0013-7227/92/1306-3411$03.00/O Endocrinology Copyright 0 1992 by The Endocrine Society

Vol. 130, No. 6 Printed

Electrophysiological Properties Gonadotrope Lineage MARTHA

M. BOSMA

AND

BERTIL

Department of Physiology and Biophysics, Seattle, Washington 98195

of a Cell Line

in U.S.A.

of the

HILLE University

of

Washington

ABSTRACT. The role of ion channels in the secretion of gonadotropins from anterior pituitary gonadotropes has been difficult to study at the single cell level because the cells are difficult to distinguish from other pituitary cell types. Recently, a cell line, aT3-1, has been generated that makes and secretes the a-subunit of gonadotropins. These cells have GnRH receptors, but not TRH receptors, and are, thus, specific to the gonadotrope lineage. We have used the patch clamp technique to investigate the types of ion channels expressed in (uT3-1 cells and to test for electrophysiological responses to GnRH and a phorbol ester. These cells express TTX-sensitive sodium chan-

Mail

Stop SJ-40,

nels with rapid kinetics, several types of potassium channels, including Ca*+-sensitive ones, and two types of calcium channels. The currents through calcium channels are augmented by application of 100 nM GnRH or 10 nM phorbol 12-myristate 13acetate, a phorbol ester. The augmentation by GnRH and phorbol 12-myristate 13-acetate is consistent with other reports that a portion of stimulated gonadotropin release is dependent on external calcium and sensitive to block by dihydropyridine antagonists. Thus, this cell line may be useful for studies of mechanisms underlying responses to GnRH. (Endocrinology 130: 3411-3420,1992)

G

nRH secreted in a pulsatile manner from hypothalamic neurons acts at GnRH receptors on pituitary gonadotropes to elicit secretion of the gonadotropins LH and FSH. The cellular signals underlying GnRH regulation of secretion from gonadotropes have been investigated at the biochemical level (1). There are two phases of GnRH-stimulated LH release. The first is independent of extracellular Ca’+, lasts 2-3 min, and is correlated with activation of phospholipase-C, generation of inositol 1,4,5trisphosphate (IP,), and a rise in internal Ca*+ (2, 3). The second phase lasts for 8-10 min or longer in the presence of a continuous stimulation by GnRH; it corresponds to a slower and smaller elevation of internal Ca*+ and is abolished by removal of external Ca*+ or application of dihydropyridine Ca channel antagonists. Some evidence also implicates activation of protein kinase-C (PKC) in the second phase of LH secretion. This enzyme is activated by diacylglycerols produced by the action of phospholipase-C at the same time as IP3 is made (4). Exogenous stimulators of PKC have been shown to induce LH release, and a major portion of this release is sensitive to external calcium (5). It has been suggested that voltage-gated Ca channels are affected Received November 13, 1991. Address all correspondence and requests for reprints Bosma, Department- of Pharmacology, University of School of Medicine. Mail Stan SJ-30. Seattle. Washington * This work was supported by NiH Grants NS-08174, and NS-07097 and a Research Award from the McKnight

School of Medicine,

(2).

Electrophysiological experiments have been less common than biochemical ones because gonadotropes are difficult to identify; only a small percentage of the cells obtained from the anterior pituitary are gonadotropes (6). Voltage-gated ion channels have been studied in several kinds of gonadotropes: ovine pars tuberalis secretory cells, which are almost exclusively gonadotropes (7, 8); rat anterior pituitary preparations enriched for gonadotropes (9-12); and identified rat gonadotropes (12, 13). However, few of these studies have asked what role the channels might play in gonadotropin secretion. Recently, a cell line was generated by targeted oncogenesis, fusing the gene for the simian virus-40 T-antigen to the promoter for the glycoprotein a-chain, injecting the construct into mouse eggs to produce transgenic mice, and allowing tumors to develop (14). One such tumor gave rise to the (uT3-1 cell line, which has GnRH receptors, responds to GnRH with an increase in IP9 turnover and PKC translocation, and seems to be of the gonadotrope lineage (15). Our study describes the ion channels of (YT~-1 cells and demonstrates modulation of their Ca channels by GnRH and phorbol ester. Materials

to: Dr. M. Washington 98195. ND-12629, Foundation.

and Methods

Cell culture

(YT~-1 cells were kindly given to us by Dr. Pamela Mellon. They were grown in Dulbecco’s Modified Eagle’s Medium sup3411

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 09:54 For personal use only. No other uses without permission. . All rights reserved.

3412

GONADOTROPE

CELL

LINE

plemented with 5% horse serum and 5% fetal calf serum, with 1% penicillin-streptomycin. Cell culture reagents were obtained from Gibco (Grand Island, NY). Stock tissue culture dishes (100 mm) were split every 3-4 days when cells reached 50% confluency. Cells were grown at low density on 35mm culture dishes for electrophysiology experiments. Electrophysiological

recording

Whole cell electrophysiological experiments were performed l-2 days after the cells were plated. Dishes were placed on the stage of a Nikon Diaphot inverted microscope (Meridian Instrument Corp., Kent, WA) and viewed under Hoffman Modulation Contrast optics (Modulation Optics, Greenvale, NY). The cells were round or spindle-shaped and sometimes had small processes. Patch-clamp measurements were made only on cells that did not touch each other and had no processes to avoid possible cell to cell coupling artifacts and maintain good space clamp. Tight seals between the recording pipette and cell could be obtained in most cases, although some cells from later passage numbers (more than 10 passages after thawing) were difficult to work with. Usually, if seals could be formed, the patch could be broken to obtain whole cell recording. Recordings could last for up to 30 min, although the more usual duration was lo-12 min. The measured membrane capacitance of the cells (an*electrical measure of their membrane surface area) was 6 + 1.5 picofarad (pF; mean + SD; equivalent to 600 pm* of membrane), with a range of 2.5-12 pF (n = 245). Capacitance was routinely determined at the beginning and end of the experiment and did not change. Currents were recorded using a List EPC-7 patch clamp amplifier (Medical Systems Corp., Greenvale, NY). Voltage pulses were generated and currents recorded in real time, using the BASIC-FASTLAB system of software and hardware (Indec Systems, Sunnyvale, CA). In some experiments, leak and capacity currents were subtracted automatically using a P/4 pulse protocol (16). A chamber was constructed inside the tissue culture dish to reduce total bath volume by sealing a Sylgard (Dow Corp., Midland, MI) ring to the bottom of the dish. The total bath volume was 150-200 ~1. Bath solutions were exchanged by perfusing 15-25 vol solution through the chamber or changing the input line on a continuous flow system. The results obtained were similar in the two conditions. All reported membrane potentials were corrected for junction potentials of -3 to -14 mV between the pipette and bath solutions. To indicate the range of single cell properties encountered in the actively dividing and growing population of this new cell type, measurements are given as the mean f SD, except as indicated. Statistics were calculated using Student’s two-tailed t test; significance (P) is given in Results. Solutions For measuring Na’ currents, the solutions were: external: 150 mM NaCl, 2.5 mM KCl, 1 mM MgC12, 10 mM HEPES, 8 mM glucose, and 3 mM CaCl, (for 0 Ca experiments, the 3 mM CaC12 was replaced by 3 mM MgCl& internal: 120 mM CsCl, 3 mM MgCl*, 10 mM EGTA, 25 mM HEPES, 2.5 mM ATP, 0.2 mM GTP, and 0.1 mM leupeptin. For recording K’ currents, the solutions were: external: 2.5 mM KCl, 150 mM N-methylD-glucamine (NMDG), 1 mM MgC&, 3 mM CaCL, 10 mM

ELECTROPHYSIOLOGY

Endo. Voll30.

1992 No 6

HEPES, and 8 mM glucose (for 0 Ca experiments, the 3 mM CaC& was replaced by 3 mM MgCl,); internal: 100 mM K aspartate, 20 mM KCl, 25 mM HEPES, 10 mM EGTA, 3 mM MgCl*, 2.5 mM ATP, 0.2 mM GTP, and 0.1 mM leupeptin. In some experiments, the EGTA concentration was reduced from 10 to 0.1 or 0 mM, and the K aspartate concentration was increased to keep the osmolarity constant. For K’ selectivity experiments, K’ was increased, with a concomitant decrease in NMDG. For recording Ba2+ currents in Ca channels, the solutions were: external: 30 mM BaC12, 109 mM NMDG, 2.5 mM KCl, 1 mM MgC12, 10 mM HEPES, and 8 mM glucose (for permeability-sequence experiments, the BaCl, was replaced by equimolar SrC12 or CaCl,); internal: 120 mM CsCl, 3 mM MgC12, 25 mM HEPES, 10 mM EGTA, 2.5 mM ATP, 0.2 mM GTP, and 0.1 mM leupeptin. All solutions were titrated to pH 7.4 using NaOH or HCl for external solutions and CsOH or KOH for internal solutions, as appropriate. GnRH was obtained from Peninsula Laboratories (Belmont, CA), and 100 PM stock SOlution was stored at -20 C; phorbol 12-myristate 13-acetate (PMA), staurosporine, tetraethylammonium, 4-aminopyridine, and most reagent salts were obtained from Sigma. PN200-110 was kindly given to us by Dr. W. Catterall, Department of Pharmacology, University of Washington. All experiments were performed at 22-24 C.

Results Using whole cell voltage clamp, we identified components of current carried in different voltage-gated ionic channels by manipulating the ionic conditions and voltage-clamp command pulses and adding channel blockers. Sodium

channels

Ionic conditions appropriate for looking for currents in Na channels include 150 mM Na+ in the bathing solution and 120 mM Cs+ in the (internal) pipette solution (to block current in K channels). Under such conditions, depolarizing voltage steps from a negative holding potential elicit fast transient inward currents in the aT3-1 cells (Fig. 1A). They have many of the characteristics of Na+ currents; appreciable current begins to appear at -45 mV, and the current reaches a maximum between -15 and -5 mV (Fig. 1B). Activation and inactivation are complete within a few milliseconds near 0 mV. The fast inward current is not seen when Na+ in the bath is replaced by NMDG. The current is blocked by tetrodotoxin (TTX) in the bath (Fig. lC), with a Ki of 39 nM and a Hill coefficient of 1.05 (n = 13). At 1000 nM TTX, no fast transient inward current remained. The Na channels were readily inactivated by lOO-msec depolarizing prepulses. Figure 1D shows that the voltage dependence of this steady state inactivation is steep and has a midpoint at -85 + 5 mV (n = 13). Replacing the 3 mM Ca2+of the bath with 3 mM Mg2+ did not appreciably affect the amplitude, activation (Fig. lB), or inactivation (Fig. 1D) of the Na+ current. On the average, the maxi-

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 09:54 For personal use only. No other uses without permission. . All rights reserved.

GONADOTROPE

CELL

LINE

FIG. 1. Sodium current in otT3-1 cells. Currents are recorded using the P/4 subtraction procedure. A, Currents elicited by stepping for 12.5 msec from a holding potential of -125 mV to test potentials between -45 and -5 mV. A diagram of the voltage steps is shown aboue. B, Peak current-voltage relation for a different cell, recorded with (0) and without (0) 3 mM external Ca’+. C, Block by TTX. Currents recorded at a test potential of -5 mV, showing control (C), 25 nM TTX (+), and 100 nM TTX (++). D, Steady state inactivation curve, with lOO-msec prepulses to the indicated potentials. The peak Na’ current is recorded during a step to -5 mV from a cell bathed in 3 IIIM external Ca2+ solution (0) or 0 mM external Ca2+ solution (Cl). The solid line is a fit of the Boltzmann relation I/I,,, = l.l/(l + exp[(E - EiJ/k]}, with values of El,* = -92 mV and k = 14 mV.

ELECTROPHYSIOLOGY

0.8

2 ms

1

3413

I/lmax

50 pA 0.4

0.0 -150

mum inward Na+ current was -710 + 617 picoampere (PA; n = 31). The density of current normalized to the surface capacitance was -116 + 82 pA/pF. Calcium channels

We looked at Ba2+ current in Ca channels using a bath solution containing 30 mM of the permeant Ba2+ ion. To reduce Na+ currents, Na+ ions were removed from the bath, and to reduce KC currents, there was Cs+ and no K+ in the pipette. Depolarizing steps from a holding potential of -94 mV elicited slowly developing and slowly inactivating inward currents (Fig. 2A). They had the characteristics of inward Ba*+ currents in a combination of conventional low voltage-activated (LVA; also known as T-type Ca channels) and high voltage-activated (HVA; also known as L-type Ca channels) Ca channels. The inward current began to appear negative to -50 mV and reached a maximum near -5 mV (Fig. 2B, 0). This voltage dependence could be altered by reducing the holding potential to -54 mV (Fig. 2B, Cl). Activation of inward current then required depolarization to about -30 mV, and the maximum current was reached at 5 mV. Furthermore, with the less negative holding potential, the time course of the current was changed. The transient component was gone, and only a noninactivating sustained component remained (Fig. 2C). The dashed line in Fig. 2B (0; obtained by subtraction of the other two measurements) shows the current-voltage relation of the component of current that is lost by changing the holding potential from -94 to -54 mV. These results suggest that LVA Ca channels generate a transient current that begins to activate near -50 mV and becomes inactivated

-50

-100

Prepulse

Potential

0 (mV)

when the holding potential is -54 mV. In addition, HVA channels that are not inactivated at holding potentials of -54 mV begin to activate near -30 mV and generate sustained current in Ba2+ solutions. The two channel types were differentially expressed in different cells, and in a few cells only the transient or the sustained component of current was present. Even though the recording pipette contained ATP, leupeptin, and GTP, the HVA channels were subject to rundown or washout during long recordings (17), i.e. the peak HVA current gradually fell during the course of dialysis of the recorded cell. The rate of rundown was on the order of 50% in 15 min. The LVA channels were less sensitive to dialysis and could be studied conveniently after the rundown of HVA channels. On the average, the peak Ba2+ current measured at 6 mV from a holding potential of -94 mV was -99 f 54 PA/cell, with a current density of -19 f 10 pA/pF (n = 148). The cell shown in Fig. 2 had a relatively large transient component and was chosen to demonstrate the effect of holding potential on the two types of channels. Activation of the transient component was evident at -44 mV and peaked at -14 mV when isolated from the sustained component (Fig. 2B, 0). The sustained current, which peaked at 6 mV, was usually smaller (-66 + 45 PA/cell; -13 + 9 pA/pF). When the holding potential was -54 mV, the sustained current was slightly reduced (-51 + 22 PA/cell; -10 + 4 pA/pF; n = 53), and the transient component was absent. The transient component had a fairly steep dependence on holding potential, beginning to inactivate at about -84 mV, and was 50% inactivated at -71 mV. The sustained current showed

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 09:54 For personal use only. No other uses without permission. . All rights reserved.

3414

GONADOTROPE

CELL

LINE

ELECTROPHYSIOLOGY

Endo. Voll30.

1992 No 6

C -44

FIG. 2. Two types of Ca channels in (uT3-1 cells. A, Current traces recorded in 30 IIIM Ba2+ external solution, stepping for 90 msec from a holding potential of -94 mV to test potentials between -54 and -4 mV. B, Current-voltage relation for Ba2+ current. 0, Peak of total current (in first 30 msec) from a holding potential of -94 mV; Cl, sustained current (averaged for the last 30 msec) from a holding potential of -54 mV; 0, the isolated peak transient component obtained by subtracting currents recorded when holding at -54 mV from those when holding at -94 mV. C, Comparison of current traces recorded using the -94mV (0) or -54-mV (0) holding potential. The three panels show steps to -44, -24, or -4 mV.

little dependence on holding potential up to -54 mV. The two Ca channels could also be distinguished on the basis of ionic selectivity and pharmacology. When the current carrier was changed, the sizes of both currents followed the sequence Ba2+ > Sr2+ > Ca*+; the amplitude ratios for the LVA component were 1.9:1.3:1, and those for the HVA component were 8:4:1. The HVA channel was sensitive to the dihydropyridine blocker PN200-110, with half-block at 10 nM (n = 2), 60% block at 50 nM (n = 2) and total block at 100 nM (n = 3). The LVA channel was not as sensitive. The top pair of traces in Fig. 3A shows Ba*+ current elicited from a holding potential of -54 mV by stepping to 6 mV, a paradigm that isolates the sustained current. Under control conditions (Fig. 3, 0), approximately 100 pA of current is elicited. Upon application of 100 nM PN200-110, the current is completely blocked (Fig. 3A, n ). Holding at -94 mV and stepping to the same potential (bottom pair of traces) activates the transient current, which is still present in 100 nM PN200-110 (Fig. 3A, n ). The channels had different sensitivities to Cd*+, with the HVA channel being completely blocked by 20 PM Cd*+ (n = 2), and the LVA channel being only partially blocked by 100 PM (n = 3; mean block, 47%). The top pair of traces in Fig. 3B show the complete block of isolated sustained current (holding potential, -54 mV) in 100 PM Cd*+, while most of the transient current still remained (bottom traces, n ). At 500 ~.LM Cd*+, the LVA channel was 92% blocked (n = 5). Potassium channels

Ionic conditions useful for studying K’ currents include filling the pipette with K aspartate and replacing

-4

mV

mV

B, CdZ+

A PNZOO-110

I

Vh=-54

mV

Vh=-94

mV

0 FIG. 3. Differential block of sustained and transient Ca channels by a dihydropyridine antagonist and Cd ‘+ . A, The top pair of traces shows currents elicited from a holding potential of -54 mV to a potential of 6 mV. 0, Control conditions; H, after bath application of 100 nM PN200-110. The bottom pair of truces shows voltage steps to the same potential, but from a holding potential of -94 mV, before and after PN200-110. B, In a different cell, current traces before and after bath application of 100 pM Cd” are shown. Symbols and voltage paradigms are explained in A. Calibration bars are 40 ms and 50 pA (A) or 30 pA (B).

all of the extracellular Na+ ion with the larger impermeant cation NMDG to eliminate Na’ currents. Under these conditions, depolarizing test pulses from negative holding potentials (typically -98 mV) elicited several components of outward current. As would be expected

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 09:54 For personal use only. No other uses without permission. . All rights reserved.

GONADOTROPE

CELL

LINE

for currents carried by K+ ions, the reversal potential for this current was negative to -85 mV in the standard 2.5 IIIM K bath solution and changed to -43 + 4 mV (n = 8) when the K’ concentration was raised to 25 mM (the Nernst equation predicts an equilibrium potential for K+, Ex, of -41.7 mV for 132.5 mM K+ inside and 25 mM outside). Small depolarizations from -98 mV elicited a slowly rising outward current that inactivated only slightly during a 180-msec test pulse (e.g. the -8 mV trace in Fig. 4A). Large depolarizations elicited a more rapidly activating current, which reached a peak in less than 10 msec and then decayed partially during the test pulse (e.g. the 52 mV trace in Fig. 4A). From a holding potential of -98 mV, the peak current at 52 mV was 1180 + 500 pA (n = 71), with a current density of 200 f 83 pA/pF (n = 66), and the sustained level after 180 msec was 685 + 370 pA, with a density of 117 + 71 pA/pF. Is this kinetic behavior due to a single type of channel that inactivates only partly at positive potentials, or is it due to a combination of channels, some with transient and others with sustained gating kinetics? Several observations favor the latter explanation. The relative sizes of transient and sustained K+ currents varied from cell

A

+52 mV

n

ELECTROPHYSIOLOGY

3415

to cell, with the sustained level ranging from 25-90% of the peak level at 52 mV. There was a difference in sensitivity to holding potential; holding the cell at -58 mV instead of -98 mV diminished the transient current much more than the sustained current (Fig. 4B), and subtracting the two records gives a clearer picture of the transient component (Fig. 4C). The transient and sustained currents developed at different test potentials; at 0 mV, the conductance of the sustained current was almost fully activated, whereas the conductance of the peak current was only half of its maximal value (Fig. 4D). The peak current had a steady state half-inactivation point of -74 mV (measured after 5-set prepulses in three cells). However, the sustained component inactivated so slowly (two-pulse experiments showed that at least 30 set were required to remove inactivation) that it was not feasible to determine its inactivation curve on the time scale of these experiments. The currents could also be separated by pharmacological means. The peak K+ current was more than 75% blocked by 4-aminopyridine (4-AP) at a concentration of 0.5-l mM, whereas the sustained component was less than 25% blocked (Fig. 4E). Both components were strongly blocked by 10 mM tetraethylammonium (TEA; Fig. 4F). Both components

B Vh=-98

mV WI=-58

FIG. 4. Multiple potassium channels in c~T3-1 cells. The pipette solution includes 10 mM EGTA with no added Ca2+. A and B, Currents recorded at the indicated potentials from a holding potential of -98 mV (A) or -58 mV (B). C, Difference currents made by subtracting traces in B from those in A to show more clearly the transignt current component that is inactivated when the holding potential is -58 mV. D, Peak and sustained conductance-voltage curves for a different cell from a holding potential of -98 mV. The solid lines are fitted Boltzmann relations, with midpoints of -2 mV for the peak current and -18 mV for the sustained current. E, Block of transient current by 1 mM 4-AP, showing the control trace (C), exposure to 4-AP (4-AP), and recovery after wash-off of 4-AP (R). F, Block by 10 mM TEA, showing control trace (C), during TEA exposure (TEA), and recovery after wash-off of TEA (R).

mV

-8 mv -2Em3

-

f

C

peak

-50

-30

-10

10

50

(I$

Test potential

E

30mr

F

30mr

200 pA

4-AP

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 09:54 For personal use only. No other uses without permission. . All rights reserved.

200 PA

GONADOTROPE

3416

CELL

LINE

of K+ current decreased gradually during the course of an experiment as if some substance(s) essential to the maintenance of the current was being washed out (analogous to the washout of Ca*+ currents). The rate of this rundown of current was different in each cell, probably due to variation in pipette and cell sizes, but, on the average, it was 4.6 times faster for the peak component than for the sustained component in the same cell (n = 17). Some kinds of K channels open in response to a rise in intracellular free Ca*+ and, therefore, may become activated during depolarizing steps that open voltagegated Ca channels. Evidence that only a small and variable part of the K’ current in aT3-1 cells is Ca*+ sensitive came from experiments reducing the external or internal Ca*+. When the recording pipette contained 10 mM EGTA to prevent changes in intracellular Ca2+, none of the K’ current was sensitive to removal of extracellular Ca*‘, but when the EGTA in the pipette was reduced to 0.1 or 0 mM to avoid buffering intracellular Ca*+, a small component of sustained K+ current could be reversibly eliminated by removal of extracellular Ca*+. This Ca*+-sensitive current is shown in Fig. 5A (inset) as the difference (+) between records taken in 3 InM (0) and 0 InM external Ca*+ solutions. The current-voltage relation for the difference current is plotted in Fig. 5A (+). The difference currents obtained when external Ca*’ was removed ranged from -9 to 64% (mean, 21%) of the total outward current at a test potential of 2 mV and from 7-79% (mean, 27%; n = 11) at a test potential of 42 mV. To further characterize the Ca*+-dependent channel, we used apamin, a bee venom toxin that blocks the small conductance type of Ca*+-sensitive K’ channels (18). Of seven cells with Ca*+-sensitive K+ current, only two had convincing block with 0.5 or 1 PM apamin. One of these cells is shown in Fig. 5B. The top trace is the current in the control solution. Apamin suppressed a portion of this K’ current, and there was very little recovery after a rinse with control solution. Removal of extracellular Ca*+ reduced the K’ current a bit more. We conclude that Ca*+-sensitive K’ channels are only minor contributors to outward currents in this cell line and, furthermore, that only a minority of the cells express the small conductance apamin-sensitive type of channel. Actions of peptide hormones

We examined the effects of GnRH, the physiological releasing hormone for pituitary gonadotropes, on currents in Ca and K channels. All tests on K+ current proved negative, whether the pipette contained 10, 0.1, or 0 mM EGTA (n = 9). However, in 32 of 46 cells, GnRH augmented the Ba*+ current (Fig. 6A), affecting LVA and HVA channels about equally. The modulation was first studied in 28 cells held at -94 mV. When 100

ELECTROPHYSIOLOGY

Endo. Voll30.

1992 No 6

6OOr I(PA)

-40

-20

0

Test

potential

20

40

60

(mV)

B 50

DA

FIG. 5. Evidence for Ca*+-stimulated K channels in aT3-1 cells. Recorded with 0.1 mM EGTA in pipette and holding at -98 mV. A, Current-voltage relation for sustained K+ current (averaged for the last 30 msec of pulse) in 3 mM external Ca2+ (O), 0 mM Ca2+ (O), and the difference current (4- -+). The inset shows current traces from a different cell, during steps to 52 mV, with 3 mM Ca2+ (O), 0 mM Ca*+, and the difference current (+; trace with 0 mM Ca*+ subtracted from trace with 3 mM Ca”). B, Block by apamin. Currents are elicited by stepping to a test potential of 32 mV from a holding potential of -98 mV. Traces are (top to bottom): control solution (3 mM Ca*+), 0.5 jtM apamin, rinse for 3 min with control (no apamin) solution, and after 3 min with 0 Ca’+. nM GnRH

was added to the bathing medium, the peak Ba*+ current at 6 mV was increased, on the average, by 15% (f14; P < 0.001; range, 2-59%), and the sustained current was increased by 18% (+20; P < 0.001; range, 380%; Fig. 6A). Currents at negative test potentials (-45 to -35 mV), where LVA channels are selectively activated, were augmented as much as those at more positive potentials, as shown in a different cell in Fig. 6B. In five cells, GnRH was tested with a holding potential of -54 mV to inactivate LVA channels, and there was a 14% (k 7; P < 0.01; range, 5-22%) increase in the sustained component of current at 6 mV. Responses to GnRH were variable between batches of aT3-1 cells, a variability that seemed not to be correlated with culture conditions or the length of time the stock of cells had been frozen. Much biochemical evidence points to the importance of the phospholipase-C/PKC pathway in LH secretion

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 09:54 For personal use only. No other uses without permission. . All rights reserved.

GONADOTROPE

-751

CELL

LINE

ELECTROPHYSIOLOGY

3417

I(pA)

+PMA

FIG. 6. Augmentation of Ba2+ current in Ca channels by GnRH. A, Current-voltage relations for Ba2+ currents before (0 and Cl) and after (0 and W) the addition of 100 nM GnRH to the bath solution. Current traces show steps to -34, -14, and 6 mV from a holding potential of -94 mV before and after (+) GnRH. The current-voltage relation shows both the sustained current (0 and n ), measured as the average of the last 30 msec of the pulse, and the isolated transient component of current (0 and l ), calculated as the peak current from a holding potential of -94 mV minus the current from a holding potential of -54 mV. B, In a different cell with a large transient current, current traces and current-voltage relations before (0 and 0) and after (a and 0) the addition of 100 nM GnRH to the bathing solution. Symbols are explained in A.

(19, 20). Therefore, we tested the effects of stimulators of PKC on Ba2+ current. The addition of PMA augmented the Ba2+ current (Fig. 7) in all 11 cells tested. A concentration of 10 nM PMA increased the peak current recorded at 6 mV by 15% (fll; P < 0.01; range, O-31%), and the sustained current by 14% (+12; P < 0.01; range, 2-42%). In three cells tested from a holding potential of -54 mV, the sustained current at 6 mV increased 14% (f13; P = NS). This effect of PMA on the amplitude of the currents was similar to that of GnRH. However, unlike GnRH, PMA also caused a negative shift of the current-voltage relation of the sustained current in six of seven cells in which a full current-voltage relation was recorded under both control and PMA conditions. The shift makes more current available for activation at negative potentials and might contribute to the reported

+PMA

+GnRH

FIG. 7. Augmentation of Ba*+ currents by PMA and the combination of PMA and GnRH. A, Current traces and current-voltage relations before (0 and 0) and after (0 and n ) the addition of 10 nM PMA to the bathing medium. Symbols and lines are explained in Fig. 6A. B, Current-voltage relation for Ba2+ currents in control solution (O), after the addition of 10 nM PMA (m), and after the addition of 10 nM PMA with 100 nM GnRH (+). Only the sustained current, measured as the average of the last 30 msec of the pulse from a holding potential of -94 mV, is shown in the current-voltage relation. The pairs of current traces at three different potentials show control conditions compared to PMA addition (left paneki) and PMA alone compared to PMA with GnRH (right panels). Calibration bars are 30 msec and 25 pA.

stimulatory effects of PMA on gonadotropin secretion (5). This implies that in addition to augmenting the current, PMA also changes the gating of the Ca2’ channels. Higher concentrations (100 nM) of PMA did not further augment the current.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 09:54 For personal use only. No other uses without permission. . All rights reserved.

3418

GONADOTROPE

CELL

LINE

When PMA was present, further addition of GnRH augmented the Ba2+ current more than PMA alone. Figure 7B shows the augmentation of current and a shift of the current-voltage relation to more negative potentials when 10 nM PMA was added to the bath solution (m). When 100 nM GnRH was added with the 10 nM PMA, a small further augmentation of the current was seen in 3 cells (+); however, this was not accompanied by a further shift of the current-voltage relation. Because the c-u-subunit of the gonadotropins is the same as that for TSH, it seemed possible that the aT3-1 cell line generated by using the a-chain promoter might have some characteristics of thyrotopes. For this reason we tested TRH, the physiological releasing hormone for TSH, in aT3-1 cells. No effect of TRH was seen on either K+ (n = 3) or Ba*+ currents (n = 3). Discussion Several types of voltage-gated ion channels are expressed in (uT3-1 cells; these include a fast Na channel, two Ca channels, and numerous types of K channels. The addition of GnRH augments the currents in Ca channels, as does PMA, a stimulator of PKC. The Na channels in the mouse-derived aT3-1 cells are similar to those recorded from ovine (7) and rat gonadotropes (10, 12), except that the steady state inactivation curve has a more negative midpoint in (rT3-1 cells. In our experiments we used a more negative holding potential than others, tested a more negative range of prepulses, and subtracted a correction for junction potentials. We confirm previous reports that l-10 pM TTX blocks Na+ current and now show that the TTX sensitivity is high, with half-block at 39 nM. The Na channel is not believed to be essential for LH release from gonadotropes, since adding TTX or removing external Na+ does not change the amount of LH secreted in response to GnRH (7, 21), and removal of external Na+ is reported not to change the waveform of spontaneous action potentials (12). At the level of resting membrane potentials recorded in unstimulated gonadotropes (-70 to -40 mV) (12, 13, 22), the majority of Na channels would be inactivated; they would require periods of hyperpolarization to become available for opening. In identified rat gonadotropes, Chen et al. (12) reported three types of outward K’ current, similar to those seen in (YT~-1 cells. Some K+ current(s) in gonadotropes can be modulated by GnRH stimulation. Sikdar et al. (23) have reported on-cell recording of a large conductance K channel with activity that is modulated by GnRH. This might be a Ca*+- activated K channel. Work from our laboratory using identified rat gonadotropes has shown

ELECTROPHYSIOLOGY

Endo. Voll30.

1992 No 6

that GnRH activates large oscillatory currents in apamin-sensitive Ca*+-activated K+ channels via IPB-mediated oscillatory Ca*+ release (13). The lack of such prominent modulation of K channels in (YT~-1 cells suggests that transformation of these cells has changed the repertoire of intracellular signaling, perhaps by attenuating the rise of intracellular Ca*+ that normally is associated with GnRH responses (l-3) as well as by reducing the expression of apamin-sensitive Ca*+-activated channels. Another possible explanation for the lack of full responses to GnRH is that the cell line may be arrested at an early stage of differentiation of gonadotropes in which this signalling is not yet developed (14), or arrested in one of the less responsive subforms that are found in the normal adult pituitary population (24). Two types of Ca channels, comprising an inactivating LVA component and a sustained HVA component, have been reported in all types of gonadotropes studied (812), and as we confirm, the sustained HVA channel is sensitive to dihydropyridine blockers (8). Recording Ca*+ current from enriched rat gonadotrope preparations, Marchetti et al. (10) reported that in half of the cells, 20 nM GnRH suppressed both types of currents by about 20% and caused a lo-mV negative shift in the currentvoltage relation. In contrast, we found an increase in Ba*+ current with GnRH and both an increase and a shift in gating with PMA. Their experiments differ from ours in not including ATP, GTP, Mg*+, or leupeptin in the whole cell pipettes and in using acutely obtained gonadotropes and a lower concentration of GnRH (20 us. 100 nM). One report (25) invokes a role for Ca channels in the desensitization of LH release during sustained administration of GnRH. Long term GnRH applications (100 nM for 40 min) appeared to reduce both types of Ca current. These experiments compared populations of cells rather than individual cells. When applied to cultured pituitary cells or to (YT~-1 cells, GnRH causes translocation of PKC from the cytoplasm to the membrane (15, 26,27). However, there is a lack of agreement about whether PKC is needed in GnRH-stimulated LH release. In some reports, PKC depletion does not affect levels of GnRH-stimulated LH release (28). Stojilkovic et al. (19) have shown that GnRH and PMA stimulate LH release in both a Ca*+-dependent and -independent manner, and that in PKC-depleted cells (10% enzyme activity remaining), the release stimulated by both agents is much reduced. In addition, there is one report that staurosporine, a broad spectrum inhibitor of protein kinases, completely inhibits PMA-provoked LH release, while only partially reducing GnRHinduced release (20). Thus, GnRH may be able to act through two (or more) distinct pathways to elicit LH release; one is dependent on PKC, while others are independent of PKC (such as the IP3/Ca2+ pathway).

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 09:54 For personal use only. No other uses without permission. . All rights reserved.

GONADOTROPE

CELL

LINE

GnRH causes a biphasic rise in intracellular Ca2+ levels, with an initial rapid spike that may be oscillatory, followed by a slower smaller second rise of Ca2+ (2, 3, 29). These two phases correspond to the two phases of LH release measured in suspensions of mixed pituitary cells (3). Shangold et al. (2) showed, using measurement of intracellular Ca2+ in identified gonadotropes, that the secondary rise of Ca2+ was dependent on external Ca2+, augmented by PMA, and sensitive to block by nifedipine, a dihydropyridine antagonist. In addition, more than one rapid increase in internal Ca2+ could not be elicited unless external Ca2+ was available; this external Ca2+ enters in part through dihydropyridine-sensitive Ca channels. Our work in aT3-1 cells shows that a dihydropyridine-sensitive HVA Ca channel is augmented by GnRH, and that a second LVA Ca channel type may be enhanced as well. Our results also demonstrate the augmentation of currents in Ca channels by PKC stimulation, which would be a consequence of phospholipase-C activation by GnRH. This augmentation may be an important mechanism for maintenance of Ca2+ levels available for use in LH secretion in gonadotropes. The (YT~-1 cell line shows important similarities to and differences from primary gonadotropes. This cell line synthesizes and secretes the CYportion of the LH molecule, expresses voltage-gated currents similar to those recorded from primary gonadotopes, and responds to GnRH. However, because the cells do not synthesize the P-chain of the hormone, the processing and control of secretion may be different from those in primary gonadotropes. Finally, the absence of electrical signs of GnRH-induced intracellular Ca2+ oscillations suggests that the IP3/Ca2+ signaling system is attenuated.

Acknowledgments We thank Dr. G. S. McKnight for advice and use of cell culture facilities; Dr. R. A. Steiner for discussions;L. Miller and D. G. Anderson for technical help; and K. Mackie, W. J. Moody, M. Shapiro, A. Tse, Y. B. Park, and L. Wollmuth for helpful commentson the manuscript.

References Huckle WR, Conn PM 1988 Molecular mechanism of gonadotropin releasing hormone action. II. The effector system. Endocr Rev 9:387-385 Shangold GA, Murphy SN, Miller RJ 1988 Gonadotropin-releasing hormone-induced Ca*+ transients in single identified gonadotropes and Ca2+ influx. Proc require both intracellular Ca*+ mobilization Nat1 Acad Sci USA 85:6566-6570 Tasaka K, Stojilkovic SS, Izumi S-I, Catt KJ 1988 Biphasic activation of cytosolic free calcium and LH responses by gonadotropinreleasing hormone. Biochem Biophys Res Commun 154:398-403 Berridge MJ 1984 Inositol trisphosphate and diacylglycerol as second messengers. Biochem J 220:659-666

ELECTROPHYSIOLOGY

3419

5. Stojilkovic SS, Chang JP, Izumi S-I, Tasaka K, Catt KJ 1988 Mechanisms of secretory responses to gonadotropin-releasing hormone and phorbol esters in cultured pituitary cell. Participation of protein kinase C and extracellular calcium mobilization. J Biol Chem 263:17301-17306 6. Childs GV 1986 Functional ultrastructure of gonadotropes: a review. Curr Top Neuroendocrinol7:49-97 7. Mason WT, Sikdar SK 1988 Characterization of voltage-gated sodium channels in ovine gonadotrophs: relationship to hormone secretion. J Physiol (Lond) 399:493-517 8. Mason WT, Sikdar SK 1989 Characterization of voltage-gated Ca*+ currents in ovine gonadotrophs. J Physiol (Land) 415:367-391 9. Marchetti C, Childs GV, AM Brown 1987 Membrane currents of identified isolated rat corticotropes and gonadotropes. Am J Physiol252:E340-E346 10. Marchetti C, Childs GV, Brown AM 1990 Voltage-dependent calcium currents in rat gonadotropes separated by centrifugal elutriation. Am J Physiol258:E589-E596 11. Stutzin A, Stojilkovic SS, Catt KJ, Rojas E 1988 Characteristics of two types of Ca channels in rat pituitary gonadotrophs. Am J Physiol257:C865-C874 12. Chen C, Zhang J, Dayanithi G, Vincent J-D, Israel J-M 1989 Cationic currents on identified rat gonadotroph cells maintained in primary culture. Neurochem Int 15:265-275 13. Tse A, Hille B 1992 GnRH-induced Ca*’ oscillations and rhythmic hyperpolarizations of pituitary gonadotropes. Science 255:462-464 14. Windle JJ, Weiner RI, Mellon PM 1990 Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice. Mol Endocrinol 4:587-603 15. Horn F, Bilezikjian LM, Perrin MH, Bosma MM, Windle JJ, Huber KS, Blount AL, Hille B, Vale W, Mellon PL 1991 Intracellular responses to GnRH in a clonal cell line of the gonadotrope lineage. Mol Endocrinol5:357-355 F, Armstrong CM 1977 Inactivation of the sodium chan16. Bezanilla nel. I. Sodium current experiments. J Gen Physiol 70:549-566 17. Byerly L, Hagiwara S 1982 Ca currents in internally perfused nerve cell bodies of Limnea staganalis. J Physiol (Land) 322:503-528 18. Romey G, Lazdunski M 1984 The coexistence in rat muscle cells of two distinct classes of Ca’+-dependent K+ channels with different pharmacological properties and different physiological functions. Biochem Biophys Res Commun 118:669-674 SS, Chang JP, Ngo D, Catt KJ 1988 Evidence for a role 19. Stojilkovic of protein kinase C in luteinizing hormone synthesis and secretion. Impaired responses to gonadotropin-releasing hormone in protein kinase C-depleted pituitary cells. J Biol Chem 263:17307-17311 20. Dan-Cohen H, Naor Z 1990 Mechanism of action of gonadotropin releasing hormone upon gonadotropin secretion: involvement of protein kinase C as revealed by staurosporine inhibition and enzyme depletion. Mol Cell Endocrinol 69:135-144 21. Conn PM, Rogers DC 1980 Gonadotropin release from pituitary cultures following activation of endogenous ion channels. Endocrinology 107:2133-2144 22. Mason WT, Waring, DW 1985 Electrophysiological recordings from gonadotrophs. Evidence for Ca*+ channels mediated by gonadotrophin-releasing hormone. Neuroendocrinology 41:258-268 RP, Mason WT 1989 Differential modulation 23. Sikdar SK, McIntosh of Ca’+-activated K+ channels in ovine pituitary gonadotrophs by GnRH, Ca’+ and cyclic AMP. Brain Res 496:113-123 24. Leong DA 1991 A model for intracellular calcium signalling and the coordinate regulation of hormone biosynthesis, receptors and secretion. Cell Calcium 12:255-268 25. Stojilkovic SS, Rojas E, Stutzin A, Izumi S-I, Catt, KJ 1989 Desensitization of pituitary gonadotropin secretion by agonistinduced inactivation of voltage-sensitive Ca channels. J Biol Chem 264:10939-10942 26. Hirota K, Hirota T, Aguilera G, Catt KJ 1985 Hormone-induced redistribution of calcium-activated phospholipid-dependent protein kinase in pituitary gonadotrophs. J Biol Chem 260:3243-3246

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 09:54 For personal use only. No other uses without permission. . All rights reserved.

3420

GONADOTROPE

CELL

LINE

27. Naor Z, Zer J, Zakut H, Hermon J 1985 Characterization of pituitary calcium-activated, phospholipid-dependent protein kinase: redistribution by gonadotropin-releasing hormone. Proc Nat1 Acad Sci USA 82:8203-8207 28. McArdle CA, Huckle WR, Conn PM 1987 Phorbol esters reduced gonadotrope responsiveness to protein kinase C activators but not

ELECTROPHYSIOLOGY

Endo. Voll30.

1992 No 6

to Ca*+-mobilizing secretagogues. J Biol Chem 262:5028-5035 29. Naor Z, Capponi AM, Rossier MF, Ayalon D, Limor R 1988 Gonadotropin-releasing hormone-induced rise in cytosolic free Ca2+ levels: mobilization of cellular and extracellular Ca*+ pools and relationship to gonadotropin secretion. Mol Endocrinol2:512520

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 09:54 For personal use only. No other uses without permission. . All rights reserved.