© 1996 American Heart Association, Inc.
Articles |
Modulatory Effects of Arachidonic Acid on the Delayed Rectifier K+ Current in Rat Pulmonary Arterial Myocytes
Structural Aspects and Involvement of Protein Kinase C
the Department of Pharmacology, United Medical and Dental Schools of Guy's and St Thomas's Hospitals, London, UK.
Correspondence to Dr S.V. Smirnov, Department of Pharmacology, United Medical and Dental Schools of Guy's and St Thomas's Hospitals, St Thomas's Campus, Lambeth Palace Rd, London SE1 7EH, UK. E-mail s.smirnov@umds.ac.uk.
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Abstract |
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The effect of arachidonic acid (AA) on the delayed rectifier K+ current (IK) was evaluated in rat pulmonary myocytes by using the whole-cell patch-clamp technique. Externally applied AA (50 µmol/L) caused a membrane depolarization, averaging 16 mV in six cells. AA (1 to 50 µmol/L) caused a dual effect on IK. First, AA accelerated the rate of IK activation, increasing current amplitude at the beginning of voltage step. Second, AA caused a marked acceleration of current decay, thereby reducing IK amplitude measured toward the end of the depolarizing steps. These effects were not prevented by indomethacin or nordihydroguaiaretic acid, blockers of cyclooxygenase and lipoxygenase, respectively. AA did not affect the voltage dependence of current activation or inactivation. The magnitude of the inhibitory effect on IK was correlated with the number of double bonds but was independent of tail length in fatty acids containing between 14 and 22 carbons. Linoleic acid (18:2, cis-9,12) inhibited IK much more than did its trans-stereoisomer, linolelaidic acid. Arachidonyl alcohol, which is uncharged, and arachidonyl coenzyme A, which does not `flip' across the cell membrane, were less effective than AA in inhibiting IK; this effect of fatty acids may therefore require passage across the cell membrane. The enhancement of early IK was mimicked by the protein kinase C (PKC) stimulator 1-oleoyl-2-acetyl-sn-glycerol (10 µmol/L), was suppressed by ATP removal from the pipette solution, and was blocked by PKC inhibitors chelerythrine (10 µmol/L) and staurosporine (100 nmol/L). This effect may therefore require PKC-dependent phosphorylation.
Key Words: vascular smooth muscle • pulmonary artery • K+ channel • arachidonic acid • protein kinase C
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Introduction |
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Arachidonic acid is normally released from membrane phospholipids by either cellular damage or receptor-mediated stimulation of phospholipases A2, C, or D (eg, see References 1 and 2). In addition to serving as a substrate for oxidative metabolism to the eicosanoids, AA itself may act as a second messenger and is capable of stimulating PKC,3 NADPH oxidase,4 guanylate cyclase,5 p21ras,2 and mitogen-activated protein kinases.6 Furthermore, there is extensive evidence that AA and other long-chain unsaturated fatty acids can modify the activities of a wide range of ion channels.7 8 9 10 With regard to smooth muscle, for example, AA and similar fatty acids have been shown to inhibit Ca2+ currents in rabbit ileal cells11 and to open stretch- and Ca2+-activated K+ channels in both vascular and intestinal myocytes.12 13 14 Moreover, it has been proposed recently that AA or an as-yet-unidentified metabolite of AA may act as an endothelium-derived hyperpolarizing factor.15
AA is known to exert a striking effect on several types of IK from nonvascular cells, causing an acceleration of both current activation and decay.16 17 18 The mechanisms underlying this complex effect remain obscure. In preliminary work, we observed similar responses to AA in rat pulmonary arterial myocytes.19 20 In view of the important role of voltage-gated K+ currents in setting the membrane potential in vascular smooth muscle in general,21 and in rat pulmonary artery smooth muscle cells in particular,22 23 in the present study we characterize and explore the mechanistic basis of the effects of AA and other fatty acids on IK in these cells.
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Materials and Methods |
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Cell Isolation and Electrophysiology
Male Wistar rats (250 to 300 g) were either killed by cervical dislocation or anesthetized with ether; the heart and lungs were removed; and small pulmonary arteries (internal diameter, 300 to 700 µm) were isolated, dissected free of connective tissue, cut into pieces (
1x1 mm), and allowed to recover for 60 minutes in PSS. Tissue was then placed in Ca2+-free PSS for 25 minutes at 37°C, before enzymatic digestion for 20 to 25 minutes in low-Ca2+ PSS (15 µmol/L Ca2+ added to nominally Ca2+-free PSS) containing 2 mg/mL collagenase (type XI), 1 mg/mL papain, and 1 mmol/L dithiothreitol (all from Sigma Chemical Co). Tissue was then transferred to enzyme-free Ca2+-free PSS and triturated with a Pasteur pipette in order to disperse cells. The suspension was stored in low-Ca2+ PSS at 4°C for use within 4 to 6 hours. PSS contained (mmol/L) NaCl 130, KCl 5, MgCl2 1.2, CaCl2 1.5, HEPES 10, and glucose 10; pH was adjusted to 7.2 by NaOH. TEA (10 mmol/L) was also present in all external solutions used in voltage-clamp experiments in order to block the Ca2+-activated K+ current.22 24 The pipette solution contained (mmol/L) KCl 110, MgCl2 0.5, Na2ATP 5.0, HEPES 10, EGTA 10, and Ca2+ 0.5 (giving an estimated free [Ca2+] of 8 nmol/L) and was buffered to pH 7.2 using KOH.
For electrophysiological recording, cells were placed in a chamber with a volume of 50 to 100 µL and were continually superfused with PSS or a test solution via a five-barrel pipette at a rate of 0.9 to 1 mL/min. The rate of the exchange of external solutions in the experimental chamber was estimated using distilled water as a control solution and normal PSS as a test solution. A small voltage step was applied at 0.5 Hz to a patch pipette immersed in the bath, and the alteration in the pipette resistance was measured during solution exchange. Data from five such recordings were normalized and averaged (see Fig 3B
). Experiments were carried out at room temperature. Electrophysiological recordings and data analysis have been previously described in detail.24
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Means were compared using Student's two-tailed paired or unpaired t tests as appropriate; differences were deemed significant at P<.05 (unless otherwise stated). All data are presented as mean±SEM.
Drugs
All fatty acids, eicosanoids, ethoxyresorufin, indomethacin, NDGA, and PKC inhibitors (chelerythrine and staurosporine) were purchased from Sigma. All drugs were dissolved in 100% dimethyl sulfoxide. Eicosanoids were aliquoted into small vials under nitrogen, stored at -20°C, and used immediately before each experiment. 11,12-EET was stored at -70°C. Dimethyl sulfoxide at the maximal concentration of 0.1% used in the present experiments did not produce any effect on IK.
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Results |
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Effect of AA on Membrane Potential in Single Pulmonary Arterial Myocytes
Two to 3 minutes after formation of the whole-cell configuration, a stable Em could be recorded in current-clamp mode in rat pulmonary arterial cells superfused with normal (TEA-free) PSS. Em ranged from -34 to -60 mV in the six cells studied, with a mean value of -44±4 mV. Under these conditions, the external application of 50 µmol/L AA consistently caused a membrane depolarization of up to 24 mV (Fig 1
). Em began to fall after a 30- to 60-second delay, and the membrane slowly depolarized to a sustained level, which differed from the resting potential by 16±2 mV (n=6). The effect of AA under these conditions was only partly reversible. In three of six cells, a transient hyperpolarization of 7 to 13 mV preceded the membrane depolarization (not shown). A membrane depolarization of 14 mV was also observed in one cell in the presence of 10 mmol/L TEA in the external solution.
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Time- and Concentration-Dependent Effects of AA on IK
A standard voltage protocol was used to study the effect of AA on the IK present in these cells.24 Cells were held at -60 mV and stepped to +60 mV every 10 s for 300 ms to activate IK completely. This resulted in a rapid activation of IK, which then inactivated relatively slowly (Fig 2, current traces marked C). The bath application of 3 µmol/L AA caused an increase in the rate of decay of IK, which developed progressively over 3 to 4 minutes (Fig 2A). This resulted in a fall in the amplitude of IK measured at the end of the voltage step. This response was concentration dependent between 1 and 50 µmol/L AA (Figs 2 and 3
). At the same time, each concentration of AA appeared to increase the rate of activation of IK, resulting in an increase in the current amplitude during the first few milliseconds after the start of depolarization, as shown using an expanded time scale (Fig 2, right panels).
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In order to further characterize these effects of AA on IK, the current amplitude in the presence and absence of AA was measured, first, at the time when IK reached its peak in the presence of AA (between 4 and 8 ms, hereafter referred to as early IK) and, second, at the end of the 300-ms voltage step. Fig 3A shows that externally applied AA caused a small but consistent enhancement of early IK, which developed with a delay of 30 to 60 s. At the highest concentration of AA tested (50 µmol/L), the increase in early IK was rapidly succeeded by its decrease. A slower late decrease in early IK was also observed at lower concentrations of AA (Fig 3A). The enhancement of early IK (measured as the maximal increase of the early current in each individual cell) was significant at 3 and 10 µmol/L (P<.001 and P<.003, respectively) and was not significant at 1 µmol/L (Fig 3C). The enhancement of the early current at 50 µmol/L was of borderline significance (P<.03, one-tailed t test), and it is possible that the enhancement of early IK at this AA concentration is partially masked by the rapid development of current inhibition.
The inhibitory effect of AA on IK measured at 300 ms showed a clear monophasic time and concentration dependence on the concentration of the fatty acid. Suppression of IK developed more rapidly and to a greater extent at higher AA concentrations (Fig 3B). These effects were not rate-limited by the increase in the AA concentration during bath turnover, since a 95% exchange of the bath solution was effected within 10 s (Fig 3B, solid circles). When the inhibitory effect was measured after 3 to 4 minutes in AA, 50% inhibition was observed at 3 µmol/L (Fig 3D). The extent of inhibition by 1 and 3 µmol/L AA, as measured using this protocol, was probably somewhat underestimated, since the effect of AA developed relatively slowly at these concentrations (Fig 3B) and since the decay of IK was not complete within 300 ms (Fig 2A). Recovery from the inhibitory effect after removal of AA from the bath was slow and incomplete (50% to 80%, not shown).
A similar dual modulation of the cloned mouse cardiac Kv1.5 IK has been previously reported by Honore et al16 in 1994 and was associated with sizable hyperpolarizing shifts in both the I-V and inactivation-potential relationships. Therefore, we sought similar responses in rat pulmonary arterial myocytes. In the absence of AA, the apparent threshold of IK appeared at membrane potentials near -30 mV (Fig 4). With increasing depolarization, both the amplitude and activation rate of IK were enhanced. Application of 10 µmol/L AA did not result in a leftward shift of the apparent current threshold, although a substantial enhancement of the activation rate of IK occurred; this was especially noticeable at negative membrane potentials, where IK was activated slowly (Fig 4A). The effect was less prominent at very positive voltages, where the activation rate was already rapid. When the current amplitude at each membrane potential (measured at the end of the 120-ms depolarizing step) was normalized to that at +100 mV, no significant shift in I-V relationships was observed (Fig 4C). Similar results were obtained when a longer (300-ms) membrane depolarization was used (not shown).
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) and presence (
) of AA. Current amplitude in panel C was normalized with respect to the current at +100 mV. Data were obtained from five cells.
To study the effect of AA (10 µmol/L) on the potential dependence of IK availability, 10-s conditioning steps (applied over a range of potentials from -100 to +20 mV), followed by a step to +60 mV, were applied under control conditions and in the presence of 10 µmol/L AA. Panels C and D of Fig 5 illustrate that AA did not alter the resulting inactivation-potential relationships for either early IK or the current measured at the end of 120 ms, at the test potential of +60 mV. The average half-inactivation potential and slope factor were, respectively, -31.6±3 and 6.6±1 mV (absence of AA) and -33.7±3 and 5±0.4 mV (presence of AA) for the early current and -23.9±3 and 9.1±1 mV (absence of AA) and -28.3±3 and 7.9±2 mV (presence of AA) for IK at 120 mV (n=5). No significant effect of AA was recorded.
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Effect of Inhibitors of AA Metabolism
AA can be oxidized to a variety of biologically active metabolites via three major pathways: cyclooxygenase, lipoxygenase, and monooxygenase/cytochrome P-450.25 In order to determine whether the effects of AA on IK in rat pulmonary myocytes were dependent on the cyclooxygenase pathway, indomethacin (20 µmol/L), a widely used cyclooxygenase inhibitor, was applied to cells for 5 to 8 minutes, and then 50 µmol/L AA was added (Fig 6A). Indomethacin at this concentration very slightly decreased the amplitude of the delayed rectifier. It did not, however, prevent either inhibition or potentiation of IK by AA in five cells studied. Early IK was increased by a factor of 1.2±0.14, and IK at 300 ms was decreased to 0.4±0.18 compared with the control value, and these values were not significantly different from those obtained without indomethacin (1.05±0.03 and 0.21±0.03, n=8, respectively). In addition, application of 0.2 µmol/L U44169, a stable thromboxane A2 analogue, did not produce any effect on IK, whereas prostaglandin F2
at a very high concentration of 200 µmol/L only slightly (5% and 11% in two cells studied) inhibited IK (data not shown). NDGA (a nonselective lipoxygenase inhibitor) applied at a concentration of 10 µmol/L itself caused a substantial inhibition of IK (Fig 6B), the amplitude of which measured at 300 ms was reduced to 0.37±0.1 compared with the control value. Application of 50 µmol/L AA in the presence of NDGA further significantly suppressed IK to 0.17±0.1 (n=3).
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Incubation of cells (beginning 65 to 195 minutes before currents were recorded) in ethoxyresorufin (10 µmol/L), a selective inhibitor of cytochrome P-450 epoxygenase, had no significant effect on either potentiation or inhibition of IK by 10 µmol/L AA (1.08±0.04 and 0.21±0.07 [n=5] for early IK and IK measured at 300 ms in the presence of ethoxyresorufin compared with 1.11±0.04 and 0.39±0.04 [n=12] in the control condition). On the other hand, application of 11,12-EET, a cytochrome P-450 metabolite, caused a reversible diminution of IK (Fig 6D). IK measured at 300 ms was reduced by 16±1% (n=3) and 24±7% (n=2) in the presence of 0.8 and 3 µmol/L 11,12-EET, respectively. 11,12-EET did not, however, enhance early IK (Fig 6D).
Although the effect of 11,12-EET raised the possibility that AA might in part be inhibiting IK via its cytochrome P-450 metabolites, subsequent experiments with the AA analogue ETYA (50 µmol/L) suggested that this was unlikely. This substance, which is not a substrate for metabolism, also inhibited the delayed rectifier measured at 300 ms by 84±5% in four cells (Fig 6C). However, it is noteworthy that ETYA did not mimic AA in stimulating early IK (Fig 6C).
Structural Requirements for the Inhibitory Effect of Fatty Acids on IK
The results presented above suggested that although metabolites generated as a result of adding exogenous AA could play some role in modulating IK, the fatty acid itself is likely to be mainly responsible for the alterations in IK recorded. We subsequently focused on the inhibitory effects on IK of a number of fatty acids and related substances that differ from AA either with respect to the tail or head groups. Fatty acids were externally applied at a concentration of 50 µmol/L, cells were stepped from -60 to +60 mV, and the current amplitude was measured at 300-ms depolarization as previously described. Data, expressed as percentage of current inhibition by fatty acid, are summarized in Fig 7.
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The inhibition of IK showed several indications of structural dependence. Saturated fatty acids with no double bonds, including myristic (14:0), palmitic (16:0), and stearic (18:0) acids, had similar and minor (14% to 20%) inhibitory effects on IK. A somewhat larger, but also fairly consistent, degree of inhibition (19% to 42%) was observed for monoenoic (one double bond) fatty acids with tails ranging from 14 to 22 carbons. No relationship between chain length and inhibition of IK was apparent. Further increases in the number of double bonds in the fatty acid molecule then greatly potentiated the inhibition of IK, which reached its saturation level of 90% when fatty acids with five or six double bonds were applied. The clear pattern that emerged was therefore that the degree of unsaturation of the fatty acid tail, but not its length, was important in determining the inhibition of IK. The importance of the degree of unsaturation is further illustrated in Fig 8, which shows the time course of development of IK inhibition by different fatty acids having the same length (eg, 22 carbons) but a variable number of double bonds. Fig 8 also demonstrates that the inhibitory effect developed more rapidly when the number of double bonds was increased. A similar effect was observed when fatty acids containing 18 and 20 carbons and different numbers of double bonds were used (data not shown).
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), 3 (
), and 4 (
) cells studied. Current calibration bars are 0.3 nA.
The protocol used in these experiments (50 µmol/L fatty acid; test potential, +60 mV) was designed specifically to explore inhibitory effects, since the potentiating effect was more prominent at less positive potentials and at the lower concentration of 10 µmol/L (Fig 3). Even so, a significant enhancement (4% to 30%) of early IK was observed with myristic, palmitoleic, oleic, linoleic, eicosapentaenoic, and docosahexaenoic acids.
The configuration of the fatty acid tail group also influenced both of its effects on IK. Fig 9 shows a comparison of the effect of linoleic acid, which has 18 carbons and two double bonds in the cis configuration, and linolelaidic acid, its structural stereoisomer, which has the same number of carbons and two double bonds, both, however, in the trans position. Application of 50 µmol/L linoleic acid caused a substantial increase of early IK that was followed by a significant acceleration of IK decay (Fig 9A, 9B, and 9D [circles]). However, in the presence of the same concentration of linolelaidic acid, no substantial potentiation of early IK was observed (Fig 9B and 9C [inverted triangles]), and IK at 300 ms was inhibited by only 22% (Fig 9B and 9D).
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To determine whether the head group of the fatty acid molecule might also be important in determining the effects on IK, we examined the effects of arachidonyl alcohol and arachidonyl coenzyme A (both at 50 µmol/L). Arachidonyl alcohol has a structure similar to that of AA, but its head group contains a hydroxyl, rather than a carboxylic, group; therefore, it is a neutral derivative of AA. In contrast, arachidonyl coenzyme A has a large negatively charged head group, and evidence has been presented that it therefore cannot "flip" across the cell membrane.14 Both arachidonyl alcohol and arachidonoyl coenzyme A, when applied externally at concentrations of 50 µmol/L, had similar and relatively limited inhibitory effects on IK. Arachidonyl alcohol and arachidonoyl coenzyme A reduced IK by 35±2% (n=5) and 36±7% (n=4), respectively (Fig 10A through 10C). These effects were substantially and significantly smaller than the effect of 50 µmol/L AA (78±2%, Figs 7 and 10C). In addition, early IK was not significantly enhanced by these substances.
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Evidence for an Involvement of PKC in the Activation of IK by AA
Since fatty acids are able to activate PKC, we explored whether the effects of AA on IK in rat pulmonary myocytes were PKC dependent. In order to measure the enhancement of early IK, as well as the inhibition, we applied 120-ms voltage steps to 0 mV, since the AA-mediated increase in early IK was larger at 0 mV than at +60 mV (Fig 4A). Application of 10 µmol/L AA caused a slowly developing (with 1-minute delay) acceleration of activation of IK and the enhancement of its early amplitude (Fig 11A). The time to peak for IK at this test potential in the presence of 10 µmol/L AA was 10 ms, and the amplitude of the current (referred to as IK,10 below) was therefore measured at this time both in the presence and absence of AA.
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The enhancement of IK,10 measured after incubation in 10 µmol/L AA for 4 minutes was observed in each of seven rat pulmonary arterial cells studied and amounted to 33±7% (range, 9% to 62%; P<.02; Fig 12A). AA also caused a diminution of IK measured at the end of the 120-ms depolarization to 0 mV (current referred to as IK,120 below). IK,120 decreased by 56±5% (n=7, P<.02, Fig 12B).
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These experiments were conducted using our standard pipette solution, which contained 5 mmol/L ATP. However, when we omitted ATP from the pipette solution in an effort to reduce nonspecifically the activity of cellular kinases, application of 10 µmol/L AA for a similar time resulted in an increase in IK,10 in only two of six cells studied (9% and 16%). In the other four cells, there was only a progressive decrease in IK,10, which developed in parallel with the inhibition of IK,120 (Fig 11B). Therefore, the mean amplitude of IK,10 measured in the nominal absence of ATP fell significantly (by 14±11%, n=6, P<.003) (Fig 12A). However, no effect of ATP omission on the inhibition of IK,120 was observed (Fig 12B).
Evidence for the possible involvement of PKC in the enhancement of IK emerged from experiments in which pharmacological inhibitors were used, with 5 mmol/L ATP included in the pipette solution. Preincubation (2 minutes) of rat pulmonary arterial cells with either 10 µmol/L chelerythrine, a selective PKC inhibitor acting at the substrate binding site,26 or 100 nmol/L staurosporine, which blocks the ATP-binding site of PKC,27 prevented AA-mediated enhancement of early IK (Fig 12A; P<.002 and P<.01, respectively, compared with the absence of PKC inhibitor). In the presence of staurosporine, a decrease in IK,10 was observed in four of six cells. In two other cells, a 26% and 27% enhancement of IK,10 occurred. In the presence of chelerythrine, only a decrease in IK,10 was observed in each of the five cells studied. Neither PKC inhibitor itself had any substantial effect on IK (eg, compare control IK and the current in chelerythrine in Fig 11C). No significant effect of either drug on the inhibition of IK,120 by AA was observed (Fig 12B).
Fig 11D further illustrates that OAG (10 µmol/L), a membrane-permeable diacylglycerol used to activate PKC,28 markedly enhanced IK,10, as well as significantly inhibiting IK,120 (P<.02). The amplitudes of these responses in five cells are summarized in Fig 12. Fig 11D also illustrates that the current observed in the presence of OAG was completely blocked by 4-AP (5 mmol/L), a blocker of voltage-gated K+ channels. Similarly, IK elicited by a 300-ms step to +60 mV in the presence of AA (10 µmol/L) was abolished in two cells by 4-AP (not shown).
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Discussion |
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Arachidonate and other fatty acids have been shown to exert effects on a diverse array of channels gated by voltage and other factors in many types of cell. Channels/currents activated by micromolar concentrations of AA include a class of 50-pS K+ channels in toad gastric myocytes,29 two types of K+ current in rat neonatal atrial cells,30 Ca2+-activated K+ channels in rabbit pulmonary arterial myocytes,12 stretch-activated K+ channels in gastric smooth muscle cells,14 the M current in bullfrog sympathetic neurons,8 L-type and T-type Ca2+ currents in guinea pig ventricular myocytes,9 T-type Ca2+ currents in rat osteoblastic cells,31 and transient A-type K+ and voltage-dependent Ca2+ currents in GH3 pituitary cells.32 Conversely, fatty acids inhibited Na+ currents in squid giant axon,33 secretory Cl- channels in cultured airway smooth muscle cells,10 L-type Ca2+ channels in rabbit intestinal smooth muscle11 and rat osteoblastic31 cells, the A current in sympathetic neurons,8 and IK in GH3 cells.32
Although the effect of AA has therefore been found to vary between different channels, in most cases it seems to have a unidirectional effect, either enhancing or reducing channel activity or the whole-cell current amplitude. On the other hand, there have now been several reports that underscore the complexity of the effects of AA on IK. In the present case, AA accelerated the rate of IK activation and increased the amplitude of the current measured during the early part of the depolarizing step. At the same time, AA greatly accelerated the decay of IK, which under control conditions was relatively slow. This led to a pronounced reduction in the amplitude of IK measured later on during the depolarizing step. This type of dual effect of AA has been observed in cloned and expressed K+ channels of the Kv1.5,16 Kv1.1,17 and Kv1.218 classes. Vacher et al32 in 1989 also reported an analogous effect on the voltage-gated K+ currents in GH3 cells. The work of Honore et al16 in 1994 indicated that hyperpolarizing shifts in both the activation and inactivation of a single K+ channel, as well as its use-dependent block, were the basis of the effects of AA and its analogues. In contrast to these observations, we did not record any effect of AA on the voltage dependence of either the I-V or inactivation relationships of IK, a current that has the pharmacological and kinetic characteristics expected for a Kv1.5 channel (authors' unpublished data). Instead, our experiments suggest that the enhancement of early IK is likely to be PKC-mediated, whereas the subsequent suppression of late IK has some other basis.
It is well known that fatty acids can directly activate PKC.3 Moreover, it has been shown previously that the pseudosubstrate inhibitor for PKC, 19-31 peptide, inhibited the reduction of the muscarinic K+ current by AA in mouse neuroblastoma and rat glioma hybrid cells34 and the suppression of the N-type Ca2+ current by oleoylacetylglycerol in chick sensory neurons.35 We investigated the possible involvement of PKC using the following approaches. First, OAG, a selective activator of PKC,28 mimicked the effect of AA on the early IK. Second, ATP was removed from the pipette solution to minimize its concentration within the cell. Finally, two PKC inhibitors with different sites of action were used. Staurosporine is non–PKC selective and competes with ATP for its binding site on the enzyme,27 whereas chelerythrine selectively interacts with the catalytic domain of PKC.26 Both omission of ATP and pharmacological PKC inhibition significantly reduced the potentiation of early IK by AA, suggesting that activation of PKC was required for this effect. No effect of these maneuvers on the AA-mediated inhibition of late IK was observed. Honore et al16 also reported no effect of staurosporine or H-7 on the inhibitory effects of AA on the cloned and expressed Kv1.5. Conversely, the fact that OAG inhibited late IK might suggest a role for PKC in this response. It is noteworthy, however, that the OAG molecule contains an oleic acid moiety, which would be expected to inhibit late IK. It has also been shown by Bowlby and Levitan36 in 1995 that 1,2-dioctanoyl-sn-glycerol suppressed the Kv1.3 delayed rectifier via a PKC-independent pathway.
Although a dual effect of AA similar to that which we have observed has been reported for several types of cloned and presumably homogeneously expressed delayed rectifier K+ channels,16 17 18 an analogous effect on the voltage-gated K+ current in GH3 cells32 was interpreted as resulting from an increase in the A current coupled with a decrease in a delayed rectifier. In 1995, Yuan23 used differential block by 4-AP to detect the presence of "transient" and "steady state" components of IK in cultured rat pulmonary myocytes. However, a similar approach does not reveal the presence of such components in freshly isolated cells.24 Therefore, it is unclear at present whether the dual effect of AA on IK in these cells involves a complex effect on one class of K+ channels or differential effects on a heterogeneous channel population. The complete abolition by 4-AP of IK in the presence of OAG or AA indicates, however, that their enhancement of the early current is likely to be due to the opening of some type of voltage-gated K+ channel.
Structural Requirements for the Inhibitory Effect of AA on IK
In an attempt to define the possible mechanism(s) underlying the inhibitory effects of AA on IK in rat pulmonary arterial myocytes, the effect of fatty acids having a different structure of the "tail" and "head" groups of the molecule was investigated. We found that the inhibitory effect of fatty acids on IK correlated with several structural characteristics.
Although the length of the fatty acid chain, at least in the range we investigated, had no obvious effect on IK inhibition, the inhibition of IK was quite small with saturated fatty acids and increased in proportion to the number of double bonds in the fatty acid molecule. A similar pattern has also been reported for suppression of Na+ currents in squid axon,33 Cl- channels in epithelial cells,10 and suppression of Ca2+ currents in rabbit ileum.11 Conversely, both saturated and unsaturated fatty acids activated Ca2+ currents in guinea pig ventricular myocytes,9 and Ca2+-activated K+ channels in gastric myocytes.14 IK inhibition was also more prominent when fatty acid was in a cis, compared to a trans, configuration. Analogous observations have previously been made in studies of the action of fatty acids on a number of types of ion channels.10 11
It is well known that a variety of functions in many types of cells are altered by cis-unsaturated, but not trans-unsaturated and saturated, fatty acids.37 These effects have been ascribed to a perturbation of the bilayer lipid acyl chain order, which only the unsaturated fatty acids elicit.38 Although it was proposed on theoretical grounds that the lack of effect of saturated and trans-unsaturated fatty acids might reflect an inability to penetrate lipid bilayers,39 the coefficient for partition of saturated fatty acids into cell membranes has recently been measured to be much higher than that for unsaturated fatty acids.38 The correlation between double bond number and effect on IK might also reflect differences in the free concentrations of the various fatty acids in solution; the critical micellar concentration (the concentration above which the fatty acid forms micelles), which effectively sets the free fatty acid concentration, is likely to increase with the degree of unsaturation.40 However, it should be pointed out that the effect of AA on IK increased monotonically between 1 and 50 µmol/L, a range that includes the critical micellar concentration (measured as 6.5 µmol/L under similar conditions40 ). Since the free fatty acid concentration should remain constant as the total concentration is raised above the critical micellar concentration, these results suggest that micellar as well as free fatty acid can enter the membrane and affect IK. It is clear that resolution of this question will await further study.
Also, the presence of an uncharged (alcohol), rather than an acidic, head group greatly reduced the inhibition of IK. Other work has shown that neutral alcohol and methyl ester fatty acid analogues did not modulate channel function in cases where the fatty acids themselves did.9 10 14 17 Thus, the need for a charged head group may be a widespread property of the interaction of fatty acids and ion channels.
Finally, the external application of arachidonyl coenzyme A, which has a large head group and does not "flip" across the cell membrane,14 produced a markedly smaller inhibition of IK than did AA. This observation suggested that these substances may act from the cytosolic leaflet of the membrane bilayer, and it is consistent with previous observations made regarding the action of fatty acids on other types of channels.14 On the other hand, application of AA in the pipette solution appeared not to affect the kinetics of the current, implying an action from the extracellular side of the membrane. Honore et al16 found that although AA was effective in blocking the Kv1.5 channel when placed onto either the cytoplasmic or extracellular surfaces of appropriately isolated membrane patches, the whole-cell Kv1.5 current was not affected when AA was added to the pipette solution. They interpreted these observations as implying an action of AA from the extracellular side of the membrane. Alternatively, the lack of an action of AA added to the pipette solution might reflect the existence of intracellular barriers restricting its access to the membrane.
Fatty acids are known to stimulate a number of enzymes other than PKC, which might affect K+ channel function. These include NADPH oxidase,4 guanylate cyclase,5 p21ras,2 and mitogen-activated protein kinases.6 The application of 60 U/mL of superoxide dismutase did not change the effects of AA (10 µmol/L) on IK in three cells studied under our experimental conditions (data not shown), suggesting that production of free radicals via the NADPH-dependent pathway is unlikely to be involved in inhibitory effects of AA. Alternatively, fatty acids might directly interact with K+ channels, as proposed by Ordway et al7 in 1991. Honore et al16 in 1994 also proposed a direct interaction of fatty acids with delayed rectifier K+ channels. They found that the inhibitory effect of docosahexaenoic acid on the Kv1.5 delayed rectifier was use dependent. Bowlby and Levitan36 in 1995 similarly demonstrated that diacylglycerols caused a dramatic and use-dependent acceleration of the inactivation of Shaker IR, Kv1.3, and Kv1.6 K+ channels and suggested that open channel block was occurring. Although we have not examined the frequency dependence of the block of the delayed rectifier in pulmonary myocytes, it is notable that the accelerated decay of this current caused by fatty acids was less prominent when PKC was inhibited and when ETYA, which has been shown not to stimulate PKC,41 was used. These results suggest that preventing PKC activation unmasks a "pure" inhibitory effect of fatty acids that does not involve a very pronounced acceleration of current decay. The nature of any direct interaction between fatty acids and the delayed rectifier K+ channel in these cells therefore remains to be elucidated.
Does AA Modulate IK Via Its Metabolites?
Although AA and other fatty acids can themselves alter the activity of ionic channels, it is also possible that they might act via metabolites generated by any of three metabolic pathways. These include prostaglandins and thromboxanes (cyclooxygenase), 12- and 15-hydroperoxyeicosanoids and leukotrienes (12- and 15- or 5-lipoxygenases, respectively), and various hydroxyeicosanoids and epoxyeicosanoids (cytochrome P-450 epoxygenase).25 External application of AA as well as leukotrienes A4 and C4 activated muscarinic K+ channels in cardiac myocytes. This effect of AA was blocked by the lipoxygenase inhibitors NDGA and AA-861 but not by the cyclooxygenase inhibitor indomethacin.42 43 Eddlestone44 in 1995 also showed that AA (5 to 50 µmol/L) caused two effects on the activity of ATP-sensitive K+ channels in insulin-secreting HIT tumor cells. AA inhibited ATP-sensitive channels in excised patches (inside-out mode) and caused the activation of these channels in the cell-attached configuration. The latter effect was abolished by indomethacin.44 In our experiments, indomethacin did not prevent the effects of AA, and neither prostaglandin F2 nor the thromboxane analogue U44169 affected IK, suggesting that cyclooxygenase metabolites were not involved. Experiments designed to explore whether lipoxygenase activity was involved in the generation of the effects of AA were more difficult to interpret, since the lipoxygenase inhibitor NDGA itself produced a substantial inhibition of IK; however, NDGA did not prevent further inhibition of IK upon the application of AA. Ethoxyresorufin, a selective inhibitor of cytochrome P-450 epoxygenase, did not substantially affect IK, and preincubation of cells with this drug failed to eliminate the effects of AA. On the other hand, low concentrations of the cytochrome P-450 metabolite 11,12-EET did inhibit IK, although not to a greater extent than did similar concentrations of AA. Finally, ETYA, an AA analogue that is not metabolized, strongly inhibited IK. Taken together, these results suggest that AA itself has an inhibitory effect on IK that may also be manifested in products of its metabolic oxidation by cytochrome P-450. It is of interest that 11,12-EET and three other epoxyeicosatrienoic acids also increased the opening of Ca2+-activated K+ channels in vascular smooth muscle cells.45 The role of these metabolites in the regulation of vascular K+ channels clearly deserves additional study.
The effects of AA and other fatty acids on ion channels in smooth muscle, which have previously been described, include both the inhibition of Ca2+ currents11 and the opening of Ca2+- and stretch-activated K+ channels.12 13 14 These properties are consistent with a role for AA as a vasodilator and with its possible function as an endothelium-derived hyperpolarizing factor.15 On the other hand, an extensive literature suggests that the effects of exogenous AA on vascular tension development are mainly mediated by its metabolites.46 Prostaglandin I2 and thromboxane A2 seem to be particularly important in the pulmonary vasculature, such that pharmacological interference with their production reveals little residual vasoactive effect when AA is applied.47 48 49 Our observations that AA causes membrane depolarization and a modification of the properties of IK that are likely to cause its steady state suppression suggest that this and other similar fatty acids may also have electrophysiological effects that favor vasoconstriction. An indication that the concentrations of AA used in the present study may be physiologically relevant is that they fall into the range of Km values for membrane enzymes that utilize AA as a substrate. Thus, for example, cyclooxygenase and 12-lipoxygenase both have Km values for AA of 5 µmol/L,25 whereas cytochrome P-450s have Km values 10-fold higher.50 Also, it has recently been shown that the increase in high conductance Ca2+-activated K+ channel activity (BKCa) caused by membrane stretch may be secondary to the release of fatty acids within the plasmalemma and that the magnitude of this increase is similar to that evoked by 20 µmol/L myristic acid.13
Considering that AA exerts effects on a number of types of K+ conductance, it is possible that the net effect of AA release on the membrane potential may depend on the relative importance of these various K+ channels in controlling Em under specific conditions and in different blood vessels.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Special Trustees of St Thomas's Hospital, the British Heart Foundation (grant BS/95001), and the Wellcome Trust (grant 038048/2/93).
Received December 11, 1995; accepted April 9, 1996.
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