pH buffering of single rat skeletal muscle fibers in the in vivo environment

Homeostasis of intracellular pH (pH_i) has a crucial role for the maintenance of cellular function. Several membrane transporters such as lactate/H⁺ cotransporter (MCT), Na⁺/H⁺ exchange transporter (NHE), and Na⁺/HCO₃^– cotransporter (NBC) are thought to contribute to pH_i regulation. However, the relative importance of each of these membrane transporters to the in vivo recovery from the low pH_i condition is unknown. Using an in vivo bioimaging model, we pharmacologically inhibited each transporter separately and all transporters together and then evaluated the pH_i recovery profiles following imposition of a discrete H⁺ challenge loaded into single muscle fibers by microinjection. The intact spinotrapezius muscle of adult male Wistar rats (n = 72) was exteriorized and loaded with the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (10 μM). A single muscle fiber was then loaded with low-pH solution [piperazine-N,N'-bis(2-ethanesulfonic acid) buffer, pH 6.5, ~2.33 x 10^–3 μl] by microinjection over 3 s. The rats were divided into groups for the following treatments: 1) no inhibitor (CONT), 2) MCT inhibition (by α-Cyano-4-hydroxyciannamic acid; 4 mM), 3) NHE inhibition (by ethylisopropyl amiloride; 0.5 mM), 4) NBC inhibition (by DIDS; 1 mM), and 5) MCT, NHE, and NBC inhibition (All blockade). The fluorescence ratio (F500 nm/F445 nm) was determined from images captured during 1 min (60 images/min) and at 5, 10, 15, and 20 min after injection. The pH_i at 1–2 s after injection significantly decreased from resting pH_i (pH_i = –0.73 ± 0.03) in CONT. The recovery response profile was biphasic, with an initial rapid and close-to-exponential pH_i increase (time constant, : 60.0 ± 7.9 s). This initial rapid profile was not affected by any pharmacological blockade but was significantly delayed by carbonic anhydrase inhibition. In contrast, the secondary, more gradual, return toward baseline that restored CONT pH_i to 84.2% of baseline was unimpeded by MCT, NHE, and NBC blockade separately but abolished by All blockade (pH_i = –0.60 ± 0.07, 72.8% initial pH_i, P < 0.05 vs. CONT). After injection of H⁺ into, or superfusion onto, an adjacent fiber pH_i of the surrounding fibers decreased progressively for the 20-min observation period (~7.0, P < 0.05 vs. preinjection/superfusion). In conclusion, these results support that, after an imposed H⁺ load, the MCT, NHE, and NBC transporters are not involved in the initial rapid phase of pH_i recovery. In contrast, the gradual recovery phase was abolished by inhibiting all three membrane transporter systems simultaneously. The alteration of pH_i in surrounding fibers suggest that H⁺ uptake by neighboring fibers can help alleviate the pH consequences of myocyte H⁺ exudation.

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