Search

A sufficiently high intracellular pH (Phi) is permissive for cell survival, growth, function and proliferation. High metabolic production of H+ ions is compensated for by acid extrusion on ion transport proteins such as Na/H exchange (NHE) and Na-HCO3 co-transport (NBC). This helps to regularise pHi in individual cells. In addition, H+-ion cross-talk between adjacent cells helps to co-ordinate pHi throughout the tissue. The talk will contrast two different pHi-co-ordination strategies. One is common in poorly vascularised developing tumours and relies on the activity of exofacial carbonic anhyrase enzymes, notably HIF-activated CAIX, which facilitates the venting of cellular CO2. The other, which is common in the heart, is the buffer-shuttling of acid via histidyl dipeptides (HDPs) and CO2/HCO3-- between cells, through gap junctions (predominantly Cx43 channels in the myocardium). Extracellular CAIX operates in concert with membrane HCO3- influx on NBC. CAIX up-regulation in hypoxic zones is associated with down-regulation of cytoplasmic CAs such as CAII. NBCs are constitutively expressed at high basal activity in a wide range of tumour cell-lines, unlike NHE1 that can be at either high or low functional level. By facilitating CO2 diffusion out of cells, CAIX raises pHi and reduces pHo, producing the functional hall-mark of aggressive cancer. Down regulation of CAIX activity, either by genetic manipulation or by pharma- cological inhibition, typically results in a more acidic core pHi in 3-D spheroid growths (eg. RT112; HCT116), a more heterogeneous spatial pHi distribution, and a more alkaline pH in the restricted extracellular spaces. Gap-junctional channels tend to be repressed in developing tumours, but they are commonly expressed in most healthy tissues. Cx43 channels in the myocardium mediate high passive H+ flux whenever [H+]i differences are presented across junctional regions. High H+-flux is achieved by reversible binding to intrinsic HDPs (carnsoine, anserine, homocarnosine) that are present in cytoplasm (~15mM), and which readily permeate the junction. HCO3 anion flux through the channels also enhances cell-cell H+ transmission. The H+ and HCO3- flux is gated by moderate [H+]i and high [H+]i which increases and reduces Cx43 channel permeability respectively. These are acute channel-gating phenomena, also observed for Cx43 channels stably transfected into Hela or N2A cell-pairs. Because buffer permeation of Cx43 channels is high, so is cell-cell H+ ion transmission. The system provides for spatial H+ communication and permits slow H+ diffusive equilibration of pHi in tissue, resulting in a pHi syncitium that regularises cell function. H+-gating of cell-cell H+ flux (autoregulation) is absent when the cytoplasmic tail of Cx43 sub-units is truncated. The talk will compare the kinetics and spatial outreach of CAIX and Cx43 mediated pHi control, highlighting the potential advantages of each mechanism. [ABSTRACT FROM AUTHOR]

Intracellular acidosis is an important modulator of cardiac excitation and contraction, and a potent trigger of electrical arrhythmia, partly through its ability to stimulate acid extruders, which indirectly raises sarcoplasmic reticulum (SR) Ca2+. When this effect is large (a condition known as Ca2+-overload), cardiac myocytes engage in a mode of Ca2+ signaling in which Ca2+ release from the SR to myoplasm occurs in self-propagating succession along the length of the cell. This event is called a Ca2+ wave and is fundamentally a diffusion-reaction phenomenon that is best described with a mathematical model. Wave properties, e.g. frequency, velocity, amplitude and relaxation time are useful parameters to infer how acidic pH affects Ca2+-related components inside the cell, such as the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), myoplasmic Ca2+ buffers and the ryanodine receptor (RyR). We present a new, continuum mathematical model, based on previous work (1), that simulates spontaneous Ca2+ waves generated during intracellular acidosis and Ca2+-overload. The model, developed with a user-friendly interface in Matlab, is capable of reproducing Ca2+ sparks and wave failure, collision and annihilation. In particular, Ca2+ sparks are modeled by a random function that regulates the opening and closing probability of RyRs, which in turn controls the likelihood of wave generation. Numerical simulations predict that, during Ca2+-overloading conditions, spontaneous Ca2+ wave generation significantly helps the cell to control and maintain at a constant level the myoplasmic and luminal Ca2+ concentration, as suggested in (2). Increasing Ca2+ wave frequency and speed enhances this mechanism, thus giving a simple explanation of what observed experimentally in rat isolated ventricular myocytes, where reducing pHi increased to 360% (by 60 s) wave frequency, together with increasing wave velocity up to 140%. Inhibiting the sarcolemmal Na2+/H2+ exchanger (NHE) suppressed wave frequency but not the increased velocity. In the model, this behaviour can be reproduced by decreasing KON of the lumped Ca2+ buffers, which is experimentally expected being independent of NHE activity. Wave frequency reduction during NHE inhibition can be explained by a fall in SR Ca2+ content mediated by H+-reduced SERCA activity no longer supported by NHE-driven Ca2+ import on NCX. The model shows that reducing SERCA maximum flux instead of its affinity best accounts for the observed experimental change in wave amplitude and relaxation time. Our model successfully reproduces Ca2+ waves using experimentally-derived variables and highlights the importance of SR Ca2+ load on wave propagation. At present, we are developing a model to describe a cardiac multicellular system connected by gap junctions, providing Ca2+ and H+ intercellular diffusion. [ABSTRACT FROM AUTHOR]

Na+/H+ exchanger 1 (NHE1) is a ubiquitous membrane transporter that regulates pHi, [Na Na+]i and cell volume. In the heart, stretch-activation of NHE1 has been claimed to be a key element of the slow force response to stretch 1. By raising pHi and [Na+], NHE1 activation may initiate hypertrophy 2. We examined osmotic activation of NHE1 and the related Na+/HCO3- cotransporter (NBC) in rat isolated ventricular myocytes. Adult rat ventricular myocytes were enzymatically isolated. pHi was imaged using cSNARF-1. 160 mM sucrose was added to increase osmolarity. Data are mean ± SEM, compared with unpaired Student's t-test. Intrinsic buffering capacity (βint) was measured by ammonium removal. Hyperosmotic shrinkage increased βint 1.6-fold (26.3 ± 1.6mM (n=17) vs. 16.1 ± 1.9mM (n=6) at pH 7.3, P<0.01). pHi was increased using an ammonium prepulse and the slope of pHi recovery was used to calculate acid-efflux (J=dpH/dt* β). NHE-mediated acid-efflux was greatly increased in hyperosmotic medium (11.0+0.5mM/min (n=25) vs. 2.7 ± 0.3mM/min (n=8) at pH 7.0, P<0.001), producing an alkaline shift of 0.25 pH units in the pHi-flux relationship. Upon addition of 160 mM sucrose, myocytes showed gradual NHE-mediated alkalinization of 0.3 pH units over 8 min (n=5). This was abolished by the NHE-inhibitor DMA (5-(N,N-dimethyl)amiloride, 30µM, n=7). To elucidate the osmotic regulation of NBC, bicarbonate-buffered solutions were used (NHE inhibited with 30µM DMA). NBC-mediated acid-efflux was 1.8-fold increased in hyperosmotic solution (6.3 ± 0.7mM/min (n=17) vs. 3.4 ± 0.3mM/min (n=25) at pH 7.0, P<0.001). Testing a possible role of carbonic anhydrase (CA) in the osmotic activation of NHE1 and NBC, we found that acid-efflux was not altered by addition of ETZ (100µM) in isosmotic or hyperosmotic bicarbonate-buffered media (isosmotic: 10.5 ± 0.9mM/min (n=21) vs. ETZ 14.1 ± 1.6mM/min (n=6) at pH 6.6, P=0.065; hyperosmotic: 25.9 ± 1.7mM/min (n=15) vs. ETZ 26.7 ± 1.8mM/min (n=5) at pH 6.7, P>0.8). This rules out the necessity of CA activity for the combined osmotic effect on NHE and NBC. Given the strong osmotic effect on NHE, we examined its role in myocyte volume regulation. Both video-imaging and z-stack techniques 3 showed a rapid shrinkage in hyperosmotic solution within the first minute without subsequent regulatory volume increase (z-stack data as normalized volume: after 1 min, 81.8 ± 0.7%; 5 min, 81.1 ± 1.0%; 10 min, 79.5 ± 1.3%, n=7). The contribution of NHE to cellular volume maintenance during hyperosmotic shock was found to be negligible (30µM DMA added, compared to above data: after 1 min, 86.6 ± 1.2%, P<0.01; 5 min, 83.9 ± 0.7%, P>0.065; 10 min, 81.2 ± 1.0%, P>0.36, n=5). We conclude that strong activation by hyperosmotic stimuli is a common characteristic of the cardiac acid-extruders NHE1 and NBC, but does not directly involve activation of catalytic activity from native carbonic anhydrase. [ABSTRACT FROM AUTHOR]

The heart's intensive pumping activity is powered by a uniquely high rate of mitochondrial respiration. This generates vast quantities of the waste-product CO2, which must be removed efficiently from mitochondria. Carbonic anhydrase (CA) enzymes, expressed at various sites in ventricular myocytes, may affect mitochondrial CO2-clearance by catalyzing CO2 hydration (to H+ and HCO3-) thereby changing the gradient for CO2 venting. In this study, we measured CA activity in cardiac myocytes, and investigated the effects of CA inhibition on cardiac energetics and function. CA activity in the cytoplasm of isolated ventricular myocytes was measured by fluorescence imaging of cells loaded with the pH reporter dye, cSNARF1 (10 μM, AM-loading for 5 min). Rapid exposure to CO2-containing solution evoked CO2 entry and its intracellular hydration which was only modestly accelerated by CA activity (2.7±0.3[SEM]-fold above spontaneous kinetics determined in the presence of the broad-spectrum CA inhibitor acetazolamide, ATZ, 100 μM). A similar experiment performed on isolated ventricular mitochondria demonstrated negligible intra-mitochondrial CA activity. CA activity was also investigated in intact hearts by 13C magnetic resonance spec-troscopy (MRS) from the rate of H13CO3-production from 13CO2 released specifically from mitochondria by pyruvate dehydrogenase-mediated metabolism of hyperpolarized [1-13C]pyruvate. Under these conditions, the steady-state between de novo production and capillary wash-out produces a gradient of 13CO2 from mitochondria to the sarcolemma. CA activity measured upon [1-13C] pyruvate infusion was 11.4±0.9 fold above spontaneous kinetics (i.e. four-fold higher than the cytoplasm-averaged value). Afluorescent CA-ligand (fluorescein-thioureido-homosulfanil-amide) co-localized with the mitochondrial markerTM RE (Pearson's coefficient 0.735±0.06), indicating that mitochondria are near a CA-rich domain. Based on immunoreactivity, this domain comprises the nominally cytoplasmic CAII and sar-coplasmic reticulum-associated CAXIV. Inhibition of extra-mitochondrial CA activity with ATZ acidified the matrix by up to 0.1 unit (pH 7.73±0.03 to 7.62±0.02 measured by fluorescence imaging of permeabilized myocytes and isolated mitochondria by flow cytometry), impaired cardiac energetics (phos-phocreatine-to-ATP ratio decreased from 2.02±0.13 to 1.58±0.14, measured by 31P-MRS of perfused hearts) and reduced contractility (developed pressure in perfused hearts decreased by 18±5%). These data provide evidence for a functional domain of high CA activity around mitochondria to support CO2-venting, particularly during elevated and fluctuating respiratory activity. Aberrant distribution of CA activity may therefore reduce the heart's energetic efficiency. [ABSTRACT FROM AUTHOR]

An abstract of the article "Biphasic modulation of Na+/H+ exchange by exogenous and endogenous nitric oxide in the rat ventricular myocyte" by M. Richards, D. J. Paterson, R. D. Vaughan-Jones and P. Swietach is presented.

An abstract of the article "Spatial pHi heterogeneity as a substrate for Ca2+ wave initiation in the ventricular myocyte" by K. L. Ford, P. Swietach and R. D. Vaughan-Jones is presented.

An abstract of the article "Carbonic anhydrase activity regulates intracellular pH in 3D pancreatic cancer growths" by T. H. Dovmark, A. Hulikova, R. D. Vaughan-Jones and P. Swietach is presented.

An abstract of the article "Independent high and low pH block of connexin 43 gap junctions" by C. D. Garciarena, K. W. Spitzer, P. Swietach, A. P. Moreno, and R. D. Vaughan-Jones is presented.

Intracellular acidosis is a feature of both myocardialischaemia, where it has been linked to ischaemia-reperfusion injury1, and heart failure2. A decrease in pHi has been shown to have multiple effects on the calcium handling apparatus, leading to the hypothesis that acidosis may drive myocardial pathology via effects on calcium. Here, we show that altering pHi globally and locally affects multiple parameters of spontaneous Ca2+ waves, which may be an arrhythmogenic risk factor. pHi was altered globally by superfusing with 20-80 mM acetate (acidosis) or 20 mM trimethylamine (alkalosis). Spontaneous Ca2+ waves, induced by raising superfusate Ca2+, were lines-can-imaged at 37°C in rat isolated ventricular myocytes AM-loaded with f luo-3. Parallel pHi measurements were made using AM-loaded SNARF-1. Data are mean±SE, P values calculated using a paired Student's ttest. Intracellular acidosis increased spontaneous wave frequency up to 360±62% of control (P=0.000, n=18). This effect was prevented by inhibiting the sarcolemmal Na+/H+ exchanger (NHE) with 30 μM 5-(N,N-dimethyl)amiloride, demonstrating Na+-coupling to intracellular Ca2+ dynamics. Under these conditions, waves were suppressed relative to control (to 61±22% of control; P=0.002, n=11), indicating that H+ may have cardioprotective properties. Intracellular alkalosis also decreased wave frequency to 60% of control after 60 seconds (P=0.0048, n=10). Decreasing pHi increased wave velocity up to 140±3% of control (P<0.000, n=17), while an intracellular alkalosis decreased velocity to 80% of control (P<0.000, n=12). Analysis over a range of pHi values demonstrated a linear relationship between pHi and wave velocity. Computational modelling4 suggests that this may be due to a decrease in cytoplasmic Ca2+ buffering; consistent with this, inhibiting NHE had no further significant effect during acidosis. It is likely that pHi in the ischaemic heart is heterogeneous, therefore to investigate whether differences in pHi affect local Ca2+ properties, we induced a stable pHi gradient (pHi 6.6-7.3) in single ventricular myocytes by superfusing two parallel microstreams perpendicular to the cell, one containing normal Tyrode, the other 80 mM acetate5. With NHE inhibited, wave initiation was inhibited in the acidic microdomain (31±7% of control;P<0.05, n=13) and stimulated in the more alkaline microdomain (164±25%; P<0.05, n=13), while wave velocity was increased in the acidic microdomain and decreased in the more alkaline microdomain. Other effects on Ca2+ dynamics, including wave decay, also mapped on to the pHi gradient. We conclude that pHi locally regulates Ca2+ handling and dynamics, and thus pHi heterogeneity may provide a substrate for aberrant Ca2+ signalling in cardiac pathology. [ABSTRACT FROM AUTHOR]