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Apparent positive cooperativity of ACTH action on adrenocortical cells: The effect of hormone degradation
Molecular and Cellular Endocrinology, 6 (1977) 211-216 0 Elsevier/North-Holland Scientific Publishers Ltd.
APPARENT POSITIVE COOPERATIVITY OF ACTH ACTION ON ADRENOCORTICAL CELLS: THE EFFECT OF HORMONE DEGRADATION Dermot M.F. COOPER and Dennis SCHULSTER Biochemistry Laboratory, BNI 9QG, UK Received
School of Biological Sciences,
31 March 1976; accepted
University of Sussex, Falmer, Brighton
15 June 1976
The ability of adrenocortical cells to degrade ACTHt_39 and [ 1251]ACTH has been assessed under various conditions. Under conditions leading to increased hormone degradation there was an elevation of both the EDso and the value of the Hill coefficient derived from concentration-effect curves for ACTH-stimulated steroidogenesis. Such degradative mechanisms offer a simple explanation for the apparent positive cooperativity proposed by others for ACTH-receptor-adenylate cyclase interactions.
Keywords:
ACTH; adrenal;
positive
cooperativity;
degradation.
The similarity in shape between concentration-effect curves for hormones and plots of allosteric enzyme activity against substrate concentration has led to many attempts to imbue hormone interactions with the allosteric properties originally proposed for regulatory enzymes by Monod and colleagues (1963) and Koshland and colleagues (1966). The sigmoid-shaped concentration-effect curves are commonly fitted to some form of the Hill equation (Brown and Hill, 1923) and a value for the Hill coefficient (n) extracted as an index of cooperative effects. A value of y1> 1 is taken to indicate ‘positive cooperativity’ (Newsholme and Start, 1973). Using a weighted curve-fitting procedure, Rodbard (1974) found elevated Hill coefficients in concentration-effect curves of ACTH stimulation of cyclic AMP and corticosterone output by isolated adrenal cells (data of Seelig and Sayers, 1973). No evidence for positive cooperativity has been found in the binding of ACTH to adrenal cells (McIlhinney and Schulster, 1974) leading to the suggestion that positive cooperative effects might lie in the interaction of the occupied receptor with the adenylate cyclase of the membrane. Wenke (1970) has, however, pointed out that sometimes a shift from a hyperbolic to a sigmoid curve may be achieved by the existence of a hormone-degrading 211
212
D.M.F.
Cooper, D. Schulster
system *. We present results here which indicate that hormonal degradation may be the mechanism whereby sigmoidal concentration-effect curves are obtained for ACTH stimulation of adrenal cells.
MATERIALS
AND METHODS
Purified porcine ACTH,_sa (140 IU/mg) was a gift from Armour Pharmaceuticals, Eastbourne, UK. Isolated rat adrenal cells were prepared by collagenase dispersion of decapsulated glands and corticosterone production measured as described by Richardson and Schulster (1972). ‘251-Labelled ACTH was prepared by adding 2 pg of pure ACTH to 1 mCi Na 125I (14 mCi/pg I; Radiochemical Centre, Amersham, Bucks, UK) followed by 1 pg lactoperoxidase (80 U/mg; Sigma Chemical Co.), 0.5 pg glucose oxidase (110 U/mg; Worthington Biochemical Corp.) and 10 pg glucose in 0.1 M phosphate buffer, pH 7.0. Each reagent was added in a volume of 10 ~1 giving a total of 50 ~1. Iodination was allowed to proceed for 3 min at room temperature, resulting in around 90% incorporation of iodine equivalent to 0.9 I atom per ACTH molecule. The reaction was stopped by addition of 1.9 mg sodium metabisulphite and 10 ml of a 0.1% human serum albumin solution (pH 7.0,O.Ol M phosphate). [‘251]ACTH was adsorbed to Spherosil XOA-400 (10 mg: JJ’s (Chromatography) Ltd., Kings Lynn, Norfolk, UK), washed and then eluted in 0.7 N HCl : ethanol (1 : 3; v/v). Chromatoelectrophoresis was performed on Whatman No. 3MM paper, as described by Yalow and Berson (1960).
RESULTS AND DISCUSSION The patterns obtained following chromatoelectrophoresis of ACTH, after it had been incubated with various cell concentrations, are shown in fig. 1. It can be seen that in the control sample (in the absence of cells) 92% of the total radioactivity applied is found in the first 2 cm. By contrast, in the sample which was incubated with 1.2 X lo6 cells/ml, only about 50% of the radioactivity is in this region with the rest of the counts distributed along the paper. This leads to the conclusion that increasing cell concentration increases [ ‘25I] ACTH degradation. In order to determine whether native ACTH was degraded to a similar extent, at a range of concentrations normally employed in dose-response experiments, adrenal cells were incubated with various concentrations of hormone. The cells were
* This discussion refers to linear plots of hormone concentration versus effect. However hormone concentration-effect curves are commonly plotted with semilogarithmic axes, which usually yield sigmoid-shaped curves. In order to determine whether the underlying relationship is ‘hyperbolic’ or ‘sigmoid’, the Hill equation is commonly used as outlined above.
213
Positive cooperativity and ACTH degradation
Fig. 1. Degradation of [ 1251]ACTH incubated with adrenal cells as assessed by chromatoelectrophoresis. [ ‘*‘I]ACTH (10 ng/ml) was incubated (1 ml; 20 min; 37°C) with the cell concentrations indicated. Aliquots (20 ~1) of the incubation medium were then run at 500 V on paper (Whatman No. 3MM) in barbitone buffer (pH 8.6; I = 0.1; 4°C). Peak a (0.2 cm) represents undegraded ACTH; Peak b, peptide fragments (degradation products), and Peak c, free ‘251-.
then spun down, and aliquots of the supernatant were taken for estimation of corticosterone and bioassay of ACTH (method of Richardson and Schulster, 1972). This ACTH assay was performed using fresh adrenal cells, and comparing its activity with that of 3rd International Standard ACTH. The results of three of these experiments are shown in table 1. It should be pointed out that these values are not absolute since the standard ACTH will also have been degraded to a similar extent. The unusual relationship between hormone concentrations and degradation may be a consequence of the low concentrations of hormone employed (160 pU/ml, equal to approx. 1.5 X 10-*” M). It is quite likely that under these conditions the Km of the
Table 1. Degradation of ACTH following incubation with isolated adrenal cells. Different concentrations of ACTH were incubated in duplicate with adrenal cells (0.5 ml; 150,000 cells/ml; 37°C; 1 h). After centrifugation (6OOg; 4°C; 5 min) aliquots (200 ~1) of the supernatant were taken for assay of corticosterone and ACTH. Corticosterone was assayed fluorimetrically (see text). ACTH was bioassayed using fresh adrenal cells, allowance being made for the fluorogenic material produced during the first incubation. The data shown is from three experiments in which every effort was made to keep cell number, preparative methodology and other conditions constant. ACT11 added 20 40 80 160
(pU/ml)
ACTH degraded 3.7 16.7 42.0 96.7
+ 1.2 + 2.3 + 8.2 f 19.2
(uU/ml
f SEM)
ACTH degraded 18.5 41.8 52.5 60.4
f 6.0 f 5.8 + 10.3 ? 12.0
i SEM (%)
D.M.F. Cooper, D. Schulster
214
degrading system is not approached and equilibrium rates may not have been established. The observation of ACTH1_s9 degradation by adrenal cell suspensions, concurs with the findings of Bennett et al. (1974) who showed considerable degradation of ACTH1_-24 and its analogues by this system. In order to establish whether the amount of degradation by a fixed number of cells could be varied, a further type of experiment was performed. Gentle mechanical agitation was employed using a rotator at 25 rpm for varying time lengths with identical cell concentrations. This treatment resulted in considerable cell damage, as can be seen from the decrease in maximal steroid output from 1.6 pg/rat/h to 1.25 and 0.62 pg/rat/h after 9 and 22 min rotation, respectively. The concentrationeffect curves obtained are shown in fig. 2. In a simple analysis of the data obtained from this experiment, Hill plots (log (u - Q/V,,, - U) against log (H)) were performed by unweighted linear regression. For the control data (no rotation) a Hill coefficient of 1.9 with an EDse of 7.2 X lop5 IU/ml was obtained compared with a Hill coefficient of 2.3 .and an EDse of 1 .l X lop4 IU/ml from the data for the concentration-effect curve of the cells rotated for 22 min. We interpret this increase in ED se in terms if increased hormone degradation and note that these increases are also associated with elevated Hill coefficients.
u/
0
I
5.10-5
:
v--Y
1.10-’ ACTH
2s10-4
4x10-’
1 xl o-2
( tu/nI)
Fig. 2. Concentration-effect curves for ACTH-stimulated steroidogenesis after subjecting adrenal cells to different periods of rotation. Cells were rotated (25 rpm) in 12 ml plastic tubes for 0 min (e), 9 min (X), and 22 min (A), after which steroid output in a l-h incubation was measured in response to a range of ACTH concentrations. The cell damage caused by rotation lowered the maximum steroid output from 1.6 to 1.25 pg/rat/h (after 9 min) and 0.62 pg/rat/h after 22 min. Results (mean f S.E.M.) shown above are expressed as a percentage of the maximum output after subtraction of basal; i.e. (u - uo) X lOO/(V,,, - uo).
215
Positive cooperativity and ACTH degradation
Increasing degradation
Constant rate
degradation
rate c
11
1
I
I
LOG
HORMONE
CONCENTRATION
Fig. 3. Schematic presentation of effect of changes in the hormone degrading system on the shape of concentration-effect curves. It is suggested that increasing the degradation capacity (broken lines) from low to high, results in increased slope and higher EDso values for the hormone concentration-effect curves (solid lines). Thin and thick lines refer to systems with low and high hormone-degrading capacity, respectively.
Rodbard (1974) presented results obtained by fitting the Hill equation to concentration-effect curves of ACTHr_a4-stimulated steroidogenesis. He found Hill coefficients varying between 1 .17 and 1.98 and EDso values which varied between 23.02 and 92.26 pg/ml. Examination of his results demonstrates a close correlation between Hill coefficient and EDso (correlation coefficient = 0.89; P< 0.001); i.e. the higher Hill coefficients were associated with the higher ED,, values. Our interpretation of these and our own results are illustrated in fig. 3, in which we assume normal Michaelis-Menten behaviour for the degrading system. Increasing amounts of the degrading system will result in a potentially serious distortion of the lower end of the concentration-effect curve, leading to elevated Hill coefficients in addition to shifted ED,, values. Therefore, since we have shown that degradation can be related to both number and treatment of the cells and that this may result in altered kinetic parameters, we reel there are insufficient grounds for invoking allosteric phenomena in the mechanism of hormonal activation until the contribution by any degrading system has been quantified. ACKNOWLEDGEMENTS We are grateful to Armour Pharm. Co. and the M.R.C. for financial assistance. Generous gifts of 3rd Inst. Standard ACTH from W.H.O., porcine ACTHr_sa from
216
D.M.F.Cooper, L2. Schrdster
Mr. P. Lloyd, Armour Pharmaceutics Co. and human serum atbumin from Dr. W. d’A. Maycock, of the Lister Institute of Preventive Medicine are also gratefully acknowledged.
REFERENCES Bennett, H.P.J.,‘Bullock, G., Lowry, P.J., McMartin, C. and Peters, J. (1974) Biochem. J. 138, 185-194. Brown, W.E.L. and Hill, A.V. (1923) Proc. R. Sot. Lond. (Bioi.) 94,297. Koshland, DE., Nemethy, G. and Filmer, D. (1976) Biochemistry 5,365-385. McI~inney, R.A.J. and Schulster, D. (1974) 3. ~ndoc~nol. 64, 175 - 184. Monod, J., Changeux, J.P. and Jacob, F. (1963) J. Mol. Biot. 6 306-329. Newsho~e, F.A. and Start, C. (1973) Reguiation in Metabolism, (John Wiley and Sons, London, New York, Sydney, Toronto). Richardson, MC. and Schulster, D. (1972) J. Endocrinol. 55, 127-139. Rodbard, D. (1974) Endocrinology 94,1427-1437. Seelig, S. and Sayers, G. (1973) Arch. Biochem. Biophys. 154,230-239. Wenke, M. (1970) In: Adipose Tissue: Its Regulation and Metabolic Function. Eds.: B. Jeanrenaud and D. Hepp, (Geoig Thieme-Verlag, Stuttgart) Horm. Metab. Res., Suppl. 2, pp. 55-62. Yalow, R.S. and Berson,S.A. (1960) J. Clin. Invest. 39,11.57-1175.
APPARENT POSITIVE COOPERATIVITY OF ACTH ACTION ON ADRENOCORTICAL CELLS: THE EFFECT OF HORMONE DEGRADATION Dermot M.F. COOPER and Dennis SCHULSTER Biochemistry Laboratory, BNI 9QG, UK Received
School of Biological Sciences,
31 March 1976; accepted
University of Sussex, Falmer, Brighton
15 June 1976
The ability of adrenocortical cells to degrade ACTHt_39 and [ 1251]ACTH has been assessed under various conditions. Under conditions leading to increased hormone degradation there was an elevation of both the EDso and the value of the Hill coefficient derived from concentration-effect curves for ACTH-stimulated steroidogenesis. Such degradative mechanisms offer a simple explanation for the apparent positive cooperativity proposed by others for ACTH-receptor-adenylate cyclase interactions.
Keywords:
ACTH; adrenal;
positive
cooperativity;
degradation.
The similarity in shape between concentration-effect curves for hormones and plots of allosteric enzyme activity against substrate concentration has led to many attempts to imbue hormone interactions with the allosteric properties originally proposed for regulatory enzymes by Monod and colleagues (1963) and Koshland and colleagues (1966). The sigmoid-shaped concentration-effect curves are commonly fitted to some form of the Hill equation (Brown and Hill, 1923) and a value for the Hill coefficient (n) extracted as an index of cooperative effects. A value of y1> 1 is taken to indicate ‘positive cooperativity’ (Newsholme and Start, 1973). Using a weighted curve-fitting procedure, Rodbard (1974) found elevated Hill coefficients in concentration-effect curves of ACTH stimulation of cyclic AMP and corticosterone output by isolated adrenal cells (data of Seelig and Sayers, 1973). No evidence for positive cooperativity has been found in the binding of ACTH to adrenal cells (McIlhinney and Schulster, 1974) leading to the suggestion that positive cooperative effects might lie in the interaction of the occupied receptor with the adenylate cyclase of the membrane. Wenke (1970) has, however, pointed out that sometimes a shift from a hyperbolic to a sigmoid curve may be achieved by the existence of a hormone-degrading 211
212
D.M.F.
Cooper, D. Schulster
system *. We present results here which indicate that hormonal degradation may be the mechanism whereby sigmoidal concentration-effect curves are obtained for ACTH stimulation of adrenal cells.
MATERIALS
AND METHODS
Purified porcine ACTH,_sa (140 IU/mg) was a gift from Armour Pharmaceuticals, Eastbourne, UK. Isolated rat adrenal cells were prepared by collagenase dispersion of decapsulated glands and corticosterone production measured as described by Richardson and Schulster (1972). ‘251-Labelled ACTH was prepared by adding 2 pg of pure ACTH to 1 mCi Na 125I (14 mCi/pg I; Radiochemical Centre, Amersham, Bucks, UK) followed by 1 pg lactoperoxidase (80 U/mg; Sigma Chemical Co.), 0.5 pg glucose oxidase (110 U/mg; Worthington Biochemical Corp.) and 10 pg glucose in 0.1 M phosphate buffer, pH 7.0. Each reagent was added in a volume of 10 ~1 giving a total of 50 ~1. Iodination was allowed to proceed for 3 min at room temperature, resulting in around 90% incorporation of iodine equivalent to 0.9 I atom per ACTH molecule. The reaction was stopped by addition of 1.9 mg sodium metabisulphite and 10 ml of a 0.1% human serum albumin solution (pH 7.0,O.Ol M phosphate). [‘251]ACTH was adsorbed to Spherosil XOA-400 (10 mg: JJ’s (Chromatography) Ltd., Kings Lynn, Norfolk, UK), washed and then eluted in 0.7 N HCl : ethanol (1 : 3; v/v). Chromatoelectrophoresis was performed on Whatman No. 3MM paper, as described by Yalow and Berson (1960).
RESULTS AND DISCUSSION The patterns obtained following chromatoelectrophoresis of ACTH, after it had been incubated with various cell concentrations, are shown in fig. 1. It can be seen that in the control sample (in the absence of cells) 92% of the total radioactivity applied is found in the first 2 cm. By contrast, in the sample which was incubated with 1.2 X lo6 cells/ml, only about 50% of the radioactivity is in this region with the rest of the counts distributed along the paper. This leads to the conclusion that increasing cell concentration increases [ ‘25I] ACTH degradation. In order to determine whether native ACTH was degraded to a similar extent, at a range of concentrations normally employed in dose-response experiments, adrenal cells were incubated with various concentrations of hormone. The cells were
* This discussion refers to linear plots of hormone concentration versus effect. However hormone concentration-effect curves are commonly plotted with semilogarithmic axes, which usually yield sigmoid-shaped curves. In order to determine whether the underlying relationship is ‘hyperbolic’ or ‘sigmoid’, the Hill equation is commonly used as outlined above.
213
Positive cooperativity and ACTH degradation
Fig. 1. Degradation of [ 1251]ACTH incubated with adrenal cells as assessed by chromatoelectrophoresis. [ ‘*‘I]ACTH (10 ng/ml) was incubated (1 ml; 20 min; 37°C) with the cell concentrations indicated. Aliquots (20 ~1) of the incubation medium were then run at 500 V on paper (Whatman No. 3MM) in barbitone buffer (pH 8.6; I = 0.1; 4°C). Peak a (0.2 cm) represents undegraded ACTH; Peak b, peptide fragments (degradation products), and Peak c, free ‘251-.
then spun down, and aliquots of the supernatant were taken for estimation of corticosterone and bioassay of ACTH (method of Richardson and Schulster, 1972). This ACTH assay was performed using fresh adrenal cells, and comparing its activity with that of 3rd International Standard ACTH. The results of three of these experiments are shown in table 1. It should be pointed out that these values are not absolute since the standard ACTH will also have been degraded to a similar extent. The unusual relationship between hormone concentrations and degradation may be a consequence of the low concentrations of hormone employed (160 pU/ml, equal to approx. 1.5 X 10-*” M). It is quite likely that under these conditions the Km of the
Table 1. Degradation of ACTH following incubation with isolated adrenal cells. Different concentrations of ACTH were incubated in duplicate with adrenal cells (0.5 ml; 150,000 cells/ml; 37°C; 1 h). After centrifugation (6OOg; 4°C; 5 min) aliquots (200 ~1) of the supernatant were taken for assay of corticosterone and ACTH. Corticosterone was assayed fluorimetrically (see text). ACTH was bioassayed using fresh adrenal cells, allowance being made for the fluorogenic material produced during the first incubation. The data shown is from three experiments in which every effort was made to keep cell number, preparative methodology and other conditions constant. ACT11 added 20 40 80 160
(pU/ml)
ACTH degraded 3.7 16.7 42.0 96.7
+ 1.2 + 2.3 + 8.2 f 19.2
(uU/ml
f SEM)
ACTH degraded 18.5 41.8 52.5 60.4
f 6.0 f 5.8 + 10.3 ? 12.0
i SEM (%)
D.M.F. Cooper, D. Schulster
214
degrading system is not approached and equilibrium rates may not have been established. The observation of ACTH1_s9 degradation by adrenal cell suspensions, concurs with the findings of Bennett et al. (1974) who showed considerable degradation of ACTH1_-24 and its analogues by this system. In order to establish whether the amount of degradation by a fixed number of cells could be varied, a further type of experiment was performed. Gentle mechanical agitation was employed using a rotator at 25 rpm for varying time lengths with identical cell concentrations. This treatment resulted in considerable cell damage, as can be seen from the decrease in maximal steroid output from 1.6 pg/rat/h to 1.25 and 0.62 pg/rat/h after 9 and 22 min rotation, respectively. The concentrationeffect curves obtained are shown in fig. 2. In a simple analysis of the data obtained from this experiment, Hill plots (log (u - Q/V,,, - U) against log (H)) were performed by unweighted linear regression. For the control data (no rotation) a Hill coefficient of 1.9 with an EDse of 7.2 X lop5 IU/ml was obtained compared with a Hill coefficient of 2.3 .and an EDse of 1 .l X lop4 IU/ml from the data for the concentration-effect curve of the cells rotated for 22 min. We interpret this increase in ED se in terms if increased hormone degradation and note that these increases are also associated with elevated Hill coefficients.
u/
0
I
5.10-5
:
v--Y
1.10-’ ACTH
2s10-4
4x10-’
1 xl o-2
( tu/nI)
Fig. 2. Concentration-effect curves for ACTH-stimulated steroidogenesis after subjecting adrenal cells to different periods of rotation. Cells were rotated (25 rpm) in 12 ml plastic tubes for 0 min (e), 9 min (X), and 22 min (A), after which steroid output in a l-h incubation was measured in response to a range of ACTH concentrations. The cell damage caused by rotation lowered the maximum steroid output from 1.6 to 1.25 pg/rat/h (after 9 min) and 0.62 pg/rat/h after 22 min. Results (mean f S.E.M.) shown above are expressed as a percentage of the maximum output after subtraction of basal; i.e. (u - uo) X lOO/(V,,, - uo).
215
Positive cooperativity and ACTH degradation
Increasing degradation
Constant rate
degradation
rate c
11
1
I
I
LOG
HORMONE
CONCENTRATION
Fig. 3. Schematic presentation of effect of changes in the hormone degrading system on the shape of concentration-effect curves. It is suggested that increasing the degradation capacity (broken lines) from low to high, results in increased slope and higher EDso values for the hormone concentration-effect curves (solid lines). Thin and thick lines refer to systems with low and high hormone-degrading capacity, respectively.
Rodbard (1974) presented results obtained by fitting the Hill equation to concentration-effect curves of ACTHr_a4-stimulated steroidogenesis. He found Hill coefficients varying between 1 .17 and 1.98 and EDso values which varied between 23.02 and 92.26 pg/ml. Examination of his results demonstrates a close correlation between Hill coefficient and EDso (correlation coefficient = 0.89; P< 0.001); i.e. the higher Hill coefficients were associated with the higher ED,, values. Our interpretation of these and our own results are illustrated in fig. 3, in which we assume normal Michaelis-Menten behaviour for the degrading system. Increasing amounts of the degrading system will result in a potentially serious distortion of the lower end of the concentration-effect curve, leading to elevated Hill coefficients in addition to shifted ED,, values. Therefore, since we have shown that degradation can be related to both number and treatment of the cells and that this may result in altered kinetic parameters, we reel there are insufficient grounds for invoking allosteric phenomena in the mechanism of hormonal activation until the contribution by any degrading system has been quantified. ACKNOWLEDGEMENTS We are grateful to Armour Pharm. Co. and the M.R.C. for financial assistance. Generous gifts of 3rd Inst. Standard ACTH from W.H.O., porcine ACTHr_sa from
216
D.M.F.Cooper, L2. Schrdster
Mr. P. Lloyd, Armour Pharmaceutics Co. and human serum atbumin from Dr. W. d’A. Maycock, of the Lister Institute of Preventive Medicine are also gratefully acknowledged.
REFERENCES Bennett, H.P.J.,‘Bullock, G., Lowry, P.J., McMartin, C. and Peters, J. (1974) Biochem. J. 138, 185-194. Brown, W.E.L. and Hill, A.V. (1923) Proc. R. Sot. Lond. (Bioi.) 94,297. Koshland, DE., Nemethy, G. and Filmer, D. (1976) Biochemistry 5,365-385. McI~inney, R.A.J. and Schulster, D. (1974) 3. ~ndoc~nol. 64, 175 - 184. Monod, J., Changeux, J.P. and Jacob, F. (1963) J. Mol. Biot. 6 306-329. Newsho~e, F.A. and Start, C. (1973) Reguiation in Metabolism, (John Wiley and Sons, London, New York, Sydney, Toronto). Richardson, MC. and Schulster, D. (1972) J. Endocrinol. 55, 127-139. Rodbard, D. (1974) Endocrinology 94,1427-1437. Seelig, S. and Sayers, G. (1973) Arch. Biochem. Biophys. 154,230-239. Wenke, M. (1970) In: Adipose Tissue: Its Regulation and Metabolic Function. Eds.: B. Jeanrenaud and D. Hepp, (Geoig Thieme-Verlag, Stuttgart) Horm. Metab. Res., Suppl. 2, pp. 55-62. Yalow, R.S. and Berson,S.A. (1960) J. Clin. Invest. 39,11.57-1175.