291
Biochimica et Biophysica Acta, 399 (1975) 2 9 1 - - 3 0 1 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s
BBA 27708
ACTIVATION OF A D E N Y L A T E CYCLASE IN R E N A L M E D U L L A BY BOVINE GROWTH H O R M O N E AN A R T I F A C T A T T R I B U T A B L E TO VASOPRESSIN*
S T E V E N B. L E I C H T E R and LEWIS R. C H A S E * *
Metabolism Division, Department of Medicine, Washington University School of Medicine, St. Louis, Mo. (U.S.A.) (Received F e b r u a r y 25th, 1975)
Summary The effect of bovine growth hormone on adenylate cyclase activity was studied in bovine and rat renal medulla. Highly purified growth hormone (lot B1003A) increased adenylate cyclase activity in plasma membranes from bovine renal medulla from 132 + 6 pmol cyclic AMP formed/mg protein per 10 min to 364 + 10 pmol cyclic AMP formed/mg protein per 10 min. Similar results were seen with homogenates of rat renal medulla. The minimum effective concentration of bovine growth hormone required to activate adenylate cyclase was 0.5 pg/ml and maximum activation was detected at 500 pg/ml. The amount of vasopressin determined by radioimmunoassay to contaminate the growth hormone caused an increase in adenylate cyclase activity comparable to that of the corresponding concentration of growth hormone that was tested. Dialysis of growth hormone and vasopressin resulted in parallel reductions in the effect of each hormone on adenylate cyclase activity. Similarly, both growth hormone and vasopressin produced increases in short circuit current in isolated toad bladders b u t these effects were no~ detectable after dialysis of the hormones. In contrast, the effect of growth hormone on the uptake of 3 s SO~by cartilage from hypophysectomized rats was not decreased after dialysis. These results indicate that available preparations of growth hormone are contaminated by small but physiologically significant amounts of vasopressin and that the activation of adenylate cyclase activity in renal medulla in response to growth hormone can be explained by this contamination rather than by an effect of growth hormone per se.
* Presented in part at the annual m e e t i n g o f The Endocrine S o c i e t y , Atlanta, Georgia, June 1974. ** Investigator~ Howa.rd Hughes Medical Institute.
292 Introduction Previous studies have indicated that growth hormone causes physiological changes in renal function. Administration of growth hormone [1--7] to humans, rats, and cats results in hypercalciuria and retention of Na ÷, water, K ÷ and phosphate. In vitro, the hormone stimulates salt and water transport across toad skin [8] and increases the uptake of poly-acyl R N A by ribosomes from rat renal cortex [9]. Although these changes have been attributed to a direct interaction between growth hormone and renal tissue, it has not been established with certainty whether some or all of these effects may be secondary to effects of growth hormone per se or to a minor contaminant in the preparation of the hormone. The latter has been suggested previously [8,10]. To date growth hormone, unlike most peptide hormones, has n o t been shown to cause rapid changes in the adenylate cyclase-cyclic AMP system in target tissues. However, previous studies have demonstrated that the hormone causes an increased concentration of cyclic AMP in adipose tissue after prolonged periods of incubation [11]. In view of the previous reports showing that growth hormone affects renal function, studies were carried out to determine if this hormone activates adenylate cyclase in the kidney, and if so, whether this activation could be attributed directly to growth hormone or to a contaminant in the preparations that are currently available. Methods and Materials Bovine kidneys were chilled on ice immediately after removal, and the cortex and medulla were separated within 2 h by blunt dissection. Tissues were minced and then homogenized in 0.25 M sucrose, 0.001 M EDTA, and 0.05 M Tris • HC1, pH 7.5, in a Dounce homogenizer with the loose fitting glass pestle. The crude homogenates were fractionated to the partially pure stage according to the method of Marx and Aurbach [12]. The membrane fractions were frozen in liquid nitrogen and stored at --80°C without significant loss of adenylate cyclase activity over a 2-week period. Adult male Sprague-Dawley rats in the fed state were killed by cervical fracture and homogenates prepared from renal medulla, renal cortex, brain, heart, liver and adrenal as described above. Hemibladders removed from adult male toads, Bufo Marinus, were washed in lactated Ringer's solution and placed immediately in an Ussing chamber [13]. Tissues were equilibrated for at least 20 min before test substances were added to the serosal chamber. 21-day-old hypophysectomized Sprague-Dawley rats were obtained from Hormone Assay Laboratories. The rats were fed ad libitum and were given supplemental salt. Adenylate cyclase activity was determined by the method of Krishna et al. [14] using the modification of Ramachandran [15]. Protein concentration was determined by the m e t h o d of Lowry et al. [16] using bovine serum albumin as the standard. Short circuit current across the toad bladders was measured by the method of Ussing and Zerahn [13]. Uptake of 3 s SO~- by costal cartilage from hypophysectomized rats was determined according to the method of
293 Daughaday et al. [17]. Vasopressin was quantitated by radioimmunoassay according to Robertson et al. [ 1 8 ] . 12 s I-labeled bovine growth hormone was prepared by the m e t h o d of Thorell and Johansonn [19] using lactoperoxidase and was separated from unreacted iodide on Sephadex G-50. 12 s I-labeled vasopressin was prepared with chloramine-T and was purified on Sephadex G-25 [18]. Both iodinated hormones were used immediately after preparation or were stored at --80°C. The 12 s I-labeled growth hormone was repurified over Sephadex G-100 after thawing. Dialyses were carried out either in Bio-Rad-50 minibeakers with a molecular weight cut-off of 6000 daltons, or in a standard dialysis system using cellulose membranes with a molecular weight cut-off of 12 000 daltons. Purified bovine growth hormone lots B-16 and B-17, 0.92 unit/mg and highly purified growth hormone, lot B1003A, 2 units/mg were obtained from the NIAMDD. The hormones were stored in desiccated form a t - - 2 0 ° C . For each experiment, unless noted, the growth hormone was dissolved initially in 0.005 M NaOH, pH 10.5, and the solution was buffered to a final pH of 7.5 with 0.5% bovine serum albumin, 12.5 mM Tris. HC1, pH 7.3. Bovine luteinizing hormone NIH lot ]3-9, 0.7 unit/mg, ovine follicle stimulating hormone, NIH lot S-10, 1.10 units/mg, bovine thyrotropin, NIH lot B-6, 2.54 units/mg, commercial adrenocorticotropin, 20 units/mg all dissolved in distilled water, and arginine vasopressin, 173 units/ml, dissolved in acetic acid, were diluted with bovine serum albumin/Tris • HC1 buffer as described above. Bovine parathyroid hormone, 970 units/mg, and beef-pork glucagon were purchased from Wilson and Company and Eli Lilly and Company, respectively, and were also diluted with the bovine serum albumin/Tris. HC1 buffer. Controls in-which equivalent amounts of the bovine serum albumin/Tris • HC1 buffer alone were added to the assay mixture were included in each experiment. Each experiment was carried o u t on at least t w o separate occasions. Representative experiments have been chosen for presentation. [a.32 p] ATP, 15 Ci/mmol, was purchased from New England Nuclear Corporation and cyclic[3H]AMP, 14.2 Ci/mmol from Schwarz/Mann, 3sSO~from Amersham-Searle, and ~ 2 s I, from International Nuclear. Non-radioactive nucleotides were obtained from Sigma Chemical and neutral aluminum oxide, very pure grade, from M. Woelm. Results
Effect o f bovine growth hormone on adenylate cyclase in kidney Bovine growth hormone, 50 pg/ml, caused significant activation of adenylate cyclase in partially purified membranes from bovine renal medulla (Table I). All three preparations of growth hormone, including the highly purified lot B1003A, were effective in stimulating adenylate cyclase activity. The activation of adenylate cyclase by growth hormone was significant within 1 min of incubation (p < 0.01) and the rate was maximal and linear from 5 to 15 min (Fig. 1). Bovine growth hormone produced relatively greater stimulation of adenylate cyclase in partially purified plasma membranes from bovine renal medulla than in membranes from the cortex prepared in parallel from the
294
TABLE I A C T I V A T I O N OF A D E N Y L A T E C Y C L A S E GROWTH HORMONE AND VASOPRESSIN
FROM
BOVINE
Condition
RENAL
MEDULLA
BY B O V I N E
A d e n y l a t e cyclase a c t i v i t y * ( p m o l cyclic AMP f o r m e d / m g p r o t e i n p e r 10 m i n )
E x p e r i m e n t No. 1 Control Bovine g r o w t h h o r m o n e (lot B-16), 50 p g / m l Vasopressin, 20 m u n i t s / m l Vasopressin, 2 0 0 m u n i t s / m l Vasopressin, 20 m u n i t s / m l + b o v i n e g r o w t h h o r m o n e , 50 ~ g / m l V a s o p r e s s i n 2 0 0 m u n i t s / m l + b o v i n e g r o w t h h o r m o n e , 50 p g / m l
37 114 105 119 111 115
+++++ +-
E x p e r i m e n t No. 2 Control Bovine g r o w t h h o r m o n e (lot B-17), 50 p g ] m l V a s o p r e s s i n , 20 m u n i t s / m l N a F , 1.5 - 10 -2 M
133 272 241 265
+- 12 -+ 9 -+ 6 -+ 16
E x p e r i m e n t No. 3 Control B o v i n e g r o w t h h o r m o n e (lot B 1 0 0 3 A ) , 50 p g / m l Vasopressin, 20 m u m t s / m l Vasopressin, 2 0 0 m u n i t s / m l NaF, 1.5 • 10 -2 M
132 364 263 284 958
-+ 6 + 10 +- 12 +- 9 + 40
4 8 2 3 7 4
* Mean + S.E. o f t r i p l i c a t e d e t e r m i n a t i o n s .
same kidney (Fig. 2). The effect of vasopressin, 20 munits/ml, was similar, whereas the stimulation produced by parathyroid hormone, 20 pg/ml, was greater in renal cortex than in medulla, as reported previously [20]. Bovine growth hormone also caused activation of adenylate cyclase in homogenates of 200
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Biochimica et Biophysica Acta, 399 (1975) 2 9 1 - - 3 0 1 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s
BBA 27708
ACTIVATION OF A D E N Y L A T E CYCLASE IN R E N A L M E D U L L A BY BOVINE GROWTH H O R M O N E AN A R T I F A C T A T T R I B U T A B L E TO VASOPRESSIN*
S T E V E N B. L E I C H T E R and LEWIS R. C H A S E * *
Metabolism Division, Department of Medicine, Washington University School of Medicine, St. Louis, Mo. (U.S.A.) (Received F e b r u a r y 25th, 1975)
Summary The effect of bovine growth hormone on adenylate cyclase activity was studied in bovine and rat renal medulla. Highly purified growth hormone (lot B1003A) increased adenylate cyclase activity in plasma membranes from bovine renal medulla from 132 + 6 pmol cyclic AMP formed/mg protein per 10 min to 364 + 10 pmol cyclic AMP formed/mg protein per 10 min. Similar results were seen with homogenates of rat renal medulla. The minimum effective concentration of bovine growth hormone required to activate adenylate cyclase was 0.5 pg/ml and maximum activation was detected at 500 pg/ml. The amount of vasopressin determined by radioimmunoassay to contaminate the growth hormone caused an increase in adenylate cyclase activity comparable to that of the corresponding concentration of growth hormone that was tested. Dialysis of growth hormone and vasopressin resulted in parallel reductions in the effect of each hormone on adenylate cyclase activity. Similarly, both growth hormone and vasopressin produced increases in short circuit current in isolated toad bladders b u t these effects were no~ detectable after dialysis of the hormones. In contrast, the effect of growth hormone on the uptake of 3 s SO~by cartilage from hypophysectomized rats was not decreased after dialysis. These results indicate that available preparations of growth hormone are contaminated by small but physiologically significant amounts of vasopressin and that the activation of adenylate cyclase activity in renal medulla in response to growth hormone can be explained by this contamination rather than by an effect of growth hormone per se.
* Presented in part at the annual m e e t i n g o f The Endocrine S o c i e t y , Atlanta, Georgia, June 1974. ** Investigator~ Howa.rd Hughes Medical Institute.
292 Introduction Previous studies have indicated that growth hormone causes physiological changes in renal function. Administration of growth hormone [1--7] to humans, rats, and cats results in hypercalciuria and retention of Na ÷, water, K ÷ and phosphate. In vitro, the hormone stimulates salt and water transport across toad skin [8] and increases the uptake of poly-acyl R N A by ribosomes from rat renal cortex [9]. Although these changes have been attributed to a direct interaction between growth hormone and renal tissue, it has not been established with certainty whether some or all of these effects may be secondary to effects of growth hormone per se or to a minor contaminant in the preparation of the hormone. The latter has been suggested previously [8,10]. To date growth hormone, unlike most peptide hormones, has n o t been shown to cause rapid changes in the adenylate cyclase-cyclic AMP system in target tissues. However, previous studies have demonstrated that the hormone causes an increased concentration of cyclic AMP in adipose tissue after prolonged periods of incubation [11]. In view of the previous reports showing that growth hormone affects renal function, studies were carried out to determine if this hormone activates adenylate cyclase in the kidney, and if so, whether this activation could be attributed directly to growth hormone or to a contaminant in the preparations that are currently available. Methods and Materials Bovine kidneys were chilled on ice immediately after removal, and the cortex and medulla were separated within 2 h by blunt dissection. Tissues were minced and then homogenized in 0.25 M sucrose, 0.001 M EDTA, and 0.05 M Tris • HC1, pH 7.5, in a Dounce homogenizer with the loose fitting glass pestle. The crude homogenates were fractionated to the partially pure stage according to the method of Marx and Aurbach [12]. The membrane fractions were frozen in liquid nitrogen and stored at --80°C without significant loss of adenylate cyclase activity over a 2-week period. Adult male Sprague-Dawley rats in the fed state were killed by cervical fracture and homogenates prepared from renal medulla, renal cortex, brain, heart, liver and adrenal as described above. Hemibladders removed from adult male toads, Bufo Marinus, were washed in lactated Ringer's solution and placed immediately in an Ussing chamber [13]. Tissues were equilibrated for at least 20 min before test substances were added to the serosal chamber. 21-day-old hypophysectomized Sprague-Dawley rats were obtained from Hormone Assay Laboratories. The rats were fed ad libitum and were given supplemental salt. Adenylate cyclase activity was determined by the method of Krishna et al. [14] using the modification of Ramachandran [15]. Protein concentration was determined by the m e t h o d of Lowry et al. [16] using bovine serum albumin as the standard. Short circuit current across the toad bladders was measured by the method of Ussing and Zerahn [13]. Uptake of 3 s SO~- by costal cartilage from hypophysectomized rats was determined according to the method of
293 Daughaday et al. [17]. Vasopressin was quantitated by radioimmunoassay according to Robertson et al. [ 1 8 ] . 12 s I-labeled bovine growth hormone was prepared by the m e t h o d of Thorell and Johansonn [19] using lactoperoxidase and was separated from unreacted iodide on Sephadex G-50. 12 s I-labeled vasopressin was prepared with chloramine-T and was purified on Sephadex G-25 [18]. Both iodinated hormones were used immediately after preparation or were stored at --80°C. The 12 s I-labeled growth hormone was repurified over Sephadex G-100 after thawing. Dialyses were carried out either in Bio-Rad-50 minibeakers with a molecular weight cut-off of 6000 daltons, or in a standard dialysis system using cellulose membranes with a molecular weight cut-off of 12 000 daltons. Purified bovine growth hormone lots B-16 and B-17, 0.92 unit/mg and highly purified growth hormone, lot B1003A, 2 units/mg were obtained from the NIAMDD. The hormones were stored in desiccated form a t - - 2 0 ° C . For each experiment, unless noted, the growth hormone was dissolved initially in 0.005 M NaOH, pH 10.5, and the solution was buffered to a final pH of 7.5 with 0.5% bovine serum albumin, 12.5 mM Tris. HC1, pH 7.3. Bovine luteinizing hormone NIH lot ]3-9, 0.7 unit/mg, ovine follicle stimulating hormone, NIH lot S-10, 1.10 units/mg, bovine thyrotropin, NIH lot B-6, 2.54 units/mg, commercial adrenocorticotropin, 20 units/mg all dissolved in distilled water, and arginine vasopressin, 173 units/ml, dissolved in acetic acid, were diluted with bovine serum albumin/Tris • HC1 buffer as described above. Bovine parathyroid hormone, 970 units/mg, and beef-pork glucagon were purchased from Wilson and Company and Eli Lilly and Company, respectively, and were also diluted with the bovine serum albumin/Tris. HC1 buffer. Controls in-which equivalent amounts of the bovine serum albumin/Tris • HC1 buffer alone were added to the assay mixture were included in each experiment. Each experiment was carried o u t on at least t w o separate occasions. Representative experiments have been chosen for presentation. [a.32 p] ATP, 15 Ci/mmol, was purchased from New England Nuclear Corporation and cyclic[3H]AMP, 14.2 Ci/mmol from Schwarz/Mann, 3sSO~from Amersham-Searle, and ~ 2 s I, from International Nuclear. Non-radioactive nucleotides were obtained from Sigma Chemical and neutral aluminum oxide, very pure grade, from M. Woelm. Results
Effect o f bovine growth hormone on adenylate cyclase in kidney Bovine growth hormone, 50 pg/ml, caused significant activation of adenylate cyclase in partially purified membranes from bovine renal medulla (Table I). All three preparations of growth hormone, including the highly purified lot B1003A, were effective in stimulating adenylate cyclase activity. The activation of adenylate cyclase by growth hormone was significant within 1 min of incubation (p < 0.01) and the rate was maximal and linear from 5 to 15 min (Fig. 1). Bovine growth hormone produced relatively greater stimulation of adenylate cyclase in partially purified plasma membranes from bovine renal medulla than in membranes from the cortex prepared in parallel from the
294
TABLE I A C T I V A T I O N OF A D E N Y L A T E C Y C L A S E GROWTH HORMONE AND VASOPRESSIN
FROM
BOVINE
Condition
RENAL
MEDULLA
BY B O V I N E
A d e n y l a t e cyclase a c t i v i t y * ( p m o l cyclic AMP f o r m e d / m g p r o t e i n p e r 10 m i n )
E x p e r i m e n t No. 1 Control Bovine g r o w t h h o r m o n e (lot B-16), 50 p g / m l Vasopressin, 20 m u n i t s / m l Vasopressin, 2 0 0 m u n i t s / m l Vasopressin, 20 m u n i t s / m l + b o v i n e g r o w t h h o r m o n e , 50 ~ g / m l V a s o p r e s s i n 2 0 0 m u n i t s / m l + b o v i n e g r o w t h h o r m o n e , 50 p g / m l
37 114 105 119 111 115
+++++ +-
E x p e r i m e n t No. 2 Control Bovine g r o w t h h o r m o n e (lot B-17), 50 p g ] m l V a s o p r e s s i n , 20 m u n i t s / m l N a F , 1.5 - 10 -2 M
133 272 241 265
+- 12 -+ 9 -+ 6 -+ 16
E x p e r i m e n t No. 3 Control B o v i n e g r o w t h h o r m o n e (lot B 1 0 0 3 A ) , 50 p g / m l Vasopressin, 20 m u m t s / m l Vasopressin, 2 0 0 m u n i t s / m l NaF, 1.5 • 10 -2 M
132 364 263 284 958
-+ 6 + 10 +- 12 +- 9 + 40
4 8 2 3 7 4
* Mean + S.E. o f t r i p l i c a t e d e t e r m i n a t i o n s .
same kidney (Fig. 2). The effect of vasopressin, 20 munits/ml, was similar, whereas the stimulation produced by parathyroid hormone, 20 pg/ml, was greater in renal cortex than in medulla, as reported previously [20]. Bovine growth hormone also caused activation of adenylate cyclase in homogenates of 200
A ¢
--- -
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BGH Control
~- ~. 150 :> c
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