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Effect of hydrocortisone on beta-adrenergic receptors in lung membranes
Life Sciences, Vol. 25, pp . 1925-1930 Printed in the U.S .A .
Pergamon Press
EFFECT OF HYDROCORTISONE ON BETA-ADRENERGIC RECEPTORS IN LUNG MEMBRANES Kenji Mano, Abdolah Akbarzadeh and Robert G. Townley Allergic Disease Center Creighton University School of Medicine Omaha, Nebraska 68178 (Received in final form October 22, 1979)
Summary It has been observed that glucocorticoids potentiate beta-adrenergic stimulation of cardiovascular and airway tissues. In order to investigate the mechanism of this potentiating action, we examined the effect of glucocorticoids on the number and affinity of beta-adrenergic receptors in animal lung tissues, by a direct binding technique using [125]I-Iodohydroxybenzylpindolol ([125]2-HYP), a potent beta-adrenergic receptor antagonist . Specific binding of [1251I-HYP to rat lung membranes was saturable with 386 fmol of [125]2-HYP/mg protein at saturation . The apparent equilibrium dissociation constant of [1251I-HYP for beta-receptors was 221 nM . Chronic administration of hydrocortisone increased the density of beta-adrenergic receptors by 70% from 386 fmol to 657 fmol/mg with some decrease in the affinity of [1251I-HYP for beta-adrenergic receptors . By contrast, adrenalectomy produced a 29% fall in the number of beta-adrenergic receptors without altering the affinity of [1251I-HYP for beta-receptors, and this change was reversed by exogenous administration of hydrocortisone . The present study suggests that glucocorticoids may participate in regulating the density of beta-adrenergic receptors, and may potentiate beta-adrenergic receptors stimulation, at least in part by increasing beta-receptor density in tissue membranes . In the past several years, great progress has been made in developing direct binding techniques for assessing the properties of beta-adrenergic receptors using radiolabeled ligand . Recent studies utilizing these techniques, have demonstrated that pharmacological treatment with various hormones (1-3) and surgical inervention (4) can cause alterations in the number of beta-adrenergic receptors, and that this alteration may play an important role in tissue responsiveness to catecholamines . Glucocorticoids have been shown to exert a potentiating effect on adrenergic stimulation in several tissues such as airway smooth muscle (5-7), and cardiovascular tissue (8) ; however, the mechanism is unclear . In order to investigate the mechanism of the potentiating effect of glucocorticoids on adrenergic stimulation, we tested the modulating effect of glucocorticoids on the density and affinity of beta-adrenergic receptors in rat lung tissues, using [125]2-Iodohydroxybenzylpindolol, a potent beta-adrenergic antagonist (9) . Materials and Methods Animal experiments . Male Sprague-Dawley rats (250-300 g) received subcutaneous injections of hydrocortisone (50 ma/ka) or 0 .3 ml of isotonic saline once every 0024-3205/79/221925-06$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd.
1926
Hydrocortisone on ß-Receptors in Lung
Vol . 25, No . 22, 1979
day for 9 days . The rats were killed by decapitation 24 hours after the last injection . In a separate experiment, the rats were bilaterally adrenalectomized or sham-operated . Four of the adrenalectomized rats received subcutaneous injections of hydrocortisone (50 mg/kg) daily for 6 days after adrenalectomy . Adrenalectomized rats treated with hydrocortisone were sacrificed 24 hours after the last injections . Tissue preparation . Lungs of the rats were removed immediately after sacrifice, 3íssected ee o large vessels and bronchi, chilled on ice, and homogenized in 11 .3% (w/v) sucrose in 5 mM Tris-HC1 buffer pH 7 .4 (20 ml/g wet tissue) containing 1 mM MgC1[21 with a Brinlamann Polytron homogenizer at full speed for 15 seconds . The homogenate was centrifuged at 90 g . for 5 min . a t 4 degrees C . The resultant supernate was centrifuged at 30,000 g . for 15 min . a t 4 degrees C (1) . The final pellets enriched in plasma membranes were resuspended in Tris-HC1 incubation buffer (50 mM Tris, 10 mM MgC1[21, pH 7 .4) using a Brinkmann Polytron . This suspension was used for the binding assays . The yields of final membrane preparation per gram of initial lung wet weight from each group were not statistically significantly different and were about 25 mg of membrane protein/g of initial wet weight . Protein concentrations were determined by the method of Lowry (10) with human serum albumin as standard . Iodination and paperchromatography for ligand . Hydroxybenzylpindolol (HYP) was iodinatedand extracted by the meth6of Harden et al (11) . HYP (30 ug in 20 ul of 10 mM HC1), Na[1251I (2 mCi in 20 ul of distilled water) and potassium phosphate buffer (0 .3 M, 20 ul, pH 7 .4) were combined . Chloramine T (20 ul of 0 .17 mg/ml in distilled water) was then added and allowed to stand at room temperature for 3 min . The reaction was stopped by adding 300 ul of 1 M acetic acid containing 1 mg/ml sodium metabisulfite . Using a Pasteur pipette as a separation funnel this solution was washed three times with 300 ul of ethyl acetate containing 0 .01% phenol . The three washes were combined and washed twice with 400 ul of acetic acid containing 1 mg/ml sodium meta- bisulfite . The upper layer (ethyl acetate) which contains [1251I-HYP and HYP was kept for paperchromatography . A significant amount of [1251I-HYP can be recovered by repeatedly washing the lower layer with ethyl acetate containing 0 .01% phenol, at least 4 times . Following iodination, the ethyl acetate extract was spotted on Whatman No . 3MM paper . Descending paperchromatography was performed at room temperature with 0 .1 M ammonium formate, pH 8 .5, containing 0 .01% phenol . After development for 12-16 hours the paper was cut, while still wet, into strips 1 cm wide . The [1251I-HYP is found 4-7 am from the origin, while the HYP has run completely off the paper . Each strip was placed in a vial, and [1251I-HYP was eluted with 5 ml of methyl alcohol containing 0 .01% phenol . The eluted [1251I-HYP was stored at -20 degrees C . [1251I-HYP was chemically stable for at least 4 weeks . (The final specific activity of [1251I-HYP was 2,200 Ci/mmol .) Binding assay . [1251I-HYP binding was assayed by incubating the membrane particulate fractions (120-150 ug of protein in 0 .2 ml incubation buffer) with [1251I-HYP (0 .1 ml) for 60 min . . at 37 degrees C either in the absence (incubation buffer alone 0 .1 ml) or presence of 1 uM (-)-propranolol (0 .1 ml) in a final volume of 0 .4 ml . The aliquots were then vacuum filtered through Gelman (Type A-E) glass fiber filters with 10 ml Tris incubation buffer . The time required to filter and wash the aliquots was generally between 10 and 20 sec . The radioactivity that remained on the filters was then counted in a gamma counter . In the absence of tissue, radioactivity retained on the glass fiber filters was usually about 2% of the radioactivity added . Specific binding was defined as the difference between counts per min observed in the
Vol . 25, No . 22, 1979
Hydrocortisone on ß-Receptors in Lung
absence (total binding) and presence (nonspecific binding) (-)-propranolol .
1927
of 1 uM
Results Specific binding of [125]I-HYP was generally 90% of the total binding . The number and The binding sites were saturable with [125]I-HYP (Fig 1) . 1 affinity of the binding sites were determined by Scatchard analysis (Fig . Inset) (12) .
c 0
500,
â
-125 1-HYP BOUND
(fmol/mg protein)
E 400ó E
300-
c z
0 200 m a
100
N 0.1
0.2
0.4
.3 0
CONCENTRATION
FIG .
OF
125
0.5
1-HYP
(Le
0.7
(nM )
1
Saturation curves of [125]I-HYP specific binding to lung membranes from Membrane rats treated with saline (control) or hydrocortisone . particulate fractions of lung were incubated with increasing concentraticins of [125]I-HYP under standard conditions described in methods . Each point is the mean of duplicate determinations from a Inset : Data for saturation curves were representative experiment .
1928
Hydrocortisone on ß-Receptors in Lung
Vol . 25, No . 22, 1979
plotted by the method of Scatchard . The straight lines are the least squares linear regression fit of these data . The negative reciprocals of the slopes of the lines yield the equilibrium dissociation constants, and intercepts on the abscissa give the maximal number of [125]1-HYp binding sites . Data are summarized on TABLE I . :'ABLE I Effect of hydrocortisone on the number and affinity of [125]1-HYP binding sites in lung membranes
[125]1-HYP binding (flnol/mg protein)
Control (saline) Hydrocortisone
386 _+ 28 657 + 45 (p < 0.0005)
Dissociaion Constant(Kd) (nM)
Number of animals
0 .222 + 0 .015 0 .282 + 0 .023 (p =-0 .051)
8 9
The number and affinity of [125]1-HYP binding sites for each animal was determined by Scatchard plot each of which had 5 to 7 duplicate points . The results shown are the means + 1 S .E .M . Student's 't-test' was used to test for differences between means (for binding t = 4 .94; for Kd t = 2 .12 ; df = 15) ; 'p' values are two-tailed .
Chronic administration of hydrocortisone increased the density of beta-adrenergic receptors by 70% from 386 fmol to 657 fmol/mg membrane protein with some increase in the dissociation constant of [125]1-HYP for beta-adrenergic receptors. (TABLE I) . TABLE II Effect of adrenalectomy on the number and affinity of [125]1-HYP binding sites in rat lung membranes [125]1-HYP binding fmol/mg protein) 1. 2. 3. 4.
Non-treated 300 + 14 Sham-operated 262 + 17 Adrenalectomized 187 + 16 Adrenalectomized 338 _+ 49 plus hydrocortisone (4)=(1) > (3) P<0 .01 (1)=(2) > (3) p<0 .01
Dissociation Constant (Kd) (nM) 0 .161 0 .156 0 .138 0 .161
+ + + _+
0 .010 0 .010 0 .007 0 .016
Kd ;no significant difference
Number of
5 6 7 4
Vol. 25, No . 22, 1979
Hydrocortisone on B-Receptors in Lung
1929
The number and affinity of [125]I-HYP binding sites were determined by Scatchard plot . The results are mean + SEM of the indicated number of One-way analysis of variance and experiments each done in duplication . Duncan's multiple range test were used to test for differences among means . For maximum binding, (F=8 .0, df=3,18, p=0 .002), Kd means are not different (F=1 .5, df---3,18, p=0 .28) . In the separate experiment (TABLE II), the effect of adrenalectomy on the binding sites in the rat membranes was evaluated . Adrenalectomy decreased the density of beta-receptors by 29% from 262 to 187 fmol/mg protein without altering the affinity . This change was reversed by the exogenous administration of hydrocortisone . By contrast, sham-operation did not significantly alter the density or the affinity of beta-receptors, as compared to non-treated rats . The [125]I-HYP used in TABLES I and II were obtained from different iodination and paperchromatography runs which produced the slight variation in the values for beta-receptor densities and dissociation constants seen in control between TABLES I and II . lliscussion Recent studies have shown an increase in the number of beta-receptors in cardiac membranes from rats treated with thyroid hormone (1-3) ; this increase may be responsible for the beta-adrenergic hyperreactivity seen in hyperthyroid states . Conversely, thyroid hormone has been shown to decrease alpha-adrenergic receptors in rat heart (2) . Malbon et al (13) reported that thyroid hormone did not alter the number of beta-receptors in rat fat cells . These findings would suggest . that the effects of thyroid hormone on adrenergic receptors are tissue- and receptor-specific . A recently published study has . also demonstrated that the effect of aging on beta-receptors is tissue-specific (14) . The steroselectivity and beta-adrenergic order of potency of IHYP binding in rat lung has recently been reported by Minneman et al . (15) . In view of a possible role of beta-receptor density in tissue responsiveness to catecholamine, it is of interest to test whether hydrocortisone can regulate the beta-receptor density in lung membranes . We have demonstrated that chronic administration of hydrocortisone increased the density of beta-receptors in rat lung by 70%. These results are consistent with the observations that glucocorticoids potentiate beta-adrenergic stimulation, and they suggest that glucocorticoids may exert a potentiating effect by increasing the density of receptors in the tissue . In the separate experiment, adrenalectomy in rats decreased the density of beta-receptors by 29% and this change was reversed by the exogenous administration of hydrocortisone without altering the affinity of receptors . These results also suggest that endogenous glucocorticoids are involved in regulating the receptor density. By contrast, Wolfe et al (4) have demonstrated that adrenalectomy increased beta-receptors in rat liver membranes by 3-5 fold, and that this change was reversed by administration of hydrocortisone . These findings conflict with ours . The reason for this discrepancy is not clear . One possible explanatión is that the effect of glucocorticoids on beta-receptor density may be tissue dependent, like thyroid hormone, thus producing different effects in different tissues. 111e mechanism by which glucocorticoids increases beta-receptor density in lung membranes is unknown at this point . A recent study (15) suggests that the stimulating effect of chronic thyroid hormone on beta-receptor density in rat heart is a transcriptional-translational event which leads to the synthesis of
Vol . 25, No . 22, 1979
Hydrocortisone on ß-Receptors in Lung
1930
new beta-receptors by thyroid homone, because this effect is inhibited by cycloheximide . Since glucocorticoids have been shown to stimulate the synthesis of protein (16), they may help to provide the proteins necessary for receptor formation . However, because thyroid hormone increases beta-receptors, but decreases alpha-receptors in the same heart tissue (2), the observed increase in the density of receptors may not be due simply to a general gain in proteins . Other mechanisms may also be involved in the increase of tissue receptors induced by hydrocortisone . Holgate et al (5) have shorn that in subjects resistant to the betaagonist, salbutamol sensitivity could be restored after administration of hydrocortisone . The present study shows that administration of hydrocortisone increased the density of beta-receptors in lung tissues . These findings suggest that glucocorticoids treatment in bronchial asthma may restore sensitivity to catecholamines by increasing the density of beta-receptors in airway tissues . Acknowledgment We thank Koesnander Koesnadi and Gavin Watt for technical assistance, and Dr . Hans Ruprecht for advice on the assay procedure . This work was supported by grants from the NIH (2-P50-AI-12029 AIRC) and American Lung Association of Nebraska . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 " 14 . 15 . 16 . 17 .
L . T . WILLIAMS, R . J . LEFKOWITZ, A . M . WATNABE, D . R . HATHAWAY, and H . R . JR . BESCH, J . Biol . Chem . 252 2787-2789 (1977) . T . CIARALDI, G . V . MARINETTI, Biochem .Biopys . Res . Commun . 7 4 984-991 (1977) . S. P . BANERJEE, and L . S . KUNG, Eur . J . Pharmacol . 43 207-208 (1977) . B . B . WOLFE, T . K . HARDEN, and P . B . MOLINOFF, Pros . Natl . Acad . Sci . 7 3 1343-1347 (1976) . S . T .HOLGATE, C . J . BALDWIN, and A . E . TATTERSFIELD, Lancet 11 375-377 (1977) . G. M . SHENFIELD, M . E . HODSON, S . W . CLARKE, and J . W . PATERSON, Thorax 30 430-435 (1975) . R . G . TOWNII,EY, R . REEB, T FITZGIBBONS, and R . L . ADOLPHSON, J . Allergy . 45 118 (1970) . S . KALSNER, Circulation Res . 24 383-295 (1969) . G . D . AURBACH, S . A . FEDAK, C.J . WOODARD, J . S . PALMER, D . HAUSER, and F . TROXLER, Science . 186 1223-1223 (1974) . 0 . H . LOWRY, H . J . ROSEBROGH, A . L . FARR, and R . J . RANDALL, J . Biol . Chem . 193 265-275 (1951) . .T . K . HARDEN, B . B . WOLFE, and P . B . MOLINOFF, Mol . Pharacol . 12 1-15 (1976) . G . SCATCHARD, Ann . N .Y . Acad . Sci . 5 1 660-672 (1949) . C . C . MALBON, F . J . MORENO, R . J . CA-BELLI, and J . N . FAIN, J . Biol . Chem 253 671-678 (1978) . J . P . BILEZIKIAN, and D . E . GAMMON, Life Sci . 23 253-260 (1978) . MINNEMAN, HEGSTRAND and MOLINOFF, Mol . Pharma6ol . 16 21-33, (1979) . S . KEMPSON, G . V . MARINETTI, and A SHAW, Biochem . Biophys . Acta . 540 320-329 (1978) . .C C . CHING-LING, and P . FEIGELSON, J . Biol . Chem . 253 7880-7885 (1978) .
Pergamon Press
EFFECT OF HYDROCORTISONE ON BETA-ADRENERGIC RECEPTORS IN LUNG MEMBRANES Kenji Mano, Abdolah Akbarzadeh and Robert G. Townley Allergic Disease Center Creighton University School of Medicine Omaha, Nebraska 68178 (Received in final form October 22, 1979)
Summary It has been observed that glucocorticoids potentiate beta-adrenergic stimulation of cardiovascular and airway tissues. In order to investigate the mechanism of this potentiating action, we examined the effect of glucocorticoids on the number and affinity of beta-adrenergic receptors in animal lung tissues, by a direct binding technique using [125]I-Iodohydroxybenzylpindolol ([125]2-HYP), a potent beta-adrenergic receptor antagonist . Specific binding of [1251I-HYP to rat lung membranes was saturable with 386 fmol of [125]2-HYP/mg protein at saturation . The apparent equilibrium dissociation constant of [1251I-HYP for beta-receptors was 221 nM . Chronic administration of hydrocortisone increased the density of beta-adrenergic receptors by 70% from 386 fmol to 657 fmol/mg with some decrease in the affinity of [1251I-HYP for beta-adrenergic receptors . By contrast, adrenalectomy produced a 29% fall in the number of beta-adrenergic receptors without altering the affinity of [1251I-HYP for beta-receptors, and this change was reversed by exogenous administration of hydrocortisone . The present study suggests that glucocorticoids may participate in regulating the density of beta-adrenergic receptors, and may potentiate beta-adrenergic receptors stimulation, at least in part by increasing beta-receptor density in tissue membranes . In the past several years, great progress has been made in developing direct binding techniques for assessing the properties of beta-adrenergic receptors using radiolabeled ligand . Recent studies utilizing these techniques, have demonstrated that pharmacological treatment with various hormones (1-3) and surgical inervention (4) can cause alterations in the number of beta-adrenergic receptors, and that this alteration may play an important role in tissue responsiveness to catecholamines . Glucocorticoids have been shown to exert a potentiating effect on adrenergic stimulation in several tissues such as airway smooth muscle (5-7), and cardiovascular tissue (8) ; however, the mechanism is unclear . In order to investigate the mechanism of the potentiating effect of glucocorticoids on adrenergic stimulation, we tested the modulating effect of glucocorticoids on the density and affinity of beta-adrenergic receptors in rat lung tissues, using [125]2-Iodohydroxybenzylpindolol, a potent beta-adrenergic antagonist (9) . Materials and Methods Animal experiments . Male Sprague-Dawley rats (250-300 g) received subcutaneous injections of hydrocortisone (50 ma/ka) or 0 .3 ml of isotonic saline once every 0024-3205/79/221925-06$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd.
1926
Hydrocortisone on ß-Receptors in Lung
Vol . 25, No . 22, 1979
day for 9 days . The rats were killed by decapitation 24 hours after the last injection . In a separate experiment, the rats were bilaterally adrenalectomized or sham-operated . Four of the adrenalectomized rats received subcutaneous injections of hydrocortisone (50 mg/kg) daily for 6 days after adrenalectomy . Adrenalectomized rats treated with hydrocortisone were sacrificed 24 hours after the last injections . Tissue preparation . Lungs of the rats were removed immediately after sacrifice, 3íssected ee o large vessels and bronchi, chilled on ice, and homogenized in 11 .3% (w/v) sucrose in 5 mM Tris-HC1 buffer pH 7 .4 (20 ml/g wet tissue) containing 1 mM MgC1[21 with a Brinlamann Polytron homogenizer at full speed for 15 seconds . The homogenate was centrifuged at 90 g . for 5 min . a t 4 degrees C . The resultant supernate was centrifuged at 30,000 g . for 15 min . a t 4 degrees C (1) . The final pellets enriched in plasma membranes were resuspended in Tris-HC1 incubation buffer (50 mM Tris, 10 mM MgC1[21, pH 7 .4) using a Brinkmann Polytron . This suspension was used for the binding assays . The yields of final membrane preparation per gram of initial lung wet weight from each group were not statistically significantly different and were about 25 mg of membrane protein/g of initial wet weight . Protein concentrations were determined by the method of Lowry (10) with human serum albumin as standard . Iodination and paperchromatography for ligand . Hydroxybenzylpindolol (HYP) was iodinatedand extracted by the meth6of Harden et al (11) . HYP (30 ug in 20 ul of 10 mM HC1), Na[1251I (2 mCi in 20 ul of distilled water) and potassium phosphate buffer (0 .3 M, 20 ul, pH 7 .4) were combined . Chloramine T (20 ul of 0 .17 mg/ml in distilled water) was then added and allowed to stand at room temperature for 3 min . The reaction was stopped by adding 300 ul of 1 M acetic acid containing 1 mg/ml sodium metabisulfite . Using a Pasteur pipette as a separation funnel this solution was washed three times with 300 ul of ethyl acetate containing 0 .01% phenol . The three washes were combined and washed twice with 400 ul of acetic acid containing 1 mg/ml sodium meta- bisulfite . The upper layer (ethyl acetate) which contains [1251I-HYP and HYP was kept for paperchromatography . A significant amount of [1251I-HYP can be recovered by repeatedly washing the lower layer with ethyl acetate containing 0 .01% phenol, at least 4 times . Following iodination, the ethyl acetate extract was spotted on Whatman No . 3MM paper . Descending paperchromatography was performed at room temperature with 0 .1 M ammonium formate, pH 8 .5, containing 0 .01% phenol . After development for 12-16 hours the paper was cut, while still wet, into strips 1 cm wide . The [1251I-HYP is found 4-7 am from the origin, while the HYP has run completely off the paper . Each strip was placed in a vial, and [1251I-HYP was eluted with 5 ml of methyl alcohol containing 0 .01% phenol . The eluted [1251I-HYP was stored at -20 degrees C . [1251I-HYP was chemically stable for at least 4 weeks . (The final specific activity of [1251I-HYP was 2,200 Ci/mmol .) Binding assay . [1251I-HYP binding was assayed by incubating the membrane particulate fractions (120-150 ug of protein in 0 .2 ml incubation buffer) with [1251I-HYP (0 .1 ml) for 60 min . . at 37 degrees C either in the absence (incubation buffer alone 0 .1 ml) or presence of 1 uM (-)-propranolol (0 .1 ml) in a final volume of 0 .4 ml . The aliquots were then vacuum filtered through Gelman (Type A-E) glass fiber filters with 10 ml Tris incubation buffer . The time required to filter and wash the aliquots was generally between 10 and 20 sec . The radioactivity that remained on the filters was then counted in a gamma counter . In the absence of tissue, radioactivity retained on the glass fiber filters was usually about 2% of the radioactivity added . Specific binding was defined as the difference between counts per min observed in the
Vol . 25, No . 22, 1979
Hydrocortisone on ß-Receptors in Lung
absence (total binding) and presence (nonspecific binding) (-)-propranolol .
1927
of 1 uM
Results Specific binding of [125]I-HYP was generally 90% of the total binding . The number and The binding sites were saturable with [125]I-HYP (Fig 1) . 1 affinity of the binding sites were determined by Scatchard analysis (Fig . Inset) (12) .
c 0
500,
â
-125 1-HYP BOUND
(fmol/mg protein)
E 400ó E
300-
c z
0 200 m a
100
N 0.1
0.2
0.4
.3 0
CONCENTRATION
FIG .
OF
125
0.5
1-HYP
(Le
0.7
(nM )
1
Saturation curves of [125]I-HYP specific binding to lung membranes from Membrane rats treated with saline (control) or hydrocortisone . particulate fractions of lung were incubated with increasing concentraticins of [125]I-HYP under standard conditions described in methods . Each point is the mean of duplicate determinations from a Inset : Data for saturation curves were representative experiment .
1928
Hydrocortisone on ß-Receptors in Lung
Vol . 25, No . 22, 1979
plotted by the method of Scatchard . The straight lines are the least squares linear regression fit of these data . The negative reciprocals of the slopes of the lines yield the equilibrium dissociation constants, and intercepts on the abscissa give the maximal number of [125]1-HYp binding sites . Data are summarized on TABLE I . :'ABLE I Effect of hydrocortisone on the number and affinity of [125]1-HYP binding sites in lung membranes
[125]1-HYP binding (flnol/mg protein)
Control (saline) Hydrocortisone
386 _+ 28 657 + 45 (p < 0.0005)
Dissociaion Constant(Kd) (nM)
Number of animals
0 .222 + 0 .015 0 .282 + 0 .023 (p =-0 .051)
8 9
The number and affinity of [125]1-HYP binding sites for each animal was determined by Scatchard plot each of which had 5 to 7 duplicate points . The results shown are the means + 1 S .E .M . Student's 't-test' was used to test for differences between means (for binding t = 4 .94; for Kd t = 2 .12 ; df = 15) ; 'p' values are two-tailed .
Chronic administration of hydrocortisone increased the density of beta-adrenergic receptors by 70% from 386 fmol to 657 fmol/mg membrane protein with some increase in the dissociation constant of [125]1-HYP for beta-adrenergic receptors. (TABLE I) . TABLE II Effect of adrenalectomy on the number and affinity of [125]1-HYP binding sites in rat lung membranes [125]1-HYP binding fmol/mg protein) 1. 2. 3. 4.
Non-treated 300 + 14 Sham-operated 262 + 17 Adrenalectomized 187 + 16 Adrenalectomized 338 _+ 49 plus hydrocortisone (4)=(1) > (3) P<0 .01 (1)=(2) > (3) p<0 .01
Dissociation Constant (Kd) (nM) 0 .161 0 .156 0 .138 0 .161
+ + + _+
0 .010 0 .010 0 .007 0 .016
Kd ;no significant difference
Number of
5 6 7 4
Vol. 25, No . 22, 1979
Hydrocortisone on B-Receptors in Lung
1929
The number and affinity of [125]I-HYP binding sites were determined by Scatchard plot . The results are mean + SEM of the indicated number of One-way analysis of variance and experiments each done in duplication . Duncan's multiple range test were used to test for differences among means . For maximum binding, (F=8 .0, df=3,18, p=0 .002), Kd means are not different (F=1 .5, df---3,18, p=0 .28) . In the separate experiment (TABLE II), the effect of adrenalectomy on the binding sites in the rat membranes was evaluated . Adrenalectomy decreased the density of beta-receptors by 29% from 262 to 187 fmol/mg protein without altering the affinity . This change was reversed by the exogenous administration of hydrocortisone . By contrast, sham-operation did not significantly alter the density or the affinity of beta-receptors, as compared to non-treated rats . The [125]I-HYP used in TABLES I and II were obtained from different iodination and paperchromatography runs which produced the slight variation in the values for beta-receptor densities and dissociation constants seen in control between TABLES I and II . lliscussion Recent studies have shown an increase in the number of beta-receptors in cardiac membranes from rats treated with thyroid hormone (1-3) ; this increase may be responsible for the beta-adrenergic hyperreactivity seen in hyperthyroid states . Conversely, thyroid hormone has been shown to decrease alpha-adrenergic receptors in rat heart (2) . Malbon et al (13) reported that thyroid hormone did not alter the number of beta-receptors in rat fat cells . These findings would suggest . that the effects of thyroid hormone on adrenergic receptors are tissue- and receptor-specific . A recently published study has . also demonstrated that the effect of aging on beta-receptors is tissue-specific (14) . The steroselectivity and beta-adrenergic order of potency of IHYP binding in rat lung has recently been reported by Minneman et al . (15) . In view of a possible role of beta-receptor density in tissue responsiveness to catecholamine, it is of interest to test whether hydrocortisone can regulate the beta-receptor density in lung membranes . We have demonstrated that chronic administration of hydrocortisone increased the density of beta-receptors in rat lung by 70%. These results are consistent with the observations that glucocorticoids potentiate beta-adrenergic stimulation, and they suggest that glucocorticoids may exert a potentiating effect by increasing the density of receptors in the tissue . In the separate experiment, adrenalectomy in rats decreased the density of beta-receptors by 29% and this change was reversed by the exogenous administration of hydrocortisone without altering the affinity of receptors . These results also suggest that endogenous glucocorticoids are involved in regulating the receptor density. By contrast, Wolfe et al (4) have demonstrated that adrenalectomy increased beta-receptors in rat liver membranes by 3-5 fold, and that this change was reversed by administration of hydrocortisone . These findings conflict with ours . The reason for this discrepancy is not clear . One possible explanatión is that the effect of glucocorticoids on beta-receptor density may be tissue dependent, like thyroid hormone, thus producing different effects in different tissues. 111e mechanism by which glucocorticoids increases beta-receptor density in lung membranes is unknown at this point . A recent study (15) suggests that the stimulating effect of chronic thyroid hormone on beta-receptor density in rat heart is a transcriptional-translational event which leads to the synthesis of
Vol . 25, No . 22, 1979
Hydrocortisone on ß-Receptors in Lung
1930
new beta-receptors by thyroid homone, because this effect is inhibited by cycloheximide . Since glucocorticoids have been shown to stimulate the synthesis of protein (16), they may help to provide the proteins necessary for receptor formation . However, because thyroid hormone increases beta-receptors, but decreases alpha-receptors in the same heart tissue (2), the observed increase in the density of receptors may not be due simply to a general gain in proteins . Other mechanisms may also be involved in the increase of tissue receptors induced by hydrocortisone . Holgate et al (5) have shorn that in subjects resistant to the betaagonist, salbutamol sensitivity could be restored after administration of hydrocortisone . The present study shows that administration of hydrocortisone increased the density of beta-receptors in lung tissues . These findings suggest that glucocorticoids treatment in bronchial asthma may restore sensitivity to catecholamines by increasing the density of beta-receptors in airway tissues . Acknowledgment We thank Koesnander Koesnadi and Gavin Watt for technical assistance, and Dr . Hans Ruprecht for advice on the assay procedure . This work was supported by grants from the NIH (2-P50-AI-12029 AIRC) and American Lung Association of Nebraska . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 " 14 . 15 . 16 . 17 .
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