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Forskolin: Unique diterpene activator of adenylate cyclase in pregnant and nonpregnant guinea pig myometrial membranes
Forskolin: Unique diterpene activator of adenylate cyclase in pregnant and nonpregnant guinea pig myometrial membranes Christos G. Hatjis, M.D. Winston-Salem, North Carolina In guinea pig myometrium, ~-adrenergic receptors are functionally coupled to adenylate cyclase. ~-Adrenergic receptor agonists in the presence of guanosine triphosphate stimulate adenylate cyclase
activity, thus increasing 3'5'-cyclic adenosine monophosphate synthesis and promoting myometrial relaxation. In pregnant animals close to term (65 days), ~-adrenergic receptor density as well as basal and (-)isoproterenol-dependent (in the presence of guanosine triphosphate) adenylate cyclase activity is significantly higher than that in nonpregnant animals or those in early pregnancy. Since this system appears to be made up of at least three components (~·adrenergic receptor, guanosine triphosphatebinding protein, and a catalytic component), these observations on total adenylate cyclase activity may reflect alterations in one or more of these components. To answer the question whether the catalytic unit of this system can be directly assayed and whether its activity is influenced by pregnancy, we have performed in vitro experiments to measure the enzymatic activity of the catalytic component of the ~-adrenergic receptor-adenylate cyclase complex in guinea pig myometrial membranes. We have used two compounds that stimulate the catalytic component: forskolin and manganese chloride. Forskolin, regardless of the presence or absence of guanosine triphosphate, is the most potent stimulator of adenylate cyclase activity in myometrial membranes from nonpregnant and pregnant animals; manganese chloride is a less potent activator. The degree of adenylate cyclase stimulation by forskolin tends to be higher in uteri from pregnant (;>0.5 gestation) than from nonpregnant or postpartum animals. It was concluded: (1) that adenylate cyclase stimulation by forskolin does not depend on the presence of ~-adrenergic receptor agonists or guanosine triphosphate and (2) that with advancing gestation there might be a qualitative or quantitative change with regard to the interaction between forskolin and the presumed catalytic component of the ~-adrenergic receptor-adenylate cyclase complex. (AM J OBSTET GYNECOL 1986;155:1202·8.)
Key words: Forskolin, adenylate cyclase, myometrial membranes
Myometrial contractility is influenced by the relative concentrations of a- and 13-adrenergic receptors. 1 Stimulation of 13-adrenergic receptors in human or animal myometrium by 13-adrenergic receptor agonists results in increased adenylate cyclase activity and high levels of intracellular 3'5'-cyclic adenosine monophosphate (3'5'-cAMP). The latter is the second messenger that initiates a series of events leading to smooth muscle relaxation. 2· 3 This system is composed of at least three distinct protein components 4 : a catalytic subunit that converts the substrate magnesium-adenosine triphosphate to 3'5'-cAMP, a guanine-nucleotide-binding protein that Frm the Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Bowman Gray School of Medicine of Wake Forest University. Supported in part by Basil O'Connor Grant 5-362, The National Foundation March of Dimes, BGSM United Way Starter Grant, and National Institutes of Health IR23 HD20796-0J. Presented in part at the Thirty-second Annual Meeting of the Society for Gynecologic Investigation, Phoenix, Arizona, March 20-23, 1985. Reprint requests: Christos G. Hatjis, M.D., Department of Obstetrics and Gynecology, Bowman Gray School of Medicine, 300 South Hawthorne Road, Winston-Salem, NC 27103.
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mediates hormonal responsiveness and activation, and a hormone receptor. Each of these components is most likely made up of more than one polypeptide subunit. My colleagues and I have been studying the properties of the 13-adrenergic receptor-adenylate cyclase system in myometrial membranes from nonpregnant and pregnant guinea pigs. P have shown that the concentration of myometrial 13-adrenergic receptor is dramatically increased in late pregnancy. Moreover, basal and (-)isoproterenol-stimulated adenylate cyclase activity, which depends on the presence of guanosine triphosphate, was significantly increased in parallel with the 13-adrenergic receptor changes. However, these initial experiments studied only a physiologically important aspect of this system. Since the latter is a multicomponent complex, it is important to examine the properties of each of these components in both the nonpregnant and the pregnant state. Thus, to further elucidate the molecular relationship between the various components of the 13-adrenergic receptor-adenylate cyclase system in guinea pig myometrial membranes, we performed experiments to determine the activity of the catalytic component of the 13-adrenergic
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receptor-adenylate cyclase complex and answer the question whether the catalytic component is regulated independently of 13-adrenergic receptor activity and hormone-dependent adenylate cyclase activity in pregnant and nonpregnant animals. For this purpose, two compounds were used that, in most systems studied, have been shown to directly stimulate the catalytic component forskolin, a diterpene extacted from the roots of Coleus forskohlii, a potent hypotensive vasodilatory and antispasmolytic agent,6- 9 and manganese chloride. 10" 16 Material and methods
Materials. o:- 32 P-adenosine triphosphate (o:- 32 P-ATP) (20 to 40 Ci/mmol) and [3',8''H]3'5'-cAMP (15 Ci/ mmol) were obtained from New England Nuclear. Other drugs and materials were obtained from commercial sources and were of reagent grade. Forskolin was purchased from Calbiochem and dissolved in 95% ethanol (10 mmol/L). Animals. Virgin female guinea pigs (weighing 400 to 600 gm) and guinea pigs with time-dated pregnancies (gravida 1, para 0, weighing 600 to 1200 gm) were obtained from Perfection Breeders. Before being put to death, they were housed in our animal care facility for 2 to 20 days. Pregnant animals were studied at 0.39 (early), 0.69 (middle), and 0.9 to 1.0 (late) gestation (term 65 days). Some pregnant guinea pigs were allowed to deliver and were studied in the postpartum period (1 to 5 days). Animals were anesthetized with 40 to 55 mg/kg of pentobarbital intraperitoneally. At laparotomy, a hysterectomy was performed. The uterus was cleaned of attached adipose and vascular tissue and the cervical region was removed. If the animal was pregnant, hysterotomy was performed and the endometrial cavity cleaned of the products of conception. Tissue was snapfrozen in liquid nitrogen and stored at -70° C until used. Tissue preparation. Uterine tissue was thawed in icecold buffer A containing 0.005 mol/L ethylenediaminetetraacetic acid, 0.025 mol/L sucrose, 0.9% sodium chloride, and 0.02 mol/L Tris hydrochloride buffer, pH 7.5. The thawed uterus was then split along each horn and the endometrium was scraped. The uterus was placed in five to 10 volumes per gram of wet weight of buffer A, minced finely with scissors, and sonicated with a Brinkman Polytron (setting No. 7, twice for 30 seconds). The homogenate was then filtered through eight layers of cheesecloth. An aliquot of the homogenate to be used for adenylate cyclase experiments was centrifuged at 80,000 X g for 15 minutes (4° C) and the pellet resuspended in 2 to 4 ml of ice-cold buffer B (0.9% sodium chloride and 0.02 mol/L Tris hydrochloride, pH 7.5). It was
Forskolin: Diterpene activator of adenylate cyclase
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preincubated for 10 minutes at 4o C in the presence of 0.5 mmol/L dithiothreitol, 0.17 mmol/L ethyleneglycol-bis(l3-aminoethyl ether)-N ,N'-tetraacetic acid, l mmol/L hydrochloric acid, 0.2% bovine serum albumin, and 5 mmoi/L magnesium chloride, pH 7.45. The suspension was then recentrifuged as previously outlined. The resulting pellets were resuspended in icecold buffer B (100 to 150 volumes per gram of wet weight) and used in the assay. Adenylate cyclase assay. Adeny1ate cyclase activity was determined by measuring the conversion of o:- 32 PATP to [' 2 P]-cAMP and isolating the product by a modification of a method developed by Salomon et al. 17 The reaction was carried out in triplicate at 30° C for 15 minutes in a final volume of 0.2 ml containing 0.1 ml of membrane fraction (8 to 60 J.Lg of protein per tube) and 50 mmol/L Tris hydrochloride, pH 7 .5, 0.45% sodium chloride, 0.5 mol/L adenosine triphosphate, 5 mmol/L magnesium chloride, 0.17 mmol/L ethyleneglycol-bis(l3-aminoethyl ether)-N ,N'-tetraacetic acid, 0.05 mmol/L cAMP, 0.5 mmol/L dithiothreitol, 0.75 mmol/L 3-isobutylmethylxanthine, 0.1 mg/ml of creatine kinase, 10 mmol/L creatine phosphate, and 1 to 2 X 106 cpm of o:- 32 P-ATP. Appropriate drugs were added as required. The reaction was initiated by addition of the membrane fraction to the incubation tubes. It was linear with respect to time (up to 20 minutes) and protein concentration. The reaction was terminated by the addition of 0.25 ml of a solution containing 50 mmol/L Tris hydrochloride (pH 7.5), 5 mmol/L adenosine triphosphate, 1 mmol/L 3'5'-cAMP, and 5% sodium dodecyl sulfate. 'H-cAMP (10,000 to 25,000 cpm per tube) was added to all tubes to serve as an internal standard and monitor the efficiency of recovery. The tubes were then placed in boiling water for 10 minutes and allowed to cool down to room temperature. [32 P]-cAMP was isolated with sequential Dowex (AG 50W-X4, 200 to 400 mesh, Bio-Rad) and neutral alumina columns essentially as described by Salomon et al. and counted in a scintillation counter (Packard). Recovery of 3'5'-cAMP was determined by the percent recovery of 3 H-cAMP external standard in each sam pie. Results have been corrected for percent recovery (routinely greater than 40% to 60%). Enzymatic activity was expressed as picomoles of 3'5' -cAMP produced per milligram of protein per minute per uterus. The normalization per uterus was necessary in order to account for the differences in the amount of tissue used per experiment (that is, for nonpregnant animals, the entire uterus had to be used while for pregnant animals only an aliquot was required). Protein assay. Protein concentrations were determined according to the method of Bradford 18 with bovine serum albumin used as a standard.
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December 1986 Am J Obstet Gynecol
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li:.lJ Post-Partum, 1-3 Days Fig. I. Basal adenylate cyclase activity as a function of reproductive state. Late pregnant animals (0. 9 to 1.0 gestation) show increased basal activity either in the presence or in the absence of guanosine triphosphate (GTP)or ethanol (ETOH) (p < 0.001). The percent stimulation by guanosine triphosphate over basal activity was higher in animais in late pregnancy than in the other groups (p < 0.02). Numbers in bars refer to the number of animals studied in each group.
Statistical analysis. Results are expressed as mean ± SEM. We utilized analysis of variance to test the null hypothesis that there is no difference in basal and drugstimulated enzymatic activity with advancing gestation. Since absolute values of adenylate cyclase activity varied considerably, depending on the reproductive state of the animal, a log 10 transformation was carried out to satisfy the prerequisite of homogeneity of variance. 19 A p value of <0.05 was considered significant.
Results First, we determined basal adenylate cyclase activity under three experimental conditions: no addition and addition of ethanol and guanosine triphosphate (Fig. 1). Basal, basal plus guanosine triphosphate, and basal plus ethanol adenylate cyclase activities increased significantly with advancing gestation. Moreover, the percent increase in adenylate cyclase activity in the presence of guanosine triphosphate was higher in myometrial membranes from guinea pigs in late pregnancy than in the other groups. In contrast, ethanol increased basal adenylate cyclase levels to the same degree in all animals. Since forskolin is dissolved in ethanol, it was crucial to include basal activity in the presence of an equivalent dose of ethanol. The observation that eth-
anol stimulates basal adenylate cyclase actlVlty is in agreement with findings reported in other systems. Although the mode of action is not established, it has been suggested that perturbation in the structure-fluidity of plasma membranes might be involved in the process? 0- 22 Second, we examined adenylate cyclase activity in the presence offorskolin and manganese chloride (Fig. 2). Of the agents we have tested to date, 5 forskolin has been the most potent stimulator of adenylate cyclase activity (range tenfold to sixtyfold over basal level) in all groups. Maximal stimulation occurred at a forskolin concentration of 100 ~J.mol/L. The addition of guanosine triphosphate did not result in any significant differences in the absolute values of adenylate cyclase stimulation by forskolin (Fig. 2). There was a gradual increase in forskolin-stimulated adenylate cyclase activity with advancing gestation. Uterine membranes from animals in late pregnancy demonstrated the highest absolute levels of enzymatic activity in the presence of forskolin regardless of the presence or absence of guanosine triphosphate (Fig. 2). Similarly, when adenylate cyclase stimulation in the presence of manganese chloride was studied, animals in late pregnancy showed higher absolute levels when compared to those of the
Forskolin: Diterpene activator of adenylate cyclase
Volume 155 Number 6
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fll!l Post-Partum, 1-3 Days Fig. 2. Stimulated adenylate cyclase activity. Groups and number of animals per group are the same as described in legend to Fig. l.
other groups. Addition of guanosine triphosphate did not influence manganese chloride-dependent stimulation (data not shown). Fig. 3 shows the degree of stimulation of adenylate cyclase activity over basal enzymatic activity. With regard to the addition of forskolin compared with basal levels (panel A) the only significant difference was between animals in late pregnancy and nonpregnant animals (p < 0.05). When forskolin was compared with the basal level plus ethanol (panel B), there were no significant differences between individual groups although there was a trend for pregnant animals to show higher stimulation. When forskolin plus guanosine triphosphate was compared with the basal level plus guanosine triphosphate (panel C), the highest degree of stimulation was seen in the early pregnant group (0.39 gestation, p < 0.05). finally the degree of stimulation by manganese chloride was equivalent in all groups.
Comment The regulation of cellular responses by cyclic nucleotides continues to be an area of intense investigation. 3'5'-cAMP, as a second messenger, mediates hormonal effects ranging from intermediary metabolism to smooth muscle relaxation. Stimulation of uterine
smooth muscle by 13-adrenergic receptor agonists results in an increase in 3'5'-cAMP production and secondary smooth muscle relaxation. 1• 3 It has been shown that 13-adrenergic receptors and the catalytic components of adenylate cyclase are coupled with a stimulatory guanosine triphosphate-binding protein (G, protein).'· 23 • 24 Binding of 13-adrenergic receptor agonists but not antagonists to the 13-adrenergic receptor results in a high-affinity state of the 13-adrenergic receptor, which in turn interacts with the G, protein and activates it. Guanosine triphosphate can then bind to the 13-adrenergic receptor-G, protein complex, dissociate the agonist from the 13-adrenergic receptor, and stimulate the catalytic component of the complex. Guanosine triphosphate is subsequently hydrolyzed and dissociates from the guanosine triphosphate-binding protein. P have previously shown that in guinea pig uteri advancing gestation results in a remarkable increase in the density of 13 2-adrenergic receptors as well as basal and (-)isoproterenol-stimulated adenylate cyclase activity. Stimulation of the enzyme by (-)isoproterenol depended on the presence of guanosine triphosphate. Since this system is composed of at least three components, the question was whether properties of one or more of these are altered by the pregnant state. Since
1206 Hatjis
December 1986 Am J Obstet Gynecol
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•Pregnant, 0.39 Gestation ElPregnant, 0.9-1.0 Gestation Fig. 3. Degree of adenylate cyclase stimulation over basal level. Data shown in Figs. I and 2 have been used for the calculations. Group and number of animals per group are the same as described in legend to Fig. I. Panel A: Forskolin (basal). Panel B: Forskolin/basal plus ethanol (6%). Panel C: Forskolin plus guanosine triphosphate (100 fLmol/L)/basal plus guanosine triphosphate (100 fLmol/L).
the density and perhaps the affinity of the 13-adrenergic receptor have been shown to change with pregnancy,' I next sought to examine the catalytic component of this system as well as the interaction of (-)isoproterenol with the 13-adrenergic receptor as a function of guanosine triphosphate. In this article I present data regarding the catalytic component. To characterize its activity, two agents were used that have previously been shown to directly stimulate the catalytic components of the 13-adrenergic receptoradenylate cyclase complex, forskolin ~ and manganese chloride. ~ In general, forskolin activation of adenylate cyclase does not appear to depend on the presence of guanosine triphosphate or 13-adrenergic receptor. In cell-free reconstitution experiments in lipid micelles, the presence of the catalytic component was the only prerequisite to show forskolin stimulation. 6 Addition of guanosine triphosphate-binding protein and guanosine triphosphate did not alter the response. Although the latter observation confirms the unique properties of forskolin, it does not rule out the presence of other factor(s) associated with the catalytic component that may modify the interaction between the latter and forskolin. Moreover, in some systems, hormonal and guanosine triphosphate-dependent adenylate cyclase stimulation can be augmented in the presence of submaximal concentrations of forskolin. Thus it has been suggested that there might be an interaction between guanosine triphosphate-binding protein and forskolin-induced stimulation of the catalytic component. 25" 28 To date the precise nature of this interaction is not 6 9
10 16
known and further experiments are needed to elucidate this point. We have found that, in myometrial membranes of both nonpregnant and pregnant guinea pigs, forskolin is the most potent stimulator of adenylate cyclase activity. Its action is independent of guanosine triphosphate suggesting that under these conditions forskolin bypasses the G, protein. This observation is in contrast to previous experiments in nonpregnant rat uteri! 9 which have suggested that guanosine triphosphate is involved in adenylate cyclase stimulation by forskolin. Differences in species and experimental design may be partly responsible for these conflicting observations. More important, since the percent increase in adenylate cyclase activity by forskolin compared with the basal level in uteri from animals in late pregnancy might be higher than in the other groups tested, it is possible that the interaction between forskolin and the catalytic component is qualitatively and/or quantitatively different in late pregnancy compared with the nonpregnant state or early and middle pregnancy. Alternatively, similar changes might apply to other component(s) of this complex that is (are) closely linked to the catalytic component and have not been separated to date. However, these "derived" observations should be interpreted with caution since they depend on the choice of basal activity. For example, when the basal plus guanosine triphosphate experiment was used, it was the group in early pregnancy that showed a higher degree of stimulation. ·
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Manganese chloride directly stimulates the catalytic component of the complex. 10• 16 The precise mode of action is not known and interaction with an inhibitory guanosine triphosphate-binding protein cannot be ruled out. 30· 3 ' In our system, manganese chloride significantly increased adenylate cyclase activity but to a lesser extent than that seen with forskolin. Although the percent increase of manganese chloride-dependent stimulation is higher in animals in late pregnancy, the difference did not reach statistical significance. This is the first instance where a direct assay of the catalytic activity of the 13-adrenergic receptor-adenylate cyclase complex has been reported in uterine membranes from pregnant and nonpregnant animals. Our results demonstrate that the presence of the 13-adrenergic receptor is not an absolute requirement for stimulation of adenylate cyclase to occur. Moreover, it appears that the catalytic activity of the 13-adrenergic receptor-adenylate cyclase complex may be regulated independently of the other components. If that is indeed the case, then our approach to study of the properties of each component of the system at the molecular level is necessary if we are to gain a better understanding of the factors controlling adenylate cyclase activity and, secondarily, myometrial contractility. Furthermore, the fact that forskolin is a stronger stimulator of adenylate cyclase activity than (- )isoproterenol suggests that the former may be a more potent agent with regard to uterine muscle relaxation. 32 Further work should provide an approach to selective manipulation of this adenylate cyclase system in intact cells and intact organisms that will significantly improve our understanding of the in vivo control of the myometrial contractility. Since the guinea pig model appears to be very similar to that of the human,33 • 34 it provides a very valuable tool for studying the myometrial 13-adrenergic receptor-adenylate cyclase system in vivo and in vitro. With the recognition of the potential pitfalls associated with extending in vitro observations to in vivo systems, these and similar studies may provide a valuable insight to clinically significant areas such as pregnancy maintenance, onset of term or preterm labor, and others.
1. 2. 3. 4.
REFERENCES Marshall JM. Modulation of smooth muscle activity by catecholamines. Fed Proc 1977;36:2450. Roberts JM. Receptors and transfer of myometrial contractility and cervical maturation. Semin Perinatol 1981; 5:266. Huszar G. Biology and biochemistry of myometrial contractility and cervical maturation. Semin Perina to! 1981; 5:216. Birnbaumer L, Codinaj, Mattera R, et al. Regulation of hormone receptors and adenylyl cyclases by guanine nucleotide binding N proteins. Recent Prog Horm Res 1986;41 :41.
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5. Hatjis CG. 13-Adrenoreceptor and adeny~ate cy~lase properties in pregnant and nonpregnant gumea p1g myometrium. AMj 0BSTET GYNECOL 1985;151:943. 6. Cerione RA, Sibley DR, CodinaJ, et al. Reconstitution of a hormone-sensitive adenylate cyclase system. J Bioi Chern 1984;259:9979. 7. Seamon K, Daly JW. Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein. J Bioi Chern 1981 ;256:9799. 8. Seamon KB, Daly JW. Forskolin: a unique diterpene activator of cyclic AMP-generating systems. J Cyclic Nucleotide Res 1981 ;7:20 1. 9. Daly JW. Forskolin, adenylate cyclase, and cell physiology: an overview. In: Greengard P, Robison GA, Paoletti R, Nicosia S, eds. Advances in cyclic nucleotide and protein phosphorylation research. New York: Raven Press, 1984, voll7:81. 10. Braun T, Frank H, Dods R, Sepsenwol S. Mn 2 • sensitive, soluble adenylate cyclase in rat testis: differentiation from other testicular nucleotide cyclases. Biochim Biophys Acta 1977;481:227. 11. Ross EM, Howlett AC, Ferguson KM, Gilman AG. Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. J Bioi Chern 1978;253:6401. 12. Neer EJ. Interaction of soluble brain adenylate cyclase with manganese. J Bioi Chern 1979;254:2089. 13. Somkuti SG, Hildebrandt JD, Herberg JT, Iyengar R. Divalent cation regulation of adenylyl cyclase. J Bioi Chern 1982;257:6387. 14. Bender JL, Neer EJ. Properties of the adenylate cyclase catalytic unit from caudate nucleus. J Bioi Chern 1983; 258:2432. 15. Hildebrandt JD, Sekura RD, Codina J, Iyengar R, Manclark CR, Brinbaumer L. Stimulation and inhibition of adenylyl cyclases mediated by distinct regulatory proteins. Nature 1983;302:706. 16. Kurokawa T, Dantura T, Ishibashi S. Mode of interaction between forskolin and manganese ion in activating catalytic unit of adenylate cyclase from rat brain. J Pharmacobiodyn 1984;7:665. 17. Salomon Y, Londos C, Rod bell M. A highly sensitive adenylate cyclase assay. Anal Biochem 1974;58:541. 18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248. 19. Sokal RP, Rohlf FJ. Biometry. 2d ed. New York: WH Freeman, 1981. 20. Huang R, Smith MF, Zahler WL. Inhibition of forskolinactivated adenylate cyclase by ethanol and other solvents. J Cyclic Nucleotide Res 1982;8:385. 21. Whetton AD, Needham L, Dodd NJF, Heyworth CM, Houslay MD. Forskolin and ethanol both perturb the structure of liver plasma membranes and activate adenylate cyclase activity. Biochem Pharmacol 1983;32: 1601. 22. Robberecht P, Waelbroeck M, Chatelain P, Camus J-C, Christophe ]. Inhibition of forskolin-stimulated cardiac adenylate cyclase activity by short-chain alcohols. Fed Eur Biochem Soc 1983; 154:205. 23. Smigel M, Katada T, Northup JK, Bokoch GM, Ui M, Gilman AG. Mechanisms of guanine nucleotide-mediated regulation of adenylate cyclase activity. In: Greengard P, Robison GA, Paoletti R, Nicosia S, eds. Advances in cyclic nucleotide and protein phosphorylation research. New York: Raven Press, 1984, vol 17:1. 24. Stiles GL, Caron MG, Lefkowitz RJ. 13-Adrenergic receptors: biochemical mechanisms of physiological regulation. Physiol Rev 1984;64:661. 25. Stengel D, Guenet L, Desmier M, Insel P, Hanoune]. Forskolin requires more than the catalytic unit to activate adenylate cyclase. Mol Cell Endocrinol 1982;28:681.
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26. Darfler FJ, Mahan LC, Koachman AM, Insel PA. Stimulation of forskolin of intact S49 lymphoma cells involves the nucleotide regulatory protein of adenylate cyclase. J Bioi Chern 1982;257:11901. 27. Seamon KB, Padgett W, Daly JW. Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc Natl Acad Sci USA 1981;78:3363. 28. Morris SA, BilezikianJP. Evidence that forskolin activates turkey erythrocyte adenylate cyclase through a noncatalytic site. Arch Biochem Biophys 1983;220:628. 29. Krall JF. A kinetic analysis of activation of smooth muscle adenylate cyclase by forskolin. Arch Biochem Biophys 1984;229:492. 30. Hoffman BB, Yim S, Tsai BS, Lefkowitz RJ. Preferential uncoupling by manganese of alpha adrenergic receptor mediated inhibition of adenylate cyclase in human platelets. Biochem Biophys Res Commun 1981;100:724.
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31. Limbird LE, Macmillan ST. Mn2+ uncoupling of the catecholamine-sensitive adenylate cyclase system of rat reticulocytes: parallel effects on cholera toxin-catalyzed ADPribosylation of the system. Biochim Biophys Acta 1981 ;677 :408. 32. Lindner E, Metzger H. The action of forskolin on muscle cells is modified by hormones, calcium ions and calcium antagonists. Arzneimittelforschung 1983;33: 1437. 33. Csapo AI, Eskola J, Tarro S. Gestational changes in the progesterone and prostaglandin F levels of the guinea pig. Prostaglandins 1981 ;21 :53. 34. Thorbert G, Aim P, Bjorklund AB, Owman C, Sjoberg N-0. Adrenergic innervation of the human uterus. AM J 0BSTET GYNECOL 1979;135:223.
activity, thus increasing 3'5'-cyclic adenosine monophosphate synthesis and promoting myometrial relaxation. In pregnant animals close to term (65 days), ~-adrenergic receptor density as well as basal and (-)isoproterenol-dependent (in the presence of guanosine triphosphate) adenylate cyclase activity is significantly higher than that in nonpregnant animals or those in early pregnancy. Since this system appears to be made up of at least three components (~·adrenergic receptor, guanosine triphosphatebinding protein, and a catalytic component), these observations on total adenylate cyclase activity may reflect alterations in one or more of these components. To answer the question whether the catalytic unit of this system can be directly assayed and whether its activity is influenced by pregnancy, we have performed in vitro experiments to measure the enzymatic activity of the catalytic component of the ~-adrenergic receptor-adenylate cyclase complex in guinea pig myometrial membranes. We have used two compounds that stimulate the catalytic component: forskolin and manganese chloride. Forskolin, regardless of the presence or absence of guanosine triphosphate, is the most potent stimulator of adenylate cyclase activity in myometrial membranes from nonpregnant and pregnant animals; manganese chloride is a less potent activator. The degree of adenylate cyclase stimulation by forskolin tends to be higher in uteri from pregnant (;>0.5 gestation) than from nonpregnant or postpartum animals. It was concluded: (1) that adenylate cyclase stimulation by forskolin does not depend on the presence of ~-adrenergic receptor agonists or guanosine triphosphate and (2) that with advancing gestation there might be a qualitative or quantitative change with regard to the interaction between forskolin and the presumed catalytic component of the ~-adrenergic receptor-adenylate cyclase complex. (AM J OBSTET GYNECOL 1986;155:1202·8.)
Key words: Forskolin, adenylate cyclase, myometrial membranes
Myometrial contractility is influenced by the relative concentrations of a- and 13-adrenergic receptors. 1 Stimulation of 13-adrenergic receptors in human or animal myometrium by 13-adrenergic receptor agonists results in increased adenylate cyclase activity and high levels of intracellular 3'5'-cyclic adenosine monophosphate (3'5'-cAMP). The latter is the second messenger that initiates a series of events leading to smooth muscle relaxation. 2· 3 This system is composed of at least three distinct protein components 4 : a catalytic subunit that converts the substrate magnesium-adenosine triphosphate to 3'5'-cAMP, a guanine-nucleotide-binding protein that Frm the Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Bowman Gray School of Medicine of Wake Forest University. Supported in part by Basil O'Connor Grant 5-362, The National Foundation March of Dimes, BGSM United Way Starter Grant, and National Institutes of Health IR23 HD20796-0J. Presented in part at the Thirty-second Annual Meeting of the Society for Gynecologic Investigation, Phoenix, Arizona, March 20-23, 1985. Reprint requests: Christos G. Hatjis, M.D., Department of Obstetrics and Gynecology, Bowman Gray School of Medicine, 300 South Hawthorne Road, Winston-Salem, NC 27103.
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mediates hormonal responsiveness and activation, and a hormone receptor. Each of these components is most likely made up of more than one polypeptide subunit. My colleagues and I have been studying the properties of the 13-adrenergic receptor-adenylate cyclase system in myometrial membranes from nonpregnant and pregnant guinea pigs. P have shown that the concentration of myometrial 13-adrenergic receptor is dramatically increased in late pregnancy. Moreover, basal and (-)isoproterenol-stimulated adenylate cyclase activity, which depends on the presence of guanosine triphosphate, was significantly increased in parallel with the 13-adrenergic receptor changes. However, these initial experiments studied only a physiologically important aspect of this system. Since the latter is a multicomponent complex, it is important to examine the properties of each of these components in both the nonpregnant and the pregnant state. Thus, to further elucidate the molecular relationship between the various components of the 13-adrenergic receptor-adenylate cyclase system in guinea pig myometrial membranes, we performed experiments to determine the activity of the catalytic component of the 13-adrenergic
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receptor-adenylate cyclase complex and answer the question whether the catalytic component is regulated independently of 13-adrenergic receptor activity and hormone-dependent adenylate cyclase activity in pregnant and nonpregnant animals. For this purpose, two compounds were used that, in most systems studied, have been shown to directly stimulate the catalytic component forskolin, a diterpene extacted from the roots of Coleus forskohlii, a potent hypotensive vasodilatory and antispasmolytic agent,6- 9 and manganese chloride. 10" 16 Material and methods
Materials. o:- 32 P-adenosine triphosphate (o:- 32 P-ATP) (20 to 40 Ci/mmol) and [3',8''H]3'5'-cAMP (15 Ci/ mmol) were obtained from New England Nuclear. Other drugs and materials were obtained from commercial sources and were of reagent grade. Forskolin was purchased from Calbiochem and dissolved in 95% ethanol (10 mmol/L). Animals. Virgin female guinea pigs (weighing 400 to 600 gm) and guinea pigs with time-dated pregnancies (gravida 1, para 0, weighing 600 to 1200 gm) were obtained from Perfection Breeders. Before being put to death, they were housed in our animal care facility for 2 to 20 days. Pregnant animals were studied at 0.39 (early), 0.69 (middle), and 0.9 to 1.0 (late) gestation (term 65 days). Some pregnant guinea pigs were allowed to deliver and were studied in the postpartum period (1 to 5 days). Animals were anesthetized with 40 to 55 mg/kg of pentobarbital intraperitoneally. At laparotomy, a hysterectomy was performed. The uterus was cleaned of attached adipose and vascular tissue and the cervical region was removed. If the animal was pregnant, hysterotomy was performed and the endometrial cavity cleaned of the products of conception. Tissue was snapfrozen in liquid nitrogen and stored at -70° C until used. Tissue preparation. Uterine tissue was thawed in icecold buffer A containing 0.005 mol/L ethylenediaminetetraacetic acid, 0.025 mol/L sucrose, 0.9% sodium chloride, and 0.02 mol/L Tris hydrochloride buffer, pH 7.5. The thawed uterus was then split along each horn and the endometrium was scraped. The uterus was placed in five to 10 volumes per gram of wet weight of buffer A, minced finely with scissors, and sonicated with a Brinkman Polytron (setting No. 7, twice for 30 seconds). The homogenate was then filtered through eight layers of cheesecloth. An aliquot of the homogenate to be used for adenylate cyclase experiments was centrifuged at 80,000 X g for 15 minutes (4° C) and the pellet resuspended in 2 to 4 ml of ice-cold buffer B (0.9% sodium chloride and 0.02 mol/L Tris hydrochloride, pH 7.5). It was
Forskolin: Diterpene activator of adenylate cyclase
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preincubated for 10 minutes at 4o C in the presence of 0.5 mmol/L dithiothreitol, 0.17 mmol/L ethyleneglycol-bis(l3-aminoethyl ether)-N ,N'-tetraacetic acid, l mmol/L hydrochloric acid, 0.2% bovine serum albumin, and 5 mmoi/L magnesium chloride, pH 7.45. The suspension was then recentrifuged as previously outlined. The resulting pellets were resuspended in icecold buffer B (100 to 150 volumes per gram of wet weight) and used in the assay. Adenylate cyclase assay. Adeny1ate cyclase activity was determined by measuring the conversion of o:- 32 PATP to [' 2 P]-cAMP and isolating the product by a modification of a method developed by Salomon et al. 17 The reaction was carried out in triplicate at 30° C for 15 minutes in a final volume of 0.2 ml containing 0.1 ml of membrane fraction (8 to 60 J.Lg of protein per tube) and 50 mmol/L Tris hydrochloride, pH 7 .5, 0.45% sodium chloride, 0.5 mol/L adenosine triphosphate, 5 mmol/L magnesium chloride, 0.17 mmol/L ethyleneglycol-bis(l3-aminoethyl ether)-N ,N'-tetraacetic acid, 0.05 mmol/L cAMP, 0.5 mmol/L dithiothreitol, 0.75 mmol/L 3-isobutylmethylxanthine, 0.1 mg/ml of creatine kinase, 10 mmol/L creatine phosphate, and 1 to 2 X 106 cpm of o:- 32 P-ATP. Appropriate drugs were added as required. The reaction was initiated by addition of the membrane fraction to the incubation tubes. It was linear with respect to time (up to 20 minutes) and protein concentration. The reaction was terminated by the addition of 0.25 ml of a solution containing 50 mmol/L Tris hydrochloride (pH 7.5), 5 mmol/L adenosine triphosphate, 1 mmol/L 3'5'-cAMP, and 5% sodium dodecyl sulfate. 'H-cAMP (10,000 to 25,000 cpm per tube) was added to all tubes to serve as an internal standard and monitor the efficiency of recovery. The tubes were then placed in boiling water for 10 minutes and allowed to cool down to room temperature. [32 P]-cAMP was isolated with sequential Dowex (AG 50W-X4, 200 to 400 mesh, Bio-Rad) and neutral alumina columns essentially as described by Salomon et al. and counted in a scintillation counter (Packard). Recovery of 3'5'-cAMP was determined by the percent recovery of 3 H-cAMP external standard in each sam pie. Results have been corrected for percent recovery (routinely greater than 40% to 60%). Enzymatic activity was expressed as picomoles of 3'5' -cAMP produced per milligram of protein per minute per uterus. The normalization per uterus was necessary in order to account for the differences in the amount of tissue used per experiment (that is, for nonpregnant animals, the entire uterus had to be used while for pregnant animals only an aliquot was required). Protein assay. Protein concentrations were determined according to the method of Bradford 18 with bovine serum albumin used as a standard.
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li:.lJ Post-Partum, 1-3 Days Fig. I. Basal adenylate cyclase activity as a function of reproductive state. Late pregnant animals (0. 9 to 1.0 gestation) show increased basal activity either in the presence or in the absence of guanosine triphosphate (GTP)or ethanol (ETOH) (p < 0.001). The percent stimulation by guanosine triphosphate over basal activity was higher in animais in late pregnancy than in the other groups (p < 0.02). Numbers in bars refer to the number of animals studied in each group.
Statistical analysis. Results are expressed as mean ± SEM. We utilized analysis of variance to test the null hypothesis that there is no difference in basal and drugstimulated enzymatic activity with advancing gestation. Since absolute values of adenylate cyclase activity varied considerably, depending on the reproductive state of the animal, a log 10 transformation was carried out to satisfy the prerequisite of homogeneity of variance. 19 A p value of <0.05 was considered significant.
Results First, we determined basal adenylate cyclase activity under three experimental conditions: no addition and addition of ethanol and guanosine triphosphate (Fig. 1). Basal, basal plus guanosine triphosphate, and basal plus ethanol adenylate cyclase activities increased significantly with advancing gestation. Moreover, the percent increase in adenylate cyclase activity in the presence of guanosine triphosphate was higher in myometrial membranes from guinea pigs in late pregnancy than in the other groups. In contrast, ethanol increased basal adenylate cyclase levels to the same degree in all animals. Since forskolin is dissolved in ethanol, it was crucial to include basal activity in the presence of an equivalent dose of ethanol. The observation that eth-
anol stimulates basal adenylate cyclase actlVlty is in agreement with findings reported in other systems. Although the mode of action is not established, it has been suggested that perturbation in the structure-fluidity of plasma membranes might be involved in the process? 0- 22 Second, we examined adenylate cyclase activity in the presence offorskolin and manganese chloride (Fig. 2). Of the agents we have tested to date, 5 forskolin has been the most potent stimulator of adenylate cyclase activity (range tenfold to sixtyfold over basal level) in all groups. Maximal stimulation occurred at a forskolin concentration of 100 ~J.mol/L. The addition of guanosine triphosphate did not result in any significant differences in the absolute values of adenylate cyclase stimulation by forskolin (Fig. 2). There was a gradual increase in forskolin-stimulated adenylate cyclase activity with advancing gestation. Uterine membranes from animals in late pregnancy demonstrated the highest absolute levels of enzymatic activity in the presence of forskolin regardless of the presence or absence of guanosine triphosphate (Fig. 2). Similarly, when adenylate cyclase stimulation in the presence of manganese chloride was studied, animals in late pregnancy showed higher absolute levels when compared to those of the
Forskolin: Diterpene activator of adenylate cyclase
Volume 155 Number 6
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other groups. Addition of guanosine triphosphate did not influence manganese chloride-dependent stimulation (data not shown). Fig. 3 shows the degree of stimulation of adenylate cyclase activity over basal enzymatic activity. With regard to the addition of forskolin compared with basal levels (panel A) the only significant difference was between animals in late pregnancy and nonpregnant animals (p < 0.05). When forskolin was compared with the basal level plus ethanol (panel B), there were no significant differences between individual groups although there was a trend for pregnant animals to show higher stimulation. When forskolin plus guanosine triphosphate was compared with the basal level plus guanosine triphosphate (panel C), the highest degree of stimulation was seen in the early pregnant group (0.39 gestation, p < 0.05). finally the degree of stimulation by manganese chloride was equivalent in all groups.
Comment The regulation of cellular responses by cyclic nucleotides continues to be an area of intense investigation. 3'5'-cAMP, as a second messenger, mediates hormonal effects ranging from intermediary metabolism to smooth muscle relaxation. Stimulation of uterine
smooth muscle by 13-adrenergic receptor agonists results in an increase in 3'5'-cAMP production and secondary smooth muscle relaxation. 1• 3 It has been shown that 13-adrenergic receptors and the catalytic components of adenylate cyclase are coupled with a stimulatory guanosine triphosphate-binding protein (G, protein).'· 23 • 24 Binding of 13-adrenergic receptor agonists but not antagonists to the 13-adrenergic receptor results in a high-affinity state of the 13-adrenergic receptor, which in turn interacts with the G, protein and activates it. Guanosine triphosphate can then bind to the 13-adrenergic receptor-G, protein complex, dissociate the agonist from the 13-adrenergic receptor, and stimulate the catalytic component of the complex. Guanosine triphosphate is subsequently hydrolyzed and dissociates from the guanosine triphosphate-binding protein. P have previously shown that in guinea pig uteri advancing gestation results in a remarkable increase in the density of 13 2-adrenergic receptors as well as basal and (-)isoproterenol-stimulated adenylate cyclase activity. Stimulation of the enzyme by (-)isoproterenol depended on the presence of guanosine triphosphate. Since this system is composed of at least three components, the question was whether properties of one or more of these are altered by the pregnant state. Since
1206 Hatjis
December 1986 Am J Obstet Gynecol
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•Pregnant, 0.39 Gestation ElPregnant, 0.9-1.0 Gestation Fig. 3. Degree of adenylate cyclase stimulation over basal level. Data shown in Figs. I and 2 have been used for the calculations. Group and number of animals per group are the same as described in legend to Fig. I. Panel A: Forskolin (basal). Panel B: Forskolin/basal plus ethanol (6%). Panel C: Forskolin plus guanosine triphosphate (100 fLmol/L)/basal plus guanosine triphosphate (100 fLmol/L).
the density and perhaps the affinity of the 13-adrenergic receptor have been shown to change with pregnancy,' I next sought to examine the catalytic component of this system as well as the interaction of (-)isoproterenol with the 13-adrenergic receptor as a function of guanosine triphosphate. In this article I present data regarding the catalytic component. To characterize its activity, two agents were used that have previously been shown to directly stimulate the catalytic components of the 13-adrenergic receptoradenylate cyclase complex, forskolin ~ and manganese chloride. ~ In general, forskolin activation of adenylate cyclase does not appear to depend on the presence of guanosine triphosphate or 13-adrenergic receptor. In cell-free reconstitution experiments in lipid micelles, the presence of the catalytic component was the only prerequisite to show forskolin stimulation. 6 Addition of guanosine triphosphate-binding protein and guanosine triphosphate did not alter the response. Although the latter observation confirms the unique properties of forskolin, it does not rule out the presence of other factor(s) associated with the catalytic component that may modify the interaction between the latter and forskolin. Moreover, in some systems, hormonal and guanosine triphosphate-dependent adenylate cyclase stimulation can be augmented in the presence of submaximal concentrations of forskolin. Thus it has been suggested that there might be an interaction between guanosine triphosphate-binding protein and forskolin-induced stimulation of the catalytic component. 25" 28 To date the precise nature of this interaction is not 6 9
10 16
known and further experiments are needed to elucidate this point. We have found that, in myometrial membranes of both nonpregnant and pregnant guinea pigs, forskolin is the most potent stimulator of adenylate cyclase activity. Its action is independent of guanosine triphosphate suggesting that under these conditions forskolin bypasses the G, protein. This observation is in contrast to previous experiments in nonpregnant rat uteri! 9 which have suggested that guanosine triphosphate is involved in adenylate cyclase stimulation by forskolin. Differences in species and experimental design may be partly responsible for these conflicting observations. More important, since the percent increase in adenylate cyclase activity by forskolin compared with the basal level in uteri from animals in late pregnancy might be higher than in the other groups tested, it is possible that the interaction between forskolin and the catalytic component is qualitatively and/or quantitatively different in late pregnancy compared with the nonpregnant state or early and middle pregnancy. Alternatively, similar changes might apply to other component(s) of this complex that is (are) closely linked to the catalytic component and have not been separated to date. However, these "derived" observations should be interpreted with caution since they depend on the choice of basal activity. For example, when the basal plus guanosine triphosphate experiment was used, it was the group in early pregnancy that showed a higher degree of stimulation. ·
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Manganese chloride directly stimulates the catalytic component of the complex. 10• 16 The precise mode of action is not known and interaction with an inhibitory guanosine triphosphate-binding protein cannot be ruled out. 30· 3 ' In our system, manganese chloride significantly increased adenylate cyclase activity but to a lesser extent than that seen with forskolin. Although the percent increase of manganese chloride-dependent stimulation is higher in animals in late pregnancy, the difference did not reach statistical significance. This is the first instance where a direct assay of the catalytic activity of the 13-adrenergic receptor-adenylate cyclase complex has been reported in uterine membranes from pregnant and nonpregnant animals. Our results demonstrate that the presence of the 13-adrenergic receptor is not an absolute requirement for stimulation of adenylate cyclase to occur. Moreover, it appears that the catalytic activity of the 13-adrenergic receptor-adenylate cyclase complex may be regulated independently of the other components. If that is indeed the case, then our approach to study of the properties of each component of the system at the molecular level is necessary if we are to gain a better understanding of the factors controlling adenylate cyclase activity and, secondarily, myometrial contractility. Furthermore, the fact that forskolin is a stronger stimulator of adenylate cyclase activity than (- )isoproterenol suggests that the former may be a more potent agent with regard to uterine muscle relaxation. 32 Further work should provide an approach to selective manipulation of this adenylate cyclase system in intact cells and intact organisms that will significantly improve our understanding of the in vivo control of the myometrial contractility. Since the guinea pig model appears to be very similar to that of the human,33 • 34 it provides a very valuable tool for studying the myometrial 13-adrenergic receptor-adenylate cyclase system in vivo and in vitro. With the recognition of the potential pitfalls associated with extending in vitro observations to in vivo systems, these and similar studies may provide a valuable insight to clinically significant areas such as pregnancy maintenance, onset of term or preterm labor, and others.
1. 2. 3. 4.
REFERENCES Marshall JM. Modulation of smooth muscle activity by catecholamines. Fed Proc 1977;36:2450. Roberts JM. Receptors and transfer of myometrial contractility and cervical maturation. Semin Perinatol 1981; 5:266. Huszar G. Biology and biochemistry of myometrial contractility and cervical maturation. Semin Perina to! 1981; 5:216. Birnbaumer L, Codinaj, Mattera R, et al. Regulation of hormone receptors and adenylyl cyclases by guanine nucleotide binding N proteins. Recent Prog Horm Res 1986;41 :41.
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5. Hatjis CG. 13-Adrenoreceptor and adeny~ate cy~lase properties in pregnant and nonpregnant gumea p1g myometrium. AMj 0BSTET GYNECOL 1985;151:943. 6. Cerione RA, Sibley DR, CodinaJ, et al. Reconstitution of a hormone-sensitive adenylate cyclase system. J Bioi Chern 1984;259:9979. 7. Seamon K, Daly JW. Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein. J Bioi Chern 1981 ;256:9799. 8. Seamon KB, Daly JW. Forskolin: a unique diterpene activator of cyclic AMP-generating systems. J Cyclic Nucleotide Res 1981 ;7:20 1. 9. Daly JW. Forskolin, adenylate cyclase, and cell physiology: an overview. In: Greengard P, Robison GA, Paoletti R, Nicosia S, eds. Advances in cyclic nucleotide and protein phosphorylation research. New York: Raven Press, 1984, voll7:81. 10. Braun T, Frank H, Dods R, Sepsenwol S. Mn 2 • sensitive, soluble adenylate cyclase in rat testis: differentiation from other testicular nucleotide cyclases. Biochim Biophys Acta 1977;481:227. 11. Ross EM, Howlett AC, Ferguson KM, Gilman AG. Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. J Bioi Chern 1978;253:6401. 12. Neer EJ. Interaction of soluble brain adenylate cyclase with manganese. J Bioi Chern 1979;254:2089. 13. Somkuti SG, Hildebrandt JD, Herberg JT, Iyengar R. Divalent cation regulation of adenylyl cyclase. J Bioi Chern 1982;257:6387. 14. Bender JL, Neer EJ. Properties of the adenylate cyclase catalytic unit from caudate nucleus. J Bioi Chern 1983; 258:2432. 15. Hildebrandt JD, Sekura RD, Codina J, Iyengar R, Manclark CR, Brinbaumer L. Stimulation and inhibition of adenylyl cyclases mediated by distinct regulatory proteins. Nature 1983;302:706. 16. Kurokawa T, Dantura T, Ishibashi S. Mode of interaction between forskolin and manganese ion in activating catalytic unit of adenylate cyclase from rat brain. J Pharmacobiodyn 1984;7:665. 17. Salomon Y, Londos C, Rod bell M. A highly sensitive adenylate cyclase assay. Anal Biochem 1974;58:541. 18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248. 19. Sokal RP, Rohlf FJ. Biometry. 2d ed. New York: WH Freeman, 1981. 20. Huang R, Smith MF, Zahler WL. Inhibition of forskolinactivated adenylate cyclase by ethanol and other solvents. J Cyclic Nucleotide Res 1982;8:385. 21. Whetton AD, Needham L, Dodd NJF, Heyworth CM, Houslay MD. Forskolin and ethanol both perturb the structure of liver plasma membranes and activate adenylate cyclase activity. Biochem Pharmacol 1983;32: 1601. 22. Robberecht P, Waelbroeck M, Chatelain P, Camus J-C, Christophe ]. Inhibition of forskolin-stimulated cardiac adenylate cyclase activity by short-chain alcohols. Fed Eur Biochem Soc 1983; 154:205. 23. Smigel M, Katada T, Northup JK, Bokoch GM, Ui M, Gilman AG. Mechanisms of guanine nucleotide-mediated regulation of adenylate cyclase activity. In: Greengard P, Robison GA, Paoletti R, Nicosia S, eds. Advances in cyclic nucleotide and protein phosphorylation research. New York: Raven Press, 1984, vol 17:1. 24. Stiles GL, Caron MG, Lefkowitz RJ. 13-Adrenergic receptors: biochemical mechanisms of physiological regulation. Physiol Rev 1984;64:661. 25. Stengel D, Guenet L, Desmier M, Insel P, Hanoune]. Forskolin requires more than the catalytic unit to activate adenylate cyclase. Mol Cell Endocrinol 1982;28:681.
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26. Darfler FJ, Mahan LC, Koachman AM, Insel PA. Stimulation of forskolin of intact S49 lymphoma cells involves the nucleotide regulatory protein of adenylate cyclase. J Bioi Chern 1982;257:11901. 27. Seamon KB, Padgett W, Daly JW. Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc Natl Acad Sci USA 1981;78:3363. 28. Morris SA, BilezikianJP. Evidence that forskolin activates turkey erythrocyte adenylate cyclase through a noncatalytic site. Arch Biochem Biophys 1983;220:628. 29. Krall JF. A kinetic analysis of activation of smooth muscle adenylate cyclase by forskolin. Arch Biochem Biophys 1984;229:492. 30. Hoffman BB, Yim S, Tsai BS, Lefkowitz RJ. Preferential uncoupling by manganese of alpha adrenergic receptor mediated inhibition of adenylate cyclase in human platelets. Biochem Biophys Res Commun 1981;100:724.
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31. Limbird LE, Macmillan ST. Mn2+ uncoupling of the catecholamine-sensitive adenylate cyclase system of rat reticulocytes: parallel effects on cholera toxin-catalyzed ADPribosylation of the system. Biochim Biophys Acta 1981 ;677 :408. 32. Lindner E, Metzger H. The action of forskolin on muscle cells is modified by hormones, calcium ions and calcium antagonists. Arzneimittelforschung 1983;33: 1437. 33. Csapo AI, Eskola J, Tarro S. Gestational changes in the progesterone and prostaglandin F levels of the guinea pig. Prostaglandins 1981 ;21 :53. 34. Thorbert G, Aim P, Bjorklund AB, Owman C, Sjoberg N-0. Adrenergic innervation of the human uterus. AM J 0BSTET GYNECOL 1979;135:223.