Arachidonic acid reversibly enhances N-type calcium current at an extracellular site

Curtis F. Barrett1,2, Liwang Liu2,3, and Ann R. Rittenhouse1,2,3

1 Program in Cellular and Molecular Physiology, 3 Program in Neuroscience, and 2 Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the effects of arachidonic acid (AA) on whole cell Ca2+ channel activity in rat superior cervical ganglion neurons. Our companion paper (Liu L, Barrett CF, and Rittenhouse AR. Am J Physiol Cell Physiol 280: C1293-C1305, 2001) demonstrates that AA induces several effects, including enhancement of current amplitude at negative voltages, and increased activation kinetics. This study examines the mechanisms underlying these effects. First, enhancement is rapidly reversible by bath application of BSA. Second, enhancement appears to occur extracellularly, since intracellular albumin was without effect on enhancement, and bath-applied arachidonoyl coenzyme A, an amphiphilic AA analog that cannot cross the cell membrane, mimicked enhancement. In addition, enhancement is voltage dependent, in that currents were enhanced to the greatest degree at -10 mV, whereas virtually no enhancement occurred positive of +30 mV. We also demonstrate that AA-induced increases in activation kinetics are correlated with enhancement of current amplitude. An observed increase in the voltage sensitivity may underlie these effects. Finally, the majority of enhancement is mediated through N-type current, thus providing the first demonstration that this current type can be enhanced by AA.

calcium channel; eicosatetraynoic acid; fatty acids; arachidonoyl coenzyme A; voltage dependence


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN RAT SUPERIOR CERVICAL GANGLION (SCG) neurons, the polyunsaturated fatty acid arachidonic acid (AA; 20:4, n-6) is a potent inhibitor of Ca2+ channel activity. When unitary currents were elicited in cell-attached patches, using Ba2+ as the charge carrier, applying 5 µM AA led to a significant decrease in the activity of both L- and N-type Ca2+ channels (13). In our companion paper (12), we examined the effects of exogenous AA on whole cell currents in SCG neurons. In doing so, we discovered that AA induces several effects in addition to inhibiting L- and N-type currents. Consistent with inhibition, AA increased holding potential-dependent inactivation, in agreement with our observation that AA increases the occurrence of null sweeps in cell-attached patch recordings of single L- and N- type Ca2+ channels (13). In addition, AA increased the rate of whole cell current activation. This effect was largely selective for N-type Ca2+ channels and was independent of G protein activity.

AA also induced a significant increase in current amplitude at negative voltages (12). This enhancement could be separated from inhibition, since dialyzing the cell with bovine serum albumin (BSA), a protein that can bind fatty acids (19), blocked the majority of inhibition but was without effect on enhancement. Finally, as with both inhibition and faster activation, enhancement is independent of G protein activity.

Although AA-induced enhancement of Ca2+ currents has been reported in other preparations (5, 8, 22), this effect has not been observed previously in neurons. Because Ca2+ entry through neuronal voltage-gated Ca2+ channels can play an important role in coordinating electrical signaling with cellular processes like neurotransmitter release and enzyme activation (3, 17), a pathway that induces enhanced Ca2+ influx in neurons is of particular interest. Therefore, the purpose of this study was to examine the mechanism and properties of AA-induced enhancement of Ca2+ currents in SCG neurons. In this report, we present the first evidence that AA reversibly enhances whole cell N-type current by a mechanism distinct from its inhibitory effects on L- and N-type currents. This effect is voltage dependent, with the greatest enhancement observed at -10 mV. In addition, enhancement does not require AA metabolism, and appears to be mediated extracellularly. Finally, our results indicate that the mechanism of AA-induced enhancement involves both an increase in activation rate and an increase in voltage sensitivity. Together with the accompanying paper (12), our findings add to the growing body of evidence that AA can exert a diverse range of effects on neuronal Ca2+ currents.


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INTRODUCTION
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Preparation and culture of SCG neurons. Following decapitation, the SCG were removed from 1- to 4-day-old Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), and neurons were dissociated by trituration through a 22-gauge, 1.5-in. needle. Following dissociation, cells were plated on poly-L-lysine-coated glass coverslips in 35-mm culture dishes and incubated at least 4 h before recording (7). Cells were maintained in 5% CO2 at 37°C, in DMEM supplemented with 7.5% fetal bovine serum, 7.5% calf serum, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 4 mM L-glutamine (all from Sigma Chemical, St. Louis, MO), and 0.2 µg/ml nerve growth factor (Bioproducts for Science, Indianapolis, IN); cells were used within 12 h of preparation. Spherical neurons lacking visible processes were selected for recording.

Preparation and culture of tsA201 cells. The simian virus 40 (SV40) large T antigen-transformed HEK-293 subclone tsA201, transfected to stably express the rat N-type Ca2+ channel alpha 1B-a splice variant (Delta A415/Delta SFMG/+ET; see Table 2 in Ref. 10) together with rat neuronal-derived beta 3 and alpha 2/delta -1 subunits, was a generous gift from Y. Lin and D. Lipscombe (Brown University; Ref. 9). Cells were maintained in 5% CO2 at 37°C in DMEM supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 1× antibiotic-antimycotic (100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B; GIBCO BRL, Rockville, MD), 25 µg/ml Zeocin (Invitrogen, Carlsbad, CA), and 5 µg/ml Blasticidin (Invitrogen). For recording, semiconfluent cells were trypsinized, plated on poly-L-lysine-coated glass coverslips, and cultured for at least 1 h before recording. Cells were used for recording within 6 h of preparation.

Electrophysiology. Ba2+ currents were recorded at room temperature (20-24°C) using the whole cell configuration of an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA). Except where noted, voltage steps of 100 ms duration were applied every 4 s from a holding potential of -90 mV. Currents were passed through the amplifier's four-pole low-pass Bessel filter set at 2 kHz, then digitized at 20 kHz with a 1401plus interface (Cambridge Electronic Design, Cambridge, UK). Data were collected using the Patch software suite, version 6.3 (Cambridge Electronic Design) and stored on a personal computer. Before analysis, capacitive and leak currents were subtracted using a scaled-up current elicited with a test pulse to -115 mV. Recording pipettes were pulled from borosilicate capillary tubes (Drummond Scientific, Broomall, PA; #2-000-210) and heat polished just before use; when filled with internal solution, pipette resistance ranged from 2.5 to 3 MOmega . Drugs were applied via gravity-driven bath perfusion, with an estimated time to complete bath exchange of 5-10 s. For SCG experiments using the irreversible N-type Ca2+ channel blocker omega -conotoxin GVIA (omega -CgTx), cells were placed in Tyrode solution (145 mM NaCl, 5.4 mM KCl, 10 mM HEPES, pH 7.5) containing 1 µM omega -CgTx for at least 10 min before recording. Recording solutions containing dihydropyridines were protected from light until use.

The control bath solution contained (in mM) 125 HEPES, 20 barium acetate, and 0.0005 tetrodotoxin (TTX), pH 7.5; for tsA201 cell recordings, TTX was excluded. The pipette solution contained (in mM) 122 cesium aspartate, 10 HEPES, 0.1 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 5 MgCl2, 4 ATP (disodium salt), and 0.1 mM guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S; trilithium salt), pH 7.5.

For experiments with BSA, 0.5 mg/ml BSA (essentially fatty acid free; Sigma Chemical) was directly dissolved into the recording solutions. When internal BSA was used, the pipette tip was first filled with BSA-free pipette solution and then backfilled with BSA solution.

Pharmacology. AA was obtained from Nu-Check-Prep (Elysian, MN). GDPbeta S was obtained from either Research Biochemicals (Natick, MA) or Sigma Chemical. omega -CgTx was from Bachem Bioscience (King of Prussia, PA), and (+)-202-791 was a gift from Sandoz Pharmaceuticals (Sandoz, Switzerland). FPL-64176 and nimodipine (NMN) were both obtained from Research Biochemicals. Except where noted, all other chemicals and reagents were obtained from Sigma Chemical.

Stock solutions of TTX, FPL-64176, omega -CgTx, arachidonoyl coenzyme A, and palmitoyl coenzyme A were prepared in double-distilled water and stored at -25°C. Stock solutions of (+)-202-791 and NMN were prepared in 100% ethanol and stored at -25°C. Stock solutions of AA were prepared in 100% ethanol and stored under nitrogen in glass vials at -95°C. Stock solutions of 5,8,11,14-eicosatetraynoic acid (ETYA) were prepared in DMSO and used within 1 day. Neither ethanol nor DMSO, at the maximal concentrations used in these experiments (0.05% and 0.1%, respectively), had a significant effect on whole cell currents (not shown).

Data analysis. Analysis software included the Patch software suite, version 6.3 (Cambridge Electronic Design), and Origin 5.0 (Microcal Software, Northampton, MA). Data are presented as means ± SE where applicable. Statistical significance was determined using a two-way paired or unpaired t-test, as appropriate. Sample size (n) represents the number of cells recorded under each condition. Percent inhibition and percent enhancement were calculated by the equations [1 - (Idrug/Icon)]100 and [(Idrug/Icon- 1]100, respectively, where Idrug is the current amplitude measured at a given time after drug application, and Icon is the current amplitude before drug application. The Boltzmann fits of the activation data presented in Fig. 5D were calculated using the equation I/Imax = {(I1 - I2)/[1 + <IT>e</IT><SUP>(<IT>V</IT> − <IT>V</IT><SUB>h</SUB>)/<IT>k</IT></SUP>] I2}, where I/Imax is normalized tail current amplitude, I1 and I2 are the minimum and maximum values of I/Imax, respectively, V is test potential in mV, Vh is the voltage at half-maximal I/Imax, and k is the slope factor of activation in mV/e-fold change in I/Imax.


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AA-induced enhancement and inhibition of whole cell Ba2+ currents follow distinctly different time courses. The first step in our analysis was to test whether current enhancement by AA is mediated at a site different from inhibition. If enhancement and inhibition are mediated at the same site, then these effects should develop along a similar time course. Therefore, in the same recording, we applied alternating test pulses to +10 and -10 mV, to monitor inhibition and enhancement, respectively (Fig. 1, A and B). Both inhibition and enhancement were then fit to single-exponential time constants (Fig. 1C). Bath application of 5 µM AA led to a rapid increase in current amplitude at both voltages (Fig. 1A); this increase was observed in six of six recordings. However, after the initial increase in amplitude, currents elicited at +10 mV then began to decrease (Fig. 1A), with a mean time constant of 4.30 ± 0.51 min (Fig. 1C). Thus, after 5 min, the overall effect of AA on currents elicited at +10 mV was an inhibition of 40.5 ± 9.7% (see Figs. 1D and 12A). In contrast, enhancement of currents elicited at -10 mV occurred with a mean time constant of 0.69 ± 0.10 min (Fig. 1C); after 5 min, AA enhanced current amplitude at -10 mV by 155.5 ± 28.1% (see Figs. 1D and 12B). These results demonstrate that enhancement develops significantly faster than inhibition, providing evidence that AA may have separate sites of action for these two effects.


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Fig. 1.   Arachidonic acid (AA)-induced enhancement and inhibition follow distinct time courses. A: currents were elicited by applying alternating 100-ms test pulses to +10 and -10 mV, and then peak inward current was plotted against time. AA (5 µM) was applied to the bath as indicated by the solid bar. The solid lines represent single-exponential fits of the data. B: sweeps from the experiment in A, obtained before (triangle ) and after (black-triangle) bath application of AA. C: summary of the time constants of inhibition and enhancement (see A). Bath application of 5 µM AA inhibited current amplitude, measured at +10 mV, with a time constant of 4.30 ± 0.51 min (n = 15). In contrast, AA increased current amplitude, measured at -10 mV, with a time constant of 0.69 ± 0.10 min (n = 5; **P < 0.001 compared with +10 mV). When cells were first dialyzed for >= 1 min with 0.5 mg/ml BSA, bath application of AA increased current amplitude with a time constant of 0.82 ± 0.13 min (n = 7, P > 0.1 compared with without BSA). D: current-voltage (I-V) plots were generated by applying 100-ms test pulses at 10-mV increments. Peak inward current was then plotted against voltage for control (CON; n = 7) and 5 min following bath application of 5 µM AA (n = 7). In this and subsequent figures, error bars not visible are contained within the symbols. *P < 0.05.

Our companion paper (12) shows that dialysis with BSA (a protein that can bind fatty acids; Ref. 19) blocked the majority of AA-induced inhibition but did not decrease the magnitude of AA-induced enhancement. To examine whether intracellular BSA affects the time course of AA-induced enhancement, we measured the time constant of enhancement after dialysis with 0.5 mg/ml BSA. Even in the presence of cytoplasmic BSA, AA enhanced currents elicited at -10 mV with an average time constant of 0.82 ± 0.13 min (Fig. 1C). This value was not significantly different from that observed in the absence of BSA (P > 0.1). Thus intracellular BSA has no effect on the rate of AA-induced enhancement.

Enhancement of whole cell current amplitude by AA is reversible. AA-induced inhibition was largely reversed by bath application of BSA (12, 13). In this study, we employed a similar approach to determine whether enhancement is also reversible. We first examined whether enhancement could be reversed by washing control solution (without BSA) into the recording chamber. Washing with control solution for 1 min reversed 24.6 ± 8.2% of AA-induced enhancement (n = 3). Although significant (P < 0.05), this reversal was far from complete. Therefore, 0.5 mg/ml BSA was added to the wash solution. A typical time course is shown in Fig. 2A. After 1 min in AA, the introduction of wash solution containing BSA rapidly reduced current amplitude to control levels. AA's effect on current amplitude at -10 mV, and its reversibility by BSA, are summarized in Fig. 2C. Washing out AA with BSA reversed 97.2 ± 6.1% of AA-induced enhancement (n = 6).


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Fig. 2.   External BSA reverses AA-induced enhancement of whole cell currents. A: currents were elicited at -10 mV and plotted against time. AA (5 µM) was applied to the bath as indicated by the solid bar, and wash solution containing 0.5 mg/ml BSA was applied to the bath as indicated by the open bar. B: traces from the experiment in A, taken at the times indicated. C: summary of the effect of AA at -10 mV and its reversibility by BSA (n = 6). In control solution, current amplitude was 35.3 ± 2.8 pA. One minute after application of AA, amplitude had increased to 106.4 ± 11.4 pA. After AA treatment, bath application of BSA for 1 min reduced current amplitude to 38.2 ± 3.1 pA (P > 0.5 vs. control). *P < 0.005 vs. control and vs. BSA.

AA-induced enhancement occurs extracellularly. The observation that intracellular BSA was without effect on AA-induced enhancement suggests that this effect may be mediated from the outside of the cell. To test this, we applied the fatty acid analog palmitoyl coenzyme A (PCoA), which contains a negatively charged moiety that prevents "flipping" across cell membranes (4), to the bath. This molecule has been shown previously to mimic the effects of AA in increasing the activity of potassium channels in smooth muscle cells (16). In SCG neurons, bath application of 10 µM PCoA led to a rapid, sustained enhancement of current amplitude (Fig. 3A). This enhancement was over a narrow range of test potentials, with significant enhancement observed at 0 mV (Fig. 3, B and C), providing evidence that fatty acid-induced enhancement is voltage dependent. In addition, unlike AA, PCoA caused no inhibition of current amplitude at any voltage tested, suggesting that inhibition is likely mediated from the cytoplasmic side, consistent with our intracellular BSA data.


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Fig. 3.   Palmitoyl coenzyme A (PCoA) and arachidonoyl coenzyme A (ACoA) mimic AA-induced enhancement of whole cell currents. A and D: peak inward current was elicited at +10 mV and plotted against time; 10 µM PCoA (A) or 10 µM ACoA (D) was applied to the bath as indicated by the solid bars. The extended breaks in the time courses represent time periods during which I-V data were collected. B and E: I-V plots were generated before (n = 4-5) and after (n = 4-5) bath application of 10 µM PCoA (B) or 10 µM ACoA (E). *P < 0.05. C and F: percent enhancement by PCoA (C) or ACoA (F) was calculated and plotted against voltage (n = 4-5).

Palmitate (the derivative of PCoA) is a 16-carbon fatty acid, whereas AA is a 20-carbon fatty acid. Thus we also tested arachidonoyl coenzyme A (ACoA), an analog of AA containing a CoA group, for its ability to enhance whole cell currents. As with PCoA, ACoA caused enhancement, yet no inhibition at any voltage (Fig. 3, D and E). ACoA-induced enhancement occurred over a similar range of voltages as with PCoA, with significance observed at both -10 and 0 mV (Fig. 3, E and F). Taken together, these findings provide additional support for our hypothesis that AA enhances currents by acting on the outside of SCG neurons, either within the outer leaflet of the cell membrane or at the extracellular surface.

Enhancement does not require AA metabolism. Modulation of ion currents by AA can occur either by direct interaction with the channels or indirectly by a downstream pathway, sometimes involving the generation of biologically active AA metabolites (for review, see Refs. 14 and 15). However, there are several arguments against an AA metabolite mediating enhancement of Ba2+ currents in SCG neurons. First, enhancement was observed even in the presence of intracellular BSA, a condition that should prevent AA metabolism. Moreover, its immediate onset, rapid development, and rapid reversibility suggest that enhancement might be mediated directly by AA. Finally, the fatty acid analogs PCoA and ACoA, which are unable to enter the cell, mimicked AA-induced enhancement of current amplitude. Therefore, it is highly unlikely that enhancement of current involves AA metabolism.

Despite these arguments, we could not, based on our experiments thus far, exclude the possibility that enhancement involves an AA metabolite. Therefore, to address this possibility, we examined the effect of ETYA, an AA analog that cannot be metabolized by the three common pathways (the lipoxygenase, cyclooxygenase, and epoxygenase pathways) known to generate biologically active products (20, 21). Bath application of 30 µM ETYA led to a rapid, sustained enhancement of whole cell current amplitude (Fig. 4, A and B). As with PCoA and ACoA, this enhancement was voltage dependent (Fig. 4, C and D). ETYA caused no significant inhibition at any voltage tested, consistent with the results in our companion paper (12). These findings support the hypothesis that AA-induced enhancement of current amplitude does not require metabolism. Moreover, because ETYA mimics enhancement, but not inhibition, these results provide further evidence that these two effects are likely mediated by distinct mechanisms.


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Fig. 4.   AA-induced enhancement does not require AA metabolism. A: peak inward current was elicited at +10 mV and plotted against time; 30 µM eicosatetraynoic acid (ETYA) was applied to the bath as indicated by the solid bar. The extended breaks in the time course represent the time periods during which I-V data were collected. B: sweeps from the experiment in A, taken before (triangle ) and after (black-triangle) application of ETYA. C: I-V plots were generated before and after bath application of 30 µM ETYA (n = 7). *P < 0.05. D: percent enhancement by ETYA was calculated and plotted against voltage (n = 7).

AA increases the voltage dependence of activation. In our companion paper (12), we examined the effect of AA on voltage-dependent activation. Consistent with AA's effects on current amplitude, application of AA for 5 min induced a slight increase in normalized tail current amplitude at negative voltages. However, possibly due to the influence of inhibition in the presence of AA, no significant change in voltage-dependent activation was noted. Therefore, we investigated the effect of AA on activation under recording conditions designed to maximize enhancement and minimize inhibition: 0.5 mg/ml BSA was included in the pipette solution, and the effect of 5 µM AA was examined at 1 min. We first measured the whole cell current-voltage (I-V) relationship under these recording conditions before and after application of AA. Currents were elicited by applying 200-ms voltage ramps from -60 to +80 mV. Figure 5A shows that application of AA increased current amplitude between -30 and +30 mV, but was without effect on the reversal potential.


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Fig. 5.   AA increases the voltage sensitivity of current activation. For these recordings, the pipette solution contained 0.5 mg/ml BSA. A: whole cell currents were elicited by applying a linear ramp pulse from -60 through +80 mV (slope = 0.75 mV/ms) in control solution and ~1 min after bath application of AA. Shown are mean ramp currents generated from 4 recordings; error bars were omitted for clarity. B: I-V curves were generated by applying 15-ms test pulses to incremental voltages at 1-s intervals. Current amplitude was measured 13.3 ms into the test pulse and plotted against voltage. open circle , Currents elicited in control solution (n = 5); , currents elicited 1 min after bath application of 5 µM AA (n = 4). *P < 0.05. C: percent of AA-induced enhancement in the recordings shown in B was calculated and plotted against voltage. D: for the recordings shown in B, tail current amplitude, induced by stepping the membrane potential back to -90 mV following the test pulse, was measured and then normalized to maximum tail current amplitude and plotted against test potential to generate activation-voltage curves. The solid lines represent Boltzmann fits to the data. In control solution, voltage at half-maximal normalized tail current (Vh) was 25.6 ± 9.4 mV and k was 12.2 ± 3.1 mV/e-fold change in activation; after AA application, Vh was -3.53 ± 1.44 mV (P < 0.05 vs. control) and k was 8.08 ± 1.79 mV/e-fold change (P > 0.1 vs. control).

Having confirmed that including BSA in the pipette solution was without effect on the I-V properties of whole cell currents, we next examined the effect of AA on the voltage dependence of activation. For these experiments, 15-ms test pulses were applied at incremental voltages to generate I-V and activation-voltage plots (Fig. 5, B-D). Consistent with previous results, application of AA induced a significant increase in current amplitude at negative voltages, but was without effect on currents elicited at positive voltages (Fig. 5B). Moreover, in agreement with the results obtained with PCoA, ACoA, and ETYA, maximum AA-induced enhancement occurred at -10 mV and decreased as more positive test pulses were applied (Fig. 5C), confirming that AA-induced enhancement has voltage-dependent properties.

To measure the effect of AA on activation, tail current amplitude from the recordings in Fig. 5B was measured at each voltage and normalized to maximum amplitude to generate activation-voltage plots (Fig. 5D). Application of AA had no significant effect on the slope of activation but induced a negative shift of ~30 mV in Vh (the voltage that elicits half-maximal activation). These results suggest that AA enhances currents, at least in part, by increasing the channels' sensitivity to voltage.

AA-induced enhancement is associated with faster activation. In our examination of AA's effects on whole cell currents elicited at +10 mV, we observed that, in addition to changing current amplitude, AA appeared to cause an increase in the rate of activation at this voltage (12). This increase in activation is not likely associated with inhibition, since, in cell-attached patch recordings, channels inhibited by AA exhibited increased first latency (13). Nevertheless, we explored whether this effect occurs in association with either inhibition or enhancement.

Whole cell activation was examined by normalizing inward current at each time point to the peak inward current obtained during a 100-ms voltage step to +10 mV. Normalized data from several experiments were then averaged for each condition (Fig. 6). Figure 6A shows that bath application of 5 µM AA for 1 min led to a significant increase in the rate of activation. Because AA-induced enhancement of current amplitude appears to reach steady-state within ~1 min, whereas inhibition takes several minutes to reach steady-state (see Fig. 1, A and C), we tested whether the increase in activation rate also reached steady state within 1 min. Activation was therefore examined after treatment for 5 min with AA for comparison (Fig. 6B). At 5 min, the rate of activation was not noticeably different from at 1 min, as the two curves superimposed (compare the AA curves in Fig. 6, A and B). Therefore, as with enhancement, AA-induced changes in activation reached steady state within 1 min, suggesting that increased activation kinetics may be associated with enhancement, rather than inhibition.


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Fig. 6.   The AA-induced increased rate of activation shares characteristics with AA-induced enhancement of current amplitude. Whole cell currents were elicited by applying a voltage step to +10 mV, and, for each experiment, average current traces were generated before (CON) and after drug application. For each average trace, peak inward current was determined, and currents were normalized to that value. The first ~8.5 ms of mean, normalized currents (±SE) were then plotted against time from the onset of the voltage step. A: currents were elicited before and 1 min after bath application of 5 µM AA (n = 5). B: the same recordings shown in A, before and 5 min after application of AA. C: cells were first dialyzed for >1 min with 0.5 mg/ml BSA, and then currents were elicited before and 1 min after bath application of 5 µM AA (n = 5). D: currents were elicited before and 1 min after bath application of 10 µM ACoA (n = 6).

We showed that intracellular BSA was sufficient to block the majority of AA-induced inhibition, but was without effect on enhancement. Therefore, to confirm that the AA-induced increase in activation is not a component of inhibition, we examined whether AA could induce a change in activation kinetics in the presence of intracellular BSA. When cells were dialyzed for at least 1 min with 0.5 mg/ml BSA, bath application of 5 µM AA for 1 min still led to a significant increase in activation (Fig. 6C), confirming that this effect is not associated with AA's inhibitory effects on whole cell currents.

We next looked for further evidence that the increase in activation kinetics is associated with enhancement of current amplitude. Because both ACoA and ETYA mimic enhancement of current amplitude, we examined whether these compounds could also induce faster activation kinetics. Bath application of 10 µM ACoA for 1 min induced faster activation (Fig. 6D). In separate experiments, a similar increase in activation rate was observed 1 min after application of 30 µM ETYA (not shown). Taken together, these results suggest that the increased activation rate is associated with AA-induced enhancement of current amplitude.

If faster activation kinetics is associated with enhancement, the changes in activation should be correlated to the increase in amplitude observed following application of AA. Therefore, we compared the change in activation with the change in amplitude on application of AA. In four cell recordings, we examined the first 10 sweeps (applied at 4-s intervals) following bath application of 5 µM AA. For amplitude, current in each sweep was normalized to the peak current obtained in the 10th sweep. Superimposed, mean normalized currents are shown in Fig. 7A. Activation was then calculated for the same 10 sweeps per experiment by normalizing each sweep to the peak inward current of that sweep, as in Fig. 6. (Fig. 7B). We then plotted the mean, normalized values obtained in Fig. 7A against the mean, normalized values obtained in Fig. 7B; this plot is shown in Fig. 7C. A linear regression analysis of these data shows a strong correlation in time between AA-induced enhancement of current amplitude and AA-induced increased kinetics of activation, indicating that these two effects are directly associated.


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Fig. 7.   AA-induced faster activation is correlated with AA-induced enhancement of current amplitude. A: whole cell currents were elicited for 10 consecutive sweeps after application of 5 µM AA; sweeps were applied at 4-s intervals. The current at each sweep was then normalized to the maximum current elicited by the 10th sweep. Each trace represents the average of 4 normalized cell recordings (error bars were omitted for clarity, and fast tail currents were clipped). Values at 8 ms (dashed line) were used for the x-coordinates in C as a measure of relative current amplitude. B: normalized currents were generated for the same sweeps in A, using the method in Fig. 6. Error bars were again omitted. Values at 3 ms (dashed line) were used for the y-coordinates in C as a measure of activation rate. C: normalized inward current from sweeps 1-10 in A was plotted along the abscissa, and normalized current from sweeps 1-10 in B was plotted along the ordinate. The solid line represents the linear regression of the data points, and the Pearson correlation coefficient (r) is given.

AA enhances N-type current. Our analysis of AA's effects revealed that the majority of increased activation kinetics is selective for N-type current, as it was still observed in the presence of the L-type Ca2+ channel antagonist NMN, but was largely lost when cells were pretreated with the N-type Ca2+ channel blocker omega -CgTx (12). Given that this effect appears to be associated with AA-induced enhancement, it seems likely that enhancement should be mediated, at least in part, by N-type current. Therefore, if AA enhances N-type current, then application of AA, in the continued presence of NMN, should still lead to enhancement. Bath application of 5 µM AA, in the presence of 1 µM NMN, led to an initial enhancement of current amplitude at both -10 and +10 mV (Fig. 8A; n = 7). As observed in the absence of NMN, currents elicited at +10 mV began to decrease after ~1 min, leading to a net result of significant inhibition after treatment for 5 min with AA (see Fig. 12A). Also, as seen in the absence of NMN, the enhancement observed at -10 mV was sustained throughout the recording (see Figs. 8 and 12B). AA-induced enhancement at -10 mV, in the presence of NMN, was 92.9 ± 22.7%, compared with 155.5 ± 28.1% in the absence of NMN (see Fig. 12B). This decreased level of enhancement observed in the presence of NMN raises the possibility that NMN-sensitive (e.g., L-type) current may also be enhanced by AA.


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Fig. 8.   The majority of AA-induced enhancement is nimodipine (NMN) insensitive. A: time course of whole cell Ba2+ currents elicited at +10 and -10 mV. NMN (1 µM) was present throughout the recording (open bar), and 5 µM AA was applied to the bath as indicated by the solid bar. The extended break in the time course represents the time period during which I-V data were collected. B: I-V curves were generated, in the continued presence of 1 µM NMN, before (n = 8) and 5 min after (n = 5) bath application of 5 µM AA. *P < 0.05.

If AA-induced enhancement is mediated by N-type current, then blocking this current should preclude at least part of the enhancement. Therefore, cells were incubated with omega -CgTX (1 µM) before whole cell recording. After omega -CgTX treatment, 5 µM AA induced a slight initial enhancement of current at both -10 and +10 mV (Fig. 9A). This enhancement was considerably less than that observed in the absence of omega -CgTX, suggesting that the majority of enhancement is mediated by N-type current. After 5 min in AA, significant inhibition was observed at both positive and negative voltages (see Figs. 9, A and B, and 12), suggesting that inhibition of L-type current is similar at +10 and -10 mV. Similar results were obtained when the dihydropyridine L-type channel agonist (+)-202-791 was included in the recording solutions to enhance L-type current (see Figs. 9, C and D, and 12). Taken together, these findings suggest that enhancement is mediated in large part by N-type current. Because treatment with omega -CgTX did not completely abolish AA-induced enhancement, these results also raise the possibility that AA also may cause an enhancement of non-N-type current. This finding was not completely surprising, as Huang et al. (8) demonstrated that fatty acids enhance nifedipine-sensitive (i.e., L-type) Ca2+ currents in guinea pig ventricular myocytes.


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Fig. 9.   N-type Ca2+ current mediates the majority of AA-induced enhancement. A-D: cells were pretreated with 1 µM omega -conotoxin GVIA (omega -CgTX) before recording. A: whole cell currents were elicited at +10 and -10 mV and plotted against time; 5 µM AA was applied to the bath as indicated by the solid bar. B: I-V curves were generated before (n = 11) and 5 min after (n = 9) bath application of 5 µM AA. C: whole cell currents were elicited at +10 and -10 mV and plotted against time; 1 µM (+)-202-791 [(+)-202] was present throughout the recording, and 5 µM AA was applied to the bath as indicated by the solid bar. The extended breaks in the time course represent the time period during which I-V data were collected. D: I-V curves were generated, in the continued presence of 1 µM (+)-202-791, before (n = 13) and 5 min after (n = 6) bath application of 5 µM AA. *P < 0.05.

To directly demonstrate that AA induces enhancement of N-type currents, we examined whole cell currents in tsA201 cells expressing rat Ca2+ channel alpha 1B-a, beta 3, and alpha 2/delta -1 subunits (9). The alpha 1B-a subunit is the principal splice variant in rat SCG neurons (10), allowing a comparison with our SCG results. Expression of N-type channels was confirmed by bath application of 1 µM omega -CgTX, which eliminated >95% of whole cell Ba2+ currents elicited at both -10 and +10 mV (Fig. 10, A and B). In separate experiments, the effect of AA on whole cell currents was then examined. In four of four cells, currents elicited at both voltages were initially enhanced following bath application of 5 µM AA (Fig. 10C). After this initial enhancement, currents elicited at +10 mV began to decrease in amplitude, resulting in significant inhibition by 5 min (see Figs. 10D and 12A). In contrast, currents elicited at -10 mV remained significantly enhanced throughout the recording (see Figs. 10, C and D, and 12B). These data are in agreement with those collected from SCG neurons and confirm that N-type current can be enhanced by bath application of AA.


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Fig. 10.   AA both enhances and inhibits whole cell recombinant N-type Ca2+ current. A: time course of whole cell Ba2+ currents in tsA201 cells expressing the rat Ca2+ channel alpha 1B-a, beta 3, and alpha 2/delta -1 subunits. Bath application of 1 µM omega -CgTX (solid bar) virtually eliminated inward current at +10 mV. B: I-V plots were generated before (n = 5) and 5 min after (n = 5) bath application of 1 µM omega -CgTX. C: whole cell currents were elicited at +10 and -10 mV and plotted against time; 5 µM AA was applied to the bath as indicated by the solid bar. D: I-V plots were generated before (n = 4) and 5 min after (n = 4) bath application of 5 µM AA. *P < 0.05. In A and C, the extended breaks in the time courses represent the time periods during which I-V data were collected.

L-type Ca2+ channel agonists do not preclude AA-induced enhancement. Our lab previously presented whole cell I-V plots generated before and several minutes after bath application of AA, in the continued presence of (+)-202-791 (see Ref. 13). Under those conditions, the dominant observed effect of AA was an inhibition of current amplitude; no AA-induced enhancement was observed at any voltage, suggesting that the presence of (+)-202-791 may have precluded AA-induced enhancement. Since the data in the present study indicate that the presence of L-type Ca2+ channel ligands should not affect AA-induced enhancement, we sought to resolve this discrepancy.

The I-V plots presented by Liu and Rittenhouse (13) represented data collected in control solution and again after at least 7 min of AA treatment. Thus, if enhancement did initially occur, but was then offset by inhibition, this would not be observed simply by comparing I-V plots. Therefore, in addition to I-V plots, we also examined time courses of current amplitude during application of AA in the continued presence of L-type Ca2+ channel agonists.

When whole cell currents were elicited in the continued presence of 1 µM (+)-202-791, bath application of 5 µM AA had an initial effect similar to that observed without an agonist: currents elicited at both +10 and -10 mV were initially enhanced by AA (Fig. 11A; n = 6). At +10 mV, inhibition became the dominant effect after several min (Figs. 11, A and B, and 12A). At -10 mV, current also decreased, but only to approximately control levels (Figs. 11, A and B, and 12B), thereby explaining why I-V plots generated before and several minutes after AA showed no significant enhancement or inhibition at negative voltages. Similar I-V results were obtained with the nondihydropyridine L-type channel agonist FPL-64176 (1 µM; Figs. 11C and 12). These findings suggest that, in recording conditions designed to enhance L-type current, inhibition can be observed at -10 mV, and this inhibition can eventually obscure enhancement.


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Fig. 11.   L-type Ca2+ channel agonists do not preclude AA-induced enhancement. A: time course of whole cell Ba2+ currents elicited at +10 and -10 mV. (+)-202-791 (1 µM) was present throughout the recording (open bar), and 5 µM AA was applied to the bath as indicated by the solid bar. The extended breaks in the time course represent the time periods over which I-V data were collected. B and C: I-V curves were generated before (open circle , n = 5-11) and 5 min after (, n = 4-5) bath application of 5 µM AA. Recordings were conducted in the continued presence of either 1 µM (+)-202-791 (B) or 1 µM FPL-64176 (C). *P < 0.05. D: summary of the effect of AA on currents elicited at -10 mV, in the absence (open bars) and presence (solid bars) of 1 µM (+)-202-791. Before AA application (-AA), current amplitude in control and (+)-202-791 was 34.4 ± 4.5 pA (n = 6) and 53.6 ± 5.0 pA (n = 6), respectively; 1 min after application of AA, current amplitude had increased to 104.1 ± 15.2 and 114.9 ± 16.2 pA, respectively. By 5 min, control current was 99.2 ± 14.1 pA, and current in the presence of (+)-202-791 was 78.6 ± 9.4 pA.



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Fig. 12.   Summary of the effects of AA on the amplitude of whole cell currents elicited at +10 mV (A) and -10 mV (B). Percent inhibition and percent enhancement were calculated by comparing current amplitude 5 min after bath application of AA to amplitude before application of AA; "---" indicates application of AA in the absence of any other drugs. For all experiments, AA concentration was 5 µM; NMN, omega -CgTX, (+)-202-791, and FPL-64176 were each used at 1 µM. *P < 0.05, compared with before AA treatment; dagger P < 0.01, compared with -. Sample size (n) is given in parentheses, and refers to both A and B.

We next tested whether the presence of L-type Ca2+ channel agonists affects AA-induced enhancement at -10 mV. Under control conditions (with no agonist present), application of AA for 1 min increased current amplitude by 69.7 ± 11.2 pA (n = 6; Fig. 11D). In the presence of 1 µM (+)-202-791, application of AA increased amplitude by 61.3 ± 13.6 pA (n = 6; P > 0.5, compared with control). Thus AA is able to induce similar levels of enhancement at -10 mV in the presence or absence of an L-type Ca2+ channel agonist. After 5 min of treatment with AA, control currents were still significantly enhanced, whereas currents recorded in the presence of (+)-202-791 had decreased, further supporting our hypothesis that inhibition can be observed under conditions designed to enhance L-type current.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the accompanying paper (12), we presented evidence that exogenous AA induces several effects on whole cell Ba2+ currents in neonatal rat SCG neurons, including inhibition of both L- and N-type currents and increased holding potential-dependent inactivation. AA also induces faster activation kinetics, largely selective for N-type current. Finally, we found that application of AA causes an enhancement of whole cell current amplitude. This enhancement appeared distinct from inhibition, as including BSA in the pipette solution reduced inhibition but did not affect enhancement. The purpose of this study was to investigate the mechanism and properties of this enhancement.

Our results show that AA reversibly enhances N-type current by inducing an increase in voltage-dependent activation, as well as an increase in activation kinetics. Blocking L-type current with NMN did not affect AA's capacity to enhance current amplitude, suggesting that the remaining current type is enhanced by AA. Moreover, when cells were pretreated with the irreversible N-type Ca2+ channel blocker omega -CgTX, most, but not all AA-induced enhancement was abolished, also raising the possibility that AA may enhance non-N-type current. This is consistent with our finding that AA induced a lower level of enhancement in the presence of NMN. Finally, when AA was applied to tsA201 cells expressing recombinant N-type channels, currents elicited at negative voltages increased in amplitude to the same degree as SCG neurons recorded under similar conditions.

Because AA-induced enhancement is mediated, for the most part, through N-type channels, then AA, in the continued presence of L-type channel agonists, should still cause an enhancement at negative voltages; i.e., the agonist's effects and AA-induced enhancement should sum. Our data demonstrate exactly that: AA caused the same increase in current amplitude at -10 mV whether the L-type channel agonist (+)-202-791 was present or not. In addition, the enhanced current elicited in the presence of (+)-202-791 decreased over time, possibly explaining why, in the presence of L-type Ca2+ channel agonists, no apparent change in current amplitude was observed at negative voltages after several minutes in AA (13).

Our findings suggest that AA has at least two distinct sites of action, one mediating inhibition and one mediating enhancement. Dialyzing the cytoplasm with BSA largely blocked AA-induced inhibition but was without effect on either the magnitude or rate of enhancement. In addition, bath-applied PCoA and ACoA, which cannot cross the cell membrane, each caused enhancement but not inhibition.

AA-induced enhancement of Ca2+ currents has been observed in GH3/B6 pituitary cells (22), rat osteoblasts (5), and guinea pig ventricular myocytes (8). In pituitary cells and osteoblasts, this enhancement was voltage dependent. When we examined enhancement in SCG neurons, we found that enhancement of Ba2+ currents by AA in SCG currents also is voltage dependent.

Under our recording conditions, AA-induced enhancement of whole cell currents was most readily observed at negative test potentials. In addition, at voltages more positive than 0 mV, where neuronal Ca2+ currents are often examined, inhibition became the dominant effect over time, thus occluding enhancement. Finally, our analysis suggests that AA appears to enhance N-type current to the greatest degree. Taken together, these findings may explain why AA-induced enhancement of currents has not been previously identified in other neuronal preparations.

In ventricular myocytes, L-type current appeared to reach peak more rapidly after AA treatment than control (8), consistent with AA-induced faster activation. In contrast, no change in activation kinetics was noted in association with AA-induced enhancement of low-voltage-activated Ca2+ currents in GH3/B6 pituitary cells (22) or rat osteoblasts (5), although this property was not directly examined. In neurons, changes in activation kinetics could have a profound influence on Ca2+ influx, which could in turn influence excitability. Therefore, it was important to determine whether the increased activation kinetics observed in SCG neurons is a component of AA-induced inhibition or enhancement. Our results indicate that increased activation is not associated with inhibition, consistent with cell-attached patch recordings of unitary Ca2+ channels, in which AA increased first latency (13). However, our findings do support the conclusion that increased activation is a component of AA-induced enhancement, as the development of both effects was strongly correlated.

Changes in surface charge can sometimes lead to changes in ion channel permeation and gating (6, 23). Therefore, fatty acid-induced enhancement might be the result of altered surface charge. This is unlikely because the coenzyme A head groups of PCoA and ACoA contain several negative charges, whereas ETYA and AA lack these charges. Although these compounds contain different charges, they induce a similar effect on whole cell currents. In addition, we would predict that changes in surface charge should occur very rapidly following perfusion of the recording chamber. In contrast, enhancement of current amplitude by AA occurred with a time constant of ~41 s, too slowly to be accounted for by a surface charge effect.

AA can be liberated from cell membrane phospholipids by several pathways (2). G protein-coupled receptors can activate phospholipases, which in turn can catalyze the release of AA (1). A number of neurotransmitters can activate receptor-mediated AA liberation (1), and SCG neurons are known to express receptors for these agonists, including muscarinic receptors (11, 18). Therefore, trans-synaptic stimulation of these neurons may lead to diverse modulation of Ca2+ currents by AA.

Together with our accompanying paper (12), we show that AA can induce several effects on Ca2+ currents in sympathetic neurons. Which effects may be induced may depend on the source of AA and the length of exposure to AA. Our results suggest that a brief external exposure to AA may be sufficient to enhance Ca2+ influx, even with very brief depolarizations, thereby potentiating neuronal excitability. In contrast, exposure to intracellular AA may lead to inactivation of Ca2+ channels, which would inhibit excitability.


    ACKNOWLEDGEMENTS

We thank Drs. Diane Lipscombe and Yingxin Lin for generously providing the tsA201 cell line expressing rat sympathetic N-type Ca2+ channels. We thank Drs. Alex Dopico, Thomas W. Honeyman, José Lemos, and Joshua J. Singer for helpful discussions and critical reading of this manuscript.


    FOOTNOTES

This study was supported by National Institutes of Health Grant NS-34195 and American Heart Association Grant 9940225. Its contents are the responsibility of the authors and do not necessarily reflect the official view of these granting agencies.

A. R. Rittenhouse is the recipient of an Established Investigator Award from the American Heart Association.

Address for reprint requests and other correspondence: A. R. Rittenhouse, Rm. S4-221, Dept. of Physiology, Univ. of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655 (E-mail: Ann.Rittenhouse{at}umassmed.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 August 2000; accepted in final form 13 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Axelrod, J, Burch RM, and Jelsema CL. Receptor-mediated activation of phospholipase A2 via GTP-binding proteins: arachidonic acid and its metabolites as second messengers. Trends Neurosci 11: 117-123, 1988[ISI][Medline].

2.   Bazan, NG. Arachidonic acid in the modulation of excitable membrane function and at the onset of brain damage. Ann NY Acad Sci 559: 1-16, 1989[ISI].

3.   Berridge, MJ. Neuronal calcium sgnaling. Neuron 21: 13-26, 1998[ISI][Medline].

4.   Boylan, JG, and Hamilton JA. Interactions of acyl-coenzyme A with phosphatidylcholine bilayers and serum albumin. Biochemistry 31: 557-567, 1992[ISI][Medline].

5.   Chesnoy-Marchais, D, and Fritsch J. Concentration-dependent modulations of potassium and calcium currents of rat osteoblastic cells by arachidonic acid. J Membr Biol 138: 159-170, 1994[ISI][Medline].

6.   Green, WN, and Anderson OS. Surface charges and ion channel function. Annu Rev Physiol 41: 341-359, 1991.

7.   Hawrot, E, and Patterson PH. Long-term culture of dissociated sympathetic neurons. Methods Enzymol 58: 574-584, 1979[Medline].

8.   Huang, JM, Xian H, and Bacaner M. Long-chain fatty acids activate calcium channels in ventricular myoctyes. Proc Natl Acad Sci USA 89: 6452-6456, 1992[Abstract].

9.   Lin, Y, and Lipscombe D. Stable expression of N-type Ca channel splice variants in tsA201 cells. Soc Neurosci Abstracts 26: 829, 2000.

10.   Lin, Z, Haus S, Edgerton J, and Lipscombe D. Identification of functionally distinct isoforms of the N-type Ca2+ channel in rat sympathetic ganglia and brain. Neuron 18: 153-166, 1997[ISI][Medline].

11.   Lindl, T, Teufel E, and Cramer H. 3':5'-Nucleotide phosphodiesterase in the superior cervical ganglion of the rat. Evidence for multiple forms and influence of the beta -adrenergic receptor system in the electrophoretic pattern of the enzyme. Hoppe Seylers Z Physiol Chem 357: 983-989, 1976[ISI][Medline].

12.   Liu, L, Barrett CF, and Rittenhouse AR. Arachidonic acid both inhibits and enhances whole cell calcium currents in rat sympathetic neurons. Am J Physiol Cell Physiol 280: C1293-C1305, 2001[Abstract/Free Full Text].

13.   Liu, L, and Rittenhouse AR. Effects of arachidonic acid on unitary calcium currents in rat sympathetic neurons. J Physiol (Lond) 525: 391-404, 2000[Abstract/Free Full Text].

14.   Meves, H. Modulation of ion channels by arachidonic acid. Prog Neurobiol 43: 175-186, 1994[ISI][Medline].

15.   Ordway, RW, Singer JJ, and Walsh JV. Direct regulation of ion channels by fatty acids. Trends Neurosci 14: 96-100, 1991[ISI][Medline].

16.   Petrou, S, Ordway RW, Hamilton JA, Walsh JV, and Singer JJ. Structural requirements for charged lipid molecules to directly increase or supress K+ channel activity in smooth muscle cells: effects of fatty acids, lysophosphatidate, acyl coenzyme A and sphingosine. J Gen Physiol 103: 471-486, 1994[Abstract].

17.   Rittenhouse, AR, and Zigmond RE. Role of N- and L-type calcium channels in depolarization-induced activation of tyrosine hydroxylase and release of norepinephrine by sympathetic cell bodies and nerve terminals. J Neurobiol 40: 137-148, 1999[ISI][Medline].

18.   Skok, MV, Voitenko LP, Voitenko SV, Lykhmus EY, Kalashnik EN, Litvin TI, Tzartos SJ, and Skok VI. Alpha subunit composition of nicotinic acetylcholine receptors in the rat autonomic ganglia neurons as determined with subunit-specific anti-alpha(181-192) peptide antibodies. Neuroscience 93: 1427-1436, 1999[ISI][Medline].

19.   Spector, AA. Fatty acid binding to plasma albumin. J Lipid Res 16: 165-179, 1975[Abstract].

20.   Taylor, AS, Morrison AR, and Russell JH. Incorporation of 5,8,11,14-eicosatetraynoic acid (ETYA) into cell lipids: competition with arachidonic acid for esterification. Prostaglandins 29: 449-458, 1985[Medline].

21.   Tobias, LD, and Hamilton JG. The effect of 5,8,11,14-eicosatetraynoic acid on metabolism. Lipids 14: 181-193, 1979[ISI][Medline].

22.   Vacher, P, McKenzie J, and Duffy B. Arachidonic acid affects membrane ionic conductances of GH3 pituitary cells. J Physiol (Lond) 257: E203-E211, 1989.

23.   Zhou, W, and Jones SW. Surface charge and calcium channel saturation in bullfrog sympathetic neurons. J Gen Physiol 105: 441-462, 1995[Abstract].


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