptor Expression during the Progression of Atherosclerosis.

Cardiovascular disease remains the most frequent cause of death worldwide. Atherosclerosis, an underlying cause of cardiovascular disease, is an inflammatory disorder associated with endothelial dysfunction. The endothelin system plays a crucial role in the pathogenesis of endothelial dysfunction and is involved in the development of atherosclerosis. We aimed to reveal the expression levels of the endothelin-A receptor (ETAR) in the course of atherogenesis to reveal possible time frames for targeted imaging and interventions. We used the ApoE−/− mice model and human specimens and evaluated ETAR expression by quantitative rtPCR (qPCR), histology and fluorescence molecular imaging. We found a significant upregulation of ETAR after 22 weeks of high-fat diet in the aortae of ApoE−/− mice. With regard to translation to human disease, we applied the fluorescent probe to fresh explants of human carotid and femoral artery specimens. The findings were correlated with qPCR and histology. While ETAR is upregulated during the progression of early atherosclerosis in the ApoE−/− mouse model, we found that ETAR expression is substantially reduced in advanced human atherosclerotic plaques. Moreover, those expression changes were clearly depicted by fluorescence imaging using our in-house designed ETAR-Cy 5.5 probe confirming its specificity and potential use in future studies.

Targeting endothelin 1 receptor-miR-200b/c-ZEB1 circuitry blunts metastatic progression in ovarian cancer

Identification of regulatory mechanisms underlying the poor prognosis of ovarian cancer is necessary for diagnostic and therapeutic implications. Here we show that endothelin A receptor (ETAR) and ZEB1 expression is upregulated in mesenchymal ovarian cancer and correlates with poor prognosis. Notably, the expression of ETAR and ZEB1 negatively correlates with miR-200b/c. These miRNAs, besides targeting ZEB1, impair ETAR expression through the 3’UTR binding. ZEB1, in turn, restores ETAR levels by transcriptionally repressing miR-200b/c. Activation of ETAR drives the expression of ZEB1 integrating the miR-200/ZEB1 double negative feedback loop. The ETAR-miR-200b/c-ZEB1 circuit promotes epithelial-mesenchymal transition, cell plasticity, invasiveness and metastasis. Of therapeutic interest, ETAR blockade with macitentan, a dual ETAR and ETBR antagonist, increases miR-200b/c and reduces ZEB1 expression with the concomitant inhibition of metastatic dissemination. Collectively, these findings highlight the reciprocal network that integrates ETAR and ZEB1 axes with the miR-200b/c regulatory circuit to favour metastatic progression in ovarian cancer.

Endothelin receptors promote schistosomiasis-induced hepatic fibrosis via splenic B cells.

Endothelin receptors (ETRs) are activated by vasoactive peptide endothelins and involved in the pathogenesis of hepatic fibrosis. However, less is known about the role of ETRs in Schistosoma (S.) japonicum-induced hepatic fibrosis. Here, we show that the expression of ETRs is markedly enhanced in the liver and spleen tissues of patients with schistosome-induced fibrosis, as well as in murine models. Additional analyses have indicated that the expression levels of ETRs in schistosomiasis patients are highly correlated with the portal vein and spleen thickness diameter, both of which represent the severity of fibrosis. Splenomegaly is a characteristic symptom of schistosome infection, and splenic abnormality may promote the progression of hepatic fibrosis. We further demonstrate that elevated levels of ETRs are predominantly expressed on splenic B cells in spleen tissues during infection. Importantly, using a well-studied model of human schistosomiasis, we demonstrate that endothelin receptor antagonists can partially reverse schistosome-induced hepatic fibrosis by suppressing the activation of splenic B cells characterized by interleukin-10 (IL-10) secretion and regulatory T (Treg) cell-inducing capacity. Our study provides insights into the mechanisms by which ETRs regulate schistosomiasis hepatic fibrosis and highlights the potential of endothelin receptor antagonist as a therapeutic intervention for fibrotic diseases.

Exposure to Morphine and Caffeine Induces Apoptosis and Mitochondrial Dysfunction in a Neonatal Rat Brain.

Background: Preterm infants experience rapid brain growth during early post-natal life making them vulnerable to drugs acting on central nervous system. Morphine is administered to premature neonates for pain control and caffeine for apnea of prematurity. Simultaneous use of morphine and caffeine is common in the neonatal intensive care unit. Prior studies have shown acute neurotoxicity with this combination, however, little information is available on the mechanisms mediating the neurotoxic effects. The objective of this study was to determine the effects of morphine and caffeine, independently and in combination on mitochondrial dysfunction (Drp1 and Mfn2), neural apoptosis (Bcl-2, Bax, and cell damage) and endothelin (ET) receptors (ETA and ETB) in neonatal rat brain.Methods: Male and female rat pups were grouped separately and were divided into four different subgroups on the basis of treatments–saline (Control), morphine (MOR), caffeine (CAFF), and morphine + caffeine (M+C) treatment. Pups in MOR group were injected with 2 mg/kg morphine, CAFF group received 100 mg/kg caffeine, and M+C group received both morphine (2 mg/kg) and caffeine (100 mg/kg), subcutaneously on postnatal days (PND) 3–6. Pups were euthanized at PND 7, 14, or 28. Brains were isolated and analyzed for mitochondrial dysfunction, apoptosis markers, cell damage, and ET receptor expression via immunofluorescence and western blot analyses.Results: M+C showed a significantly higher expression of Bax compared to CAFF or MOR alone at PND 7, 14, 28 in female pups (p < 0.05) and at PND 7, 14 in male pups (p < 0.05). Significantly (p < 0.05) increased expression of Drp1, Bax, and suppressed expression of Mfn2, Bcl-2 at PND 7, 14, 28 in all the treatment groups compared to the control was observed in both genders. No significant difference in the expression of ETA and ETB receptors in male or female pups was seen at PND 7, 14, and 28.Conclusion: Concurrent use of morphine and caffeine during the first week of life increases apoptosis and cell damage in the developing brain compared to individual use of caffeine and morphine.

Dibutyryl cAMP down-regulates gene expression of endothelin receptor A subtype in rat astrocytoma C6 cells

In this study, we have investigated the gene expression of endothelin receptors in rat astrocytoma C6 cells regulated by the increase of intracellular cAMP. Following treatment with 1 mM dibutyryl cAMP, the specific binding of [125I]-endothelin-1 to the C6 cells was down-regulated to about 40 % of the original level. Radioligand binding assay and Northern blot analysis indicated that endothelin receptors on C6 cells, which were either treated or untreated with 1 mM dibutyryl cAMP, belong to subtype A. Analysis of RNA by Northern blotting and slot hybridization demonstrated that the presence of dibutyryl cAMP induced the down regulation of ET(A) endothelin receptor gene in C6 cells. The level of ET(A) mRNA was rapidly reduced to about 10 % of its initial level after 4 hour treatment with dibutyryl cAMP and then was slightly elevated to about 40 % after one day treatment. All the findings suggest that the down regulation of ET(A) receptor in C6 cells by the treatment of dibutyryl cAMP occurs not only at the receptor binding level but also at the mRNA level.

Secondhand cigarette smoke induces increased expression of contractile endothelin receptors in rat coronary arteries via a MEK1/2 sensitive mechanism

Objectives Cigarette smoke, a strong risk factor for cardiovascular diseases, upregulates contractile endothelin (ET) receptors in coronary arteries. The present study examined the effects of second hand cigarette smoke exposure on the contractile endothelin receptors and the role of the MEK1/2 pathway in rat coronary arteries. Design: Rats were exposed to secondhand smoke (SHS) for 8 weeks followed by intraperitoneal injection of a MEK1/2 inhibitor, U0126 daily for another 4 weeks. Contractile responses of isolated coronary arteries were recorded by a sensitive wire myograph. The receptor protein expression levels were examined by Western blotting. Results: The results showed that SHS in vivo caused increased expression of ET receptors ETA and ETB, and that the MEK1/2 blocker U0126 significantly reversed SHS exposure-increased ETA-mediated contractile responses and protein levels. Similar alterations were observed in ETB receptors. U0126 showed dose-dependent effects on SHS-induced response on contractile property and protein levels of the ETB receptor. However, only the higher dose U0126 (15 mg/kg) had inhibitory effects on the ETA receptor. Conclusions: Taken together, our data show that SHS increases contractile ET receptors and MEK1/2 pathway inhibitor offsets SHS exposure-induced ETA and ETB receptor upregulation in rat coronary arteries.