Proc. Nati. Acad. Sci. USA Vol. 89, pp. 6452-6456, July 1992 Medical Sciences Long-chain fatty acids activate calcium channels in ventricular myocytes (free fatty acids//olekic add) JAMES MIN-CHE HUANG, Hu XIAN, AND MARVIN BACANER* Department of Physiology, University of Minnesota, Minneapolis, MN 55455 Communicated by James Serrin, March 30, 1992 (receivedfor review September 15, 1991)

ABSTRACT Nonesterified fatty acids accumulate at sites down (>70%o decrease in ICa) during the 10- to 15-min control of tissue injury and necrosis. In cardiac tissue the concentra- period were discarded. Whole-cell voltage-clamp experi- tions of , arachidonic acid, leukotrienes, and other ments with myocytes were done at 320C with 2- to 3-MU glass fatty acids increase greatly during ischemia due to receptor or pipettes (Narishige PB 7 puller). Dagan 3900A patch-clamp nonreceptor-mediated activation of phospholipases and/or circuit, axon DMA interface, IBM cloned AT 386 computer, diminished reacylation. In ischemic myocardium, the time and P-CLAMP software were used for command pulses, data course of increase in fatty acids and tissue calcium closely acquisition, and analysis. All lipophilic agents were dissolved parallels irreversible cardiac damage. We postulated that fatty in 95% ethanol to make stock solutions and then diluted to acids released from membrane phospholipids may be involved <0.1% ethanol concentration before being applied by con- in the increase ofintraceilular calcium. We report here that low tinuous bath perfusion. In control studies, 0.1% ethanol in the concentrations (3-30 ,AM) of each long-chain unsaturated buffer solution had no influence on ICa or membrane response (oleic, linoleic, linolenic, and arachidonic) and saturated to fatty acids (see text and Fig. 4). ICa was measured with 2.5 (paimitic, stearic, and arachidic) tested induced mM Ba2+ as the charge carrier. From a holding potential of multifold increases in voltage-dependent calcium currents (Ic.) -85 mV, sodium current was inactivated by prepulsing to in cardiac myocytes. In contrast, neither short-chain fatty acids -40 mV for 60 msec. Then a series of depolarization pulses (<12 carbons) or fatty acid esters (oleic and palmitic methyl ranging from -60 to +50 mV in 10-mV increments were sent esters) had any effect on IcS, indicating that activation of at 10-sec intervals. Except where otherwise specified, each calcium channels depended on chain length and required a free family of control ICa pulses was recorded repeatedly with a carboxyl group. Inhibition of protein kinases C and A, G 2-min rest period in between until several sequential families proteins, eicosanoid production, or nonenzymatic oxidation of stable ICa traces were obtained before the bath was did not block the fatty acid-induced increase in Ica. Thus, changed. Potassium currents were blocked with internal Cs long-chain fatty acids appear to directly activate Ica, possibly and external Cs and tetraethylammonium solutions. The bath by acting at some sites near the channels or directly on the solution contained 65 mM NaCl, 2.5 mM BaCl2, 80 mM channel protein itself. We suggest that the combined effects of tetraethylammonium chloride, 5 mM CsCl, 1 mM MgCl2, 5 fatty acids released during ishemla on Ica may contribute to mM Hepes, and 5 mM glucose (pH 7.45). The pipette solution ischemia-induced pathogenic events on the heart that involve contained 145 mM CsCl, 5 mM NaCl, 1 mM MgCl2, 6 mM calcium, such as arrhythmlas, conduction disturbances, and EGTA, 5 mM Hepes, 3 mM Na2ATP, 3 mM creatine phos- myocardial damage due to cytotoxic calcium overload. phate, and 5 mM glucose (pH 7.3). Junction potentials were subtracted by adjusting voltage-offset compensation to zero with pipette in the bath. LaCl3 (0.1 mM), which has no effect Considerable evidence suggests that the accumulation of on sodium current but completely blocks calcium channels, membrane-derived free fatty acids and lysophospholipids are was added as a final step in each study to block ICa for important in the pathogenic events caused by myocardial leakage-current subtraction. Alternatively, nifedipine (3 ,uM) ischemia (1-8). The onset of disturbed membrane function, was used to specifically block L-type calcium channels to proceeding on to irreversible cardiac damage, closely paral- assess the ICa component carried via T-type channels. lels the time course of accumulation of fatty acids and an All chemicals were from Sigma except arachidonic acid associated increase in tissue calcium (3, 8). We postulated (Nu-Chek, Austin, MN), A63162 (Abbott), WY50295 that the increase in fatty acids might be involved in the altered (Wyeth), and U74500A (Upjohn). membrane function that leads to increased tissue calcium. We report that fatty acids have powerful effects on voltage- dependent calcium currents (ICa) in cardiac cells, which could RESULTS AND DISCUSSION trigger ischemia-induced pathogenic actions on the heart that The -10 mV traces shown in Fig. 1 (Left) indicate that involve calcium-such as arrhythmias, coronary vasocon- perfusion of myocytes with 10 ,uM arachidonic acid, 10 gM striction, and cytotoxic calcium overload. oleic acid, or 30 uM caused a large increase in Ica. The respective current-voltage relationships (Fig. ld) show MATERIALS AND METHODS an increase in ICa with no shift in activation or maximal peak current potential. Nor was there a significant potential shift The effect of fatty acids on calcium currents was studied in in steady-state activation and inactivation curves (Fig. 2). single guinea pig ventricular myocytes using a standard The current was completely blocked by LaCl3, which blocks whole-cell voltage-clamp technique. Ventricular myocytes both L- and T-type channels. Blockade of L-type channels were isolated as described (9). Only calcium-tolerant myo- with nifedipine suppressed 95-98% of the current, indicating cytes with clear striations were used. Cells with rapid run- Abbreviations: Ica. voltage-dependent calcium currents; PKA and The publication costs ofthis article were defrayed in part by page charge PKC, protein kinase A and C, respectively; NDGA, nordihydrogua- payment. This article must therefore be hereby marked "advertisement" iaretic acid; GDP[,6S], guanosine 5'-[-thio]diphosphate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

6452 Downloaded by guest on September 26, 2021 Medical Sciences: Huang et al. Proc. Natl. Acad. Sci. USA 89 (1992) 6453

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-100 -80 -60 -40 -20 0 20 SA 4ms -5.0 Em (mV)

FIG. 1. acid oleic acid (OA), and stearic acid FIG. 2. Steady-state activation and inactivation curves of cal- Arachidonic (AA), cium channels in control and after application of fatty acid (linole- (SA) increase calcium currents in guinea pig ventricular myo- (IC.) laidic acid, 30 MM). Both activation and steady-state inactivation cytes. (a-c) ICa (before leakage-current subtraction) during - 10-mV curves were from the same representative cell. The steady-state test pulse before (middle traces) and after (lower traces), showing a multifold increase in ICa after perfusion with arachidonic acid (10 inactivation curves were obtained with a double-pulse protocol. From a holding potential of -85 mV, a prepulse (-100 to +20 mV) 16 oleic acid 14 or stearic acid AM, min) (a), (10MAM, min) (b), (30MuM, of 5-sec duration was followed by a test pulse at 0 mV. Sodium 17 min) (c). Upper trace in a-c shows that lanthanum (0.1 mM) added after the fatty acids completely suppressed the calcium current. (d) current was blocked by 30 MM tetrodotoxin. Peak amplitudes of ICa Respective current-voltage relationships after subtraction ofleakage of the test pulses were normalized to the amplitude of the test pulse with the -100-mV prepulse and plotted as a function of prepulse current. Open symbols are controls. After addition of arachidonic potential. Activation curves were obtained by normalizing conduc- acid (e), oleic acid (U), or stearic acid (A), ICa increased significantly tances (g) to the value at 0 mV (g max). Current amplitudes at 0-mV with no shift in activation or maximal peak currents. Em, membrane potential. test pulses in control and after application of 30 AM linolelaidic acid were 664 pA and 1250 pA, respectively. Em, membrane potential; I, current; hoc, steady-state inactivation; moo, steady-state activation. that not more than 5% was carried via T channels. After nifedipine blockade, fatty acids increased ICa up to 5%, and linolenic acids. Oleic acid produced maximal increases in indicating a small component was carried via T-type chan- at nels. The increase in peak ICa induced by fatty acids was ICa 10 uM concentration, whereas arachidonic, linoleic, associated with accelerated inactivation. In the control and linolenic acids (3-50 uM) increased ICa in a dose- concen- -10-mV trace (Fig. la) ICa inactivation fit a single exponen- dependent manner (Table 1). In contrast, at 100 MLM the relative increases in ICa by oleic acid and tial (Trs0w = 264 msec), whereas after arachidonic acid there trations, were + = was a fast component (Tfast = 24 msec) as well as a slow arachidonic acid decreased to 446 207% (n 3) and ± = the component (Trjow = 278 msec) in the -10-mV trace. The 213 117% (n 4), respectively. (30 MkM), effect offatty acids on ICa was similar to the kinetics seen with most potent saturated fatty acid, increased ICa 6-fold, fol- the calcium-channel agonist Bay K 8644 and may be similarly lowed by stearic and arachidic acids. Fatty acids applied related to altered channel gating and a partial antagonist internally had an identical effect on 'Ca, but the effects were effect on inactivation attributed to Bay K 8644 (10, 11). slower in onset because of slow diffusion from the pipette. The relative activation of 'Ca by various unsaturated fatty The sodium salt of oleic acid, which dissociates when dis- acids shows that at each concentration tested (3-50 AM), solved in water, also increased ICa (n = 3), whereas fatty acid oleic acid (10 MLM) had the greatest effect, increasing ICa more esters (oleic and palmitic methyl esters) had no effect on 'Ca, than 7-fold, followed (in order) by arachidonic acid, linoleic, clearly indicating that activation ofcalcium channels required Table 1. Effect of fatty acids on Ica ICa, mean % increase + SD (n) Fatty acid 3 1uM 10 ,M 30 jLM Long chain Unsaturated Oleic acid (18:1 cis-A9) 337 ± 130 (3) 713 ± 556 (9) 681 ± 281 (4) (18:2 cis-A9'12) 104 70 (3) 158 ± 63 (5) 407 ± 237 (6) Linolelaidic acid (18:2 trans-A9'12) 251 ± 152 (7) Linolenic acid (18:3 cis-A9'12"15) 48 ± 29 (3) 149 ± 120 (6) 275 ± 178 (8) Arachidonic acid (20:4 cis-A&58'11"14) 112 ± 47 (3) 207 ± 183 (22) 287 ± 142 (4)* Saturated Palmitic acid (16:0) 110± 83 (4) 644 ± 496 (7) Stearic acid (18:0) 306 ± 162 (6) (20:0) 345 ± 93 (6) Short chain Heptanoic acid (7:0) No effect (3) Valproic acid (8:0) No effect (3) (8:0) No effect (3) Undecanoic acid (11:0) No effect (3) Esterified Oleic acid methyl ester No effect (4) Palmitic acid methyl ester No effect (3) *50 MM arachidonic acid. Downloaded by guest on September 26, 2021 6454 Medical Sciences: Huang et al. Proc. NatL Acad. Sci. USA 89 (1992)

-4.0 - a b OA 1dA) -60 -40 ? 3.0-3 Em (mV) AA LA -2.0- \1-l~

-1.0 FIG. 4. Effect of arachidonic acid and washout of arachidonic acid on ICa after blockade of PKC and PKA with staurosporine. (a) CTL Membrane current traces of the -10-mV clamp step. Traces: 1, control; 2, 15 min after superfusion of staurosporine (300 nM), ICa is 0 o lb 20 30 40 50I sharply suppressed; 3, 20 min after addition of arachidonic acid (10 ,uM) and staurosporine, Ica is increased, reversing staurosporine Time (min) suppression and increasing ICa above original control levels; 4, 11 min after washout of arachidonic acid and staurosporine with control FIG. 3. Time course of change in Ica shown by the -10-mV test perfusion medium containing 0.1% ethanol, showing that the ara- pulse recorded for 45 min in a representative control cell and in three chidonic acid-induced peak increase in ICa is reversed but is not representative cells after addition of fatty acids (10 AM) (arrows). reversed completely because inactivation remains faster than initial The control cell (CTL) typically had a slow decrease in ICa over 45 control; 5, 6 min after reapplication of arachidonic acid (10 1sM), Ica min. After perfusion with oleic acid (OA), Ica began to increase in 2 is again increased with the arachidonic acid traces (3 and 5) virtually min, reached a plateau in 14 min, and started to decline after 25 min. superimposed. (b) Current-voltage relationship for each family of In the cell perfused with arachidonic acid (AA), ICa began to increase test pulses after subtraction of leakage current. A, Control; o, in 3 min and reached a plateau in 17 min that was sustained for 10 staurosporine; o, arachidonic acid plus staurosporine; A, washout; *, min. Linoleic acid (LA) added 10 min after breaking the membrane reapplication of arachidonic acid. Em, membrane potential. caused a progressive increase in ICa, which lasted for 31 min before declining. the bath or the pipette solution suppressed ICa (Fig. 4) but did not prevent a large increase in ICa by arachidonic acid or other the free carboxyl group (Table 1). Short-chain fatty acids fatty acids tested (Table 2). Fig. 4 also shows that washout (<12 carbons) also had no effect on ICa (Table 1). and reapplication of arachidonic acid induced reproducible Fig. 3 shows the time course of change in ICa over 45 min. increases in 'Ca, even with staurosporine. The consistent 70% In the control cell ICa decreased slowly; in cells perfused with suppression of ICa by staurosporine alone (Table 2) suggests 10 ,M oleic acid, arachidonic acid, or linoleic acid ICa that it may suppress background calcium currents activated increased to a peak or plateau over 15 up to 24 min before via both PKA and PKC pathways. Specific inhibition ofPKA declining. The fatty acid-induced increase in ICa could be alone decreased ICa 20% (16), whereas the 70% suppression reversed by washout with buffer (containing 0.1% ethanol) observed with staurosporine suggests that it may also sup- and restored by reperfusing the cell with the fatty acids (Fig. press an additional PKC-pathway-dependent background 4). The increase in ICa was not a detergent effect because 30 component. However, failure of staurosporine to block an A&M deoxycholate, a potent detergent, had no effect on 'Ca (n increase in ICa by fatty acids indicates that the increase did = 3). not require activation of PKA or PKC pathways. With To explore mechanism(s) and determine whether fatty staurosporine, the increase in peak ICa by fatty acids was acids activated ICa directly or acted indirectly via activation associated with rapid inactivation (Figs. 4 and 5). The effect of protein kinases, we inhibited protein kinase A (PKA) and of fatty acids on 'Ca kinetics differs from that induced by protein kinase C (PKC) pathways before adding the fatty P-adrenergic agonists that are associated with delayed inac- acids. Phorbol esters were reported to increase ICa by acti- tivation of calcium channels (10, 11). It is also possible that vation of PKC (12). Because fatty acids can also activate inhibition of protein kinases by staurosporine removes a PKC (13), cells were pretreated with 300 nM staurosporine background effect of cAMP-dependent delayed inactivation. (14, 15), a potent inhibitor of both PKC and PKA (IC50 for G proteins have been shown to directly modulate voltage- PKC is 3 nM; IC50 for PKA is 8 nM). Staurosporine added to dependent calcium channels in myocytes and neurons (17, Table 2. Effect of fatty acids on ICa after inhibitors Inhibition Effectj mean % 'Ca after fatty acid,§ Fatty acid* Inhibitor(s)t inhibition + SD mean % change ± SD (P, n) Oleic acid (10 AM) Staurosporine -72 ± 15 919 ± 610 (P < 0.01, n = 5) Arachidonic acid (10 ,uM) Staurosporine -71 ± 9 328 ± 209 (P < 0.001, n = 7) Linoleic acid (30,M) Staurosporine -65 ± 14 628 ± 478 (P < 0.01, n = 5) Linolenic acid (30 AM) Staurosporine -69 ± 16 142 ± 90 (P < 0.025, n = 4) Palmitic acid (30 ,uM) Staurosporine -71 ± 4 242 ± 35 (P < 0.001, n = 3) Oleic acid (10 ,uM) Staurosporine and -79 ± 2 659 ± 394 (P < 0.01, n = 4) GDP[LS]l Arachidonic acid (10 AM) Staurosporine and -83 ± 10 251 + 194 (P < 0.01, n = 5) GDPBpS]¶ *Fatty acid was added after treatment with inhibitor(s). tStaurosporine (300 nM), a protein kinase inhibitor, was externally applied (IC50 for PKC, 3 nM; IC50 for PKA, 8 nM). tMean percent change from control ICa. §Percent change in ICa from values after suppression by inhibitor(s). $Internal application of GDP[,BS] (500 ,AM), a G protein inhibitor. Downloaded by guest on September 26, 2021 Medical Sciences: Huang et al. Proc. Natl. Acad. Sci. USA 89 (1992) 6455 a

200 PAL 40 ms

FIG. 5. Effects ofarachidonic acid on ICa after blockade ofG proteins with GDP[13S] internally (alone) and combined with inhibition ofprotein kinases (PKC, PKA) with staurosporine externally. Only the -10-mV test pulse traces are shown in a. Trace: 1, control trace right after membrane rupture; 2, decrease in ICa 6 min later as GDPLBS] (500 ,AM) contained in the pipette solution diffused into the cell; 3, 15 min after application of staurosporine (300 nM) externally in the presence of GDPLfS1 for 21 min, ICa was suppressed further; 4, after blockade of both G proteins and protein kinases (PKC, PKA), the addition of arachidonic acid (10 ,uM) still greatly increased Ica. Other traces are omitted for clarity. The respective current-voltage curves are shown after leakage-current subtraction in b. Em, membrane potential. 18). To prevent possible activation of ICa by G proteins, trans configuration) long-chain fatty acids can directly acti- guanosine 5'-[,f-thio]diphosphate (GDP[13S]; 500 ,uM) was vate calcium channels at some lipid sites near the channel or included in the pipette solution (19). ICa fell as GDP[,8S] on the channel protein itself. The effect appears to reside in diffused into the cell (19), but its presence did not block a the free carboxyl group because esterified fatty acids have no large increase in ICa when fatty acids were subsequently effect and depends on chain length because short-chain fatty added (n = 6). Even pretreatment with GDP[j3S] (internally) acids also have no effect (Table 1). A direct effect of fatty together with staurosporine (externally) to block both G acids on ICa is in accord with earlier reports that arachidonic proteins and activation of protein kinases failed to prevent a acid and other fatty acids directly modulate outward recti- fatty acid-induced increase in ICa (Table 2 and Fig. 5). fying potassium channels in smooth muscle (28) as well as Arachidonic acid differs from other fatty acids in that it is PKC (29) and Ca2+-ATPase (30). However, an indirect action a substrate for cyclooxygenase, lipoxygenase, and cy- via bioactive eicosanoid production may also occur because tochrome P450 pathway enzymes that synthesize many bio- both indomethacin and eicosatetraynoic acid attenuated active eicosanoids (20-22). The arachidonic acid-mediated rather than prevented the increase in ICa by arachidonic acid. increase in ICa was independent of leukotriene or prostaglan- There is evidence that arachidonic acid metabolic interme- din production because it was not blocked by cyclooxygenase diates are synthesized by myocytes (31). Moreover, we have inhibition with indomethacin (23) (30 ,uM, n = 11) or eico- recently reported that leukotrienes A4, B4, C4, D4, and satetraynoic acid (10,tM, n = 7), which was reported to block prostaglandins E2 and F2< are potent activators of ICa in cytochrome P450 as well as 12-, and 15-lipoxygenase path- ventricular myocytes (32, 33). ways (24, 25). Moreover, two putatively specific 5-lipoxyge- We suggest that by increasing ICa, the combined accumu- nase blockers (A-63162, n = 6; WY-50295, n = 5) as well as lation of many fatty acids during myocardial ischemia (1-8) butylated hydroxytoluene (BHT, n = 2) and U-74500A (n = can increase intracellular calcium, which may trigger calci- 3) (inhibitors of nonspecific lipid peroxidation) failed to um-dependent arrhythmogenic afterdepolarizations (34-36), diminish the increase in ICa by arachidonic acid or oleic acid. as well as induce cytotoxic calcium overload (37, 38). Direct Surprisingly, however, nordihydroguaiaretic acid evidence that fatty acids induced calcium-mediated physio- (NDGA), an inhibitor of both lipoxygenase-mediated and logic and pathologic actions on ventricular myocytes was nonspecific lipid peroxidation (26), not only suppressed 'Ca reported in a recent abstract (39). Using calcium fluorescence itself but in its presence arachidonic acid suppressed ICa measurements in cells loaded with indo or fura-2 together further (n = 8). The effect of NDGA was seemingly at odds with a cell-edge motion detector, we found that fatty acids with the failure to block the arachidonic acid-induced in- increased intracellular calcium as well as the amplitude ofthe crease in ICa by other agents that specifically inhibit lipoxy- calcium-fluorescence transients. This increase was associ- genase activity (A-63162, WY-50295) as well as nonenzy- ated with positive inotropic and chronotropic effects, which matic lipid oxidation (butylated hydroxytoluene, U-74500A). were followed in 30 to 60 min by arrhythmias and cell Although NDGA is widely used to inhibit lipoxygenase contracture (39). By modulating both ICa, as shown here, as activity, it has also recently been shown to have a second well as potassium (20, 28, 40, 41) and chloride currents (42), action directly blocking calcium channels in pituitary cells the accumulation offatty acids can greatly disturb membrane independent of lipoxygenase inhibition (27). We found that functions that regulate cardiac excitability, conduction, pretreatment with NDGA (50 ,uM) in myocytes completely rhythm, contraction, and viability. blocked an increase in ICa by the calcium-channel agonist Bay K 8644 (10, 11) (n = 5) as well as by oleic acid, which is not 1. Katz, A. M. (1982) J. Mol. Cell Cardiol. 14, 627-632. a substrate for lipoxygenase enzymes (n = 2). This result 2. Prinzen, F. W., Van Der Vusse, G. J., Arts, T., Roemen, suggests that in myocytes (as in pituitary cells) NDGA T. H. M., Coumans, W. A. & Reneman, R. S. (1984) Am. 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