Neuronal TRH synthesis: Developmental and circadian TRH mRNA levels

Vol.

151,

February

No.

1, 1988

29,

1988

BIOCHEMICAL

AND

BIOPHYSICAL

COMMUNICATIONS Pages

NEURONAL TRH SYNTHESIS: L. Covarrubias*,

R.M.

DEVELOPMENTAL

Uribe,

AND CIRCADIAN

M. MGndez,

J.L.

Centro de Investigaci6n sobre Ingenieria *Centro de Investigaci6n en Reproducci6n Divisi6n de Estudios Especiales, Institute Received

RESEARCH

December

23,

Charli

Gene'tica Animal, Mexican0

615-622

TRH mRNA LEVELS

and P. Joseph-Bravo y Biotecnologla, CINVESTAV-IPN de Psiquiatria,

UNAM; and Me'xico

1987

SUMMARY: Peptide biosynthesis within a neuron involves several steps occurring at the soma and during its travel to the nerve terminal, where it accumulates to be released under stimulatory conditions. We have measured hypothalamic observed TRH and TRH mRNA during ontogeny and circadian cycle and that TRH mRNA variations are more prominent than TRH ones. On the basis of these results and in vitro release experiments, we propose a compensatory mechanism 0 1988 Academrc working at the nerve terminal which is activated after release. Press, Inc. When a peptide stimuli,

is

a mechanism

Although

many complex

and/or

are

in peptide level

other

modifications TRH see 6).

affected

a tripeptide

with

The sequence

for

and TRH mode of degradation focus

on

different to study for

TRH

and

its

in vivo

a coupling

intracellular

be

might where

mRNA levels

been

shown

has same effecters as

an

that

must

important

Several

role.

can be a limiting be

to

stimulate

postranslational

exist.

biosynthesis

regulated release

processing

at (1,2,3);

and

peptide

(4,5). both

endocrine

TRH precursor has also

has been been

mRNA (11)

and neural

the

cycle

kinetics

of TRH metabolism. and release

from

(8,9,10). in

and circadian

functions

deduced

studied levels

biosynthesis

compensatory

environmental

involved,

biosynthesis

specific

overall between

levels

can play

ontogeny

conditions,

or

intracellular

rate

such

also

to endogeneous

basal

processes

by the

steps

are is

the

biosynthesis

Peptide

transcriptional POMC,

response

degradation

involved

component.

in

recovering regulatory

intracellular

steps

for

released for

In this (12),

gives

review

a cDNA clone report,

hypothalamus

Our data and

(for

as

in

(7) we two

an

approach

show

evidence

insights

for

an

mechanism.

MATERIALS AND METHODS: Male Wistar rata, fed ad libitum (Purina Chow) were maintained in a 12 h light-dark cycle (light on 6:00-18:OO). All rats at each time were sacrificed within an hour and tissues were dissected and kept frozen (-70 "C) until assayed.

*Correspondence

to:

L. Covarrubias, Centro de Investigaci6n sobre Ingenieria Gene'tica y Biotecnologia, Apartado Postal 510-3, Cal. ML raval, Cuernavaca, Mor., 62270, MEXICO. 0006-291x/88 615

$1.50

Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Each hypothalamus was homogeneized in 200 ul SOmM Tris-HCl pH 7.4, 25 mM NaCl, 5mM MgCl; 8ul of 25% Triton X-100 and 100 ul of 72% sacarose were added and the homogenate centrifuged 10 min at 4'C; the supernatant (cytosolic fraction) where 100 ul of 6% SDS, 0.4M NaCl, 40mM EDTA solution were added, was extracted 3x with phenol-chloroform (vol./vol); to the aqueous phase, 5M NaCl (1/25th of total vol.) and 2 vol. of ethanol were added and left at -7O'C for 12 h to precipitate total RNA. RNA samples were verified on 1% agarose minigels (containing 2.2M formaldehyde, 1OmM sodium phosphate buffer, pH 7). TRH cDNA kindly donated by Dr. R. Goodman (7) was [32P]-labeled by Nick Translation to 1-5~10~ cpm/ug specific activity. RNA samples were run in formaldehyde minigels and transfered to nitrocellulose paper. Hybridization 20mM sodium phosphate pH 6.5, was performed at 42'C in 5x SSC, lx Denhart, 10% dextran sulphate and 50% formamide (13). Ribosomal RNA bands (stained with ethidium bromide) and TRH-mRNA autoradiographic band were quantified All data were by densitometry in a Hoeffer scanning densitometer GS300. measured within the linear range of detection. TRH was quantified by specific radioimmunoassay according to Mender. et al (14). RESULTS AND DISCUSSION To determine within

the

whether

hypothalamus,

TRH mRNA changes we measured

both

concomitantly

parameters

with

during

TRH

ontogeny

levels (Fig. 1).

TRH mRNA levels showed same increasing tendency as TRH within the first 20 days postnatal but, later in development, some differences were seen between TRH and TRH mRNA behaviour. responsible

for

Several

those

TRH levels authors

(11,15,16). of

Therefore, during

have

Looking

age of Martin0

an analogy

et al

(11)

in ontogenesis

seen

to

two

specifically

TRH-ergic

TRH biosynthesis accumulation

of TRH,

how

much

prolactin

secreted

to the

median

eminence.

can be due to different rat

(e.g.

regulatory population

rhythm of is

this

is

hormones.

have made Within

TRH mRNA levels

feedback

of might

the

can be

due

hypothalamus,

by thyroid

on day 45

resemble

authors

development

is

well

in

documented,

other

hormones

be due

other

neurons

pattern

for

phenomena

C?fiCh

nuclei

will

on

to

the

not

functions

clear (e.g.

functions

do not

direct

axons

TRH and TRH mRNA observed as

grollp help

is

non-endocrine which

occurring

puberty)

in

it

endocrine

TRH can have TRH-ergic the

results

last

days

period.

maturation, working

hypothalamic

of TRH-ergic

TRH release

those

Therefore,

mechanisms

Microdissection

at

up to 20-30

our

These in

mRNA fall

physiological

circadian

mRNA

of TRH content

similar

thyroid

negative

involved

Hypothalamic from

(15).

and

on TSH secretion is

TRH

pattern

are

cellular

The TRH

TRH

secretion).

reports

variations

and

hormones

TRH role

neurotrnnsmitter)

(e.g.

axis neurons,

of thyroid

part,

in development;

TSH

processes:

(17.18).

Although

all

later

and Gayo et al

hypothalamus-hypophysis-thyroid essentially

in

the ontogenetic

pattern

are

least

ontogeny.

reported

at curve

and differences

at

in well 0r

to get

the

developing

as

different

neurons

involved.

a more

homogeneous

neurons.

a rapid

event

causing 616

a depletion

of intracellular

TRH and

BIOCHEMICAL

Vol. 151, No. 1, 1988

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

28s 18S-.

-

aogm* 1

5

10

RNAm TRH

20 30 45

B

Days after birth

the study

Fig.

1.

need

for

the

TRH mRNA

TRH and TRH mRNA during ontogeny. Hypothalamic TRH and TRH mRNA were measured at different days after birth as indicated in Materials and (A) Autoradiography of representative RNA samples hybridized Methods. with TRH cDNA [32P]-labeled; note that expected size (1.6 Kb) is seen at all ages. (B) TRH and TRH mRNA were normalized Per mg of protein Each value is the mean? SEM (n=3). and ug of total RNA respectively. a mechanism

role together

of

respocsible

mRNA

on

with

TRY

adjustment during

for of

recovering

basal

hypothalamic

circadian

617

cycle

TRR (Fig.

In

levels. levels, 2).

order

we measured The

observed

to

Vol. 151, No. 1, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

-

RNAm TRH

B

0 I 1

0 Fig.

2.

6

12

18 24 Day hour

TRH and TRH mRNA during circadian cycle. Hypothalamic TRH and TRH mRNA of adult rats were measured at different time of the day as indicated in Materials and Methods. (A) Autoradiography of RNA samples hybridized with TRH cDNA [32P] labeled. (B) TRH and TRH mRNA were normalized per mg of protein and ug of total RNA respectively. nean* SEM (n-33.

618

Vol.

151,

No.

BIOCHEMICAL

1, 1988

circadian

rhythm

highest

of

levels

light-dark

at

cycle

TRH

and

18 hrs.

is

AND

its

BIOPHYSICAL

mRNA were

These

results

closely

coupled

circadian

pattern

observed

TRH peak

appears

RESEARCH

similar suggest

with

COMMUNICATIONS

in

adult

that

TRH

TRH mRNA levels

animals,

with

release

within

during

the

24

hrs.

period. TRH (12,19). near

its

end.

This

mentioned

before,

functions,

some

rhythm

in

nuclei

are

(19)

showing

just

result of which

the

well TRH

might

areas

with

described

period

cycles most

and falls

rhythm, no

too.

but

well

defined

TRH-ergic

cycling

by Kerdelhue’s

maximum values

at

as

defined

The well

of

supported

before

of rats

in other

that is

ones

TSH circadian

circadian

hypothesis

two hypothalamic

activity

involved

suggests

this

to the

with

is

have

hypothalamus

syncronized;

similar

before

correlates

hypothalamic

the whole

is

results

around

the

same

time. Since

we found

circadian

cycle,

and Martin0

rhythm,

we decided

rhythm

and postnatal

shows

a variety

ranging

from

to analyze

data

they

are

stage,

that achieved,

to speculative (such

it

to

biosynthesis

which

As shown,

amidation)

.

increased

10 fold

increased

3x compared

to the

although

correlations

the

phenomena

described,

the

final and

in mRNA levels

depend

is

attained

is

difficult,

mRNA

on

both

neurons; processes.

only

at

influences

no all)

ages

(TRH

TRH-ergic

3

adult

that

only

and would

factors

be

can affect

steps

of

protein

synthesis

(pGlu

formation

modifications between and,

days

during

and

circadian

increase

of TRH. intracellular

mechanisms

1

20

cycle,

These

and

TRH

mRNA

TRH

mRNA

observations TRH and

might

plus

also

imply

TRH

be

mRNA

involved

in in

TRH levels.

steps

translation

(if

between

compensatory

different

and physiological

It

Fig.

behavioural).

all

bare

exists

of

which

circadian

one peak

of multifactorial

puberty-or

5 fold

than

behaviour

sinchronized.

ontogeny,

and TRH only

that

to more

rhythm

and terminal

during

between

of TRH

and mRNA levels.

TRH and TRH mRNA levels

rhythm

involves

exist

during rise

TRH and TRH mRNA at

of different

i.e.

levels

a developmental

peptide

of biological

becomes

processing

Several

that

is a consequence it

hormonal-

postranslational

setting

indicate

stress

as neuronal, TRH

for

of TRH circadian

are

shown

(TRH at 5 days)

a combination

suggest

have

defining

and circadian

The development when all

for

patterns

no fluctuations These

(12)

as TRH mRNA

how much dependency

development

development

therefore,

et al

of circadian

at 30 days). postnatal

in TRH as well

fluctuations

could

postranslational

observed

stability;

direct

dominate

mRNA levels

be regulated are

measurement in our

after events.

due to the

transcription: We have

transcription

of transcription conditions.

619

not

established

process rate

On the

mRNA

other

will

hand,

if

itself

define

stability,

which

experiments

changes or

mRNA process should

Vol. 151, No. 1, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

100

m z x

4-

f

3-

3 : 3 E

2-

91 a

’ -

‘:,

0-

24 Day

2DD

-

0 Fig.

3.

be adressed events

in

m I! Y

V30D

0

24 Day

hour

6

12

hour

IS 24 Day hour

Development of circadian TRH and TRH mRNA variations. Circadian TRH and TRH nRNA variations ware measured at different days after birth. Observe that in most cases TRH and TRH mRNA peaks occur at the same time. Mean + SEM (x7=3). to estimate this

We do not

the

regulatory

role

of translation

and postranslational

system. know when

release

is

ocurring 620

in

the

two

phenomena

analyzed,

Vol.

151,

No.

BIOCHEMICAL

1, 1988

therefore,

synthesis

of in vivo

release

during

and release is

circadian

Accordingly,

cycie

mRNA of paraventricular axons rats

whereas

(14), not

that

released

in

47.8+

is

suggests min

experiments,

release.

terminal

TRH from involved,

might

could

of intracellular intracellular hormone

degrading status

affect

from

same TRH-precursor

the

since

a proportion

peptides

and be regulated

In conclusion, establishing

of

TRH levels

TRH

part

by pyroglutamate spinal

initial and therefore

activated

do not

affect activity

that

other

(25).

Although

TRH mRNA measured

(21).

is

working

the

(22) ;

give mechanism actively

forms enzymes

Regulation

of the

two soluble nor level

could also

at the

(5) TRH (23)

hypothesis

60 or 70 that

hypothalamic

might

This

process

acid

are

activity

after

reaction

peptides

could

regulatory terminal

released

inhibition at

this

differentially, 2

II

endogeneous

this

levels

membranes

MSH acetylation

active

chord), (n= 8) and

peptidase

amidation

since

spinal

amino

by ascorbic for

enzyme

of

do

content

tissue

a biosynthetic i.e.

chord

chord

content

seems unlikely

at nerve

of

described

of spinal

released

as reported

we propose

part

TRH

to release,

postulated

cervical

as

now that

enzymes

et al have

performed

9.9 +1.8%

degrading

Segerson

(20).

were

and is

degradation

hypothyroid

(cervical

total

TRH

sending

in

56mM KCl,

that

that

neurons

with

be coupled

be regulated

synthesis.

show

70 min

tissue

pglu-his-pro-gly

of

(18)

or

of

either

variations

time

increases

percentage

We suspect

measure

TRH

period

in hypothalamic

at leastsLO% from

or e.g.

Mean + SEM

(hypothalamus)

higher

incubation

nerve

unchanged

in in vitro

due to degradation

that

hypothalamus

in hypothalamus

Direct

TRH-ergic

TRH remains

6) respectively.

5-fold

at

most

COMMUNICATIONS

bare

own observations

eminence

60min

underestimated which

release

and our (containing

a 10 min stimulation 4.9% (n=

however,

we observed

despite

including

coupled.

or whole

TRH content

change

be directly

eminence)

median

In addition

not

active

(17)

nucleus

to the median

RESEARCH

difficult;

suggest

report

BIOPHYSICAL

might

experimentally

a recent

AND

be

our

to other

still

is

(24).

synthesized

explain

rise

thyroid

results active

speculative.

responsible after

of

release.

ACKNOWLEDGMENTS We would like to thank Dr. R.H. Goodman for providing his TRH cDNA clone, the technical assistance of E. Mata and the typing This work was partially supported by CONACyT grants ICSAXNA Aldama. and PCEXCNA-051014.

us with by V. 030915

REFERENCES 1 .-

Roberts, J.L., Eberwine, J.H. and Gee, C.E. (1985) Synposia on Quantitative Biology Vol. XLIX, 385-391. 2.- Eiden, L.E., Giraud, P., Dave, J.R., Hotchkiss, A.J. and Affolter, H.-U. (1984) Nature 312, 661-663. 3 .- Shupnik, M.A., Greenspan, S.L. and Ridgway, E.C. (1986) J. Biol. Chem. 261- 12675-12679. H., Watson, S.J., Kelsey, 4 .- Shiomi, J.E. and Akil, H. (1986) Endocrinology 119, 1793-1799. 621

Vol. 151, No. 1, 1988

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5 .- Millington, W.R., O'Donohue, T.L., Chappell, M.C., Roberts, J.L. and Mueller, G.P. (1986) Endocrinology 118, 2024-2033. 6.- Prasad, C. (1985) In: Handbook of Neurochemistry (Lajtha, A. ed.) Vol. 8. Plenum Publishing Corporation, New York, 175-200. 7 .- Lechan, R.M., Wu, P., Jackson, I.M.D., Wolf, H., Cooperman, S., Mandel, G. and Goodman, R.H. (1986) Science 231, 159-161. 8 .- Bauer, K. and Kleinkauf, H. (1980) Eur. J. Biochem. 106, 107-117. 9 ,- Wilk, S. (1986) Life Sciences 39, 1487-1492. lO.- Garat, B., Miranda, J., Charli, .J-L. and Joseph-Bravo P. (1985) Neuropeptides 6, 27-40. ll.- Martino, E., Seo, l-l., Lernmark, A. and Refetoff, S. (1980) Proc. Natl. Acad. Sci. USA 77, 4345-4348. 12.- Martino, E., Bambini, G., Bartalena, L., Aghini-Lombardi, F., Breccia, M. Baschieri, L. and Pinchera, A. (1986) Endocrinology 119, 232-235. 13.- Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory, New York. 14.- Mendez, M., Joseph-Bravo, P., Cisneros, M., Vargas, M.A. and Charli J-L. (1987) Peptides 8, 291-298. 15.- Gayo, L., Bonet, B., Herranz, A.S., Iglesias, R., Toro, M.J. and Montoya E. (1986) Acta Endocrinologica 112, 7-11. 16.- Lamberton, R.P., Lechan, R.M. and Jackson, I.M.D. (1984) Endocrinology 115, 2400-2405. 17.- Segerson, T.P., Kauer, J., Wolfe, H.C., Mobtaker, H., Wu, P., Jackson, I.M.D. and Lechan, R.M. (1987) Science 238, 78-80. 18.- Covarrubias, L., Uribe, R,M., Mendez, M., Charli, J-L. and Joseph-Bravo, P. (1987) In: Recent Advances in the Biomedical Significance of Thyrotropin Releasing Hormone, Annals of the New York Academy of Sciences, New York (in press). 19.- Kerdelhue, B., Palkovits, M., Karteszi, M. and Reinberg, A. (1981) Brain Research 206, 405-413. 20.- Kardon, F., Marcus, R.J., Winokur, A. and Utiger, R.D. (1977) Endocrinology 100, 1604-1609. 21.- Vargas, M.A., Mendez, M., Cisneros, M., Joseph-Bravo, P. and Charli J-L. (1987) Neuroscience Letters 79, 311-314. 22.- Glembotski, C.C., Manaker, S., Winokur, A. and Gibson, T.R. (1986) J. Neurosc. 6, 1796-1802. J-L., Mendez, M., Joseph-Bravo, P. and Wilk, S. (1987) 23.- Charli, Neuropeptides 9, 373-378. 24.- Ponce, G., Charli, J-L., Pasten, .I., Aceves, C. and Joseph-Bravo, P. (1987). 25.- Segerson, T.P,, Hoefler, H., Childers, H., Wolfe, H.J., Wu, P., Jackson, I.M.D. and Lechan (1987) Endocrinology 121, 98-107.

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