Cyclic AMP and Amino Acid Transport
81
BrtIllerTTll1n, L.E., A.E. Foster, s.H. lngbar: Thyroid hormone transport in the serum of patients with thyrotoxic Graves' Disease before and after treatment. J.Clin.lnvest. 47: 1349-1357 (1968) Cuaron, A.: Relationship between the in vitro uptake of 131 I triiodothyronine by erythrocytes and its binding by serum proteins in thyroid disease. J.Clin.EndocrinoI.Metab. 26: 53-64 (1966) Furth, E.D., K. Rives, D. V. Beliger: Non thyroidal action of propylthiouracile in euthyroid and hyperthyroid man. J.Clin.EndocrinoI.Metab. 26: 239-246 (1966) lnada, M., K. Sterling: Thyroxine transport in thyrotoxicosis and hypothyroidism. J.Clin.Invest. 46: 1442-1450 (1967) lngbar, L., L.E. Braverman, NA. Dawber, G. Y. Lee: A new method for measuring the free thyroid hormone in human serum and an analysis of the factors that influence its concentration. J.Clin.lnvest. 44: 1679-1689 (1965) lngbar, S.H., N Freinkel: Regulation of the peripheral metabolism of the thyroid hormones. Rec.Progr, in Horm.Res. 16: 353-397 (1960) laffiol, c., L. Baldet, Y. Vierne, M. Robin, I. Mirouze: Comparaison du dosage chimique par automation de )'jode hormonal plasmatique et de la thyroxim:mie meSUfC!e par competition (Test T4). Rev.Franc.Endocrinol.c1in. 13: 363-368 (1972) NauTTll1n, I.A., A. Nauman, s.c. Werner: Total and free triiodothyronine in human serum. J.Clin.lnvest. 46: 13461355 (1967) Oppenheimer, I.H., R. Squef, I. Surks, H. Hauer: Binding of thyroxine by serum proteins evaluated by equilibrium dialysis and electrophoretic techniques. Alterations in nonthyroidal illness. J.Clin.lnvest. 42: 1769-1782 (1963)
Richards, I.B., I. T. Dowling, S.H. Ingbar: Alterations in the plasma transport of thyroxine in sick patients and theu relation to the abnormality in Graves' disease. Abstracts of the 51 annual meeting of the American Society for clinical investigation. J.Clin.lnvest. 38: 1035 (1959) Robbins, J., J.E. Roll: Proteins associated with the thyroid hormones. Physiol.Rev. 40: 415-489 (1960) Robin, M., L. Baldet, C. Jaffiol, I. Mirouze: Etude comparee de la thyroxine libre meSUfC!e par dialyse et de l'index de thyroxine libre Sephadex pour le diagnostic et la surveillance therapeutique des malades thyroidiens. Ann.Endocrinol. 35: 298-300 (1974) Schussler, G.c.: Thyroxine binding globulin (TBG) in thyrotoxicosis and non thyroidal illness. Abstracts of the 48th meeting of the Endocrine Society Chicago, no. 113 (1966) Silverstein, I.N, H.L. Schwartz , E.B. Feldman, D.M. Kydd, C. Cartera: Correlation of the red blood cell uptake of 1311-L-triiodothyronine and thyroxine binding globulin capacity in man. J.Clin.EndocrinoI.Metab. 22: 1002-1006 (1962) Sterling, K., A. Hegedus: Measurement of free thyroxine concentration in human serum. J.Clin.lnvest. 41: 10311040 (1962) Sterling, K., M.A. Brenner: Free thyroxine in human serum: simplified measurement with the aid of magnesium precipitation. J.Clin.lnvest. 45: 153-163 (1966) Walfish, P.G., A. Britton, R. Volpe, C. Ezrin: Quantitative plasma binding capacity studies of thyroxine binding globulin by use of a modification of the erythrocyte 1-triiodothyronine-J131 uptake test. J.Clin.Endocrinol. 22: 178-186 (1962)
Requests for reprints should be addressed to: Professeur C. Jaffiol, Clinique des Maladies Metaboliques et Endocriniennes, Hopital Saint-Eloi, F-34059 Montpellier Cedex (France)
Horm. Metab. Res. 9 (1977) 81-85
© Georg Thieme Verlag Stuttgart
Effects of Cyclic AMP and Dibutyryl Cyclic AMP on Amino Acid Transport in the Isolated Rat Ovary H. Herlitz, L. Hamberger and K. Ahren Department of Physiology, University of Göteborg, Göteborg, Sweden
Summary Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids a-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine- 14 C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5 mM cyclic AMP (cAMP) caused a slight stimulation of the AlB-transport but in higher concentrations 00-25 mM) an inhibition was noted. With dibutyryl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0,5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AlB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied Received: 20 Apr, 1976
Accepted: 28 June 1976
(30-240 min). The phosphodiester&se inhibitor aminophylline in lower concentrations did not influence AlB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotrophins on ovarian amino acid transport. Key-Words: Ovary - Cyclic AMP - Amino Acid Transport
Introduction In a previous study it was reported that addition of cyclic AMP (cAMP) or the dibutyryl derivative of
Downloaded by: University of British Columbia. Copyrighted material.
References
82
H. Herlitz, L. Hamberger and K. Ahn!n
this nucleotide (dbcAMP) markedly influenced the cellular space, the ovaries were incubated in the same manrate of lactic acid production by isolated ovaries of ner in medium containing sucrose- 3 H. prepubertal rats (Herlitz, Hamberger and Ahren 1974). Measurements and calculations: The radioaetivity of ineubaThe effect of the nucleotide was compared to the tion m~dia andTCA extracts was assayed in a Packard T:~._ weil established influence of gonadotrophins on ovar- Carb LIquId Semtdla,lIon Spectr?met~r as descnbe,d m pr I
, o u s papers (e.g. Ahren and Rubmstem 1965, Ahren. Hamberger and Hartford 1967). Determination of total and extracellular tissue water contents under various experimental conditions were performed as described in previous papers (Ahren and Rubinstein 1965). In the presence of 1 mM dbcAMP or cAMP there was a slight but significant inerease in the sucrose.3H space from 29.6 ± 0.6% to 31.7 ± 0.6% or 32.5 ± 0.6%, respectively. The intracellular accumulation of AIB-14C and cycloleucine- 14 C is expressed as distribution ratio (= cpm/ml intracellular water: cpm/ml incubation mein the isolated rat ovary has been investigated. The dium) which indicated the extent of which the amino acid observations have relevance both in relation to earlier is concentrated in the cell water. It has been shown in previdescribed effects on gonadotrophins (FSH and LH) ous experiments (Ahren. Hamberger and Hartford 1967) that on ovarian amino acid transport (e.g. Ahren and AIB-14C and cycloleucine- 14 C are not metabolized by the ovarian tissue. .
.
Kostyo 1963, Nilsson and Se/stam 1975) and in relation to reported effects of cAMP and dbcAMP on amino acid transport in other tissues, e.g. liver (Tews, Woodcock and Harper 1970), uterus (Gri//in and Szego 1968), thyroid (Wilson, Raghupatlzy, Tonoue and Tong 1968), bone and kidney cortex (Phang, Downing and Weiss 1970) and jejunal mucosa (Kinzie, Ferrendelli and A/pers 1973).
Statistical procedures: Mean value and standard error of the means (SEM) were calculated and the results were compared by analysis of variance (with one criterion of elassification) followed by Student-Newman-Keul's multiple range test (Woolf 1968). In presence of only two groups a simple Student's t-test was used. A p-value of 0.05 or less was considercd significant in this study. 25
Methods Animals: Female rats of the Sprague-Dawley strain were used. 20 The rats were used for acute experiments when 24-26 days 0 old. All animals were kept in rooms with eonstant tempera~ ture (25 0 C) and relative humidity (55-65%) and given a semic synthetic diet (Gustafsson 1959) and water ad Iibitum. 20-24 h .g 15 be fore saerifiee the rats were deprived of food. .c '" .;: Chemieals: two radioaetive non-utilizable amino acids were used: ~aminoisobutyric acid (AlB) and I-aminocyclopentane carboxylic acid (cycloleucine). AIB-1-14C (specific activity 3.0 JJ.C/fJrnole) was obtained from the Radioehemical Center, Amersham, England and cycloleucine-I-14C (specific activity 1.87 JlC/fJrnole) from New England Nuclear Corporation, Boston, Mass., USA. For extracellular space determinations, sucrose-U-14C (specific activity 5.0 JlC/fJrnole) from the Radiochemical Center, Amersham, England was used. Adenosine 3',S'-cyclic monophosphate (cAMP), puromycin dihydrochloride and aminophylline were purchased from Sigma Chemical Co., SI. Louis, Mo., USA. Two different preparations of N6-2'-0-dibutyryl-adenosine-3' ,S'-monophosphate (dbcAMP) were used; one monopotassium saH from Calbiochem, LId., San Diego, Calif., USA, and one monosodium salt from Boehringer LId., Mannheim, West-Germany. No qualitative or quantitative differences in effects of these two preparations were seen.
-..
6
r-------I
.------. cAMP ...-............... dbcAMP
l. .-.....
·i·........
I
.... 1
',.., ......,.,..••."
~, 10 CD
81
BrtIllerTTll1n, L.E., A.E. Foster, s.H. lngbar: Thyroid hormone transport in the serum of patients with thyrotoxic Graves' Disease before and after treatment. J.Clin.lnvest. 47: 1349-1357 (1968) Cuaron, A.: Relationship between the in vitro uptake of 131 I triiodothyronine by erythrocytes and its binding by serum proteins in thyroid disease. J.Clin.EndocrinoI.Metab. 26: 53-64 (1966) Furth, E.D., K. Rives, D. V. Beliger: Non thyroidal action of propylthiouracile in euthyroid and hyperthyroid man. J.Clin.EndocrinoI.Metab. 26: 239-246 (1966) lnada, M., K. Sterling: Thyroxine transport in thyrotoxicosis and hypothyroidism. J.Clin.Invest. 46: 1442-1450 (1967) lngbar, L., L.E. Braverman, NA. Dawber, G. Y. Lee: A new method for measuring the free thyroid hormone in human serum and an analysis of the factors that influence its concentration. J.Clin.lnvest. 44: 1679-1689 (1965) lngbar, S.H., N Freinkel: Regulation of the peripheral metabolism of the thyroid hormones. Rec.Progr, in Horm.Res. 16: 353-397 (1960) laffiol, c., L. Baldet, Y. Vierne, M. Robin, I. Mirouze: Comparaison du dosage chimique par automation de )'jode hormonal plasmatique et de la thyroxim:mie meSUfC!e par competition (Test T4). Rev.Franc.Endocrinol.c1in. 13: 363-368 (1972) NauTTll1n, I.A., A. Nauman, s.c. Werner: Total and free triiodothyronine in human serum. J.Clin.lnvest. 46: 13461355 (1967) Oppenheimer, I.H., R. Squef, I. Surks, H. Hauer: Binding of thyroxine by serum proteins evaluated by equilibrium dialysis and electrophoretic techniques. Alterations in nonthyroidal illness. J.Clin.lnvest. 42: 1769-1782 (1963)
Richards, I.B., I. T. Dowling, S.H. Ingbar: Alterations in the plasma transport of thyroxine in sick patients and theu relation to the abnormality in Graves' disease. Abstracts of the 51 annual meeting of the American Society for clinical investigation. J.Clin.lnvest. 38: 1035 (1959) Robbins, J., J.E. Roll: Proteins associated with the thyroid hormones. Physiol.Rev. 40: 415-489 (1960) Robin, M., L. Baldet, C. Jaffiol, I. Mirouze: Etude comparee de la thyroxine libre meSUfC!e par dialyse et de l'index de thyroxine libre Sephadex pour le diagnostic et la surveillance therapeutique des malades thyroidiens. Ann.Endocrinol. 35: 298-300 (1974) Schussler, G.c.: Thyroxine binding globulin (TBG) in thyrotoxicosis and non thyroidal illness. Abstracts of the 48th meeting of the Endocrine Society Chicago, no. 113 (1966) Silverstein, I.N, H.L. Schwartz , E.B. Feldman, D.M. Kydd, C. Cartera: Correlation of the red blood cell uptake of 1311-L-triiodothyronine and thyroxine binding globulin capacity in man. J.Clin.EndocrinoI.Metab. 22: 1002-1006 (1962) Sterling, K., A. Hegedus: Measurement of free thyroxine concentration in human serum. J.Clin.lnvest. 41: 10311040 (1962) Sterling, K., M.A. Brenner: Free thyroxine in human serum: simplified measurement with the aid of magnesium precipitation. J.Clin.lnvest. 45: 153-163 (1966) Walfish, P.G., A. Britton, R. Volpe, C. Ezrin: Quantitative plasma binding capacity studies of thyroxine binding globulin by use of a modification of the erythrocyte 1-triiodothyronine-J131 uptake test. J.Clin.Endocrinol. 22: 178-186 (1962)
Requests for reprints should be addressed to: Professeur C. Jaffiol, Clinique des Maladies Metaboliques et Endocriniennes, Hopital Saint-Eloi, F-34059 Montpellier Cedex (France)
Horm. Metab. Res. 9 (1977) 81-85
© Georg Thieme Verlag Stuttgart
Effects of Cyclic AMP and Dibutyryl Cyclic AMP on Amino Acid Transport in the Isolated Rat Ovary H. Herlitz, L. Hamberger and K. Ahren Department of Physiology, University of Göteborg, Göteborg, Sweden
Summary Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids a-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine- 14 C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5 mM cyclic AMP (cAMP) caused a slight stimulation of the AlB-transport but in higher concentrations 00-25 mM) an inhibition was noted. With dibutyryl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0,5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AlB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied Received: 20 Apr, 1976
Accepted: 28 June 1976
(30-240 min). The phosphodiester&se inhibitor aminophylline in lower concentrations did not influence AlB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotrophins on ovarian amino acid transport. Key-Words: Ovary - Cyclic AMP - Amino Acid Transport
Introduction In a previous study it was reported that addition of cyclic AMP (cAMP) or the dibutyryl derivative of
Downloaded by: University of British Columbia. Copyrighted material.
References
82
H. Herlitz, L. Hamberger and K. Ahn!n
this nucleotide (dbcAMP) markedly influenced the cellular space, the ovaries were incubated in the same manrate of lactic acid production by isolated ovaries of ner in medium containing sucrose- 3 H. prepubertal rats (Herlitz, Hamberger and Ahren 1974). Measurements and calculations: The radioaetivity of ineubaThe effect of the nucleotide was compared to the tion m~dia andTCA extracts was assayed in a Packard T:~._ weil established influence of gonadotrophins on ovar- Carb LIquId Semtdla,lIon Spectr?met~r as descnbe,d m pr I
, o u s papers (e.g. Ahren and Rubmstem 1965, Ahren. Hamberger and Hartford 1967). Determination of total and extracellular tissue water contents under various experimental conditions were performed as described in previous papers (Ahren and Rubinstein 1965). In the presence of 1 mM dbcAMP or cAMP there was a slight but significant inerease in the sucrose.3H space from 29.6 ± 0.6% to 31.7 ± 0.6% or 32.5 ± 0.6%, respectively. The intracellular accumulation of AIB-14C and cycloleucine- 14 C is expressed as distribution ratio (= cpm/ml intracellular water: cpm/ml incubation mein the isolated rat ovary has been investigated. The dium) which indicated the extent of which the amino acid observations have relevance both in relation to earlier is concentrated in the cell water. It has been shown in previdescribed effects on gonadotrophins (FSH and LH) ous experiments (Ahren. Hamberger and Hartford 1967) that on ovarian amino acid transport (e.g. Ahren and AIB-14C and cycloleucine- 14 C are not metabolized by the ovarian tissue. .
.
Kostyo 1963, Nilsson and Se/stam 1975) and in relation to reported effects of cAMP and dbcAMP on amino acid transport in other tissues, e.g. liver (Tews, Woodcock and Harper 1970), uterus (Gri//in and Szego 1968), thyroid (Wilson, Raghupatlzy, Tonoue and Tong 1968), bone and kidney cortex (Phang, Downing and Weiss 1970) and jejunal mucosa (Kinzie, Ferrendelli and A/pers 1973).
Statistical procedures: Mean value and standard error of the means (SEM) were calculated and the results were compared by analysis of variance (with one criterion of elassification) followed by Student-Newman-Keul's multiple range test (Woolf 1968). In presence of only two groups a simple Student's t-test was used. A p-value of 0.05 or less was considercd significant in this study. 25
Methods Animals: Female rats of the Sprague-Dawley strain were used. 20 The rats were used for acute experiments when 24-26 days 0 old. All animals were kept in rooms with eonstant tempera~ ture (25 0 C) and relative humidity (55-65%) and given a semic synthetic diet (Gustafsson 1959) and water ad Iibitum. 20-24 h .g 15 be fore saerifiee the rats were deprived of food. .c '" .;: Chemieals: two radioaetive non-utilizable amino acids were used: ~aminoisobutyric acid (AlB) and I-aminocyclopentane carboxylic acid (cycloleucine). AIB-1-14C (specific activity 3.0 JJ.C/fJrnole) was obtained from the Radioehemical Center, Amersham, England and cycloleucine-I-14C (specific activity 1.87 JlC/fJrnole) from New England Nuclear Corporation, Boston, Mass., USA. For extracellular space determinations, sucrose-U-14C (specific activity 5.0 JlC/fJrnole) from the Radiochemical Center, Amersham, England was used. Adenosine 3',S'-cyclic monophosphate (cAMP), puromycin dihydrochloride and aminophylline were purchased from Sigma Chemical Co., SI. Louis, Mo., USA. Two different preparations of N6-2'-0-dibutyryl-adenosine-3' ,S'-monophosphate (dbcAMP) were used; one monopotassium saH from Calbiochem, LId., San Diego, Calif., USA, and one monosodium salt from Boehringer LId., Mannheim, West-Germany. No qualitative or quantitative differences in effects of these two preparations were seen.
-..
6
r-------I
.------. cAMP ...-............... dbcAMP
l. .-.....
·i·........
I
.... 1
',.., ......,.,..••."
~, 10 CD
