0021-972X/78/4702-0319$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 47, No. 2 Printed in U.S.A.
"* Adenylate Cyclase of Human Parathyroid Gland* HECTOR J. RODRIGUEZ, AUBREY MORRISON, EDUARDO SLATOPOLSKY, AND SAULO KLAHR Renal Division, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 ABSTRACT. Experiments were performed on a particulate fraction from human parathyroid glands. A high activity of adenylate cyclase was detected which was linear with time and protein concentration. The enzyme had an optimum pH in the range of 7-8 and a Km for ATP of 0.44 X 10~3 M. Ca++ had a profound inhibitory effect; a concentration of 0.5 mM Ca++ reduced enzyme activity by 60%. Maximal enzyme activity was obtained
A
LTHOUGH the factors controlling paraL thyroid hormone (PTH) secretion are well defined, little is known regarding the cellular mechanisms involved in release of the hormone. Recently, evidence has been presented suggesting that the release of hormone from the parathyroid gland may be regulated via the adenylate cyclase system (1, 2). At least two reported studies have shown a profound inhibitory effect of calcium on the adenylate cyclase activity of dog (3) and horse (4) parathyroid glands. In addition, Sherwood and Abe have demonstrated that the inhibitory effect of magnesium on the release of PTH in vitro is associated with a decrease in cAMP production (5). There is also evidence suggesting that glucagon (6), calcitonin (6, 7), and the catecholamines (8, 9) stimulate PTH secretion in vitro (6-8) and in vivo (9), but the effects of these hormones on human parathyroid gland adenylate cyclase have not been explored. Characterization of the parathyroid gland adenylate cyclase system may provide additional information regarding the control of PTH secretion. We report here multiple exReceived August 31, 1977. Address requests for reprints to: Dr. Saulo Klahr, Renal Division, Department of Internal Medicine, Washington University School of Medicine, 4550 Scott Avenue, St. Louis, Missouri 63110. •This work was supported by USPHS NIAMDD Grants AM-05248 and AM-09976.
with 5 mM Mg++; higher concentrations of this cation also inhibited enzyme activity. The effect of Mn++ was similar to that of Mg++. Enzyme activity was stimulated by NaF, catecholamines, glucagon, and calcitonin. The effect of catecholamines seems to be mediated through /?-adrenergic receptors. (J Clin Endocrinol Metab 47: 319, 1978) -
periments performed with a large parathyroid gland obtained from a subject with primary hyperparathyroidism and additional experiments on the effects of Ca++, Mg++, and hormones performed on two parathyroid adenomas and three hyperplastic glands from hyperparathyroid subjects. A high activity of adenylate cyclase was detected in a particulate fraction prepared from these human parathyroid glands. The enzyme was found to be magnesium dependent; it was inhibited by calcium and high magnesium concentrations and stimulated by sodium fluoride. In addition, catecholamines, glucagon, and calcitonin stimulated enzyme activity. Materials and Methods A section of the tissues obtained at surgery was immediately fixed and processed for light microscopy. The remainder was placed in ice-cold saline and used for enzyme preparation. All subsequent steps were conducted at 4 C. Light microscopic examination of the tissue sections disclosed the presence of only occasional fat cells and virtually no white blood cells or red blood cells. The predominant cell type in the glands employed in our studies was the chief cell. The tissue was meticulously cleaned of fat, sliced, and homogenized in 50 mM Tris-HCl, pH 7.4, with 10% (vol/vol) dimethylsulfoxide (DMSO) using a motor-driven pestle. The particulate fractions were prepared by the method described by Marcus and Aurbach for rat kidney (10). The homogenate was centrifuged at 2200 X g 319
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320
JCE&M i 1978 VoU7 No 2
RODRIGUEZ ET AL.
for 10 min, and the pellet was suspended in the Tris-DMSO buffer and centrifuged again. This pellet was resuspended in the same buffer and stored at —60 C. Storage for as long as 8 weeks did not result in significant loss of enzyme activity. Adenylate cyclase activity was assayed by a modification of the method of White and Zenser (11). The reaction mixture contained Tris-HCl (25 mM, pH 7.4), MgCl2 (5 mM), EDTA (1 mM), cAMP (1 mM), phosphocreatine (20 mM), creatine phosphokinase (1000 IU/ml), ATP (1.2 mM), theophylline (5 mM), a-[32P]ATP (1 X 106 cpm/tube), bovine serum albumin (BSA; 0.11%), appropriate amounts of enzyme protein, and hormone or NaF, as indicated, in a total volume of 0.05 ml. Incubation was carried out at 37 C and the reaction was stopped by adding 0.1 ml carrier solution containing ATP (40 mM), cAMP (12 mM), [3H]cAMP (20,000-30,000 cpm), and sodium dodecyl sulfate (10 mg/ml), after which the tubes were immersed in ice. The cAMP formed was separated by applying the content of the tubes to 0.5 x 15.0-cm columns packed with 1 g neutral aluminum oxide. Elution was carried out with 2 ml 50 mM Tris-HCl, pH 7.6. The tritium and 32P radioactivity were counted in a Packard scintillation counter. Double precipitation with Ba(OH)2/ZnSO4 (12) followed by treatment with phosphodiesterase demonstrated that 95% of the 32P radioactivity eluted from the column was [32P]cAMP, as previously reported (11). All assays were run in duplicate or triplicate and the results always agreed within 5-10%. Appropriate corrections were made in the final calculations for recovery of tritium-labeled cAMP, which in 225 samples examined averaged 81% ± 3. Protein concentrations were determined by the method of Lowry et al. (13). The protein from the Tris-DMSO enzyme suspension was precipitated with 50% trichloroacetic acid and redissolved in 1 N NaOH before assay in order to eliminate interference of the Tris-HCl buffer with the Lowry method (13). For the experiments on the effect of calcium on the activity of adenylate cyclase, EDTA and BSA were omitted from the reaction mixture and replaced by identical volumes of 25 mM Tris-HCl. Tritium-labeled cAMP was purchased from New England Nuclear (Boston, MA), a-[32P]ATP was obtained from International Chemical and Nuclear Corp. (Cleveland, OH), and aluminum oxide was purchased from M. Woelm (Eschwege, West Germany). Dr. Lewis Chase kindly provided us with bovine GH and calcitonin and Dr. Lawrence Jacobs provided the TSH and PRL. Other reagents utilized were reagent grade and were purchased from stan-
dard suppliers. Statistical analyses were done utilizing the Student t test for paired data. Results The assay proved to be highly reproducible. Formation of cAMP was linear with time for as long as 20 min. Formation of the nucleotide was also linear with varying protein concentrations in the range of 5-50 jug. In view of these findings, all subsequent experiments were done using 15-25 jug enzyme protein/tube and an incubation time of 10 min. Effect ofpH on parathyroid gland adenylate cyclase activity The enzyme had a rather broad pH optimum between 6-8 with an apparent maximum at pH 7.5 (Fig. 1). This profile of pH optimum is similar to that reported for the adenylate cyclase of rat kidney (14) and guinea pig heart (15), but different from that reported for the enzyme of frog erythrocytes (16). Effects of sodium fluoride Rail and Sutherland first reported the activation of adenylate cyclase by F~ in 1962 (17). The mechanism of this effect has remained unclear. We found that NaF stimulated the adenylate cyclase from human parathyroid gland in a dose-dependent manner. Maximal stimulation was observed at a concentration of 10 mM. This concentration of NaF also produces maximal stimulation of adenylate cyclase in other tissues (14-16, 18-20). The degree of stimulation observed in our study is
FIG. 1. Effect of pH on activity of adenylate cyclase. Protein concentration was 25 fig/tube; incubation time was 10 min. Each point represents the mean of three determinations.
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PARATHYROID GLAND ADENYLATE CYCLASE comparable to that reported for the enzyme of bovine adrenal medulla (19), rat cerebral cortex (20), and horse parathyroid gland (4) but considerably less than that reported for rat kidney (14) and guinea pig heart adenyl cyclase (15). Effect of divalent cations There was an absolute requirement for magnesium. This is most likely the result of the fact that at pH 7.5 the substrate for the enzyme is a Mg++-ATP complex (15). Activity was detected at a Mg++ concentration of 2 HIM; it was maximal at 5 mM and decreased considerably at higher Mg++ concentrations. Manganese could partially replace Mg++. The pattern of enzyme activity was similar to the one obtained with Mg++, but maximal activity was observed at a Mn ++ concentration of 2.5 mM. These results are depicted in Fig. 2. Similar results were obtained when the concentration of ATP was changed together with the concentration of Mg++. Additional experiments performed on two parathyroid adenomas and three hyperplastic parathyroid glands are shown in Table 1. Activity was detected at Mg++ concentrations of 1 mM, was maximal at 5 mM, and decreased at Mg++ concentrations of 25 mM. As shown in Fig. 3, calcium had a profound inhibitory effect on the activity of human parathyroid gland adenylate cyclase. Maximal activity (158 pmol/mg protein/min) was obtained when calcium was not included in the incubation mixture and the Mg++ concentration was 5 mM in the presence of EDTA and
321
BSA. Removing EDTA and BSA from the incubation mixture did not change the activity (156 pmol/mg protein/min). The lack of effect of EDTA is similar to that reported for the enzyme from horse parathyroid gland (4) but different from the results obtained with the adenylate cyclase of guinea pig heart (15). However, when 0.5 mM Ca was added in the absence of EDTA and BSA, the activity of the enzyme markedly decreased to 36 pmol/mg protein/min, which represented 76% inhibition of the basal activity. Ca++ concentrations of 0.2 and 0.3 mM resulted in a comparable inhibitory effect. Raising the Ca++ concentration in the incubation mixture to 5 mM resulted in virtually complete abolition (96%) of the adenylate cyclase activity. Additional experiments performed on the adenylate cyclase of two parathyroid adenomas and three hyperplastic parathyroid glands are shown in Table 2. Again, a profound inhibitory TABLE 1. Effect of increasing concentrations of Mg ++ on adenylate cyclase activity Mg ++
Tissue histology
Hyperplasia Adenoma Hyperplasia Adenoma Adenoma Mean SEM
1.0
2.5
5.0
25.0
9 28
95 88 17 120
189 193 51 181
7
156
297
229 277 88 236 326
208 202 39 173 292
182.2 ±39.1
231.2 ±39.8
182.8 ±41.1
0 8
13.0
±5.0
95.2 ±22.9
Values are given in picomoles per mg protein/min.
200-
0 05
FIG. 2. Effect of varying concentrations of Mg ++ and Mn ++ on activity of adenylate cyclase. Protein concentration was 25 /ig. Each point represents the mean of triplicate determinations.
FIG. 3. Effect of Ca ++ on activity of adenylate cyclase. Protein concentration was 25 /xg. Mg ++ concentration was 5 mM. Incubation time was 10 min. In these experiments, EDTA and BSA were replaced in the incubation mixture by identical volumes of Tris-HCl buffer. Each point represents the mean of three determinations.
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JCE&M • 1978 Vol 47 • No 2
RODRIGUEZ ET AL.
322
effect of Ca++ on the activity of parathyroid gland adenylate cyclase was observed. Effects of changes in ATP concentration Formation of cAMP was maximal at an ATP concentration of 1 mM. A LineweaverBurke plot, depicted in Fig. 4, revealed a Km for ATP of 0.444 X 10~3 M and a maximum velocity (Vmax) of 238 pmol/mg protein/min. The Km value obtained is of the same order of magnitude as the one reported for the enzymes of guinea pig heart (0.08 X 10~3 M) (15), fat cell ghosts (0.259 X 10~3 M) (21), frog erythrocyte (0.159 X 10"3 M) (16), and rat kidney (0.2 X 10~3 M) (14) but considerably greater than the value for the enzyme of frog bladder epithelial cells (1.7 X 10~7 M) (22). Effects of hormones on the activity of adenyl cyclase Table 3 summarizes the results of these experiments. Of the hormones tested, only the catecholamines, glucagon, and calcitonin stimulated the activity of parathyroid adenylate cyclase. The stimulatory effect of catecholamines was further characterized, as indicated in Table 4. The /?-adrenergic agonist, isoproterenol, produced a substantially greater stimulation of adenylate cyclase activity than arterenol, a substance with predominant a-adrenergic activity. Propranolol, a /?-adrenergic antagonist, blocked the stimulation of isoproterenol, whereas phentolamine, an a-adrenergic antagonist, had no effect on the stimulation produced by arterenol. This probably indicates that the small stimulatory effect of TABLE 2. Effect of increasing Ca+
concentrations on
adenylate cyclase activity Ca ++ (mM) 0
0.5
1.0
2.5
Hyperplasia Adenoma Hyerplasia Adenoma Adenoma
441 453 72 332
156 170 25 123 210
74 135 14 68 124
56 66 8 17 53
Mean
324.5 ±88.5
136.8 ±31.2
83.8 ±22.2
40.0 ±11.5
Values are given in picomoles per mg protein/min.
0
2
a
6
8
10
1/ATP mM
FIG. 4. Lineweaver-Burke plot of the effect of ATP concentration on activity of adenylate cyclase. Protein concentration 25.5 /xg. Incubation time was 10 min. Mg ++ concentration was 5 mM. Each point is the mean of triplicate determinations. TABLE 3. Effect of hormones on activity of parathyroid adenylate cyclase cAMP (pmol/mg protein/ min)
P values
Control 210 ± 3° Arterenol (10~4 M) 239 ± 4
Vol. 47, No. 2 Printed in U.S.A.
"* Adenylate Cyclase of Human Parathyroid Gland* HECTOR J. RODRIGUEZ, AUBREY MORRISON, EDUARDO SLATOPOLSKY, AND SAULO KLAHR Renal Division, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 ABSTRACT. Experiments were performed on a particulate fraction from human parathyroid glands. A high activity of adenylate cyclase was detected which was linear with time and protein concentration. The enzyme had an optimum pH in the range of 7-8 and a Km for ATP of 0.44 X 10~3 M. Ca++ had a profound inhibitory effect; a concentration of 0.5 mM Ca++ reduced enzyme activity by 60%. Maximal enzyme activity was obtained
A
LTHOUGH the factors controlling paraL thyroid hormone (PTH) secretion are well defined, little is known regarding the cellular mechanisms involved in release of the hormone. Recently, evidence has been presented suggesting that the release of hormone from the parathyroid gland may be regulated via the adenylate cyclase system (1, 2). At least two reported studies have shown a profound inhibitory effect of calcium on the adenylate cyclase activity of dog (3) and horse (4) parathyroid glands. In addition, Sherwood and Abe have demonstrated that the inhibitory effect of magnesium on the release of PTH in vitro is associated with a decrease in cAMP production (5). There is also evidence suggesting that glucagon (6), calcitonin (6, 7), and the catecholamines (8, 9) stimulate PTH secretion in vitro (6-8) and in vivo (9), but the effects of these hormones on human parathyroid gland adenylate cyclase have not been explored. Characterization of the parathyroid gland adenylate cyclase system may provide additional information regarding the control of PTH secretion. We report here multiple exReceived August 31, 1977. Address requests for reprints to: Dr. Saulo Klahr, Renal Division, Department of Internal Medicine, Washington University School of Medicine, 4550 Scott Avenue, St. Louis, Missouri 63110. •This work was supported by USPHS NIAMDD Grants AM-05248 and AM-09976.
with 5 mM Mg++; higher concentrations of this cation also inhibited enzyme activity. The effect of Mn++ was similar to that of Mg++. Enzyme activity was stimulated by NaF, catecholamines, glucagon, and calcitonin. The effect of catecholamines seems to be mediated through /?-adrenergic receptors. (J Clin Endocrinol Metab 47: 319, 1978) -
periments performed with a large parathyroid gland obtained from a subject with primary hyperparathyroidism and additional experiments on the effects of Ca++, Mg++, and hormones performed on two parathyroid adenomas and three hyperplastic glands from hyperparathyroid subjects. A high activity of adenylate cyclase was detected in a particulate fraction prepared from these human parathyroid glands. The enzyme was found to be magnesium dependent; it was inhibited by calcium and high magnesium concentrations and stimulated by sodium fluoride. In addition, catecholamines, glucagon, and calcitonin stimulated enzyme activity. Materials and Methods A section of the tissues obtained at surgery was immediately fixed and processed for light microscopy. The remainder was placed in ice-cold saline and used for enzyme preparation. All subsequent steps were conducted at 4 C. Light microscopic examination of the tissue sections disclosed the presence of only occasional fat cells and virtually no white blood cells or red blood cells. The predominant cell type in the glands employed in our studies was the chief cell. The tissue was meticulously cleaned of fat, sliced, and homogenized in 50 mM Tris-HCl, pH 7.4, with 10% (vol/vol) dimethylsulfoxide (DMSO) using a motor-driven pestle. The particulate fractions were prepared by the method described by Marcus and Aurbach for rat kidney (10). The homogenate was centrifuged at 2200 X g 319
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320
JCE&M i 1978 VoU7 No 2
RODRIGUEZ ET AL.
for 10 min, and the pellet was suspended in the Tris-DMSO buffer and centrifuged again. This pellet was resuspended in the same buffer and stored at —60 C. Storage for as long as 8 weeks did not result in significant loss of enzyme activity. Adenylate cyclase activity was assayed by a modification of the method of White and Zenser (11). The reaction mixture contained Tris-HCl (25 mM, pH 7.4), MgCl2 (5 mM), EDTA (1 mM), cAMP (1 mM), phosphocreatine (20 mM), creatine phosphokinase (1000 IU/ml), ATP (1.2 mM), theophylline (5 mM), a-[32P]ATP (1 X 106 cpm/tube), bovine serum albumin (BSA; 0.11%), appropriate amounts of enzyme protein, and hormone or NaF, as indicated, in a total volume of 0.05 ml. Incubation was carried out at 37 C and the reaction was stopped by adding 0.1 ml carrier solution containing ATP (40 mM), cAMP (12 mM), [3H]cAMP (20,000-30,000 cpm), and sodium dodecyl sulfate (10 mg/ml), after which the tubes were immersed in ice. The cAMP formed was separated by applying the content of the tubes to 0.5 x 15.0-cm columns packed with 1 g neutral aluminum oxide. Elution was carried out with 2 ml 50 mM Tris-HCl, pH 7.6. The tritium and 32P radioactivity were counted in a Packard scintillation counter. Double precipitation with Ba(OH)2/ZnSO4 (12) followed by treatment with phosphodiesterase demonstrated that 95% of the 32P radioactivity eluted from the column was [32P]cAMP, as previously reported (11). All assays were run in duplicate or triplicate and the results always agreed within 5-10%. Appropriate corrections were made in the final calculations for recovery of tritium-labeled cAMP, which in 225 samples examined averaged 81% ± 3. Protein concentrations were determined by the method of Lowry et al. (13). The protein from the Tris-DMSO enzyme suspension was precipitated with 50% trichloroacetic acid and redissolved in 1 N NaOH before assay in order to eliminate interference of the Tris-HCl buffer with the Lowry method (13). For the experiments on the effect of calcium on the activity of adenylate cyclase, EDTA and BSA were omitted from the reaction mixture and replaced by identical volumes of 25 mM Tris-HCl. Tritium-labeled cAMP was purchased from New England Nuclear (Boston, MA), a-[32P]ATP was obtained from International Chemical and Nuclear Corp. (Cleveland, OH), and aluminum oxide was purchased from M. Woelm (Eschwege, West Germany). Dr. Lewis Chase kindly provided us with bovine GH and calcitonin and Dr. Lawrence Jacobs provided the TSH and PRL. Other reagents utilized were reagent grade and were purchased from stan-
dard suppliers. Statistical analyses were done utilizing the Student t test for paired data. Results The assay proved to be highly reproducible. Formation of cAMP was linear with time for as long as 20 min. Formation of the nucleotide was also linear with varying protein concentrations in the range of 5-50 jug. In view of these findings, all subsequent experiments were done using 15-25 jug enzyme protein/tube and an incubation time of 10 min. Effect ofpH on parathyroid gland adenylate cyclase activity The enzyme had a rather broad pH optimum between 6-8 with an apparent maximum at pH 7.5 (Fig. 1). This profile of pH optimum is similar to that reported for the adenylate cyclase of rat kidney (14) and guinea pig heart (15), but different from that reported for the enzyme of frog erythrocytes (16). Effects of sodium fluoride Rail and Sutherland first reported the activation of adenylate cyclase by F~ in 1962 (17). The mechanism of this effect has remained unclear. We found that NaF stimulated the adenylate cyclase from human parathyroid gland in a dose-dependent manner. Maximal stimulation was observed at a concentration of 10 mM. This concentration of NaF also produces maximal stimulation of adenylate cyclase in other tissues (14-16, 18-20). The degree of stimulation observed in our study is
FIG. 1. Effect of pH on activity of adenylate cyclase. Protein concentration was 25 fig/tube; incubation time was 10 min. Each point represents the mean of three determinations.
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PARATHYROID GLAND ADENYLATE CYCLASE comparable to that reported for the enzyme of bovine adrenal medulla (19), rat cerebral cortex (20), and horse parathyroid gland (4) but considerably less than that reported for rat kidney (14) and guinea pig heart adenyl cyclase (15). Effect of divalent cations There was an absolute requirement for magnesium. This is most likely the result of the fact that at pH 7.5 the substrate for the enzyme is a Mg++-ATP complex (15). Activity was detected at a Mg++ concentration of 2 HIM; it was maximal at 5 mM and decreased considerably at higher Mg++ concentrations. Manganese could partially replace Mg++. The pattern of enzyme activity was similar to the one obtained with Mg++, but maximal activity was observed at a Mn ++ concentration of 2.5 mM. These results are depicted in Fig. 2. Similar results were obtained when the concentration of ATP was changed together with the concentration of Mg++. Additional experiments performed on two parathyroid adenomas and three hyperplastic parathyroid glands are shown in Table 1. Activity was detected at Mg++ concentrations of 1 mM, was maximal at 5 mM, and decreased at Mg++ concentrations of 25 mM. As shown in Fig. 3, calcium had a profound inhibitory effect on the activity of human parathyroid gland adenylate cyclase. Maximal activity (158 pmol/mg protein/min) was obtained when calcium was not included in the incubation mixture and the Mg++ concentration was 5 mM in the presence of EDTA and
321
BSA. Removing EDTA and BSA from the incubation mixture did not change the activity (156 pmol/mg protein/min). The lack of effect of EDTA is similar to that reported for the enzyme from horse parathyroid gland (4) but different from the results obtained with the adenylate cyclase of guinea pig heart (15). However, when 0.5 mM Ca was added in the absence of EDTA and BSA, the activity of the enzyme markedly decreased to 36 pmol/mg protein/min, which represented 76% inhibition of the basal activity. Ca++ concentrations of 0.2 and 0.3 mM resulted in a comparable inhibitory effect. Raising the Ca++ concentration in the incubation mixture to 5 mM resulted in virtually complete abolition (96%) of the adenylate cyclase activity. Additional experiments performed on the adenylate cyclase of two parathyroid adenomas and three hyperplastic parathyroid glands are shown in Table 2. Again, a profound inhibitory TABLE 1. Effect of increasing concentrations of Mg ++ on adenylate cyclase activity Mg ++
Tissue histology
Hyperplasia Adenoma Hyperplasia Adenoma Adenoma Mean SEM
1.0
2.5
5.0
25.0
9 28
95 88 17 120
189 193 51 181
7
156
297
229 277 88 236 326
208 202 39 173 292
182.2 ±39.1
231.2 ±39.8
182.8 ±41.1
0 8
13.0
±5.0
95.2 ±22.9
Values are given in picomoles per mg protein/min.
200-
0 05
FIG. 2. Effect of varying concentrations of Mg ++ and Mn ++ on activity of adenylate cyclase. Protein concentration was 25 /ig. Each point represents the mean of triplicate determinations.
FIG. 3. Effect of Ca ++ on activity of adenylate cyclase. Protein concentration was 25 /xg. Mg ++ concentration was 5 mM. Incubation time was 10 min. In these experiments, EDTA and BSA were replaced in the incubation mixture by identical volumes of Tris-HCl buffer. Each point represents the mean of three determinations.
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JCE&M • 1978 Vol 47 • No 2
RODRIGUEZ ET AL.
322
effect of Ca++ on the activity of parathyroid gland adenylate cyclase was observed. Effects of changes in ATP concentration Formation of cAMP was maximal at an ATP concentration of 1 mM. A LineweaverBurke plot, depicted in Fig. 4, revealed a Km for ATP of 0.444 X 10~3 M and a maximum velocity (Vmax) of 238 pmol/mg protein/min. The Km value obtained is of the same order of magnitude as the one reported for the enzymes of guinea pig heart (0.08 X 10~3 M) (15), fat cell ghosts (0.259 X 10~3 M) (21), frog erythrocyte (0.159 X 10"3 M) (16), and rat kidney (0.2 X 10~3 M) (14) but considerably greater than the value for the enzyme of frog bladder epithelial cells (1.7 X 10~7 M) (22). Effects of hormones on the activity of adenyl cyclase Table 3 summarizes the results of these experiments. Of the hormones tested, only the catecholamines, glucagon, and calcitonin stimulated the activity of parathyroid adenylate cyclase. The stimulatory effect of catecholamines was further characterized, as indicated in Table 4. The /?-adrenergic agonist, isoproterenol, produced a substantially greater stimulation of adenylate cyclase activity than arterenol, a substance with predominant a-adrenergic activity. Propranolol, a /?-adrenergic antagonist, blocked the stimulation of isoproterenol, whereas phentolamine, an a-adrenergic antagonist, had no effect on the stimulation produced by arterenol. This probably indicates that the small stimulatory effect of TABLE 2. Effect of increasing Ca+
concentrations on
adenylate cyclase activity Ca ++ (mM) 0
0.5
1.0
2.5
Hyperplasia Adenoma Hyerplasia Adenoma Adenoma
441 453 72 332
156 170 25 123 210
74 135 14 68 124
56 66 8 17 53
Mean
324.5 ±88.5
136.8 ±31.2
83.8 ±22.2
40.0 ±11.5
Values are given in picomoles per mg protein/min.
0
2
a
6
8
10
1/ATP mM
FIG. 4. Lineweaver-Burke plot of the effect of ATP concentration on activity of adenylate cyclase. Protein concentration 25.5 /xg. Incubation time was 10 min. Mg ++ concentration was 5 mM. Each point is the mean of triplicate determinations. TABLE 3. Effect of hormones on activity of parathyroid adenylate cyclase cAMP (pmol/mg protein/ min)
P values
Control 210 ± 3° Arterenol (10~4 M) 239 ± 4
