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Biochemical evidence for peripheral neural regulation of adrenocortical regeneration in response to bilateral adrenal enucleation
Life Sciences, Vol. 53, pp. 275-282 Printed in the USA
Pergamon Press
BIOCHEMICAL EVIDENCE FOR PERIPHERAL NEURAL REGULATION OF ADRENOCORTICAL REGENERATION IN RESPONSE TO BILATERAL ADRENAL ENUCLEATION Richard D. Gragg and Karam F.A. Soliman * College of Pharmacy and Pharmaceutical Sciences Florida A&M University, Tailahassee, Florida 32307 (Received in final form May 4, 1993) Summary. Male Sprague-Dawley rats (130-150 g) with bilateral adrenal enucleation were used in this study. Animals were sacrificed at 2, 7, and 11 days post enucleation and plasma corticosterone and adrenal gland acetylcholin-esterase (ACHE), choline acetyltransferase (CHAT), corticosterone, epinephrine (EP) and norepinephrine (NE) were assayed. The results show a progressive and gradual increase in plasma and adrenal corticosterone levels, ACHE, CHAT, EP, and NE levels in the regenerating adrenal cortex from day 2 to day l 1 post bilateral adrenal enucleation. At day 11 post surgery, the activities of ChAT and AChE were 60% and 25%, respectively when compared to sham operated control. The EP and NE levels returned to normal levels after 11 days post surgery in the regenerating gland when compared to the sham control. The presence of both cholinergic enzymes and the availability of high levels of catecholamine strongly suggest a progressive development of sympathetic innervation in the regenerating adrenal cortex.
Evidence suggests that hypothalamic nuclei of the autonomic nervous system may influence the adrenal cortex directly, via its autonomic innervation (1-4). Adrenocortical innervation may provide an extrapituitary mechanism regulating the adrenal cortical rhythmicity in rats, (5). The adrenal gland has been postulated to be innervated by three different systems. These included the afferent and the efferent postganglionic sympathetic neurons which have their cell bodies outside the adrenal cortex (6). These fibers run with arteries to terminate or originate in the adrenal capsule or the zona glomerulosa (6). The third neuronal system is intrinsic to the adrenal and it originates in the neuronal cell bodies of the adrenal medulla. This third system has neuronal fibers which connect with both medulla and cortex (6). Afferent and efferent pathways between the hypothalamus and the adrenal gland have been shown to mediate compensatory adrenocortical growth (1). Adrenocortical regeneration
*Corresponding and reprints request 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
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after bilateral enucleation occurs by different mechanisms and differ from that of the compensatory adrenocortical growth after unilateral adrenalectomy (7). In vitro addition of epinephrine, 11 days post enucleation to the regenerating adrenal gland resulted in a significant increase in corticosterone levels output (8). In addition epinephrine also restored the diurnal fluctuation of corticosterone output in the regenerating adrenal cortex in vitro (8). The involvement of peripheral cholinergic pathway in the regulation of adrenal cortex function has been reported (8). Neostigmine, the peripherally active cholinomimetic agent, caused a significant increase in plasma corticosterone in intact or hypophysectomized animals (9). However, the centrally and peripherally active cholinomimetic agent physostigmine, caused a significant increase in the level of plasma corticosterone in intact, hypophysectomized or bilaterally enucleated rats (9). The process of adrenocortical regeneration is initiated by removal of the medulla and most of the cortex (enucleation) of the adrenal gland in-situ, (10). Immediately after enucleation, the empty capsule fills with blood which is gradually resolved (7). Regeneration of the three cortical zones occurs from the mitotic activity and proliferation of the remaining cells in the subcapsular region of the enucleated gland. Due to the nature of the operation procedure, the vascular and neural connections remain attached to the capsule in the enucleated gland (7). Between days 1015 post enucleation, ACTH level is twice that of the sham operated control with no significant difference in corticosteroid levels (7,10). Several humoral factors have been implicated in the process of adrenal regeneration. For instance the N-terminal of pro-opiomelanocortin was found to be involved in the rat adrenal regeneration (11) as well as the thyroid gland (12). The present study was designed to provide biochemical evidence for the neural development of the regenerating adrenal cortex by measuring the adrenal cholinergic enzymes activity, adrenal catecholamines and steroid hormone levels. Methods
Male Sprague-Dawley rats obtained from Southern Animal Farms (Prattville, AL) were maintained in a controlled environmental chamber with 12:12 light/dark cycle (lights on at 08:00 h), and a temperature of 21+1°C, for a period of three weeks. Feed and water were available ad libitum. Bilateral enucleation was performed by the dorsal approach. The capsule of the adrenal gland was slit and the gland was gently squeezed with a pair of forceps, removing the medulla and most of the cortex except for the capsule and cells of subcapsular region. Sham-operated controls underwent the same surgical procedure except that the adrenal glands were touched only. Animals were returned to the environmental chamber after the surgery and saline solution (0.9%) was used for drinking in the enucleated animals. At days 2,7, and 11 post surgery, 8 animals were sacrificed by decapitation at 16:00 h. Trunk blood was collected in heparinized tubes and the adrenal glands were removed, cleaned and weighed. Plasma and adrenal corticosterone levels were determined using the method of Vernikos-DaneUis et al (13). For acetylcholinesterase (ACHE) activity, adrenal glands were homogenized (1% w/v) in 0.1 M ice-cold phosphate buffer (pH 7.4) containing 0.1% Triton X 100 (Sigma Chemical Co., St. Louis, MO). AChE activity in the homogenate was determined spectrophotometrically and expressed as micromole of substrate (acetylcholine iodide; Sigma)
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hydrolyzed per minute per gram of tissue (14). For choline acetyltransferase (CHAT) activity, adrenal glands were homogenized (1% w/v) in ice-cold 0.5 M phosphate buffer (pH 7.0). ChAT activity in the homogenate was determined spectrophotometrically and expressed as micromoles of coenzyme-A sulr'hydryl (CoASH) formed per minute per milligram of tissue (15). The detection of catecholamines were carried out by high pressure liquid chromatography (HPLC) procedure (16) using the BAS electrochemical detector (Bioanalytical System Inc., West Lafayette, IN) mounted with a glassy carbon working electrode set at an oxidation potential of 0.55 V. For the separation of catecholamines, each pair of adrenal glands were homogenized in 2 ml of mobile phase: 0.1 M citric acid monohydrate, sodium phosphate dibasic buffer containing 50 mg EDTA 162.4 mM sodium octyl sulfate, and 6.5% (v/v) methanol. The homogenate was centrifuged for 15 minutes and the supernatant was filtered for injection onto a p.-bondapack C-I 8 reverse phase column at flow rate of 1 ml/min.The AgCI reference electrode was set at sensitivity rate of 5 nA/V. Standards for EP, NE, and the internal standard dihydroxybenzylamine (DHBA) were obtained from Sigma. Peaks were analyzed using a Perkin Elmer Sigma 1-B data console. Data were statistically analyzed using one-way analysis of variance with significance level set at p<0.05. The separation of means was determined using the LSD-Test (17).
Results
The results of this experiment show a gradual increase in plasma (Fig. 1) and adrenal (Fig. 2) corticosterone levels over the period of days 2, 7, and 11 post enucleation in the regenerating adrenal cortex. There was no significant difference between sham- operated rats and enucleated animals at day 11 post surgery in plasma or adrenal corticosterone levels. Over the period of 11 days post surgery there was a gradual increase in AChE and ChAT activity in the regenerating adrenal cortex. At days 11 post enucleation, AChE activity (Fig. 3) was about 25% while ChAT activity (Fig. 4) was about 60% when compared to the intact sham operated animals. There was a significant (p < 0.01) increase in NE (Fig. 5) and EP (Fig. 6) levels in the regenerating adrenal gland at days 11 post enucleation. No significant differences in NE or EP were detected between the intact sham-operated gland and enucleated groups at 11 days post surgery.
Discussion
The present work examined biochemical parameters in plasma and adrenal glands as indicative of functional activity of the adrenal cortex (corticosterone), adrenal medulla (EP) and neural innervation (ACHE, ChAT and NE) at various time intervals following bilateral adrenal enucleation. These biochemical measurements indirectly provide evidence for the contribution of sympathetic innervation to the regenerating adrenal cortex following bilateral removal of the medulla and most of the cortex. The vascular and neural connection to the capsule remain intact in the enucleated gland (7). The gradual increase in corticosterone levels equivalent to that of
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sham operated controls over the 11 days post enucleation period is in accord with the established recovery period of 10-15 days for the regenerating adrenal cortex (7,18).
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Fig. 1. Plasma corticosterone levels at 2, 7 and 11 days post bilateral adrenal enucleation. Each bar represents the average + SEM of 8 animals. *p<0.05, ** p<0.01: Significant difference from sham-operated group.
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Fig. 2. Adrenal corticosterone levels at 2, 7 and 11 days post bilateral adrenal enucleation. Each bar represents the average + SEM of 8 animals. *p<0.05, ** p<0.01: Significant difference from sham-operated group.
Vol. 53, No. 3, 1993
Regulation of Adrenal Regeneration
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Fig. 3. Adrenal acetylcholinesterase activity, at 2, 7 and 11 days, post bilateral adrenal enucleation. Each bar represents the average + SEM of 8 animals. *p<0.05, ** p<0.01' Significant difference from sham-operated group.
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Fig. 4. Adrenal choline acetyltransferase activity, at 2, 7 and 11 days, post bilateral adrenal enucleation. Each bar represents the average + SEM of 8 animals. *p<0.05, **p<0.01: Significant difference from sham-operated group.
279
280
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Fig. 5. Adrenal norepinephrine levels at, 2, 7 and 11 days post bilateral adrenal enucleation. Each bar represents the average + SEM of 8 animals. *p<0.05, **p <0.01: Significant difference from sham-operated group
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Fig. 6. Adrenal epinephrine levels at 2, 7 and 11 days post bilateral adrenal enucleation. Each bar represents the average + SEM of 8 animals. *p<0.05, ** p<0.01: Significant difference from sham-operated group.
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The autonomic nervous system has been reported to participate in the direct modulation or regulation of cell proliferation (19). In the adrenal gland, evidence was provided for neural afferent and efferent pathways in CNS and spinal cord which mediate compensatory adrenal growth of the remaining adrenal gland in response to unilateral adrenalectomy (1). Using histofluorescence techniques and electron microscopy, catecholamine nerve fibers were found to be present in the capsule and zona glomerulosa (20). The proximity of these fibers to the glomerulosa cells provide evidence for morphological substrate responsible for direct neural interaction (6). Furthermore, it was found that the catecholamine content of these neurons appears to be unaffected by splanchnic nerve input to the adrenal medulla, but disappears following chemical sympathectomy and is reduced when the animals are stressed (6). In the present study, it can be assumed that the enucleated adrenal gland is devoid of the majority of the intrinsic neuronal system. The established increase in NE contents of the regenerating gland as reported in the present study indicate that neuronal fibers innervating the regenerating gland might be sympathetic fibers. It has been shown that hypothalamic hemi-island lesions did not prevent adrenal regeneration (18). However, such lesions prevented normal compensatory growth response to unilateral adrenalectomy provided that they were placed on the same side of the remaining adrenal glands (1). It is of interest to note that animals with regenerating glands showed compensatory growth after unilateral adrenalectomy, 6 weeks post enucleation. This might indicate the need of intrinsic neural supply to reestablish (1) and before adrenal compensatory growth, which is neurally mediated, can take place. The results of these studies also show a progressive increase in the adrenal content of CHAT, EP, NE and with the catecholamines retuming to sham levels by day 11 post surgery. The data clearly show that AChE activity does not increase progressively to sham levels by 11 day but only reaches 25% of the sham-operated levels. Since this enzyme is typically associated with target sites for cholinergic nerves, it appears that these cells have not regenerated as readily as those cells synthesizing catecholamines. The incomplete development of the cholinergic system might be related to the inability of the young regenerating gland (0-6 weeks) to show compensatory growth after unilateral adrenalectomy enucleation. It can also be assumed that the presence of a fully functioning cholinergic system is required for adrenal compensatory growtJa. The gradual increase in the cholinergic enzyme activity during the 11 day period supports evidence for the role of the peripheral cholinergic fibers in adrenocortical function in intact, hypophysectomized or enucleated rats (9). Recent evidence indicated the participation of the cholinergic receptors in the modulation of the cortical function (21). Thus the gradual increase in catecholamine levels over the same period further supports evidence for the proposed role of epinephrine in the regulation of adrenal cortical synthesis, release, and rhythmicity in the regenerating adrenal cortex (18). Diurnal variation in plasma and adrenocortical corticosterone within the first 21 days after bilateral adrenal transplantation or enucleation (19) support the possibility of neural regulation. Based on evidence for adrenocortical rhythmicity in hypophysectomized rats and the data which indicate that disruption of adrenal innervation suppresses adrenocortical activity, such innervation may be an extrapituitary mechanism involved in the regulation of adrenocortical rhythms (4). The presence of significant levels of cholinergic enzymes and catecholamine 11 days post enucleation provide biochemical evidence for the reinnervation of the regenerating adrenal cortex. This is supported by other results showing adrenocortical rhythms and compensatory adrenocortical growth, post bilateral enucleation (7,22).
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Acknowledgements This work was supported by grants from the National Aeronautics and Space Administration (NSG 2183 and NAG 2-411), from the National Institutes of Health (NIH Grant RR0811), and from the Division of Research Resources, National Institutes of Health (NIH Grant RR 03020).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
M.A HOLZWARTH and M.F. DALLMAN, Brain Res. 162 33-43 (1979) N. MIGALLY, Anat. Rec. 194 105-112 (1979). K. UNSICKER, ZELFORSCH 146 403-416 (1973). J.E. OTTENWELLER and A.H. MEIER, Endocrinology 111 1334-1338 (1982). H.L. FEHM, E. KLEIN, R. HOLL and K.H. VOLIGHT, Endocrin. Metab. 58 410-414 (1984). M.A. HOLZWARTH, L.A. CUNNINGHAM and N. KEITMAN, Ann. NY, Acad. Sci 51_22449-464 (1985). M.A. HOLZWARTH, J. SHINSAKO, and M.F. DALLMAN, Neuroendocrinology 31, 168-178 (1980). K.F.A. SOLIMAN, and M.G. KOLTA, Res. Comm. Chem. Pathol. 32 373-376 (1981). M.G. KOLTA, and K.F.A. SOLIMAN, Endocrin. Res. Comm. 8 239-246 (1981). T.M. Taki, and P.A. Nickerson, Lab Invest. 53 & 91-100 (1985) F.E. ESTIVARIZ, M.I. MORANO, M. CARINO, S. JACKSON, and P.J. LOWRY, J. Endocrinol. 116 207-216 (1988). R.M. CONRAN, P.A. NICKERSON, Am. J. Pathol. 100 411-426 (1980). J. VERNIKOS-DANELLIS, E. ANDERSON, and TRIGG, Endocrinolgy 7_29624-630 (1966). G.L. ELLMAN, K.K. COURTNEY, V. ANDRES, JR., and R.M. FEATHERSTONE, Biochem. Pharmacol. 7 88-95 (1961). L.P. CHAO and F. WOLFGRAM, Anal. Biochem. 46 114-118 (1972). I.M.J. MEFFORD, Neuroscience Meth. 13 207-224 (1981). R.G.D. STEEL, and J.H. TORR1E, Principles and Procedures of Statistics, ~ Hill, New York (1960). M.A. HOLZWARTH, C.A. WILKINSON, and M.F. DALLMAN, Neuroendocrinology 31 34-38 (1980). J.F. WHITEFIELD, In Handbook Exp. Pharmacol. Szerkeres Ed. Berlin 54 267-317 (1980). N. KEITMAN, AND M.A. HOLZWARTH, Cell Tissue Res. 241 139-149 (1985). J.R. TOBIN, M.J. BRESLOW, and R.J. TRAYSTMAN, Am. J. Physiol. 263 H1208H1212 (1992). C.W. WILKINSON, J. SHINSAKO and M.F. DALLMAN, Endocrinology 109 162-169 (1981).
Pergamon Press
BIOCHEMICAL EVIDENCE FOR PERIPHERAL NEURAL REGULATION OF ADRENOCORTICAL REGENERATION IN RESPONSE TO BILATERAL ADRENAL ENUCLEATION Richard D. Gragg and Karam F.A. Soliman * College of Pharmacy and Pharmaceutical Sciences Florida A&M University, Tailahassee, Florida 32307 (Received in final form May 4, 1993) Summary. Male Sprague-Dawley rats (130-150 g) with bilateral adrenal enucleation were used in this study. Animals were sacrificed at 2, 7, and 11 days post enucleation and plasma corticosterone and adrenal gland acetylcholin-esterase (ACHE), choline acetyltransferase (CHAT), corticosterone, epinephrine (EP) and norepinephrine (NE) were assayed. The results show a progressive and gradual increase in plasma and adrenal corticosterone levels, ACHE, CHAT, EP, and NE levels in the regenerating adrenal cortex from day 2 to day l 1 post bilateral adrenal enucleation. At day 11 post surgery, the activities of ChAT and AChE were 60% and 25%, respectively when compared to sham operated control. The EP and NE levels returned to normal levels after 11 days post surgery in the regenerating gland when compared to the sham control. The presence of both cholinergic enzymes and the availability of high levels of catecholamine strongly suggest a progressive development of sympathetic innervation in the regenerating adrenal cortex.
Evidence suggests that hypothalamic nuclei of the autonomic nervous system may influence the adrenal cortex directly, via its autonomic innervation (1-4). Adrenocortical innervation may provide an extrapituitary mechanism regulating the adrenal cortical rhythmicity in rats, (5). The adrenal gland has been postulated to be innervated by three different systems. These included the afferent and the efferent postganglionic sympathetic neurons which have their cell bodies outside the adrenal cortex (6). These fibers run with arteries to terminate or originate in the adrenal capsule or the zona glomerulosa (6). The third neuronal system is intrinsic to the adrenal and it originates in the neuronal cell bodies of the adrenal medulla. This third system has neuronal fibers which connect with both medulla and cortex (6). Afferent and efferent pathways between the hypothalamus and the adrenal gland have been shown to mediate compensatory adrenocortical growth (1). Adrenocortical regeneration
*Corresponding and reprints request 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
276
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after bilateral enucleation occurs by different mechanisms and differ from that of the compensatory adrenocortical growth after unilateral adrenalectomy (7). In vitro addition of epinephrine, 11 days post enucleation to the regenerating adrenal gland resulted in a significant increase in corticosterone levels output (8). In addition epinephrine also restored the diurnal fluctuation of corticosterone output in the regenerating adrenal cortex in vitro (8). The involvement of peripheral cholinergic pathway in the regulation of adrenal cortex function has been reported (8). Neostigmine, the peripherally active cholinomimetic agent, caused a significant increase in plasma corticosterone in intact or hypophysectomized animals (9). However, the centrally and peripherally active cholinomimetic agent physostigmine, caused a significant increase in the level of plasma corticosterone in intact, hypophysectomized or bilaterally enucleated rats (9). The process of adrenocortical regeneration is initiated by removal of the medulla and most of the cortex (enucleation) of the adrenal gland in-situ, (10). Immediately after enucleation, the empty capsule fills with blood which is gradually resolved (7). Regeneration of the three cortical zones occurs from the mitotic activity and proliferation of the remaining cells in the subcapsular region of the enucleated gland. Due to the nature of the operation procedure, the vascular and neural connections remain attached to the capsule in the enucleated gland (7). Between days 1015 post enucleation, ACTH level is twice that of the sham operated control with no significant difference in corticosteroid levels (7,10). Several humoral factors have been implicated in the process of adrenal regeneration. For instance the N-terminal of pro-opiomelanocortin was found to be involved in the rat adrenal regeneration (11) as well as the thyroid gland (12). The present study was designed to provide biochemical evidence for the neural development of the regenerating adrenal cortex by measuring the adrenal cholinergic enzymes activity, adrenal catecholamines and steroid hormone levels. Methods
Male Sprague-Dawley rats obtained from Southern Animal Farms (Prattville, AL) were maintained in a controlled environmental chamber with 12:12 light/dark cycle (lights on at 08:00 h), and a temperature of 21+1°C, for a period of three weeks. Feed and water were available ad libitum. Bilateral enucleation was performed by the dorsal approach. The capsule of the adrenal gland was slit and the gland was gently squeezed with a pair of forceps, removing the medulla and most of the cortex except for the capsule and cells of subcapsular region. Sham-operated controls underwent the same surgical procedure except that the adrenal glands were touched only. Animals were returned to the environmental chamber after the surgery and saline solution (0.9%) was used for drinking in the enucleated animals. At days 2,7, and 11 post surgery, 8 animals were sacrificed by decapitation at 16:00 h. Trunk blood was collected in heparinized tubes and the adrenal glands were removed, cleaned and weighed. Plasma and adrenal corticosterone levels were determined using the method of Vernikos-DaneUis et al (13). For acetylcholinesterase (ACHE) activity, adrenal glands were homogenized (1% w/v) in 0.1 M ice-cold phosphate buffer (pH 7.4) containing 0.1% Triton X 100 (Sigma Chemical Co., St. Louis, MO). AChE activity in the homogenate was determined spectrophotometrically and expressed as micromole of substrate (acetylcholine iodide; Sigma)
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hydrolyzed per minute per gram of tissue (14). For choline acetyltransferase (CHAT) activity, adrenal glands were homogenized (1% w/v) in ice-cold 0.5 M phosphate buffer (pH 7.0). ChAT activity in the homogenate was determined spectrophotometrically and expressed as micromoles of coenzyme-A sulr'hydryl (CoASH) formed per minute per milligram of tissue (15). The detection of catecholamines were carried out by high pressure liquid chromatography (HPLC) procedure (16) using the BAS electrochemical detector (Bioanalytical System Inc., West Lafayette, IN) mounted with a glassy carbon working electrode set at an oxidation potential of 0.55 V. For the separation of catecholamines, each pair of adrenal glands were homogenized in 2 ml of mobile phase: 0.1 M citric acid monohydrate, sodium phosphate dibasic buffer containing 50 mg EDTA 162.4 mM sodium octyl sulfate, and 6.5% (v/v) methanol. The homogenate was centrifuged for 15 minutes and the supernatant was filtered for injection onto a p.-bondapack C-I 8 reverse phase column at flow rate of 1 ml/min.The AgCI reference electrode was set at sensitivity rate of 5 nA/V. Standards for EP, NE, and the internal standard dihydroxybenzylamine (DHBA) were obtained from Sigma. Peaks were analyzed using a Perkin Elmer Sigma 1-B data console. Data were statistically analyzed using one-way analysis of variance with significance level set at p<0.05. The separation of means was determined using the LSD-Test (17).
Results
The results of this experiment show a gradual increase in plasma (Fig. 1) and adrenal (Fig. 2) corticosterone levels over the period of days 2, 7, and 11 post enucleation in the regenerating adrenal cortex. There was no significant difference between sham- operated rats and enucleated animals at day 11 post surgery in plasma or adrenal corticosterone levels. Over the period of 11 days post surgery there was a gradual increase in AChE and ChAT activity in the regenerating adrenal cortex. At days 11 post enucleation, AChE activity (Fig. 3) was about 25% while ChAT activity (Fig. 4) was about 60% when compared to the intact sham operated animals. There was a significant (p < 0.01) increase in NE (Fig. 5) and EP (Fig. 6) levels in the regenerating adrenal gland at days 11 post enucleation. No significant differences in NE or EP were detected between the intact sham-operated gland and enucleated groups at 11 days post surgery.
Discussion
The present work examined biochemical parameters in plasma and adrenal glands as indicative of functional activity of the adrenal cortex (corticosterone), adrenal medulla (EP) and neural innervation (ACHE, ChAT and NE) at various time intervals following bilateral adrenal enucleation. These biochemical measurements indirectly provide evidence for the contribution of sympathetic innervation to the regenerating adrenal cortex following bilateral removal of the medulla and most of the cortex. The vascular and neural connection to the capsule remain intact in the enucleated gland (7). The gradual increase in corticosterone levels equivalent to that of
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sham operated controls over the 11 days post enucleation period is in accord with the established recovery period of 10-15 days for the regenerating adrenal cortex (7,18).
E
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40
T
IX
E
30
0 0
.~
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ID
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Fig. 1. Plasma corticosterone levels at 2, 7 and 11 days post bilateral adrenal enucleation. Each bar represents the average + SEM of 8 animals. *p<0.05, ** p<0.01: Significant difference from sham-operated group.
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Fig. 2. Adrenal corticosterone levels at 2, 7 and 11 days post bilateral adrenal enucleation. Each bar represents the average + SEM of 8 animals. *p<0.05, ** p<0.01: Significant difference from sham-operated group.
Vol. 53, No. 3, 1993
Regulation of Adrenal Regeneration
"[:
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Fig. 3. Adrenal acetylcholinesterase activity, at 2, 7 and 11 days, post bilateral adrenal enucleation. Each bar represents the average + SEM of 8 animals. *p<0.05, ** p<0.01' Significant difference from sham-operated group.
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Fig. 4. Adrenal choline acetyltransferase activity, at 2, 7 and 11 days, post bilateral adrenal enucleation. Each bar represents the average + SEM of 8 animals. *p<0.05, **p<0.01: Significant difference from sham-operated group.
279
280
Regulation of Adrenal Regeneration
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Vol. 53, No. 3, 1993
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Fig. 5. Adrenal norepinephrine levels at, 2, 7 and 11 days post bilateral adrenal enucleation. Each bar represents the average + SEM of 8 animals. *p<0.05, **p <0.01: Significant difference from sham-operated group
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Fig. 6. Adrenal epinephrine levels at 2, 7 and 11 days post bilateral adrenal enucleation. Each bar represents the average + SEM of 8 animals. *p<0.05, ** p<0.01: Significant difference from sham-operated group.
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The autonomic nervous system has been reported to participate in the direct modulation or regulation of cell proliferation (19). In the adrenal gland, evidence was provided for neural afferent and efferent pathways in CNS and spinal cord which mediate compensatory adrenal growth of the remaining adrenal gland in response to unilateral adrenalectomy (1). Using histofluorescence techniques and electron microscopy, catecholamine nerve fibers were found to be present in the capsule and zona glomerulosa (20). The proximity of these fibers to the glomerulosa cells provide evidence for morphological substrate responsible for direct neural interaction (6). Furthermore, it was found that the catecholamine content of these neurons appears to be unaffected by splanchnic nerve input to the adrenal medulla, but disappears following chemical sympathectomy and is reduced when the animals are stressed (6). In the present study, it can be assumed that the enucleated adrenal gland is devoid of the majority of the intrinsic neuronal system. The established increase in NE contents of the regenerating gland as reported in the present study indicate that neuronal fibers innervating the regenerating gland might be sympathetic fibers. It has been shown that hypothalamic hemi-island lesions did not prevent adrenal regeneration (18). However, such lesions prevented normal compensatory growth response to unilateral adrenalectomy provided that they were placed on the same side of the remaining adrenal glands (1). It is of interest to note that animals with regenerating glands showed compensatory growth after unilateral adrenalectomy, 6 weeks post enucleation. This might indicate the need of intrinsic neural supply to reestablish (1) and before adrenal compensatory growth, which is neurally mediated, can take place. The results of these studies also show a progressive increase in the adrenal content of CHAT, EP, NE and with the catecholamines retuming to sham levels by day 11 post surgery. The data clearly show that AChE activity does not increase progressively to sham levels by 11 day but only reaches 25% of the sham-operated levels. Since this enzyme is typically associated with target sites for cholinergic nerves, it appears that these cells have not regenerated as readily as those cells synthesizing catecholamines. The incomplete development of the cholinergic system might be related to the inability of the young regenerating gland (0-6 weeks) to show compensatory growth after unilateral adrenalectomy enucleation. It can also be assumed that the presence of a fully functioning cholinergic system is required for adrenal compensatory growtJa. The gradual increase in the cholinergic enzyme activity during the 11 day period supports evidence for the role of the peripheral cholinergic fibers in adrenocortical function in intact, hypophysectomized or enucleated rats (9). Recent evidence indicated the participation of the cholinergic receptors in the modulation of the cortical function (21). Thus the gradual increase in catecholamine levels over the same period further supports evidence for the proposed role of epinephrine in the regulation of adrenal cortical synthesis, release, and rhythmicity in the regenerating adrenal cortex (18). Diurnal variation in plasma and adrenocortical corticosterone within the first 21 days after bilateral adrenal transplantation or enucleation (19) support the possibility of neural regulation. Based on evidence for adrenocortical rhythmicity in hypophysectomized rats and the data which indicate that disruption of adrenal innervation suppresses adrenocortical activity, such innervation may be an extrapituitary mechanism involved in the regulation of adrenocortical rhythms (4). The presence of significant levels of cholinergic enzymes and catecholamine 11 days post enucleation provide biochemical evidence for the reinnervation of the regenerating adrenal cortex. This is supported by other results showing adrenocortical rhythms and compensatory adrenocortical growth, post bilateral enucleation (7,22).
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Acknowledgements This work was supported by grants from the National Aeronautics and Space Administration (NSG 2183 and NAG 2-411), from the National Institutes of Health (NIH Grant RR0811), and from the Division of Research Resources, National Institutes of Health (NIH Grant RR 03020).
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