0022- 1554/78/2607-0581$02.00/0 THE

JOURNAL

OF

HISTOCHEMISTRY

AND

© 1978 by The Histochemical

Copyright

Vol. 26, No.

CYTOCHEMISTRY

Society,

IMMUNOCYTOCHEMICAL

EVIDENCE

FOR

MONA Department

7,

Inc.

of Zoology,

Hebrew (MS

VASOPRESSIN

pp. 581-592, 1978 Printed in U.S.A.

RECEPTORS’

CASTEL University

of Jerusalem,

Israel2

78-146)

An electron microscopic study was made of mouse pituitaries immunocytochemically stained with anti-lysine vasopressin (LVP) as the primary antiserum in the unlabeled antibody peroxidase-anti-peroxidase procedure. Vasopressin (VP) was identified in the neurosecretory granules of the neural lobe which stained with peroxidase anti-peroxidase molecules. Electron density was induced in secretory granules of the pars intermedia (P1), both in the melanocyte stimulating hormone and ACTH cell types, probably indicating VP molecules attached to binding (receptor) sites. Omission of anti-LVP abolished staining both in the neural lobe and the P1. Anti-LVP absorbed with antigen, by admixing with LVP, abolished staining in the neural lobe but not in the P1; according to optical density measurements the P1 showed a ± 22% staining increase over controls. Staining intensity in the P1 probably reflects occupancy of binding (receptor) sites for VP. Exposure of P1 granules to LVP before the usual staining sequence resulted in ± 48% increased staining. In water-deprived mice with high endogenous VP titers, staining was ± 33% and ± 40% more intense than in normal mice. Solid phase absorbed and eluted antibodies to LVP provided additional proof that staining in both neural lobe and P1 could be attributed to anti-LYP. Results indicate that binding or receptor sites for VP are located on secretory granules in the P1. Possible physiological significance is discussed.

The

proliferation

of

studies on neurohormones has rendered illuminating insights into their precise

of secretion.

a tissue

during recent years and often unexpected origins and pathways

the tissue molecule its receptor

Immunocytochemical

neurohormones organs largely

immunocytochemical

attached

detection to receptors

has now become feasible to increasing refmement

of current

techniques,

such

of

on

and

(36-39), due sensitivity

controls,

but target

un-

A different

slant

teria

has

tiate

detection

evolved of an

antigen

in order that

‘Research Binational

supported by the Science Foundation,

Anatomy, University,

Physiology and Pharmacology, Ames, Iowa, U.S.A.

2Temporary

address:

United grant

Department

than

cri-

hinder

nous

to

not

reaction.

measurable ogenous

of Veterinary State

reaction

applied

application

cytochemical increase in states in the

simply

the

(Fig.

1).

that serves to increase may lead to enhancement. fixation, receptors may

antigen

sequent

States-Israel no. 200. Iowa

a receptor

such an antigen of attaching to time binding its

be relevant to yenA classic immunologic

may

of this

treatment cupancy histological

to substanis not

via

action; but as regards antigen molecules attached to receptor sites, the entire antigen-antibody complex may bind to receptor, so that this very procedure may serve to enhance rather

to be judged knowledge of clues may be

on immunocytochemical

necessarily

bound

Provided competent at the same

control is admixture of antigen and antibody, and subsequent suppressing of the staining re-

may also challenge organs may reveal

themselves, their validity needing in the context of our integrative neuroendocrine systems. New found to old controversies.

however,

fication

labeled antibody peroxidase-antiperoxidase (PAP) procedure. The localization of receptor sites is expected to underscore physiological and biochemical fmdings, them. Unsought for

but

(8, 36-39). is sterically site and

antibody, it is a candidate for immunocytochemical detection. The conventional experimental

target

as Sternberger’s

component,

detected

cally

(37).

tissue

so that and

on

any

sub-

immuno-

reagents may render a many-fold staining. Different physiological intact animal may be reflected

variance neurohormone

and

to the

of antibody

Indeed,

receptor ocEven after bind exoge-

in

of receptor occupancy. Exmay be injected in vivo receptors

581

Downloaded from jhc.sagepub.com at NORTHERN KENTUCKY UNIV on March 23, 2015

immunocytochemi-

582

CASTEL

Incttation

with

anti-vasopreuln

kic.AatIon absorbed

FIG.

receptor

1. Diagrammatic sites on pars

representation of possible mode intermedia (P1) secretory granules.

Specificity of receptor sites for the mone luteinizing hormone-releasing (LH-RH) has been demonstrated with antigen analogs, selected from the antiserum by sorbance, and rigorous serum even at the cost The detection

of cells. Likewise, (PD) also stained

of the (39).

deals primarily (VP) in the

both in hormone

the so-called (MSH) and

ACTH with

neurohorhormone the aid of

removal of antibodies solid phase immunoabpurification of low titer

present study of vasopressin

media (P1), stimulating

anti-

with the pars inter-

melanocyte ACTH types

cells in the pars distalis anti-LVP. Immunocyto-

chemical evidence at the electron microscopic (EM) level indicates that there are receptor sites for VP on the secretory granules of these cells. This suggests a role for VP apart from its welldocumented

renal

with anUserum

and

hemodynamic

effects.

of action Key 0

=

of vasopressin (VP) and anti-VP VP; = anti-VP.

with

specific

Unconventional receptor sites, however, prone to suspicion of nonspecific binding their validity is fortified by physiological vance. the

Granted limited

significance ACTH counts:

that only parameters of VP

cells (a)

availability the P1 via

surmise in hand, receptor

can prevail with yet the possible

sites

on

warrants consideration Circumstantial evidence of neurohypophysial peptidergic nerve

MSH on

lobe

(29).

(b)

VP

may

23). other

(c) A relevant neurohypophysial

precedent

Downloaded from jhc.sagepub.com at NORTHERN KENTUCKY UNIV on March 23, 2015

exists hormone,

in in

portal from the

potentiate

effect of the elusive hypothalamic releasing factor (CRF) on ACTH

and

several indicates

hormones (1, 2), and

fibers

the PD via the hypothalamic-pituitary system (44) as well as via portal vessels neural

are until rele-

corticotropin secretion

the (17,

in that anoxytocin,

VASOPRESSIN

also

serves

as a second melanocyte

tripeptide

IN

order prohormone stimulating

THE

26-28,

While

significance

pigmentary

control

of

acknowledged sparse knowledge tra-pigmentary effects only

factors

P1

in

and and

controlling

PT peptides

are

imperfectly understood (42). Thus the enigma the mammalian P1 remains largely unsolved, that all clues to function deserve attention. A preliminary published (8).

report

of this

MATERIALS

Animals:

albinos

School

AND

Adult

strain,

male

of so

has

been

METHODS

mice

of the

Hebrew

seven

common

and

study

were

used:

10

Mus

University

Medical

spiny

Acomys

mice,

from the Dead Sea region of Israel. Control mice were supplied purina chow and agar cubes (in lieu of water) ad libitum. Water deprivation implied

cahirinus,

reduction

of pre-weighed

Antiserum: in the

PAP

scribed ther

On

purified

two

by

and

and

the

fur-

was

PAP

Laboratories,

and

Synthetic

was

of

of stan-

measured, with

LVP

Parke-Davis,

and

Mo.

were

and

hyde,

0.1

pituitaries

AVP

from

were

Sigma

(oxytocin) and

prolactin

of Arthritis

M

phosphate

were

fixed

buffer,

in pH

on

of nuclear

modified

40

kV

LVP

or

the

chromatin

subtracted

from

was

granule

and

2.5% 7.4,

at

was was

a

Meta-

glutaralderoom

observations:

PAP molecules observed that

while (Fig.

Diseases. Standard Pitressin powder was a gift from Dr. Frederick Armstrong, Parke-Davis. Immuno-electronmicroscopy: Mice were swiftly decapitated with minimum stress between 9 and 10 and

at

superimposed

with was

ules

Cappel

bolic

AM.,

The

unlabeled

of Sternberger

excess

staining in and Acomys,

Sternberger.

Miles-Yeda,

Mich.,

Institutes

with

in granules

immuno-

Pa. or from Syntocinon

Detroit,

National

grids.

examined

cific Mus

from

Ludwig

sera

treat-

was

followed

in a Philips

AVP.

ie.

10,000

scale

of the

exposure

taken

readings Optical

as background

readings.

RESULTS

bioas-

activity

Dr.

were

10 was

called

other

nickel procedure

admixture

(25) and

rou-

dilution

established

Downingtown,

Co., St Louis,

was

density

capacity.

was a gift from PAP

Grids

Initial were

of an anti-diuretic

AVP

on PAP

yielded

a particular

or

subsequent

immunoabsorbed

anti-diuretic

LVP

all

meter in order to provide relative positive (photographic negatives give negative values).

vasopressin

of anti-LVP

aid

and

jiU/ml (for bioassay purposes far lower doses of antigen sufficed to absorb antibody activity). In one series of experiments, relative optical density (OD) of P1 secretory granules was measured from electron-micrograph negatives, projected by a photographic enlarger at constant magnification and illumination. An Olympus EMM-6 exposure meter was adapted to give readings for single granules. The window of the photoelectric cell was covered by an opaque disc perforated by a small central hole, so that a thin beam could be focused on individual granules for measuring relative OD. An arbitrary scale ranging from 1 to

col-

of the

which

the

staining

from

was

This treatment of low titer.

to

correlation

Lyophiized

gift

de-

acid.

with

of

cytochemical

Israel.

used

as previously a lysine

efficacy

extent

solutions

reasonable

from

elution

abolished

Liquid

3 weeks.

antiserum

antiserum

through

albeit

monitored

anti-LVP

Chem

this

biological

(7). The

dard

occasions

acetic

anti-LVP,

tinely say

treated

subsequent with

Titer

during

bromide-activated-Sepharose

antibodies

purified

was

passing

(LVP)-cyanogen

cubes

the primary

procedure,

(9).

umn,

agar

Anti-LVP,

Fixation

300 electronmicroscope, and photographed at a magmfication of about X 22,000. Anti-LVP was used at dilutions ranging from 1:50 to 1:3,200, generally at 1:800. Exposure of tissue to LVP, AVP, Syntocinon or prolactin before immunocytochemical staining was for 90 min at room temperature. The concentration of LVP or AVP used for this pretreatment was equivalent to 10,000 U/ml buffer (10-20 tU/ml LVP or AVP elicited an adequate antidiuretic response in bioassay rats); other substances were at similar concentrations. Anti-LVP absorption (30).

is com-

the

other

1 hr.

sections

antibody

dermal

vertebrates

significance

of PI-ACTH

musculus

thin

and vastly documented, exists concerning the exofMSH in mammals. The

monly

physiological

the

of lower

for

ment of tissue were carried out on a Penetron Rotary Shaker. Postembedding staining was done on Araldite

31-33).

the

583

INTERMEDIA

perature

for the hormone-

release inhibiting factor (MSH-RIF) that influences P1 secretion (10, 40). (d) Functional reciprocity between neural lobe and P1, associated with salt and water balance, has been reported (18-21,

PARS

tem-

Anti-LVP the

of the

caused

neurohypophysis covering most

(9). electron

On the density

adjacent

P1,

spe-

of both of the NSG same was both

grids, induced in the

it so-

MSH and ACTH cells (Figs. 2, 3). Granin ACTH cells of the PD also darkened, those in other cell types remained pale 4). As staining dissociation between MSH

and

ACTH

the

experiments

granules

was

undertaken,

ules” infers both types. At anti-LVP dilutions 1:1,600, electron density vious, even without OD

not

observed

in any

the

“P1

term

of

gran-

ranging from 1:50 to of P1 granules was obmeasurements, yet PAP

molecules were rarely discerned in the P1 (Fig. 8), although they abounded in the neural lobe (Fig. 9). Staining of the pleomorphic P1 granules

Downloaded from jhc.sagepub.com at NORTHERN KENTUCKY UNIV on March 23, 2015

584

CASTEL

I,

S. I-.

J

#{149} -

?;

.J,,

-

0#{149}

1

‘PC 0’

IL .

-4,

#{149}

-I*

-3

J.

-e FIGS.

2-9.

#{149}

4

Electronmicrographs

of pituitary

(PAP) technique. FIG. 2. Anti-lysine vasopressin (MSH)-type granules in P1 (left),

of Mus

musculus

albino,

unlabeled

antibody

peroxidase

anti-

peroxidase

FIG.

lobe

3. Anti-LVP

(right);

1:1,600.

(LVP) 1:1,600. Immunoreactive and of neurosecretory granules Immunoreactive staining of ACTH-type

staining

of melanocyte

stimulating

(NSG) in neural lobe (right); granules in P1 (left), and

x15,200.

Downloaded from jhc.sagepub.com at NORTHERN KENTUCKY UNIV on March 23, 2015

hormone

x14,400.

of NSG

in neural

VASOPRESSIN

IN

THE

PARS

585

INTERMEDIA

.. ‘1JL

‘

#{149}Pe

1’

S

-

-

I’,.-

,

.-

.b

-.

#{149}o ..,‘‘

-:

.

;:

#{149}#{149}#{149} ‘‘ #{149}#{149}lt’

FIG. peripheral

was

.-‘:

‘

4. Anti-LVP 1:1,600. array. Unstained

fairly

uniform,

*

Distal lobe gonadotroph

except

for

the

granules

described

of pituitary. ACTH cell granules (lower); x14,000.

some

ing

sites.

could

There cross-react

is scant with

granular appeared dense rim. structures

by Moriarty

Halmi (24). This unexpected electron ules was the first indication detected in the P1, presumably

.4.

-

structures in the MSH cells that doughnut shaped with an electron These may be similar to the vesicular among

:-‘-..

!#{149}

and

density of P1 granthat VP had been attached to bind-

likelihood that anti-LVP any of the several pep-

(upper) with electron dense granules From Castel and Hochman 1976(8).

granules absorbed tron that

(NSG) antiserum

in

the neural persisted

density ofPI granules the antigen-antibody

in

lobe. Yet in inducing

this elec-

(Figs. 5, 6), suggesting complex could be im-

mobilized on free VP-binding sites (Fig. 1). There may also have been some dissociation of the VP-anti-VP complex, and binding of liberated antibody receptors on only

by VP already attached in vivo to P1 granules. However, this could accounted for part of the staining intensified in comparison to that in-

have

which

was

tides claimed to be endogenous to the P1 (22). Interestingly, anti-LVP used by Silverman and Zimmermann (34) also induced darkening of P1 granules in the guinea pig, whereas anti-neuro-

duced by unabsorbed In order to determine plication of exogenous

anti-LVP. whether sequential apantigen followed by anti-

body

would

to identify

physin

sites AVP

(38), tissue before the

did

Control LVP by serum

not. experiments: buffer, pre-immune

left

both

ules unstained staining could

Substitution serum

neurohypophysial (Fig. 7), be attributed

of antinormal

or and

P1 gran-

thus indicating to the primary

that an-

also

serve

was exposed usual PAP

duced. This phenomenon it was found that

was more

(1:3,200), which produced on untreated P1 granules, visibly stronger staining ment. Neither Syntocinon

granules ules have

tin pretreatment of P1 granules

in controls that induced

Although electron

ACTH density

graneven

(Fig. 7), it is never comparable by the primary antiserum (Figs.

to 3,

Much

depended

antiserum

antigen

Our Table

admixed

(38).

Thus,

on the

control

and

absorbed

anti-LVP

was

using with

absorbed

primary excess

with

excess LVP or AVP, losing all biological activity and abolishing staining of the neurosecretory

of NSG

cannot

of P1 granules, for vious PAP molecules

the

emphasized when dilute antiserum

subsequent are noted

that

be compared

with

former demonstrate (Fig. 9), while the

by estimations

diffuse electron for each type

ules

relative

to its own

made

staining

summarized in the staining

are characterized (Fig. 8). Thus, are

Inin-

only a weak reaction was able to induce after LVP pre-treat(oxytocin) nor prolac-

enhanced by LVP.

initial observations 1. It should be

intensity

4,8).

binding LVP or by using

anti-LVP (1:800) as the primary antiserum. tense electron density of P1 granules was

tiserum used in the immunocytochemical procedure. Various method controls, leaving out goat anti-rabbit immunoglobulin, PAP or diaminobenzidine, likewise resulted in unstained in both lobes. some inherent

specific to either sequence

Downloaded from jhc.sagepub.com at NORTHERN KENTUCKY UNIV on March 23, 2015

control.

that oblatter

density of gran-

CASTEL

586

-

.5

5

S

.5 S

4) S

-

5. C

5)

-

C

#{149}

S C .C

NL C

P.S.

4.

S

#{149} -

..S-_ S

#{149}

-C

-

-

P1

-

-5

S.

4

s’

r.

*

-v.p

,.

,.

-“‘

/

--

--‘4 -C

4.-’

5’. 55

Li-.._j : .

-

4!#{149}

#{149};‘E...

r’

-.‘.‘

#{149}...

%

:

.

.

‘\

‘

e

u’

-

,

-

:

-‘

-

.

.d -

.,

,,

-

*75

-.iF

.

#{149}..p

-..

-

%f .,.

I#{149}”.

...

-

.-.

.

.

C-

:

#{149}

r. #{149}

FIG. 5. Anti-LVP Staining of MSH-type FIG. 6. Anti-LVP No staining of NSG

admixed granules

and absorbed in P1 (lower);

admixed

and

absorbed

in neural

lobe

(lower);

with excess x13,500.

with

excess

LVP.

LVP.

No

Staining

staining

of

NSG

of ACTH-type

xll,200.

Downloaded from jhc.sagepub.com at NORTHERN KENTUCKY UNIV on March 23, 2015

in

neural

granules

lobe

(upper).

in P1 (upper).

VASOPRESSIN

IN

THE

PARS

587

INTERMEDIA

-7 C

:

.5 C

S

-

C

:TL)%

-:

-

.I;-,

..

‘-S #{149}

5,. a-*

q

-

..

‘---

o

P1

---‘:-‘#{149}--

....1

-

-.: #{149}

C.

-

a

.

j_

p.. T-’t45#{149}’ .

#{149} :f-

row

FIt;. 7. Normal rabbit serum instead of anti-LVP. No immunoreactive staining. NSG in neural lobe of ACTH granules in P1 (center) with slight inherent electron density; MSH-type granules (left);

FIG. 8. Anti-LVP 1:1,600. Higher magnification of immunoreactive P1 granules; on left MSH-type reticular or doughnut-shaped structures; on right row of ACTH-type granules; x32,000. FIG. 9. Anti-LVP 1:800. Demonstrates peroxidase anti-peroxidase (PAP) molecules in neural lobe, NSG, but also some extragranular; x32,000.

(upper); x 13,000. granules

and

Downloaded from jhc.sagepub.com at NORTHERN KENTUCKY UNIV on March 23, 2015

mostly

on

588

CASTEL TABLE

Immunoreactive

Staining

Relating

to

Water

I of NSG

Various

and

Treatments

P1

Anti-

Involving

LVP

mice,

Staining Intensity Treatment

P1

NSG Buffer, normal serum Anti-LVP 1:800, 1:1600 Purified anti-LVP 1:20 Absorbed anti-LVP (excess

antigen)

Pretreated

with

Key

on the

P1 granules,

+

+

Mus

+

water-deprived tory strain.

++

none;

-

+

may

and attempts with acetic

were made to elute the antibodies acid. Such purified antibodies were

been imcolumn,

of low titer

and very labile, loosing effectiveness, biological and immunocytochemical, 2 days of storage at -4#{176}C.Nevertheless, occasions succeeded

staining with at a dilution

ing immunoreactive staining P1 granules, equivalent to 1:1,600 employed.

of the usual partly Thus, additional

NSG and of about

purified proof

is

antiserum provided

body-antigen

granules

were These

measured OD

in

be used as an index or receptor On an arbitrary scale of 1-10 ing

in the

P1 of normal

Mus

the

P1

measurements

rates

±

5.6,

of

more electron dense, not unequivocally

both

strains

of mice

is about

the

staining staining

of VP

dehydrating laboratory

deprivation,

saturated pretreatment

VP

P1. by

in the

rat

con(6,

Acomys

receptors on PT of the tissue with

with anti-LVP intensity, implying

occupancy) admixed

of and

stained the normal than unabsorbed

failed lack

water more

absorbed absorbed

to of

depVP

antiserum. with LVP

Mus PT ± 22% more antiserum, proving

complex

capable

antigen reason

followed for only

intensely the anti-

of binding

by antiserum. sub-maximal

anti-LVP

antigen

(LVP)

while

bound by free receptors. nation, it is not surprising ing of VP to the receptor

in vivo bindspecimens of same.

water

in the than

than in the laborahave proved resilient

under to the

sorbed

the OD measurements reflect this impression,

thus leading to the conclusion that ing of VP on P1 granules in normal

deprivation

availability

stain-

that of normal Acomys rates ± 6.0. Although subjective appraisal in the electron microscope gives the impression that the granules of Acomys are do

These receptor

to re-

ceptor. Yet despite the great excess of this complex available for binding, many receptors may have remained free, for a further 24% OD increase could be induced by pretreatment with

may

occupancy. anti-LVP

a dif-

available, had not been sufficient to saturate VP receptors in the PT, as free receptor was still available for VP later applied in vitro. We have confirmed our initial observations regarding the enhancing effects on staining (=

indicated. OD measurements: In one series of experiments, the relative OD was measured for MSHtype granules in the P1 of both Mus and Acomys (Fig. 10). In each experimental situation about specimens.

water

tissue. This again shows that in Mus, although making

receptor Anti-LVP

three

out

free receptor. In water-deprived Mus, however, pretreatment of the P1 with LVP increased receptor occupancy (= staining intensity) to maximal values, (± 8.4), about 20% more than un-

that the staining in question should indeed be attributed to anti-LVP, not to a possible unidentified autoimmune factor in the antiserum. Specificity for the binding or receptor sites is also

300-400

to

have since

LVP before increase the

treated rivation

these purified of 1:20, induc-

in both a dilution

greater

response to

that

xenc strain Both, however,

7).

anti-LVP: on an

on two antibodies

imply

moderate;

Purified munoabsorbed

both within

and

as regards VP turnover ditions in comparison In

had

brings

that alters VP binding VP was bound by Acomys

+++

seems granules, Anti-LVP LVP-Sepharose

however,

in Acomys a ± 40% increase. that there had been free

show

+

+

intensity: intense.

+++

and

results

is a stimulus That more

-

-

LVP

to staining

enhanced;

++

granules

-

deprivation,

ference between Mus and Acomys, for while Mus rates ± 7.2, Acomys rates ± 8.4. In Mus, this is a ± 33% increase over normally hydrated

Granules

may that

Another staining

be the tends

body shows greater stability more cumbersome complex. Staining with anti-LVP sorbed nor on pretreated those VP molecules that granules. The enhancing

Downloaded from jhc.sagepub.com at NORTHERN KENTUCKY UNIV on March 23, 2015

possible with ab-

presence to

be

of excess

preferentially

Whatever the explathat sequential bindand then to the antithan alone,

binding neither

of the ab-

tissue, identifies only bound in vivo to the P1 effects of pretreatment

VASOPRESSIN

Nonn

IN

THE

PARS

Normal

I#.is

Nonn

Mus

sorbed

-

±

Water

-

Mus

Water

deprived

Mus

4 pretrsatsd

=

4

Water

deprived

Acomys

Water

deprived

Momys

0

±

=

pretreated

U

U

U

U

U

U

U

U

U

U

1

2

3

4

5

6

7

8

9

10

Staining 10.

589

M

NormAcomys

Fi;.

INTERMEDIA

intensity

of

PU granules

with anti-LVP

showing semi-quantitative optical density measurements of MSH-type under various experimental conditions, by using anti-LVP as primary unlabeled antibody PAP method. Absorbed = admixture of anti-LVP and LVP. Pretreated tissue before anti-LVP. Water deprived = reduction of water supply during 3 weeks.

P1 of Mus

Histogram

and Acomys

with

antigen

have

been

(LVP) amply

for

receptor

displayed

identification

in this

series

cell

of ex-

periments. DISCUSSION

Why receptors

on

granules?: are traditionally

membrane,

yet

we

Polypeptide found

located

hormone on the cell

VP receptors on the P1. In all probability

membrane

(38,

15),

free

granules antiserum =

LVP

receptor

from the in the

applied

seeming

abound at the latter location (38). TRH other neuropeptide located on intracellular ceptors (4, 13). In vitro it has been found that several thalamic releasing and inhibiting factors isolated

pituitary

granules,

in

the

to

is anrehypoact on

presence

of

but as lack of visible membranes is one of the banes of immunocytochemical postembedding staining, we were unable to detect them. Inter-

calcium (12). TRH and prolactin-releasing factor applied directly to secretory granules promoted release of prolactin and growth hormone, while somatostatin and prolactin release-inhibiting factor inhibited release. It may be assumed that this direct action of hypothalamic neurohor-

nalization of polypeptide been reported. Both

has and

mones tional

on a variety of cell surface membrane,

In may

secretory

there

insulin organelles

granules

are

VP

bind

with as

of

the

receptors

well

also

the

receptors, nerve growth

receptors as

at

at

the

cell

surface,

however, factor

this dual localization possibly reflecting dual function (3). LH-RH receptors are found both on gonadotroph secretory granules and on the

on the receptors vivo bypass

isolated on the

intracellular the need

granules granule

was surface.

receptors for a second

for

via

to

func-

hormone messenger.

Bradshaw and Frazier (3), in connection with nerve growth factor and insulin, speculate that intracellular receptors for polypeptides may in-

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CASTEL

590 dicate

that

may

be “to

the

role

of the

specifically

hormone

regulate

This is an attractive secretory granules,

explain

one

granule several facilitates

contents at theories (5) attachment

the cell granule

membrane. receptors

nism cell

of

whereby surface,

the

crucial

steps

Releasing could account

release

of

factors bound to for the mechatowards the and liberate

their contents. tionship between

“The potential regulatory intraand extracellular

tors

a new

dimension

tory action of hormones Why no PAP molecules?:

One

relarecep-

in the

regula-

of the

advan-

then

be taken

as a criterion.

degree quately

of immunocytochemical measured by electron

ecules

are

more

reliable

for

Although

the

PAP

is usually its

seen

when

site of secretion, is bound to receptor

sification

was

The failure cytochemistry

crucial

referring be

to VP

considered

studies,

of

“receptors”

on

premature.

the

This

the

most

dif-

P1 granules

At

circumstantial

active ACTH,

Protein P1

this

may

stage

evidence

substances LPH, CLIP

P1 may

tissue

parameter

of our

presented

also

(16).

be a major

Thus

future

for

identified in the (22), endorphin

even

the

component choice

investigations

of

presents

PT we have found VP receptors, while et al. (11) have detected receptors for angiotensin I. Both these findings may indicate that hormonal factors regulating salt and water Changaris

balance events.

evaluating

cies (20), obliged to contend with the chronic challenge of negative water balance, may reflect this involvement. The P1 hypertrophies when VP secretion is intensified by the stress of water

site

of

a hormone

is local-

but seldom when (38). Sternberger

on P1 granules, only staining

even inten-

could involve The exceptionally

the

desert

species

sebotropic environment.

to

an

augmented

indicate

that

basic

clue

desert to the

ularly, a small peptide such as VP may be engulfed by the receptor (43), and thus be sterically hindered from displaying its PAP molecule.

indebeted to my colleague of the Hebrew University and to Dr. Dieter Dellmann

Even

versity

for

with their of thought

animals enigma

and as nephbalance

sebotropic from MSHlarge P1 of need

factor in contending Thus several lines

Sincere thanks are due berger for guidance and

to a

in their cycle of P1 of desert spe-

(26, 27). Thody (41) favors the cutaneous function of P1 lipotropin, originating type granules. He attributes the

to

endogenous

P1 large

deprivation (unpublished observations), also under pathological conditions such rogenic DI which impairs normal water

some P1.

of

a a

dilemma. In the

to see PAP in receptor immunomay be due to the orientation

of hormone

and

for VP binding in the P1 leaves reactions and effects to speculation. The “MSH-type” secretory granules on which VP was located may contain one or more of the

discerned.

staining

proportion

(Fig. 9). granules?:

question,

the PAP molecule. Possibly PAP molecules may only be discerned when the tissue antigen juts out from the section, while antigen attached to receptor may be more deeply embedded. Partic-

the

a small

staining is adeopacity, PAP mol-

(36) maintains that endogenous hormone bound in vivo does not show PAP molecules, but in vitro exogenously applied and bound hormone may display PAP. Thus when free receptors on LH granules are filled by pretreatment with LH, and then application of LH-RH, moderate PAP is visualized. However, regarding immunocytochemical localization of VP with antigen pretreatment,

only

PAP complexes, while the rest embedded in the granule, visu-

ficult to answer. If “binding to a receptor triggers the first of a series of reactions eventually leading to observed effects”, then we concede that

(14).

localization.

ized at hormone

the

biologically P1: MSH,

tages of the unlabeled antibody PAP procedure is that it facilitates EM visualization of distinctive PAP molecules that may be relied upon to circumscribe true antigenic sites (35). However, the PAP structure is not always discerned, so that electron opacity, monitored by controls, must

to reveal

alized only as electron density Why VP on P1 secretory is really

the cell surface. Despite it is still not clear what of a secretory granule to

granules orientate initiate attachment

represents

only at

hypothesis for it could in

seems

the existing are presumably of

metabolism

of its receptor, which may ultimately act through its position on the plasma membrane the cell surface.” in relation to

granule

in question the

may

of the

the arid seem

provide

mammalian

ACKNOWLEDGMENTS

for

many

stimulating

Downloaded from jhc.sagepub.com at NORTHERN KENTUCKY UNIV on March 23, 2015

to Dr. Ludwig encouragement.

SternI am

Dr. Jacob Hochman for his collaboration, of Iowa State Unidiscussions.

The

VASOPRESSIN consistently Mrs.

excellent

Ilana

technical

Sabanay

THE

assistance

is gratefully

LITERATURE

IN

of

Comp

Endocr

3. Bradshaw regulators in Cellular

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