Calcium regulation of endothelin-1 synthesis in rat inner medullary collecting duct

Abstract

Collecting duct-derived endothelin-1 (ET-1) reduces blood pressure and inhibits Na and water reabsorption. Collecting duct ET-1 production is increased by volume expansion; however, the mechanism by which this occurs is unknown. We hypothesized that intracellular calcium, which is likely to be increased by volume expansion, regulates collecting duct ET-1 synthesis. Rat inner medullary collecting ducts (IMCD) were studied in primary culture. ET-1 release was decreased by 50-70% after chelation of intracellular calcium (BAPTA) or inhibition of CaM (W7) or CaMK (KN-93). These agents reduced ET-1 mRNA to a similar degree. CaM inhibition did not affect ET-1 mRNA stability. Transfection of IMCD with rat ET-1 promoter-luciferase constructs revealed maximal activity within 1.7 kb 5' to the transcription start site; 5, 20, 35, and 90% of this activity were in the 0.08-, 0.37-, 1.0-, and 3.0-kb promoter regions, respectively. W7 markedly inhibited activity of the 3.0-kb but not 0.37- or 1.0-kb promoter regions. In contrast, W7 did not affect ET-1 release by rat aortic endothelial cells. Furthermore, transfected endothelial cells had maximal activity in the 0.37-kb region (as compared with the 1.7- and 3.0-kb regions), whereas W-7 had no effect on the activity of any of these promoter regions. In summary, IMCD ET-1 synthesis is regulated by calcium/CaM/CaMK-dependent pathways. The calcium/CaM-sensitive pathway is active in IMCD, but not endothelial cells. This suggests that IMCD-specific enhancer elements exist within the ET-1 promoter that confer unique calcium responsiveness.

Characterization of vasopressin-responsive collecting duct adenylyl cyclases in the mouse

“…Primary isolates of mouse (C57BL/6, 25 g) or rat (Sprague-Dawley, 200 -250 g) IMCD cells were obtained with a modification of previously described procedures (50,51). Institutional Animal Care and Use Committee approval was obtained for the use of mice and rats for these purposes.…”

“…The final pellet containing primarily tubules was resuspended in HBSS-HEPES. This procedure has previously been shown to yield predominantly collecting ducts (50,51).…”

“…Freshly isolated mouse IMCD were grown in primary culture as previously described (51). The cell suspension was plated in 24-well plates in Renal Epithelial Cell Growth Medium (REGM, Cambrex, Walkersville, MD) containing epidermal growth factor, insulin, hydrocortisone, gentamicin, amphotericin, 0.5% fetal bovine serum, epinephrine, tri-iodothyronine, and transferrin (concentrations are proprietary).…”

Identification of Two Nuclear Factor of Activated T-cells (NFAT)-response Elements in the 5′-Upstream Regulatory Region of the ET-1 Promoter

“…ET-1 Promoter-Luciferase Constructs-5Ј-Serial deletion constructs of the rat ET-1 upstream promoter sequences were generated using our previously described 3.2-kb rat ET-1 promoter construct (22), which contains 3048 bp of the 5Ј-flanking sequence and 189 bp of the untranslated region of the first exon. Briefly, our 3.2-kb construct (containing Ϫ3048 bp of 5Ј-flanking sequence) was generated using high fidelity Platinum Taq (Invitrogen) PCR from rat genomic DNA and ligated into the XhoI/NheI sites in the pGL3 basic vector (Ϫ3048 ET-1 pGL3).…”

“…In a previous study from our laboratory (22), we examined the regulation of the ET-1 promoter in endothelial as compared with renal IMCD 2 cells. As anticipated from the previous studies cited above, we showed that maximal transcriptional activity of the rat ET-1 promoter in primary cultures of aortic endothelial cells was confined to the first Ϫ366 bp of 5Ј-upstream sequence; additional sequences up to 3.0 kb 5Ј-upstream produced no further enhancement of promoter activity.…”