Evidence that endogenous cyclic AMP does not modulate serum sulfation factor action on embryonic chicken cartilage

Vol. 52, No. 4, 1973

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

EVTDBNCE THAT EMK)GENOUS CYCLICAMP DOESNOTWDULATESERUMSULFATIONFACTOR ACTION ONEMBRYONIC CHICKENCARTILAGE Bruce M. Birch, Harry K. Delcher, John L. Rendall, George S. Eisenbarth and Harold E. Lebovitz Division of Endocrinology Departments of Medicine and Physiology Duke University Medical Center Durham, North Carolina Received

April

26,

1973

!5UMMARY The role of cartilage cyclic AMP as a mediator or modulator of serum sulfation factor (SSF) action on embryonic chicken cartilage was assessed. Media with concentrations of rat serum (7.5%) sufficient to maximally stimulate chondromucoprotein synthesis as measured by 35~4 incorporation did not change cartilage cyclic AMP levels. Theophylline (2.5&l) doubled cyclic AMP in cartilage incubated in media but had no effect on 35SO4incorporation. In media containing 5%rat serum, theophylline at 0.5, 1.5 and 2.5mM caused a similar and si ificant rise in tissue cyclic AMP but only 2.5mM inhibited SSF stimulated $ 5S0, incorporation. The data indicate that cartilage cyclic AMP neither mediates nor modulates SSF action on cartilage chondromucoprotein synthesis. Cartilage sulfation uridine,

growth is in part mediated by a circulating

factor, sulfate

or more recently,

related

called

somatomedin (1) which stimulates thymidine,

and amino acid incorporation

chondromucoproteins and proteins

serum factor

respectively

in vitro (2,3).

into cartilage This sulfation

DNA, RNA, factor

to growth hormone in that it is markedly decreased in the serum of

hypophysectomized animals and is specifically

restored to normal with growth

hormone treatment

has no effect

in

vitro

(2).

Growth hormone itself

on cartilage

growth

(2).

Recently we reported that serum sulfation

factor

(SSF) activity

onic chicken cartilage

as measured by 35SOt, incorporation

teins is significantly

inhibited

incubation media (4).

This inhibitory

by the addition effect

of exogenous cyclic

of cyclic

theophylline

to the incubation media produced a similar

These results suggested that endogenouscartilage

0 19 73 bJ# Academic

Press. ltzc

1184

cyclic

on embry-

into chondromucoproAMP to the

AMP was dose related.

In the sameseries of experiments we showed that the addition

Cop.vright

is

of 2.5 to 5.@nM

inhibitory

effect.

AMP might be involved

Vol.

52,

No.

4, 1973

BIOCHEMICAL

The follo~ine Searle:

radioactive

[l- 14Clacetate

mole),

[l-l4

(48 .uCi/pmo?e).

BIOPHYSICAL

substrates

were

(62 :ICi/fltmole),

Clhexadecanoic

Schwarz-Yann

AND

acid

[1-"'Cloleic and [U-l4

RESEARCH

purchased

(51.2

acid

(15.4

pCi/

and [l- 14 Cloctadecanoic

pCi/Hmole)

acid

Clpalmitoleic

from Amersham-

[1-14C]tetradecanoic

(55 pCi/pmole)

acid

COMMUNICATIONS

was purchased

(272 PCi/smole)

acid

from

from Trace

Lab.

R"SUJ>TS APT! DISCUSSIOV our

In

initial

the capacity

of diarunted

linolenate

he sharply

hours

at

hand,

stromal

reduced

fractions acid

normally

first

released

acid.

Earlier

work

been directly

added

fraction

would

the stroma

appear

that

of the pressate

to po%nt out

incubation

medium the

acid

be in

the

critical umolar

out

that

at micelle

region.

the

ionic

In this

1192

of has now

14 C-acetate

in the

and the

fatty

14 C- a-linolenic

these

with is

conditions, require-

necessary. this

quite

concentration for

acid

by

and the

no longer

acid

concentration

was

as a substrate

under

Km measurements

hexadecatrienoic

lipids,

to a-linolenic

the pressate was

other

a precursor

was increased

acid; than

svstem

that

systems

the synthesis

of hexadecatrienoic

to synthesize

the precursor

acid

or pressate

several

as the precursor

1, when both

Furthermore,

the Km for

free

variability

unable

elongated

acid

C-a-

On the

memhrane

was indeed

in Table

we have not carried

important

would

hexadecatrienoic

this

in

employed.

suggested

and then

the

14

preincubated

were

lamellar

was now more effective

pre-incubation

Although

also

to either

the concentration

ment for

activity

by employinn

synthesized.

the stroma

in pressate

As indicated

system.

data

hexadecatrienoate

demonstrated

was readily

it

That

(1).

were

present

were

membranes

These

that

were

concentrations

C-acetate.

suggested

however,

pressates

of lamellar

was observed

to synthesize

We observed,

pressate

bv galactolipase

a-linolenate

pressate

14

variahilitp

preparations

when chloronlast

free

from

of a-linolenate

raising

(1).

2' and when hiph

a-linolenic

considerable

chloroplast

14C-acetate

from

could

acid

experi.ments,

crude low.

used in

extract, It

the

hexadecatrienoic

case the apparent

saturation

is

Vol. 52, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

^D 120 E \ E ; 80

G E 5 0.3 t

CYCLIC

AMP.

f

NO SERUM

Figure 1:

0.5 % SERUM

7.5% SERUM

Effect of sulfation factor serum on 35SO~uptake and cyclic AMP concentration in embryonic chicken cartilages. One group was incubated in mediumwithout sulfation factor serum (rat serum) and compared to cartilages incubated with 0.5% rat serum and 7.5% rat serum. Incubation time was 12 hours for the 35SO~incorporation and 2 hours for the cartilage cyclic AMP levels. The 35SOt+and cyclic AMP assays were done as described. The vertical bars represent the mean + S.E. of five observations.

phodiesterase (Sigma Lot lZOC-7760) using the method of Butcher and Sutherland (81. Using our modification causes a linear

of Hall’s

system, normal rat serum containing

increase in 35SO~ incorporation

SSF

into embryonic chicken carti-

lage over the dose range of 0.5 to 5.0% rat serum added to the incubation media. Maximal 35S04 incorporation

occurs at a serum concentration

upper panel of Figure 1 shows the effect cubation media on 3sSQ,+incorporation The lower panel depicts the results lage cyclic

Al@ levels after

has no significant

effect

more than a two-fold

of 0.5 and 7.5% rat serum in the in-

into cartilage

after

a 12 hour incubation.

of the sameserum concentrations

2 hours of incubation.

on cartilage

cyclic

on carti-

The data indicate

AMP in concentrations

that SSF

that cause

increase in chondromucoprotein synthesis.

Since it was possible that SSF effects evanescent, cartilages

of 7.5 to 10%. The

on cartilage

cyclic

AMP might be

were incubated for from 5 minutes to 240 minutes in me-

1186

BIOCHEMICAL

Vol. 52, No. 4, 1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 1 EFFECTOF RAT SERUMONEMBRYONIC CARTILAGECYCLICAMF’LEVELS Embryonic chick cartilage was incubated for the indicated times in mediumcontaining no rat serum and 7.5% rat serum. Results represent the mean + S.E. based on five samples for each time. TIME OF INCUBATION (minutes)

CARTILAGECYCLICAME’ (p moles/mg cartilage) no serum

dia containing

5

0.20 f 0.02

0.27 + 0.02

10

0.28 ?; 0.05

0.28 -?:0.03

30

0.27 + 0.03

0.36 f 0.09

60

0.31 f 0.03

0.31 + 0.03

120

0.23 c 0.03

0.25 +- 0.03

0.17 2 0.03

0.23 +_0.02

no serum or 7.5% rat serum and tissue cyclic

Table 1 shows that media containing cartilage

7.5% rat serum

cyclic

7.5% rat serum does not significantly

AMP levels as compared to their

fect of SSF in stimulating

cartilage

incorporation

parable cartilages

2 hours of incubation.

spite of a two-fold

increase in the intracellular

AMP. The tissue cyclic

by 0.5, 1.5 and 2.SmMtheophylline.

The inhibitory attributed

on SSF stimulated effect

to its

on 35SO~incorporation

cyclic

AMP levels.

caused a significant

In contrast,

35SObincorporation

in elevating

in com-

in

In the

elevation

of

AMP level was elevated to the sameextent

of 2.5mM theophylline

effect

AMP levels

on 35S0,+

In the absence of added rat

had no effect

presence of 5%added rat serum, theophylline

concentrations

of theophylline

12 hours of incubation and tissue cyclic after

we next

Cartilages were incu-

5%rat serum, to which varying

serum to the media, 2.5 I&I theophylline

had no effect

AMP levels,

AMP levels would influence the ef-

were added. Table 2 shows the effects after

tissue cyclic

cyclic

chondromucoprotein synthesis.

bated in media, with or without of theophylline

cyclic

alter

controls.

Since SSF did not appear to change cartilage assessed whether altering

AMP was measured.

into chondromucoprotein.

on 35SOr incorporation

intracellular 1187

0.5 and 1.5 mMtheophylline

cyclic

AMP.

can not be

BIOCHEMICAL

Vol. 52, No. 4, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

?‘ABLE2 AND TISSUE CYCLICAMP LEVELSIN EFFECTOF THEOPHYLLINE ON 35S04 INCORPORATION EMBRYONIC CHICKENCARTILAGE Tissues were incubated as described in the text. Data are mean + S.E. of five cartilages. 35S0304 is incorporation into chondromucoproteins and is expressed as cpm/min. Cyclic AMP is p moles/mg cartilage. MEDIA WITH NO SERUM.-

MEDIA WITH 5%RAT SERUM

Control

35sol+ 203 k 11

cyclic AMP 0.40 it 0.04

35sQl+ 510 f 61

cyclic AMP 0.56 + 0.07

Theophylline 0.5 mM 1.5 mM

283 * 33 231 + 12

0.53 f 0.05 0.53 * 0.02

540 + 28 526 t 24

1.02 f 0.12 0.91 f 0.11

2.5 JnM

228 + 16

0.79 i 0.12

344 f 25

0.97 f 0.10

To be certain was cyclic taining

that the material measured by the competitive

AMP, extracts

7.5% rat serum, and media containing

were assayed for cyclic cyclic

of pools of cartilages

binding assay

incubated in media, media con-

7.5% rat serum plus theophylline

AMP, digested with phosphodiesterase and reassayed for

AMP. In every instance greater than 99%of the measureable cyclic

AMP

was destroyed by the digestion. The results presented here indicate

that SSF does not significantly

intracellular

cyclic

AMP levels in cartilage.

intracellular

cyclic

AMP has no effect

synthesis. clic

Additionally,

it appears that

on SSF stimulated chondromucoprotein

The previous data that we reported on the effects

of exogenous cy-

AMPmust be regarded as a pharmacological action and the effects

concentrations

of theophylline

other than its

ability

It is of interest

cyclic

embryonic chicken cartilage

had no effect until

These concentrations

of high

of the molecule

3’5’AMP phosphodiesterase.

that Tell et al recently

fied somatomedinpreparation

achieved (9) .

as being mediated by a property

to inhibit

alter

reported that a partially

on adenylate cyclase activity

concentrations

of 10 to 20 units/ml

puriin

were

are 10 to 20 times that present in undilu-

ted plasma and about 200 to 400 times the concentration

1188

present in the media

Vol. 52, No. 4, 1973

containing

5% rat serum used in this

reproducible likely

BIOCHEMICAL

stimulation

study.

of adenylate

to its actions

RESEARCH COMMUNICATIONS

The latter

of chondromucoprotein

that the inhibition

port is related

AND BIOPHYSICAL

synthesis.

causes a striking Thus it

and

seems un-

cyclase by somatomedin which they re-

in stimulating

cartilage

anabolic

processes.

ACKNOWLEDGEMENTS This work was supported Metabolic

and Digestive

by grants from the National

Diseases of the N.I.H.

17,954) and the Veterans Administration Presented Washington,

Institute

of Arthritis,

(AM 01324, ST1 AM 5074, K3 AM

(H.K.D.).

in part at the IV International

Congress of Endocrinology,

D.C., June 1972. REFERENCES

Daughaday, W.H., Hall, K., Raben, M.S., Salmon, W.D., Van den Brande, J.L., and Van Wyk, J.J.: Nature 235, 107 (1972). 2. Salmon, W.D. and Daughaday, W.H.: J. Lab. Clin. Med. 48, 825 (1957). Salmon, W.D. and Duvall, M.R.: Endocrinology 86, 721 v970). 43: Rendall, J.L., Delcher, H.K. and Lebovitz, H.E.: Biochem. Biophys. Res. Comm. 5, 1425 (1972). Hall, K.: Acta Endocrinol. 63, 338 (1970). :: Delcher, H.K., Eisenbarth, ES. and Lebovitz, H.E.: J. Biol. Chem. 248, 1901 (1973). 7. Gilman, A.G.: Proc. Nat. Acad. Sci. (USA) 67, 305 (1970). Butcher, R.W. and Sutherland, E.W.: J. Biol. Chem. 237, 1244 (1962). i: Tell, G.P.E., Cautrecasas, P., Van Wyk, J.J. and Hi=, R.L.: Science 180, 312 (1973). 1.

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