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Pain Medicine  |   June 2001
Propofol Increases Myofilament Ca2+Sensitivity and Intracellular pH via  Activation of Na+–H+Exchange in Rat Ventricular Myocytes
Author Affiliations & Notes
  • Noriaki Kanaya, M.D.
    *
  • Paul A. Murray, Ph.D.
  • Derek S. Damron, Ph.D.
  • * Research Fellow, ‡ Project Scientist, † Carl E. Wasmuth Endowed Chair and Director.
  • Received from the Center for Anesthesiology Research, Division of Anesthesiology and Critical Care Medicine, The Cleveland Clinic Foundation, Cleveland, Ohio.
Article Information
Pain Medicine
Pain Medicine   |   June 2001
Propofol Increases Myofilament Ca2+Sensitivity and Intracellular pH via  Activation of Na+–H+Exchange in Rat Ventricular Myocytes
Anesthesiology 6 2001, Vol.94, 1096-1104. doi:
Anesthesiology 6 2001, Vol.94, 1096-1104. doi:
MOST studies have reported that the intravenous anesthetic propofol has either no direct effect on cardiac contractile function 1–4 or has a modest negative inotropic effect. 5–8 However, propofol is known to inhibit action potential duration, 3 sarcolemmal L-type Ca2+channels, 9–11 K+channels, 11 and sarcoplasmic reticulum Ca2+handling, 2,5,12 which indicates that propofol has multiple sites of action in the cardiac cell. It is conceivable that propofol may alter some mechanism of cardiomyocyte function that normally increases contractile activity. This effect would offset or mask a negative inotropic effect of propofol.
We recently reported indirect evidence suggesting that propofol increases myofilament Ca2+sensitivity in rat ventricular myocytes. 5 Similar indirect findings have been reported in intact beating guinea pig hearts. 13 However, direct measurements of propofol-induced changes in myofilament Ca2+sensitivity have not been assessed in cardiac muscle. Myofilament Ca2+sensitivity can be altered by changes in intracellular pH (pHi) 14,15 or via  phosphorylation of contractile proteins. In the current study, our objective was to examine the direct effects of propofol on myofilament Ca2+sensitivity by measuring myofibrillar actomyosin adenosine triphosphatase (ATPase) activity. A second goal was to identify the cellular mechanism of action by which propofol alters myofilament Ca2+sensitivity. Specifically, we tested the hypothesis that propofol increases myofilament Ca2+sensitivity by enhancing Na+–H+exchange via  a protein kinase C (PKC)-dependent pathway.
Materials and Methods
All experimental procedures were approved by the Cleveland Clinic Institutional Animal Care and Use Committee (Cleveland, OH).
Ventricular Myocyte Preparation
Adult ventricular myocytes from rat hearts were isolated as previously described. 5 In brief, the hearts were excised, cannulated via  the aorta, attached to a modified Langendorff perfusion apparatus, and perfused with oxygenated (95% O2, 5% CO2) Krebs-Henseleit buffer (37°C) containing 118 mm NaCl, 4.8 mm KCl, 1.2 mm MgCl2, 1.2 mm KH2PO4, 1.2 mm CaCl2, 37.5 mm NaHCO3, and 16.5 mm dextrose, pH 7.35. After a 5-min equilibration period, the perfusion buffer was changed to a Ca2+-free Krebs-Henseleit buffer containing 30 mg collagenase type II (347 U/ml). After collagenase digestion (20 min), the ventricles were minced and shaken in Krebs-Henseleit buffer, and the resulting cellular digest was washed, filtered, and resuspended in phosphate-free HEPES-buffered saline (HBS) containing 118 mm NaCl, 4.8 mm KCl, 1.2 mm MgCl2, 1.25 mm CaCl2, 11 mm dextrose, 25 mm HEPES, and 5 mm pyruvate, pH 7.35, and vigorously bubbled immediately before use with 100% O2. Typically, 6–8 × 106cells per rat heart were obtained using this procedure. Viability, as assessed by the percentage of cells retaining a rod-shaped morphology, was routinely between 80 and 90%. Myocytes were suspended in HBS (1 × 106cells/ml) and stored in an oxygen hood until used.
Preparation of Cardiac Myofibrils
Two-milliliter aliquots of the freshly isolated myocyte suspension were incubated in the presence or absence of propofol (10, 30, 100 μm) for 10 min at 37°C with gentle agitation. The myocytes were immediately washed twice in ice-cold HBS containing protease and phosphatase inhibitors and pelleted at 400 g  for 3 min, after which an equivalent volume of extraction buffer, 50 mm Tris (pH 7.5) containing Triton-X 100 (0.1%), NaF (20 mm), dithiothreitol (0.5 mm), MgCl2(0.5 mm), EDTA (0.125 mm), antipain (5 μg/ml), leupeptin (10 μg/ml), pepstatin A (5 μg/ml), and paramethylsulfonic acid (43 μg/ml) was added to the suspension. The cells were homogenized and kept on ice for 30 min. The triton-extracted myofibrils were pelleted at 10,000 g  (5 min, 4°C). The detergent solubilized supernatant was set aside and the pellet was resuspended in an equivalent volume of extraction buffer and washed twice again. The resultant myofibrillar fraction was resuspended in Ca2+-free extraction buffer and stored at −20°C. Examination of the pellet under the microscope indicated that it was enriched in myofibrils.
Actomyosin Adenosine Triphosphatase Activity
The Ca2+-stimulated actomyosin ATPase activity of the myofibrillar fraction was measured from the rate of decrease of nicotinamide adenine dinucleotide (NADH) absorbance (340-nm excitation wavelength) in a reaction coupled to the pyruvate kinase and lactate dehydrogenase reactions. 16 Triton-X 100–extracted myofibrils were solubilized in a buffer containing 25 mm BES (N,N  -bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid, pH 7.4), 85 mm potassium methanol sulfonic acid, 3 mm MgCl2, 2 mm EGTA, 10 mm NaF, 0.5 mm dithiothreitol, and 0.5 mm leupeptin. The protein concentration of the extracted myofibrils was determined using the Lowry protein assay. The reaction mixture consisted of 25 mm BES (pH 7.0), 2.7 mm MgCl2, 2 mm EGTA, 10 mm NaF, and 126 mm potassium methanol sulfonic acid and varying free CaCl2concentrations, giving free Ca2+concentrations from pCa 9 to 4, according to an iterative computer program provided by Dr. Frank Brozovich (Case Western Reserve University School of Medicine, Cleveland, OH). The reaction mixture also contained 200 mm phosphoenolpyruvate, 10 mm NADH, 0.5 mg/ml lactate dehydrogenase, 12.5 mg/ml pyruvate kinase, and 1 ml of one of the calcium buffers (pCa 4–9) containing myofibrillar fractions. The reaction was initiated by addition of 2 mm ATP and allowed to continue for up to 10 min, although the reaction was usually complete within 5 min. Ca2+-stimulated actomyosin ATPase activity was monitored by the formation of adenosine diphosphate, coupled to the oxidation of NADH, and recorded by the change in absorption at 340 nm. The enzyme activity was determined from the rate of ATP hydrolysis and expressed as the percent of maximal actomyosin ATPase activity per milligram of protein. Because crude myofibrillar fractions were used for these assays, we determined whether there was a contribution to the activity measured from other ATPases present in the sample. We found no significant difference in ATPase activity measured in the presence or absence of ATPase inhibitors, including 2 mm thapsigargin, 200 mm ouabain, 2 mm rotenone, and 2 mm oligomycin.
Contractility and Intracellular pH Measurements
For simultaneous measurement of shortening and pHi, ventricular myocytes (0.5 × 106cells/ml) were incubated in HBS containing 2 μm 2′,7′-bis-(2-carboxyethyl)-5 (6′)-carboxyfluorescein–acetoxymethyl ester (BCECF–AM) at 37°C for 20 min. BCECF-loaded ventricular myocytes were placed in a temperature-regulated (30°C) chamber mounted on the stage of an inverted fluorescence microscope. The volume of the chamber was 1.5 ml. The cells were superfused continuously with HBS at a flow rate of 2 ml/min and field-stimulated via  bipolar platinum electrodes at a frequency of 0.3 Hz and a duration of 5 ms using a stimulator.
Fluorescence measurements were performed on single ventricular myocytes using a dual-wavelength spectrofluorometer at excitation wavelengths of 440 and 500 nm and an emission wavelength of 530 nm. 17 The cells were also illuminated with red light at a wavelength greater than 600 nm for simultaneous video edge detection. An additional postspecimen dichroic mirror deflects light at wavelengths greater than 600 nm into a charge coupled device video camera for measurement of myocyte shortening. The fluorescence sampling frequency was 10 Hz, and data were collected using a software package. Background fluorescence was determined from the blank dish and subtracted from fluorescence at each wavelength. To estimate the pHivalue from the ratio of 500/440 nm fluorescence, we used an in situ  calibration procedure. 17,18 At the end of each experiment, the fluorescence ratio from each cell was calibrated in situ  by exposing the cell to solutions of varying pH. Each solution contained 140 mm K+, M1.0 mm gCl2, 4.0 mm HEPES, 2.0 mm EGTA, 30 mm 2,3-butanedione monoxime, 50 μm 1,2-bis(o-Aminophenoxy)ethane-N,N,N′,N′- tetraacetic acid, and 14 μm nigericin and was titrated to varying pH values (6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8) using 1.0 N KOH. The pHifor each cell was then determined from a linear regression of fluorescence ratio versus  the pH value of the calibration buffer. We previously determined that a linear relation exists between the 500/440 nm ratio and pHiin the physiologic range (pH 6.6–7.8). 17 
Simultaneous measurement of cell shortening was monitored using a video edge detector with 16-ms temporal resolution. The video edge detector was calibrated using a stage micrometer so that cell lengths during shortening could be monitored. LabVIEW (National Instruments, Austin, TX) was used for data acquisition of cell shortening using a sampling rate of 100 Hz.
Analysis of Intracellular pH and Contractile Data
Fluorescence data for the pHimeasurements were imported into LabVIEW, which allowed the simultaneous analysis of pHiand shortening. Myocyte length in response to field stimulation was measured (micrometers) and expressed as the change from resting cell length (twitch amplitude). Changes in twitch amplitude in response to the interventions are expressed as a percent of baseline shortening. Contractile parameters from 15 contractions were averaged to obtain mean values at baseline and in response to the various interventions. Averaging the parameters over time minimizes beat-to-beat variation. Changes in pHiwere measured as the change in the 500/440 ratio from baseline.
Experimental Protocols
Where applicable, protocols were designed so that each cell could serve as its own control.
Protocol 1: Effect of Propofol on Myofibrillar Actomyosin Adenosine Triphosphatase Activity.
To determine whether propofol alters myofilament Ca2+sensitivity, changes in actomyosin ATPase activity were measured in myofibrils isolated from control and propofol-treated cardiomyocytes. The myocyte suspension was divided into separate aliquots, and each aliquot was treated with one concentration of propofol (10, 30, 100 μm) or intralipid equivalent for 10 min at 37°C with gentle agitation. Propofol had no effect on extracellular pH at the concentrations used in this study and did not appear to alter the assay conditions. Activity is expressed as a percentage of the maximum rate per milligram of protein.
Protocol 2: Effect of Alkalinization and Acidification on Myocyte Shortening.
To identify the effects of changing pHion myocyte shortening, changes in myocyte shortening and pHiin response to 10 mm NH4Cl (2 min) followed by washout were determined. Baseline measurements were collected from individual myocytes for 1 min in the absence of any intervention.
Protocol 3: Effect of Propofol on Intracellular pH.
To determine whether propofol alters pHi, we measured pHifor 15 min after addition of propofol. Baseline pHiwas collected from individual myocytes for 1 min. Propofol (10, 30, 100 μm) or intralipid equivalent was added by exchanging the superfusion buffer in the dish with new buffer containing propofol at the desired concentration. Each myocyte was exposed to only one concentration for propofol.
Protocol 4: Effect of Propofol on Intracellular pH and Shortening—Role of Na+–H+Exchange and Protein Kinase C.
To examine the role of the Na+–H+exchange and PKC in modulating propofol-induced changes in pHiand shortening, we assessed propofol-induced intracellular alkalinization and shortening in myocytes pretreated (5 min) with a Na+–H+exchange inhibitor, 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA, 1 μm), or a PKC inhibitor, bisindolylmaleimide I (Bis, 1 μm). We have verified that propofol, EIPA, and Bis have no effect on the BCECF signal at the concentrations used in these studies.
Protocol 5: Effect of Propofol on NH4Cl-induced Changes in Intracellular pH and Contractility.
To determine whether propofol alters NH4Cl-induced changes in pHiand contractility, we simultaneously measured changes in pHiand myocyte shortening in the presence or absence of propofol. Baseline pHiand shortening were measured in individual myocytes for 1 min. In the propofol group, the cells were superfused with buffer containing propofol (30 μm) 5 min before exposure to NH4Cl. Washout was performed with HBS containing propofol.
Protocol 6: Effect of Propofol on Recovery from Acid Load.
To determine whether propofol alters pHirecovery from acid load, we measured the changes in pHifrom intracellular acidosis after washout with 10 mm NH4Cl in the presence or absence of propofol. Baseline pHiwas measured in individual myocytes for 1 min. At the time of NH4Cl washout, each myocyte was superfused with buffer either in the presence or absence of propofol (10, 30, 100 μm). Rate of recovery of pHifrom NH4Cl-induced acidosis was assessed by calculating the derivative of the slope of the pHitime course trace for 10-s intervals through the first 120 s of recovery.
Protocol 7: Effect of Propofol and Ethylisopropyl Amiloride on the Extracellular Ca2+-shortening Relation.
We previously reported that propofol caused a leftward shift in the extracellular Ca2+concentration ([Ca2+]o)–shortening relation, with no concomitant effect on the intracellular Ca2+transient. 5 Baseline parameters were collected from individual myocytes for 1.5 min. Dose–response curves to [Ca2+]owere performed by exchanging the buffer in the dish with a new buffer containing the desired [Ca2+]o. Data were acquired for 1.5 min after establishment of a new steady state. Dose–response curves to [Ca2+]owere then performed in the presence of propofol (30 μm). Cells were allowed to stabilize for 5 min after each intervention. The relative contribution of the Na+–H+exchanger in mediating changes in the [Ca2+]o–shortening relation was assessed by pretreating the cells (5 min) with 1 μm EIPA.
Materials
Collagenase type II was obtained from Worthington Biochemical (Freehold, NJ). Propofol and intralipid were obtained from The Cleveland Clinic Pharmacy. BCECF–AM was obtained from Texas Fluorescence Labs (Austin, TX). Nigericin, EIPA, and Bis were purchased from Sigma Chemical Co. (St. Louis, MO).
Statistical Analysis
Each experimental protocol was performed on multiple myocytes from the same heart and repeated in at least four hearts. Results obtained from myocytes in each heart were averaged so that all hearts were weighted equally. The effects of each concentration of propofol on myocyte shortening and pHiwere assessed using one-way analysis of variance with repeated measures and the Bonferonni correction for multiple comparisons. Comparisons between groups were made by two-way analysis of variance and unpaired t  test. Results are expressed as mean ± SD. P  < 0.05 was considered significant.
Results
Effect of Propofol on Actomyosin ATPase Activity
To directly test the hypothesis that propofol increases myofilament Ca2+sensitivity, we assessed the effects of propofol on myofibrillar actomyosin ATPase activity in myocytes pretreated with propofol (10–100 μm). Propofol caused a concentration-dependent leftward shift in the actomyosin ATPase activation curve (fig. 1A). Propofol increased the EC50(pCa) value (i.e.  , decreased the Ca2+requirement) for Ca2+-activated actomyosin ATPase (fig. 1B). Propofol (30 and 100 μm) reduced (P  < 0.05) the maximal activation of actomyosin ATPase (Vmax) by 11 ± 4% and 17 ± 5%, respectively, from a control value of 175 ± 8 nmol · min1· mg1. The intralipid vehicle had no effect on actomyosin ATPase activity (EC50= 5.6 ± 0.1; Vmax= 169 ± 9 nmol · min1· mg1) at a concentration equivalent to 100 μm propofol.
Fig. 1. (Top  ) Summarized data depicting the leftward shift induced by propofol on actomyosin adenosine triphosphatase activity in isolated myofibrils. (Bottom  ) Summarized data depicting the increase (* P  < 0.05 vs.  control) in the EC50value for Ca2+after propofol treatment.
Fig. 1. (Top 
	) Summarized data depicting the leftward shift induced by propofol on actomyosin adenosine triphosphatase activity in isolated myofibrils. (Bottom 
	) Summarized data depicting the increase (* P 
	< 0.05 vs. 
	control) in the EC50value for Ca2+after propofol treatment.
Fig. 1. (Top  ) Summarized data depicting the leftward shift induced by propofol on actomyosin adenosine triphosphatase activity in isolated myofibrils. (Bottom  ) Summarized data depicting the increase (* P  < 0.05 vs.  control) in the EC50value for Ca2+after propofol treatment.
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Effect of NH4Cl on Intracellular pH and Myocyte Shortening
Baseline pHiand diastolic cell length were 7.05 ± 0.03 and 130 ± 15 μm, respectively. Twitch amplitude was 9 ± 1% (11.7 ± 2.8 μm) of the resting cell length. To test the hypothesis that changes in pHialter the contractile properties of ventricular myocytes, we measured pHiand shortening simultaneously in response to NH4Cl followed by washout to cause intracellular alkalinization and acidification, respectively. Figure 2illustrates the effects of NH4Cl (10 mm) on shortening and pHiin an individual cardiomyocyte. Field stimulation alone had no effect on baseline pHi. NH4Cl initially caused intracellular alkalinization (7.39 ± 0.06), a 4.4 ± 1.7% decrease (P  < 0.05) in diastolic cell length, and an increase (P  < 0.05) in myocyte shortening (135 ± 10% of baseline). Washout of NH4Cl caused intracellular acidification (6.92 ± 0.02), a return of diastolic cell length to baseline (1.0 ± 0.1%), and a decrease (P  < 0.05) in myocyte shortening (48 ± 8% of baseline). Myocyte shortening gradually increased after washout of NH4Cl and reached 95 ± 8% of the baseline value after 10 min.
Fig. 2. Representative trace demonstrating the effect of exposure to and washout of 10 mm NH4Cl on myocyte shortening and intracellular pH (pHi). Exposure to NH4Cl for 2–3 min induced an intracellular alkalosis accompanied by a decrease in diastolic cell length and an increase in myocyte shortening. Subsequent washout of NH4Cl produced a transient intracellular acidosis accompanied by a return in diastolic cell length to baseline values and a decrease in myocyte shortening. Shortening and pHigradually returned to baseline values after washout.
Fig. 2. Representative trace demonstrating the effect of exposure to and washout of 10 mm NH4Cl on myocyte shortening and intracellular pH (pHi). Exposure to NH4Cl for 2–3 min induced an intracellular alkalosis accompanied by a decrease in diastolic cell length and an increase in myocyte shortening. Subsequent washout of NH4Cl produced a transient intracellular acidosis accompanied by a return in diastolic cell length to baseline values and a decrease in myocyte shortening. Shortening and pHigradually returned to baseline values after washout.
Fig. 2. Representative trace demonstrating the effect of exposure to and washout of 10 mm NH4Cl on myocyte shortening and intracellular pH (pHi). Exposure to NH4Cl for 2–3 min induced an intracellular alkalosis accompanied by a decrease in diastolic cell length and an increase in myocyte shortening. Subsequent washout of NH4Cl produced a transient intracellular acidosis accompanied by a return in diastolic cell length to baseline values and a decrease in myocyte shortening. Shortening and pHigradually returned to baseline values after washout.
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Effect of Propofol on Intracellular pH and Myocyte Shortening
To test the hypothesis that the propofol-induced increase in myofilament Ca2+sensitivity was caused by intracellular alkalinization, we assessed the extent to which propofol altered pHi. Propofol at 10 μm had no significant effect on pHior shortening. Propofol at 30 and 100 μm caused a concentration- and time-dependent increase in pHiof 0.03 ± 0.01 and 0.07 ± 0.02, respectively, after a 15-min exposure (fig. 3). Despite the increase in pHi, propofol (30 and 100 μm) reduced (P  < 0.05) shortening by 10 ± 3% and 33 ± 7%, respectively. The intralipid vehicle had no effect on pHi(7.03 ± 0.03) or shortening at a concentration equivalent to 100 μm propofol. In some cells (6 of 16), 30 μm propofol decreased resting cell length from 127 ± 10 μm to 124 ± 7 μm. Similarly, 100 μm propofol decreased resting cell length to 122 ± 9 μm in 10 of 16 cells studied.
Fig. 3. Summarized data for the effects of propofol on intracellular pH (pHi) in rat ventricular myocytes. Results are expressed as changes from baseline pHiin the absence of any intervention. *Significant change from baseline value (P  < 0.05). n = 16 cells per 4 hearts in each group.
Fig. 3. Summarized data for the effects of propofol on intracellular pH (pHi) in rat ventricular myocytes. Results are expressed as changes from baseline pHiin the absence of any intervention. *Significant change from baseline value (P 
	< 0.05). n = 16 cells per 4 hearts in each group.
Fig. 3. Summarized data for the effects of propofol on intracellular pH (pHi) in rat ventricular myocytes. Results are expressed as changes from baseline pHiin the absence of any intervention. *Significant change from baseline value (P  < 0.05). n = 16 cells per 4 hearts in each group.
×
Role of Na+–H+Exchange and Protein Kinase C on Propofol-induced Changes in Intracellular pH and Myocyte Shortening
To test the hypothesis that the propofol-induced increase in pHiwas mediated by an increase in Na+–H+exchange, we assessed the effects of propofol in cells pretreated with EIPA, a Na+–H+exchange inhibitor. Pretreatment with EIPA had no effect on baseline pHior shortening. Figure 4summarizes changes in pHiafter 15-min exposure to 100 μm propofol. The propofol-induced intracellular alkalinization was inhibited by pretreatment with EIPA. PKC inhibition with Bis also inhibited the propofol-induced intracellular alkalinization (fig. 4). In the presence of EIPA, 30 μm propofol decreased (P  < 0.05) cell shortening by an additional 17 ± 3% compared with propofol alone. In the presence of Bis, propofol (30 μm) decreased (P  < 0.05) cell shortening by an additional 24 ± 4% compared with propofol alone.
Fig. 4. Summarized data for the effect of 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA), a Na+–H+exchange inhibitor, and bisindolylmaleimide I (Bis), a specific protein kinase C inhibitor, on propofol-induced (100 μm) increase in baseline intracellular pH (pHI). *Significant difference compared with propofol alone (P  < 0.05). n = 16 cells per 4 hearts in each group.
Fig. 4. Summarized data for the effect of 5-(N 
	-ethyl-N 
	-isopropyl)-amiloride (EIPA), a Na+–H+exchange inhibitor, and bisindolylmaleimide I (Bis), a specific protein kinase C inhibitor, on propofol-induced (100 μm) increase in baseline intracellular pH (pHI). *Significant difference compared with propofol alone (P 
	< 0.05). n = 16 cells per 4 hearts in each group.
Fig. 4. Summarized data for the effect of 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA), a Na+–H+exchange inhibitor, and bisindolylmaleimide I (Bis), a specific protein kinase C inhibitor, on propofol-induced (100 μm) increase in baseline intracellular pH (pHI). *Significant difference compared with propofol alone (P  < 0.05). n = 16 cells per 4 hearts in each group.
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Effect of Propofol on NH4Cl-induced Changes in Intracellular pH and Myocyte Shortening
Administration of NH4Cl results in activation of Na+–H+exchange without the involvement of second messengers. We used NH4Cl to test the hypothesis that propofol-induced changes in pHiare mediated by changes in Na+–H+exchange activity. Baseline pHiand myocyte shortening were 7.03 ± 0.02 and 8 ± 1%, respectively. Pretreatment with propofol (30 μm, 10 min) increased (P  < 0.05) pHi(+0.03 ± 0.01), whereas shortening decreased (P  < 0.05) to 92 ± 4% of control. As expected, pretreatment with propofol had no effect on NH4Cl-induced increases in pHiand cell shortening (fig. 5), because these changes are not mediated by Na+–H+exchange activation. However, the acidification-induced decreases in pHi(mediated by an increase in Na+–H+exchange) and myocyte shortening in response to NH4Cl were attenuated by propofol (fig. 5).
Fig. 5. Effect of 30 μm propofol on NH4Cl exposure- and washout-induced changes in intracellular pH (pHi) (A  ) and myocyte shortening (B  ). W/O = peak of acidosis after washout of NH4Cl; W/O 3 = 3 min after washout of NH4Cl. *Significant difference compared with control (P  < 0.05). n = 16 cells per 4 hearts in each group.
Fig. 5. Effect of 30 μm propofol on NH4Cl exposure- and washout-induced changes in intracellular pH (pHi) (A 
	) and myocyte shortening (B 
	). W/O = peak of acidosis after washout of NH4Cl; W/O 3 = 3 min after washout of NH4Cl. *Significant difference compared with control (P 
	< 0.05). n = 16 cells per 4 hearts in each group.
Fig. 5. Effect of 30 μm propofol on NH4Cl exposure- and washout-induced changes in intracellular pH (pHi) (A  ) and myocyte shortening (B  ). W/O = peak of acidosis after washout of NH4Cl; W/O 3 = 3 min after washout of NH4Cl. *Significant difference compared with control (P  < 0.05). n = 16 cells per 4 hearts in each group.
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Effect of Propofol on Recovery Rate from NH4Cl-induced Acid Load
To further test the hypothesis that propofol enhances the activity of the Na+–H+exchanger, we assessed the extent to which propofol altered the rate of recovery after acidosis (dpHi/dt) induced by a 2–3-min exposure to 10 mm NH4Cl. NH4Cl produced an intracellular alkalosis, whereas subsequent washout produced a transient intracellular acidosis (fig. 6A). EIPA abolished the recovery from acidosis induced by NH4CI (fig. 6A). The time course of pHirecovery from acidosis was compared in the presence or absence of propofol. Propofol at 30 μm accelerated the rate of recovery from NH4Cl-induced intracellular acidosis (fig. 6B). This effect of propofol on the rate of recovery is even more apparent when the data are normalized to peak acidosis (fig. 6C). As summarized in figure 7, propofol increased the recovery rate from acidosis in a concentration-dependent manner.
Fig. 6. Representative traces demonstrating the effects of propofol on the rate of recovery from intracellular acidosis after washout of NH4Cl. (A  ) 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA) abolished the recovery from intracellular acidosis induced by washout of NH4CI. (B  ) Propofol (30 μm) attenuated the magnitude of the NH4CI acidosis and increased the rate of recovery. (C  ) Data are normalized to peak acidosis values to illustrate the effect of propofol on rate of recovery.
Fig. 6. Representative traces demonstrating the effects of propofol on the rate of recovery from intracellular acidosis after washout of NH4Cl. (A 
	) 5-(N 
	-ethyl-N 
	-isopropyl)-amiloride (EIPA) abolished the recovery from intracellular acidosis induced by washout of NH4CI. (B 
	) Propofol (30 μm) attenuated the magnitude of the NH4CI acidosis and increased the rate of recovery. (C 
	) Data are normalized to peak acidosis values to illustrate the effect of propofol on rate of recovery.
Fig. 6. Representative traces demonstrating the effects of propofol on the rate of recovery from intracellular acidosis after washout of NH4Cl. (A  ) 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA) abolished the recovery from intracellular acidosis induced by washout of NH4CI. (B  ) Propofol (30 μm) attenuated the magnitude of the NH4CI acidosis and increased the rate of recovery. (C  ) Data are normalized to peak acidosis values to illustrate the effect of propofol on rate of recovery.
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Fig. 7. Summarized data for the effects of propofol on the rate of recovery (dpHi/dt) from acid load. *Significant difference compared with control (P  < 0.05). n = 16 cells per 4 hearts in each group.
Fig. 7. Summarized data for the effects of propofol on the rate of recovery (dpHi/dt) from acid load. *Significant difference compared with control (P 
	< 0.05). n = 16 cells per 4 hearts in each group.
Fig. 7. Summarized data for the effects of propofol on the rate of recovery (dpHi/dt) from acid load. *Significant difference compared with control (P  < 0.05). n = 16 cells per 4 hearts in each group.
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Effect of Ethylisopropyl Amiloride on the Propofol-induced Leftward Shift in the Dose–Response Curve to Extracellular Ca2+Concentration
Confirming our previous results, 5 the propofol-induced increase in myofilament Ca2+sensitivity is manifested as a leftward shift in the [Ca2+]o–shortening relation (fig. 8). To test the hypothesis that this effect is caused by an increase in Na+–H+exchange, we assessed the effect of 1 μm EIPA on the propofol-induced change in the [Ca2+]o–shortening relation. EIPA attenuated, but did not abolish, the propofol-induced leftward shift in the [Ca2+]o–shortening relation (fig. 8).
Fig. 8. Summarized data for the effect of 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA) on the propofol-induced (30 μm) leftward shift in the [Ca2+]o–shortening relation. *Significant change from control (P  < 0.05). †Significant change from propofol (P  < 0.05). n = 13 cells per 4 hearts.
Fig. 8. Summarized data for the effect of 5-(N 
	-ethyl-N 
	-isopropyl)-amiloride (EIPA) on the propofol-induced (30 μm) leftward shift in the [Ca2+]o–shortening relation. *Significant change from control (P 
	< 0.05). †Significant change from propofol (P 
	< 0.05). n = 13 cells per 4 hearts.
Fig. 8. Summarized data for the effect of 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA) on the propofol-induced (30 μm) leftward shift in the [Ca2+]o–shortening relation. *Significant change from control (P  < 0.05). †Significant change from propofol (P  < 0.05). n = 13 cells per 4 hearts.
×
Discussion
This is the first study to directly assess the effects of propofol on myofilament Ca2+sensitivity, Na+–H+exchange, and pHiin isolated cardiac muscle cells. A previous study from our laboratory using isolated rat ventricular myocytes, as well as a recent study using intact beating guinea pig hearts, provided indirect evidence that propofol may increase the sensitivity of the contractile machinery to Ca2+. 5,13 In contrast, a recent study using phase-loop diagrams depicting the continuous relation between cell length and the fura-2 fluorescence ratio in rat ventricular myocytes has suggested that propofol may reduce myofilament Ca2+sensitivity. 19 In addition, several previous studies have indicated that propofol does not alter myofilament Ca2+sensitivity. 2,6 In this study, we measured changes in Ca2+-activated myofibrillar actomyosin ATPase activity in response to propofol to directly assess changes in myofilament Ca2+sensitivity. The key finding of this study is that propofol markedly reduced the Ca2+requirement for activation of myofibrillar actomyosin ATPase. Moreover, the mechanism responsible for this effect involves, at least in part, a propofol-induced increase in pHimediated via  PKC-dependent activation of the Na+–H+exchanger.
Propofol and Actomyosin ATPase Activity
Cardiac myofilaments contain an intrinsic actomyosin ATPase that represents the molecular basis for contraction. Sensitivity of the actomyosin ATPase to Ca2+is mediated by the troponin complex in the thin filament. This multiprotein complex is comprised of the Ca2+binding TnC subunit, the ATPase inhibiting TnI subunit, and the tropomyosin binding TnT subunit. It is well established that changes in pHiand/or direct phosphorylation of the contractile proteins, TnI, TnT, and myosin light chain 2, can alter the sensitivity of the contractile machinery to Ca2+. Although propofol had no direct effect on actomyosin ATPase activity in isolated myofibrils, it caused a leftward shift in the Ca2+-activated actomyosin ATPase activity curve (i.e.  , a decrease in the amount of Ca2+required for activation) with a small decrease in maximal activity (Vmax) in myofibrils isolated from propofol-treated cardiomyocytes. These results imply the requirement of a cytosolic mediator for the actions of propofol on actomyosin ATPase activity. Taken together, it appears that propofol may alter multiple cellular mechanisms that regulate calcium sensitivity. PKC activators have similar effects on actomyosin ATPase activity. 20–22 
Regulation of Intracellular pH in Cardiomyocytes
Control of pHiwithin the physiologic range is essential for the optimal function of enzymes, cell growth, and protein synthesis. 23,24 In cardiac muscle, pHiis a key factor regulating myocardial contractility, because changes in pHialter the sensitivity of the contractile apparatus to Ca2+. 15 The Na+–H+exchanger represents the most important mechanism for regulation of cardiomyocyte pHi. Baseline pHiin this study was 7.05 ± 0.03, which is similar to that in other reports. 18,25 
Myocyte Contractility and Intracellular pH
Both the negative and positive inotropic effects of acidosis and alkalosis, respectively, have been documented. 26 These effects appear to result almost entirely from changes in pHiand not from changes in extracellular pH. 27 However, the precise mechanisms underlying the changes in myocardial contractility in response to changes in pHiare not known, because the development of force on Ca2+activation involves many pH-dependent biochemical processes. To date, several mechanisms have been postulated to contribute to pHi-mediated changes in myocardial contractility, including changes in the affinity of myofibrillar troponin C for Ca2+, 28 pH dependence of the myofibrillar actomyosin ATPase activity, 29 and changes in sarcoplasmic reticulum Ca2+handling. 15 
Effects of Propofol on Intracellular pH and Myocyte Shortening
Propofol caused a time- and dose-dependent increase in pHi. The magnitude of the increase in pHiwas comparable to that observed in response to α1-adrenergic agonists, endothelin, and angiotensin II. 25,30,31 Despite the increase in pHi, propofol did not exert a positive inotropic effect. However, propofol caused a slight decrease in resting cell length in approximately half of the cells studied. Propofol likely alters multiple cellular mechanisms that negatively regulate contractility (e.g.  , sarcoplasmic reticulum Ca2+handling, L-type Ca2+channel activity), which could offset the effects of increasing myofilament Ca2+sensitivity. In fact, a propofol-induced depression of the intracellular Ca2+transient has been observed in beating guinea pig hearts with no concomitant decrease in contractility, implying enhanced myofilament Ca2+sensitivity. 13 Moreover, intracellular alkalosis can cause a decrease in peak intracellular Ca2+concentration, 14 which could partially offset the effect of increased pHion myofilament Ca2+sensitivity. Propofol may also alter myofilament Ca2+sensitivity via  PKC-dependent phosphorylation of contractile proteins. 32 
Role of Na+–H+Exchanger and Protein Kinase C on Propofol-induced Intracellular Alkalinization
Protein kinase C activation is the primary regulator of Na+–H+exchange in cardiomyocytes. We hypothesized that inhibition of Na+–H+exchange and/or PKC should attenuate the ability of propofol to induce intracellular alkalinization. Consistent with this, the propofol-induced increase in pHiwas markedly attenuated by EIPA and Bis, indicating that the effects are mediated by a PKC-dependent activation of Na+–H+exchange. Pretreatment with EIPA or Bis also resulted in an even greater propofol-induced reduction in shortening. Moreover, the propofol-induced decrease in shortening was greater in Bis-treated myocytes compared with EIPA-treated myocytes. Taken together, these results suggest that PKC-dependent intracellular alkalinization offsets the negative inotropic effect of propofol (likely because of a decrease in intracellular Ca2+availability) 13 via  an increase in myofilament Ca2+sensitivity.
Effect of Propofol on NH4Cl-induced Changes in Intracellular pH
We used NH4Cl as a tool to assess the effects of changing pHion myocardial contractility in our experimental model. NH4Cl initially causes intracellular alkalosis because of the highly permeant NH3crossing the membrane more quickly than NH4+. Once inside the myocyte, NH3combines with H+to form NH4+, which increases pHiand shortening. With continued exposure to NH4Cl, pHigradually decreases as NH4+enters the cell. The subsequent abrupt removal of external NH4Cl produces an intracellular acidosis. This occurs because NH4+ions that had accumulated in the cytosol dissociate to form NH3, which rapidly leaves the cell, resulting in intracellular retention of H+and a decrease in shortening. We hypothesized that NH4Cl-induced alkalinization (independent of Na+–H+exchange) would not be affected by propofol, whereas the acidification (dependent on Na+–H+exchange) would be attenuated if propofol caused intracellular alkalinization via  activation of Na+–H+exchange. We found that propofol had no effect on NH4Cl-induced intracellular alkalinization but attenuated the acidification. In addition, propofol attenuated the decrease in shortening induced by acidification. These data give further support to the idea that propofol activates the Na+–H+exchange mechanism in ventricular myocytes.
Effect of Propofol on Recovery Rate from NH4Cl-induced Acid Load
Because these experiments were performed in the absence of HCO3and carbon dioxide, the acid-load recovery is likely mediated by Na+–H+exchange. 33 We hypothesized that if propofol activated the Na+–H+exchanger as a mechanism to increase pHi, propofol should stimulate a faster rate of recovery from an acid load in response to NH4Cl washout. At concentrations greater than 10 μm, propofol enhanced the rate of recovery from an acid load. Because the recovery from an acid load is dependent on Na+–H+exchange activity, these data give further support to the hypothesis that propofol enhances the activity of the Na+–H+exchanger during acid-load conditions.
Effect of Ethylisopropyl Amiloride on the Propofol-induced Leftward Shift in the Extracellular Ca2+Concentration–Shortening Relation
If intracellular alkalinization mediates the propofol-induced leftward shift in the [Ca2+]o–shortening relation, then this effect should be blocked by inhibiting Na+–H+exchange. Our results indicate that inhibition of Na+–H+exchange with EIPA attenuates the propofol-induced increase in myofilament Ca2+sensitivity by approximately 50%. It is possible that phosphorylation of contractile proteins by propofol also plays a role in mediating the increase in myofilament Ca2+sensitivity. 32 Inhibition of Na+–H+exchange with amiloride has been reported to attenuate the increase in myofilament Ca2+sensitivity in response to endothelin 31 and phenylephrine 14 in rat ventricular myocytes. In contrast to the present study, EIPA abolished the leftward shift in the [Ca2+]o–shortening relation induced by thiopental. 17 
Limitations
Because propofol partitions in vivo  between serum proteins, lipid microsomes, and into tissue, it is difficult to know the precise concentration of free and active propofol at the tissue level. The concentrations of propofol used in these in vitro  studies are supraclinical. Because we used isolated cells rather than intact tissue, the effects of propofol may be enhanced because of optimal solute diffusion distances between the cytosol and extracellular medium. In addition, this in vitro  study only deals with intrinsic myocardial contractility, whereas changes in cardiac function in vivo  after propofol administration also depend on venous return, afterload, and compensatory mechanisms. We also acknowledge that the experimental conditions (temperature, stimulation frequency, unloaded cells) do not perfectly simulate the in vivo  heart. However, the strength of this model is that we can directly assess the effects of propofol on the fundamental contractile unit, the individual cardiomyocyte. Finally, we now have direct evidence that propofol increases the Ca2+sensitivity of the actomyosin ATPase, causes intracellular alkalosis, and stimulates both PKC-dependent phosphorylation of contractile proteins 32 and Na+–H+exchange activity in rat cardiomyocytes. However, we have not directly demonstrated that intracellular alkalosis increases the Ca2+sensitivity of actomyosin ATPase activity.
In summary, our results provide the first direct evidence that propofol increases the sensitivity of myofibrillar actomyosin ATPase to Ca2+(i.e.  , increases myofilament Ca2+sensitivity), at least in part by increasing pHivia  PKC-dependent activation of Na+–H+exchange.
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Fig. 1. (Top  ) Summarized data depicting the leftward shift induced by propofol on actomyosin adenosine triphosphatase activity in isolated myofibrils. (Bottom  ) Summarized data depicting the increase (* P  < 0.05 vs.  control) in the EC50value for Ca2+after propofol treatment.
Fig. 1. (Top 
	) Summarized data depicting the leftward shift induced by propofol on actomyosin adenosine triphosphatase activity in isolated myofibrils. (Bottom 
	) Summarized data depicting the increase (* P 
	< 0.05 vs. 
	control) in the EC50value for Ca2+after propofol treatment.
Fig. 1. (Top  ) Summarized data depicting the leftward shift induced by propofol on actomyosin adenosine triphosphatase activity in isolated myofibrils. (Bottom  ) Summarized data depicting the increase (* P  < 0.05 vs.  control) in the EC50value for Ca2+after propofol treatment.
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Fig. 2. Representative trace demonstrating the effect of exposure to and washout of 10 mm NH4Cl on myocyte shortening and intracellular pH (pHi). Exposure to NH4Cl for 2–3 min induced an intracellular alkalosis accompanied by a decrease in diastolic cell length and an increase in myocyte shortening. Subsequent washout of NH4Cl produced a transient intracellular acidosis accompanied by a return in diastolic cell length to baseline values and a decrease in myocyte shortening. Shortening and pHigradually returned to baseline values after washout.
Fig. 2. Representative trace demonstrating the effect of exposure to and washout of 10 mm NH4Cl on myocyte shortening and intracellular pH (pHi). Exposure to NH4Cl for 2–3 min induced an intracellular alkalosis accompanied by a decrease in diastolic cell length and an increase in myocyte shortening. Subsequent washout of NH4Cl produced a transient intracellular acidosis accompanied by a return in diastolic cell length to baseline values and a decrease in myocyte shortening. Shortening and pHigradually returned to baseline values after washout.
Fig. 2. Representative trace demonstrating the effect of exposure to and washout of 10 mm NH4Cl on myocyte shortening and intracellular pH (pHi). Exposure to NH4Cl for 2–3 min induced an intracellular alkalosis accompanied by a decrease in diastolic cell length and an increase in myocyte shortening. Subsequent washout of NH4Cl produced a transient intracellular acidosis accompanied by a return in diastolic cell length to baseline values and a decrease in myocyte shortening. Shortening and pHigradually returned to baseline values after washout.
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Fig. 3. Summarized data for the effects of propofol on intracellular pH (pHi) in rat ventricular myocytes. Results are expressed as changes from baseline pHiin the absence of any intervention. *Significant change from baseline value (P  < 0.05). n = 16 cells per 4 hearts in each group.
Fig. 3. Summarized data for the effects of propofol on intracellular pH (pHi) in rat ventricular myocytes. Results are expressed as changes from baseline pHiin the absence of any intervention. *Significant change from baseline value (P 
	< 0.05). n = 16 cells per 4 hearts in each group.
Fig. 3. Summarized data for the effects of propofol on intracellular pH (pHi) in rat ventricular myocytes. Results are expressed as changes from baseline pHiin the absence of any intervention. *Significant change from baseline value (P  < 0.05). n = 16 cells per 4 hearts in each group.
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Fig. 4. Summarized data for the effect of 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA), a Na+–H+exchange inhibitor, and bisindolylmaleimide I (Bis), a specific protein kinase C inhibitor, on propofol-induced (100 μm) increase in baseline intracellular pH (pHI). *Significant difference compared with propofol alone (P  < 0.05). n = 16 cells per 4 hearts in each group.
Fig. 4. Summarized data for the effect of 5-(N 
	-ethyl-N 
	-isopropyl)-amiloride (EIPA), a Na+–H+exchange inhibitor, and bisindolylmaleimide I (Bis), a specific protein kinase C inhibitor, on propofol-induced (100 μm) increase in baseline intracellular pH (pHI). *Significant difference compared with propofol alone (P 
	< 0.05). n = 16 cells per 4 hearts in each group.
Fig. 4. Summarized data for the effect of 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA), a Na+–H+exchange inhibitor, and bisindolylmaleimide I (Bis), a specific protein kinase C inhibitor, on propofol-induced (100 μm) increase in baseline intracellular pH (pHI). *Significant difference compared with propofol alone (P  < 0.05). n = 16 cells per 4 hearts in each group.
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Fig. 5. Effect of 30 μm propofol on NH4Cl exposure- and washout-induced changes in intracellular pH (pHi) (A  ) and myocyte shortening (B  ). W/O = peak of acidosis after washout of NH4Cl; W/O 3 = 3 min after washout of NH4Cl. *Significant difference compared with control (P  < 0.05). n = 16 cells per 4 hearts in each group.
Fig. 5. Effect of 30 μm propofol on NH4Cl exposure- and washout-induced changes in intracellular pH (pHi) (A 
	) and myocyte shortening (B 
	). W/O = peak of acidosis after washout of NH4Cl; W/O 3 = 3 min after washout of NH4Cl. *Significant difference compared with control (P 
	< 0.05). n = 16 cells per 4 hearts in each group.
Fig. 5. Effect of 30 μm propofol on NH4Cl exposure- and washout-induced changes in intracellular pH (pHi) (A  ) and myocyte shortening (B  ). W/O = peak of acidosis after washout of NH4Cl; W/O 3 = 3 min after washout of NH4Cl. *Significant difference compared with control (P  < 0.05). n = 16 cells per 4 hearts in each group.
×
Fig. 6. Representative traces demonstrating the effects of propofol on the rate of recovery from intracellular acidosis after washout of NH4Cl. (A  ) 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA) abolished the recovery from intracellular acidosis induced by washout of NH4CI. (B  ) Propofol (30 μm) attenuated the magnitude of the NH4CI acidosis and increased the rate of recovery. (C  ) Data are normalized to peak acidosis values to illustrate the effect of propofol on rate of recovery.
Fig. 6. Representative traces demonstrating the effects of propofol on the rate of recovery from intracellular acidosis after washout of NH4Cl. (A 
	) 5-(N 
	-ethyl-N 
	-isopropyl)-amiloride (EIPA) abolished the recovery from intracellular acidosis induced by washout of NH4CI. (B 
	) Propofol (30 μm) attenuated the magnitude of the NH4CI acidosis and increased the rate of recovery. (C 
	) Data are normalized to peak acidosis values to illustrate the effect of propofol on rate of recovery.
Fig. 6. Representative traces demonstrating the effects of propofol on the rate of recovery from intracellular acidosis after washout of NH4Cl. (A  ) 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA) abolished the recovery from intracellular acidosis induced by washout of NH4CI. (B  ) Propofol (30 μm) attenuated the magnitude of the NH4CI acidosis and increased the rate of recovery. (C  ) Data are normalized to peak acidosis values to illustrate the effect of propofol on rate of recovery.
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Fig. 7. Summarized data for the effects of propofol on the rate of recovery (dpHi/dt) from acid load. *Significant difference compared with control (P  < 0.05). n = 16 cells per 4 hearts in each group.
Fig. 7. Summarized data for the effects of propofol on the rate of recovery (dpHi/dt) from acid load. *Significant difference compared with control (P 
	< 0.05). n = 16 cells per 4 hearts in each group.
Fig. 7. Summarized data for the effects of propofol on the rate of recovery (dpHi/dt) from acid load. *Significant difference compared with control (P  < 0.05). n = 16 cells per 4 hearts in each group.
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Fig. 8. Summarized data for the effect of 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA) on the propofol-induced (30 μm) leftward shift in the [Ca2+]o–shortening relation. *Significant change from control (P  < 0.05). †Significant change from propofol (P  < 0.05). n = 13 cells per 4 hearts.
Fig. 8. Summarized data for the effect of 5-(N 
	-ethyl-N 
	-isopropyl)-amiloride (EIPA) on the propofol-induced (30 μm) leftward shift in the [Ca2+]o–shortening relation. *Significant change from control (P 
	< 0.05). †Significant change from propofol (P 
	< 0.05). n = 13 cells per 4 hearts.
Fig. 8. Summarized data for the effect of 5-(N  -ethyl-N  -isopropyl)-amiloride (EIPA) on the propofol-induced (30 μm) leftward shift in the [Ca2+]o–shortening relation. *Significant change from control (P  < 0.05). †Significant change from propofol (P  < 0.05). n = 13 cells per 4 hearts.
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