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Am J Physiol Cell Physiol 280: C1293-C1305, 2001;
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Vol. 280, Issue 5, C1293-C1305, May 2001

Arachidonic acid both inhibits and enhances whole cell calcium currents in rat sympathetic neurons

Liwang Liu1,2, Curtis F. Barrett2,3, and Ann R. Rittenhouse1,2,3

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We recently reported that arachidonic acid (AA) inhibits L- and N-type Ca2+ currents at positive test potentials in the presence of the dihydropyridine L-type Ca2+ channel agonist (+)-202-791 in dissociated neonatal rat superior cervical ganglion neurons [Liu L and Rittenhouse AR. J Physiol (Lond) 525: 291-404, 2000]. In this first of two companion papers, we characterized the mechanism of inhibition by AA at the whole cell level. In the presence of either omega -conotoxin GVIA or nimodipine, AA decreased current amplitude, confirming that L- and N-type currents, respectively, were inhibited. AA-induced inhibition was concentration dependent and reversible with an albumin-containing wash solution, but appears independent of AA metabolism and G protein activity. In characterizing inhibition, an AA-induced enhancement of current amplitude was revealed that occurred primarily at negative test potentials. Cell dialysis with albumin minimized inhibition but had little effect on enhancement, suggesting that AA has distinct sites of action. We examined AA's actions on current kinetics and found that AA increased holding potential-dependent inactivation. AA also enhanced the rate of N-type current activation. These findings indicate that AA causes multiple changes in sympathetic Ca2+ currents.

calcium channel; 5,8,1,14-eicosatetraynoic acid; FPL-64176; fatty acid; oleic acid


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ARACHIDONIC ACID (AA; C20:4, n-6), a cis-polyunsaturated fatty acid, appears to serve as an intracellular messenger in a variety of receptor-mediated signal transduction cascades (3, 43). After the stimulation of G protein-coupled receptors, activated phospholipases liberate AA from phospholipids in the plasma membrane (35). A major effect of increased free AA is the modulation of ion channel activity including voltage- and ligand-gated channels and intracellular Ca2+ release channels (24, 30, 46, 48). The coordinated modulation of these channels by AA can result in changes in membrane excitability (12). AA appears to exert its actions by either direct binding to channel proteins or indirectly via molecules downstream of AA, including AA metabolites, free radicals, AA-sensitive phosphatases, and/or protein kinases (24, 30, 46, 48).

We are interested in understanding how AA modulates voltage-gated Ca2+ currents in neurons, since Ca2+ entry plays important roles in coordinating electrical activity with many cellular processes, such as neurotransmitter release, enzyme activation, and gene expression (9). Few studies have examined the effects of AA on Ca2+ currents in neurons; however, in each case, AA inhibited high threshold-activated whole cell Ca2+ currents (16, 25, 26, 44). We previously examined the effects of AA on Ca2+ currents in neonatal rat superior cervical ganglion (SCG) neurons (29). In these neurons, the majority of the whole cell Ba2+ current is N-type current. The remaining current is mostly L-type current; a small residual current appears to be non-L- or N-type (36, 39). At the whole cell level, we found that AA decreases both L- and N-type Ca2+ currents (29). From cell-attached patch recordings, we found that AA has no effect on unitary current amplitude, but inhibits the activity of both L- and N-type channels. Decreased activity is due in part to an increase in the incidence of null sweeps, suggesting that AA promotes inactivation (29). While inhibition of Ca2+ currents appears to predominate in neurons, enhancement as well as inhibition of Ca2+ currents by AA has been reported in non-neuronal cells (11, 19, 41, 52). These observed differences in action raise the possibility that AA modulates Ca2+ currents by more than one mechanism; however, whether both of these processes are active in neurons is unknown.

In this study, we investigated the mechanism by which AA inhibits whole cell Ca2+ currents in neonatal rat SCG neurons using Ba2+ as the charge carrier. We report here that AA-induced inhibition is reversible and concentration dependent. Furthermore, in the presence of the N-type Ca2+ channel blocker omega -conotoxin GVIA (omega -CgTx) or in the presence of the L-type Ca2+ channel antagonist nimodipine (NMN), AA decreased the whole cell current, confirming that AA inhibits both L- and N-type currents. In characterizing the inhibitory actions of AA, we found that, while AA inhibited currents at positive test potentials, AA enhanced currents at negative potentials. Cell dialysis with bovine serum albumin (BSA) minimized the inhibitory actions of AA, while enhancement remained, indicating that AA may have more than one site of action in SCG neurons. We also examined whether AA modulated whole cell current kinetics. We found that AA increased holding potential-dependent inactivation and selectively increased the activation kinetics of N-type current. The accompanying paper describes an association of enhancement with the increase in activation of N-type current by AA acting either at the extracellular surface or within the outer leaflet of the cell membrane (6).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SCG neuron preparation. SCG were removed from 1- to 4-day old Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) following decapitation. Neurons were mechanically dissociated by trituration (18) and plated on poly-L-lysine (Sigma, St. Louis, MO)-coated glass coverslips and incubated at 37°C in a 5% CO2 environment. Cells were maintained in DMEM supplemented with 7.5% calf serum, 7.5% fetal bovine serum, 4 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Sigma), and 0.2 µg/ml nerve growth factor (Bioproducts for Science, Indianapolis, IN). Cells were used within 12 h to avoid recording from neurons with processes.

Whole cell current recording conditions. Whole cell Ba2+ currents were measured by the method of Hamill et al. (15) with an Axopatch 200A or 200B (Axon Instruments, Foster City, CA) or Dagan 3900 (Dagan, Minneapolis, MN) patch-clamp amplifier at room temperature (20-24°C). Pipette capacitance was zeroed on sealing. Whole cell capacitive transients were compensated by ~70% in most experiments. Currents were low-pass filtered at 2 or 5 kHz using the four-pole Bessel filter in the clamp amplifier and sampled at 20 kHz except where noted. Current traces were stored and later analyzed on a personal computer using CED Patch 6.3 acquisition and analysis programs (Cambridge Electronic Design, Cambridge, UK) or a PDP-11 computer using custom-written software. Electrodes were made from borosilicate glass capillaries (Drummond Scientific, Broomall, PA) and heat-polished to a tip diameter of ~1 µm. When filled with internal solution, the pipette resistance ranged from 2.0 to 3.0 MOmega . During the recording, changes in the bath solution were made by gravity-driven perfusion.

Solutions and drugs. The external solution was composed of (in mM) of 20 barium acetate, 125 N-methyl-D-glucamine (NMG)-aspartate, 10 HEPES, and 0.0005 tetrodotoxin (TTX; 293 mosmol/l; Research Biochemicals, Natick, MA or Sigma). The pipette solution was composed of (in mM) 123 cesium aspartate, 10 HEPES, 0.1 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 5 MgCl2, and 4 ATP (264 mosmol/l; Sigma). For experiments measuring the effects of AA in the presence of EGTA (Aldrich, Milwaukee, WI), the pipette solution was composed of (in mM) 123 cesium aspartate, 10 EGTA, 10 HEPES, 5 MgCl2, and 4 ATP (296 mosmol/l); the NMG aspartate in the external solution was raised to 135 mM unless indicated otherwise. In some experiments, 0.4 mM GTP (Sigma) or 0.1 mM guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S; Research Biochemicals or Sigma) was also included in the pipette solution. The pH of all solutions was adjusted to 7.5 with CsOH.

NMN (Miles, New Haven, CT or Research Biochemicals), (+)-202-791, (a gift from Sandoz, Switzerland), FPL-64176 (Research Biochemicals), 5,8,11,14-eicosatetraenoic acid (AA), 5,8,11,14-ecoisatetraynoic acid (ETYA), myristic acid, and oleic acid (all from Nu-Check-Prep, Elysian, MN), indomethacin, and 5,8,11-eicosatriynoic acid (ETI; Biomol, Plymouth Meeting, PA) were prepared from stock solutions made up in 100% ethanol and diluted with the bath solution to a final ethanol concentration of <0.17%. This concentration of ethanol had no significant effect of its own on whole cell currents (data not shown). Stock solutions of all fatty acids were kept under nitrogen in sealed glass vials at -90°C. Hydrophobic compounds were considered in solution if solutions were transparent, and all solutions used were clear. Stock solutions of omega -CgTx (List Biological Laboratories, Campbell, CA) and TTX, made up in water, were diluted with bath solution at least 1,000-fold. 1-Aminobenzotriazole (Biomol) and BSA (essentially fatty acid free; Sigma) were added directly to the bath or pipette solution.

Data analysis. Before analysis, leak and residual capacitive transients were minimized by subtracting from each trace a scaled up current elicited with a hyperpolarizing test pulse. In some figures, residual transients that remained after leak subtraction were digitally removed. Whole cell current amplitudes, defined as the peak current, were measured 15 ms after the start of the test pulse. For experiments where long-lasting tail currents were elicited in the presence of the L-type Ca2+ channel agonist FPL-64176, tail currents were measured ~13 ms after the membrane was stepped from +10 mV to a tail potential of -40 mV. Data analysis began 1-2 min following breakthrough to ensure complete dialysis of the cell with the nucleotides contained in the pipette solution, a time delay shown to be sufficient to maximally affect G protein activity in these cells (5).

Summarized data are expressed as means ± SE. Sample size (n) indicates the number of cells. Statistical significance was determined by either a two-way unpaired or paired t-test. The activation data in Fig. 7 were fitted using the Boltzmann equation: Y = {(I1 - I2)/[1 + <IT>e</IT><SUP>(<IT>V</IT> − <IT>V</IT><SUB>h</SUB>)/<IT>k</IT></SUP>] + I2}, where Y is either Itail (tail current amplitude) or I/Imax (normalized tail current amplitude), I1 and I2 are the minimum and maximum values of Y, respectively, V is test potential in mV, Vh is the voltage at half-maximal Y, and k is the slope factor of activation in mV/e-fold change in Y.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AA inhibits whole cell L- and N-type currents in SCG neurons. To characterize the inhibitory effects of AA on whole cell L- and N-type currents in SCG neurons, a voltage protocol originally developed by Plummer et al. (36, 37) was used to isolate L-type from N-type currents. Membrane voltage was held at -90 mV, stepped to +10 mV for 20 ms, and then stepped back to an intermediate potential of -40 mV. Under control conditions, only 4.3 ± 0.7 pA of current was present 13 ms following the step from +10 mV to the tail potential (n = 12). When the nondihydropyridine L-type Ca2+ channel agonist FPL-64176 (1 µM) was present in the bath, a long-lasting component of the tail current made up entirely of L-type current was elicited (Fig. 1A) and averaged 225.7 ± 42.9 pA (n = 12). This component of current was monitored as a measure of L-type current. The peak current was monitored as a measure of N-type current since the majority of it is inhibited by omega -CgTx (36). Furthermore, in contrast to the tail current, FPL-64176 increased the peak current only modestly (Fig. 1A): on average 25.8 ± 5.9% (n = 12).


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  		Fig. 1.   Inhibition of whole cell Ba2+ currents by arachidonic acid (AA) is concentration dependent and reversible. A: an example of individual sweeps illustrates the inhibition of the peak and the long-lasting tail current by 5 µM AA in the presence of 1 µM FPL-64176 (FPL), a nondihydropyridine L-type Ca2+ channel agonist. Dashed lines indicate where the peak and the long-lasting tail current amplitudes were measured in this and subsequent figures when agonist was used. In this example, peak current increased from 170 to 206 pA and the long-lasting tail current increased from 2 to 145 pA with FPL. B: summary of the percent of current remaining following bath application of different concentrations of AA for at least 5 min compared with current amplitude in the presence of FPL alone (%FPL). Open bars, peak current; hatched bars, long-lasting tail current (n = 3-5/group). C: an example of reversible inhibition of the peak and long-lasting tail currents by 10 µM AA. Bars indicate the times of drug application; 1.0 mg/ml BSA (essentially fatty acid free) was washed into the bath as indicated. For these recordings, the pipette solution contained 0.4 mM GTP.

We observed a concentration-dependent effect of AA on whole cell currents when measured ~7 min after bath application of AA. In the presence of 1 µM FPL-64176, application of 1 µM AA to the bath had little inhibition of either the peak (5 ± 6%) or the long-lasting tail current (18 ± 3%), while 5 µM AA significantly (P < 0.05) inhibited both the peak (51 ± 3%) and the long-lasting tail current (42 ± 6%; Fig. 1, A and B). Exposure to 10 µM AA inhibited the peak (58 ± 15%) and the long-lasting tail current (57 ± 7%) to an equal extent (Fig. 1, B and C); however, inhibition was not significantly greater than with 5 µM AA. Application of 100 µM AA to the bath inhibited both the peak and the long-lasting tail currents by 76 ± 15% and 71 ± 7%, respectively (Fig. 1B). The magnitude and time to 50% inhibition (data not shown) of the peak and the long-lasting tail current with 100 µM AA were not significantly different (P > 0.2) than with 10 µM AA. These results demonstrate that the inhibition of whole cell current by AA, measured at +10 mV, is concentration dependent.

We next examined the reversibility of the actions of AA under these conditions. Whole cell current inhibition could be only partially reversed when AA was washed from the bath (data not shown). When 1.0 mg/ml of BSA (essentially fatty acid free), which rapidly binds fatty acids (47), was included in the wash solution, the majority of the inhibition by AA could be reversed (n = 3). An example of the time course of reversibility is shown in Fig. 1C. Bath application of BSA alone had no effect of its own on whole cell currents (data not shown). The reversibility of AA's effects, as observed previously in the presence of (+)-202-791 (29), indicates that AA is not causing some irreversible disruption of channel activity.

When either the dihydropyridine L-type Ca2+ channel agonist (+)-202-791 (29) or FPL-64176 (Fig. 1) was included in the bath solution to isolate L-type current, AA inhibited the slow component of the tail current. However, it is possible that under these conditions the inhibition of whole cell currents by AA was simply due to the displacement of agonist. This is unlikely since AA caused no change in mean open time of unitary L- and N-type channel activity in the presence of (+)-202-791 (29), suggesting that the inhibitory actions of AA are independent of (+)-202-791. Furthermore, FPL-64176 and another dihydropyridine agonist, BAY K 8664, appear to bind to distinct sites on L-type Ca2+ channels (38), making it unlikely that AA's only action is to displace these agonists. Nevertheless, to rule out this possibility, L-type current was isolated from N-type current and tested for its sensitivity to AA in the absence of agonist. Cells were preincubated 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 to block N-type current selectively and irreversibly. The whole cell recording configuration was then established, and 5 µM AA was applied to the bath. An example of current inhibition by AA under these conditions is shown in Fig. 2, A and B. AA (5 µM) inhibited the remaining peak current by 51 ± 6% (Fig. 2C), indicating that AA-induced inhibition of L-type current is independent of agonists.


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  		Fig. 2.   AA inhibits whole cell L-type currents. Cells were preincubated in Tyrode solution containing 1 µM omega -conotoxin GVIA (omega -CgTx) for at least 10 min before recording to inhibit N-type Ca2+ channel activity. A: time course of the inhibition by 5 µM AA of omega -CgTx-insensitive currents. Bars indicate time of drug application. B: individual sweeps taken from the times indicated in A. C: summary of the effect of AA on peak current amplitude (n = 5). Mean current amplitude following omega -CgTx treatment was 91 ± 11 pA. Bath application of AA reduced current amplitude to 51 ± 9 pA. For these recordings, the pipette solution contained 10 mM EGTA and 0.1 mM guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S) and the bath solution contained 125 mM N-methyl-D-glucamine aspartate. *P < 0.005, compared with omega -CgTx. Ipeak, peak current.

The large decrease in the amplitude of the peak current in Fig. 1C indicates that N-type current is inhibited by AA. To verify this finding under conditions that isolate N-type current from L-type, recordings were performed in the presence of the selective L-type Ca2+ channel antagonist NMN. Cells were held at -50 mV to enhance NMN binding to the channels; however, at this potential, a greater percentage of N-type Ca2+ channels inactivate than at -90 mV (36). Thus the contribution of L- and N-type current to the whole cell current is altered; peak current amplitude tended to be smaller than when holding at a more negative potential, and the amount of current sensitive to NMN appeared larger than the 10-15% reported previously for SCG neurons (36). In the presence of 1 µM NMN, 5 µM AA significantly inhibited the peak current by 74 ± 3% (Fig. 3C), confirming our earlier findings that AA inhibits N-type current (29). Taken together, these results are consistent with our previous whole cell and single channel data (29) and confirm that AA inhibits both L- and N-type currents.


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  		Fig. 3.   AA inhibits whole cell N-type currents. A: the time-course of the AA-induced inhibition of the peak current remaining after bath application of nimodipine (NMN), a selective dihydropyridine antagonist of L-type Ca2+ currents. Bars indicate the times of drug application. B: individual sweeps were taken from A where indicated. C: summary of the effect of AA on the peak current at +20 mV. Mean current amplitude following NMN treatment was 125 ± 18 pA. Bath application of AA reduced current amplitude to 32 ± 5 pA (*P < 0.01 compared with NMN levels; n = 5). For these recordings, the pipette solution contained 10 mM EGTA and 0.4 mM GTP. Currents were sampled at 10 kHz.

Other fatty acids have been shown to mimic AA's effects on ion channels. Therefore, we examined whether oleic acid (C18:1, n-9), another unsaturated fatty acid, or myristic acid (C14:0), a fatty acid with the same effective carbon length, can mimic the inhibitory actions of AA. FPL-64176 (1 µM) was present throughout these recordings to monitor the peak and the long-lasting tail current. Both oleic acid and myristic acid were unable to significantly decrease either the peak or the long-lasting tail current after at least 7 min in the bath (Fig. 4). In addition, ETYA, a polyunsaturated fatty acid AA analog, which can mimic the direct actions of AA on some ion channels (2, 10, 50, 53), was tested for its ability to inhibit whole cell currents. ETYA (30 and 100 µM) also had no significant effect on either the peak or the long-lasting tail current amplitude when applied to the bath for at least 2 min (Fig. 4). In a separate set of experiments, ETYA (30 µM) was applied to the bath for up to 4 min in the absence of FPL-64176, and again no significant inhibition occurred (data not shown).


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  		Fig. 4.   Summary of the effects of other fatty acids on the peak and long-lasting tail currents. In the continued presence of 1 µM FPL, the peak (open bars) and the long-lasting tail (hatched bars) current amplitudes were measured 7 min after oleic acid (OA) or myristic acid (MA) or 2 min after 5,8,11,14-eicosatetraynoic acid (ETYA) was applied to the bath (n = 4-11/group). Data are expressed as the percent of current remaining following bath application of different fatty acids compared with current amplitude in the presence of FPL alone (%FPL); 0.4 mM GTP was present in the pipette solution.

Current inhibition appears independent of AA metabolism. AA can be metabolized by several pathways to generate biologically active products, some of which have been shown to modulate ion channel activity (24, 30, 35). To examine the possible involvement of a metabolite in current inhibition, selective inhibitors were used to block the three common pathways of AA metabolism (24). The cyclooxygenase pathway was inhibited by indomethacin (4), the lipoxygenase pathway was inhibited by ETI (4), and the cytochrome P-450 oxygenase or "epoxygenase" pathway was inhibited by the suicide substrate 1-aminobanzotriazole (1-ABT) (16, 17). Each inhibitor was used at a concentration shown previously to block a particular metabolic pathway (see Fig. 5). FPL-64176 was included in the bath solution so that both the peak and long-lasting tail currents could be monitored simultaneously. We first examined whether bath application of any of the inhibitors for at least 2 min altered whole cell currents, and found no effect on either the peak or the long-lasting tail current (data not shown).


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  		Fig. 5.   Inhibition of the peak and the long-lasting tail currents by 10 µM AA appears independent of AA metabolism. The cyclooxygenase pathway was inhibited by preincubation for >= 20 min with 10 µM indomethacin (Indo; n = 6). The cytochrome P-450 oxygenase pathway was inhibited by preincubation for >= 60 min with 3 mM of the suicide substrate 1-aminobanzotriazole (1-ABT; n = 5). The lipoxygenase pathway was inhibited by preincubation for >= 30 min with 5 µM 5,8,11-eicosatriynoic acid (ETI; n = 6). FPL (1 µM) was included in the bath solution. To block all 3 pathways simultaneously, cells were preincubated with all 3 inhibitors (Indo, 1-ABT, and ETI) for at least 60 min (n = 4). Cells were preincubated with drugs in Tyrode solution. 100 µM ETYA was added to the bath 2 min before AA (n = 6). Data are expressed as the percent of current remaining following bath application of AA compared with current amplitude in the presence of FPL and inhibitor(s) (%FPL). In every case, application of AA to the bath in the continued presence of inhibitor(s) failed to block significantly (P > 0.05, compared with the AA group) the decrease in either the peak (open bar) or the long-lasting tail (hatched bar) current; 0.4 mM GTP was present in the pipette solution. *P < 0.05, compared with the AA group.

To test whether any of these inhibitors can block the actions of AA, cells were preincubated with an inhibitor and then the whole cell recording configuration was established. Adequate preincubation times and concentrations were determined from studies where an inhibitor blocked the actions of AA (see Fig. 5 for details). AA (10 µM) was then applied to the bath in the continued presence of an inhibitor. Indomethacin, 1-ABT, and ETI each failed to block the AA-induced decrease in both the peak and the long-lasting tail current (Fig. 5). Furthermore, simultaneous preincubation with, and in the continued presence of, all three inhibitors failed to block AA-induced current inhibition (Fig. 5). Indeed, in the presence of ETI or all three inhibitors, the long-lasting tail current inhibition by AA increased significantly over the inhibition observed with AA alone. These results suggest that, under the conditions used, some metabolism of AA by the lipoxygenase pathway occurs, but the resultant metabolites do not participate in current inhibition. Lastly, we found that AA inhibited currents when cells were treated with 100 µM ETYA, which, in addition to mimicking some direct effects of AA on other ion channels, blocks the formation of bioactive metabolites from AA (34). These results suggest that the mechanism of L- and N-type current inhibition by AA is independent of the generation of AA metabolites.

AA increases holding potential-dependent inactivation of whole cell currents. In our cell-attached patch experiments, AA increased the incidence of null sweeps for both L- and N-type Ca2+ channel activity but had no effect on fast inactivation in sweeps with activity (29). These findings suggest that AA enhances a slow form of inactivation, one that develops over many seconds, such as holding potential-dependent inactivation. To investigate whether AA has any effects on holding potential-dependent inactivation that can be observed at the whole cell level, inactivation curves were generated in the absence and presence of 5 µM AA using the protocol shown in Fig. 6A. We found that AA significantly increased the level of inactivation compared with controls at positive holding potentials (Fig. 6B).


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  		Fig. 6.   AA increases holding potential-dependent inactivation. A: voltage protocol used to collect data for the holding potential-dependent inactivation curves. Currents were elicited by applying a 2.2-s prepulse in 10-mV increments, starting at -110 mV, followed 5 ms later by a 100-ms test pulse to +10 mV; the break during the prepulse was 2.15 s. The current traces shown were elicited with a prepulse to -90 mV (down-triangle) and to +30 mV (black-triangle). B and C: maximal inward current was measured with a trough-seeking function and occurred at ~14 ms. Current amplitudes were normalized to the maximum inward Current and plotted against prepulse potential. Inactivation curves before (open circle ) and after () bath application of 5 µM AA were generated from currents elicited without a GTP analog in the pipette solution (n = 4) in B and with 0.1 mM GDPbeta S in the pipette solution (n = 5-7) in C.

These experiments were performed in the absence of a guanosine nucleotide analog in the pipette solution. It was possible that some of the differences in inactivation between control and AA conditions occurred when G protein activity decreased over time due to endogenous GTP moving out of the cell and into the pipette, rather than to the actions of AA. Therefore, to ensure that voltage-dependent relief of tonic G protein inhibition or possible G protein effects on inactivation (5) did not contaminate any effects of AA on inactivation, the experiment was repeated with 0.1 mM GDPbeta S present in the pipette solution (Fig. 6C). Under these conditions, AA again caused a significant increase in inactivation at positive holding potentials, demonstrating that the change in inactivation was independent of G proteins. These results indicate that AA enhances holding potential-dependent inactivation and are consistent with AA-induced increases in the incidence of null sweeps previously observed at the single channel level (29).

The actions of AA on whole cell currents are voltage dependent. To determine whether the effects of AA on whole cell currents are sensitive to test potential, current amplitude (Fig. 7A) was measured at test potentials from -60 to +80 mV in 10-mV increments in the absence and presence of 5 µM AA. No Ca2+ channel ligand (i.e., blocker or agonist) was present in the bath during these experiments. The current-voltage (I-V) plots (Fig. 7B) show that the threshold of current activation was similar in the absence and presence of AA (approximately -30 mV). In addition, the reversal potentials were similar (around +60 mV). However, we did find that the effects of AA on current amplitude varied with the test potential. AA significantly decreased the currents elicited from +10 to +50 mV (P < 0.05). Surprisingly, the I-V curves also revealed that AA significantly enhanced current amplitude at negative potentials (-20 and -10 mV, P < 0.05), raising the possibility that AA has another effect on whole cell currents in addition to inhibition.


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  		Fig. 7.   AA both inhibits and enhances whole cell currents. A: voltage protocol used to collect the data shown in B and C. Currents were elicited by applying 15-ms test pulses in 10-mV increments, ranging from -60 to +80 mV; for these recordings, 0.1 mM GDPbeta S was included in the pipette solution. Shown on the left is a typical current elicited at +10 mV. On the right is an expansion of the end of the test pulse. Peak inward current amplitude, measured ~14 ms into the test pulse (trace 1), was plotted against voltage to produce the current-voltage relationship shown in B. The amplitude of the fast component of the tail current (trace 2) shown in A was plotted against various test potentials to produce the activation curves shown in C. B: mean current-voltage relationships were generated before (open circle , n = 7) and after (, n = 4) bath application of 5 µM AA. The symbols and sample sizes pertain to B-D. C: mean tail current amplitude is plotted against voltage. *P < 0.05, control vs. AA. D: activation curves were generated by normalizing the data presented in C. Symbols (black-down-triangle ) indicate voltages where AA increased activation. Boltzmann fits were applied to the data in C and D. For these experiments, 0.1 mM BAPTA or 10 mM EGTA was included in the pipette solution.

To examine whether the voltage-dependent actions of AA were due in part to a change in the sensitivity of channel activation to voltage, activation was examined in the absence and presence of AA (Fig. 7, C and D). Activation curves were generated by measuring the amplitude of the fast tail current following whole cell currents elicited at 10-mV increments (Fig. 7A). Tail current amplitude was plotted against test potential (Fig. 7C). The threshold for activation occurred around -30 mV for both AA and control conditions in agreement with the I-V relationship shown in Fig. 7B. At negative voltages, AA caused no obvious change in current amplitude or voltage sensitivity (Fig. 7C). However, at positive voltages, inhibition by AA was prominent and the percent decrease in current appeared constant, suggesting that, at least at positive potentials, inhibition by AA is voltage insensitive (Fig. 7C).

To examine further the voltage dependence of activation, the data shown in Fig. 7C were normalized and fitted with Boltzmann curves. Normalized data (Fig. 7D) show that the half-maximal activation (Vh) for control and AA were not significantly different (9.6 ± 3.6 and 6.7 ± 3.0 mV, respectively; P > 0.05). However, Fig. 7D also shows that the slopes of activation (k) for AA appears to deviate from control (11.2 ± 1.0 and 7.6 ± 1.5 mV/e-fold change, respectively), in that activation occurs over a greater range of voltages in the presence of AA compared with control. Most notable is the increase in activation at voltages similar to those in the I-V relationship where AA enhanced current amplitude.

Internal BSA blocks AA-induced inhibition but not enhancement. The I-V relationships and activation curves indicate that AA inhibits currents at positive test potentials and may enhance currents at negative test potentials (Fig. 7, B and D). To verify that enhancement of current by AA is stable and reproducible, and not an artifact of the experimental protocol, the time courses of the development of current enhancement and inhibition were examined concurrently. Currents were measured by applying alternating test pulses to +10 or -10 mV from a holding potential of -90 mV, as shown in Fig. 8A. When 5 µM AA was applied, currents elicited at +10 mV were initially enhanced. At this voltage, enhancement was then offset by a decrease in current amplitude, leading to significant inhibition (40.5 ± 9.7%) measured after 7 min (Fig. 8B, left bars). In contrast, at -10 mV enhancement dominated after 5 min of AA, such that the current was increased by 155 ± 28.1% (Fig. 8C, left bars).


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  		Fig. 8.   Internal BSA decreases AA-induced inhibition, but not enhancement. A: whole cell currents were elicited from a holding potential of -90 mV by applying alternating test pulses to +10 mV () and -10 mV (open circle ). Solid bar, bath application of 5 µM AA. B: summary of AA's effects at +10 mV. Mean current amplitude was 228.2 ± 41.6 pA in control (Con) and 149.8 ± 28.8 pA 7 min after the application of AA. When cells were dialyzed for 3 min with 0.5 mg/ml BSA, mean current amplitude was 351.5 ± 95.7 pA in control and 347.0 ± 82.8 pA after application of AA. C: summary of AA's effects at -10 mV. Mean current amplitude was 37.3 ± 7.3 pA in control and 84.8 ± 16.9 pA after application of AA. With BSA, mean current amplitude was 39.0 ± 11.3 pA in control and 140.0 ± 39.7 pA after application of AA. For these experiments, GDPbeta S was included in the pipette solution; n = 5 recordings for each group. *P < 0.05, compared with paired control.

AA-induced inhibition of the peak and the long-lasting tail current develops slowly, taking several minutes to reach steady-state levels (Figs. 1C and 8A). In contrast, AA-induced enhancement measured at a test potential of -10 mV reached steady-state levels much more rapidly, suggesting different sites of action. To determine whether either effect is mediated from the cytoplasmic side of the membrane, 0.5 mg/ml BSA was included in the pipette solution where it will diffuse into the cell and bind intracellular AA (47). Dialysis of BSA into the cell had no obvious effect of its own on current amplitude (Fig. 8, B and C). Under these conditions, bath application of AA failed to produce significant inhibition at +10 mV (Fig. 8B, right bars), suggesting that this effect is mediated from the inside of the cell. In contrast, significant enhancement at -10 mV remained (Fig. 8C, right bars). Thus AA-induced current inhibition can be separated from enhancement, raising the possibility that the sites of action are distinct.

AA increases the activation kinetics of N-type currents. Last, we examined whether AA changes the activation kinetics of currents elicited at a test potential of +10 mV. To more clearly visualize any changes in activation kinetics, sweeps collected 5 min after the application of 5 µM AA were normalized so that the plateau phase of the current was superimposed onto that of the control sweep. Using this procedure, we found that AA (5 µM) accelerated the rate of activation in five of five cells (Fig. 9A). A similar increase in the rate of activation was also observed in three of three cells when FPL-64176 was present in the bath solution (Fig. 10A). In contrast, 5 µM myristic acid (data not shown; n = 3) and 5 µM oleic acid (Fig. 9B; n = 3) had no obvious effect on the activation kinetics when FPL-64176 was included in the bath solution, indicating that, like inhibition, the kinetic change shows some specificity for AA.


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  		Fig. 9.   AA increases whole cell activation kinetics independently of AA metabolism and G protein activity. Currents were elicited before and after bath application of 5 µM AA (A, C, D) or 5 µM OA (B), and raw sweeps are shown superimposed in the top traces in each panel. The current elicited in the presence of AA or OA was normalized to the control sweep and superimposed onto the control sweep shown in the bottom traces in each panel. Arrowheads (black-triangle) indicate a change in the rate of activation. Tail currents in A-D have been truncated. A: 0.4 mM GTP was included in the pipette solution; FPL was absent from the bath. B: 0.4 mM GTP was included in the pipette solution and 1 µM FPL was present in the bath throughout the recording. C: to test whether the change in activation kinetics by AA requires active G proteins, 0.1 mM GDPbeta S was included in the pipette solution to inhibit G protein activity. FPL was absent from the bath. Voltage protocol used is shown in A. D: an example of a cell preincubated for 60 min in 5 mM Ca2+-Tyrode solution containing 5 µM ETI, 3 mM ABT, and 10 µM indomethacin; 0.4 mM GTP was included in the pipette solution, and 1 µM FPL along with the inhibitors were present in the bath throughout the recording. Currents were elicited with the voltage protocol shown in B.



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  		Fig. 10.   AA-induced increases in activation kinetics is associated with N-type current. Currents were recorded and displayed using the methods described in Fig. 9; 0.4 GTP was included in the pipette solution. Arrowheads (black-triangle) indicate a change in the rate of activation. Tail currents in A-D have been truncated. A: 1 µM FPL was present in the bath. B: an example of the effects of AA in the presence of 1 µM NMN. C: an example of currents recorded from a cell pretreated with 1 µM omega -CgTx for at least 10 min; 1 µM FPL was present in the bath. Voltage protocol used is shown in A. D: an example of currents recorded from a cell pretreated with 1 µM omega -CgTx for at least 10 min. FPL was absent from the bath. Voltage protocol used is shown in B.

Increased activation kinetics of whole cell N-type current in neonatal SCG neurons occurs with the relief of tonic G protein-mediated inhibition (5). Therefore, one possible means by which AA may increase activation kinetics is by relieving G protein-mediated inhibition. To rule out this possibility, the GTP in the pipette solution was substituted with 0.1 mM GDPbeta S. In the presence of GDPbeta S, AA still induced an increase in activation kinetics in five of five cells, indicating that the effect is independent of G protein activity (Fig. 9C). In these cells, AA was still able to inhibit the current 43.8 ± 4.4%. This inhibition is not significantly different from the inhibition when GTP is in the pipette solution. Thus, as with the change in activation kinetics, the inhibition of whole cell currents by AA also appears to be independent of G protein activity as was observed under similar conditions in the I-V relationship (Fig. 7B) and voltage dependence of activation (Fig. 7C).

To determine whether the increased rate of activation is due to bioactive metabolites, AA was tested for its ability to enhance the activation kinetics in the presence of inhibitors of its metabolism. FPL-64176 (1 µM) was included in the bath solution. Preincubation of cells with 10 µM indomethacin (n = 3), 5 µM ETI (n = 3), or 3 mM ABT (n = 3) did not block the effects of AA on the activation kinetics (data not shown). Moreover, when cells were preincubated with all three inhibitors, the increase in activation remained (Fig. 9D). These results suggest that the increased rate of current activation by AA does not require its metabolism.

The change in activation appears to be associated primarily with N-type current. The AA-induced increased rate of activation occurred when 1 µM NMN was present in the bath (n = 4; Fig. 10B), but was lost when cells were treated with 1 µM omega -CgTx and FPL-64176 (n = 4; Fig. 10C). In the presence of omega -CgTx and FPL-64176, conditions where the ability to observe changes in L-type current is optimal, AA still inhibited the peak current by 54.3 ± 11.8% (n = 5), suggesting that the AA-induced kinetic change is independent of the inhibition of L-type current. When AA was tested in cells preincubated with omega -CgTx (3 µM) alone, no effect was observed in 10 of 15 cells. In the remaining five cells, only a small increase in activation kinetics could be observed (Fig. 10D), confirming that the increase in the rate of activation is due primarily to changes in N-type current. However, these data raise the possibility that N-type current does not exclusively mediate this effect. Similar inhibition and changes in kinetics were observed in the presence of NMN, FPL-64176, and (+)-202-791 when GDPbeta S was substituted with GTP in the pipette solution (data not shown). These findings suggest that G protein activity also is not important for the actions of AA observed with these conditions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Modulation of Ca2+ currents by AA has been described in a number of different cell types (24, 30). However, studies examining the effects of AA on Ca2+ currents in neurons are few. Here, we used pharmacological and biophysical methods to isolate whole cell L- and N-type Ca2+ currents in SCG neurons to characterize the effects of AA on them. We confirmed that AA inhibits both L- and N-type currents at positive test potentials and revealed an enhancement of whole cell currents at negative test potentials. In addition, AA produced two kinetic changes: an increase in holding-potential-dependent inactivation and a selective increase in activation kinetics of N-type current.

In characterizing the inhibition by AA of whole cell L- and N-type Ca2+ currents in SCG neurons, we have found that it is sensitive to micromolar concentrations of AA, a concentration range that is considered physiological (see Ref. 29 for further discussion). The effects of AA are partially reversible and at least somewhat specific for AA since three structurally similar fatty acids failed to inhibit the current. In addition, inhibition appears unaffected by the level of G protein activity. Whole cell current inhibition by AA has been observed in other neuronal preparations, although the channel types affected were not identified (17, 25, 26, 44). Whether these reported actions of AA are physiological was questioned, since the 25 or 50 µM concentrations of AA used in these studies were considered high. At these concentrations, AA may be above its critical micellar concentration, which has been estimated to be ~10 µM in a balanced salt solution containing 1 mM Ca2+ (40). If so, the actual AA concentration in solution in these studies may have been lower. Another concern with the use of higher AA concentrations is that the presence of micelles might interfere with channel gating, obscuring the physiological actions of AA. It is unknown whether micelles do form at high concentrations of AA under our recording conditions. Empirically, we (data not shown) and others (33, 54) have found that bath application of AA does destabilize whole cell current recordings at concentrations >= 50 µM. However, the inhibition of L- and N-type currents in SCG neurons by AA occurs at lower concentrations (5 µM) where we have not had this problem.

In addition to inhibiting L- and N-type currents, AA-induced enhancement of whole cell currents could be observed at negative voltages in the I-V relationships (Fig. 7B) and in plots of current amplitude vs. time (Fig. 8A). When cells were dialyzed with BSA, enhancement remained while inhibition was minimized, suggesting that the site of inhibition may be at the internal leaflet of the membrane or may occur at an intracellular location. Moreover, these results suggest that AA may enhance whole cell currents by acting either on external portions of a transmembrane protein, such as the Ca2+ channel itself, or in the outer leaflet of the membrane. It is also possible that enhancement occurs by AA acting with much higher affinity at an intracellular site such that BSA's affinity for AA is insufficient to block enhancement. Either mechanism argues for distinct sites of action for enhancement and inhibition. In our companion paper, we have characterized whole cell current enhancement by AA. We have confirmed that AA's site of action appears to be on the extracellular surface or outer leaflet of the cell membrane since bath application of an AA analog that cannot cross the membrane mimicked enhancement but not inhibition (6). We also have found that application of AA causes no observable enhancement of unitary L- or N-type Ca2+ channel activity when recorded at +30 mV in the cell-attached patch configuration (29), consistent with whole cell data where inhibition dominates at positive test potentials.

AA increased the amount of inactivation that occurs at positive holding potentials. In other cell types, AA decreased whole cell L-type Ca2+ currents (32, 45, 51), at least in part by shifting the inactivation curve more negative (33, 44, 45, 55). These results suggest that, although AA increases inactivation in a number of cell types, the exact mechanism of action may vary. At the cell-attached level, AA inhibits both L- and N-type Ca2+ channel activity similarly by increasing the incidence of null sweeps (29), consistent with the AA-induced increase in holding potential inactivation observed at the whole cell level (Fig. 6). Thus increases in channel inactivation are most likely associated with inhibition. In addition to the increase in null sweeps, we found that, in sweeps with activity, mean closed time increased (29). This change may also contribute to AA-induced decreases in whole cell current amplitude.

In addition to changes in inactivation, AA increases the activation kinetics of whole cell N-type currents. These data are in contrast to our single channel results where AA increased the first latency (29). This discrepancy may be due to the observation that, when L- and N-type channels did open in the presence of AA, they did so on average with a first latency >50 ms followed by quite low activity (29). Therefore, at the whole cell level, we would predict that AA-inhibited channels contribute little to the whole cell current because so few of these channels will have opened by the end of the 20-ms test pulse. Furthermore, the increase in whole cell activation kinetics by AA may be independent of AA's inhibitory effects since current inhibition is largely eliminated when BSA is dialyzed into the cell (Fig. 8), whereas faster activation kinetics remain unchanged (6). Indeed, regression analysis performed in our companion paper (6) indicates that the increased rate of current activation is directly correlated with the magnitude of current enhancement. Thus it appears that, with inhibition, AA increases first latency (29), whereas with enhancement activation kinetics increase. Thus, in contrast to inhibition, we would expect the increased rate of activation, which is associated with enhancement, to be observed at the single channel level as a decrease in first latency.

Our findings may resolve some of the controversy in the field concerning the differing effects of AA on Ca2+ currents. Previous reports of AA-induced current enhancement vs. inhibition (24) appear to show conflicting results; this may be due to one or more of the actions reported here, rather than to nonspecific effects. Whether AA exerts its actions on Ca2+ currents in SCG neurons directly or indirectly remains unanswered. The data presented in this study and in our companion paper (6) found little evidence for metabolites of AA mediating inhibition, enhancement, or the increase in activation kinetics. These results are consistent with previous findings in mammalian neurons that, when examined, the inhibitory actions of AA on whole cell Ca2+ currents appear independent of AA metabolism (16, 25, 44). However, these studies do not rule out an independent modulatory role for AA metabolites since exogenously applied prostaglandin E2 can inhibit N-type currents in SCG neurons via a membrane-delimited, G protein-coupled pathway (20). We have ruled out a direct role for G proteins since GDP-beta -S in the pipette had no effect on any of the AA-induced changes in whole cell currents described in this study. Whether protein kinases and/or phosphatases play a role in mediating any of the actions of AA in SCG neurons, as has been proposed for other cells (25, 33), has not yet been examined.

The role of AA and its metabolites in cellular signaling has received increasing attention due to their ability to modulate a wide variety of ionic currents. The brain is particularly rich in AA-containing phospholipids. Stimulation of certain neurotransmitter receptors, a number of which are found in the SCG, as well as ischemic conditions increase the release of AA and its eicosanoid metabolites (3, 13, 14, 22-24). The characterization of the effects of AA on whole cell L- and N-type currents in SCG neurons in this and the companion report (6) raises the prospect that one of the primary mechanisms for neuronal Ca2+ current modulation is by receptor-mediated liberation of AA from membranes. Moreover, our data predict that, depending on the types of Ca2+ channels present in a cell type and the recording conditions used, the observed effect of AA modulation of Ca2+ currents could vary widely. Fatty acids, once released from neurons, have been hypothesized to play a role both in physiological and pathophysiological conditions, such as synaptic plasticity, ischemia/reperfusion-induced cell death, and seizures (1, 7, 43). Thus Ca2+ current modulation by AA may participate at the cellular level in changes in synaptic plasticity; this possibility awaits further investigation.


    ACKNOWLEDGEMENTS

We thank John F. Heneghan, Thomas W. Honeyman, and Joshua J. Singer for reading various versions of the paper and H. Maurice Goodman and José Lemos for helpful discussion.


    FOOTNOTES

This publication was made possible by a Grant-In-Aid from the American Heart Association and a First Award from the National Institutes of Health.

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

Present address of C. F. Barrett: Dept. of Molecular and Cellular Physiology, Stanford University School of Medicine, Beckman Center, Stanford, CA 94305-5345.

Address for reprint requests and other correspondence: A. R. Rittenhouse, Room 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 1 December 2000.


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N-Arachidonoyl L-Serine, a Putative Endocannabinoid, Alters the Activation of N-Type Ca2+ Channels in Sympathetic Neurons
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Lipid Bilayer-mediated Regulation of Ion Channel Function by Amphiphilic Drugs
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Inhibition of L-type Ca2+ channel current and negative inotropy induced by arachidonic acid in adult rat ventricular myocytes
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