Proc. Nati. Acad. Sci. USA Vol. 87, pp. 8855-8859, November 1990
Physiology/Pharmacology
Dependence of hormone secretion on activation-inactivation kinetics of voltage-sensitive Ca2+ channels in pituitary gonadotrophs STANKO S. STOJILKOVI(*t, ToSHIHIKO IIDA*, MOHAMED A. VIRMANI*, SHUN-ICHIRO IZUMI*, EDUARDO ROJASI, AND KEVIN J. CATT* *Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, and *Laboratory of Cell Biology and Genetics, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Communicated by G. D. Aurbach, July 23, 1990
The relationships between the activation staABSTRACT tus of voltage-sensitive Ca2+ channels and secretory responses were analyzed in perifused rat gonadotrophs during stimulation by high extraceflular K+ concentration ([K+]k) or the physiological agonist, gonadotropin-releasing hormone (GnRH). Increase of [Kk to 50 mM evokes an on-offsecretory response, with a rapid rise in luteinizing hormone (LH) secretion to a peak at 35 sec (on response) followed by an exponential decrease to the steady-state level. Cessation of K+ stimulation elicits a transient (off) response followed by an exponential decrease to the basal level. The LH response to high [K+lJ is nifedipine-sensitive and its amplitude depends on membrane potential. There is a close relationship between the LH secretory response to high [K+Je and the amplitude of the inward Ca2+ current measured at 100 msec in whole-cell patch damp experiments. In addition, the prorfle of the LH secretory response is similar to that of the response of intracellular Ca2+ concentration ([Ca2+]O) in KV-stimulated cells. In Ca2+deficient medium, the effect of high [K+]J is abolished; subsequent elevation of [Ca2eIe during the K+ pulse is followed by restoration of the on response, but with reduced magnitude. Agonist stimulation during the steady-state phase of the [K+] pulse or after repetitive stimulation by high [K+le elicited biphasic [Ca2+]J and secretory responses with a significantly reduced plateau phase; conversely, KV-induced LH release was reduced in cells treated with desensitizing doses of GnRH. These findings indicate that depolarization-induced changes in the status of voltage-sensitive Ca2+ channels determine the profiles of [Ca2+]1 and LH responses to stimulation by high [K+]k; the initial activation of dihydropyridine-sensitive Ca2+ channels is clearly dependent on membrane potential, whereas their subsequent inactivation depends on increased [Ca2+1. Such inactivation of voltage-sensitive Ca2+ channels also occurs during GnRH action and may represent an additional regulatory mechanism to limit the entry of extracellular Ca2+ during prolonged or frequent agonist stimulation. The stimulatory effects of high extracellular K+ concentration ([Kk]e) on intracellular Ca2+ concentration ([Ca2 ]) and luteinizing hormone (LH) release (1, 2), and the sensitivity of these effects to dihydropyridine agonist and antagonist analogs (1-3), have provided indirect evidence for the presence of voltage-sensitive Ca2" channels (VSCCs) in pituitary gonadotrophs. Electrophysiological studies (4) have confirmed that pituitary gonadotrophs, like corticotrophs (5) and pituitary tumor cells (6), possess a fast, transient (T-type) channel and a slower channel that is dihydropyridinesensitive. The threshold for activation of the transient channels is around -50 mV. The dihydropyridine-sensitive chan-
nels activate at more positive potentials (-25 mV) and exhibit a very slow inactivating phase; they are regarded as longlasting (L-type) channels. The characteristic properties of T-type channels are appropriate for the generation of action potentials (7), which are present in pituitary gonadotrophs (8). On the other hand, the slower activation and delayed inactivation properties of L-type channels are more appropriate for mediating Ca2+ influx during agonist stimulation. Agonist-induced elevation of [Ca2+]j is a primary intracellular signal mediating the stimulation of LH secretion from pituitary gonadotrophs by gonadotropin-releasing hormone (GnRH). In addition to the mobilization of intracellular Ca2 , GnRH-induced secretion also depends on the entry of external Ca2+ (for review, see ref. 9). The combined operation of these two mechanisms is responsible for the characteristic biphasic increase in [Ca2J]i during GnRH stimulation, with an early rapid phase followed by a prolonged plateau phase (3). Under extracellular Ca2+-deficient conditions, the size of the early phase of the [Ca2+]j response is decreased and the sustained increase in [Ca2+], is abolished (3), indicating that influx of Ca2' participates in both phases. The stimulatory actions of GnRH on [Ca2+]1 and LH release depend on Ca2+ entry by at least two pathways, the best-defined of which is through VSCCs that are modulated by dihydropyridine agonist and antagonist analogs (1, 3, 9). The residual, dihydropyridine-insensitive entry of Ca2+ may occur through another type of VSCC or through receptor-operated Ca2" channels. To further delineate the properties and the role of these channels in stimulus-secretion coupling, we have defined the temporal course of LH secretion evoked by high [K+]e and/or GnRH in perifused pituitary cells. A detailed analysis of LH release during rapid sample collection has provided quantitative kinetic profiles of these secretory responses suitable for comparison with the electrophysiological properties of the voltage-sensitive Ca2+ current and the cytosolic [Ca2 ] response. Such analyses have shown that activation of voltage-sensitive L-type channels by high [K+]e is responsible for increased [Ca2 ]j and the concomitant LH release. The results have also shown the presence of [Ca2+]I-mediated inactivation of L-type of channels in gonadotrophs, a response that may represent the primary factor in the onset of desensitization by GnRH (10).
MATERIALS AND METHODS Anterior pituitary glands from normal or 2-week ovariectomized female rats were enzymatically dissociated and the Abbreviations: GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; [K+]e, extracellular K+ concentration; [Ca2+]i, intracellular Ca2+ concentration; VSCC, voltage-sensitive Ca2+ channel. tTo whom reprint requests should be addressed at: Building 10, Room B1-L400, National Institutes of Health, Bethesda, MD 20892.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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dispersed cells were cultured for 3 days (1) on Cytodex-1 beads (Pharmacia; 1-2 x 107 cells per 250 1.L of beads). The cells were then perifused (0.6 ml/min) for 2 hr at 370C with medium 199 supplemented with 20 mM Hepes, 0.1% bovine serum albumin, and antibiotics. Cell stimulation by high [K+] was performed in the presence of Ca2` (1.25 mM) or in Ca2+-deficient medium (total Ca2', 5 gM) keeping the sum of [K+] plus [Nal] constant at 140 mM. Fractions were collected every 5 sec and stored at -20'C prior to radioimmunoassay using the RP-3 rat LH standard provided by the National Pituitary Agency (Baltimore). Measurehents of Ca2+ current were performed with 6- to 8-fold enriched gonadotrophs (4). Whole-cell currents were measured at room temperature under voltage-clamp conditions, with holding potential adjusted to -80 mV and other conditions as previously described (4, 11). Measurements of [Ca2+]i in cell suspensions or in single gonadotrophs were performed as described (3, 12). Cell suspensions were loaded with fura-2 by incubation with 1 jgM fura-2 acetoxymethyl ester for 30 min at 37TC. Two million cells were used for [Ca2+]i assay by fluorescence analysis in a 3-ml cuvette in a Delta Scan spectrofluorimeter (Photon Technology International, Princeton, NJ). For [Ca2+]i measurements in single cells, pituitary cells plated on 25-mm coverslips coated with poly(L-lysine) were loaded with 2 ,M indo-1 acetoxymethyl ester for 60 min at 37°C and mounted on the stage of an inverted Diaphot microscope attached to an intracellular calcium-analysis system (Nikon). Cellular diacylglycerol content was assayed as described (13).
RESULTS AND DISCUSSION VSCCs Involved in K+-Induced LH Secretion. Activation of VSCCs by [K+]e-induced depolarization stimulates pituitary hormone secretion (14) and induces prominent [Ca2i1 and secretory responses in cultured gonadotrophs (2). In the present study, rapid cell perifusion with 5-sec resolution time permitted an accurate comparison of the profiles of both LH release and [Ca2W]i response, as well as the activationinactivation profiles of voltage-sensitive Ca2` currents. Application of 50 mM [K+ie evoked typical on-off secretory responses in the presence of 1.25 mM Ca2+ (Fig. LA) but had no effect on Ca2+-deficient medium (Fig. 1C). The rapid rise in LH secretion (referred to as the on response) occurred within 35 sec and the peak was followed by an exponential decrease in LH release to a steady-state level over the next 2.5 min. LH release then remained significantly higher than basal until the stimulation was withdrawn (Fig. 1 legend). Return of [K+It to 5.4 mM elicited a further rapid rise in LH secretion (the off response) followed by an exponential
-80 40-
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U
20 r
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10 Time, min
FIG. 1. Stimulation of LH release by high [K+]e. Pituitary cells were perifused with high [K+] in normal Ca2+ (1.25 mM) (A) or Ca24-deficient medium ([Ca2], = 5 ,uM) (C). Each point represents a 5-sec collection of perifusion medium (0.6 ml/min). The results are typical of 20 separate experiments. LH concentrations in ng/ml (mean ± SE): basal, 3.6 ± 0.3; K+ (50 mM)-induced on-response peak value, 82 ± 17; steady-state response, 22 ± 3; off response, 71 ± 12; P < 0.001 between basal and steady-state level. For single-cell Ca2+ measurements, gonadotrophs were identified by their Ca2+ responses to transient exposure to 10 pM GnRH. After washing, the cell was depolarized by high K+ added by pipette to the chamber in the presence (B) or absence (D) of Ca2+; partial repolarization was performed by adding K+-free medium 199 (3-fold dilution; final concentration of K+, =15 mM).
decrease to the basal level of LH secretion. The profile of the [KI].-induced rise in [Ca24h in single gonadotrophs was similar to that of LH release, with a rapid increase to a peak value, followed by an exponential decrease to a steady-state level (Fig. 1B). Three-fold dilution of the 50 mM K+ medium caused a further rapid off rise in [Ca2+]i. The on, off, and steady-state responses were not seen under Ca24-deficient conditions (Fig. 1D). A comparison of the whole-cell Ca2+ current and K+induced increase in [Ca2+]i and LH release revealed marked similarities among these responses. Both the amplitude of the current and the peak value of [Ca2+]i and LH release increased progressively with depolarization of the cell membrane (Figs. 2 and 3). A family of Ca2+-current records from a pituitary gonadotroph is shown in Fig. 2A. Application of B
0MV~~
FIG. 2. Effects of depolarizing voltage and
B A
0.8
A
[K4Ie pulses on inward current and LH release.
[
Vm, mV
-4't
C U
Time, min 4
8
'
10
100 -10
0.1
j0
f 0 / \ (A) Time course ofthe inward current at different -' membrane potentials (Vm). Superimposed membrane current records from a pituitary gonadot-20 50 55 roph internally dialyzed with Cs4 (75mM cesium _______i V'4 glutamate/75 mM CsCl/2.5 mM Mg2+4ATP/10 J -30 03 45 mM Na Hepes/0.1 mM Na EGTA) and bathed in modified Krebs buffer (140 mM choline chlo-\ ride/lO mM Na Hepes/1.3 mM MgCl2/5.2 mM -40 0______] ' \' CaCl2/25 AM tetrodotoxin, pH 7.4). (B) CurrentnA _ _ voltage relationship. o, Peak inward current;*, 100 msec inward current at 100 msec. (C) Dose-dependent effects of increasing [K+] (KCI: 15, 45, 55, and 100 mM) on the on, steady-state, and off values of LH release. Perifusion experiments were performed on individual columns containing the same number of cells (20 x 106 per column) derived from a single batch of pituitary cells.
Physiology/Pharmacology: Stojilkovic' et al.
1-
+ s
u
K+ (60 mM)
200 sec
50 msec
FIG. 3. Effects of high K+ on cytosolic [Ca2+] and LH release and washout effect. (A) Comparison between [Ca2+]i response in single gonadotroph and LH responses induced by K+. Results shown are representative of 10 similar experiments. (Inset) Dose-dependent effect of [K+], on [Ca2+]j. (B) Whole-cell Ca2+ current before (trace a) and after (trace b) washout. Voltage steps are shown above current records. About 70o of the current was washed out. Note that the residual, washout-resistant current was completely inactivated during 100 msec.
depolarizing voltage pulses of increasing amplitude activated inward Ca2+ currents with two components. The currentvoltage (I-V) relationships for the peak and steady-state value of each record and that measured at 100 msec (Fig. 2B) permitted a comparison of the voltage dependence of the two types of Ca2' channels with the secretory response of the gonadotrophs. The dose-dependent effects of [K+]e on LH release and [Ca2+j] levels are shown in Figs. 2C and 3A. The peaks and steady-state levels of both parameters were dependent on the degree of depolarization, being greater in higher K+ than in lower K+, while the times required to reach the peak and steady-state or basal levels were similar. In addition, [Ca2+] and LH secretion are probably linearly related; the profiles of [Ca2+j] and LH release in K+stimulated cells show a striking temporal similarity (Fig. 3B). GnRH, the physiological agonist for gonadotropin secretion, stimulated Ca2+ mobilization and increased diacylglycerol production (188 ± 16 vs. 101 ± 1 pmol per 106 cells; 100 nM GnRH for 10 min). The GnRH-induced increase in diacylglycerol, and the consequent activation of protein kinase C, may mediate the amplification of the Ca2+ signal seen in agonist-stimulated cells (15). In contrast, high K+ did not increase diacylglycerol production (101 ± 0.8 vs. 104.5 ± 5 pmol per 106 cells; 50 mM KCI for 10 min), indicating that its secretory actions do not involve phospholipid hydrolysis and depend solely on Ca2+-mediated mechanisms.
Proc. Nati. Acad. Sci. USA 87 (1990)
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When the membrane potential calculated for different K+ concentrations was compared with the concomitant release of LH (Fig. 4), the amplitude of the on LH response (A) was closely correlated with the steady-state amplitude of the Ca2+ current at 100 msec (0). This finding indicates that L-type channels are responsible for the K+-induced increases in [Ca2+]i and LH release. Only a washout-resistant (T) component (17) was present when no Ca2+ buffers were included in the patch pipette solution (Fig. 3B), and inactivation of this current occurred within 50 msec, in agreement with Ca2+current measurements in pituitary gonadotrophs by the double-pulse protocol (4). In other words, the residual current measured at 100 msec represents only the L-type component of Ca2+ current. In addition to the above-noted difference in kinetics, the voltage sensitivity of the gating system of the two types of Ca2+ channels, and of the activation curves, are fundamentally different. Thus, the two types of Ca2+ channels have different midpoint potentials and slopes; for the transient component the midpoint of the activation curve was about -30 mV, whereas for the long-lasting component it was -13 mV. The I-V curves clearly show that LH secretion follows the activation curve of the long-lasting component (Fig. 4A), with similar voltage sensitivity and midpoint potential. Furthermore, by using normalized values of the secretory responses to [K+Ie (maximum = 1.0), both peak and steadystate values of the on responses can be fitted by the open probability functions of the two Ca2+ channels (Fig. 4B; ref. 4). The estimated midpoint for the best fit fell within the range characteristic of the L-type Ca2+ current (-15 and -17 mV for the peak and steady-state values of LH responses, respectively). This finding is further supported by the observation that K+-induced LH release is dihydropyridinesensitive (1, 2). These results suggest that VSCCs are activated and subsequently inactivated in the presence of high [K+Je and that the [Ca2+j] and LH secretory profiles follow the activation status of VSCCs in K+-stimulated gonadotrophs. The ascending part of the on phase of LH secretion correlates with the increasing number of VSCCs in the open state, and the subsequent decrease in LH release is associated with the inactivation of these channels. The steady-state level of LH secretion should, therefore, reflect the equilibrium between the open and the inactivated state of the channels. The initiation of the second peak of LH secretion that occurs during repolarization after the return to 5 mM [K+Ie, analogous to the tail current following repolarization, is attributable to transient reactivation of the Ca2+ channels. Finally, K+-induced increases in
FIG. 4. Relationship between LH release and A B Vm, mV current. (A) Correlation between Ca2+ current -40 0 40 1.00 and LH release at different voltages. The symbols 7 LHp/LHPM for the peak (0) and Ca2+ current at 100 msec (a) ° represent the mean + SE of 27 experiments on\ -0.1 different cells. LH release (A) was expressed as the 100oo -0-5 \ peak values during the on response. Each value of = [K+], has been corrected to an equivalent mem- ^ 200 A _ '.A --0.2 brane potential (Vm) according to the Goldman= / \ Hodgkin-Katz equation: Vm (RT/F)ln({[K], + 300 E LHss/LHsM (PNa/PK)[Na~e}/Ki) = 61 log({[K], + (0.037)(150)}/ \Z 160). The estimated values for membrane potential \ ..5/ closely correlate with the measured values of rest.1 ing potential (V0) in GH4C1 pituitary cells treated with increasing concentrations of external K+ (16). The correlation coefficient (r) for a linear average 0 50 0 -50 relationship between current and LH release was . Vnm, mV 0.98, P < 0.001. (B) Steady-state m variables. (Upper) ml.. calculated from LHp/LHpM. (Lower) m2,- calculated from LHss/LHsM. LHp and LHSS, peak and steady-state values of LH release during the K+ stimulation.=LHpM and LHSM, the maximum for LH peak and steady-state values. Continuous curves were calculated with the following equation: mi.. 1/{1 + exp[a(Vm + VO)kTJ}, where kTwas taken as 25.4 meV. The parameters for the curves are as follows. For ml,.: a = -2.74, VO = -14.9 mV. For m2,-: a = 2.2, VO = -17.5 mV.
Ca2l
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Physiology/Pharmacology: Stojilkovic et al.
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[Ca2+]j and LH release occur predominantly through activation of L-type channels in gonadotrophs. Since T-type Ca2' channels exhibit voltage-dependent inactivation, they would not significantly contribute to the rise in [Ca2+]i during prolonged depolarization by K+. Inactivation of L-Type Ca2' Channels. Inactivation of L-type channels during membrane depolarization results from a change in channel conformation caused by the depolarization and/or the ensuing rise in [Ca2W]i (17, 18). However, the inward Ca2' current towards the end of either single (Fig. 2A) or double (4) voltage pulses resembles the current at the steady-state level and predicts that L-type VSCCs do not show inactivation at this time point (100 msec). These experiments exclude significant participation of a voltage-dependent mechanism in the inactivation of L-type of channels in gonadotrophs. However, they do not exclude [Ca2+]i-dependent inactivation, because Ca2' buffers (ATP and EGTA) were present in the pipette solution and inactivation develops more slowly when internal Ca2+ accumulation is prevented (19). The presence of a "washout" effect in patch-clamp gonadotrophs indirectly supports this conclusion. When a gonadotroph was dialyzed against a low-Ca2' saline solution, L-type channels "washed out" whereas T-type currents were more stable (Fig. 3B). The washout of Ca2+ current can be greatly reduced by adding Mg2+-ATP, indicating that dihydropyridine-sensitive channels are second messengerdependent (18, 19), while the insensitivity of T channels is in agreement with their voltage-dependent inactivation. Dialyzing patch-clamp cells with pipette buffer is attended by problems concerning the exact [Ca2+] in the buffer solution and potential loss of cytosolic kinases or phosphatases, or other factors that may influence the inactivation kinetics of the L-type channels. To determine whether inactivation of dihydropyridine-sensitive channels is [Ca2+]j-dependent, but to circumvent the problems associated with dialysis, we employed [K+Ie pulses to depolarize perifused cells in the presence or absence of Ca2+. Application of a depolarizing [K+]e pulse for 5 min under extracellular Ca2+-deficient conditions ([Ca2+1, = 5 AM) had no effect on basal LH release (0.5-2.0 ng/ml) (Fig. 5A). However, elevation of Ca2+ to 1.25 mM during depolarization by high [K+Ie caused the appearance of a prominent LH response, followed by an exponential decrease (typical on response). Fig. 5A shows five examples of differences in the amplitudes of the on responses but no change in the profile of the LH response or in the time required to reach the peak
Proc. Natl. Acad. Sci. USA 87 (1990)
value of each on response. These data indicate that inactivation of L-type channels by K+ is dependent on [Ca2+], or on Ca2+-dependent mechanisms, rather than on membrane potential. However, changes in membrane potential can exert secondary effects on the magnitude of the on response, which was significantly reduced when elicited during K+ infusion (Fig. SB). Otherwise, the peak time and the profile of decrease of the peak value to the steady-state level were unchanged. The declining phases of the [Ca2+]i and LH responses during elevation of [K+]e represent the balance between influx and efflux of Ca2l and could reflect activation of the efflux mechanisms with time. However, this is unlikely since addition of nifedipine and EGTA during the declining phase of the [Ca2+]i response to high K+ results in an immediate return of [Ca2+]i and LH release to their basal levels (3). Further, the subsequent addition of Ca2+ to cells stimulated by GnRH (15) and K+ (present data) caused a marked increase in LH release, reflecting the activity of the stimulated Ca2' influx pathway. These results suggest that the mechanisms responsible for Ca2' efflux are not rate-limiting (20) and that the declining [Ca2+]i level reflects inactivation of plasma membrane Ca2+ channels that occurs in a [Ca2+]1dependent manner, but with secondary effects of membrane potential on the magnitude of the response. Role of L-Type Channels in Agonist-Stimulated Cells. The presence of VSCCs in the plasma membrane of an individual cell type does not necessarily mean that such channels play a role in the agonist-induced influx of Ca2W. However, studies on [K+]e-induced inactivation of L-type channels have provided evidence for the physiological significance of these channels in GnRH-stimulated cells. An example of this is shown in Fig. 6A, where 50 mM K+ was applied throughout the experiment and a 10 mM GnRH pulse (8 min) was
11'1:!1
*
I; I'
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FIG. 5. Dependence of K+-induced LH release on [Ca2+le. (A) Depolarization by high [K+], in Ca2+-deficient medium had no effect on basal release (0.5-2.0 ng/ml). Addition of Ca2+ to the medium during the depolarizing K+ pulse caused immediate release of LH, followed by an exponential decrease. Tracings a-e represent the responses observed in five separate experiments. (B) Comparison between on responses mediated by Ca2+ at the beginning or during the [K+]e pulse. Data are representative of three similar experiments.
FIG. 6. Inactivation of long-lasting channels and agonist-induced gonadotropin release. (A) A GnRH pulse was applied during the steady-state phase of a 50 mM K+ pulse (solid line). The control GnRH pulse was obtained in the presence of 5.4 mM K+. Only 2% of the total LH content was released during the K+ infusion. Mean LH values during the plateau phases (ng/ml; mean + SE from 60 points obtained during 5-min stimulation) were as follows: controls, 19.5 ± 2.2; K+-stimulated, 2.1 ± 0.8, P < 0.001, estimated by Student's t test). Data are representative of three experiments with similar statistics. (B) Cytosolic Ca2+ profile in K+-stimulated single gonadotrophs. Data are typical of 12 similar experiments.
*t.
Proc. Natl. Acad. Sci. USA 87 (1990)
Physiology/Pharmacology: Stojilkovid et al. A 80
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40
*
:
5
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B
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FIG. 7. Prolongedinactivation of long-lasting
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stimulation with K+ (50mM for 10 min, followed by 10-min washing period) was followed by decreases in the peak and steady-state levels. (B) Response to a GnRH pulse (50 nM, 10 min) applied 10 min after l>=0 o the fourth K+pulse was significantly reduced (plateau-phase LH, ng/ml; mean ± SE from four ex50 periments: controls, 62 ± 8, treated, 37 ± 5, P < min 0.01, Student's t test). (C) Repetitive stimulation with GnRH (100 nM, 30 min, followed by 30 min 1 washing period), was accompanied by the develop-25 = ment of desensitization. (D) Response to Ki pulse | (50 mM, 10 min) applied 30 min after the fourth GnRH pulse was greatly reduced in comparison i with the control pulse. About 20% of the total LH content was released during GnRH stimulation. KJ '"-..- 0 Results in C and D are representative of three similar experiments. to
:.
*
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80
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Ca2l channels and LH responses. (A) Repetitive
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superimposed after 5 min. Initiation of the K+ infusion was followed by the appearance of on and steady-state responses. Application of the GnRH pulse during the steady-state phase of the response to [K'], was followed by a modified LH response in which the second phase was markedly reduced in comparison with that of the control GnRH pulse ([K+] = 5.4 mM). In parallel experiments on single gonadotrophs, the plateau phase of the GnRH-induced [Ca2+]i response was abolished, while the peak (predominantly extracellular Ca2+independent) phase was only slightly reduced (Fig. 6B), revealing the inability of GnRH to operate through VSCCs under these conditions. The plateau region of the biphasic [Ca2+] and LH responses to agonist stimulation is predominantly dependent on extracellular Ca2+. The participation of L-type channels in the Ca2+ entry pathway during the plateau phase has been confirmed by several reports based on the dihydropyridine sensitivity of GnRH action (for review, see ref. 9). GnRH does not modulate the frequency of spontaneous action potentials in gonadotrophs (8), and the mechanism by which it causes L-type channel opening is still unclear. However, it is possible that second-messenger systems, protein kinases, or guanine nucleotide-binding regulatory proteins participate in the modulation of L-type channel activity in pituitary cells. [Ca2+]i-dependent inactivation and washout of L-type currents appear to result from dephosphorylation of a cAMPdependent site in snail neurons (21), and Ca2+ current in cardiac muscle (22) and GH3 pituitary cells (17) is enhanced by cAMP-dependent phosphorylation. Thus, Ca2+ channels could be activated by protein kinases and inactivated by phosphatases (19, 22). GnRH receptors are coupled to the phospholipase C pathway and activation of protein kinase C, which could promote phosphorylation of L-type channels as suggested by the ability of phorbol esters to stimulate LH release and modulate Ca2' entry in gonadotrophs (23, 24). Our recent proposal that inactivation of VSCCs could participate in agonist-induced desensitization of gonadotrophs (10) was based on the effects of [Ca2+le during prolonged stimulation with GnRH, which reduced the voltage-sensitive Ca2+ current and attenuated the plateau phase of GnRH-induced LH release. This phenomenon was further analyzed during repetitive stimulation with high K+ (50 mM), which was accompanied by progressive reduction in the amplitudes of the on and steady-state responses. In such cells, a GnRH infusion given 30 min after the last K+ pulse was much less effective than the control pulse in releasing LH (Fig. 7 A and B). Conversely, the LH response to high K+ was markedly reduced in cells desensitized by repeated exposure to GnRH (Fig. 7C). A 50 mM K+ pulse, applied 30 min after the last agonist stimulation, elicited typical on-off responses
with magnitudes reduced to 25% of the control values and complete loss of the plateau phase (Fig. 7D). In summary, the L-type channels of pituitary gonadotrophs are highly susceptible to [Ca2+]i-dependent inactivation during K+-induced depolarization; the concomitant attenuation of GnRH-induced LH release supports the conclusion that these channels are involved in agonist-induced mobilization of extracellular Ca2 . Such Ca2+-dependent inactivation of L-type channels correlates with GnRH-stimulated Ca2' entry and probably initiates GnRH-induced desensitization of gonadotrophs (10). Such inactivation of VSCCs during agonist action may supplement the action of plasma-membrane Ca2+ pumps by preventing excessive entry of Ca2+ during prolonged or frequent agonist stimulation by GnRH. 1. Chang, J. P., Stojilkovid, S. S., Graeter, J. S. & Catt, K. J. (1988) Endocrinology 123, 87-97. 2. Stojilkovid, S. S., Izumi, S.-I. & Catt, K. J. (1988) J. Biol. Chem. 263, 13054-13061. 3. Izumi, S.-I., Stojilkovid, S. S. & Catt, K. J. (1989) Arch. Biophys. Biochem. 275, 410-428. 4. Stutzin, A., Stojilkovid, S. S., Catt, K. J. & Rojas, E. (1989) Am. J. Physiol. 257, C865-C874. 5. Marchetti, C., Childs, G. V. & Brown, A. M. (1987) Am. J. Physiol. 252, E340-E346. 6. Simasko, S. M., Weiland, G. A. & Oswald, R. E. (1988) Am. J. Physiol. 254, E328-E336. 7. Miller, R. J. (1987) Science 235, 46-52. 8. Croxton, T. L., Ben-Jonathan, N. & Armstrong, N. McD. (1988) Endocrinology 123, 1783-1791. 9. Catt, K. J. & Stojilkovid, S. S. (1989) Trends Endocrinol. Metabol. 1, 15-20. 10. Stojilkovid, S. S., Rojas, E., Stutzin, A., Izumi, S.-I. & Catt, K. J. (1989) J. Biol. Chem. 264, 10939-10942. 11. Collins, C. A., Rojas, E. & Suarez-Isla, B. A. (1982)J. Physiol. (London) 324, 297-318. 12. Stojilkovid, S. S., Merelli, F., fida, T., Krsmanovid, L. Z. & Catt, K. J.
(1990) Science 248, 1663-1666.
13. Hunyady, L., Baukal, A. J., Bor, M., Ely, J. E. & Catt, K. J. (1990) Endocrinology 126, 1001-1008. 14. Wakabayashi, K., Kamberi, I. A. & McCann, S. M. (1969) Endocrinology 85, 1046-1056. 15. Stojilkovid, S. S., Stutzin, A., Izumi, S.-I., Dufour, S., Torsello, A., Virmani, M., Rojas, E. & Catt, K. J. (1990) New Biol. 2, 272-283. 16. Tan, K. N. & Tashjian, A. H. (1983) J. Biol. Chem. 259, 418-426. 17. Kalman, D., O'Lague, P. H., Erxleben, C. & Armstrong, D. L. (1988) J. Gen. Physiol. 92, 531-548. 18. Eckert, R. & Chad, J. E. (1984) Prog. Biophys. Mol. Biol. 44, 215-267. 19. Byerly, L. & Hagiwara, S. (1988) in Calcium and Ion Channel Modulation, eds. Grinnell, A. D., Armstrong, D. & Jackson, M. B. (Plenum, New York), pp. 3-18. 20. Schilling, W., Rajan, L. & Strobl-Jager, E. (1989) J. Biol. Chem. 264, 12838-12848. 21. Doroshenko, P. A., Kostyuk, P. G. & Martynyuk, A. E. (1982) Neuroscience 7, 2125-2134. 22. Reuter, M. (1983) Nature (London) 301, 569-774. 23. Stojilkovid, S. S., Chang, J. P., Ngo, D. & Catt, K. J. (1988) J. Biol. Chem. 263, 17307-17311. 24. Shangold, G. A., Murphy, S. W. & Miller, R. J. (1988) Proc. Natd. Acad. Sci. USA 85, 6566-6570.
Physiology/Pharmacology
Dependence of hormone secretion on activation-inactivation kinetics of voltage-sensitive Ca2+ channels in pituitary gonadotrophs STANKO S. STOJILKOVI(*t, ToSHIHIKO IIDA*, MOHAMED A. VIRMANI*, SHUN-ICHIRO IZUMI*, EDUARDO ROJASI, AND KEVIN J. CATT* *Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, and *Laboratory of Cell Biology and Genetics, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Communicated by G. D. Aurbach, July 23, 1990
The relationships between the activation staABSTRACT tus of voltage-sensitive Ca2+ channels and secretory responses were analyzed in perifused rat gonadotrophs during stimulation by high extraceflular K+ concentration ([K+]k) or the physiological agonist, gonadotropin-releasing hormone (GnRH). Increase of [Kk to 50 mM evokes an on-offsecretory response, with a rapid rise in luteinizing hormone (LH) secretion to a peak at 35 sec (on response) followed by an exponential decrease to the steady-state level. Cessation of K+ stimulation elicits a transient (off) response followed by an exponential decrease to the basal level. The LH response to high [K+lJ is nifedipine-sensitive and its amplitude depends on membrane potential. There is a close relationship between the LH secretory response to high [K+Je and the amplitude of the inward Ca2+ current measured at 100 msec in whole-cell patch damp experiments. In addition, the prorfle of the LH secretory response is similar to that of the response of intracellular Ca2+ concentration ([Ca2+]O) in KV-stimulated cells. In Ca2+deficient medium, the effect of high [K+]J is abolished; subsequent elevation of [Ca2eIe during the K+ pulse is followed by restoration of the on response, but with reduced magnitude. Agonist stimulation during the steady-state phase of the [K+] pulse or after repetitive stimulation by high [K+le elicited biphasic [Ca2+]J and secretory responses with a significantly reduced plateau phase; conversely, KV-induced LH release was reduced in cells treated with desensitizing doses of GnRH. These findings indicate that depolarization-induced changes in the status of voltage-sensitive Ca2+ channels determine the profiles of [Ca2+]1 and LH responses to stimulation by high [K+]k; the initial activation of dihydropyridine-sensitive Ca2+ channels is clearly dependent on membrane potential, whereas their subsequent inactivation depends on increased [Ca2+1. Such inactivation of voltage-sensitive Ca2+ channels also occurs during GnRH action and may represent an additional regulatory mechanism to limit the entry of extracellular Ca2+ during prolonged or frequent agonist stimulation. The stimulatory effects of high extracellular K+ concentration ([Kk]e) on intracellular Ca2+ concentration ([Ca2 ]) and luteinizing hormone (LH) release (1, 2), and the sensitivity of these effects to dihydropyridine agonist and antagonist analogs (1-3), have provided indirect evidence for the presence of voltage-sensitive Ca2" channels (VSCCs) in pituitary gonadotrophs. Electrophysiological studies (4) have confirmed that pituitary gonadotrophs, like corticotrophs (5) and pituitary tumor cells (6), possess a fast, transient (T-type) channel and a slower channel that is dihydropyridinesensitive. The threshold for activation of the transient channels is around -50 mV. The dihydropyridine-sensitive chan-
nels activate at more positive potentials (-25 mV) and exhibit a very slow inactivating phase; they are regarded as longlasting (L-type) channels. The characteristic properties of T-type channels are appropriate for the generation of action potentials (7), which are present in pituitary gonadotrophs (8). On the other hand, the slower activation and delayed inactivation properties of L-type channels are more appropriate for mediating Ca2+ influx during agonist stimulation. Agonist-induced elevation of [Ca2+]j is a primary intracellular signal mediating the stimulation of LH secretion from pituitary gonadotrophs by gonadotropin-releasing hormone (GnRH). In addition to the mobilization of intracellular Ca2 , GnRH-induced secretion also depends on the entry of external Ca2+ (for review, see ref. 9). The combined operation of these two mechanisms is responsible for the characteristic biphasic increase in [Ca2J]i during GnRH stimulation, with an early rapid phase followed by a prolonged plateau phase (3). Under extracellular Ca2+-deficient conditions, the size of the early phase of the [Ca2+]j response is decreased and the sustained increase in [Ca2+], is abolished (3), indicating that influx of Ca2' participates in both phases. The stimulatory actions of GnRH on [Ca2+]1 and LH release depend on Ca2+ entry by at least two pathways, the best-defined of which is through VSCCs that are modulated by dihydropyridine agonist and antagonist analogs (1, 3, 9). The residual, dihydropyridine-insensitive entry of Ca2+ may occur through another type of VSCC or through receptor-operated Ca2" channels. To further delineate the properties and the role of these channels in stimulus-secretion coupling, we have defined the temporal course of LH secretion evoked by high [K+]e and/or GnRH in perifused pituitary cells. A detailed analysis of LH release during rapid sample collection has provided quantitative kinetic profiles of these secretory responses suitable for comparison with the electrophysiological properties of the voltage-sensitive Ca2+ current and the cytosolic [Ca2 ] response. Such analyses have shown that activation of voltage-sensitive L-type channels by high [K+]e is responsible for increased [Ca2 ]j and the concomitant LH release. The results have also shown the presence of [Ca2+]I-mediated inactivation of L-type of channels in gonadotrophs, a response that may represent the primary factor in the onset of desensitization by GnRH (10).
MATERIALS AND METHODS Anterior pituitary glands from normal or 2-week ovariectomized female rats were enzymatically dissociated and the Abbreviations: GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; [K+]e, extracellular K+ concentration; [Ca2+]i, intracellular Ca2+ concentration; VSCC, voltage-sensitive Ca2+ channel. tTo whom reprint requests should be addressed at: Building 10, Room B1-L400, National Institutes of Health, Bethesda, MD 20892.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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dispersed cells were cultured for 3 days (1) on Cytodex-1 beads (Pharmacia; 1-2 x 107 cells per 250 1.L of beads). The cells were then perifused (0.6 ml/min) for 2 hr at 370C with medium 199 supplemented with 20 mM Hepes, 0.1% bovine serum albumin, and antibiotics. Cell stimulation by high [K+] was performed in the presence of Ca2` (1.25 mM) or in Ca2+-deficient medium (total Ca2', 5 gM) keeping the sum of [K+] plus [Nal] constant at 140 mM. Fractions were collected every 5 sec and stored at -20'C prior to radioimmunoassay using the RP-3 rat LH standard provided by the National Pituitary Agency (Baltimore). Measurehents of Ca2+ current were performed with 6- to 8-fold enriched gonadotrophs (4). Whole-cell currents were measured at room temperature under voltage-clamp conditions, with holding potential adjusted to -80 mV and other conditions as previously described (4, 11). Measurements of [Ca2+]i in cell suspensions or in single gonadotrophs were performed as described (3, 12). Cell suspensions were loaded with fura-2 by incubation with 1 jgM fura-2 acetoxymethyl ester for 30 min at 37TC. Two million cells were used for [Ca2+]i assay by fluorescence analysis in a 3-ml cuvette in a Delta Scan spectrofluorimeter (Photon Technology International, Princeton, NJ). For [Ca2+]i measurements in single cells, pituitary cells plated on 25-mm coverslips coated with poly(L-lysine) were loaded with 2 ,M indo-1 acetoxymethyl ester for 60 min at 37°C and mounted on the stage of an inverted Diaphot microscope attached to an intracellular calcium-analysis system (Nikon). Cellular diacylglycerol content was assayed as described (13).
RESULTS AND DISCUSSION VSCCs Involved in K+-Induced LH Secretion. Activation of VSCCs by [K+]e-induced depolarization stimulates pituitary hormone secretion (14) and induces prominent [Ca2i1 and secretory responses in cultured gonadotrophs (2). In the present study, rapid cell perifusion with 5-sec resolution time permitted an accurate comparison of the profiles of both LH release and [Ca2W]i response, as well as the activationinactivation profiles of voltage-sensitive Ca2` currents. Application of 50 mM [K+ie evoked typical on-off secretory responses in the presence of 1.25 mM Ca2+ (Fig. LA) but had no effect on Ca2+-deficient medium (Fig. 1C). The rapid rise in LH secretion (referred to as the on response) occurred within 35 sec and the peak was followed by an exponential decrease in LH release to a steady-state level over the next 2.5 min. LH release then remained significantly higher than basal until the stimulation was withdrawn (Fig. 1 legend). Return of [K+It to 5.4 mM elicited a further rapid rise in LH secretion (the off response) followed by an exponential
-80 40-
0
+
0.4
U
20 r
0
5
10 Time, min
FIG. 1. Stimulation of LH release by high [K+]e. Pituitary cells were perifused with high [K+] in normal Ca2+ (1.25 mM) (A) or Ca24-deficient medium ([Ca2], = 5 ,uM) (C). Each point represents a 5-sec collection of perifusion medium (0.6 ml/min). The results are typical of 20 separate experiments. LH concentrations in ng/ml (mean ± SE): basal, 3.6 ± 0.3; K+ (50 mM)-induced on-response peak value, 82 ± 17; steady-state response, 22 ± 3; off response, 71 ± 12; P < 0.001 between basal and steady-state level. For single-cell Ca2+ measurements, gonadotrophs were identified by their Ca2+ responses to transient exposure to 10 pM GnRH. After washing, the cell was depolarized by high K+ added by pipette to the chamber in the presence (B) or absence (D) of Ca2+; partial repolarization was performed by adding K+-free medium 199 (3-fold dilution; final concentration of K+, =15 mM).
decrease to the basal level of LH secretion. The profile of the [KI].-induced rise in [Ca24h in single gonadotrophs was similar to that of LH release, with a rapid increase to a peak value, followed by an exponential decrease to a steady-state level (Fig. 1B). Three-fold dilution of the 50 mM K+ medium caused a further rapid off rise in [Ca2+]i. The on, off, and steady-state responses were not seen under Ca24-deficient conditions (Fig. 1D). A comparison of the whole-cell Ca2+ current and K+induced increase in [Ca2+]i and LH release revealed marked similarities among these responses. Both the amplitude of the current and the peak value of [Ca2+]i and LH release increased progressively with depolarization of the cell membrane (Figs. 2 and 3). A family of Ca2+-current records from a pituitary gonadotroph is shown in Fig. 2A. Application of B
0MV~~
FIG. 2. Effects of depolarizing voltage and
B A
0.8
A
[K4Ie pulses on inward current and LH release.
[
Vm, mV
-4't
C U
Time, min 4
8
'
10
100 -10
0.1
j0
f 0 / \ (A) Time course ofthe inward current at different -' membrane potentials (Vm). Superimposed membrane current records from a pituitary gonadot-20 50 55 roph internally dialyzed with Cs4 (75mM cesium _______i V'4 glutamate/75 mM CsCl/2.5 mM Mg2+4ATP/10 J -30 03 45 mM Na Hepes/0.1 mM Na EGTA) and bathed in modified Krebs buffer (140 mM choline chlo-\ ride/lO mM Na Hepes/1.3 mM MgCl2/5.2 mM -40 0______] ' \' CaCl2/25 AM tetrodotoxin, pH 7.4). (B) CurrentnA _ _ voltage relationship. o, Peak inward current;*, 100 msec inward current at 100 msec. (C) Dose-dependent effects of increasing [K+] (KCI: 15, 45, 55, and 100 mM) on the on, steady-state, and off values of LH release. Perifusion experiments were performed on individual columns containing the same number of cells (20 x 106 per column) derived from a single batch of pituitary cells.
Physiology/Pharmacology: Stojilkovic' et al.
1-
+ s
u
K+ (60 mM)
200 sec
50 msec
FIG. 3. Effects of high K+ on cytosolic [Ca2+] and LH release and washout effect. (A) Comparison between [Ca2+]i response in single gonadotroph and LH responses induced by K+. Results shown are representative of 10 similar experiments. (Inset) Dose-dependent effect of [K+], on [Ca2+]j. (B) Whole-cell Ca2+ current before (trace a) and after (trace b) washout. Voltage steps are shown above current records. About 70o of the current was washed out. Note that the residual, washout-resistant current was completely inactivated during 100 msec.
depolarizing voltage pulses of increasing amplitude activated inward Ca2+ currents with two components. The currentvoltage (I-V) relationships for the peak and steady-state value of each record and that measured at 100 msec (Fig. 2B) permitted a comparison of the voltage dependence of the two types of Ca2' channels with the secretory response of the gonadotrophs. The dose-dependent effects of [K+]e on LH release and [Ca2+j] levels are shown in Figs. 2C and 3A. The peaks and steady-state levels of both parameters were dependent on the degree of depolarization, being greater in higher K+ than in lower K+, while the times required to reach the peak and steady-state or basal levels were similar. In addition, [Ca2+] and LH secretion are probably linearly related; the profiles of [Ca2+j] and LH release in K+stimulated cells show a striking temporal similarity (Fig. 3B). GnRH, the physiological agonist for gonadotropin secretion, stimulated Ca2+ mobilization and increased diacylglycerol production (188 ± 16 vs. 101 ± 1 pmol per 106 cells; 100 nM GnRH for 10 min). The GnRH-induced increase in diacylglycerol, and the consequent activation of protein kinase C, may mediate the amplification of the Ca2+ signal seen in agonist-stimulated cells (15). In contrast, high K+ did not increase diacylglycerol production (101 ± 0.8 vs. 104.5 ± 5 pmol per 106 cells; 50 mM KCI for 10 min), indicating that its secretory actions do not involve phospholipid hydrolysis and depend solely on Ca2+-mediated mechanisms.
Proc. Nati. Acad. Sci. USA 87 (1990)
8857
When the membrane potential calculated for different K+ concentrations was compared with the concomitant release of LH (Fig. 4), the amplitude of the on LH response (A) was closely correlated with the steady-state amplitude of the Ca2+ current at 100 msec (0). This finding indicates that L-type channels are responsible for the K+-induced increases in [Ca2+]i and LH release. Only a washout-resistant (T) component (17) was present when no Ca2+ buffers were included in the patch pipette solution (Fig. 3B), and inactivation of this current occurred within 50 msec, in agreement with Ca2+current measurements in pituitary gonadotrophs by the double-pulse protocol (4). In other words, the residual current measured at 100 msec represents only the L-type component of Ca2+ current. In addition to the above-noted difference in kinetics, the voltage sensitivity of the gating system of the two types of Ca2+ channels, and of the activation curves, are fundamentally different. Thus, the two types of Ca2+ channels have different midpoint potentials and slopes; for the transient component the midpoint of the activation curve was about -30 mV, whereas for the long-lasting component it was -13 mV. The I-V curves clearly show that LH secretion follows the activation curve of the long-lasting component (Fig. 4A), with similar voltage sensitivity and midpoint potential. Furthermore, by using normalized values of the secretory responses to [K+Ie (maximum = 1.0), both peak and steadystate values of the on responses can be fitted by the open probability functions of the two Ca2+ channels (Fig. 4B; ref. 4). The estimated midpoint for the best fit fell within the range characteristic of the L-type Ca2+ current (-15 and -17 mV for the peak and steady-state values of LH responses, respectively). This finding is further supported by the observation that K+-induced LH release is dihydropyridinesensitive (1, 2). These results suggest that VSCCs are activated and subsequently inactivated in the presence of high [K+Je and that the [Ca2+j] and LH secretory profiles follow the activation status of VSCCs in K+-stimulated gonadotrophs. The ascending part of the on phase of LH secretion correlates with the increasing number of VSCCs in the open state, and the subsequent decrease in LH release is associated with the inactivation of these channels. The steady-state level of LH secretion should, therefore, reflect the equilibrium between the open and the inactivated state of the channels. The initiation of the second peak of LH secretion that occurs during repolarization after the return to 5 mM [K+Ie, analogous to the tail current following repolarization, is attributable to transient reactivation of the Ca2+ channels. Finally, K+-induced increases in
FIG. 4. Relationship between LH release and A B Vm, mV current. (A) Correlation between Ca2+ current -40 0 40 1.00 and LH release at different voltages. The symbols 7 LHp/LHPM for the peak (0) and Ca2+ current at 100 msec (a) ° represent the mean + SE of 27 experiments on\ -0.1 different cells. LH release (A) was expressed as the 100oo -0-5 \ peak values during the on response. Each value of = [K+], has been corrected to an equivalent mem- ^ 200 A _ '.A --0.2 brane potential (Vm) according to the Goldman= / \ Hodgkin-Katz equation: Vm (RT/F)ln({[K], + 300 E LHss/LHsM (PNa/PK)[Na~e}/Ki) = 61 log({[K], + (0.037)(150)}/ \Z 160). The estimated values for membrane potential \ ..5/ closely correlate with the measured values of rest.1 ing potential (V0) in GH4C1 pituitary cells treated with increasing concentrations of external K+ (16). The correlation coefficient (r) for a linear average 0 50 0 -50 relationship between current and LH release was . Vnm, mV 0.98, P < 0.001. (B) Steady-state m variables. (Upper) ml.. calculated from LHp/LHpM. (Lower) m2,- calculated from LHss/LHsM. LHp and LHSS, peak and steady-state values of LH release during the K+ stimulation.=LHpM and LHSM, the maximum for LH peak and steady-state values. Continuous curves were calculated with the following equation: mi.. 1/{1 + exp[a(Vm + VO)kTJ}, where kTwas taken as 25.4 meV. The parameters for the curves are as follows. For ml,.: a = -2.74, VO = -14.9 mV. For m2,-: a = 2.2, VO = -17.5 mV.
Ca2l
/1
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[Ca2+]j and LH release occur predominantly through activation of L-type channels in gonadotrophs. Since T-type Ca2' channels exhibit voltage-dependent inactivation, they would not significantly contribute to the rise in [Ca2+]i during prolonged depolarization by K+. Inactivation of L-Type Ca2' Channels. Inactivation of L-type channels during membrane depolarization results from a change in channel conformation caused by the depolarization and/or the ensuing rise in [Ca2W]i (17, 18). However, the inward Ca2' current towards the end of either single (Fig. 2A) or double (4) voltage pulses resembles the current at the steady-state level and predicts that L-type VSCCs do not show inactivation at this time point (100 msec). These experiments exclude significant participation of a voltage-dependent mechanism in the inactivation of L-type of channels in gonadotrophs. However, they do not exclude [Ca2+]i-dependent inactivation, because Ca2' buffers (ATP and EGTA) were present in the pipette solution and inactivation develops more slowly when internal Ca2+ accumulation is prevented (19). The presence of a "washout" effect in patch-clamp gonadotrophs indirectly supports this conclusion. When a gonadotroph was dialyzed against a low-Ca2' saline solution, L-type channels "washed out" whereas T-type currents were more stable (Fig. 3B). The washout of Ca2+ current can be greatly reduced by adding Mg2+-ATP, indicating that dihydropyridine-sensitive channels are second messengerdependent (18, 19), while the insensitivity of T channels is in agreement with their voltage-dependent inactivation. Dialyzing patch-clamp cells with pipette buffer is attended by problems concerning the exact [Ca2+] in the buffer solution and potential loss of cytosolic kinases or phosphatases, or other factors that may influence the inactivation kinetics of the L-type channels. To determine whether inactivation of dihydropyridine-sensitive channels is [Ca2+]j-dependent, but to circumvent the problems associated with dialysis, we employed [K+Ie pulses to depolarize perifused cells in the presence or absence of Ca2+. Application of a depolarizing [K+]e pulse for 5 min under extracellular Ca2+-deficient conditions ([Ca2+1, = 5 AM) had no effect on basal LH release (0.5-2.0 ng/ml) (Fig. 5A). However, elevation of Ca2+ to 1.25 mM during depolarization by high [K+Ie caused the appearance of a prominent LH response, followed by an exponential decrease (typical on response). Fig. 5A shows five examples of differences in the amplitudes of the on responses but no change in the profile of the LH response or in the time required to reach the peak
Proc. Natl. Acad. Sci. USA 87 (1990)
value of each on response. These data indicate that inactivation of L-type channels by K+ is dependent on [Ca2+], or on Ca2+-dependent mechanisms, rather than on membrane potential. However, changes in membrane potential can exert secondary effects on the magnitude of the on response, which was significantly reduced when elicited during K+ infusion (Fig. SB). Otherwise, the peak time and the profile of decrease of the peak value to the steady-state level were unchanged. The declining phases of the [Ca2+]i and LH responses during elevation of [K+]e represent the balance between influx and efflux of Ca2l and could reflect activation of the efflux mechanisms with time. However, this is unlikely since addition of nifedipine and EGTA during the declining phase of the [Ca2+]i response to high K+ results in an immediate return of [Ca2+]i and LH release to their basal levels (3). Further, the subsequent addition of Ca2+ to cells stimulated by GnRH (15) and K+ (present data) caused a marked increase in LH release, reflecting the activity of the stimulated Ca2' influx pathway. These results suggest that the mechanisms responsible for Ca2' efflux are not rate-limiting (20) and that the declining [Ca2+]i level reflects inactivation of plasma membrane Ca2+ channels that occurs in a [Ca2+]1dependent manner, but with secondary effects of membrane potential on the magnitude of the response. Role of L-Type Channels in Agonist-Stimulated Cells. The presence of VSCCs in the plasma membrane of an individual cell type does not necessarily mean that such channels play a role in the agonist-induced influx of Ca2W. However, studies on [K+]e-induced inactivation of L-type channels have provided evidence for the physiological significance of these channels in GnRH-stimulated cells. An example of this is shown in Fig. 6A, where 50 mM K+ was applied throughout the experiment and a 10 mM GnRH pulse (8 min) was
11'1:!1
*
I; I'
J.r .... .. .....-
FIG. 5. Dependence of K+-induced LH release on [Ca2+le. (A) Depolarization by high [K+], in Ca2+-deficient medium had no effect on basal release (0.5-2.0 ng/ml). Addition of Ca2+ to the medium during the depolarizing K+ pulse caused immediate release of LH, followed by an exponential decrease. Tracings a-e represent the responses observed in five separate experiments. (B) Comparison between on responses mediated by Ca2+ at the beginning or during the [K+]e pulse. Data are representative of three similar experiments.
FIG. 6. Inactivation of long-lasting channels and agonist-induced gonadotropin release. (A) A GnRH pulse was applied during the steady-state phase of a 50 mM K+ pulse (solid line). The control GnRH pulse was obtained in the presence of 5.4 mM K+. Only 2% of the total LH content was released during the K+ infusion. Mean LH values during the plateau phases (ng/ml; mean + SE from 60 points obtained during 5-min stimulation) were as follows: controls, 19.5 ± 2.2; K+-stimulated, 2.1 ± 0.8, P < 0.001, estimated by Student's t test). Data are representative of three experiments with similar statistics. (B) Cytosolic Ca2+ profile in K+-stimulated single gonadotrophs. Data are typical of 12 similar experiments.
*t.
Proc. Natl. Acad. Sci. USA 87 (1990)
Physiology/Pharmacology: Stojilkovid et al. A 80
r-
A1-.
40
*
:
5
5 min
B
I
FIG. 7. Prolongedinactivation of long-lasting
g
stimulation with K+ (50mM for 10 min, followed by 10-min washing period) was followed by decreases in the peak and steady-state levels. (B) Response to a GnRH pulse (50 nM, 10 min) applied 10 min after l>=0 o the fourth K+pulse was significantly reduced (plateau-phase LH, ng/ml; mean ± SE from four ex50 periments: controls, 62 ± 8, treated, 37 ± 5, P < min 0.01, Student's t test). (C) Repetitive stimulation with GnRH (100 nM, 30 min, followed by 30 min 1 washing period), was accompanied by the develop-25 = ment of desensitization. (D) Response to Ki pulse | (50 mM, 10 min) applied 30 min after the fourth GnRH pulse was greatly reduced in comparison i with the control pulse. About 20% of the total LH content was released during GnRH stimulation. KJ '"-..- 0 Results in C and D are representative of three similar experiments. to
:.
*
KK+(50 mM) 30 min
80
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Ca2l channels and LH responses. (A) Repetitive
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8859
(100 nM)
superimposed after 5 min. Initiation of the K+ infusion was followed by the appearance of on and steady-state responses. Application of the GnRH pulse during the steady-state phase of the response to [K'], was followed by a modified LH response in which the second phase was markedly reduced in comparison with that of the control GnRH pulse ([K+] = 5.4 mM). In parallel experiments on single gonadotrophs, the plateau phase of the GnRH-induced [Ca2+]i response was abolished, while the peak (predominantly extracellular Ca2+independent) phase was only slightly reduced (Fig. 6B), revealing the inability of GnRH to operate through VSCCs under these conditions. The plateau region of the biphasic [Ca2+] and LH responses to agonist stimulation is predominantly dependent on extracellular Ca2+. The participation of L-type channels in the Ca2+ entry pathway during the plateau phase has been confirmed by several reports based on the dihydropyridine sensitivity of GnRH action (for review, see ref. 9). GnRH does not modulate the frequency of spontaneous action potentials in gonadotrophs (8), and the mechanism by which it causes L-type channel opening is still unclear. However, it is possible that second-messenger systems, protein kinases, or guanine nucleotide-binding regulatory proteins participate in the modulation of L-type channel activity in pituitary cells. [Ca2+]i-dependent inactivation and washout of L-type currents appear to result from dephosphorylation of a cAMPdependent site in snail neurons (21), and Ca2+ current in cardiac muscle (22) and GH3 pituitary cells (17) is enhanced by cAMP-dependent phosphorylation. Thus, Ca2+ channels could be activated by protein kinases and inactivated by phosphatases (19, 22). GnRH receptors are coupled to the phospholipase C pathway and activation of protein kinase C, which could promote phosphorylation of L-type channels as suggested by the ability of phorbol esters to stimulate LH release and modulate Ca2' entry in gonadotrophs (23, 24). Our recent proposal that inactivation of VSCCs could participate in agonist-induced desensitization of gonadotrophs (10) was based on the effects of [Ca2+le during prolonged stimulation with GnRH, which reduced the voltage-sensitive Ca2+ current and attenuated the plateau phase of GnRH-induced LH release. This phenomenon was further analyzed during repetitive stimulation with high K+ (50 mM), which was accompanied by progressive reduction in the amplitudes of the on and steady-state responses. In such cells, a GnRH infusion given 30 min after the last K+ pulse was much less effective than the control pulse in releasing LH (Fig. 7 A and B). Conversely, the LH response to high K+ was markedly reduced in cells desensitized by repeated exposure to GnRH (Fig. 7C). A 50 mM K+ pulse, applied 30 min after the last agonist stimulation, elicited typical on-off responses
with magnitudes reduced to 25% of the control values and complete loss of the plateau phase (Fig. 7D). In summary, the L-type channels of pituitary gonadotrophs are highly susceptible to [Ca2+]i-dependent inactivation during K+-induced depolarization; the concomitant attenuation of GnRH-induced LH release supports the conclusion that these channels are involved in agonist-induced mobilization of extracellular Ca2 . Such Ca2+-dependent inactivation of L-type channels correlates with GnRH-stimulated Ca2' entry and probably initiates GnRH-induced desensitization of gonadotrophs (10). Such inactivation of VSCCs during agonist action may supplement the action of plasma-membrane Ca2+ pumps by preventing excessive entry of Ca2+ during prolonged or frequent agonist stimulation by GnRH. 1. Chang, J. P., Stojilkovid, S. S., Graeter, J. S. & Catt, K. J. (1988) Endocrinology 123, 87-97. 2. Stojilkovid, S. S., Izumi, S.-I. & Catt, K. J. (1988) J. Biol. Chem. 263, 13054-13061. 3. Izumi, S.-I., Stojilkovid, S. S. & Catt, K. J. (1989) Arch. Biophys. Biochem. 275, 410-428. 4. Stutzin, A., Stojilkovid, S. S., Catt, K. J. & Rojas, E. (1989) Am. J. Physiol. 257, C865-C874. 5. Marchetti, C., Childs, G. V. & Brown, A. M. (1987) Am. J. Physiol. 252, E340-E346. 6. Simasko, S. M., Weiland, G. A. & Oswald, R. E. (1988) Am. J. Physiol. 254, E328-E336. 7. Miller, R. J. (1987) Science 235, 46-52. 8. Croxton, T. L., Ben-Jonathan, N. & Armstrong, N. McD. (1988) Endocrinology 123, 1783-1791. 9. Catt, K. J. & Stojilkovid, S. S. (1989) Trends Endocrinol. Metabol. 1, 15-20. 10. Stojilkovid, S. S., Rojas, E., Stutzin, A., Izumi, S.-I. & Catt, K. J. (1989) J. Biol. Chem. 264, 10939-10942. 11. Collins, C. A., Rojas, E. & Suarez-Isla, B. A. (1982)J. Physiol. (London) 324, 297-318. 12. Stojilkovid, S. S., Merelli, F., fida, T., Krsmanovid, L. Z. & Catt, K. J.
(1990) Science 248, 1663-1666.
13. Hunyady, L., Baukal, A. J., Bor, M., Ely, J. E. & Catt, K. J. (1990) Endocrinology 126, 1001-1008. 14. Wakabayashi, K., Kamberi, I. A. & McCann, S. M. (1969) Endocrinology 85, 1046-1056. 15. Stojilkovid, S. S., Stutzin, A., Izumi, S.-I., Dufour, S., Torsello, A., Virmani, M., Rojas, E. & Catt, K. J. (1990) New Biol. 2, 272-283. 16. Tan, K. N. & Tashjian, A. H. (1983) J. Biol. Chem. 259, 418-426. 17. Kalman, D., O'Lague, P. H., Erxleben, C. & Armstrong, D. L. (1988) J. Gen. Physiol. 92, 531-548. 18. Eckert, R. & Chad, J. E. (1984) Prog. Biophys. Mol. Biol. 44, 215-267. 19. Byerly, L. & Hagiwara, S. (1988) in Calcium and Ion Channel Modulation, eds. Grinnell, A. D., Armstrong, D. & Jackson, M. B. (Plenum, New York), pp. 3-18. 20. Schilling, W., Rajan, L. & Strobl-Jager, E. (1989) J. Biol. Chem. 264, 12838-12848. 21. Doroshenko, P. A., Kostyuk, P. G. & Martynyuk, A. E. (1982) Neuroscience 7, 2125-2134. 22. Reuter, M. (1983) Nature (London) 301, 569-774. 23. Stojilkovid, S. S., Chang, J. P., Ngo, D. & Catt, K. J. (1988) J. Biol. Chem. 263, 17307-17311. 24. Shangold, G. A., Murphy, S. W. & Miller, R. J. (1988) Proc. Natd. Acad. Sci. USA 85, 6566-6570.
