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.     

Intracellular osmotic gradient generation in rat ventricle myocyte by using a micro-perfusion system

This experimental study describes the fabrication and analysis of a micro-perfusion system that can be used in many bioengineering experiments to create rapid, large regional intracellular changes within single ventricular myocytes. The myocyte was a kind of osmometer since the cell volume was found to be strongly dependent on the perfusion solution osmolarity. This volume change was measured, indirectly, by measuring the cell width change using video-microscopy and image analysis software. Jacob's equation was used to model these results successfully. Some dual perfusion experiments to see the effects of the localized perfusion of different osmotic solutions to generate an osmotic gradient inside myocytes were also investigated. This device can be useful for studying the effects of localized pH or osmotic gradients inside myocytes, estimating intracellular ion diffusion rates, and inducing regional changes in other important intracellular ions.     

Intracellular osmotic gradient generation in rat ventricle myocyte by using a micro-perfusion system

This experimental study describes the fabrication and analysis of a micro-perfusion system that can be used in many bioengineering experiments to create rapid, large regional intracellular changes within single ventricular myocytes. The myocyte was a kind of osmometer since the cell volume was found to be strongly dependent on the perfusion solution osmolarity. This volume change was measured, indirectly, by measuring the cell width change using video-microscopy and image analysis software. Jacob's equation was used to model these results successfully. Some dual perfusion experiments to see the effects of the localized perfusion of different osmotic solutions to generate an osmotic gradient inside myocytes were also investigated. This device can be useful for studying the effects of localized pH or osmotic gradients inside myocytes, estimating intracellular ion diffusion rates, and inducing regional changes in other important intracellular ions.     

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.     

Beta-myosin heavy chain myocytes are more resistant to changes in power output induced by ischemic conditions

During ischemia intracellular concentrations of P(i) and H+ increase. Also, changes in myosin heavy chain (MHC) isoform toward beta-MHC have been reported after ischemia and infarction associated with coronary artery disease. The purpose of this study was to investigate the effects of myoplasmic changes of P(i) and H+ on the loaded shortening velocity and power output of cardiac myocytes expressing either alpha- or beta-MHC. Skinned cardiac myocyte preparations were obtained from adult male Sprague-Dawley rats (control or treated with 5-n-propyl-2-thiouracil to induce beta-MHC) and mounted between a force transducer and servomotor system. Myocyte preparations were subjected to a series of isotonic force clamps to determine shortening velocity and power output during Ca2+ activations in each of the following solutions: 1) pCa 4.5 and pH 7.0; 2) pCa 4.5, pH 7.0, and 5 mM P(i); 3) pCa 4.5 and pH 6.6; and 4) pCa 4.5, pH 6.6, and 5 mM P(i). Added P(i) and lowered pH each caused isometric force to decline to the same extent in alpha-MHC and beta-MHC myocytes; however, beta-MHC myocytes were more resistant to changes in absolute power output. For example, peak absolute power output fell 53% in alpha-MHC myocytes, whereas power fell only 38% in beta-MHC myocytes in response to elevated P(i) and lowered pH (i.e., solution 4). The reduced effect on power output was the result of a greater increase in loaded shortening velocity induced by P(i) in beta-MHC myocytes and an increase in loaded shortening velocity at pH 6.6 that occurred only in beta-MHC myocytes. We conclude that the functional response to elevated P(i) and lowered pH during ischemia is MHC isoform-dependent with beta-MHC myocytes being more resistant to declines in power output.     

Na/K pump-induced [Na](i) gradients in rat ventricular myocytes measured with two-photon microscopy

Via the Na/Ca and Na/H exchange, intracellular Na concentration ([Na](i)) is important in regulating cardiac Ca and contractility. Functional data suggest that [Na](i) might be heterogeneous in myocytes that are not in steady state, but little direct spatial information is available. Here we used two-photon microscopy of SBFI to spatially resolve [Na](i) in rat ventricular myocytes. In vivo calibration yielded an apparent K(d) of 27 +/- 2 mM Na. Similar resting [Na](i) was found using two-photon or single-photon ratiometric measurements with SBFI (10.8 +/- 0.7 vs. 11.1 +/- 0.7 mM). To assess longitudinal [Na](i) gradients, Na/K pumps were blocked at one end of the myocyte (locally pipette-applied K-free extracellular solution) and active in the rest of the cell. This led to a marked increase in [Na](i) at sites downstream of the pipette (where Na enters the myocyte and Na/K pumps are blocked). [Na](i) rise was smaller at upstream sites. This resulted in sustained [Na](i) gradients (up to approximately 17 mM/120 microm cell length). This implies that Na diffusion in cardiac myocytes is slow with respect to trans-sarcolemmal Na transport rates, although the mechanisms responsible are unclear. A simple diffusion model indicated that such gradients require a Na diffusion coefficient of 10-12 microm(2)/s, significantly lower than in aqueous solutions.     

alpha(4)-integrin mediates neutrophil-induced free radical injury to cardiac myocytes

Previous work has demonstrated that circulating neutrophils (polymorphonuclear leukocytes [PMNs]) adhere to cardiac myocytes via beta(2)-integrins and cause cellular injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. Since PMNs induced to leave the vasculature (emigrated PMNs) express the alpha(4)-integrin, we asked whether (a) these PMNs also induce myocyte injury via NADPH oxidase; (b) beta(2)-integrins (CD18) still signal oxidant production, or if this process is now coupled to the alpha(4)-integrin; and (c) dysfunction is superoxide dependent within the myocyte or at the myocyte-PMN interface. Emigrated PMNs exposed to cardiac myocytes quickly induced significant changes in myocyte function. Myocyte shortening was decreased by 30-50% and rates of contraction and relaxation were reduced by 30% within the first 10 min. Both alpha(4)-integrin antibody (Ab)-treated PMNs and NADPH oxidase-deficient PMNs were unable to reduce myocyte shortening. An increased level of oxidative stress was detected in myocytes within 5 min of PMN adhesion. Addition of an anti-alpha(4)-integrin Ab, but not an anti-CD18 Ab, prevented oxidant production, suggesting that in emigrated PMNs the NADPH oxidase system is uncoupled from CD18 and can be activated via the alpha(4)-integrin. Addition of exogenous superoxide dismutase (SOD) inhibited all parameters of dysfunction measured, whereas overexpression of intracellular SOD within the myocytes did not inhibit the oxidative stress or the myocyte dysfunction caused by the emigrated PMNs. These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed alpha(4)-integrin functions as the ligand that signals oxidant production. The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the alpha(4)-integrin-coupled NADPH oxidase pathway that can be inhibited by extracellular, but not intracellular SOD.     

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.     

Coverslip hypoxia: a novel method for studying cardiac myocyte hypoxia and ischemia in vitro

In vitro experimental models designed to study the effects of hypoxia and ischemia typically employ oxygen-depleted media and/or hypoxic chambers. These approaches, however, allow for metabolites to diffuse away into a large volume and may not replicate the high local concentrations that occur in ischemic myocardium in vivo. We describe herein a novel and simple method for creating regional hypoxic and ischemic conditions in neonatal rat cardiac myocyte monolayers. This method consists of creating a localized diffusion barrier by placing a glass coverslip over a portion of the monolayer. The coverslip restricts covered myocytes to a thin film of media while leaving uncovered myocytes free to access the surrounding bulk media volume. Myocytes under the coverslip undergo marked morphology changes over time as assessed by video microscopy. Fluorescence microscopy shows that these changes are accompanied by alterations in mitochondrial membrane potential and plasma membrane dynamics and eventually result in myocyte death. We also show that the metabolic activity of myocytes drives cell necrosis under the coverslip. In addition, the intracellular pH of synchronously contracting myocytes under the coverslip drops rapidly, which further implicates metabolic activity in regulating cell death under the coverslip. In contrast with existing models of hypoxia/ischemia, this technique provides a simple and effective way to create hypoxic/ischemic conditions in vitro. Moreover, we conclude that myocyte death is hastened by the combination of hypoxia, metabolites, and acidosis and is facilitated by a reduction in media volume, which may better represent ischemic conditions in vivo.     

Effects of acidosis on ventricular myocyte shortening and intracellular Ca2+ in streptozotocin-induced diabetic rats

We have investigated the effects of acute acidosis on ventricular myocyte shortening and intracellular Ca2+ in streptozotocin (STZ)-induced diabetic rat. Shortening and intracellular Ca2+ were measured in electrically stimulated myocytes superfused with either normal Tyrode solution pH adjusted to either 7.4 (control solution) or 6.4 (acid solution). Experiments were performed at 35-36 degrees C. At 8-12 weeks after treatment, the rats that received STZ had lower body and heart weights compared to controls, and blood glucose was characteristically increased. Contractile defects in myocytes from diabetic rat were characterized by prolonged time to peak shortening. Superfusion of myocytes from control and diabetic rats with acid solution caused a significant reduction in the amplitude of shortening; however, the magnitude of the response was not altered by STZ treatment. Acid solution also caused significant and quantitatively similar reductions in the amplitude of Ca2+ transients in myocytes from control and diabetic rats. Effects of acute acidosis on amplitude of myocyte contraction and Ca2+ transient were not significantly altered by STZ treatment. Altered myofilament sensitivity to Ca2+ and altered mechanisms of sarcoplasmic reticulum Ca2+ transport might partly underlie the acidosis-evoked reduction in amplitude of shortening in myocytes from control and STZ-induced diabetic rat.     

Enhanced activity of ventricular Na+-HCO3- cotransport in pressure overload hypertrophy

The Na(+)-HCO(3)(-) cotransporter (NBC) plays a key role in intracellular pH (pH(i)) regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. pH(i) was measured in single cells using the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)5-(6)carboxyfluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCe1 (SLC4A4) and electroneutral NBCn1 (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC (J(NBC)) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via Na(+)/H(+) exchange was also increased in hypertrophied myocytes, the relative enhancement of J(NBC) was larger, 6) membrane depolarization markedly increased J(NBC) in hypertrophied myocytes, and 7) losartan, an ANG II AT(1) receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular Na(+) overload, which may render the ventricles more vulnerable to Ca(2+) overload during ischemia-reperfusion.     

Morphological and functional changes in cardiac myocytes isolated from mice overexpressing TNF-alpha

Transgenic (TG) TNF1.6 mice, which cardiac specifically overexpress tumor necrosis factor-alpha (TNF-alpha), exhibit heart failure (HF) and increased mortality, which is markedly higher in young (<20 wk) males (TG-M) than females (TG-F). HF in this model may be partly caused by remodeling of the extracellular matrix and/or structure/function alterations at the single myocyte level. We studied left ventricular (LV) structure and function using echocardiography and LV myocyte morphometry, contractile function, and intracellular Ca(2+) (Ca(i)(2+)) handling using cell edge detection and fura 2 fluorescence, respectively, in 12-wk-old TG-M and TG-F mice and their wild-type (WT) littermates. TG-F mice showed LV hypertrophy without dilatation and only a small reduction of basal fractional shortening (FS) and response to isoproterenol (Iso). TG-M mice showed a large LV dilatation, higher mRNA levels of beta-myosin heavy chain and atrial natriuretic factor versus TG-F mice, reduced FS relative to both WT and TG-F mice, and minimal response to Iso. TG-F and TG-M myocytes were similarly elongated (by approximately 20%). The amplitude of Ca(i)(2+) transients and contractions and the response to Iso were comparable in WT and TG-F myocytes, whereas the time to 50% decline (TD(50%)) of the Ca(i)(2+) transient, an index of the rate of sarcoplasmic reticulum Ca(2+) uptake, was prolonged in TG-F myocytes. In TG-M myocytes, the amplitudes of Ca(i)(2+) transients and contractions were reduced, TD(50%) of the Ca(i)(2+) transient was prolonged, and the inotropic effect of Iso on Ca(i)(2+) transients was reduced approximately twofold versus WT myocytes. Protein expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and phospholamban was unaltered in TG versus WT hearts, suggesting functional origins of impaired Ca(2+) handling in the former. These results indicate that cardiac-specific overexpression of TNF-alpha induces myocyte hypertrophy and gender-dependent alterations in Ca(i)(2+) handling and contractile function, which may at least partly account for changes in LV geometry and in vivo cardiac function in this model.     

Nonesterified fatty acid accumulation and release during heart muscle-cell (myocyte) injury: modulation by extracellular "acceptor"

Long-chain nonesterified fatty acid (NEFA) accumulation in the heart muscle cell (myocyte) and NEFA release to the extracellular milieu are considered contributors to the pathogenesis of myocardial injury in a number of cardiovascular disease states. Reported here is a study of the factors which influence and control the interactions among NEFA formation, intracellular NEFA accumulation, and NEFA release to the extracellular compartment by the irreversibly injured myocyte. Under conditions of metabolic inhibition, neonatal rat myocytes in primary monolayer culture became virtually depleted of ATP within 8 h. The metabolically inhibited myocytes evidenced membrane phospholipid degradation and a resultant net accumulation of NEFA produced thereby in the extracellular medium. However, under conditions of nutrient deprivation, the injured myocytes retained the NEFA produced from phospholipid catabolism intracellularly and did not release it to the culture medium, although the extent of myocyte ATP depletion was the same as it had been from metabolic inhibition. Serum could elicit, in a concentration-dependent fashion, the quantitative release of NEFA from metabolically inhibited myocytes to the culture medium but did not influence the net production of NEFA by the injured cells. Similarly, NEFA release from nutrient-deprived myocytes incubated in serum-free, substrate-free medium or in physiological buffer could be induced by supplementing the medium or buffer with bovine serum albumin (BSA), and the extent of NEFA release, but not NEFA formation, was dependent upon the extracellular BSA concentration. No manipulations to media other than changing their serum content or supplementing them with BSA were found to influence the disposition of NEFA produced during phospholipid catabolism in the irreversibly injured, ATP-depleted myocyte. Therefore, although progressive metabolic compromise in the myocyte was correlated with increasing, net NEFA formation, the distribution of the NEFA between the intracellular and the extracellular compartments was not determined by the magnitude of ATP loss or by the nature or duration of at least two injury stimuli, metabolic inhibition and nutrient deprivation. Rather, the net release of NEFA from the ATP-depleted myocyte to the culture medium and the consequent reduction of intracellular myocyte NEFA overload were critically and causally dependent upon the presence and concentration of extracellular NEFA "acceptor". The influence of acceptor on the mobilization of NEFA from the injured myocyte has implications regarding the use of NEFA release as an index of myocyte pathology and could serve to modify the progression and extent of myocardial injury in vivo.     

Stimulation of single isolated adult ventricular myocytes within a low volume using a planar microelectrode array

Microchannels (40- microm wide, 10- microm high, 10-mm long, 70- microm pitch) were patterned in the silicone elastomer, polydimethylsiloxane on a microscope coverslip base. Integrated within each microchamber were individually addressable stimulation electrodes (40- microm wide, 20- microm long, 100-nm thick) and a common central pseudo-reference electrode (60- microm wide, 500- microm long, 100-nm thick). Isolated rabbit ventricular myocytes were introduced into the chamber by micropipetting and subsequently capped with a layer of mineral oil, thus creating limited volumes of saline around individual myocytes that could be varied from 5 nL to 100 pL. Excitation contraction coupling was studied by monitoring myocyte shortening and intracellular Ca(2+) transients (using Fluo-3 fluorescence). The amplitude of stimulated myocyte shortening and Ca(2+) transients remained constant for 90 min in the larger volume (5 nL) configuration, although the shortening (but not the Ca(2+) transient) amplitude gradually decreased to 20% of control within 60 min in the low volume (100 pL) arrangement. These studies indicate a lower limit for the extracellular volume required to stimulate isolated adult cardiac myocytes. Whereas this arrangement could be used to create a screening assay for drugs, individual microchannels (100 pL) can also be used to study the effects of limited extracellular volume on the contractility of single cardiac myocytes.     

Effects of acidosis on ventricular myocyte shortening and intracellular Ca2+ in streptozotocin-induced diabetic rats

We have investigated the effects of acute acidosis on ventricular myocyte shortening and intracellular Ca²⁺ in streptozotocin (STZ)-induced diabetic rat. Shortening and intracellular Ca²⁺ were measured in electrically stimulated myocytes superfused with either normal Tyrode solution pH adjusted to either 7.4 (control solution) or 6.4 (acid solution). Experiments were performed at 35–36°C. At 8–12 weeks after treatment, the rats that received STZ had lower body and heart weights compared to controls, and blood glucose was characteristically increased. Contractile defects in myocytes from diabetic rat were characterized by prolonged time to peak shortening. Superfusion of myocytes from control and diabetic rats with acid solution caused a significant reduction in the amplitude of shortening; however, the magnitude of the response was not altered by STZ treatment. Acid solution also caused significant and quantitatively similar reductions in the amplitude of Ca²⁺ transients in myocytes from control and diabetic rats. Effects of acute acidosis on amplitude of myocyte contraction and Ca²⁺ transient were not significantly altered by STZ treatment. Altered myofilament sensitivity to Ca²⁺ and altered mechanisms of sarcoplasmic reticulum Ca²⁺ transport might partly underlie the acidosis-evoked reduction in amplitude of shortening in myocytes from control and STZ-induced diabetic rat. (Mol Cell Biochem 261: 227–233, 2004)     

Uptake of basic amino acids and peptides into liposomes in response to transmembrane pH gradients.

The uptake of derivatives of lysine and a pentapeptide (ala-met-leu-trp-ala) into large unilamellar vesicle (LUV) systems in response to transmembrane pH gradients has been examined. In these derivatives, the C-terminal carboxyl functions have been converted to methyl esters or amides. It is shown that the presence of a pH gradient (interior acidic) results in the rapid and efficient accumulation of these weak base amino acid and peptide derivatives into LUVs in a manner consistent with permeation of the neutral (deprotonated) form. It is suggested that this property may have general implications for mechanisms of transbilayer translocation of peptides, such as signal sequences, which exhibit weak base characteristics.     

pH(i) responses to osmotic cell shrinkage in the presence of open-system buffers.

Changes in plasma volume in vivo cause rapid changes in extracellular pH by altering the plasma bicarbonate concentration at a constant Pco(2) (Garella S, Chang BS, and Kahn SI. Kidney Int 8: 279, 1975). Few studies have examined the possibility that changes in cell volume produce comparable changes in intracellular pH (pH(i)). In the present study, alveolar macrophages were exposed to hyperosmotic medium in the absence or presence of the open-system buffers CO(2)-HCO(3)(-), propionic acid-propionate, or NH(3)-NH(4)(+). In the absence of open-system buffers, exposure to twice-normal osmolarity (2T) produced a slow cellular alkalinization [change in pH(i) (DeltapH(i)) approximately 0.38; exponential time constant (tau) approximately 120 s]. In the presence of 5% CO(2), 2T caused a biphasic pH(i) response: a rapid increase (DeltapH(i) approximately 0.10, tau approximately 15 s) followed by a slower pH(i) increase. Identical rapid pH(i) increases were produced by 2T in the presence of propionic acid (20 mM). Conversely, 2T caused a rapid pH(i) decrease (DeltapH(i) approximately -0.21, tau approximately 10 s) in the presence of NH(3) (20 mM). Thus osmotic cell shrinkage caused rapid pH(i) changes of opposite direction in the presence of a weak acid buffer (contraction alkalosis with CO(2) or propionic acid) vs. a weak base buffer (contraction acidosis with NH(3)). Graded DeltapH(i) were produced by varying extracellular osmolarity in the presence of open-system buffers; osmolarity increases of as little as 5-10% produced significant DeltapH(i). The rapid pH(i) responses to 2T were insensitive to inhibitors of membrane H(+) transport (ethylisopropylamiloride and bafilomycin A(1)). The results are consistent with shrinkage-induced disequilibria in the total cellular buffer system (i.e., intrinsic buffers plus added weak acid-base buffer).     

Distribution of isomyosin in cultured cardiac myocytes as determined by monoclonal antibodies and adenosine triphosphatase activity

The distribution of isomyosin in cardiac muscle cells in culture has been investigated with monoclonal antibodies and Ca2+-activated myosin ATPase cytochemical staining. With immunofluorescent studies using monoclonal antibodies to isomyosins V1 and V3, the cardiac myocytes grown in a serum-free and thyroxine (T4)-free medium for 7 days contained a predominant population of cells which were strongly reactive to anti-V3 antibody. A small population of myocytes in this culture exhibited weak or no reaction to anti-V3 antibody. When cultures were exposed to anti-V1 antibody, the predominant cardiac myocyte population showed little or no reactivity to this antibody, whereas a small population of the myocytes were strongly reactive. The myosin ATPase staining reaction of the positive myocyte population was significantly less pronounced than that of the V3-negative population which showed a strong reaction. The staining pattern changed dramatically after exposure of cultured myocytes to thyroid hormone for 7 days. Most of the cells were found to react strongly with anti-V1 antibody, while some cells showed little reactivity and some were not stained at all. A small number of cardiac myocytes in this culture showed little or no reactivity to anti-V1 antibody but were strongly reactive to anti-V3 antibody. The predominant anti-V1-positive myocyte population exhibited strong myosin ATPase staining as compared to a smaller V3-positive myocyte population which showed very weak staining. The cytochemical results of ATPase staining in cardiac myocytes agreed well with ATPase activity as determined on pyrophosphate gels containing isomyosin derived from cultured cardiac myocytes with or without T4. This study has demonstrated that cultured myocytes contain a small population of muscle cells which is not responsive to thyroid hormone or to the lack of it.     

Regulation of AE2-mediated Cl- transport by intracellular or by extracellular pH requires highly conserved amino acid residues of the AE2 NH2-terminal cytoplasmic domain

We reported recently that regulation by intracellular pH (pH(i)) of the murine Cl-/HCO(3)(-) exchanger AE2 requires amino acid residues 310-347 of the polypeptide's NH(2)-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pH(o)) with unclamped or clamped pH(i), or during variation of pH(i) at constant pH(o). Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited by acid pH(o), with a value of pH(o50) = 6.87 +/- 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312-347 identified the greatest acid shift in pH(o(50)) value, approximately 0.8 pH units in the mutant (A)6 342-347, but only a modest acid-shift in the mutant (A)6 336-341. Two of the six (A)6 mutants retained normal pH(i) sensitivity of 36Cl- efflux, whereas the (A)6 mutants 318-323, 336-341, and 342-347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336-347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH(o) and to pH(i) were found independently and in concert. The E346A mutation acid-shifted the pH(o(0) value to the same extent whether pH(i) was unclamped or held constant during variation of pH(o). Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pH(o) as well as pH(i).     

Ouabain interaction with cardiac Na+/K+-ATPase initiates signal cascades independent of changes in intracellular Na+ and Ca2+ concentrations

We have shown previously that partial inhibition of the cardiac myocyte Na(+)/K(+)-ATPase activates signal pathways that regulate myocyte growth and growth-related genes and that increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and reactive oxygen species (ROS) are two essential second messengers within these pathways. The aim of this work was to explore the relation between [Ca(2+)](i) and ROS. When myocytes were in a Ca(2+)-free medium, ouabain caused no change in [Ca(2+)](i), but it increased ROS as it did when the cells were in a Ca(2+)-containing medium. Ouabain-induced increase in ROS also occurred under conditions where there was little or no change in [Na(+)](i). Exposure of myocytes in Ca(2+)-free medium to monensin did not increase ROS. Increase in protein tyrosine phosphorylation, an early event induced by ouabain, was also independent of changes in [Ca(2+)](i) and [Na(+)](i). Ouabain-induced generation of ROS in myocytes was antagonized by genistein, a dominant negative Ras, and myxothiazol/diphenyleneiodonium, indicating a mitochondrial origin for the Ras-dependent ROS generation. These findings, along with our previous data, indicate that increases in [Ca(2+)](i) and ROS in cardiac myocytes are induced by two parallel pathways initiated at the plasma membrane: One being the ouabain-altered transient interactions of a fraction of the Na(+)/K(+)-ATPase with neighboring proteins (Src, growth factor receptors, adaptor proteins, and Ras) leading to ROS generation, and the other, inhibition of the transport function of another fraction of the Na(+)/K(+)-ATPase leading to rise in [Ca(2+)](i). Evidently, the gene regulatory effects of ouabain in cardiac myocytes require the downstream collaborations of ROS and [Ca(2+)](i).