Biomedical subjects

Pawel Swietach

Publications and source records attributed to Pawel Swietach.

13 recordsLinked to original sources

PubMed2006

pH-Dependence of extrinsic and intrinsic H(+)-ion mobility in the rat ventricular myocyte, investigated using flash photolysis of a caged-H(+) compound.

Passive H(+)-ion mobility within eukaryotic cells is low, due to H(+)-ion binding to cytoplasmic buffers. A localized intracellular acidosis can therefore persist for seconds or even minutes. Because H(+)-ions modulate so many biological processes, spatial intracellular pH (pH(i))-regulation becomes important for coordinating cellular activity. We have investigated spatial pH(i)-regulation in single and paired ventricular myocytes from rat heart by inducing a localized intracellular acid-load, while confocally imaging pH(i) using the pH-fluorophore, carboxy-SNARF-1. We present a novel method for localizing the acid-load. This involves the intracellular photolytic uncaging of H(+)-ions from a membrane-permeant acid-donor, 2-nitrobenzaldehyde. The subsequent spatial pH(i)-changes are consistent with intracellular H(+)-mobility and cell-to-cell H(+)-permeability constants measured using more conventional acid-loading techniques. We use the method to investigate the effect of reducing pH(i) on intrinsic (non-CO(2)/HCO(3)(-) buffer-dependent) and extrinsic (CO(2)/HCO(3)(-) buffer-dependent) components of H(i)(+)-mobility. We find that although both components mediate spatial regulation of pH within the cell, their ability to do so declines sharply at low pH(i). Thus acidosis severely slows intracellular H(+)-ion movement. This can result in spatial pH(i) nonuniformity, particularly during the stimulation of sarcolemmal Na(+)-H(+) exchange. Intracellular acidosis thus presents a window of vulnerability in the spatial coordination of cellular function.

Animals↗

PubMed2006

pH-Regulated Na(+) influx into the mammalian ventricular myocyte: the relative role of Na(+)-H(+) exchange and Na(+)-HCO Co-transport.

In the heart, intracellular Na(+) concentration (Na(+) (i)) is a controller of intracellular Ca(2+) signaling, and hence of key aspects of cell contractility and rhythm. Na(+) (i) will be influenced by variation in Na(+) influx. In the present work, we consider one source of Na(+) influx, sarcolemmal acid extrusion. Acid extrusion is accomplished by sarcolemmal H(+) and HCO(3) (-) transporters that import Na(+) ions while exporting H(+) or importing HCO(3) (-). The capacity of this system to import Na(+) is enormous, up to four times the maximum capacity of the Na(+)-K(+) ATPase to extrude Na(+) ions from the cell. In this review we consider the role of Na(+)-H(+) exchange (NHE) and Na(+)-HCO(3) (-)co-transport (NBC) in mediating Na(+) influx into cardiac myocytes. We consider, in particular, the role of NBC, as so little is known about Na(+) influx through this transporter. We show that both proteins mediate significant Na(+) influx and that although, in the ventricular myocyte, NBC-mediated Na(+) influx is less than through NHE, the proportions may be altered under a variety of conditions, including exposure to catecholamines, membrane depolarization, and interference with activity of the enzyme, carbonic anhydrase.

Adenosine Triphosphate↗

PubMed2005

Spatial aspects of intracellular pH regulation in heart muscle.

Intracellular pH (pH(i)) is an important modulator of cardiac function. Because it is readily influenced by metabolic processes, pH(i) is controlled physiologically. Classical models of intracellular pH regulation comprise acid/base transport proteins expressed in the sarcolemma, acting in concert with intracellular buffers. These two processes are coupled via a diffusive movement of protons. Because intracellular H(+) buffering is high, H(i)(+)-diffusion occurs through a passive shuttling on intrinsic mobile buffers such as acetylated carnosine, anserine and homocarnosine: low molecular weight imidazole compounds. This mechanism is assisted by carbonic buffer, a system regulated biochemically by the enzyme carbonic anhydrase. H(i)(+)-mobility via the buffer shuttles is low, and this can result in significant pH(i) non-uniformity under conditions of high proton flux across the sarcolemma or within the cell. Spatial regulation of pH(i) is complemented by passive H(+) permeation between cells through gap junctions. This permeation is also mediated via protonated buffers. The control of pH(i) is therefore dependent on carrier molecules that spatially shuttle protons within and between cells. In this review, we consider the physiological regulation of H(i)(+)-mobility and permeation, and its relevance to pH(i)-control in normal and pathophysiological states such as myocardial ischaemia, a clinical condition associated with severe intracellular acidosis.

Animals↗

PubMed2005

Relationship between intracellular pH and proton mobility in rat and guinea-pig ventricular myocytes.

Intracellular H+ ion mobility in eukaryotic cells is low because of intracellular buffering. We have investigated whether Hi+ mobility varies with pHi. A dual microperfusion apparatus was used to expose guinea-pig or rat myocytes to small localized doses (3-5 mm) of ammonium chloride (applied in Hepes-buffered solution). Intracellular pH (pHi) was monitored confocally using the fluorescent dye, carboxy-SNARF-1. Local ammonium exposure produced a stable, longitudinal pHi gradient. Its size was fed into a look-up table (LUT) to give an estimate of the apparent intracellular proton diffusion coefficient (D(app)H). LUTs were generated using a diffusion-reaction model of Hi+ mobility based on intracellular buffer diffusion. To examine the pHi sensitivity of D(app)H, whole-cell pHi was initially displaced using a whole-cell ammonium or acetate prepulse, before locally applying the low dose of ammonium. In both rat and guinea-pig, D(app)H decreased with pHi over the range 7.5-6.5. In separate pipette-loading experiments, the intracellular diffusion coefficient for carboxy-SNARF-1 (a mobile-buffer analogue) exhibited no significant pHi dependence. The pHi sensitivity of D(app)H is thus likely to be governed by the mobile fraction of intrinsic buffering capacity. These results reinforce the buffer hypothesis of Hi+ mobility. The pHi dependence of D(app)H was used to characterize the mobile and fixed buffer components, and to estimate D(mob) (the average diffusion coefficient for intracellular mobile buffer). One consequence of a decline in Hi+ mobility at low pHi is that it will predispose the myocardium to pHi nonuniformity. The physiological relevance of this is discussed.

Animals↗

PubMed2005

Experimental generation and computational modeling of intracellular pH gradients in cardiac myocytes.

It is often assumed that pH(i) is spatially uniform within cells. A double-barreled microperfusion system was used to apply solutions of weak acid (acetic acid, CO(2)) or base (ammonia) to localized regions of an isolated ventricular myocyte (guinea pig). A stable, longitudinal pH(i) gradient (up to 1 pH(i) unit) was observed (using confocal imaging of SNARF-1 fluorescence). Changing the fractional exposure of the cell to weak acid/base altered the gradient, as did changing the concentration and type of weak acid/base applied. A diffusion-reaction computational model accurately simulated this behavior of pH(i). The model assumes that H(i)(+) movement occurs via diffusive shuttling on mobile buffers, with little free H(+) diffusion. The average diffusion constant for mobile buffer was estimated as 33 x 10(-7) cm(2)/s, consistent with an apparent H(i)(+) diffusion coefficient, D(H)(app), of 14.4 x 10(-7) cm(2)/s (at pH(i) 7.07), a value two orders of magnitude lower than for H(+) ions in water but similar to that estimated recently from local acid injection via a cell-attached glass micropipette. We conclude that, because H(i)(+) mobility is so low, an extracellular concentration gradient of permeant weak acid readily induces pH(i) nonuniformity. Similar concentration gradients for weak acid (e.g., CO(2)) occur across border zones during regional myocardial ischemia, raising the possibility of steep pH(i) gradients within the heart under some pathophysiological conditions.

Acetates↗

PubMed2005

Spatial regulation of intracellular pH in the ventricular myocyte.

In the cardiac myocyte, an adequate intracellular proton mobility is necessary for coupling sarcolemmal proton transport to bulk cytoplasm during intracellular pH regulation. It is also important for dissipating intracellular pH nonuniformity in response to local acid/base disturbances. Because cardiac myocytes have a high buffering capacity, intracellular H(+) mobility is low. Spatial H(i)+ movements occur via a mobile buffer shuttle that most likely involves intracellular dipeptides. In the present work with isolated ventricular myocytes, it is demonstrated that stimulating a large acid efflux on sarcolemmal Na(+)-H(+) exchange, results in spatial pH(i) gradients of up to 0.1 U. These may have important implications for pH-sensitive processes within the cell, such as contraction. By using computational modeling, it is shown that the gradients can be attributed to the low H(i)+ mobility. Computational modeling is also used to assess the importance of H(i)+ mobility in mediating local recovery of pH following an acidosis in a small region of the cell. Results indicate that local mechanisms for H(i)+ movement will be important in determining the global regulation of intracellular pH.

Animals↗

PubMed2004

Functional diversity of electrogenic Na+-HCO3- cotransport in ventricular myocytes from rat, rabbit and guinea pig.

The Na(+)-HCO(3)(-) cotransporter (NBC) is an important sarcolemmal acid extruder in cardiac muscle. The characteristics of NBC expressed functionally in heart are controversial, with reports suggesting electroneutral (NBCn; 1HCO(3)(-) : 1Na(+); coupling coefficient N= 1) or electrogenic forms of the transporter (NBCe; equivalent to 2HCO(3)(-) : 1Na(+); N= 2). We have used voltage-clamp and epifluorescence techniques to compare NBC activity in isolated ventricular myocytes from rabbit, rat and guinea pig. Depolarization (by voltage clamp or hyperkalaemia) reversibly increased steady-state pH(i) while hyperpolarization decreased it, effects seen only in CO(2)/HCO(3)(-)-buffered solutions, and blocked by S0859 (cardiac NBC inhibitor). Species differences in amplitude of these pH(i) changes were rat > guinea pig approximately rabbit. Tonic depolarization (-140 mV to -0 mV) accelerated NBC-mediated pH(i) recovery from an intracellular acid load. At 0 mV, NBC-mediated outward current at resting pH(i) was +0.52 +/- 0.05 pA pF(-1) (rat, n= 5), +0.26 +/- 0.05 pA pF(-1) (guinea pig, n= 5) and +0.10 +/- 0.03 pA pF(-1) (rabbit, n= 9), with reversal potentials near -100 mV, consistent with N= 2. The above results indicate a functionally active voltage-sensitive NBCe in these species. Voltage-clamp hyperpolarization negative to the reversal potential for NBCe failed, however, to terminate or reverse NBC-mediated pH(i)-recovery from an acid load although it was slowed significantly, suggesting electroneutral NBC may also be operational. NBC-mediated pH(i) recovery was associated with a rise of [Na(+)](i) at a rate approximately 25% of that mediated via NHE, and consistent with an apparent NBC stoichiometry between N= 1 and N= 2. In conclusion, NBCe in the ventricular myocyte displays considerable functional variation among the three species tested (greatest in rat, least in rabbit) and may coexist with some NBCn activity.

Acidosis↗

PubMed2004

Novel method for measuring junctional proton permeation in isolated ventricular myocyte cell pairs.

Partial exposure of single ventricular myocytes to membrane-permeant weak acids or bases, using a dual-microperfusion technique, generates large and stable intracellular pH (pHi) gradients. In this study, we have investigated the feasibility of using the technique to estimate junctional proton permeability. This was done by recording the pHi gradient developed across the junctional region of a pair of conjoined ventricular myocytes, isolated enzymically from a guinea pig heart when one of the cells was partially exposed to acetate or ammonium. We show that under HEPES-buffered conditions, the junctional discontinuity in the pHi profile can be used to derive an apparent proton permeability coefficient (PHapp). The mean PHapp obtained was 4.45 +/- 0.21.10(-4) cm/s (n=43) at an average junctional pHi of 7.04 +/- 0.02. In the presence of the junctional inhibitor alpha-glycyrrhetinic acid, exposure of the proximal cell to weak acid or base produced no pHi change in the distal cell, confirming that distal changes were normally caused by acid-base flux through connexons assembled into junctional channels. The validity of the dual-microperfusion method was tested further by using a diffusion-permeation-reaction model for intracellular protons, designed to highlight possible errors in the estimates of PHapp. Our technique for measuring PHapp provides a useful alternative to the previous, more invasive technique of locally loading acid through a cell-attached patch pipette. The technique may provide a simple method for investigating the factors regulating cell-to-cell proton transmission.

Animals↗

PubMed2003

Proton permeation through the myocardial gap junction.

Although protons can directly or indirectly gate solute permeability of the myocardial gap junction, there is little information regarding their own permeation, despite their importance in the regulation of myocardial contractility and rhythm. By pipette-loading of acid into guinea pig isolated, ventricular myocyte pairs while imaging pH(i) confocally using SNARF fluorescence, we have observed that protons permeate the junctional region. Permeation is inhibited by glycyrrhetinic acid, an agent that also increases intercellular electrical resistance, suggesting H+ permeation via gap junctions. The rate of spread of acid between cells appears to be limited by junctional permeation rather than by cytoplasmic diffusion. Mathematical analyses, combined with experiments using SNARF as a proton carrier, suggest that gap junctional H+ transmission may be accomplished physiologically by the permeation of intrinsic "proton-porter" molecules. We propose that proton flux through gap junctions will contribute to the dissipation of regional acid loads within the myocardium. This represents a mechanism for the local control of myocardial pH(i).

Animals↗

PubMed2003

Temperature dependence of Na+-H+ exchange, Na+-HCO3- co-transport, intracellular buffering and intracellular pH in guinea-pig ventricular myocytes.

Almost all aspects of cardiac function are sensitive to modest changes of temperature. We have examined the thermal sensitivity of intracellular pH regulation in the heart. To do this we determined the temperature sensitivity of pHi, intracellular buffering capacity, and the activity of sarcolemmal acid-extrusion proteins, Na+-H+ exchange (NHE) and Na+-HCO3- co-transport (NBC) in guinea-pig isolated ventricular myocytes. pHi was recorded fluorimetrically with acetoxymethyl (AM)-loaded carboxy-SNARF-1 at either 27 or 37 degrees C. At 27 degrees C, intrinsic (non-CO2-dependent) buffering power (betai) was approximately 60% of that at 37 degrees C. Acid-extrusion (Je) through NHE was approximately 50% slower than at 37 degrees C, consistent with a Q10 of approximately 2. In 5% CO2/HCO3--buffered conditions, in the presence of 30 microM cariporide to inhibit NHE, acid extrusion via NBC was also slowed at 27 degrees C, suggestive of a comparable Q10. Resting pHi at 27 degrees C was similar in Hepes- or 5% CO2/HCO3--buffered superfusates but, in both cases, was approximately 0.1 pH units lower at 37 degrees C. The higher the starting pHi, the larger was the thermally induced fall of pHi, consistent with a mathematical model where intrinsic buffers with a low principal pKa (e.g. close to 6.0) are less temperature-sensitive than those with a higher pKa. The high temperature sensitivity of pHi regulation in mammalian cardiac cells has implications for experimental work conducted at room temperature. It also has implications for the ability of intracellular acidosis to generate intracellular Na+ and Ca2+ overload, cardiac injury and arrhythmia in the heart.

Animals↗

PubMed2003

Intracellular proton mobility and buffering power in cardiac ventricular myocytes from rat, rabbit, and guinea pig.

Intracellular pH (pHi) is an important modulator of cardiac function. The spatial regulation of pH within the cytoplasm depends, in part, on intracellular H+ (Hi+) mobility. The apparent diffusion coefficient for Hi+, DHapp, was estimated in single ventricular myocytes isolated from the rat, guinea pig, and rabbit. DHapp was derived by best-fitting predictions of a two-dimensional model of H+ diffusion to the local rise of intracellular [H+], recorded confocally (ratiometric seminaphthorhodafluor fluorescence) downstream from an acid-filled, whole cell patch pipette. Under CO2/HCO3--free conditions, DHapp was similar in all three species (mean values: 8-12.5 x 10-7 cm2/s) and was over 200-fold lower than that for H+ in water. In guinea pig myocytes, DHapp was increased 2.5-fold in the presence of CO2/HCO3- buffer, in agreement with previous observations in rabbit myocytes. Hi+ mobility is therefore low in cardiac cells, a feature that may predispose them to the generation of pHi gradients in response to sarcolemmal acid/base transport or local cytoplasmic acid production. Low Hi+ mobility most likely results from H+ shuttling among cytoplasmic mobile and fixed buffers. This hypothesis was explored by comparing the pHi dependence of intrinsic, intracellular buffering capacity, measured for all three species, and subdividing buffering into mobile and fixed fractions. The proportion of buffer that is mobile will be the main determinant of DHapp. At a given pHi, this proportion appeared to be similar in all three species, consistent with a common value for DHapp. Over the pHi range of 6.0-8.0, the proportion is expected to change, predicting that DHapp may display some pHi sensitivity.

Acid-Base Equilibrium↗

PubMed2003

Modelling intracellular H(+) ion diffusion.

Intracellular pH, an important modulator of cell function, is regulated by plasmalemmal proteins that transport H(+), or its equivalent, into or out of the cell. The pH(i) is also stabilised by high-capacity, intrinsic buffering on cytoplasmic proteins, oligopeptides and other solutes, and by the extrinsic CO(2)/HCO(3)(-) (carbonic) buffer. As mobility of these buffers is lower than for the H(+) ion, they restrict proton diffusion. In this paper we use computational approaches, based on the finite difference and finite element methods (FDM and FEM, respectively), for analysing the spatio-temporal behaviour of [H(+)] when it is locally perturbed. We analyse experimental data obtained for various cell-types (cardiac myocytes, duodenal enterocytes, molluscan neurons) where pH(i) has been imaged confocally using intracellular pH-sensitive dyes. We design mathematical algorithms to generate solutions for two-dimensional diffusion that fit data in terms of an apparent intracellular H(+) diffusion coefficient, D(H)(app). The models are used to explore how the spatial distribution of [H(+)](i) is affected by membrane H(+)-equivalent transport and by cell geometry. We then develop a mechanistic model, describing spatio-temporal changes of [H(+)](i) in a cardiac ventricular myocyte in terms of H(+)-shuttling on mobile buffers and H(+)-anchoring on fixed buffers. We also discuss how modelling may include the effects of extrinsic carbonic-buffering. Overall, our computational approach provides a framework for future analyses of the physiological consequences of pH(i) non-uniformity.

Animals↗

PubMed2002

Functional and molecular determination of carbonic anhydrase levels in bovine and cultured human chondrocytes.

In this study, bovine articular and human chondrocytes from the C-20/A4 cell line were tested for the functional activity and molecular presence of the enzyme carbonic anhydrase. This enzyme is classically considered to be important in the maintenance of high cellular buffering capacity by catalysing the slow attainment of equilibrium between CO(2) and HCO(3)(-). The first functional assay measured the rate of pH equilibration after administration of a fixed dose of CO(2) solution to cell lysates. Compared to positive controls (human erythrocytes, murine M1 cells and purified carbonic anhydrase), chondrocyte lysates attained equilibrium at a significantly slower rate, similar to the rate obtained with a negative control (Xenopus oocytes). A second functional assay studied CO(2) hydration kinetics in intact C-20/A4 cells, using a pH-sensitive fluorescent dye, as the CO(2) content of the extracellular solution was changed. It was shown that C-20/A4 cells accelerate hydration only to a small degree. Hydration kinetics were reduced to the spontaneous rate in the presence of acetazolamide. Western immunoblotting with isoform-nonspecific antibodies to carbonic anhydrase demonstrated weak staining in both bovine and human chondrocytes.

Acetazolamide↗