330
M.A. Moxley and D.O. Allen
Nieschlag, E., H.K. Kley, W. Wiegelmann, H.G. Solbach, H.L. Kroskemper: Lebensalter und endokrine Funktion der Testes des erwachsenen Mannes. Dtsch.med.Wschr. 98: 1281-1284 (1973a) Nieschlag, E., D.L. Lorillux, H.J. Ruder, J.R. Zucker, M. Kirschner, M.B. Lipsett: The secretion of dehydroepiandrosterone and dehydroepiandrosterone sulfate in man. J.Endocrinol. 57: 123-134 (1973b) -Niesch/ag, E., J. Mauss, H. Coert, P. Kicovic: Plasma androgens in men after oral administration of free testosterone and testosterone undecanoate. Acta endocrinologica 79: 366-314 (1975) Oertel, G. W., K.B. Eik Nes: Plasma levels of dehydroepiandrosterone in the dog. Endocrinology 65: 766-776 (1959)
Raheja, M.C., O.J. Lucis: Role of dehydroepiandrosterone and its sulfate in synthesis of testosterone by human testes in vivo and in vitro. J.Endocrinol. 46: 21-28 (1970) Siiteri, P. K., P.L. MacDonald: The utilization of circulating dehydroepiandrosterone sulfate for estrogen synthesis during human pregnancy. Steroids 2: 713-730 (1963) Wang, D. Y., R.D. Bulbrook: Steroid Sulphates. In: A. McLaren (Ed.): Advances in reproductive physiology. Vol. 3. Logos Press, London (1968) 113 Wassermann, G.F., K.B. Eik Nes: Interrelation between adrenal function and formation of testosterone in vivo in the testis of the dog. Acta endocrinologica 61: 33-47 (1969)
Horm. Metab. Res. 7 (1975) 330-334
© Georg Thieme Verlag Stuttgart Effects of Glucagon, Phosphodiesterase Inhibitors, and Trypsin Treatment on Cyclic 3',5' Adenosine Monophosphate Levels in Isolated Hepatocytes* M.A. Moxley** and D.O. Allen*** Department of Pharmacology, Indiana University School of Medicine. Indianapolis. Indiana. USA Summary
Introduction
Cyclic 3',5' adenosine monophosphate (cyclic AMP) levels were measured in isolated hepatocytes und er several conditions. Following the addition of glucagon cyclic AMP levels increased rapidly with peak values occurring at three minutes. The increase in cyclic AMP was dose dependent. Significant increases were found with \O-loM glucagon and a maximum increase of twenty fold was produced by \0-11 M glucagon. This action of glucagon was augmented by the phosphodiesterase inhibitors, theophylline, SQ 20,009, and papaverine. Treatment of the hepatocytes with trypsin markedly reduced the response to glucagon.
Previous studies of the relationship between hormonal control of hepatic metabolism and cyclic 3',5'-adenosine monophosphate (cyclic AMP) have been conducted mainly using the isolated perfused liver (Exton, Robison, Sutherland and Park 1971). This technique has the disadvantages that a limited number of sampies can be obtained from a single liver and that cell types other than hepatocytes are present in the organ and may contribute to the response under observation. Other investigators have studied the relationship of cyc1ic AMP and metabolism using the liver slice preparation (Ingebretsen, Clark, Allen and Ashmore 1974). This preparation has the disadvantage that cells in the interior of the slice faH to receive adequate oxygenation and nutrients and only a small proportion of the cell population is viable and responsive. The isolated hepatocyte preparation allows multiple sampling from a single population of cells made up almost exc1usively of the functional unit of the liver, the hepatocyte. The investigations described here entail an examination of the effects of glucagon on cyclic AMP levels and the modification of that effect by inhibitors of phosphodiesterase and by trypsin treatment.
Key-Words: Hepatocytes - Cyclic AMP - Glucagon - Tryp· sin - Phosphodiesterase Inhibitors Abbreviations Cyclic 3',5' adenosine monophosphate will be abbreviated as cyclic AMP. l-ethyl-4-(isopropylidenehydrazine-lH-pyrazole-(3,4-b)-pyridine-5-carboxylic acid, ethylester-HCl will be abbreviated as SQ 20,009. *This work was supported by United States Public Health Service Grant AM-14070. **Recipient of Meade-Johnson Fellowship. Present address: Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104. *"Recipient of United States Public Health Service Career Development Award 1 K04-AM-50316. Received: I Nov. 1914
Accepted: 21 Apr. 1915
Downloaded by: NYU. Copyrighted material.
Requests for reprints should be addressed to: Priv.-Doz. Dr. E. Nieschlag, 2. Medizinische Univ.-Klinik, Moorenstr. 5, D 4 Düsseldorf (Germany)
Cyclic AMP Levels in Isolated Hepatocytes
331
40~~~--~~--~~------------~------------~
E 20
.. :11
~
10
10 TIME (min)
15
Fig. 1. Time course of cyclic AMP accumulation in hepatocytes after addition of glucagon (5 x lO-sM). All values are expressed as Mean ± SEM of the number of experiments in parentheses. * denotes p 0.05 when compared to zero time.
M.A. Moxley and D.O. Allen
Nieschlag, E., H.K. Kley, W. Wiegelmann, H.G. Solbach, H.L. Kroskemper: Lebensalter und endokrine Funktion der Testes des erwachsenen Mannes. Dtsch.med.Wschr. 98: 1281-1284 (1973a) Nieschlag, E., D.L. Lorillux, H.J. Ruder, J.R. Zucker, M. Kirschner, M.B. Lipsett: The secretion of dehydroepiandrosterone and dehydroepiandrosterone sulfate in man. J.Endocrinol. 57: 123-134 (1973b) -Niesch/ag, E., J. Mauss, H. Coert, P. Kicovic: Plasma androgens in men after oral administration of free testosterone and testosterone undecanoate. Acta endocrinologica 79: 366-314 (1975) Oertel, G. W., K.B. Eik Nes: Plasma levels of dehydroepiandrosterone in the dog. Endocrinology 65: 766-776 (1959)
Raheja, M.C., O.J. Lucis: Role of dehydroepiandrosterone and its sulfate in synthesis of testosterone by human testes in vivo and in vitro. J.Endocrinol. 46: 21-28 (1970) Siiteri, P. K., P.L. MacDonald: The utilization of circulating dehydroepiandrosterone sulfate for estrogen synthesis during human pregnancy. Steroids 2: 713-730 (1963) Wang, D. Y., R.D. Bulbrook: Steroid Sulphates. In: A. McLaren (Ed.): Advances in reproductive physiology. Vol. 3. Logos Press, London (1968) 113 Wassermann, G.F., K.B. Eik Nes: Interrelation between adrenal function and formation of testosterone in vivo in the testis of the dog. Acta endocrinologica 61: 33-47 (1969)
Horm. Metab. Res. 7 (1975) 330-334
© Georg Thieme Verlag Stuttgart Effects of Glucagon, Phosphodiesterase Inhibitors, and Trypsin Treatment on Cyclic 3',5' Adenosine Monophosphate Levels in Isolated Hepatocytes* M.A. Moxley** and D.O. Allen*** Department of Pharmacology, Indiana University School of Medicine. Indianapolis. Indiana. USA Summary
Introduction
Cyclic 3',5' adenosine monophosphate (cyclic AMP) levels were measured in isolated hepatocytes und er several conditions. Following the addition of glucagon cyclic AMP levels increased rapidly with peak values occurring at three minutes. The increase in cyclic AMP was dose dependent. Significant increases were found with \O-loM glucagon and a maximum increase of twenty fold was produced by \0-11 M glucagon. This action of glucagon was augmented by the phosphodiesterase inhibitors, theophylline, SQ 20,009, and papaverine. Treatment of the hepatocytes with trypsin markedly reduced the response to glucagon.
Previous studies of the relationship between hormonal control of hepatic metabolism and cyclic 3',5'-adenosine monophosphate (cyclic AMP) have been conducted mainly using the isolated perfused liver (Exton, Robison, Sutherland and Park 1971). This technique has the disadvantages that a limited number of sampies can be obtained from a single liver and that cell types other than hepatocytes are present in the organ and may contribute to the response under observation. Other investigators have studied the relationship of cyc1ic AMP and metabolism using the liver slice preparation (Ingebretsen, Clark, Allen and Ashmore 1974). This preparation has the disadvantage that cells in the interior of the slice faH to receive adequate oxygenation and nutrients and only a small proportion of the cell population is viable and responsive. The isolated hepatocyte preparation allows multiple sampling from a single population of cells made up almost exc1usively of the functional unit of the liver, the hepatocyte. The investigations described here entail an examination of the effects of glucagon on cyclic AMP levels and the modification of that effect by inhibitors of phosphodiesterase and by trypsin treatment.
Key-Words: Hepatocytes - Cyclic AMP - Glucagon - Tryp· sin - Phosphodiesterase Inhibitors Abbreviations Cyclic 3',5' adenosine monophosphate will be abbreviated as cyclic AMP. l-ethyl-4-(isopropylidenehydrazine-lH-pyrazole-(3,4-b)-pyridine-5-carboxylic acid, ethylester-HCl will be abbreviated as SQ 20,009. *This work was supported by United States Public Health Service Grant AM-14070. **Recipient of Meade-Johnson Fellowship. Present address: Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104. *"Recipient of United States Public Health Service Career Development Award 1 K04-AM-50316. Received: I Nov. 1914
Accepted: 21 Apr. 1915
Downloaded by: NYU. Copyrighted material.
Requests for reprints should be addressed to: Priv.-Doz. Dr. E. Nieschlag, 2. Medizinische Univ.-Klinik, Moorenstr. 5, D 4 Düsseldorf (Germany)
Cyclic AMP Levels in Isolated Hepatocytes
331
40~~~--~~--~~------------~------------~
E 20
.. :11
~
10
10 TIME (min)
15
Fig. 1. Time course of cyclic AMP accumulation in hepatocytes after addition of glucagon (5 x lO-sM). All values are expressed as Mean ± SEM of the number of experiments in parentheses. * denotes p 0.05 when compared to zero time.
