Characterization of Na(+)-H+ exchange in segments of rat mesenteric artery

Abstract

To develop a technique for measuring Na(+)-H+ exchange activity and intracellular pH (pH(i)) “on line” in resistance vessels, we utilized strips of rat mesenteric arteries loaded with the pH-sensitive dye 2',7”-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Strips were held at a fixed length within a 3-ml cuvette, and fluorescence emission was monitored at 530 nm. The spectrofluorimeter was monitored in the ratio mode, and the excitation wavelength was alternated between 440 and 505 nm. Tissues were maintained by perfusing with N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid containing buffers. The introduction of ammonium chloride produced a rapid alkalinization. Washout of ammonium caused rapid acidification. Restoration of pH(i) was Na+ dependent and inhibited by dimethyl amiloride (concentration that produces half-maximal inhibition, K0.5 = 30 microM), features characteristic of Na(+)-H+ exchange. Further studies assessed the transport rate of the exchanger, which averaged 0.19 +/- 0.02 pH U/min (means +/- SE, n = 8). An estimate of the dependence of Na(+)-H+ exchange on external Na+ gave an apparent Michaelis constant for external Na+ of 10 mM and an apparent maximal velocity of 0.1 mM H+/s. Intracellular H+ was found to have a cooperative effect (Hill coefficient = 4) on Na(+)-H+ exchange.

Diabetes-Induced Vascular Hypertrophy Is Accompanied by Activation of Na + -H + Exchange and Prevented by Na + -H + Exchange Inhibition

“…[23][24][25] The level of acidification was sufficient to cause maximum activation of the pH recovery process (see Figure 3, which shows a distinctive sigmoidal shape with a plateau at maximal recovery). Hence, and importantly, our methodology ensured that the measurement represents maximal NHE activity for all groups of vessels studied.…”

Role of 11β-Hydroxysteroid Dehydrogenase in Nongenomic Aldosterone Effects in Human Arteries

“…pH i was determined by monitoring the fluorescence of acetoxymethyl ester of the pH-sensitive dye 2Ј,7Ј-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM; Texas Fluorescence Laboratories) as described by Foster et al 23 Chorionic and uterine radial arteries were prepared in oxygenated physiological salt solution (PSS) containing (in mmol/L) 140 NaCl, 4 KCl, 10 d-glucose, 20 HEPES, 1.8 CaCl 2 , and 1.0 MgCl 2 , adjusted to pH 7.35 to 7.40. Vascular strips were incubated for 1.5 hours with 10 mol/L BCECF-AM in HEPES-PSS with gentle agitation at 35°C.…”

Use of flow cytometry and SNARF to calibrate and measure intracellular pH in NS0 cells

“…The fluoroprobe is usually loaded into cells as an acetoxymethyl ester; this enters the cells readily and is hydrolysed to yield the free fluorescent dye. The fluorescent ratio can then be determined by one of several methods based on a spectrofluorometer, a fluorescent microscope, or a flow cytometer (1–3).…”