Comparison of cumulative and individual dose-response curves on the rat uterus of neurohypophyseal peptides

Pharmacological

Research Communications,

81

Vol. 8, No. I, 1976

COi‘lPARISON OF CUMULATIVE AND INDIVIDUAL

DOSE-RESPONSE CURVES ON THE RAT

UTERUS OF MEUROHYPOPHYSEAL PEPTIDES*

Roderich

University

Walter

and M. Wahrenburg+

Department of Physiology of Illinois at the Medical Center P. 0. Box 6998 Chicago, Illinois 60680, USA and

tCharles Received

28 August

SUMMARY

1975

The dose-response

:

neurohypophyseal

hormones

analogs

was compared

dure

the

or

method

tides

only

hypophyseal

on the *

This

work

This

was supported

studies

and synthetic

or inhibiting

analogs.

caution

of our

hormones

potentiating

response

observed effect phenomenon

isolated

cumulative method. it

the

this

category

certain

doubts

uterine

peptides

of pep-

effects

on

tissue exert

a

on the hormones as to

No. AM-18399.

the

of

cumulative

of neuro-

on rat

on themselves,

by USPHS Grant

the

studied.

on the action

the

proce-

by the adverse

analogs

of

and a number

Although

peptides

created

uterus

dose-response

can with

that

rat

vasopressin

as revealed

of some of

we repeatedly

in vitro,

the

advantages,

activity course

lysine

either

dose

be used with

In the

on the

oxytocin,

has certain

intrinsic

behavior

using

individual

assay

the

Pfizer, Inc., Research Laboratories Easton Point Road Groton, Connecticut 06040

general

or

Pharmacological

82

applicability analog

of

might

additively. tration

cumulative

have

an effect

This

effect

would

where this

number

of neurohypophyseal

curves

obtained

each

by the

cumulative

normal

doses

significant is

were

method

where

at

the

determined.

t!le

concen-

In order curves

compared

with

Vol.

administered

dose-response

peptides

with

to

of a

dose-response

draining

and washing

after

Sprague-Dawley

rats

ob-

injection.

MATERIALS AND METHODS: tained

from

180 to

200 g.

vaginal were

be most activity

the

tecllnique

on subsequent

intrinsic

point

Communicaticns,

dose-response

also

range

investigate

the

Research

Marland

smears

Farms,

The stage

a 10 ml overflow fluid

recorded

with

the

at

To obtain

dose-response individual

the

corresponding

was set

at

P values

of

the

(OT)

acidloxytocin

(Manning,

in

springs

tension method

of

1 g.

as well

increasing

activities

obtained

by the

The limit

crystalline

(deamino-oxytocin,

bath

conjunction

(FT03C,

statistically

1968),

in

Contractions

bath.

geometrically

obtained.

1960)

the ambient

cumulative

in

of van Rossum

in intrinsic

evaluated

(Munsick,

to a baseline

procedure

in estrus

immediately

polygraph

the

of by

of

t-test significance

P = 0.05.

Oxytocin propionic

the

choosing

were

suspended

transducer

with

weight

Rats

by a circulating

curves

were

was determined

solution

was subjected

The differences

two methods

horns

on a Grass

doses,

(19631, was followed,

the

experiment.

force-displacement

The tissue

sequence.

the

The temperature 37'C

an average

cycle

of

isometrically

Grass

at

estrus

uterine

chamber.

removed).

as with

the

N.J.,

van Dyke-Hastings

was maintained

were

of

and the

Mg +I -free

virgin

Wayne,

on the morning

decapitated,

modified

Female

[1-B-mercaptoDe-OT)

(Ferrier

dose with and

8, No.

1, 1976

Pharmacological et al.,

Research

1965),

1968)

were

thesis

and lysine

prepared

acidloxytocin

(Walter

[1-B-mercaptopropionic

suberic

(Chiu

[4-ornithineloxytocin [1-S-mercaptopropionic (Havran,

tivated

method

peptides

tested;

equal

response Kelley,

to 1.00)

6 uterine

Each value horns

specific

tissue

compound

in terms

the

of

by Arigns

and de Groot

context.

Rudinger

this

procedure

synthesis

using

curve

injection

by the ac-

to oxytocin

of all

other

intrinsic

by the

activity

cumulative method

dose(Ghan and

the average

toward

interaction is

the

"affinity"

The cumulative

prepared

of results

6 rats.

the

1954)

were

an identical

One approach

receptor

(Ariefis,

other.

of

1970),

(De-Orn4-OT)

curves

represents

from

crystal-

1969),

dose-response

or individual

the mechanism

activity"

peptide

shows

RESULTS AND DISCUSSION: into

unpublished)

when determined

procedure

et al.,

dose-response

oxytocin

1967).

from

for

al -et -.-L)

4-ornithine]oxytocin

The cumulative

as reference

1968))

[1,6-amino-

(Yamanaka (Havran

of stepwise

was used

[l-B-selenopropionic

crystalline

-0T)

and Walter,

esters.

(set

4

acid,

Schwartz

conventional

1969).

(Orn

syn-

6-selenocysteineloxytocin

(Asu 196 -0T)

acidloxytocin

and Sano,

of peptide

and Schwartz,

acid,

et al.,

(Meienhofer

method

Crystalline

(De-Se'-OT)

(De-Se 6 -0T)

(LVP)

solid-phase

1969).

83

Vol. 8, No. 1, 1976

vasopressin

by the

(Merrifield,

line

Communications,

evaluation

or "efficacy"

and Krejcf,

(1962)

to make an experimental

with

of potency

of

its the

one hand and "intrinsic (Stephenson,

1956)

technique

introduced

dose-response plays

insight

of an agonist

on the

(1954)

gaining

an important were

the

distinction

role first

in

on

this

to adopt between

the

Pharmacological

84

intrinsic its

oxytocic

affinity

method nism

for

thetic

Somlyo

frequently

not

still

enough

analogs for

these

potentiation

with

factors

intrinsic

effects

may express

activities, method.

to remove

dose

a cumulative horn.

1967;

no detectable by either as reference

dose-response Confirming

Krejci difference

in

method in

the

and this

subsequent

assay

by the

cumulative behavior

curve

compared

(tissue

sufficiently with

each

of oxytocin

studies we found

dose-response

studies.

on

dose-response

to rest

hormone

con-

dose-response

curve

1967),

in

effect

earlier

et al -2)

possible

a significant

and allowed were

oxytocin

neglected.

dose-response

effect)

ap-

and often

of

that

hormone,

themselves

a cumulative

dose"

each

the

when obtained

where

any residual

same uterine

have

fact

Although

particular

A comparison

and an "individual after

the

understood

e.g.,

curve

was washed

under

however,

1) was performed,

used

which

the

between

extensively,

too well

of

activities

However

in an analog

profile.

are discussed

(Fig.

tained

response

1972).

to the

organ

Bentley,

Walter

al -et 2)

may result

target

in intrinsic

might,

dose-response

Kelley,

the

syn-

1964;

1967;

is given

hormone

or inhibition are not

and with

the

has a different

and its

These

of

the mecha-

al -et ---23

Budinger

consideration

change

differences

ditions

1970;

and

this

and their

Chan and Kelley,

Altura,

time

studying

Krejci

Vol.

peptide

that

hormones

example,

1967;

interacts

which

parent

for

1969;

chemical

which

Since

tissue.

by many researchers

et al.,

1968,

et al.,

bases

uterine

(see

Communications,

of a neurohypophyseal

of neurohypophyseal

analogs

1965;

but

the

has been used of action

each

activity

Research

(Chan with

on the and

oxytocin

pattern

was therefore Both

other

procedures

ob-

8, No.

7, 1976

Pharmacological

Research

Communications,

Vol. 8, No. 1, 1976

De-OT

85

De-Se6-OT

De-Orn4-

32xlO%O

De-Se’-OT

Om4-OT

DOSE

Fig.

gave

1

also

very

similar

results

for

when

the

(Table

1).

This

seems plausible

on the

reference in other

shown a definite

were

De-Se'-OT

evaluated since

no secondary

assays.

However,

De-Se'-OT,

autopotentiating

produced

lative

dose-response

doses

a higher

curves. (Yamanaka,

effect

curves On the et al.,

with

with

other 1970),

-5-

these

which

had

and Schwartz,

activity

than

effect

and a potentiating

of OT (Walter intrinsic

and

by t-tests

observed

1968),

-0T

results

De-OT,

had been

on subsequent

AS&~

(Ml

compound

effect

dose-response

AGONIST

66~16~

LVP

Comparison of the dose-response behavior obtained by the cumulative method (o---o) or the individual injection procedure (o--o) of oxytocin and a series of neurohypophyseal peptides on the rat uterus Oxytocin (OT), deamino-oxytocinl(De-OT), --in vitro. [deamino-1-selenocysteine]oxytgcin, (De-Se -0T) and its 6-seleno iso,&og (De-Se -OT), [4-orni(Orn -0T) and its deamino analog thine]-Exytocin (De-prg -OT), [1,6- aminosuberic acidloxytocin (Asu ' -OT), and lysine vasopressin (LVP).

De-Orn4-OT,

analogs

OF

‘-,

OT

during

individual hand,

cumudose

analogs

LVP and Orn4-OT,

such

as

which

have

of

0.80

0.68

1.02

0.85

0.79

De-Or*'-OT

Asu"~-OT

De-Se'-OT

Orn4-OT

LVP

+ 0.05

f 0.02

+ 0.02

f 0.03

f 0.04

+ 0.02

cP = 0.05

bCalculated

is

N.S.

- aindividual)

significant;

("cumulative

considered

from

of qhe Rmax analog is the maximum effect effect of the reference compound oxytocin.

0.87

De-Se6-OT

_.~

of

significant.

t (acumulative)*

-11.8 ~-

+29.4

+12.5

-3.4

+17.7

Centb

Activity "cumulati

-

-

---

ve --I-

Studies

and the E oxytocin eo%?s u. oxytocin

-

0 -6.8

Per

Intrinsic verSuS

Dose-Response

+9.4 --__--__

Change in a individual

or Individual

under investigation Emax analog/E,,,

k 0.01

2 0.02

t 0.03

+ 0.08

zk 0.02

f 0.02

_+ 0.06

f 0.02

agonist The ratio

= not

the

0.90

0.94

0.90

0.88

0.90

0.84

0.95

1.00

Individual

Dose

Cumulative

Rat Uterus.

from

Intrinsic Activity (a f S.E.)a

+ 0.06

1.0

Obtained Isolated

Cumulative

on the

Activities

1.02

Peptides

Intrinsic

De-OT

OT

Compound

Neurohypophyseal

Comparison

is

(N.S.)

(N.S.)

(N.S.)

PC

maximum

0.009

0.014

0.010

0.047

0.08

0.19

0.45

the

of

Pharmacological a tendency

Research Communications, to relax

concentrations

of

dose-response

the peptide

curves

We therefore response

studies

peptides,

but

maximal

level

analog,

followed

determine produce vitro pressin

in response

during

than

in response

feel

that

that

the

with

individual

the

the

true

uterus derivatives

maximal

response

this

will

and oxytocin

curves

high

doses.

of cumulative

dose-

many neurohypophyseal must be checked

concentration

washout

to a given assay

convenience

activity high

dose-response

to individual

with

intrinsic

by complete

to increasing

cumulative

may be retained

in response rat

tissue

Vol. 8, No. I, 1976

of the

compound.

the

doses effect,

tissue

is able

important

analogs

of low

for

to the

the

the

in order

In case of

be most

of

at

-in

vaso-

affinity.

REFERENCES: ALTURA, B. M. (1970). Significance of amino acid residues in vasopressin on contraction in vascular muscle. Amer. J. Physiol. 219, 222-229. ARIENS, E. J. (1954). Affinity and intrinsic activity in the theory of competitive inhibition. I. Problems and theory. Arch. Int. Pharmacodyn. 99, 32-49. -ARIENS, E. J., AND DE GROOT,W. M. (1954). Affinity and intrinsic-activity in the theory of competitive inhibition. III. Homologous decamethonium-derivatives and succinyl-choline-esters. Arch. Int. Pharmacodyn. 2, -193-205. BENTLEY, P. J. (1965). The potentiating action of magnesium and manganese on the oxytocic effect of some oxytocin analogues. 215-222. -J. Endocrinol. 32, CHAN, w. Y., AND KELLN, N. (1967). A pharmacologic analysis on the significance of the chemical functional groups of oxytocin to its oxytocic activity and on the effect of magnesium on the --in vitro and -in vivo oxytocic activity of neurohypophyseal hormones. -J. Pharm. Exper. Therap. 156, 150-158. -

to

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AND WALTER, R. (1969). Oxytoch: CHID, c., SCHWARTZ,I.L., Science 163, 925-926. Crystal data of a seleno analog. -FERRIER, B. EI., JARVIS, D., AND DU VIGNEAUD, V. (1965). Deamino-oxytocin. Its isolation by partition chromatography on Sephadex and crystallization from water, J. Biol. Chem. 240 and its biological activities. ----* 4264-4266. SCHWARTZ, I. L., AND WALTER, R. (1969). Oxytocin analogs with basic amino acid residues in positions 4 and 5: Synthesis and pharmacological properties of [4-ornithineland [Eornithineloxytocin. J. Amer. Chem. Sot. 91 1836-1840. ----_) -

HAVRAN, R. T.,

KREJCI', I., POLkK, I., KUPKOVA,B., AND RDDINGER, J. (1964). Dose-response analysis of the action of some oxytocin The effect of ions. analogues on the isolated uterus: In "Oxytocin, Vasopressin and Their Structural Analogs" n. Rudinger, ea.), pp. 117-123. Pergamon Press, Oxford. KREJ%., I., POLAEEK,I., AN! RIJDINGER,J. (1967). The action of 2-0-methyltyrosine-oxytocin on the rat and rabbit Effect of some experimental conditions on change uterus: from agonism to antagonism, Brit. J. Pharm. Chemother. --30, 506-517. = MANNING, M. (1968). Synthesis by the Merrifield method of a protected nonapeptide amide with the amino acid sequence of oxytocin. J. Amer. Chem. Sot. 90 1348-1349. -----) MEIENHOFER, J., AND SANO, Y. of [lysinel-vasopressin nonapeptide intermediate. 2997.

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MERRIFIELD, R. B. (1969). Solid Adv. Enzym. =32, 221-296.

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