Biomedical subjects

Kenneth W Spitzer

Publications and source records attributed to Kenneth W Spitzer.

14 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

Cell-to-cell electrical interactions during early and late repolarization.

Cardiac electrical activity is significantly affected by variations in the conductance of gap junctions that connect myocytes to one another. To better understand how intrinsic (single cell) electrical activity is modulated by junctional conductance, we used a two-myocyte coupling system in which physically separate cells were electrically coupled via a variable resistance set by the investigator. This brief review summarizes our findings regarding: (1) the effect of the early phase of action potential repolarization (phase 1) and transient outward current (I(to)) on action potential conduction, and (2) the effect of coupling on the action potential plateau (late repolarization). We found that inhibition of I(to) markedly increased the ability of action potentials to propagate from cell-to-cell when junctional conductance was low. Electrically coupling two myocytes together also suppressed their beat-to-beat variability in action potential duration and contraction. Similarly, early afterdepolarizations (EADS) were readily suppressed by connecting a normal myocyte to one generating EADs. This high sensitivity of the plateau to variations in junctional interactions arises from the large increase in membrane resistance that occurs during this phase of the action potential.

Action Potentials↗

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

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

Propagation of Ca2+ release in cardiac myocytes: role of mitochondria.

Factors contributing to "local control" of Ca2+ release in cardiac myocytes are incompletely understood. We induced local release of Ca2+ by regional exposure of mouse atrial and ventricular myocytes to 10mM caffeine for 500 ms using a rapid solution switcher. Propagation of Ca2+ release was imaged by means of a Nipkow confocal microscope, and fluo-3. Under physiologic conditions, a local release of Ca2+ propagated in atrial myocytes, not in ventricular myocytes. Inhibition of SR Ca2+ uptake (500 nM thapsigargin), and of Ca2+ extrusion via Na/Ca exchange (5mM Ni2+), did not result in propagation in ventricular myocytes. The density of mitochondria was greater in ventricular than in atrial myocytes, although the abundance of ryanodine receptors and myofilaments was similar. Partial inhibition of Ca2+ uptake via the mitochondrial Ca2+ uniporter (5 microM Ru360) caused an increase in the [Ca2+]i transient in paced ventricular myocytes, and consistently resulted in propagation of Ca2+ release. This effect of Ru360 did not appear to be due to altered SR Ca2+ content. These data indicate that Ca2+ uptake via the mitochondrial uniporter occurs on a beat-to-beat basis, and may contribute to local control of Ca2+ release. Propagation of Ca2+ release in atrial myocytes may result in part from the relatively low density of mitochondria present.

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↗

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

Spontaneous activity induced in rabbit Purkinje myocytes during coupling to a depolarized model cell.

OBJECTIVE: The development of an "injury current" secondary to heterogeneous ion accumulation and cellular uncoupling across the ischemic border zone has been implicated as a trigger for arrhythmias arising during acute ischemia. The purpose of the present study was to determine the effects of injury current across the Purkinje-ventricular interface in the development of abnormal automaticity. METHODS: Patch clamp and electronic cell coupling techniques were used to record action potentials from and to apply injury current to isolated rabbit Purkinje myocytes. Injury current was dependent upon: (1) a coupling resistance, which was varied to simulate different degrees of cellular uncoupling, and (2) the difference in Purkinje membrane potential and depolarized ischemic myocardium, which was represented by a passive resistor-capacitor circuit with initial voltages of -70, -60, or -50 mV. RESULTS: During coupling to the moderately depolarized cell (-60 or -50 mV), Purkinje myocytes developed repetitive, spontaneous activity within a window of coupling resistances. This abnormal automaticity was dependent upon L-type calcium current, as cadmium or nifedipine completely suppressed coupling-induced spontaneous activity. CONCLUSIONS: Our results demonstrate that injury current alone can induce spontaneous activity in normal Purkinje myocytes. The level of myocardial depolarization and the degree of cellular uncoupling required to induce this activity suggest spontaneous Purkinje activity induced by injury current as a potent trigger for acute ischemic arrhythmias.

Action Potentials↗

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

Comparison of sarcoplasmic reticulum Ca2+-ATPase function in human, dog, rabbit, and mouse ventricular myocytes.

It has been reported that sarcoplasmic reticulum (SR) Ca(2+) uptake is more rapid in rat than rabbit ventricular myocytes, but little information is available on the relative SR Ca(2+) uptake activity in others species, including humans. We induced Ca(2+) transients with a short caffeine pulse protocol (rapid solution switcher, 10 mM caffeine, 100 ms) in single ventricular myocytes voltage clamped (-80 mV) with pipettes containing 100 microM fluo-3 and nominal 0 Ca(2+), in 0 Na(+)(o)/0 Ca(2+)(o) solution to inhibit Na/Ca exchange. SR in non-paced human, dog, rabbit, and mouse ventricular myocytes could be readily loaded with Ca(2+) under our experimental conditions with a pipette [Ca(2+)] = 100 nM. Resting [Ca(2+)](i) was similar in four types of ventricular myocytes. Activation of the Ca(2+)-release channel with a 100-ms caffeine pulse produced a rise in [caffeine](i) to slightly above 2 mM, the threshold for caffeine activation of Ca(2+) release. This caused a similar initial rate of rise and peak [Ca(2+)](i) in the four types of ventricular myocytes. However, there were significant differences in the duration of the plateau (top 10%) [Ca(2+)](i) transients and the time constant of the [Ca(2+)](i) decline (reflecting activity of the SR Ca(2+)-ATPase), with values for human > dog > rabbit > mouse. In paced myocytes under physiologic conditions, SR Ca(2+) content was greater in mouse than in rabbit myocytes, while peak I(Ca,L) was smaller in mouse. These findings confirm substantial species difference in SR Ca(2+)-ATPase activity, and suggest that the smaller the animal and the more rapid the heart rate, greater the activity of the SR Ca(2+)-ATPase. In addition, it appears that substantial species differences exist in the degree of SR Ca(2+) loading and I(Ca,L) under physiologic conditions.

Animals↗

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↗

PubMed2003

Ca2+ sparks induced by Na/Ca exchange.

Whether Ca(2+) influx on the Na/Ca exchanger (NCX) can trigger elementary sarcoplasmic reticulum (SR) Ca(2+) release events (Ca(2+) sparks) is controversial. We imaged [Ca(2+)](i) (Nipkow confocal microscope and fluo-3) in left ventricular myocytes isolated from wild type (WT) and transgenic (TG) mice overexpressing NCX 2.5-fold. Sudden activation of Ca(2+) influx via NCX induced by abrupt exposure to "0" [Na(+)](o)/normal [Ca(2+)](o) solution by means of a rapid solution switcher-induced Ca(2+) sparks in NCX TG myocytes in 425+/-17 ms, n=21. The diameter and amplitude (F/F(0)) of these sparks (2.74+/-0.14 microm, F/F(0)=2.16+/-0.06, n=18) were similar to those induced by field stimulation of myocytes in the presence of 20 microM nifedipine (2.70+/-0.10 microm, F/F(0)=1.98+/-0.08, n=17). In WT myocytes no Ca(2+) sparks were observed within the first 600 ms after abrupt removal of extracellular Na. In parallel experiments, voltage clamp current measurements (-80 mV) showed that the Na/Ca exchange current (I(NCX)) began within 60 ms of activation of the switcher, and peaked at 312+/-57 pA in TG myocytes within 300-500 ms. I(Ca,L) in 20 microM nifedipine was 10.3+/-4.3 pA, n=7. These results indicate that Ca(2+) entering the myocyte via NCX can cause Ca(2+) sparks which are similar to those elicited by electrical stimulation. However, Ca(2+) influx on NCX is much less efficient in inducing Ca(2+) sparks than Ca(2+) influx via I(Ca,L).

Animals↗

PubMed2002

Role of bicarbonate in the regulation of intracellular pH in the mammalian ventricular myocyte.

Bicarbonate is important for pHi control in cardiac cells. It is a major part of the intracellular buffer apparatus, it is a substrate for sarcolemmal acid-equivalent transporters that regulate intracellular pH, and it contributes to the pHo sensitivity of steady-state pHi, a phenomenon that may form part of a whole-body response to acid/base disturbances. Both bicarbonate and H+/OH- transporters participate in the sarcolemmal regulation of pHi, namely Na(+)-HCO3-cotransport (NBC), Cl(-)-HCO3- exchange (i.e., anion exchange, AE), Na(+)-H+ exchange (NHE), and Cl(-)-OH- exchange (CHE). These transporters are coupled functionally through changes of pHi, while pHi is linked to [Ca2+]i through secondary changes in [Na+] mediated by NBC and NHE. Via such coupling, decreases of pHo and pHi can ultimately lead to an elevation of [Ca2+]i, thereby influencing cardiac contractility and electrical rhythm. Bicarbonate is also an essential component of an intracellular carbonic buffer shuttle that diffusively couples cytoplasmic pH to the sarcolemma and minimises the formation of intracellular pH microdomains. The importance of bicarbonate is closely linked to the activity of the enzyme carbonic anhydrase (CA). Without CA activity, intracellular bicarbonate-dependent buffering, membrane bicarbonate transport, and the carbonic shuttle are severely compromised. There is a functional partnership between CA and HCO3- transport. Based on our observations on intracellular acid mobility, we propose that one physiological role for CA is to act as a pH-coupling protein, linking bulk pH to the allosteric H+ control sites on sarcolemmal acid/base transporters.

Acid-Base Equilibrium↗