Journal of Endocrinology - uni-muenchen.de · Journal of Endocrinology ... a comparison between relaxin, ... 459-468 Expression of the growth hormone receptor gene in chicken K. L

Journal of Endocrinology

Volume I 3 5 • 1992 E D I T O R G. P. Vinson A S S O C I A T E E D I T O R J. A. Franklyn

E D I T O R I A L B O A R D

P. H. Baylis A. Logan Newcastle upon Tyne Birmingham

J. R. G. Challis A. M . McGregor London, Ontario London

J. K. Findlay I . D. Morris Melbourne Manchester

D. J. Flint E. E. Müller Ayr Milan

G. E. Gillies J. A. Russell London Edinburgh

C. Goddard S. I . Said Edinburgh Chicago

I . W. Henderson S. M . Shalet Sheffield Manchester

J. Herbert M . C. Sheppard Cambridge Birmingham

M. B. Hodgins D. C. Wathes Glasgow Cambridge

F. J. Karsch B. J. Whitehouse Ann Arbor London

P. G. Knight B. C. Williams Reading Edinburgh

K. Kohmoto Tokyo

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C O U N C I L O F M A N A G E M E N T I 9 9 1 / 9 2

P R O D U C T I O N E D I T O R D. E. Follett

E D I T O R I A L A S S I S T A N T C. V. Brewser

I . W. Henderson (Chairman), D . T. Baird, R. J. Balment, P. H . Baylis, J. C. Buckingham, R. N . Clayton, R. Fräser, B. J. A . Furr (Secretary), D. A . Heath (Treasurer), S. G. Hillier, M . C. Sheppard, A. White

Journal of Endocrinology • volume 135 contents

Number 1 • October 1992

l-< 4 Commentary: Chemical signalling systems: the rules of the game

M . Peaker

5- 16 Specific binding sites for 1 2 5I-labelled epidermal growth factor in the ovine corpus luteum and human placenta

T. A. Bramley & G. S. Menzies

17 28 Influence of ovarian steroids on myometrial sensitivity and tolerance to relaxin in the rat in vivo: lack of cross-tolerance between relaxin, salbutamol and cromakalim

S. J. Downing & M . Hollingsworth

29 -36 Interaction between myometrial relaxants and oxytocin: a comparison between relaxin, cromakalim and salbutamol

S. J. Downing & M . Hollingsworth

37- 43 Atrial natriuretic peptide is involved in the A C T H response to stress and glucocorticoid negative feedback in the rat

G. Fink, R. C. Dow, D. Casley, C. I . Johnston, J. Bennie, S. Carroll & H . Dick

45- 51 Relationship between the resting metabolic rate and hepatic metabolism in rats: effect of hyperthyroidism and fasting for 24 hours

S. Iossa, G. Liverini & A. Barletta

53 58 11 ß-Hydroxysteroid dehydrogenase in the rat ovary: high expression in the oocyte

R. Benediktsson, J. L. W. Yau, S. Low, L . P. Brett, B. E. Cooke, C R. W. Edwards & J. R. Seckl

59 67 Growth hormone promotes early initiation of hepatocyte growth factor gene expression in the liver of hypophysectomized rats after partial hepatectomy

S. Ekberg, M . Luther, T. Nakamura & J.-O. Jansson

69 75 Activation of the hypothalamo-pituitary-adrenocortical axis in the conscious rabbit by the pyrogen polyinosinic:polycytidylic acid is dependent on corticotrophin-releasing factor-41

N . G. N . Mil ton, E. W. Hillhouse & A. S. Mil ton

77 -83 Epidermal growth factor concentrations in pig tissues and body fluids measured using a homologous radioimmunoassay

T. J. Vaughan, C. J. Littlewood, J. C Pascall & K. D. Brown

85 90 Salivary insulin-like growth factor-I originates from local synthesis

J. Ryan, T. Mantle, S. McQuaid & D. C. Costigan

91 101 Fluid balance, electrolyte profiles and plasma parathyroid hormone concentrations in ewes treated with epidermal growth factor

C. B. Gow, M . Wilkinson, M . J. Silvapulle & G P. M . Moore

103 114 Effects of basic fibroblast growth factor, transforming growth factor-ß and nerve growth factor on the secretory function of the bovine corpus luteum in vitro

A. Miyamoto, K . Okuda, F. J. Schweigert & D. Schams

115-123 Effects of tri-iodothyronine and insulin-like growth factor-I ( IGF-I) on alkaline phosphatase activity, [ H]thymidine incorporation and I G F - I receptor m R N A in cultured rat epiphyseal chondrocytes

C. Ohlsson, A. Nilsson, O. Isaksson, J. Bentham & A. Lindahl

Journal of Endocrinology (1992) 135

iv Contents of volume 135

125--133 Origin of aldosterone in trypsin-stimulated rat adrenal zona glomerulosa incubations

G P. Vinson, S. M . Laird, J. P. Hinson & R. Teja

135--145 The induction of specific proteases for insulin-like growth factor-binding proteins following major heart surgery

S. C. Cwyfan Hughes, A. M . Cotterill, A. R. Molloy, T. B. Cassell, N . Braude, C. J. Hinds, J. A . H . Wass & J. M . P. Holly

147--152 Glycyrrhizin induces mineralocorticoid activity through alterations in Cortisol metabolism in the human kidney

Y. Kageyama, H . Suzuki & T. Saruta

153--159 Carbachol increases intracellular free calcium concentrations in human granulosa-lutein cells

A. Mayerhofer, K. J. Föhr , K . Sterzik & M . Gratzl

161--174 Comparison of specific immunoassays for detection of the ß-core human chorionic gonadotrophin fragment in body fluids

S. F. de Medeiros, F. Amato, C. D . Matthews & R. J. Norman

175--188 Distribution of the ß-core human chorionic gonadotrophin fragment in human body fluids

S. F. de Medeiros, F. Amato, D . Bacich, L . Wang, C. D. Matthews & R. J. Norman

189 Forthcoming papers and Announcements

Number 2 November 1992

191 -194 Commentary: Specificity of nuclear hormone receptor action: who conducts the orchestra?

G. R. Williams & G A. Brent

195-202 The role of growth hormone and insulin-like growth factors in the regulation of rat mammary gland and adipose tissue metabolism during lactation

M . C. Barber, R. A . Clegg, E. Finley, R. G Vernon & D. J. Flint

203--211 In-vivo regulation of messenger R N A encoding insulin-like growth factor-I ( IGF-I) and its receptor by diabetes, insulin and IGF- I in rat muscle

K. E. Bornfeldt, A. Skottner & H . J. Arnqvist

213--219 Effects of insulin, glucocorticoids and thyroid hormones on the activities of key enzymes of glycolysis, glutaminolysis, the pentose-phosphate pathway and the Krebs cycle in rat macrophages

L. F. B. P. Costa Rosa, Y . Cury & R. Curi

221--227 Gonadotrophin surge-attenuating factor attenuates in-vitro L H secretion induced by gonadotrophin-releasing hormone from cultured ovine pituitary cells only during the breeding season

P. A. Fowler, C. Townsend, I . E. Messinis, P. Cunningham & A. Templeton

229--237 Regulation of adrenal steroidogenesis by adrenaline: expression of cytochrome P450 genes

H . Güse-Behling, M . Ehrhart-Bornstein, S. R. Bornstein, M . R. Waterman, W. A . Scherbaum & G. Adler

239--247 Antiuterotrophic effects of a pure antioestrogen, I C I 182,780: magnetic resonance imaging of the uterus in ovariectomized monkeys

M . Dukes, D . Miller, A . E. Wakeling & J. C. Waterton


Contents of volume J 35 v

249-256 Ectopic transcription of the parathyroid hormone gene in O. Handt, A. Reis & J. Schmidtke lymphocytes, lymphoblastoid cells and tumour tissue

257--261 The role of thromboxane in the uterotrophic response in the gravid normotensive and spontaneously hypertensive rat

M . B. Kerr, K . Marshall & J. Senior

263- 269 A comparison of the uterotrophic response in cyclic and ovariectomized normotensive and spontaneously hypertensive rats

M . B. Kerr, K . Marshall & J. Senior

271--278 Fine structure of adrenal cortex in rats harbouring a medullary thyroid carcinoma transfected with a corticotrophin-releasing hormone cDNA expression vector

M . Feinmesser, S. L . Asa, K . Kovacs & M . J. Low

279--284 Passive immunization against circulating insulin-like growth factor-I ( IGF-I ) increases protein catabolism in lambs: evidence for a physiological role for circulating IGF- I

J. B. Koea, B. W. Gallaher, B. H . Breier, R. G. Douglas, S. Hodgkinson, J. H . F. Shaw & P. D . Gluckman

285--292 A role for di-acetyl a-melanocyte-stimulating hormone in the control of Cortisol release in the teleost Oreochromis mossambicus

A. E. Lamers, G. Flik, W. Atsma & S. E. Wendelaar Bonga

293--302 Zona radiata proteins are synthesized by rainbow trout (Oncorhynchus my kiss) hepatocytes in response to oestradiol-17ß

D. O. Oppen-Berntsen, E. Gram-Jensen & B. T. Walther

303--309 Immunochemical detection of the major vitelline envelope proteins in the plasma and oocytes of the maturing female rainbow trout, Oncorhynchus mykiss

S. J. Hyllner & C. Haux

311--316 Milking frequency alters the milk yield and mammary blood flow response to intra-mammary infusion of insulin-like growth factor-I in the goat

C. G Prosser & S. R. Davis

317--323 Different molecular forms of C-type natriuretic peptide isolated from the brain and heart of an elasmobranch, Triakis scyllia

R. Suzuki, A. Takahashi & Y. Takei

325--331 Atrial natriuretic peptide in eel plasma, heart and brain characterized by homologous radioimmunoassay

Y. Takei, K . Ando & M . Kawakami

333- 341 A polyclonal antibody to the rat oestrogen receptor expressed in Escherichia coli: characterization and application to immunohistochemistry

H . Okamura, K . Yamamoto, S. Hayashi, A . Kuroiwa & M . Muramatsu

343--352 Intracellular p H and growth hormone-releasing factor-stimulated adenosine 3'5'-monophosphate, intracellular calcium and growth hormone release from rat anterior pituitary cells

A. L . Puttagunta, C. L . Chik, M . Girard, L . O'Brien & A. K. Ho

353--359 The identification and characterization of 1 2 5I-labelled iodomelatonin-binding sites in the duck kidney

Y. Song, E. A. Ayre & S. F. Pang

361--369 Inhibition of bovine adrenocortical steroidogenesis by benzodiazepines: a direct effect on microsomal hydroxylation or an inhibition of calcium uptake?

I . Thomson, R. Fraser & C. J. Kenyon

371--382 Restoration of juvenile baseline growth hormone secretion with preservation of the ultradian growth hormone rhythm by continuous delivery of growth hormone-releasing factor

R. Vasilatos-Younken, P. H . Tsao, D. N . Foster, D . L . Smiley, H . Bryant & M . L . Heiman


vi Contents of volume 135

383-390 Activation of phospholipase A 2 and phospholipase C by endothelin-1 in human endometrium

A. Ahmed, I . T. Cameron, R. A. Ferriani & S. K . Smith

391-401 Intracellular pH regulation in human thyrocytes: evidence of both N a + / H + exchange and Na+-dependent CI7HCO3

L. Kayser, P. E. H0yer, H . Perrild, A. M . Wood & W. R. Robertson

403 Forthcoming papers and Announcements

R1-R4 Immunohistochemical identification of androgen receptors in germ cell neoplasia

E. Rajpert-De Meyts & N . E. Skakkebaek

R5-R8 Direct stimulation of bovine ovarian progesterone secretion by low concentrations of a-interferon

M . R. Luck, J. A. Shale & J. H . Payne

Number 3 • December 1992

405-406 Commentary: Growth hormone and puberty A. L . Ogilvy-Stuart & S. M . Shalet

407-413 Testosterone-induced suppression of self-healing Plasmodium chabaudi malaria: an effect not mediated by androgen receptors?

W. P. M . Benten, F. Wunderlich & H . Mossmann

415-420 Noradrenergic peripheral denervation of the female rat accelerates the positive feedback mechanisms resulting in pubertal ovulation, and blocks the modifications induced by administration of testosterone propionate at birth

A. Flores & R. Dominguez

421-430 Oestradiol increases the extracellular levels of amine metabolites in the ewe hypothalamus during anoestrus: a microdialysis study

V. Gayrard, B. Malpaux & J.-C. Thiery

431-438 Plasma atrial natriuretic peptide in vasopressin deficiency: the effects of acute water deprivation in rats

W. J. Burgess & R. J. Balment

439-446 Prepubertal reduction of the ovarian follicle population by W. A. van Cappellen, combined LH-releasing hormone antagonist treatment E. C. M . van Leeuwen, and unilateral ovariectomy influences follicle characteristics P. Kramer & but not the ovulation rate at first oestrus H . M . A. Meijs-Roelofs

447-457 Growth hormone (GH)-binding protein in normal and D. F. Carmignac, T. Wells, GH-deficient dwarf rats L. M . S. Carlsson, R. G. Clark &

I . C. A. F. Robinson

459-468 Expression of the growth hormone receptor gene in chicken K. L. Hull , R. A. Fraser & pituitary glands S. Harvey

469-478 The processing of ß-endorphin and a-melanotrophin in the K. Maruthainar, Y . Peng-Loh & pars intermedia of Xenopus laevis is influenced by D. G. Smyth background adaptation

479-484 Immunization with human thyrotrophin receptor peptide M . Ohmori, T. Endo, M . Ikeda & induces an increase in thyroid hormone in rabbits T. Onaya

485-493 Site-dependent effects of experimental hypo- and E. D . Schmidt, G van Hogerwou, hyperthyroidism on resident macrophages in extraocular R. van der Gaag, W. M . Wiersinga, muscles of rats: a quantitative immunohistochemical study G. Asmussen & L . Koornneef


Contents of volume 135 vii

495-505 Paradoxical hypertrophy and plasticity of the testis in rats E. Meisami, T. J. Sendera & recovering from early thyroid deficiency: a growth study L. B. Clay including effects of age and duration of hypothyroidism

507- 515 Age-related discrepancies between serum and pituitary gonadotrophin, and pituitary gonadotrophin subunit m R N A responses to castration and testosterone replacement in male rats

P. Pakarinen & I . Huhtaniemi

517--525 Effects of FSH on Leydig cell morphology and function in the hypogonadal mouse

P. J. O'Shaughnessy, M . K . Bennett, I . S. Scott & H . M . Charlton

527--533 Expansion of plasma volume by intragastric isotonic saline inhibits supraoptic neurones in rats

S. T. Pendlebury, R. E. J. Dyball & K. Honda

535--542 Pituitary-dependent hyperadrenocorticism in a family of Dandie Dinmont terriers

B. E. Scholten-Sloof, B. W. Knol , A. Rijnberk, J. A. Mol , D. J. Middleton & G. J. Ubbink

543--550 Transforming growth factor-ß is a potent inhibitor of basal and stimulated relaxin release by porcine luteal cells maintained in monolayer culture

M . J. Taylor & C. L. Clark

551--561 Catecholamines released from local adrenergic axon terminals are possibly involved in fine tuning of steroid secretion from zona glomerulosa cells: functional and morphological evidence

E. S. Vizi, I . E. Töth, K. Sz. Szalay, K. Windisch, E. Orsö, D. Szabö & G. P. Vinson

563--569 Human chorionic gonadotrophin and subunit composition of maternal serum and coelomic and amniotic fluids in the first trimester of pregnancy

R. K . lies, N . C. Wathen, D. J. Campbell & T. Chard

571--578 Evidence for lipopolysaccharide binding in human granulosa-luteal cells

M . Sancho-Tello, T.-Y. Chen, T. K . Clinton, R. Lyles, R. F. Moreno, L. Tilzer, K . Imakawa & P. F. Terranova

579--588 Distribution and properties of endo-ogliopeptidases A and B in the human neuroendocrine system

M . S. Medeiros, N . Iazigi, A. C. M . Camargo & E. B. Oliveira

589--595 Immunohistochemical localization of 17a-hydroxylase/ CI7-20 lyase and aromatase cytochrome P-450 in the human ovary during the menstrual cycle

T. Tamura, J. Kitawaki, T. Yamamoto, Y . Osawa, S. Kominami, S. Takemori & H . Okada

597--602 Retention of normal ovarian function after hysterectomy M . G. Metealf, V. Braiden & J. H . Livesey

603 -604 Forthcoming papers and Announcements

605--623 Indexes to Volumes 132, 133, 134 and 135


153

Carbachol increases intracellular free calcium concentrations in human granulosa-lutein cells

A. Mayerhofer, K. J . Föhr*, K. Sterzikf and M. Gratzl Abteilung Anatomie und Zellbiologie, Universität Ulm, Albert-Einstein Allee 11, D-7900 Ulm, Germany *Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen in Reutlingen,

Gustav Werner Strasse 3, D-7410 Reutlingen, Germany tUniversitätsfrauenklink Ulm, Prittwitzstrasse 43, D-7900 Ulm, Germany

R E C E I V E D 13 January 1992

ABSTRACT

We investigated whether the stimulation of human granulosa-lutein cells with muscarinic and nicotinic receptor agonists can cause increases in intracellular free calcium (Ca 2 + ) , using Fura-2 microfluorimetry. The addition of carbachol (a non-selective muscarinic and nicotinic receptor agonist) to cultured human granulosa-lutein cells increased intracellular free Ca 2 +

levels. Concentrations as low as 10 nmol/1 were effective. In contrast, nicotine did not evoke elevations of intracellular free Ca 2 + . Basal Ca 2 + levels ranged around 70-140 nmol/1 and maximal, carbachol-induced peaks reached M umol/1. The carbachol-induced Ca 2 + signal was abolished after preincubation of the cells with the muscarinic receptor antagonists quinuclidinyl benzilate or atropine, but it was not affected by removal of extracellular Ca 2 + . Further evi

dence for the involvement of intracellular Ca 2 + stores is provided by experiments in the absence of extracellular Ca 2 + . While thapsigargin (a blocker of ATP-driven Ca 2 + uptake by intracellular stores) and iono-mycin (an ionophore by which Ca 2 + is released from intracellular stores) evoked small Ca 2 + transients, cells pretreated with these agents did not respond to carbachol any more. These data suggest the presence of a functional muscarinic receptor on human granulosa-lutein cells and imply the involvement of intracellular Ca 2 + stores during the cellular response. These results also suggest the participation of the nervous system, acting through muscarinic receptors, in the control of the function of human granulosa-lutein cells. Journal of Endocrinology (1992) 135, 153-159

INTRODUCTION

The support of luteinizing hormone (LH) or human chorionic gonadotrophin (hCG) is essential for the survival and function of the primate corpus luteum (Hsueh, Adashi, Jones & Welsh, 1984; Niswender & Nett, 1988). However, there is also increasing evidence for the functional involvement of ovarian innervation by adrenergic and cholinergic nerves (see Burden, 1978; Burden & Lawrence, 1978, Tsafriri, 1988; Luck, 1990). Stimulation by acetylcholine of bovine granulosa cells, which were undergoing luteini-zation in culture, has been reported to cause secretion of oxytocin and progesterone (Luck, 1990) and the muscarinic receptor antagonists scopolamine and atropine have been shown to inhibit the effect of acetylcholine (Luck, 1990). The author therefore proposed the presence of muscarinic receptor on bovine granulosa-lutein cells. Based on these results, we have

examined, in the present study, whether cultured human granulosa-lutein cells possess a muscarinic receptor coupled to intracellular Ca 2 + .

M A T E R I A L S AND METHODS

Materials

Fura-2 and Fura-2/penta-acetoxymethyl ester ( A M ) were purchased from Calbiochem (La Jolla, CA, U.S.A.), EGTA and DMSO from Fluka (Neu Ulm, Germany) and medium DMEM-F12 from Sigma (Munich, Germany). Fetal calf serum (FCS) was bought from Gibco (Berlin, Germany). Quinuclininyl benzilate (QNB) was a generous gift from Thomae Pharmaceutical Company (Biberach, Germany). A l l other chemicals including atropine, thapsigargin and ionomycin nicotine-di-tartrate-HCl were from Sigma.

Journal of Endocrinology (1992) 135, 153-159 © 1992 Journal of Endocrinology Ltd Printed in Great Britain 0022-0795/92/0135 -0153 $02.00/0

A. M A Y E R H O F E R and others Evidence for muscarinic receptors on human granulosa-lutein cells

Methods

Culture of human granulosa-lutein cells Human granulosa cells were isolated from the follicular fluid (after the cumulus oopherus cells and the oocyte were separated) obtained from 23 women undergoing aspiration of follicular fluid for in-vitro fertilization at the Universitätsfrauenklink Ulm. The project was approved by the local ethical committee and the women gave written consent. Follicular growth was stimulated prior to follicular puncture by treatment of the women with human menopausal gonadot ropin . Follicular growth was monitored by ultrasound and daily measurements of oestradiol, progesterone and L H . Ovulation was induced with 10 000 I U hCG as the follicles reached a diameter of 17 mm. Follicular aspiration was performed 32-36 h after the induction of ovulation under sonographic control in all women who showed a continuous rise in serum levels of oestradiol (Sterzik, Grab, Sasse et al 1989). Cells were mechanically dispersed by repeated aspiration into a 5 ml pipette and through a 20 gauge needle. Follicular fluid was subsequently mixed with dextran T250 (4-5%, w/v) in 0-9% (w/v) NaCl; 5 : 1 ; Roth, Karlsruhe, Germany) and placed in an incubator (37 °C) for 30 min to allow the contaminating red blood cells to settle. Granulosa cells in the supernatant were removed and centrifuged (800 r.p.m., approximately 1000#; Heraeus Digifuge; 3 min). The resulting pellets were repeatedly washed with D M E M : Ham's F12 ( 1 : 1 ; v /v) and centrifuged. Cells were plated with DMEM:Ham' s F12 (1:1) with 10% fetal calf serum in 60 mm Falcon (Becton-Dickinson, Heidelberg, Germany) culture dishes and kept in an incubator at a humidified atmosphere with 5% C 0 2

at 37 °C. One day after plating, media were replaced and all non-adherent cells (mainly red blood cells) removed by careful washing. Subsequently, medium was changed every 2 days and cells were used 2 and 4 days after plating. Granulosa cells in culture, as well as in vivo, undergo luteinization (see Webley, Luck & Hearn, 1988). Luteinization of the cells in the present study is indicated by the ability of the cells to produce progesterone (K. Sterzik & A. Mayerhofer, unpublished results) and the typical morphology of these cells, which resembled the morphology reported previously (McAllister, Mason, Byrd et al. 1990).

Fura-2 loading The cells were loaded with Fura-2/AM (1-5 ^imol/1; dissolved in DMSO) in culture medium without serum for 15-30 min at 37 °C. The cells were then washed with solution A (concentrations given in mmol/1: 140 NaCl, 4- 7 KCl , 1 • 2 K H 2 P 0 4 , 1-2 M g S 0 4 , 1 CaCl 2 , 0-5 ascorbic acid, 11 glucose, 15 Pipes; pH7-2) and used for Ca 2 + measurements

Journal of Endocrinology (1992) 135, 153-159

within 1-3 h. Solution A was modified by adding 1 mmol EGTA/1 instead of 1 mmol CaCl 2 / l to carry out measurements in the absence of extracellular Ca 2 + .

Microscopic measurements of single cells Fluorescence measurements were performed using a Zeiss Microscope Photometer System (fast fluorescence photometry; Zeiss, Oberkochen, Germany), which is based on an inverted microscope (Axiovert 35), equipped for epifluorescence. Measurements at 340/10 nm and 380/10 nm were performed. Fluorescence light from an area adjusted to the cell size was collected (usually approximately 400 nm 2 ) . The autofluorescence of unloaded cells (less than 1% of the fluorescence of dye-loaded cells) was ignored. Background was determined in a cell-free area which contributed to about 5-10% of the signal obtained from Fura-2-loaded cells. Cell signals were corrected for this value and the value of the dark current from the photomultiplier (noise of the photomultiplier in the complete dark, depending on the high-voltage adjustment). Ratio calculations of the 340 nm recording divided by the 380 nm recordings were carried out according to Grynkiewicz, Poenie & Tsien (1985). The calculations are based on the formula:

[Ca 2 + ] = Kö x (R - R m i n / R m a x - R) x ß

(R, ratio of fluorescence [ F ] ; R m a x , 340 nm/F380 nm (fluorescence ratio at [Ca 2 + ] saturation); R m j n , F340 nm/F380 nm (fluorescence ratio in the absence of [ C a 2 + ] ) ; ß = F380 (at zero [Ca 2 + ] ) /F380 (at [Ca 2 + ] saturation); Kd = 224.nmol/l).

Calibration of the Fura-2 signal The Fura-2 signals were calibrated with solution B (150 mmol KCl/1 , 20 mmol Mops/1, pH 7-2; see Föhr, Scott, Ahnert-Hilger & Gratzl, 1989). Solution B, supplemented with 5 jimol/1 of the free acid of Fura 2 and either 5 mmol Ca 2 + /1 or 5 mmol E G T A / 1 was used to determine R m a x , Rmin and ß. With the experimental setup, R m a x was 8-5, R m i n 0-25 and ß was 8-5. For the calculation of free C a 2 + concentrations, an association constant of the Fura-2/calcium of 224 nmol/1 was used (Grynkiewicz et al. 1990).

Addition of test substances The substances were added to the culture dishes with a motor driven syringe (Hamilton microlab P system, speed setting 9) connected with tubings, ending via an Eppendorf comfort tip approximately 3 mm from the cell, or by hand pipetting. A l l test substances were diluted in solution A (exception: Q N B and atropine were dissolved in DMSO; final dilution 0-01%) and were added in a total volume of 50 j i l . The concentrations of the test substances given below always refer

Evidence for muscarinic receptors on human granulosa-lutein cells A. M A Y E R H O F E R and others

800

~ 600 "6 B 3 400

200

(a)

c o

(b)

o e

(d)

0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120 0

Time (s)

20 40 60 80 100 120

F I G U R E 1. Intracellular free C a 2 + transient in a single human granulosa-lutein cell after adding increasing doses of carbachol. (a) The effects of 10 nmol carbachol/1 (Carb; all concentrations inside the pipette). Note that an increased level of approximately 300 nmol Ca 2 + / 1 was achieved, after which the C a 2 f levels returned to basal. (b and c) Higher concentrations of carbachol caused higher and sustained elevation of intracellular C a 2 + levels, which took several minutes to reach (d) baseline level again. Dotted lines between the panels indicate intervals of approximately 5 min (between a and b, and b and c) and approximately 30 s (between c and d). Cells were in culture for 2 days.

to the concentrations of the stock which was added, because the actual effective concentration reaching the cells cannot be given (estimate dilution 1-4).

R E S U L T S

After Fura-2 loading, the granulosa-lutein cells showed a homogeneous distribution of the dye. The basal values of intracellular free Ca 2 + ranged between 70 and 140 nmol/1 (n = 183 individual cells; see Figs 1-4 before stimulation). Wi th the exception of one single cell, the basal C a 2 + value was stable and spontaneous changes of intracellular free Ca 2 + were not

observed (observation periods of individual cells ranging from 2 min to 20 min).

The addition of carbachol produced with 1-5 s an increase in the levels of cytoplasmic free Ca 2 + (with the exception of one cell in all cells tested). The form and maximal peak levels of the Ca 2 + transients appeared to depend on the carbachol concentration: low concentrations produced a small, single transient characterized by a rapid return to baseline values (Fig. \a). Higher concentrations (mmol range) evoked a greater signal with sustained elevations of Ca 2 + levels and a prolonged return period to baseline levels (Fig. \b, c, and especially d). At high concentrations a distinct second, but smaller Ca 2 + transient was

600 -

400

200

Ui)

0 20 40 60

EGTA (1 mmol/1)

— i 1 1

80 100 120

(b)

o u

<

J O

CO

u 1 1 — r — 20

(c)

40 60 Time (s)

80 100 120 0 20 40 60 i i 1

80 100 120

F I G U R E 2. The effect of carbachol (Carb) was not abolished by performing the experiments in the absence of extracellular C a 2 + . (a) The medium contained 1 mmol EGTA/1 (see Materials and Methods). Note sustained elevation of the C a 2 + levels, (b) After adding atropine (100 umol/1) to the cell, carbachol was not effective any more (time interval between a and b approximately 3 min). However, (c) ionomycin (Iono; 1 umol/1) was still able to cause a small increase of free intracellular C a 2 + . Cells were in culture for 2 days.



1000 -(c)

if)

I 0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 %0 100 120

Time (s)

F I G U R E 3. Effects of ionomycin on carbachol-triggered Ca2 +-transients. (a) Time-course of changes in intracellular free C a 2 + levels in response to 10 umol carbachol/1 (Carb) of a single human granulosa-lutein cell (2 days in culture) is depicted. Note that calcium in the medium was replaced by 1 mmol E G T A/1. This cell showed an immediate, strong response to the addition of carbachol (initial large peak), followed by a second, smaller C a 2 + transient, (b) After approximately 3 min the C a 2 + levels had returned to baseline, (c) Ionomycin (Iono, 1 umol/1) was given to the cells and caused a small increase in C a 2 + levels, which returned to (d) basal levels. (e,f) Carbachol, even at high concentration was subsequently not effective in inducing an increase in C a 2 +

levels.

occasionally observed (Figs 3 and 4). Intracellular free C a 2 + stayed elevated (around 350-400 nmol/1) for 3-5 min and then returned to values near the baseline. Maximal peaks of intracellular free Ca 2 + reached maximally 11 umol Ca 2 + /1 (Figs 3 and 4). One single cell showed oscillations for 5 min after carbachol (1 mmol/1) was added. Replacing 1 mmol Ca 2 + /1 in the medium by EGT A (1 mmol/1; Figs 2, 3 and 4) did not abolish the carbachol-induced effect on intracellular free C a 2 + and did not change the described prolonged return period to baseline values. I t should be mentioned that occasionally, as in the presence of C a 2 + in the medium, a second peak following the initial transient was also observed (carbachol at 200 umol/1, 22/23 cells; carbachol at 100 umol/1, 6/ 8 cells; carbachol at 10 umol/1, 10/10 cells; carbachol at 1 mmol/1, 16/16 cells). Preincubation of the cells with the muscarinic antagonist QNB for 30 min prior to carbachol stimulation (carbachol at 10 umol/1 and 100 umol/1), completely abolished increases in intracellular free C a 2 + (QNB at 10 umol/1, 15/15 cells;


QNB at 100 umol/1, 8/8 cells). In addition, atropine (100 umol/1) given to the cells prior to carbachol stimulation also prevented the carbachol-induced rise in free intracellular Ca 2 + (Fig. 2; 9/9 cells examined). Addition of DMSO (001%, in which QNB and atropine were diluted) to the cells did not interfere with their ability to respond to carbachol (four cells tested). When, instead of carbachol, nicotine (1 mmol/1) was used, 13/14 tested cells did not respond with elevation of intracellular free Ca 2 + . To examine the involvement of intracellular Ca 2 + stores further, additional studies with thapsigargin, which inhibits Ca 2 + uptake by the intracellular stores (tested at 10 and 100 nmol/1), and the ionophore ionomycin (tested at 1 and 10 umol/1) were performed in the absence of Ca 2 + in the medium (Figs 3 and 4). For this purpose carbachol (at 100 umol/1 or 1 umol/1; Fig. 3a) was added to a total of 13 cells. Then ionomycin was added (Fig. 3c) and in all 13 cases (at either concentration) caused a small increase of intracellular free Ca 2 + (Fig. 3d) on addition. Ionomycin-treated


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F I G U R E 4. Effects of thapsigargin on carbachol-triggered C a 2 + transients. The measurement of intracellular free C a 2 + in a single human granulosa-lutein cell is seen (compare Fig. 3, cells were cultured for 2 days), (a, b) The response of this cell to carbachol (Carb; 10 umol/1) and the experimental protocol was comparable with the experiment depicted in Fig. 3. (c) Instead of ionomy-cin, thapsigargin ( T G ; at 10 nmol/1) was added. After this treatment, the ability of carbachol to induce a C a 2 + increase was partially blocked (note the difference in the height of the signal between a and d). After (e) additional thapsigargin addition (100 nmol/1) the cell ( / ) did not respond to carbachol any more.

cells were subsequently not responsive to carbachol, even at 100 x higher concentration (Fig. 3e , / ) . The same protocol was used for thapsigargin (Fig 4; 16 cells tested). However, only the higher dose completely abolished the ability of the cells to respond to carbachol, while the lower dose caused only a gradual decrease of the C a 2 + signal (Fig. 4c).

DISCUSSION

The present study provides clear evidence, for the first time, for the presence of muscarinic receptors on human granulosa-lutein cells in culture. These receptors are most probably coupled to the phosphoinosi-t ide /Ca 2 + pathway and use intracellular Ca~+ stores.

Stimulation of oxytocin and progesterone secretion by bovine granulosa-lutein cells in culture in response to acetylcholine, but not in response to carbachol or nicotine, has been reported by Luck (1990), who, using the muscarinic receptor antagonists atropine

and scopolamine, concluded that a muscarinic receptor is probably present on the cells in this species. In contrast to the present work, however, Luck (1990) did not examine the second messenger(s) coupled to the assumed muscarinic receptors. Interestingly, a recent preliminary report in abstract form (Morley, Tsang, Whitfield, & Schwartz, 1991) indicates that, in chicken granulosa cells, carbachol can increase intracellular free Ca 2 + levels four- to eightfold over basal levels. A detailed study by the same authors has now been published (Morley, Tsang, Whitfield, & Schwartz, 1992): These investigators found that carbachol at a maximally stimulating concentration of 2 mmol/1 evoked Ca 2 + elevations in 85% of all examined cells. In their study, the Ca 2 + signal appeared to consist of two components: a large initial Ca2t~ spike, followed by a sustained but lower elevation, with superimposed oscillations and prolonged return to baseline values within several minutes. The initial spike appeared to be derived from release of C a 2 +

from intracellular stores, because it was not abolished



in the absence of extracellular Ca 2 + nor by Ca 2 + channel blockers. In contrast, the sustained elevation is reportedly due to influx of extracellular Ca 2 + (Morley et al. 1992).

The similarities and the discrepancies between the study by Morley et al. (1992) and our present study are obvious. Both studies come to the conclusion that granulosa-lutein cells from different species possess functional, Ca 2 +-activating muscarinic receptors. The main difference is that Morley et al. (1992) found that the second component of the carbachol-induced C a 2 +

signal was due to influx of extracellular Ca 2 + . We do not come to such a conclusion. We found that even in the absence of extracellular Ca 2 + , a second C a 2 +

peak is observed, clearly indicating that solely intracellular Ca 2 + stores are involved in the response of human granulosa-luteal cells to carbachol. In the present study, the Ca 2 + signal showed sustained elevations and a second peak only at high carbachol concentrations (in the range as also used by Morley et al. 1992), while at lower concentrations the signal obtained lacked these features. Although we have no clear explanation for this, one may speculate that ^over-stimulation' of the cell by high carbachol doses may interfere with the capability of the intracellular stores to take up the excess of intracellular Ca24". We substantiated the nature of the intracellular source of C a 2 f by experiments with thapsigargin and ionomycin (see Fasolato, Zottini, Clementi et al. 1991, Zacchetti, Clementi, Fasolato et al. 1991). Indeed, blockage of Ca 2 + uptake by the stores (with thapsigargin) or depletion of the stores (by the ionophore ionomycin) completely abolished the response of the cells upon carbachol stimulation.

Despite the differences of the two studies, our results with human granulosa-lutein cells are in agreement with the reports by Morley et al. (1992) in the chicken and Luck (1990) in the bovine species in respect of the presence of a Ca 2 +-activating muscarinic receptor on granulosa-lutein cells. Currently two muscarinic receptor subtypes, termed M l and M3 (see Watson & Abbott, 1991) are known to be coupled to phospholipase C, which, after activation, hydrolyses phosphatidylinositol 4,5-biphosphate which generates diacylglycerol and inositol 1,4,5-triphosphate (see Nishizuka, 1986). The latter causes an increase in cytosolic free Ca 2 + by releasing intracellular^ stored Ca 2 + . Our results permit the conclusion that this is very probably the case in human granulosa-lutein cells, since the effect of carbachol was not affected in the absence of extracellular Ca 2 + and only disrupting of intracellular stores by thapsigargin and ionomycin (see also Fasolato et al. 1991; Zacchetti et al. 1991) abolished the carbachol signal.

The ovary is densely innervated by the autonomous nervous system and acetylcholine and its synthesizing

enzymes are present in ovarian nerves (Burden, 1978; Burden & Lawrence, 1978). Although Morley et al. (1992) did not observe stimulation of progesterone secretion by carbachol in cultures of chicken granulosa cells, Luck (1990) reported that acetylcholine stimulated oxytocin and progesterone secretion by bovine granulosa-luteal cells. Therefore, the functional relevance of our results can be assumed, but this point requires further investigation. The species-differences in the ability of acetylcholine/carbachol to stimulate Ca2^-dependent secretion of products in luteal cells (e.g. by exocytosis; see Aimers, 1990) may indicate that this is not the sole function of muscarinic receptor activation and that the nervous system may participate in the regulation of ovarian function in other ways.

In conclusion, we have shown that single cell Ca 2 +

measurement can be used as a tool to examine receptor-mediated effects. Using this method we have obtained evidence for the presence of a functional, Ca 2 + -mobilizing muscarinic receptor on human granulosa-lutein cells. Clearly, more work is required to elucidate the nature of the muscarinic receptor subtype on human granulosa-lutein cells and details of the involvement of this receptor in the regulation of specific functions within this cell type.

A C K N O W L E D G E M E N T S

This work was supported by a grant from Deutsche Forschungsgemeinschaft (DFG) Ma 1080/2-1. We thank Zeiss (Oberkochen, Germany) for providing the microfluorometric equipment and Dr B. Rosen-busch for his help in co-ordinating the work. The expert technical assistance of Mrs U . Frölich, Mrs M . Rudolf and M r W. Podschuweit is gratefully acknowledged.

R E F E R E N C E S

Almers, W. (1990). Exocytosis. Annual Review of Physiology 52, 607-673.

Burden, H. W. (1978). Ovarian innervation. In The Vertebrate Ovary, pp. 615-638. Ed. R. E. Jones. New York: Plenum Press.

Burden, H. & Lawrence, I. E. Jr. (1978). Experimental studies on the acetylcholinesterase-positive nerves in the ovary of the rat. Anatomical Record 190, 233-242.

Fasolato, C , Zottini, M , Clementi, E. , Zacchetti, D., Meldolesi, J. & Pozzan, T. (1991). Intracellular C a 2 + pools in PC 12 cells. Three intracellular pools are distinguished by their turnover and mechanism of C a 2 4 accumulation, storage and release. Journal of Biological Chemistry 266, 20159-20167.

Föhr, K. J. , Scott, J., Ahnert-Hilger, G. & Gratzl, M. (1989). Characterization of the inositol 1,4,5-triphosphate-induced calcium release from permeabilized endocrine cells and its inhibition by decavanate and p-hydroxymercuribenzoate. Biochemical Journal 262, 83-89.



Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985). A new generation of C a 2 + indicators with greatly improved fluorescent properties. Journal of Biological Chemistry 260, 3440-3450.

Hsueh, A. J. W., Adashi, E. Y. , Jones, P. B. C. & Welsh, T. H. (1984). Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocrine Reviews 5, 76-127.

Luck, M. (1990). Cholinergic stimulation through muscarinic receptors, of oxytocin and progesterone secretion from bovine granulosa cells undergoing spontaneous luteinization in serum-free culture. Endocrinology 126, 1256-1263.

McAllister, J. M., Mason, J. I., Byrd, W., Trant, J. M., Waterman, M. R. & Simpson, E . R. (1990). Proliferating human granulosa-lutein cells in long term monolayer culture: Expression of aromatose, cholesterol side-chain cleavage and 3ß-hydroxysteroid dehydrogenase. Journal of Clinical Endocrinology and Metabolism 71, 26-33.

Morley, P., Tsang, B. K., Whitfield, J. F. & Schwartz, J. L . (1991). Carbachol increases chicken granulosa cell intracellular calcium ( C a + + ) , but does not affect cyclic AMP or progesterone production. Biology of Reproduction 44, (Suppl. 1), 119 (Abstract No. 265).

Morley, P., Tsang, B. K., Whitfield, J. F. & Schwartz, J. L . (1992). The effect of muscarinic cholinergic agonists on intracellular calcium and progesterone production by chicken granulosa cells. Endocrinology 130, 663-670.

Nishizuka, Y. (1986). Studies and perspectives of protein kinase C. Science 233, 305-331.

Niswender, G. D. & Nett, T. M. (1988). The corpus luteum and its control. In The Physiology of Reproduction, pp. 489-525. Eds E . Knobil, J. Neill et ai New York: Raven Press.

Sterzik, K., Grab, D., Sasse, V., Hüttner, W., Rosenbusch, B. & Terinde, R. (1989). Doppler sonographic findings and their correlation with implantation in an in vitro fertilization program. Sterility and Fertility 52, 825-828.

Tsafriri, A. (1988). Local nonsteroidal regulation of ovarian function. In The Physiology of Reproduction, pp. 527-565. Eds E. Knobil, J. Neill et al. New York; Raven Press.

Watson, S. & Abbott, A. (1991). Muscarinic acetylcholine receptors. Trends in Pharmacological Sciences (January 1991 Suppl.) p. 19.

Webley, G. E. , Luck, M. R. & Hearn, J. P. (1988). Stimulation of progesterone secretion by cultured human granulosa cells with melatonin and catecholamines. Journal of Reproduction and Fertility 84, 669-677.

Zacchetti, D., Celmenti, E. , Fasolata, C , Lorenzon, P., Zottini, M., Grohovaz, F., Fumagalli, G., Pozzan, T. & Meldolesi, J. (1991). Intracellular C a 2 f pools in PC 12 cells. A unique, rapidly exchanging pool is sensitive to both inositol 1,4,5-triphosphate and caffeine-ryanodine. Journal of Biological Chemistry 266, 20152-20158.


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