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Evidence for A2 adenosine receptor-mediated effects on adenylate cyclase activity in rat ovarian membranes
Molecular and Cellular Endocrinology, 56 (1988) 205-210 Elsevier Scientific Publishers Ireland. Ltd.
205
MCE 01825
Evidence for A, adenosine receptor-mediated effects on adenylate cyclase activity in rat ovarian membranes H. Billig and S. Rosberg Department
of Physiology, University of Giiteborg S-400 33 Giiteborg Sweden
(Received 18 September 1987; accepted 8 December 1987)
Key work:
Adenylate cyclase; Adenosine receptor; Ovarian membrane
Effects of adenosine analogues on adenylate cyclase activity in preovulatory rat ovarian membranes were studied. Adenosine analogues stimulated adenylate cyclase activity in the following rank order of potency: NECA (5’-( N-ethyl)carboxamidoadenosine) > 2-chloroadenosine > N6-( R-phenylisopropyl)adenosine > N6-( S-phenylisopropyl)adenosine. The apparent EC,, for NECA was 0.28 PM. The adenosine receptor antagonist 8-phenyltheophylline (10 PM) displaced the dose-response curve for NECA to the right, increasing the EC,,, for NECA about one order of magnitude. NECA also additively increased maximally FSH-stimulated adenylate cyclase activity. These results suggest that adenosine stimulates adenylate cyclase in rat ovarian membranes via adenosine receptors of the A, type.
Introduction
Adenosine receptor-mediated effects have been shown in a number of tissues and adenosine has been proposed to have a role as a local regulator in many of these tissues (Londos et al., 1981). Extracellular adenosine receptors are divided into two subclasses based on the affinity of different adenosine analogues to the receptors and on their effect on adenylate cyclase activity. Activation of A, receptors inhibits and activation of A, recep-
Address for correspondence: H. Billig, Department of Physiology, University of Gbteborg, S-400 33 GBteborg, Sweden. Abbreviations: ADA, adenosine deaminase; 2-Clado, 2chloroadenosine; FSH, follicle stimulating hormone; NECA, 5 ‘-( N-ethyl)carboxamidoadenosine; I-PHT, 8-phenyltheophylline; R-PIA, N6-( R-phenylisopropyl)adenosine; S-PIA, N6-( Sphenylisopropyl)adenosine. 0303-7207/88/$03.50
tors stimulates adenylate cyclase (Van Calker et al., 1979; Londos et al., 1980). Adenosine receptors have been reported also in endocrine tissues. For instance, in the thyroid, adenosine and adenosine analogues increased CAMP accumulation (Fradkin et al., 1982). ACTH-stimulated CAMP production and steroidogenesis in the adrenal cortex was inhibited by adenosine (Shima, 1986). In the testis, activation of inhibitory adenosine receptors (A,) has been shown to inhibit adenylate cyclase activity in Sertoli cells (Monaco et al., 1984). Non-metabolizable adenosine analogues increased CAMP accumulation to a small extent, but potentiated the maximal stimulation of CAMP accumulation by FSH in granulosa cells (Billig et al., 1987), suggesting the presence of adenosine receptors of the A, type in granulosa cells. However, the estimated EC,, was found to be at least one order of potency higher than that found for A,
0 1988 Elsevier Scientific Publishers Ireland, Ltd.
206 receptors in other cell types (Daly, 1983). The present investigation was performed to further characterize the effects of adenosine analogues on adenylate cyclase activity in an ovarian membrane preparation. Materials and methods
Animals Immature SpragueDawley rats (Alab, Stockholm, Sweden) were kept under standardized conditions with lights on between 05.00 and 19.00 h. They were given 10 IU pregnant mare’s serum gonadotropin (PMSG, NIDDK), s.c., on day 26 of life to induce fol~cul~ growth and were killed by cervical dislocation 48-50 h later, before the endogenous LH/FSH surge (Hillensjij et ai.,1974). Hormones and chemicals Stock solution of ovine FSH (S15, NIDDK; 0.1 mg/ml) was kept frozen at -2O’C in sterile PBS with 0.1% BSA until use. NECA (5’-(Nethyl)carboxamidoadenosine), R-PIA (&-( Rphenylisopropyl)adenosine) and S-PIA ( N6-(Sph~nylisopropyl)adenosine) (Boehringer-Mannheim, Ma~eim, F.R.G.) and 2-Clad0 (2-chloroadenosine; Sigma, St. Louis, MO, U.S.A.) were dissolved in the assay medium by mixing and 5 s sonication. Stock solutions of adenosine deaminase (ADA, Boehringer-Mannheim; 400 U/ml in glycerol), X-phenyltheophylline (8PHT, Sigma; 20 mM in 0.1 M NaOH) and Ro 20-1724 (a gift from Hoffmann-La Roche, Basel, Switzerland; 500 mM in 95% ethanol) were diluted in assay medium. [a- 32P]ATP and [ 3H]cAMP were purchased from Amersham International (Buckinghamshire, U.K.). Adenylate cyclase assay Ovarian adenylate cyclase activity was determined essentially according to Bimbaumer and coworkers (1976). The preovulatory ovaries containing IO-15 preovulatory follicles/ovary were isolated and trimmed from adhesive tissues and kept frozen at -70’ C until use within 2 weeks. The frozen ovaries were homogenized in ice-cold Tris-sucrose buffer (25 mM Tris-HCl, 5 mM MgCl,, 1 mM EGTA, 27% sucrose, pH 7.5) with an all-glass Dounce homogenizer. The homogenate was centrifuged for 5 min at 160 x g to
remove debris, filtered through two layers cheese cloth and centrifuged again for 50 min at 10000 x g. The crude membrane fraction was resuspended in the Tris-sucrose buffer and aliquots of this suspension were used in the adenylate cyclase assay. The final concentrations in the adenylate cyclase assay were: 0.1 mM ATP (with approximately 2 X lo6 cpm [a!-32P]ATP), 0.1 mM CAMP, 0.05 mM GTP, 5 mM creatine phosphate, 25 U/ml creatine phosphokinase, 5 mM MgCl, and 1 U/ml adenosine deaminase in 25 mM Tris-HCl at pH 7.5. The reaction was initiated by the addition of 100 ~1 membrane suspension to 100 ~1 assay medium. After 10 rnin at 37” C, the reaction was terminated by the addition of 100 ~1 of stopping solution (5 mM CAMP, 20 mM ATP and 1% sodium dodecylsulfate). The [ 3’P]cAMP formed was isolated by Dowex and alumina column chromatography (Salomon et al., 1974) with added [3H]cAMP for recovery correction. The eluates from the alumina columns were collected directly into scintillation vials, mixed with scintillation fluid and counted for radioactivity. Protein contents of membrane aliquots (80-120 pg/sample) were determined with the Lowry method (1951) after precipitating the aliquots with ice-cold 10% trichloroacetic acid. Statistics Each experiment was repeated three or more times with similar results. Experimental values are given as mean rf: SEM of triplicate samples and calculated EC,, and slope values are given with mean f SD. Differences between groups were calculated by analysis of variance, followed by Student-Newman-Keuls’ multiple range test (Woolf, 1968). A P-value less than 0.05 was considered significant. The dose-response curves were fitted with non-linear regression to cl-parametric dose-response curves (McIntosh and McIntosh, 1975). Results
The adenosine analogue NECA dose-dependently stimulated adenylate cyclase in the ovarian membrane preparation, with an apparent EC,, of 0.28 FM (SD 0.14 FM; Fig. 1). Basal adenylate
207
130
120
110
100
SC
I
0
I
.Ol
“““1
,“““-I
‘-“““I
.l
1
NECA
(FM)
’
10
““1
100
FSH Fig. 1. NECA dose-dependent stimulation of ovarian membrane adenylate cyclase. Membranes were incubated as described in Materials and Methods. Each point represents the mean&SE of five experiments with triplicate samples. The EC,, for NECA was calculated to 0.28 +0.14 pM (SD) and the slope factor was 1.44 f 0.58 (SD).
cyclase activity was 10 pmol/min/mg protein and maximal stimulation by FSH increased the CAMP production by 2.5fold (Fig. 2). In the presence of a maximal concentration of NECA (100 PM) together with increasing concentrations of FSH, NECA additively increased CAMP production over the whole dose range of FSH (Fig. 2). Furthermore, when the adenylate cyclase was stimulated by a maximally stimulatory concentration of FSH (1000 ng/ml) together with increasing concentrations of NECA, the adenylate cyclase was also stimulated in a dose-dependent manner (Fig. 3). The stimulatory effect of NECA was antagonized by the adenosine receptor antagonist 8phenyltheophylline (8-PHT; Fig. 3). 8-PHT (10 PM) increased the EC,, for NECA with one order of magnitude. The antagonizing effect of 8-PHT on NECA-stimulated adenylate cyclase was also evident when NECA was present in combination with FSH (1000 ng/ml; Fig. 3).
(ngiml)
Fig. 2. FSH dose-dependent stimulation of ovarian membrane adenylate cyclase in the presence @) and absence (*) of NECA (100 pM). Membranes were incubated as described in Materials and Methods. Each point represents the mean* SE of triplicate samples from two different experiments. The points were fitted with non-linear regression to Cparametric dose-response curves. The curves were significantly different (P < 0.01) when tested with two-way analysis of variance.
In order to further characterize the type of adenosine receptor three other adenosine analogues, apart from NECA, were also tested for activation of adenylate cyclase activity in the TABLE 1 EFFECT OF ADENOSINE MEMBRANE ADENYLATE
ANALOGUES CYCLASE
Ovarian membranes were incubated and Methods. The EC,, and slope calculated with non-linear regression. response for NECA was used when other substances.
NECA 2-Clado R-PIA S-PIA
ON OVARIAN
as described in Materials values (mean* SD) were The calculated maximal calculating EC,, for the
EGO (PM)
Slope
0.26+ 0.16 35 k24 96 k40 239 k214
0.84 + 0.36 0.53 + 0.21 1.46*1.54 0.53 * 0.30
208
NECA
(pi-l)
Fig. 3. Displacement of NECA dose-dependent stimulation of ovarian membrane adenylate cyclase with 8-PHT. Membranes were incubated as described in Materials and Methods with (0) and without (A) FSH (1000 ng/ml) in the absence (filled symbols) or presence (open symbols) of 8-PHT (10 CM). Each point represents the mean*SEM of triplicate samples. The EC,s values (mean + SD) for NECA were calculated to 0.95 k 0.54 PM and 1.6k2.86 PM in the absence and presence of I-PHT, respectively. In the presence of FSH the corresponding values were 0.03 f 0.02 pM and 3.1+ 1.2 FM, respectively.
ovarian membrane preparation. All of the analogues stimulated adenylate cyclase in a dose-dependent manner, but NECA was found to be the most potent of the four with the following rank order of potency: NECA > 2Xlado > R-PIA > SPIA (Table 1). Discussion
In the present study we demonstrate that adenosine analogues stimulate adenylate cyclase activity in an ovarian membrane preparation from PMSG-treated immature rats. The EC,, for NECA (0.28 FM) in the ovarian membrane preparation is comparable with other reports (Daly, 1983). The order of potency, NECA > 2-Clad0 > R-PIA > S-
PIA, indicates an A, receptor (Londos et al., 1981). In presence of the adenosine receptor antago~s~ 8-PHT ~G~ffith et al., 1981) the doseresponse curve of NECA was displaced to the right and increased the EC,, by one order of magnitude. These data are compatible with the conception that A, receptors (Londos et al., 1981) are present in rat ovarian membranes. Furthermore, NECA additively increased FSH-stimulated adenylate cyclase activity demonstrating stimulation of adenylate cyclase by an adenosine receptor agonist in concert with a classical hormone. Also in the presence of FSH, S-PHT shifted the doseresponse for NECA to the right, but the EC,, for NECA in presence of FSH seems to be subject to large variations. A dual stimulation has also been reported for adenosine analogues and LH in Leydig tumour cells (Dix et al., 1985). In the ovary, exogenously added adenosine has, a number of effects. Adenosine is transported into granulosa (Billig and Rosberg, 1986) and luteal cells (Behrman et al., 1983). Furthermore, adenosine increases the ATP and CAMP levels in granulosa cells (Polan et al., 1983; Okhawa et al., 1985; Billig and Rosberg, 1986), in oocyte-cumulus complexes (Billig and Ma~usson, 1985) and in luteal cells (Hall et al., 1981). The progesterone synthesis in luteal cells (Hall et al., 1981) and to a lesser degree in granulosa cells (Polan et al., 1983; Billig and Rosberg, 1986) is also increased by adenosine, but not to the extent that would be anticipated considering the increase in cAMP levels. Adenosine normalizes the decrease in oxygen consumption and the increase in lactate production caused by FSH in oocyte cumulus complexes (Billig and Magnusson, 1985). The above-mentioned effects of adenosine in the ovary have largely been ascribed to adenosine being an intracellular substrate to further metabolization. However, we have suggested that adenosine also might exert its effects as a receptor agonist, since the adenosine analogue NECA, alone and especially in combination with FSH, increased CAMP production in granulosa cells (Billig et al., 1987). It is not likely that the effects of NECA are due to metabolism of the analogue in follicular cells, since the ATP levels were not changed in granulosa cells by NECA in contrast to metabohzable adenosine (Billig et al., 1987). However, it cannot
be excluded that NECA ~rnpet~ with or interferes with the action of adenosine at intraceIlular adenosine-sensitive or -dependent processes in granulosa eel&, since for instance NECA, R-PIA and S-PIA all compete with the uptake of adenosine in these cells (Bill@ et al., 1987). In conclusion, the data from the present and previous studies indicate that adenosine, apart from serving as an ~~ace~~~ substrate, also can exert effects via extrace~ul~ A, adenosine receptors in the preo~iato~ ovary. The ovary consists of several cell types and the ceflular 1ocaIization of the senator adenosine receptors evident in ovarian membranes is at present not specified. However, we have earlier reported that NECA don-de~ndently potentiates FSH-stimulatd cAMP accumulation in rat 8mrmlosa cells, even though a discrepancy in the potency of NEC!A in ovarian membr~es versus that in granulosa cells is evident. The EC& was reported to be one order of ma~tude higher in the c&Is (Big et al., 1987), compared to the ovarian membr~e in the present study. Further investigations are needed to establish the cellular of the stimulatory A2 receptors in ovarian membr~es. The physiolo~cal relevance of these stimulatory A2 receptors in the ovary is at present unclear. However, an autocrine or paracrine model can be proposed. It is well known that striation of ovarian ceils with FSW results is an increased cAMP production and a marked release of CAMP into the culture me~um (Selst~ et al., 1976). Since ovarian cells possess the CAMP de~adative edges phosph~iesterase and 5’“~ucl~tidase (Rosberg et al., 19751, the released CAMP will rapidly be degraded to AMP and adenosine (Bruns, 1980; Pearson et al., 1980). FSH also increases both ~x~a~~ul~ (Se&am and Rosberg, 1976) and intra~~ul~ (Conti et al., 1984) phosthe pho~esterase activity, further enh~c~g cAMP de~adation to AMP and adenosine. Furthe~ore, gonado~op~s decrease the ATP levels in whole ovaries (Al&n et al., 1968) and in gr~~osa cells (O~awa et al., 1985; ping and Rosberg, 1986), and a decrease in ATP levels is almost always associated with a release of adenosine (Arch and Newshohne, 1978). It is thus conceivable that locally produced and released
adenosine potentiates the surrountig fo~cul~ cells via adenosine receptors in a placate and/or auto~ne manner, as has been su ted for adenosine in the testis (Monaco et al., 19841,
We wish to thank Prof. Kurt Ah&n and Dr. Torbjom H~~sj~ for valuable su tions and the NEDDK and NHPP, U~versity of Maryland School of Me~cine for generous’~fts of FSH and PMSG, This work was supported by grants from the Swedish Medical Research Council (Nos. 27 and 73141, the Royal Swedish Academy of Sciences, the Gijteborg and the Swedish Medical Societies, the Royal Society of Arts and Sciences in G~teborg, the Handl. Hj. Svensson Research Fund and the Faculty of Medicine, diversity of G~teborg, Sweden.
Ain& K., Beviz, L., Hamberger, L. and Lundholm, L. (1968) Acta Pbysioi, Stand. 73,23A-24A. Arch, JRS. and ~~w$~o~~, EA. (1978) in Essays in Biochemistry (CampeR, P.N. and Akirid8e, W.N., e&s.), Vol. 14, pp. 82-123, Academic Press, London. Behrman, H.R., Okhawa, R., Preston, S.L. and MacDonald, G.J. (1983) adroit 113,1132-114Q. Big, H. and ~a~u~on, C. (1985) Biol. Reprod. 33,890-898. BiBig, H. and Rosberg S. (1986) ~d~~oIogy 118,645~652. Billig, H., Tbetander, H. and Rosberg, S. (1987) ~nd~~ol~ (in press). ~i~baumer, L., Yang, P.C., ~~cker-~, M., Bockaert, J. and Duran, J.M. (1976) EndocrinoIogy 99,163-l&t. Bruns, RF. (1980) Naunyn-Scbmied. Arch Pbarmacoi. 315, 5-13. Conti, M., Kasson, B.G. and Hsueh, A.J.W. (1984) Endocrmology 114,2361-2368. Daly, J.W. (1983) in Pby~o~ogy and Ph~a~lo~ of Adenosine Derivates (Daly, J.W., Kuroda, Y., Phi&s, J.W., Sb& miza, H. and Ui, M., e&s.), pp. 275-290, Raven press, New York. Dix, C.J., Habbe~~eld~ AD. and Cooke, B.A. (1985) Bi~b~istry 230, 211-216. F&kin, J.E., Hardy, WC. and Wolff, J. (1982) ~d~~ology 110, 2018-2023. Freon, B.B. (1978) Acta Physiol. Stand, 102,191-198. Griffhb, S.G., Meghji, P., Moody, C.J. and Burnstock, G. (1981) Eur. J. pb~~l. 75,61-64. Hall, AK., Preston, S.L. and Behrman, H.R.(1981) J. Biol. Chem. 256,10390-10398.
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Hillensjii, T., Bamea, A., Niisson, L., He&z. H. and Abren, K. (1974) Endocrinology 95, 1762-1766. Londos, C., Cooper, D.M.F. and Wolff, J. (1980) Proc. N&l. Acad. Sci. U.S.A. 77, 2551-2554. Londos, C., Wolff, J. and Cooper, D.M.F. (1981) in Purinergic Receptors: Receptors and Recognition (Burnstock, G., ed.), Series B, Vol. 12, pp. 289-323, Chapman and Hall, London. Lowry, OH., Rosebrough, N.J., Farr, AL. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. McIntosh, J.E.A. and McIntosh, R.P. (1980) Monographs on End~~nology: Mathematical M~elling and Computers in Endocrinology, Vol. 16, Sponger-Ver~ag, Berlin. Monaco, L., Toscano, M.V. and Conti, M. (1984) Endocrinology 115, 1616-1624. Ohkawa, R., Polan, M.L. and Behrman, H.R. (1985) Endocrinology 11’7,248-254. Pearson, J.D., Carleton, J.S. and Gordon, J.L. (1980) Biochem. J. 190, 421-429.
Polan, M.L.. DeChemey, A.H., Haseltine, F.P., Muer, H.C. and Behrman, H.R. (1983) J. Clin. Endocrinol. Metab. 56, 288-294. Rosberg, S., Selstam, G. and Isaksson, 0. (1975) Acta Physiol. &and. 94, 522-535. Salomon, Y., Londos, C. and Rodbell, M. (1974) Anal. Biothem. 58, 541-548. Selstam, G. and Rosberg, S. (1976) Acta Endocrinol. 81, 563-573. Selstam, G., Rosberg, S., Liljekvist, J., Gronquist, L., Perklev, T. and Ah&r, K. (1976) Acta Endocrinol. 81, 150-164. Shima. S. (1986) Mol. Cell. Endocrinol. 47, 35-42. Van Calker, M., Miiller, M. and Hamrecht, B. (1979) J. Neurothem. 33, 999-1005. Woolf, C.M. (1968) Principles of Biometry. Statistics for Bioiogists, Van Nostrand, Princeton, NJ.
205
MCE 01825
Evidence for A, adenosine receptor-mediated effects on adenylate cyclase activity in rat ovarian membranes H. Billig and S. Rosberg Department
of Physiology, University of Giiteborg S-400 33 Giiteborg Sweden
(Received 18 September 1987; accepted 8 December 1987)
Key work:
Adenylate cyclase; Adenosine receptor; Ovarian membrane
Effects of adenosine analogues on adenylate cyclase activity in preovulatory rat ovarian membranes were studied. Adenosine analogues stimulated adenylate cyclase activity in the following rank order of potency: NECA (5’-( N-ethyl)carboxamidoadenosine) > 2-chloroadenosine > N6-( R-phenylisopropyl)adenosine > N6-( S-phenylisopropyl)adenosine. The apparent EC,, for NECA was 0.28 PM. The adenosine receptor antagonist 8-phenyltheophylline (10 PM) displaced the dose-response curve for NECA to the right, increasing the EC,,, for NECA about one order of magnitude. NECA also additively increased maximally FSH-stimulated adenylate cyclase activity. These results suggest that adenosine stimulates adenylate cyclase in rat ovarian membranes via adenosine receptors of the A, type.
Introduction
Adenosine receptor-mediated effects have been shown in a number of tissues and adenosine has been proposed to have a role as a local regulator in many of these tissues (Londos et al., 1981). Extracellular adenosine receptors are divided into two subclasses based on the affinity of different adenosine analogues to the receptors and on their effect on adenylate cyclase activity. Activation of A, receptors inhibits and activation of A, recep-
Address for correspondence: H. Billig, Department of Physiology, University of Gbteborg, S-400 33 GBteborg, Sweden. Abbreviations: ADA, adenosine deaminase; 2-Clado, 2chloroadenosine; FSH, follicle stimulating hormone; NECA, 5 ‘-( N-ethyl)carboxamidoadenosine; I-PHT, 8-phenyltheophylline; R-PIA, N6-( R-phenylisopropyl)adenosine; S-PIA, N6-( Sphenylisopropyl)adenosine. 0303-7207/88/$03.50
tors stimulates adenylate cyclase (Van Calker et al., 1979; Londos et al., 1980). Adenosine receptors have been reported also in endocrine tissues. For instance, in the thyroid, adenosine and adenosine analogues increased CAMP accumulation (Fradkin et al., 1982). ACTH-stimulated CAMP production and steroidogenesis in the adrenal cortex was inhibited by adenosine (Shima, 1986). In the testis, activation of inhibitory adenosine receptors (A,) has been shown to inhibit adenylate cyclase activity in Sertoli cells (Monaco et al., 1984). Non-metabolizable adenosine analogues increased CAMP accumulation to a small extent, but potentiated the maximal stimulation of CAMP accumulation by FSH in granulosa cells (Billig et al., 1987), suggesting the presence of adenosine receptors of the A, type in granulosa cells. However, the estimated EC,, was found to be at least one order of potency higher than that found for A,
0 1988 Elsevier Scientific Publishers Ireland, Ltd.
206 receptors in other cell types (Daly, 1983). The present investigation was performed to further characterize the effects of adenosine analogues on adenylate cyclase activity in an ovarian membrane preparation. Materials and methods
Animals Immature SpragueDawley rats (Alab, Stockholm, Sweden) were kept under standardized conditions with lights on between 05.00 and 19.00 h. They were given 10 IU pregnant mare’s serum gonadotropin (PMSG, NIDDK), s.c., on day 26 of life to induce fol~cul~ growth and were killed by cervical dislocation 48-50 h later, before the endogenous LH/FSH surge (Hillensjij et ai.,1974). Hormones and chemicals Stock solution of ovine FSH (S15, NIDDK; 0.1 mg/ml) was kept frozen at -2O’C in sterile PBS with 0.1% BSA until use. NECA (5’-(Nethyl)carboxamidoadenosine), R-PIA (&-( Rphenylisopropyl)adenosine) and S-PIA ( N6-(Sph~nylisopropyl)adenosine) (Boehringer-Mannheim, Ma~eim, F.R.G.) and 2-Clad0 (2-chloroadenosine; Sigma, St. Louis, MO, U.S.A.) were dissolved in the assay medium by mixing and 5 s sonication. Stock solutions of adenosine deaminase (ADA, Boehringer-Mannheim; 400 U/ml in glycerol), X-phenyltheophylline (8PHT, Sigma; 20 mM in 0.1 M NaOH) and Ro 20-1724 (a gift from Hoffmann-La Roche, Basel, Switzerland; 500 mM in 95% ethanol) were diluted in assay medium. [a- 32P]ATP and [ 3H]cAMP were purchased from Amersham International (Buckinghamshire, U.K.). Adenylate cyclase assay Ovarian adenylate cyclase activity was determined essentially according to Bimbaumer and coworkers (1976). The preovulatory ovaries containing IO-15 preovulatory follicles/ovary were isolated and trimmed from adhesive tissues and kept frozen at -70’ C until use within 2 weeks. The frozen ovaries were homogenized in ice-cold Tris-sucrose buffer (25 mM Tris-HCl, 5 mM MgCl,, 1 mM EGTA, 27% sucrose, pH 7.5) with an all-glass Dounce homogenizer. The homogenate was centrifuged for 5 min at 160 x g to
remove debris, filtered through two layers cheese cloth and centrifuged again for 50 min at 10000 x g. The crude membrane fraction was resuspended in the Tris-sucrose buffer and aliquots of this suspension were used in the adenylate cyclase assay. The final concentrations in the adenylate cyclase assay were: 0.1 mM ATP (with approximately 2 X lo6 cpm [a!-32P]ATP), 0.1 mM CAMP, 0.05 mM GTP, 5 mM creatine phosphate, 25 U/ml creatine phosphokinase, 5 mM MgCl, and 1 U/ml adenosine deaminase in 25 mM Tris-HCl at pH 7.5. The reaction was initiated by the addition of 100 ~1 membrane suspension to 100 ~1 assay medium. After 10 rnin at 37” C, the reaction was terminated by the addition of 100 ~1 of stopping solution (5 mM CAMP, 20 mM ATP and 1% sodium dodecylsulfate). The [ 3’P]cAMP formed was isolated by Dowex and alumina column chromatography (Salomon et al., 1974) with added [3H]cAMP for recovery correction. The eluates from the alumina columns were collected directly into scintillation vials, mixed with scintillation fluid and counted for radioactivity. Protein contents of membrane aliquots (80-120 pg/sample) were determined with the Lowry method (1951) after precipitating the aliquots with ice-cold 10% trichloroacetic acid. Statistics Each experiment was repeated three or more times with similar results. Experimental values are given as mean rf: SEM of triplicate samples and calculated EC,, and slope values are given with mean f SD. Differences between groups were calculated by analysis of variance, followed by Student-Newman-Keuls’ multiple range test (Woolf, 1968). A P-value less than 0.05 was considered significant. The dose-response curves were fitted with non-linear regression to cl-parametric dose-response curves (McIntosh and McIntosh, 1975). Results
The adenosine analogue NECA dose-dependently stimulated adenylate cyclase in the ovarian membrane preparation, with an apparent EC,, of 0.28 FM (SD 0.14 FM; Fig. 1). Basal adenylate
207
130
120
110
100
SC
I
0
I
.Ol
“““1
,“““-I
‘-“““I
.l
1
NECA
(FM)
’
10
““1
100
FSH Fig. 1. NECA dose-dependent stimulation of ovarian membrane adenylate cyclase. Membranes were incubated as described in Materials and Methods. Each point represents the mean&SE of five experiments with triplicate samples. The EC,, for NECA was calculated to 0.28 +0.14 pM (SD) and the slope factor was 1.44 f 0.58 (SD).
cyclase activity was 10 pmol/min/mg protein and maximal stimulation by FSH increased the CAMP production by 2.5fold (Fig. 2). In the presence of a maximal concentration of NECA (100 PM) together with increasing concentrations of FSH, NECA additively increased CAMP production over the whole dose range of FSH (Fig. 2). Furthermore, when the adenylate cyclase was stimulated by a maximally stimulatory concentration of FSH (1000 ng/ml) together with increasing concentrations of NECA, the adenylate cyclase was also stimulated in a dose-dependent manner (Fig. 3). The stimulatory effect of NECA was antagonized by the adenosine receptor antagonist 8phenyltheophylline (8-PHT; Fig. 3). 8-PHT (10 PM) increased the EC,, for NECA with one order of magnitude. The antagonizing effect of 8-PHT on NECA-stimulated adenylate cyclase was also evident when NECA was present in combination with FSH (1000 ng/ml; Fig. 3).
(ngiml)
Fig. 2. FSH dose-dependent stimulation of ovarian membrane adenylate cyclase in the presence @) and absence (*) of NECA (100 pM). Membranes were incubated as described in Materials and Methods. Each point represents the mean* SE of triplicate samples from two different experiments. The points were fitted with non-linear regression to Cparametric dose-response curves. The curves were significantly different (P < 0.01) when tested with two-way analysis of variance.
In order to further characterize the type of adenosine receptor three other adenosine analogues, apart from NECA, were also tested for activation of adenylate cyclase activity in the TABLE 1 EFFECT OF ADENOSINE MEMBRANE ADENYLATE
ANALOGUES CYCLASE
Ovarian membranes were incubated and Methods. The EC,, and slope calculated with non-linear regression. response for NECA was used when other substances.
NECA 2-Clado R-PIA S-PIA
ON OVARIAN
as described in Materials values (mean* SD) were The calculated maximal calculating EC,, for the
EGO (PM)
Slope
0.26+ 0.16 35 k24 96 k40 239 k214
0.84 + 0.36 0.53 + 0.21 1.46*1.54 0.53 * 0.30
208
NECA
(pi-l)
Fig. 3. Displacement of NECA dose-dependent stimulation of ovarian membrane adenylate cyclase with 8-PHT. Membranes were incubated as described in Materials and Methods with (0) and without (A) FSH (1000 ng/ml) in the absence (filled symbols) or presence (open symbols) of 8-PHT (10 CM). Each point represents the mean*SEM of triplicate samples. The EC,s values (mean + SD) for NECA were calculated to 0.95 k 0.54 PM and 1.6k2.86 PM in the absence and presence of I-PHT, respectively. In the presence of FSH the corresponding values were 0.03 f 0.02 pM and 3.1+ 1.2 FM, respectively.
ovarian membrane preparation. All of the analogues stimulated adenylate cyclase in a dose-dependent manner, but NECA was found to be the most potent of the four with the following rank order of potency: NECA > 2Xlado > R-PIA > SPIA (Table 1). Discussion
In the present study we demonstrate that adenosine analogues stimulate adenylate cyclase activity in an ovarian membrane preparation from PMSG-treated immature rats. The EC,, for NECA (0.28 FM) in the ovarian membrane preparation is comparable with other reports (Daly, 1983). The order of potency, NECA > 2-Clad0 > R-PIA > S-
PIA, indicates an A, receptor (Londos et al., 1981). In presence of the adenosine receptor antago~s~ 8-PHT ~G~ffith et al., 1981) the doseresponse curve of NECA was displaced to the right and increased the EC,, by one order of magnitude. These data are compatible with the conception that A, receptors (Londos et al., 1981) are present in rat ovarian membranes. Furthermore, NECA additively increased FSH-stimulated adenylate cyclase activity demonstrating stimulation of adenylate cyclase by an adenosine receptor agonist in concert with a classical hormone. Also in the presence of FSH, S-PHT shifted the doseresponse for NECA to the right, but the EC,, for NECA in presence of FSH seems to be subject to large variations. A dual stimulation has also been reported for adenosine analogues and LH in Leydig tumour cells (Dix et al., 1985). In the ovary, exogenously added adenosine has, a number of effects. Adenosine is transported into granulosa (Billig and Rosberg, 1986) and luteal cells (Behrman et al., 1983). Furthermore, adenosine increases the ATP and CAMP levels in granulosa cells (Polan et al., 1983; Okhawa et al., 1985; Billig and Rosberg, 1986), in oocyte-cumulus complexes (Billig and Ma~usson, 1985) and in luteal cells (Hall et al., 1981). The progesterone synthesis in luteal cells (Hall et al., 1981) and to a lesser degree in granulosa cells (Polan et al., 1983; Billig and Rosberg, 1986) is also increased by adenosine, but not to the extent that would be anticipated considering the increase in cAMP levels. Adenosine normalizes the decrease in oxygen consumption and the increase in lactate production caused by FSH in oocyte cumulus complexes (Billig and Magnusson, 1985). The above-mentioned effects of adenosine in the ovary have largely been ascribed to adenosine being an intracellular substrate to further metabolization. However, we have suggested that adenosine also might exert its effects as a receptor agonist, since the adenosine analogue NECA, alone and especially in combination with FSH, increased CAMP production in granulosa cells (Billig et al., 1987). It is not likely that the effects of NECA are due to metabolism of the analogue in follicular cells, since the ATP levels were not changed in granulosa cells by NECA in contrast to metabohzable adenosine (Billig et al., 1987). However, it cannot
be excluded that NECA ~rnpet~ with or interferes with the action of adenosine at intraceIlular adenosine-sensitive or -dependent processes in granulosa eel&, since for instance NECA, R-PIA and S-PIA all compete with the uptake of adenosine in these cells (Bill@ et al., 1987). In conclusion, the data from the present and previous studies indicate that adenosine, apart from serving as an ~~ace~~~ substrate, also can exert effects via extrace~ul~ A, adenosine receptors in the preo~iato~ ovary. The ovary consists of several cell types and the ceflular 1ocaIization of the senator adenosine receptors evident in ovarian membranes is at present not specified. However, we have earlier reported that NECA don-de~ndently potentiates FSH-stimulatd cAMP accumulation in rat 8mrmlosa cells, even though a discrepancy in the potency of NEC!A in ovarian membr~es versus that in granulosa cells is evident. The EC& was reported to be one order of ma~tude higher in the c&Is (Big et al., 1987), compared to the ovarian membr~e in the present study. Further investigations are needed to establish the cellular of the stimulatory A2 receptors in ovarian membr~es. The physiolo~cal relevance of these stimulatory A2 receptors in the ovary is at present unclear. However, an autocrine or paracrine model can be proposed. It is well known that striation of ovarian ceils with FSW results is an increased cAMP production and a marked release of CAMP into the culture me~um (Selst~ et al., 1976). Since ovarian cells possess the CAMP de~adative edges phosph~iesterase and 5’“~ucl~tidase (Rosberg et al., 19751, the released CAMP will rapidly be degraded to AMP and adenosine (Bruns, 1980; Pearson et al., 1980). FSH also increases both ~x~a~~ul~ (Se&am and Rosberg, 1976) and intra~~ul~ (Conti et al., 1984) phosthe pho~esterase activity, further enh~c~g cAMP de~adation to AMP and adenosine. Furthe~ore, gonado~op~s decrease the ATP levels in whole ovaries (Al&n et al., 1968) and in gr~~osa cells (O~awa et al., 1985; ping and Rosberg, 1986), and a decrease in ATP levels is almost always associated with a release of adenosine (Arch and Newshohne, 1978). It is thus conceivable that locally produced and released
adenosine potentiates the surrountig fo~cul~ cells via adenosine receptors in a placate and/or auto~ne manner, as has been su ted for adenosine in the testis (Monaco et al., 19841,
We wish to thank Prof. Kurt Ah&n and Dr. Torbjom H~~sj~ for valuable su tions and the NEDDK and NHPP, U~versity of Maryland School of Me~cine for generous’~fts of FSH and PMSG, This work was supported by grants from the Swedish Medical Research Council (Nos. 27 and 73141, the Royal Swedish Academy of Sciences, the Gijteborg and the Swedish Medical Societies, the Royal Society of Arts and Sciences in G~teborg, the Handl. Hj. Svensson Research Fund and the Faculty of Medicine, diversity of G~teborg, Sweden.
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