ETA Receptor mRNA Levels Research Articles

Altered expression and signal transduction of endothelin‐1 receptors in heritable and idiopathic pulmonary arterial hypertension

Human pulmonary arterial smooth muscle cells (PASMC) were isolated from elastic pulmonary arteries dissected from lungs of individuals with and without pulmonary arterial hypertension (PAH). Reflecting increased smooth muscle constriction in cells from PAH subject, Ca(2+) influx in response to endothelin-1 (ET-1) increased in all the PAH PASMC populations relative to the normal donor control cells. The ETA receptor mRNA levels remained unchanged, whereas the ETB receptor mRNA levels decreased in both heritable and idiopathic PAH-derived PASMC. All the PASMC populations expressed considerably higher ETA compared to ETB receptor number. Both ETA and ETB receptor numbers were reduced in bone morphogenetic protein receptor type II (BMPR2) mutation PAH. ETB receptors showed a particular reduction in number. Phospho-antibody array analysis of normal and BMPR2 deletion PASMC illustrated ERK and Akt activation to be the most prominent and to be taking place principally through ETB receptors in normal PASMC, but primarily through ETA receptors in PASMC from BMPR2 PAH subjects. Additionally in the PAH cells the total relative ET-1 signal response was markedly reduced. Western analysis from the BMPR2 PASMC duplicated the array results, whereas PASMC from iPAH subjects showed variability with most samples continuing to signal through ETB. In sum, these results indicate that generally both receptors are reduced in PAH particularly ETB, and that ETB signaling through protein kinases becomes markedly reduced in BMPR2 PASMC, while it continues in IPAH. Importantly, the data suggest that caution must be taken when applying ET-1 receptor antagonist therapy to PAH patients.

ACTIVATED PROTEIN C REDUCES HEPATIC ET-1 TRANSCRIPT LEVELS AFTER TRAUMA AND SEPSIS

Introduction: Activated protein C (aPC) confers survival benefit in patients with sepsis; yet its protective mechanisms remain unclear. Recently, we found that aPC ameliorates ET-1 induced alterations in the hepatic microcirculation after femur fracture (FFx) and cecal ligation and puncture (CLP). Here, we hypothesize that the protective effects of aPC are mediated by a reduction in the vasoconstrictor ET-1 and its receptor, ETA. Methods: Male Sprague Dawley rats underwent control, sham, FFx, CLP, or FFx followed 24 hours by CLP. Subsequently, animals received either aPC (1 mg/kg) or saline intravenously, twice daily for four days. Twenty four hours after the final aPC or saline treatment, liver tissue was harvested for RT-PCR. Results: CLP and FFx + CLP resulted in a significant upregulation in ET-1 and ETA receptor transcripts when compared to control and sham. Treatment with aPC resulted in a reduction in ET-1 and ETA receptor mRNA levels after CLP and FFx+CLP (see Figure); no effect was seen in control, sham, and FFx animals.FigureConclusion: Treatment with aPC reduced the vasoconstrictor ET-1 and ETA receptor mRNA levels in CLP and FFx+CLP animals, which plays an important role in the protective effects of aPC on the observed hepatic microcirculation in response to ET-1. Figure: Saline treated vs aPC treated RT-PCR bands for ET-1 and ETA receptor. 1 = CLP and 2 = FFx + CLP

Involvement of Protein Kinases on the Upregulation of Endothelin Receptors in Rat Basilar and Mesenteric Arteries

Endothelin(B) (ET(B)) receptors are upregulated in experimental stroke or after 24 hrs of organ culture. This upregulation is manifested both as stronger contraction and as an increase in ET(B) receptor messenger RNA (mRNA) levels. The present study was designed to evaluate the importance of protein kinases (c-Jun N-terminal kinase [JNK], protein kinase C [PKC], and extracellular signal-regulated kinase [ERK1/2]) in ET(B) receptor upregulation after organ culture. Rat basilar and mesenteric arteries were incubated for 24 hrs in Dulbecco's modified Eagle's medium (DMEM) with or without the PKC inhibitor, RO-31-7549; the ERK1/2 inhibitor, SB386023; or the JNK inhibitor, SP600125, added 3, 6, or 12 hrs after initiation of incubation. Subsequently, vessel segments were mounted in myographs and the contractile responses to ET-1 and sarafotoxin 6c were studied. The ET(B) and ET(A) receptor mRNA levels were determined with a real-time polymerase chain reaction (PCR). The cellular localization and protein level of ET(B) receptors were evaluated by immunohistochemistry. The PKC and ERK1/2 inhibitors attenuated the contraction induced by S6c in the basilar arteries more than in the mesenteric arteries. The efficiency of the inhibitors was proportional to the incubation time. Real-time PCR showed a decrease in the ET(B) receptor mRNA levels in arteries treated with PKC or ERK inhibitors. The JNK inhibitor had a significant inhibitory effect on ET(B) receptor upregulation in the basilar arteries. Immunohistochemistry revealed that the ET(B) receptor upregulation occured in the smooth-muscle cells and that it had the same pattern as in the quantitative PCR. Our results show that the PKC, ERK1/2, and JNK are more important for the upregulation of contractile ET(B) receptors in cerebral arteries compared with mesenteric arteries. ERK1/2 seems to be more important for the ET(B) receptor upregulation, as compared with PKC and JNK. The evaluation of the time dependency suggests that the phenomenon can be reversed even after its initiation.

Fibroblast Matrix Gene Expression and Connective Tissue Remodeling: Role of Endothelin-1

This study examines endothelin-induced modulation of extracellular matrix synthesis and remodeling by fibroblasts, and its potential role in the pathogenesis of systemic sclerosis (scleroderma). Endothelin-1 promoted fibroblast synthesis of collagen types I and III, but not fibronectin, by a mechanism dependent upon both ETA and ETB receptors. Conversely, endothelin-1 inhibited both protein expression of matrix metalloproteinase 1 and zymographic activity exclusively via ETA receptors. A dual regulatory role for endothelin-1 in transcriptional regulation was suggested by the ability of endothelin-1 to enhance steady-state levels of collagen mRNA and activate the proalpha2(I) collagen (Col1a2) promoter, but in contrast to reduce matrix metalloproteinase 1 transcript expression and suppress transcription of a human matrix metalloproteinase 1 promoter reporter construct in transient transfection assays. Although endothelin-1 significantly enhanced remodeling of three-dimensional collagen lattices populated by normal fibroblasts, this was not observed for lattices populated by systemic sclerosis fibroblasts. Promotion of matrix remodeling was dependent upon ETA receptor expression and was blocked by specific inhibitors of tyrosine kinases or protein kinase C. Reverse transcriptase polymerase chain reaction, S1 nuclease, and functional cell surface binding studies showed that normal and systemic sclerosis fibroblasts express both ETA and ETB receptors (predominantly ETA), but that ETA receptor mRNA levels and ETA binding sites on fibroblasts cultured from systemic sclerosis skin biopsies are reduced by almost 50%. Endothelin-1 is thus able to induce a fibrogenic phenotype in normal fibroblasts that is similar to that of lesional systemic sclerosis fibroblasts. Moreover, reduced responsiveness to exogenous endothelin-1 in systemic sclerosis suggests that downstream pathways may have already been activated in vivo. These data further implicate dysregulated endothelin-receptor pathways in fibroblasts in the pathogenesis of connective tissue fibrosis.

Characterization and regulation of type A endothelin receptor gene expression in bovine luteal cell types.

Our previous studies demonstrated that endothelin-1 (ET-1), a 21-amino acid vasoconstrictor peptide, has a paracrine regulatory role in bovine corpus luteum (CL). The peptide is produced within the gland where it inhibits progesterone production by acting via the selective type A endothelin (ETA) receptors. The present study was designed to characterize ETA receptor gene expression in different ovarian cell types and its hormonal regulation. ETA receptor messenger RNA (mRNA) levels were high in follicular cells as well as in CL during luteal regression. At this latter stage, high ETA receptor expression concurred with low prostaglandin F2alpha receptor mRNA. The ETA receptor gene was expressed by all three major cell populations of the bovine CL; i.e. small and large luteal cells, as well as in luteal endothelial cells. Among these various cell populations, the highest ETA receptor mRNA levels were found in endothelial cells. cAMP elevating agents, forskolin and LH, suppressed ETA receptor mRNA expression in luteinized theca cells (LTC). This inhibition was dose dependent and was evident already after 24 h of incubation. In luteinized granulosa cells (LGC), 10 and 100 ng/ml of insulin-like growth factor I and insulin (only at a concentration of 2000 ng/ml) markedly decreased ETA receptor mRNA levels. In both LGC and LTC there was an inverse relationship between ETA receptor gene expression and progesterone production; insulin (in LGC) and forskolin (in LTC) enhanced progesterone production while inhibiting ETA receptor mRNA levels. Our findings may therefore suggest that, during early stages of luteinization when peak levels of both LH and insulin-like growth factor I exist, the expression of ETA receptors in the gland are suppressed. This study demonstrates physiologically relevant regulatory mechanisms controlling ETA receptor gene expression and further supports the inhibitory role of ET-1 in CL function.

Modulation of glomerular endothelin and endothelin receptor gene expression in aminonucleoside-induced nephrosis.

This study assessed glomerular endothelin (ET)-1, ET-3, and ET-receptor A and B mRNA levels in puromycin aminonucleoside (PAN)-induced nephrosis. During the nephrotic stage, 8 days after PAN injection, ET-1 and ETB receptor mRNA were elevated by 2.8 +/- 0.8-fold (P < 0.01) and 2.4 +/- 0.9-fold (P < 0.01), respectively, as compared with controls. These mRNA levels decreased to control levels by Day 20, when the nephrosis was in remission. In contrast, glomerular ETA receptor mRNA levels did not change in PAN nephrosis or control rats during the experimental period. ET-3 mRNA was not detected in the glomeruli of PAN nephrosis or control rats. Additionally, plasma ET concentration and glomerular ET production were measured in PAN nephrosis and control rats by radio-immunoassay. Eight days after PAN injection, ET-1 levels in plasma and glomeruli were not significantly altered in rats with PAN-induced nephrosis (glomeruli, 104.68 +/- 16.46 pg/mg of protein versus 98.24 +/- 13.68 pg/mg of protein; plasma, 2.68 +/- 1.10 versus 2.52 +/- 0.98 pg/mL). The administration of methylprednisolone to PAN rats resulted in the rapid disappearance of proteinuria and partially attenuated the increased ET-1 and ETB receptor gene expression in the glomeruli. These data indicate that glomerular ET-1 and ETB receptor expression in PAN nephrosis in increased at the mRNA level and that methylprednisolone treatment results in an attenuated increase.

Enhanced endothelin-1 and endothelin receptor gene expression in chronic hypoxia.

To test the hypothesis that endothelin (ET)-1 synthesis and ET receptor levels are increased selectively in the lung of rats with chronic hypoxic pulmonary hypertension, the current study examined the effects of exposure to chronic hypoxia (10% O2, 1 atm, 4 wk) on pulmonary arterial pressure, ET-1 levels in plasma and lung, and ET-1 and ETA and ETB receptor mRNA levels in lung, heart, pulmonary artery, aorta, kidney, spleen, and liver. Hypoxic exposure was associated with increases in pulmonary arterial pressure, plasma ET-1 levels, ET-1 mRNA in lung and pulmonary artery, and ET-1 stores and ETA and ETB receptor mRNA levels in lung. In thoracic aorta and the four heart chambers, ETA and ETB receptor mRNA levels were increased, but ET-1 mRNA levels were unchanged from air control levels. No change in ET-1 or ET receptor mRNA levels was seen in organs perfused by the systemic vascular bed, except in liver, where ETA receptor mRNA levels were decreased. The findings of concomitant increases in gene transcript levels for ET-1 and the ETA and ETB receptors in lung, but not in the great vessels or any other organ examined, are consistent with the hypothesis that increased ET-1 synthesis in the lung contributes to pulmonary vascular remodeling and the maintenance of chronic hypoxic pulmonary hypertension.