Regulation of oxytocin secretion by the ovine corpus luteum: effect of activators of protein kinase C J. J.

Hirst, G. E. Rice, G. Jenkin and G. D. Thorburn

Department of Physiology, Monash University, Clayton, Victoria 3168, Australia (Requests for offprints should be addressed to J. J. Hirst, Oregon Regional Primate 505 N.W. 185th Avenue, Beaverton, Oregon 97006, U.S.A.) REVISED MANUSCRIPT RECEIVED 22 August 1989

Research Center,

ABSTRACT

The effect of protein kinase C activation and dibutyryl cyclic AMP on oxytocin secretion by ovine luteal tissue slices was investigated. Several putative regulators of luteal oxytocin secretion were also examined. Oxytocin was secreted by luteal tissue slices at a basal rate of 234\m=.\4\m=+-\32\m=.\8pmol/g per h (n 24) during 60-min incubations.Activators of protein kinase C: phorbol =

12,13-dibutyrate (n 8), phorbol 12-myristate,13\x=req-\ acetate (n 4) and 1,2-didecanoylglycerol (n 5), caused a dose-dependent stimulation of oxytocin secretion in the presence of a calcium ionophore (A23187; 0\m=.\2\g=m\mol/l).Phospholipase C (PLC; 50\p=n-\250 units/l) also caused a dose-dependent stimulation of =

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INTRODUCTION

The corpus luteum of a number of mammalian species contains substantial amounts of the nonapeptide oxytocin (Wathes, Swann, Porter & Pickering, 1986).

Oxytocin is synthesized by the corpus luteum of cattle and sheep, and is secreted into the ovarian vein (Flint & Sheldrick, 1982; Rodgers, O'Shea, Findlay et al. 1983; Ivell & Richter, 1984; Walters, Schams & Schallenberger, 1984). The mechanisms involved in the control of the secretion of oxytocin by the corpus luteum remain equivocal. Flint & Sheldrick (1983a) reported that systemic injection of a prostaglandin F2a

analogue into sheep, during the oestrous cycle, stimu¬ lated luteal oxytocin secretion and that prostaglandin E2 had similar effects (Sheldrick & Flint, 1984). These prostaglandins did not, however, stimulate oxytocin

secretion, either from luteal slices incubated in vitro from isolated luteal cells (Hirst, Rice, Jenkin & Thorburn, 1986, 1988; Harrison, Kenny & Niswender, or

1987).

We have previously demonstrated that phospho¬ lipase C (PLC) stimulates oxytocin secretion from

oxytocin secretion by luteal slices. Phospholipase C-stimulated oxytocin secretion was potentiated by the addition of an inhibitor of diacylglycerol kinase (R59 022; n 4). These data suggest that the activation of protein kinase C has a role in the stimulation of luteal oxytocin =

secretion. The results are also consistent with the involvement of protein kinase C in PLC-stimulated oxytocin secretion. The cyclic AMP second messenger system does not appear to be involved in the control of oxytocin secretion by the corpus luteum. Journal of Endocrinology (1990) 124, 225\p=n-\232

luteal tissue slices incubated in vitro (Hirst et al. 1988). We have also shown that oxytocin secretion is enhanced by an increase in the influx of calcium into luteal cells, and is inhibited by removal of calcium from the extra¬ cellular fluid (Hirst et al. 1986). These findings are consistent with the involvement of mechanisms leading to phospholipid breakdown and calcium mobilization in the control of luteal oxytocin secretion. Enhanced plasma membrane phospholipid metab¬ olism has been implicated in secretory responses that result from agonist stimulation (Nishizuka, 1984). These processes are initiated by activation of PLC, resulting from an agonist-receptor interaction. The hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol is catalysed by PLC. Inositol 1,4,5-trisphosphate stimu¬ lates the release of calcium from intracellular stores, whilst diacylglycerol has been shown to activate the

calcium-dependent phospolipid-dependent protein kinase (protein kinase C; Berridge, 1984). Protein kinase C has been identified in many secretory cell types, including bovine luteal cells (Davis & Clark, 1983; Brunswig, Mukhopadhyay, Budnik et al. 1986).

Alila, Dowd, Corradino et al. (1988) have shown that

activators of protein kinase C stimulate progesterone secretion by a purified population of bovine small, but not large, luteal cells. Large luteal cells are, however, the source of luteal oxytocin in the sheep (Watkins, 1983; Theodosis, Wooding, Sheldrick & Flint, 1986). In the present study, we have investigated the effect of protein kinase C activation in the control of luteal oxytocin secretion using phorbol 12,13-dibutyrate, phorbol 12-myristate, 13-acetate and a synthetic diacylglycerol 1,2-didecanoylglycerol. The effect of increasing the endogenous concentration of diacylgly¬ cerol on luteal oxytocin secretion was also examined using a recently synthesized inhibitor of diacylglycerol kinase (R59 022). It has been shown that the cyclic AMP (cAMP) second messenger system regulates steroidogenesis by ovine luteal cells (for review see Hoyer & Niswender, 1985). We have examined the effect of cAMP on oxytocin secretion. The involve¬ ment of protein kinase C and cAMP in the control of oxytocin secretion by the corpus luteum has not previously been investigated. To identify endogenous regulators of oxytocin secretion further, we examined the effect of several putative secretagogues on oxytocin secretion by luteal tissue slices. MATERIALS AND METHODS

Ovine luteal tissue was prepared and incubated by the method previously described (Hirst et al. 1988). Briefly, ovine corpora lutea were obtained from ovaries col¬ lected at local abattoirs. The ovaries were placed in cold saline within 15 min of slaughter of the ewes. Corpora lutea, between days 6 and 9 of the oestrous cycle, were selected for use in the study on the basis of size-age charts (Restall, 1964) and gross appearance with reference to the photographic series of Oldham & Lindsay (1980). Corpora lutea were excised from the ovaries at 4°C and cut into slices of < 0-5 mm thick with a wet weight of 20-40 mg, using a hand microtome, incorporating a skin graft knife blade. The first and last slices of each corpus luteum were discarded and tissue processing was always completed within 90 min of the time of collection of the ovaries. Luteal slices were placed in Hepes-buffered medium 199 (Flow Laboratories, Sydney, Australia) for up to 30 min until incubations were commenced. Slices from each corpus luteum were divided equally between treatment groups. Each slice was incubated individually in 5 ml open-topped wells, in multi-well trays (Flow Laboratories) which contained 3 ml Hepes-buffered Krebs-Henseleit solution (pH 7-4; NaCl, 120 mmol/1; KC1,4-6 mmol/1; CaCl2,2-0 mmol/1;

MgS04, 1-1 3-0 mmol/1;

mmol/1; KH2P04, 1-1 mmol/1; NaHC03, glucose, 11-0 mmol/1; Hepes, 20 mmol/1;

Sigma Chemical Co., St Louis, MO, U.S.A.), supple¬ mented as stated below. Incubations were performed at 37 °C, under an atmosphere of 02, in a water bath shaking at 30 oscillations per min. Before commencing an experiment, slices were preincubated for three incubation periods of 20 min and the medium was changed between each incubation. In order to determine the concentration of oxytocin in luteal tissue, nine corpora lutea were collected in the manner described above. The weights of the corpora lutea were recorded and cross-sections, weighing 6080 mg, were cut from each corpus luteum. Oxytocin was extracted from the luteal sections by the method of Wathes, Swann & Pickering (1984). The dried luteal extract was resuspended in oxytocin assay buffer and stored at —20 °C until assayed. Phorbol 12,13-dibutyrate, phorbol 12-myristate, 13-acetate (Sigma Chemical Co.) and 6-[2-[4-[(4floropheny 1) phenylmethylene]-1 -piperidinyl]ethyl]-7methyl-5H-thiazolo[3,2-a] pyrimidin-5-one (R59 022;

Janssen Pharmaceutica Research Laboratories, Beerse, Belgium), were diluted and stored in ethanol. 1,2Didecanoylglycerol was obtained, in hexane, from Serdary Research Biochemicals (London, Ontario, Canada). Aliquots of this solution were evaporated under nitrogen and resuspended in ethanol. All ethanol stock solutions were stored at 20 °C and aliquots of stock solutions were added to incubations to the concentrations detailed below. A fine suspension of 1,2didecanoylglycerol, in incubation medium, was made by vigorous vortexing for three periods of 10 s. The final concentration of ethanol in the incubation medium was 0-1% and the same concentration of ethanol was —

present in control incubations. Phospholipase C (from

Clostridiumperfringens, > 300 units/mg protein; Sigma Chemicals Co.) was purified chromatographically and added to the incubation medium and stored as a sus¬ pension in (NH4)2S04 (3-2 mol/1; pH 6-0) at 4 °C. The concentration of (NH4)2S04 in control and PLC incu¬ bations was 101 mmol/1. Neither oxytocin secretion nor the pH of the incubation medium were affected by this concentration of (NH4)2S04 (Hirst et al. 1988).

Dibutyryl cAMP, phenylephrine, isoprenaline, ßendorphin, naloxone and calcium ionophore (A23187) were obtained from Sigma Chemical Co. and were diluted in incubation medium immediately before the commencement of incubations. Ovine prolactin (NIADDK-oPRL-I-1, NIH, Bethesda, MD, U.S.A.) and epidermal growth factor (EGF), purified from mouse submaxillary glands by the method of Elson, Browne & Thorburn (1984), were also diluted in incu¬ bation medium. The pH of the incubation medium

monitored after the inclusion of all additions and adjusted to 7-4 when necessary. Following preincubation, luteal slices were incu¬ bated in test solutions or in medium containing the was

appropriate

vehicle for 60 min. At the conclusion of the incubations, samples of medium were immediately frozen in a —70 °C bath and, once frozen, were stored at —20 °C until assayed.

Oxytocin assay Oxytocin, present in medium or in resuspended luteal tissue extracts, was quantified by radioimmunoassay as described by Rice & Thorburn (1985) using an antiserum to synthetic oxytocin (GJ 137/1) generously provided by Dr A. P. F. Flint (Institute of Zoology, Regent's Park, London, U.K.). The sensitivity of the assay was 9 fmol/tube and the intra- and interassay coefficients of variation, estimated from six assays, were 5-1 and 8-7% respectively. The cross-reactivity of the antiserum was less than 0-02% with arginine vasopressin, lysine vasopressin, mesotocin, pressinole acid and prolactin; less than 0-4% with melanocytestimulating hormone inhibiting factor and less than 0-7% with isotocin. Statistical

analysis

The secretion of oxytocin by luteal slices into the incu¬ bation medium was corrected for wet weight of luteal tissue and is expressed as secretion per g of luteal tissue during 60 min of incubation. All incubations of luteal slices were performed in duplicate and the secretion rates from the duplicate incubations were averaged. Within each experiment, luteal slices from each corpus luteum used were distributed equally to control and all treatment incubations. The results presented are expressed as the mean ± s.e.m. for the

numbers of corpora lutea used in each experiment. Differences between means were analysed using Student's /-test or, when multiple comparisons were made, by analysis of variance and Fisher's least sig¬ nificant difference test. The data presented in Fig. 3 were analysed by a three-way factorial analysis of variance as described by Sokal & Rohlf (1969). RESULTS

Luteal slices secreted oxytocin into the incubation medium at a mean rate of 234-4+ 32-8 pmol/g per h (« 24) during 60-min incubation periods. This rate of secretion was not significantly different to that observed in a previous study (312-5±44-9 pmol/g per h, =18; Hirst et al. 1988). The concentration of oxytocin in luteal tissue, obtained at a similar stage of the oestrous cycle to that used for secretion studies, was 2-6±0-4nmol/g luteal tissue (wet weight, 9). The mean weights of the corpora lutea used for deter¬ mination of concentration was 0-425 + 0-018 g and the calculated total oxytocin content of the corpora lutea was IT ±0-4nmol. In the absence of calcium ionophore, oxytocin secretion by luteal slices was stimulated by phorbol 12,13-dibutyrate at a concentration of 1000 nmol/1 (Table 1). Phorbol 12-myristate,13-acetate had no effect on oxytocin secretion over a concentration range of 10-1000 nmol/1. Both phorbol esters, however, stimulated oxytocin secretion in a dose-dependent manner in the presence of a low concentration of the calcium ionophore A23187 (0-2 pmol/1; Table 1). =

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1. Effect of phorbol 12,13-dibutyrate and phorbol 12-myristate,13acetate, in the presence and absence of the calcium ionophore A23187, on oxytocin secretion by ovine luteal tissue slices incubated for 60 min. Values table

are means ± s.e.m. The numbers of corpora lutea used in the investigation are indicated in parentheses

Oxytocin secretion (% of control) Phorbol

(nmol/1)

without A23187

with A23187

100(n 9)a

107-2+ 13-3 (« 8)6

12,13-dibutyrate

0 10 100 1000 Phorbol l2-mvristate,13-acetate

(nmol/1) 0 10 100 1000

=

=

1210+13-2 1280+18-6 132-6+12-5*

125-9+11-6 135-6 + 15-4*

100(« 5)c

I08-0 + 8-7(n

=

98-0 + 4-5 95-7 + 9-4 80-0 ±16-4

151-4+12-5**

110-1+9-4

117-2+1-3*

144-2+10-3*

*/>