Effectors of ATP-sensitive K+ channels inhibit the regulatory effects of somatostatin and GH-releasing factor on growth hormone secretion

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Effecters of ATP-sensitive K+ channels inhibit the regulatory effects of somatostatin and GH-releasing factor on growth hormone secretion Jan R. De WEILLE,

Michel Michel

FOSSET,

Jacques

EPELBAUM*

and

LAZDUNSKI§

Institut de Pharmacologic MolCculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis 06560 Valbonne, France *U. 1.59INSERM, Unite de Neuroendocrinologie, Centre Paul Broca, 2 ter rue d’AlCsia, 75014 Paris, France Received

August

4,

1992

SUMMARY.

Somatostatin inhibition of growth hormone (GH) secretion from adenohypophysiscells in culture was antagonized by the antidiabetic sulfonylurea glipizide (Ko.5 = 10 f 5 nM). Although all cells that hyperpolarize with somatostatin have ATPsensitiveK+ channels,the antagonisticactionsof the hormoneand of the antidiabetic drug are due to effects on different types of K+ channels. Diazoxide, an opener of ATP-sensitive K+ channels, abolished the increase of intracellular Ca2+provoked by growth hormonereleasingfactor (GRF) and induced inhibition of GRF stimulated GH secretion (Q.5 = 138 PM). This inhibition by diazoxide was largely suppressed by glipizide which blocked the ATP-sensitive K+ channelsopenedby diazoxide. In summary, hormonal activation of GH secretion is inhibited by openers of ATPsensitiveK+ channels,while hormonalinhibition of GH secretionis suppressed by blockersof ATP-sensitive K+ channels. o 1~ ~~~~~~~~ press,I”~.

INTRODUCTION,

ATP-sensitive K+ (KATP) channels are key channelsin insulin secreting

cells (l-3). They close when the pancreaticB-cell is extracellularly perfusedwith glucose(via an increaseof the ATP/ADP ratio) and this closureprovokes a depolarizationthat leadsto Ca2+ entry via voltage-dependentCaz+channelsandto insulin secretion. KAT channelshave recently been identified in adenohypophysiscells (Bemardi et al., submitted).As KAT channelsin pancreaticB-cells (2,3), KAY channelsin adenohypophysis areblocked by antidiabeticsulfonylureasandare activated by the K+ channelopenerdiazoxide.

STo whom correspondence should be addressed at Institut de Pharmacologic Molkculaire et Cellulaire. 660 route des Lucioles. Sophia Antipolis 06560 Valbonne, France.

1007

Copyight 0 1992 .411 rights of reprodttctiort

0006-291 X/92 $4.00 by Academic Press, Inc. in any form reserved.

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The release of growth

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hormone (GH) from somatotrophs

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of the anterior pituitary

is

controlled by two hypothalamic peptides, GH-releasing factor (GRF) and somatostatin (SRIF). GRF stimulates GH release (4-6) while SRIF inhibits basal and GRF-induced 9). GRF-stimulated

GH release (7-

GH release results from an increased Ca2+ influx (6, lo), while SRIF

inhibits GH release by reducing Caz+ influx (11). Ca 2+ entry occurs via voltage-dependent Ca2+ channels (IO), the activity of which is itself controlled by the membrane potential and thus by the activity of K+ channels. The purpose of this work is to determine whether the actions of SRIF and GRF on GH secretion are modulated by effecters of KAp

channels such

as antidiabetic sulfonylureas and d&oxide.

MATERIALS

AND METHODS

Chemicals. Glipizide was from Laboratoires Pfizer (Paris, France). SRIF and GRF were obtained from Peninsula Laboratories (Paris, France). Dulbecco’s Modified Eagle’s Medium (DMEM, 074-02100, containing 22 mM glucose) was from Gibco (Paris, France), fetal calf serum and glutamine were obtained from Boehringer (Mannheim, Germany). Cromakalim was from Beecham Pharmaceuticals (UK), pinacidil from Leo Pharmaceutical Products (Denmark), diazoxide from Laboratoires Cetrane (Paris, France) and RP 49356 was from RhBne-Poulenc Rorer (Paris, France). Deoxyribonuclease, trypsin, soybean trypsin inhibitor, Norit-A charcoal, bovine serum albumine (BSA, fraction V), penicillin, streptomycin, estradiol, forskolin, Indo-l and bacitracin were obtained from Sigma . Cell culture. Adenohypophysis lobes were rapidly obtained after decapitation of 10 adult female rats. Cell dispersion and culture were carried out as previously described (12, 13). Cells were counted and plated at a density of 300 000 per well in 24 -multiwell plates for the studies of hormone secretion.The density was 100 000 cells per 35 mm Petri dish for electrophysiology. For Ca2+ measurements, cells were plated at a density of 100 000 cells per cover glasses. Cells were maintained for 4 days in DMEM supplemented with 10% fetal calf serum, 1% glutamine, 1% penicillin and 5 mg/ml streptomycin, at 37’C in a standard humidified incubator. Hormone release. On the fourth day of culture the cells were washed once and incubated for 2 h at 37’C in serum-free DMEM supplemented with 10 mM HEPES-NaOH at pH 7.5. Hormone release was determined after 3 h of incubation in the presence or absence of effecters. At the end of the incubation, the medium was removed and stored at -2O’C until GH was determined by radioimmunoassay (13). GH was estimated by radioimmunoassay using reagents supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, Bethesda, MD). Electrophysiology. Current-clamp experiments were carried out on adenohypophysis cells after 2 to 3 days of culture using the whole-cell suction-pipette technique (14). In all experiments, the intracellular solution contained KC1 150 mM, MgC12 1 mM, EGTA 2 mM, HEPES-KOH 10 mM, pH 7.2. The extracellular solution was: NaCl 140 mM, KC1 5mM, MgC12 2mM, CaC12 2 mM, HEPES-NaOH 10 &I, pH 7.3. Pipettes were coated with Sylgard resin to reduce pipet capacity. Electrical signals were digitized by a digital oscilloscope (Nicolet, Madison, Wisconsin, USA) and stored on hard-disk by computer (Hewlett Packard, Fort Collins, Colorado, USA) for further analysis. Experiments were carried out at room temperature (24 Z!Z2°C). Variations of intracellular Ca 2+ levels. Variations of cytosolic Ca2+ in isolated hypophysis cells were measured using the fluorescent Ca2+ chelator Indo-1. Cells were 1008

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incubated at room temperature for l-2 h in Ringer solution : 140 mM NaCl, 5 mM KCl, 2 mM MgC12, 2 mM CaC12, 10 mM HEPES-NaOH, pH 7.3, containing 3 mM glucose and 5 PM of the indo-ester. The medium was then replaced by the same solution lacking the indicator. A Petri dish with the cells was mounted on a fluorescence microscope (Nikon, France) and lit by 100 W Mercury light source via a 40x oil-immersion objective. A N-128 neutral filter was placed in the light path to reduce excitation. Fluorescent light was filtered at 405 and 480 nm, converted to electrical signals by photomultiplier tubes and recorded on digital tape (DAT, Sony, Japan). The ratio of the signals at 405 and 480 nm was taken as a measure of cytosolic Ca2+ and calibrated in vitro, using EGTA-buffered solutions.

RESULTS Fig. 1A shows the classical inhibition of GH release by SRIF (100 nM). The inhibition amounted 70% and was suppressed by glipizide which is one of the most potent antidiabetic sulfonylureas

(15). The dose-response curve of glipizide inhibition of SRIF action is shown in

Fig. 1B. Half-maximal

inhibition was observed at 10 + 5 nM glipizide. This Ko.5 value is

similar to the dissociation

constant of 5 nM found for glipizide from binding data with a

tritiated ligand (Bernardi et al., submitted). Because antidiabetic sulfonylureas largely abolish SRIF-induced inhibition of GH release and because in pancreatic B-cells SRIF activates the KATP channel (16), it was essential to determine whether

KATP channels in adenohypophysis

are also activated by SRIF. SRIF

(100 nM) was found to induce a 20 to 30 mV hyperpolarization

with a reduction of membrane

resistance in 20 out of 68 cells, as previously described by other authors (17, 18). The cells that

10

9

7

-log [g&i&),

6

M

Fig. 1. Inhibition of basal GH secretion from cultured rat female adenohypophysis cells by SRIF and reversal by increasing concentrations of glipizide. (A) values represent + SEM of three samples. 100% GH : 67.0 2~ 16.8 rig/ml. * P < 0.05 versus control; Ooo P < 0.001 versus SRIF. (B) values represent + SEM of three samples. GH control : 76.5 + 3.7 @ml ; SRIF : 19.5 5~ 1.1 @ml. SRIF : 100 nM. 1009

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responded to SRIP application shared burst-like electrical activity and the presence of voltagedependent

Na+ channels,

characteristics.

delayed rectifier

K+ and A-type

K+ currents

as common

Out of 25 cells that were tested both for their response to SRIF and for the

presence of a KAP current, induced by intracellular perfusion with a solution devoid of ATP, 7 were positive in both respects. None was found to have one of the responses without showing the other. Hence, SRIF-sensitive

adenohypophysis cells contain KAP channels.

Fig. 2A shows the effect of a series of SRIF (100 nM) applications to a cell intracellularly perfused with a K+-rich solution including 2 mM ATP. In the experiments carried out with SRIF, 100 PM GTP was also systematically applications,

included in the pipette. During three successive

about 1 min apart, the SRIF-induced

hyperpolarization

tended to diminish,

possibly due to desensitization, but was restored completely following

3 min of wash-out (Fig.

2A). When ATP was absent in the dialyzing solution, cells hyperpolarized due to KAP channel activation (Fig. 2B, n=7). Glipizide (1 PM) antagonized this membrane hyperpolarization, could not prevent subsequent Kf channel activation by SRIF. Adenohypophysis

but

cells were also

intracellularly perfused with 2 mM ATP and their responses to SRIF before and after glipizide application were compared (Fig. ZC). In these conditions 13 out of 43 cells hyperpolarized upon application of 100 nM SRIF. The amplitude of the SRIF-induced

hyperpolarization

was

not attenuated by glipizide. Hence, SRIF and glipizide apparently act on two distinct K+ conductances. Variations in cytosolic free calcium in single adenohypophysis

cells were measured by

fluorimetry, using the Caz+ indicator, indo-1. The type of cell being studied was identified by the responses observed to SRIF, GRF and/or thyrotropin

releasing hormone (TRH). It was

found that most cells responded either to SRIF with reduction of the frequency of smallamplitude Ca2+ transients (53%, n=15) or to TRH with a large Ca2+ spike, often followed by Ca2+ oscillations of smaller size (53%, n=38). A small fraction responded to neither one of the hormones (1 I%, n=9), while an other fraction responded to both (1 l%, n=9). GRF increased the frequency of small-amplitude with a single exception, adenohypophysis

Ca2+ transients in 11 out of 20 cells tested (55%), which,

were also sensitive

to SRIF. Application

of 10 nM GRF to

cells increased the frequency of spontaneously occurring Ca2+ transients.

These transients were due to the influx of Ca2+ via voltage-dependent Ca2+ channels, as they disappeared upon application of 100 nM of the dihydropyridine 1010

PN200-110 (not shown). Figs.

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Or

ITI\’

SRIF-

-

0 mV -20

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-

glipiride SRIF -

I3 -JO

SO J glipiride -SRlF

GRF

SRlF

SRlF

-

nM C

0

150

w

100 SO iA

60s

GRF

diazoxide

Fig. 2. Absence of effect of sulfonylureas on the SRIF-induced hyperpolarization. (A) The cell was intracellularly dialyzed with a K+-rich solution containing 2 mM of ATP to inhibit KAp channel activity. Application of 100 nM SRIF induced membrane hyperpolarization and a decrease in membrane resistance, which amplitude decreased with subsequent applications of the peptide. Following 3 min of wash out, a full-sized response to SRIF was again obtained. (B) Cells that were intracellularly perfused with an ATP-free solution, hyperpolarized after a few minutes due to the opening of KAn channels, as the hyperpolarization was antagonized by 1 l.tM glipizide. Subsequent extracellular application of 100 nM SRIF again induced membrane hyperpolarization, despite the continuous presence of the sulfonyurea. (C) The amplitude of the SRIF-induced hyperpolarization (with 2 mM ATP intracellularly) varies little, if at all, if glipizide is present or not. Fig. 3. SRIF and diazoxide inhibit GRF-activated Ca2+ influx. In most somatotrophs, wit free Ca2+ varied spontaneously due to the electrical activity. (A) GRF (10 nM) stimulated spontaneous Ca2+ transients, which were inhibited by 100 nM SRIF. (B) Diazoxide (300 pM) also inhibited GRF-induced Ca2+ entry. The effects of this high concentration of diazoxide was not easily reversed over the short experimental period.

3A and 3B show that both SRIF and diazoxide antagonizedthe GRF-induced

rise of cytosolic

Ca2+.The sameresultswere obtained with forskolin-stimulated somatotrophs. Since the KATZ channel opener diazoxide abolishedGRF induced [Ca2+]in increaseas SRIF does, the effect of diazoxide on GRF-stimulatedGH releasewas studied.

GRP stimulatedGH secretionby a factor of 4 (Fig. 4A). A nearly aspotent stimulationof GH secretionwas observedwith forskolin (Fig. 4A) which, like GRP, increasesCAMP levels in these cells (4, 9, 19). In both cases, stimulatory effects on GH secretion were nearly completely abolishedby the KAW channel openerdiazoxide (Fig. 4A). The inhibitory effects of diazoxide were abolishedby glipizide (Pig. 4A). 1011

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Al

800 .E ; 600 L? B 400 P

200

0 GRF Forskolin Diazoxide Glipizide

-

+ -

+ + -

+ + +

_ + -

+ + -

+ + +

Fig. 4. Inhibition of GRF- and forskolin- activated GH secretion from adenohypophysis cells byoxide and reversal by glipizide. (A) Values represent SEM of 12 sampIes(* P < 0.05 versus control; ** P < 0.01 versus GRF and versus forskolin; *** P c 0.001 versus diazoxide). GRF: 10 nM; forskolin: 10 PM, diazoxide: 300 KM, and glipizide: 100 nM. (B) Inhibition curve by diazoxide. Values represent SEM of 12 samples. 100 % GH: 805 f 20 rig/ml ; 0% GH: 350 k 30 @ml. GRF : 10 nM.

The concentration dependenceof the inhibitory effect of diazoxide is shownin Fig. 4B. Half-maximal inhibition was found at a Q.5 value of 138 f 26 PM. Maximal inhibition of GRF stimulated GH releasewas 69 _C8% for 500 FM diazoxide and 93 + 6% for 100 nM SRIF. With forskolin the extents of inhibition were 90% by SRIF and 65% by diazoxide. Other K+ channel openers(20, 21) such as cromakalim, RP 49356 and pinacidil used at 300 PM, were without effect on basalandGRF-activated GH secretion(not shown).

DISCUSSION

SRIF activates KAP channelsin insulin-secretingcells via a G protein that is sensitiveto pertussistoxin (16) and this activation is believed to play an important role in SRIF-induced inhibition of insulin secretion. This work showsthat SRIF-induced inhibition of GH secretion from adenohypophysis cells is antagonizedby the antidiabetic sulfonylureaglipizide. However, whereasSRIF-induced 1012

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hyperpolarization

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of insulin secreting cells was reversed by antidiabetic sulfonylureas,

induced hyperpolarization

SRE-

of adenohypophysis cells was insensitive to glipizide. It is therefore

clear that, in our experimental conditions, SRIF did not activate KATP channels. It has been shown that SRIF activates an inwardly rectifying K+ current (22) as well as voltage-dependent K+ currents

(17) in somatotrophs.

hyperpolarization

experimental

of the inward

rectifier

produces

leading to inhibition of Ca2+ entry through the voltage-sensitive

channels and to inhibition hyperpolarization

SRIF activation

of GH secretion. It is proposed that a significant

is reversed

by closing

KATP channels, which

Ca2+

part of this

are still open in our

conditions, with glipizide so that the membrane potential reaches again the

threshold for activation of voltage-dependent channels leading to restored GH secretion. The action of GRF is mediated by intracellular CAMP (4,9) and by Ca2+ (6, 10). GRFstimulated GH release is due to activation of dihydropyridine-sensitive

L-type Ca2+ channels

(10). GRF stimulation of [Ca2+]in is inhibited by dihydropyridines

(lo), it is inhibited in the

same way by SRIF but also by diazoxide (Fig. 3). Therefore inhibition of L-type Ca2+ channel activity by direct action of Ca 2+ channel blockers, by SRIF-induced diazoxide-induced surprising

hyperpolarization

hyperpolarization

or by

(Fig. 3) all induce a decrease of [Ca2+]in. It is not

then to find that diazoxide, like SRIF (Fig. 4), behaves as an inhibitor of GRF-

induced GH secretion. The same effect of diazoxide was seen if GH secretion was stimulated by forskolin. All these results taken together show that pharmacological modifications of the activity of KAp channels have drastic effects on the hormonal regulation of GH secretion: KAp channels are probably important channels for the physiology of adenohypophysis cells.

Acknowledgments. This work was supported by the Centre National de la Recherche Scientifique and the Minis&e de la Recherche et de la Technologie (grant 89.C.0883). We thank Laboratoires Pfizer for a gift of glipizide, Beecham Pharmaceuticals for a gift of cromakalim, Leo Pharmaceutical Products for a gift of pinacidil, Rhone-Poulenc Rorer for a gift of RP 49356 and Laboratoires Cetrane for a gift of diazoxide. We thank the NIDDK (Bethesda, MD, USA) and the National Hormone and Pituitary Program (U. of Maryland, School of Medicine, MD, USA) for gifts of radioimmunoassay reagents. Thanks are due to F. Aguila, C. Roulinat and C. Videau for expert assistance. REFERENCES 1. 2.

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