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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Inhibition of L-type Ca²⁺ channel current and negative inotropy induced by arachidonic acid in adult rat ventricular myocytes

Department of Pharmaceutical Sciences and Department of Pharmacology & Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas

Submitted 6 July 2007 ; accepted in final form 31 August 2007

	ABSTRACT

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We have previously shown an increase in arachidonic acid (AA) release in response to proinflammatory cytokines in adult rat ventricular myocytes (ARVM). AA is known to alter channel activities; however, its effects on cardiac L-type Ca²⁺ channel current (I_Ca,L) and excitation-contraction coupling remain unclear. The present study examined effects of AA on I_Ca,L, using the whole cell patch-clamp technique, and on cell shortening (CS) and the Ca²⁺ transient of ARVM. I_Ca,L was monitored in myocytes held at –70 mV and internally equilibrated and externally perfused with Na⁺- and K⁺-free solutions. Exposure to AA caused a voltage-dependent block of I_Ca,L concentration dependently (IC₅₀ 8.5 µM). The AA-induced inhibition of I_Ca,L is consistent with its hyperpolarizing shift in the voltage-dependent properties and reduction in maximum slope conductance. In the presence of AA, BSA completely blocked the AA-induced suppression of I_Ca,L and CS. Intracellular load with AA had no effect on the current density but caused a small depolarizing shift in the I_Ca,L activation curve, suggesting a site-specific action of AA. Moreover, intracellular AA had no effect on the extracellular AA-induced decrease in I_Ca,L. Pretreatment with indomethacin, an inhibitor of cyclooxygenase, or addition of nordihydroguaiaretic acid, an inhibitor of lipoxygenase, had no effect on AA-induced changes in I_Ca,L. Furthermore, AA suppressed CS and Ca²⁺ transients of intact ARVM with no significant effect on SR function and myofilament Ca²⁺ sensitivity. Therefore, these results suggest that AA inhibits contractile function of ARVM, primarily due to its direct inhibition of I_Ca,L at an extracellular site.

excitation-contraction coupling; cell shortening; contractility; Ca²⁺ transient

The effect of AA on contractile function of adult ventricular myocytes is also unclear. AA has been shown to enhance cell shortening (CS) and the Ca²⁺ transient in ARVM (10) or to reduce CS in adult guinea pig ventricular myocytes (36). It also has been reported that AA mediates a biphasic effect of TNF- on contraction and the Ca²⁺ transient in ARVM (1). Regardless, results from measurements of cardiac contractile function do not correlate well with the above-mentioned electrophysiological findings.

We (38) previously showed that during activation of Ca²⁺-independent PLA₂, hydrolysis of membrane plasmalogen phospholipids causes an increase in AA release and lysoplasmenylcholine (LPlasC) accumulation (38). It was then demonstrated that exposure to LPlasC exerts a positive inotropic and an arrhythmogenic effect on adult rabbit ventricular myocytes by enhancing Ca²⁺ transients and I_Ca,L (34). Thus it is important to know what the effect is of AA on the electrical and contractile function of adult ventricular myocytes. This study was designed to determine the effect of exposure of ARVM to extracellular AA on I_Ca,L, CS, and the Ca²⁺ transient. I found that exposure to AA directly altered the voltage dependence of gating properties of L-type Ca²⁺ channel and thereby reduced I_Ca,L, which accounts for AA inhibition of contraction and the Ca²⁺ transient in ARVM.

	METHODS

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Ventricular myocyte preparation. Single ventricular myocytes were isolated from hearts of adult male Sprague-Dawley rats (250–300 g) by using enzymatic dissociation as described previously (34). Briefly, rats were deeply anesthetized with isoflurane (Vedco, St. Joseph, MO), and hearts were rapidly excised and perfused at 37°C via the aorta with a control buffer solution, followed by enzymatic and mechanical dissociation. Isolated single ARVM were resuspended in culture medium containing antibiotic-free, bicarbonate-buffered culture medium 199 (60%; GIBCO, Grand Island, NY) with 36% Earle's balanced salt solution composed of (in mM) 116 NaCl, 4.7 KCl, 0.9 NaH₂PO₄, 0.8 MgSO₄, 26 NaHCO₃, and 5.6 glucose and containing 4% fetal bovine serum (GIBCO) (pH 7.40 in 5% CO₂-95% air at 37°C). After incubation for 3–4 h to allow recovery, ARVM were plated in culture dishes with serum-free culture medium overnight before experiments. Quiescent rod-shaped ARVM with clear striations were used for I_Ca,L, Ca²⁺ transient, and CS measurements. All experiments were performed at 36–37°C. The use of animals was carried out under a protocol approved by the Animal Care and Use Committee at the University of Arkansas for Medical Sciences.

Measurement of I_Ca,L. Whole cell I_Ca,L of ARVM was measured in Na⁺- and K⁺-free solutions with 2 mM Ca²⁺ with the use of conventional whole cell patch techniques (15) using a patch-clamp amplifier (Axopatch 200A; Axon Instruments, Foster City, CA) as described previously (34). Briefly, patch electrodes were filled with the following pipette solution and had a tip resistance typically of 1–3 M. The pipette solution consisted of (in mM) 100 CsOH, 70 aspartate, 11 CsCl, 15 tetraethylammonium chloride, 2 MgC1₂, 5 MgATP, 5 EGTA, 0.1 CaC1₂, 5 pyruvic acid, 5.6 glucose, 5 Tris₂-phosphocreatine, 0.4 Li₄-GTP, and 10 HEPES/Tris (pH adjusted to 7.20 with CsOH). The bath solution contained (in mM) 145 N-methyl-D-glucamine chloride, 0.8 MgC1₂, 2 CaC1₂, 2 4-aminopyridine, and 10 HEPES/Tris (pH adjusted to 7.40 with CsOH) to minimize membrane currents associated with Na⁺ and K⁺. The time-dependent effect of AA on I_Ca,L was assessed by applying 160- or 200-ms single voltage pulses to +10 mV from a holding potential (E_h) of –70 mV every 15 s or double pulses to +10 mV with a depolarization of E_h to –40 mV for 400 ms before application of the second pulse. The kinetics of I_Ca,L inactivation were analyzed using a double-exponential equation of y(t) = A₀ + A₁e^–t/_f + A₂e^–t/_s, where _f and _s are the fast and slow time constants, respectively, and A_x is the amplitude scalar. The current-voltage (I-V) relationship of I_Ca,L was constructed by applying 250-ms voltage pulses to potentials between –60 and +60 mV from an E_h of –70 mV in 10-mV increments at 0.2 Hz. The voltage dependence of steady-state inactivation and activation of I_Ca,L were determined using a gapped double-pulse protocol; a 1-s prepulse to potentials between –90 and +60 mV was followed by a 10-ms return to the holding potential and then a fixed 250-ms test pulse to +10 mV every 10 s. All raw data obtained for steady-state inactivation and activation and for the kinetics of I_Ca,L inactivation were fit to the Boltzmann distribution and a double-exponential function, respectively, using the nonlinear least-squares curve-fitting algorithm in Origin software (OriginLab, Northampton, MA).

After the whole cell configuration was achieved, a period of 10–15 min was allowed before the experiment so that rundown of I_Ca,L was minimal. Series resistance was 3–6 M and electronically compensated (90%), whereas the recorded currents were filtered at 1–2 kHz and sampled at 5 kHz using pCLAMP 8.0 software (Axon Instruments). The current density of I_Ca,L in each myocyte was obtained by normalizing the current amplitude to membrane capacitance (C_m) that was calculated from uncompensated capacity current transients recorded in response to 5-mV hyperpolarizing pulses from the holding potential.

Contractile function or CS. Unloaded CS of intact ARVM was elicited and measured as described previously (34). Briefly, CS was elicited by field stimulation (1–2 ms in duration, 1.5-fold threshold voltage) at 0.5 Hz in normal Tyrode's solution containing (in mM) 140 NaCl, 5.4 KCl, 1 CaCl₂, 0.8 MgCl₂, 10 HEPES/Tris, and 5.6 glucose (pH 7.40 at 37°C). CS was monitored with a video edge-motion detector system (Crescent Electronics, Sandy, UT). Data for CS were not expressed as percentages of original cell length, because the degree of CS could be affected by the degree of cell attachment to the dish. The measured parameters of contractile function in single ARVM included the peak magnitude of CS and rise time and decay time (duration between 10 and 90% of the peak amplitude) during contraction and relaxation, respectively. The protocol of post-rest potentiation (PRP) was used as an indicator to estimate Ca²⁺ handling by the sarcoplasmic reticulum (SR) (3, 52). PRP30 was measured as the ratio of the amplitude of the first potentiated contraction after a 30-s rest interval to that of the steady-state CS before the rest.

Measurement of intracellular free Ca²⁺ concentration. Intracellular free Ca²⁺ concentration in ventricular myocytes was measured as described previously (34). Briefly, ventricular myocytes seeded on 25-mm coverslips in culture medium were loaded with 2 µM fura-PE3 AM for 30 min in a culture incubator at 37°C. Myocytes were then transferred to a perfusion chamber on the stage of an inverted microscope (Nikon TE300; Irving, TX) and superfused with normal Tyrode's solution. After subtraction of the background signal, Ca²⁺ signals were recorded as an intensity ratio (R or F₃₄₀/F₃₈₀), i.e., the fluorescent intensity excited at 340 nm (F₃₄₀) divided by that at 380 nm (F₃₈₀). In some experiments, myocyte contraction was recorded simultaneously with Ca²⁺ transients when the cells were illuminated with a halogen lamp (Nikon) through a long-wavelength pass filter (640 nm; Chroma).

Chemicals. Fura-PE3 AM was purchased from TEFLABS (Austin, TX). AA, fatty acid-free BSA, and other reagents were purchased from Sigma Chemical (St. Louis, MO). The stock solutions of AA (1 M) and indomethacin (0.1 M) were made in 100% ethanol. The stock solution of nordihydroguaiaretic acid (NDGA; 1 M) was prepared in dimethyl sulfoxide (DMSO). The final concentration of ethanol and DMSO in experimental control solutions (i.e., Na⁺-, K⁺-free and 2 mM Ca²⁺ solution) was < 0.01% and had no significant effect on I_Ca,L (data not shown).

Statistics. In all experiments, data recorded in response to AA were compared with that of the steady-state control or the level in the presence of metabolic inhibitors before exposure to AA in each individual cell. Thus data are expressed as a percentage or fraction of each control value before combining for statistical analysis. Values are means ± SE. Statistical significance (P < 0.05) was evaluated using Student's paired t-test or one-way ANOVA with Duncan's multiple range test (when more than two conditions were compared).

	RESULTS

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Effect of AA on whole cell I_Ca,L in ventricular myocytes. The acute effect of AA on I_Ca,L was first examined by monitoring peak I_Ca,L elicited by repeatedly applying a single 160-ms voltage pulse to +10 mV from an E_h of –70 mV (Fig. 1A, inset) before, during, and after exposure to AA in superfusion solutions. In some experiments, I-V curves were obtained before and during exposure to AA. Figure 1A shows that AA elicited a concentration-dependent inhibition of peak I_Ca,L. In this cell, the amplitude of I_Ca,L was reduced to 87% of the control level after 9–10 min in the presence of 1 µM AA. Upon exposure to 10 µM AA, I_Ca,L was transiently increased by 8% in the first couple of minutes, followed by a decrease to 33% of the control level. The transient increase in I_Ca,L was observed in 60% of tested cells (Fig. 1A). Table 1 shows summarized data of concentration-dependent effects of AA on the amplitude and time constants of I_Ca,L inactivation. To examine the voltage-dependent block of AA, cells were given a double 160-ms test pulse to +10 mV every 15 s from two different holding potentials; i.e., after the first test pulse, E_h was switched from –70 mV to –40 mV before the second test pulse (Fig. 1B, inset). Figure 1B shows that AA-induced inhibition of I_Ca,L was enhanced when cells were voltage-clamped at –40 mV. This phenomenon became more evident at higher concentrations of 3 and 10 µM AA (Table 1), suggesting a voltage-dependent block of L-type Ca²⁺ channels. Note that exposure to 100 µM AA caused many cells to become shortened with loss of voltage control within a few minutes. Thus only a few data could be obtained, and the values shown in Table 1 could underestimate the maximal effect of 100 µM AA on I_Ca,L.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Effect of arachidonic acid (AA) on cardiac L-type Ca2+ channel current (ICa,L) in adult rat ventricular myocytes (ARVM). A: whole cell ICa,L of ARVM was measured at a holding potential (Eh) of –70 mV in Na+- and K+-free bath solution containing 2 mM Ca2+. The effect of AA on peak ICa,L was examined using a single voltage-pulse protocol (inset) before and during exposure to 1 and 10 µM AA. Upon exposure to 10 µM AA, there was an initial transient increase in ICa,L before inhibition, which was observed in 60% of tested cells. AA caused a concentration-dependent suppression of ICa,L. B: AA-induced inhibition of ICa,L was potentiated when Eh was depolarized to –40 mV (b) from –70 mV (a) following the protocol shown in the inset. The insets in A and B show individual current traces (numbered) where indicated. Membrane capacitance (Cm) was 206 (A) and 190 pF (B). The dashed line in A represents the zero current level. The positive capacitance transient in each curve trace was truncated. Gaps in the current traces in the presence of 1 µM AA in A and B indicate when a current-voltage (I-V) relationship was assessed.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Concentration-response curve and I-V relationship of AA-induced inhibition of ICa,L. Raw data of ICa,L current levels relative to each control in the presence of various concentrations of AA at Eh of –40 and –70 mV in A were curve fit with the Hill equation with a fixed Hill coefficient of 1 to obtain the concentration producing a half-maximal effect (IC50). The I-V curve in B was constructed in the absence and presence of AA by applying 250-ms voltage pulses to potentials between –60 and +60 mV from a holding potential of –80 mV in 10-mV increments. Data are means ± SE, and numbers in parentheses in A indicate the number of cells. *P < 0.05; **P < 0.01 compared with Eh of –70 mV (paired Student's t-test).

View larger version (12K): [in this window] [in a new window]   Fig. 3. Effect of AA on voltage dependency of steady-state activation and inactivation of ICa,L. The steady-state activation (A) and inactivation (B) of ICa,L were determined using a gapped double-pulse protocol; a 1-s prepulse to potentials between –90 and +60 mV was followed by a 10-ms return to the holding potential and then a fixed 250-ms test pulse to +10 mV. Raw data were fit by the Boltzmann distribution to obtain half-maximum activation or inactivation potential (Vh) and the slope factor by using the nonlinear least-squares curve-fitting algorithm. Data are means ± SE. G/Gmax, normalized conductance; I/Imax, normalized inactivated current.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Effect of BSA on AA-induced inhibition of ICa,L. A: upon removal of 10 µM AA, ICa,L slowly returned toward the control level. Extracellular application of BSA (0.1%) facilitated the recovery of ICa,L. B: BSA in the presence of AA reversibly blocked the inhibition of peak ICa,L induced by 10 µM AA. BSA also reversibly inhibited the AA effect on ICa,L inactivation (inset, individual current traces where indicated). Cm was 118 (A) and 205 pF (B).

View larger version (12K): [in this window] [in a new window]   Fig. 5. Effect of intracellular application of AA on ICa,L. Myocytes were internally equilibrated for 15–20 min with a pipette solution containing 3 µM AA before the I-V curve (A) and steady-state activation and inactivation (B) of ICa,L were assessed. Except for a rightward shift in Vh of activation curve, intracellular application with AA had no significant effect on the maximum current density, the I-V curve, and steady-state inactivation of ICa,L. C: ICa,L was decreased in a myocyte internally loaded with 3 µM AA in response to extracellular AA (10 µM). Cm was 115 pF (C).

View larger version (17K): [in this window] [in a new window]   Fig. 6. Effect of indomethacin on AA-induced inhibition of ICa,L. A: a myocyte was first exposed to 20 µM indomethacin, followed by cotreatment with 10 µM and then 100 µM AA. In the presence of indomethacin, AA still reduced ICa,L in a concentration- and voltage-dependent manner. Inset shows individual current traces (numbered) where indicated. Cm was 118 pF. B: concentration-response curves were obtained in the presence of 20 µM indomethacin. C: kinetics of ICa,L inactivation were assessed in the presence of indomethacin and coexposure to various concentrations of AA. Data in B and C are means ± SE; numbers in parentheses indicate the number of cells. *P < 0.01 compared with indomethacin (ANOVA). IDM, indomethacin; f and s, fast and slow time constants of ICa,L inactivation, respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Effect of nordihydroguaiaretic acid (NDGA) on AA-induced inhibition of ICa,L. A: a myocyte was first exposed to 20 µM indomethacin for 3 min and then to indomethacin plus 10 µM NDGA for another 5 min before cotreatment with 10 µM AA. NDGA slightly decreased peak ICa,L. In the presence of indomethacin plus NDGA, exposure to 10 µM AA reduced ICa,L in a voltage-dependent manner, the same as that in the absence of inhibitors. Inset shows individual current traces (numbered) where indicated. Cm was 180 pF. B: NDGA in the presence of indomethacin had no significant effect on AA-induced concentration- and voltage-dependent inhibition on the maximum amplitude of peak ICa,L. *P < 0.01 compared with 3 µM AA. C: ICa,L inactivation kinetics were assessed in the presence of indomethacin plus NDGA and cotreatments with 3 and 10 µM AA. *P < 0.01; P < 0.05 compared with indomethacin plus NDGA (ANOVA).

View larger version (17K): [in this window] [in a new window]   Fig. 8. Effect of AA on cell shortening (CS) and Ca2+ transients in intact ventricular myocytes. CS (A and B) and Ca2+ transients (C) of ventricular myocytes were monitored independently when cells were exposed to 1 µM AA followed by 3 µM (A, top) or 10 µM AA (A, bottom) and to 30 µM AA alone (B). Removal of AA restored CS and Ca2+ transients. Addition of 0.1% BSA (B) completely blocked suppression of CS induced by 30 µM AA. Subsequent removal of BSA resumed AA inhibition of CS. Post-rest potential (PRP) was also assessed before and during exposure to AA by monitoring CS (A) and Ca2+ transients (C) after a 30-s rest period (PRP30). D: summarized data of peak amplitude and PRP30 of CS (top) and the Ca2+ transient (bottom) during exposure to AA relative to those before AA exposure. Note that the amplitude of CS and the Ca2+ transient in the presence of 3 µM at 30 s before AA removal was used. *P < 0.01 compared with control. The vertical bar in A and B is an arbitrary unit.

View larger version (17K): [in this window] [in a new window]   Fig. 9. AA has no significant effect on the relationship of CS and the Ca2+ transient in ventricular myocytes. A: CS (top) and the Ca2+ transient (bottom) were simultaneously recorded from a myocyte before and during exposure to various concentrations of AA. B: single traces of CS (top) and the Ca2+ transient (bottom) are shown (numbered) where indicated. C: a phase-plan plot of CS as a function of the Ca2+ transient was constructed after normalizing the traces (shown in B) to their peak amplitude to minimize any influence due to the difference in the amplitude in different conditions.

	DISCUSSION

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Inhibition of I_Ca,L induced by AA. The present study showed that exposure to AA (<100 µM) reduced I_Ca,L with no change in the holding current at E_h of –70 or –40 mV (Figs. 1 and 5–7). The recovery of I_Ca,L upon AA removal was facilitated by BSA (Fig. 4). Thus AA-induced inhibitory effect on I_Ca,L is not nonspecific and consistent with findings reported by others (16, 30, 35, 46, 49). The voltage-dependent inhibition of I_Ca,L induced by AA suggests that AA interferes with both the closed state (i.e., at –70 mV) and inactivated state (i.e., at –40 mV) of L-type Ca²⁺ channels. This voltage-dependent block is supported by the fact that AA caused a hyperpolarizing shift in the steady-state inactivation of I_Ca,L, which resulted in 70 and 7% reduction in channel availability at –40 and –70 mV, respectively. The AA-induced reduction of macroscopic I_Ca,L presented is compatible with its reduction in open probability of single-channel activity of L-type Ca²⁺ channel with an increase in the number of nulls observed in rat sympathetic neurons (31). Moreover, AA (at 10 µM) elicited a hyperpolarizing shift in V_h of I_Ca,L activation curve (Fig. 3A), which is accountable for the initial transient increase in I_Ca,L (Fig. 2B). Interestingly, similar transient increase before inhibition of Ba²⁺ current and hyperpolarizing shift in the I-V curve induced by AA were observed in sympathetic neurons (30). The AA-induced decrease in amplitude of I_Ca,L is consistent with findings in neonatal and adult ventricular myocytes reported previously by other investigators (36, 41, 49). However, those studies showed less inhibition of I_Ca,L induced by AA (e.g., <50% at 10 µM) than the present study and did not determine the effect of AA on gating properties of basal I_Ca,L (36, 41, 49). In noncardiac cells, AA was also shown to decrease L-type (30), T-type (46) and N-type Ca²⁺ channels (30), indicative of AA acting on primarily the ₁-subunit of Ca²⁺ channels.

The present study showed that AA slowed I_Ca,L inactivation with a stronger effect on _f in a voltage-dependent manner. This could be due to greater relief of Ca²⁺-dependent inactivation of I_Ca,L and/or a smaller degree of voltage-dependent inactivation at more negative potentials (14). In fact, inactivation of I_Ca,L that transiently increases is faster than those of reduced I_Ca,L in the presence of 10 µM AA under the same voltage conditions (data not shown). Thus relief of Ca²⁺-dependent inactivation likely causes an increase in time constants of I_Ca,L inactivation induced by AA. Other possible mechanisms, which require further investigations, include AA facilitating the transition to the open state from its proximate closed state, thereby delaying channel entry into the inactivated state from the open state.

Direct action at the extracellular site. BSA, a protein that does not cross the cell membrane, has been widely used to identify membrane receptor-mediated nongenomic actions of estrogen (39). BSA also is known to have multiple binding sites (6, 42) with a K_d of 16 nM for AA (6). Thus the antagonizing effect of BSA on AA-induced changes in I_Ca,L is attributable to BSA dissociation of AA from the surface of the cell membrane. The present study showed that extracellular BSA not only facilitates the recovery of I_Ca,L following AA removal but also reversibly abolished AA-induced suppression of I_Ca,L and CS (Figs. 4B and 8B). With the molar ratio of AA (10–30 µM) to BSA (15 µM), BSA can remove AA rapidly (22) and thereby leave no partition of AA to the cell membrane (42). Thus these results strongly suggest that AA acts on ion channels primarily at the extracellular side of the cell membrane (30, 35).

If inhibition of I_Ca,L induced by extracellular application of AA were mediated by AA metabolites, one would expect that internal load cells with AA would yield the same or stronger inhibitory effect than extracellular AA. This is apparently not the case, because internal application of AA had no effect on basal I_Ca,L amplitude or the inhibitory effect induced by extracellular AA (Fig. 5). Interestingly, internal application of AA altered I_Ca,L activation curve, a small effect opposite to that induced by extracellular AA, indicating a site-specific action of AA on Ca²⁺ channels. When applied extracellularly, the hydrocarbon tail of AA incorporates readily into the hydrophobic domains of the membrane or channel because of its amphipathic property. This would increase membrane fluidity and disorder as expected from four cis carbon double bounds of AA. Membrane fluidity has been shown to increase steeply when AA is increased from 10 to 100 µM (2). This can account for the membrane disruption observed at 100 µM AA described previously in the present study. In addition, the carboxylic head of AA can interact with hydrophilic head groups of the membrane or the polar portion of the channel. Thus AA could alter readily the configuration of membrane microdomains containing Ca²⁺ channels via hydrophilic (e.g., when the channel is opened) and hydrophobic (i.e., closed- and inactivated-state block) pathways as proposed for free polyunsaturated fatty acids interacting with cardiac Na⁺ channels (24). Moreover, the pH gradient across the cell membrane can affect the distribution of unionized form and the degree of hydrophobicity of AA within the cell membrane. This can account at least in part for the site-specific direct effect of AA on Ca²⁺ channels. In addition, neither indomethacin nor NDGA affects extracellular AA-induced changes in I_Ca,L, suggesting no involvement of AA metabolites. Together, these results strongly suggest that AA-induced inhibition of I_Ca,L is mediated by its direct action primarily on the proximity of the extracellular side of the cell membrane.

It has been reported that a certain percentage of long-chain fatty acids (including AA) could release protons to the cytosol, thereby causing a transient acidification (22, 23). A study using nonsuperfused, quiescent ARVM and neonatal cardiomyocytes reported an intracellular acidification by 0.42 pH unit 20 min after exposure to 10 µM AA (48). The sustained intracellular acidification reported in that study is, however, unexpected, because such acidification should be recovered by effective pH regulatory mechanisms and buffer capacity (28, 32, 47). Nevertheless, decreased intracellular pH either has no effect (26) or causes a small hyperpolarizing shift in voltage-dependent gating of I_Ca,L (e.g., by 2.0 pH units) (21). Thus a transient decrease in intracellular pH cannot account for AA-induced changes in I_Ca,L.

Negative inotropy and inhibition of Ca²⁺ transients induced by AA. AA-induced inhibition of I_Ca,L would lead to suppression of excitation-contraction (E-C) coupling in intact cardiac myocytes. The present study showed that concentration-dependent suppression of CS and Ca²⁺ transients with an initial increase before cessation (at high concentrations) parallels the observed biphasic change in I_Ca,L described above. The response of CS and the Ca²⁺ transient to AA stimulation is relatively more rapid than that of I_Ca,L, primarily due to well-established voltage, ionic, and buffer control to optimize I_Ca,L measurement under patch clamping. My results showing AA-induced suppression of the Ca²⁺ transient are also comparable to those observed in neonatal cardiomyocytes (18) in which, however, complete inhibition was achieved at 30 µM AA. This could result from different lipid composition in the cell membrane between adult and neonatal cardiomyocytes. By contrast, one study (10) in ARVM showed that AA (>10 µM) enhanced CS and Ca²⁺ transients, which were suggested to be mediated by cyclooxygenase metabolites via PKC-dependent inhibition of K⁺ channels. It is unclear whether this discrepancy could be explained by differences in experimental conditions; e.g., only cells that responded to AA were selected for study (10). Furthermore, the effect of AA on K⁺ channels is controversial. AA has been shown to block cardiac transient outward current (5) and delayed-rectifier K⁺ channel with IC₅₀ > 20 µM (19) or to enhance cloned human inward-rectified K⁺ channel (35) and K⁺ current in guinea pig ventricular myocytes (36). Thus the effect of AA-induced change in K⁺ channels on the action potential and CS is still unclear. Moreover, although AA metabolites acting in concert on Ca²⁺ channels could facilitate inhibition of E-C coupling, inhibitors of AA metabolism were shown to have no effect on AA-induced suppression of Ca²⁺ transients (18) or CS (36). Thus the AA-induced suppression of CS and the Ca²⁺ transient is most consistent with its direct inhibition of I_Ca,L.

The present study also found no apparent change in relaxation kinetics or PRP of CS and Ca²⁺ transients induced by AA (Fig. 8), suggesting no significant effect on SR function. This result is also consistent with findings of no effect of AA on Ca²⁺ uptake of the Ca²⁺ pump in isolated cardiac SR vesicles (13) or single-channel activity of ryanodine receptor Ca²⁺ release channels in planar lipid bilayer (45). In addition, although there was a trend of reduction in the Ca²⁺ sensitivity of myofilaments (Fig. 9C), it was too small to account for the negative inotropic effect of AA. Together, these findings indicate that AA-induced negative inotropy parallels its suppression of the Ca²⁺ transient, primarily resulting from its direct inhibition of I_Ca,L.

Physiological and pathophysiological relevance and significance. AA is liberated from membrane phospholipids primarily via activation of PLA₂s (12) under pathophysiological conditions such as in response to thrombin (4), acetylcholine (9), hypoxia (38), inflammation (40, 51), and myocardial ischemia (8). We (33) have previously shown that AA is released to culture medium in response to stimulation by IL-1β and TNF-, cytokines closely associated with inflammation- and injury-mediated changes in brain and cardiac function (29, 43). The released AA, which could readily reach the levels used in the present study (7), directly acts on membrane targets such as ion channels in an autocrine/paracrine fashion. Meanwhile, after reentering the cell, AA can be oxidized to various eicosanoid metabolites that alter various cellular functions (7, 11). During hypoxia or ischemia, the direct action of AA on membrane proteins becomes more dominant because of low oxygen tension. We (33, 37, 38) previously showed that LPlasC accumulation occurs concomitantly with AA release under hypoxia or cytokine stimulation. We (34) also showed that LPlasC increases I_Ca,L and SR function, thereby inducing arrhythmias in ventricular myocytes. The direct inhibitory effect of AA on I_Ca,L can counterbalance the deleterious effect of LPlasC to prevent intracellular Ca²⁺ overload and consequently protect the cell from injury. Thus AA and –6 polyunsaturated free fatty acids have been considered as antiarrhythmic agents (36, 49). Similarly, AA counteracting the stimulatory effect of β-adrenergic stimulation (36, 41) can also be cardiac protective. However, high concentrations of AA (e.g., 100 µM) become cytotoxic because of disruption of membrane integrity.

	GRANTS

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This work was supported in part by National Heart, Lung, and Blood Institute Grant R01 HL-62226.

	ACKNOWLEDGMENTS

 
I thank Dr. Joseph Stimers for comments and editorial assistance and Meei-Yueh Liu and Daniel SD Liu for excellent technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. J. Liu, Dept. of Pharmaceutical Sciences and Dept. of Pharmacology & Toxicology, Univ. of Arkansas for Medical Sciences, 4301 West Markham St. MS 522-3, Little Rock, AR 72205 (e-mail: sliu{at}uams.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.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
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