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Acetaldehyde Inhibits Current through Voltage-Dependent Calcium Channels
TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.
143, 70–74 (1997)
TO968072
Acetaldehyde Inhibits Current through Voltage-Dependent Calcium Channels JUAN A. MORALES, JEFFREY L. RAM, JIANBEN SONG,
AND
RICARDO A. BROWN
Department of Physiology, Wayne State University, Detroit, Michigan 48201 Received April 4, 1996; accepted November 12, 1996
1983). The acute effects of ACA on aortic and portal vein contractility and reactivity have been studied in detail by Altura et al. (1978). It was demonstrated that ACA attenuates aortic contractures induced by a variety of agonists, including epinephrine, angiotensin, vasopressin, serotonin, and KCl. Recently, Brown and Savage (1996) compared the effects of acute versus chronic ACA exposure, on KCl- and norepinephrine (NE)-elicited contractions of isolated aortic rings and examined the influence of the endothelium on these responses. Chronic exposure to ACA attenuated the inotropic response to KCl and NE in endothelium-denuded preparations. Moreover, an acute pharmacological dose of ACA (30 mM) relaxed KCl-induced contractures of aortic rings. Because KCl causes contraction by depolarizing smooth muscle cell membranes their results suggest that ACA exposure impairs the inotropic response of aortic vascular smooth muscle to membrane depolarization. Modification of responses to depolarization could be mediated by changes in voltage-dependent ion channels, especially voltage-dependent Ca2/ channels. Contraction of muscle is ultimately dependent on the extent to which intracellular calcium concentration is elevated. Intracellular calcium concentration is increased by the opening of both sarcolemmal voltage-dependent calcium channels and calcium release channels on the sarcoplasmic reticulum. However, to date the effect of acute ACA exposure on vascular smooth muscle intracellular calcium homeostasis has not been examined in detail. The goal of the present investigation was to determine the mechanism underlying the acute vasorelaxant effect of ACA by examining its influence on the magnitude and voltage dependence of current recorded through vascular smooth muscle sarcolemmal L-type voltage-dependent Ca2/ channels.
Acetaldehyde Inhibits Current through Voltage-Dependent Calcium Channels. MORALES, J. A., RAM, J. L., SONG, J., AND BROWN, R. A. (1997). Toxicol. Appl. Pharmacol. 143, 70–74. Ethanol consumption is often accompanied by an increase in both cardiac and vascular dysfunction. Underlying mechanisms may include direct actions of acetaldehyde (ACA), the principal by-product of ethanol metabolism, which has previously been shown to decrease both KCl- and nonrepinephrine-elicited contractions of isolated aortic rings. To determine whether ACA reduces vascular contractility through a direct action on sarcolemmal Ca2/ currents of vascular smooth muscle cells, Ca2/ channel currents in an aortic smooth muscle cell line (A7r5) were studied using the whole-cell patch clamping technique. With Ba2/ as the major charge carrier, Ca/ in the electrode, and TEA to block K/ currents, ramp depolarization activated an inward current consisting mostly of current through L-type Ca2/ channels. ACA caused a progressive decline in inward current, causing a significant reduction in 30 mM ACA of 21.2 { 4.3% (n Å 6 cells; p õ 0.01) within 4 min and 39.4 { 6.8% (n Å 5 cells, p õ 0.001) reduction within 8 min. Although the decline in inward current in 10 mM ACA was not significant at 4 min, significant (p õ 0.05) reductions in 10 mM ACA were present at 8 min (15.5 { 3.5%, n Å 9 cells) and 12 min (25.2 { 6.7%, n Å 3 cells). There was no apparent shift in the voltage dependence of the current in response to ACA. The results of this study support the hypothesis that one of the underlying causes of ACA inhibition of potassium-elicited contraction is inhibition of voltage-dependent Ca2/ currents in smooth muscle cells. q 1997 Academic Press
The effects of ethanol exposure on vascular smooth muscle contractility are well documented, and its action is dependent not only on concentration but also on the vascular bed being studied (Regan, 1982). For example, while acute ethanol exposure vasoconstricts cerebral, coronary, pulmonary, and renal vessels, it also dilates both mesenteric and cutaneous vessels (Altura and Altura, 1982). Acetaldehyde (ACA), the principal metabolite of ethanol, also has actions on vascular smooth muscle, and has been implicated as a potential mediator in the development of vascular pathology associated with excessive ethanol consumption (Shepard and Vanhoutte, 1972; Takeda and Momose, 1978; Toda et al.,
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Cell culture. A7r5 cells, a vascular smooth muscle cell (VSMC) line derived from embryonic DB1X rat thoracic aorta, were obtained from American Type Culture Collection (Rockville, MD). As described previously by Standley et al. (1991), cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 9% fetal bovine serum, 0.2% tylosin (an antibacterial antibiotic), 100 U/ml penicillin, and 0.1 mg/ml streptomycin and 70
0041-008X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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grown in a 377C water-jacketed incubator under 5% CO2 and 100% humidity. Whole-cell patch clamp. Confluent A7r5 cells were released with 0.2% trypsin, 1 mM ethylene glycol bis(b-aminoethyl ether)-N,N *-tetraacetic acid (EGTA) in Ca2/ –Mg 2/-free Hanks’ balanced salt solution for 5 min at 377C. Released cells were centrifuged at 1000 rpm for 3 min, and the pellet was resuspended in growth medium. Then cells were transferred to 35 1 10-mm Petri dishes containing rectangular glass chips (dimensions approximately 9 1 4 mm), and incubated in growth medium for 1 hr before recording. After 1 hr incubation, one glass chip to which VSMC had adhered was transferred to the recording chamber. The recording chamber was 0.8 ml in volume and it was perfused at a rate of 0.8 ml/min throughout recording. Whole-cell voltage-clamp experiments were performed on attached cells with a Dagan 8900 patch-clamp amplifier, controlled by a Labmaster TL-1 DMA A/D-D/A interface, using the pClamp (Axon Instruments) suite of programs. Voltage protocols for analysis of L-type Ca2/ channels, described below, were run at 20-sec intervals. To study L-type Ca2/ channels, Ba2/ was substituted for Ca2/ in the extracellular medium. The extracellular medium (Ba-ext) contained (in mM) 20 BaCl2 , 83 NaCl, 30 tetraethylammonium (TEA), 4 KCl, 2 MgCl2 , and 10 N-2-hydroxyethylpiperazine-N *-2-ethanesulfonic acid (Hepes), pH 7.4, at Ç280 mOsm/liter. The electrode solution contained (in mM) 120 CsCl, 10 Cs-EGTA, 1.4 MgCl2 , 3.6 Mg-ATP, and 10 Hepes, pH 7.2, at Ç280 mOsm/liter. Membrane current was elicited using a ramp protocol. From a holding potential of 080 mV, the membrane potential was stepped down to 0100 mV for 50 msec, and then was depolarized by a ramp going from 0100 to 50 mV in 300 msec with a digital resolution of 1 mV. Linear leakage and holding current were removed from the entire current response by subtracting the linear current trend and average holding current between 080 and 060 mV, using the pClamp Clampan program and Excel macro programs, as described by Song et al. (1996). Previous studies (Zhang et al., 1994a,b; Song et al., 1996) have shown that the major current peak elicited during this ramp protocol is L-type current, as evidenced by the appropriate ion selectivity, dihydropyridine sensitivity, and voltage dependence, and because neither T-type or sodium currents are contained in the peak current. The voltage dependence of the current generated by the ramp protocol is nearly identical to the L-type current voltage dependence measured with more traditional pulse-type voltage protocols (Song et al., 1996). In addition, the ramp is of suitable speed such that the L-type current can activate almost completely and yet hardly inactivate at all during the ramp. The advantage of the ramp protocol is that the magnitude of the current and its entire I-V curve can be measured in less than 1 sec and as frequently as once every 20 sec, whereas measurement of the I-V curve using pulses takes more than a minute, during which time properties of the cell may be changing in response to drug application. To examine effects of ACA, ACA was dissolved in Ba-extr solution (composition described previously), at concentrations of 10 and 30 mM. Currents were recorded in Ba-extr medium for 2.5 to 10 min followed by application of ACA-containing solutions for 5 to 15 min, followed by a wash out with Ba-extr medium. Results were considered statistically significant for p õ 0.05, as determined by ANOVA, followed by pairwise multiple comparisons (Student– Newman–Keuls or Dunnett’s methods as appropriate; Sigmastat).
RESULTS
An inward L-type current was elicited in A7r5 cells by the ramp protocol under control conditions (Ba-extr solution) and was reduced in magnitude by 10 and 30 mM ACA. This is illustrated by data from representative cells in Figs. 1 and 2 and summary data from several cells in Figs. 2C and 3. Under control conditions during 4 min preceding ACA
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FIG. 1. Effect of 30 mM acetaldehyde (ACA) on current through Ltype Ca2/ channels in a vascular smooth muscle cell. Data from a representative A7r5 cell are shown. Cells were voltage-clamped in whole-cell configuration and held at 080 mV, and the response to a depolarizing ramp (0100 to /50 mV in 300 msec) was elicited at 20-sec intervals, as described under Materials and Methods. (A) Currents elicited by the voltage ramps during superfusion in normal medium prior to ACA application (control) and 4 and 8 min after beginning superfusion with 30 mM ACA. The traces shown were recorded at the times indicated by filled circles in (B). (B) Time course of changes of peak current during superfusion with control medium (C), 30 mM ACA (ACA 30), and control medium again (Wash out).
application, the magnitude of the peak current averaged 1019 { 150 pA (mean { SEM, n Å 9 cells) and did not change significantly during the control period (0 mM ACA, Fig. 3). Upon exposure of cells to ACA, the current usually began an abrupt and progressive decline in magnitude within a minute of the entry of ACA into the recording chamber. This is illustrated by Figs. 1B and 2B showing typical time courses of the effects of 30 and 10 mM ACA, respectively. For 30 mM ACA, the average decrease in peak current magnitude was by 21.2 { 4.3% within 4 min (n Å 6 cells, p õ 0.01) and 39.4 { 6.8% within 8 min (n Å 5 cells; p õ 0.001, compared to control). Although exposure of A7r5 cells to 10 mM ACA for 4 min did not produce a significant change in the magnitude of the peak current (reduction of 6.3 { 1.4%, n Å 12 cells), the decline was significant at 8 min (16.5 { 3.5%, n Å 9 cells; p õ 0.05). In a subset of 3 cells exposed to 10 mM ACA for more than 12 min, summarized in Fig. 2C, the current continued to decline, exhibiting a reduction of 25.2 { 6.7% (ANOVA, p õ 0.01) at 12 min. Despite the progressive and significant reduction in magnitude of inward current caused by ACA, there was no effect on the voltage dependence of the current. This is apparent in the representative current traces in Figs. 1A and 2A, which
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FIG. 2. Effects of 10 mM acetaldehyde (ACA) on current through Ltype Ca2/ channels in vascular smooth muscle cells. Command potentials, recording, and abbreviations are the same as in Fig. 1. (A and B) Data from a representative A7r5 cell. (C) Summary of data from 3 A7r5 cells which were exposed to 10 mM ACA for ú12 min. To normalize currents before averaging, Ipeak(t), the peak current at time t after beginning ACA superfusion, was divided by Ipeak(0), the peak current just before the cells were exposed to ACA. Error bars are SEM for selected time points. Repeated measures ANOVA, p õ 0.01. Pairwise multiple comparisons to control (current prior to ACA) by Dunnett’s method gave p õ 0.05 at 4, 8, and 12 min.
show no shift in the voltage eliciting peak inward current. Furthermore, the effects of ACA were often reversible. This is illustrated in the time course recording (Fig. 1B) of a cell that exhibited recovery to approximately 80% of control within 15 min. Recovery was Ç90% complete in 6 of 7 cells studied, whereas 1 cell failed to recover from the acute effects of acetaldehyde. DISCUSSION
Our results demonstrate that ACA can inhibit voltagedependent L-type Ca2/ channels, and consequently this
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might be responsible in part for the vasodepressant and inhibitory actions of ACA in aortic vascular smooth muscle observed by Altura et al. (1978) and Brown and Savage (1996). Brown and Savage (1996) observed that potassium chloride-elicited contractions in rat aortic rings were significantly inhibited by 30 mM ACA but not by 10 mM ACA. Correspondingly, our results show that 30 mM ACA inhibited current through Ca2/ channels in vascular smooth muscle cells, and that 10 mM ACA was less effective at reducing the current than was 30 mM ACA. Since potassium chloride depolarizes vascular smooth muscle cells and causes contraction by activating voltage-dependent Ca2/ channels in the cell membrane, the inhibition of L-type Ca2/ channels by ACA would explain its inhibitory effect on contraction. Recording of currents through Ca2/ channels, however, is a more direct and sensitive way of demonstrating effects of ACA on ion channels. These direct measurements of currents through Ca2/ channels support the idea, proposed by Altura et al. (1978a,b) and Brown and Savage (1996), that inhibition of aortic contraction by ACA depends to some extent on the inhibition of calcium ion influx. Nevertheless, other types of mechanisms also might be involved. For instance, ACA might interfere somewhere intracellularly with the interactions of Ca2/ and the contractile proteins (Altura et al., 1976). It was shown by Puszkin and Rubin (1975) that ACA can produce inhibitory effects on superprecipitation of human skeletal muscle actomyosin. Additionally, ACA could interfere with the mobility of cellular Ca2/ reservoirs since it is lipophilic and can penetrate membranes and cells rapidly (Truitt and Walsh, 1971). Moreover, it has been proposed by Altura et al. (1978a,b) that ACA might induce its depressant actions through some effect on metabolism, although they later concluded that this mechanism is unlikely to be responsible for ACA’s inhibitory actions.
FIG. 3. Summary of changes in peak current through L-type Ca2/ channels in response to acetaldehyde (ACA). The bar for 0 mM ACA (n Å 9 cells) shows the percentage change in current during the 4 min in control medium and indicates no significant change with time under control conditions. Bars for 10 mM ACA (n Å 6 cells) and 30 mM ACA (n Å 6 cells) are the percentage change of the current 4 min (open bars) and 8 min (filled bars) after application of the indicated solution, as a percentage of the current just prior to ACA application. Bars represent mean { SEM; ANOVA, p õ 0.001. *p õ 0.05, compared to 0 mM ACA control (Student– Newman–Keuls method).
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Acetaldehyde metabolism is quite rapid, almost 5 times that of ethanol, and low blood concentrations (0.5 mM) are maintained after short-term administration of 0.5 g of ethanol/kg in a normal individual (Lubin and Westerfeld, 1945; Leiber, 1987). However, higher acetaldehyde levels, approaching 1.8 mM, are achieved in alcoholic compared to nonalcoholic subjects when given the same dose of ethanol (Leiber, 1987). The higher acetaldehyde concentration among alcoholic subjects is thought to be due to hepatic inhibition of acetaldehyde dehydrogenase activity. Furthermore, acetaldehyde levels approach 30–53 mM in approximately 52% of individuals of Asian decent who lack a low Km aldehyde dehydrogenase and at least one individual survived a plasma concentration of 0.5 mM following severe ethanol intoxication (Watanabe et al., 1985). These isozyme variations which have enhanced affinity for ethanol, but diminished specificity toward acetaldehyde, enable blood levels of the latter to exceed that of normal individuals. However, the concentration of acetaldehyde in cardiac myocytes might be up to 10-fold higher than the plasma concentrations seen in the rat (Espinet and Argiles, 1984). Therefore, although the high concentration used in this study may be irrelevant to clinical arterial blood concentrations, they may be indicative of the concentration seen in vivo by the heart cells. Studying the acute effects of acetaldehyde on membrane ionic currents provides a means of assessing its potential role in the development of hypertension as a result of long-term ethanol consumption. Since ACA is the major metabolite of ethanol, these results raise the question of whether ACA may play a role in the pathological effects of chronic alcohol ingestion. Ethanol can produce dose-dependent relaxant, vasodilator as well as inhibitory actions on agonist-induced contractions on a variety of tissue preparations including rat arterioles, venules, arteries, and veins both in situ and in vitro (Altura et al., 1976, 1978a,b; Altura and Edgarian, 1976; Edgarian and Altura, 1976; Altura and Altura, 1978). These actions have been implicated in the development of cardiovascular and neurological disorders related to excessive ethanol consumption as in the case of alcoholics. Although the inhibitory actions of ACA on smooth muscle L-type currents demonstrated here suggest a possible mechanism by which alcohol metabolism could result in an inhibitory action on blood vessel contractility, it is important to keep in mind that the concentrations of ACA used here and in previous contraction studies (10 and 30 mM; Brown and Savage, 1996) are very high. While there are many instances in which plasma ACA concentrations may be dramatically elevated following ethanol ingestion (Tabakoff et al., 1989), this concentration of ACA would not usually be encountered in the plasma of even an extreme alcoholic as it would be expected to cause death first. On the other hand, these experiments showed that the effect of ACA increased pro-
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gressively even at the lowest concentration tested, suggesting that ACA applied for longer time periods may have inhibitory effects at lower concentrations, more relevant to those occurring in response to ethanol ingestion. Furthermore, these results demonstrate that when toxicological levels of ACA are encountered, one expected effect is inhibition of blood vessel contractility through a direct effect on membrane Ca2/ currents. ACKNOWLEDGMENTS The authors gratefully acknowledge support for this research from NIH Grant HL-02748 (to R.A.B.) and NIH Grant GM-08167 (to J.A.M. and J.L.R.). We also thank Ms. Linda McCraw for assistance in preparing the manuscript.
REFERENCES Altura, B. M., and Edgarian, H. (1976). Ethanol-prostaglandin interactions in contraction of vascular smooth muscle. Proc. Soc. Exp. Biol. Med. 152, 334–336. Altura, B. M., Edgarian, H., and Altura, B. T. (1976). Differential effects of ethanol and mannitol on contraction of arterial smooth muscle. J. Pharmacol. Exp. Ther. 197, 352–361. Altura, B. M., Ogunkoya, A., Gebrewald, A., and Altura, B. T. (1978a). Effects of ethanol on terminal arterioles and muscular venules: Direct observations on the microcirculation. J. Cardiovasc. Pharmacol. 1, 97– 113. Altura, B. M., Carella, A., and Altura, B. T. (1978b). Acetaldehyde on vascular smooth muscle: Possible role in vasodilator action of ethanol. Eur. J. Pharmacol. 52, 73–83. Altura, B. T., and Altura, B. M. (1978). Intravenous anesthetic agents and vascular smooth muscle function. In Mechanisms of Vasodilation (Vanhoutte, P. M., Ed.), p. 165. Karger, Basel. Brown, R. A., and Savage, A. O. (1995). Effects of acute acetaldehyde, chronic ethanol and pargyline treatment on agonist responses of the rat aorta. Toxicol. Appl. Pharmacol. 136, 170–178. Edgarian, H., and Altura, B. M. (1976). Ethanol and contraction of venous smooth muscle. Anesthesiology 44, 311–317. Espinet, C., and Argiles, J. M. (1984). Ethanol and acetaldehyde levels in rat blood tissues after an acute ethanol administration. IRCS Med. Sci. 12, 830–840. Lieber, C., Barona, E., Leo, M., and Garro, A. (1987). Effects of chronic alcohol consumption on the metabolism of ethanol. Prog. Clin. Biochem. Res. 241, 161–172. Lubin, M., and Westerfield, W. W. (1945). Metabolism of acetaldehyde. J. Biol. Chem. 161, 503–512. Pawlak, D., Morelowska-Spierzak, D., Arsalan, A., Kazimierez, W., and Wlodzimierz, B. (1993). Lack of effect of acetaldehyde on the cardiovascular system in rats. Alcohol Alcoholism 28, 529–533. Preedy, V. R., and Richardson, P. J. (1994). Ethanol induced cardiovascular disease. Br. Med. Bull. 50, 152–163. Puszkin, S., and Rubin, E. (1975). Adenosine diphosphate effect on contractility of human muscle actomyosin: Inhibition by ethanol and acetaldehyde. Science 188, 1319–1320. Savage, A. O., Dunbar, J. C., and Brown, R. A. (1995). Effects of acetaldehyde on the isolated papillary muscle of diabetic rats. J. Cardiovasc. Pharmacol. 26, 251–258. Song, J., Standley, P. R., Zhang, F., Joshi, D., Gappy, S., Sowers, J. R.,
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and Ram, J. L. (1996). Tamoxifen (estrogen antagonist) inhibits voltagegated calcium current and contractility in vascular smooth muscle from rats. J. Pharmacol. Exp. Ther. 277, 1444–1453.
Von Burg, R. (1991). Toxicology update (acetaldehyde). J. Appl. Toxicol. 11, 373–376.
Tabakoff, B., Eriksson, C. J. P., and von Wartburg, J. P. (1989). Methionine lowers circulating levels of acetaldehyde after acetaldehyde ingestion. Alcoholism Clin. Exp. Res. 13, 164–171.
Watanabe, A., Kobayashi, M., Hobara, N., Nakatsukasa, H., Nagashima, H., and Fugimoto, A. (1985). A report of unusually high blood ethanol and acetaldehyde levels in two surviving patients. Alcoholism Clin. Exp. Res. 9, 14–16.
Truitt, E. B., Jr., and Walsh, M. J. (1971). The role of acetaldehyde in the actions of ethanol. In The Biology of Alcoholism, Vol. 1 (Kissin, B., and Begleiter, H., Eds.), p. 165. Plenum, New York.
Zhang, F., Ram, J. L., Standley, P. R., and Sowers, J. R. (1994). 17b-Estradiol attenuates voltage-dependent Ca2/ currents in A7r5 vascular smooth muscle cell line. Am. J. Physiol. 266 (Cell Physiol. 35), C975–C980.
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TO968072
Acetaldehyde Inhibits Current through Voltage-Dependent Calcium Channels JUAN A. MORALES, JEFFREY L. RAM, JIANBEN SONG,
AND
RICARDO A. BROWN
Department of Physiology, Wayne State University, Detroit, Michigan 48201 Received April 4, 1996; accepted November 12, 1996
1983). The acute effects of ACA on aortic and portal vein contractility and reactivity have been studied in detail by Altura et al. (1978). It was demonstrated that ACA attenuates aortic contractures induced by a variety of agonists, including epinephrine, angiotensin, vasopressin, serotonin, and KCl. Recently, Brown and Savage (1996) compared the effects of acute versus chronic ACA exposure, on KCl- and norepinephrine (NE)-elicited contractions of isolated aortic rings and examined the influence of the endothelium on these responses. Chronic exposure to ACA attenuated the inotropic response to KCl and NE in endothelium-denuded preparations. Moreover, an acute pharmacological dose of ACA (30 mM) relaxed KCl-induced contractures of aortic rings. Because KCl causes contraction by depolarizing smooth muscle cell membranes their results suggest that ACA exposure impairs the inotropic response of aortic vascular smooth muscle to membrane depolarization. Modification of responses to depolarization could be mediated by changes in voltage-dependent ion channels, especially voltage-dependent Ca2/ channels. Contraction of muscle is ultimately dependent on the extent to which intracellular calcium concentration is elevated. Intracellular calcium concentration is increased by the opening of both sarcolemmal voltage-dependent calcium channels and calcium release channels on the sarcoplasmic reticulum. However, to date the effect of acute ACA exposure on vascular smooth muscle intracellular calcium homeostasis has not been examined in detail. The goal of the present investigation was to determine the mechanism underlying the acute vasorelaxant effect of ACA by examining its influence on the magnitude and voltage dependence of current recorded through vascular smooth muscle sarcolemmal L-type voltage-dependent Ca2/ channels.
Acetaldehyde Inhibits Current through Voltage-Dependent Calcium Channels. MORALES, J. A., RAM, J. L., SONG, J., AND BROWN, R. A. (1997). Toxicol. Appl. Pharmacol. 143, 70–74. Ethanol consumption is often accompanied by an increase in both cardiac and vascular dysfunction. Underlying mechanisms may include direct actions of acetaldehyde (ACA), the principal by-product of ethanol metabolism, which has previously been shown to decrease both KCl- and nonrepinephrine-elicited contractions of isolated aortic rings. To determine whether ACA reduces vascular contractility through a direct action on sarcolemmal Ca2/ currents of vascular smooth muscle cells, Ca2/ channel currents in an aortic smooth muscle cell line (A7r5) were studied using the whole-cell patch clamping technique. With Ba2/ as the major charge carrier, Ca/ in the electrode, and TEA to block K/ currents, ramp depolarization activated an inward current consisting mostly of current through L-type Ca2/ channels. ACA caused a progressive decline in inward current, causing a significant reduction in 30 mM ACA of 21.2 { 4.3% (n Å 6 cells; p õ 0.01) within 4 min and 39.4 { 6.8% (n Å 5 cells, p õ 0.001) reduction within 8 min. Although the decline in inward current in 10 mM ACA was not significant at 4 min, significant (p õ 0.05) reductions in 10 mM ACA were present at 8 min (15.5 { 3.5%, n Å 9 cells) and 12 min (25.2 { 6.7%, n Å 3 cells). There was no apparent shift in the voltage dependence of the current in response to ACA. The results of this study support the hypothesis that one of the underlying causes of ACA inhibition of potassium-elicited contraction is inhibition of voltage-dependent Ca2/ currents in smooth muscle cells. q 1997 Academic Press
The effects of ethanol exposure on vascular smooth muscle contractility are well documented, and its action is dependent not only on concentration but also on the vascular bed being studied (Regan, 1982). For example, while acute ethanol exposure vasoconstricts cerebral, coronary, pulmonary, and renal vessels, it also dilates both mesenteric and cutaneous vessels (Altura and Altura, 1982). Acetaldehyde (ACA), the principal metabolite of ethanol, also has actions on vascular smooth muscle, and has been implicated as a potential mediator in the development of vascular pathology associated with excessive ethanol consumption (Shepard and Vanhoutte, 1972; Takeda and Momose, 1978; Toda et al.,
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Cell culture. A7r5 cells, a vascular smooth muscle cell (VSMC) line derived from embryonic DB1X rat thoracic aorta, were obtained from American Type Culture Collection (Rockville, MD). As described previously by Standley et al. (1991), cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 9% fetal bovine serum, 0.2% tylosin (an antibacterial antibiotic), 100 U/ml penicillin, and 0.1 mg/ml streptomycin and 70
0041-008X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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grown in a 377C water-jacketed incubator under 5% CO2 and 100% humidity. Whole-cell patch clamp. Confluent A7r5 cells were released with 0.2% trypsin, 1 mM ethylene glycol bis(b-aminoethyl ether)-N,N *-tetraacetic acid (EGTA) in Ca2/ –Mg 2/-free Hanks’ balanced salt solution for 5 min at 377C. Released cells were centrifuged at 1000 rpm for 3 min, and the pellet was resuspended in growth medium. Then cells were transferred to 35 1 10-mm Petri dishes containing rectangular glass chips (dimensions approximately 9 1 4 mm), and incubated in growth medium for 1 hr before recording. After 1 hr incubation, one glass chip to which VSMC had adhered was transferred to the recording chamber. The recording chamber was 0.8 ml in volume and it was perfused at a rate of 0.8 ml/min throughout recording. Whole-cell voltage-clamp experiments were performed on attached cells with a Dagan 8900 patch-clamp amplifier, controlled by a Labmaster TL-1 DMA A/D-D/A interface, using the pClamp (Axon Instruments) suite of programs. Voltage protocols for analysis of L-type Ca2/ channels, described below, were run at 20-sec intervals. To study L-type Ca2/ channels, Ba2/ was substituted for Ca2/ in the extracellular medium. The extracellular medium (Ba-ext) contained (in mM) 20 BaCl2 , 83 NaCl, 30 tetraethylammonium (TEA), 4 KCl, 2 MgCl2 , and 10 N-2-hydroxyethylpiperazine-N *-2-ethanesulfonic acid (Hepes), pH 7.4, at Ç280 mOsm/liter. The electrode solution contained (in mM) 120 CsCl, 10 Cs-EGTA, 1.4 MgCl2 , 3.6 Mg-ATP, and 10 Hepes, pH 7.2, at Ç280 mOsm/liter. Membrane current was elicited using a ramp protocol. From a holding potential of 080 mV, the membrane potential was stepped down to 0100 mV for 50 msec, and then was depolarized by a ramp going from 0100 to 50 mV in 300 msec with a digital resolution of 1 mV. Linear leakage and holding current were removed from the entire current response by subtracting the linear current trend and average holding current between 080 and 060 mV, using the pClamp Clampan program and Excel macro programs, as described by Song et al. (1996). Previous studies (Zhang et al., 1994a,b; Song et al., 1996) have shown that the major current peak elicited during this ramp protocol is L-type current, as evidenced by the appropriate ion selectivity, dihydropyridine sensitivity, and voltage dependence, and because neither T-type or sodium currents are contained in the peak current. The voltage dependence of the current generated by the ramp protocol is nearly identical to the L-type current voltage dependence measured with more traditional pulse-type voltage protocols (Song et al., 1996). In addition, the ramp is of suitable speed such that the L-type current can activate almost completely and yet hardly inactivate at all during the ramp. The advantage of the ramp protocol is that the magnitude of the current and its entire I-V curve can be measured in less than 1 sec and as frequently as once every 20 sec, whereas measurement of the I-V curve using pulses takes more than a minute, during which time properties of the cell may be changing in response to drug application. To examine effects of ACA, ACA was dissolved in Ba-extr solution (composition described previously), at concentrations of 10 and 30 mM. Currents were recorded in Ba-extr medium for 2.5 to 10 min followed by application of ACA-containing solutions for 5 to 15 min, followed by a wash out with Ba-extr medium. Results were considered statistically significant for p õ 0.05, as determined by ANOVA, followed by pairwise multiple comparisons (Student– Newman–Keuls or Dunnett’s methods as appropriate; Sigmastat).
RESULTS
An inward L-type current was elicited in A7r5 cells by the ramp protocol under control conditions (Ba-extr solution) and was reduced in magnitude by 10 and 30 mM ACA. This is illustrated by data from representative cells in Figs. 1 and 2 and summary data from several cells in Figs. 2C and 3. Under control conditions during 4 min preceding ACA
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FIG. 1. Effect of 30 mM acetaldehyde (ACA) on current through Ltype Ca2/ channels in a vascular smooth muscle cell. Data from a representative A7r5 cell are shown. Cells were voltage-clamped in whole-cell configuration and held at 080 mV, and the response to a depolarizing ramp (0100 to /50 mV in 300 msec) was elicited at 20-sec intervals, as described under Materials and Methods. (A) Currents elicited by the voltage ramps during superfusion in normal medium prior to ACA application (control) and 4 and 8 min after beginning superfusion with 30 mM ACA. The traces shown were recorded at the times indicated by filled circles in (B). (B) Time course of changes of peak current during superfusion with control medium (C), 30 mM ACA (ACA 30), and control medium again (Wash out).
application, the magnitude of the peak current averaged 1019 { 150 pA (mean { SEM, n Å 9 cells) and did not change significantly during the control period (0 mM ACA, Fig. 3). Upon exposure of cells to ACA, the current usually began an abrupt and progressive decline in magnitude within a minute of the entry of ACA into the recording chamber. This is illustrated by Figs. 1B and 2B showing typical time courses of the effects of 30 and 10 mM ACA, respectively. For 30 mM ACA, the average decrease in peak current magnitude was by 21.2 { 4.3% within 4 min (n Å 6 cells, p õ 0.01) and 39.4 { 6.8% within 8 min (n Å 5 cells; p õ 0.001, compared to control). Although exposure of A7r5 cells to 10 mM ACA for 4 min did not produce a significant change in the magnitude of the peak current (reduction of 6.3 { 1.4%, n Å 12 cells), the decline was significant at 8 min (16.5 { 3.5%, n Å 9 cells; p õ 0.05). In a subset of 3 cells exposed to 10 mM ACA for more than 12 min, summarized in Fig. 2C, the current continued to decline, exhibiting a reduction of 25.2 { 6.7% (ANOVA, p õ 0.01) at 12 min. Despite the progressive and significant reduction in magnitude of inward current caused by ACA, there was no effect on the voltage dependence of the current. This is apparent in the representative current traces in Figs. 1A and 2A, which
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FIG. 2. Effects of 10 mM acetaldehyde (ACA) on current through Ltype Ca2/ channels in vascular smooth muscle cells. Command potentials, recording, and abbreviations are the same as in Fig. 1. (A and B) Data from a representative A7r5 cell. (C) Summary of data from 3 A7r5 cells which were exposed to 10 mM ACA for ú12 min. To normalize currents before averaging, Ipeak(t), the peak current at time t after beginning ACA superfusion, was divided by Ipeak(0), the peak current just before the cells were exposed to ACA. Error bars are SEM for selected time points. Repeated measures ANOVA, p õ 0.01. Pairwise multiple comparisons to control (current prior to ACA) by Dunnett’s method gave p õ 0.05 at 4, 8, and 12 min.
show no shift in the voltage eliciting peak inward current. Furthermore, the effects of ACA were often reversible. This is illustrated in the time course recording (Fig. 1B) of a cell that exhibited recovery to approximately 80% of control within 15 min. Recovery was Ç90% complete in 6 of 7 cells studied, whereas 1 cell failed to recover from the acute effects of acetaldehyde. DISCUSSION
Our results demonstrate that ACA can inhibit voltagedependent L-type Ca2/ channels, and consequently this
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might be responsible in part for the vasodepressant and inhibitory actions of ACA in aortic vascular smooth muscle observed by Altura et al. (1978) and Brown and Savage (1996). Brown and Savage (1996) observed that potassium chloride-elicited contractions in rat aortic rings were significantly inhibited by 30 mM ACA but not by 10 mM ACA. Correspondingly, our results show that 30 mM ACA inhibited current through Ca2/ channels in vascular smooth muscle cells, and that 10 mM ACA was less effective at reducing the current than was 30 mM ACA. Since potassium chloride depolarizes vascular smooth muscle cells and causes contraction by activating voltage-dependent Ca2/ channels in the cell membrane, the inhibition of L-type Ca2/ channels by ACA would explain its inhibitory effect on contraction. Recording of currents through Ca2/ channels, however, is a more direct and sensitive way of demonstrating effects of ACA on ion channels. These direct measurements of currents through Ca2/ channels support the idea, proposed by Altura et al. (1978a,b) and Brown and Savage (1996), that inhibition of aortic contraction by ACA depends to some extent on the inhibition of calcium ion influx. Nevertheless, other types of mechanisms also might be involved. For instance, ACA might interfere somewhere intracellularly with the interactions of Ca2/ and the contractile proteins (Altura et al., 1976). It was shown by Puszkin and Rubin (1975) that ACA can produce inhibitory effects on superprecipitation of human skeletal muscle actomyosin. Additionally, ACA could interfere with the mobility of cellular Ca2/ reservoirs since it is lipophilic and can penetrate membranes and cells rapidly (Truitt and Walsh, 1971). Moreover, it has been proposed by Altura et al. (1978a,b) that ACA might induce its depressant actions through some effect on metabolism, although they later concluded that this mechanism is unlikely to be responsible for ACA’s inhibitory actions.
FIG. 3. Summary of changes in peak current through L-type Ca2/ channels in response to acetaldehyde (ACA). The bar for 0 mM ACA (n Å 9 cells) shows the percentage change in current during the 4 min in control medium and indicates no significant change with time under control conditions. Bars for 10 mM ACA (n Å 6 cells) and 30 mM ACA (n Å 6 cells) are the percentage change of the current 4 min (open bars) and 8 min (filled bars) after application of the indicated solution, as a percentage of the current just prior to ACA application. Bars represent mean { SEM; ANOVA, p õ 0.001. *p õ 0.05, compared to 0 mM ACA control (Student– Newman–Keuls method).
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ACETALDEHYDE INHIBITS CURRENT
Acetaldehyde metabolism is quite rapid, almost 5 times that of ethanol, and low blood concentrations (0.5 mM) are maintained after short-term administration of 0.5 g of ethanol/kg in a normal individual (Lubin and Westerfeld, 1945; Leiber, 1987). However, higher acetaldehyde levels, approaching 1.8 mM, are achieved in alcoholic compared to nonalcoholic subjects when given the same dose of ethanol (Leiber, 1987). The higher acetaldehyde concentration among alcoholic subjects is thought to be due to hepatic inhibition of acetaldehyde dehydrogenase activity. Furthermore, acetaldehyde levels approach 30–53 mM in approximately 52% of individuals of Asian decent who lack a low Km aldehyde dehydrogenase and at least one individual survived a plasma concentration of 0.5 mM following severe ethanol intoxication (Watanabe et al., 1985). These isozyme variations which have enhanced affinity for ethanol, but diminished specificity toward acetaldehyde, enable blood levels of the latter to exceed that of normal individuals. However, the concentration of acetaldehyde in cardiac myocytes might be up to 10-fold higher than the plasma concentrations seen in the rat (Espinet and Argiles, 1984). Therefore, although the high concentration used in this study may be irrelevant to clinical arterial blood concentrations, they may be indicative of the concentration seen in vivo by the heart cells. Studying the acute effects of acetaldehyde on membrane ionic currents provides a means of assessing its potential role in the development of hypertension as a result of long-term ethanol consumption. Since ACA is the major metabolite of ethanol, these results raise the question of whether ACA may play a role in the pathological effects of chronic alcohol ingestion. Ethanol can produce dose-dependent relaxant, vasodilator as well as inhibitory actions on agonist-induced contractions on a variety of tissue preparations including rat arterioles, venules, arteries, and veins both in situ and in vitro (Altura et al., 1976, 1978a,b; Altura and Edgarian, 1976; Edgarian and Altura, 1976; Altura and Altura, 1978). These actions have been implicated in the development of cardiovascular and neurological disorders related to excessive ethanol consumption as in the case of alcoholics. Although the inhibitory actions of ACA on smooth muscle L-type currents demonstrated here suggest a possible mechanism by which alcohol metabolism could result in an inhibitory action on blood vessel contractility, it is important to keep in mind that the concentrations of ACA used here and in previous contraction studies (10 and 30 mM; Brown and Savage, 1996) are very high. While there are many instances in which plasma ACA concentrations may be dramatically elevated following ethanol ingestion (Tabakoff et al., 1989), this concentration of ACA would not usually be encountered in the plasma of even an extreme alcoholic as it would be expected to cause death first. On the other hand, these experiments showed that the effect of ACA increased pro-
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gressively even at the lowest concentration tested, suggesting that ACA applied for longer time periods may have inhibitory effects at lower concentrations, more relevant to those occurring in response to ethanol ingestion. Furthermore, these results demonstrate that when toxicological levels of ACA are encountered, one expected effect is inhibition of blood vessel contractility through a direct effect on membrane Ca2/ currents. ACKNOWLEDGMENTS The authors gratefully acknowledge support for this research from NIH Grant HL-02748 (to R.A.B.) and NIH Grant GM-08167 (to J.A.M. and J.L.R.). We also thank Ms. Linda McCraw for assistance in preparing the manuscript.
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and Ram, J. L. (1996). Tamoxifen (estrogen antagonist) inhibits voltagegated calcium current and contractility in vascular smooth muscle from rats. J. Pharmacol. Exp. Ther. 277, 1444–1453.
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Tabakoff, B., Eriksson, C. J. P., and von Wartburg, J. P. (1989). Methionine lowers circulating levels of acetaldehyde after acetaldehyde ingestion. Alcoholism Clin. Exp. Res. 13, 164–171.
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Zhang, F., Ram, J. L., Standley, P. R., and Sowers, J. R. (1994). 17b-Estradiol attenuates voltage-dependent Ca2/ currents in A7r5 vascular smooth muscle cell line. Am. J. Physiol. 266 (Cell Physiol. 35), C975–C980.
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