Richard D. Vaughan-Jones

Regulation of tumor pH and the role of carbonic anhydrase 9

The high metabolic rate required for tumor growth often leads to hypoxia in poorly-perfused regions. Hypoxia activates a complex gene expression program, mediated by hypoxia inducible factor 1 (HIF1α). One of the consequences of HIF1α activation is up-regulation of glycolysis and hence the production of lactic acid. In addition to the lactic acid-output, intracellular titration of acid with bicarbonate and the engagement of the pentose phosphate shunt release CO2 from cells. Expression of the enzyme carbonic anhydrase 9 on the tumor cell surface catalyses the extracellular trapping of acid by hydrating cell-generated CO2 into

Intracellular pH regulation in heart

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APPLICATION OF A NEW PH-SENSITIVE FLUOROPROBE (CARBOXY-SNARF-1) FOR INTRACELLULAR PH MEASUREMENT IN SMALL, ISOLATED CELLS

and H+. These mechanisms contribute towards an acidic extracellular milieu favoring tumor growth, invasion and development. The lactic acid released by tumor cells is further metabolized by the tumor stroma. Low extracellular pH may adversely affect the intracellular milieu, possibly triggering apoptosis. Therefore, primary and secondary active transporters operate in the tumor cell membrane to protect the cytosol from acidosis. We review mechanisms regulating tumor intracellular and extracellular pH, with a focus on carbonic anhydrase 9. We also review recent evidence that may suggest a role for CA9 in coordinating pHi among cells of large, unvascularized cell-clusters.

Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells.

1 Intracellular pH was recorded fluorimetrically by using carboxy‐SNARF‐1, AM‐loaded into superfused ventricular myocytes isolated from guinea‐pig heart. Intracellular acid and base loads were induced experimentally and the changes of pHi used to estimate intracellular buffering power (β). The rate of pHi recovery from acid or base loads was used, in conjunction with the measurements of β, to estimate sarcolemmal transporter fluxes of acid equivalents. A combination of ion substitution and pharmacological inhibitors was used to dissect acid effluxes carried on Na+‐H+ exchange (NHE) and Na+‐HCO3− cotransport (NBC), and acid influxes carried on Cl−‐HCO3− exchange (AE) and Cl−‐OH− exchange (CHE). 2 The intracellular intrinsic buffering power (βi), estimated under CO2/HCO3−‐free conditions, varied inversely with pHi in a manner consistent with two principal intracellular buffers of differing concentration and pK. In CO2/HCO3−‐buffered conditions, intracellular buffering was roughly doubled. The size of the CO2‐dependent component (βCO2) was consistent with buffering in a cell fully open to CO2. Because the full value of βCO2 develops slowly (2·5 min), it had to be measured under equilibrium conditions. The value of βCO2 increased monotonically with pHi. 3 In 5 % CO2/HCO3−‐buffered conditions (pHo 7·40), acid extrusion on NHE and NBC increased as pHi was reduced, with the greater increase occurring through NHE at pHi < 6·90. Acid influx on AE and CHE increased as pHi was raised, with the greater increase occurring through AE at pHi > 7·15. At resting pHi (7·04‐7·07), all four carriers were activated equally, albeit at a low rate (about 0·15 mM min−1). 4 The pHi dependence of flux through the transporters, in combination with the pHi and time dependence of intracellular buffering (βi+βCO2), was used to predict mathematically the recovery of pHi following an intracellular acid or base load. Under several conditions the mathematical predictions compared well with experimental recordings, suggesting that the model of dual acid influx and acid efflux transporters is sufficient to account for pHi regulation in the cardiac cell. Key properties of the pHi control system are discussed.

New insights into the physiological role of carbonic anhydrase IX in tumour pH regulation

Intracellular pH (pHi) is an important modulator of cardiac excitation and contraction, and a potent trigger of electrical arrhythmia. This review outlines the intracellular and membrane mechanisms that control pHi in the cardiac myocyte. We consider the kinetic regulation of sarcolemmal H+, OH- and HCO3- transporters by pH, and by receptor-coupled intracellular signalling systems. We also consider how activity of these pHi effector proteins is coordinated spatially in the myocardium by intracellular mobile buffer shuttles, gap junctional channels and carbonic anhydrase enzymes. Finally, we review the impact of pHi regulatory proteins on intracellular Ca2+ signalling, and their participation in clinical disorders such as myocardial ischaemia, maladaptive hypertrophy and heart failure. Such multiple effects emphasise the fundamental role that pHi regulation plays in the heart.

Tumor-associated Carbonic Anhydrase 9 Spatially Coordinates Intracellular pH in Three-dimensional Multicellular Growths

We have studied the role of carbonic anhydrase 9 (CA9), a cancer-associated extracellular isoform of the enzyme carbonic anhydrase in multicellular spheroid growths (radius of ∼300 μm) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pHi) and pH in the restricted extracellular spaces (pHe). With no CA9 expression, spheroids developed very low pHi (∼6.3) and reduced pHe (∼6.9) at their core, associated with a diminishing gradient of acidity extending out to the periphery. With CA9 expression, core intracellular acidity was less prominent (pHi = ∼6.6), whereas extracellular acidity was enhanced (pHe = ∼6.6), so that radial pHi gradients were smaller and radial pHe gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pHe indicates the enzyme is facilitating CO2 excretion from cells (by converting vented CO2 to extracellular H+), rather than facilitating membrane H+ transport (such as H+ associated with metabolically generated lactic acid). This latter process requires titration of exported H+ ions with extracellular HCO3−, which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pHe will depend on the cellular CO2/lactic acid emission ratio (set by local oxygenation and membrane HCO3− uptake). Our results suggest that CO2-producing tumors may express CA9 to facilitate CO2 excretion, thus raising pHi and reducing pHe, which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity.

Effects of hypercapnia on membrane potential and intracellular calcium in rat carotid body type I cells.

We report the use of a new pH-sensitive dualemission fluoroprobe, carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1) for ratiometric recording of intracellular pH (pHi) in small isolated cells. The method is illustrated with pHi measurement in single type-1 cells (cell diameter ∼10 μm) isolated from the carotid body of the neonatal rat. Carboxy-SNARF-1 is loaded using bath application of the acetoxymethyl ester. When excited at 540 nm, the fluoroprobe gives strong, inversely related emission signals at 590 nm and 640 nm. Stable ratiometric recordings of pHi can be achieved from a single cell (pHi 8.5-6.5) for up to 50 min. Photobleaching of the probe is minimised by illuminating at relatively low light intensity (50 W xenon lamp with 0.2% transmission neutral density filter). The probe can be calibrated in situ using the nigericin technique and this is in good quantitative agreement with the independent null-point technique (extracellular weak acid/weak base application) of Eisner et al. (1989). This fluoroprobe offers certain advantages over the other commonly used probe for pHi 2′,7′-bis-(2-carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF): (i) because of its two strong pH-sensitive peak emissions, SNARF displays a good signal-to-noise ratio for ratiometric recording at low light intensities; (ii) unlike BCECF, the dual emisson of SNARF requires no sequential mechanical switching of excitation filters, thus simplifying the epifluorescence set-up; (iii) because carboxy-SNARF-1 emission signals are at the yellow/red end of the visible spectrum, fluorescent drugs like amiloride, ethyl-isopropyl-amibride (EIPA), 4,4′-diisothiocyanostilbene 2,2′-disulphonic acid (DIDS) and cinnamate analogues do not interfere with the pHi recording, even when used at high concentrations.

Mechanism of potassium efflux and action potential shortening during ischaemia in isolated mammalian cardiac muscle

1 Mitochondrial uncouplers are potent stimulants of the carotid body. We have therefore investigated their effects upon isolated type I cells. Both 2,4‐dinitrophenol (DNP) and carbonyl cyanide p‐trifluoromethoxyphenyl hydrazone (FCCP) caused an increase in [Ca2+]i which was largely inhibited by removal of extracellular Ca2+ or Na+, or by the addition of 2 mm Ni2+. Methoxyverapamil (D600) also partially inhibited the [Ca2+]i response. 2 In perforated‐patch recordings, the rise in [Ca2+]i coincided with membrane depolarization and was greatly reduced by voltage clamping the cell to −70 mV. Uncouplers also inhibited a background K+ current and induced a small inward current. 3 Uncouplers reduced pHi by 0.1 unit. Alkaline media diminished this acidification but had no effect on the [Ca2+]i response. 4 FCCP and DNP also depolarized type I cell mitochondria. The onset of mitochondrial depolarization preceded changes in cell membrane conductance by 3–4 s. 5 We conclude that uncouplers excite the carotid body by inhibiting a background K+ conductance and inducing a small inward current, both of which lead to membrane depolarization and voltage‐gated Ca2+ entry. These effects are unlikely to be caused by cell acidification. The inhibition of background K+ current may be related to the uncoupling of oxidative phosphorylation.