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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:959-966.)
© 1999 American Heart Association, Inc.

Original Contributions

Farnesol Blocks the L-Type Ca²⁺ Channel by Targeting the _1C Subunit

Ulrich C. Luft; Rostislav Bychkov; Maik Gollasch; Volkmar Gross; Jean-Baptiste Roullet; David A. McCarron; Christian Ried; Franz Hofmann; Yoram Yagil; Chana Yagil; Hermann Haller; Friedrich C. Luft

From the Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Virchow Klinikum-Charité, Humboldt University of Berlin, Berlin, Germany (U.C.L., R.B., M.G., V.G., C.R., H.H., F.C.L.); the Division of Nephrology, Hypertension, and Clinical Pharmacology, Oregon Health Sciences University, Portland (J.-B.R., D.A.M.); the Institute of Pharmacology, Technical University of Munich, Munich, Germany (F.H.); and the Barzilai Medical Center, Faculty of Health Sciences, Ben Gurion University, Ashkelon, Israel (Y.Y., C.Y.).

Correspondence to Friedrich C. Luft, MD, Franz Volhard Clinic, Wiltberg Strasse 50, 13122 Berlin, Germany. E-mail luft{at}fvk-berlin.de

	Abstract

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Abstract—We recently demonstrated that farnesol, a 15-carbon isoprenoid, blocks L-type Ca²⁺ channels in vascular smooth muscle cells. To elucidate farnesol's mechanism of action, we performed whole-cell and perforated-patch clamp experiments in rat aortic A7r5 cells and in Chinese hamster ovary (CHO) C9 cells expressing smooth muscle Ca²⁺ channel _1C subunits. Farnesol dose-dependently and voltage-independently inhibited Ba²⁺ currents in both A7r5 and CHOC9 cells, with similar half-maximal inhibitions at 2.6 and 4.3 mmol/L, respectively (P=NS). In both cell lines, current inhibition by farnesol was prominent over the whole voltage range without changes in the current-voltage relationship peaks. Neither intracellular infusion of the stable GDP analogue guanosine-5'-O-(2-thiodiphosphate) (100 mmol/L) via the patch pipette nor strong conditioning membrane depolarization prevented the inhibitory effect of farnesol, which indicates G protein–independent inhibition of Ca²⁺ channels. In an analysis of the steady-state inactivation curve for voltage dependence, farnesol induced a significant, negative shift (10 mV) of the potential causing 50% channel inactivation in both cell lines (P<0.001). In contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unlike pharmacological Ca²⁺ channel blockers, farnesol blocked Ca²⁺ currents in the resting state: initial block was 63±8% in A7r5 cells and 50±9% in CHOC9 cells at a holding potential of -80 mV. We then gave 500 mg/kg body weight farnesol by gavage to Sabra hypertensive and normotensive rats and found that farnesol reduced blood pressure significantly in the hypertensive strain for at least 48 hours. We conclude that farnesol may represent an endogenous smooth muscle L-type Ca²⁺ channel antagonist. Because farnesol is active in cells expressing only the pore-forming ₁ subunit, the data further suggest that this subunit represents the molecular target for farnesol binding and principal action. Finally, farnesol has a blood pressure–lowering action that may be relevant in vivo.

Key Words: smooth muscle cells • farnesol • patch clamp • calcium channel blockers • L-class channels

	Introduction

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Farnesol is 1 of several nonsterol mevalonate derivatives in the cholesterol pathway.¹ Farnesol is derived from farnesyl pyrophosphate, a C₁₅ isoprenoid lipid implicated in the regulation of G protein activity.² Some studies suggest that farnesol participates in the control of cholesterol synthesis by increasing 3-hydroxy-3-methylglutaryl coenzyme A reductase degradation.^{3 4} Others suggest that farnesol is implicated in the regulation of cell growth.⁵ Roullet et al⁶ recently showed that farnesol inhibited contraction in both human and animal arteries. This effect explained earlier reports of a mevalonate-dependent positive regulation of vascular tone.^{7 8} These studies featured a series of experiments that showed that farnesol blunted the KCl-induced increase in intracellular Ca²⁺ concentration in intact arteries and vascular smooth muscle cells (VSMCs) in culture.⁹ The patch-clamp technique revealed that micromolar concentrations of farnesol inhibited depolarization-elicited L-type Ca²⁺ channel currents in 2 VSMC lines, A10 and A7r5 cells.⁹ The inhibition of SMC Ca²⁺ signaling by farnesol was reversible and structure specific, for neither geraniol nor geranylgeraniol, 2 structurally related isoprenoids, was active. In another recent communication,¹⁰ we further showed that the action of farnesol on Ca²⁺ channels was not likely to be the consequence of a nonspecific effect on membrane dynamics, because farnesol did not alter VSMC fluorescence anisotropy. Altogether, these studies suggested that farnesol may act directly on voltage-dependent Ca²⁺ channels. However, the mechanism of this action remained speculative, and comparison of Ca²⁺ current inhibition by farnesol with those of classic Ca²⁺ channel blockers was lacking.

In this study, we sought to characterize the effects of farnesol on vascular L-type Ca²⁺ channels. L-type Ca²⁺ channels are heteroligomeric complexes consisting of 5 subunits (₁, ₂, ß, , and ). The ₁ subunit functions as a voltage sensor, a drug receptor, and a Ca²⁺-selective pore. The other subunits serve regulatory purposes.¹¹ To investigate farnesol's site of action, we used 2 cell lines, the A7r5 cell line, which expresses multisubunit complex (C-class) L-type Ca²⁺ channels, and the Chinese hamster ovary (CHO) C9 cell line, which stably expresses the smooth muscle, L-type Ca²⁺ channel pore–forming _1C subunit and displays L-type Ca²⁺ currents despite the lack of regulatory subunits.^{12 13} G protein dependence and state-of-the-channel dependence on the inhibitory effect of farnesol were also evaluated using guanosine-5'-O-(2-thiodiphosphate) (GDPßS) and various voltage protocols. Together, these analyses allowed us to compare the inhibitory effects of farnesol with previously characterized pharmacological Ca²⁺ channel blockers and better define the mechanism of its action. Finally, we performed preliminary experiments to explore the hypothesis that farnesol can lower blood pressure in a hypertensive animal model.

	Methods

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Cell Culture
Rat aortic A7r5 cells (passages 10 to 25) were purchased from the American Type Culture Collection (Manassas, Va). The cells were grown in Dulbecco's modified Eagle's medium under 95% O₂ and 5% CO₂ in the presence of 10% dialyzed BSA (Sigma-Aldrich), 1.7 mmol/L L-glutamine, streptomycin (30 mg/mL), penicillin (30 U/mL), and nonessential amino acids.⁹ The CHOC9 cells, which do not contain endogenous Ca²⁺ channel ß, 2, and subunits, were grown as previously described.^{13 14}

Patch Clamp
Ca²⁺ channel currents were recorded in the whole-cell (WC) or perforated-patch (PP) configuration with nystatin^{15 16} and with the use of a List patch-clamp amplifier EPC7. Data acquisition and command potentials were controlled with commercial software and a CED1401 interface (Cambridge Electronic Design Ltd). Currents were recorded from holding potentials of -80 mV (-100 mV) during linear voltage ramps at 0.67 V/s from -100 mV to +100 mV or 300-ms step pulses to different potentials; pulse frequency was 0.2 Hz. Analysis of WC currents was performed using CED patch and voltage clamp software version 6.08 (Cambridge Electronic Design Ltd). Ba²⁺ was used as the charge carrier; K⁺ currents were blocked by Cs⁺. In WC experiments, the extracellular solution contained (in mmol/L) NaCl 125, BaCl₂ 10.8, CsCl 5.4, glucose 10, and Na-HEPES 10 (pH 7.4 at 37°C). The patch pipette was filled with a solution containing (in mmol/L) CsCl 120, MgCl₂ 1, Mg-ATP 3, EGTA 10, and Cs-HEPES 10 (pH 7.4 at 22°C). For PP experiments, the bath solution contained (in mmol/L) NaCl 125, BaCl₂ 10.8, CsCl 5.4, glucose 10, and Na-HEPES 10 (pH 7.4 at 22°C); the pipette was filled with a solution containing (in mmol/L) CsCl 75, MgCl₂ 5, HEPES 5, and aspartate 75 (pH 7.4 at 22°C). The resistance of the pipettes was 4 to 6 M. Experiments were performed at room temperature, 22°C.

To test a possible involvement of G proteins in the effects of farnesol, the GDP analogue GDPßS was intracellularly infused via the patch pipette at a concentration of 100 mmol/L by using the WC patch clamp configuration. In control experiments, 100 µmol/L GDPßS (5-minute infusion) prevented G protein–activated cation currents induced by vasopressin (1 µmol/L) in A7r5 cells, which was consistent with the observations of Krautwurst et al.¹⁷ Furthermore, our previous studies demonstrated that 100 µmol/L GDPßS abolished G protein–dependent inhibition of voltage-gated, L-type Ca²⁺ channels by several receptor agonists.^{18 19 20 21}

Reagents
Bay K 8644 and nimodipine were obtained from RBI. EGTA, HEPES, and all other reagents were purchased from Sigma-Aldrich. Farnesol was obtained from Aldrich. All salts were obtained from Merck.

Animal Model
We relied on the Sabra desoxycorticosterone acetate (DOCA) –salt genetic model. The rats used in the current study were subbred from the original colony and are referred to as SBH/yr or SBN/yr.²² Experiments were performed in male, salt-resistant SBN/yr rats weighing 286±7 g and male, salt-sensitive SBH/yr rats weighing 309±7 g. The animals were bred in the animal facility of the Barzilai Medical Center, Ashkelon, Israel. The rats were allowed free access to standard chow (0.3% NaCl, SSNIFF Specialitäten GmbH) and drinking water. The experimental protocol was approved by the local council on animal care, which corresponds to the standards of the American Physiological Society. A 25-mg DOCA tablet (Innovative Research of America) was implanted into SBN/yr and SBH/yr rats below the skin at the nape of the neck. The animals were provided with 1% NaCl solution to drink. After 3 weeks (20±1 days) of DOCA salt treatment, the rats were surgically prepared and outfitted with a radiotelemetry device (DSI) to continuously record mean arterial blood pressure and heart rate. To test the oral effects of farnesol on blood pressure, farnesol (500 mg/kg) was mixed with olive oil and instilled by gavage feeding at a rate of 2 mL/min g body weight. Control rats received only vehicle. Blood pressure and heart rate were recorded for 48 hours thereafter.

Statistics
All values are given as mean±SEM. The term n represents the number of cells tested. For group comparisons, paired and unpaired Student' s t tests or nonparametric tests were used as appropriate. A value of P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Inhibition of Multisubunit, Complex L-Type Ca²⁺ Channels by Farnesol
Calcium currents in A7r5 cells were characterized by using 10.8 mmol/L Ba²⁺ as a divalent charge carrier with the use of the WC or PP configuration of the patch clamp technique. The inward Ba²⁺ currents were reversibly blocked by 1 mmol/L nimodipine or 100 mmol/L Cd²⁺; they were reversibly increased by 1 mmol/L (±)Bay K 8644 (not shown). A fast inactivating (T-type) current was observed in only 10% of all cells tested (N=54). To estimate the current-voltage relationship (IV curves), the test potential was linearly varied from -100 to +100 mV (ramp), or 300-ms step pulses to test potentials between -70 and +100 mV were applied from a holding potential of -80 mV (Figure 1A through 1C). The current recorded during voltage-ramp pulses (Figure 1C) was U-shaped and exhibited a maximum at 2±8 mV (N=54). The currents corresponded to those measured during step pulses to the respective potentials (Figure 1B). The apparent threshold occurred at -33±7 mV and the reversal potential at 55±9 mV (N=54).

View larger version (24K): [in this window] [in a new window]   Figure 1. Effects of farnesol on inward Ba2+ currents (IBa) in A7r5 cells.A, Superimposed current records at different test potentials of a representative cell before (open circles) and after (closed circles) application of 5 mmol/L farnesol. Ba2+ currents were evoked by 300-ms depolarizing voltage-step pulses to test potentials of -15, -10, -5, 0, 5, 10, 15, and 20 mV (holding potential, -80 mV; pulse frequency, 0.33 Hz). The dotted lines represent the zero-current levels. B, IV relation of peak Ba2+ currents recorded during step pulses (A) before (open circles) and after (closed circles) application of 5 mmol/L farnesol (n=5). Solid lines were drawn according to Equation 1 for the data points (see text). C, Superimposed current records of a representative cell before (control) and after application of 5 mmol/L farnesol. Ba2+ currents were evoked by linear voltage ramps at 0.67 V/s from -100 to +100 mV (pulse frequency, 0.2 Hz). Currents either are expressed in pA (IBa, top) or have been normalized (Inorm, bottom).

Figure 1A shows the effect of farnesol (5 mmol/L) on Ca²⁺ channel currents evoked by voltage steps to test potentials between -15 and 20 mV from a holding potential of -80 mV in a representative A7r5 cell. Farnesol significantly reduced the Ba²⁺ current at all potentials, and the currents at relatively positive potentials (0 mV) showed a faster decay in the presence of farnesol. Figure 1B shows the corresponding IV curves determined from peak Ba²⁺ channel currents recorded before and after farnesol application. The IV curves were fitted using the following equation and assuming a Boltzmann type of channel activation:

(1)

where I_Ba (pA) is the peak Ba²⁺ current, V_0.5 (mV) is the potential of half-maximal activation, V (mV) is the test potential, g_max (pS) is the maximal conductance, V_rev (mV) is the extrapolated reversal potential, and h is the slope factor of Ca²⁺ channel activation. Statistical analysis of the fits revealed that only g_max was significantly decreased by farnesol, 8.08±1.1 and 2.6±0.48 pS for control and farnesol-treated cells, respectively (P<0.05, n_PP=5). The other variables were not significantly altered (V_0.5, -13.8±2.2 and -9.3±2.3 mV; h, 4.2±0.5 and 5.1±0.41 mV; and V_rev, 38.2±1.7 and 35.3±1.9 mV for control and farnesol-treated cells, respectively; n=5).

To further substantiate this point, we recorded Ca²⁺ channel currents by using linear voltage-ramp pulses at 0.67 V/s from -100 to +100 mV; the holding potential was -80 mV. As shown for the normalized data in Figure 1C, farnesol reduced the current without affecting the potential at which peak Ba²⁺ currents occurred. The current was proportionally inhibited at all potentials where Ba²⁺ currents could be evoked; the apparent threshold and the reversal potential were not affected by farnesol. Thus, the data demonstrate that farnesol induces a voltage-independent inhibition of multisubunit, complex Ca²⁺ channels in VSMCs.

The effect of farnesol on steady-state inactivation was also tested. As shown in Figure 2, farnesol affected the voltage dependence of Ca²⁺ channel inactivation in A7r5 cells. The Ca²⁺ channel availability was determined by measuring Ba²⁺ currents during test pulses to 0 mV after 3-second conditioning voltage steps. Representative pulse recordings in control and farnesol-treated cells are shown in Figure 2A. The data were fitted with a Boltzmann equation of the form:

(2)

where I (pA) is the amplitude of the inward Ba²⁺ current elicited by a test pulse after the conditioning prepulse V (mV), I_max (pA) is the amplitude of the inward Ca²⁺ current elicited by a test pulse from a conditioning prepulse of -80 mV, V_h (mV) is the value of the conditioning potential causing 50% inactivation of the Ca²⁺ current, and k (mV) is the steepness factor characterizing the voltage sensitivity of the channels. Farnesol (5 mmol/L) significantly shifted the midpoint (V_h) of the normalized steady-state inactivation curve by 10 mV to more negative membrane potentials in comparison with control conditions; ie, from -29.9±2.0 to -41.4±1.7 mV (P<0.001, n_PP=5; Figure 2B). The slope factor k was not significantly altered; k values were 10.2±0.2 and 9.7±0.63 mV for control and farnesol-treated cells, respectively.

View larger version (19K): [in this window] [in a new window]   Figure 2. Effects of farnesol on steady-state inactivation of Ca2+ channels in A7r5 cells. A, Superimposed records of Ba2+ currents of a representative cell in the absence (left) and presence (right) of 5 mmol/L farnesol. The currents were recorded during a 300-ms test pulse to 0 mV after a 3-second conditioning prepulse to voltages between -80 and 0 mV. Holding potential was -80 mV. B, Corresponding voltage dependence of steady-state inactivation of Ca2+ channels averaged from 5 cells in the absence (open circles) and presence (closed circles) of 5 mmol/L farnesol. Normalized peak Ca2+ channel currents (I/Imax) were plotted vs the voltage of the conditioning prepulses. Pulse protocol was the same as that used in A. Solid lines were drawn according to Equation 2 for the data points.

G Protein–Independent Ca²⁺ Channel Inhibition
In these experiments, we tested the hypothesis that the effect of farnesol was secondary to the activation of a putative G protein. In a first set of experiments, we applied the GDP analogue GDPßS intracellularly, which stabilizes G proteins in the inactive GDP-bound form^{23 24} and thus, prevents receptor-activated, G protein–dependent regulation of ion channels.^{18 19 20 21} GDPßS was intracellularly infused via the patch pipette at a concentration of 100 mmol/L by using the WC patch-clamp configuration. Estimation of the rate of GDPßS diffusion²⁵ and patch-clamp experiments¹⁸ showed that the cytoplasmic concentration of GDPßS should have reached 80% of the concentration in the pipette solution within 5 minutes after membrane disruption. With the use of this protocol, the farnesol block was compared with that obtained in the absence of GDPßS in the recording pipette. As illustrated in Figure 3A and 3B, GDPßS did not modify farnesol-induced inhibition of Ca²⁺ channels. After 5 minutes of intracellular infusion of GDPßS, farnesol (10 mmol/L) inhibited the peak Ba²⁺ current by 66.1±3.7% (n=6). This result was not significantly different from the value obtained in control experiments with standard pipette solution (77.0±11.2%; P=NS, n_WC=6).

View larger version (24K): [in this window] [in a new window]   Figure 3. G protein–independent inhibition of L-type Ca2+ channels by farnesol in A7r5 cells. A and B, Effect of GDPßS on the inhibition of Ba2+ currents by farnesol. Ba2+ currents (IBa) were evoked by linear voltage ramps at 0.67 V/s from -100 to +100 mV (pulse frequency, 0.2 Hz). A, The pipette solution was filled with standard pipette solution; B, the pipette solution contained 100 mmol/L GDPßS. Currents were recorded after 5 minutes of intracellular infusion, under control conditions, with 10 mmol/L farnesol and after washout (w.o.). C, Superimposed Ba2+ current traces evoked in the presence or absence of 10 mmol/L farnesol in a representative cell by using the double-pulse voltage protocol shown above the traces. In this cell, application of farnesol decreased the current to 22% and 17% of control values before and after the conditioning prepulse to +80 mV, respectively.

Voltage-gated Ca²⁺ channels can be directly inhibited by receptor-activated G proteins via a membrane-delimited pathway independent of soluble messengers. This inhibition results from the interaction of G protein ß/ subunits with the Ca²⁺ channel protein and is typically relieved by strong membrane depolarizations.^{25 26 27} We compared inhibitions of Ca²⁺ currents by farnesol before and after strong membrane depolarization. As shown in Figure 3C, a large conditioning depolarization (+80 mV) inserted between 2 identical test pulses to 0 mV ("double-pulse" protocol) did not affect the amplitude of the control Ba²⁺ currents. Similarly, it did not affect the amplitude of the Ba²⁺ currents in the presence of farnesol (10 mmol/L): inhibition was 71±3% and 75±4% before and after the conditioning prepulse, respectively (n_PP=5, P=NS). These results suggest that farnesol blocks L-type channels by a G protein–independent mechanism.

Effect of Farnesol on _1C Subunit Currents
As previously described,¹³ the CHOC9 cells exhibited a typical, slowly inactivating Ca²⁺ current (L-type) and no fast inactivating (T-type) current (n=49). The recorded IV curves (Figure 4A) were U-shaped, with a maximum current at a potential of 4±5 mV, an apparent threshold potential at -35±6 mV, and a reversal potential at 52±6 mV (n=49). Ba²⁺ currents were reversibly blocked by 1 mmol/L nimodipine or 100 mmol/L Cd²⁺ and were reversibly increased by 1 mmol/L (±)-Bay K 8644 (not shown).

View larger version (24K): [in this window] [in a new window]   Figure 4. Effects of farnesol on Ca2+ currents in CHOC9 cells. A, Original current records of a representative cell under control conditions, with 5 mmol/L farnesol, and after washout (w.o.). Ba2+ currents were evoked by linear voltage ramps at 0.67 V/s from -100 to +100 mV with a 0.2-Hz pulse frequency. The insert shows normalized Ba2+ currents (Inorm) in the presence of farnesol and under control conditions. B, Dose-response curves of Ba2+ currents and farnesol concentration in CHOC9 (open circles) and A7r5 (closed circles) cells. Inward Ba2+ currents were recorded as peak currents at test potentials of 0 mV in A7r5 cells (n=5) and CHOC9 cells (n= 4). If represents the percent current in the presence of farnesol as a fraction of its control value at 0 mV. Solid lines are drawn according to Equation 3 for the data points (see text). Symbols represent mean±SEM. Holding potential, -80 mV. C, Corresponding voltage dependence of steady-state inactivation of the channels averaged from 5 cells in the absence (open circles) and presence (closed circles) of 5 mmol/L farnesol. Normalized peak Ba2+ currents (I/Imax) were plotted vs voltage of the conditioning prepulses. Pulse protocols are as described in the legend to Figure 2A. Solid lines were drawn according to Equation 2 for the data points.

The same figure illustrates the effect of 5 mmol/L farnesol on CHOC9 cell Ca²⁺ channel currents evoked by linear voltage-ramp pulses at 0.67 V/s from -100 to +100 mV. The holding potential was -80 mV. As shown for the normalized data (Figure 4A, insert), farnesol reduced the current without affecting the potential at which peak currents occurred. Ba²⁺ currents were proportionally inhibited at all potentials where they could be evoked; however, neither the threshold potential nor the reversal potential was affected by farnesol. The effect of farnesol was reversible on washout. The onset and offset of the inhibitory effect of farnesol were similar to those seen in A7r5 cells, ie, 1 minute. Thus, the inhibition of ₁ subunits by farnesol occurred in a similar voltage-independent manner as the inhibition of complex, multisubunit, native L-type channels.

Figure 4B shows the average dose-response curve for inhibition of Ca²⁺ channel currents by farnesol in CHOC9 cells. The curve was fitted to the following equation:

(3)

where I_f is the percent Ba²⁺ current in the presence of farnesol as a fraction of its control value at 0 mV, [farnesol] is the concentration of farnesol (mmol/L), IC₅₀ is the concentration of farnesol yielding half-maximal inhibition, and n is the Hill coefficient. The results indicate that IC₅₀ was 4.3±1.2 mmol/L and n was 1.18±0.18 (n_PP=4). The dose-response curve to farnesol in A7r5 cells is given for comparison on the same figure. Fitting the data points with Equation 3 revealed that IC₅₀ was 2.6±1.1 mmol/L (n_PP=5) and n was 0.99±0.13 in A7r5 cells (P=NS versus CHOC9 cells).

Farnesol also affected the voltage dependence of the steady-state inactivation of Ba²⁺ currents through ₁ subunits in CHOC9 cells (Figure 4C). The kinetic protocol used with CHOC9 cells was the same as that used with A7r5 cells; the data were fit with Equation 2. Farnesol (5 mmol/L) significantly shifted the midpoint (V_h) of the normalized steady-state inactivation curve by 7 mV to more negative membrane potentials: V_h was -30.5±1.4 and -37.2±2.5 mV for control and farnesol, respectively (P<0.05, n_PP=5). Although there was a trend for the inactivation shift in CHOC9 cells to be less than that in A7r5 cells, the difference was not statistically significant. The slope factor k was not significantly altered by farnesol: 12.6±0.6 and 13.1±1.1 mV in control and farnesol-treated cells, respectively.

Initial Block of Multisubunit, Complex Channels and ₁ Subunits
These experiments were designed to determine whether the farnesol-induced inhibition of Ca²⁺ channel currents depended on the opening of L-type channels; ie, whether farnesol could induce an "initial block." A7r5 cells were patched and held at -80 mV. Control Ba²⁺ currents were recorded during 300-ms pulses to 0 mV (frequency of 0.2 Hz). After 10 pulses, the cells were left unstimulated at resting potential (-80 mV). Farnesol (10 mmol/L) was then applied extracellularly; after 2 minutes, pulsing to 0 mV (frequency of 0.2 Hz) was resumed. The first depolarization evoked a current whose amplitude was reduced to 37±8% (n_PP=5) of control values before farnesol application. Thus, the initial block of the current by farnesol was 63%. Subsequent pulsing did not further reduce Ba²⁺ currents. This effect of farnesol on initial block is illustrated in Figure 5A for a representative A7r5 cell. These experiments were repeated in CHOC9 cells, and similar results were obtained (Figure 5B). For CHOC9 cells, the initial block with 10 mmol/L farnesol was 50±9% (n_PP=7). As observed in A7r5 cells, subsequent pulsing did not yield a significant further decrease in current magnitude (Figure 5B).

View larger version (25K): [in this window] [in a new window]   Figure 5. Farnesol-induced initial block of L-type Ca2+ channels in A7r5 and CHOC9 cells. A, Shown are Ba2+ current traces of a representative A7r5 cell. The initial-block protocol is represented below the trace: currents were elicited by applying 6 test pulses to 0 mV (300-ms duration; 0.2-Hz frequency) from a holding potential of -80 mV. Farnesol (10 mmol/L) was then applied to the unstimulated cell, ie, clamped at a holding potential of -80 mV. After 2 minutes, pulsing was resumed. In this cell, the first postfarnesol depolarization evoked a Ca2+ current whose amplitude was reduced to 25% of the control current before farnesol (initial block of 75%). Further pulsing (pulse frequency, 0.2 Hz) did not significantly decrease the initial block. B, Shown are Ba2+ currents evoked by the same initial-block protocol as shown in A for a representative CHOC9 cell. In this cell, farnesol induced an initial block of 45%. Subsequent pulsing did not significantly decrease Ba2+ currents.

Effect of Farnesol on Blood Pressure in Hypertensive Rats
Farnesol administration (500 mg/kg by gavage to SBH/yr rats) reduced blood pressure at 12 hours (Figure 6A) to levels lower than in vehicle-treated rats. This difference in blood pressure persisted and was still detectable at 48 hours. The cumulative 48-hour decrease in blood pressure (Figure 6B) was 12 mm Hg, whereas no decrease was observed in vehicle-treated SBH/yr rats (P<0.05). We also tested the effect of farnesol 500 mg/kg on salt-resistant SBN/yr rats. Farnesol had no effect on the blood pressure of the normotensive strain. Farnesol did not influence the heart rate of either strain (data not shown).

View larger version (12K): [in this window] [in a new window]   Figure 6. A, Farnesol given by gavage reduced arterial blood pressure to lower levels in SBH/yr rats than in vehicle-treated SBH/yr rats by 12 hours. This decrease persisted for 48 hours. B, The cumulative difference in blood pressure in farnesol-treated, SBH/yr rats compared with vehicle-treated SBH/yr rats was 16 mm Hg (*P<0.05).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The important observation in this study is that farnesol, a mevalonate derivative produced during cholesterol metabolism in all cells, inhibits multisubunit, complex (C-class) L-type Ca²⁺ channels in VSMCs. This inhibition is G protein independent and may be explained by direct inhibition of the pore-forming Ca²⁺ channel _1C subunit. It is unlikely that protein kinase C was involved in the response of Ca²⁺ channels to farnesol because activators of protein kinase C, such as phorbol 12-myristate-13-acetate (100 nmol/L), do not affect Ca²⁺ channel currents in A7r5 cells (data not shown, but also see References 28 through 30^{28 29 30} ) and currents through Ca²⁺ channel _1C subunit in CHOC9 cells.³⁰

To compare the mechanisms of L-type channel block by farnesol with those of pharmacological L-type Ca²⁺ channel blockers, we analyzed the effects of farnesol on channel activation and inactivation and determined whether farnesol induced an initial channel block. We performed the experiments on native, multisubunit, complex C-class, L-type channels in A7r5 cells and on Ca²⁺ channel _1C subunits stably expressed in CHOC9 cells. Farnesol did not alter the voltage dependence of the activation process of both multisubunit, complex Ca²⁺ channels and _1C subunits alone. This characteristic is similar to that of verapamil-type Ca²⁺ channel blockers^{31 32} but contrasts with the action of dihydropyridines. These compounds modify the IV relationship by shifting the voltage of half-maximal activation leftward.^{33 34} Farnesol was found to shift the steady-state inactivation curves of both multisubunit, complex Ca²⁺ channels and _1C subunits to more negative membrane potentials. Similar effects were reported for dihydropyridines, benzothiazepines, and verapamil-type Ca²⁺ antagonists on both complex L-type channels and the pore-forming ₁ subunits alone.^{35 36 37 38 39}

We also showed that farnesol induced a pronounced initial block (63% at a concentration of 5 mmol/L) of multisubunit, complex Ca²⁺ channels when the membrane potential was held at resting potential (-80 mV). On repetitive pulsing, farnesol did not further decrease the peak Ca²⁺ channel current through the channels. Thus, our data suggest that farnesol is capable of inducing calcium channel blockade without the need for prior channel opening or inactivation. Phenylalkylamines and benzothiazepines bind preferentially to the L-type Ca²⁺ channel in the open state,^{33 40 41 42} whereas dihydropyridines preferentially bind to the channel in the inactivated state.^{43 44} Thus, all 3 pharmacological Ca²⁺ channel blockers share the unique property of inhibiting L-type channels by binding to a voltage-sensitive portion of the ₁ subunit.^{31 45 46} As a consequence, phenylalkylamines, benzothiazepines, and dihydropyridines either are inactive³² or have a small inhibitory effect at negative potentials (-80 mV), as reported for a dihydropyridine in the rabbit ear artery,⁴⁷ and are unable to induce a significant "initial" channel block. Therefore, the high efficacy of the farnesol block at negative membrane potentials (-80 mV), ie, without the need for prior channel opening or inactivation, suggests the possibility of a different binding site on the Ca2+ channel, perhaps at a point less affected by membrane potential than the ones recognized by currently known Ca²⁺ channel blockers. Interestingly, a similar blocking mechanism of L-type channels was identified for mibefradil, a recently developed Ca²⁺ channel blocker.^{48 49} This synthetic Ca²⁺ channel blocker causes resting-state block of VSMC L-type channels with an IC₅₀ of 2.3 mmol/L. Possibly, mibefradil and farnesol bind to the same site. Furthermore, this site may represent the regulatory unit of mammalian natural endogenous Ca²⁺ channel blockade.

Finally, we determined the Ca²⁺ channel subunit requirements for farnesol's activity on Ca²⁺ currents. These experiments were conducted in CHOC9 cells, which stably express the pore-forming _1c subunit of VSMC L-type Ca²⁺ channels. As reported for cells transiently transfected with cardiac _1c subunits,⁵⁰ the CHOC9 _1c channel subunit is functional, and the cells exhibit voltage-activated, dihydropyridine-sensitive Ba²⁺ currents. These currents were also sensitive to the inhibitory action of farnesol. Therefore, the _1c subunit probably represents the molecular target for farnesol's binding and principal action. This suggestion is based on several considerations. First, farnesol inhibited currents through Ca²⁺ channel ₁ subunits with an IC₅₀ that was not significantly different from that found with multisubunit, complex Ca²⁺ channels. Second, the farnesol block of the ₁ subunits was voltage independent like that of multisubunit, complex Ca²⁺ channels. Third, the farnesol block of ₁ subunits involved a significant shift of the inactivation curve to more negative potentials, which was also observed in multisubunit, complex Ca²⁺ channels. Finally, similar levels of initial, resting-state blockade were seen in both CHOC9 and A7r5 cells. The putative binding site of farnesol on the ₁ subunit remains to be characterized. However, it is possible that the hydrophobicity of farnesol causes binding at the channel entrance or at a transmembrane segment in the plasma membrane. Further studies are needed to clarify this point.

Finally, we conducted preliminary experiments to determine whether or not orally administered farnesol 500 mg/kg can lower blood pressure in a hypertensive animal model. We selected this dose because Keller et al⁵¹ found that farnesol 500 mg/kg in olive oil increased hepatic farnesol levels 100-fold. We reasoned that at this dose, farnesol might have a systemic effect. We were able to detect a significant and persistent blood pressure–lowering effect in hypertensive but not in normotensive Sabra rats. We selected the Sabra rat because earlier we had characterized pressure natriuresis in this model and found that extrarenal regulatory mechanisms, rather than an intrinsic renal defect, were responsible for the pressure natriuresis shift in this strain.⁵² The mechanisms may involve NO.⁵³ The present data are highly preliminary; however, they support the possibility that our electrophysiological results may have a functional significance in living organisms. We do not view farnesol as a drug and have not performed dose-response curves or bioavailability studies. We would speculate that if farnesol influences blood pressure in vivo, its local production will be important in that regard. Mevalonate availability has been shown to be important in blood pressure regulation in earlier studies.^{7 8} To our knowledge, these are the first data to show that orally administered farnesol can lower blood pressure in a hypertensive animal model.

In conclusion, our results demonstrate that farnesol, a natural and endogenous metabolite present in all mammalian cells, is a potent blocker of smooth muscle (C-class) L-type channels. Furthermore, the blockade mechanisms involved are different from those of classic synthetic L-type Ca²⁺ channel blockers. Our study further demonstrates that the pore-forming ₁ subunit is the main channel subunit for channel block and suggests the existence of previously unrecognized regulatory sites on L-type Ca²⁺ channels. Elucidation of the role of farnesol as an in vivo modulator of ion channels may provide new insight into the control of blood vessel function in physiological and pathophysiological conditions and may also have therapeutic, pharmacological implications.

	Acknowledgments

 
This work was supported by grants-in-aid from the Bundesministerium für Bildung und Forschung to Friedrich C. Luft and from the Deutsche Forschungsgemeinschaft to Maik Gollasch. Hermann Haller, Volkmar Gross, and Friedrich C. Luft are supported by the Deutsche Forschungsgemeinschaft. Maik Gollasch is the recipient of a Humboldt Fellowship.

Received May 29, 1998; accepted September 4, 1998.

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