Am J Physiol Regul Integr Comp Physiol 293: R1684R1692, 2007. First published July 18, 2007; doi:10.1152/ajpregu.00219.2007.

Burn injury decreases myocardial Na-K-ATPase activity: role of PKC inhibition

Jureta W. Horton, Jing Tan, D. Jean White, and David L. Maass
Department of Surgery, University of Texas Southwestern Medical Center, Dallas, Texas
Submitted 30 March 2007; accepted in nal form 16 July 2007

Horton JW, Tan J, White DJ, Maass DL. Burn injury decreases myocardial Na-K-ATPase activity: role of PKC inhibition. Am J Physiol Regul Integr Comp Physiol 293: R1684R1692, 2007. First published July 18, 2007; doi:10.1152/ajpregu.00219.2007.Cardiomyocyte sodium accumulation after burn injury precedes the development of myocardial contractile dysfunction. The present study examined the effects of burn injury on Na-K-ATPase activity in adult rat hearts after major burn injury and explored the hypothesis that burn-related changes in myocardial Na-K-ATPase activity are PKC dependent. A third-degree burn injury (or sham burn) was given over 40% total body surface area, and rats received lactated Ringer solution (4 ml kg 1 % burn 1). Subgroups of rats were killed 2, 4, or 24 h after burn (n 6 rats/time period), hearts were homogenized, and Na-K-ATPase activity was determined from ouabain-sensitive phosphate generation from ATP by cardiac sarcolemmal vesicles. Additional groups of rats were studied at several times after burn to determine the time course of myocyte sodium loading and the time course of myocardial dysfunction. Additional groups of sham burninjured and burn-injured rats were given calphostin, an inhibitor of PKC, and Na-K-ATPase activity, cell Na , and myocardial function were measured. Burn injury caused a progressive rise in cardiomyocyte Na , and myocardial Na-K-ATPase activity progressively decreased after burn, while PKC activity progressively rose. Administration of calphostin to inhibit PKC activity prevented both the burn-related decrease in myocardial Na-K-ATPase and the rise in intracellular Na and improved postburn myocardial contractile performance. We conclude that burn-related inhibition of Na-K-ATPase likely contributes to the cardiomyocyte accumulation of intracellular Na . Since intracellular Na is one determinant of electrical-mechanical recovery after insults such as burn injury, burn-related inhibition of Na-K-ATPase may be critical in postburn recovery of myocardial contractile function. myocyte sodium; ventricular function; calphostin; protein kinase C inhibitor; rat burn injury model; protein kinase C activity

and disease states are associated with a rise in intracellular Na that, in turn, promotes Na /Ca2 exchange, a rise in cellular Ca2 , and cell injury (21, 29, 34, 47, 58). Pharmacological interventions that limit cellular accumulation of Na (for example, amiloride inhibits the Na /H exchanger and correction of intracellular acidosis eliminates the stimulus for Na /H exchange) have been shown to improve cardiac performance (26, 40, 42, 43). Recent data have suggested that activity of the Na-K-ATPase during injury and disease is regulated by PKC, which produces phosphorylation of serine and threonine residues on the Na-K-ATPase, reducing ion transport function (6, 11, 16, 59). Since major burn injury increases myocardial PKC activity, increases cardiac myocyte Na levels, and impairs myocardial contraction and relaxation (21, 48, 58), we postulated that postburn inhibition of PKC would alter burn-related changes in myocardial Na-K-ATPase activity, reducing myocyte Na accumulation and improving myocardial contractile performance. To examine this hypothesis, we used a rat model of burn injury over 40% of the total body surface area (TBSA) and inhibited PKC activity with a specic inhibitor, calphostin. Na-K-ATPase activity, PKC activity, myocyte Na , and left ventricular performance were measured in hearts collected at several time points after burn injury.
MATERIALS AND METHODS

ion exchange protein functions to transport Na out of and K into the cardiac myocyte. The Na-K-ATPase transporter plays a major role in electricalmechanical activity of cardiac muscle, establishing a transarcolemmal Na gradient that, in turn, generates the rapid rise of the action potential and contributes to numerous ion transport functions that maintain cell homeostasis (19, 36). Na-KATPase function is dependent on ATP availability and is regulated, in part, by phosphorylation by protein kinase C (PKC) (29). Myocardial contractile dysfunction has been linked to defects in the functional activity of several ion exchange proteins, including Na-K-ATPase (5, 14, 15, 31). In this regard, myocardial ischemia and dysfunction that occur in several injury
Address for reprint requests and other correspondence: J. W. Horton, Dept. of Surgery, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9160 (e-mail: jureta.horton@utsouthwestern.edu). Aug. 20, 2007. R1684

THE MYOCARDIAL NA-K-ATPASE

Experimental model. Adult Sprague-Dawley male rats (320 350 g) were obtained from Harlan Laboratories (Houston, TX) and housed in the animal care facility. Commercial rat chow and tap water were available ad libitum, and rats were maintained at a constant temperature with a 12:12-h light-dark cycle. All work was performed under a protocol that was approved by the University of Texas Southwestern Medical Center Institutional Animal Care and Research Advisory Committee. The work conformed to the guidelines outlined in the Guiding Principles in the Care and Use of Animals of the American Physiological Society and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Burn procedure. Rats were deeply anesthetized with isourane and secured in a constructed template device as described previously (1). The skin exposed through the template was immersed in 100C water for 12 s on each side to produce a full-thickness dermal burn over 40% TBSA. This burn technique produces complete destruction of the underlying neural tissue. After immersion the rats were immediately dried, and each animal was placed in an individual cage. All burninjured animals received standard uid resuscitation consisting of 4 ml kg 1 % burn 1 lactated Ringer solution, with one-half of this calculated volume given intraperitoneally immediately after the burn injury was completed and the remaining volume given 8 h after burn injury. Buprenorphine (0.05 0.1 mg/kg) was given every 12 h during
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. http://www.ajpregu.org

0363-6119/07 $8.00 Copyright 2007 the American Physiological Society

PKC MODULATES NA-K-ATPASE ACTIVITY

R1685

the postburn period. Burn-injured rats did not display discomfort or pain, moved freely about the cage, and consumed food and water within 15 min after recovering from isourane anesthesia. Sham burn-injured rats also received identical regimens of analgesics (buprenorphine) throughout the study period. Protein extraction. Protein extraction of sarcolemmal membranes from rat heart tissue fractionation was carried out as described by Fuller et al. (19). All procedures were performed at 4C. Rat heart tissue was homogenized with a glass homogenizer in buffer containing (in mM) 20 HEPES, 250 sucrose, 2 EDTA, and 1 MgCl2, pH 7.4. To isolate a puried sarcolemmal/particulate (SLP) fraction, the resulting homogenate was centrifuged at 10,000 rpm for 20 min. The supernatant containing partially puried SLP fraction was centrifuged again for 30 min at 20,000 rpm, and the resulting pellet was suspended in lysis buffer. This SLP fraction contained sarcolemmal membranes as well as other subcellular components that do not contain Na-KATPase, including sarcoplasmic reticulum, nuclei, residual myolaments, and mitochondria. Measurement of PKC activity. PKC activity was measured in myocardial membrane with a PKC assay kit (Upstate Biotechnology, Lake Placid, NY). Following the manufacturers protocol, [ -32P]ATP (6,000 Ci/mmol stock solution) was used in this study. The reaction mixture included 2.5 l of 10 reaction buffer, 2.5 l of PKC substrate, 2.5 l of inhibitor cocktail, 2.5 l of freshly sonicated PKC lipid activator, and 50 g of sample protein. The reaction was initiated by adding 7 l of the Mg2 -ATP cocktail containing [ -32P]ATP, followed by incubation. The reaction was stopped by transferring 25 l of the reaction mixture onto the center of a P81 paper square (provided in the assay kit from Upstate Biotechnology). The assay squares were then washed three times for 5 min each with 0.75% phosphoric acid. Finally, the assay squares were washed once with acetone for 3 min and then transferred into scintillation vials; 5 ml of scintillation cocktail was added, and samples were counted with a scintillation counter. PKC activity was calculated as picomoles of phosphate per microgram of protein according to the manufacturers suggested protocol. Na-K-ATPase methods. Na-K-ATPase activity was measured in triplicate by the inorganic phosphate (Pi) released from ATP as described by Tsakiris (52). Cardiac sarcolemmal membrane protein (50 g) was used to measure total ATPase activity (Na,K,Mgdependent ATPase and Mg2 -dependent ATPase activity). The incubation medium contained (in mM) 120 NaCl, 20 KCl, 1 K-EDTA, 240 sucrose, 4 MgCl2, and 50 Tris HCl, pH 7.4, in a nal volume of 200 l. The reaction was initiated by addition of ATP to a nal concentration of 3.0 mM. Ouabain-insensitive Mg-ATPase was assayed under the same conditions after addition of 2 mM ouabain and without NaCl and KCl. The reaction was initiated by adding ATP at 37C and stopped after an incubation period of 15 min by addition of 60 l of an ice-cold mixture of 1% ammonium molybdate in 0.9 M H2SO4 (7, 52, 53). Released Pi was colorimetrically measured at 390 nm by the method of Chen and colleagues (10). ATPase activity of these preparations ranged from 0.8 to 1.5 mol Pi mg protein 1 min 1. Enzyme activity was determined after a 20-min preincubation with various concentrations of each myocardial protein sample. Each sample was assayed in triplicate, and Na-K-ATPase activity was calculated by the difference in values measured with and without ouabain. Specic activities of the enzymes were expressed as micromoles of Pi released per minute per milligram of protein. Experimental groups. To examine the time course of burn-related changes in myocardial PKC and Na-K-ATPase activities, burn-injured rats were killed at several times after burn (2, 4, and 24 h; n 5 or 6 rats/time period); hearts were collected and freeze clamped for in vitro study. Sham burn-injured rats were included for controls. To examine the time course of changes in cardiac contractile performance after burn injury and to correlate these changes in left ventricular performance with changes in myocyte Na levels, additional groups of burn-injured rats were killed 2, 4, 8, 12, 24, 48, or 72 h or 8 days after burn over 40% TBSA. Hearts were either perfused in vitro
AJP-Regul Integr Comp Physiol VOL

(Langendorff) to assess contraction and relaxation defects (n 79 rats per group per time) or perfused to isolate cardiac myocytes to measure intracellular sodium (n 4 or 5 rats per group per time). The effects of PKC inhibition on myocardial PKC and Na-KATPase activities, myocyte Na levels, and cardiac function were also examined in additional groups of rats treated with either vehicle (saline) or calphostin (0.1 mg/kg injected iv over 5 min, 30 min, 6 h, and 21 h after burn injury). The vehicle- and calphostin-treated groups were randomized and killed at baseline (control), 2, 4, or 24 h after burn injury to examine the effects of PKC inhibition on myocardial PKC and Na-K-ATPase activity (n 4 rats per experimental groups per time period). The effects of PKC inhibition on left ventricular function (n 79 rats per group) and myocyte sodium accumulation (n 4 rats/group) were studied 24 h after burn injury (a time when burn-related myocardial contractile decits were maximal); groups included group 1, sham burn vehicle; group 2, sham burn calphostin; group 3, burn injury vehicle; group 4, burn calphostin (n 79 rats/experimental group). The experimental protocol and times for tissue harvest are summarized in Fig. 1. Isolated heart perfusion. To examine the time course of cardiac contraction and relaxation responses to burn injury in the absence of calphostin therapy, subgroups of rats were anticoagulated with sodium heparin (200 U; Elkins-Sinn, Cherry Hill, NJ) and decapitated 2, 4, 8, 12, 24, 48, or 72 h or 8 days after burn over 40% TBSA (n 79 rats/time period). To examine the effects of PKC inhibition on myocardial performance, additional burn-injured rats (and shaminjured rats) treated with calphostin or vehicle after injury were anticoagulated and killed 24 h after burn injury, a time when myocardial defects were maximal after burn injury. The heart from each experimental animal was rapidly removed and placed in a petri dish containing ice-cold (4C) Krebs-Henseleit bicarbonate-buffered solution (in mM: 118 NaCl, 4.7 KCl, 21 NaHCO3, 1.25 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 11 glucose). All solutions were prepared

Fig. 1. Summary of experimental study design. SD, Sprague-Dawley; PKC, protein kinase C; LV, left ventricular.
293 OCTOBER 2007

www.ajpregu.org

R1686

PKC MODULATES NA-K-ATPASE ACTIVITY

Table 1. Time course of changes in left ventricular function and cardiac myocyte sodium levels
After Burn Sham 2h 4h 8h 12 h 18 h 24 h 48 h 72 h 8 Days

Na , mM LVP, mmHg dP/dtmax, mmHg/s dP/dtmax, mmHg/s

11 96 2,104 1,750

1 11 1 16 1 18 2 21 2* 2 97 4 94 6 80 5* 75 5* 56 2,167 88 2,146 156 1,819 112 1,613 130* 37 1,800 58 1,783 1,483 1,471 151 1,275 151*

24 67 1,500 1,260

1* 5* 163* 1,663*

39 63 1,309 1,025

2* 24 3* 15 1 12 4 4* 87 3* 87 4* 85 2* 59* 1,772 96 1,889 84 1,965 45 98* 1,593 83 1,683 78 1,710 49

All values are means SE. Sham, sham burn injury; Na , sodium measured in cardiac myocytes isolated at several times after injury; LVP, left ventricular pressure; dP/dtmax, maximum rise in LVP; dP/dtmax, maximum fall in LVP. *Signicant difference from sham burn at P 0.05 (ANOVA, multiplecomparison procedure).

each day with demineralized, deionized water and bubbled with 95% O2-5% CO2 (pH 7.4, PO2 550 mmHg, PCO2 38 mmHg). A cannula placed in the ascending aorta was connected via glass tubing to a buffer-lled reservoir for perfusion of the coronary circulation at a constant ow rate. Hearts were suspended in a temperature-controlled chamber maintained at 38C, and a constant ow pump (Ismatec, model 7335-30, Cole-Parmer Instrument, Chicago, IL) was used to maintain perfusion of the coronary artery (ml/min) by retrograde perfusion of the aortic stump cannula. Coronary perfusion pressure was measured, and efuent was collected to conrm coronary ow rate. Contractile function was assessed by measuring intraventricular pressure with a water-lled latex balloon attached to a polyethylene tube and threaded through the apex of the left ventricular chamber. Peak systolic left ventricular pressure (LVP) was measured with a Statham pressure transducer (model P23ID, Gould Instruments, Oxnard, CA) attached to the balloon cannula, and the rates of LVP rise ( dP/dt) and fall ( dP/dt) were obtained with an electronic differentiator (model 7P20C, Grass Instruments, Quincy, MA) and recorded (Grass model 7DWL8P). Left ventricular developed pressure was calculated from peak systolic LVP and left ventricular end-diastolic pressure. Data from the Grass recorder were input into a Dell Pentium computer, and a Grass PolyVIEW Data Acquisition System was used to convert acquired data into digital form. Cardiac myocyte isolation. After burn injury (or sham burn), animals from each experimental group (n 4 6 rats/group) were heparinized, blood samples were collected, and rats were decapitated; hearts were harvested and placed in a petri dish containing ice-cold heart medium [in mM: 113 NaCl, 4.7 KCl, 0.6 KH2PO4, 0.6 Na2HPO4, 1.2 MgSO4, 12 NaHCO3, 10 KHCO3, 20 D-glucose, 10 HEPES; 30 taurine, 2.0 carnitine, and 2.0 creatine with 0.5 minimum essential medium (MEM) and amino acids (50 , GIBCO/BRL 11130-051)]. Hearts were cannulated via the aorta and perfused with heart medium at a rate of 12 ml/min for a total of 5 min in a nonrecirculating mode. Enzymatic digestion was initiated by perfusing the heart with digestion solution, which contained 34.5 ml of the heart medium described above plus 50 mg of collagenase II (Worthington 4177, lot no. MOB3771), 50 mg of bovine serum albumin (BSA, endotoxin free), fraction V (GIBCO/BRL 11018-025), 0.5 ml of trypsin (2.5%, 10 , GIBCO/BRL 15090-046), and 15 M CaCl2. Enzymatic digestion was accomplished by recirculating this solution through the heart at a ow rate of 12 ml/min for 20 min. All solutions perfusing the heart were maintained at a constant temperature of 37C. At the end of the enzymatic digestion, the ventricles were removed and mechanically disassociated in 6 ml of enzymatic digestion solution containing a 6-ml aliquot of 2 BSA solution (2 mg BSA fraction V to 100 ml of heart medium). After mechanical dissociation with ne forceps, the tissue homogenate was ltered through a mesh lter into a conical tube. The cells adhering to the lter were collected by washing with an additional 10-ml aliquot of 1 BSA solution (100 ml of heart medium described above and 1 g of BSA fraction V). Cells were then allowed to pellet in the conical tube for 10 min. The supernatant was removed, and the pellet was resuspended in 10 ml of 1 BSA. The cells were washed and pelleted further in BSA buffer
AJP-Regul Integr Comp Physiol VOL

with increasing increments of calcium (100, 200, 500 M, to a nal concentration of 1,000 M). After the nal pelleting step the supernatant was removed, and the pellet was resuspended in MEM [prepared by adding 10.8 g of 1 MEM (Sigma M-1018), 11.9 mM NaHCO3, 10 mM HEPES, and 10 ml penicillin-streptomycin, 100 , GIBCO/BRL 1540-122 with 950 ml of MilliQ water]; total volume was adjusted to 1 liter. At the time of MEM preparation, the medium was bubbled with 95% O2-5% CO2 for 15 min and the pH was adjusted to 7.1 with 1 M NaOH. The solution was then lter sterilized and stored at 4C until use. At the nal concentration of calcium, the cardiomyocyte cell number was calculated and myocyte viability was determined. Measurement of myocyte-derived cytokines. Myocytes were pipetted into microtiter plates at 5 104 cells ml 1 well 1 (12-well cell culture cluster, Corning, Corning NY) and incubated for 18 h (CO2 incubator at 37C). Supernatants were collected to measure myocytesecreted TNF- , IL-1 , IL-6, and IL-10 (rat ELISA, Endogen, Woburn, MA). We previously examined (22) the contribution of contaminating cells (nonmyocytes) in our cardiomyocyte preparations with ow cytometry, cell staining (hematoxylin and eosin), and light microscopy. We conrmed that 2% of the total cell number in a myocyte preparation was noncardiomyocytes. Since immediately after myocyte isolation our preparations are 98% cardiomyocytes, we concluded that a majority of the inammatory cytokines measured in the cardiomyocyte supernatant were indeed cardiomyocyte derived (22). Myocyte viability after 24-h incubation was 96 97%. Cardiac myocyte Na levels. Additional aliquots of cardiac myocytes were loaded with sodium-binding benzofuran isophthalate

Fig. 2. Time course of PKC activity measured in hearts from vehicle-treated sham burn-injured animals and in groups of hearts harvested 2, 4, or 24 h after vehicle-treated burn over 40% total body surface area (open bars) and effect of calphostin treatment (lled bars). All values are means SE. *Signicant difference from sham burn (P 0.05); signicant difference between vehicle-treated burn and calphostin-treated burn at each time point (P 0.05).
293 OCTOBER 2007

www.ajpregu.org

PKC MODULATES NA-K-ATPASE ACTIVITY

R1687

length the uorescence emissions were collected for 1-min intervals, and the time between data collection was 12 min. Since quiescent or noncontracting myocytes were used in these studies, the Na levels measured reect diastolic levels. Statistical analysis. All values are expressed as means SE. Analysis of variance (ANOVA) was used to assess an overall difference among the groups for each of the variables. Cardiac function determined by the Langendorff preparation (including stabilization data) is expressed as the mean SE, and separate analysis were performed for each LVP, maximum dP/dt ( dP/dtmax), and maximum dP/dt ( dP/dtmax) as a function of treatment group and coronary ow rate with a repeated-measures ANOVA. Levenes test for equality of variance was used to suggest the multiple comparison procedure to be used. If equality of variance among the four groups was suggested, multiple-comparison procedures were performed (Bonferroni). Probability values 0.05 were considered statistically signicant (analysis was performed with SPSS for Windows, version 7.5.1).
Fig. 3. Na-K-ATPase activity measured in hearts harvested from vehicletreated sham-burned rats and in separate groups of hearts harvested 2, 4, or 24 h after vehicle-treated burn injury (open bars) and effects of PKC inhibitor calphostin on myocardial Na-K-ATPase activity (lled bars). All values are means SE. *Signicant difference from sham burn (P 0.05); signicant difference between vehicle-treated burn and calphostin-treated burn at each time point (P 0.05). RESULTS

(SBFI) for 1 h at room temperature in the dark. Myocytes were then suspended in 1.0 mM calcium-containing MEM and washed to remove extracellular dye; myocytes were placed on a glass slide on the stage of a Nikon inverted microscope. The microscope was interfaced with Grooney optics for epi-illumination, a triocular head, phase optics, and 30 phase-contrast objective and mechanical stage. This InCyt Im2 Fluorescence Imaging System (Intracellular Imaging, Cincinnati, OH) included an imaging workstation and computer. The computer-controlled lter changer allowed alternation between the 340- and 380-nm excitation wavelengths, and images were captured by monochrome charge-coupled device camera equipped with a TV relay lens. InCyt Im2 Image software allowed measurement of intracellular sodium concentrations from the ratio of the two uorescent signals generated from the two excitation wavelengths (340 nm/380 nm); background was removed by the InCyt IM2 software. The calibration procedure included measuring uorescence ratio with buffers containing different concentrations of sodium. At each wave-

Time course of myocyte sodium accumulation and time course of myocardial contractile dysfunction. Burn injury caused a progressive rise in cardiac myocyte Na over the rst 24 h after burn as measured by the uorescent indicator SBFI. As shown in Table 1, cardiomyocyte Na levels were significantly elevated as early as 12 h after burn over values measured in sham burn-injured animals (P 0.05), and a maximal increase in cardiac myocyte Na levels was evident 24 h after burn. Myocyte Na levels measured 72 h after burn returned to values that were comparable to those measured in sham burn-injured rats and remained at this basal level 8 days after burn injury (Table 1). Our time course studies examining cardiac contractile function showed signicant burn-mediated cardiac contraction and relaxation decits that were evident 12 h after burn injury, a time consistent with signicant myocyte Na loading (Table 1). LVP and dP/dtmax progressively fell after burn, achieving a nadir 24 h after burn. Left ventricular performance improved 48 h after burn compared with values measured 24 h

Table 2. Hemodynamic response to burn measured 24 h after burn in presence and absence of PKC inhibition
Group 1 Sham Vehicle Sham Group 2 Calphostin Group 3 Burn Vehicle Burn Group 4 Calphostin

MAP, mmHg HR, beats/min pH PCO2, mmHg PO2, mmHg Hct HCO3, mEq/l O2 Sat, % Lactate, mM/l Base excess, mM/l Ca2 , mM/l Na , mM/l Plasma TNF- , pg/ml Plasma IL-1 , pg/ml Plasma IL-6, pg/ml Plasma IL-10, pg/ml

150.6 523 7.47 23.5 116.0 43 19.5 99.8 2.20 1.64 1.29 141.2 4.7 2.3 5.96 3.9

4.9 12 0.04 1.2 4.5 3 0.7 0.2 0.3 0.4 0.01 0.6 0.3 0.3 0.4 0.3

150.5 491 7.49 29.8 96.8 38 22.0 99.4 2.4 1.58 1.2 145.3 5.5 3.2 5.7 2.8

4.3 12 0.01 1.4 3.0 0.5 0.4 0.2 0.3 0.5 0.02 0.3 1 0.4 3.0 .0.5*

111.6 500 7.48 26.2 112.8 32 21.4 99.6 3.3 2.17 0.87 138.8 12.4 8.3 24.9 13.8

8.3* 9 0.02 1.2 4.3 2.5* 0.2 0.1 0.4* 0.68* 0.04* 1.4 1.2* 0.6* 3.2* 1.0*

136.8 500 7.50 28.4 105.0 36 21.9 99.9 2.2 0.55 0.69 142.3 7.1 3.6 14 5.6

4* 7 0.01 0.7 7.3 1* 0.4 0.3 0.1 0.3* 0.05* 0.4 0.4* 0.3* 0.94* .0.2*

All values are means SE. PKC, protein kinase C; MAP, mean arterial pressure; HR, heart rate; Hct, hematocrit; HCO3, serum bicarbonate; O2 Sat, oxygen saturation; Ca2 , serum ionized calcium; Na , serum ionized sodium. *Signicant difference from sham burn at P 0.05. (ANOVA, multiple comparison procedure); signicant difference from burn vehicle (group 3 vs. group 4) at P 0.05. AJP-Regul Integr Comp Physiol VOL
293 OCTOBER 2007

www.ajpregu.org

R1688

PKC MODULATES NA-K-ATPASE ACTIVITY

after burn, but LVP measured 8 days after burn failed to return to values measured in sham burn-injured rats (P 0.05). Myocardial PKC activity and Na-K-ATPase activity. In this study, vehicle-treated burn injury caused a progressive rise in myocardial PKC activity in tissue harvested 2, 4, or 24 h after burn over 40% TBSA (Fig. 2). This progressive change in PKC activity was paralleled by a progressive decrease in Na-KATPase activity 2, 4, and 24 h after burn (Fig. 3). Administration of calphostin, a specic inhibitor of PKC, after burn injury attenuated the burn-related increases in myocardial PKC (Fig. 2) and blunted the burn-related decrease in Na-K-ATPase activity (Fig. 3). Recovery of Na-K-ATPase activity during the postburn period also attenuated the burnrelated accumulation of Na by cardiomyocytes. Effects of burn injury on hemodynamic and metabolic function. All animals survived the experimental period. Twentyfour hours after burn injury, mean arterial blood pressure was lower in group 3 (burn injury vehicle; 112 8 mmHg) compared with that measured in sham burn-injured rats (group 1, 151 5 mmHg; P 0.002). Metabolic acidosis occurred after vehicle-treated burn injury, as indicated by the increase in whole blood lactate (3.3 0.4 mM) compared with values measured in sham burn-injured rats (2.2 0.3 mM; P 0.04). Hematocrit levels fell 24 h after burn injury, likely because of the aggressive uid resuscitation (Table 2). Plasma cytokines were signicantly elevated 24 h after vehicle-treated burn injury (group 3) compared with values measured in sham burn-injured rats (group 1; P 0.05). While administration of calphostin in burns (group 4) improved mean arterial blood pressure and improved metabolic acidosis compared with values measured in vehicle-treated burns (group 3), these values did not return to the levels measured in sham burn-injured rats. Calphostin administration after burn injury lowered plasma cytokine levels compared with values that were measured in vehicle-treated burns (P 0.05), but plasma cytokine levels in calphostin-treated burns remained above those measured in sham burns (P 0.05; Table 2). Effects of burn injury and calphostin on cardiac inammation. Burn injury produced signicant pro- and anti-inammatory responses in the myocardium. as indicated by increased secretion of TNF- , IL-1 , IL-6, and IL-10. Calphostin therapy after burn injury attenuated cardiac myocyte secretion of the proinammatory cytokines TNF- (Fig. 4A), IL-1 (Fig. 4B), and IL-6 (Fig. 4C), as well as the anti-inammatory cytokine IL-10 (Fig. 4D). Effects of calphostin treatment on cardiac myocyte Na levels and myocardial function. As shown in Fig. 5, the burn-related rise in myocyte Na levels measured 24 h after vehicle-treated burn injury was signicantly attenuated by calphostin treatment of burns (P 0.05). The attenuation of cardiomyocyte Na overload and restoration of intracellular Na homeostasis in calphostin-treated burns was associated with improved myocardial contraction and relaxation in group 4 compared with left ventricular performance measured in vehicle-treated burn (group 3; P 0.05). Left ventricular developed pressure (LVP) and dP/dt responses (Table 3) were measured in all four experimental groups as the hearts were perfused at constant preload, constant heart rate, and constant coronary ow rate. Additional studies included in vitro perfusion of the heart 24 h after burn injury (Langendorff approach), providing further evidence of cardiac contracAJP-Regul Integr Comp Physiol VOL

Fig. 4. Secretion of cytokines by cardiac myocytes. TNF- (A), IL-1 (B), IL-6 (C), and IL-10 (D) were measured 24 h after either vehicle-treated sham burn or vehicle-treated burn injury in the presence (light and dark gray bars) or absence (open and lled bars) of calphostin treatment. All values are means SE. *Signicant difference from sham burn (P 0.05); signicant difference between vehicle-treated burn and calphostin-treated burn (P 0.05).

tile depression as indicated by the reduced LVP and dP/dt responses to incremental increases in either preload (Fig. 6A) or increases in perfusate Ca2 (Fig. 6B). Hearts harvested from calphostin-treated burn-injured rats generated signicantly greater levels of LVP and dP/dt at all levels of left ventricular preload (Fig. 6A) and better left ventricular performance in response to incremental increases in perfusate Ca2 (Fig. 6B) compared with values generated by hearts harvested from vehicle-treated burn-injured rats (P 0.05).
DISCUSSION

The present study conrmed that administration of a specic PKC inhibitor, calphostin, attenuated the burn-related increase in myocardial PKC activity. Furthermore, calphostin attenuated burn-related myocardial contraction and relaxation defects, and this improved myocardial performance was associated with increased Na-K-ATPase activity and signicant attenuation of the burn-related increase in cardiomyocyte intracellular Na loading. These data are consistent with our
293 OCTOBER 2007

www.ajpregu.org

PKC MODULATES NA-K-ATPASE ACTIVITY

R1689

Fig. 5. Intracellular Na levels ([Na ]i) measured in isolated cardiomyocytes 24 h after burn injury with a uorescent indicator technique (sodium-binding benzofuran isophthalate). Groups included vehicle-treated sham burn, calphostin-treated sham burn, vehicle-treated burn injury, and calphostin-treated burn injury. Calphostin (PKC inhibitor) administration attenuated burn-related increase in cardiomyocyte Na loading. All values are means SE. *Signicant difference from sham burn (P 0.05); signicant difference between vehicle-treated burn and calphostin-treated burn at each time point (P 0.05).

hypothesis that PKC activation in burn injury decreases NaK-ATPase activity, interrupting Na efux during the postburn course and contributing to cardiomyocyte Na overload. These factors contribute, in part, to burn-related myocardial contraction and relaxation defects. In the present study, intracellular Na rose after burn injury, likely because of an increase in anaerobic metabolism, a decrease in intracellular pH, and activation of the Na /H exchange mechanism (2, 18, 30, 41, 44, 57). A rise in intracellular Na has been shown to activate the Na-K-ATPase enzyme, and the Na gradient regulates the Na /Ca2 exchanger, an ion transport system that couples cellular Na and Ca2 transport (11, 16, 20, 30, 41, 44, 50, 54). Aggressive uid resuscitation from burn injury likely reactivates Na /H exchange, correcting intracellular acidosis but exacerbating cellular Na /Ca2 overload. Altered Na and Ca2 handling by the myocyte have been shown to play a pivotal role in myocardial injury and dysfunction in a number of experimental

models (21, 44, 45, 47, 49, 51, 58). Further support for the role of myocyte Na accumulation in postburn myocardial dysfunction was provided by the time course data in the present study. There was no cardiac contractile depression 2 h after burn, when there was little change in myocyte intracellular Na levels. However, progressive Na accumulation was followed by progressive myocardial contraction and relaxation defects. These data suggest that Na accumulation by the cardiac myocyte likely initiated a cellular signaling cascade that perhaps includes increased myocyte inammation, as indicated by increased myocyte secretion of TNF- , IL-1 , and IL-6, culminating in myocardial contractile depression. Pharmacological interventions that limit the rise in intracellular Na have been shown to reduce myocardial injury and improve myocardial performance in several experimental models (12, 26, 37, 43). Since the Na-K-ATPase exchanger is the primary regulator of sodium efux from the cell, a pharmacological agent that increases Na-K-ATPase activity should promote Na efux from the cell, limit injury-related Na accumulation, and provide organ protection (13). Data from the present study support this hypothesis with increased myocardial Na-K-ATPase activity after calphostin treatment of burn injury paralleled by reduced myocyte Na levels, reduced myocardial inammation, and improved cardiac contractile performance. While the Na-K-ATPase transporter plays a pivotal role in regulating cellular ionic homeostasis through its transmembrane transport of Na and K , Na-K-ATPase function is modulated in several cell types by phosphorylation by either PKA or PKC (3, 8, 17, 27, 32). In this regard, PKC activity has been shown to inhibit Na-K-ATPase activity in noncardiac tissue by phosphorylating the enzyme, decreasing afnity of the Na-K-ATPase for sodium, and producing a decrease in activity of the enzyme (4, 6, 28, 59). However, the present study is the rst, to our knowledge, to suggest that burn-related changes in myocardial Na-K-ATPase activity are PKC dependent. In our study, the PKC inhibitor calphostin increased myocardial Na-K-ATPase after burn injury, and these data are consistent with studies by Lundmark and colleagues (29), who described that chelerythrine increased Na-K-ATPase activity after ischemic injury in isolated rat hearts. While increased PKC activity after burn injury in our study was paralleled by a fall in Na-K-ATPase activity and impaired cardiac function,

Table 3. Stabilization of perfused heart at constant left ventricular volume (preload), constant coronary ow rate, and constant heart rate
Group 1 Sham Vehicle Sham Group 2 Calphostin Group 3 Burn Vehicle Burn Group 4 Calphostin

LVP, mmHg dP/dtmax, mmHg/s dP/dtmax, mmHg/s DP40, mmHg/s Time to peak pressure, ms Time to 90% relaxation, ms Time to dP/dtmax, ms Time to dP/dtmax, ms Coronary perfusion pressure, mmHg Coronary vascular resistance, mmHg Heart rate, beats/min

93 2,055 1,725 1,792 85.6 83.7 56.3 56.8 55.0 11.0 262

4 120 126 123 1.9 1.3 1.3 1.6 2.8 0.6 16

90 1,910 1,640 1,600 97.2 101.0 51.0 58.2 51.2 10.3 258

4 74 67 114 3.6 5.6 5.6 0.9 5.4 1.1 3

61 1,170 989 986 94.3 92.6 56.1 54.0 56.3 11.3 265

3* 67* 52* 51* 2.4 5.5 1.4 1.1 5.6 1.1 9

89 1,788 1,542 1,572 105.3 102.8 59.2 59.2 46.5 9.3 253

3 65 92 68 5.0* 5.2* 4.6 3.3 4.1 0.8 6

All values are means SE. DP40, developed pressure at 40 mmHg. *Signicant difference from respective sham group at P in group 4 (calphostin-treated burns) compared to values measured in group 3 (vehicle-treated burns) at P 0.05. AJP-Regul Integr Comp Physiol VOL
293 OCTOBER 2007

0.05. signicant difference

www.ajpregu.org

R1690

PKC MODULATES NA-K-ATPASE ACTIVITY

Fig. 6. A: LV developed pressure (LVP) and maximum rate of rise and fall of LVP ( dP/dtmax) responses to increases in left ventricular volume or preload assessed 24 h after burn. B: LVP and dP/dtmax responses to increases in perfusate Ca2 in all experimental groups. All values are means SE. *Signicant difference from respective sham group (P 0.05, ANOVA, StudentNewman-Keuls).

increased PKC activity has been associated with both protective and detrimental effects. While PKC translocation from the cytosol to the cell membrane has been shown to play a role in ischemic preconditioning (33, 46), PKC inhibition has been shown to lessen myocardial injury and dysfunction, and these benecial effects were linked to downregulation of proinammatory responses (23, 25). Similarly, PKC inhibition has been shown to afford cardioprotection in models of coronary occlusion (25). While burn-related PKC activation likely contributed to the progressive decrease in myocardial Na-K-ATPase in our study, other factors should be considered. Alterations in membrane lipid content clearly alter the function of membrane-bound enzymes (38). In this regard, Pierce and Dhalla (39) described an association between increased cholesterol content of the
AJP-Regul Integr Comp Physiol VOL

sarcolemmal membranes and a decrease in a sarcolemmal Na-K-ATPase activity. Similarly, feeding a cholesterolenriched diet increased sarcolemmal cholesterol content and depressed Na-K-ATPase activity in adult rats (35). The rise in plasma cholesterol and triglycerides that occurs after burn injury may provide another mechanism by which burn injury alters Na-K-ATPase activity (9, 24, 56). Alternatively, increased proinammatory activities after burn injury as indicated in our study by a rise in plasma and tissue TNF- , IL-1 , and IL-6 levels may exert direct inhibitory effects on Na-K-ATPase enzymatic activity, promoting myocyte accumulation of sodium and cellular injury. We showed previously (44) that increased myocyte secretion of TNF- and IL-1 parallel a rise in myocyte sodium. Several limitations of the present study must be considered. The changes in myocardial Na-K-ATPase, cardiac myocyte
293 OCTOBER 2007

www.ajpregu.org

PKC MODULATES NA-K-ATPASE ACTIVITY

R1691

Na levels, myocyte cytokine secretion, and left ventricular function parallel one another, but do not indicate causality. We proposed that burn injury initiates a sequence of events that include increased PKC activity, which in turn impaired Na-KATPase expression and activity, promoting a subsequent rise in myocyte Na . The rise in cellular Na likely triggered local inammatory responses, culminating in impaired left ventricular function. Our nding that calphostin (a PKC inhibitor) improved Na-K-ATPase expression and pump activity, lowered myocyte Na , and improved left ventricular function supports this proposal. However, calphostin may have directly altered cytokine secretion by myocytes, improving cardiac function regardless of calphostins effects on either PKC or Na-K-ATPase activity or myocyte Na levels. Other limitations include the fact that calphostin may have altered several aspects of cellular function in other organs as well as in the heart. While the administration of calphostin in vivo after major burn injury allowed us to examine several aspects of the burn-related sequelae (cell signaling, inammation, as well as organ or heart function), this in vivo approach was complicated by the potential effects of calphostin on many cell types and organ systems. In summary, burn injury over 40% of the total body surface area in adult rats increased myocardial PKC activity and decreased Na-K-ATPase activity. These biochemical changes were paralleled by cardiac myocyte accumulation of sodium and myocardial contraction and relaxation decits that were evident by 12 h after burn. Administration of calphostin, a specic PKC inhibitor, increased postburn myocardial Na-KATPase activity, decreased burn-related myocardial sodium overload, and improved myocardial performance. These data suggest that burn-related changes in Na-K-ATPase activity promoted sodium accumulation by the myocyte, which likely initiated a cellular signaling cascade that culminated in myocardial contractile depression. Our data further suggest that inhibition of PKC activity after burn injury provides cardiac protection by preventing myocyte sodium loading that occurs via the PKC-related decrease in Na-K-ATPase activity.
GRANTS This work was supported by National Institute of General Medical Sciences Grant R01 GM-57054. REFERENCES 1. Adams HR, Baxter CR, Parker JL. Contractile function of heart muscle from burned guinea pigs. Circ Shock 9: 6373, 1982. 2. Anderson SE, Murphy E, Steenbergen C, London RE, Cala PM. Na-H exchange in myocardium: effects of hypoxia and acidication on Na and Ca. Am J Physiol Cell Physiol 259: C940 C948, 1990. 3. Aperia A, Holtbakc U, Syren ML, Svensson LB, Fryckstedt J, Greengard P. Activation/deactivation of renal Na ,K -ATPase: a nal common pathway for regulation of natriuresis. FASEB J 8: 436 539, 1994. 4. Asghar M, Kansra V, Hussain T, Lokhandwala MF. Hyperphosphorylation of Na-pump contributes to defective renal dopamine response in old rats. J Am Soc Nephrol 12: 226 232, 2001. 5. Bers DM. Isolation and characterization of cardiac sarcolemma. Biochim Biophys Acta 555: 131146, 1979. 6. Bertorello AM, Katz AI. Short-term regulation of renal Na-K-ATPase activity: physiological relevance and cellular mechanisms. Am J Physiol Renal Fluid Electrolyte Physiol 265: F743F755, 1993. 7. Bowler K, Tirri R. The temperature characteristics of synaptic membrane ATPases from immature and adult rat brain. J Neurochem 23: 611 613, 1974. AJP-Regul Integr Comp Physiol VOL

8. Buhagiar KA, Hansen PS, Bewick NI, Rasmussen HH. Protein kinase C contributes to regulation of the sarcolemmal Na -K pump. Am J Physiol Cell Physiol 281: C1059 C1063, 2001. 9. Cetinkale O, Yazici Z. Early postburn fatty acid prole in burn patients. Burns 23:392399, 1997. 10. Chen KM, Defer D, Junger KD. A direct colorimetric assay for Ca2 stimulated ATPase activity. Anal Biochem 157: 374 380, 1986. 11. Chilbalin AV, Vasilets LA, Hennekes H, Pralong D, Geering K. Phosphorylation of Na-K-ATPase -subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C. J Biol Chem 267: 23378 22384, 1992. 12. Cox CS Jr, Allen SJ, Sauer H, Laine GA. Improved myocardial function using a Na /H exchanger inhibitor during cardioplegic arrest and cardiopulmonary bypass. Chest 123: 187194, 2003. 13. Cross HR, Clarke K, Radda GK. The role of Na /K ATPase activity during low ow ischemia in preventing myocardial injury: a 31P, 23Na, and 87 Rb NMR spectroscopic study. Magn Reson Med 34: 673 685, 1995. 14. Dhalla NS, Das PK, Sharma GP. Subcellular basis of cardiac contractile failure. J Mol Cell Cardiol 10: 363385, 1978. 15. Dhalla NS, Ziegelhoffer A, Harrow JAC. Regulatory role of membrane systems in heart function. Can J Physiol Pharmacol 55: 12111234, 1977. 16. Feraille E, Carranza ML, Bufn-Meyer B, Rousselot M, Doucet A, Favre H. Protein kinase C-dependent stimulation of Na -K -ATPase in rat proximal convoluted tubules. Am J Physiol Cell Physiol 268: C1277 C1288, 1995. 17. Fisone G, Cheng SX, Nairn AC, Czernik AJ, Hemmings HC Jr, Hoog JO, Bertorello AM, Kaiser R, Bergman T, Jornvall H, Aperia A, Greengard P. Identication of the phosphorylation site for cAMP-dependent protein kinase on Na ,K -ATPase and effects of site-directed mutagenesis. J Biol Chem 269: 9368 9373, 1994. 18. Frelin C, Vigne P, Lazdunski M. The role of the Na /H exchange system in the regulation of the internal pH in cultured cardiac cells. Eur J Biochem 149: 1 4, 1985. 19. Fuller W, Parmar V, Eaton P, Bell JR, Shattock MJ. Cardiac ischemia causes inhibition of the Na/K ATPase by a labile cytosolic compound whose production is linked to oxidant stress. Cardiovasc Res 57: 1044 1051, 2003. 20. Haigney MCP, Miyata H, Lakatta EG, Stern MD, Silverman HS. Dependence of hypoxic cellular calcium loading on Na -Ca2 exchange. Circ Res 71: 547557, 1992. 21. Horton JW, Lin C, Maass DL. Burn trauma and TNF- alter calcium handling by cardiomyocytes. Shock 10: 270 277, 1998. 22. Horton JW, Maass DL, White J, Sanders B. Hypertonic saline-dextran suppresses burn-related cytokine secretion by cardiomyocytes. Am J Physiol Heart Circ Physiol 280: H1591H1601, 2001. 23. Horton JW, White J, Maass D. Protein kinase C inhibition improves ventricular function after thermal trauma. J Trauma 44: 254 265, 1998. 24. Kamolz LP, Andel H, Mittlbock M, Winter W, Haslik kW, Meissl G, Frey M. Serum cholesterol and triglycerides: potential role in mortality prediction. Burns 29: 810 815, 2003. 25. Lasley RD, Noble MA, Mentzer RMJ. Effects of protein kinase C inhibitors in in situ and isolated ischemic rabbit myocardium. J Mol Cell Cardiol 29: 33453356, 1997. 26. Linakis JG, Raymond RM. Effect of amiloride on age-dependent cardiac dysfunction after ischemia/reperfusion in the isolated, perfused rat heart. Shock 11: 218 223, 1999. 27. Lowndes JM, Hokin-Neaverson M, Bertics PJ. Kinetics of phosphorylation of Na /K -ATPase by protein kinase C. Biochim Biophys Acta 1052: 143151, 1990. 28. Lundmark JA, Trueblood N, Wang LF, Ramasamy R, Schaefer S. Repetitive acidosis protects the ischemic heart: implications for mechanisms in preconditioned hearts. J Mol Cell Cardiol 31: 907917, 1999. 29. Lundmark JL, Ramasamy R, Vulliet PR, Schaefer S. Chelerythrine increases Na-K-ATPase activity and limits ischemic injury in isolated rat hearts. Am J Physiol Heart Circ Physiol 277: H999 H1006, 1999. 30. Malloy CR, Buster DC, Margarida M, Castro M, Geraldes CF, Jeffrey FM, Sherry AD. Inuence of global ischemia on intracellular sodium in the perfused rat heart. Magn Reson Med 14: 33 44, 1990. 31. McNamara DB, Sulakhe PV, Singh JN, Dhalla NS. Properties of heart sarcolemmal Na -K ATPase. J Biochem Tokyo 75: 795 803, 1974. 32. Middleton JP, Khan WA, Collinsworth G, Hannu YA, Medord RM. Heterogeneity of protein kinase C-mediated rapid regulation of Na/KATPase in kidney epithelial cells. J Biol Chem 268: 15958 15964, 1993.
293 OCTOBER 2007

www.ajpregu.org

R1692

PKC MODULATES NA-K-ATPASE ACTIVITY 47. Steenbergen C, Murphy E, Watts JA, London RE. Correlation between cytosolic free calcium, contracture, ATP, and irreversible ischemic injury in perfused rat heart. Circ Res 66: 135146, 1990. 48. Tan J, Maass DL, White DJ, Horton JW. Effects of burn injury on myocardial signaling and cytokine secretion. Possible role of PKC. Am J Physiol Regul Integr Comp Physiol 292: R887R896, 2007. 49. Tani M, Neely J. Na accumulation increases Ca2 overload and impairs function in anoxic rat heart. J Mol Cell Cardiol 22: 5772, 1990. 50. Tani M, Neely JR. Role of intracellular Na in Ca2 overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Circ Res 65: 10451056, 1989. 51. Tani M. Mechanisms of Ca2 overload in reperfused ischemic myocardium. Annu Rev Physiol 52: 543559, 1990. 52. Tsakiris S. Effects of L-phenylalanine on acetylcholinesterase and Na ,K -ATPase activities in adult and aged rat brain. Mech Ageing Dev 122: 491501, 2001. 53. Tsakiris S, Deliconstantinos G. Inuence of phosphatidylserine on (Na K )-stimulated ATPase and acetylcholinesterase activities of dog brain synaptosomal plasma membranes. Biochem J 220: 301307, 1984. 54. VanEmous JG, Nederhoff MGJ, Ruigrok TJC, van Echteld CJA. The role of the Na channel in the accumulation of intracellular Na during myocardial ischemia: consequences for post ischemic recovery. J Mol Cell Cardiol 29: 8596, 1997. 55. VanEmous JG, Schreur JHM, Ruigrok TJC, van Echteld CJA. Both Na -K -ATPase and Na -H exchanger are immediately active upon post-ischemic reperfusion in isolated rat hearts. J Mol Cell Cardiol 30:337348, 1998. 56. Vanni HE, Gordon BR, Levine DM, Sloan BJ, Stein DR, Yurt RW, Saal SD, Parker TS. Cholesterol and interleukin-6 concentrations relate to outcomes in burn-injured patients. J Burn Care Rehabil 24: 133141, 2003. 57. Wallert MA, Frohlich O. Na -H exchange in isolated myocytes from adult rat hearts. Am J Physiol Cell Physiol 257: C207C213, 1989. 58. White DJ, Maass DL, Sanders B, Horton JW. Cardiomyocyte intracellular calcium and cardiac dysfunction after burn trauma. Crit Care Med 30:14 22, 2002. 59. Xia P, Kramer RM, King GL. Identication of the mechanism for the inhibition of Na ,K -adenosine triphosphatase by hyperglycemia involving activation of protein kinase C and cytosolic phospholipase A2. J Clin Invest 96: 733740, 1995.

33. Mitchell MG, Meng X, Ao L, Brown JM, Harken AH, Banerjee A. Preconditioning of isolated rat heart is mediated by protein kinase C. Circ Res 76: 73 81, 1995. 34. Miyata H, Lakatta EG, Stern MD, Silverman HS. Relation of mitochondrial and cytosolic free calcium to cardiac myocyte recovery after exposure to anoxia. Circ Res 71: 604 613, 1992. 35. Moffat MP, Dhalla NS. Cardiac sarcolemmal ATPase activities and calcium binding in rats fed a high cholesterol diet (Abstract). Fed Proc 41: 1467, 1982. 36. Mullins LJ. Ion Transport in the Heart. New York: Raven, 1981. 37. Ohara F, Ohkubo K, Maeda K, Seki J, Goto T. FR183998, a Na /H exchange inhibitor, suppresses both IL-8 content and myocardial infarct size in a cardiac ischaemia-reperfusion model in rats. J Pharm Pharmacol 54: 263268, 2002. 38. Papahadjopoulos D, Cowden M, Kimelberg H. Role of cholesterol in membranes: effects on phospholipid-protein interactions, membrane permeability and enzymatic activity. Biochim Biophys Acta 330: 8 26, 1973. 39. Pierce GN, Dhalla NS. Sarcolemmal Na -K -ATPase activity in diabetic rat heart. Am J Physiol Cell Physiol 245: C241C247, 1983. 40. Pike MM, Luo CS, Clark MD, Kirk KA, Kitakaze M, Madden MC, Cragoe EJ, Phost GM. NMR measurements of Na and cellular energy in ischemic rat heart: role of Na -H exchange. Am J Physiol Heart Circ Physiol 256: H2017H2026, 1989. 41. Rasmussen HH, Cragoe EJ, Ten Eick RE. Na -dependent activation of Na -K pump in human myocardium during recovery from acidosis. Am J Physiol Heart Circ Physiol 256: H256 H264, 1989. 42. Scholz W, Albus U, Linz AW, Martorana P, Lang HJ, Scholkens BA. Effects of Na /H exchange inhibitors in cardiac ischemia. J Mol Cell Cardiol 24: 731740, 1992. 43. Sikes P, Zhao P, Maass DL, White DJ, Horton JW. Sodium/hydrogen exchange activity in sepsis and in sepsis complicated by previous injury: 31 P and 23Na NMR study. Crit Care Med 33: 605 615, 2005. 44. Sikes PJ, Zhao P, Maass DL, Horton JW. Time course of myocardial sodium accumulation after burn trauma: a 31P- and 23Na-NMR study. J Appl Physiol 91: 26952702, 2001. 45. Silverman HS, Stern MD. Ionic basis of ischaemic cardiac injury: insights from cellular studies. Cardiovasc Res 28: 581597, 1994. 46. Speechly-Dick ME, Mocanu MM, Yellon DM. Protein kinase C: its role in ischemic preconditioning in the rat. Circ Res 75:586 590, 1994.

AJP-Regul Integr Comp Physiol VOL

293 OCTOBER 2007

www.ajpregu.org