BIOLOGY

OF

REPRODUCTION

16,

Effect

200-211

(1977)

of Adrenergic

Oviduct:

Nerve

Correlation

DAVID

Stimulation

on the Rabbit

with Norepinephrine Turnover Rate’

R. KENNEDY Division

and

of

JEAN

Sciences,

University,

and

Endocrinology Department

and

M. MARSHALL

Bio-Medical

Brown

Content

Section, of Medicine,

Rhode

Island

Hospital,

Rhode

Island

Providence,

02912

ABSTRACT The

effects of adrenergic rabbit were correlated with hormonal environment of grouped as follows: 1 = 12 estradiol-1713, i.,.; III = 12 i.m. on final 4 days. After and the contralateral isthmus cular muscle activity during

nerve stimulation on the motility of the oviductal isthmus of the certain parameters of norepinephrine (NE) metabolism and with the the muscle. Mature, virgin, Dutch rabbits were ovariectomized and days post-ovariectomy (p.o.); II = 12 days p.o. + 8 days 0.25 pg/kg days p.o. + 8 days 0.25 ag/kg estradiol-1 713 + 2.5 mg/kg progesterone treatment one oviductal isthmus was removed for NE determinations was arranged for simultaneous recording of the longitudinal and cirstimulation of adrenergic (perivascular) nerves. Stimulation (5-50 Hz) III but only in the and tetrodotoxin was significantly ± 0.14; III = 1.42 among the three

caused contractions of both circular and longitudinal muscles in Groups II and circular muscles of castrates (I). Contractions were prevented by phentolamine but unaffected by propanolol or atropine. The NE content (nmoles/isthmus) lower in castrates than in hormonally-treated animals (I = 1.03 ± 0.11; II = 1.55 ± 0.10), but the concentration of NE (nmoles/g wet weight) was not different groups (I = 13.43 ± 1.6; II were also significantly lower Turnover of NE was much hormone treatment. These transmitter during adrenergic layers. Progesterone, in the nervous activity is probably suggested by the respective adrenergic tone is considerably

15.54 ± 2.2; Ill 13.85 ± 2.2). Turnover rates (nmoles/g/h) for NE in the castrates (I = 0.54 ± 0.05; II = 1.29 ± 0.15; III = 1.35 ± 0.07). higher in atria than in oviducts and not influenced by castration or findin suggest that estrogen is essential for adequate release of stimulation and/or for electrical communication between the muscle concentration used here, has little additional effect. In vivo, tonic, quite low in castrates compared with hormonally-treated animals as rates of turnover of NE. However, even in the latter animals the less in oviducts than in atria.

INTRODUCTION

The

oviduct

rabbit

has

nerves

whose

an

of

Black,

The tubal

are

prein

and the

“long” smooth

of

them

in

al.,

paravertebral

neurons muscle

1965;

supply while

of

both the

The

vascular

muscular

wall

the

man

and

anatomic

higher

levels

Grant

innervation

HD-

chemical

200

well-developed

On

the

isthmus,

and

fluorescence

show

around

NE as

mucosa

are

oviduct

(Owof

the

significantly

neurotransmitter

Estrogen

increases density

the hand,

substantiate

adrenergic

especially junction.

richly

estimates

contents

the

of

other

the the

Chemical

the

is

region

and

findings of

to

layer

throughout

(NE)

general-

contrast

the

muscle

1976).

these

progesterone

in

The

and

a

circular

junction.

al.,

1974).

In

has

whose

(Owman

al.,

is sparse

vasculature.

innervated

the

et

especially

et

musculature

isthmus

norepinephrine

fibers

tubal

ampulla

longitudinal

poorly

lary-isthmic

USPHS

the the

the

in Accepted September 16, 1976. Received August 30, 1976. ‘This study was supported by 06963.

to

ampulla,

the

cervix.

“short”

of

related

the

Pauerstein

ampullary-isthmic

and

and

the

to

1966;

innervated

originating

ganglia

ganglia

ly

regula-

“long”

former

al.,

innervation dis-

1974).

so-called the

peripheral

the

(Brindin, et

the

the

and

primarily

et

adrenergic

arrangement

postganglionics,

the

including

network

Pauerstein nerves

“short”

latter

extensive anatomical

1974;

adrenergic

in

mammals

most

have implicated oviductal motility

tribution tion

of

run

the with

content

determined microscopy

ampul-

or

without

the

and by

histo-

(Brundin,

ADRENERGIC

1965;

Owman

1973).

The

electrical

et

represent

of

the

cells

Hodgson

of

et

1974;

al.,

sphincter

under

1965;

effects

the

motility of

of

the

well

as

castrates

or

estrogen

studied.

The

results

exert

renergic

actions

(Kennedy

certain

and

and

the

with

progesterone

were

that

the

ovarian

influence

on

ad-

oviduct.

account and

with Castrated

supplemented

a significant the

on parame-

muscle.

indicate

in

preliminary

given

the

correlated

stimulation

with metabolism

estrogen

hormones

unclear.

nerve

of

this

Marshall,

MATERIALS

AND

work

has

been

1976).

METHODS

The 2-3 distally

nerve

Mature, Dutch rabbits (2.0-2.5 kg body weight) were used. All animals were separated from males after weaning and were obtained from the same breeder. They were ovariectomized under pentobarbital-ether anesthesia and divided into three groups. Group 1: 12 days postovariectomy (p.o.); Group II: 12 days p.o. followed by 8 daily intramuscular injections of 0.25 1.Lg/kg estradiol-1713 in sesame oil; Group III: 12 days p.o. followed by 8 daily i.m. injections of 0.25 Mg/kg estradiol-1 7j3 plus 2.5 mg/kg/day progesterone i.m. on the final 4 days. The dose of estrogen approximates the daily (24-h) ovarian output of this hormone in estrous rabbits. Similarly, 2.5 mg/kg/day progesterone is within the range of ovarian output (24-h) of this hormone in pseudopregnant rabbits (Eaton and Hilhard, 1971; Brundin and Tab, 1972). Vaginal smears and inspection of the uterus showed that Groups II and III were estrogenor progesterone-dominated respectively at the end of the treatment periods.

Rabbits

(Perivascular) from

each

Nerve of

the

Stimulation three

groups

were

isthmus refers mm above 2-3 mm

stimulation

together horizontally

with

nutrient

solution

the

mI/mm.

The

in

analyzed for isthmuses were or biochemical

to 2-3 cm of oviduct the ampullary-isthmic above the utero-tubal

experiments

attached a 10-mi

flowed

mesentery organ bath

0.01.

It

was

CO2

and

aerated

maintained

killed

and and

both oviducts dissected from the surrounding fat connective tissue. They were placed in a dish containing oxygenated nutrient solution at room temperature and the isthmus and ampulla identified. One isthmus together with its attached mesentery containing blood vessels was cut off and transferred to the muscle bath containing oxygenated nutrient solution at 37#{176}Cand prepared for physiological experiments. The contralateral isthmus, without attached

the

isthmus

was suspended through which

continuously

composition

of

the

at a rate of 5.0 solution was (mM): 16.3; NaH2PO4 1.37;

with

95

7.7; Na2 EDTA 02-5 percent

percent

at 37#{176} C. The contractions

of the

longitudinal musculature were recorded by securing the ampullary end of the isthmus to a glass hook on the bottom of the organ bath and attaching the uterine end to a Statham force transducer. The activity of the circular muscles was monitored by recording changes in intraluminal pressure with a saline-filled, open-ended polyethylene catheter (Clay Adams, PE1O) inserted into the lumen and secured by a thread near the ampullary-isthmic junction. The catheter was attached to a Statham pressure transducer and care was taken not to overdilate the oviduct. Both force and pressure transducers were connected to a Brush ink-writing oscilbograph, Model 280. The mesentery including blood vessels and penvascular nerves immediately adjacent to the oviduct was threaded through insulated bipolar platinum, ring electrodes which were connected via an isolation

transformer Animals

Adrenergic

201

NaCI 133.4; KC1 4.67; NaHCO3 CaCI2 2.52; MgCl2 0.1; D-glucose

present nerves

here

oviduct

of

the

adrenergic

remains

a

(Brundin,

At

the

the as

OVIDUCT

was weighed and immediately below). Right and left selected for physiological

junction. For

1973;

Moawad,

function

THE

experiments. cut proximally junction and

smooth al.,

supports

1974).

environment as

and

reported

norepinephrine

rabbits

Higgs

may

adrenergic

of

hormonal

et

which

of

physiology

the

control

al., role

experiments

the

A

b)

that

capable

(Ueda

isthmus

of

show

of

1975;

et the

oviductal The

nerves

adrenergic

Pauerstein

effects

ON

mesentery, NE (see randomly

Harper,

the

innervation

oviduct

the

however,

ters

these

1976a, that

and

on

activity

the 1973,

Paton,

suggestion

time,

Bodkhe

studies

a functional

influencing

muscle

in

1966; of

stimulation

they of

al.,

results

INFLUENCES

to

lated

for

30

msec

duration

at frequencies was begun mm during muscle was

between

a stimulator.

seconds

with

The

nerves

rectangular

were pulses

stimuof

0.5

and

1.5 mA (supra-maximal) intensity between 5 and 50 Hz. Nerve stimulation after an initial equilibration period of 30 which time the resting adjusted to 0.5 grams.

stimulation

periods

was

tension The

about

of the interval

5 mm

and

always after the effects of the previous stimulation had disappeared. We assume that the intraluminal pressure changes reflect alterations in the internal diameter of the oviduct which, in turn, is determined by the contraction of the circular muscle layer (Brundin, 1965). Although the open-ended catheter technique has certain disadvantages, such as the possibility of occlusion from oviductal movements or interference with tubal secretions (Daniel, 1976), the technique in the present experiments sufficed to illustrate a frequencyresponse relationship between nerve stimulation and rise of intraluminal pressure. This relationship was consistently reproducible. Furthermore, in certain instances (see Results) we could record changes in intraluminal pressure when the contractions of the longitudinally oriented muscles were abolished. Therefore, we assume here that the intraluminal pressure measures primarily the activity of circularly-oriented muscle fibers although this activity is monitored in a relatively circumscribed area and may include some contribution from the longitudinal muscle layers. The procedure was to record first the contractions

of the

longitudinal

of nerve stimulation. was inserted and

muscle

at the various

frequencies

Then the intraluminal catheter the simultaneous registration of

KENNEDY

202

AND

longitudinal was made. was markedly

contractions and intraluminal pressure If the response of the longitudinal muscle altered by the insertion of the catheter, experiment was discarded. This rarely occurred.

MARSHALL

the The reported results were taken from simultaneously recorded traces of both longitudinal force and intraluminal pressure. After determining the frequency-re-

transducer. The resting tension was adjusted 0.2 gram. After an equilibration period utes, cumulative dose-response curves increasing amounts of norepinephrine structed for both circular and longitudinal The response was taken as the average muscle tension and normalized in terms of

sponse

normal nutrient solution, propranolol (3 X 106M) was added to the perfusion medium. About 30 mm later (when the inhibitory response to isoproterenol, 106M, was abolished) the frequency-response relation was again measured. At the end of some of the experiments the response of the muscle to transmural stimulation (rectangular pulses, 0.5 msec duration supra-maximal intensity 30 mA) was checked in order to compare the effects of extrinsic and intrinsic nerve stimulation. The magnitude of the muscle response to both types of stimulation was essentially the same and was prevented by phentolamine (5 X 10-6M) or tetrodotoxin (10-6 gm/mI) but unaffected by hexamethonium (5.5 X 10-5M) suggesting that postganglionic adrenergic neurons were activated in both situations. In a separate set of experiments the effects of

of the

extrinsic

that prevented the inhibitory effects of maximal doses of isoproterenol). Thirty mm later a second dose-response curve to norepinephrine was determined. Propranolol was then removed from the bathing medium and when the inhibitory response to isoproterenol returned, a third dose-response curve for norepinephrine was determined. The “control” curve was taken as the average of curves one and three.

relationship

solution 10-5M, uptake In

nerve

on

stimulation

were

tested

first

in normal

and then in the presence of cocaine, 3 X a concentration that inhibits the neuronal of NE in the isthmus (Johns and Paton, 1975). all experiments the response to nerve stimula-

tion was taken as the area under the contraction curve measured with a compensating polar planimeter calibrated in terms of mg-mm for the force traces and mm Hg-mm for the pressure traces. The area was measured for a 1-mm period at the beginning of nerve stimulation. The baseline was taken as the “resting” tension or pressure when the muscle was quiescent. All muscles had varying amounts of spontaneous activity and this was also integrated for 1 mm just before nerve

to

the

bath

and

registered. gram. To

The record

circularly-oriented mm long were These

uterine

maximal, the next higher dose dose-response curve took about 15 The sensitivity of the musde was period of 4 h, although desensitization at doses of NE above 6 X 10-6M.

not tested

above

wet weight dose was added. Each to complete.

to each

was mm unchanged often Therefore

over a occurred NE was

this concentration.

Cocaine (3 X 105M) was present in the nutrient solution throughout each experiment to prevent uptake of norepinephnine into adrenergic nerve terminals within the muscle (Trendelenburg, 1972; Johns and

Paton, 1975) concentration um.

After

an

dose-response and then containing

the

and thus ensure a relatively constant of exogenous NE in the bathing medi-

ED5,, values were log concentration-response

The

in a 10-mi at 37#{176}Cand

percent

CO2.

One

organ aerated

end

bath with

of

the

to a glass

resting tension contractions

of

muscles, rin cut from the remaining isthmic portion. ringlets were randomly taken from either the or ampullary ends of the isthmus. Two small

platinum hooks were inserted through the wall of the ringlets, one on either side. One hook was fastened to a glass hook in the bottom of a 10-mi organ bath and the other was attached to a Statham force

20

30-mm

propranolol

Norepinephrine

in

initial

equilibration

curve for norepinephnine the bathing solution was (3

X 106M,

obtained

period,

a

was obtained switched to one a concentration

by interpolation curves.

from

Determinations

isthmus

was weighed

and

immediately

placed

the alumina was transferred into glass columns (0.5 X 50 cm), washed with 20 ml 0.2 M NaAc and 4 ml H2O, followed by elution of the amines with 2mb 0.3

NE

vertically solution

attached

Statham force longitudinally-oriented

response

was adjusted to 0.5 the predominantly of muscle about 5

were suspended containing nutrient 95 percent 02-5 was

the

hook in the bottom of the other end was connected to a transducer, so that contractions of the muscle bundles were primarily

Exogenous

In other experiments on animals from each of the three groups, isthmic portions of both oviducts (without attached mesentery) were excised as described above. Segments 1-1.5 cm long from each isthmus

segment

When

ice-cold 0.4 M perchboric acid (PCA) and homogenized in a Virtis homogenizer (Model 60K) at 20,000 RPM for 5 mm. The homogenate was transferred to a 50-mb polyethylene centrifuge tube in an ice bucket. The blade of the homogenizer was rinsed in approximately 20 ml of ice-cold PCA and rotated at 20,000 RPM for 2 mm, and this additional 20 ml was then added to the original homogenate. Catecholamines were extracted by swirling the homogenate every 10 mm for 30 mm. After extraction of catecholamines the homogenate was centrifuged at 0#{176}C and 5000 X g and filtered through disposable Nalgene filters (0.2 M). To the filtrate was added 0.5 ml Na2 EDTA (0.2 M), 10mb NaAc (0.2 M) and 0.5 g of alumina (Sigma neutral activity), which had been prepared by the method of Crout (1961). This mixture was titrated to pH 8.3 with 5 M NH4 OH and, during 8 mm of stirring, catecholamines were adsorbed onto the alumina. After the overlay was aspirated off,

stimulation. The magnitude of the spontaneous activity was insignificant as compared to the nerve-induced activity (see legend to Fig 2). All responses were normalized with respect to wet weight of the tissue.

Response

muscle.

to about of 30 minto serially were conmuscles. increase in

ml

M acetic

NE

by

acid.

The

determining

acetic

its

acid

relative

eluate

was

fluorescence

assayed

for

against

known standards using the tnihydroxyindole procedure of Lund (1950). A reverse blank was run with each sample and fluorescence was determined with an Aminco Bowman spectrophotofluorometer at 400 nM excitation and 510 nM emission (uncorrected) wave-

ADRENERGIC

lengths.

of

Recovery

L-norepinephrine-7,83H Ci/mmol)

before percent.

as

an

NE was

homogenization.

Atria “controls”

determined DPM,

(2,200,000 internal standard

The

were also analyzed since they are

to

recovery

INFLUENCES by S.A. each

adding = 32.7 sample

averaged

for NE innervated

and by

75

served adrenergic

as

neurons whose transmitter content is not altered by ovarian steroids (Brundin, 1965; Owman et al., 1966). The amount of NE/sample was calculated and expressed as content (nmoles/isthmus) and as concentration

have

been

Estimation

(nmoles/g corrected

of NE

wet for

weight recovery.

Turnover

of

tissue).

All

results

Rate

Whole

oviducts from each animal were pooled for these determinations since the NE content of a single oviduct from castrates after several hours exposure to o-MPT was at the limit of sensitivity of our assay. The turnover rate of NE was estimated by measuring the rate of decline of this amine in the tissue after blockade of synthesis by intravenous injections of

a-methyl-p-tyrosine methyl ester according to the method of Brodie et al. (1966). After inhibition of synthesis, the amine levels decline exponentially. When the tissue NE concentration is plotted as a log1 0 function against time, the rate constant (k) for the decline in tissue NE can be determined from the slope of the disappearance curve, according to the relationship k = slope/0.434. The slope is calculated using two variable linear regression analysis. The product of k times the NE concentration at t = 0 yields the initial rate of efflux. During steady state, the rate of NE efflux equals the rate of NE synthesis, and hence is an estimate of the turnover rate of NE (Costa et al., 1972; Brodie et al., 1966). In these experiments we assume that within the time constant of our sampling the NE compartment remains at steady state and that o-MPT does not interfere with the rate of NE efflux (Costa et al., 1972). At the end of each treatment period, rabbits from each of the three groups were given an initial

intravenous injection ester (o-MPT, 0.81

of ts-methyl-p-tyrosine nmol/kg), followed

by

methyl mainte-

nance doses of o-MPT (0.41 nmol/kg) 2 and 4 h later. -MPT was dissolved in H2 0 to form a 3 percent solution, which was adjusted to pH 6.0 with 2 or 3 drops of 5 M NaOH. The solution, warmed to 37#{176}C, was injected into the marginal ear vein at a rate of 0.5 mllmin. The total volume injected never exceeded 5 ml. The rabbits were killed at 0 and 2, 4 and 8 h after the administration of a-MPT. Both oviducts and atria were excised, weighed and analyzed for NE content as described previously. Log,,, NE concentrations were plotted as a function of time after administration of o-MPT. To verify that our n-MPT dose regime blocked the synthesis of NE, L-tyrosine-’4C(U), 0.165 I2moles/kg

ON THE

OVIDUCT

203

labeled

amine 30 mm before sacrifice at the end of the 8 h exposure periods to u-MPT. After sacrifice, atria and oviducts were removed and immediately homogenized in 20 ml ice-cold 0.4 M PCA. Catechoiamines were extracted as described above and radioactive NE was partially separated from L-tyorosmne2 and

‘4C on alumina columns (Crout, 1961) and then completely separated on Dowex-50W columns (Na form) according to the method of Hughes and Roth (1974). 3H-NE of the eluate was counted by liquid scintillation spectrometry. To determine percent recovery, L-norepinephrmne-73 H (2,200,000 DPM, S.A. = 32.7 Ci/mmole) was used as an external tracer and carried through all procedures. Results are corrected for recovery which averaged about 60 percent. In untreated rabbits the radioactivity in NE (DPM/organ) were for the oviduct: castrate, 262, estrogen, 584, estrogen + progesterone, 476; for ama: castrate, 988, estrogem, 1000, estrogen + progesterone, 952 (one animal each a-MPT, the indistinguishable

group). DPM5

After both

in

from

2 and oviducts

average

and

Drugs

RESULTS

Effects

of

Adrenergic

Nerve

Representation sponses each

of of

the

Fig.

1.

the

three

trates

isthmus to three groups most

The

only but

that

the

incorporation

is

the

control animals were and killed 30 mm later.

injected

Treated

with labeled animals were

tyrosmne

given

the

atropine

(4.3

the

refor in

among stimulation

muscle

in

of

the

both

caslongi-

muscle in the hormonallymagnitude of this response

blocked (3 x

x

nerve

contraction

and circular animals, the

of

difference

that

circular

causes

effects were phentolamine

traces

nerve stimulation of rabbits appear

prominent

groups

activates

Stimulation

polygraph

the

being essentially the same estrogen + progesterone-treated

showed

of

Chemicals

Preliminary

experiments

counts

Drugs used were L-norepinephrine HU, DL-alphamethyl-p-tyrosine methyl ester HCI (Sigma Chemical Company, St. Louis, Mo.); estradiol-17j3 (Calbiochem, San Diego, Cal.); progesterone, hexamethonium chloride (Nutritional Biochemicals Corporation, aeveland, Ohio); phentolamine HC1 (Ciba Pharmaceutical Company, Summit, N. J.); L-propanobol (Ayerst Laboratories Incorporated, New York, N.Y.); atropine sulfate (K & K Laboratories, Inc., Plainview, N.Y.). Chemicals used were L-norepinephrine-7,8-3 H (32.7 Ci/mM), L-tyrosine-’ 4C(U) (380 mCi/mM) (New England Nuclear, Boston, Mass.); L-tyrosine sodium-pentahydrate (Nutritional Biochemicals Corporation, Cleveland, Ohio); aluminum oxide, neutral (Sigma Chemical Company, St. Louis, Mo.). Statistical evaluation of the results was done using Student’s t test to determine the significance of differences between groups of animals following group comparison analysis.

tudinal treated

amount of labeled tyrosine into NE increased linearly over a period of 60 mm in untreated animals from each of the three groups. Therefore, untreated

background

to were

20 DPM/sample.

(S.A. = 138 MCi/Mmol) was injected into the marginal ear vein of untreated rabbits and rabbits given cs-MPT.

of this

8 h exposure and atria

by 106M)

10-7M)

in the the but

estrogenrabbits.

and These

alpha antagonist unaffected

indicating

that

by

they

KENNEDY

204

CASTRATE

a

I

a

I

AND

MARSHALL

ESTROGEN

ESTROGEN

#{163} PROGESTERONE

-__

20

PPS

I MINUTE

FIG.

1. Typical

oviducts

from

polygraph

each

of

traces

three

groups

of contractions of

pulses/sec for a period of 30 seconds. in terms of mm Hg intraluminal pressure.

were

mediated

by

epinephrine.

neurotransmitter

Furthermore,

hexamethonium the that

the

the

x

(5.5

nerve-mediated the postganglionic

of longitudinal and circular muscle in the isolated isthmus of rabbits in response to perivascular nerve stimulation at a frequency of 20 Longitudinal contractions measured in terms of grams tension and circular

10-5M)

contractions, adrenergic

stimulation.

nor-

ganglion

The

blocker

did

not

alter

absence

layer

of

reduction

suggesting neurons were

muscle

since

direct

A summary of the results from 5 similar experiments in each of the three groups appears in Fig. 2. The magnitude of the responses of

to that of hormonally-treated

and increasing

stimulation

the

In the a

in

castrates

similar

did

not

circular muscle layers frequency of nerve

hormonally-treated

the

circular

muscle

fashion

but

the

longitudinal

contract

at

any

frequency

tors

to

since

in nerve

exogenous

lower

NE

a

frequencies.

it

or elicequal

was

in

not

beta-adrenocep-

response

increased 50

the

was

(propranolol, the

being most At

muscle

Also

the

a

this

to 10-7M) the

of

significantly stimulation,

to

of

muscle

blockade

to influence

due

magnitude

longitudinal groups.

predominance

beta

(106 of

whose

the

longitudinal

not

ability

stimulation

failed

Cocaine to nerve

muscle

in the was

contractile

contraction

10-6M)

responds of

a

related

groups.

the

electrical

ited

response

castrates

in

activated.

both longitudinal increases with

of

the

3 (Fig. response

effective

Hz

x 2).

there

at the was

no

#{149} LONOITUOINA,

o ISTROORN

0

40

10

ESTROGEN

40

LONGITUDINALOP8OP

#{149} CIRCULAR o CIRCULAR+P5Op

PROGESTERONE 8

-

32i’

E

8 E

2

24

6

l6

Z4

08

0

20 H,

FIG.

0

10

20 H,

H,

Effects of various frequencies of nerve stimulation on the longitudinal and circular muscle contractions of the isthmus of the rabbit oviduct. Propranobol, 3 X 106M. Magnitudes of the spontaneous contractions (not graphed) for longitudinal muscle were (mg-mn/mg tissue): castrate = 0.40 ± 0.01; estrogen = 0.65 ± 0.10; estrogen and progesterone = 0.60 ± 0.30. For circular muscle (mmHg/mg tissue): castrate = 0.015 ± 0.002; estrogen = 0.038 ± 0.012; estrogen and progesterone = 0.020 ± 0.01. Means ± SEM of 5 experiments each group.

2.

2

ADRENERGIC

INFLUENCES

ON THE

OVIDUCT

cc

‘0 I/

WI

A

#{149}

0

t:en

en

CC

205

e-l

r

T’4

V

C 0

V 0

#{149}

0.

‘4 Q 0

0L)

*

r’ien

*1

0’

T”2

-I-I

-H

;-1zi-

T-

©. -

II

‘-‘s -

-I-I -.-

-4+1

+

+1

+1

V 0

‘4 -

0

0

z

‘fI

+1

‘0C

-

4 *1

N

N

IfSWI

C

‘-4 +I

+1

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en

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KENNEDY

206

potentiation. the

The

circular

in

agreement

more

potentiation for

than with

dense

(Owman

anatomical

al.,

longitudinal

muscle

activated

and

of

by nerve

the

the

a

pharma-

was

(Table

by along

parable

values

not

The

and Response

to Exogenous

The

reduced

muscle

to

nerve

due

to

may

be

that

muscle

in

a

cles

for of

these

the

of

exogenous

experiments,

and

from

in

each

the

The of

dose-re-

NE

cocaine

present

castrates

sensitivity

circular

of (3

were

the

x

bathing

groups. 10-5M)

medium.

cle

the

were

mus-

and

10-7M

The

(estrogen),

10-7M

±

changed

of

mus-

propranolol

x

1.5 ± 0.9

ED50’s

for

x

3.5

± 2.0

10-7M

(estrogen the

circular

propranolol

10-7M

and

longitudinal

10-7M

of

1.0

(estrogen

(castrate),

(castrate),

x

±

significantly For

x

The

4.0

0.8

x

2.5

10-7M

group.

presence

presence 1.5

x

± each

not

2.0

in the

x

circular,

3).

progesterone).

was

x 10-7M (estrogen

the 2.5

the

(Fig.

(castrate), 2.0

for

± 0.4

(estrogen),

muscle

In

x

2.0

0.4

groups

muscle

10-7M

in

‘5

the

± 1.0

were

as

longitudinal

10-7M

(Fig.

ED5

groups

dose-response by the com-

x

6 for

=

responses

propranolol

±

x n

by

con-

± 1.5

2.5

the circular

three

among the

(castrate),

2.5

and

the

ED50 for

0.8

progesterone);

Therefore,

longitudinal

isthmus

experiments

always

the

the

neurotransmitter.

to the

longitudinal

in in

series

curves

structed

the

stimulation

to the

for

(estrogen), of

a reduction

separate

sponse

NE

response

of

progesterone); ±

essentially

position of the dose axes and

ED50’s

10-7M

was

longitudinal

each

the the

were 3.0 x 10-7M ± 0.6 (estrogen),

1).

NE

the

from

indicated curves 3).

exogenous

both

in

muscles

Paton (1975). however, the

castrates

to

same

layer

the

with

stimulation

sensitivity

for

showing circular

Johns and of cocaine,

MARSHALL

response

studies of

1966)

studies of the presence

greater

longitudinal

innervation et

cological Even in

was

the

AND

were 10-M

4.0 ±

(estrogen

1.0

and

CIRCULAR 20 LONGITUDINAL ESTROGEN

16

16 ESTROGEN #{163} PROGESTERONE

12

#{163} PROGESTERONE

12

48

8

01

4

a

16

1-’

06

‘

12

-

12

S ------

ESTROGEN

8

8

z0

/

ESTROGEN

a-

4

--1> 0 16

4

:

12

CASTRATE

2’ ------9-

9

8 -Log

7 M

FIG. 3. Response L-norepinephrine with bathing medium during

NOREPINEPHRINE

of

6 CONCENTRATION

5

9 -Log

8 M

7 NOREPINEPURINE

longitudinal and circular muscles of the isthmus of and without (x-x) propranolol, 3 X 106M. Cocaine, all experiments. Means ± SEM of 6 experiments each group. (0--

-0)

6 CONCENTRATION

the rabbit 3 X 105M,

oviduct present

5

to in

ADRENERGIC

O

9 C

INFLUENCES

ON THE

OVIDUCT

progesterone);

en

*

.

*

_

*

+4

+1

+

n

207

6 for

=

Norepinephrine

each

Content

group.

and

Concentration

‘CtSen

-z o

and

2 lists the concentrations

0’

three groups reduces the hormonally-treated

o

changes

+1

those

u C-i

Table NE

NE

contents, in isthmuses

wet

weights from the

of animals. Castration significantly content of NE below that of groups. However, since

the the

0’NQ’

.

+1

9 0

+1

CC C-I ‘COt-I

N

wet

NE

difference

.0 C

in

for

In

,

atrial

C-I

WI

in

the

tissue

was

concentration was

contents,

among

paralleled

no

significant

among

there

NE

concentrations

CS

of

there

muscle

difference .9

NE

in

groups.

-

weight

content,

the

no

significant

tissue

the

three

weights

three

groups

or

(Table

2).

000

.0 +1+1+1

0’

WI

NE

CC

Efflux

and

Turnover

Rates

9 00

000

The

‘4

rate

tissue ‘4-

NE

synthesis

O ‘0

-

‘0

0

9

0

..‘

C-I

+1

+1

*1

en . *WICC ee

WI

was

from treated

WI

constant

were

the

decline

inhibition

reduced

compared (Fig. 4).

of

efflux

castrate

the

after

significantly

castrates animals

and

for

(K)

concentration

with The

in

the rates

NE

oviducts

hormonallyof turnover

neurotransmitter

significantly

of of

in

lower

the

than

those

of

There

was

no

of

NE

C

the

‘-4

hormonally-treated

groups.

0

o

significant

difference

in

the

rate

constant

.9

efflux 0. *

* .

0 ‘0

*

gen-

0

animals.

or

NE

and

the

Similar

0 © +1+1+1

en

WI

,-

..

same

turnover

rate

estrogen

between

and

in

showed

estro-

progesterone-treated

determinations

rabbits

the

atria

no

significant

either

rate

from

these

difference

O.c .

among

,-

the

efflux

C

groups

in

or turnover

rate

(Fig.

constant

of

NE

5).

.9 ,_

SI

DISCUSSION

-

*

U C

Our

0 U

‘0 C ‘4

results

show

that

adrenergic

nerve

stim-

+1+1+1

0 be

-

ulation and

‘

causes circular

oviducts V

0

U C-i C

en en

--.4*

-

U C

V

V

C

‘4

2 U

0. + U

V

0

0

bObe

c.

00

2 ‘I

U

the longitudinal isthmus of

the

+ C

U :12

C ‘4

V

estrogen-

and

rabbits,

but

in castrates. findings for

the

estrogen-progester-

only

of

the

circular

hormonally-treated

ani-

agree with those of previous workers who showed that in the oviduct the neurotransmitter, norepinephrine, stimulates primarily alpha

C V

‘ *

adrenoceptors

in

one-dominated

both

states

estrogen-

or

(Higgs

progester-

and

Moawad,

0.

1973;

Although

Hodgson

et

the

at.,

activity

1973;

of beta

Paton,

1976a,

b).

(inhibitory)

re-

U

00

2

“2’-

.00

i-

C

.

0

cc

of

mats v

0

C-i

the

U

-

.0 0.

C ‘0 C

from

one-treated muscle The

V

C

a contraction muscles in

0

000

* *

ceptors has

may little

oviduct Hodgson

be affinity

(Hodgson et

al.,

increased for

and 1973).

by

progesterone,

these

NE in

receptors

Pauerstein,

1974,

Therefore,

the

the

1975; response

KENNEDY

208

AND

MARSHALL

CASWATETATE

CASTRATE

201

01

K

0053±

US

066

0 54

K

r

0087±011

US

r

161

h,1

nmol/g/hr

±017

o,e’oI/g/h,

±005

I

I

I

I

I 20

K

ESTROGEN

10

ESTROGEN

K

13

0 123k

TR

I 29

032

hr1

0118±026

TR

191

,moI/g/hr

hr1

nmol/g/h,

±036

±01S

ESTROGEN

& PROGESTERONE

20 ESTROGENS

PROGESTERONE

10 K

10

0146±0013

r

± 00 7

I

L 0

FIG.

L 2

4

t

t

4. Decline

6

8

cMPT

=

of oviductal

norepinephrine

(NE)

after an intravenous injection of o-methyl-p-tyrosine (ci-MPT). 0.81 mM/kg at t = 0, 0.41 mM/kg at 2 and 4 h. K = rate constant of concentration

at

various

0101±

TR

1.3SomoI/g/hr

TR

Kr

-

times

TR = turnover rate for NE. Each point mean ± SEM of 4 animals. For K, castrate vs. estrogen, P