f’hcmtrr (1992), 13, 123-133

Dual Regulation of Human Syncytial Adenylyl Cyclase MARK M. JACOBSalb, XIN LI” & NICHOLAS P. ILLSLEY”,” u Laboratoy for Perinatal Biolo~, Departmentof Obstetrics,Gvnecolony6 ReproductiveSciences b The ReproductiveEndocrinologyCenter, Universityof California, San Francisco ’ To whom correspondenceshould be addressed at: 1462 Health SciencesEast, Universiy of California, San Francisco, C4 941430550, USA Paper accepted16. IO.1991

SUMMARY The dual fitimulato y and inkibito y) regulation of adenylyl cyclase was studied in syncytiotrophoblast basal membranes prepared from term human placenta. Stimulation of adenylyl cyclase activity with GTP, non-hydrolyzable GTP analogs, isoproterenol and PGE, was observed, confirming thepresence ofan intact stimulato y pathway in these membranes. Investigations ofthe inhibitorypathway revealed tight coupling of the G-protein, Gi,, to catalytic adenylyl cyclase, with high doses of GTP producing 80 per cent inhibition of GTP/‘rskolin-stimulated activity, Confirming Gio involvement, pertussis toxin (Pm) treatment ofbasal membranes augmented the responses of adenylyl cyclase to both GTP andforskolin. In addition, immunoblotting ofbasal membraneproteins revealed thepresence ofthe G-protein subunits, G,,, G,,, and GolY The response of adenylyl cyclase was measured to a series of agonists known to inhibit adenylyl cyclase in other tissues, however a reproducible inhibitory eflect was produced only by somatostatin (-80 per cent). Treatment of basal membranes with PTX caused a degree of reversal of the somatostatin-mediated adenylyl cyclase inhibition. However, the intoxication was insuficient to restore GTP/firskolin-stimulated activity,

INTRODUCTION Investigations of the adenylyl cyclase signalling pathway have revealed a stimulatory pathway which includes plasma membrane-spanning receptors, the stimulatory guanine nucleotide binding protein, G,, and a parallel inhibitory pathway comprised of another set of receptors and an inhibitory guanine nucleotide binding protein, Gi (Gilman, 1986). The molecular basis of adenylyl cyclase stimulation appears to be the receptor-mediated binding of GTP to 0143-4004/92/020123

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G, and its dissociation into G,, and GfijY with subsequent activation of catalytic adenylyl cyclase by G,, (Lefkowitz and Caron, 1988). Although analogous events occur with inhibitory receptor binding to Gi, there is evidence that cyclase inhibition may result both by release of Gp,y subunits that bind the dissociated G,, (thus preventing G,,-mediated stimulation), and by direct inhibitory interaction of Gi, with catalytic adenylyl cyclase (Mattera et al, 1988; Newton and Klee, 1990; Birnbaumer, 1990). While Gi, has been purified from membranes prepared from homogenized placental tissue (Codina et al, 1985), the presence of a coupled inhibitory pathway regulating the basal membrane adenylyl cyclase has not previously been reported in the syncytiotrophoblast. The adenylyl cyclase second messenger signalling system of human syncytiotrophoblast has been localized to the basal or fetal-facing surface (Whitsett et al, 1980; Figgs, 1988), whereas the microvillous surface, which faces the intervillous space and maternal blood, appears to be devoid of adenylyl cyclase (Matsubara, Tamada and Saito, 1987; Spreca et al, 1988). Previous reports have described stimulation of cyclase activity in placental membranes by several agonists (Ferre, 1986), magnesium, guanine nucleotides and forskolin (Whitsett et al, 1980; Milewich et al, 1982). Cyclic AMP produced by adenylyl cyclase has been shown to regulate a number of functions in the syncytiotrophoblast including stimulation of hCG secretion, inhibition of estradiol secretion and more recently, inhibition of the microvillous membrane chloride conductance (Feinman et al, 1986; Benoit et al, 1988; Piacchi et al, 199 1). Given the number of cellular functions regulated by cyclic AMP in other tissues, it is probable that many more roles will be described for adenylyl cyclase and cyclic AMP in the placental syncytium. A method has been described recently that permits preparation of microvillous and basal membranes from the same initial placental material (Illsley et al, 1990). This method yields both membrane fractions, without significant contamination by non-syncytial plasma membranes such as fibroblasts, macrophages, endothelial cells and cytotrophoblast. Previous studies of syncytial adenylyl cyclase have used membrane preparations contaminated with microvillous particulates as well as possible contamination by plasma membranes from other cell types (e.g. vascular endothelium, fibroblasts) which are known to contain cyclase activity. The new placental membrane preparation generated a 16-fold enrichment of the basal membrane marker, adenylyl cyclase in the absence of significant contamination by microvillous or other membranes. The hypothesis we tested in these studies was that syncytial adenylyl cyclase, localized to the basal membranes, is under both stimulatory and inhibitory control. We also re-examined adenylyl cyclase responses to various effecters to confirm that previously reported effects were not artifacts of contamination by other membranes.

MATERIALS

AND

METHODS

Placental membrane preparation Microvillous and basal membranes were prepared simultaneously from normal term (38-41 week gestation) placental tissue by a method developed in this laboratory (Illsley et al, 1990). Briefly, after removal of the chorionic plate and a decidual layer, the tissue was washed, homogenized and centrifuged to remove mitochondria and cellular debris then the supernatant was centrifuged to pellet the plasma membranes. After resuspension of the pellet, 12 mu MgClz was added to aggregate non-microvillous membranes and the suspension was stirred on ice and centrifuged to remove the M$+-precipitated membranes. M$+precipitated membranes were layered on to a two-step, sucrose density gradient (1.165 and

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1.190 g/ml), centrifuged at 141000 g for 60 min and the membrane fraction obtained at the interface between the lower two steps was collected and centrifuged to produce a basal membrane (BM) fraction. The supernatant obtained from the magnesium precipitation step u-as centrifuged to pellet the microvillous membranes (MVM). Both membrane fractions pH 7.0, frozen in were washed and resuspended in 250 mM sucrose, 10 mM HEPEWTris, liquid N2 and stored at -70°C. BM were enriched -16-fold in the basal marker adenylyl cyclase while MVM were enriched -20-fold in the marker alkaline phosphatase. Both fractions were free from cross contamination and from contamination by mitochondria, microsomes and lysosomes as measured by marker enzyme analysis. The contamination of microvillous and basal membranes by non-syncytial plasma membranes as measured by immunoblotting has been determined to be minimal (Illsley et al, 1990). Protein concentration was determined by the method of Bradford (1976). ,Idenylyl cyclase assay Adenylyl cyclase activity was quantified by a modification (Maier, Roberts and Jacobs, 1989) of the method of Salomon Londos and Rodbell (1974) which measures the conversion of rx[“*P]ATP to [32P]cAMP. Placental membranes were incubated with 0.4 rn.M ATP, an ATP generation system composed of creatine phosphate and creatine phosphokinase, 5 rn\t MgC12, and effecters. The reaction was started by addition of Q[~*P]ATP, followed by incubation at 37°C for 10 min, and stopped by addition of 5 per cent SDS, 1 mhl CAMP, and 2 ml,1 ATP. Separation of CAMP from ATP was accomplished by sequential chromatography on Dowex and alumina and the [32P] CAMP was quantified by liquid scintillation counting. Recovery of CAMP was monitored individually with [3H]cAMP and averaged 80 per cent. Gel electrophoresis and immunoblotting MV’hl and BM samples were tested for the presence of G proteins by immunoblotting. Samples were solubilized by sonication in 2 per cent SDS and single dimension SDS-PAGE was carried out using 50 ,ug of membrane protein by the method of Laemmli (197(l), on 5 x 10 cm, 10 per cent gels (0.75 mm thick). All samples were run on the same gel and the experiment repeated at least three times. Proteins from the SDS gels were electrophoretically transferred to nitrocellulose sheets (0.2 pm pore size) (Towbin et al, 1979). Primary antibodies were: U9, an affinity purified antibody to a defined region of the C terminus ot G,,, A.569 which recognizes the alpha subunit of G,, Gi and G,,, and S217, raised against Giill, (both 35 and 36kD forms), obtained from Drs H. R. Bourne and S. Mumby (Mumby et al, 1986). A second antibody, goat anti-rabbit [t*‘I]F(ab), ( lo6 ct/min/ml) was used as a secondary probe. Pertussus toxin treatment PTX treatment (Codina et al, 1983) was performed at 37°C for 30 min by mixing basal membranes (0.5 mg/ml) with activated PTX (1 mg/40 mg membrane protein), 10 rnh4 NAD, 10 m\l DTT, 200 ~14 GTP, 400 puhl ATP, 20 ph#i thymidine and an ATP regeneration system. Materials Radioisotopes were obtained from Amersham Corp. (Arlington Heights, IL), nitrocellulose from Schlier and Schuell (Keene, NH), pertussis toxin from List Biological Laboratories Inc. (Campbell, CA) and peptide hormones from Peninsula Laboratories Inc. (Belmont,

Phcenta(1992), Cd. 13 24

(a)

(b)

2.0

1.2

J Basa I

1.0

GwNHP

F&m I. Stimulation of basal memhranc adenylyl cyclase. Stimulation ofadenyl$ cyclase was pcrformcd in basal membranes (a), in the absence of effectar (basal), by Cr’l‘l’ (10 !!\I) and non-hydrolysable &many1 nucleotide analogucs ((;TP+ and (;ppNI Ip; lO/r\~). Data are the mean k s.e.m. ofthrec placental preparations performed in triplicate. Stimulation of adenylyl cyclasc was also pcrformcd with the stimulatory agonists prostaglandin 1.1,(closed bar) and isoproterenol (open bar) in the prcsencc of (;TP (IO 1r\1) (h). Data is expressed as fold increase (mean + s.e.m.) compared to (;TP alone (20 % 1 pmols c.A\lP/mg protein/min) lin three placental preparations assayed in triplicate.

CA). Anti-G,, antibody and anti-Gi,and anti-G, GppNHp > GTP (P < 0.05, ANOVA). Isoproterenol and prostaglandin Et, effecters acting at the membrane-receptor level, enhanced stimulation by 10 P-\I GTP, the prostaglandin being significantly more effective than the adrenergic agonist [P < 0.05, ANOVA; Figure l(b)]. Thus adenylyl cyclase is stimulated both by guanine nucleotides and by /3-adrenergic agonists. Inhibitory effects of guanine nucleotides and pertussis toxin To test the direct coupling of the inhibitory guanine nucleotide binding protein, Gin, to the catalytic component of adenylyl cyclase, the effects of increasing doses of GTP on forskolin-

,~~uvbset al: Regulation

ofplacentala&n$$

127

gdase

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~i~zwe 1. GTP effects on forskolin-stimulated adenylyl cyclase activity. The effect of increasing doses of GTP on stimulation of adenylyl cyclase by 100 /AMforskolin in basal membranes. The activity in the presence of 100 !(\I forskolin was 155 i 33 pmols cAMP/mg protein/min. Data are expressed as fold forskolin response for each experiment. Data are given as the mean + s.e.m. of three experiments assayed in triplicate.

stimulated adenylyl cyclase activity were measured (Figure 2). Because GTP binds to G,,, with greater affinity than to Gi, (Cooper, 1982), it can be predicted that activation of cyclase due to GTP/G,, binding would predominate at lower [GTP], followed by increasing inhibition at higher [GTP] due to increased GTP/Gi, binding. At low doses of GTP an augmentation of forskolin-stimulation was observed, consistent with a GTP/G,, interaction. By contrast, at higher doses of GTP there was almost 80 per cent inhibition of dually (forskolin/GTP) stimulated adenylyl cyclase activity, indicating activation of Gi,,. Nonspecific inhibition by high nucleotide triphosphate concentrations (GTP) was deemed unlikely since the final ATP concentration in these experiments was 0.4 mu. Additionally, 1 mhl cytosine triphosphate (CTP) added to the assay mix did not decrease the stimulation produced by 10,~~ GTP plus 100~~~ forskolin (252 * 53 pmol/mg/min minus CTP versus 232 + 45 pmol/mg/min plus CTP; II = 3). To ensure that these effects were mediated via Gi,, adenylyl cyclase stimulation with forskolin/GTP was performed with and without PTX treatment, testing the effect of inhibiting Gi, (Table 1). Compared to untreated preparations, basal membranes treated with PTX demonstrated an increased stimulation of adenylyl cyclase in the presence of GTP plus forskolin or forskolin alone. The experiment shown in Table 1 was repeated three times with Tablr 1. Inhibition

by pertussus

Basal membrane (pmols cAMP/mg protein/min,

toxin of basal membrane stimulation

adenylyl cyclase aCtkit\ mean + s.e.m. of a typical experiment) Control

Forskolin Forskolin

(100 1~) (100 ,UM)

“PTX-treated

+

adenylyl cyclase

GTP (1 mxt)

> control, P < 0.05; t-test.

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Placenta (1992), Vol. I3

128

similar results. This data supports these membranes.

the contention

that Gi, is coupled to adenylyl cyclase in

Inhibitory effects of agonists A series of agonists which have been reported to inhibit adenylyl cyclase in other tissues were tested for their effect on syncytial adenylyl cyclase (Table 2). The concentrations of effecters used were lOO-lOOO-fold greater than the reported IC50 for adenylyl cyclase inhibition in other tissues. To maximize detection of inhibitory responses, experiments were performed with two types of stimulation; in the presence of 100,~~~ forskolin plus either 10,~~ or 100,1~~ GTP. The first of these two concentrations of GTP was chosen because it was the concentration at which maximal activation had been observed, (see Figure 2) while the latter concentration was chosen since it had displayed the inhibitory effects induced by higher GTP concentrations (see Figure 2). Under the conditions of these experiments neither adenosinergic (adenosine), cL-adrenergic (norepinephrine and propranolol), muscarinic (carbachol), opioid (dynorphin l-l 3), nor dopaminergic (dopamine), agonists inhibited adenylyl cyclase after stimulation by GTP plus forskolin. Of the two peptidergic compounds tested, neuropeptide Y had no inhibitory effect, but inhibition was observed with somatostatin. The degree of somatostatin inhibition was higher at the lower (10 ,LL~M) dose of GTP, presumably because of the preexisting inhibition of adenylyl cyclase activity by GTP at the higher (100 ,LV) GTP dose. Somatostatin was ineffective as an inhibitor in the absence of guanine nucleotides (data not shown). Because the efficiency of inhibitory receptor coupling to Gi,, has been shown to be altered by Na+ concentration, agonist-mediated inhibition was tested in the presence of 10 and 100 rn,v NaCl. These experiments demonstrated no differences in inhibitory responses (or lack thereof) between 10 and 100 mM NaCl for the agonists tested. Analysis of basal membrane G-proteins To confirm the presence of the relevant G-protein subunits in the basal membrane, immunoblotting was performed using antibodies to the specific G protein subunits (Mumby et al, 1986). In order to use immunoblots to determine relative protein concentration, Tub/r 2. Inhibition

of basal membrane

adenylyl qclase

stimulation

by agonists at high and low NaCl concentration

Basal membrane adenylyl cyclase activity (fraction of the forskolin/GTP stimulated activity) Forskolin (100 kltht) + GTP (100 /l.hl) Low NaCl (10 m>l) Dynorphin (1 ,u,M) Somatostatin (10 ~51) Neuropeptide Y (100 nh%) Carbachol(lO0 pM) Adenosine (10 ,unr) Norepinephrine (10 PM) + Propranol(l0 Dopamine (1 +I) N.D., not done. Mean f s.e.m., n = 3. u = P < 0.05, r-test.

PM)

0.94 0.79 0.93 1.11 1.12 1.07 0.99

f + f f f f f

0.02 0.06” 0.09 0.06 0.10 0.02 0.04

High NaCl (100 m\t) 0.98 0.81 0.93 1.01 0.95 1.05 0.94

t 0.08 t 0.05” +- 0.06 ?z 0.10 f 0.05 + 0.05 + 0.06

Forskolin (100 p&l) GTP (10 /ch~) Low NaCl (10 m\l) 1.08 * 0.12 0.21 * 0.07” 1.14 + 0.08 1.06 + 0.05 N.D. N.D. N.D.

+

129 1”

6-

6-

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Gia

II

F&ye 3. Relative concentration of G-protein subunits in syncytial basal and microvillous membranes. G-proteins were analvsed using specific antibodies in microvillous membranes (closed bars) and basal membranes (open bars). Solubilized extracts of microvillous and basal membranes were electrophoresed on polyacylamide gels, the proteins were transferred to nitrocellulose and probed with antibodies specific to G,,, G,,z and G/i,;,. After binding nf a [‘251] labeled IgG, autoradiograms were obtained and scanned with a laser densitometer. Data are expressed as the mean If s.e.m. for three sets of MV~M and BM.

experimental conditions were established to assure antibody excess and thus not underestimate subunit concentration. Densitometry of autoradiograms made from the blots showed linearly increasing density up to 200 pug of membrane protein per lane. Subsequent immunoblotting experiments were performed with 50,ug protein per lane. This method does not allow measurement of the absolute value of G protein subunit concentration in the membranes in the absence of purified samples of G protein. However, it will reveal the presence of the G-protein subtypes. Autoradiograms made from immunoblots showed the presence of G,,, Gi,, and Gp,:, subunits in the syncytiotrophoblast basal plasma membranes and in microvillous membranes from the same source (Figure 3). Reversal of somatostatin inhibition of adenylyl cyclase by pertussis toxin Previous experiments using PTX (Table 1) demonstrated that the inhibitory effect of GTP was mediated by Gi,, however it was thought possible that the effects of somatostatin might take place via another pathway, as has been suggested for S49 lymphocytes (Hildebrandt and Kohnken, 1990). To test this, basal membrane adenylyl cyclase was activated by 100 ;C.ZI forskolin plus 10 ,~unlGTP and the effects of 10 ,UM somatostatin and/or PTX (10 &ml) on the dually stimulated cyclase were measured (Table 3). It is apparent PTX was not very effective in reversing the effects of somatostatin on GTP/forskolin-stimulated adenylyl cyclase activity, despite the fact that PTX treatment produces an increase in the forskolin/ GTP-stimulated activity in the absence of somatostatin. PTX treatment reduced the somatostatin inhibition from 80 to 65 per cent of the GTP/forskolin-stimulated activity.

DISCUSSION Very little is known about the function of placental second messenger signalling systems including those of the syncytial adenylyl cyclase-coupled pathway. Hecause the placenta

Placenta (1992), Vol. 13

130 Table 3. Pertussis toxin reversal of somatostatin-mediated inhibition of adenylyl cyclase Basal membrane adenylyl cyclase activi? (fraction of the forskolin/GTP stimulated activity) Forskolin (100 &/GTP (10 PU~I) + PTX (lOpg/ml) + Soma (10 j,~ull) + PTX (lO,~g/ml) + Soma (lo/~)

1.00 3.55 + 0.11” 0.19 f 0.02” 0.36 + 0.05”

PTX, pertussis toxin; Soma, somatostatin. ForskolinIGTP-stimulated activity = 149 2 46 pmols cAMP/mg protein/min, mean ? s.e.m., n = 3. ’ = p < 0.05, /-test.

serves as an epithelial transport organ like the gut or kidney and also as a metabolic organ, like the liver, adenylyl cyclase may play a regulatory role in placental transport and metabolism (Freissmuth, Casey and Gilman, 1989; Wheeler and Yudilevich, 1989; Piacchi et al, 1991). These studies were designed to define the pathways by which external factors modulate the activity of the adenylyl cyclase located in the syncytiotrophoblast basal membrane. This report confirms the stimulation of basal membrane adenylyl cyclase by P-adrenergic agonists (Whitsett et al, 1980; Milewich et al, 1982). This is useful information since the previous studies employed methods of basal membrane preparation in which, although contamination with microvillous membranes was measured, there was no systematic search for adenylyl cyclase-containing plasma membranes from other cell types, such as fibroblasts and endothelial cells. Since the method used in these studies produces basal membranes essentially free from such contamination, it can now be stated with certainty that basal membranes contain an active adenylyl cyclase. This paper also demonstrates that PGEi will stimulate basal membrane adenylyl cyclase activity, providing a mechanism for the effect of PGEr seen previously in perfused placental tissue (Levilliers et al, 1974). In a previous study, subcellular fractions frozen at -70°C while retaining adenylyl cyclase activity, lost their response to /&adrenergic agonists (Milewich et al, 1982). In our hands, this preparation retained not only adenylyl cyclase activity, but also P-adrenergic responsiveness after freezing. It will be essential that these experiments be repeated in intact syncytiotrophoblast cells to confirm their validity, however, the only syncytiotrophoblast cells currently available are those derived in culture from the fusion of primary cytotrophoblast (Kliman et al, 1986). Most of the structural and functional attributes of these fused cells remain to be defined and, since they are secondary cells cultured in vitro rather than primary cells, the quantification of adenylyl cyclase signalling pathway components in the in vitro culture may not resemble that observed in vivo. The results reported here are important therefore because they describe the presence of the inhibitory arm of this signalling pathway in membranes obtained from primary tissue. Stimulation of cyclase activity was greater in the presence of non-hydrolysable guanine nucleotide analogs than in the presence of GTP, consistent with an active, resident G,, GTP-ase. Of the two analogs tested, GTPyS stimulated adenylyl cyclase significantly more than GppNHp. This may be due to greater efficiency of GTPyS in activating G,,, or conversely, greater efficiency of GppNHp in activating Girl. These studies do not directly address which of these possibilities is valid. The presence of functional, coupled Gi,, and thus an inhibitory pathway, is indicated by

jhobs the

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biphasic GTP dose response of forskolin-activated adenylyl cyclase. Stimulation of cyclase activity occurred at low GTP concentrations most probably because of the higher affinity of G,, compared to Gi,, for GTP (Cooper, 1982). At higher GTP doses Gi, was fully activated, resulting in a high degree of inhibition of adenylyl cyclase (>80 per cent at 1 rn%l GTP). This biphasic dose response was also observed by Milewich in placental basal membranes (Milewich et al, 1982), although the degree of inhibition at high [GTP] was lower than that observed here, and the reason for the decreased activity at high [GTP] was not explained. Confirmation that GTP-mediated inhibition was due to Gi, activation was provided by experiments demonstrating that pertussis intoxication, which inactivates Gi ~OPM), GTP binding to the Giu-G~/I, complex causes release inhibition of cyclase. It is also possible that residual guanine of Gi,,, and consequent nucleotide binding to Gi was the cause of adenylyl cyclase inhibition in the nominal absence of GTP, although the extensive preparative procedure suggests this is unlikely. The demonstration that the maximal adenylyl cyclase activity observed after PTX treatment is not significantly different in the presence or absence of GTP suggests that the limiting element in the stimulation of adenylyl cyclase may be the quantity of cyclase present in the membrane. The search for inhibitory agonists produced mixed results. Although somatostatin demonstrated consistent and reproducible inhibition of forskolin-stimulated adenylyl cyclase (-80 per cent), no other agonist tested was able to inhibit adenylyl cyclase. The coupling of plasma membrane receptors to the inhibition of adenylyl cyclase in other tissues is altered by Na+ concentration (Limbird, 1988; Limbird et al, 1985). When we examined the effect of Na+ on inhibition we found neither enhancement of somatostatin effects nor the appearance of inhibition using other agonists. The reasons for the lack of other inhibitory agonists is unknown although the most obvious is lack of receptors on the basal membrane. However, the intention in this research was not to catalog the stimulatory and inhibitory agonists but rather to establish the existence of both pathways in the syncytiotrophoblast basal membrane, and we have not therefore pursued this further. Treatment of basal membranes with PTX prior to the measurement of somatostatin inhibition only reduced the inhibition from 80 to 65 per cent. One possible explanation is incomplete intoxication by PTX. Attempts to measure the extent of ADP-ribosylation (intoxication) resulting from PTX treatment revealed a continuing degree ofde-ribosylation/ re-ribosylation as demonstrated by [32P]NAD labeling (data not shown), preventing the quantitation of ribosylation. We have noted, however, that in similar experiments using cultured syncytiotrophoblast, PTX treatment fully reversed somatostatin inhibition of isoproterenol-stimulated adenylyl cyclase activity (Grullon et al, 1991). The ineffectiveness of PTX in reversing somatostatin inhibition of adenylyl cyclase in basal membrane vesicles was therefore most probably due to incomplete intoxication. We conclude that the adenylyl cyclase shown to be present in the basal membranes from

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human syncytiotrophoblast is subject to stimulation by both external factors and guanine nucleotides, suggesting G-protein mediated signal transduction. The adenylyl cyclase is also subject to inhibitory modulation by somatostatin via a guanine nucleotide-sensitive pathway which is at least partially if not completely mediated by Gi.

ACKNOWLEDGEMENTS This work was supported by grants HD23498 and HD26392 from the National Institute of Child Health and Human Development. The authors would lie to thank Dr H. R. Boume and S. Mumby for providing the anti-G protein antibodies, and the labor and delivery staff at U.C. Medical Center for their help in obtaining placental tissue.

REFERENCES Benoit, J., Rodway, M., Ho Yuen, B. & Lang, P. C. K. (1988) Effects of cyclic adenosine monophosphate on human chorionic gonadotropin and estradiol output by cultured human placental cells. American journal of Obstetrics and Gynecology, 158, 328-332. Bimbaumer, L. (1990) G proteins in signal transduction. Annual Review ofPhamzacology and Toxicology, 30, 675705. Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. Codina, J., Hildebrt, J., Iyengar, R., Bimbaumer, L., Sekura, R. & Manclark, C. (1983) Pertussis toxin substrate, the putative Ni component of adenylyl cyclases, is an a/l heterodimer regulated by guanine nucleotide and magnesium. Proceedings ofthe NationalAcadumy ofSciences of the USA, 80,4276-4280. Codina, J., Rosenthal, A., Hildebrandt, J., Bimbaumer, L. & Sekura, R. (1985) Purification of N, and Ni, the coupling proteins of hormone-sensitive adenylyl cyclases without intervention of activating regulatory ligands. Methods in Enzymology, 109,446-465. Cooper, D. M. F. (1982) Bimodal regulation of adenylate cyclase. FEBS Letters, 138,157-163. Evans, T., Brown, M., Fraser, E. & Northup, J. (1986) Purification of the major GTP-binding proteins from human placental membranes.3obumal ofBiological Chemistry, 261,7052-7059. Evans, T., Fawzi, A., Fraser, E., Brown, M. & Northup, J. (1987) Purification of a B35 form of the py complex common to G-proteins from human placental membranes.3oumal ofBiological Chemistry, 262, 176-181. Feinman, M. A., KIiman, H. J., Caitaliano, S. & Strauss, J, F., III (1986) 8-Bromo-3’,5’-adenosine monophosphate stimulates the endocrine activity of human cytotrophoblasts in culture. Journal of Clinical Ena’ocritzologyandMetabolism, 63,1211-1216. Ferre, F. (1986) Dopamine-stimulated adenylate cyclase in human term placenta. Life Sciences, 39, 1893-1900. Figgs, L. W. (1988) Guanine nucleotide binding in human placental syncytiotrophoblast membranes and comparative regulation of adenylate cyclase in syncytiotrophoblast, turkey erythrocyte and bovine calf testes membranes by guanosine-5’-triphosphate. Comparative Biochemistry and Physiology, 89, 119-125. Freissmuth, M., Casey, P. & Gilman, A. (1989) G proteins control diverse pathways oftransmembrane signaling. FASEB3oumal,3,2125-2131. Gilman, A. (1986) G proteins and regulation of adenylyl cyclase.ym, 262, 1819-1825. Grullon, K. E., Jacobs, M. M., Sellers, M. C., Li, X. & Illsley N. P. (1991) Regulation of cyclic AMP generation in cultured human syncytiotrophoblast by the stimulatory and inhibitory arms of the receptor-coupled adenylyl cyclase. Ena’utinology (submitted for publication). Hildebrandt, J. D. & Kohnken, R. E. (1990) Hormone inhibition of adenylyl cyclase. 3obumaJ of Biological Chemistry, 265,9825-9830. Illsley, N. P., Wang, Z. Q., Gray, A., Sellers, M. C. &Jacobs, M. M. (1990) Simultaneous preparation of paired microvillous and basal membranes from human placental syncytiotrophoblast. Biochimica et Bioph.ysicaActa, 1029, 218-226. Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M. & Strauss, J. F. I. (1986) Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endotinology, 118,1567-1582. Laemmli, U. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature, 277,680-689. Letkowitz, R. & Caron, M. (1988) Adrenergic receptors. Advances in Second Messenger Phosphoprotein Research, 21, l-10. Levilliers, J., Alsat, E., Laudat, P. & Cedard, L. (1974) Hormone-stimulated CAMP production in human placenta perfused in vitro. FEBS Lettm, 477, 146-148. Levitzki, A. (1988) Signal transduction in hormone-dependent adenylate cyclase. Oil Bioph,ysics, 12, 133-143.

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Dual regulation of human syncytial adenylyl cyclase.

The dual (stimulatory and inhibitory) regulation of adenylyl cyclase was studied in syncytiotrophoblast basal membranes prepared from term human place...
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