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Receptors for insulin and epidermal growth factor-urogastrone in adult human fibroblasts do not change with donor age
Mechanisms of Ageing and Development, 11 (1979) 37-43
37
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
RECEPTORS FOR INSULIN AND EPIDERMAL GROWTH FACTORUROGASTRONE IN ADULT HUMAN FIBROBLASTS DO NOT CHANGE WITH DONOR AGE*
MORLEY D. HOLLENBERG Division o f Clinical Pharmacology, Department of Medicine and Department o f Pharmacology and Experimental Therapeutics, The Johns Hopkins University School o f Medicine, Baltimore, Maryland 21205 :U.S.A.)
EDWARD L. SCHNEIDER Gerontology Research Center, National Institute on Aging, National Institutes o f Health, Baltimore, Maryland 21224 {U.S.A.)
(Received July 7, 1978; in revised form February 21, 1979)
SUMMARY The ligand binding and biological responsiveness of cultured fibroblast monolayers were measured for porcine insulin and for murine epidermal growth factor-urogastrone in skin fibroblast cultures derived from young (22 to 31 years) and old (65 to 80 years ) normal male volunteer donors. The receptor characteristics of the cells studied from the two groups of donors were not found to differ. In contrast with previous data, suggesting that there may be a genetically programmed aging of the insulin binding system, it is concluded that for both insulin and epidermal growth factor, receptor characteristics do not change with adult donor age.
INTRODUCTION Considerable interest has been focused on the role of hormone receptors in development and aging [1-3] as well as in the pathogenesis of certain diseases such as diabetes. Cultured skin fibroblasts provide an attractive model system for the study of the effects of aging on receptors. Large quantities of cells can be readily obtained so as to compare the receptor characteristics with other cellular parameters which change as a function of the age of the human donor [3]. Recent work has established that for insulin [4-10] and for both human and murine epidermal growth factor-urogastrone (EGF-URO) [5, 8, 1114], cultured human fibroblasts possess receptors that can be related to the stimulation *Portions of these data were reported in preliminary form to the American Society for Clinical Investigation [ 24 ].
38 of both thymidine incorporation and the uptake of the amino acid analogue, c~-amint> isobutyric acid (AIB) [5, 8, 9]. Tile relative potency of insulin and insulin analogues in stimulating fibroblast AIB uptake parallels the relative activity measured in other insulinresponsive tissues [9]. Studies with fibroblast monolayers provide the distinct advantage that binding measurements can be interpreted in the context of biological responsiveness so as to minimize the risk of observing non-receptor binding artifacts [15]. Furthermore. measurements of receptors for two active polypeptides in the same sample permit a broader interpretation of the data than might be possible from measurements with either ligand alone. In a previous study [16], data obtained with skin samples from children and from adults predominantly under the age of 50 have been presented to suggest that there may be an increase in fibroblast insulin affinity as a function of normal human aging. In this report, we have focused on the characteristics of receptors for both insulin and EGFURO in skin fibroblast samples obtained from young and old adult males. In contrast to the conclusions of the previous study [16] we observe that the receptor characteristics do not differ appreciably with the age of the adult donor.
MATERIALS AND METHODS After formal consent had been obtained, skin biopsies were performed on five young (age range 22-31) and four old (age range 65-80) non-hospitalized male volunteer members of the Baltimore Longitudinal Study [3]. Skin fibrobtast cultures derived from those biopsies were studied at early passage in vitro, between 7 and 13 cumulative cell population doublings. Patients with diabetes mellitus or abnormal glucose tolerance were excluded from this study. Cells were routinely propagated in 75 cm 2 T-flasks using Eagle's minimal essential medium supplemented with glutamine, non-essential amino acids, 10cS, fetal calf serum and 50 /~g/ml chlorotetracycline (aureomycin, GIBCO), as previously described [3]. For the studies of tile binding and biological activity of insulin and EGF-URO, cells were subcultured in 1.5 cm diam. multidish trays (Linbro, New Haven, CT) and measurements were performed essentially as previously described [5, 8, 9] and detailed in the figure legends. Murine EGF-URO prepared from fresh-frozen mouse submaxillary glands [17], was isolated for these studies; highly purified porcine insulin (lot 6151082B108-I) was generously provided through the courtesy of Dr. R. E. Chance, Eli Lilly and Co., Indianapolis, Ind. Donor cell strains were examined in pairs (one from tire young group; one from the old), in a double-blind protocol. Cell cultures were screened and found free from mycoplasma [ 18].
RESULTS Stimulation of AIB uptake provided the most convenient and reliable index of biological responsiveness for insulin activity (reported as the concentration causing a half-
39 10~ x MOLARITY _ 1;(]
_20
30
4.0
108 x MOLAREY 50
60
1
20f
25~
2
3
4
I
~
~ (c)
I (a)
20F o
, ~0
10
1(]
20
30
20
30 50
40
, / ,I-----. ~ ~' o ....
~~~~ 1'o
0~. 3
10
•
• '
40
8(]
~ ~o
INSULIN,ng./ml
'
Jo
20
"
30
-
t
t ~ 5~,ILlo~7 ~''-'° "~'~ 4o
oo
~
~6
3o~
EGF,ng/ml
Fig. 1. Representative binding isotherms and dose-response curves for insulin and EGF-URO in intact fibrobtast monolayers. Cell monolayers were grown to confluency in multiple 1.5 cm diam. multidish trays and assayed in parallel by previously described methods [5, 8, 9]. Data obtained for two young (©, o) and two old (•, I) cell strains are shown. (a) Specific binding of 12Slqabeled insulin (100-500 c.p.m./pg) was measured at 24 °C in monolayers containing, on average, 110,000 cells. The protein content of each monolayer for which binding was measured was used to calculate the binding per cell; the correlation of protein content with cell number was determined separately in identically prepared cell monolayers. Non-specific binding, measured in the presence of 2 ~g/ml unlabeled insulin, was subtracted from the total binding to yield the figure for specific binding; no "specific" binding to the multidish trays was detected. (b) The increased uptake of AIB (440 c.p.m./pmol) in response to increasing amounts of insulin was measured during a 12 min pulse with 3H-labeled A1B after a 2.5 h preincubation with hormone. (c) Specific binding of 12Sl-labeled mouse EGF URO (100-250 c.p.m./ pg) to cell monolayers was measured as for insulin; non-specific binding was determined in the presence of 1 ug/ml unlabeled EFG-URO. (d) EGI:-URO-stimulated incorporation of 3H-thymidine (1 t~Ci/ml; 6 Ci/mmol; counting efficiency, 13%) into trichloroacetic acid insoluble material was measured during a 2 h period at 37 °C begun 22 h after the addition of increasing amounts of EGFURO to triplicate cell monolayers.
maximal effect, EDso) (Fig. l ( b ) and Table I). Measurements o f the specific binding o f 12SI-labeled insulin over the range o f biological responsiveness (AIB uptake) yielded isotherms which approach saturation in the c o n c e n t r a t i o n range between 3 and 6 n M (Fig. l(a)). Values for the m a x i m u m b i n d i n g per cell (Bma~) and the c o n c e n t r a t i o n at which insulin b i n d i n g was estimated to be ½-maximal (C1/,ma~) were determined directly from these isotherms (Table I). S t i m u l a t i o n of t h y m i d i n e i n c o r p o r a t i o n provided the best index o f biological responsiveness for E G F - U R O ; E D s o values were determined from the dose-response curves (Fig. l ( d ) and Table I). As in the case of insulin, the binding of 12Sllabeled E G F yielded isotherms (Fig. l(c)) from which the Cv, max and Bma ~ could be estimated (Table I). Receptor b i n d i n g isotherms and biological dose-response curves for b o t h insulin and E G F were reproducible at both early and middle passage in vitro, as well as in frozen cell stocks after return to tissue culture. As in previous studies [5, 8, 9 ] , the
11.6 5.8 2.3 7.6 6.2 6.7 + 3.4
1.6 +- 0.5
1.9 1.4 1.4 1.1 2.4
1.6 +- 0.5
Average:
31 28 28 26 22
Average:
9.5 -+ 3.4
0.75 -+ 0.55
1.6 0.97 0.30 0.53 0.33
0.74 +- 0.23
0.80 0.62 1.0 0.50
12.3 8.6 5.0 12.0
2.2 1.4 1.7 1.0
80 73 73 65
0.70 -+ 0.11
0.70 0.68 0.65 0.58 0.87
0.77 +- 0.12
0.60 0.83 0.75 0.88
(nM)
Clhrna x
ED 5o (nM)
(nM)
(10 -3 X sites~cell)
C~hmax
"
B max
EGF-URO
Insulin
Age o f Cell Culture Donor ~vearsJ
78 +- 58
160 116 19 52 43
106 -+ 90
238 83 59 42
(10 - 3 × sites~cell)
B max
0.046 + 0.020
0.067 0.067 0.042 0.030 0.025
0.030 +- 0.014
0.050 0.022 0.022 0.025
(nM)
ED 5o
Parameters for the biological responsiveness (EDs0) and ligand binding (C~hrnax, Bmax) were estimated for both insulin and E G F - U R O from dose-response curves and ligand binding isotherms as depicted in Fig. I. The average, -+S.D. were calculated for cell strains derived from the groups of old (age 6 5 - 8 0 ) and young (age 22-31) donors.
CHARACTERISTICS OF RECEPTOR BINDING AND BIOLOGICAL RESPONSIVENESS
TABLE I
41 degree of cellular responsiveness (stimulation of either AIB uptake (t.5 to 2.2-fold) or thymidine incorporation (5 to 20-fold) was observed to vary considerably in an individual cell strain, without appreciably affecting the values of the EDso'S. However, even for an individual cell strain, the maximum cellular binding capacity for both EGF-URO and insulin was observed to vary markedly from one sample to another, although all measurements were performed on quiescent (DNA synthesis) confluent cell monolayers. Variations in maximum binding capacity do not appear to correlate with variations in maximal biological responsiveness. Unfortunately, it is not possible to examine this correlation rigorously, since in a given cell strain, measurements of binding and responsiveness could not be performed on the same cell monolayers.
DISCUSSION
From the data summarized in Table I and illustrated in Fig. 2, it is evident that for both insulin and EGF-URO the receptor binding parameters studied in fibroblast cultures do not differ appreciably with adult donor age. The values for the Cv, ma x of specific insulin binding obtained in this study (1.6 -+ 0.5 nM) are in good agreement with measurements of insulin affinity from previous studies of fibroblasts from different donors [16]. Additionally, the affinity of EGF for fibroblasts, as indicated by the C1/, values in this stUdy is in excellent agreement with values obtained by other laboratories under different experimental conditions [11]. The relatively large variation in the maximum cellular binding capacity cannot be readily explained, but appears to be similar in cells from both young and old donors. From this and from previous work [5, 8, 9] it is evident that such variations may be expected for measurements of the kind we have performed and that a very large number of donor samples would be needed to detect any small differences.
E
0.10f == 0,05 0
=~
i
i
•
m
:~ 0.5 0
E
=
w
2I---1 o
•
C.
,e,
z
oo
20
40
60
80
DONORAGE I YEARS I
Fig. 2. Receptor parameters as a function o f donor age. Values for the EDso (A, C) and for the 12Slligand concentration corresponding to Vz maximal saturation o f binding (Clhmax, B.D.) were estimated fo~ E G F - U R O (o, I ) and for insulin (o, e) in individual cell strains from data like those depicted in Fig. 1.
42 Most importantly, variation in maximum binding capacity does not appear to affect either the biological sensitivity (EDs0) or the cellular ligand affinity (Cv2m~x). The lack of change with donor age of the EDso'S for the cellular responses for both E G F - U R O and insulin substantiates the data obtained in the present study by ligand binding measurements. In contrast with the report proposing a genetically programmed aging of the insulin binding system [16], our data indicate that during adult life the characteristics of the receptors for both insulin and E G F - U R O do not change with age. The previously reported differences in insulin binding [16] in fibroblast samples are most striking when the data from children are compared with those from adult samples. These differences may therefore reflect developmental changes rather than adult aging. It should be pointed out, however, that our measurements on adult fibroblasts are in excellent accord with previous measurements performed on fibroblasts from newborn males [5, 8]. It may be also of interest in this regard that fibroblasts from a child with a Progeria-like syndrome possess characteristics of binding and biological responsiveness for EGF which are similar to those o f adult fibroblasts [19]. It is important to note that the measurements reported here may not reflect receptor characteristics in vivo where humoral factors such as the presence of antireceptor antibodies [20] or elevated hormone levels [21, 22] may alter receptor function. These measurements on confluent monolayers also do not eliminate possible differences in receptor numbers which may be present during the active phases of cell growth. However, it has been observed that insulin receptors increase with cell density [23]. Thus, our measurements should reflect the maximum numbers of receptors.
ACKNOWLEDGEMENTS We are grateful for the expert technical assistance of W. H. Shackelford and are indebted to Dr. J. H. Helderman for help in initiating these studies. Support came in part from a Basil O'Connor Research Starter Grant of the National Foundation-March of Dimes and from the Maryland Division of the American Cancer Society. M. D. Hollenberg is an investigator of the Howard Hughes Medical Institute.
REFERENCES 1 G. S. Roth, Reduced glucocorticoid responsiveness and receptor concentration in splenic leukocytes of senescent rats, Bioehim. Biophys. Aeta, 399 (1975) 145-156. 2 G. S. Roth and J. N. Livingston, Reduction in glucocorticoid inhibition of glucose oxidation and presumptive glucocorticoid receptor content in rat adipocytes during aging, Endocrinology, 99 (1976) 831-839. 3 E. L. Schneider and Y. Mitsui, The relationship between in vitro cellular aging and in vivo human age, Proc. Nat. Acad. Sci. U.S.A., 73 (1976) 3584-3588. 4 J. R. Gavin lIl, J. Roth, P. Jen and P. Freychet, Insulin receptors in human circulating cells and fibroblasts, Proc. Nat. Acad. SeL U.S.A., 69 (1972) 747-751.
43 5 M. D. ttollenberg and P. Cuatrecasas, Epidermal growth factor receptors in human fibroblasts and modulation of action by cholera toxin, Proc. Nat. Aead. ScL U.S.A., 70 (1973) 2964-2968. 6 W. Y. Fujimoto and R. H. Williams, Insulin action on cultured human fibroblasts. Glucose uptake, protein synthesis, RNA synthesis, Diabetes, 24 (1974) 443-448. 7 M. M. Rechler, J. Podskalny, I. D. Goldfine and C. A. Wells, DNA synthesis in human fibroblasts: stimulation by insulin and by nonsuppressible insulin like activity (NSILA-S), J. Clin. Endocrinol. Metab., 39 (1974) 512-521. 8 M. D. ttollenberg and P. Cuatrecasas, Insulin and epidermal growth factor: human fibroblast receptors related to DNA synthesis and amino acid transport, J. BioL Chem.. 250 (1975) 3845-3853. 9 M. D. Hollenberg, Action of insulin analogues on cultured human fibroblasts reflects biological potency, Life Sci., 18 (1976) 521-528. 10 M. M. Rechler and J. Podskalny, Insulin receptors in cultured human fibroblasts, Diabetes, 25 (1976) 250-255. 11 G. Carpenter, K. J. Lembach, M. M. Morrison and S. Cohen, Characterization of the binding of 1251-labeled epidermal growth factor to human fibroblasts, J. Biol. Chem., 250 (1975) 4297-4304. 12 G. Carpenter and S. Cohen, 125I-labeled human epidermal growth factor binding, internalization, and degradation in human fibroblast, J. CellBiol., 71 (1976) 159-171. 13 K. Lembach, Enhanced synthesis and extracellular accumulation of hyaluronic acid during stimulation of quiescent human fibroblast by mouse epidermal growth factor, J. CellPhysiol., 89 (1976) 277--288. 14 M. D. Flollcnberg and tt. Gregory, Human urogastrone and mouse epidermal growth factor share a common receptor site in cultured human fibroblasts, LiJe Sci., 20 (1977) 267-274. 15 P. Cuatrecasas and M. D. Hollenberg, Binding of insulin and other hormones to non-receptor materials: saturability, specificity and apparent "negative cooperativity", Biochem. Biophys. Res. Commun., 62 (1975) 31-41. 16 A. L. Rosenbloom, S. Goldstein and C. C. Yip, Insulin binding to cultured human fibroblasts increases with normal and precocious aging, Science, 193 (1976) 412-415. 17 C. R. Savage Jr. and S. Cohen, Epidermal growth factor and a new derivative, J. Biol. Chem., 247 (1972) 7609-7611. 18 E. L. Schneider, E. J. Stanbridge and C. J. Epstein, Incorporation of 3Houridine and 3H-uracil into RNA. A simple technique for the detection of mycoplasma contamination of cultured cells, Exp. CellRes., 84 (1974) 311-318. 19 M. D. Hollenberg and K. Sharp, Receptors for epidermal growth factor: relationship to growth in normal and progeroid human fibroblasts, Clin. Pharmacol. Therapeut., 17 (1975) 236. 20 C. R. Kahn, J. S. Flier, R. S. Bar, J. A. Archer, P. Gorden, M. M. Martin and J. Roth, Tile syndromes of insulin resistance and Acanthosis nigrieans insulin-receptor disorders in man, New Engl. J. Med., 294 (1976) 739-745. 21 J. R. Gavin, I11, J. Roth, D. M. Neville, Jr., P. Demeyts and D. N. Buell, Insulin-dependent regulation of insulin receptor concentrations: a direct demonstration in cell culture, Proc. Nat. Acad. Sci. U.S.A., 71 (1974) 84-88. 22 D. Huang and P. Cuatrecasas, Insulin-induced reduction of membrane receptor concentrations in isolated fat cells and lymphocytes. Independence from receptor occupation and possible relation to proteolytic activity of insulin, J. Biol. Chem., 250 (1975) 8251. 23 P. Thomopoulos, J. Roth, E. Lovelace and 1. Pastan, Insulin receptors in normal and transformed fibroblasts: relationship to growth and transformation, Cell, 8 (1976) 417. 24 M. D. tlollenberg, E. L. Schneider and J. H. Helderman, Insulin and epidermal growth factor: receptors in cultured skin fibroblasts from young and old adult males, Clin. Res., 24 (1976) 423A.
37
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
RECEPTORS FOR INSULIN AND EPIDERMAL GROWTH FACTORUROGASTRONE IN ADULT HUMAN FIBROBLASTS DO NOT CHANGE WITH DONOR AGE*
MORLEY D. HOLLENBERG Division o f Clinical Pharmacology, Department of Medicine and Department o f Pharmacology and Experimental Therapeutics, The Johns Hopkins University School o f Medicine, Baltimore, Maryland 21205 :U.S.A.)
EDWARD L. SCHNEIDER Gerontology Research Center, National Institute on Aging, National Institutes o f Health, Baltimore, Maryland 21224 {U.S.A.)
(Received July 7, 1978; in revised form February 21, 1979)
SUMMARY The ligand binding and biological responsiveness of cultured fibroblast monolayers were measured for porcine insulin and for murine epidermal growth factor-urogastrone in skin fibroblast cultures derived from young (22 to 31 years) and old (65 to 80 years ) normal male volunteer donors. The receptor characteristics of the cells studied from the two groups of donors were not found to differ. In contrast with previous data, suggesting that there may be a genetically programmed aging of the insulin binding system, it is concluded that for both insulin and epidermal growth factor, receptor characteristics do not change with adult donor age.
INTRODUCTION Considerable interest has been focused on the role of hormone receptors in development and aging [1-3] as well as in the pathogenesis of certain diseases such as diabetes. Cultured skin fibroblasts provide an attractive model system for the study of the effects of aging on receptors. Large quantities of cells can be readily obtained so as to compare the receptor characteristics with other cellular parameters which change as a function of the age of the human donor [3]. Recent work has established that for insulin [4-10] and for both human and murine epidermal growth factor-urogastrone (EGF-URO) [5, 8, 1114], cultured human fibroblasts possess receptors that can be related to the stimulation *Portions of these data were reported in preliminary form to the American Society for Clinical Investigation [ 24 ].
38 of both thymidine incorporation and the uptake of the amino acid analogue, c~-amint> isobutyric acid (AIB) [5, 8, 9]. Tile relative potency of insulin and insulin analogues in stimulating fibroblast AIB uptake parallels the relative activity measured in other insulinresponsive tissues [9]. Studies with fibroblast monolayers provide the distinct advantage that binding measurements can be interpreted in the context of biological responsiveness so as to minimize the risk of observing non-receptor binding artifacts [15]. Furthermore. measurements of receptors for two active polypeptides in the same sample permit a broader interpretation of the data than might be possible from measurements with either ligand alone. In a previous study [16], data obtained with skin samples from children and from adults predominantly under the age of 50 have been presented to suggest that there may be an increase in fibroblast insulin affinity as a function of normal human aging. In this report, we have focused on the characteristics of receptors for both insulin and EGFURO in skin fibroblast samples obtained from young and old adult males. In contrast to the conclusions of the previous study [16] we observe that the receptor characteristics do not differ appreciably with the age of the adult donor.
MATERIALS AND METHODS After formal consent had been obtained, skin biopsies were performed on five young (age range 22-31) and four old (age range 65-80) non-hospitalized male volunteer members of the Baltimore Longitudinal Study [3]. Skin fibrobtast cultures derived from those biopsies were studied at early passage in vitro, between 7 and 13 cumulative cell population doublings. Patients with diabetes mellitus or abnormal glucose tolerance were excluded from this study. Cells were routinely propagated in 75 cm 2 T-flasks using Eagle's minimal essential medium supplemented with glutamine, non-essential amino acids, 10cS, fetal calf serum and 50 /~g/ml chlorotetracycline (aureomycin, GIBCO), as previously described [3]. For the studies of tile binding and biological activity of insulin and EGF-URO, cells were subcultured in 1.5 cm diam. multidish trays (Linbro, New Haven, CT) and measurements were performed essentially as previously described [5, 8, 9] and detailed in the figure legends. Murine EGF-URO prepared from fresh-frozen mouse submaxillary glands [17], was isolated for these studies; highly purified porcine insulin (lot 6151082B108-I) was generously provided through the courtesy of Dr. R. E. Chance, Eli Lilly and Co., Indianapolis, Ind. Donor cell strains were examined in pairs (one from tire young group; one from the old), in a double-blind protocol. Cell cultures were screened and found free from mycoplasma [ 18].
RESULTS Stimulation of AIB uptake provided the most convenient and reliable index of biological responsiveness for insulin activity (reported as the concentration causing a half-
39 10~ x MOLARITY _ 1;(]
_20
30
4.0
108 x MOLAREY 50
60
1
20f
25~
2
3
4
I
~
~ (c)
I (a)
20F o
, ~0
10
1(]
20
30
20
30 50
40
, / ,I-----. ~ ~' o ....
~~~~ 1'o
0~. 3
10
•
• '
40
8(]
~ ~o
INSULIN,ng./ml
'
Jo
20
"
30
-
t
t ~ 5~,ILlo~7 ~''-'° "~'~ 4o
oo
~
~6
3o~
EGF,ng/ml
Fig. 1. Representative binding isotherms and dose-response curves for insulin and EGF-URO in intact fibrobtast monolayers. Cell monolayers were grown to confluency in multiple 1.5 cm diam. multidish trays and assayed in parallel by previously described methods [5, 8, 9]. Data obtained for two young (©, o) and two old (•, I) cell strains are shown. (a) Specific binding of 12Slqabeled insulin (100-500 c.p.m./pg) was measured at 24 °C in monolayers containing, on average, 110,000 cells. The protein content of each monolayer for which binding was measured was used to calculate the binding per cell; the correlation of protein content with cell number was determined separately in identically prepared cell monolayers. Non-specific binding, measured in the presence of 2 ~g/ml unlabeled insulin, was subtracted from the total binding to yield the figure for specific binding; no "specific" binding to the multidish trays was detected. (b) The increased uptake of AIB (440 c.p.m./pmol) in response to increasing amounts of insulin was measured during a 12 min pulse with 3H-labeled A1B after a 2.5 h preincubation with hormone. (c) Specific binding of 12Sl-labeled mouse EGF URO (100-250 c.p.m./ pg) to cell monolayers was measured as for insulin; non-specific binding was determined in the presence of 1 ug/ml unlabeled EFG-URO. (d) EGI:-URO-stimulated incorporation of 3H-thymidine (1 t~Ci/ml; 6 Ci/mmol; counting efficiency, 13%) into trichloroacetic acid insoluble material was measured during a 2 h period at 37 °C begun 22 h after the addition of increasing amounts of EGFURO to triplicate cell monolayers.
maximal effect, EDso) (Fig. l ( b ) and Table I). Measurements o f the specific binding o f 12SI-labeled insulin over the range o f biological responsiveness (AIB uptake) yielded isotherms which approach saturation in the c o n c e n t r a t i o n range between 3 and 6 n M (Fig. l(a)). Values for the m a x i m u m b i n d i n g per cell (Bma~) and the c o n c e n t r a t i o n at which insulin b i n d i n g was estimated to be ½-maximal (C1/,ma~) were determined directly from these isotherms (Table I). S t i m u l a t i o n of t h y m i d i n e i n c o r p o r a t i o n provided the best index o f biological responsiveness for E G F - U R O ; E D s o values were determined from the dose-response curves (Fig. l ( d ) and Table I). As in the case of insulin, the binding of 12Sllabeled E G F yielded isotherms (Fig. l(c)) from which the Cv, max and Bma ~ could be estimated (Table I). Receptor b i n d i n g isotherms and biological dose-response curves for b o t h insulin and E G F were reproducible at both early and middle passage in vitro, as well as in frozen cell stocks after return to tissue culture. As in previous studies [5, 8, 9 ] , the
11.6 5.8 2.3 7.6 6.2 6.7 + 3.4
1.6 +- 0.5
1.9 1.4 1.4 1.1 2.4
1.6 +- 0.5
Average:
31 28 28 26 22
Average:
9.5 -+ 3.4
0.75 -+ 0.55
1.6 0.97 0.30 0.53 0.33
0.74 +- 0.23
0.80 0.62 1.0 0.50
12.3 8.6 5.0 12.0
2.2 1.4 1.7 1.0
80 73 73 65
0.70 -+ 0.11
0.70 0.68 0.65 0.58 0.87
0.77 +- 0.12
0.60 0.83 0.75 0.88
(nM)
Clhrna x
ED 5o (nM)
(nM)
(10 -3 X sites~cell)
C~hmax
"
B max
EGF-URO
Insulin
Age o f Cell Culture Donor ~vearsJ
78 +- 58
160 116 19 52 43
106 -+ 90
238 83 59 42
(10 - 3 × sites~cell)
B max
0.046 + 0.020
0.067 0.067 0.042 0.030 0.025
0.030 +- 0.014
0.050 0.022 0.022 0.025
(nM)
ED 5o
Parameters for the biological responsiveness (EDs0) and ligand binding (C~hrnax, Bmax) were estimated for both insulin and E G F - U R O from dose-response curves and ligand binding isotherms as depicted in Fig. I. The average, -+S.D. were calculated for cell strains derived from the groups of old (age 6 5 - 8 0 ) and young (age 22-31) donors.
CHARACTERISTICS OF RECEPTOR BINDING AND BIOLOGICAL RESPONSIVENESS
TABLE I
41 degree of cellular responsiveness (stimulation of either AIB uptake (t.5 to 2.2-fold) or thymidine incorporation (5 to 20-fold) was observed to vary considerably in an individual cell strain, without appreciably affecting the values of the EDso'S. However, even for an individual cell strain, the maximum cellular binding capacity for both EGF-URO and insulin was observed to vary markedly from one sample to another, although all measurements were performed on quiescent (DNA synthesis) confluent cell monolayers. Variations in maximum binding capacity do not appear to correlate with variations in maximal biological responsiveness. Unfortunately, it is not possible to examine this correlation rigorously, since in a given cell strain, measurements of binding and responsiveness could not be performed on the same cell monolayers.
DISCUSSION
From the data summarized in Table I and illustrated in Fig. 2, it is evident that for both insulin and EGF-URO the receptor binding parameters studied in fibroblast cultures do not differ appreciably with adult donor age. The values for the Cv, ma x of specific insulin binding obtained in this study (1.6 -+ 0.5 nM) are in good agreement with measurements of insulin affinity from previous studies of fibroblasts from different donors [16]. Additionally, the affinity of EGF for fibroblasts, as indicated by the C1/, values in this stUdy is in excellent agreement with values obtained by other laboratories under different experimental conditions [11]. The relatively large variation in the maximum cellular binding capacity cannot be readily explained, but appears to be similar in cells from both young and old donors. From this and from previous work [5, 8, 9] it is evident that such variations may be expected for measurements of the kind we have performed and that a very large number of donor samples would be needed to detect any small differences.
E
0.10f == 0,05 0
=~
i
i
•
m
:~ 0.5 0
E
=
w
2I---1 o
•
C.
,e,
z
oo
20
40
60
80
DONORAGE I YEARS I
Fig. 2. Receptor parameters as a function o f donor age. Values for the EDso (A, C) and for the 12Slligand concentration corresponding to Vz maximal saturation o f binding (Clhmax, B.D.) were estimated fo~ E G F - U R O (o, I ) and for insulin (o, e) in individual cell strains from data like those depicted in Fig. 1.
42 Most importantly, variation in maximum binding capacity does not appear to affect either the biological sensitivity (EDs0) or the cellular ligand affinity (Cv2m~x). The lack of change with donor age of the EDso'S for the cellular responses for both E G F - U R O and insulin substantiates the data obtained in the present study by ligand binding measurements. In contrast with the report proposing a genetically programmed aging of the insulin binding system [16], our data indicate that during adult life the characteristics of the receptors for both insulin and E G F - U R O do not change with age. The previously reported differences in insulin binding [16] in fibroblast samples are most striking when the data from children are compared with those from adult samples. These differences may therefore reflect developmental changes rather than adult aging. It should be pointed out, however, that our measurements on adult fibroblasts are in excellent accord with previous measurements performed on fibroblasts from newborn males [5, 8]. It may be also of interest in this regard that fibroblasts from a child with a Progeria-like syndrome possess characteristics of binding and biological responsiveness for EGF which are similar to those o f adult fibroblasts [19]. It is important to note that the measurements reported here may not reflect receptor characteristics in vivo where humoral factors such as the presence of antireceptor antibodies [20] or elevated hormone levels [21, 22] may alter receptor function. These measurements on confluent monolayers also do not eliminate possible differences in receptor numbers which may be present during the active phases of cell growth. However, it has been observed that insulin receptors increase with cell density [23]. Thus, our measurements should reflect the maximum numbers of receptors.
ACKNOWLEDGEMENTS We are grateful for the expert technical assistance of W. H. Shackelford and are indebted to Dr. J. H. Helderman for help in initiating these studies. Support came in part from a Basil O'Connor Research Starter Grant of the National Foundation-March of Dimes and from the Maryland Division of the American Cancer Society. M. D. Hollenberg is an investigator of the Howard Hughes Medical Institute.
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