Hormonal Regulation of Renal Ornithine Decarboxylase Activity in the Rat1 WENDELL E. NICHOLSON, JON H. LEVINE,2-3 AND DAVID N. ORTH4 Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 growth hormone, ACTH and cortisol each increased renal omithine decarboxylase activity in the hypophysectomized rat, with the highest levels of activity being achieved with growth hormone. Other pituitary hormones (FSH, LH, TSH and prolactin) were ineffective. After bilateral adrenalectomy, renal omithine decarboxylase activity retained a rhythmical pattern similar to that observed in the intact rat, but the levels were increased. Growth hormone and cortisol increased renal ornithine decarboxylase activity in the adrenalectomized-hypophysectomized animal to the same extent as in the hypophysectomized animal, but ACTH was almost totally ineffective. These data suggest that the pituitary plays a major role in the regulation of renal ornithine decarboxylase activity in the rat, primarily through the rhythmical secretion of growth hormone and ACTH. (Endocrinology 98: 123, 1976)
ABSTRACT. The regulation of the activity of the renal enzyme omithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was examined in the rat. In the intact animal adapted to a light/dark cycle of 14 hours and 10 hours, respectively, the level of renal omithine decarboxylase activity was rhythmical and paralleled the diurnal rhythm in plasma corticosteroid concentration. Renal omithine decarboxylase activity and plasma corticosterone were highest during the early hours of darkness and lowest during the hours of light. Following hypophysectomy, the level of renal omithine decarboxylase activity declined rapidly and remained low and without a demonstrable diurnal rhythm. When pituitary hormone levels were temporarily restored in the hypophysectomized rat by the injection of pituitary extract, renal ornithine decarboxylase activity increased rapidly, reached a peak within 8 hours, and returned toward pre-injection levels by 12 hours. Exogenous
T
HE regulation of the enzyme ornithine decarboxylase (L-ornithine carboxylyase, EC 4.1.1.17) has attracted considerable attention in the past few years because of its possible role in the early events associated with growth. It has been suggested that the conversion of ornithine to putrescine by ornithine decarboxylase may be the ratelimiting step in the synthesis of the polyamines. Ornithine decarboxylase activity has been reported to increase during compensatory growth of the remaining kidney following unilateral nephrectomy (1), and both cortisol and growth hormone administered to rats have been found to increase the levels
of renal enzyme activity (1,2,3). It has been shown that there is a diurnal rhythm in hepatic ornithine decarboxylase activity which is altered both by hypophysectomy and by adrenalectomy (4,5). In this report, we have studied the effects of several pituitary hormones and cortisol on renal ornithine decarboxylase activity and have demonstrated a possible physiological role for growth hormone and ACTH-dependent corticosteroid secretion in the maintenance and regulation of the level of enzyme activity in the rat kidney. Materials and Methods
Received July 14, 1975. 1 These studies were supported in part by the following grants-in-aid from The National Institutes of Health, United States Public Health Service: 5-T01AM05092 and 5-R01-AM05318. 2 Canadian Medical Research Council Fellow at the time these studies were performed. 3 Present address: Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29401. 4 Investigator, Howard Hughes Medical Institute.
The ACTH preparation, generously provided by Armour Laboratories, was dissolved in 4% gelatin and administered subcutaneously. The other pituitary hormones were kindly provided by the Endocrinology Study Section of the National Institutes of Health: FSH (NIH-FSHS9), LH (NIH-LH-S18), TSH (NIH-TSH-S7), prolactin (NIH-P-B4) and GH (NIH-GH-S10) were dissolved in 0.154M NaCl (pH adjusted to 9.0) and administered intraperitoneally. Cortisol hemisuccinate (SolucortefR) was dissolved in
123
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124
NaCl and administered intraperitoneally. Ovine pituitary tissue which had been lyophilized, pulverized and stored at - 2 0 C was kindly provided by Dr. Robert W. Bates, Hormone Distribution Officer, National Institute of Arthritis, Metabolism, and Digestive Diseases. The pituitary powder was extracted with 0. 1M (NH4)HCO3, 10 ml per g of powder, in a Waring blender during 2 successive 30-second homogenizations at 4 C. Followingcentrifugationat50,000 x gfor20min, the precipitate was again extracted. The two supernatants were pooled and stored at —56 C. Male Sprague-Dawley rats weighing 130-180 g were used for the study. Animals used for investigations of diurnal rhythm were kept in a quiet room at 22 C and adapted to light/dark cycles of 14 and 10 hours, respectively (06002000 light). Food and water were provided ad libitum. In the experiments requiring hypophysectomized animals, noncycled rats were hypophysectomized under ether anesthesia by the transaural approach. Adrenalectomy of noncycled rats was performed under sodium pentobarbital anesthesia. Some of these animals were then cycled as above, but were kept at 26 C and were given 0.077M NaCl as their drinking solution. The rats were sacrificed by decapitation, and, in some experiments, trunk blood was collected at this time for corticosterone determination as previously described (6). In all groups studied, alternate right and left kidneys were rapidly removed, the capsule was stripped, and the kidney was then bisected in a longitudinal plane. The pelvis and the major calyces were resected, and a 150-200 mg wedge of tissue with its base at the cortex was obtained. This tissue was placed
0.154M
Endo • 1976 Vol 98 • No 1
NICHOLSON, LEVINE AND ORTH
in 1 ml of chilled 0.067M sodium-potassium phosphate buffer (pH 7.2) containing5 mMEDTA and 1 mM dithiothreitol and homogenized for 10-15sat4 C withaPolytron P-10 tissue grinder. The homogenates were centrifuged at 20,000 x g for 20 min at 4 C and the supernates were stored at —56 C until assayed for ornithine decarboxylase activity as previously described (7). Samples could be kept frozen for as long as two weeks without detectable losses in enzyme activity. However, approximately 33% of the enzyme activity was lost each time the samples were thawed and refrozen.
Results Renal ornithine decarboxylase activity in the intact rat Two hormones, growth hormone and cortisol, have been shown to increase the levels of renal ornithine decarboxylase activity in the rat (1-3). While the plasma level of corticosterone, the physiological glucocorticoid in the rat, displays a circadian rhythm (8), the evidence for a single daily peak in growth hormone secretion in rodents remains equivocal (9-12). A diurnal rhythm in ACTH secretion was present in intact cycled rats as reflected by plasma corticosterone concentration, which reached a peak at the time of onset of darkness and then declined and remained low throughout the remainder of the day (Fig. 1). Renal ornithine decarboxylase activity also appeared rhythmic, with peak levels of enzyme activity reached dur-
FlG. 1. Renal ornithine decarboxylase activity and plasma corticosterone in the intact rat. Normal male rats were adapted to light/dark cycles for 7 days and PLASMA RENAL CORTICOODC then were sacrificed at the times STERONE ACTIVITY indicated. The 2000 and 2400 h nmoles l 4 yug/lOOml o -o levels of omithine decarboxylase activity, which are not different from each other, are significantly higher than all other levels (Duncan's multiple range 1200 1600 2000 2400 0400 0800 1200 test,P = .05). The 2000 h plasma corticosterone concentration is TIME significantly higher than all others, and the 1600 and 2400 h levels, which are not significantly different from each other, are greater than the rest (Duncan's multiple range test, P = .05). In this and subsequent figures, the brackets represent the standard errors of the mean responses of several animals. N = 6.
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RENAL ORNITHINE DECARBOXYLASE ACTIVITY
125
ing the hours when corticosterone and ACTH secretion were highest and low levels when corticosterone and ACTH were lowest (Fig. 1). Renal ornithine decarboxylase activity in the hypophysectomized rat The apparent influence of pituitary hormones on renal ornithine decarboxylase activity was examined further by studying hypophysectomized animals. There was an immediate fall in renal ornithine decarboxylase activity following hypophysectomy, with an estimated half-life of 1.9 h; there was no further fall from 8 to 24 h (Fig. 2). Furthermore, the level of renal ornithine decarboxylase activity observed in other animals that were hypophysectomized for 4 days was only slightly lower than the level found just 8 hours after hypophysectomy. In these chronically hypophysectomized animals, which were maintained on regular light/dark cycles, renal ornithine decarboxylase activity and plasma corticosterone remained low and failed to demonstrate significant diurnal rhythmicity. When temporary restoration of pituitary hormone levels was attempted by intraperi100
RENAL ODC ACTIVITY nmoles
l4
FiG. 3. Effect of pituitary extract on renal omithine decarboxylase activity in the hypophysectomized rat. Sixteen hours following hypophysectomy, the animals were given an intraperitoneal injection of 0.5 ml of crude pituitary extract. They were sacrificed at the times indicated for determination of renal omithine decarboxylase activity. N = 5.
toneal injection of pituitary extract 16 hours after hypophysectomy, renal ornithine decarboxylase activity increased within 2 hours of the injection, reached a peak at 8 hours, and fell nearly to preinjection levels by 12 hours (Fig. 3). Growth hormone, ACTH, FSH, LH, TSH, prolactin and cortisol were also injected into rats 16 hours after hypophysectomy. Only growth hormone, ACTH and cortisol stimulated increased renal ornithine decarboxylase activity (Fig. 4). The effect of growth hormone persisted for at least 8 hours, while that of ACTH and cortisol had disappeared by 8 hours. Growth hormone appeared to be the most effective stimulus for increasing renal ornithine decarboxylase activity (Table 1). Renal ornithine decarboxylase activity in the adrenalectomized rat
24
FiG. 2. Renal omithine decarboxylase activity in the acutely hypophysectomized rat. Noncycled rats were hypophysectomized by the transaural approach and were then sacrificed at the times indicated. A regression line was fitted to the data obtained during the first 8 h following hypophysectomy (T4 = 1.9 h). N = 5.
The role of adrenal hormones in the regulation of renal ornithine decarboxylase activity was examined in bilaterally adrenalectomized rats maintained on regular light/dark cycles. In these animals, plasma ACTH levels are elevated and rhythmical, with peak levels observed at about the middle of the light period (13). In contrast to the report that adrenalectomy nearly abolished the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 November 2015. at 12:08 For personal use only. No other uses without permission. . All rights reserved.
Endo i 1976 Vol 98 . No 1
NICHOLSON, LEVINE AND ORTH
126 90
TABLE 1. Effect of ACTH, cortisol and growth hormone on renal ODC activity in the hypophysectomized rat
80
Hormone 0
60
ACTH
50
Cortisol 40
30
Growth hormone
20
10
FlG. 4. Time course of pituitary and adrenal hormone action in the hypophysectomized rat. Sixteen hours following hypophysectomy, the animals received either 0.5 mg of growth hormone, LH, FSH, TSH, or prolactin, 50 IU of ACTH, or 5 mg of cortisol. They were sacrificed at the times indicated for determination of renal ornithine decarboxylase activity. (* significantly different from control. Student's t test, F < .05 for ACTH and cortisol at 4 h, P < .02 for growth hormone at 4 h and 8 h)
diurnal rhythm in hepatic ornithine decarboxylase activity (3), we observed that renal ornithine decarboxylase activity was markedly elevated after adrenalectomy, with most levels exceeding the highest level of activity observed in intact, cycled animals (Fig. 5). Renal ornithine decarboxylase activity in these adrenalectomized cycled rats also demonstrated a diurnal rhythm, but the peak levels of activity appeared to be delayed about 4 hours (Fig. 5). Renal ornithine decarboxylase activity in the adrenalectomized-hypophysectomized rat The parallel diurnal rhythms in renal ornithine decarboxylase activity and plasma
Dose
F*
—
3.2 ±
0.7
—
0.4 IU 2 10 50 0.04 mg 0.2 1 5 0.04 mg 0.2 1 5
18.8 ± 24.2 ± 45.3 ± 74.7 ± 5.4 ± 4.8 ± 8.3 ± 30.3 ±
5.6 9.1 19.1 10.4 1.0 0.4 2.6 7.0
A A A A ©bob o to to to
70
Renal ODC Activity nmoles u C(Vg (mean ± SE)
7.3 ± 2.0 56.5 ± 18.6 212.4 ± 54.1 346.7 ± 35.0
NS
ABSTRACT. The regulation of the activity of the renal enzyme omithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was examined in the rat. In the intact animal adapted to a light/dark cycle of 14 hours and 10 hours, respectively, the level of renal omithine decarboxylase activity was rhythmical and paralleled the diurnal rhythm in plasma corticosteroid concentration. Renal omithine decarboxylase activity and plasma corticosterone were highest during the early hours of darkness and lowest during the hours of light. Following hypophysectomy, the level of renal omithine decarboxylase activity declined rapidly and remained low and without a demonstrable diurnal rhythm. When pituitary hormone levels were temporarily restored in the hypophysectomized rat by the injection of pituitary extract, renal ornithine decarboxylase activity increased rapidly, reached a peak within 8 hours, and returned toward pre-injection levels by 12 hours. Exogenous
T
HE regulation of the enzyme ornithine decarboxylase (L-ornithine carboxylyase, EC 4.1.1.17) has attracted considerable attention in the past few years because of its possible role in the early events associated with growth. It has been suggested that the conversion of ornithine to putrescine by ornithine decarboxylase may be the ratelimiting step in the synthesis of the polyamines. Ornithine decarboxylase activity has been reported to increase during compensatory growth of the remaining kidney following unilateral nephrectomy (1), and both cortisol and growth hormone administered to rats have been found to increase the levels
of renal enzyme activity (1,2,3). It has been shown that there is a diurnal rhythm in hepatic ornithine decarboxylase activity which is altered both by hypophysectomy and by adrenalectomy (4,5). In this report, we have studied the effects of several pituitary hormones and cortisol on renal ornithine decarboxylase activity and have demonstrated a possible physiological role for growth hormone and ACTH-dependent corticosteroid secretion in the maintenance and regulation of the level of enzyme activity in the rat kidney. Materials and Methods
Received July 14, 1975. 1 These studies were supported in part by the following grants-in-aid from The National Institutes of Health, United States Public Health Service: 5-T01AM05092 and 5-R01-AM05318. 2 Canadian Medical Research Council Fellow at the time these studies were performed. 3 Present address: Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29401. 4 Investigator, Howard Hughes Medical Institute.
The ACTH preparation, generously provided by Armour Laboratories, was dissolved in 4% gelatin and administered subcutaneously. The other pituitary hormones were kindly provided by the Endocrinology Study Section of the National Institutes of Health: FSH (NIH-FSHS9), LH (NIH-LH-S18), TSH (NIH-TSH-S7), prolactin (NIH-P-B4) and GH (NIH-GH-S10) were dissolved in 0.154M NaCl (pH adjusted to 9.0) and administered intraperitoneally. Cortisol hemisuccinate (SolucortefR) was dissolved in
123
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124
NaCl and administered intraperitoneally. Ovine pituitary tissue which had been lyophilized, pulverized and stored at - 2 0 C was kindly provided by Dr. Robert W. Bates, Hormone Distribution Officer, National Institute of Arthritis, Metabolism, and Digestive Diseases. The pituitary powder was extracted with 0. 1M (NH4)HCO3, 10 ml per g of powder, in a Waring blender during 2 successive 30-second homogenizations at 4 C. Followingcentrifugationat50,000 x gfor20min, the precipitate was again extracted. The two supernatants were pooled and stored at —56 C. Male Sprague-Dawley rats weighing 130-180 g were used for the study. Animals used for investigations of diurnal rhythm were kept in a quiet room at 22 C and adapted to light/dark cycles of 14 and 10 hours, respectively (06002000 light). Food and water were provided ad libitum. In the experiments requiring hypophysectomized animals, noncycled rats were hypophysectomized under ether anesthesia by the transaural approach. Adrenalectomy of noncycled rats was performed under sodium pentobarbital anesthesia. Some of these animals were then cycled as above, but were kept at 26 C and were given 0.077M NaCl as their drinking solution. The rats were sacrificed by decapitation, and, in some experiments, trunk blood was collected at this time for corticosterone determination as previously described (6). In all groups studied, alternate right and left kidneys were rapidly removed, the capsule was stripped, and the kidney was then bisected in a longitudinal plane. The pelvis and the major calyces were resected, and a 150-200 mg wedge of tissue with its base at the cortex was obtained. This tissue was placed
0.154M
Endo • 1976 Vol 98 • No 1
NICHOLSON, LEVINE AND ORTH
in 1 ml of chilled 0.067M sodium-potassium phosphate buffer (pH 7.2) containing5 mMEDTA and 1 mM dithiothreitol and homogenized for 10-15sat4 C withaPolytron P-10 tissue grinder. The homogenates were centrifuged at 20,000 x g for 20 min at 4 C and the supernates were stored at —56 C until assayed for ornithine decarboxylase activity as previously described (7). Samples could be kept frozen for as long as two weeks without detectable losses in enzyme activity. However, approximately 33% of the enzyme activity was lost each time the samples were thawed and refrozen.
Results Renal ornithine decarboxylase activity in the intact rat Two hormones, growth hormone and cortisol, have been shown to increase the levels of renal ornithine decarboxylase activity in the rat (1-3). While the plasma level of corticosterone, the physiological glucocorticoid in the rat, displays a circadian rhythm (8), the evidence for a single daily peak in growth hormone secretion in rodents remains equivocal (9-12). A diurnal rhythm in ACTH secretion was present in intact cycled rats as reflected by plasma corticosterone concentration, which reached a peak at the time of onset of darkness and then declined and remained low throughout the remainder of the day (Fig. 1). Renal ornithine decarboxylase activity also appeared rhythmic, with peak levels of enzyme activity reached dur-
FlG. 1. Renal ornithine decarboxylase activity and plasma corticosterone in the intact rat. Normal male rats were adapted to light/dark cycles for 7 days and PLASMA RENAL CORTICOODC then were sacrificed at the times STERONE ACTIVITY indicated. The 2000 and 2400 h nmoles l 4 yug/lOOml o -o levels of omithine decarboxylase activity, which are not different from each other, are significantly higher than all other levels (Duncan's multiple range 1200 1600 2000 2400 0400 0800 1200 test,P = .05). The 2000 h plasma corticosterone concentration is TIME significantly higher than all others, and the 1600 and 2400 h levels, which are not significantly different from each other, are greater than the rest (Duncan's multiple range test, P = .05). In this and subsequent figures, the brackets represent the standard errors of the mean responses of several animals. N = 6.
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RENAL ORNITHINE DECARBOXYLASE ACTIVITY
125
ing the hours when corticosterone and ACTH secretion were highest and low levels when corticosterone and ACTH were lowest (Fig. 1). Renal ornithine decarboxylase activity in the hypophysectomized rat The apparent influence of pituitary hormones on renal ornithine decarboxylase activity was examined further by studying hypophysectomized animals. There was an immediate fall in renal ornithine decarboxylase activity following hypophysectomy, with an estimated half-life of 1.9 h; there was no further fall from 8 to 24 h (Fig. 2). Furthermore, the level of renal ornithine decarboxylase activity observed in other animals that were hypophysectomized for 4 days was only slightly lower than the level found just 8 hours after hypophysectomy. In these chronically hypophysectomized animals, which were maintained on regular light/dark cycles, renal ornithine decarboxylase activity and plasma corticosterone remained low and failed to demonstrate significant diurnal rhythmicity. When temporary restoration of pituitary hormone levels was attempted by intraperi100
RENAL ODC ACTIVITY nmoles
l4
FiG. 3. Effect of pituitary extract on renal omithine decarboxylase activity in the hypophysectomized rat. Sixteen hours following hypophysectomy, the animals were given an intraperitoneal injection of 0.5 ml of crude pituitary extract. They were sacrificed at the times indicated for determination of renal omithine decarboxylase activity. N = 5.
toneal injection of pituitary extract 16 hours after hypophysectomy, renal ornithine decarboxylase activity increased within 2 hours of the injection, reached a peak at 8 hours, and fell nearly to preinjection levels by 12 hours (Fig. 3). Growth hormone, ACTH, FSH, LH, TSH, prolactin and cortisol were also injected into rats 16 hours after hypophysectomy. Only growth hormone, ACTH and cortisol stimulated increased renal ornithine decarboxylase activity (Fig. 4). The effect of growth hormone persisted for at least 8 hours, while that of ACTH and cortisol had disappeared by 8 hours. Growth hormone appeared to be the most effective stimulus for increasing renal ornithine decarboxylase activity (Table 1). Renal ornithine decarboxylase activity in the adrenalectomized rat
24
FiG. 2. Renal omithine decarboxylase activity in the acutely hypophysectomized rat. Noncycled rats were hypophysectomized by the transaural approach and were then sacrificed at the times indicated. A regression line was fitted to the data obtained during the first 8 h following hypophysectomy (T4 = 1.9 h). N = 5.
The role of adrenal hormones in the regulation of renal ornithine decarboxylase activity was examined in bilaterally adrenalectomized rats maintained on regular light/dark cycles. In these animals, plasma ACTH levels are elevated and rhythmical, with peak levels observed at about the middle of the light period (13). In contrast to the report that adrenalectomy nearly abolished the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 November 2015. at 12:08 For personal use only. No other uses without permission. . All rights reserved.
Endo i 1976 Vol 98 . No 1
NICHOLSON, LEVINE AND ORTH
126 90
TABLE 1. Effect of ACTH, cortisol and growth hormone on renal ODC activity in the hypophysectomized rat
80
Hormone 0
60
ACTH
50
Cortisol 40
30
Growth hormone
20
10
FlG. 4. Time course of pituitary and adrenal hormone action in the hypophysectomized rat. Sixteen hours following hypophysectomy, the animals received either 0.5 mg of growth hormone, LH, FSH, TSH, or prolactin, 50 IU of ACTH, or 5 mg of cortisol. They were sacrificed at the times indicated for determination of renal ornithine decarboxylase activity. (* significantly different from control. Student's t test, F < .05 for ACTH and cortisol at 4 h, P < .02 for growth hormone at 4 h and 8 h)
diurnal rhythm in hepatic ornithine decarboxylase activity (3), we observed that renal ornithine decarboxylase activity was markedly elevated after adrenalectomy, with most levels exceeding the highest level of activity observed in intact, cycled animals (Fig. 5). Renal ornithine decarboxylase activity in these adrenalectomized cycled rats also demonstrated a diurnal rhythm, but the peak levels of activity appeared to be delayed about 4 hours (Fig. 5). Renal ornithine decarboxylase activity in the adrenalectomized-hypophysectomized rat The parallel diurnal rhythms in renal ornithine decarboxylase activity and plasma
Dose
F*
—
3.2 ±
0.7
—
0.4 IU 2 10 50 0.04 mg 0.2 1 5 0.04 mg 0.2 1 5
18.8 ± 24.2 ± 45.3 ± 74.7 ± 5.4 ± 4.8 ± 8.3 ± 30.3 ±
5.6 9.1 19.1 10.4 1.0 0.4 2.6 7.0
A A A A ©bob o to to to
70
Renal ODC Activity nmoles u C(Vg (mean ± SE)
7.3 ± 2.0 56.5 ± 18.6 212.4 ± 54.1 346.7 ± 35.0
NS
