334
I . Erbil, K. M., Jones, J. D. & Klee. G. G. (1985) (’wicer 5 5 .
404-409
2. Debray, H.. Qin. Z., Delannoy, P., Montreuil, J., Dus, D.,
Radzikowski, C., Christensen, H. & Kieler, J. ( 1 986) lnf. J .
Ciincer 3 7 , 6 0 7 - 6 I I
3. Moss, A. J., Bissada, N. K., Boyd, C. M. & Hunter, W. C. ( 1979)
Urology 13. 182- I 84
4. Santamaria, L., Galioto, G. B., Benazzo, M., Pizzala. R.,
Bianchi, A. & Santagati, G. ( 1987)J . Tumoirr Marker Oncol. 2 .
41-49
5 . Colli, A.. Buccino. G., Cocciolo, M., Parravicini, R., Mariani, F.
& Scaltrini,G.(1989)Chncrr63,912-916
BIOCHEMICAL SOCIETY TRANSACTIONS
6 . Erbil. K. M., Sen, S. E., Zincke, H. & Jones, J. D. ( 1986) (iincrr
5 7 , 1 3 8 9 - 1394
7. Warren, L. ( 1 959) J. B i d . C’hem. 234. I97 I - 1975
8 . Katopodis, N. & Stock, C. C. ( I 980) Hex C’ommrcn. (’kern.
I’clrhol. I’harmucol. 3 0 , 1 7 I - I80
9. Saha. S. & Chattopadhyay, U. (1988) I n r . J . C’uncer 41,
432-435
10. Berrd, B., Rapelli, S., Monticelli, G., Fighetti, M. A,, Mea, 1. D.,
Raspagliesi, F.. Di Re, E.. Ringhini. R. & Bombardieri. E.
(19x6) Inr. J. Biol. Markers 1.39-46
Received 15 September 1 989
Growth stimulatory effects of extracellular adenine nucleotides on the proliferation of human lung
fibroblasts
significant at 24 h but ranged bctwccn 145 f 17‘%, and
1 6 3 f 19% of control values at 48 h (1’50.05. t i = 10).This
stimulation was maintained for 72 h. There appeard to be no
order of potency in the ability of the four nucleotides to
stimulate proliferation. A similar result was seen upon
lritroductioti
nucleotide stimulation of quiescent fibroblasts. However, in
The extracellular adenine nucleotides ATP, ADP and 0.4% FCS the time course of stimulation appeared to be different with greatest stimulation observed at 24 h (range
AMP have been shown to inhibit the proliferation of a
variety of mammalian cell types in vifro, including trans- 145 k 19% to 205 f 39%, 1’20.05, n = 5). The proliferative
effect was sustained into the later time periods and there
formed mouse fibroblasts [ 1, 21. However, recent studies
appeared once again to be no distinction between the four
have also shown that ATP in serum-free medium can stimunucleotides in their ability to stimulate proliferation.
late the proliferation of quiescent chick embryo fibroblasts
The growth-inducing ability of Ca2’ in resting fibroblastic
/3]. This study was undertaken to investigate the effects of
extracellular adenine nucleotides on [ 3H]thymidine incor- cell cultures has been demonstrated [ 4 Jand ATP has been
shown to induce Ca2+ mobilization and inositol 1,4,5-triporation by normal human lung fibroblasts. The effect on
both actively dividing cells and on quiescent cells were phosphate formation in various cell types 151. We investigated
a possible role for nucleotide-mediated Ca?’ influx in the
examined and the possible role of CaZ+in the nucleotide/
proliferative effects seen. Pretreatment of a population of
fibroblast interaction was studied.
actively growing cells with verapamil yielded a population of
cells impermeable to extracellular Ca’+ influx. These cells
along with a control untreated population were stimulated
Methods
A primary cell line of normal human fibroblasts was estab- with the four nucleotides for 48 h. Proliferation of control
lished by explant culture of finely minced normal human cells (i.e. no added verapamil) was stimulated to between
lung tissue. Cells between passage 3 and 15 were used in 121 f 12% and 1 8 3 + 33% of control by the nucleotides
these experiments. Characterization by light microscopy and (1’10.05, n = 4). However, no stimulation of cell growth was
electron microscopy showed these cells to be truly fibro- observed with these nucleotides when C a ? + influx was
blasts. The cells were rendered quiescent by growing to con- blocked with verapamil (range 92 _+ 24% to 1 10 k 2 1% of
fluence and maintaining for a further 48 h in Dulbecco’s control, tz = 4). A similar pattern was observed with quiemodified Eagle’s medium (DMEM) containing 0.4% (v/v) scent cells, where stimulation was completely abolished
foetal calf serum (FCS). To examine the effects of adenine when the cell population was pretreated wtih verapamil
nucleotides cells were plated at a density of 5 x 1O2 cells per before incubation with the nucleotides.
The Ca” ionophore A23187 stimulates the entry of
well in 96-well tissue culture plates in DMEM containing
extracellular
Ca’+ into cells. Two hour pretreatment of fibroeither 10% or 0.4% FCS. After 24 h the medium was replaced
with fresh medium containing 10% or 0.4% FCS, 150 PM- blasts wth ionophore resulted in an increase in [>H]thymidine
ATP, -ADP, -AMP or -cyclic AMP, and 0.5 ,uCi of [3H]thy- incorporation to 131% after 48 h in actively growing cells
and to 137% of control after 24 h in quiescent cells, although
midine. In experiments examining the effects of Ca” influx
this did not reach significance. Ionophore-treated cells
this was preceded by a 2 h incubation with either 0.1 mMshowed a greater proliferative response to the nucleotides. In
verapamil (blocks Ca” channels) or 3 m ~ - A 2 3 1 8 7(stimuquiescent cells nucleotides alone stimulated [,’H]thymidine
lates Ca2+influx). The cells were incubated for 24,48 or 72 incorporation (range 192% to 267% of control) while in cells
h before the medium was removed, the monolayers washed
pretreated with ionophore greater stimulation was observed
with Hanks balanced salt solution and solubilized with 2% (range 262% to 392% of control). A similar pattern was seen
(w/v) SDS overnight at room temperature. Aliquots from
each of quadruplicate wells were counted in a B-scintillation in actively growing cells after 4 8 h.
counter. In each case results were expressed as percentage of
control c.p.m. Paired t tests comparing c.p.m. incorporated in Conclusion
control cells compared with c.p.m. incorporated in test cells
These results indicate that extracellular ATP, ADP, AMP
were used for statistical analysis.
and cyclic AMP can stimulate proliferation of both quiescent
and actively growing human lung fibroblasts. As this effect is
Results
abolished by blocking C a 2 + channels it would appear that
Adenine nucleotides stimulated [ 3H]thymidine incorpora- Ca2+ mobilization plays an important role in the effects of
tion in actively growing fibroblasts. The increase was not adenine nucleotides on fibroblasts. Although the activation
MARTINA M. DEMPSEY, IONA S. PRATT and
CLARE OCONNOR
Departments of I’hurmacology and Medicitre, University
College Dublin, Relfield. Dublin 4, Republic of Ireland
1990
632nd MEETING, CORK
33s
of puriergic receptors by the nucleotides cannot be ruled out,
the mobilization of Ca2+ and inositol triphosphate generation are probably involved in the proliferative effects
Observed
since these events exactly correspond to those
observed after the action of serum and growth factors upon
resting cells [ 31.
I . Weisman, G. A. & De. B. K. ( 1984) Fed. I'roc. Fed. Am. Soc.
Exp. Riol. 43,520
2. Belzer, 1. tk Friedberg, 1. ( 19x4) h.J . Med. Sci. 20.483
3 . Pietzkowski, Z. tk Kavohoda. W. ( I U X X ) FOI;(i / / I . S I O ( ' ~ O W I . C:VlO263143-154
4. Praeger, F. C. & Cristofolo. V. J. ( 1986) I n Virro 22. 3SS-35Y
5 , Horstman, D, A,, Tennes, K, A, & Puktney, J, W. ( 1 9 x 6 ) F[i[jS
Lerr,204, I 89- I c)2
Received 18 September 19x9
Association of plasma insulin-like growth factor 1 carrier protein in vivo
JENNIFER RYAN,* TIM MANTLE+ and
D. COLM COSTIGAN*
* Children S Reseurch Centre9Oirr Lady's Hospitul for Sick
Children, Crumlin, Dublin 12 und t Depurtmenl of
Biochemistry, Trinity College, Dublin, Repirhlic of Ireland
The majority of steroid and thryoid hormones are carried in
plasma as macromolecular complexes with specific carrier
proteins. Carrier proteins are not associated, however, with
peptide hormones. An exception t o this generalization are
the somatomedins or insulin-like growth factors (IGFs),first
described by Daughaday & Kipnis [ I ] . IGF-1 is a growthstimulating peptide of molecular mass 7640 Da, which circulates in normal adult plasma in the concentration range
100-180 pg/ml [2]. Some 80°/o of normal plasma IGF-1 is
carried as part of a 150 kDa complex [ 3 ] .The percentage
unbound IGF-1 is exceedingly low ( < 1%) relative to the
total plasma concentration 14,sI. Proposed functions of IGF1-carrier proteins include extending the plasma half-life ( l , , ? )
and prevention of uncontrolled expression of their insulinlike effects. In this study the ability of IGF-binding protein to
Abbreviation used: IGF, insulin-like growth factor.
stabilize large fluctuations of free peptide addition to the
plasma pool was investigated by intracardial administration
of I2'I-IGF-1 in rats and subsequent plasma elution through
a Sephacryl S-200 slurry.
IGF-1 was iodinated t o a specific activity of 130 pCi/pg
using a modification of the chloramine-T method [6]. I z 5 1 IGF-1 (0.6 pg), osmotically adjusted t o 280-300 mOsM with
NaCI, was administered intracardially t o S O 0 g SpragueDawley rats using a 23-gauge butterfly needle. Animals
anaesthetized initially with 0.4 ml o f intraperitoneal sagatal
were administered additional 0.2 ml volumes on signs of
recovery. Blood samples (400 p l ) were collected at staggered
time intervals via aortic cannulation using a 23-gauge needle.
'''1 measurement of plasma samples reflected a biphasic
clearance of administered '2sI-IGF-1: an initial rapid phase
with a biological lIjz
of 10 min and a subsequent lower rate
of decline. Chromatographic separation of plasma samples
through a Sephacryl S-200 filled column (10 mm x 40 cm)
eluting at a flow rate of 1 0 ml/h with 0.2 M-Tris/HCI, pH 7.4,
resulted in three '%associated peaks. The largest of the
three peaks was of approximate molecular mass 140- 1SO
kDa. This peak dominated plasma elution profiles within the
experimental time frame remaining consistent in magnitude
4500
4000
3500
-
-
3
3000
0
0
b
2500
Ei
-2
2000
I
N
w'
-
1500
1000
500
0
15
I
150 kDa
150 kDa
L
45 kDa
35
n
Iodine
55
75
Fraction no. (0.5 ml vol)
\
45 kDa
Iodine
95
15
35
55
75
95
Fraction no. (0.5 ml vol.)
Fig. 1. Sephuctyl S-2M)plustna elution profile
Plasma from time-staggered blood samples, collected from Sprague-Dawley rats
administered 1251-IGF-1(0.6 pg, 130 pCi/pg), were separated by Sephacryl S-200
chromatography. Elution profiles of plasma samples taken at 3 min ( u ) and 3 1 min ( h )
are shown.
Vol. 18