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Human myometrial adrenergic receptors: Identification of the beta-adrenergic receptor by [3H] dihydroalprenolol binding
Human myometrial adrenergic receptors: Identification of the beta-adrenergic receptor by 3 [ H] dihydroalprenolol binding DAVID N. HAYASHIDA, B.S. RAYMOND LEUNG, M.D.
ALAN GOLDFIEN, M.D . .JAMES M. ROBERTS, M.D.* San Francisco, Caiifomia The radioactive beta-adrenergic antagonist [ 3H] dihydroalprenolol (DHA) binds to particulate preparations of human myometrium in a manner compatible with binding to the beta-adrenergic
receptor. The binding of DHA is rapid (attaining equilibrium in 12 minutes), readily reversible (ha!f time= 16 minutes), high affinity (Ko = 0.50 nM), low capacity (Bmax = 70 fmoles/mg of protein), and stereoselective ([-]-propranolol is 100 times as potent as [ +] -propranolol in inhibiting DHA binding). Adrenergic agonists competed for DHA binding sites in a manner compatible with beta-adrenergic interactions and mirrored {3 2 pharmacologic potencies: isoproterenol > epinephrine>> norepinephrine. Studies in which zinteroi, a,B 2-adrenergic agonist, competed for DHA binding sites in human myometrial particulate indicated that at least 87% of the beta-adrenergic receptors present are fkadrenergic receptors. Binding of DHA to human myometrial beta-adrenergic receptors provides a tool which may be used in the examination of gonadal hormonal modification of adrenergic response in human uterus as well as in the analysis of beta-adrenergic agents as potentially useful tocolytic agents. (AM.
J.
OBSTET. GYNECOL. 142:389, 1982.)
BoTH ALPHA- and beta-adrenergic receptors can be demonstrated pharmacologically in the human uterus. Alpha-adrenergic stimulation increases frequency and intensity of uterine contractions, 1 while beta adrenergic stimulation decreases either spontaneous or induced uterine contractility. 2 At present, beta-adrenergic agonists are the most useful clinical agents to prevent or reduce uterine activity. 3 From the Departments of Obstetrics, Gynecology, and Reproductive Sciences, and Medicine, and the Cardiovascular Research Institute, Universitv of California, San Francisco. ' " Supported by a Basal O'Conner Research Starter Grant from the March of Dimes Birth Defect Foundation. Presented at the Twenty-sixth Annual Meeting of the Society for Gynecologic Investigation, San Diegu. California, March 21-24, 1979. Reprint requests: James M. Roberts, M.D., Department of Obstetrics, Gynecology, and Reproductive Sciences, Room 1489 Moffit Hospital, University of California, San Francisco, San Francisco, California 94143. *Recipient of National Institutes of Health Research Career Development Award HD-00267. 0002-9378/82/040389+05$00.50/0 © 1982 The C. V. Mosby Co.
The adrenergic response of the human uterus is modified by the hormonal environment. During the follicular phase of the menstrual cycle, in which serum estrogen concentrations are elevated, the response of human uterus to epinephrine is augmented contractility-an effect which can be prevented by alphaadrenergic antagonists. However, if serum progesterone concentrations are increased, as during the luteal phase of the menstrual cycle or during late pregnancy, inhibition of contractile response by epinephrine is puleulialed; Lhis is a bela-adrenergic response blocked by beta-adrenergic antagonists_4- 7 We have previously demonstrated in rabbit myometrium, in which a similar hormonal modification of adrenergic response is present, that alteration of adrenergic predominance is associated with changes in adrenergic receptor concentration as measured by radioligand binding." To provide a tool to directly examine beta-adrenergic receptors in human uterus, we examined the binding of[:lH]dihydroalprenolol (DHA) to myometrial particulates and report that this agent
389
390
February 15, 191'1;1 Am. J. Obstet. Gynecol.
Hayashida et al.
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Fig. LA. Time course and reversal of DHA binding. Percentage of specific binding(X/Xeq) is plotted as a function of time. X specific binding at timet andXeq specific binding at equilibrium. A total of 2.5 nM DHA was incubated at 3{)• C with myometrialparti<;ulate (final protein concentration I mg/ml) with or without 10 ~M (- )-alprenolol for the time pe~s indicated (o). Specific binding was determined as discussed in Methods. At t I 0 minutes, (- )-alpren!>fel was added (final concentration"" 10 ILM) to an aliquot of particulate not containing (- )-alprenolol and binding was terminated at the times indicated (t,). B, First-order kinetic plot of the reversal of DHA binding. From the mathematical model of a unimolecular dissociation: In X/Xeq = - k011t; the slope of the plot defines korr. the reverse rate constant, which was 0.043 min- 1 • C, Pseudo-first-order kinetic plot of DHA binding. From the mathematical model of a pseudofirst order association 11 : ln [Xeq/(Xeq- X)]= k01,(t), where kub = [DHA]kon + k0 ff; the slope of the plot defines k0 "' which was 0.24 min~ 1 • [DHA) is the concentration of free radioligand (2.5 nM) which was sufficiently large to guarantee it did not change over the course of the experiment. k01r From 1, B was 0.043 min- 1 : thus k00 = 0.079 nM~'min- 1 • Kd = kofflkon = 0.54 nM.
binds with interactions consistent with binding to the beta-adrenergic receptor. Material and methods Material. (3 H]Dihydroalprenolol with specific activity of 15 to 20 Ci/mmole was obtained from New England Nuclear. (-)-Propranolol was a gift from Ayerst Laboratories. Other drugs and chemicals were from commercial sources. Particulate preparation. Human myometrium was obtained from uteri removed for obstetric indications or from strips of myometrium removed at the time of cesarean section. Myometrium was sharply dissected free of endometrium and serosa. The tissue was minced, suspended in cold 5 mM Tris HCI, pH 7.4, 0.25M sucrose, in a 4-to-l (volume-to-weight) ratio, and then homogenized in a Waring Blendor. The homogenate was filtered through four layers of cheesecloth and centrifuged at 600 and I 0,000 X g for 15 minutes each. The pellets were discarded after each centrifugation and the resulting supernate was centrifuged at 30,000 x g for 20 minutes. The pellet was suspended in cold 50 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid (HEPES), pH 7.5, 4 mM MgS0 4 , rapidly frozen, and stored at -70° C until used for DHA binding studies. Binding of DHA was similar
in fresh particulates and in those stored for up to ~l months. Particulate protein concentration was determined by the method of Bradford, 9 with bovine serum albumin used as the standard. Binding assay. DHA was diluted in 50 mM HEPES, pH 7.5, 4 mM Mgso•. I mM ascorbic acid, and 20\)[ ethanol. Ethanol was included to standardize binding with subsequent alpha adrenergic receptor assays with [:JH)dihydroergocryptine. Ethanol in a final concentration of 2% had no effect on DHA binding. Two tenths milliliter of particulate (protein concentration "' 1.25 mg/ml) was added to 0.025 ml of the radioligand solution and 0.025 ml of l mM HCI or adrenergic agents in 1 mM HCl. Samples were incubated at 30° C for 15 minutes in equilibrium studies; the incubation was stopped by adding 5 ml of 5 mM HE PES pH 7 .5, at 4° C and immediately filtering through Whatman GF/C filters to separate bound from free radioligand. Filters were washed with an additional 15 ml of cold 5 mM HEPES under low vacuum (1 ml/sec). The filters were dried under high vacuum and counted in a liquid scintillation counter at 54% efficiency. Data aaelysis. Specific binding was detecmined by an iterative analysis of Scatchard plots whkh detennined a best fit for data at different estimates of nonsaturable binding. Specific binding was approximately 90% at
Volume H2 Number 4
Human myometrial adrenergic receptors
.14~
100
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•
_lL
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Bound (nM)
trations of D HA fronl 0.125 Htvlto 10 nrvl
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391
incubated with
particulate (final protein concentration = l mg/ml) for 15 minutes at 30° C. B =specific DHA bound; F =free DHA. Specific binding was determined as discussed in Methods. The intercept of this plot is the receptor concentration (Bmax). The slope, determined by linear regression analysis, is the negative reciprocal of the dissociation constant (K 0 ). Bmax = 70 fmob,/mg of protein; K0 = 0.50 nM.lnset, Direct plot of bound versus free DHA. D HA concentrations equal to the dissociation constant (0.50 nM). Similar dissociation constants and binding site concentrations were obtained if specific binding was defmed as binding in the absence minus binding in the presence of i 0 ,uM (- )-alprenolol. This technique was used to dehne specific binding in competition experiments in which adrenergic compounds competed for D HA binding sites. The inhibition constant (K 1) was determined for each competing adrenergic agent in competition experiments by the relationship described by Cheng and Prusoffl": K1 = I.-, 0 /[1 + (L!Kn)]; where 130 = the concentration of competing adrenergic agent which inhibits binding of DHA by 50%, L = the concentration of DHA used in the assay and Kn = the dissociation constant of D HA for its binding sites. In determining the percentages of {3 1- and {3 2 -adrenergic receptors present in human myometrium, we used an iterative, nonlinear, curve-fitting program which determined the number of sites and their affinities and the relative concentration of each site which best described the experimental data.
Results Kinetics of binding. D HA hound rapidly and reversibly to human myometrial particulate (Fig. I. A). At :100 C, equilibrium was attained in 12 minutes using 2.5
8
7
6
5
4
- log [ propranolol]
Fig. 3. Stereoselective competition for DHA binding sites. DHA, 2 nM, was incubated with myometrial particulate (final protein concentration = l mg/m]) and the indicated concentrations of the beta antagonist propranolol. Binding is expressed as percentage of total binding in the absence of proprano!o!. The (-)-stereoisomer of propranolol '.-vas 100 times
as potent as the (+)-stereoisomer. K1( - )-propranolol = 0.6 nM, K1 (+)-propranolol= 60 nM. nM DHA. With the addition of excess nonradioactive (- )-alprenolol, binding was rapidly reversed; D HA dissociated from the binding sites with a half time (ty2 ) of 16 minutes. The forward rate constant (kon) and the reverse rate constant (korr) were calculated to be 0.079 nM- 1min- 1 and 0.043 min- 1 , respectively (Fig 1, B and C). The dissociation constant (Kn), caicuiated from the ratio of korr to k., 11 , was 0.54 nM. Equilibrium studies. DHA bound to human myometrium saturably and with high affinity (Fig. 2). Binding \Vas to a single class of receptors with a binding site concentration of 70 fmoles/mg of protein. The Kn determined in equilibrium experiments was 0.50 nM, which is in excellent agreement with the kinetically determined Kn. DHA binding sites were competed for stereoselectively (Fig. 3). The active (-)-stereoisomer of the beta antagonist propranolol was I 00 times as potent as the inactive (+)-stereoisomer. Adrenergic agonists competed in a manner compatible with beta-adrenergic interactions and mirrored beta 2 pharmacologic potencies (Fig. 4): isoproteronol (K 1 = 0.12 ,uM) >epinephrine (K 1 = 1.1 ,uM) >>norepinephrine (K 1 = 50 ,uM). To determine the proportion of {3 2 -adrenergic receptors in the myometrial particulate, we examined the competition of zinterol, a highly specific {3 2 -adrenergic agonist of high affinity, for DHA binding sites (Fig. 5). Zinterol competes for {3 1-adrenergic receptors with approximately 70 times the potency that it competes for
Februarv 1S. Hli\C/
392 Hayashida et al. Am
-----e-e
100
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80 Percent Specific Binding
't:;.
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!-epinephrine !-norepinephrine 1- isoproterenol
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%
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K1L 2,100 nM
% RL
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5
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Percent specific binding 60
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40f-
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- log [ Agonist J 0
Fig. 4. Rank order of potency of agonists competing lor DHA binding sites. DHA. 2.4 nM. was incubated with myometrial particulate (final protein concentration = I mg/ml) and I 0 ~-tM (- )-alprenolol or increasing concentrations of agonist. Binding is expressed as percentage of specific binding in the absence of agonist. K1 isoproteronol = 0.12 ~-tM: K1 epinephrine= 1.1 ~-tM; K1 norepinephrine= 5.0 ~-tM.
,8 1-adrenergic receptors. 12 Thus, the competition of zinterol with DHA (which binds to ,8 1- and ,82-adrenergic receptors with equal affinity) can be analyzed.by computer-curve fitting to define the affinity and proportion. of high-atfinity (,8 2 ) and low-affinity (,8 1) sitesY When beta-adrenergic agonists such as zinterol bind to membrane preparations in the absence of guanylnucleotides, a very high-affinity ,8 2-binding component is present. 14 To eliminate the contribution of this very high-affinity component and therefore allow identification of.B 1- and .B~-adrenergic receptors by their differing affinities for zinterol, we included guanosine 5'-triphosphate (GTP) at a concentration of 0 .I m M in our assay. This concentration of GTP has bee11 demonstrated in several systems to prevent this very high-affinity bindingY · Analysis of data demonstrated that the findings were most consistent with zinterol competing with high affinity (K 1 13.4 nM, which is compatible with zinterol's potency in other ,8 2-adrenergic systems 12 ) for H7% of the DHA binding sites. Zinterol competed with a lower affinity for the remaining 13% of the DHA binding sites.
Comment DHA binds to particulate preparations of human myometrium with interactions consistent with binding to the beta-adrenergic receptor. Binding is high affinity, low capacity, saturable, and stereoselective. The kinetically determined dissociation constant is in excellent agreement with that obtained in equilibrium experiments. Even at 30° C, the reversal of DHA bind-
'---..1.-.....-...l.__
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9
8
~
_l__...L_ )::....,_.
7
6
5
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-log [Zlnteroi] Fig. 5. Competition of zinterul for DHA binding sites. DHA. 0.6 nM, was incubated with myometrial particulate (final protein concentration = 0.9 mg/ml) and 10 ~-tM (- )-alprennlol01 the indicated concentrations of zinterol. Binding is expressed as percentage of specific binding in the absern:c of zinterol. The curve is the curve of best fit as defi0€d by the iterative, nonlinear, curve-fitting program and was determined by the following parameters: KIH = K1 of zinterol for the ,8 2-adrenergic receptor (13.4nM); K11 • K1 of zinterol for the {3 1-adrellergic receptor (2,1 00 nM): q RH percemage nf beta-adrenergic receptors which are ,82 -adrenergic receptors (87% ); %RL = percentage of beta-adrenergic receptors which are {3 1adrenergic receptors (13%).
ing has a ty2 of 16 minutes; thus separation of bound from free radioligand by filtration at 4° C is an appropriate technique for this study . Competition studies indicate a rank order of potencies consistent with ,8 2-adrenergic potencies. Epinephrine is a mllch more potent competitor than norepinephrine. The great majority ot beta-adrenergic receptors present are ,8 2-adrenergic receptors. This is compatible with indirect pharmacologic determinations which indicate that inhibition of myometrial o>ntractility is determined by ,8 2-adrenergic receptors. 1:; Quantitation ot beta-adrenergic receptor subtypes in myometrial particulate indicates that at least 87% of the beta-adrenergic receptors present are ,8 2-adrenergic receptors. Since the particulate fraction we examined ('.Ontained .several cell types, that is, myometrium, blood veS~~els, andconnective tissue, it is possible that all of the beta-adrener~ gic receptors on myometrial cells are ,8 2-adrenergk re~ ceptors. The possibility that both receptor subtypes are present on myometrial cells, or even on an indi\'idual cell, cannot be excluded by these studies. The ability to directly measure beta-adrenergic rc-
Volume 142 Number 4
Human myometrial adrenergic receptors
393
ceptors in human myometrium provides a tool which
interaction of pharmacologic agents with myometrial
may be used in the examination of gonadal hormonal modification of adrenergic response in the human
beta-adrenergic receptors should aid in the analysis of
bela-adrenergic agents as potentially useful tocolytic
uterus. Furthermore, the ability to directly examine the
agents.
REFERENCES I. Althabe, 0., Jr., Schwarcz, R. L., Jr., Sala, N. L., and Fisch, L.: Effect of phentolamine methanesulfonate upon
2.
3.
4.
5. 6. 7. 8.
uterine contractility induced by !-norepinephrine pregnancy, AM. J. 0BSTET. GYNECOL. 101:1083, 1968. Mahon, W. A., Reid, D. W.J., and Day, R. A.: The in vivo effects of beta-adrenergic stimulation and blockade on the human uterus at term, J. Pharmacal. Exp. Ther. 156:178, 1967. Merkatz, I. R., Peter,J. B., and Barden, T. P.: Ritodrine hydrochloride: A betamimetic agent for use in preterm labor. II. Evidence of efficacy, Obstet. Gynecol. 56:7, 1980. Garret, W. J .: The effects of adrenalin and noradrenalin on the intact non-pregnant human uterus, J. Obstet. Gynaecol. Br. Emp. 62:876, 1955. Wansbrough, H., Nakanishi, H., and Wood, C.: Effect of epinephrine on human uterine activity in vitro and in vivo, Obstet. Gynecol. 30:779, 1967. Garret, W. J .: The effects of adrenalin and noradrenalin on the intact human uterus in late pregnancy and labour, J. Obstet. Gynaecol. Br. Emp. 61:586, 1954. Barden, R. P., and Stander, R. W.: Effects of adrenergic blocking agents and catet:hulanlines in hutnan pregnancy, AM.j. 0BSTET. GYNECOL. 102:226, 1968. Roberts,]. M., Insel, P. A., Goldfien, R. D., and Goldfien, A.: a-Adrenoreceptors but not !3-adrenoreceptors increase in rabbit uterus with oestrogen, Nature 270:624, 1977.
9. Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anai. Biochem. 72:248, 1976. 10. Cheng, Y., and Prusoff, W. H.: Relationship between the inhibition constant ( K 1) and the concentration of inhibitor which causes 50 percent inhibition (1 50 ) of an enzymatic reaction, Biochem. Pharmacal. 22:3099, 1973. 11. Williams, L. T., Mullikan, D., and Lefkowitz, R. J.: Identification of a-adrenergic receptors in uterine smooth muscle membranes by [ 3 H]dihydroergocryptine binding, J. Bioi. Chern. 251:6915, 1976. I2. Pittman, R.N., Minneman, K. P., and Mollinoff, P. B.: Ontogeny of /3 1- and {3 2-adrenergic receptors in rat cerebellum and cerebral cortex, Brain Res. 188:357, 1980. 13. Hoffman, B. B., De Lean, A., Wood, C. L., Schocken, D. D., and Lefko,Nitz, R.
J.:
A..!pha-adrenergic receptor
subtypes: Quantitative assessment by ligand binding, Life Sci. 24:1739, 1979. 14. Lefkowitz, R. J., Mullikin, D., and Caron, M.G.: Regulation of {3-adrenergic receptors by guanyl-5'-yl imidodiphosphate and other purine nucleotides, J. Bioi. Chern. Clil!"' .AC:OC.
~OJJ.O'"tUOU,
1
li'"H~
.1;:1/U,
15. Lands, A.M., Luduena, F. L., and Buzzo. H. P.: Differentiation of receptors responsive to isoproteronol, Life Sci. 6:224i, i967.
ALAN GOLDFIEN, M.D . .JAMES M. ROBERTS, M.D.* San Francisco, Caiifomia The radioactive beta-adrenergic antagonist [ 3H] dihydroalprenolol (DHA) binds to particulate preparations of human myometrium in a manner compatible with binding to the beta-adrenergic
receptor. The binding of DHA is rapid (attaining equilibrium in 12 minutes), readily reversible (ha!f time= 16 minutes), high affinity (Ko = 0.50 nM), low capacity (Bmax = 70 fmoles/mg of protein), and stereoselective ([-]-propranolol is 100 times as potent as [ +] -propranolol in inhibiting DHA binding). Adrenergic agonists competed for DHA binding sites in a manner compatible with beta-adrenergic interactions and mirrored {3 2 pharmacologic potencies: isoproterenol > epinephrine>> norepinephrine. Studies in which zinteroi, a,B 2-adrenergic agonist, competed for DHA binding sites in human myometrial particulate indicated that at least 87% of the beta-adrenergic receptors present are fkadrenergic receptors. Binding of DHA to human myometrial beta-adrenergic receptors provides a tool which may be used in the examination of gonadal hormonal modification of adrenergic response in human uterus as well as in the analysis of beta-adrenergic agents as potentially useful tocolytic agents. (AM.
J.
OBSTET. GYNECOL. 142:389, 1982.)
BoTH ALPHA- and beta-adrenergic receptors can be demonstrated pharmacologically in the human uterus. Alpha-adrenergic stimulation increases frequency and intensity of uterine contractions, 1 while beta adrenergic stimulation decreases either spontaneous or induced uterine contractility. 2 At present, beta-adrenergic agonists are the most useful clinical agents to prevent or reduce uterine activity. 3 From the Departments of Obstetrics, Gynecology, and Reproductive Sciences, and Medicine, and the Cardiovascular Research Institute, Universitv of California, San Francisco. ' " Supported by a Basal O'Conner Research Starter Grant from the March of Dimes Birth Defect Foundation. Presented at the Twenty-sixth Annual Meeting of the Society for Gynecologic Investigation, San Diegu. California, March 21-24, 1979. Reprint requests: James M. Roberts, M.D., Department of Obstetrics, Gynecology, and Reproductive Sciences, Room 1489 Moffit Hospital, University of California, San Francisco, San Francisco, California 94143. *Recipient of National Institutes of Health Research Career Development Award HD-00267. 0002-9378/82/040389+05$00.50/0 © 1982 The C. V. Mosby Co.
The adrenergic response of the human uterus is modified by the hormonal environment. During the follicular phase of the menstrual cycle, in which serum estrogen concentrations are elevated, the response of human uterus to epinephrine is augmented contractility-an effect which can be prevented by alphaadrenergic antagonists. However, if serum progesterone concentrations are increased, as during the luteal phase of the menstrual cycle or during late pregnancy, inhibition of contractile response by epinephrine is puleulialed; Lhis is a bela-adrenergic response blocked by beta-adrenergic antagonists_4- 7 We have previously demonstrated in rabbit myometrium, in which a similar hormonal modification of adrenergic response is present, that alteration of adrenergic predominance is associated with changes in adrenergic receptor concentration as measured by radioligand binding." To provide a tool to directly examine beta-adrenergic receptors in human uterus, we examined the binding of[:lH]dihydroalprenolol (DHA) to myometrial particulates and report that this agent
389
390
February 15, 191'1;1 Am. J. Obstet. Gynecol.
Hayashida et al.
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100
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Fig. LA. Time course and reversal of DHA binding. Percentage of specific binding(X/Xeq) is plotted as a function of time. X specific binding at timet andXeq specific binding at equilibrium. A total of 2.5 nM DHA was incubated at 3{)• C with myometrialparti<;ulate (final protein concentration I mg/ml) with or without 10 ~M (- )-alprenolol for the time pe~s indicated (o). Specific binding was determined as discussed in Methods. At t I 0 minutes, (- )-alpren!>fel was added (final concentration"" 10 ILM) to an aliquot of particulate not containing (- )-alprenolol and binding was terminated at the times indicated (t,). B, First-order kinetic plot of the reversal of DHA binding. From the mathematical model of a unimolecular dissociation: In X/Xeq = - k011t; the slope of the plot defines korr. the reverse rate constant, which was 0.043 min- 1 • C, Pseudo-first-order kinetic plot of DHA binding. From the mathematical model of a pseudofirst order association 11 : ln [Xeq/(Xeq- X)]= k01,(t), where kub = [DHA]kon + k0 ff; the slope of the plot defines k0 "' which was 0.24 min~ 1 • [DHA) is the concentration of free radioligand (2.5 nM) which was sufficiently large to guarantee it did not change over the course of the experiment. k01r From 1, B was 0.043 min- 1 : thus k00 = 0.079 nM~'min- 1 • Kd = kofflkon = 0.54 nM.
binds with interactions consistent with binding to the beta-adrenergic receptor. Material and methods Material. (3 H]Dihydroalprenolol with specific activity of 15 to 20 Ci/mmole was obtained from New England Nuclear. (-)-Propranolol was a gift from Ayerst Laboratories. Other drugs and chemicals were from commercial sources. Particulate preparation. Human myometrium was obtained from uteri removed for obstetric indications or from strips of myometrium removed at the time of cesarean section. Myometrium was sharply dissected free of endometrium and serosa. The tissue was minced, suspended in cold 5 mM Tris HCI, pH 7.4, 0.25M sucrose, in a 4-to-l (volume-to-weight) ratio, and then homogenized in a Waring Blendor. The homogenate was filtered through four layers of cheesecloth and centrifuged at 600 and I 0,000 X g for 15 minutes each. The pellets were discarded after each centrifugation and the resulting supernate was centrifuged at 30,000 x g for 20 minutes. The pellet was suspended in cold 50 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid (HEPES), pH 7.5, 4 mM MgS0 4 , rapidly frozen, and stored at -70° C until used for DHA binding studies. Binding of DHA was similar
in fresh particulates and in those stored for up to ~l months. Particulate protein concentration was determined by the method of Bradford, 9 with bovine serum albumin used as the standard. Binding assay. DHA was diluted in 50 mM HEPES, pH 7.5, 4 mM Mgso•. I mM ascorbic acid, and 20\)[ ethanol. Ethanol was included to standardize binding with subsequent alpha adrenergic receptor assays with [:JH)dihydroergocryptine. Ethanol in a final concentration of 2% had no effect on DHA binding. Two tenths milliliter of particulate (protein concentration "' 1.25 mg/ml) was added to 0.025 ml of the radioligand solution and 0.025 ml of l mM HCI or adrenergic agents in 1 mM HCl. Samples were incubated at 30° C for 15 minutes in equilibrium studies; the incubation was stopped by adding 5 ml of 5 mM HE PES pH 7 .5, at 4° C and immediately filtering through Whatman GF/C filters to separate bound from free radioligand. Filters were washed with an additional 15 ml of cold 5 mM HEPES under low vacuum (1 ml/sec). The filters were dried under high vacuum and counted in a liquid scintillation counter at 54% efficiency. Data aaelysis. Specific binding was detecmined by an iterative analysis of Scatchard plots whkh detennined a best fit for data at different estimates of nonsaturable binding. Specific binding was approximately 90% at
Volume H2 Number 4
Human myometrial adrenergic receptors
.14~
100
.12~ ~;::;: ~:L ~.--.--·-· .10
•
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Fig. 2. Scatchard analysis of specific binding of DHA. ConcenWtTe
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391
incubated with
particulate (final protein concentration = l mg/ml) for 15 minutes at 30° C. B =specific DHA bound; F =free DHA. Specific binding was determined as discussed in Methods. The intercept of this plot is the receptor concentration (Bmax). The slope, determined by linear regression analysis, is the negative reciprocal of the dissociation constant (K 0 ). Bmax = 70 fmob,/mg of protein; K0 = 0.50 nM.lnset, Direct plot of bound versus free DHA. D HA concentrations equal to the dissociation constant (0.50 nM). Similar dissociation constants and binding site concentrations were obtained if specific binding was defmed as binding in the absence minus binding in the presence of i 0 ,uM (- )-alprenolol. This technique was used to dehne specific binding in competition experiments in which adrenergic compounds competed for D HA binding sites. The inhibition constant (K 1) was determined for each competing adrenergic agent in competition experiments by the relationship described by Cheng and Prusoffl": K1 = I.-, 0 /[1 + (L!Kn)]; where 130 = the concentration of competing adrenergic agent which inhibits binding of DHA by 50%, L = the concentration of DHA used in the assay and Kn = the dissociation constant of D HA for its binding sites. In determining the percentages of {3 1- and {3 2 -adrenergic receptors present in human myometrium, we used an iterative, nonlinear, curve-fitting program which determined the number of sites and their affinities and the relative concentration of each site which best described the experimental data.
Results Kinetics of binding. D HA hound rapidly and reversibly to human myometrial particulate (Fig. I. A). At :100 C, equilibrium was attained in 12 minutes using 2.5
8
7
6
5
4
- log [ propranolol]
Fig. 3. Stereoselective competition for DHA binding sites. DHA, 2 nM, was incubated with myometrial particulate (final protein concentration = l mg/m]) and the indicated concentrations of the beta antagonist propranolol. Binding is expressed as percentage of total binding in the absence of proprano!o!. The (-)-stereoisomer of propranolol '.-vas 100 times
as potent as the (+)-stereoisomer. K1( - )-propranolol = 0.6 nM, K1 (+)-propranolol= 60 nM. nM DHA. With the addition of excess nonradioactive (- )-alprenolol, binding was rapidly reversed; D HA dissociated from the binding sites with a half time (ty2 ) of 16 minutes. The forward rate constant (kon) and the reverse rate constant (korr) were calculated to be 0.079 nM- 1min- 1 and 0.043 min- 1 , respectively (Fig 1, B and C). The dissociation constant (Kn), caicuiated from the ratio of korr to k., 11 , was 0.54 nM. Equilibrium studies. DHA bound to human myometrium saturably and with high affinity (Fig. 2). Binding \Vas to a single class of receptors with a binding site concentration of 70 fmoles/mg of protein. The Kn determined in equilibrium experiments was 0.50 nM, which is in excellent agreement with the kinetically determined Kn. DHA binding sites were competed for stereoselectively (Fig. 3). The active (-)-stereoisomer of the beta antagonist propranolol was I 00 times as potent as the inactive (+)-stereoisomer. Adrenergic agonists competed in a manner compatible with beta-adrenergic interactions and mirrored beta 2 pharmacologic potencies (Fig. 4): isoproteronol (K 1 = 0.12 ,uM) >epinephrine (K 1 = 1.1 ,uM) >>norepinephrine (K 1 = 50 ,uM). To determine the proportion of {3 2 -adrenergic receptors in the myometrial particulate, we examined the competition of zinterol, a highly specific {3 2 -adrenergic agonist of high affinity, for DHA binding sites (Fig. 5). Zinterol competes for {3 1-adrenergic receptors with approximately 70 times the potency that it competes for
Februarv 1S. Hli\C/
392 Hayashida et al. Am
-----e-e
100
,,
80 Percent Specific Binding
't:;.
"'e.
• 0
40
•e. 0
!-epinephrine !-norepinephrine 1- isoproterenol
8
7
•
"\~ 6
K1H 13.4 nM
%
RH
87
K1L 2,100 nM
% RL
13
80
.""'
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60
20
\
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.J. Obsrc:-i. Gynecol.
5
4
Percent specific binding 60
\
40f-
20
- log [ Agonist J 0
Fig. 4. Rank order of potency of agonists competing lor DHA binding sites. DHA. 2.4 nM. was incubated with myometrial particulate (final protein concentration = I mg/ml) and I 0 ~-tM (- )-alprenolol or increasing concentrations of agonist. Binding is expressed as percentage of specific binding in the absence of agonist. K1 isoproteronol = 0.12 ~-tM: K1 epinephrine= 1.1 ~-tM; K1 norepinephrine= 5.0 ~-tM.
,8 1-adrenergic receptors. 12 Thus, the competition of zinterol with DHA (which binds to ,8 1- and ,82-adrenergic receptors with equal affinity) can be analyzed.by computer-curve fitting to define the affinity and proportion. of high-atfinity (,8 2 ) and low-affinity (,8 1) sitesY When beta-adrenergic agonists such as zinterol bind to membrane preparations in the absence of guanylnucleotides, a very high-affinity ,8 2-binding component is present. 14 To eliminate the contribution of this very high-affinity component and therefore allow identification of.B 1- and .B~-adrenergic receptors by their differing affinities for zinterol, we included guanosine 5'-triphosphate (GTP) at a concentration of 0 .I m M in our assay. This concentration of GTP has bee11 demonstrated in several systems to prevent this very high-affinity bindingY · Analysis of data demonstrated that the findings were most consistent with zinterol competing with high affinity (K 1 13.4 nM, which is compatible with zinterol's potency in other ,8 2-adrenergic systems 12 ) for H7% of the DHA binding sites. Zinterol competed with a lower affinity for the remaining 13% of the DHA binding sites.
Comment DHA binds to particulate preparations of human myometrium with interactions consistent with binding to the beta-adrenergic receptor. Binding is high affinity, low capacity, saturable, and stereoselective. The kinetically determined dissociation constant is in excellent agreement with that obtained in equilibrium experiments. Even at 30° C, the reversal of DHA bind-
'---..1.-.....-...l.__
10
9
8
~
_l__...L_ )::....,_.
7
6
5
4
-log [Zlnteroi] Fig. 5. Competition of zinterul for DHA binding sites. DHA. 0.6 nM, was incubated with myometrial particulate (final protein concentration = 0.9 mg/ml) and 10 ~-tM (- )-alprennlol01 the indicated concentrations of zinterol. Binding is expressed as percentage of specific binding in the absern:c of zinterol. The curve is the curve of best fit as defi0€d by the iterative, nonlinear, curve-fitting program and was determined by the following parameters: KIH = K1 of zinterol for the ,8 2-adrenergic receptor (13.4nM); K11 • K1 of zinterol for the {3 1-adrellergic receptor (2,1 00 nM): q RH percemage nf beta-adrenergic receptors which are ,82 -adrenergic receptors (87% ); %RL = percentage of beta-adrenergic receptors which are {3 1adrenergic receptors (13%).
ing has a ty2 of 16 minutes; thus separation of bound from free radioligand by filtration at 4° C is an appropriate technique for this study . Competition studies indicate a rank order of potencies consistent with ,8 2-adrenergic potencies. Epinephrine is a mllch more potent competitor than norepinephrine. The great majority ot beta-adrenergic receptors present are ,8 2-adrenergic receptors. This is compatible with indirect pharmacologic determinations which indicate that inhibition of myometrial o>ntractility is determined by ,8 2-adrenergic receptors. 1:; Quantitation ot beta-adrenergic receptor subtypes in myometrial particulate indicates that at least 87% of the beta-adrenergic receptors present are ,8 2-adrenergic receptors. Since the particulate fraction we examined ('.Ontained .several cell types, that is, myometrium, blood veS~~els, andconnective tissue, it is possible that all of the beta-adrener~ gic receptors on myometrial cells are ,8 2-adrenergk re~ ceptors. The possibility that both receptor subtypes are present on myometrial cells, or even on an indi\'idual cell, cannot be excluded by these studies. The ability to directly measure beta-adrenergic rc-
Volume 142 Number 4
Human myometrial adrenergic receptors
393
ceptors in human myometrium provides a tool which
interaction of pharmacologic agents with myometrial
may be used in the examination of gonadal hormonal modification of adrenergic response in the human
beta-adrenergic receptors should aid in the analysis of
bela-adrenergic agents as potentially useful tocolytic
uterus. Furthermore, the ability to directly examine the
agents.
REFERENCES I. Althabe, 0., Jr., Schwarcz, R. L., Jr., Sala, N. L., and Fisch, L.: Effect of phentolamine methanesulfonate upon
2.
3.
4.
5. 6. 7. 8.
uterine contractility induced by !-norepinephrine pregnancy, AM. J. 0BSTET. GYNECOL. 101:1083, 1968. Mahon, W. A., Reid, D. W.J., and Day, R. A.: The in vivo effects of beta-adrenergic stimulation and blockade on the human uterus at term, J. Pharmacal. Exp. Ther. 156:178, 1967. Merkatz, I. R., Peter,J. B., and Barden, T. P.: Ritodrine hydrochloride: A betamimetic agent for use in preterm labor. II. Evidence of efficacy, Obstet. Gynecol. 56:7, 1980. Garret, W. J .: The effects of adrenalin and noradrenalin on the intact non-pregnant human uterus, J. Obstet. Gynaecol. Br. Emp. 62:876, 1955. Wansbrough, H., Nakanishi, H., and Wood, C.: Effect of epinephrine on human uterine activity in vitro and in vivo, Obstet. Gynecol. 30:779, 1967. Garret, W. J .: The effects of adrenalin and noradrenalin on the intact human uterus in late pregnancy and labour, J. Obstet. Gynaecol. Br. Emp. 61:586, 1954. Barden, R. P., and Stander, R. W.: Effects of adrenergic blocking agents and catet:hulanlines in hutnan pregnancy, AM.j. 0BSTET. GYNECOL. 102:226, 1968. Roberts,]. M., Insel, P. A., Goldfien, R. D., and Goldfien, A.: a-Adrenoreceptors but not !3-adrenoreceptors increase in rabbit uterus with oestrogen, Nature 270:624, 1977.
9. Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anai. Biochem. 72:248, 1976. 10. Cheng, Y., and Prusoff, W. H.: Relationship between the inhibition constant ( K 1) and the concentration of inhibitor which causes 50 percent inhibition (1 50 ) of an enzymatic reaction, Biochem. Pharmacal. 22:3099, 1973. 11. Williams, L. T., Mullikan, D., and Lefkowitz, R. J.: Identification of a-adrenergic receptors in uterine smooth muscle membranes by [ 3 H]dihydroergocryptine binding, J. Bioi. Chern. 251:6915, 1976. I2. Pittman, R.N., Minneman, K. P., and Mollinoff, P. B.: Ontogeny of /3 1- and {3 2-adrenergic receptors in rat cerebellum and cerebral cortex, Brain Res. 188:357, 1980. 13. Hoffman, B. B., De Lean, A., Wood, C. L., Schocken, D. D., and Lefko,Nitz, R.
J.:
A..!pha-adrenergic receptor
subtypes: Quantitative assessment by ligand binding, Life Sci. 24:1739, 1979. 14. Lefkowitz, R. J., Mullikin, D., and Caron, M.G.: Regulation of {3-adrenergic receptors by guanyl-5'-yl imidodiphosphate and other purine nucleotides, J. Bioi. Chern. Clil!"' .AC:OC.
~OJJ.O'"tUOU,
1
li'"H~
.1;:1/U,
15. Lands, A.M., Luduena, F. L., and Buzzo. H. P.: Differentiation of receptors responsive to isoproteronol, Life Sci. 6:224i, i967.