Identification of Neurohypophysial

Hormones with Their Antisera

HELMUTHVoiumR and ROBERTA. MUNsICm

From the Departments of Obstetrics-Gynecology & Pharmacology, Universityof NewMexico School of Medicine, Albuquerque, NewMexico 87106

A B S T R A C T Identifying posterior pituitary hormonesin body fluids or neurohypophysial extracts was hereto-fore partially achieved by using pharmacologic potencyratios or semispecific inactivation by thioglycolate orenzymes. Production of antisera against oxytocin andlysine-vasopressin has prompted us to test their speci-ficity against lysine-vasopressin, arginine-vasopressin,arginine-vasotocin, and oxytocin. In ethanol anesthe-tized rats, antidiuretic and milk-ejection activities wereassayed for each peptide-antiserum combination after0, 30, 60, and 90 min of incubation. Results indicatethat (a) oxytocin antiserum inactivates oxytocin, butnot arginine-vasopressin, lysine-vasopressin, or argi-nine-vasotocin; vasopressin antiserum inactivates ar-ginine-vasopressin and lysine-vasopressin, but neitheroxytocin nor arginine-vasotocin; (b) ) an identifiableantigenic site exists for each hormone; (c) relativelyspecific identifications of natural neurohypophysial pep-tides are possible using antisera and bioassays; (d) thismethod is promising for identifying neurohypophysialpeptides in body fluids and pituitary extracts; and (e)active and passive immunization against oxytocin andvasopressin may increase our understanding of theirphysiologic functions.

INTRODUCTION

E.ccept for the Suina, all mammals so far studied secrete8-arginine-vasopressin (AVP) and oxytocin from theirposterior pituitary glands. In most members of theSuina, 8-lysine-vasopressin (LVP) replaces AVP as theantidiuretic hormone. Among nonmammalian verte-brates, almost all secrete 8-arginine-oxytocin (arginine-vasotocin, AVT) as well as oxytocin or peptides closelyresembling oxytocin (1).

These neurohypophysial hormones are produced inhypothalamic nuclei, transported via axons to the poste-

Received for publication 2 September 1969 and in revisedform 5 December 1969.

rior pituitary gland for storage and are released fromthere into the systemic circulation. Their basic struc-tures are very similar, differing only in the amino acidresidues at positions 3 and 8. Therefore, it is not sur-prising that they have similar pharmacologic effectssuch as antidiuretic, milk-ejection, oxytocic, avian vaso-depressor, and rat vasopressor activities, or that theseeffects are absolutely and relatively different dependingupon the structure of the peptide (2).

Naturally occurring substances other than the neuro-hypophysial hormones can cause some or all of theaforementioned pharmacologic effects. These substancesare histamine, catecholamines, 5-hydroxytryptamine,acetylcholine, angiotensin, bradykinin, and other non-specific peptides (3, 4). Hence, to identify tentativelya neurohypophysial peptide by its pharmacologic ac-tions, one must use a highly purified preparation or onewith so much of the hormone present that other con-taminating, extraneous, pharmacologically active sub-stances can be diluted out of consideration. However, solittle hormone is present in body fluids that neither ofthese techniques is entirely satisfactory or possible.

For absolute identification of a neurohypophysial pep-tide, chemical identification of all the amino acid resi-dues and their sequence are required, and ultimate proofrequires synthesis of the proposed peptide molecule anddemonstration of biological activity identical to that ofthe natural hormone (5). On a weight basis, the neuro-hypophysial hormones are the most biologically activepeptide hormones known; as little as 0.01 ng of AVPor oxytocin can be detected in suitable antidiuretic ormilk-ejection assays, respectively. Because of the minis-cule concentration of these hormones in body fluids,chemical identification of them is impossible by cur-rently known techniques.

Another approach used for tentative identification ofthe neurohypophysial hormones utilizes relatively spe-cific destructive techniques. For example, oxytocin, butnot AVP, LVP, or AVT is destroyed by chymotrypsin,

828 The Journal of Clinical Investigation Volume 49 1970

which acts at the glycinamide-leucine bond. AVP, LVP,and AVT, but not oxytocin, are destroyed by trypsindigestion, which acts at the glycinamide-arginine orglycinamide-lysine bond. Oxytocin, AVP, LVP, andAVT are inactivated by incubation with each of the fol-lowing: the oxytocinase-vasopressinase system of humanpregnancy plasma, tyrosinase, and thioglycolate. Theseagents act by disrupting the peptide bonds betweencystine and tyrosine, by the probable formation of anO-quinone group on tyrosine, and by disruption of thedisulfide bond between the two cystine half-moieties,respectively (6). Although considerable evidence re-garding structure can be adduced from such destructionexperiments, a specific identification is never possible.Also, inadequate amounts of material are present forsuch identification experiments, especially when ana-lyzing biological fluids such as blood plasma or urine.For these reasons simpler and more specific means ofidentifying neurohypophysial hormones in body fluidshave been sought. Recently, antisera against LVP (7)and oxytocin (8) have been produced in rabbits andused in immunoassays for the estimation and identifica-tion of neurohypophysial hormones.

In some preliminary studies (9-11) it was found thatthese antisera abolished the activities of their corre-sponding antigenic hormones, and it also appeared thattheir inactivating properties were rather potent andspecific. Therefore, it seemed important to evaluate thedegree of structural specificity of these antisera againstdifferent neurohypophysial octapeptide substrates.Should high specificity be demonstrated, a quick andaccurate test for tentatively identifying neurohypo-physial peptides might be achieved.

In the present study we have established that rabbitantisera prepared against LVP and oxytocin are potentinactivators of their corresponding antigenic hormones.The specificity of these antisera was demonstrated bythe absence of cross-reaction, i.e., LVP antiserum couldnot inactivate oxytocin and oxytocin antiserum couldnot inactivate LVP. Neither antiserum abolished theantidiuretic or milk-ejection activities of the structurallysimilar AVT.

METHODSBioassays. For the measurement of antidiuretic activity,

ethanol anesthetized albino rats (160-230 g) under waterdiuresis were used. The test solutions were injected intra-venously (injection volume 0.5 ml) and changes in urineflow were related to the base line flow, (average of pre-injection and postresponse flow). The lowest assayable dosein this bioassay is 2.5-5 ,uU/dose of AVP. This assayprocedure has been previously described in detail (12).

For the measurement of milk-ejection activity in ethanol-anesthetized lactating rats, a teat duct of an abdominalmammary gland was cannulated. Ipsilateral injections (in-jection volume 0.2 ml) were given retrograde via thefemoral artery and changes in intraductal pressure were

recorded. In this assay the maximal sensitivity to oxytocinis 2.5-5 ,uU/dose. The assay procedure used has alreadybeen described (9).

Hormone substrates. Natural AVP1 (200, 500, and 1000,uU/ml), synthetic LVP 2 (200 and 400 ,AU/ml), syntheticAVT2 (1 ng/ml), and synthetic oxytocin2 (50, 100, and200 ,U/ml) were separately dissolved in 0.9% NaCl-0.1 Mphosphate buffer, pH 7.4, and served as substrates for allincubations.

Inactivating agents. As inactivating agents, 0.01 ml of8-lysine-vasopressin antiserum,3 0.01 ml of oxytocin anti-serum,4 or 0.1 ml of human near-term pregnancy plasmawere used for each milliliter of substrate incubation medium.Equal volumes of normal rabbit plasma or plasma fromnonpregnant women incubated with corresponding sub-strates served as controls.

For the production of antibodies against oxytocin andLVP, the hormones are usually coupled in vitro with carbo-diimide in order to facilitate their aggregation to serumproteins and thus to enhance the formation of antibodies(7, 13, 14). Then the antigenic octapeptide is mixed with anequal volume of complete Freund's adjuvant and the emul-sion is injected in the footpads of rabbits. In general, weeklydoses of 02 mg of either oxytocin or LVP given with com-plete Freund's adjuvant for 3-6 wk will cause productionof the respective antibodies. Additional injections in 4- to8-wk intervals with similar hormone doses will maintainantibody formation and occasionally increase serum anti-body titers.

Human pregnancy plasma and plasma of pregnant anthro-pomorphous apes contain a specific oxytocinase-vasopressin-ase enzyme system which is thought to be produced in thesyncytial cells of the trophoblast. This plasma enzyme(s)increases as pregnancy advances. It is not excreted in theurine and it disappears from the bloodstream within 2-3wk after delivery. The physiologic function of this enzymesystem is not understood (6).

Inactivating capacities of these agents are shown in Figs.1 and 2, indicating that 0.01 ml of either. vasopressin or oxy-tocin antiserum was able to inactivate 200 ,U/ml of AVPor oxytocin, respectively, within 15-20 min; 0.05 ml ofhuman pregnancy plasma was able to inactivate the anti-diuretic effect of 200 ,U/ml of vasopressin within 15 minof incubation. With 200 AU/ml of oxytocin as incubationmedium, 0.1 ml of human pregnancy plasma was able todestroy the milk-ejection activity after 60 min of incubation.

Incubation experiments. After 30, 60, and 90 min incu-bation of the various mixtures at 37°C, they were testedfor antidiuretic and milk-ejection activities. These activities

'Purified bovine 8-arginine-vasopressin (52 pressor units/mg) was kindly supplied by Dr. F. Armstrong, Parke, Davis& Co., Detroit, Mich.

2 Synthetic 8-lysine-vasopressin (270 pressor units/mg),synthetic 8-arginine-oxytocin (245 pressor units and 200milk-ejection units/mg), and synthetic oxytocin (450 milk-ejection units/mg) were kindly supplied by Sandoz, Inc.,Hanover, N. J.

s8-Lysine-vasopressin antiserum from rabbits was pre-pared after the method described by Permutt, Parker, andUtiger (7) and was kindly supplied by Dr. R. Utiger,Department of Medicine, Washington University, St. Louis,Mo.

4Oxytocin antiserum from rabbits was prepared after themethod described by Gilliland and Prout (8) and was kindlysupplied by Dr. R. Bashore, Department of Obstetrics andGynecology, University of California at Los Angeles, Calif.

Identification of Neurohypophysial Hormones 829

0----o VASOPRESSINANTISERUM O.Olml0-no PREGNANCYPLASMA 0.05ml0-e* PREGNANCYPLASMA 0.025 mlAnd SODIUM THIOGLYCOLATE 0.05M

%VASOPRESSIN _INACTIVATION 1

60

1000 20 40 60 80 100 120 140 160 ISO

INCUBATION TIME- MINUTES

FIGURE 1 Vasopressin inactivation (rat antidiuresis assay). Inactivation of bovinearginine-vasopressin, 200 /AU/ml by vasopressin antiserum, near-term pregnancyplasma, and thioglycolate. Each point represents the mean ±SE from four to seveninjections in different rats.Per cent of vasopressin inactivation =

E l - Antidiuretic activity after incubation X 100Antidiuretic activity without incubation J

were compared with those of similar solutions of zero timeincubations, i.e., inactivating agents and substrates keptseparately at 370C for 30-90 min were then mixed togetherand immediately injected. Antidiuretic and milk-ejectionactivities measured at zero time incubation were defined

0

%OXYTOCININACTIVATION

arbitrarily as 100% and the activities obtained at differenttimes of incubation with different agents and media wererelated, percentagewise, to this base line value. Only zerotime activities of corresponding AVP/oxytocin-antisera mix-tures were 10-15% less than the controls with normal

0*- OXYTOCIN ANTISERUM 0.01 ml

- ° 0 PREGNANCYPLASMA O.I ml

60INCUBATION TIME - MINUTES

FIGURE 2 Oxytocin inactivation (rat milk-ejection assay). Inactivation of synthetic oxytocin,200 ,uU/ml by oxytocin antiserum and near-term pregnancy plasma. Each point represents themean ±SE from three to four injections in different rats.Per cent of oxytocin inactivation =

I _ Milk-ejetion activity after incubation X 100L Milk-ejection activity without incubationX

830 H. Vorherr and R. A. Munsick

151

INTRADUCTALPRESSURE 5

cMH200

14OxlJ t f to.!.2ng t0.2ngVASOPRESSINVa ncnMotocin OMYTOCIN+0X.0 ml +001 ml +O0lml +Oolml +0.1 mlNORMAL OXYTOCIN A hl V n PREGNANCYRA88IT ANTISERUMANTE A PLASMAPLASMA

FIGURE 3 Identification of vasopressin and vasotocin (rat milk-ejection assay). Identi-fication experiment with arginine-vasopressin and arginine-vasotocin as substrates andoxytocin antiserum, lysine-vasopressin antiserum, and near-term pregnancy plasma asinactivating agents. Vasopressin antiserum destroys the milk-ejection activity of vaso-pressin but not that of vasotocin. Oxytocin antiserum cannot block the milk-ejectionactivity of vasopressin. Pregnancy plasma eliminates the milk-ejection activity of vaso-tocin. Vasopressin has milk-ejection activity equivalent to 10-20%o of oxytocin on a

molar basis.

plasma. Also, solutions tested before and after incubationwere always compared with similar antidiuretic and milk-ejection effects elicited with AVP and oxytocin standardsolutions, respectively. Administration of different inacti-vating agents as contained in the various incubation solu-tions did not influence the response to subsequent injectionof AVP or oxytocin standards. The latter hormones werealso incubated on each assay day with their respectiveantiserum and tested in order to ensure the persistingpotency of the antisera.

25*U AVP 0.5ng VASOTOCIN 0.5nc+0. lml NORMAL +0. lml NORMAL +0.11RABBIT PLASMA RABBIT PLASMA PREG

+20

+101-

In another series of experiments 1 ml of a 1000 uU/mlsolution of AVP was incubated with 0.1 ml of vasopressinantiserum and 1 ml of a 1.0 ng/ml solution of AVT wasincubated separately with 0.1 ml of either vasopressin oroxytocin antiserum.

Characterization of neurohypophysial hormone-like ac-tivities in biologic fluids and neurohypophysial extracts. Inthis final part of the study the practical application ofidentifying neurohypophysial peptides was investigated. (a)Cerebrospinal fluid (CSF) was obtained from a dog anes-

g VASOTOCIN[ml HUMAN -GNANCY PLASMA

0.5ng VASOTOCIN+QIml VASOPRESSINANTI SERUM

0.5ng VASOTOCIN+0.lml OXYTOCINANTI SERUM

% CHANGE -10IN -20

URINE FLOW-30

-40

-50

-60

-70

-80

-90

FFI 1hFHFTTTl1FFFlT1lT FFT1lFTFlT1T1TIME - 2 MINUTE INTERVALS

FIGURE 4 Identification experiment with vasotocin (rat antidiuresis assay). Incubation studywith arginine-vasotocin as substrate, antisera and pregnancy plasma as inactivating agents. Theantidiuretic activity of the different incubation mixtures is compared with bovine arginine-vasopressin. Antisera incubated with vasotocin did not alter the hormone's antidiuretic activitywhile after incubation with pregnancy plasma the octapeptide was inactivated-.

Identification of Neurohypophysial Hormones 831

TABLE I

Inactivation Studies with Vasopressin, Vasotocin,Oxytocin Antiserum (0.01 mi/ml of In-

(0.1 ml/ml of Incubation Medium)

Inactivating

Vasopressin antiserum

Time ofSubstrates 0 30 60 90

8-Arginine-vasopressinAntidiuretic activity: 200 p U/ml 100 <5

[8] [8](P < 0.01)Milk-ejection activity: 200 ,U/ml 100 <25

[5] [5](P <0.01)500 MAU/ml 100 <25

[2] [2](P < 0.01)

8-Lysine-vasopressinAntidiuretic activity: 200 IAUlml 100 <5

[3] [3] (P < 0.01)Milk-ejection activity: 400 IAU/ml 100 <25

[5] [5](P <0.01)

8-Arginine-oxytocin (vasotocin)Antidiuretic activity: 1 ng/ml 100 106 ±34 75 17

[6] [2](P > 0.9) [4] (P > 0.4)Milk-ejection activity: 1 ng/ml 100 120 416 112 ±23

[6] [4](P > 0.4) [4](P > 0.7)

Synthetic oxytocinMilk-ejection activity: 50 ,uU/ml 100 92 ±6 94 ±t16

[8] [4](P > 0.4) [3](P > 0.8)100 MuU/ml

200 MuU/ml

Zero time activities are considered 100%. Comparative activities at different incubation times are expressedas percentage of zero time activity ±SE. Tests of significance were performed using Student's t test; values inparentheses are considered significant if P < 0.05. When percentage value is shown as <, this indicates thatno activity was demonstrable and must therefore have been less than the threshold level in that assay. Shownin brackets is the number of animals used in each study.

thetized with sodium pentobarbital during an intravenousinfusion of acetylcholine. The antidiuretic and milk-ejectionactivities correspond to 109 ±18 ,U/ml of AVP and 69 ±8MU/ml of oxytocin, respectively. Aliquots of this CSFwere separately incubated for 30 min with 0.01 ml of oxy-tocin antiserum, vasopressin antiserum, and 0.1 ml of preg-nancy plasma and tested again for antidiuretic and milk-ejection activity. (b) Blood plasma from a dog anesthetizedwith sodium pentobarbital was obtained during hemorrhagicshock. Plasma antidiuretic and milk-ejection activities werefound corresponding to 368 ±27 MU/ml of AVP and 62+12 MU/ml of oxytocin, respectively. Separate aliquots ofplasma were incubated for 30 min with 0.01 ml of oxytocinantiserum, vasopressin antiserum, and 0.1 ml of pregnancyplasma and tested again for antidiuretic and milk-ejectionactivity. (c) A diluted rat pituitary extract with anti-diuretic and milk-ejection activities corresponding to 70±+10 MU/ml of AVP and 30 ±9 MU/ml of oxytocin, re-

spectively, was separately incubated with 0.01 ml of eitheroxytocin or vasopressin antiserum for 30 min at 37°C andthen reassayed.

RESULTSIncubation studies with vasopressin antiserum as in-

activating agent. LVP antiserum (0.01 ml) completelyblocked the antidiuretic and milk-ejection activity ofAVP and LVP after 30 min of incubation (Table Iand Fig. 3). With 0.1 ml of LVP antiserum, theactivity of 1000 MU/ml of AVP was abolished after 30min of incubation.

Neither 0.01 ml nor 0.1 ml of LVP antiserum couldinactivate the antidiuretic activity of AVT or the milk-ejection activity of AVT or oxytocin (Table I, Figs.

832 H. Vorherr and R. A. Munsick

and Oxytocin as Substrates, and with Vasopressin Antiserum,cubation Medium), and Pregnancy Plasmaas Inactivating Agents

agents

Oxytocin antiserum Pregnancy plasma

incubation, min0 30 60 90 0 30 60

100 95 ±19 100 <5[4] [4](P > 0.9) [5] [77J(P < 0.01)100 102 ±33 100 <25[6] [5](P > 0.9) [5] [4](P < 0.01)

100 92 415[3] [3](P > 0.8)100 101 ±7 100 <25[4] [4](P > 0.9) [3] [3](P < 0.01)

100 104 ±11 82 ±12 100 <5[6] [3](P > 0.8) [3](P > 0.2) [6] [7](P <0.01)100 86 47 114 413 100 <25 <25[6] [4](P > 0.4) [5](P > 0.5) [7] [4](P < 0.01) [5](P < 0.01)

100 <25 100 <25 <25[3] [3](P <0.02) [6] [3](P <0.05) [3](P <0.05)100 13 ±13 100 26 ±13 <25[4] [4](P < 0.01) [7] [3](P < 0.05) [5](P < 0.01)100 8 ±8 100 30 ±13 <25[6] [6](P <0.01) [4] [3](P <0.01) [4](P <0.01)

3-5). The complete inactivation of the milk-ejectionactivity of 1000 iU of arginine vasopressin with8-lysine-vasopressin antiserum indicates that the puri-fied beef vasopressin contained very little or no oxy-tocin. Since the milk-ejection method is sensitive to2.5-5 mU of oxytocin per dose, the oxytocin impurityof the 1000 /AU of purified beef vasopressin must liebelow 12.5-25 &U.

Incubation studies with oxytocin antiserum as inac-tivating agent. After 30-90 min incubation of AVP,LVP, and AVT with 0.01 ml or 0.1 ml of oxytocinantiserum, no changes in hormonal activities were ob-served as compared with the unincubated control solu-tions. With 0.01 ml of oxytocin antiserum the milk-

ejection activity of 50 uU/ml oxytocin was completelyabolished. In three out of four experiments with 100;&U/ml and in five out of six experiments with 200 i&U/ml oxytocin, complete inactivation occurred after 30min of incubation (Table I, Fig. 5).

Incubation studies with pregnancy plasma as inac-tivating agent. Within 30-60 min of incubation withpregnancy plasma the antidiuretic and milk-ejectionactivities of AVP, LVP, AVT, and oxytocin were alldestroyed (Table I, Figs. 3 and 4).

Characterization of neurohypophysial hormone-likeactivities in biologic fluids and hypophysial extracts.(a) The antidiuretic activity of CSF was eliminatedafter 30 min incubation with pregnancy plasma and

Identification of Neurohypophysial Hormones 833

15-

10-INTRADUCTAL

PRESSURE 5CnH2O -

0-' -

120$U a 1 !.2Zng 0.2ngOYtWOCIN * a * VASOTOCN VASOTOCIN+00I ml +001 ml +0.01 ml +0.01 ml +0.01 ml +0.01.mNORMAL OXYTOCIN NORMAL VASOPRES1SN NORMAL OXY OCINRABBIT ANTISERUM RABBIT ANTISERUM RABBIT ANTISERUMPLASMA PLASMA PLASMA

FIGURE 5 Identification of oxytocin and vasotocin (rat milk-ejection assay). Incu-bation study with oxytocin and arginine-vasotocin as substrates and antisera as in-activating agents. Oxytocin antiserum blocks the milk-ejection activity of oxytocinbut not that of vasotocin. Vasopressin antiserum does not suppress the milk-ejectionactivity of oxytocin.

LVP antiserum, but not with oxytocin antiserum. Themilk-ejection activity of the CSF was abolished byincubation with pregnancy plasma and oxytocin anti-serum but not with LVP antiserum (Fig. 6). There-fore, we concluded that the CSF contained AVP andoxytocin. (b) The antidiuretic and milk-ejection activi-ties of the plasma from a dog in hemorrhagic shockwere destroyed after incubation with LVP antiserum andpregnancy plasma (Fig. 7). Incubation of the plasmawith oxytocin antiserum did not influence the anti-diuretic or milk-ejection activities. Wetherefore assumethat the measured activities were due entirely or pre-dominantly to AVP. (c) The antidiuretic and milk-ejection activities of the diluted rat pituitary extractwere abolished only after separate incubations withLVP and oxytocin antisera, respectively. In Fig. 8 theselective suppression of the extract's milk-ejection ac-tivity after incubation with oxytocin antiserum is de-picted; after incubation with LVP antiserum, themilk-ejection activity was not significantly diminished.

DISCUSSIONChemical identification of vasopressin or oxytocin inextracted, purified, and concentrated test material is

required for absolute proof of the existence of anyneurohypophysial hormone (5). Since the content ofneurohypophysial hormones extracted from body fluidsis insufficient by present-day techniques to identify thesehormones chemically, crude biologic procedures havegenerally been utilized.

In the present study, it was demonstrated that oxy-tocin, the two vasopressins, and AVT could be differ-entiated one from the other by incubation with vaso-pressin and oxytocin antisera. However, it was impos-sible to differentiate the two mammalian antidiuretichormones, AVP and LVP, one from the other. Withthe exception of AVP, the activity of which is blockedby antibodies produced against lysine vasopressin, itwas found in confirmation of earlier reports (8, 13)that antisera against vasopressin or oxytocin do notcross-react. In addition, the structurally similar AVTresisted both of these antisera even when tenfold con-centrations of antisera were employed. Because LVPantiserum blocks the biologic activity of AVP andLVP, but not AVT or oxytocin, it seems that the3-phenylalanine moiety in the ring forms a key portionof the antigenic site of the molecule and is responsiblefor the specificity of this antiserum. When 3-phenyl-

151

101INTRADUCTAL 1

PRESSUREcmH2O 5-

tOXYTOCIN

t t t tCSF 20OU CSFafter 30min l0jU

OXYTOCIN incubation with OXYUOINOxytocin antierum

FIGURE 6 Identification of dog cerebrospinal fluid activity (CSF) (rat milk-ejection assay). The milk-ejection activity of CSF was blocked by incubation withoxytocin antiserum but not with vasopressin antiserum. This suggests that thesample contained oxytocin in measurable amounts.

834 H. Vorherr and R. A. Munsick

INTRADUCTALPRESSURE

OnH20

15-

O0-

t It t f20OU H oa 40U Hmorrhoge 4OpU

i Plasm in Plasm after 30min aeincubafton with

- anirmm

FIGURE 7 Identification of dog hemorrhage plasma activity (rat milk-ejection assay). Theplasma's milk-ejection activity is destroyed after incubation with vasopressin antiserum butnot with oxytocin antiserum. This suggests that the neurohypophysial hormone-like activityof the plasma is due to vasopressin.

alanine is replaced by 3-isoleucine as in AVT or oxy-tocin, the vasopressin antiserum fails to inhibit theirbiologic activities. On the other hand, oxytocin anti-serum is incapable of destroying AVT, AVP, or LVPindicating that replacement of the 8-leucine moiety byarginine or lysine is sufficient to prevent inactivationby oxytocin antiserum. It is probable, therefore, that akey portion of the antigenic site of the oxytocin mole-cule is represented by leucine. If these hypotheses arecorrect, then oxypressin (2) (3-phenylalanine-oxytocinor 8-leucine-vasopressin) should be inactivated by bothvasopressin and oxytocin antisera, and mesotocin(8-isoleucine-oxytocin), isotocin (4-serine-8-isoleucine-oxytocin), and glumitocin (4-serine-8-glutamine-oxy-tocin), all naturally occurring oxytocin-like peptidesin nonmammalian vertebrates (1, 2), should be re-sistant to both oxytocin and vasopressin antisera. Withthe limited number of antisera and peptides so farstudied, no sweeping conclusions can be reached con-cerning the manner by which certain amino acids andtheir locations may or may not alter antigenicity andspecificity. Nevertheless, with several more hormoneanalogues and antisera to study, these questions shourdbe readily answerable.

Use of antisera and biological assays does allow oneto conclude that, tentatively, a biologic solution con-tains a definite amount of a specific neurohypophysialhormone. As demonstrated in Fig. 8, the milk-ejectionactivity of the rat pituitary extract was eliminated afterincubation with oxytocin antiserum thus indicating thatit contained oxytocin in amounts exceeding 12.5-25'OU/ml.

The tentative identification of AVP and oxytocin inCSF by using antisera and antidiuretic and milk-ejection assays provides evidence for the first time thatboth neurohypophysial hormones can be released intothe CSF. Likewise, by incubating oxytocin and vaso-pressin antisera with plasma from hemorrhaged dogs,we have confirmed the observations of others (15)which suggest that under the stimulus of hemorrhagevasopressin but little or no oxytocin is released fromthe posterior pituitary gland into the circulation.

The rather specific behavior of these antisera suggeststheir potential use for the biologic identification ofnaturally occurring octapeptides. Despite much researchit is still not clear which oxytocin-like peptides or com-binations of them are secreted by various nonmam-malian species (1, 16). This problem and the perplex-

10-

INTRADUCTALPRESSURE 5-

cmH20

t5plU 10.2m1 PITUITARY ! 1p 0.2 mlPITIJITAR'rOXYTOCIN E 0T00C . OCYTCIN EXTRACT 1:5000DILUTED Mi 20mnDILUTEDAFER20 ml.

INCUBATIONWITH OXYTOCINANTISERUM

FIGURE 8 Identification of rat pituitary extract activity (rat milk-ejection assay). Theextract's milk-ejection activity is blocked after incubation with oxytocin antiserum butnot with vasopressin antiserum. This suggests that this sample contains oxytocin.

Identification of Neurohypophysial Hormones 835

ing, controversial finding of AVT in extracts frombeef pineal glands (17) may be assessed and reevalu-ated by utilizing different antisera combined with phar-macologic studies.

Utilizing suitable extraction and assay techniquescombined with antiserum identification studies it seemspossible now to follow more accurately the physiologyand pathophysiology of neurohypophysial hormones.Also, immunization of experimental animals againstneurohypophysial octapeptides may increase our knowl-edge in this respect. Thus, Miller and Moses (18) haverecently found that active immunization of rabbits toLVP can result in anti-LVP antibody-induced diabetesinsipidus. If active or passive immunization against oxy-tocin can be accomplished in various species, it may bepossible to delineate further the effects of selective oxy-tocin depletion on the subsequent reproductive per-formance of such animals.

ACKNOWLEDGMENTSThe senior author is deeply grateful to Dr. Charles R.Kleeman, Director, Divisions of Medicine Cedars-SinaiMedical Center and Professor of Medicine, University ofCalifornia School of Medicine at Los Angeles, for hisencouragement and support of this research work.

This work was supported by NIH Grant Number1 R01 HD04028-01 and a grant from Damon RunyonMemorial Fund Number DRG1002 (Dr. Vorherr).

REFERENCES1. Sawyer, W. H. 1968. Phylogenetic aspects of the neuro-

hypophysial hormones. In Handbook of ExperimentalPharmacology. B. Berde, editor. Springer-Verlag OHG.,Berlin.

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836 H. Vorherr and R. A. Munsick