Angiotensin II receptor blockers differentially affect CYP11B2 expression in human adrenal H295R cells.

We generated a stable H295R cell line expressing aldosterone synthase gene (CYP11B2) promoter/luciferase chimeric reporter construct that is highly sensitive to angiotensin II (AII) and potassium, and defined AII receptor blocker (ARB) effects. In the presence of AII, all ARBs suppressed AII-induced CYP11B2 transcription. However, telmisartan alone increased CYP11B2 transcription in the absence of AII. Telmisartan dose-dependently increased CYP11B2 transcription/mRNA expression and aldosterone secretion. Experiments using CYP11B2 promoter mutants indicated that the Ad5 element was responsible. Among transcription factors involved in the element, telmisartan significantly induced NGFIB/NURR1 expression. KN-93, a CaMK inhibitor, abrogated the telmisartan-mediated increase of CYP11B2 transcription/mRNA expression and NURR1 mRNA expression, but not NGFIB mRNA expression. NURR1 over-expression significantly augmented the telmisartan-mediated CYP11B2 transcription, while high-dose olmesartan did not affect it. Taken together, telmisartan may stimulate CYP11B2 transcription via NGFIB and the CaMK-mediated induction of NURR1 that activates the Ad5 element, independent of AII type 1 receptor.

Analysis of nucleotides by pressure-assisted capillary electrophoresis-mass spectrometry using silanol mask technique.

A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis-electrospray ionization mass spectrometry (PACE-MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions, 14 phosphorylated species such as nucleotides, nicotinamide-adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations (n=6) of the method were better than 0.9% for migration times and between 1.7% and 8.1% for peak areas. The detection limits for these species were between 0.5 and 1.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme.