Cyclic AMP-induced release of [14C]taurine from pinealocytes

Vol. 90, No. 1, 1979 September

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

12, 1979

Pages

CYCLIC AMP-INDUCED RELEASE OF [14C]TAu~I~E G.H.T.

June

and D.C.

FROM PINEALOCYTES

Klein

Section on Neuroendocrinology Laboratory of Developmental Neurobiology Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20205

National

Received

Wheler

22-27

29,1979

SUMMARY: Pinealocytes were prelabelled with [14C]taurine. Twenty-four hours later they were treated with derivatives of cyclic AMP. It was found that dibutyryl cyclic AMP and p-chloro-phenyl-thio cyclic AMP treatment caused a large increase in the release of [14C]taurine. The effect of dibutryl cyclic AMP on [14C]taurine release was near maximal fifteen minutes after treatment started. In view of the known stimulatory effects of norepinephrine on pineal cyclic AMP and the recent discovery that norepinephrine causes the release of taurine from pinealocytes, one can conclude that norepinephrine stimulates [14C]taurine release from pinealocytes by acting through a cyclic AMP mechanism. INTRODUCTION Taurine

occurs

and is apparently recently

found are

mitter

of pineal

the

activity

in pineal igated

treated

is

pineal

entirely

nerves. to increase

gland

is

rapidly

culture

with

One other

rapid

cyclic

second

the enzyme which

metabolism of whether

(3),

(7-9). cyclic

In the present AMP may also

We have pineal

the neurotrans-

of norepinephrine This

20 mM,

(1,2).

when denervated

norepinephrine effect

of about

structures

released

AMP (4,5).

of N-acetyltransferase,

question

at concentrations

in postsynaptic

[14C]taurine in organ

indoleamine the

rat

located that

glands

pinealocyte

in the

on the

messenger

regulates report

stimulate

controls

large

changes

we have the release

investof

taurine. MATERIALS Sprague-Dawley commercially.

male

rats,

AND METHODS

80-100

g, were

used.

Chemicals

were

obtained

Pineal glands were cultured for a total period of 48 to 54 hours as previously described (I). During the first 24 hours the glands were incubated in 0.25 mM [14C]taurine(5.04 Ci/mol)-containing culture medium. Culture medium was then replaced with taurine-free medium and glands were incubated under control conditions for a second 24-hour period. During this 48-hour culture period pineal nerves degenerate. 0006-291X/79/170022-06$01.00/0 22

BIOCHEMICAL

Vol. 90, No. 1, 1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

To test the effects of drugs, glands were transferred into medium containing test compounds after the first 48 hours of culture. The test period was no longer than 8 hours. At the end of the incubation period, glands were removed and homogenized in 0.1 M sodium phosphate buffer, pH 6.8. Samples of culture medium were analyzed for total radioactivity, and glands were analyzed for N-acetyltransferase activity. Thin-layer chromatographic analysis indicated that all the radioactivity released into the medium migrated with authentic taurine. The four chromatographic systems used were (taurine Rf values appear in parentheses): n-propanol:H20, 1:l (0.5); n-propanol:NH40H, 7:3 (0.3); isopropanol:methyl acetate:NH40H, 7:9:4 (0.12); n-butanol:acetic acid:H20, 12:3:5 (0.2). RESULTS [14C]Taurine as previously release

observed

two-fold

dibutyryl the

was released

The effects activity

similar

doses

maximal

stimulation

were

to be a potent

tially

was not

inactive

N-acetyltransferase stimulating

(9,lO).

Finally,

taurine

release

N-acetyltransferase stimulation)

agent.

was found

0.01

[14C]taurine

that

this

effect

seen with

and it

required

taurine

The relative which cell

treatment

activity

was submaximally release

was not

23

AMP, was

indicating

active

in elevating

of cyclic

a result

adenosine

half

AMP was essen-

ineffectiveness

has been

that

l),

cyclic

membranes,

the

apparent

cyclic

and weakly

is probably

that

release.

(Table

In contrast, release

N-acetyl-

to produce

release

of

was found

[14C]taurine

In

1 VM

AMP on serotonin 1),

mM p-chloro-phenyl-thio

activity but

(Fig.

the active

or N-acetyltransferase

In two cases,

cyclic

of taurine

through it

was found

AMP, p-chloro-phenyl-thio

N-acetyltransferase, penetration

of 1 mM.

the same as that

stimulator

activity.

and poor

at a concentration

the enzyme and the

in stimulating

increased at 0.1 ml1

AMP were

of cyclic

conditions,

detectable

it

examined

cyclic

control

AMP treatment

was not

data)

also

of both

derivative

acid

effect

of dibutyryl

of dibutyryl

Another

butyric

This

under

cyclic

AMP (1 mM) was about

norepinephrine.

found

1).

(unpublished

cyclic

transferase

Dibutyryl

glands

AMP, but was detectable

same experiment

dibutyryl

(3).

(Fig.

cyclic

by pineal

of degredation

previously did

AMP in

reported

not

stimulate

(Table

1).

cyclic

AMP and 10 mM cyclic

elevated increased.

(less

than

AMP,

25% of maximal

A reasonable

expla-

BIOCHEMICAL

Vol. 90, No. 1, 1979

Table

1.

Experiment

Effects

Test

I

of cyclic AMP and on N-acetyltransferase Compound1

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

related

on taurine

Concentration (UN

Medium [14C]taurine (nCi/gland)

None pC1 $SH CAMP 1000

II

None L-Norepinephrine Cyclic AMP Adenosine

Pineal glands, were treated test period. 'The abbreviation 2Significantly

Figure 1. transferase

0.1 10,000 100 1000

preincubated with culture in organ culture with the Values are mean + SE for used different

is

Dose response activity with

compounds activity.

and

N-Acetyltransferase activity (nmol/gland/hr)

13.9 14.7 21.2 31.8

+ T 7 5

2.8 0.5 0.2 0.32

0.19 0.46 6.83 10.74

+ T T 5

0.06 0.09 l.352 1.942

15.7 28.4 16.4 13.6 19.4

+ T T T 5

4.3 l.22 1.2 0.8 1.3

0.02 6.62 1.09 0.08 0.02

+ T T 7 -

0.012 0.61 0.162 0.03 0.01

medium containing 0.25 mM [14C]taurine, chemical listed below for a four hour six glands or three media.

pC1 $SH CAMP, p-chloro-phenyl-thio from control values (~~0.05).

relationships dibutyryl

release

of cyclic

24

[14C]taurine AMP (DB-CAMP).

cyclic

release

AMP;

and Dibutyryl

of

N-acetylcyclic

AMP

Vol. 90, No. 1, 1979

BIOCHEMICAL

nation

discrepancy

of this

release

apparent

was undetectable The time

course

Near-maximal

2).

of the

is

effect

release

RESEARCH COMMUNICATIONS

a submaximal

increase

in

[14C]taurine

(2,11,12).

cyclic

AMP was examined

(Figure

was evident

fifteen

minutes

6 hours.

The time

course

for

[14C]taurine

of released

of dibutyryl

and was sustained

cyclic

that

of re-uptake

[14C]taurine

ment had started, of dibutyryl

because

AND BIOPHYSICAL

AMP on N-acetyltransferase

activity

is also

after for

treat-

the

effect

presented.

DISCUSSION Our purpose AMP might

mediate

pinealocytes. it

occurring

derivatives of dibutyryl

because tic

second

of norepinephrine

on [14C]taurine

provided

could

release (3).

support.

could

[14C]taurine

stimulate

cyclic

AMP were

dibutyryl during

findings

cyclic the

messenger

Thus,

convincing

One possibility

is

Second,

evidence

arise that

release.

it

that

acts

via

this.

within in

two cyclic maximal

AMP effects

The possibil-

structures

seems remote

treatment, report

AMP does act

presynapand preas the

on [14C]taurine First,

how is

a phosphorylation

was present for the entire four hour test period. for six glands or three media at four hours.

values

30 minutes.

of norepinephrine regarding

peak

presented

near

in this

cyclic

First,

maximal

that

drug

the observations

the effects

innnediately

was found

preceeding

this.

was known that

the experiments

on presynaptic

period

it

was near

within

from

for

Second,

cyclic

release

AMP, with

it

detectable

culture

mediating

from

First,

easily

disintegrate.

cyclic

(4,5,9).

AMP is acting

48 hour

provide

stimulate

The evidence

whether

some support

by norepinephrine

adds additional

Two questions acting?

was to determine

of treatment

of taurine

structures

vious

studies

available

20 minutes

of treatment

report

that

effect

these

norepinephrine

within

15 minutes

ity

that

stimulation

this

the

The evidence

was known

the

in performing

Values

cyclic

mechanism are

release.

the means

AMP to (+ SE)

Figure 2. Time course of dibutyryl cyclic AMP stimulated [14C]taurine release and N-acetvltransferase activitv. Glands were incubated for two hours in control medium. Dibutyryl cyclic AMP (final concentration 1 mM) was added in 15 ul of 10x concentrated solution and samples of medium were removed at the times indicated. Diluent was added to control cultures. Several sets of glands were used to prevent removal of more than a total of 30 ul from each dish. Values are the means (+SE) for six media at each time point.

25

Vol. 90, No. 1, 1979

BIOCHEMICAL

activate

a mechanism

Although

cyclic

neural

tissue

AMP is (12),

(8,13),

no direct

lation

of membrane

This

does,

question

Taurine

is

out

ger

are

It

cations.

is

ability

and that

that

with

also

reuptake

arises

this,

the gland.

the effects membrane to fit

interact

with

of norepinephrine

bound into other

possibility

might

act

transynaptic

either

pre-

release is

or postsynaptic

infers

compound,

intimately

involved

with

as an extracellular

found

that on the

(1).

(14).

high

capable

sites

binding

messen-

involved These

function

of

by direct

apparent

we suspect

in the extracellular

of pineal

the A second

gland

Based on this receptor,

ions

concentrations

pineal

of reuptake.

adrenergic

of taurine.

question.

group

hydrated

binding

contol

first

is acting

taurine

phosphory-

Answers

membranes

regulation

gland

is a means of transporting

B-adrenergic

that

released?

of pineal

B-receptors

adrenergic

that

in other

to pursue.

sulfonate

of norepinephrine

the

proteins

the release

to the

a highly

taurine

or the

the interesting to modulate

the

of taurine.

in the pineal

in

taurine

those

We have previously

raise

with

in that

release

to indicate

involved

than

in turn,

released

time

is why is

taurine

protein

hypothesis

hyperpolarization

of taurine

might

is

makes taurine

Perhaps

can mimick

interaction

at this

the

of membrane

of nuclear

pineal

compound

mediating

phosphorylation

no more definitive

an interesting

in the pineal

taurine

in the

which

norepinephrine-induced possibility

to cause

seem to be a reasonable

question

of the cell,

membrane

is available

proteins

characteristics.

of carrying

cell

and phosphorylation

however,

to this

known

evidence

The second

unique

in the pineal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

taurine

in neuronal considerations space

by interacting

sites.

REFERENCES 1. 2. 3. 4. ;: 7.

Wheler, G.H.T., Weller, J.L., and Klein, D.C. (1979) Brain Research, 166, 65-74. Grosso, D.G., Bressler, R. and Benson, B. (1978) Life Sci. 22, 1789-1798. Wheler, G.H.T. and Klein, D.C. (1979) Brain Research (submitted). Strada, S., Klein, D.C., Weller, J., and Weiss, B. (1972) Endocrinology 90, 1470-1476. Weiss, B. and Costa, E. (1968) J. Pharmacol. Exp. Ther. 161, 310-319. Klein, D.C. and Weller, J.L. (1973) J. Phannacol. Exp. Ther. 186, 516-527. Klein, D.C. (1978) in The Hypothalamus (Reichlin, S., Baldessarini, R.J. and Martin, J.B. , eds.), pp. 303-327, Raven Press, New York.

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Vol. 90, No. 1, 1979

8. 9. 10. 11. 12. 13. 14.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Axelrod, J. and Katz, M. (1977) in Biochemical Actions of Hormones, Volume 4 (Littwack, G., ed.), pp. 248-268, Academic Press, New York. Deguchi, T. (1973) Mol. Pharm. 9, 184-190. Klein, D.C. and Berg, G.R. (1970) in Role of Cyclic AMP in Cell Function, Volume 3 (Greengard, P. and Costa, E., eds.), pp. 241-263, Raven Press, New York Krusz, J.C., Dix, R.K. and Baskin, S.I. (1977) Fed. Proc. 37, 907. Greengard, P. (1976) Nature, 260, 101-108. Winters, K.E., Morrissey, J.J, Loos, P.J., and Lovenberg, W. (1977) Proc. Natl. Acad. Sci., USA, 79, 1928-1931. Parfitt, A., Weller, J.L., Klein, D.C., Sakai, K.K. and Marks, B.H. (1975) Molec. Pharmacol. 11, 241-255.

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