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Stimulation of ovine choriomammotrophin release, in vitro, by phospholipase C
Placenta (1986),7, 533-54z
Stimulation of Ovine Choriomammotrophin Release, in Vitro, by Phospholipase C
G. E. RICE & G. D. T H O R B U R N Department of Physiology, Monash University, Clayton, Victoria 3168, Australia Paper accepted 7.4.1986 INTRODUCTION Ovine choriomammotrophin (oCM, previously placental lactogen) is a protein hormone synthesized and secreted by fetal trophoblast cells throughout gestation (Dubois, Martal and Djianne, 1976; Martal and Djianne, 1977; Reddy and Watkins, 1978). Choriomammotrophins display considerable structural homology with growth hormone and prolactin (Hurley et al, 1977) but, despite extensive in vitro and in vivo studies, the factors regulating the synthesis and secretion of this hormone have yet to be established (Thorburn, 1979). Recently, it has been recognized that the control of protein hormone secretion may be intimately involved with plasma membrane phospholipid metabolism (Handwerger et al, 198Ia; Leung, Raymond and Labrie, 1982; Sutton and Martin, i982 , Canonico, Valdenergo and MacLeod, t983; Canonico et al, 1985). It has been suggested that the phospholipid metabolites diacylglycerol and inositol trisphosphate act as second messengers in the process of receptormediated cell activation (for reviews see Farese, I983; Berridge and Irvine, 1984). This process is dependent upon the hydrolysis of membrane phospholipid by the phosphodiesterase phospholipase C, and the liberation of the polar head group and sn-I,a-diacylglycerol. Diacylglycerol may act directly to activate protein kinase C, an enzyme implicated in hormone secretion and granule exocytosis (Rittenhouse-Simmons, 1979, Bell and Majerus, 198o; Kawahara et al, 198o; Michell, 1983; Nishizuka, 1983; Sano et al, 1983; Takai et al, i979; Di Virgilo, Lew and Pozzan, 1984; Swann and Whitaker, 1985). Alternatively, diacylglycerol may be metabolized by diacylglycerol lipase, which has recently been shown to be present in fetal membranes and uterine decidua (Di Renzo et al, i98i , Okazaki et al, I98I), liberating arachidonic acid, the substrate for cyclo-oxygenase and lipoxygenase pathways-and itself a putative second messenger in the regulation of cellular function (McPhail, Clayton and Snyderman, 1984). There is a paucity of information concerning factors which regulate the release of choriomammotrophin from the placenta, and the aim of the present study was to assess the potential involvement ofa phospholipase C- (PLC-)mediated pathway in the regulation ofoCM release from the ovine placenta. To further characterize PLC-stimulated oCM release, we investigated the effects of altering the availability of extracellular calcium. Membrane calciumchannel blocking agents (verapamil, nifedipine, cobalt chloride and lanthanum chloride) and nominally calcium-free medium (prepared as calcium-free and containing I mM EDTA) were used as pharmacological tools for this purpose. Inhibitors of arachidonic acid metabolism 533
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(ibuprofen, naproxen and nordihydroguaiaretic acid (NDGA)) were used to assess the involvement of arachidonic acid metabolites in PLC-stimulated oCM release.
MATERIALS AND METHODS Materials Phospholipase C (PLC; Bacillus cereus, Type V), verapamil, nordihydroguaiaretic acid (NDGA), naproxen and nifedipine were purchased from Sigma Chemical Co., St Louis, MO, USA. Ibuprofen was obtained from The Upjohn Co., Kalamazoo, MI, USA. Stock solutions of N D G A , naproxen and ibuprofen were prepared using dimethyl sulphoxide as solvent. Verapamil and nifedipine solutions were prepared in ethanol. The final concentration of either solvent in explant incubations did not exceed i per cent, and at this concentration neither of these solvents affect oCM release. Tissue p r e p a r a t i o n a n d i n c u b a t i o n Placentomes were obtained, at post mortem, from Merino-Border Leicester ewes (n = 20) at i oo to 14~ days' gestation. Four or five placentomes were selected at random from the pregnant horn of the uterus and immediately placed in ice-cold saline. T h e placentomes were decapsulated, the haemophagous zone was removed, and the remaining tissue was washed with Krebs-Ringer (NaC! I23 mmol/l; KCI 4.6mmol/l; MgSO4.7H20 i.I mmol/l; KHzPO 4 i.i mmol/l; CaCI 2 1.9mmol/q; and glucose Ii mmol/I) buffer containing 2omM N-2hydroxyethylpiperazine-N'-2-ethanesulphonic acid ( K R - H E P E S , p H 7.4). Explants (4 to 8) of approximately I to 2 mm 3, with a combined wet weight of 45 to 55 mg (5-3-I-o.6 ~tg oCM/5 o mg), were placed in each well of multi-well culture plates (Linbro, i2-well culture plates, 2.4 x 1. 7 cm; Flow Laboratories Inc., McLean, VA, USA) containing 5 ml of K R - H E P E S buffer. T h e tissue explants were incubated at 37~ in a metabolic shaking water bath (3o to 4 ~ cycles/minute) under an oxygen atmosphere. The medium in each well was changed after the first and second hour of incubation. The tissue was then incubated for a further i to 3 h (experimental incubation period) in 3 ml of control medium or medium containing one of the variables to be tested. At the end of the incubation period an aliquot (o. 5 to i .o ml) of supernatant was removed and immediately placed in an ethanol bath ( - 4o~ In those experiments in which the time course of oCM release was assessed, 200 #1 of medium were removed from control and experimental incubation wells at specified time intervals and immediately frozen. All samples were assayed for oCM content within three days of collection. The release of oCM from placental explants was expressed as either/~g oCM released per 50 mg wet weight tissue/h, or as a percentage of the release of oCM during control incubations run in parallel. The statistical differences between treatment means were assessed by analysis of variance and Student-Newman-Keuls test (Sokal and Rohlf, i969). To elucidate the effects of extracellular calcium on oCM release, placental explants were incubated in K R - H E P E S buffer or K R - H E P E S buffer prepared without CaCI 2 and containing i mM E D T A (calcium-free medium). Explants were incubated in calcium-free medium throughout the preincubation and experimental incubation periods. The effects of perturbing cellular calcium flux, on basal and stimulated oCM release, were also assessed by the inclusion of the organic calcium-channel blocking agents verapamil 0oo/~M) and nifedipine (ioo/~M), and the inorganic calcium-channel blocking agents cobalt chloride (4 mM) and lanthanum chloride (i mM), in the preincubation and experimental incubation medium.
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To elucidate further the mechanism of PLC-stimulated oCM release, the affects of cyclooxygenase and lipoxygenase inhibitors were examined. Placental explants were pre-incubated for I h in a medium containing one of the following agents: ibuprofen (io/~M); naproxen (ioo/ZM); or N D G A (5/ZM). The medium was then replaced with buffer containing the same concentration of inhibitor to which was added o.2 units PLC/ml (4 #1). The incubations were then conducted as described above. oCM radioimmunoassay oCM was assayed according to the method of Chan, Robertson and Friesen (1978) as modified by Leach Harper, Ford and Thorburn (1984). The oCM standard and antiserum were generously provided by Dr M. J. Waters (Department of Physiology, University of Queensland, Australia). Iodination of oCM was performed using the chloramine T method of Hunter and Greenwood (i962). The cross-reactivity of the antiserum with ovine growth hormone, luteinizing hormone, follicle-stimulating hormone, prolactin and thyroid-stimulating hormone was less than I per cent. The intra- and inter-assay coefficients of variation were 3-4 and 2o. i per cent, respectively. Samples were diluted I : 8o or I : 1oo with buffer prior to assay, and at this concentration none of the experimental reagents interfered with the assay. P r o t e i n assay Tissue protein content was estimated using a protein dye-binding method (Bradford, 1976) and bovine serum albumin as the reference standard. Tissue samples (5 ~ to ioo mg wet weight tissue) were homogenized in I M N a O H and centrifuged at 2ooo g for 2o min. The pH of the supernatant was adjusted to 7.o with o.5 M HCI. An aliquot (2oo/~1) was mixed with 6oo/~1 distilled water and 2oo/zl of dye reagent (Bio-Rad Laboratories, Richmond, CA, USA). The optical density (at 595 nm) was measured within 3o min from the addition of dye reagent to protein standards and samples, using a Varian spectrophotometer (Series 634 ). Protein concentration was calculated by linear regression analysis. L a c t a t e d e h y d r o g e n a s e (LDH) L D H released into incubation medium during control and PLC-treatment incubations was quantified spectrophotometrically by the method of Pickup and Hope (I97 0.
RESULTS Basal release o f o C M in vitro The release of oCM from placental explants incubated in K R - H E P E S buffer averaged 2.93 + o.25/~g/5 o mg wet weight tissue/h (n = 2o). This rate of release was maintained for at least 4 h in in vitro incubation. The tissue content of oCM before incubation was 5.3 + o.6 #g/5 o mg tissue wet weight (n = 12). The release of oCM during extended incubation periods thus represents the release of newly synthesized protein and is indicative of sustained tissue viability during the incubation period. Effects o f p h o s p h o l i p a s e C (PLC) on o C M release in vitro Incubation of placental explants in the presence of P L C caused a dose-dependent stimulation of oCM release. As little as o.2 units PLC/ml caused oCM release from tissue to approximately double that observed in control incubations (3.26_+o.3ktg/5omg/h ( n = I I ) and 7.o8 +o.39/~g/5 o mg/h (n = I I), respectively: Figure 1). P L C treatment did not increase,
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Placenta (~986), Vol. 7 DOSE RESPONSE: PLC-STIMULATED oCM RELEASE ~-00-
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Figure I. Doseresponseof phospholipaseC--(PLC-)stimulatedovinechoriomammotrophin(oCM) release.The release of oCM from placentalexplants, incubatedfor 2 h at 37~ is presented as the percentageof oCM released during control incubation(KR-HEPES buffer). Values + s.e. are presented;, = 3 or 4- * signifies a significantdifference (P < o.os) between the mean ovine choriomammotrophin(oCM) release from control and treatment incubations.
significantly, the release of total protein or lactate dehydrogenase (LDH) into the medium when compared with release during control incubations. Total protein and L D H concentrations in incubation medium following control or P L C treatment were 4 . 8 + 1.9 #g/ml and 6.2 + 2.o #g/ml (n = I2), respectively, for protein, and 6o.6 + 9.2 mU/ml and 83.8 + i9.9mU/ml (n = i I), respectively, for LDH. A significant ( P < o.oi) increase in oCM release occurred within 5 min after the addition of PLC (0.2 units/ml) and release continued throughout the incubation period (Figure 2). T o assess whether P L C treatment stimulated oCM release by irreversibly altering the permeability of the plasma membrane, explants were incubated for 15~ min in the presence of excess P L C (io units PLC/ml) and then incubated in control medium for a further 6o min. At this concentration PLC stimulated oCM release throughout the initial i so-min incubation, when compared with control incubations. When the medium was replaced with control medium, oCM release in PLC-treated tissues returned to basal levels and was not significantly different from oCM release observed in control incubations (Figure 3). Effects o f c a l c i u m o n b a s a l a n d P L C - s t i m u l a t e d o C M release T o determine the effects of extracellular calcium concentration on basal and phospholipase C- (PLC-)stimulated ovine choriomammotrophin (oCM) release, placental explants were incubated for 2 h in calcium-free medium. As shown in Figure 4, the basal release o f o C M from
Rice, Thorburn: Ovine Choriomammotrophin Release in Vitro
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TIME COURSE : PLC-STIMULATED oCM RELEASE
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TIME ; min Figure 2, Time course of ovine choriomammotrophin (oCM) release. The release of oCM from control and phospholipase C- (PLC-)treated (o.2 unit/ml) explants, incubated in vitro, is presented as ng oCM released per ml incubation buffer. Values + s.e. are presented; n = 3- * signifies a significant difference ( P < o.o5) between the mean ovine choriomammotrophin (oCM) release from control and treatment incubations.
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TIME ; min Figure 3. The effect on basal ovine choriomammotrophin (oCM) release of preincubation for ] 5~ min of placental explants with io units/ml phospholipase C (PLC). oCM released from control (KR-HEPES buffer) and PLC--treated incubations is presented as ng oCM per ml incubation buffer. The incubation medium in both control and treatment wells was replaced at i5o rain with KR-HEPES buffer. The tissue was then incubated for a further I h and the oCM release was measured. The mean of two experiments is presented.
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EFFECTS OF CALCIUM CHANNEL BLOCKING AGENTS AND EDTA ~350Z
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Figure 4. The effect of reduced extracellularcalciumavailabilityon basal and phospholipase (i- (PLC-)stimulated ( + PLC) ovinechoriomammotrophin(oCM) release,oCM released from placentalexplantsincubatedin KR HEPES bufferat 37~ for 2 h is presentedas a percentageofoCM releasedduringcontrolincubations.The effectof incubating tissue in KR-HEPES buffer (CONT) or buffer containingverapamil(VERAP), nifedipine(NIFED), lanthanum chloride (LaCI3), cobaltchloride(CoCl2) or nominallycalcium-freemedium(EDTA i mM) are shown. Values + s.e. are presented;* = P < o.ooi; n = 3 to 7.
explants incubated in calcium-free medium ( E D T A i mM) was not significantly different from oCM released from control incubations. Incubation of placental explants in calcium-free medium, however, completely abolished the increase in oCM release following the addition of 0.2 units/ml PLC. Neither the organic nor inorganic calcium--channel blocking agents affected basal oCM release. PLC-stimulated release of oCM, also, was not affected by the inclusion of either verapamil or nifedipine at a concentration of ioo/aM. T h e effects of P L C on oCM release, however, were abolished by the inclusion of either CoCI 2 (4 mM) or LaCI 3 (i mM). T o examine the potential involvement of voltage-dependent calcium channels in oCM release, explants were incubated in medium containing IOO mM potassium chloride. This depolarizing stimulus had no significant effect on basal oCM release, oCM release during the 2-h incubation period averaged 84. 4 + 14. 4 (n = 3) per cent of oCM released during control incubations. Effects o f i n h i b i t o r s o f a r a c h i d o n i c acid m e t a b o l i s m o n P L C - s t i m u l a t e d o C M release The basal release of oCM was not significantly affected by inhibitors of arachidonic acid metabolism (Figure 5). T h e effects of the cyclo-oxygenase inhibitors ibuprofen and naproxen, and tfle 5-1ipoxygenase inhibitor N D G A , on PLC-stimulated release o f o C M are presented in Figure 5. Ibuprofen, naproxen and N D G A all inhibited PLC-stimulated release ofoCM. In the presence of-these inhibitors, oCM was not significantly different from oCM released during control incubations.
539
Rice, Thorburn: Ovine Chorioraammotrophin Release in Vitro
EFFECTS OF INHIBITORS OF ARACHIDONIC ACID METABOLISM 350 me l--
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Figure 5. The effects of inhibitors of arachidonic acid metabolism on basal and phospholipaseC- (PLC-)stimulated ovine choriomammotrophin(oCM) release (+ PLC). oCM released from placental explants as a percentage of that releasedfrom controlexplants incubated for 2 h at 37~ is presented. The effectsof incubatingtissue in buffer(CONT) or buffer containing ibuprofen (IBUPR), naproxen (NAPRO) or nordihydroguaiareticacid (NDGA) are shown. Values _+_+ s.e. are presented; * = P < o.ol; n = 4 to 6.
DISCUSSION In the present study, we demonstrated that phospholipase C (PLC) causes a dose-dependent release ofovine choriomammotrophin (oCM) from ovine placental explants incubated in vitro. P L C has been previously implicated in secretion-coupling in other tissues (Nishizuka, i983). P L C causes a dose-dependent release of growth hormone (GH), a protein with considerable structural and, possibly, functional homology with oCM, from rat pituitary primary cell cultures (Ohmura and Friesen, 1985). PLC-stimulated G H release may occur by a similar mechanism to that which results in increased release of oCM. T h e concentration of P L C required to achieve a doubling of the basal rate of G H secretion was, however, 2o-fold higher than the concentration used in the present study to achieve the same increase in o C M release. A dependence of PLC-stimulated release of o C M upon the availability of extracellular calcium was demonstrated. T h e omissionof calciumand the inclusion of ethylenediaminetetraacetic acid ( E D T A ) (i mM) in the incubation buffer completely abolished the stimulatory action of PLC. The inhibition of PLC-stimulation by the inclusion of the inorganic calciumchannel blocking agents lanthanum chloride (i mM) and cobalt chloride (4mM) in the incubation buffer further supports this suggestion. These ions have been reported to competitively displace calcium from the extracellular surface of the plasma membrane and prevent its subsequent influx (Cervetto and Piccolino, T974; Weiss, 1974; Martin and Richardson, 1979). T h e organic calcium-channel blocking agents verapamil (ioo #M) and nifedipine (i oo #M) were ineffective in blocking P L C stimulation of o C M release. These agents have been reported to antagonize voltage-dependent calcium channels (Fleckenstein, 1977; Bolton, I979) and it is thought that they interfere with the action of calcium in linking electrical
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Placenta (1986), Vol. 7
membrane activity with intracellular biochemical events, which results in cellular activation. The inability of these agents to prevent PLC-stimulated oCM release suggests that voltagedependent calcium channels are not involved in this response. The observation by Handwerger et al (198ib) that a depolarizing concentration of potassium chloride (ioo raM) failed to elicit significant release of human placental lactogen (choriomammotrophin) from placental explants incubated in vitro supports the suggested lack of involvement of voltage-dependent calcium channels in the release of this protein hormone. Similar results, using Ioo mM potassium chloride, were obtained in the present study of oCM release. It is of interest to note that basal oCM release was not suppressed by reduced extracellular calcium availability (Figure 4). Calcium is essential for release in a number of secretory systems involving the exocytosis of hormone-containing granules (Douglas and Poisner, x964; Douglas, 1968; Rasmussen and Goodman, i977; Trifaro, x977; Moriarty, I978). Although the mechanism involved in the paradoxical effect of calcium on basal oCM release remains unclear, our results are consistent with those of Handwerger et al (I98Ib) who demonstrated that the release of human placental lactogen in vitro is not suppressed by decreased extracellular calcium concentrations. In human placental tissue, stimulation of placental lactogen was observed in response to decreased extracellular calcium. Our results further suggest that PLC-stimulated oCM release involves arachidonic acid metabolites since the stimulatory effects of PLC were blocked by three inhibitors of arachidonic acid metabolism: nordihydroguaiaretic acid (NDGA), ibuprofen and naproxen. From the present study, it is not possible to elucidate further the mechanism of action of PLC-stimulated oCM release. Arachidonic acid, however, has been previously shown to stimulate the release, in vitro, of human placental lactogen (Handwerger et al, I98Ia) and rat prolactin (Canonico et al, x985) and the release, in vivo, of oCM (Huyler et al, I985). In conclusion, the results of the present study, that PLC stimulates the release of oCM in a dose-dependent manner, and that PLC-stimulated oCM release is suppressed by reduced extracellular calcium availability and inhibitors of arachidonic acid metabolism, provide some insight into a possible mechanism of action. We suggest that PLC hydrolyses plasma membrane phospholipids causing the release of polar head group moieties" and increasing the plasma membrane concentration of diacylglycerol. The polar head groups liberated by PLC hydrolysis have been implicated in initiating cell activation (see Berridge and Irvine, i984, for review). It is unlikely, however, that these water-soluble products participate in the release of oCM stimulated by exogenous PLC. These products would disperse in the incubation medium and thus not reach their intracellular site of action. Diacylglycerol, however, may initiate cell activation, directly, by acting as a second messenger, or indirectly, by being metabolized to arachidonic acid (Nishizuka, i983). Further study of this proposed mechanism is required to elucidate the action of PLC.
SUMMARY Phospholipid metabolites have previously been implicated in receptor-mediated stimulation of protein hormone secretion. As the factors which regulate the release of choriomammotrophin remain to be elucidated, we investigated the potential involvement of phospholipase C-induced phospholipid metabolism in the release of this placental hormone. Phospholipase C (PLC) caused a dose-dependent release of choriomammotrophin from ovine placenta, incubated in vitro. At a concentration of o.a units/ml (o.z 5 #g protein/ml), PLC caused the release of choriomammotrophin from placental tissue to approximately double that observed in control
Rice, Thorburn: Ovine Choriomammotrophin Release in Vitro
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i n c u b a t i o n s (7.o8 + o. 4/~g/5 o m g / h a n d 3.z6 + o. 3 gg/5 o m g / h , respectively). P L C t r e a t m e n t did n o t significantly alter p l a s m a m e m b r a n e p e r m e a b i l i t y , as i n d i c a t e d by t h e release o f lactate d e h y d r o g e n a s e a n d protein. P L C - s t i m u l a t e d release o f o C M was c o m p l e t e l y a b o l i s h e d b y i n c u b a t i o n in c a l c i u m - f r e e m e d i u m or b y p r e i n c u b a t i o n w i t h the i n o r g a n i c c a l c i u m - c h a n n e l b l o c k i n g agents cobalt c h l o r i d e (4 raM) a n d l a n t h a n u m c h l o r i d e (I mM). T h e effects o f P L C t r e a t m e n t o n ovine c h o r i o m a m m o t r o p h i n ( o C M ) release were also i n h i b i t e d b y p r e i n c u b a t i o n o f placental tissue w i t h i n h i b i t o r s o f a r a c h i d o n i c acid m e t a b o l i s m : i b u p r o f e n ( I O - 5 M), n a p r o x e n ( i o TM M) or n o r d i h y d r o g u a i a r e t i c acid ( N D G A ) (5 x i o -6 M). T h e s e results suggest t h a t t h e effects o f P L C o n t h e release o f c h o r i o m a m m o t r o p h i n are m e d i a t e d via m e t a b o l i t e s o f a r a c h i d o n i c acid.
ACKNOWLEDGEMENTS This work was supported by an NH and MRC Program Grant to Professor G. D. Thorburn. Excellent technical support was provided by Miss R. Poxon. The authors are grateful to Dr G. Jenkin for his critical discussion of the manuscript and to Mrs A. Woods for editorial assistance. REFERENCES Bell, R. L. & Majerus, P. W. ( I980 ) Thrombin-induced hydrolysis of phosphatidylinositol in human platelets.Journal of Biological Chemistry, 255, I79O-I79 z. Berridge, M. J. & Irvine, R. F. (1984) Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature, 312, 315-3zi. Bolton, T. B. (1979) Mechanisms of action of transmitters and other substances on smooth muscle. Physiological Reviews, 59, 6o6-718. Bradford, M. M. (i976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 7z, z48-z54. Canonico, P. L., Valdenergo, C. A. & Macleod, R. M. (1983) The inhibition of phosphatidylinositol turnover: a possible postreceptor mechanism for the prolactin secretion-inhibitingeffect of dopamine. Endocrinology, I I3, 7-14. Canonico, P. L., Judd, A. M., Koike, K. et al (1985) Arachidonate stimulates prolactin release in vitro: a role for the fatty acid and its metabolites as intraceilular regulator(s) in mammotrophs. Endocrinology, 116, zx8-2z5. Cervetto, L. & Piccolino, M. (1974) Synaptic transmission between photoreceptors and horizontal cells in the turtle retina. Science, 183, 417-419 . Cimn, J. S. D., Robertson, H. A. & Friesen, H. G. (1978) Maternal and fetal concentrations of ovine placental lactogen measured by radioimmunoassay. Endocrinology, IO2, I6O6-1613. Di Renzo, G. C., Johnston, J. M., Okazaki, T. et al (I98I) Phosphatidylinositol-specific phospholipase C in fetal membranes and uterine decidua. Journal of Clinical lnvestsgation, 67, 847-856. Di Virgilio, F., Lew, D. P. & Pozzan, T. (i984) Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cytosolic Ca~+ levels. Nature, 31o, 691-693. Douglas, W. W. (1968) Stimulus~ecretion coupling: the concept and clues from chromattin and other cells. British Journal of Pharmacology, z4, 451-474. Douglas, W. W. & Poisner, A. M. (1964) Stimulus~ecretion coupling in a neurosecretory organ: the role of calcium in the release of vasopressin from the neurohypophysis. Journal of Physiology, I72, l-x8. Dubois, M. P., Martal, J. & Djianne, J. (1976) Immunofluorescence localization of placental lactogen. Proceedings of the 5th International Congress on Endocrinology, Hamburg, p. z5 (Abstract). Farese, R. V. (i983) Phospboinositide metabolism and hormone action. Endocrine Reviews, 4, 78~95Fleckenstein, A. (1977) Specific pharmacology of calcium in myocardium, cardiac pacemakers and vascular smooth muscle. Annual Review of Pharmacology and Toxicology, 17, I49-166. Handwerger, S., Barrett, J., Markoff, E. et al (1981a) Stimulation of human placental lactogen release by arachidonic acid. Molecular Pharmacology, 2o, 6o9-613. Handwerger, S., Coma, P. M., Barrett, J. et al (i98ib) Human placental lactogen release in vitro: paradoxical effects of calcium. American Journal of Physiology, 24o, E55o-E555. Hunter, W. M. & Greenwood, F. C. (196z) Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature, 144, 495-496. Hurley, T. W., D'Ercole, A. J., Handwerger, S. et al (I977) Ovine placental lactogen induces somatomedin: a possible role in fetal growth. Endocrinology, IOX, I635-1638. Huyler, S. E., Butler, W. R., Grandis, A. et al (i985) Stimulation of ovine placental lactogen secretion by arachidonic acid. Journal of Endocrinology, IO6, 43-47.
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Kaibuchi, K., Takai, Y., Sawamura, M. et al (i983) Synergistic |unctions of protein phosphorylation and calcium mobilization in platelet activation. Journal of Biological Chemistry, 258 , 67oi~7o 4. Kawahara, Y., Takai, Y., Minakuchi, R. et al (i 98o) Phospholipid turnover as a possible transmembrane signal for protein phosphorylation during human platelet activation by thrombin. Biochemical and Biophysical Research Communications, 97, 3o9-317 . Leach Harper, C. M., Ford V. L. & Thorburn, G. D. (1984) Isolation of choriomammotropin. AustralianJournal of Biological Sciences. 37, 63-7o. Leung, P. C. K., Raymond, V. & Labrie, F. (1982) Mechanism of action of TRH: involvement of the phosphatidylinositol (PI) response in the action of TRH in rat anterior pituitary cells. Life Sciences, 31, 3o37-3o41. Martal, J. & Djianne, J. 0977) Immunofluorescent localization of ovine placental lactogen. Cell and Tissue Research, I84, 427 435Martin, R. B. & Richardson, F. S. (i979) Lanthanides as probes for calcium in biological systems. Quarterly Review of Biophysics, i2, I8I-2O9. McPhail, C., Clayton, C. C. & Snyderman, R. (i984) A potential second messenger role for unsaturated fatty acids: activation of Ca2+--dependent protein kinase. Science, 224, 622~25. Michell, R. H. (1983) Ca 2+ and protein kinase C: two synergistic cellular signals. Trends in Neuroscjences. 8, 263-265. Moriarty, C. M. (I978) Role of calcium in the regulation of adenohypophyseal hormone release. Life Sciences, 23, 185-x94. Nishizuka, Y. (i983) Calcium, phospholipid turnover and transmembrane signalling. Philosophical Transactions of the Royal Society of London, Series B, 302, IOl-I12. Ohmura, E. & Friesen, H. G. (1985) 12-O-Tetradecanoyl phorbol-I3-acetate stimulates rat growth hormone (GH) release through different pathways from that of human pancreatic GH-releasing factor. Endocrinology, I I6, 728-733. Okazaki, T., Sagawa, N., Okita, J. et al (x98 i) Diacylglycerol metabolism and arachidonic acid release in human fetal membranes and decidua vera. Journal of Biological Chemistry, 256 , 7316-7321. Pickup, J. C. & Hope, D. B. (197 I) Protease and ribonuclease activities in bovine pituitary lobes. BiochemicalJournal, I23, I53-I62. Rasmussen, H. & Goodman, D. B. P. (1977) Relationships between calcium and cyclic nucleotides in cell activation. Physiological Reviews, 57, 421-5o9. Reddy, S. & Watkins, W. B. (1978) tmmunofluorescence localization of ovine placental lactogen. Journal of Reproduction and Fertility, 52, i73-174. Reddy, S. & Watkins, W. B. (1983) Plasma clearance and distribution of lzsI-ovine placental lactogen in sheep. Australian Journal of Experimental Biology and Medical Sciences, 6x, 3Ol-31 I. Rittenhouse-Simmons, S. (x979) Production of diglyceride from phosphatidylinositol in activated human platelets. Journal of Clinical Investigation, 63, 58o-587 . Sano, K., Takai, Y., Yamanishi, J. & Nishizuka, Y. (1983) A role of calcium-activated phospholipid-dependent protein kinase in human platelet activation. Journal of Biological Chemistry, 258 , 2oio--2Ol 3. Sokal, R. R. & Rohlf, F. J. (1969) Biometry. p. 239. San Francisco, CA: W. F. Freeman. Sutton, C. A. & Martin, T. F. J. (1982) Thyrotropin-releasing hormone (TRH) selectively and rapidly stimulates phosphatidylinositol turnover in GH pituitary cells: a possible second step of TRH action. Endocrinology, Ho, I273. Swarm, K. & Whitaker, M. (x985) Stimulation of the Na/H exchanger of sea urchin eggs by phorbol ester. Nature, 3 I4, 274-277. Takai, Y., Kishimoto, A., Kikkawa, U. et al (1979) Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, pbospholipid-dependent protein kinase system. Biochemicaland BiophysicalResearch Communications, 9I, 1218-I224. Thorburn, G. D. (x 979) Physiology and control of parturition: reflections on the past and ideas for the future. Animal Reproduction Science, a, i-27. Trifaro, J. M. (1977) Common mechanisms of hormone secretion. Annual Review of Pharmacology and Toxicology, 17, 27-47. Weiss, G. B. (I974) Cellular pharmacology of lanthanum. Annual Review of Pharmacology, 14s 343-354.
Stimulation of Ovine Choriomammotrophin Release, in Vitro, by Phospholipase C
G. E. RICE & G. D. T H O R B U R N Department of Physiology, Monash University, Clayton, Victoria 3168, Australia Paper accepted 7.4.1986 INTRODUCTION Ovine choriomammotrophin (oCM, previously placental lactogen) is a protein hormone synthesized and secreted by fetal trophoblast cells throughout gestation (Dubois, Martal and Djianne, 1976; Martal and Djianne, 1977; Reddy and Watkins, 1978). Choriomammotrophins display considerable structural homology with growth hormone and prolactin (Hurley et al, 1977) but, despite extensive in vitro and in vivo studies, the factors regulating the synthesis and secretion of this hormone have yet to be established (Thorburn, 1979). Recently, it has been recognized that the control of protein hormone secretion may be intimately involved with plasma membrane phospholipid metabolism (Handwerger et al, 198Ia; Leung, Raymond and Labrie, 1982; Sutton and Martin, i982 , Canonico, Valdenergo and MacLeod, t983; Canonico et al, 1985). It has been suggested that the phospholipid metabolites diacylglycerol and inositol trisphosphate act as second messengers in the process of receptormediated cell activation (for reviews see Farese, I983; Berridge and Irvine, 1984). This process is dependent upon the hydrolysis of membrane phospholipid by the phosphodiesterase phospholipase C, and the liberation of the polar head group and sn-I,a-diacylglycerol. Diacylglycerol may act directly to activate protein kinase C, an enzyme implicated in hormone secretion and granule exocytosis (Rittenhouse-Simmons, 1979, Bell and Majerus, 198o; Kawahara et al, 198o; Michell, 1983; Nishizuka, 1983; Sano et al, 1983; Takai et al, i979; Di Virgilo, Lew and Pozzan, 1984; Swann and Whitaker, 1985). Alternatively, diacylglycerol may be metabolized by diacylglycerol lipase, which has recently been shown to be present in fetal membranes and uterine decidua (Di Renzo et al, i98i , Okazaki et al, I98I), liberating arachidonic acid, the substrate for cyclo-oxygenase and lipoxygenase pathways-and itself a putative second messenger in the regulation of cellular function (McPhail, Clayton and Snyderman, 1984). There is a paucity of information concerning factors which regulate the release of choriomammotrophin from the placenta, and the aim of the present study was to assess the potential involvement ofa phospholipase C- (PLC-)mediated pathway in the regulation ofoCM release from the ovine placenta. To further characterize PLC-stimulated oCM release, we investigated the effects of altering the availability of extracellular calcium. Membrane calciumchannel blocking agents (verapamil, nifedipine, cobalt chloride and lanthanum chloride) and nominally calcium-free medium (prepared as calcium-free and containing I mM EDTA) were used as pharmacological tools for this purpose. Inhibitors of arachidonic acid metabolism 533
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(ibuprofen, naproxen and nordihydroguaiaretic acid (NDGA)) were used to assess the involvement of arachidonic acid metabolites in PLC-stimulated oCM release.
MATERIALS AND METHODS Materials Phospholipase C (PLC; Bacillus cereus, Type V), verapamil, nordihydroguaiaretic acid (NDGA), naproxen and nifedipine were purchased from Sigma Chemical Co., St Louis, MO, USA. Ibuprofen was obtained from The Upjohn Co., Kalamazoo, MI, USA. Stock solutions of N D G A , naproxen and ibuprofen were prepared using dimethyl sulphoxide as solvent. Verapamil and nifedipine solutions were prepared in ethanol. The final concentration of either solvent in explant incubations did not exceed i per cent, and at this concentration neither of these solvents affect oCM release. Tissue p r e p a r a t i o n a n d i n c u b a t i o n Placentomes were obtained, at post mortem, from Merino-Border Leicester ewes (n = 20) at i oo to 14~ days' gestation. Four or five placentomes were selected at random from the pregnant horn of the uterus and immediately placed in ice-cold saline. T h e placentomes were decapsulated, the haemophagous zone was removed, and the remaining tissue was washed with Krebs-Ringer (NaC! I23 mmol/l; KCI 4.6mmol/l; MgSO4.7H20 i.I mmol/l; KHzPO 4 i.i mmol/l; CaCI 2 1.9mmol/q; and glucose Ii mmol/I) buffer containing 2omM N-2hydroxyethylpiperazine-N'-2-ethanesulphonic acid ( K R - H E P E S , p H 7.4). Explants (4 to 8) of approximately I to 2 mm 3, with a combined wet weight of 45 to 55 mg (5-3-I-o.6 ~tg oCM/5 o mg), were placed in each well of multi-well culture plates (Linbro, i2-well culture plates, 2.4 x 1. 7 cm; Flow Laboratories Inc., McLean, VA, USA) containing 5 ml of K R - H E P E S buffer. T h e tissue explants were incubated at 37~ in a metabolic shaking water bath (3o to 4 ~ cycles/minute) under an oxygen atmosphere. The medium in each well was changed after the first and second hour of incubation. The tissue was then incubated for a further i to 3 h (experimental incubation period) in 3 ml of control medium or medium containing one of the variables to be tested. At the end of the incubation period an aliquot (o. 5 to i .o ml) of supernatant was removed and immediately placed in an ethanol bath ( - 4o~ In those experiments in which the time course of oCM release was assessed, 200 #1 of medium were removed from control and experimental incubation wells at specified time intervals and immediately frozen. All samples were assayed for oCM content within three days of collection. The release of oCM from placental explants was expressed as either/~g oCM released per 50 mg wet weight tissue/h, or as a percentage of the release of oCM during control incubations run in parallel. The statistical differences between treatment means were assessed by analysis of variance and Student-Newman-Keuls test (Sokal and Rohlf, i969). To elucidate the effects of extracellular calcium on oCM release, placental explants were incubated in K R - H E P E S buffer or K R - H E P E S buffer prepared without CaCI 2 and containing i mM E D T A (calcium-free medium). Explants were incubated in calcium-free medium throughout the preincubation and experimental incubation periods. The effects of perturbing cellular calcium flux, on basal and stimulated oCM release, were also assessed by the inclusion of the organic calcium-channel blocking agents verapamil 0oo/~M) and nifedipine (ioo/~M), and the inorganic calcium-channel blocking agents cobalt chloride (4 mM) and lanthanum chloride (i mM), in the preincubation and experimental incubation medium.
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To elucidate further the mechanism of PLC-stimulated oCM release, the affects of cyclooxygenase and lipoxygenase inhibitors were examined. Placental explants were pre-incubated for I h in a medium containing one of the following agents: ibuprofen (io/~M); naproxen (ioo/ZM); or N D G A (5/ZM). The medium was then replaced with buffer containing the same concentration of inhibitor to which was added o.2 units PLC/ml (4 #1). The incubations were then conducted as described above. oCM radioimmunoassay oCM was assayed according to the method of Chan, Robertson and Friesen (1978) as modified by Leach Harper, Ford and Thorburn (1984). The oCM standard and antiserum were generously provided by Dr M. J. Waters (Department of Physiology, University of Queensland, Australia). Iodination of oCM was performed using the chloramine T method of Hunter and Greenwood (i962). The cross-reactivity of the antiserum with ovine growth hormone, luteinizing hormone, follicle-stimulating hormone, prolactin and thyroid-stimulating hormone was less than I per cent. The intra- and inter-assay coefficients of variation were 3-4 and 2o. i per cent, respectively. Samples were diluted I : 8o or I : 1oo with buffer prior to assay, and at this concentration none of the experimental reagents interfered with the assay. P r o t e i n assay Tissue protein content was estimated using a protein dye-binding method (Bradford, 1976) and bovine serum albumin as the reference standard. Tissue samples (5 ~ to ioo mg wet weight tissue) were homogenized in I M N a O H and centrifuged at 2ooo g for 2o min. The pH of the supernatant was adjusted to 7.o with o.5 M HCI. An aliquot (2oo/~1) was mixed with 6oo/~1 distilled water and 2oo/zl of dye reagent (Bio-Rad Laboratories, Richmond, CA, USA). The optical density (at 595 nm) was measured within 3o min from the addition of dye reagent to protein standards and samples, using a Varian spectrophotometer (Series 634 ). Protein concentration was calculated by linear regression analysis. L a c t a t e d e h y d r o g e n a s e (LDH) L D H released into incubation medium during control and PLC-treatment incubations was quantified spectrophotometrically by the method of Pickup and Hope (I97 0.
RESULTS Basal release o f o C M in vitro The release of oCM from placental explants incubated in K R - H E P E S buffer averaged 2.93 + o.25/~g/5 o mg wet weight tissue/h (n = 2o). This rate of release was maintained for at least 4 h in in vitro incubation. The tissue content of oCM before incubation was 5.3 + o.6 #g/5 o mg tissue wet weight (n = 12). The release of oCM during extended incubation periods thus represents the release of newly synthesized protein and is indicative of sustained tissue viability during the incubation period. Effects o f p h o s p h o l i p a s e C (PLC) on o C M release in vitro Incubation of placental explants in the presence of P L C caused a dose-dependent stimulation of oCM release. As little as o.2 units PLC/ml caused oCM release from tissue to approximately double that observed in control incubations (3.26_+o.3ktg/5omg/h ( n = I I ) and 7.o8 +o.39/~g/5 o mg/h (n = I I), respectively: Figure 1). P L C treatment did not increase,
536
Placenta (~986), Vol. 7 DOSE RESPONSE: PLC-STIMULATED oCM RELEASE ~-00-
rr
(3)
300
o h 0
W
200 1.1.1 -_1 LI..I
I00
-
i
,
002 02
2
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Figure I. Doseresponseof phospholipaseC--(PLC-)stimulatedovinechoriomammotrophin(oCM) release.The release of oCM from placentalexplants, incubatedfor 2 h at 37~ is presented as the percentageof oCM released during control incubation(KR-HEPES buffer). Values + s.e. are presented;, = 3 or 4- * signifies a significantdifference (P < o.os) between the mean ovine choriomammotrophin(oCM) release from control and treatment incubations.
significantly, the release of total protein or lactate dehydrogenase (LDH) into the medium when compared with release during control incubations. Total protein and L D H concentrations in incubation medium following control or P L C treatment were 4 . 8 + 1.9 #g/ml and 6.2 + 2.o #g/ml (n = I2), respectively, for protein, and 6o.6 + 9.2 mU/ml and 83.8 + i9.9mU/ml (n = i I), respectively, for LDH. A significant ( P < o.oi) increase in oCM release occurred within 5 min after the addition of PLC (0.2 units/ml) and release continued throughout the incubation period (Figure 2). T o assess whether P L C treatment stimulated oCM release by irreversibly altering the permeability of the plasma membrane, explants were incubated for 15~ min in the presence of excess P L C (io units PLC/ml) and then incubated in control medium for a further 6o min. At this concentration PLC stimulated oCM release throughout the initial i so-min incubation, when compared with control incubations. When the medium was replaced with control medium, oCM release in PLC-treated tissues returned to basal levels and was not significantly different from oCM release observed in control incubations (Figure 3). Effects o f c a l c i u m o n b a s a l a n d P L C - s t i m u l a t e d o C M release T o determine the effects of extracellular calcium concentration on basal and phospholipase C- (PLC-)stimulated ovine choriomammotrophin (oCM) release, placental explants were incubated for 2 h in calcium-free medium. As shown in Figure 4, the basal release o f o C M from
Rice, Thorburn: Ovine Choriomammotrophin Release in Vitro
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TIME COURSE : PLC-STIMULATED oCM RELEASE
3000
.x-
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._1 W nr
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oo
6
,6
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3'0
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s.0
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TIME ; min Figure 2, Time course of ovine choriomammotrophin (oCM) release. The release of oCM from control and phospholipase C- (PLC-)treated (o.2 unit/ml) explants, incubated in vitro, is presented as ng oCM released per ml incubation buffer. Values + s.e. are presented; n = 3- * signifies a significant difference ( P < o.o5) between the mean ovine choriomammotrophin (oCM) release from control and treatment incubations.
8000
replaced with ~control medium
/
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UJ u3
4000
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TIME ; min Figure 3. The effect on basal ovine choriomammotrophin (oCM) release of preincubation for ] 5~ min of placental explants with io units/ml phospholipase C (PLC). oCM released from control (KR-HEPES buffer) and PLC--treated incubations is presented as ng oCM per ml incubation buffer. The incubation medium in both control and treatment wells was replaced at i5o rain with KR-HEPES buffer. The tissue was then incubated for a further I h and the oCM release was measured. The mean of two experiments is presented.
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EFFECTS OF CALCIUM CHANNEL BLOCKING AGENTS AND EDTA ~350Z
o 300. I.L
o 250
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-
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j 100
'
9
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CONT
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NIFED .PLC 100 jJM
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Figure 4. The effect of reduced extracellularcalciumavailabilityon basal and phospholipase (i- (PLC-)stimulated ( + PLC) ovinechoriomammotrophin(oCM) release,oCM released from placentalexplantsincubatedin KR HEPES bufferat 37~ for 2 h is presentedas a percentageofoCM releasedduringcontrolincubations.The effectof incubating tissue in KR-HEPES buffer (CONT) or buffer containingverapamil(VERAP), nifedipine(NIFED), lanthanum chloride (LaCI3), cobaltchloride(CoCl2) or nominallycalcium-freemedium(EDTA i mM) are shown. Values + s.e. are presented;* = P < o.ooi; n = 3 to 7.
explants incubated in calcium-free medium ( E D T A i mM) was not significantly different from oCM released from control incubations. Incubation of placental explants in calcium-free medium, however, completely abolished the increase in oCM release following the addition of 0.2 units/ml PLC. Neither the organic nor inorganic calcium--channel blocking agents affected basal oCM release. PLC-stimulated release of oCM, also, was not affected by the inclusion of either verapamil or nifedipine at a concentration of ioo/aM. T h e effects of P L C on oCM release, however, were abolished by the inclusion of either CoCI 2 (4 mM) or LaCI 3 (i mM). T o examine the potential involvement of voltage-dependent calcium channels in oCM release, explants were incubated in medium containing IOO mM potassium chloride. This depolarizing stimulus had no significant effect on basal oCM release, oCM release during the 2-h incubation period averaged 84. 4 + 14. 4 (n = 3) per cent of oCM released during control incubations. Effects o f i n h i b i t o r s o f a r a c h i d o n i c acid m e t a b o l i s m o n P L C - s t i m u l a t e d o C M release The basal release of oCM was not significantly affected by inhibitors of arachidonic acid metabolism (Figure 5). T h e effects of the cyclo-oxygenase inhibitors ibuprofen and naproxen, and tfle 5-1ipoxygenase inhibitor N D G A , on PLC-stimulated release o f o C M are presented in Figure 5. Ibuprofen, naproxen and N D G A all inhibited PLC-stimulated release ofoCM. In the presence of-these inhibitors, oCM was not significantly different from oCM released during control incubations.
539
Rice, Thorburn: Ovine Chorioraammotrophin Release in Vitro
EFFECTS OF INHIBITORS OF ARACHIDONIC ACID METABOLISM 350 me l--
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Figure 5. The effects of inhibitors of arachidonic acid metabolism on basal and phospholipaseC- (PLC-)stimulated ovine choriomammotrophin(oCM) release (+ PLC). oCM released from placental explants as a percentage of that releasedfrom controlexplants incubated for 2 h at 37~ is presented. The effectsof incubatingtissue in buffer(CONT) or buffer containing ibuprofen (IBUPR), naproxen (NAPRO) or nordihydroguaiareticacid (NDGA) are shown. Values _+_+ s.e. are presented; * = P < o.ol; n = 4 to 6.
DISCUSSION In the present study, we demonstrated that phospholipase C (PLC) causes a dose-dependent release ofovine choriomammotrophin (oCM) from ovine placental explants incubated in vitro. P L C has been previously implicated in secretion-coupling in other tissues (Nishizuka, i983). P L C causes a dose-dependent release of growth hormone (GH), a protein with considerable structural and, possibly, functional homology with oCM, from rat pituitary primary cell cultures (Ohmura and Friesen, 1985). PLC-stimulated G H release may occur by a similar mechanism to that which results in increased release of oCM. T h e concentration of P L C required to achieve a doubling of the basal rate of G H secretion was, however, 2o-fold higher than the concentration used in the present study to achieve the same increase in o C M release. A dependence of PLC-stimulated release of o C M upon the availability of extracellular calcium was demonstrated. T h e omissionof calciumand the inclusion of ethylenediaminetetraacetic acid ( E D T A ) (i mM) in the incubation buffer completely abolished the stimulatory action of PLC. The inhibition of PLC-stimulation by the inclusion of the inorganic calciumchannel blocking agents lanthanum chloride (i mM) and cobalt chloride (4mM) in the incubation buffer further supports this suggestion. These ions have been reported to competitively displace calcium from the extracellular surface of the plasma membrane and prevent its subsequent influx (Cervetto and Piccolino, T974; Weiss, 1974; Martin and Richardson, 1979). T h e organic calcium-channel blocking agents verapamil (ioo #M) and nifedipine (i oo #M) were ineffective in blocking P L C stimulation of o C M release. These agents have been reported to antagonize voltage-dependent calcium channels (Fleckenstein, 1977; Bolton, I979) and it is thought that they interfere with the action of calcium in linking electrical
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Placenta (1986), Vol. 7
membrane activity with intracellular biochemical events, which results in cellular activation. The inability of these agents to prevent PLC-stimulated oCM release suggests that voltagedependent calcium channels are not involved in this response. The observation by Handwerger et al (198ib) that a depolarizing concentration of potassium chloride (ioo raM) failed to elicit significant release of human placental lactogen (choriomammotrophin) from placental explants incubated in vitro supports the suggested lack of involvement of voltage-dependent calcium channels in the release of this protein hormone. Similar results, using Ioo mM potassium chloride, were obtained in the present study of oCM release. It is of interest to note that basal oCM release was not suppressed by reduced extracellular calcium availability (Figure 4). Calcium is essential for release in a number of secretory systems involving the exocytosis of hormone-containing granules (Douglas and Poisner, x964; Douglas, 1968; Rasmussen and Goodman, i977; Trifaro, x977; Moriarty, I978). Although the mechanism involved in the paradoxical effect of calcium on basal oCM release remains unclear, our results are consistent with those of Handwerger et al (I98Ib) who demonstrated that the release of human placental lactogen in vitro is not suppressed by decreased extracellular calcium concentrations. In human placental tissue, stimulation of placental lactogen was observed in response to decreased extracellular calcium. Our results further suggest that PLC-stimulated oCM release involves arachidonic acid metabolites since the stimulatory effects of PLC were blocked by three inhibitors of arachidonic acid metabolism: nordihydroguaiaretic acid (NDGA), ibuprofen and naproxen. From the present study, it is not possible to elucidate further the mechanism of action of PLC-stimulated oCM release. Arachidonic acid, however, has been previously shown to stimulate the release, in vitro, of human placental lactogen (Handwerger et al, I98Ia) and rat prolactin (Canonico et al, x985) and the release, in vivo, of oCM (Huyler et al, I985). In conclusion, the results of the present study, that PLC stimulates the release of oCM in a dose-dependent manner, and that PLC-stimulated oCM release is suppressed by reduced extracellular calcium availability and inhibitors of arachidonic acid metabolism, provide some insight into a possible mechanism of action. We suggest that PLC hydrolyses plasma membrane phospholipids causing the release of polar head group moieties" and increasing the plasma membrane concentration of diacylglycerol. The polar head groups liberated by PLC hydrolysis have been implicated in initiating cell activation (see Berridge and Irvine, i984, for review). It is unlikely, however, that these water-soluble products participate in the release of oCM stimulated by exogenous PLC. These products would disperse in the incubation medium and thus not reach their intracellular site of action. Diacylglycerol, however, may initiate cell activation, directly, by acting as a second messenger, or indirectly, by being metabolized to arachidonic acid (Nishizuka, i983). Further study of this proposed mechanism is required to elucidate the action of PLC.
SUMMARY Phospholipid metabolites have previously been implicated in receptor-mediated stimulation of protein hormone secretion. As the factors which regulate the release of choriomammotrophin remain to be elucidated, we investigated the potential involvement of phospholipase C-induced phospholipid metabolism in the release of this placental hormone. Phospholipase C (PLC) caused a dose-dependent release of choriomammotrophin from ovine placenta, incubated in vitro. At a concentration of o.a units/ml (o.z 5 #g protein/ml), PLC caused the release of choriomammotrophin from placental tissue to approximately double that observed in control
Rice, Thorburn: Ovine Choriomammotrophin Release in Vitro
541
i n c u b a t i o n s (7.o8 + o. 4/~g/5 o m g / h a n d 3.z6 + o. 3 gg/5 o m g / h , respectively). P L C t r e a t m e n t did n o t significantly alter p l a s m a m e m b r a n e p e r m e a b i l i t y , as i n d i c a t e d by t h e release o f lactate d e h y d r o g e n a s e a n d protein. P L C - s t i m u l a t e d release o f o C M was c o m p l e t e l y a b o l i s h e d b y i n c u b a t i o n in c a l c i u m - f r e e m e d i u m or b y p r e i n c u b a t i o n w i t h the i n o r g a n i c c a l c i u m - c h a n n e l b l o c k i n g agents cobalt c h l o r i d e (4 raM) a n d l a n t h a n u m c h l o r i d e (I mM). T h e effects o f P L C t r e a t m e n t o n ovine c h o r i o m a m m o t r o p h i n ( o C M ) release were also i n h i b i t e d b y p r e i n c u b a t i o n o f placental tissue w i t h i n h i b i t o r s o f a r a c h i d o n i c acid m e t a b o l i s m : i b u p r o f e n ( I O - 5 M), n a p r o x e n ( i o TM M) or n o r d i h y d r o g u a i a r e t i c acid ( N D G A ) (5 x i o -6 M). T h e s e results suggest t h a t t h e effects o f P L C o n t h e release o f c h o r i o m a m m o t r o p h i n are m e d i a t e d via m e t a b o l i t e s o f a r a c h i d o n i c acid.
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