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The effect of ethanol and acetaldehyde on Na pump function in cultured rat heart cells
j Mol Cell Cardiol 19, 453-463 (1987)
T h e Effect o f Ethanol and A c e t a l d e h y d e on Na P u m p Function in Cultured R a t H e a r t Cells David McCall* and Kevin Ryan
The Cardiology Division, Department of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, T X 78284, USA (Received 22 September 1986, acceptedin revisedform 27 January 1987) D. MCCALL AND K. RYAN. The Effect of Ethanol and Acetaldehyde on Na Pump Function in Cultured Rat Heart Cells. Journalof Molecularand CellularCardiology(1987) 19, 453~1-63. To further define the sarcolemmal effects of ethanol and acetaldehyde, their effects on Na pump function were studied in synchronously contracting monolayers of neonatal rat myocardial cells. The effects of ethanol (10 mg/dl to 1000 mg/dl: 2 x 10 -3 i~).2 M) and acetaldehyde (10 -6 i~ to 10-4 M) on total 42K influx, ouabain-sensitive 42K influx, Na pump density (from specific 3H-ouabain binding) and pump turnover rates were measured. Applied acutely ethanol had no effect on 42K influx but after 30 min of treatment 't2K influx was decreased by 13%, 23% and 48% in 100 mg/dl, 300 mg/dl and 1000 mg/dl ethanol respectively. This primarily reflected a decrease in mean ouabain-sensitive K + influx from a control of 12.54 to 9.90, 8.95 and 6.68 (p-mol/cm2/s) in 100, 300 and 1000 mg/dl (2 x 10 -2 M, 6 x 10 -2 M and 0.2 M) ethanol. Acetaldehyde in the concentrations tested had no effect on K + influx. Ethanol treatment produced a decrease in Na pump density, maximum within 30 min and dosedependent, at concentrations of 100 mg/dl (22%), 300 mg/dl (37%) and 1000 mg/dl (55%). Acetaldehyde had no effect on Na pump density. In the presence of ethanol (300 mg/dl and 1000 mg/dl) intracellular Na + increased significantly and the Na + efflux declined in parallel with the K + influx. From the ouabain-sensitive K + and Na + fluxes and the Na pump density individual pump turnover rates were calculated at 62.5/s in control cells and 66/s and 84/s in cells treated with 300 mg/dl (6 x 10 -2 M) and 1000 mg]dl (0.2 M) respectively. We conclude that ethanol, but not acetaldehyde has a depressant effect on sarcolemmal Na pump function. The results suggest this is due primarily to a decrease in the number ofsarcolemmal Na pump sites. KEY WORDS: Ethanol; Acetaldehyde; Na Pump; Sodium; Potassium; Ion exchange; Glycoside binding.
Introduction B o t h a c u t e a n d c h r o n i c i n g e s t i o n o f a l c o h o l is associated with depression of cardiac contractility a n d w i t h c a r d i a c a r r h y t h m i a s . T h e m e c h a n i s m s o f t h e s e effects h a v e y e t to b e fully e l u c i d a t e d . E t h a n o l h a s b e e n s h o w n to p r o d u c e m a r k e d m e t a b o l i c [2, 22, 23, 29], u l t r a s t r u c t u r a l [ 6 ] , m e c h a n i c a l [2, 29, 3 6 ] a n d e l e c t r i c a l [2, 8, 24] a l t e r a t i o n s i n b o t h t h e in vivo a n d in vitro m y o c a r d i u m . N e v e r t h e l e s s , n o c l e a r d e f i n i t i o n o f t h e c a r d i o d e p r e s s a n t effects o f e t h a n o l h a s e m e r g e d a n d it a p p e a r s t h a t its effects a r e c o m p l e x a n d m u l t i p l e . Consistent with the belief that ethanol a l t e r s s a r c o l e m m a l s t r u c t u r e a n d f l u i d i t y [4,
19, 27, 34]
are observations of increased myocardial permeability, with leakage of norm a l l y i n t r a c e l l u l a r m o l e c u l e s [7, 14] to t h e e x t r a c e l l u l a r space. C h a n g e s i n m e m b r a n e p e r m e a b i l i t y c o u l d also r e s u l t i n a n a l t e r a t i o n of intracellular electrolytes in the myocardium following alcohol ingestion. It has been s h o w n [28-], for e x a m p l e , i n r a t s fed a d i e t c o n t a i n i n g e t h a n o l t h a t N a + K + C a 2+ and M g z+ t e n d to b e d i s p l a c e d d o w n t h e i r r e s p e c tive c o n c e n t r a t i o n g r a d i e n t s w i t h a r e s u l t i n g increase in myocardial Na + and Ca 2 + and a d e c r e a s e i n c e l l u l a r K + a n d M g 2+. T h e s e changes were ascribed, by the investigators [28], to a r e v e r s i b l e n o n - s p e c i f i c i n c r e a s e i n membrane permeability. However, these
Supported in part by Grant G-607 from the Texas Affiliate of the American Heart Association and by NHLBI Training Grant 5-T32-HL07350. * To whom all correspondence should be addressed. 0022-2828/87/050453 + 11 $03.00]0
9 1987 Academic Press Inc. (London) Limited
454
D. M c C a l l a n d K. R y a n
results, were not reproduced by acute alcohol intoxication in animals of the same species [20] and in the latter studies the sarcolemmat gradient for both Na + and K + actually increased rather than decreased. In view of the potential relationship between ethanolinduced changes in myocardial electrolyte distribution and ethanol-related electrocardiographic changes and arrhythmias this area requires further exploration and clarification. Although ethanol has been shown to inhibit (Na + + K +) ATPase activity in a variety of tissues in vitro [30], including cardiac sarcolemmal vesicles [37], little attention has been directed towards the effect of ethanol on Na + p u m p function in the myocardium. The present study was therefore carried out to evaluate the effects of ethanol, and its primary metabolite, acetaldehyde, on Na p u m p function in cultured myocardial cells. The studies were designed to evaluate only the effects of acute exposure (up to 1 h) of the cells to the agents on p u m p mediated fluxes, Na + p u m p density and p u m p turnover rates.
Materials
and Methods
Preparation of cultured myocardial cells The methods employed in the preparation of the neonatal rat myocyte cultures have been described in detail elsewhere [25, 26]. Using modifications of the method of H a r a r y and Farley [15] the hearts of 1- to 2-day-old rats, removed aseptically, were disaggregated to single cells by repeated trypsinizations. After separation of fibroblasts [25] the cells were seeded in 35 m m diameter petri dishes and incubated in Minimum Essential Medium, supplemented with 10% fetal calf serum, for 4 to 5 days. At the end of the incubation period each dish contained a synchronously contracting monolayer of some 0.6 to 0.75 x 106 cells of which at least 80% were myocytes. The values for cell surface area, cell volume and volume of cell water previously reported [25] were used after their applicability to the cells of each culture were established. The intrinsic contraction frequency of the cells in culture in each case lay between 120 and 140 contractions per minute. None of the concentrations of ethanol tested, up to and including 1000 mg/dl (0.2 M), affected the
contraction frequency of the cells even when present for up to 1 h. The higher concentrations of acetaldehyde (10-4M) produced some slowing of the intrinsic beating rate but, as was the case with ethanol, the cells continued to contract synchronously throughout the experiment. The cells, therefore, remained viable throughout all interventions.
Solutions used All experiments were carried out with the cells in a physiologic Balanced Salt Solution (BSS) [25, 26]. This solution, used to minimize the pH changes which would have occurred had growth medium been used, contained (mM) Na +, 136.80; K +, 5.35; Ca 2+, 2.25; Mg 1+, 1.03; C I - , 148.22; P O ] - , 0.43; glucose, 11.10; plus calf serum 5% and phenol red, 0.0002% (pH 7.2). The cells were allowed to equilibrate in this solution for 2 to 3 h before any flux measurements were made. The ethanol containing solutions were prepared by adding the desired volume of absolute ethanol, using a micropipette, to BSS (10 ml) in the petri dish. Immediately upon addition of the ethanol the petri dishes were sealed with a plexiglass plate, on the undersurface of which was a sheet of Parafilm. This effected a perfect seal with the petri dish and in essence provided a closed vessel with a head space of less than 0.5 mt. The sealed dish was thoroughly mixed to ensure even distribution of the ethanol. The ethanol levels in the petri dishes were verified (Stat-Pack Ethyl Alcohol Test, Behring Diagnostics, La Jolla, CA) and were found to remain stable in this system throughout the period of the study. The acetaldehyde-containing solutions were prepared in a similar manner. The petri dishes were filled with BSS and sealed, as described above, with a plexiglass plate. In this case, a small rubber dam was incorporated in the plate. The solutions were then prepared by injecting acetaldehyde (diluted 1 : 1000 or 1 : 100) at 4~ through the dam into the solution using a pre-cooled microsyringe. The solutions were then thoroughly mixed in the closed containers. No verification of the acetaldehyde concentrations could be obtained, but in parallel studies (unpublished observations) there was a dose-dependent decrease in contraction frequency, velocity of
Alcohol in C u l t u r e d H e a r t Cells
cell edge movement and calcium influx. These observations would tend to confirm the continued presence of acetaldehyde in this closed system and that the concentration was related to that introduced in the manner described.
Measurement of ionfluxes The methods employed to determine the K + influx have been described previously [21, 25, 26]. K + exchange in these cells can be described by a single exponential having a half-time of exchange of around 12 rain [25, 26]. Influx measurements were therefore made by exposing the cells to 42K for a period of time (2 min), short compared to the halftime of K + exchange. Following exposure for 2 min to ~2K, the cells were washed free of extracellular tracer using an isotonic Na +and K+-free Ca2+-sorbitol solution [25"] at 0~ The cellular content of 42K was then determined and the influx, as p-mol/cm2/s, calculated as described previously [25]. Na + efflux was measured by equilibrating the cells with 24Na and then observing its loss into a non-radioactive solution by measuring the amount left in the cells at various times [25]. By plotting the initial and residual tracer contents against time, efflux curves were constructed and the efllux calculated [25]. All flux studies were carried out at 37~ During the influx studies, the plates were gently agitated on a warming plate at 37~ and in the efflux studies the non-radioactive efflux solution at 37~ was continually replaced at a rate of 40 to 60 ml/min [25]. Where the effects of a drug on an ion flux was determined that drug was included in the radioactive influx solution or the non-radioactive effiux solution in the appropriate concentration. All fluxes were divided into their active, or ouabain-sensitive, and passive components by parallel measurements made in the absence and in the presence of 10 -2 ~I ouabain [25,
26]. The Na + and K + contents of the cells were determined by equilibration with 24Na and 42K respectively. Following equilibration the cells were washed free of extracellular tracer, as described above, and the contents calculated as before [-25]. This method of determination of cell ion content has previously been shown [25] to agree very closely with cell Na +
455
and K + content as determined by flame photometry.
Determination of 3H-ouabain binding The number of Na pump sites per cell under various experimental conditions, was measured using 3H-ouabain binding [25]. 3Houabain (New England Nuclear Corp., Waltham, MA, USA) was used to prepare a stock solution of 2 • 10 -6 M 3H-ouabain in BSS from which serial dilutions were made to achieve the desired final concentrations. Ouabain uptake was measured by exposing the cells to 3H-ouabain for 30 min [25], washing the cells free of extracellular tracer and measuring the amount bound by liquid scintillation spectrometry. The amount bound (molecules of 3H-ouabain per cell) was then determined from the cell number, sample counts and the specific activity of the soak solution. Duplicate determinations of 3H-ouabain bound were made in the presence and absence of 20 mM K + to provide separation of specific from non-specific binding [25]. Preliminary studies were carried out in the presence of all of the concentrations of ethanol and acetaldehyde tested to ensure that the kinetics of 3H-ouabain binding were not influenced by the presence of the test agent. Under all conditions, it was found that, as before [25"], maximum specific binding occurred in the presence of 2 x 10-7M 3H-ouabain in a K +-free solution. All isotopes were counted in a Packard Tricarb liquid scintillation spectrometer; 24Na and 42K were counted without added scintillant [10].
Materials
24Na, 42K and
3H-ouabain were purchased from New England Nuclear Corp., Waltham, MA, USA. Biologicals were from Gibco Laboratories, Grand Island, NY, USA and all chemicals from Sigma Corp., St. Louis, MO, USA were of analytical grade.
Statistical analysis Students' t-tests for paired and unpaired data were used to test for the significance of differences between groups. Linear regression
456
D. M c C a l l
and K. Ryan 20
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I0 I00 300 IO00 Ethanol (mg/dl) FIGURE 1. Total K + influx, myocardial cells at 37~ in absence (0) and presence of each of the concentrations of ethanol tested following 2 min exposure of the cells to ethanol. Bars represent 1 S.E.M.; for each column n -----6. analyses were done b y least-squares fit. All results are expressed as m e a n q- S.E.M.
Results
Effect of ethanol on If + influx A p p l i e d acutely to the cells, ethanol, in concentrations from 10 m g / d l to 1000 mg/dl, h a d no effect on the total *2K influx (Fig. 1). T h e control influx (p-mol/cm2/s) 16.08 _ 0.26 (mean -F S.E.M., n = 6) was similar to that previously r e p o r t e d [25, 26] a n d was unaffected b y a n y of the concentrations tested (Fig. 1) in these studies, in which the cells were exposed to the ethanol for a total time o f 2 mins. W h e n ceils were exposed to ethanol in the concentrations tested (10 m g / d l to 1000 m g / d l : 2 x 10 - a M to 0.2 M) for a period o f 30 mins (Fig. 2) there was a progressive, dosed e p e n d e n t decrease in total 42K influx. T h e total 42K influx (p-mol/cm2/s) decreased from a control value (mean + S.E.M.) of 15.57 + 1.98 to 15.54 + 1.64, 13.50 __+ 1.04, 11.92 + 1.30 and 8.01 _ 1.20 in 10 m g / d l (2 x 10 - 3 M), 100 mg/dl (2 x 10 - 2 U), 300 m g / d l (6 x 10 - 2 M) a n d 1000 m g / d l (0.2 u) ethanol respectively. C o m p a r e d to control, the reductions in 4ZK influx were significant in the presence of 100 m g / d l (P < 0.05), 300 m g / d l (P < 0.01) a n d 1000 m g / d l (P < 0.001) ethanol (for each n = 6-10).
'
I
0
I0 I O0 300 Ethanol (rng/dl)
I000
FIGURE 2. Constituent components of K + influx in control cells (0) and cells treated with ethanol for 30 rain. Total column represents total K + influx, shaded portion the influx in the presence of 10-2 M ouabain (ouabaininsensitive component) and the difference between the two represents the ouabain-sensitive or active K + influx. Bars represent 1 S.E.M.; for each n = 6. F u r t h e r exposure of the cells to ethanol for up to 60 mins resulted in no further decrease in the total 42K influx over those values o b t a i n e d at 30 rains ( T a b l e 1). F u r t h e r c h a r a c t e r i z a t i o n o f the effects o f ethanol on the 42K influx was o b t a i n e d from p a i r e d m e a s u r e m e n t s o f 42K influx, in the presence a n d absence of 10-2 M o u a b a i n , in cells, p r e t r e a t e d with each of the concentrations of ethanol tested, for 30 rains. As shown in Figure 2, the decline in 42K influx, in the presence of ethanol, p r i n c i p a l l y reflected a d o s e - d e p e n d e n t decrease in the active, or ouabain-sensitive c o m p o n e n t . I n control cells the ouabain-sensitive 42 K influx was measured at 12.54 _ 0.99 p-mol/cm2/s or 75% to 80% of the total influx, b o t h figures being similar to that previously r e p o r t e d for these cells [25]. I n cells treated with ethanol for 30 rains the ouabain-sensitive 42K influx (p-mol/cm2/s) was 12.12 _ 0.69 (vs control p = ns), 9.90 + 1.09 (vs control P < 0.05), 8.95 _ 52 (vs control P < 0.01) a n d 6 . 6 8 _ 0.78 (vs control P < 0.001) in 10, 100, 300 a n d 1000 m g / d l ethanol respectively (for each, n = 6). T h e r e was also small d o s e - d e p e n d e n t decrease in the ouabain-insensitive comp o n e n t to the 42K influx (Fig. 2) following 30 mins of ethanol t r e a t m e n t which did reach levels of significance in the presence of 300 m g / d l (6 x 10 - 2 M) and 1000 m g / d l (0.2 M)
Alcohol in Cultured Heart Cells
457
TABLE 1. Effect of 60 minutes' treatment with ethanol on Na:K transport in cultured myocardial cells E t h a n o l (mg/dl) (re) K influx (p-mol/cm2/s) Total O u a b Sens O u a b Insens
[Na]~ (raM) N a + efflux (p-mol/em2/s) Total O u a b Sens Na : K stoichiometry 3H o u a b a i n b i n d i n g ( x 106 molecules/cell) Pump turnover rate
0 0
15.57 12.54 3.03 13.16
• • + •
1.98 0.99 0.05 0.81
10 2 x l0 - s re
100 2 x 102 M
300 6 x 10 - 2 re
1000 0.2 M
15.94_+ 1.04 12.88 ___ 0.92 3.06 ___ 0.45
12.17+_0.47 b
10.36+_0.70 c
9.39 + 0.13 b 2.77 • 0.23
8.35 + 0.43" 2.02 ___ 0.56 b 19.39 • 0.94 c
8.22_+0.92 c 6.89 ___ 0.63 ~ 1.33 ___ 0.36 c 27.78 • 1.12"
26.30 + 0.74 b 12.52 • 1.21 b 2.7:2 0.83 • 0.09 ~
22.61 + 1.42 b 11.39 ___ 0.73" 3.4:2 0.64 _ 0.14"
66
84
30.48 • 1.28 18.78 + 1.03 3 :2 1.32 • 0.03
1.02 + 0.09 a
62.5
61
(s-I) Values given as mean _+ S.E.M. (for each n = 6-10). Compared to control (left-hand column) "P < 0.05; bp < 0.01 ; cp < 0.001.
ethanol (P < 0.01 respectively).
and
P < 0.001
Effect of acetaldehyde on K + influx None of the concentrations of acetaldehyde tested, from 10 -6 ~ to 10 -4 M, had any effect on the 42K influx either on immediate application of the drug to the cells or when the cells had been exposed to the drug for up to 60 mins. Compared to control cells, the acetaldehyde treated cells showed no change in the total 42K influx, or in its ouabainsensitive or ouabain-insensitive components. Effect of ethano ! and acetaldehyde on 3H-ouabain binding In order to further characterize the effects of ethanol on Na pump dependent K § fluxes, studies were carried out to define the maximum specific ouabain binding in ethanol treated cells. This was done to permit calculation of Na pump density (pump sites per cell) and individual pump turnover rates. Ethanol, in any of the concentrations tested, did not affect the kinetics of 3Houabain binding. In control and in ethanoltreated cells 3H-ouabain binding was rapid (T89 = 10 min) and in each case had reached saturation within 30 rain. As in prior studies [25] it was found that maximum specific binding occurred at 2 x 10 -v M 3H-ouabain in a K +-free solution. Treatment of the cells with ethanol 1001000 mg/dl (2 x 10 -2 M-0.2 M) for 30 mins
led to a progressive decline in maximum specific 3H-ouabain bound (Fig. 3). 3H-ouabain bound ( x 106 molecules 3H-ouabain per cell) declined from a control (mean + S.E.~.) of 1.32 ___0.03 tO 1.02 __+0.28, 0.83 __+0.09 and 0.64 + 0.14 in 100, 300 and 1000 mg/dl ethanol respectively. The decrease in 3Houabain bound, compared to control cells, was significant for each concentration tested, P < 0.05 at 100 mg/dl and P < 0.001 for the higher concentrations (for each n = 6). Further exposure of the cells to ethanol for up to 60 mins (Fig. 3) resulted in no further change in specific 3H-ouabain bound per cell. In contrast to the effects of ethanol, acetaldehyde (10-6M to 1 0 - 4 ~ ) even when present for 60 mins, had no effect on 3Houabain binding in the cultured myocardial cells.
Effect of ethanol on intraceUular Na + and Na +
(flux In view of the apparent inhibitory effect of ethanol on the active K + influx, and the fact that it appears to decrease the number of Na ~ump sites per cell (as measured by specific H-ouabain binding) it was felt that the effects on intracellular Na + concentration ([Nail) and the Na + effiux should be examined. This would provide confirmation of the inhibitory effect of ethanol on active transport via the Na-pump and also permit calculation of the stoichiometry of Na + : K + exchange by the pump in ethanol-treated cells.
458
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FIGURE 3. Maximum specific 3H-ouabain binding in untreated cells (0) and cells treated with ethanol for 30 min and 60 min. Bars represent 1 S.E.M.; for each n = 6. Measurement of [ N a + ] i and Na+-efflux, using 24Na as a tracer, were made in cells exposed to ethanol, 300 mg/dl (6 • 10 -2 M) and 1000 mg/dl (0.2 M) for 60 mins, at which time both the K § influx and 3H-ouabain binding were in a steady state. After 60 rains of ethanol treatment [ N a + ] i (m-mols/l cell H 2 0 ) had increased from a control of 13.16 • 0.81 to 19.39 • 0.94 and 27.78 • 1.12 in 300 mg/dl and 1000 mg/dl respectively (for each vs control P < 0.001 and n = 10). T h e increase in [ N a + ] i seen in the presence of ethanol was accompanied by a decrease in the 2r efflux. T h e control 24Na efflux (pmol/cmZ/s) from untreated cells was 30.48 _+ 1.28 (mean • S.E.M.), similar to that previously measured in these cells [25]. Following 60 mins treatment this was decreased to 26.30 • 0.74 in 300 mg/dl (6 x 10 -2 M) ethanol and 22.61 4-1.42 in 1000 mg/dl (0.2 M) ethanol (for each P < 0.01, n = 5). As in the case of the 42K influx, the reduction in 24Na efflux largely reflected a decrease in the ouabain-sensitive component, which declined from a mean control value of I8.8 p-mol/cm2/ s to a mean of 12.5 and 11.4 in 300 and 1000 mg/dl ethanol respectively (Table 1). There was no significant effect of ethanol on the ouabain-insensitive 2r effiux.
Effect of ethanol on Na pump stoichiometry and turnover rates From the ouabain-sensitive N a + and K + fluxes described above, it was possible to cal-
culate the stoichiometry of N a + : K + exchange, via the N a pump, under the various conditions tested. I n untreated cells the ratio was 3 N a + : 2 K + as previously described. This ratio did not appear to be affected by ethanol treatment and remained close to 3 : 2 (2.7 N a + : 2 K + in 300 mg/dl and 3.4 N a + : 2 K + in 1000 mg/dl). Assuming that one N a p u m p site binds one molecule of 3H-ouabain, the data presented (Fig. 3) suggests that, in the presence of ethanol, there is a decrease in the n u m b e r of Na p u m p i n g sites per cell. Using the n u m b e r of N a p u m p sites per cell after 60 mins ethanol exposure, from the 3H-ouabain studies, and the ouabain-sensitive N a + and K + fluxes under identical circumstances, individual p u m p turnover rates were calculated. I n the untreated cells, the p u m p turnover rate was 62.5/s, similar to that previously reported in these cells [25] and this was unaffected by 100 mg/dl ethanol (61/s). In the presence of 300 mg/dl and 1000 mg/dl ethanol, however, the turnover rates tended to be greater than control at 66/s and 84/s respectively.
Effect of ethanol treatment on cell volume T o ensure that the above observations of the effects of ethanol on ion fluxes and 3Houabain binding were not a reflection of osmotically induced changes in cell volume, measurements of the effect of ethanol on cell volume were made. Cell volume measurements were made using the diameters of cells
Alcohol hut C u l t u r e d H e a r t Cells
459
and guinea-pig cerebral cortex ethanol inhibited (Na § + K § ATPase [16], an observation which, in a later study [17], was correlated with a decrease in the indirectly measured active K + transport in the same preparation. It is of interest that the concentration of ethanol (0.22 M) producing halfmaximal inhibition of (Na + + K +) ATPase in these studies [16, 17] inhibits some 45% of the active K § influx in the present preparation. Other workers [33] have confirmed the inhibitory effect of ethanol on brainderived ( N a + + K +) ATPase activity and with few exceptions [5] there appears to be universal agreement [30] on this effect of ethanol. Similar observations of the inhibitory effect of ethanol on (Na + + K +) ATPase activity Discussion were made in cardiac sarcolemmal vesicles While there are several studies which show [37]. In this case, however, the concentration that ethanol decreases ( N a + - K +) ATPase of ethanol required to produce half maximal activity in a wide variety of tissues [30], inhibition (0.6 ~) was considerably higher including cardiac sarcolemma [37], this study than that required in brain tissue [16]. In the represents the first report of experiments heart [37], as in the brain [16], the inhibitory designed to define the effects of ethanol effect of ethanol seemed to be enhanced at low directly on Na pump function in intact myo- K § concentrations possibly reflecting the fact cardial cells. The concentrations of ethanol that ethanol appears to inhibit the terminal dephosphorylation step of the tested, 10 mg/dl-1000 mg/dl (2 • 10 -3 M to K§ 0.2 M), were selected to include those corre- reaction chain [31]. Consistent with inhibition of membrane sponding to the blood alcohol levels associated with minor alcohol use (10 mg/dl to 100 ( N a + + K +) ATPase are both direct and mg/dl), moderate to advanced inebriation indirect observations that ethanol, in concen(100 mg/dl to 300 mg/dl) as well as the more trations between 0.1 ~ and 0.7 M, inhibits pharmacologic concentrations of 1000 mg/dl active K + uptake by both red blood cells [32] used in prior studies [30]. The model of cul- and brain tissue [17, 18]. More recent studies tured neonatal rat myocardial cells was select- [28] showing an increase in [Na+]i and a ed because the basic properties of Na + :K + decrease in [ K § in the myocardium of rats exchange of the preparation had been defined fed alcohol are consistent with active trans[25] and had been shown to be similar in both port inhibition, but have been interpreted by properties and behaviour to that in other the authors as representing nonspecific mammalian myocardial preparations [25]. changes in membrane permeability. The present study, therefore, in some ways No part of the study was designed to examine the effects of chronic ethanol exposure but was confirms the work of others showing that, confined to an examination of the acute effects acutely, ethanol produces a dose-dependent decrease in active K + influx via the Na pump. of both ethanol and acetaldehyde. There is considerable evidence that ethanol At the highest concentration of ethanol tested, inhibits (Na + + K +) ATPase of a variety of 1000 mg/dl (0.2 M) active transport is inhibmembrane preparations, in vitro, which has ited by 50%, a finding similar to that in rat been elegantly reviewed by Roach [30]. Most brain microsomes [16]. The inhibitory effect of this work, however, has utilized brain and of ethanol on active transport of K + seen in other nerve tissue in an attempt to define the the present study did not occur immediately nature of ethanol effects in the central nervous upon addition of ethanol to the cells, but system. In microsomal preparations from rat rather took some 30 rains to fully develop.
in trypsinized suspensions prepared from monolayer cultures as previously described [-25], since this is in good agreement with the volume of the flattened cells [19, 25]. None of the concentrations of ethanol tested had any effect when present on the cells for up to 30 mins. The only measurable change in cell volume was seen in cells treated with 1000 mg/dl (0.2 M) ethanol for 60 rains. In these cells a 20% increase in cell volume was noted, from a control of 2.71 + 0.27 x 10 - 9 cm 3 to 3.22 __+0.16 x 10 - 9 c m 3 ( P < 0.01 ; for each n = 50). This increase in volume presumably represents an increase in cell water following the gain in [Na~-]i.
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D. McCall and K. Ryan
This suggests that the decrease in K + influx is not due to simple competition between ethanol and K + for a common receptor on the Na pump entity, since even the highest concentration tested, 1000 mg/dl (0.2 M), did not affect any component of the K + influx when present on the cells for only a short period of time. The increase in intracellular Na + seen in the ethanol-treated cells and the parallel decrease in the Na + effiux would further support Na pump inhibition rather than ethanol: K + competition. In addition, if the decreased K § influx were merely a reflection of ethanol competing for the K + site, then it would be extremely unlikely that the pump stoichiometry would remain 3 Na + :2K + in the ethanol treated cells. It is possible, however, that ethanol competition for the K + binding site on the Na pump could displace K + and in so doing could prevent pump turnover. It is conceivable under those circumstances that stoichiometry would he unaltered since the measured residual Na + and K + fluxes would occur via non-affected pumps. Taken together, however, the data argue very strongly that ethanol produces true inhibition of active coupled Na + and K + transport. The reason for the slow onset of the inhibition by ethanol is unclear. It is possible that the effect may be mediated by the influence of myocardial metabolites of ethanol such as fatty acid ethyl esters [22] with their capability to destroy the integrity of the phospholipid bilayer of the sarcolemma. Alternatively, it has been shown that the assembly ofglycoproreins into hepatic plasma membranes is inhibited by ethanol [34] and, therefore, the inhibitory effect of ethanol on active transport may represent inhibition of assembly of the component proteins of the transport system. Both of these processes are likely to be timedependent. It is impossible to determine whether the time course of the inhibitory effect of ethanol is different in this tissue from that in others, since in each case [16, 17, 33] ethanol was present in the reaction mixture for at least 20 to 30 mins. In only one study [5] was the effect of ethanol on microsomal (Na + + K +) ATPase activity measured over a period of less than 15 mins. In this study [5"J, however, it is of interest to note that no inhibitory effect of ethanol on N a + - K + ATPase activity was seen. Although, therefore, no
direct comparisons of the inhibitory effect of ethanol on active transport or (Na + + K +) ATPase in this tissue, with that in others can be made, the indirect evidence above suggests that it may be a time-dependent function in all. In a recent study, Green and Baron [13-] have shown that ethanol inhibits the influx of S6Rb, a K + analog, in human erythrocytes and leucocytes. Further, the inhibitory effect of ethanol was both quantitatively and qualitatively very similar to that shown in the present study. O f particular interest in this latter study [13] was the finding that ethanol in addition to inhibiting the active Rb + influx, produced a dose-dependent decrease in the ouabain-insensitive influx of that ion, a finding very similar to that of the present study. As in the present study, this did not reach levels of significance in concentrations of ethanol less than 0.16 M. The reasons for this effect of high concentrations of ethanol on the ouabain-insensitive K + influx, although of interest, are unclear. It is possible that inhibition of the Na pump by ouabain, even in concentrations of 10 -2 M, is incomplete and that the addition of a second inhibitor, ethanol, of N a + + K+-ATPase permits more complete inhibition of the pump. Although this seems unlikely, from previous studies [25, 26-J, this possibility cannot be completely discounted. The effect may be due to a decrease in passive exchange diffusion of the ion about a semipermeable membrane which is known to be dependent on the concentrations of the ion on either side of the membrane [35]. With inhibition of the active K + influx, in the presence of ethanol, intracellular K + will be decreased and the gradient favouring exchange diffusion of K § [35] decreased. It is unlikely that the effect is due to ethanol : K competition for the reasons stated previously, but this possibility cannot be completely dismissed. Inhibition of the Na pump by ethanol, resulting in changes in intracellular Na + and K + distribution, may affect the membrane potential which could in turn influence passive K § influx. Finally, a direct effect of ethanol on the phospholipids of the cell membrane could influence the passive exchange of many ions, including K+. Whatever mechanism is involved, however, it must be pointed out that inhibition of the ouabain-insensitive K +
Alcohol in C u l t u r e d Heart Cells
461
0.020 A
0.015 Q ~ 0.010
- 0 0 6 - 0 . 0 4 -0.02
I
I
I
0.02
0.04
0.06
0.I0
I/[No]i (rn.- ') FIGURE 4. Double reciprocal pl0t of [Na]i and calculated Na pump turnover rates. Data derived from control cells and cells treated with ethanol (300 mg/dl and 1000 mg/dl) for 60 min. Line shown is calculated linear regression line (y = 0.12 x +0.007; r = 0.98). Although only means are shown all contributing values for [Na]i were used in the calculation. influx is only seen at high ( > 0.1 M) concentrations of ethanol a n d is unlikely therefore to be of significance in the h e a r t in vivo. F r o m the 3 H - o u a b a i n - b i n d i n g studies it would a p p e a r t h a t the decreased active transp o r t in the presence of ethanol, in cultured h e a r t cells, is due to a decrease in the n u m b e r of available p u m p sites per cell. A l t h o u g h a possibility, it is unlikely that the decreased specific 3 H - o u a b a i n b i n d i n g seen in the etha n o l - t r e a t e d cells represents competitive inhibition of glycoside b i n d i n g by ethanol. This possibility was, however, explored in confirm a t o r y studies in which it was shown that no further increase in specific b i n d i n g occurred, in the presence of ethanol, at concentrations of 3 H - o u a b a i n of 10 - 5 M. T h e decrease in functional N a p u m p sites, therefore, a p p e a r s to be real a l t h o u g h the precise reasons for this decrease in N a p u m p density r e m a i n unclear. I t m a y reflect loss of functioning p u m p sites due to the solubilizing effect of ethanol on the p h o s p h o l i p i d m e m b r a n e . O n the other hand, there m a y be i m p a i r e d assembly of the constituent proteins of m e m b r a n e structures such as has been d e m o n s t r a t e d in hepatocytes [34]. W h a t e v e r the mechanism, however, the decrease in N a p u m p density is sufficient to account for the decreased active K + t r a n s p o r t a n d m a y well be the p r i m a r y factor in decreasing active t r a n s p o r t in e t h a n o l - t r e a t e d cells. It is well recognized that N a p u m p function is regulated by extracellular K + concentration and i n t r a c e l l u l a r N a + concentration. I n
these cells it has been shown t h a t the K"m of K + on active t r a n s p o r t is 1.8 m i [25] a value similar to that in other tissues [12]. In BSS with a [ K * ] 0 of 5.4 mM it is likely that the s t i m u l a t o r y effect of K + on the N a p u m p would be at, or n e a r a m a x i m u m . T h e increased turnover rate seen in the residual p u m p s in the e t h a n o l - t r e a t e d cells therefore p r o b a b l y reflects the influence of the increased [Na§ seen u n d e r these conditions. Stimulation of active N a + effiux via the N a p u m p b y [ N a + ] i in o t h e r tissues has been described as being either linear, showing no signs of s a t u r a t i o n [1, 3] or showing s a t u r a t i o n only at very high values of i n t r a c e l l u l a r N a + [9, 11]. I n several tissues including red blood cells [9] a n d atrial muscle [11] t h e / ( m of intracellular N a + on active N a effiux was found to be 22 to 25 m i , considerably higher t h a n the n o r m a l [ N a + ] i of a r o u n d 12 mM found in cultured n e o n a t a l rat h e a r t cells [25]. T h e modest increase in N a p u m p turnover rates seen in the ethanol treated cells is therefore, most p r o b a b l y , due to the increased driving force p r o v i d e d by the increased i n t r a c e l l u l a r N a +. I n d e e d , when a double reciprocal plot of intracellular Na + and Na pump turnover rates is constructed (Fig. 4) an a p p a r e n t / f r o of i n t r a c e l l u l a r N a + on p u m p t u r n o v e r rate of a p p r o x i m a t e l y 22 m i is obtained. I t has to be p o i n t e d out that the construct of the doublereciprocal plot of Figure 4 is o b t a i n e d from a limited a m o u n t of d a t a and that some of that d a t a is from cells p r e t r e a t e d with ethanol which could conceivably alter the affinity of
462
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Na + for its receptor on the N a p u m p . Although some caution has to be used in interpreting the data from Figure 4, it is of interest that the a p p a r e n t K m obtained is in good agreement with that in both red blood cells [9] a n d atrial muscle [11]. O n e previous report [5] showed that acetaldehyde inhibited (Na + + K +) ATPase of b r a i n microsomes. I n the present study, acetaldehyde had no measurable effect on active transport in the cultured n e o n a t a l heart cells. Although great care was taken to carry out the studies in sealed containers it is possible, that because of its extremely volatile nature, aceteldehyde evaporated from the petri dishes immediately u p o n its addition to the BSS. Although no direct measurements of acetaldehyde levels were available, it is unlikely that all of the acetaldehyde evaporated. This is supported by observations that, u n d e r identical experimental conditions, the addition of acetaldehyde to the BSS caused marked suppression of the contractile activity of the cells which was sustained for up to 60 rains. I n conclusion, therefore, ethanol, b u t not acetaldehyde, suppresses active transport by
the Na p u m p in cultured rat ventricular myocytes. T h e effect of ethanol is both timeand dose-dependent a n d appears to be mediated by an e t h a n o l - i n d u c e d decrease in the density of N a p u m p sites on the sarcolemma. T h e active transport system in these cells, i n c l u d i n g its glycoside-sensitivity (IDs0-10 -6 M), is very similar to that o f a d u h m a m m a l i a n cells [25] including those of man. I n view of this, the observation that concentrations of ethanol encountered d u r i n g acute alcoholic intoxication (300 mg/dl) significantly depress Na p u m p function in these cells m a y have considerable clinical relevance. Ethanolinduced decreases in active transport with c o n c o m i t a n t changes in [ N a i l a n d [ K + ] i m a y be i m p o r t a n t c o n t r i b u t i n g factors to the electrocardiographic changes a n d arrhythmias seen in acute alcoholic intoxication.
Acknowledgements T h e authors wish to acknowledge the expert technical assistance of M a g d a l e n a Garcia and the i n v a l u a b l e secretarial services of A n n a Fackenthall.
References 1 BAKER,P. F., BLAUSTEIN,M. P., HODGLIN,A. L., STEINHARDT,R. A. The influenceof calcium on sodium el'T[uxin squid axons.J Physiol [Lond] 200, 431~J,58 (1969). 2 BING,R.J. Cardiac metabolism: Its contribution to alcoholicheart disease and myocardial failure. Circulation 58, 965 970 (1978). 3 BRINK,F. Linear range ofNa + pump in sciatic nerve of frog. AmJ Physio1244, C 198-C204 (1983). 4 CHIN,J. H., GOLDSTEm,D. B. Membrane-disordering action of ethanol: variation with membrane cholesterol content and depth of spin label probe. Mol Pharmaco119, 425431 (1981). 5 ERWIN,V. G., KIM,J., ANDERSON,A. D. Effects of aldehydes on sodium plus potassium ion-stimulated adenosine triphosphatase of mouse brain. Biochem Pharmaco124, 2089 2095 (1975). 6 FERRANS,V.J., HIBBS, R. G., WEILBASCHER,D. G., BLACK,W. C., WALSH,J. J., BURCH,G. E. Alcoholic cardiomyopathy. A histochemical study. Am HeartJ 69, 748 765 (1965). 7 FINK,R.~ MARJOT,D. H., ROSALKI,S. B. Detection of alcoholic cardiomyopathy by serum enzyme and isoenzyme 8
determination. Ann Clin Biochem 16, 165-166 (1979). FISHER,V.J., KAVALER,F. The action ofcthanol upon thc action potential and contraction ofvcntricular muscle.
Rec Adv Study Cardiac Struct Metabol 5, 415-422 (1973). 9 GARA7,R. P., GARRAHAN,P. J. The interaction of sodium and potassium with the sodium pump in red cells.J Physiol [Lond] 231,297 325 (1973). 10 GARRAHAN,P. J., GLYNN,I. M. Measurement of 24Na and 42K with a liquid scintillation system without added scintillant.J Physiol[Lond] 186, 55P 56P (1966). 11 GLITSCH,H. G. Characteristics of active Na transport in intact cardiac cells. Am J Physiol 236, H189-H199 (1979). 12 GLITSCH,H. G., GRABOWSKI,W., THEILEN,J. Activation of the electrogenicsodium pump in guinea pig atria by external potassiumions.J Physiol[London] 276, 515-524 (1978). 13 GREEN,R.J., BARON,D. N. The acute in vitro effect of ethanol, its metabolites and other toxic alcohols on ion flux in isolated human leucocytesand erythrocytes. BiochemPharmaco135, 3457-3464 (1986). 14 H.~LLOREN,R. LUNDIN,L., ROXIN,L.-E., VENOE,P. Serum and urinary myoglobinin alcoholics. Acta Med Scand 208, 33-39 (1980). 15 HARARY,I., FARLEY,B. In vitro studies of single beating rat heart cells. I. Growth and organization. Exp Cell Res 29, 451 465 (1963).
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16 ISRAEL,Y., KALANT,H., LANFER, I. Effects of ethanol on Na, K, Mg-stimulated microsomal ATPase activity. Biochem Pharmacol 14, 1803 1814 (1965). 17 ISRAEL,Y., KALANT,H., LEBLANC,A. E. Effects of lower alcohols on potassium transport and microsomal adenosine-triphosphatase activity of rat cerebral cortex. BiochemJ 100, 27 33 (1966). 18 ISRAEL-JACARD,Y., KALANT,H. Effect of ethanol on electrolyte transport and electrogenesis in animal tissues. J Cell Comp Physio165, 127-132 (1965). 19 JOHNSON,D. A., LEE, N. M., COOKE,R., LOH, H. H. Ethanol-induced fluidization of brain lipid bilayers. Required presence of cholesterol in membranes for the expression of tolerance. Mol Pharmacol 15, 739~746 (1979). 20 KISCHUK,R. P., OTTEN,M. D., POLIMENI,P. I. Effect of acute alcoholic intoxication on myocardial electrolyte and water distributions. J Mol Cell Cardio118, 197-205 (1986). 21 LAMB,J. F., MCCALL,D. Effect of prolonged ouabain treatment on Na, K, C1 and Ca concentrations and fluxes in cultured human cells.J Physiol [Lond] 225, 599-617 (1972). 22 LANGE,L. G., SOBEL,B. E. Myocardial metabolites of ethanol. Circ Res 59, 479-482 (1983). 23 LOCHNER,A., COWLEY, R., BRINK, A.J. Effect of ethanol on metabolism and function of perfused rat heart. Am HeartJ 78, 770 779 (1969). 24 LUCA,C. Electrophysiological properties of right heart and atrioventricnlar conducting system in patients with alcoholic cardiomyopathy. Br HeartJ 49, 274-281 (1979). 25 MCCALL,D. Cation exchange and glycoside binding in cultured rat heart cells. Am J Physiol 236, C87 C95 (1979). 26 MCCALL,D., ZIMMER,L.J., KATZ, A. M. Kinetics of thallium exchange in cultured rat myocardial cells. Circ Res 56, 370 376 (1985). 27 PATERSON,S.J., BUTLER,K. W., HUANO,P., LABELLA,J.,SMITH,I. C. P., SCHNEIDER,H. The effects of alcohol on lipid bilayers : A spin-label study. Biochem Biophys Acta 266, 597 602 (1972). 28 POLIMENI,P. I., OTTEN, M. D., HOESCHEN,L. E. In vivo effects of ethanol on the rat myocardium: Evidence for a reversible, non-specificincreaseinsarcolemmalpermeability.JMol Cell Cardio115, 113 122 (1983). 29 REOAN,T.J., ETTINGER,P. O., HAIDER,B., AHMED,S. S., OLDEWURTEL,H. A., LYONS,M. M. The role of ethanol in cardiac disease. Annu Rev Med 28, 393-409 (1977). 30 ROACH,M. K. Changes in the activity of Na +, K+-ATPase during acute and chronic administration of ethanol. In Biochemistry and Pharmacology of Ethanol, vol. 2, E. Majchrowicz, E. P. Noble (Eds), pp. 67-80. New York: Plenum Press (1979). 31 RODNIOrtT,R. The effect of chemical agents on the turnover of the bound phosphate associated with the sodiumand-potassiumion-stimulated adenosinetriphosphataseinoxbrainmicrosomes.BiochemJ 120, 1 13 (1970). 32 STREETEN,D. H. P., SOLOMON,A. K. The effect of ACTH and adrenal steroids on K transport in human erythrocytes. J Gen Physio137, 643-661 (1954). 33 SUN,A. Y., SAMORAJSKI,T. The effects of age and alcohol on (Na + + K+)-ATPase activity of whole homogenate and synaptosomes prepared from mouse and human brain.J Neurochem 24, 161-164 (1975). 34 TUMA,D.J., MAILLIARD,M. E., CASEY,C. A., VOLENTXNE,G. D., SORRELL,M. F. Ethanol-induced alterations of plasma membrane assembly in the liver. BiochimBiophysActallk56,571 577 (1986). 35 USSING,H. H. The distinction by means of tracers between active transport and diffusion. Acta Physiol Scand 19, 43 56 (1950). 36 WEISHAAR,R., SARMA,J. S. M., MARUVAMA,Y., FISCHER, R., BERTUOLXA,S., BING, R.J. Reversibility of mitochondrial and contractile changes in the myocardium after cessation of prolonged ethanol intake. Am J Cardiol 40, 556-562 (1977). 37 WILLIAMS,J. W., TADA, M., KATZ, A. M., RUBIN, E. Effect of ethanol and acetaldehyde on the (Na + + K+) activated adenosine triphosphatase activity of cardiac plasma membranes. Biochem Pharmaco121, 27-32 (1975).
T h e Effect o f Ethanol and A c e t a l d e h y d e on Na P u m p Function in Cultured R a t H e a r t Cells David McCall* and Kevin Ryan
The Cardiology Division, Department of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, T X 78284, USA (Received 22 September 1986, acceptedin revisedform 27 January 1987) D. MCCALL AND K. RYAN. The Effect of Ethanol and Acetaldehyde on Na Pump Function in Cultured Rat Heart Cells. Journalof Molecularand CellularCardiology(1987) 19, 453~1-63. To further define the sarcolemmal effects of ethanol and acetaldehyde, their effects on Na pump function were studied in synchronously contracting monolayers of neonatal rat myocardial cells. The effects of ethanol (10 mg/dl to 1000 mg/dl: 2 x 10 -3 i~).2 M) and acetaldehyde (10 -6 i~ to 10-4 M) on total 42K influx, ouabain-sensitive 42K influx, Na pump density (from specific 3H-ouabain binding) and pump turnover rates were measured. Applied acutely ethanol had no effect on 42K influx but after 30 min of treatment 't2K influx was decreased by 13%, 23% and 48% in 100 mg/dl, 300 mg/dl and 1000 mg/dl ethanol respectively. This primarily reflected a decrease in mean ouabain-sensitive K + influx from a control of 12.54 to 9.90, 8.95 and 6.68 (p-mol/cm2/s) in 100, 300 and 1000 mg/dl (2 x 10 -2 M, 6 x 10 -2 M and 0.2 M) ethanol. Acetaldehyde in the concentrations tested had no effect on K + influx. Ethanol treatment produced a decrease in Na pump density, maximum within 30 min and dosedependent, at concentrations of 100 mg/dl (22%), 300 mg/dl (37%) and 1000 mg/dl (55%). Acetaldehyde had no effect on Na pump density. In the presence of ethanol (300 mg/dl and 1000 mg/dl) intracellular Na + increased significantly and the Na + efflux declined in parallel with the K + influx. From the ouabain-sensitive K + and Na + fluxes and the Na pump density individual pump turnover rates were calculated at 62.5/s in control cells and 66/s and 84/s in cells treated with 300 mg/dl (6 x 10 -2 M) and 1000 mg]dl (0.2 M) respectively. We conclude that ethanol, but not acetaldehyde has a depressant effect on sarcolemmal Na pump function. The results suggest this is due primarily to a decrease in the number ofsarcolemmal Na pump sites. KEY WORDS: Ethanol; Acetaldehyde; Na Pump; Sodium; Potassium; Ion exchange; Glycoside binding.
Introduction B o t h a c u t e a n d c h r o n i c i n g e s t i o n o f a l c o h o l is associated with depression of cardiac contractility a n d w i t h c a r d i a c a r r h y t h m i a s . T h e m e c h a n i s m s o f t h e s e effects h a v e y e t to b e fully e l u c i d a t e d . E t h a n o l h a s b e e n s h o w n to p r o d u c e m a r k e d m e t a b o l i c [2, 22, 23, 29], u l t r a s t r u c t u r a l [ 6 ] , m e c h a n i c a l [2, 29, 3 6 ] a n d e l e c t r i c a l [2, 8, 24] a l t e r a t i o n s i n b o t h t h e in vivo a n d in vitro m y o c a r d i u m . N e v e r t h e l e s s , n o c l e a r d e f i n i t i o n o f t h e c a r d i o d e p r e s s a n t effects o f e t h a n o l h a s e m e r g e d a n d it a p p e a r s t h a t its effects a r e c o m p l e x a n d m u l t i p l e . Consistent with the belief that ethanol a l t e r s s a r c o l e m m a l s t r u c t u r e a n d f l u i d i t y [4,
19, 27, 34]
are observations of increased myocardial permeability, with leakage of norm a l l y i n t r a c e l l u l a r m o l e c u l e s [7, 14] to t h e e x t r a c e l l u l a r space. C h a n g e s i n m e m b r a n e p e r m e a b i l i t y c o u l d also r e s u l t i n a n a l t e r a t i o n of intracellular electrolytes in the myocardium following alcohol ingestion. It has been s h o w n [28-], for e x a m p l e , i n r a t s fed a d i e t c o n t a i n i n g e t h a n o l t h a t N a + K + C a 2+ and M g z+ t e n d to b e d i s p l a c e d d o w n t h e i r r e s p e c tive c o n c e n t r a t i o n g r a d i e n t s w i t h a r e s u l t i n g increase in myocardial Na + and Ca 2 + and a d e c r e a s e i n c e l l u l a r K + a n d M g 2+. T h e s e changes were ascribed, by the investigators [28], to a r e v e r s i b l e n o n - s p e c i f i c i n c r e a s e i n membrane permeability. However, these
Supported in part by Grant G-607 from the Texas Affiliate of the American Heart Association and by NHLBI Training Grant 5-T32-HL07350. * To whom all correspondence should be addressed. 0022-2828/87/050453 + 11 $03.00]0
9 1987 Academic Press Inc. (London) Limited
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results, were not reproduced by acute alcohol intoxication in animals of the same species [20] and in the latter studies the sarcolemmat gradient for both Na + and K + actually increased rather than decreased. In view of the potential relationship between ethanolinduced changes in myocardial electrolyte distribution and ethanol-related electrocardiographic changes and arrhythmias this area requires further exploration and clarification. Although ethanol has been shown to inhibit (Na + + K +) ATPase activity in a variety of tissues in vitro [30], including cardiac sarcolemmal vesicles [37], little attention has been directed towards the effect of ethanol on Na + p u m p function in the myocardium. The present study was therefore carried out to evaluate the effects of ethanol, and its primary metabolite, acetaldehyde, on Na p u m p function in cultured myocardial cells. The studies were designed to evaluate only the effects of acute exposure (up to 1 h) of the cells to the agents on p u m p mediated fluxes, Na + p u m p density and p u m p turnover rates.
Materials
and Methods
Preparation of cultured myocardial cells The methods employed in the preparation of the neonatal rat myocyte cultures have been described in detail elsewhere [25, 26]. Using modifications of the method of H a r a r y and Farley [15] the hearts of 1- to 2-day-old rats, removed aseptically, were disaggregated to single cells by repeated trypsinizations. After separation of fibroblasts [25] the cells were seeded in 35 m m diameter petri dishes and incubated in Minimum Essential Medium, supplemented with 10% fetal calf serum, for 4 to 5 days. At the end of the incubation period each dish contained a synchronously contracting monolayer of some 0.6 to 0.75 x 106 cells of which at least 80% were myocytes. The values for cell surface area, cell volume and volume of cell water previously reported [25] were used after their applicability to the cells of each culture were established. The intrinsic contraction frequency of the cells in culture in each case lay between 120 and 140 contractions per minute. None of the concentrations of ethanol tested, up to and including 1000 mg/dl (0.2 M), affected the
contraction frequency of the cells even when present for up to 1 h. The higher concentrations of acetaldehyde (10-4M) produced some slowing of the intrinsic beating rate but, as was the case with ethanol, the cells continued to contract synchronously throughout the experiment. The cells, therefore, remained viable throughout all interventions.
Solutions used All experiments were carried out with the cells in a physiologic Balanced Salt Solution (BSS) [25, 26]. This solution, used to minimize the pH changes which would have occurred had growth medium been used, contained (mM) Na +, 136.80; K +, 5.35; Ca 2+, 2.25; Mg 1+, 1.03; C I - , 148.22; P O ] - , 0.43; glucose, 11.10; plus calf serum 5% and phenol red, 0.0002% (pH 7.2). The cells were allowed to equilibrate in this solution for 2 to 3 h before any flux measurements were made. The ethanol containing solutions were prepared by adding the desired volume of absolute ethanol, using a micropipette, to BSS (10 ml) in the petri dish. Immediately upon addition of the ethanol the petri dishes were sealed with a plexiglass plate, on the undersurface of which was a sheet of Parafilm. This effected a perfect seal with the petri dish and in essence provided a closed vessel with a head space of less than 0.5 mt. The sealed dish was thoroughly mixed to ensure even distribution of the ethanol. The ethanol levels in the petri dishes were verified (Stat-Pack Ethyl Alcohol Test, Behring Diagnostics, La Jolla, CA) and were found to remain stable in this system throughout the period of the study. The acetaldehyde-containing solutions were prepared in a similar manner. The petri dishes were filled with BSS and sealed, as described above, with a plexiglass plate. In this case, a small rubber dam was incorporated in the plate. The solutions were then prepared by injecting acetaldehyde (diluted 1 : 1000 or 1 : 100) at 4~ through the dam into the solution using a pre-cooled microsyringe. The solutions were then thoroughly mixed in the closed containers. No verification of the acetaldehyde concentrations could be obtained, but in parallel studies (unpublished observations) there was a dose-dependent decrease in contraction frequency, velocity of
Alcohol in C u l t u r e d H e a r t Cells
cell edge movement and calcium influx. These observations would tend to confirm the continued presence of acetaldehyde in this closed system and that the concentration was related to that introduced in the manner described.
Measurement of ionfluxes The methods employed to determine the K + influx have been described previously [21, 25, 26]. K + exchange in these cells can be described by a single exponential having a half-time of exchange of around 12 rain [25, 26]. Influx measurements were therefore made by exposing the cells to 42K for a period of time (2 min), short compared to the halftime of K + exchange. Following exposure for 2 min to ~2K, the cells were washed free of extracellular tracer using an isotonic Na +and K+-free Ca2+-sorbitol solution [25"] at 0~ The cellular content of 42K was then determined and the influx, as p-mol/cm2/s, calculated as described previously [25]. Na + efflux was measured by equilibrating the cells with 24Na and then observing its loss into a non-radioactive solution by measuring the amount left in the cells at various times [25]. By plotting the initial and residual tracer contents against time, efflux curves were constructed and the efllux calculated [25]. All flux studies were carried out at 37~ During the influx studies, the plates were gently agitated on a warming plate at 37~ and in the efflux studies the non-radioactive efflux solution at 37~ was continually replaced at a rate of 40 to 60 ml/min [25]. Where the effects of a drug on an ion flux was determined that drug was included in the radioactive influx solution or the non-radioactive effiux solution in the appropriate concentration. All fluxes were divided into their active, or ouabain-sensitive, and passive components by parallel measurements made in the absence and in the presence of 10 -2 ~I ouabain [25,
26]. The Na + and K + contents of the cells were determined by equilibration with 24Na and 42K respectively. Following equilibration the cells were washed free of extracellular tracer, as described above, and the contents calculated as before [-25]. This method of determination of cell ion content has previously been shown [25] to agree very closely with cell Na +
455
and K + content as determined by flame photometry.
Determination of 3H-ouabain binding The number of Na pump sites per cell under various experimental conditions, was measured using 3H-ouabain binding [25]. 3Houabain (New England Nuclear Corp., Waltham, MA, USA) was used to prepare a stock solution of 2 • 10 -6 M 3H-ouabain in BSS from which serial dilutions were made to achieve the desired final concentrations. Ouabain uptake was measured by exposing the cells to 3H-ouabain for 30 min [25], washing the cells free of extracellular tracer and measuring the amount bound by liquid scintillation spectrometry. The amount bound (molecules of 3H-ouabain per cell) was then determined from the cell number, sample counts and the specific activity of the soak solution. Duplicate determinations of 3H-ouabain bound were made in the presence and absence of 20 mM K + to provide separation of specific from non-specific binding [25]. Preliminary studies were carried out in the presence of all of the concentrations of ethanol and acetaldehyde tested to ensure that the kinetics of 3H-ouabain binding were not influenced by the presence of the test agent. Under all conditions, it was found that, as before [25"], maximum specific binding occurred in the presence of 2 x 10-7M 3H-ouabain in a K +-free solution. All isotopes were counted in a Packard Tricarb liquid scintillation spectrometer; 24Na and 42K were counted without added scintillant [10].
Materials
24Na, 42K and
3H-ouabain were purchased from New England Nuclear Corp., Waltham, MA, USA. Biologicals were from Gibco Laboratories, Grand Island, NY, USA and all chemicals from Sigma Corp., St. Louis, MO, USA were of analytical grade.
Statistical analysis Students' t-tests for paired and unpaired data were used to test for the significance of differences between groups. Linear regression
456
D. M c C a l l
and K. Ryan 20
2~I
16
"~ 16
i
Fi F I i
v
g
--~
l
8
l
I l
t~
4
O' 0
I0 I00 300 IO00 Ethanol (mg/dl) FIGURE 1. Total K + influx, myocardial cells at 37~ in absence (0) and presence of each of the concentrations of ethanol tested following 2 min exposure of the cells to ethanol. Bars represent 1 S.E.M.; for each column n -----6. analyses were done b y least-squares fit. All results are expressed as m e a n q- S.E.M.
Results
Effect of ethanol on If + influx A p p l i e d acutely to the cells, ethanol, in concentrations from 10 m g / d l to 1000 mg/dl, h a d no effect on the total *2K influx (Fig. 1). T h e control influx (p-mol/cm2/s) 16.08 _ 0.26 (mean -F S.E.M., n = 6) was similar to that previously r e p o r t e d [25, 26] a n d was unaffected b y a n y of the concentrations tested (Fig. 1) in these studies, in which the cells were exposed to the ethanol for a total time o f 2 mins. W h e n ceils were exposed to ethanol in the concentrations tested (10 m g / d l to 1000 m g / d l : 2 x 10 - a M to 0.2 M) for a period o f 30 mins (Fig. 2) there was a progressive, dosed e p e n d e n t decrease in total 42K influx. T h e total 42K influx (p-mol/cm2/s) decreased from a control value (mean + S.E.M.) of 15.57 + 1.98 to 15.54 + 1.64, 13.50 __+ 1.04, 11.92 + 1.30 and 8.01 _ 1.20 in 10 m g / d l (2 x 10 - 3 M), 100 mg/dl (2 x 10 - 2 U), 300 m g / d l (6 x 10 - 2 M) a n d 1000 m g / d l (0.2 u) ethanol respectively. C o m p a r e d to control, the reductions in 4ZK influx were significant in the presence of 100 m g / d l (P < 0.05), 300 m g / d l (P < 0.01) a n d 1000 m g / d l (P < 0.001) ethanol (for each n = 6-10).
'
I
0
I0 I O0 300 Ethanol (rng/dl)
I000
FIGURE 2. Constituent components of K + influx in control cells (0) and cells treated with ethanol for 30 rain. Total column represents total K + influx, shaded portion the influx in the presence of 10-2 M ouabain (ouabaininsensitive component) and the difference between the two represents the ouabain-sensitive or active K + influx. Bars represent 1 S.E.M.; for each n = 6. F u r t h e r exposure of the cells to ethanol for up to 60 mins resulted in no further decrease in the total 42K influx over those values o b t a i n e d at 30 rains ( T a b l e 1). F u r t h e r c h a r a c t e r i z a t i o n o f the effects o f ethanol on the 42K influx was o b t a i n e d from p a i r e d m e a s u r e m e n t s o f 42K influx, in the presence a n d absence of 10-2 M o u a b a i n , in cells, p r e t r e a t e d with each of the concentrations of ethanol tested, for 30 rains. As shown in Figure 2, the decline in 42K influx, in the presence of ethanol, p r i n c i p a l l y reflected a d o s e - d e p e n d e n t decrease in the active, or ouabain-sensitive c o m p o n e n t . I n control cells the ouabain-sensitive 42 K influx was measured at 12.54 _ 0.99 p-mol/cm2/s or 75% to 80% of the total influx, b o t h figures being similar to that previously r e p o r t e d for these cells [25]. I n cells treated with ethanol for 30 rains the ouabain-sensitive 42K influx (p-mol/cm2/s) was 12.12 _ 0.69 (vs control p = ns), 9.90 + 1.09 (vs control P < 0.05), 8.95 _ 52 (vs control P < 0.01) a n d 6 . 6 8 _ 0.78 (vs control P < 0.001) in 10, 100, 300 a n d 1000 m g / d l ethanol respectively (for each, n = 6). T h e r e was also small d o s e - d e p e n d e n t decrease in the ouabain-insensitive comp o n e n t to the 42K influx (Fig. 2) following 30 mins of ethanol t r e a t m e n t which did reach levels of significance in the presence of 300 m g / d l (6 x 10 - 2 M) and 1000 m g / d l (0.2 M)
Alcohol in Cultured Heart Cells
457
TABLE 1. Effect of 60 minutes' treatment with ethanol on Na:K transport in cultured myocardial cells E t h a n o l (mg/dl) (re) K influx (p-mol/cm2/s) Total O u a b Sens O u a b Insens
[Na]~ (raM) N a + efflux (p-mol/em2/s) Total O u a b Sens Na : K stoichiometry 3H o u a b a i n b i n d i n g ( x 106 molecules/cell) Pump turnover rate
0 0
15.57 12.54 3.03 13.16
• • + •
1.98 0.99 0.05 0.81
10 2 x l0 - s re
100 2 x 102 M
300 6 x 10 - 2 re
1000 0.2 M
15.94_+ 1.04 12.88 ___ 0.92 3.06 ___ 0.45
12.17+_0.47 b
10.36+_0.70 c
9.39 + 0.13 b 2.77 • 0.23
8.35 + 0.43" 2.02 ___ 0.56 b 19.39 • 0.94 c
8.22_+0.92 c 6.89 ___ 0.63 ~ 1.33 ___ 0.36 c 27.78 • 1.12"
26.30 + 0.74 b 12.52 • 1.21 b 2.7:2 0.83 • 0.09 ~
22.61 + 1.42 b 11.39 ___ 0.73" 3.4:2 0.64 _ 0.14"
66
84
30.48 • 1.28 18.78 + 1.03 3 :2 1.32 • 0.03
1.02 + 0.09 a
62.5
61
(s-I) Values given as mean _+ S.E.M. (for each n = 6-10). Compared to control (left-hand column) "P < 0.05; bp < 0.01 ; cp < 0.001.
ethanol (P < 0.01 respectively).
and
P < 0.001
Effect of acetaldehyde on K + influx None of the concentrations of acetaldehyde tested, from 10 -6 ~ to 10 -4 M, had any effect on the 42K influx either on immediate application of the drug to the cells or when the cells had been exposed to the drug for up to 60 mins. Compared to control cells, the acetaldehyde treated cells showed no change in the total 42K influx, or in its ouabainsensitive or ouabain-insensitive components. Effect of ethano ! and acetaldehyde on 3H-ouabain binding In order to further characterize the effects of ethanol on Na pump dependent K § fluxes, studies were carried out to define the maximum specific ouabain binding in ethanol treated cells. This was done to permit calculation of Na pump density (pump sites per cell) and individual pump turnover rates. Ethanol, in any of the concentrations tested, did not affect the kinetics of 3Houabain binding. In control and in ethanoltreated cells 3H-ouabain binding was rapid (T89 = 10 min) and in each case had reached saturation within 30 rain. As in prior studies [25] it was found that maximum specific binding occurred at 2 x 10 -v M 3H-ouabain in a K +-free solution. Treatment of the cells with ethanol 1001000 mg/dl (2 x 10 -2 M-0.2 M) for 30 mins
led to a progressive decline in maximum specific 3H-ouabain bound (Fig. 3). 3H-ouabain bound ( x 106 molecules 3H-ouabain per cell) declined from a control (mean + S.E.~.) of 1.32 ___0.03 tO 1.02 __+0.28, 0.83 __+0.09 and 0.64 + 0.14 in 100, 300 and 1000 mg/dl ethanol respectively. The decrease in 3Houabain bound, compared to control cells, was significant for each concentration tested, P < 0.05 at 100 mg/dl and P < 0.001 for the higher concentrations (for each n = 6). Further exposure of the cells to ethanol for up to 60 mins (Fig. 3) resulted in no further change in specific 3H-ouabain bound per cell. In contrast to the effects of ethanol, acetaldehyde (10-6M to 1 0 - 4 ~ ) even when present for 60 mins, had no effect on 3Houabain binding in the cultured myocardial cells.
Effect of ethanol on intraceUular Na + and Na +
(flux In view of the apparent inhibitory effect of ethanol on the active K + influx, and the fact that it appears to decrease the number of Na ~ump sites per cell (as measured by specific H-ouabain binding) it was felt that the effects on intracellular Na + concentration ([Nail) and the Na + effiux should be examined. This would provide confirmation of the inhibitory effect of ethanol on active transport via the Na-pump and also permit calculation of the stoichiometry of Na + : K + exchange by the pump in ethanol-treated cells.
458
D, McCall and K. R y a n 2.0 .30 rnins
1.6 .T._
C
:~_~
6 0 rnins
,.2 T
o ,
T
~EO.8
T
~o x
Rv m
0.4
0
I00
-,;, 300 I000 Ethanol (rngldl)
I00
300
I000
FIGURE 3. Maximum specific 3H-ouabain binding in untreated cells (0) and cells treated with ethanol for 30 min and 60 min. Bars represent 1 S.E.M.; for each n = 6. Measurement of [ N a + ] i and Na+-efflux, using 24Na as a tracer, were made in cells exposed to ethanol, 300 mg/dl (6 • 10 -2 M) and 1000 mg/dl (0.2 M) for 60 mins, at which time both the K § influx and 3H-ouabain binding were in a steady state. After 60 rains of ethanol treatment [ N a + ] i (m-mols/l cell H 2 0 ) had increased from a control of 13.16 • 0.81 to 19.39 • 0.94 and 27.78 • 1.12 in 300 mg/dl and 1000 mg/dl respectively (for each vs control P < 0.001 and n = 10). T h e increase in [ N a + ] i seen in the presence of ethanol was accompanied by a decrease in the 2r efflux. T h e control 24Na efflux (pmol/cmZ/s) from untreated cells was 30.48 _+ 1.28 (mean • S.E.M.), similar to that previously measured in these cells [25]. Following 60 mins treatment this was decreased to 26.30 • 0.74 in 300 mg/dl (6 x 10 -2 M) ethanol and 22.61 4-1.42 in 1000 mg/dl (0.2 M) ethanol (for each P < 0.01, n = 5). As in the case of the 42K influx, the reduction in 24Na efflux largely reflected a decrease in the ouabain-sensitive component, which declined from a mean control value of I8.8 p-mol/cm2/ s to a mean of 12.5 and 11.4 in 300 and 1000 mg/dl ethanol respectively (Table 1). There was no significant effect of ethanol on the ouabain-insensitive 2r effiux.
Effect of ethanol on Na pump stoichiometry and turnover rates From the ouabain-sensitive N a + and K + fluxes described above, it was possible to cal-
culate the stoichiometry of N a + : K + exchange, via the N a pump, under the various conditions tested. I n untreated cells the ratio was 3 N a + : 2 K + as previously described. This ratio did not appear to be affected by ethanol treatment and remained close to 3 : 2 (2.7 N a + : 2 K + in 300 mg/dl and 3.4 N a + : 2 K + in 1000 mg/dl). Assuming that one N a p u m p site binds one molecule of 3H-ouabain, the data presented (Fig. 3) suggests that, in the presence of ethanol, there is a decrease in the n u m b e r of Na p u m p i n g sites per cell. Using the n u m b e r of N a p u m p sites per cell after 60 mins ethanol exposure, from the 3H-ouabain studies, and the ouabain-sensitive N a + and K + fluxes under identical circumstances, individual p u m p turnover rates were calculated. I n the untreated cells, the p u m p turnover rate was 62.5/s, similar to that previously reported in these cells [25] and this was unaffected by 100 mg/dl ethanol (61/s). In the presence of 300 mg/dl and 1000 mg/dl ethanol, however, the turnover rates tended to be greater than control at 66/s and 84/s respectively.
Effect of ethanol treatment on cell volume T o ensure that the above observations of the effects of ethanol on ion fluxes and 3Houabain binding were not a reflection of osmotically induced changes in cell volume, measurements of the effect of ethanol on cell volume were made. Cell volume measurements were made using the diameters of cells
Alcohol hut C u l t u r e d H e a r t Cells
459
and guinea-pig cerebral cortex ethanol inhibited (Na § + K § ATPase [16], an observation which, in a later study [17], was correlated with a decrease in the indirectly measured active K + transport in the same preparation. It is of interest that the concentration of ethanol (0.22 M) producing halfmaximal inhibition of (Na + + K +) ATPase in these studies [16, 17] inhibits some 45% of the active K § influx in the present preparation. Other workers [33] have confirmed the inhibitory effect of ethanol on brainderived ( N a + + K +) ATPase activity and with few exceptions [5] there appears to be universal agreement [30] on this effect of ethanol. Similar observations of the inhibitory effect of ethanol on (Na + + K +) ATPase activity Discussion were made in cardiac sarcolemmal vesicles While there are several studies which show [37]. In this case, however, the concentration that ethanol decreases ( N a + - K +) ATPase of ethanol required to produce half maximal activity in a wide variety of tissues [30], inhibition (0.6 ~) was considerably higher including cardiac sarcolemma [37], this study than that required in brain tissue [16]. In the represents the first report of experiments heart [37], as in the brain [16], the inhibitory designed to define the effects of ethanol effect of ethanol seemed to be enhanced at low directly on Na pump function in intact myo- K § concentrations possibly reflecting the fact cardial cells. The concentrations of ethanol that ethanol appears to inhibit the terminal dephosphorylation step of the tested, 10 mg/dl-1000 mg/dl (2 • 10 -3 M to K§ 0.2 M), were selected to include those corre- reaction chain [31]. Consistent with inhibition of membrane sponding to the blood alcohol levels associated with minor alcohol use (10 mg/dl to 100 ( N a + + K +) ATPase are both direct and mg/dl), moderate to advanced inebriation indirect observations that ethanol, in concen(100 mg/dl to 300 mg/dl) as well as the more trations between 0.1 ~ and 0.7 M, inhibits pharmacologic concentrations of 1000 mg/dl active K + uptake by both red blood cells [32] used in prior studies [30]. The model of cul- and brain tissue [17, 18]. More recent studies tured neonatal rat myocardial cells was select- [28] showing an increase in [Na+]i and a ed because the basic properties of Na + :K + decrease in [ K § in the myocardium of rats exchange of the preparation had been defined fed alcohol are consistent with active trans[25] and had been shown to be similar in both port inhibition, but have been interpreted by properties and behaviour to that in other the authors as representing nonspecific mammalian myocardial preparations [25]. changes in membrane permeability. The present study, therefore, in some ways No part of the study was designed to examine the effects of chronic ethanol exposure but was confirms the work of others showing that, confined to an examination of the acute effects acutely, ethanol produces a dose-dependent decrease in active K + influx via the Na pump. of both ethanol and acetaldehyde. There is considerable evidence that ethanol At the highest concentration of ethanol tested, inhibits (Na + + K +) ATPase of a variety of 1000 mg/dl (0.2 M) active transport is inhibmembrane preparations, in vitro, which has ited by 50%, a finding similar to that in rat been elegantly reviewed by Roach [30]. Most brain microsomes [16]. The inhibitory effect of this work, however, has utilized brain and of ethanol on active transport of K + seen in other nerve tissue in an attempt to define the the present study did not occur immediately nature of ethanol effects in the central nervous upon addition of ethanol to the cells, but system. In microsomal preparations from rat rather took some 30 rains to fully develop.
in trypsinized suspensions prepared from monolayer cultures as previously described [-25], since this is in good agreement with the volume of the flattened cells [19, 25]. None of the concentrations of ethanol tested had any effect when present on the cells for up to 30 mins. The only measurable change in cell volume was seen in cells treated with 1000 mg/dl (0.2 M) ethanol for 60 rains. In these cells a 20% increase in cell volume was noted, from a control of 2.71 + 0.27 x 10 - 9 cm 3 to 3.22 __+0.16 x 10 - 9 c m 3 ( P < 0.01 ; for each n = 50). This increase in volume presumably represents an increase in cell water following the gain in [Na~-]i.
460
D. McCall and K. Ryan
This suggests that the decrease in K + influx is not due to simple competition between ethanol and K + for a common receptor on the Na pump entity, since even the highest concentration tested, 1000 mg/dl (0.2 M), did not affect any component of the K + influx when present on the cells for only a short period of time. The increase in intracellular Na + seen in the ethanol-treated cells and the parallel decrease in the Na + effiux would further support Na pump inhibition rather than ethanol: K + competition. In addition, if the decreased K § influx were merely a reflection of ethanol competing for the K + site, then it would be extremely unlikely that the pump stoichiometry would remain 3 Na + :2K + in the ethanol treated cells. It is possible, however, that ethanol competition for the K + binding site on the Na pump could displace K + and in so doing could prevent pump turnover. It is conceivable under those circumstances that stoichiometry would he unaltered since the measured residual Na + and K + fluxes would occur via non-affected pumps. Taken together, however, the data argue very strongly that ethanol produces true inhibition of active coupled Na + and K + transport. The reason for the slow onset of the inhibition by ethanol is unclear. It is possible that the effect may be mediated by the influence of myocardial metabolites of ethanol such as fatty acid ethyl esters [22] with their capability to destroy the integrity of the phospholipid bilayer of the sarcolemma. Alternatively, it has been shown that the assembly ofglycoproreins into hepatic plasma membranes is inhibited by ethanol [34] and, therefore, the inhibitory effect of ethanol on active transport may represent inhibition of assembly of the component proteins of the transport system. Both of these processes are likely to be timedependent. It is impossible to determine whether the time course of the inhibitory effect of ethanol is different in this tissue from that in others, since in each case [16, 17, 33] ethanol was present in the reaction mixture for at least 20 to 30 mins. In only one study [5] was the effect of ethanol on microsomal (Na + + K +) ATPase activity measured over a period of less than 15 mins. In this study [5"J, however, it is of interest to note that no inhibitory effect of ethanol on N a + - K + ATPase activity was seen. Although, therefore, no
direct comparisons of the inhibitory effect of ethanol on active transport or (Na + + K +) ATPase in this tissue, with that in others can be made, the indirect evidence above suggests that it may be a time-dependent function in all. In a recent study, Green and Baron [13-] have shown that ethanol inhibits the influx of S6Rb, a K + analog, in human erythrocytes and leucocytes. Further, the inhibitory effect of ethanol was both quantitatively and qualitatively very similar to that shown in the present study. O f particular interest in this latter study [13] was the finding that ethanol in addition to inhibiting the active Rb + influx, produced a dose-dependent decrease in the ouabain-insensitive influx of that ion, a finding very similar to that of the present study. As in the present study, this did not reach levels of significance in concentrations of ethanol less than 0.16 M. The reasons for this effect of high concentrations of ethanol on the ouabain-insensitive K + influx, although of interest, are unclear. It is possible that inhibition of the Na pump by ouabain, even in concentrations of 10 -2 M, is incomplete and that the addition of a second inhibitor, ethanol, of N a + + K+-ATPase permits more complete inhibition of the pump. Although this seems unlikely, from previous studies [25, 26-J, this possibility cannot be completely discounted. The effect may be due to a decrease in passive exchange diffusion of the ion about a semipermeable membrane which is known to be dependent on the concentrations of the ion on either side of the membrane [35]. With inhibition of the active K + influx, in the presence of ethanol, intracellular K + will be decreased and the gradient favouring exchange diffusion of K § [35] decreased. It is unlikely that the effect is due to ethanol : K competition for the reasons stated previously, but this possibility cannot be completely dismissed. Inhibition of the Na pump by ethanol, resulting in changes in intracellular Na + and K + distribution, may affect the membrane potential which could in turn influence passive K § influx. Finally, a direct effect of ethanol on the phospholipids of the cell membrane could influence the passive exchange of many ions, including K+. Whatever mechanism is involved, however, it must be pointed out that inhibition of the ouabain-insensitive K +
Alcohol in C u l t u r e d Heart Cells
461
0.020 A
0.015 Q ~ 0.010
- 0 0 6 - 0 . 0 4 -0.02
I
I
I
0.02
0.04
0.06
0.I0
I/[No]i (rn.- ') FIGURE 4. Double reciprocal pl0t of [Na]i and calculated Na pump turnover rates. Data derived from control cells and cells treated with ethanol (300 mg/dl and 1000 mg/dl) for 60 min. Line shown is calculated linear regression line (y = 0.12 x +0.007; r = 0.98). Although only means are shown all contributing values for [Na]i were used in the calculation. influx is only seen at high ( > 0.1 M) concentrations of ethanol a n d is unlikely therefore to be of significance in the h e a r t in vivo. F r o m the 3 H - o u a b a i n - b i n d i n g studies it would a p p e a r t h a t the decreased active transp o r t in the presence of ethanol, in cultured h e a r t cells, is due to a decrease in the n u m b e r of available p u m p sites per cell. A l t h o u g h a possibility, it is unlikely that the decreased specific 3 H - o u a b a i n b i n d i n g seen in the etha n o l - t r e a t e d cells represents competitive inhibition of glycoside b i n d i n g by ethanol. This possibility was, however, explored in confirm a t o r y studies in which it was shown that no further increase in specific b i n d i n g occurred, in the presence of ethanol, at concentrations of 3 H - o u a b a i n of 10 - 5 M. T h e decrease in functional N a p u m p sites, therefore, a p p e a r s to be real a l t h o u g h the precise reasons for this decrease in N a p u m p density r e m a i n unclear. I t m a y reflect loss of functioning p u m p sites due to the solubilizing effect of ethanol on the p h o s p h o l i p i d m e m b r a n e . O n the other hand, there m a y be i m p a i r e d assembly of the constituent proteins of m e m b r a n e structures such as has been d e m o n s t r a t e d in hepatocytes [34]. W h a t e v e r the mechanism, however, the decrease in N a p u m p density is sufficient to account for the decreased active K + t r a n s p o r t a n d m a y well be the p r i m a r y factor in decreasing active t r a n s p o r t in e t h a n o l - t r e a t e d cells. It is well recognized that N a p u m p function is regulated by extracellular K + concentration and i n t r a c e l l u l a r N a + concentration. I n
these cells it has been shown t h a t the K"m of K + on active t r a n s p o r t is 1.8 m i [25] a value similar to that in other tissues [12]. In BSS with a [ K * ] 0 of 5.4 mM it is likely that the s t i m u l a t o r y effect of K + on the N a p u m p would be at, or n e a r a m a x i m u m . T h e increased turnover rate seen in the residual p u m p s in the e t h a n o l - t r e a t e d cells therefore p r o b a b l y reflects the influence of the increased [Na§ seen u n d e r these conditions. Stimulation of active N a + effiux via the N a p u m p b y [ N a + ] i in o t h e r tissues has been described as being either linear, showing no signs of s a t u r a t i o n [1, 3] or showing s a t u r a t i o n only at very high values of i n t r a c e l l u l a r N a + [9, 11]. I n several tissues including red blood cells [9] a n d atrial muscle [11] t h e / ( m of intracellular N a + on active N a effiux was found to be 22 to 25 m i , considerably higher t h a n the n o r m a l [ N a + ] i of a r o u n d 12 mM found in cultured n e o n a t a l rat h e a r t cells [25]. T h e modest increase in N a p u m p turnover rates seen in the ethanol treated cells is therefore, most p r o b a b l y , due to the increased driving force p r o v i d e d by the increased i n t r a c e l l u l a r N a +. I n d e e d , when a double reciprocal plot of intracellular Na + and Na pump turnover rates is constructed (Fig. 4) an a p p a r e n t / f r o of i n t r a c e l l u l a r N a + on p u m p t u r n o v e r rate of a p p r o x i m a t e l y 22 m i is obtained. I t has to be p o i n t e d out that the construct of the doublereciprocal plot of Figure 4 is o b t a i n e d from a limited a m o u n t of d a t a and that some of that d a t a is from cells p r e t r e a t e d with ethanol which could conceivably alter the affinity of
462
D. McCall and K. R y a n
Na + for its receptor on the N a p u m p . Although some caution has to be used in interpreting the data from Figure 4, it is of interest that the a p p a r e n t K m obtained is in good agreement with that in both red blood cells [9] a n d atrial muscle [11]. O n e previous report [5] showed that acetaldehyde inhibited (Na + + K +) ATPase of b r a i n microsomes. I n the present study, acetaldehyde had no measurable effect on active transport in the cultured n e o n a t a l heart cells. Although great care was taken to carry out the studies in sealed containers it is possible, that because of its extremely volatile nature, aceteldehyde evaporated from the petri dishes immediately u p o n its addition to the BSS. Although no direct measurements of acetaldehyde levels were available, it is unlikely that all of the acetaldehyde evaporated. This is supported by observations that, u n d e r identical experimental conditions, the addition of acetaldehyde to the BSS caused marked suppression of the contractile activity of the cells which was sustained for up to 60 rains. I n conclusion, therefore, ethanol, b u t not acetaldehyde, suppresses active transport by
the Na p u m p in cultured rat ventricular myocytes. T h e effect of ethanol is both timeand dose-dependent a n d appears to be mediated by an e t h a n o l - i n d u c e d decrease in the density of N a p u m p sites on the sarcolemma. T h e active transport system in these cells, i n c l u d i n g its glycoside-sensitivity (IDs0-10 -6 M), is very similar to that o f a d u h m a m m a l i a n cells [25] including those of man. I n view of this, the observation that concentrations of ethanol encountered d u r i n g acute alcoholic intoxication (300 mg/dl) significantly depress Na p u m p function in these cells m a y have considerable clinical relevance. Ethanolinduced decreases in active transport with c o n c o m i t a n t changes in [ N a i l a n d [ K + ] i m a y be i m p o r t a n t c o n t r i b u t i n g factors to the electrocardiographic changes a n d arrhythmias seen in acute alcoholic intoxication.
Acknowledgements T h e authors wish to acknowledge the expert technical assistance of M a g d a l e n a Garcia and the i n v a l u a b l e secretarial services of A n n a Fackenthall.
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