Regulation of L-type Calcium Channels in Pituitary GH 4 C 1 Cells by Depolarization*

The neurosecretory anterior pituitary GH 4 C 1 cells ex- hibit the high voltage-activated dihydropyridine-sensi-tive L-type and the low voltage-activated T-type calcium currents. The activity of L-type calcium channels is tightly coupled to secretion of prolactin and other hormones in these cells. Depolarization induced by elevated extracellular K (cid:1) reduces the dihydropyridine ( (cid:1) )-[ 3 H]PN200-110 binding site density and 45 Ca 2 (cid:1) uptake in these cells (22). This study presents a functional analysis by electrophysiological techniques of short term regulation of L-type Ca 2 (cid:1) channels in GH 4 C 1 cells by membrane depolarization. Depolarization of GH 4 C 1 cells by 50 m M K (cid:1) rapidly reduced the barium currents through L-type calcium channels by (cid:1) 70% and shifted the voltage dependence of activation by 10 mV to more depolarized potentials. Down-regulation depended on the strength of the depolarizing stimuli and was reversible. The currents recovered to near control levels on repolarization. Down-regulation of the calcium channel currents was calcium-dependent but may not have been due to excessive accumulation of intracellular calcium. Membrane depolarization by voltage clamping and by veratridine also produced a down-regulation of calcium channel currents. The down-regulation of the currents

The neurosecretory anterior pituitary GH 4 C 1 cells exhibit the high voltage-activated dihydropyridine-sensitive L-type and the low voltage-activated T-type calcium currents. The activity of L-type calcium channels is tightly coupled to secretion of prolactin and other hormones in these cells. Depolarization induced by elevated extracellular K ؉ reduces the dihydropyridine (؉)-[ 3 H]PN200-110 binding site density and 45 Ca 2؉ uptake in these cells (22). This study presents a functional analysis by electrophysiological techniques of short term regulation of L-type Ca 2؉ channels in GH 4 C 1 cells by membrane depolarization. Depolarization of GH 4 C 1 cells by 50 mM K ؉ rapidly reduced the barium currents through L-type calcium channels by ϳ70% and shifted the voltage dependence of activation by 10 mV to more depolarized potentials. Down-regulation depended on the strength of the depolarizing stimuli and was reversible. The currents recovered to near control levels on repolarization. Down-regulation of the calcium channel currents was calcium-dependent but may not have been due to excessive accumulation of intracellular calcium. Membrane depolarization by voltage clamping and by veratridine also produced a down-regulation of calcium channel currents. The down-regulation of the currents hadanautocrinecomponent.Thisstudyrevealsacalciumdependent down-regulation of the L-type calcium channel currents by depolarization.
Voltage-gated Ca 2ϩ channels control the flux of Ca 2ϩ across the plasma membrane in a wide range of tissues and play a crucial role in many physiologic functions that include excitation-contraction and excitation-secretion coupling. Several major types of voltage-gated Ca 2ϩ channels have been identified that differ in their pharmacological and biophysical properties (1)(2)(3)(4). A revised classification scheme based on sequence, biophysical, and pharmacological properties has been proposed (5). L-type channels, which are sensitive to 1,4-dihydropyridine antagonists and activators, are distributed widely in the cardiovascular, central nervous, and neuroendocrine systems, and the Ca 2ϩ channel antagonists active at these channels are cardiovascular drugs of clinical significance (6).
These channels represent an important category of pharmacological receptors with discrete drug binding sites. The binding sites have been characterized, and their localization on the major ␣ 1 subunit has been well established (7,8). They are regulated by homologous and heterologous influences and are altered in their expression and function in numerous clinical and pathophysiological conditions (9 -13). Activity-dependent regulation is a common regulatory mechanism in a variety of receptor systems including ion channels, and electrical activity plays an important role in the regulation of ion channels. Thus, in many receptors of the G protein category, persistent activation by agonist ligands causes down-regulation of the receptor through a process of receptor internalization that involves an initial reversible phase in which the receptor can be recycled back to the plasma membrane and a later irreversible phase in which the receptor is degraded through the lysosomal machinery (14). In a number of systems, the membrane potential as an activating signal regulates both the function and numbers of ion channels. The membrane potential exerts two distinct categories of control over voltage-gated ion channels including Ca 2ϩ channels, both of which have the property of shutting down cellular Ca 2ϩ influx that in excess is a pathological signal. Inactivation is a rapid event with a time scale of milliseconds, and down-regulation is a slower event with a time frame of seconds to minutes or hours. Inactivation has both voltageand Ca 2ϩ -dependent mechanisms, and recovery is typically both rapid and complete after the removal of the depolarizing stimulus (15,16).
Voltage-gated Ca 2ϩ channels are also subject to a slower and more persistent regulation by depolarizing stimuli. Thus, in PC-12 cells (17,18) and chick retinal neurons (19) that contain L-type Ca 2ϩ channels, chronic depolarization with elevated extracellular K ϩ down-regulates the channel density measured by 1,4-dihydropyridine binding and channel function measured by 45 Ca 2ϩ uptake measurement. In rat myenteric neurons, persistent depolarization causes a slowly developing long term reduction in sustained 1,4-dihydropyridine-sensitive calcium channel current (20). Membrane depolarization reduces both low and high voltage-activated Ca 2ϩ currents in molluscan neurons (21). In the neuroendocrine GH 4 C 1 cells, short term depolarization (up to 2 h) with elevated K ϩ produces a decrease in L-type Ca 2ϩ channel density measured by 1,4-dihydropyridine binding and a corresponding decrease in channel function as measured by 45 Ca 2ϩ uptake into these cells. This decrease in the binding site density is ac-companied with an increase in affinity for the ligand as anticipated from voltage-dependent binding (22).
This work was designed to extend our previous study by characterizing electrophysiologically the short term regulation of L-type Ca 2ϩ channels in GH 4 C 1 cells by membrane depolar-ization. This cell line has a high density and a relatively pure population of Ca 2ϩ channels of the T-and L-types (23)(24)(25)(26), and the L-type channels are sensitive to 1,4-dihydropyridines (27). Cells of pituitary gland origin are chemically and electrically excitable, and the activation of L-type Ca 2ϩ channels is involved in the regulation of persistent secretion induced by depolarization and hormones such as thyrotropin-releasing hormone (28 -31). The activity and regulation of Ca 2ϩ channels is physiologically important for maintaining a homeostasis of hormone secretion. Ca 2ϩ influx into the cell subsequent to depolarization or hormonal response not only acts as a trigger for hormone release but also acts to inactivate the channels and prevent their sustained activation. This is analogous to the hormone-induced desensitization of receptors (32).

Materials
Cell Culture Medium F-10 medium and fetal bovine serum were purchased from Sigma. Horse serum was purchased from either Sigma or Life Technologies, Inc.

Chemicals
ATP was purchased from Sigma. BAPTA-AM 1 was purchased from Molecular Probes, Inc. (Eugene, OR).

Glass Electrodes
Electrodes were pulled from thin-walled borosilicate glass capillaries with an outer diameter of 1.2 mm (TW-120, World Precision Instruments, Sarasota, FL). The pipettes were pulled in two stages on a vertical electrode puller (Model 750, David Kopf Instruments, Tujunga, CA) and had a resistance of 8 -10 megohms when filled with internal recording solution.

Cell Culture
The rat anterior pituitary cell line, GH 4 C 1 , was obtained from Dr. Jane Chisholm (Bayer, Inc., West Haven, CT). Cells were maintained in a monolayer culture in Ham's F-10 medium supplemented with 15% horse serum and 2.5% fetal bovine serum at 37°C in a humidified incubator under an atmosphere containing 5% CO 2 . Cells were removed from flasks once a week with 0.05% trypsin and were plated either into flasks or sterile 35-mm Petri dishes (Corning) for electrophysiology experiments. The medium was changed every 2 days. Cells that were in culture for 2-6 days were used for experiments.

Electrophysiology
Whole cell voltage clamp experiments were carried out using an Axopatch 200 amplifier (Axon Instruments, Inc. Foster City, CA). Voltage commands were generated on a MacIntosh IIci computer through a 12-bit digital to analog converter using a MacADIOS II/16 board (GW Instruments, Somerville, MA). Data were acquired after 16-bit analog to digital conversion. Further analysis was performed with a program written in the laboratory by Dr. Satpal Singh in Think-C (Symantec Corporation, Cupertino, CA). Test currents were digitally sampled every 500 s, except during the examination of capacitative currents, which were sampled every 100 s. Currents were digitally corrected for linear leakage with respect to currents obtained at Ϫ60 mV from a holding potential of Ϫ40 mV. Current densities expressed as pA/pF were calculated by dividing the measured current with the cell capacitance to avoid the differences in current amplitude that arise because of the differences in cell size. Currents were filtered at 5 kHz with a Lowpass Bessel filter.
All voltage clamp traces represent an average data from several different cells. To generate the current voltage profiles (I-V plots), current amplitudes at the peak current value were measured for each voltage command. All data are shown as mean values Ϯ S.E.
Cells were rinsed with 1 ml of 5.8 mM K ϩ buffer and incubated for the desired time interval in the appropriate buffer at 37°C. At the end of the incubation, whole cell barium currents were elicited by step depolarizations to different potentials from a holding potential of Ϫ40 mV. T-type channels present in these cells inactivate by holding at Ϫ40 mV, and the L-type channels can then be studied selectively (27). The current recorded under these experimental conditions is inhibited by 1,4-dihydropyridines, which are specific L-type calcium channel blockers, in our experiments (33) done in parallel to the experiments reported here as well as in recordings made by several other groups (e.g. see Refs. [23][24][25]27).
Cells were continuously bathed in the appropriate Tyrode's solution until the whole cell configuration was achieved. Recording solution was locally applied to the cells by a gravity-driven perfusion system. Whole cell barium currents were recorded from a single cell in the dish. To avoid interference from a rundown of currents, the recordings were completed within 3-5 min of establishing the whole cell configuration unless otherwise specified. For clarity, only the currents to ϩ10 mV have been shown in all the current traces. Capacitative currents settled rapidly within 2 ms in these cells. Hence, the first and the last 3 ms have been removed from the current traces to avoid interference from capacitative currents.

RESULTS
Current Characteristics-GH 4 C 1 cells express L-type calcium channels. The currents through these channels have been previously characterized both by whole cell and single channel experiments. To establish a base line for studying the calcium channel regulation, the cells were bathed in resting Tyrode's solution (5.8 mM K ϩ ) for up to 2 h at 37°C after removing the culture medium. Barium currents were measured from the cells at the end of the incubation. The total barium current density measured after preincubation in Tyrode's solution at 37°C remained within 85-90% of the amplitude of the current observed without any preincubation. The current traces at ϩ10 mV after different incubation times are shown in Fig. 1A, and the current-voltage relationship is shown in Fig. 1B. A plot of normalized currents elicited by step depolarization to ϩ10 mV from a holding potential of Ϫ40 mV at different incubation times is shown in Fig. 1C. This shows that the preincubation of GH 4 C 1 cells in serum-free buffer at 37°C over 2-h time periods does not alter the stability of L-type calcium channel currents. However, once the whole cell configuration was established, the current rapidly ran down to smaller levels within 10 min. All recordings were therefore completed within 3-5 min after establishing the whole cell configuration at the end of any preincubation. The average L-type calcium channel current density obtained using 20 mM barium as an external charge carrier was 17-19 pA/pF. Cell capacitance measured during the experiment using a hyperpolarizing pulse to Ϫ60 mV from a holding potential of Ϫ40 mV was ϳ21-23 pF.
Effects of Cellular Depolarization on Currents-Cellular depolarization by preincubating the cells in depolarizing Tyrode's solution (50 mM K ϩ ) rapidly down-regulated the currents. The current density decreased from 17.39 Ϯ 0.82 pA/pF to 5.36 Ϯ 0.35 pA/pF within 5 min of depolarization, indicating a 69% decrease in the current amplitude ( Fig. 2A). The time course of down-regulation was established by preincubating cells in depolarizing Tyrode's solution (50 mM K ϩ ) for different time periods. Down-regulation reached a maximal level within 5 min, and longer durations of preincubations up to 1 h did not further increase the extent of down-regulation (Fig. 2, A and B). Depolarization-induced (50 mM K ϩ ) down-regulation was reversible. After 30 min of down-regulation by preincubating the cells in depolarizing 50 mM K ϩ -Tyrode's solution, the cells were allowed to recover in resting Tyrode's solution for different time periods (Fig. 2C). Currents elicited by pulses to ϩ10 mV are shown to depict the recovery from down-regulation. The cur-rents recovered to 80 -85% of control levels within 30 -45 min of repolarization (Fig. 2D). This also suggests that the phenomenon observed is down-regulation and not the calcium and voltage-dependent inactivation of calcium channels as the recovery from inactivation is relatively fast and complete within seconds.
Down-regulation of L-type calcium channel currents depended on the extent of depolarization. Increasing the concentration of K ϩ ions in the external buffer increased the depolarization stimulus and the extent of down-regulation. The resting cell potential measured in 5.8 mM K ϩ -Tyrode was  shown in A). Down-regulation of currents on depolarization was a reversible phenomenon, and the currents reverted close to control levels on repolarization. Cells were depolarized in 50 mM K ϩ buffer for 30 min and reverted to resting 5.8 mM K ϩ buffer for different time intervals. At the end of incubation, the whole cell currents were measured. C, current traces at Ϫ60 mV and ϩ10 mV in resting condition, depolarized condition, and during recovery. D, recovery from down-regulation is plotted as a function of repolarization time.
Ϫ65 to Ϫ70 mV and changed to approximately Ϫ15 mV in 50 mM K ϩ -Tyrode. The current density decreased with the increase in levels of extracellular potassium in the depolarizing buffer (Fig. 3, A and B). Down-regulation reached a maximum at 50 mM extracellular [K ϩ ], beyond which any further increase of [K ϩ ] did not produce a further decrease in the currents (Fig. 3C).
To investigate whether the down-regulation was because of cellular depolarization and not nonspecifically because of excessive potassium in the external depolarizing buffer, we explored alternate depolarization mechanisms. 50 mM K ϩ depolarizes the cell to approximately Ϫ15 mV. A similar change in membrane potential can be introduced in cells in resting Tyrode's buffer by voltage clamping them. If depolarization were to produce a down-regulation, this should produce a similar decrease in the current amplitude. We examined this hypothesis in an experiment where we measured the whole cell currents from a holding potential of Ϫ40 mV before and after voltage clamping the cell at Ϫ10 mV for 5 min in 5.8 mM K ϩ -Tyrode's solution. Voltage clamping at Ϫ10 mV for 5 min decreased the current amplitude in a manner similar to the downregulation observed with 50 mM K ϩ treatment (Fig. 3, D and E).
Another mode of depolarization also produced a down-regulation of L-type currents. Cells were exposed to the alkaloid veratridine, a sodium channel activator, for 30 min (34,35). Veratridine decreased the calcium channel currents in a concentration-dependent manner (concentration response data not shown). Treatment with 50 M veratridine for 30 min decreased the current density by 22%. The current density decreased from 15.87 Ϯ 0.61 pA/pF to 12.34 Ϯ 1.06 pA/pF (Fig. 4,  A and B). The decrease in the current density was also accompanied with an enhanced inactivation of the current. Thus, the different modes of depolarization had different effects on downregulation. Normalized current-voltage curves for cells depolarized with 50 mM K ϩ or with veratridine are shown in Fig. 4, C and D. Cells depolarized with elevated extracellular [K ϩ ] shifted the peak current to ϩ20 mV from ϩ10 mV, but cells depolarized with veratridine did not show any voltage shift in peak current. Over the 200-ms pulse duration, cells depolarized with elevated extracellular [K ϩ ] did not show any kinetic changes, but cells depolarized with veratridine showed a faster inactivation of currents (Fig. 4A).

Ca 2ϩ Dependence of K ϩ Depolarization-induced Down-regulation-The presence of [Ca 2ϩ ] in the extracellular depolarizing buffer was essential for the down-regulation of currents.
The removal of [Ca 2ϩ ] from depolarizing buffer to the contaminating levels in double distilled water inhibited the downregulation, and the current density remained the same as in the cells incubated in normal Tyrode's buffer. The current traces at ϩ10 mV and the current-voltage relationships in depolarizing buffers having different Ca 2ϩ concentrations are shown in Fig. 5, A and B. The down-regulation was enhanced with an increasing concentration of Ca 2ϩ in the depolarizing 50 mM K ϩ buffer (Fig. 5C).
BAPTA-AM, a cell permeant analog of BAPTA, chelates intracellular calcium after the hydrolysis of the acetomethoxyfunctional group (AM) inside the cell. Preloading the cells with 25 M BAPTA-AM before depolarization did not inhibit the down-regulation caused by depolarization (Fig. 5, D and E). This suggests that the down-regulation may not be attributed to the accumulation of excess Ca 2ϩ inside the cell. It might also be because of the fact that the molecular determinants of the Ca 2ϩ channel responsible for the down-regulation may be located in microdomains inaccessible to BAPTA-AM.
Autocrine Component of Down-regulation-Depolarizationinduced down-regulation appeared to have an autocrine component. When the cells were bathed in a stream of depolarizing 50 mM K ϩ buffer as opposed to stagnant buffer, a part of the down-regulation was relieved (Fig. 6). This suggests that hormones or other factors that are released by these anterior pituitary cells into the surrounding medium on depolarization may act on the channels and inhibit their activity. This could be prevented in the stream of depolarizing buffer if the factors are washed out from the vicinity of the cells. However, a substantial component of down-regulation still persisted that was independent of the factors released from the cell, suggesting alternate mechanisms for the down-regulation of the remaining component of the L-type current. DISCUSSION Membrane potential is an important regulator of ion channel activity. Whereas cellular depolarization on one hand is an important physiologic signal, it also serves as a pathophysiologic trigger. Membrane depolarization can bring about both of up to 2 h decreases the (ϩ)-[ 3 H]PN200-110 binding site density by 10-fold accompanied by a 20-fold increase in drug affinity (22). In all of these studies the regulation depended on the presence of extracellular calcium. Cellular depolarization can also cause an up-regulation of the surface channels as well. Depolarization of chick myotubes has been shown to trigger the appearance of (ϩ)-[ 3 H]PN200-110 binding sites (37). Binding studies with the radioligand 125 I--CgTx on subcellular fractions show that the intracellular pool of neuronal N-type Ca 2ϩ channels in PC-12-251 cells and IMR-32 cells constituted 60 -80% of the total calcium channels (38,39). This intracellular pool of channels is recruited to the cell membrane on depolarization with KCl. The cell surface 125 I--CgTx binding sites increased by Ͼ200% within 10 min of depolarization with 55 mM KCl. This transiently decreased over a period of 1 h and then stabilized at a level that was higher than the control level.
In this study, we show an electrophysiologic correlation of the calcium channel regulation in pituitary GH 4 C 1 cells by short term membrane depolarization. GH 4 C 1 and GH 3 pituitary clonal cells express L-type calcium channels. Currents through these channels have been characterized both by whole cell and single channel experiments (23-25, 40, 41). Short term regulation studies using radioligand binding were also done on these cells (22). Hence, we chose these cells as a model system to study the functional aspects of calcium channel regulation electrophysiologically.
We show that cellular depolarization with elevated extracellular potassium produced an ϳ70% decrease in the barium current density in GH 4 C 1 cells. This process is very rapid, and the decrease in currents is evident with depolarizations as short as 5 min. Even though the earlier studies involving (ϩ)-[ 3 H]PN200-110 binding have evaluated the regulation of the channel activity for periods shorter than half an hour, these studies may not be accurate because of the limitation posed by equilibration of the ligand with receptor. In addition and unlike the (ϩ)-[ 3 H]PN200-110 binding experiments, the electrophysiologic experiments evaluate the channel activity in the absence of any exogenous 1,4-dihydropyridines, thus precluding any possible additional regulation of the channel by the antagonist itself. Our study also shows that the down-regulation of the L-type calcium channels by depolarization is a reversible phenomenon, and that the currents recover to near control levels when the cells are placed in normal 5.8 mM K ϩ -Tyrode. The extent of down-regulation depended on the depolarizing stimuli and increased as the membrane potential was raised by increasing the extracellular potassium. Qualitatively, no appreciable changes in the channel kinetics were observed between the control cells and the cells depolarized with 50 mM K ϩ using barium as charge carrier. However, the down-regulation was accompanied with a ϩ10 mV shift in the voltage dependence of activation to a more depolarized potential. Depolarization of cells by voltage clamping at Ϫ10 mV also produced a downregulation similar to depolarization produced by 50 mM K ϩ . The depolarization of cells with veratridine, a sodium channel activator, also produced a decrease in the channel currents. However, this decrease is not as dramatic as seen with 50 mM K ϩ and also does not shift the voltage dependence of activation.
In our experiments, we observed that the down-regulation of the channel activity could be partially relieved by depolarizing the cells in a stream of depolarizing 50 mM K ϩ buffer rather than in the stagnant buffer. This finding suggests that a factor or factors may be released by these cells on depolarization with 50 mM K ϩ to act on the cell in a feedback manner under the conditions of sustained depolarization and to inhibit the channel activity. In a stream of the buffer, these factors are removed, which prevents their feedback activity on the cells. Hormone-induced modulation of channels is inherent to neurosecretory cells. The activation of calcium channels is essential for the release of hormones like prolactin, GH, and adrenocorticotropic hormone from the anterior pituitary cells (42,43). Physiologically, this can be achieved by the action of hormones like thyrotropin-releasing hormone. Depolarization with elevated potassium also leads to hormonal release in in vitro assays. On the other hand, certain hormones like somatostatin inhibit calcium channel activity and suppress the release of the pituitary hormones. This autocrine regulation of calcium channels is thought to be mediated through G proteins-G o in pituitary cells (29,42,44).
The presence of extracellular calcium ions was essential for the down-regulation, indicating that it is a calcium-dependent process. Elimination of Ca 2ϩ from depolarizing buffer abolished the down-regulation. Here we show that as the calcium concentration is increased to physiologic levels in the depolarizing buffer, the down-regulation is gradually enhanced. However, the preincubation of cells with 25 M BAPTA-AM, a cell permeant calcium chelator before depolarization, failed to relieve the down-regulation. This suggests two possibilities: (a) even though BAPTA-AM was hydrolyzed to BAPTA inside the cell, there was a constant flux of Ca 2ϩ from the extracellular medium, saturating BAPTA and thereby preventing its action, and (b) the site of action of Ca 2ϩ on the channel to produce regulation is very close to the channel pore and is inaccessible to intracellular chelators.
Calcium-and voltage-dependent inactivation of L-type calcium channels are well studied phenomena (16,(45)(46)(47)(48). In our study, the depolarizing buffer (50 mM K ϩ ) contained 2 mM Ca 2ϩ and could induce voltage-and/or Ca 2ϩ -dependent inactivation of the channel. However, the phenomenon observed in our study is not a voltage-dependent inactivation, because it does not occur with depolarization in the absence of Ca 2ϩ in the depolarizing buffer. In addition, the recovery from both voltage-and Ca 2ϩ -dependent inactivation is relatively fast and occurs within a few seconds for fast inactivation and within 1-2 min for slow inactivation (47)(48)(49). On the other hand, the recovery from depolarization-induced regulation occurred over a period of 30 -45 min in our studies, suggesting that it is down-regulation and not inactivation. This finding is also consistent with the decrease in the binding site density observed in response to depolarization in the earlier experiments (22).
In conclusion, we present evidence for depolarization-induced down-regulation of currents through L-type calcium channels in GH 4 C 1 cells. This is a reversible phenomenon. The down-regulation of L-type calcium channels is calcium-dependent and requires the presence of external calcium. The data suggest but do not prove the direct action of calcium on calcium channel to induce the down-regulation. The possible mechanisms for down-regulation may involve an autocrine component.