Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC144
Carbonic anhydrase activity in rat cardiac myocytes is extra-mitochondrial
M. Ali1, M. Schroeder1, D. Tyler1, R. D. Vaughan-Jones1, P. Swietach1
1. Physiology, Anatomy and Genetics, Oxford University, Oxford, United Kingdom.
The carbonic anhydrases (CA) catalyse the hydration of CO2 to HCO3- and H+ ions, and the reverse reaction. Their activity has been shown to facilitate fluxes of H+ ions, HCO3- ions and CO2 across membranes and optimise the pH buffering capacity of CO2/HCO3- buffer. Protein expression studies on ventricular myocytes have demonstrated immuno-reactivity for CAIV, IX and XIV in the sarcoplasmic reticulum, CAIV and XIV at the sarcolemma (Schiebe et al, 2006) and CAV in mitochondria (Vaananen et al 1991). Functional studies have demonstrated modest intracellular CA activity in guinea-pig myocytes (Leem & Vaughan-Jones, 1998). Mitochondria are a principal source of CO2, a CA substrate. Previous work on mitochondrial suspensions using radio-tracers has found no CA activity (Dodgson et al, 1980). In the present work, we have investigated the distribution of CA activity in rat ventricular myocytes using pH-fluorophores. Carboxy-SNARF-1 was AM-loaded into intact myocytes. By varying the loading time (5 min to 2 hrs), it was possible to manipulate the degree of dye partitioning between the intra- and extra-mitochondrial compartments (higher mitochondrial signal with longer loading). The fraction of dye held in mitochondria was estimated at the end of each experiment by releasing cytosolic dye with 0.005% saponin. CA activity was measured by raising CO2 in cells by switching the extracellular solution from CO2-free (Hepes-buffered at pH 7.4) to 5% CO2 (also containing 22 mM HCO3- to bring pH to 7.4). This drives intracellular CO2 hydration, lowering the pH reported by carboxy-SNARF-1 at a rate proportional to CA activity. For purely cytosolic dye loading, the CO2 hydration rate was 0.64±0.12[SEM] s-1 in controls and 0.22±0.03 s-1 in the presence of the CA inhibitor acetazolamide (ATZ; 100 µM), giving a CA activity of 2.85. A negative correlation (r=-0.23; P=0.032 for one-tailed test; n=30) was observed between CA activity and the compartmentalisation of dye in mitochondria. This result suggests that mitochondria have lower CA activity than cytosol. Further experiments were performed on isolated mitochondria (isolation according to Das et al, 2003). Mitochondria were AM-loaded with BCECF and re-suspended in 140 mM K+ solution at pH 7.2. Upon addition of CO2-saturated buffer, the mitochondrial matrix acidified. The rate of acidification was not affected by ATZ (0.017±0.0021 vs 0.017±0.0007 pH s-1; n=6), suggesting negligible CA activity. Such intracellular CA activity distribution (with a predominantly extra-mitochondrial locus) would favour extra-mitochondrial CA hydration and may accelerate CO2 venting from the mitochondrial matrix. This would reduce matrix acid-loading and hence the degree of respiratory end-product inhibition.
Where applicable, experiments conform with Society ethical requirements