Researchin
Res. exp. Med. 170, 253--257 (1977)
ExperimentalMedicine ~) Springer.Verlag 1977
Catecholamine-sensitive Adenylate Cyclase of Human Fat Cell Ghosts Inhibition of Isoproterenol-Stimulation by Dihydroergotamine Horst Kather, Bertram Vogt, and Bernd Simon Klinisches Institut ffir Herzinfarktforschung an der Medizinischen Universit~itsklinikHeidelberg, F.R.G. (Prof. Dr. Dr. h.c.G. Schettler), Bergheimer Stra6e 58, D-6900 Heidelberg
Summary. Isoproterenol-activation of the adenylate cyclase system of human fat cell ghosts was markedly inhibited by dihydroergotamine which had no effect on basal and NaF-stimulated enzyme activity. Our results indicate that the antilipolytic action of this substance in human adipose tissue is probably due to inhibition of catecholamine-sensitive adenylate cyclase activity. Key words: Adenylate cyclase activity - Catecholamines - H u m a n adipose tissue - Dihydroergotamine. Introduction In addition to its known action as an alpha-adrenergic antagonist [6], dihydroergotamine (DHE), has at higher concentrations, a variety of other effects [ 10, 15, 16]. It has been reported for instance, that this ergot alkaloide specifically depressed activation of lipolysis by catecholamines in rat fat cells via inhibition of the adenylate cyclase system [2, 8]. A catecholamine-sensitive adenylate cyclase could be demonstrated in human fat cell ghosts most recently [3, 5, 11, 12, 17]. The human enzyme differs from the rat fat cell adenylate cyclase with respect to hormone sensitivity. The r a t fat cell enzyme reacts to a variety of hormones including catecholamines and peptide hormones, such as A C T H glucagon and secretin, whereas only catecholamines have been found to stimulate the human enzyme system [1, 3, 5, 11, 12, 17]. These special differences raised the possibility that both enzymes might also differ, with respect to the action of known inhibitors of adenylate cyclase activity. Therefore, we tested the effects of D H E on basal, isoproterenol- as well as NaF-stimulated h u m a n fat cell adenylate cyclase activity.
Methods Biopsies of subcutaneous adipose tissue were obtained from 7 patients, undergoing surgical treatment. The patients were operated after an overnight fast. Anesthesia was induced with a short acting barbiturate, and maintained with halothane, nitrous oxide, and oxygen. The biopsies were usually obtained after the skin incision, at the start of the operation.
254
H. Kather et al.
Adipose tissue was cut into 20--25 mg fragments. Fat cells and fat cell ghosts were isolated essentially according to the prescription of Rodbell [18], except that higher concentrations of collagenase (3 mg/ml) were used. The ghosts were suspended in 0.1 mM KHCO3 in a final concentration of 0.25 to 2.5 mg protein/ml. The adenylate cyclase activity of fat cell ghosts, was termined according to Salomon et al. [19] at pH 8.0. The protein content of the samples was measured according to Lowry et al. [13], using bovine serum albumin as standard. Data are given as normal, of cAMP formed per mg protein per 15 min. Statistical analysis was by the Wilcoxon-test for paired samples.
Materials (a-ATp-32P) (2--6 Ci/mmol), and cyclic (3H) AMP (27 Ci/mmol) were purchased from Radiochemical Centre Amersham Bucks, U.K. L-isoproterenol bitartrate was obtained from Boehringer & S6hne, Ingelheim am Rbein, dihydroergotamine methanesulfonate from Sandoz Pharmaceutical, Ntirnberg, F.R.G. All other chemicals and reagents were of the highest grade commercially obtainable.
Results In the absence o f d i h y d r o e r g o t a m i n e , b a s a l activities a v e r a g e d 1.10 n m o l o f c A M P f o r m e d p e r m g p r o t e i n p e r 15 min (n = 6) (Table 1). I s o p r o t e r e n o l (0.1 m M ) p r o d u c e d a significant increase o f enzyme activity b y a b o u t 300%. In the presence o f N a F (20 m M ) the enzyme activity was increased 8 - - 9 fold. D H E (0.1 m M ) h a d no effect on b a s a l (98 + 2% o f control) as well as N a F s t i m u l a t e d (99 + 8% o f control) a d e n y l a t e cyclase activities. H o w e v e r , the i s o p r o t e r e n o l s t i m u l a t e d rates o f c A M P - f o r m a t i o n were red u c e d b y a b o u t 48% in these e x p e r i m e n t s (n = 6). D e p i c t e d in F i g u r e 1 are the effects o f two different c o n c e n t r a t i o n s o f D H E (0.01 a n d 0.1 m M ) , on i s o p r o t e r e n o l - s t i m u l a t e d a d e n y l a t e cyclase activities o f 7 experiments. A d d i t i o n o f 0.01 m M D H E decreased the c a t e c h o l a m i n e - a c t i v a t e d rates o f cyclic Y , 5 ' - A M P f o r m a t i o n b y as m u c h as 12% (P=< 0.05). A t the highest c o n c e n t r a t i o n o f D H E tested (0.1 m M ) , the s t i m u l a t o r y effect o f i s o p r o t e r e n o l a p p e a r e d to be r e d u c e d b y a p p r o x i m a t e l y 49% (P=< 0.05). Table 1. Effect of dihydroergotamine on basal, isoproterenol- and NaF-stimulated adenylate cyclase of human fat cell ghosts Addition
None Isoproterenol NaF
Adenylate cyclase activity nmol cAMP per mg protein per 15 min - DHE
+ DHE
1.10 + 0.05 3.85 + 0.30 11.05 + 0.70
1.05 + 0.07 2.05 + 0.18 10.90 + 0.90
Mean values + SEM of 7 separate experiments are shown
Catecholamine-sensitive Adenylate Cyclase
255
Etfect of Dihydroergotamine on Isoproterenol-sfimulated Human Fat Cell Adenylote Cyclase (p(us lsoproterenol(O,1/nM,~
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Oli~ydroergotamine
Fig. 1. Dose-dependent inhibition ofisoproterenol-stimulated adenylate cyclase activity by DHE. Isoproterenol concentration was 0.1 raM, DHE concentrations were as indicated. Means of seven experiments are shown
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Res. exp. Med. 170, 253--257 (1977)
ExperimentalMedicine ~) Springer.Verlag 1977
Catecholamine-sensitive Adenylate Cyclase of Human Fat Cell Ghosts Inhibition of Isoproterenol-Stimulation by Dihydroergotamine Horst Kather, Bertram Vogt, and Bernd Simon Klinisches Institut ffir Herzinfarktforschung an der Medizinischen Universit~itsklinikHeidelberg, F.R.G. (Prof. Dr. Dr. h.c.G. Schettler), Bergheimer Stra6e 58, D-6900 Heidelberg
Summary. Isoproterenol-activation of the adenylate cyclase system of human fat cell ghosts was markedly inhibited by dihydroergotamine which had no effect on basal and NaF-stimulated enzyme activity. Our results indicate that the antilipolytic action of this substance in human adipose tissue is probably due to inhibition of catecholamine-sensitive adenylate cyclase activity. Key words: Adenylate cyclase activity - Catecholamines - H u m a n adipose tissue - Dihydroergotamine. Introduction In addition to its known action as an alpha-adrenergic antagonist [6], dihydroergotamine (DHE), has at higher concentrations, a variety of other effects [ 10, 15, 16]. It has been reported for instance, that this ergot alkaloide specifically depressed activation of lipolysis by catecholamines in rat fat cells via inhibition of the adenylate cyclase system [2, 8]. A catecholamine-sensitive adenylate cyclase could be demonstrated in human fat cell ghosts most recently [3, 5, 11, 12, 17]. The human enzyme differs from the rat fat cell adenylate cyclase with respect to hormone sensitivity. The r a t fat cell enzyme reacts to a variety of hormones including catecholamines and peptide hormones, such as A C T H glucagon and secretin, whereas only catecholamines have been found to stimulate the human enzyme system [1, 3, 5, 11, 12, 17]. These special differences raised the possibility that both enzymes might also differ, with respect to the action of known inhibitors of adenylate cyclase activity. Therefore, we tested the effects of D H E on basal, isoproterenol- as well as NaF-stimulated h u m a n fat cell adenylate cyclase activity.
Methods Biopsies of subcutaneous adipose tissue were obtained from 7 patients, undergoing surgical treatment. The patients were operated after an overnight fast. Anesthesia was induced with a short acting barbiturate, and maintained with halothane, nitrous oxide, and oxygen. The biopsies were usually obtained after the skin incision, at the start of the operation.
254
H. Kather et al.
Adipose tissue was cut into 20--25 mg fragments. Fat cells and fat cell ghosts were isolated essentially according to the prescription of Rodbell [18], except that higher concentrations of collagenase (3 mg/ml) were used. The ghosts were suspended in 0.1 mM KHCO3 in a final concentration of 0.25 to 2.5 mg protein/ml. The adenylate cyclase activity of fat cell ghosts, was termined according to Salomon et al. [19] at pH 8.0. The protein content of the samples was measured according to Lowry et al. [13], using bovine serum albumin as standard. Data are given as normal, of cAMP formed per mg protein per 15 min. Statistical analysis was by the Wilcoxon-test for paired samples.
Materials (a-ATp-32P) (2--6 Ci/mmol), and cyclic (3H) AMP (27 Ci/mmol) were purchased from Radiochemical Centre Amersham Bucks, U.K. L-isoproterenol bitartrate was obtained from Boehringer & S6hne, Ingelheim am Rbein, dihydroergotamine methanesulfonate from Sandoz Pharmaceutical, Ntirnberg, F.R.G. All other chemicals and reagents were of the highest grade commercially obtainable.
Results In the absence o f d i h y d r o e r g o t a m i n e , b a s a l activities a v e r a g e d 1.10 n m o l o f c A M P f o r m e d p e r m g p r o t e i n p e r 15 min (n = 6) (Table 1). I s o p r o t e r e n o l (0.1 m M ) p r o d u c e d a significant increase o f enzyme activity b y a b o u t 300%. In the presence o f N a F (20 m M ) the enzyme activity was increased 8 - - 9 fold. D H E (0.1 m M ) h a d no effect on b a s a l (98 + 2% o f control) as well as N a F s t i m u l a t e d (99 + 8% o f control) a d e n y l a t e cyclase activities. H o w e v e r , the i s o p r o t e r e n o l s t i m u l a t e d rates o f c A M P - f o r m a t i o n were red u c e d b y a b o u t 48% in these e x p e r i m e n t s (n = 6). D e p i c t e d in F i g u r e 1 are the effects o f two different c o n c e n t r a t i o n s o f D H E (0.01 a n d 0.1 m M ) , on i s o p r o t e r e n o l - s t i m u l a t e d a d e n y l a t e cyclase activities o f 7 experiments. A d d i t i o n o f 0.01 m M D H E decreased the c a t e c h o l a m i n e - a c t i v a t e d rates o f cyclic Y , 5 ' - A M P f o r m a t i o n b y as m u c h as 12% (P=< 0.05). A t the highest c o n c e n t r a t i o n o f D H E tested (0.1 m M ) , the s t i m u l a t o r y effect o f i s o p r o t e r e n o l a p p e a r e d to be r e d u c e d b y a p p r o x i m a t e l y 49% (P=< 0.05). Table 1. Effect of dihydroergotamine on basal, isoproterenol- and NaF-stimulated adenylate cyclase of human fat cell ghosts Addition
None Isoproterenol NaF
Adenylate cyclase activity nmol cAMP per mg protein per 15 min - DHE
+ DHE
1.10 + 0.05 3.85 + 0.30 11.05 + 0.70
1.05 + 0.07 2.05 + 0.18 10.90 + 0.90
Mean values + SEM of 7 separate experiments are shown
Catecholamine-sensitive Adenylate Cyclase
255
Etfect of Dihydroergotamine on Isoproterenol-sfimulated Human Fat Cell Adenylote Cyclase (p(us lsoproterenol(O,1/nM,~
u3
3.0(
20( BASAL
I.OC
,T '
-
~
1
o
II
c
L
o,ot
o,I
I M
Oli~ydroergotamine
Fig. 1. Dose-dependent inhibition ofisoproterenol-stimulated adenylate cyclase activity by DHE. Isoproterenol concentration was 0.1 raM, DHE concentrations were as indicated. Means of seven experiments are shown
~1
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II
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