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Am J Physiol Heart Circ Physiol 276: H1313-H1322, 1999;
0363-6135/99 $5.00
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Vol. 276, Issue 4, H1313-H1322, April 1999

L- and T-type voltage-gated Ca2+ currents in adrenal medulla endothelial cells

Raul Vinet1,2 and Fernando F. Vargas1,3

1 Laboratory of Cell Biology and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; 2 Escuela de Quimica y Farmacia, Universidad de Valparaiso, Chile; and 3 Department of Human Physiology, School of Medicine, University of California, Davis, California 95616


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated voltage-dependent Ca2+ channels of bovine adrenal medulla endothelial cells with the whole cell version of the patch-clamp technique. Depolarization elicited an inward current that was carried by Ca2+ and was composed of a transient (T) current, present in all the cells tested, and a sustained (L) current, present in 65% of them. We separated these currents and measured their individual kinetic and gating properties. The activation threshold for T current was approximately -50 mV, and its maximum amplitude was -49.8 ± 4.8 pA (means ± SE, n = 19) at 0 mV. The time constant was 10.2 ± 1.5 ms (n = 4) for activation and 18.4 ± 2.8 ms (n = 4) for inactivation. The L current activated at -40 mV, and it reached a plateau at -20.1 ± 2.3 pA (n = 6). Its activation time course was a single exponential with an activation time contant of 26.8 ± 2.3 ms (n = 4). Current-voltage curves, kinetics, gating, response to BAY K 8644, nifedipine, amiloride, and different selectivity for Ba2+ and Ca2+ indicated that the underlying channels for the observed currents are only of the T- and L-types that resemble those of the endocrine secretory cells.

whole cell patch clamp; voltage-dependent calcium channels; single microvascular endothelial cells; BAY K 8644; dihydropyridines


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VOLTAGE-DEPENDENT Ca2+ channels (VDCCs) are pathways with high selectivity for Ca2+ that are opened by cell membrane depolarization. VDCC activation evokes a high rate of Ca2+ influx, driven by its high electrochemical gradient across the plasmalemma (12). The ensuing rise of cytosolic Ca2+ concentration ([Ca2+]i) acts as a second messenger for cell contraction and secretion (20).

VDCCs are a family of channels that exhibit differences in current amplitude, threshold, activation/inactivation kinetics, and response to drugs. They are present in neurons, muscle, and secretory cells and participate in coupling of excitation with contraction, secretion, and other cell functions (6, 19, 31).

Among the several VDCC types described, types T for transient and L for long are found together in some endocrine secretory cells, such as gonadotrophs (33), and pancreatic beta -cells (2), and in most excitable cells (11, 12, 18, 19, 25).

Microvascular endothelial cells (EC) from the bovine adrenal medulla (BAMEC) (8, 38) and from rat brain (15), but not from bovine brain (37), have been shown to contain VDCCs, whereas specific searches for these channels on EC from other vascular territories failed to reveal the presence of VDCCs (9, 14, 34, 37).

Three voltage-dependent Ca2+ currents were described in BAMEC by Bossu et al. (10), namely, a transient T-type, identified as such, for its quick inactivation and inhibition by amiloride, and two currents that were mixed and appeared as a sustained current in whole cell measurements. The sustained current was enhanced by BAY K 8644, whereas nicardipine inhibited only the BAY K 8644-enhanced part of the current but not the basic current (8). This special pharmacology and low threshold of the sustained current were attributed to the contribution of a special channel (7) that had a low threshold and responded to BAY K 8644 but not to nicardipine. The presence of an extra channel would make BAMEC sustained current different from that of other cells in which the L current has been typified as passing through only one channel (13, 33).

The main purpose of the present work was to extend the characterization of VDCCs in BAMEC. To this end, we carried out an electrophysiological and pharmacological study of single, separate T and L currents. Our investigation of gating, kinetic, and pharmacological properties that typify VDCCs indicated that BAMEC contain only two VDCC types, namely, T and L channels, which resemble those of the endocrine secretory cells (13, 28, 33).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells

We obtained bovine adrenal glands from the slaughterhouse. They were extensively washed with ice-cold Ringer solution and perfused through the adrenal vein for 20 min with 0.25% collagenase in Ringer solution at 37°C. The glands were then homogenized, and the digested material was suspended in Percoll and centrifuged at 13,000 revolutions/min in an angle-head SS-34 rotor on a Sorvall RCRB centrifuge. The band containing the highest density of EC was plated in 35-mm petri dishes (Nunc, Roskilde, Denmark) at a cell density of 5 × 105 cells per dish. BAMEC relative density in the cell mixture was increased by differential plating. This technique takes advantage of the strong adhesion of BAMEC to plastic to remove chromaffin and other cells by shaking the culture dish and washing with culture medium 2-4 h after plating (3).

Freshly dissociated BAMEC were placed in a medium that contained medium 199 supplemented with 20% fetal calf serum, 2 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (Biofluids, Rockville, MD). To enhance BAMEC growth, we added 30 g/ml EC growth supplement (Collaborative Biomedical Products, Bedford, MA) and 50 U/ml heparin (Sigma Chemical, St. Louis, MO). Experiments were run as early as 12 h but not later than 3-4 days after plating.

We identified BAMEC by their morphological characteristics, which have been described in great detail using light and electron microscopy (23, 39). We also checked morphological identification by measuring BAMEC acetylated low-density lipoprotein (LDL) uptake. To this end, BAMEC were incubated with fluorescent 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled acetylated LDL (Biomedical Technologies, Stoughton, MA) for 4 h at 37°C. The dishes were then washed with Krebs solution and placed on the stage of an epifluorescent inverted microscope (Nikon Diaphot, Tokyo, Japan). Cell fluorescence was excited with 540-nm light, and the emission through a 580-nm filter was directly observed.

Electrophysiological Experiments

Hard glass pipettes (World Precision Instruments, Sarasota, FL) were made using a two-stage puller (David Kopf Instruments, Tujunga, CA) and were heat polished with a microforge. Once filled with saline, the pipette resistance ranged from 3 to 8 MOmega .

The dish containing the cells was placed on the stage of a Nikon Diaphot inverted microscope and perfused with a gravity-driven system. A cell was selected, and a pipette mounted on an electrically driven micromanipulator (Newport, Irvine, CA) was lowered until it touched the cell surface. At this stage, a seal was formed by applying suction through the pipette. To rupture the membrane patch under the pipette tip, we used a combination of gradual suction and voltage pulses.

In some experiments we preserved the intracellular environment by perforating the membrane patch with nystatin, which is a channel former that allows only monovalent ions to pass through (29).

Membrane currents were measured with an Axopatch 1C amplifier (Axon Instruments, Burlingame, CA). Computer-generated voltage or current pulses were programmed using the pCLAMP 5.5 software, also from Axon Instruments. On-line acquired data were stored on the hard disk of a microcomputer. We used the amplifier circuits to compensate for transient currents and, at the same time, to measure series resistance and membrane capacitance. Leak currents were subtracted with a pCLAMP 5.5 p/4 subpulse program.

To isolate Ca2+ currents, we suppressed other currents by replacing both K+ and Na+ in the internal and external solutions with CsCl and tetraethylammonium chloride (TEA-Cl), respectively. Current-voltage (I-V) curves were obtained with a voltage-clamp protocol that consisted of 14 voltage pulses of 270 ms each. Holding potential was -80 mV, and the pulses spanned from -70 to 60 mV in 10-mV steps. Chord conductance was calculated from the linear part of the I-V curve. Drug effect on currents was tested using a 1-Hz single repetitive pulse to 0 mV from a holding potential of -80 mV. Drugs were added after three or four control pulses had been applied.

Gating

For activation, we first closed all the Ca2+ channels with a -120-mV, 180-ms prepulse. This was followed by a test pulse of 20-ms duration. Test pulses ranged from -70 to 40 mV in 10-mV increments. On returning to the holding potential after each test pulse, a tail current was recorded.

For inactivation, we applied 12 prepulses from -120 to -10 mV in 10-mV increments and 180-ms duration. Each of these prepulses was followed by a 20-ms, 0-mV stimulating pulse, and the tail current on returning to the holding potential was measured.

The fraction of channels open was assumed to be equivalent to the ratio of the tail current at the preceding pulse voltage over the maximum tail current measured. These experiments were run in solutions containing either 5 mM Ca2+ or 5 mM Ba2+.

Kinetics

Current rise time was described by time to peak (tp), which was measured directly from the recordings, and by an activation time constant (tau a) that was measured by fitting an exponential curve to the experimental points using Eq. 1a. Inactivation time constant (tau i) was calculated by fitting a curve to the current decay with time using Eq. 1b. For these calculations we used a subroutine of pCLAMP 5 from Axon Instruments.
<IT>I</IT> = <IT>I</IT><SUB>max</SUB> [1 − exp (−<IT>t</IT>/&tgr;)] (1a)
<IT>I</IT> = <IT>I</IT><SUB>max</SUB> exp (−<IT>t</IT>/&tgr;) (1b)

Solutions

The standard extracellular solution contained (in mM) 140 NaCl, 5 KCl, 2.6 CaCl2, 2 MgCl2, 2.5 NaHCO3, 5 HEPES-NaOH, and 10 glucose, pH 7.4 (solution 1a), and the standard pipette solution contained (in mM) 130 KCl, 1 CaCl2, 2 MgCl2, 20 HEPES-KOH, and 5 EGTA-KOH, pH 7.2 (solution 1b). The solutions used to measure Ca2+ currents contained a low external K+ concentration, with no K+ in the pipette, while external Na+ was substituted by other cations. These changes were made to prevent masking of the Ca2+ currents by other cationic currents. The extracellular solutions contained (in mM) 130 N-methyl-glucosamine chloride, 10 TEA-Cl, 4 KCl, 5 CaCl2, 2 MgCl2, 10 HEPES-NaOH, and 10 glucose, pH 7.4 (solution 2a). The K+-free pipette solution contained (in mM) 140 CsCl, 10 TEA-Cl, 1 CaCl2, 2 MgCl2, 5 EGTA-Cs, and 10 HEPES-CsOH, pH 7.2 (solution 2b). Osmolality of these solutions was adjusted to ~300 mosmol/kgH2O.

Drugs

S-(-)-BAY K 8644 and amiloride were obtained from RBI (Natick, MA), and nifedipine was obtained from Sigma Chemical. Nifedipine and BAY K 8644 were dissolved in dimethyl sulfoxide, whereas amiloride was dissolved in double-distilled water. The experiments with dihydropyridines (DHPs) were performed in semidarkness.

Statistical Analysis

Where appropriate, values were expressed as means ± SE, and statistical differences were evaluated by using one-way analysis of variance and Student's t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells

BAMEC, chromaffin cells, and fibroblasts were the main cells collected from the digested adrenal medulla, with BAMEC relative density the highest after differential plating. BAMEC were readily identified by their flat, polygonal morphology, big nucleus, and numerous vesicles. In contrast, chromaffin cells were spherical and smaller than BAMEC, whereas fibroblasts were elongated, thick cylinders. DiI-labeled acetylated LDL uptake invariably confirmed morphological identification of BAMEC.

Currents

As expected from cells that contain VDCCs, a depolarization-evoked inward current was recorded in solutions that suppressed Na+ and K+ currents (Fig. 1, A and B). The inward current increased with external Ca2+ concentration and was present after Ba2+ replaced Ca2+ in the external solution. These properties indicate that under these conditions the main current carrier was Ca2+.


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  		Fig. 1.   Inward current evoked by depolarization in acutely isolated bovine adrenal medulla endothelial cells (BAMEC). External solution contained 5 mM CaCl2. Pulse paradigms (A and B, top) show voltage steps from a holding potential of -70 mV that continue up to 60 mV in 10-mV increments. Bath and pipette solutions were designed to block K+ and Na+ currents. A: transient (T) current recorded from a cell that lacked long-lasting (L) current. B: T and L currents recorded from a cell that showed both current components. C: current-voltage (I-V) curves for T and L currents recorded from 6 cells. Data points are means ± SE. T currents were measured in cells showing only this current (naturally isolated T current). L currents were measured after inward current had reached a plateau and T currents had totally inactivated. Chord conductance was estimated from slope of linear range of I-V curve.

The inward current contained two components, a transient one (T) (Fig. 1A) and a long-lasting one (L) (Fig. 1B). The latter reached a plateau and did not inactivate during the 270-ms stimulating pulse. Whereas the T current was readily measurable in all the cells tested, the L current was apparent in 65% of them. This did not mean that the underlying L-type channels were absent in the remaining 35% of cells, but rather that their L current was too small, because it could be detected in all cells by enhancement with BAY K 8644.

Naturally isolated T current. Electrical parameters of the single, separate T current were measured in cells that did not show an L current (Fig. 1A). Threshold was -50 mV, and maximum amplitude was -48.9 ± 4.8 pA (n = 19) between -10 and 10 mV. The membrane potential at which the current reversed its direction, i.e., the reversal potential (VR), was ~55 mV (Fig. 1C). However, this value is uncertain, because VR is diminished by the outflux of ions in the internal solution through the Ca2+ channel (20). This is evidenced by the much smaller experimental VR than that calculated with the Nernst equation for the Ca2+ concentration difference across the cell membrane ([Ca2+]i = 100 nM and [Ca2+]o = 2.6 mM, where [Ca2+]o is extracellular Ca2+ concentration), which was 136 mV.

Chord conductance, measured as the slope of the linear region of the I-V curve, when divided by the cell capacitance was 40.5 ± 6.6 pS/pF (n = 6).

Both tau a and tau i decreased with membrane potential and reached a plateau near 0 mV (Fig. 2). For this reason we report both constants at 0 mV, which was approximately the membrane potential at which maximum current amplitude was recorded in this work. T current rise with time was described by a single exponential in which tau a = 10.2 ± 1.5 ms (n = 6) and r2 > 0.95, whereas tp was 13.7 ± 1.7 ms (n = 6). The inactivation time constant tau i was 21.4 ± 2.2 ms (n = 6) with r2 > 0.95. 


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  		Fig. 2.   T and L current time constants (tau ) vs. membrane potential. T current activation and inactivation and L current activation decreased with membrane potential depolarization. Data points are means ± SE (n = 4). Time constants were calculated using Eqs. 1a and 1b in text.

Current separation in cells that contained T and L currents. We separated T and L currents with a double-pulse voltage-clamp protocol that started with a 120-ms, 0-mV prepulse. The prepulse evoked an inward current with the two components shown in Fig. 3A (left), but the T current inactivated during the prepulse and remained in that state for a long period of time, because its time constant for recovery from inactivation is on the order of seconds (11). Hence, the application of test pulses after the prepulse evoked an isolated L current, shown in Fig. 3A (right). This current was then subtracted from the total current evoked by the prepulse to 0 mV to also obtain the T current at 0 mV. The total composite inward current elicited by the prepulse and the two currents separated by the above procedure are shown in Fig. 3B.


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  		Fig. 3.   Separation of T and L current components. A: pulse paradigm (top) consisted of a 120-ms prepulse that carried membrane potential to zero and was followed by 150-ms pulses from -60 to 20 mV in 10-mV increments after a brief pause (10 ms) at a holding potential of -80 mV. Prepulse evoked an inward current that contained L and T current components (left), whereas pulses evoked only L current component (right). B: total current evoked by prepulse and separated T and L currents. L current component was the only current evoked by test pulses, because T current remained inactivated during this measurement. L current was evoked by a test pulse to 0 mV. T current was obtained by subtraction of this L current component from total current evoked by prepulse, also at 0 mV.

T current. The isolated T current maximum amplitude and tp were -44.3 ± 6.1 pA (n = 4) and 12.1 ± 2.5 ms (n = 4), respectively. These values were not statistically different from those of the single, naturally isolated T current measured in this work. This indicated that the current separation procedure was successful, and, therefore, we safely assume that isolated L current parameters were obtainable with this method.

L current. The amplitude of L current in cells that exhibited both T and L current (65% of total) was measured at least 100 ms after the start of the inward current, when T current was completely inactivated (Fig. 3B). The I-V curve plotted with these measurements shows a maximum amplitude of -20 ± 2.3 pA (n = 6) at 0 mV and VR at ~50 mV (Fig. 1C).

However, current separation was needed to measure L current rise time and threshold, because at early times the two currents were mixed (Fig. 3B). The isolated L current tp was 39.2 ± 2.3 ms (n = 6), whereas tau a was 26.8 ± 2.3 ms (n = 6). tau a decreased with depolarization and reached a plateau near 0 mV (Fig. 2). The threshold was close to -40 mV, and conductance was 18.2 ± 1.6 pS/pF (n = 6). No inactivation of the L current was detected during the 270-ms stimulating pulse (Fig. 1B).

Voltage dependence of current gating. The Hodgkin-Huxley model for giant axon currents (22) was applied to evaluate activation and inactivation parameters of the T current and activation of the L current. Tail currents were recorded at a holding potential of -80 mV and ~4 ms after switching from the test pulse. Tail currents were plotted as a function of the voltage of the preceding pulses, and the experimental points were fitted by a curve calculated from a Boltzmann relationship (Eqs. 2 and 3). Measurements were carried out in either Ca2+- or Ba2+-containing external solution.

For the measurement of gating parameters, a tail current double-pulse voltage-clamp protocol was preferred over the conventional voltage-clamp protocol, because outward currents may not be fully eliminated by the use of K+-free solution. This is because Cs+ and other ions can flow through Ca2+ channels, as shown by the leftward shift of Ca2+ current reversal potential in this and other works (20).

T current activation. Gating parameters for the T current were measured in cells that did not exhibit L current. For these measurements we first restored all the T-type channels to their closed state by applying a 180-ms hyperpolarizing pulse at -120 mV. We then ran a sequence of test pulses from -120 to 50 mV and recorded the tail currents at a holding potential of -80 mV (Fig. 4A). The test pulses were 20 ms in duration, to allow the T current to reach its maximum amplitude.


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  		Fig. 4.   Voltage-dependence of current gating. Records in A and C are representative of 6 experiments performed in a solution containing 5 mM Ca2+, whereas B and D show average currents for all experiments performed in 5 mM Ca2+- or 5 mM Ba2+-containing solution. A: T current activation. Experimental protocol consisted of a -120-mV prepulse of 180-ms duration whose purpose was to close all VDCCs. This was followed by a series of pulses from -70 to 40 mV in 10-mV increments. Experiments were run with modified bath and pipette solutions 2a and 2b. Tail currents shown were recorded immediately after switch from each test pulse to holding potential at -80 mV. B: T current activation curve. Tail currents were plotted vs. voltage, and experimental points were fitted with a line calculated from a Boltzmann function (Eq. 2 in text). Curves show current increases with membrane depolarization; channels carrying T current started to open at approximately -50 mV and reached a maximum near -10 mV. T current was higher in Ca2+- than in Ba2+-containing solution. C: T current inactivation. Experimental protocol consisted of a 180-ms prepulse that varied from -120 to -10 mV in 10-mV increments. This was followed by a single pulse to 0 mV. Tail currents were recorded on repolarization to -80 mV. D: T current inactivation curve. Tail currents were plotted vs. prepulse voltage, and experimental points were fitted with a line calculated from a Boltzmann function (Eq. 3 in text). As in B, currents were higher in Ca2+ than in Ba2+, suggesting that channels carrying T current were more permeable to Ca2+ than to Ba2+.

Tail current amplitude was plotted as a function of the preceding test pulse voltage (Fig. 4B), and the experimental points were fitted by a curve calculated using Eq. 2 (33)
<IT>I</IT> = <IT>I</IT><SUB>max</SUB> {1 + exp [(<IT>V</IT><SUB>½</SUB> − <IT>V</IT>)/<IT>k</IT>]}<SUP>−1</SUP> (2)
where I is the tail current recorded at the membrane potential of the preceding test pulse (V), Imax is the maximum tail current, V1/2 is the voltage at which one-half of the channels are closed and the other half are activated (i.e., when I/Imax = 0.5), and k is the reciprocal of the maximum slope of the activation curve and is a measure of the millivolts needed to change I e-fold (steepness factor).

Six experiments run in 5 mM external Ca2+ had Imax = -34.4 ± 2.8 pA and were best fitted by a curve calculated from Eq. 2 with r2 > 0.95. The parameters giving the best fit were V1/2 = -27.6 ± 2.1 mV and k = 5.3 ± 0.4 mV (Fig. 4, A and B). When 5 mM Ca2+ was substituted by 5 mM Ba2+, Imax decreased to -17.0 ± 1.0 pA (Fig. 4B), whereas V1/2 at -27.2 ± 1.8 mV and k at 5.7 ± 0.4 mV were unchanged.

We also measured the reduction in Imax caused by Ba2+ with a single pulse to 0 mV from a holding potential of -80 mV. In six experiments Imax decreased from -25.0 ± 2.8 pA to -10.5 ± 0.5 pA when Ca2+ was replaced by Ba2+, and the difference was statistically significant (P < 0.001). The smaller Imax evoked by a pulse than that measured with tail current was probably caused by an outward Cs+ current counteracting the inward Ca2+ current at 0 mV. This current was not detected during tail current measurement carried out at a holding potential of -80 mV.

T current inactivation. These measurements were performed in either 5 mM external Ca2+ or 5 mM Ba2+. We applied a 180-ms prepulse that ranged from -80 to -10 mV. After each prepulse, a 20-ms, 0-mV test pulse was applied and the tail current after switching to the holding potential was measured (Fig. 4C). The tail current associated with each test pulse decreased as the prepulse carried the membrane potential to more depolarized levels (Fig. 4D), indicating that the fraction of closed T-type channels (ready to fire) decreased with membrane depolarization.

Tail current decay with voltage was fitted by a curve calculated from Eq. 3
<IT>I</IT> = <IT>I</IT><SUB>max</SUB> {1 + exp [(<IT>V</IT> − <IT>V</IT><SUB>½</SUB>)/<IT>k</IT>]}<SUP>−1</SUP> (3)
In six experiments Imax was -35.1 ± 3.9 pA, and the experimental points were best fitted by a curve with V1/2 = -32.8 ± 1.9 mV and k = 6.2 ± 0.08 mV per e-fold change of current. Substitution of Ba2+ for Ca2+ in the external solution reduced Imax to -13.6 ± 0.9 pA. This decrease was statistically significant (P < 0.005) (Fig. 4D). Neither V1/2 nor k was modified by the cation substitution.

L current activation. Activation parameters for the single, separate L current were measured in cells that exhibited this current spontaneously, i.e., without BAY K 8644. T current was suppressed as described earlier. In these experiments, instead of measuring tail currents, we ran a series of pulses from a holding potential of -80 mV and measured the current directly associated with each pulse. We plotted the currents against membrane potential, and the experimental points were fitted by a line calculated from Eq. 4 (25) to obtain the I-V curve shown in Fig. 5
<IT>I</IT> = <IT>g</IT><SUB>max</SUB> (<IT>V</IT> − <IT>V</IT><SUB>R</SUB>) {1 + exp [−(<IT>V</IT> − <IT>V</IT><SUB>½</SUB>)/<IT>k</IT>]}<SUP>−1</SUP> (4)
where gmax is the maximum conductance.


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  		Fig. 5.   L current activation. Activation parameters for single L current were measured in cells that exhibited this current without BAY K 8644 enhancement. T current was suppressed (see MATERIALS AND METHODS), and L currents in response to a series of pulses were recorded. Experimental points were plotted vs. voltage, and line fitting them was calculated using Eq. 4 in text. Currents were recorded in 4 experiments run in 5 mm external Ca2+ and were best fitted by a line calculated using the following parameters: gmax = 0.4 nS, membrane potential = 50 mV, V1/2 = -18.0 ± 1.9 mV, and k = 8.2 ± 0.8 mV per e-fold change of current.

The gating parameters obtained with Eq. 4 are only a good approximation because, as we previously described, VR for the Ca2+ current may be more positive than the value used for these calculations (20).

Results from four experiments carried out in 5 mM external Ca2+ were best fitted by a line obtained using the following parameters: gmax = 0.4 nS, VR = 50 mV, V1/2 = -18.0 ± 1.9 mV, and k = 8.2 ± 0.8 mV per e-fold change of current. Imax increased approximately two times when Ca2+ was substituted by Ba2+.

Pharmacology of T and L currents. To further identify the ionic channels carrying the observed currents, we used amiloride, which inhibits T currents, and DHPs, which mainly act on the L channels.

Amiloride. Amiloride (100 µM) caused a 60% inhibition of the T current amplitude in four experiments similar to the one shown in Fig. 6A. The L current was not affected by this drug, thus showing its specificity for the T channel.


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  		Fig. 6.   Effects of amiloride, nifedipine, and BAY K 8644 on Ca2+ currents. T and L currents were evoked by a voltage step to 0 mV from a holding potential of -80 mV. Pulse paradigms are shown at top of graphs. A: 100 M amiloride caused a 60% inhibition of T current amplitude, whereas L current was not affected. B: nonenhanced L current was completely inhibited by 1 µM nifedipine, whereas T current was partially inhibited. C: increase in L current was caused by 1 µM BAY K 8644, with no appreciable effect on T current. All graphs are representative of at least 4 experiments.

DHPs. Nifedipine completely blocked the L current at 1 µM, and it partially inhibited the T current (Fig. 6B). Raising nifedipine concentration to 50 µM totally blocked the T current (not shown).

BAY K 8644 at 1 µM evoked a 70-250% L current increase in cells that exhibited measurable L current, but it did not affect the T current (Fig. 6C). L current maximum amplitude increased from a control value of -20.0 ± 2.3 (n = 6) to -72.4 ± 11.9 pA (n = 6), and its conductance went from 18.2 ± 1.6 (n = 6) to 53.2 ± 4.7 pS/pF (n = 4).

L current activation V1/2 was -28.7 ± 3.8 mV (n = 4), whereas it was -18 ± 1.9 mV (n = 4) in the nonenhanced native L current. The steepness factor, k, was 3.2 ± 0.4 mV for the BAY K 8644-enhanced L current and 8.2 ± 0.8 mV for the control current. The peak was shifted to the left (Fig. 7). These changes in L-type channel gating parameters are comparable to those caused by BAY K 8644 in gonadotrophs (33) and suggest an increase in L-type channel open time probability under the drug.


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  		Fig. 7.   BAY K 8644 increased L current. I-V relationship for 6 experiments run in 5 mm external Ca2+ shows that increase in L current was caused by 1 µM BAY K 8644. Currents were evoked by a series of voltage pulses from a holding potential at -80 mV. Continuous line was fitted using Eq. 4 in text. Note marked increase in amplitude and slope of current and, also, leftward shift of maximum current under BAY K 8644.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

General

The results of our investigation of BAMEC VDCCs provide strong evidence that T- and L-type channels are present in this cell. These channels share many properties with those of endocrine cells that have been typified as being of the T and L type (28, 33). One of these properties is the L-type channel low threshold that we measured in this work and that is also found in L-type channels of other cells (25). This makes it unnecessary to postulate an extra channel that would lower the apparent threshold of the L current (7). Instead, L-type channel may not be defined as a high-threshold channel in all cells (31).

The presence of only two channel types, instead of three as previously postulated (7), is supported by our kinetics measurements, which show that only a single time constant is required for L-type channel activation, and also by our presently reported DHP inhibitory effect on the native basic L current, which indicates that the underlying channel was pharmacologically similar to other L-type channels, instead of a different channel lacking this response, as previously noted (7).

Differences between our results and those previously reported for BAMEC (7, 8) may be related to the procedures used to obtain acutely isolated single BAMEC or to our measurement of separated T and L current properties.

We next discuss in further detail the characteristics of the separate T and L currents reported in this work and their comparison with those of other cells in which similar currents have been described.

T Current

The maximum amplitude of T current in this work was ~40% higher than that previously reported for BAMEC in a similar Ca2+-containing solution (7) but smaller than that of neurons (11, 18), fibroblasts (16), and gonadotrophs (33). Differences in Ca2+ current amplitude among cells may be related with VDCC density as previously proposed (30). This is consistent with the present results showing that T current amplitude and specific conductance were smaller in BAMEC than in gonadotrophs (33).

T current activation threshold at -50 mV was similar to that previously reported for BAMEC (8), and also to that of gonadotrophs (33) and pancreatic beta -cells (30), but higher than that of neurons (11, 18). Likewise, the membrane potential at which BAMEC T current reached a maximum (-20 to 0 mV) was similar to that reported for gonadotrophs (33) and fibroblasts (16) but lower than those of neurons (11, 18) and canine atrial myocytes (5).

The higher activation V1/2 (membrane potential at which one-half of T-type channels are open) in our experiments (-27.6 mV) than that (-36 mV) previously measured in BAMEC (8) may reflect differences in the voltage-clamp protocols used to study T current gating in these two works. Whereas we measured tail currents, Bossu et al. (8) measured the currents directly elicited by pulses and used an equation similar to our Eq. 4 to fit a line to their experimental results. However, one of the limitations of this treatment, as we previously discussed, is the uncertainty about the membrane potential at which Ca2+ current reverses, because other ions such as Cs+ may flow through the same channel (20).

On the other hand, inactivation V1/2 (-32.8 mV) and tau i (21.4 ms) in the present work were quite similar to those of the T current measured in other cells (5, 11, 16, 18, 33).

The approximately similar T current kinetic and gating parameters in BAMEC and other cells and the relatively low current amplitude in the former suggest that T current function in BAMEC may be similar to that described for other cells that contain this channel, but its relative importance may be smaller. Moreover, T current transient opening, as shown by its activation/inactivation kinetics, suggests that it may not contribute much to raise [Ca2+]i. Hence, it has been proposed that its main role would be to elicit membrane potential oscillation in most cells (6) and current spikes in gonadotrophs (32).

However, our results suggest that BAMEC T currents may cause sustained Ca2+ influx, because their activation and inactivation curves in the present work overlap between -50 and 0 mV, creating a window of open channels (Fig. 8A). The open-channel probability at any membrane potential in the window is given by the product of the fraction of open channels, assumed to be equivalent to I/Imax.


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  		Fig. 8.   T current window currents in BAMEC. T current activation and inactivation curves from experiments in Fig. 4 that were run in 5 mM Ca2+-containing solution, when shown superimposed on same scale, show considerable overlap. We plotted current divided by maximum current (I/Imax), i.e., fraction of open channels, vs. membrane potential. Curves for data fitting were drawn using Eqs. 2 and 3. Continuous line shows activation and inactivation curves obtained in 5 mM Ca2+ (n = 6), whereas dotted line shows same curves obtained in 2.6 mM Ca2+ (n = 3). Product of fraction of channels open times any voltage gives probability of having channels open ("window current"). Continuous and dotted lines show window current in 5 and 2.6 mM Ca2+, respectively. Experimental data obtained in 5 mM Ca2+ are shown as means ± SE. Points obtained in 2.6 mM Ca2+ are shown only as means.

The open-channel probability reached 0.16 at -25 mV, (Fig. 8B), which is about three times higher than the maximum found in fibroblasts (16), and it was 0.06 at -40 mV. Hence, even under moderate depolarization, this high open-channel probability window may cause a sustained "window current" and, therefore, Ca2+ influx through T-type channels. Although these experiments were done in 5 mM Ca2+ [for comparison with those of Bossu et al. (8)], the results of three further experiments performed in 2.6 mM Ca2+ suggest that, under this more physiological condition, the same situation exists (Fig. 8); that is, no significant changes were observed on activation, inactivation, or window-current curves.

The higher T current in Ca2+- than in Ba2+-containing external solution suggests that BAMEC T-type channel has a higher permeability for Ca2+ than for Ba2+. Conversely, the L current was larger in Ba2+- than in Ca2+-containing medium. This may be due to either a higher L-type channel permeability for Ba2+ than for Ca2+ or to Ca2+-dependent inactivation (24). This opposite ionic selectivity of the two channels has been observed in other cells (19) and used as additional evidence to distinguish T- from L-type channels (16). Also, Imax of tail current in this work was bigger than that measured directly in response to a pulse.

L Current

Although the amplitude (13.1 pA) and the conductance per unit capacitance (182 pS/pF) of nonenhanced L currents were higher in this work than those previously reported for BAMEC (7, 8), they were much smaller than those of the L current measured in other cells in which the amplitude reached between 100 and 500 pA and the specific conductance was between 55 and 1,200 pS/pF (5, 13, 18, 28, 33). The parallelism of these two parameters in different cells supports the hypothesis that L current amplitude is mainly dependent on channel density (30).

We consider it improbable that the low amplitude of BAMEC L current, and its total absence from 35% of the cells we tested, was due to trypsin and collagenase used to digest the gland, because all the cells in this work were exposed to these enzymes and large L currents were recorded from many of them. Furthermore, no L current inhibition was observed when trypsin was added to the external solution at the same concentration used to digest the gland (not shown). Dephosphorylation was discarded as the cause of L current smallness (10), because we also measured small L currents with the perforated patch technique, which prevents cytosolic ATP dilution. Low L current amplitude in muscle cells was associated with a low opening probability (19).

The low threshold (-40 mV) that we measured for the isolated L current in BAMEC and the similarly low threshold reported for L current in beta -cells (28), ureter smooth muscle cells (25), and gonadotrophs (33) suggest that high threshold is not a sufficient identification marker for L-type channels. A special BAY K 8644-sensitive channel (SB channel) (7) that would lower L current threshold has not been reported in any of these cells.

BAMEC L current activation V1/2 (-18.0 mV) was similar to that previously reported for BAMEC (7) but more negative than the -12 mV found in gonadotrophs (33). The apparent lack of inactivation of BAMEC L-type channels during the 270-ms observation period in this work was similar to that observed in L-type channels of atrial cells (5), gonadotrophs (33), pancreatic beta -cells (28), ureter smooth muscle cells (25), and neurons (18) and differs from that of L-type channels in chromaffin cells (13), in which a relatively short inactivation time constant was measured.

The activation parameters and the apparent lack of inactivation, plus the response to DHPs and higher current in Ba2+- than in Ca2+-containing solutions, strongly suggest that the L current was carried through an L-type channel similar to those described in endocrine secretory cells (13, 28, 33).

Kinetics

The accelerated activation and inactivation of the T and L currents caused by depolarization, as shown in Fig. 2 of this work, are similar to VDCC kinetic changes with membrane potential as reported in chromaffin cells (13). Only one channel type may carry the L current that we measured in this work, because its rise with time was fitted by an exponential curve with a single time constant and very high r2. These results suggest that the proposed SB-channel current (7), if present, would be a very minor component of the whole cell L current.

Channel Pharmacology

The nifedipine concentration that inhibited L current in this work (1 µM) is in the concentration range found for L-type channel inhibition in smooth muscle cells at resting membrane potential (24). Lower nifedipine concentrations may be effective at more depolarized membrane potential levels, because DHP affinity has been shown to be higher for inactivated L-type channels (4). We could not measure nifedipine effect on depolarized BAMEC, because the small initial L current was further reduced by depolarization, making it hard to measure the remaining current.

Nifedipine inhibition of the native L current (nonenhanced by BAY K 8644) adds to the kinetic data to suggest that only one channel with pharmacological properties typical of L-type channel underlies the L current in BAMEC. On the other hand, nifedipine inhibition of T current was not unexpected because it has been reported for other cells (1, 36). However, affinity for nifedipine was stronger for L- than for T-type channel, because the former was totally blocked by 1/50 of the concentration required to block the latter (Fig. 6B).

The effect of BAY K 8644 was highly specific for the L current, because no effect on the T current was detected at the concentrations used. V1/2 was more negative, and a smaller voltage change was needed to cause an e-fold increase of the fraction of open channels in BAY K 8644-treated BAMEC than in the absence of the drug. These gating changes were similar to those caused by BAY K 8644 in chromaffin cells (13) and suggest that open-channel probability was increased by BAY K 8644 (13, 19).

Amiloride inhibition of BAMEC T current measured in this work (60%) was smaller than that previously reported for BAMEC (8). Although amiloride affinity for T-type channels is low (35), it has been used for identification purposes because it is the only drug with some inhibitory effect on this channel (31).

In conclusion, this work offers conclusive evidence that BAMEC contains two VDCCs, one of the T type and one L type. The properties of each of the single, isolated channel were in general like those of their counterparts in endocrine secretory cells (2, 13, 28, 33), neurons (11, 18), smooth muscle cells (25), and atrial cells (1, 5). This suggests that, as they do in these other types of cells, BAMEC VDCCs may participate in membrane potential and cytosolic Ca2+ regulation.

The very slow inactivation and low threshold of L-type channels in this work suggest that this channel has an important role in modulating [Ca2+]i rise under moderate depolarization. On the other hand, the fast activation/inactivation kinetics of the T-type channel suggest that its main role would be in causing membrane potential oscillation, such as that in gonadotrophs (32). In addition, the relatively high window current through this channel suggests a role in maintaining a high resting level of [Ca2+]i.

BAMEC VDCCs may be activated during adrenal medulla secretion, because chromaffin granules release catecholamines, ATP, peptides, K+, and Ca2+. K+, whose concentration in the granules in situ is as high as 83 mM (27), would induce BAMEC depolarization and thus reduce the driving force for Ca2+ influx (26). However, depolarization would also open VDCCs and cause [Ca2+]i rise, which would induce nitric oxide-mediated vasodilatation (21). The latter has been proposed as a mechanism to enhance the effect of adrenal secretion, because higher perfusion flow through the gland would shorten the distribution time of catecholamines (17).


    ACKNOWLEDGEMENTS

We thank Drs. Harvey Pollard, Mario Luxoro, and Eduardo Rojas for continuous support. We also thank the careful reading and criticism by Drs. M. Luxoro and D. Waring.


    FOOTNOTES

This work was funded by grant no. 1960302 from Fondo Nacional de Desarrollo Centifico y Tecnologico, Chile, and by the Cystic-Fibrosis Foundation.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. Vinet, Escuela de Quimica y Farmacia, Universidad de Valparaiso, Casilla 5001, Valparaiso, Chile (E-mail: rvinet{at}uv.cl).

Received 24 March 1998; accepted in final form 18 December 1998.


    REFERENCES
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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