(CANCER RESEARCH 50, 7882-7886, December 15, 1990]

Interaction of Vasopressin and Oxytocin with Human Breast Carcinoma CellsA. H. Taylor, V. T. Y. Ang, J. S. Jenkins, J. J. Silverlight, R. C. Coombes, and Y. A. Luqmani1

Divisions of Biochemical Medicine [A. H. T., K T. Y. A., J. S. J., J. J. S.] and Medical Oncology [R. C. C., Y. A. L.], and Department of Cellular and MolecularSciences, St. George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, England

ABSTRACT

The arginine vasopressin and oxytocin content of normal and canceroushuman breast tissue were measured using radioimmunoassay. Both pep-tides were present in amounts greater than that found in the circulation,but no difference between normal and malignant tissues was found.Binding of (3H|oxytocin and (3H|vasopressin were characterized in human

breast carcinoma cells (MCF7 cells). Binding of both hormones to MCF7cells was specific and saturable, the vasopressin receptor found to be ofthe V, subtype. Scatchard analyses of the data were linear, indicating asingle high affinity, low capacity binding site for each hormone (vaso-pressin: KD = 47.4 ±1.6 nmol/liter, A™,= 27,300 ±6,500 sites/cell;oxytocin: KD= 51.3 ±0.4 nmol/liter, B^ = 87,000 ±4,000 sites/cell).The effects of vasopressin and oxytocin on the growth of MCF7 cellswere assessed using protein accumulation and cell numbers. Vasopressinat 10-1000 pmol/liter was mitogenic for MCF7 cells, but higher doses(10 nmol/liter) were growth inhibitory. Oxytocin was also mitogenic forMCF7 cells but to a lesser extent than vasopressin. In conclusion, wesuggest that vasopressin and possibly oxytocin may be important modulators of the growth of some human breast carcinomas.

INTRODUCTION

Vasopressin and oxytocin are closely related nonapeptidessynthesized as part of larger neurophysin molecules in thehypothalamus and secreted from the pituitary (1). In additionto their classical roles in kidney function and smooth musclecontraction, recent observations have shown a wide variety ofactivities for both peptides in the central nervous system, aspossible modulators of adrenocorticotrophic hormone and pro-lactin secretion, maternal behavior, and memory acquisition(2-6). They are also found in peripheral tissues where they mayhave modulating activities (7, 8).

The normal breast is generally recognized as being a targetorgan for oxytocin in the process of milk ejection, but theinfluence of the hormone on breast carcinoma is not known(9). In the case of arginine vasopressin, involvement in breastfunction and growth is less certain (10). Receptors for oxytocinhave been reported in normal rat and bovine mammary tissue(11, 12). Receptors for vasopressin have been described andcharacterized in the WRK1 rat mammary tumor cell line (13,14), and vasopressin has been shown to increase lipid synthesisand protein accumulation by WRK1 cells (15).

In the present study we have investigated the presence ofoxytocin and vasopressin in normal and malignant humanbreast tissue and have sought evidence for the existence ofspecific receptors for both hormones in the human MCF7 breastcarcinoma cell line. In an attempt to establish whether theseobservations have any functional relevance we have also studiedthe effects of vasopressin and oxytocin on the growth of MCF7cells in vitro.

MATERIALS AND METHODS

Extraction of Oxytocin and Vasopressin from Breast Tissue. Tissuefrom carcinoma of the breast was obtained at the time of operation and

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from normal breast during the surgical treatment of breast reduction.All samples were histologically assessed. No macroscopically discernible differences were apparent between the normal breast tissues used,fibrous areas being avoided. All tumors were diagnosed as intraductalcarcinomas and were of approximately equivalent cellularity and didnot differ significantly in the degree of stromal invasion. Samples werefrozen immediately in liquid nitrogen. Prior to extraction, tissue wasthawed out in small pieces and homogenized in 1 mol/liter acetic acid.Fat was removed by centrifugation at 700 x g. The aggregates of toughstromal material were removed by freezing rapidly on dry ice andallowing to thaw before being centrifugea at 2000 x g for 20 min.Oxytocin and vasopressin were extracted from the supernatant withSep-Pak CIS cartridges (Waters Associates, Northwich, Cheshire, England) and assayed as described previously (16) using the first international standard for vasopressin (75/501) and the fourth internationalstandard for oxytocin (76/575). The intraassay coefficients of variationat the level of 1-10 fmol/tube were 6.5% for the oxytocin assay and7.5% for vasopressin. Interassay coefficients of variation were 12.8%for oxytocin and 13.0% for vasopressin. The limits of detection were0.6 fmol/tube for oxytocin and 0.3 fmol/tube for vasopressin. Analiquot of the extract was taken for protein measurement (17).

Characterization of Oxytocin and Vasopressin in Breast Tissue. Tissueextracts were lyophilized and subjected to HPLC2 as described before

(18). The HPLC profiles were compared with those of authentic oxytocin and vasopressin.

Culture of MCF7 Cells. MCF7 cells were maintained in DMEM(Gibco, Ltd., Paisley, Scotland) supplemented with 10% fetal calf serum(batch 2044 or 1981; Tissue Culture Services, Slough, Berkshire, UnitedKingdom), penicillin (5000 units/ml; Flow Laboratories, Ltd., Rick-mansworth, Hertfordshire, United Kingdom), streptomycin (15 mg/ml; Flow Laboratories) in monolayer cultures at 37°Cin humidified

air/5% CO2 atmosphere, in T75 flasks (Flow Laboratories).Binding Studies on MCF7 Cells. Cells from stock were removed from

the flasks by scraping with a "policeman" washed once with warm(37°C)Locke's solution consisting of 154 mmol/liter NaCl, 5.6 mmol/

liter KC1, 5.6 mmol/liter glucose, 5 mmol/liter 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer, 40 mg/liter gentamycin, 200,000units/liter penicillin, 2 g/liter BSA, and where appropriate 1 mmol/liter MgCl2 and 2.3 mmol/liter CaCl2. Cells were resuspended in thissolution to give approximately IO6 cells/assay tube. All assays werecarried out in triplicate at 4"(" and at pH 7 for 3 h in a final assay

volume of 400 Mi-Cells were incubated with [3H]vasopressin (specific activity, 60.3 Ci/

mmol) or [3H]oxytocin (specific activity, 36.6 Ci/mmol), obtained from

New England Nuclear, Stevenage, Hertfordshire., United Kingdom inthe presence of increasing concentrations of unlabeled ligands. At theend of the incubation period the tubes were placed on ice for 5 min anddiluted with 1 ml of ice-cold Locke's/BSA solution. The cells were

separated by rapid filtration on Whatman GF/B glass microfilters(BDH, Ltd., Dagenham, Essex, England), quickly washed twice withice-cold Locke's/BSA solution (2 ml), and transferred to scintillation

vials for counting. Nonspecific binding was determined by incubatingcells with labeled peptide in the presence of an excess (100 /imol/liter)of unlabeled hormone. The V|-vasopressin antagonist rf(CH2)5Tyr(Me)2 AVP was kindly supplied by Dr. M. Manning, Ohio, and theV2agonist l-deamino-8-D-AVP was supplied by Ferring AB, Malino,Sweden.

Studies on Growth of MCF7 Cells. Cells in stock culture were grown

2The abbreviations used are: HPLC, high performance liquid chromatography;DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; /?„„,maximum number of binding sites; AVP, arginine vasopressin; d(CH2)5Tyr(Me)2AVP, d-(CH2), tyrosine(methyl)2 AVP; DDAVP, l-deamino-8-D-AVP.

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VASOPRESSIN AND OXYTOCIN IN HUMAN BREAST

until confluent and removed with trypsin-EDTA (0.5 g/liter trypsin-0.2 g/liter EDTA) and replated on 35-mm Petri dishes in DMEM with2.5% fetal calf serum at an initial density of 3 x 10* or 9 x 10" cells/

dish. After 72 h the medium was changed to one with or without theaddition of vasopressin or oxytocin. Each day the cells were removedfrom the plates using 500 nl of trypsin/EDTA with vigorous pipetingand counted using a Coulter Counter.

Studies on Protein Accumulation by MCF7 Cells. Stock MCF7 cellcultures were removed from the flasks with trypsin/EDTA and replatedonto multiwell plates (Falcon, Marathon Laboratories, London, UnitedKingdom). Cells were grown for 7 days in DMEM supplemented with2.5% fetal calf serum. The medium was changed to one without serumbut containing BSA (1 g/liter) and either vasopressin or oxytocin. After24 h the cells were placed on ice, washed once with Dulbecco's phos

phate-buffered saline (Gibco), and solubili/.cd with 500 p\ of 0.5 mol/liter NaOH. Protein concentration was measured by the method ofLowry et al. (17) using BSA as standard.

Studies on the Phosphoinositide Metabolism by MCF7 Cells. Tostudy the effect of vasopressin and oxytocin on the hydrolysis ofphosphoinositides, cells in monolayer culture were incubated with myo-[3H]inositol, stimulated with peptides and the water-soluble inositolphosphates separated from nonincorporated myo-[3H]inositol usingDowex ion-exchange chromatography, as described previously (19).

RESULTS

Oxytocin and Vasopressin in Human Breast Tissue. Normalbreast tissue (n = 5) contained oxytocin in amounts rangingfrom 16.8-86 fmol/mg protein (40.3 ±11.9, mean ±SD) andbreast carcinoma tissue (n = 6) contained 6.2-43 fmol oxytocin/mg protein (28.1 ±6.9). Values for vasopressin in normalbreast ranged from 2.7-11.1 fmol/mg protein (6.0 ±1.8) andin breast carcinoma they were 4.2-13.7 (8.5 ±2.4) (Fig. 1).

Analysis of the tissue extracts by HPLC showed that theimmunoreactive oxytocin and vasopressin behaved in a manneridentical to that of the authentic peptides (Fig. 2).

Oxytocin and Vasopressin Binding to MCF7 Cells. Binding ofboth [3H]oxytocin and [3H]vasopressin to MCF7 carcinoma

cells was found to be saturable and specific (Fig. 3). Crossoverbinding experiments showed that the vasopressin and oxytocinreceptors were specific for each hormone (Fig. 3).

Scatchard plots of the data were linear, indicating a singlehigh affinity, low capacity binding site for both hormones. Theapparent KD for oxytocin was 51.3 ±0.4 nmol/liter and theBm^ was 87,000 ±4,000 sites/cell. The KD for vasopressin was47.4 ±1.6 nmol/liter and the Bm,xwas 27,300 ±6,500 bindingsites/cell (Fig. 4).

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Fig. 2. HPLC profiles of immunoreactive vasopressin (•)and immunoreactiveoxytocin (O). Top, standard peptides measured by radioimmunoassay. Bottom,human breast tissue extracts measured by radioimmunoassay. Similar profileswere found for radiolabeled peptides.

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-20 H 1-7-1 h H—(—(—I—h102 ' 1Ó4' 1Ó6' 0 ' I ' 1Ó2 104 106o 1 1er 1CT 10° o 1Unlobelled ligand added (nmol/L)

Fig. 3. Effect of vasopressin (O) and oxytocin (A) on the specific binding of[3H]vasopressin (left) and [3H]oxytocin (right) to MCF7 cells. Means (points) ±

SEM (bars) of triplicate estimations from 5 and 3 experiments, respectively.

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Fig. 4. Scatchard plots of the data shown in Fig. 3. Left, plot for oxytocin;right, plot for vasopressin. Points, means from 3 measurements for representativeexperiments.

Ça2*and Mg2* ions had no effect on the binding of [3H]vasopressin nor [3H]oxytocin to MCF7 cells (data not shown).The KD for [3H]oxytocin binding to MCF7 cells was 49.9 ±0. 15 nmol/liter in the presence of Ca2+ and Mg2+ ions and 50.3

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VASOPRESSIN AND OXYTOCIN IN HUMAN BREAST

±0.14 nmol/liter without cations. The number of sites decreased slightly from 87,000 ±4,000 to 55,000 ±1,200 sites/cell. Studies with the V, antagonist ¿(CH2)5Tyr(Me)2AVP andthe V2 agonist l-deamino-8-D-AVP showed that the vasopressinreceptor was of the V, type (Fig. 5).

Effect of Oxytocin and Vasopressin on Cell Growth. Vasopressin at concentrations of 10, 100, and 1000 pmol/litercaused a dose-dependent increase in cell number above controlincubations at 72 h after addition of the peptides (P< 0.001; n= 4). A 3- to 4-fold increase in cell number was obtained for 1nmol/liter, whereas 10 nmol/liter of vasopressin appeared tobe inhibitory (Fig. 6). Oxytocin in similar concentrations causedan increase in cell numbers but did not become statisticallysignificant until after 72-h exposure to peptides (Fig. 6). However, the difference in response between doses was not significantly different (P > 0.05; n = 6).

Effect of Oxytocin and Vasopressin on Cell Protein. Proteinsynthesis after 24-h exposure to vasopressin was biphasic. Weobserved a maximum increase of 23% in total cellular proteinfollowing exposure to 100 pmol/liter vasopressin (P < 0.001;n = 6). Oxytocin at concentrations of 1000 pmol/liter andabove caused a significant rise in total cell protein after 24 h (P< 0.002; n = 6) but did not appear to be dose related (Fig. 7).

Effect of Vasopressin and Oxytocin on Phosphoinositide Metabolism. Vasopressin in concentrations of 10 and 100 pmol/liter caused a 23% increase in accumulation of inositol phosphates (P < 0.01; n = 6) (Fig. 8). Concentrations of 1 nmol/liter vasopressin were not stimulatory. Oxytocin at concentrations which were growth stimulatory (Fig. 6) elevated phos-phoinositide metabolism by about 30% (P < 0.005; n = 6) (Fig.8).

DISCUSSION

These studies demonstrate that both oxytocin and vasopressin are present in human breast tissue. The amounts of therespective hormones between normal and carcinomatous tissuewere not statistically different. However, oxytocin was presentin amounts 3.5-7 times those of vasopressin. The amounts ofvasopressin and oxytocin found in human breast tissue arecomparable with those found in other human tissues, such asthe human adult adrenal (18).

We and others have found oxytocin and vasopressin present

100 T

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and ODA VP (V2 agonist) (A) on the specific binding of [3H]vasopressin to MCF7cells. Means (points) ±SEM (öars)of triplicate estimations from 2 experiments.

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and oxytocin (bottom) in 2.5% fetal calf serum at the concentrations shown addedon day 3. The concentrations of peptide used are: O, none; •,10 pmol/liter; A,100 pmol/liter; A, 1 nmol/liter; D, 10 nmol/liter. Mean % change in cell numbercompared with day O (points) ±SD (bars) for 4 and 3 experiments, respectively,performed in duplicate. Error bars are not shown where they are encompassed bythe data points.

in human plasma, normally in amounts <5 pg/ml (20-23),which are much less than those found in the breast tissue of thepresent study. We were unable to find vasopressin or oxytocinin MCF7 cells.3

We have found and characterized receptors which are specificfor oxytocin and vasopressin in MCF7 carcinoma cells. Oxytocin receptors have also been demonstrated in the normalmammary gland (11, 12). Mammary tissue contains a singlehigh affinity oxytocin-binding site with a A"Din the range of 1-

2 nmol/liter and Amajlof 28-60 fmol/mg protein, in the presenceof divalent cations. Our studies have demonstrated that oxytocin receptors possess a KD of about 50 nmol/liter and a fimaxof290 fmol/mg protein. Contrary to [3H]vasopressin binding and[3H]oxytocin binding in other tissues (24-26), we have foundthat divalent cations had no effect on [3H]vasopressin or [3H]

oxytocin binding to MCF7 cells (data not shown).The normal function of oxytocin in milk ejection is associated

with the myoepithelial cells where the receptors in the normalbreast are presumably located. Indeed, Van Leeuwan et al. (27)showed fairly clear localization of [3H]oxytocin binding to

myoepithelial cells. In subfractionation studies (28) most of the[3H]oxytocin binding was found associated with the fraction

3A. H. Taylor, V. T. Y. Ang, J. S. Jenkins, J. J. Silverlight, Y. A. Luqmani,

and R. C. Coombes, unpublished results.

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VASOPRESSIN AND OXYTOCIN IN HUMAN BREAST

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Fig. 7. Protein accumulation by MCF7 cells after 24-h stimulation with eithervasopressin (top) or oxytocin (bottom) in the concentrations shown. Columns, %change in protein concentration compared to control; bars, SEM. Numbers inparentheses, numbers of data points for each treatment. 'P < 0.00 \;â„¢P= 0.002(Student's I test).

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Fig. 8. Effect of vasopressin (open columns) and oxytocin (hatched columns)on the accumulation of water-soluble [3H)inositol phosphates. Data (means ±

SEM (ears) of 2 estimations performed in triplicate) are expressed as % changein total inositol phosphates compared to cells not treated with peptide. "/'•0.01:"P < 0.005; '"P < 0.001 (Student's t test).

containing myoepithelial cells, but a small amount was boundto the fraction containing epithelial cells. It is therefore ofinterest that receptors are present on the MCF7 cell line whichis thought to be derived from malignant epithelial cells.

It is interesting that oxytocin appeared to elicit a maximalmitogenic response at concentrations well below the observed

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KO, suggesting that only factional receptor occupancy may berequired. This remains to be confirmed by using oxytocin atconcentrations below 10 pmol/liter.

Our finding of distinct vasopressin receptors for the humanMCF7 cell suggests an independent role for vasopressin inbreast function. This finding is similar to the observation thatvasopressin receptors are present in the rat WRK1 mammarycarcinoma cell (13). The binding parameters found in WRK1cells (KD, 13 nmol/liter; Amax,75 fmol/106 cells) are fairly close

to those found in MCF7 cells (ATD,47 nmol/liter; AmM,45 fmol/IO6cells).

In agreement with these authors we also have identified thevasopressin receptor in the human cells as being of the V, typeand no evidence for a V2receptor was found. However, a smallamount of residual [3H]vasopressin binding remained in MCF7

cells which could be explained by the presence of more thanone class of V, receptor.

The V, receptor is generally accepted to be linked to thephosphoinositol messenger system and the V2 receptor to theadenylate cyclase system (24, 29, 30). The vasopressin receptorfound in the rat WRK1 cell line is linked to phosphoinositolmetabolism (13, 14). Our data indicate that vasopressin andoxytocin also elevate phosphoinositol metabolism in MCF7cells, which would support our conclusion that MCF7 cellshave an vasopressin receptor of the V, subtype.

The mitogenic properties of vasopressin have been describedby Rozengurt et al. (31 ) using Swiss mouse 3T3 fibroblasts, byRussell and Bucher (32) using Brattleboro rat liver, and byPayet et al. (33) using adrenal glomerulosa cells. We have nowdemonstrated for the first time that vasopressin, and to a lesserextent oxytocin, can stimulate the growth of human breastcarcinoma cells in vitro with a concentration of vasopressinranging from 10-1000 pmol/liter. Higher concentrations (10nmol/liter) were found to be nonstimulatory; this may be dueto the desensitization of the mitogenic response by prolongedhigh dosage of the peptide previously described by Collins andRozengurt (34) for 3T3 cells. These authors ascribed part ofthis effect to down regulation of vasopressin receptors. Proteinaccumulation in MCF7 cells was also increased by 10-1000pmol/liter of the peptides, an observation similar to the reportof increased protein accumulation of rat WRK1 mammarycarcinoma cells by vasopressin (15).

The concentrations of vasopressin required to obtain theseeffects on cell growth and protein accumulation were much lessthan the dissociation constant we observed for the vasopressinreceptor. This discrepancy has been emphasized by Guillon etal. (13) in the case of the WRK1 cells and may be partly due todifferent buffer conditions in the receptor and functional studies, although it is known that the concentration of vasopressin(and other ligands which act at the cell surface) which arenecessary to evoke a response will utilize only a small fractionof the receptors present (35). There may also be other factorspresent in serum which interact with vasopressin in promotingbreast growth.

Many hormones are known to interact with breast tissue. Inthe present studies we have shown that both oxytocin andvasopressin are present in the normal and carcinomatous human breast, that specific receptors are present on human breastcarcinoma cells, and that vasopressin in particular can stimulatetumor cell growth. We suggest, therefore, that in associationwith already recognized stimulatory factors, vasopressin, andpossibly oxytocin, may be important modulators of the growthof some human breast carcinomas.

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REFERENCES

1. Brownstein, M. .1., Russell, J. T., and Gainer, H. Synthesis, transport, andrelease of posterior pituitary hormones. Science (Washington DC), 207:373-398, 1980.

2. Antoni, F. A., Holmes, M. C., and Jones, M. T. Oxytocin as well asvasopressin potentiate ovine CRF in vitro. Peptides, 4:411-415, 1983.

3. Page, S. R., Ang, V. T. Y., Jackson, R., White. A., Nussey, S. S.. and Jenkins,J. S. The effect of oxytocin infusion on adenohypophyseal function in man.Clin. Endocrino!., 32: 307-313, 1990.

4. Lumpkin, M. D., Samson, W. K., and McCann, S. M. Hypothalamic andpituitary sites of action of oxytocin alter prolactin secretion in the rat.Endocrinology, 112: 1711-1717, 1983.

5. Pedersen, C. A., and Prange, A. J. Induction of maternal behavior in virginfemale rats. Science (Washington DC), 216:648-650, 1979.

6. Doris, P. A. Vasopressin and central integrative processes. Neuroendocri-nology, 38: 75-85. 1984.

7. Clements, J. A., and Funder, J. W. Arginine vasopressin and oxytocin inorgans outside the nervous system. In: L. Martini and W. F. Ganong (eds.).Frontiers in Neuroendocrinology, Vol. 10, Chap. 6, pp. 117-152. New York:Raven Press, Ltd., 1988.

8. Clements, J. A., and Funder, J. W. Arginine vasopressin (AVP) and AVP-like immunoreactivity in peripheral tissues. Endocrine Rev., 7: 449-460,1986.

9. Woll, P. J., and Rozengurt, E. Neuropeptides as growth regulators. Br. Med.Bull., 45: 492-505, 1989.

10. Monaco, M. E., Lippman, M. E.. Knazek, R., and Kidwell, W. R. Vasopressinstimulation of acetate incorporation in lipids in a dimethylbenz(o)anthracene-induced rat mammary tumor cell line. Cancer Res., 38: 4101-4104, 1978.

11. Soloff, M. S., and Schwartz, T. L. Characterization of a proposed oxytocinreceptor in rat mammary gland. J. Biol. Chem., 248: 6471-6478, 1973.

12. Zhao, X., and Gorewit, R. C. Oxytocin receptors in bovine mammary tissue.J. Receptor Res., 7: 729-741, 1987.

13. Guillon, G., Kirk, C. J., and Balestre, M-N. Characterization of specific V,.vasopressin-binding sites on a rat mammary-tumor-cell line. Biochem. J.,240: 189-196, 1986.

14. Kirk, C. J., Guillon, G., Balestre, M-N., and Jard, S. Stimulation, byvasopressin and other agonists, of inositol-lipid breakdown and inositolphosphate accumulation in WRK 1 cells. Biochem., J. 240: 197-204, 1986.

15. Monaco, M. E., Kidwell, W. R., and Lippman, M. E. Neurohypophysial-hormone-responsive cell line derived from a dimethylbenz(a)anthracene-induced rat mammary tumor. Biochem. J., 188: 437-441, 1980.

16. Jenkins, J. S., Ang, V. T. Y., Hawthorn, J., Rosser, M. N., and Iversen, L.L. Vasopressin, oxytocin and the neurophysins in the human brain and spinalcord. Brain Res., 291: 111-117, 1984.

17. Lowry, O. H., Rosebrough, N. J., Fair, A. L., and Randall, R. J. Proteinmeasurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275,1951.

18. Ang, V. T. Y., and Jenkins, J. S. Neurohypophysial hormones in the adrenalmedulla. J. Clin. Endocrinol. Metabol., 58:688-691, 1984.

19. Taylor, A. H., Whitley, G. S., and Nussey, S. S. The interaction of argininevasopressin and oxytocin with bovine adrenal medulla cells. J. Endocrinol.,121: 133-139, 1989.

20. Nussey, S. S., Page, S. R., Ang. V. T. Y., and Jenkins, J. S. The response ofplasma oxytocin to surgical stress. Clin. Endocrinol., 28: 277-282, 1988.

21. Nussey, S. S., Hawthorn. J., Page, S. R., Ang, V. T. Y., and Jenkins, J. S.Responses of plasma oxytocin and arginine vasopressin to nausea inducedby apomorphine and ipecacuanha. Clin. Endocrinol., 28: 297-304, 1988.

22. Carmichael, M. S., Humbert, R., Dixen, J., Palmisano, G., Greenleaf, W.,and Davidson, J. M. Plasma oxytocin increases in the human sexual response.J. Clin. Endocrinol. Metab., 64: 27-31, 1987.

23. Amico, J. A., Ulbrecht, J. S., and Robinson, A. G. Clearance studies ofoxytocin in humans using radioimmunoassay measurements of the hormonein plasma and urine. J. Clin. Endocrinol. Metabol., 64: 97-106, 1987.

24. Thibonnier, M.. Hinko, A., and Pearlmutter, A. F. The human plateletvasopressin receptor and its intracellular messengers: key role of divalentcations. J. Cardiovasc. Pharmacol., 10: 24-29, 1987.

25. Soloff, M. S., and Grzonka, Z. Binding studies with rat myometrial andmammary membranes on effects of manganese on relative affinities ofreceptors for oxytocin analogs. Endocrinology, 119: 1564-1569, 1986.

26. Antoni, F. A., and Chadio, S. E. Essential role of magnesium in oxytocin-receptor affinity and ligand specificity. Biochem. J., 257: 611-614, 1989.

27. Van Leeuwen, F. W., Van Heerikhuize, J., Van der Meulen, G., and Wolters,P. Light microscopic autoradiographic localization of ['Hjoxytocin bindingsites in the rat brain, pituitary and mammary gland. Brain Res., 359: 320-325, 1985.

28. Soloff, M. S., Chakraborty, J., Sadhukhan, P., Senitzer, D., Wieder, M.,Fernstrom, M. A., and Sweet, P. Purification and characterization of mammary myoepithelial and secretory cells from the lactating rat. Endocrinology,106: 887-897, 1980.

29. Bullen, D., Guillon, G., Rajerison, R. M., Jard, S., Sawyer, W. H., andManning, M. Structural requirements for activation of vasopressin-sensitiveadenylate cyclase, hormone binding, and antidiuretic actions: effects of highlypotent analogues and competitive inhibitors. Mol. Pharmacol., 14: 1006-1017, 1978.

30. Jard, S. Vasopressin: mechanisms of receptor activation. Prog. Brain Res.,(50:383-394, 1983.

31. Rosengurt, E., Legg, A., and Pettican, P. Vasopressin stimulation of mouse3T3 cell growth. Proc. Nati. Acad. Sci. USA, 76: 1284-1287, 1979.

32. Russell, W. E., and Bucher, N. L. Vasopressin modulates liver regenerationin the Brattleboro rat. Am. J. Physiol., 245: G321-G324, 1983.

33. Paye!, N., Dèziel,Y., and Lehoux, J. G. Vasopressin: a potent growth factorin adrenal glomerulosa cells in culture. J. Steroid Biochem., 20: 449-454,1984.

34. Collins, M. K. L., and Rosengurt, E. Vasopressin induces selective desensi-tization of its mitogenic response in Swiss 3T3 cells. Proc. Nati. Acad. Sci.USA, 80: 1924-1928, 1983.

35. Schroeder, B. T., Chakraborty, J., and Soloff, M. S. Binding of [3H]oxytocin

to cells isolated from the mammary gland of the lactating rat. J. Cell. Biol.,74: 428-440. 1977.

7886

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1990;50:7882-7886. Cancer Res   A. H. Taylor, V. T. Y. Ang, J. S. Jenkins, et al.   Carcinoma CellsInteraction of Vasopressin and Oxytocin with Human Breast

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