Neuropeptides (1992) 21, 157-161 0 Longman Group UK Ltd 1992
Calcitonin Gene-Related Peptide Depresses the Growth and Secretory Activity of Rat Adrenal Zona Glomerulosa G. MAZZOCCHI,
L. K. MALENDOWICZ,
V. MENEGHELLI
AND G. G. NUSSDORFER
Department of Anatomy, University of Padua, Via Gabelli 65, l-35 12 1 Padua, Italy (Correspondence and reprint requests to GGN)
Abstract-The bolus ip. injection of rat calcitonin gene-related peptide (CGRP) (5 pm. kg-‘) significantly lowered plasma aldosterone concentration (PAC) in rats, despite a mild rise in plasma renin activity. Natremia, kalaemia and the blood levels of ACTH or corticosterone were not affected. Similar results were obtained after prolonged (5 days) SC. infusion of rats with CGRP (1 pm. kg-l. h-9. Moreover, CGRP infusion caused a notable atrophy of the zona glomerulosa (ZG) and its parenchymal cells, as well as a clearcut reduction in the surge of PAC evoked by a bolus injection of a high dose of angiotensin-II (100 pg. kg-‘). From these results it is suggested that CGRP exerts an inhibitory effect on the growth and secretory activity of ZG in rats.
Introduction Calcitonin gene-related peptide (CGRP), mainly produced in the nervous system by alternative processing of the calcitonin-gene transcription (l), is a potent vasodilator somehow involved in cardiovascular regulation (2-4). CGRP was also reported to exert a strong inhibitory effect on the pancreatic insulin release (5) and gastric secretion (6-8). Recently, the possible effect of CGRP on adrenal steroidogenesis has been investigated. Bloom et al (9) reported that CGRP enhances cortisol secretion by calf adrenals in vivo, independent of any vasodilation-induced increase in the rate of ACTH pre-
Date received Date accepted
I7 September 199 28 October 199 I
1
sentation. Murakami and co-workers (10) showed that CGRP inhibits, both in vivo and in vitro, aldosterone secretion by dog zona glomerulosa (ZG) cells, especially when stimulated by angiotensin-II (ANG-II). It, therefore, seemed of interest to investigate the short- and long-term effects of CGRP on the function and morphology of rat adrenal ZG and zona fasciculata (ZF). Methods Animal
treatment
Adult male Wistar rats (300 + 30 g body weight) were employed. A group of animals (n = 16) was divided into two equal subgroups, one of which was given an ip. injection of 5 pM. kg ’ CGRP (rat; Sigma, 157
158 St. Louis, MO), dissolved in 0.2 ml 0.9% NaCl solution, 30 min before the sacrifice. The other subgroup was injected with the saline vehicle. The dose of CGRP was chosen according to Murakami et al. ( 10). Another group of rats (n = 16) was divided into two equal subgroups. One subgroup was SC.infused for 5 days (Alzet osmotic pumps; Alza, Palo Alto, CA) with CGRP (1pM. kg’. h-l), and the second one was infused with the saline vehicle. Other rats (n = 32) were again divided into two subgroups, which were SC.infused as the previous ones. Half of the rats in each subgroup received 30 min before the sacrifice an ip. injection of 100 pg. kg’ ANG-II (Sigma). The other half of animals was given an ip. injection of 0.2 ml of the saline vehicle. The rats were decapitated between 10.00 and 11 .OO h, and their trunk blood was collected and frozen. Biochemical assays Serum Na’ and K’ concentrations were measured with a flame photometer (LKB, Stockholm, Sweden), Plasma renin activity (PRA) was assayed by RIA of angiotensin-I generated after incubation of plasma (ANG-I RIA kit; Peninsula, Merseyside, UK). ACTH was extracted from plasma (1 l), and its concentration was determined by RIA (ACTHRIA kit; IRE-Sorin, Vercelli, Italy). Aldosterone and corticosterone were extracted and purified (12), and their concentrations were measured by RIA (Aldo CTK2; IRE-Sorin. Ctrx-RIA kit; Eurogenetics, Milan, Italy). Intra-assay and inter-assay variations were: angiotensin-I, 6.8% and 8.7%; ACTH, 5.5% and 7.7%; aldosterone, 4.6% and 6.3%; corticosterone, 7.1% and 9.2%.
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The volume of ZG and ZF, and the number and volume of their parenchymal cells were determined on light micrographs ofthe paraffin and 0.5~pm thick sections, using conventional morphometric methods ( 13), as described in an earlier paper (14). On electron micrographs of thin sections, the volume of nuclei and mitochondrial and lipid-droplet compartments, as well as the surface area of mitochondrial cristae and smooth endoplasmic reticulum (SER), were evaluated by the stereological techniques described by Weibel(l3), as detailed previously (14). Statistics The data obtained were averaged per experimental group and the SD of the mean was calculated. The statistical comparison of the data was done by ANOVA followed by the Multiple Range Test of Duncan. Results Neither acute nor chronic CGRP administrations evoked significant changes in the plasma levels of ACTH and corticosterone, natremia and kalaemia (Table 1). Conversely, both treatments caused a significant reduction in PAC (- 24% and -42%, respectively), and a moderate rise in PRA (about 22%) (Fig. 1).
Morphology The adrenal glands of CGRP-infused and control rats were promptly removed, freed of adherent fat, and weighed. The left adrenals were fixed in Bouin’s solution, embedded in paraffin and serially cut at 67 pm. Sliced pieces of the right glands were fixed in 3% glutaraldehyde, post-fixed in 1% osmium tetroxide and embedded in epon. Thick (0.5 pm) and thin (60-70 nm) sections were cut with LKB III ultramicrotomes at the level of the ZG and ZF. Thin sections were counterstained with lead-hydroxide, and examined and photographed in a Hitachi H-300 electron microscope.
(B) of Fig. 1 Effects of acute (A) and chronic administrations CGRP on PRA (left) and PAC (right) in rats. SD are indicated (n = 8). ’ P < 0.05 and *P < 0.01 versus control rats.
159
EFFECT OF CGRF ON ADRENALS Table 1
Effects of acute and chronic administrations of CGRP on some biochemical parameters of rats. Data are means It SD (n = 8)
Acute treatment CGRP Controls
Plasma ACTH concentration (pg. ml ‘) Natremia (mEq. 1 ‘) Kalaemia (mEq. 1-I) Plasma corticosterone concentration (pg. dl ‘)
Chronic Controls
treotment CGRP
128.6zr31.3
135.1 _+25.8
95.1 _+18.2
107.3 + 29.7
131.4_+ 15.2 4.8 f 0.9 13.9k2.1
129.5 _+19.5 5.0 _+I .o 12.2 _+3.0
136.2 _+30. I 4.7 + 0.7 10.1 + 1.9
130.4+21.4 4.8+ 0.5 9.7 t 2.6
Prolonged CGRP infusion provoked a notable decrease in the volume of ZG (-37%) and ZG cells (-35%) and nuclei (-26%), without affecting the number of ZG cells (Table 2). Stereology showed that the CGRP-induced ZG-cell atrophy was associated with significant decreases in the volume of the mitochondrial compartment (-26%) and in the surface areas per cell of mitochondrial cristae (-27%) and SER tubules (-50%). The volume of the lipid-droplet compartment underwent a striking rise (60%) (Table 2). The morphometric parameters of ZF cells were not significantly changed (Table 2). CGRP infusion caused a conspicuous reduction (about 60%) in the rise of PAC elicited by a bolus injection of ANG-II (Fig. 2). Discussion Fig. 2
PAC response to a bolus ip. injection of ANG-II in A) saline-infused and B) CGRP-infused rats. SD are indicated (n = 8). The numbers in square brackets indicate the percent increase. aP < 0.01 versus saline-infused control rats; *P < 0.01 versus the respective control rats. Table 2
Our present findings indicate that in the rat, as in the dog ( lo), a bolus administration of CGRP depresses ZG aldosterone secretion, without affecting the production of corticosterone, the main glucocorticoid secreted by inner adrenocortical layers in rodents
Effect of chronic CGRP infusion on the morphometric parameters of the rat adrenal gland. Data are means f SD (n = 8)
Zona glomerulosa CGRP Controls
Volume of zona (mm’) Number of cells (x IO>) Volume of cells @mm’) Volume of nuclei o.&) Volume of mitochondrial compartment (pm?icell) Surface area of mitochondrial cristae (u&/cell) Surface area of SER (pm’/cell) Volume of lipid-droplet compartment (pnQ/cell) + P c 0.05 and * P < 0.01 versus control rats.
Zona,fasciculato Controls CGRP
14.972 7497.8 1750.5 165.3 557 5
t 4.008 f- 1392.1 f-41 1.3 t 32.4 r 201.3
2.518f0.591 2535.8_+611.3 785.4_+ 197.2 128.5+ 15.6 156.1 ~30.6
I .586 + 0.387* 248 1.7 + 709.4 509.2 _+154.7* 95.1 * 11.7* 115.3*24.1*
15.398 + 3.81 I 7660.2 i 1406.2 1809. I i 306.2 160.8 i21.4 608.2 +_185.4
2349.3 _+401.6
1706.4 + 309.1*
12346.5 +_3608.9
11807.5 t 3198.2
5473.2+ 1218.3 44.7 f 20.2
2724.8 k 8 I I .7* 71.3 f31.4’
11350.7 f4101.4 132.2 f 51.7
10907.5 t_3218.7 142.8 !- 54.1
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160 (for references, see 15). This last result is in contrast with that reported by Bloom et al (9) in calves; interspecific differences, as well as the fact that we employed doses of CGRP more than 3 order of magnitude less than those used by Bloom and associates (PM versus nM), may easily explain this discrepancy. The slight increase in PRA, already observed by Murakami et al (lo), may conceivably be interpreted as a negative feed-back response of the renin-angiotensin system to the lowered level of circulating aldosterone. Our data also show that the prolonged exposure to CGRP evokes a notable atrophy of rat ZG and its parenchymal cells, which is mainly due to the decrease in the volume of the mitochondrial compartment and SER. These morphologic data accord well with the CGRP-elicited depression in basal and maximally ANG-II-stimulated secretory activity of ZG cells, since the enzymes of aldosterone synthesis are located in both mitochondria and SER (for references, see 15, 16), and the changes in the surface area per cell of mitochondrial cristae and SER tubules are tightly coupled with corresponding changes in the activity per cell of some of these enzymes (17,18). The lowered utilization of cholesterol in aldosterone synthesis, coupled with a presumably normal (or slightly enhanced) uptake of cholesterol from serum lipoproteins, may well account for the striking increase in the volume of the lipid-droplet compartment. In fact, it is commonly agreed that cholesterol and cholesterol esters are stored in adrenocortical lipid droplets (15, 19), and that lipoprotein uptake by adrenocortical cells is a receptor-mediated process controlled not only by ACTH (20) but also by ANG-II (21) whose blood level, according to our PRA data, should be rather elevated in CGRP-treated rats. In conclusion, our findings suggest that CGRP may cause a notable depression of the growth and secretory activity of rat ZG. This effect of CGRP probably involves a direct action on ZG cells: i) adrenocortical cells possess specific binding sites for CGRP (22); and ii) acute or chronic CGRP administrations do not cause any apparent inhibition of the three main adrenoglomerulotropic factors, i.e. ANG-II, ACTH or K’ (for review, see 15). ANG-II production is conceivably raised (see above), and the plasma concentrations of ACTH and potassium are not significantly affected.
However, the possibility cannot be ruled out that CGRP may also positively interact with inhibitors of ZG function and growth, like somatostatin (2326) and ANF (27-30). In fact, CGRP stimulates somatostatin release, at least in the gastro-intestinal apparatus (3 l), and ANF secretion by isolated atria (32,33). Parenthetically, a slight rise in ANF plasma concentration occurs in CGRP-treated dogs (10). Be that as it may, it remains to be settled whether this antiadrenoglomerulotropic effect of CGRP is only a pharmacologic one or may play a role in the physiological regulation of the ZG. In this connection, we want to recall that the levels of circulating CGRP presumably attained in our experiments are very low: lO~‘*/lO-” M (by assuming an absorption rate of 80% and 10 ml of blood per rat). Evidence is available that CGRP-immunoreactivity is present in adrenal zona medullaris and that CGRP-containing fibers (of probable medullaris origin) can reach the cortex and especially ZG (34). A good deal of data suggests that adrenal zona medullaris may exert a paracrine control on the function of the zona corticalis (35). Thus, it does not seem unreasonable to conceive that locally produced CGRP may reach in the cortex a concentration sufficient to affect ZG function.
References I.
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Amara, S. G., Jonas, V., Rosenfeld, M. G., Ong, E. S. and Evans, R. M. (1982). Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 298: 240-244. Brain, S. D., Williams, T. J., Tippins, J. R., Morris, H. R. and MacIntyre, I. (1985). Calcitonin gene-related peptide is a potent vasodilator. Nature 3 13: 54-55. Asimakis, G. K., Di Pette, D. J., Conti, V. R., Holland, 0. B. and Zwischenberger. J. B. (1987). Hemodynamic action of calcitonin gene-related peptide in the isolated rat heart. Life Sci. 41: 597-604. Siren, A. L. and Feuerstein, G. ( 1988). Cardiovascular effects of rat calcitonin gene-related peptide in the conscious rat. J. Pharmacol. Exp. Ther. 247: 69-78. Petterson, M., Ahren, B., Bottcher, G. and Sundler, F. (I 986). Calcitonin gene-related peptide: occurrence in pancreatic islets in the mouse and the rat and inhibition of insulin secretion in the mouse. Endocrinology 119: 865-869. Kraenzlin, Y., Ch’ng, J. L. C., Mulderry, P. K., Ghatei, M. A. and Bloom, S. R. ( 1985). Infusion of a novel peptide, calcitonin gene-related peptide (CGRP) in man. Pharmacokinetics and effects on gastric acid secretion and on gastrointestinal hormones. Regul. Pept. IO: 189-197. Lenz. H. J.. Mortrud. M. T.. Rivier. J. H. and Brown. M. R. ( 1985). Calcitonin gene-related peptide inhibits basal, pentagastrin, histamine and bethanechol stimulated gastric acid secretion. Gut 26: 550-557.
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Pappas, T.. Debau, H. T.. Walsh, J. H., Rivier, J. H. and Tache. Y. ( 1986). Calcitonin gene-related peptide-induced selective inhibition of gastric acid secretion in dogs. Am. J. Physiol. 250: Gl27-Gl33. Bloom. S. R., Edwards. A. V. and Jones, C. T. ( 1989). Adrenal responses to calcitonin gene-related peptide in conscious hypophysectomized calves. J. Physiol. (London) 409: 29-41. Murakami. M.. Suzuki. H.. Nakajima, S., Nakamoto, H.. Kageyama. Y. and Sanua, T. ( 1989). Calcitonin gene-related peptide IS an inhibitor of aldosterone secretion. Endocrinology 125: 2227-2229. Rees, L. H..Cook, D. M., Kendal1.J. W..Allen, C. F., Kramer, R. M.. Ratcliffe J. G. and Knight, R. A. (1971). A radioimmunoassay for rat plasma ACTH. Endocrinology 89: 254261. Sippell, W. G.. Bidlingmaier. F.. Becker, H., Briinig. T.. Dot-r. M.. Hahn, H., Golder, W., Holmann, G. and Knorr, D. ( 1978). Simultaneous radioimmunoassay of plasma aldosterone. corticosterone. I I-deoxycorticosterone, progesterone, I7hydroxyprogesterone, I I -deoxycortisol, cortisol and cortisone. J. Steroid Biochem. 9: 63-74. Weibel. E. R. (1979). Stereological methods. I. Practical methods for biological morphometry. Academic Press. London. Rebuffat. P.. Kasprzak. A.. Andreis. P. G., Mazzocchi, G.. Gottardo, G., Coi, A. and Nussdorfer, G. G. ( 1989). Effects of prolonged cyclosporine-A treatment on the morphology and function of rat adrenal cortex. Endocrinology 125: l407- 14 13. Nussdorfer, G. G. (I 986). Cytophysiology ofthe adrenal cortex. Int. Rev. Cytol. 98: l-405. Miller. W. L. ( 1988). Molecular biology of steroid hormone synthesis. Endocr. Rev. 9: 295-3 18. Nussdorfer, G. G. and Mazzocchi, G. (1983). Long-term effects ofACTH on rat adrenocortical cells: a coupled stereological and enzymological study. J. Steroid Biochem. 19: 1753-1756. Mazzocchi. G.. Malendowicz. L. K., Rebuffat, P., Robba. C.. Gottardo. G. and Nussdorfer, G. G. (1986). Short- and longterm effects of ACTH on the adrenal zona glomerulosa of the rat: a coupled stereological andenzymological study. Cell Tissue Res. 243: 303-3 IO. Moses. H. L.. Davis, W. W., Rosenthal, A. S. and Garrett, L. D. ( 1969). Adrenal cholesterol: localization by electronmicroscope autoradiography. Science 163:1203-1205. Gwynne, T. and Strauss. J. F. 111( 1982). The role of lipoproteins in steroidogenests and cholesterol metabolism in steroidogenic glands. Endocr. Rev. 3: 299-329. Leitersdorf. E.. Stein. 0. and Stein, Y. (1985). Angiotensin II stimulates receptor mediated uptake of LDL by bovine adrenal cortical cells in primary culture. Biochim. Biophys. Acta 835: 183-190.
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Goltzman. D. and Mitchel, J. ( 1985). Interaction ofcalcitonin and calcitonin gene-related peptide at receptor sites in target tissues. Science 227: 1343-1345. Aguilera, G., Harwood, J. P. and Catt, K. J. ( I98 I ). Somatostatin modulates the effects of angiotensin II in adrenal glomerulosa zona. Nature 292: 262-263. Rebuffat, P., Robba, C., Mazzocchi, G. and Nussdorfer. G. G. (I 984). Inhibitory effect of somatostatin on the growth and steroidogenic capacity of rat adrenal zona glomerulosa. J. Steroid Biochem. 2 I : 387-390. Mazzocchi. G., Robba, C., Rebuffat, P., Gottardo, Ci. and Nussdorfer, G. G. ( 1985). Effect of somatostatin on the zona glomerulosa of rats treated with angiotensin II or captopril: stereology and plasma hormone concentrations. J. Steroid Biochem. 23: 353-356. Hausdorff, W. P., Aguilera, G. and Catt, K. J. ( 19X9). Inhibitory actions of somatostatin on cyclic AMP and aldosterone production in agonist-stimulated adrenal glomerulosa cells. Cell. Signal. I: 377-386. Cantin. M. and Genest, J. (1985). The heart and the atrial natriuretic factor. Endoc. Rev. 6: 107-127. Atlas, S. A. and Maack, T. (1987). Effects ofatrial natriuretic factor on the kidney and the renin-angiotensin-aldosterone system. Endocrinol. Metab. Clin. North Am. 16: 107.143. Mazzocchi, G., Rebuffat, P. and Nussdorfer, G. G. (1987). Atrial natriuretic factor (ANF) inhibits the growth and the secretory activity of rat adrenal zona glomerulosa in viva. J. Steroid Biochem. 28: 643-646. Rebuffat, P., Mazzocchi. G., Gottardo, G.. Meneghelli, V. and Nussdorfer, G. G. ( 1988). Further investigations on the atrial natriuretic factor (ANF)-induced inhibition of the growth and steroidogenic capacity of rat adrenal zona glomerulosa in vivo. J. Steroid Biochem. 29: 605609. Dunning, B. E. and Taborsky, G. J. ( 1987). Calcitonin generelated peptide: a potent and selective stimulator of gastrointestinal somatostatin secretion. Endocrinology 120: 1774-1781. Yamamoto, A., Kimura. S., Hasui. K.. FuJisawa. Y., Tawaki. T.. Fukui. K., Iwao, H. and Abe, Y. ( 1988). Calcitonin generelated peptide stimulates the release of atrial natriuretic peptide from isolated ratatria. Biochem. Biophys. Res. Commun. 155: 1452-1458. Schiebinger, R. J. and Santora. A. C. ( 1989). Stimulation by calcitonin gene-related peptide of atrial natriuretic peptide secretion in vitro and its mechanism ofaction. Endocrinology 124: 2473-2479. Kuramoto. H.. Kondo, H. and Fujita. T. (1987). Calcitonin gene-related peptide (CGRP)-like immunoreactivity in scattered chromaffin cells and nerve fibres in the adrenal gland. Cell Tissue Res. 247: 309-3 15. Hinson. J. P. (1990). Paracrinecontrol ofadrenocortical function : a new role for the medulla? J. Endocrinol. 124: 7-9.
Calcitonin Gene-Related Peptide Depresses the Growth and Secretory Activity of Rat Adrenal Zona Glomerulosa G. MAZZOCCHI,
L. K. MALENDOWICZ,
V. MENEGHELLI
AND G. G. NUSSDORFER
Department of Anatomy, University of Padua, Via Gabelli 65, l-35 12 1 Padua, Italy (Correspondence and reprint requests to GGN)
Abstract-The bolus ip. injection of rat calcitonin gene-related peptide (CGRP) (5 pm. kg-‘) significantly lowered plasma aldosterone concentration (PAC) in rats, despite a mild rise in plasma renin activity. Natremia, kalaemia and the blood levels of ACTH or corticosterone were not affected. Similar results were obtained after prolonged (5 days) SC. infusion of rats with CGRP (1 pm. kg-l. h-9. Moreover, CGRP infusion caused a notable atrophy of the zona glomerulosa (ZG) and its parenchymal cells, as well as a clearcut reduction in the surge of PAC evoked by a bolus injection of a high dose of angiotensin-II (100 pg. kg-‘). From these results it is suggested that CGRP exerts an inhibitory effect on the growth and secretory activity of ZG in rats.
Introduction Calcitonin gene-related peptide (CGRP), mainly produced in the nervous system by alternative processing of the calcitonin-gene transcription (l), is a potent vasodilator somehow involved in cardiovascular regulation (2-4). CGRP was also reported to exert a strong inhibitory effect on the pancreatic insulin release (5) and gastric secretion (6-8). Recently, the possible effect of CGRP on adrenal steroidogenesis has been investigated. Bloom et al (9) reported that CGRP enhances cortisol secretion by calf adrenals in vivo, independent of any vasodilation-induced increase in the rate of ACTH pre-
Date received Date accepted
I7 September 199 28 October 199 I
1
sentation. Murakami and co-workers (10) showed that CGRP inhibits, both in vivo and in vitro, aldosterone secretion by dog zona glomerulosa (ZG) cells, especially when stimulated by angiotensin-II (ANG-II). It, therefore, seemed of interest to investigate the short- and long-term effects of CGRP on the function and morphology of rat adrenal ZG and zona fasciculata (ZF). Methods Animal
treatment
Adult male Wistar rats (300 + 30 g body weight) were employed. A group of animals (n = 16) was divided into two equal subgroups, one of which was given an ip. injection of 5 pM. kg ’ CGRP (rat; Sigma, 157
158 St. Louis, MO), dissolved in 0.2 ml 0.9% NaCl solution, 30 min before the sacrifice. The other subgroup was injected with the saline vehicle. The dose of CGRP was chosen according to Murakami et al. ( 10). Another group of rats (n = 16) was divided into two equal subgroups. One subgroup was SC.infused for 5 days (Alzet osmotic pumps; Alza, Palo Alto, CA) with CGRP (1pM. kg’. h-l), and the second one was infused with the saline vehicle. Other rats (n = 32) were again divided into two subgroups, which were SC.infused as the previous ones. Half of the rats in each subgroup received 30 min before the sacrifice an ip. injection of 100 pg. kg’ ANG-II (Sigma). The other half of animals was given an ip. injection of 0.2 ml of the saline vehicle. The rats were decapitated between 10.00 and 11 .OO h, and their trunk blood was collected and frozen. Biochemical assays Serum Na’ and K’ concentrations were measured with a flame photometer (LKB, Stockholm, Sweden), Plasma renin activity (PRA) was assayed by RIA of angiotensin-I generated after incubation of plasma (ANG-I RIA kit; Peninsula, Merseyside, UK). ACTH was extracted from plasma (1 l), and its concentration was determined by RIA (ACTHRIA kit; IRE-Sorin, Vercelli, Italy). Aldosterone and corticosterone were extracted and purified (12), and their concentrations were measured by RIA (Aldo CTK2; IRE-Sorin. Ctrx-RIA kit; Eurogenetics, Milan, Italy). Intra-assay and inter-assay variations were: angiotensin-I, 6.8% and 8.7%; ACTH, 5.5% and 7.7%; aldosterone, 4.6% and 6.3%; corticosterone, 7.1% and 9.2%.
NEUROPEPTIDES
The volume of ZG and ZF, and the number and volume of their parenchymal cells were determined on light micrographs ofthe paraffin and 0.5~pm thick sections, using conventional morphometric methods ( 13), as described in an earlier paper (14). On electron micrographs of thin sections, the volume of nuclei and mitochondrial and lipid-droplet compartments, as well as the surface area of mitochondrial cristae and smooth endoplasmic reticulum (SER), were evaluated by the stereological techniques described by Weibel(l3), as detailed previously (14). Statistics The data obtained were averaged per experimental group and the SD of the mean was calculated. The statistical comparison of the data was done by ANOVA followed by the Multiple Range Test of Duncan. Results Neither acute nor chronic CGRP administrations evoked significant changes in the plasma levels of ACTH and corticosterone, natremia and kalaemia (Table 1). Conversely, both treatments caused a significant reduction in PAC (- 24% and -42%, respectively), and a moderate rise in PRA (about 22%) (Fig. 1).
Morphology The adrenal glands of CGRP-infused and control rats were promptly removed, freed of adherent fat, and weighed. The left adrenals were fixed in Bouin’s solution, embedded in paraffin and serially cut at 67 pm. Sliced pieces of the right glands were fixed in 3% glutaraldehyde, post-fixed in 1% osmium tetroxide and embedded in epon. Thick (0.5 pm) and thin (60-70 nm) sections were cut with LKB III ultramicrotomes at the level of the ZG and ZF. Thin sections were counterstained with lead-hydroxide, and examined and photographed in a Hitachi H-300 electron microscope.
(B) of Fig. 1 Effects of acute (A) and chronic administrations CGRP on PRA (left) and PAC (right) in rats. SD are indicated (n = 8). ’ P < 0.05 and *P < 0.01 versus control rats.
159
EFFECT OF CGRF ON ADRENALS Table 1
Effects of acute and chronic administrations of CGRP on some biochemical parameters of rats. Data are means It SD (n = 8)
Acute treatment CGRP Controls
Plasma ACTH concentration (pg. ml ‘) Natremia (mEq. 1 ‘) Kalaemia (mEq. 1-I) Plasma corticosterone concentration (pg. dl ‘)
Chronic Controls
treotment CGRP
128.6zr31.3
135.1 _+25.8
95.1 _+18.2
107.3 + 29.7
131.4_+ 15.2 4.8 f 0.9 13.9k2.1
129.5 _+19.5 5.0 _+I .o 12.2 _+3.0
136.2 _+30. I 4.7 + 0.7 10.1 + 1.9
130.4+21.4 4.8+ 0.5 9.7 t 2.6
Prolonged CGRP infusion provoked a notable decrease in the volume of ZG (-37%) and ZG cells (-35%) and nuclei (-26%), without affecting the number of ZG cells (Table 2). Stereology showed that the CGRP-induced ZG-cell atrophy was associated with significant decreases in the volume of the mitochondrial compartment (-26%) and in the surface areas per cell of mitochondrial cristae (-27%) and SER tubules (-50%). The volume of the lipid-droplet compartment underwent a striking rise (60%) (Table 2). The morphometric parameters of ZF cells were not significantly changed (Table 2). CGRP infusion caused a conspicuous reduction (about 60%) in the rise of PAC elicited by a bolus injection of ANG-II (Fig. 2). Discussion Fig. 2
PAC response to a bolus ip. injection of ANG-II in A) saline-infused and B) CGRP-infused rats. SD are indicated (n = 8). The numbers in square brackets indicate the percent increase. aP < 0.01 versus saline-infused control rats; *P < 0.01 versus the respective control rats. Table 2
Our present findings indicate that in the rat, as in the dog ( lo), a bolus administration of CGRP depresses ZG aldosterone secretion, without affecting the production of corticosterone, the main glucocorticoid secreted by inner adrenocortical layers in rodents
Effect of chronic CGRP infusion on the morphometric parameters of the rat adrenal gland. Data are means f SD (n = 8)
Zona glomerulosa CGRP Controls
Volume of zona (mm’) Number of cells (x IO>) Volume of cells @mm’) Volume of nuclei o.&) Volume of mitochondrial compartment (pm?icell) Surface area of mitochondrial cristae (u&/cell) Surface area of SER (pm’/cell) Volume of lipid-droplet compartment (pnQ/cell) + P c 0.05 and * P < 0.01 versus control rats.
Zona,fasciculato Controls CGRP
14.972 7497.8 1750.5 165.3 557 5
t 4.008 f- 1392.1 f-41 1.3 t 32.4 r 201.3
2.518f0.591 2535.8_+611.3 785.4_+ 197.2 128.5+ 15.6 156.1 ~30.6
I .586 + 0.387* 248 1.7 + 709.4 509.2 _+154.7* 95.1 * 11.7* 115.3*24.1*
15.398 + 3.81 I 7660.2 i 1406.2 1809. I i 306.2 160.8 i21.4 608.2 +_185.4
2349.3 _+401.6
1706.4 + 309.1*
12346.5 +_3608.9
11807.5 t 3198.2
5473.2+ 1218.3 44.7 f 20.2
2724.8 k 8 I I .7* 71.3 f31.4’
11350.7 f4101.4 132.2 f 51.7
10907.5 t_3218.7 142.8 !- 54.1
NEUROPEPTIDES
160 (for references, see 15). This last result is in contrast with that reported by Bloom et al (9) in calves; interspecific differences, as well as the fact that we employed doses of CGRP more than 3 order of magnitude less than those used by Bloom and associates (PM versus nM), may easily explain this discrepancy. The slight increase in PRA, already observed by Murakami et al (lo), may conceivably be interpreted as a negative feed-back response of the renin-angiotensin system to the lowered level of circulating aldosterone. Our data also show that the prolonged exposure to CGRP evokes a notable atrophy of rat ZG and its parenchymal cells, which is mainly due to the decrease in the volume of the mitochondrial compartment and SER. These morphologic data accord well with the CGRP-elicited depression in basal and maximally ANG-II-stimulated secretory activity of ZG cells, since the enzymes of aldosterone synthesis are located in both mitochondria and SER (for references, see 15, 16), and the changes in the surface area per cell of mitochondrial cristae and SER tubules are tightly coupled with corresponding changes in the activity per cell of some of these enzymes (17,18). The lowered utilization of cholesterol in aldosterone synthesis, coupled with a presumably normal (or slightly enhanced) uptake of cholesterol from serum lipoproteins, may well account for the striking increase in the volume of the lipid-droplet compartment. In fact, it is commonly agreed that cholesterol and cholesterol esters are stored in adrenocortical lipid droplets (15, 19), and that lipoprotein uptake by adrenocortical cells is a receptor-mediated process controlled not only by ACTH (20) but also by ANG-II (21) whose blood level, according to our PRA data, should be rather elevated in CGRP-treated rats. In conclusion, our findings suggest that CGRP may cause a notable depression of the growth and secretory activity of rat ZG. This effect of CGRP probably involves a direct action on ZG cells: i) adrenocortical cells possess specific binding sites for CGRP (22); and ii) acute or chronic CGRP administrations do not cause any apparent inhibition of the three main adrenoglomerulotropic factors, i.e. ANG-II, ACTH or K’ (for review, see 15). ANG-II production is conceivably raised (see above), and the plasma concentrations of ACTH and potassium are not significantly affected.
However, the possibility cannot be ruled out that CGRP may also positively interact with inhibitors of ZG function and growth, like somatostatin (2326) and ANF (27-30). In fact, CGRP stimulates somatostatin release, at least in the gastro-intestinal apparatus (3 l), and ANF secretion by isolated atria (32,33). Parenthetically, a slight rise in ANF plasma concentration occurs in CGRP-treated dogs (10). Be that as it may, it remains to be settled whether this antiadrenoglomerulotropic effect of CGRP is only a pharmacologic one or may play a role in the physiological regulation of the ZG. In this connection, we want to recall that the levels of circulating CGRP presumably attained in our experiments are very low: lO~‘*/lO-” M (by assuming an absorption rate of 80% and 10 ml of blood per rat). Evidence is available that CGRP-immunoreactivity is present in adrenal zona medullaris and that CGRP-containing fibers (of probable medullaris origin) can reach the cortex and especially ZG (34). A good deal of data suggests that adrenal zona medullaris may exert a paracrine control on the function of the zona corticalis (35). Thus, it does not seem unreasonable to conceive that locally produced CGRP may reach in the cortex a concentration sufficient to affect ZG function.
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