Binding and structural properties of oxytocin receptors in isolated rat epididymal adipocytes

ructural properties ot lated rat epididymal

fin rtes

D o n n a Boland and H. Josept~h G o r e n Department of Medical Biochemistry, The Univesity of Calgary, Calgary, A~

'2N 4N1.

(Received 1 July 1986; revised version received and accepted a~ 4 Fe

;ummary pocytes thro~ n-specific Oxytocin initiates its insulin-like action in adipoc~ oJ ~tors with eceptors. We have studied binding and structural properties pro he radioligand [aH]oxytocin. Steady-state binding was reached after 45 rain, at 21 21'°C, nd 10 min at 37°C. Scatchard analyses of equilibrium binding data indicated a sinlgle an( lass of oxytocin binding sites at 210C (KD = 3.3 nM,, Rx = 6 × 104 sites/cell) and anc 2 class binding sites at 37°C (KD=I.5 nM, Rx=6x104 sites/cell; and Kn--20 nM, n] f~ I, and epidermal gro~ ,wth R-~ = 30 x 104 sites/cell). Insulin, insulin-like growth factor actor increased oxytocin binding (~20-40%), whereas wher adenosine, a regulator of factor xytocin action, did not affect oxytocin binding. Binding Bin OX~ activity of oxytocin was xlpaired by pretreatment of the hormone or adipocylttes with dithiothreitol. Dith im Dithiohreitol treatment of adioocvtes oreferentiallvy inactivated inactiva threitol Lt pocytes 1~ high-affinity binding sites. sit N-ethyl maleimide inhibited ited ox, oxytocin binding in adipoctyes more than dithiothreit othreitol. In contrast to the inhibitor' ory effects of dithiothreitol and N-ethyl maleimide, protea ~roteases (trypsin, chymotrypsin and nd papain) were not able to inhibit fat cell binding activity. activi These results suggested that in isolated adipocytes: (i) there are high-affinity and a low-affinity receptors, but it the low-affinity receptors are absent at 21°C; (ii) the bir binding of oxytocin can be re gulated by insulin, and growth factors; and (iii) the oxyto~ 'tocin receptors contain disulfide de bridges and free thiols that are essential for the maintcenance of oxytocin binding. N-ethyl maleimide; Dithiothreit~ iothreitol; eitol: Adenosine; [3H]oxytocin; Trypsin; Chymotryp~sin

Correspondence: H.J. Goren, Dc~epartment of Medical Biochemistry, The University of Calgary, 3330 HosH pital Drive Northwest, Calgary,¢, Alta., Canada T2N 4N1. 0167-0115/87/$03.50 O 1987 Elsevier ~,lsevier Science S( Publishers B.V. (Biomedical Division)

lin A, vitamin K, H202, and oxyto vity of intracellular glucose metal initiates its action by binding to a :eceptor [4]. To characterize the o~ the binding of 12Si.oxytocin or [3I [5-7]. 12q-oxytocin, however, is n characterization because its optim s not equivalent to oxytocin insulin-like activity [5]. [5! In con ,~quipotent to oxytocin in several oxytocin bioassays [7], [ but tt cal activity of [aH]oxytocin is much lower than the s[)ecific act 14-59 Ci/mmol for [~H]oxytocin compared to ~ 200 300 Ci/mm Soloff has employed [3H]oxytocin with spec. act. 25-36 Ci/ ;uccessfully uterine and mammary tissue oxytocin receptors re~ [8 ,'quivalent and higher specific activity is now commercially comm~ a' lestigated, therefore, adipoctye oxytocin receptors with the aH]oxytocin: (i) to confirm the binding parameters, determim md the low specific activity [3H]oxytocin (14 Ci/ml /mmol [7]); •eceptor specificity of the nonapeptide; and (iii) to characterize ch~ ,'rties of these receptors.

imic some hese insu;ma memptors that o isolated ~tisfactory ce activity ×ytocin is tdiochemi-oxytocin 3xytocin). aracterize ~ytocin of ,~have inLration of i-oxytocin rmine the ical prop-

Materials and Methods

[3H]oxytocin, 20.4-59 Ci/mmol (1 Ci= 37 GBq) was w; purchased from Amersh ~ham 2orporation, New England Nuclear Corporation, or Cambridge ( Research Biochel ~chemiCor ;als Limited. Purity of the radioligand was demonstrated demons by high-pressure liq~uid cals Bovine serum albumin albur :hromatography (Waters System, C~s-/~Bondapak column). co chr BSA~ was purchased rmrchased from Armour Pharmaceutical Pharmaceutic~ Company (fraction V, lot (BSA) La Chemical Company (A-7030, Lot 75F-0007), collagen chase W85211), or from Sigma 9oration, (type CLS, lot F2B261; 126 units/mg) from Worthington Biochemical Corporati /mg) Bio-Rad Rad Laboratories. Synthetic oxytocin (486 + 22 U/n and dithiothreitol from Bio-Rac 1 was a gift from Dr. Mauurice Manning (Medical College of Ohio, Toledo, OH). EpEGF) was a gift from Dr. Morley Hollenberg (Universityy of idermal growth factor (EGF) Mar Calgary) and insulin-like growth factor I (IGF I) was kindly provided by Dr. Marvin katchewan, hewan Saskatoon). Insulin (porcine) was provided by Bala (University of Saskatchev lenosine maught Laboratories (Toronto). Adenosine and adenos Dr. John Clement, Connau ed from Sigma. All other chemicals were of the highest quualdeaminase were purchased ity commercially available. NaC] 5 Krebs-Ringer bicarbcanate-BSA buffer (KRB-BSA) contained 118 mM NaC1, mM KC1, 1.3 mM CaCI 2, 1.2 mM MgSO,, 1.2 mM KH2PO4, 25 mM NaHCO3 and 2% w/v BSA. The buffer :r was saturated with 95% 02/5% CO2 and adjusted to pH ! 7.4. nt, male Sprague-Dawley rats (150-350 g) of similar age and In any given experiment,

al fat pads were cut into small pieq containing 3 ml Krebs-Ringer bic ir above the digestion mixture wa tles were capped and incubated d through a silk screen and the iso 0.5-5 x 10 5 cells/ml in Krebs-Rin ed [11]. The lipid content of the h 12]. Since lipid content per cell is ted from adipocyte suspension lipi

Lsferred to SA buffer with 95% min. The =ytes were nate-BSA nsion was )n rat size ad rat size

14]. 9xytocin binding Fat cell suspension (0.4 ml) was added to 12 x 75 m~ mm polyst3 ',ontaining [00 pl [3H]oxytocin (0.5-30 nM, final concentration cono bs-Ringer ~icarbonate-2% BSA buffer. Non-specific binding was vv deterr ,' presence )f 10/~M (final concentration) oxytocin. The tubes were v plac~ :ing water ~ath (60 cycles/min) at 37°C or 21°C and incubated as a noted i rods. Folowing incubation the tubes were placed on ice and two t' 200/~ ere added :o two 0.4-ml microfuge tubes containing 100 pl dinonyl din, pht[ microfuge ubes were centrifuged (Beckman Microfuge B, 45 s) and ther middle of he dinonyl phthalate layer. The top half of the tube. tub~ which he fat cell ~ellet was placed in a 20-ml glass scintillation vial containing cont~ 15 ml ACS (AmershaJ nersham). Radio~ Radioactivity was determined in an LKB 1215 Rackbeta Rack II counter. Radioacti~ lioactivity in n an aliquot of the [3H]oxytocin solution was detern determined and was used to con~ :onvert counts :ounts per minute (cpm) to molar units.

Results

Binding characteristics The time to reach stea~dy-state for adipocyte binding of oxytocin was temperat )erature dependent (Fig. 1); at 21°C ~,I°C more than 45 rain was required, while at 37°C, 10 min r was sufficient. Non-specific :ific binding per cell was less at the lower temperature (Fig. (t 1), was independent of cell num ell number umbe (0.1-0.8 × l0 s cells/0.5 ml), and increased linearly line~ with increasing weight of ~f rat from which adipocytes were obtained (approximal oximately double for 300 g rat adiipocytes compared with 150 g rat adipocytes). Steady-state specific bindin inding of [3H]oxytocin increased hyperbolically with lncreasincre ing ligand concentration.t. Fig. 2 contains 2 representative plots of several equilibri uilibrium binding experiments anal, ~lysed by the method of Scatchard [15]. At 21°C there w~ was a single class of oxytocin-bindin binding sites; KD = 3.3 nM and RT = 10.5 pmol/10 s cells (ca. ( 6 × 10'~ sites/cell). At 37°C °C, however, the Scatchard plot was curvilinear suggest ;gesting a heterogeneous population tion of oxytocin binding sites. The data was fit to a twotwo-site binding model and the binding parameters were KD = 1.5 nM and RT = 9 pmol/,10s cells for the high-affinity y binding site, and KD=20 nM and RT=50 pmol/10 s cells c (ca. 30 x 104 sites/cell) for or the low-affinity binding site.

I~ WITH RAT

ADIPOCYTES:

4PERATURE

cpm 4001-

37°C

3oo[/°

~-~

o

200 l -

loo

0

15 30 TIME (minutes)

45

• ..• ......... = .................

0

10 20 30 TIME (minutes)

•

40

Fig. 1. Total and non-specific binding of [3H]oxytocin to isolated rat adipocy incubated with adipocytes (6.6 x 105 cells/ml) from a 200 g rat rat:at 21"C, an as cells/ml) from a 130 g rat at 37"C, Bound radioligand was determined det deten Methods. Each symbol (O, total; O, non-specific) represents a single s

]oxytocin was ytes (2.5 x 105 Materials and

S t r u c t u r e - a c t i v i t y s t u d i e s [l 6,17] a n d b i n d i n g s t u d i e s [7] c o n o x y t o c i n r e c e p t o r s b i n d specifically o x y t o c i n a n d oc n l y p o o r such as v a s o p r e s s i n . F u r t h e r , o x y t o c i n c a n n o t b i n d to the in s u l i n is u n a b l e to c o m p e t e w i t h [ 3 H ] o x y t o c i n b i n d i n gLg [7], n o r It displace i z 5I - i n s u l i n f r o m a d i p o c y t e s ( d a t a n o t illustrated). illust]

Lt a d i p o c y t e analogues, o r since inM oxytocin n, h o w e v e r ,

i

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37°C

21°C

9

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1

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'tes. Left: equilibrium binding data of [3H]oxytocin to isolated rat adipocytes Fig. 2. 'Scatchard' analyses of equ ~ecifically at) were incubated with [3H]oxytocin (1-10 nM) at 21"C for 2 h. Spec adipocytes (from a 130 g rat) nined as described in Materials and Methods. Data illustrated are the mean bound oxytocin was determined ncubated with ments). Right: adipocytes (from 150 g-195 g rats) were incubatec + S.E.M. (n = 6 measurements vas deter determined [3H]oxytocin (0.85-32.7 nM[) at 37"C for 5, 10, 20 and 30 rain. Specifically bound oxytocin was Lsurements) for ~d Methods. Data illustrated are the mean + S.E.M. (n = 4 measuremen as described in Materials and Metl ~ n and 30 min incubation data gave very similar curves. the 10 min incubation, 20 rain

A

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IO

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Oxytocln

t

Ins

EGF

IGF-I

0

AdD

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'ig. 3. The effect o f insulin, growth factors and adenosine on adipoc:yte I binding : adipocytes •ore a 270 g rat were incubated 370C, 10 min with 6.6 nM [3H]oxyrtocin and ocin (0.1 or .0/~M), insulin (Ins, 1.7 nM), epidermal growth factor (EGF, 8.3 8.2 nM), or il ~¢th factor I GF-I, 20 nM). [3H]oxytocin bound was determined as described in i] Materials Results are le mean ± S.E.M. ( n = 4 measurements). Asterisks indicate signi: aificant incre trol level o f inding (P ,~ 0.1 for insulin and P ,~ 0.01 for growth factors; paireq )aired Student's ocytes from 230 g rat were incubated 10 rain, 37°C with 10 nM [3H]oxytocin ',tocin t O t , I l l tand l l l U with W l t l l I V I I I V I U A , y t l . , , ~ l rnl [Vt.)2ty)~ (Oxy), or ~th 0.5 U/ml adenosine deaminase (ADD), or with 10/zM adenosine adenosi (Ad), or with no further additi~ additions (~). 3). sSpecific binding was determined as described in Materials andJ Methods. The mean + S.E.M. (n! = 4 measu leasurements) are illustrated. Asterisks indicate significant differe~ ence from control binding (P ,~ 0. 0.01;

paired Student's t-test). wh lhether oxytocin can bind to other adipocyte peptide receptors; such as, the epidLermal aal growth factor receptor [18], or the insulin-like gro rowth factor II receptor [19]. 'To test ~st for this, adipocytes were incubated (37°C, 1.0 minlt) with 5 nM [3H]oxytocin aand epidermal pidermal growth factor (8.3 nM), or insulin-like grov rowth factor I (20 nM). (InsuL Insulinlike growth factor I, whose se KD= 9 nM for insulin-like growth factor II receptors [20], [2 was used to test [3H]oxyytocin binding to the latter receptors.) Epidermal grox~ rowth factor II receptors and ox, ,xytocin receptor binding activity may be regulated by nc)noxytocin peptide hormones. increased [3H]oxytocin bindin ~inding, 5.6 fmol/105 cells (-,~20%), whereas oxytocin ((0.1 and 1 /~M) displaced corn mpletely specifically bound [3H]oxytocin (Fig. 3A). Thus, Thl oxytocin did not bind too insulin, epidermal growth factor, or insulin-like grov~ rowth factor II receptors and ox~ xytocin receptor binding activity may be regulated by nc) n oxytocin peptide hormones. Adenosine, which is se(creted by adipocytes [21] increases adipocyte responsivem nslveness to oxytocin by increasing the magnitude of the oxytocin response [22]. The results of the effects of temperature and peptide hormones on oxytocin binding suggested that tl~ oxytocin receptor bindingg activity activit ivitv is regulatable. Thus, to determine whether aden ~r adenosine increases oxytocin responsiveness by increasing receptor binding activil activity, [3H]oxytocin binding wasts measured in the presence of adenosine deaminase (0. "0.45

ding to epididymal adipocytes acin (nM)

rapaln

.0 .7 .7 ~,.o ± 1./ .7

16.0•

9.3b

39.7 5:2.8 462 4- 0.7 40.1 4- 1.9 40.8 4- 2.8

18.0 + 0.8 21.9 4- 1.4 19.2 4- 0.9 17.2 4- 0.8

Values denote the quantities of ligand bound (pmol/liter/10s cells)). ' Isolated adipocytes from 280-290 g Sprague-Dawley rats were incubi [3H|oxytocinin the presence or absenceof protease (10/~g/ml). Bq Lg/ml).Bound ligand in Materials and Methods. Specificallybound ligand 4- S.E.M. (n = 4 mea ' Adipocytes (from 257 g Sprague-Dawley rat) were preincubal ~reincubated, l0 min ~g/ml). [3Hloxytocinwas added and then the suspensions were incubated a Specifically bound ligand ± S.E.M. (n = 6 measurements) is noted. no~ U/ml) or exogenous adenosine (10/~M). Adenosine and an adenos ncrease or decrease [3H]oxytocin binding to isolated isolate adipoc :esults indicated that adenosine did not affect the ox:ytocin re(

37"C) with as described toted. ~roteases (I0 0 rain, 37"C.

,se did not B). These

(~hernical and protease sensitivity The addition of chymotrypsin, trypsin, or papain ~am (10 #g/ml) to the adipoc5 ;tencubation medium, prior to or during [3H]oxytocin binding, 1 in( did not alter oxyto 'tocin 9inding (Table I). Increasing chymotrypsin incubation incubatio time to 30 min similarly had bindin no effect on ligand binding. The presence of dithiothreitol during adipocyte bin binding of [3H]oxytocin inhibited inhibi igand binding in a concentration-dependent manta lig~ manner (Fig, 4A). Whether dith L~hiothreitol :hreitol reduced the ligand or the receptor was determined dete~ in separate experime ~nments where [3H]oxytocin or fat 'at cells were preincubated with dithiothreitol and then e~ each were tested for binding activity. tctivity. Ten min preincubation of [3H]oxytocin with 0.6 m rt M dithiothreitol, a concentration tration sufficient to decrease steady-state binding by 50%, 50 decreased the amount ofFradioligand r~ bound (Fig. 4B). Preincubation of fat cells with v~ 0.6 m M dithiothreitol decreas tecreased 2.7 nM [3H]oxytocin binding (Fig. 4C), but it did not decrease 8.5 nM [3H]oxy t]oxytocin binding (Fig. 4D). Thus, the data in Fig. 4C md 4D indicated that the hJigh-affinity binding sites were preferentially inactivated with ,, dithiothreitol. Accordin gly, [3H]oxytocin binding to control and 0.6 mM-pretrea ~retreated adipocytes were compared :ed in Scatchard [15] plots (Fig. 5). The data supported the conclusion that dithiothreitol hreitol inactivated high-affinity receptors with little or no change in low-affinity rece •eceptors. t Further reactivation of the reduced high-affir ;h-affinity receptors would appear' to proceed slowly since the difference in binding activity acti~ ted adi between reduced and untreated Ltreated adipocytes remained constant between 2 and 10 min r (Fig. 4C). Treatment of fat cells; with N-ethyl maleimide (3 m M ) lowered oxytocin bind binding

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A: adipocytes were isolated olated from fr dithiothre Fiig. 4. Inhibition o f adipocyte [3H]oxytocin binding with dithiothreitol. pecifically 10 g rat tissue and incubated 37"C, I0 rain with 13.8 nM [3H]oxytoc ,tocin and with dithiothreitol. Specific~ 310 Percent inhibition was calcula calculated ound ligand was measured as described in Materials and Methods. Metho bound i' D is from: 'om: (Bo --BD) x 100%/Bo, where Bo is specific ligand bound in the tt absence of dithiothreitol and tB] le specific ligand bound in the presence of dithiothreitol. Illustratq Illustrated are the mean 4- S.E.M. o f 6 meam the ~rements. B: [3H]oxytocin (0.1 ml) was incubated 10 min, 37°C in i] the absence ( 0 ) or presence o f 0.6 surements. miM M dithiothreitol (A). Adipocytes (0.4 ml) from a 175 g rat were ad added to each [3H]oxytocin preparat )aration and incubation was continued for a further 10 min. Specifically bound [3H]oxytocin was determined termined as described in Materials and Methods. ,'thods. Mean 4- S.E.M. (n = 3 measurements) are illustrated. Curves wvere drawn using a polynomial expression In 'Drafter', a Hewlett Packard compatible plotting program. C : I ression in ml of KRB ( O - O ) or 1 ml 1.2 m mM M dithi dithiothreitol ithic in the same buffer ( 0 - - - 0 ) were added to 1 ml adipoc lpocyte suspensions (isolated from 240) g rat). The adipocyte mixtures were incubated I0 rain, 37°C and diluted dilu to 7 ml with KRB-BSA buffer. :r. [3H]oxytocin (2.7 nM, final concentration) was added to aliquots o f adipocyte suspension and the mixture nixture ?e w was w, ~ incubated (37°C). Specific radioligand bound was measured neasured as described in Materials and Methods. ~thods. Mean 4- S.E.M. (n = 4 measurements) are illustrated. D: 1 ml of Krebs-Ringer bicarbonate buffer, fer (©) or I ml 1.2 m M dithiothreitol in the same buffer ( 0 ) were ad( added to 1 ml adipocyte suspension (Iisolated from 265 g rat). The adipocyte mixtures were incubated I0 min, rr 37"C. Following dilution as inn C, [3H]oxytocin (8.5 nM, final concentration) was added and spec ~ecific radioligand bound was measured red as described in Materials and Methods. Mean ± S.E.M. (n = 4 meam surements) are illustrated.

much more than treatment ent with dithiothreitol; e.g. N-ethyl maleimide-treated adia, pocytes bound about 75~/o less [aH]oxytocin than untreated adipocytes (Fig. 6), while wh dithiothreitol-treated adi pocytes bound about 25% less radioligand (Fig. 4C). Thus, Thl

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Fig. 5. Seatchard plots of [3H]oxytocin binding to untreated and 0.E ).6 mM dithiothreitol-treated adipoc,. pocytes. Adi Adipocytes from a 318 g rat (open symbols) or from a 159 g rat (closed (¢ symbols) were incubated 10 min (37"C) ~37°C) in KRB-BSA buffer without ( © , O ) or with 0.6 m M dithiot] dothreitol (A,&). The fat cell suspens ~ensions we were diluted 5 fold with KRB-BSA buffer containing [3H]oxytocin (1-10 nM). After an additional 10 101rain, 37*( 37"C, specific [3H]oxytocin binding was measured. Mean + S.E.M. S.E.I~ (n = 3 measurements) are illustrated. illustra T

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[3HI OXYTOC,N (riM) Fig. 6. Inhibition of adipocytee [3H]ox [3H]oxytocin binding with N-ethyl maleimide, and with dithiothreitol reitol and N-ethyl maleimide. To 1.8 ml adi adipocyte suspensions (from a 225 g rat), 0.1 ml Krebs-Ringer bicarbonate bicarbo buffer was added (Control, NEM EM) or 0.1 ml 12 mM dithiothreitol was added (DTT/NEM). The adipo pocyte n, 37°C. Then, to 'Control' adipocytes, 0.1 ml KRB was added, and to the mixtures were incubated 5 min other 2 adipocyte mixtures 0.1 •1 ml 60 mM N-ethyl maleimide was added. The adipoeyte mixtures ~ Jxtures were incubated an additional 5 min. n, 37°C and 5 ml cold KRB-BSA buffer was added. The infranatant w a s aspirated and the adipocytes were resuspended in 6 ml KRB-BSA buffer. Specific [3Hloxytocin bin~ binding (370C, 10 min) was measured as described in Materials and Methods. Mean :t: S.E.M. (n= 4 lmeas Tleasurements) are illustrated.

xytocin binding sites markedly r~ d N-ethyl maleimide following ditJ ytocin binding further (Fig. 6).

• binding reatment

receptors ke activity in isolated adipocytes timulates [ucose oxidation and lipogenesis and inhibits catecholamine catec lipolysis tivity by ~,14,17,23,24]. The neurohypophysial peptide initiat,tes its in~, inding to a cell surface receptor. We have determined the bindi ~rs of the reviously dipocyte oxytocin receptor and these are compared in Table II site ,~ported. Adipocytes exhibited 2 types of oxytocin binding bil finity site nd a low-affinity site. At 21°C, only the high-affinity oxytocin was de:cted. (The slightly higher binding constant, KD = 3.3 nM, at 2 ed to the inding constant, KD = 1.5 nM, at 37°C may reflect the t~ slower 'tocin as~ciation at the lower temperature (Fig. 1).) The binding binc para the highffinity binding sites appear to be independent of the radiolig~ ytocin or ~SI-oxytocin) used ( K o = l . 5 to 5 nM; Rx=3 x 10" to 1 6 x 10' but they 'ere very different for the low-affinity binding sites (~KD = 20 n ,'d to 400 M (~25I-oxytocin) and R r = 3 0 x 104 sites/cell corn compared to 175x 104 sites/c'cell (~ 2 Si_ox I Si_oxytocin)). It may be suggested that the increase ased hydrophobicity of iodo-oxiodo-c tocin compared to oxytocin, allows it to associate with w ytocm non-receptor proteins, i n teasing the number of measurable binding sites and ]lowering the apparent affinity affini cre of the low-affinity site. Both oxytocin binding sites in adipocytes are probably proba oxytocin receptors whose whc ccupancy elicits insulin-like effects: the KD of the hi gh-affinity binding site is apOCCU TABLE II Binding parameters of oxytocinn bindin binding sites in rat epididymal adipocytes Radioligand

Method of analysis

Temperature (*C)

KD (nM)

Rr (sites x 10-*/cell)

Reference

125i.oxytocin

Scatchard

37

[3H]oxytocin

Kinetics Saturation"

30 37

Scatchard

21 37

5 400 2 5 n.d. 3.3 1.5 20

5 175 n.d. 3 >>21 6 6 30

6 6 7 7 7 b b b

The amount of 13H]oxytocin bound at equilibrium was plotted against the concentration of radiolig~and in the incubation medium; Ko ~o is the estimated concentration of [3H]oxytocin needed to reach 50% of the first plateau (RT). A second and plateau was not reached, b, this report.

on of oxytocin required to elicit ,17]); and the low-affinity binding ocin insulin-like activity continue aM and 100 nM, a concentration

oxytocin ~elieved to oxytocin •e binding

e.

]oxytocin binding at 21 °C was lira )arent abptors at this temperature and thei: e at 37°C, f these receptors in the plasma men :ontrolled ation is that at 21°C the low-affin s were lozated in intracellular membranes and their transloca ocation to t] nembrane zcas temperature-induced. Alternatively, the low-affinity low-affin recepl were in a :onformation unable to bind oxytocin, and raisingg the tempera d the conbrmation to allow ligand binding. A third explanation )lanation is that in mobility ~t higher temperatures may have moved the receptor ator from an e position n the plasma membrane to an accessible position, qThe data ,rt cannot fistinguish between these mechanisms. [3H]oxytocin did not occupy the receptor for insulin, insuli epider factor or nsulin-like growth factor II. Rather than inhibiting [3H]oxyt g, insulin, ,~pidermal growth factor, and insulin-like growth factor fac I ap[ crease ratioligand binding. The mechanism for the increased oxytocin c t tae growth "actors and insulin is not known, because it is not known kn wh¢ rease was tue to an increase in binding affinity or an increase increase in receptor number. Insulin, Insul however. lowever, is known to increase adipocyte insulin-like glrowth factor II binding activity acti~ 25] and plasma membrane glucose transporters [26,27]. [26,2 Both of these effects of in[25] lat insulin sulin ;ulin re~ require insulin occupancy of its receptor. It is tempting ten to speculate that insr md the growth factors may modulate oxytocin recep~tors by similar mechanisms anisms. and The phenomenon observed with insulin, epidermal growth factor, and insulin-I lsulin-like glgrowth factor I may not, however, be applicable to other adipocyte ligand-recep~tor o and adenosine deamin ase adenc S;ystems, such as adenosine receptors [28], since adenosine lid not affect [3H]oxytc ~3Hloxvtocin binding,. ,cin binding. did

oxytocin receptors Structural properties of ox) ~roteoere stable to trypsin, chymotrypsin and papain. The protq Oxytocin receptors were ected, since receptor sensitivity to dithiothreitol and N-ethyl N-et lytic stability was unexpected, maleimide indicated thatt oxytocin receptors were proteins. Possible reasons for the vity include: that bovine serum albumin in the incubatiion lack of protease sensitivit, nembrane ,ented the proteases from acting on the plasma membr~ medium may have prevented be a protein; or that carboh,ydrate side-chains on the receptor, which is likely to b~ ct the receptor against proteolysis; or that the proteases rr may glycoprotein, may protect rods in the receptors but the oxytocin binding activity w a s have cleaved peptide bonds ors whose ide chain disulfide bonds (plasma membrane receptors wh, maintained by intrapeptic ent been determined contain regions of high cysteine cont primary structure have

[29]). :~resence The effect of dithiothre'eitol on adipocyte binding of oxytocin supports the prese~ isulfides in adipocyte high-affinity oxytocin receptors. I1 It is of intrapeptide chain disulfides

inding sites also have intrapeptid~ othreitol but are then reoxidized c The latter conclusion was support ed adipocytes reduced 9 nM [3H]o ithiothreitol-reduced (Fig. 6). ThL receptors to dithiothreitol suggest~ ameters, the two oxytocin receptor

onds that ~ashing or )servation ding more ical sensiddition to e different

There appear to be two types of oxytocin receptor~ 9tors in rat el t small number with high affinity and a larger er number number with lov wtivity of these two receptors are regulated by temperature ten 'actors. Future investigations should provide insight into the~ :ion of adipocyte oxytocin receptors by temperature and pept

dipocytes; le binding td growth of regula-

~onclusions

es,

i.cknowledgement This work was supported by the Medical Research Council i c Fhe authors are grateful for the gifts of peptide hormones horm fror ~ollenberg, and Manning.

hA 7271). Clement,

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