ABSTRACT
Recent studies in our laboratory have demonstrated that bosentan, a mixed endothelin ET(A)/ET(B) receptor antagonist, prevented the upregulation of the arginine vasopressin (AVP) V(2) receptor in the inner medullary collecting duct (IMCD) of cardiomyopathic hamsters. These results suggested that endothelin-1 (ET-1) is involved in the upregulation of AVP V(2) receptors. Studies were performed to detect the effect of ET-1 on the expression of AVP V(2) receptors and the ET receptor mediating these effects within the IMCD of the rat. Rat IMCD tissue was isolated and incubated with the following: ET-1, or ET-1 in combination with ET(A) and ET(B) receptor antagonists BQ-123 and BQ-788, respectively, and sarafotoxin c (S6c), an ET(B) receptor-specific agonist. Tissue samples were then analyzed using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. ET-1 treatment resulted in increased V(2) mRNA from a control level of 186.8 +/- 15.0 amol/microg total RNA to 430.7 +/- 49.0 amol/microg total RNA (P <.003). ET-1/ET(A) treatment resulted in no significant decrease in V(2) mRNA expression 335.0 +/- 38.0 amol/microg total RNA. Whereas ET-1/ET(B), and ET-1/ET(B)/ET(A) treatment resulted in V(2) mRNA approaching control 256.0 +/- 15.0 amol/microg total RNA, and 215.6 +/- 42.3 amol/microg total RNA. However, ET-3 treatment produced no significant changes in V(2) receptor mRNA expression. Sarafotoxin treatment corroborated both the ET-1 and ET receptor antagonist data, demonstrating striking significant increases in V(2) receptor mRNA and protein expression. S6c treatment increased V(2) mRNA expression from a control level of 199 +/- 17.3 amol/microg total RNA to 284.3 +/- 42.1 amol/microg total RNA (P < 05). Western blotting revealed that changes in V(2) mRNA expression in the various treatment conditions were similar to changes in protein expression. Overall, these data indicate that in the IMCD ET-1 increases AVP V(2) receptor expression and these changes are mediated by the ET(B) receptor.
Subject(s)
Endothelin-1/pharmacology , Kidney Tubules, Collecting/metabolism , RNA, Messenger/biosynthesis , Receptors, Vasopressin/biosynthesis , Animals , Blotting, Western , Dose-Response Relationship, Drug , Endothelin Receptor Antagonists , Endothelin-1/antagonists & inhibitors , Endothelin-3/pharmacology , Female , In Vitro Techniques , Kidney Tubules, Collecting/drug effects , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Up-Regulation/drug effects , Viper Venoms/pharmacologyABSTRACT
Circulating endothelin (ET) levels are elevated in heart failure and positively correlated with severity of heart failure. Recent studies demonstrated arginine vasopressin (AVP) V2 mRNA expression was upregulated in the inner medullary collecting duct (IMCD) of cardiomyopathic hamsters (CM). The goal of the present studies was to determine if ET-1 is involved in upregulating the expression of AVP V2 mRNA in the IMCD of CM by using a mixed ETA/ETB receptor antagonist bosentan. Our results showed plasma ET-1 levels increased in CM hamsters and related with the severity of heart failure. The competitive reverse-transcriptase polymerase chain reaction (RT-PCR) method was used to quantify the expression of AVP V2 and aquaporin 2 (AQP2) mRNA in the IMCD. AVP V2 mRNA expression was elevated in placebo-treated CM hamsters and decreased significantly with 14 days of bosentan treatment. Similar results were seen with AQP2 mRNA. The effect of bosentan in normalizing the expression of AVP V2 and AQP2 mRNA in the IMCD of CM was confirmed by in situ hybridization studies. Bosentan treatments reduced the intensitites of the signals in the IMCD of CM hamsters to that seen in normal hamsters. This study demonstrated that AVP V2 and AQP2 mRNA are upregulated in CM hamsters and these upregulations are attenuated by bosentan treatment, suggesting that ET-1 plays a role in upregulating the expression of AVP V2 mRNA in CM hamsters.
