Adrenomedullin gene expression and its different regulation in human adrenocortical and medullary tumors
J Liu1, A I Kahri1, P Heikkila1 and R Voutilainen1,2
1Department of Pathology, PO Box 21, University of Helsinki, FIN-00014 Helsinki, Finland and 2Department of Pediatrics, Kuopio University Hospital, FIN-70210 Kuopio, Finland
(Requests for offprints should be addressed to ] Liu, Department of Pathology, PO Box 21, University of Helsinki, FIN-00014 Helsinki, Finland)
Abstract
Adrenomedullin (ADM) is a polypeptide originally discov- ered in a human pheochromocytoma and is also present in normal adrenal medulla. It has been proposed that ADM could be involved in the regulation of adrenal steroido- genesis via paracrine mechanisms. Our aim was to find out if ADM gene is expressed in adrenocortical tumors and how ADM gene expression is regulated in adrenal cells. ADM mRNA was detectable by Northern blotting in most normal and hyperplastic adrenals, adenomas and carcinomas. The average concentration of ADM mRNA in the hormonally active adrenocortical adenomas was about 80% and 7% of that in normal adrenal glands and separated adrenal medulla respectively. In adrenocortical carcinomas, the ADM mRNA concentration was very variable, but on average it was about six times greater than that in normal adrenal glands. In pheochromocytomas, ADM mRNA expression was about ten times greater than that in normal adrenals and three times greater than in separated adrenal medulla.
In primary cultures of normal adrenal cells, a protein kinase C inhibitor, staurosporine, reduced ADM mRNA
accumulation in a dose- and time-dependent fashion (P<0-01), whereas it simultaneously increased the expres- sion of human cholesterol side-chain cleavage enzyme (P450 scc) gene (a key gene in steroidogenesis). In cultured Cushing’s adenoma cells, adrenocorticotropin, dibutyryl cAMP ((Bu)2CAMP) and staurosporine inhibited the accumulation of ADM mRNA by 40, 50 and 70% respectively (P<0-05), whereas the protein kinase C activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA), increased it by 50% (P<0-05). In primary cultures of pheochromocytoma cells, treatment with (Bu)2CAMP for 1 and 3 days increased ADM mRNA accumulation two- to threefold (P<0-05). Our results show that ADM mRNA is present not only in adrenal medulla and pheochromocytomas, but also in adrenocortical neoplasms. Both protein kinase A- and C-dependent mechanisms regulate ADM mRNA expression in adrenocortical and pheochromocytoma cells supporting the suggested role for ADM as an autocrine or paracrine (or both) regulator of adrenal function.
Journal of Endocrinology (1997) 155, 483-490
Introduction
Adrenomedullin (ADM) is a potent hypotensive peptide originally discovered in extracts of a human pheochromo- cytoma (Kitamura et al. 1993a). ADM-immunoreactive cells are widely distributed in human tissues, including the endocrine and neuroendocrine systems: adrenal medulla, pancreatic islets, anterior pituitary, and the gastrointestinal neuroendocrine system (Kitamura et al. 1993b, Satoh et al. 1995, Washimine et al. 1995). Another peptide processed from proadrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP), has also been identified as a hypo- tensive factor (Kitamura et al. 1994). Both ADM and PAMP have been suggested to be implicated in the physiological control of many organ systems, including adrenal function (Schell et al. 1996, Montuenga et al. 1997).
Normal human adrenal medulla expresses abundantly both ADM mRNA and protein, but earlier studies failed to demonstrate ADM mRNA signal or positive immuno- staining in the adrenal cortex (Kitamura et al. 1993b, Washimine et al. 1995, Satoh et al. 1996). However, human adrenal medulla does not contribute much to the circulating concentrations of ADM, suggesting that ADM in normal adrenals is produced and metabolized as a local hormone (Nishikimi et al. 1994). In rat adrenal zona glomerulosa cell cultures, ADM inhibited aldosterone secretion induced by angiotensin II, high potassium con- centration, and an ionophore (A23187). This suggests that the secreted ADM (and PAMP) from adrenal medullary cells could modulate adrenocortical function, possibly by a paracrine mechanism. In contrast, ADM did not affect adrenocorticotropin (ACTH)- or cAMP-induced aldo- sterone secretion in vitro (Yamaguchi et al. 1995, Kapas &
Journal of Endocrinology (1997) 155, 483-490 C) 1997 Journal of Endocrinology Ltd Printed in Great Britain 0022-0795/97/0155-0483 $08.00/0
adrenal cortex
adrenal medulla
left hyperplasia
right hyperplasia
Conn’s adenoma
adjacent adrenal
Cushing’s adenoma 1
adjacent adrenal 1
Cushing’s adenoma 2
adjacent adrenal 2
pheochromocytoma 1
adjacent adrenal 1
pheochromocytoma 2 adjacent adrenal 2
28S
— 18S
ADM
28S
18S
P450scc
28S
28S
Hinson 1996). The inhibitory effect of ADM on adrenal steroidogenesis might be specific to zona glomerulosa cells, as ADM was shown to have little effect on corticosterone secretion (Yamaguchi et al. 1996, Mazzocchi et al. 1996).
In the adrenal medulla, ADM (and PAMP) is co-secreted with catecholamines in response to nicotinic receptor stimulation (Katoh et al. 1994). The locally released ADM and PAMP may also act in an autocrine fashion, feeding back to inhibit acetylcholine-induced catecholamine secretion, as cells pretreated with PAMP released less catecholamines in response to carbachol (Katoh et al. 1995). The mechanisms underlying this inhibition may involve a reduction of intracellular calcium concentration by ADM (Houchi et al. 1996, Takano et al. 1996).
Recently, expression of ADM mRNA was detected in a human adrenocortical carcinoma cell line, NCI-H295
(Miller et al. 1996). The aim of this study was to find out if the ADM gene is expressed in primary adrenocortical tumors. In addition, we aimed to clarify how ADM gene expression is regulated in adrenal cells.
Materials and Methods
Tissues and cell cultures
Normal adrenal glands were obtained from nine patients who underwent nephrectomy for kidney tumors. Patho- logical adrenocortical samples, pheochromocytomas and adrenal tissues adjacent to the tumors were obtained from 39 patients during operations performed at the Departments of Surgery, Helsinki University Central Hospital. The pathological adrenocortical tissues investigated included Cushing’s, Conn’s, virilizing and
(clinically) non-functional adenomas, bilateral and nodular hyperplasias, and Cushing’s, aldosterone-producing and (clinically) non-functional carcinomas. The adrenal tissues were processed as described previously (Liu et al. 1995). Briefly, normal adrenal cortical and medullary tissues were carefully dissected from five adrenals. Part of the normal and pathological adrenal tissues was frozen in liquid nitrogen and then stored at - 70 ℃ until required for extraction of total RNA. The remaining tissues were minced into small pieces and dissociated with collagenase- dispase and deoxyribonuclease-I. Dispersed cells were maintained in Dulbecco’s Modified Eagle’s Medium- Ham’s F-12 Medium containing 10% fetal calf serum for 5-7 days before the test agents were added. The concen- trations of the test agents used in this study were previously found to be effective in the modulation of steroidogenic gene expression, and not to be toxic to the adrenal cells in culture (Liu et al. 1996). In experiments in which more than one compound was added, the cells were exposed to the agents simultaneously. All experiments were performed in duplicate or triplicate and repeated at least twice with tissues from different patients.
RNA analysis
Isolation of total and cytoplasmic RNA, Northern blotting and hybridizations were carried out as described previously (Liu et al. 1995). A 30-mer oligonucleotide probe for ADM mRNA was synthesized at the Institute of Biotechnology, University of Helsinki. The sequence was 5’-ATC TGT GAA CTG GTA GAT CTG GTG TGC CAG-3’, corresponding to nucleotides 514-543 of the human ADM cDNA (Kitamura et al. 1993b). The human cholesterol side-chain cleaving enzyme (P450 scc) cDNA probe (Chung et al. 1986) was used as a marker of adrenocortical steroidogenesis, and mouse 28S ribosomal RNA cDNA probe (Arnheim 1979) was used as a loading control. The relative intensities of autoradiographic signals were quantitated by densitometric scanning. All mRNA data shown were normalized with the respective 28S RNA values. The correlations between ADM and P450 scc mRNA concentrations in different in vivo samples were analyzed by Spearman’s test. Differences in the RNA levels between various types of adrenal tissues in vivo or different treatments in vitro were assessed by Mann-Whitney’s test. The level of significance was chosen as P<0.05.
Results
The ADM transcript of approximately 1.6 kb in size was detectable by Northern blotting in most normal adrenals, adrenocortical hyperplasias, Conn’s, Cushing’s, virilizing and non-functional adenomas, adrenocortical carcinomas and pheochromocytomas (Figs 1 and 2). ADM mRNA
adrenal cortex 1
adrenal medulla 1
adrenal medulla 2
adrenal cortex 2
Cushing’s carcinoma 1
Cushing’s carcinoma 2
adjacent adrenal
nonfunctional adenoma
virilizing adenoma 1 adjacent adrenal 2
virilizing adenoma 2
28S
18S
ADM
28S
18S
P450scc
28S
28S
concentrations varied considerably in different adrenocor- tical samples, even within the same histopathological diagnosis. Among the adrenocortical neoplasms, carcino- mas and non-functional adenomas had the greatest average ADM mRNA expression (Table 1). The ster- oidogenic tissue marker, P450 scc, was expressed in all adrenocortical tissues, although its concentration varied remarkably from patient to patient (Figs 1 and 2); after sufficiently long exposure times, P450 scc mRNA was detectable in all adrenocortical samples. No significant correlation was found in the expression of ADM and P450 scc mRNAs in either normal or pathological adrenocortical tissues (Table 1). The mean ADM mRNA abundance in the dissected adrenal medullary samples and pheochromocytomas was respectively about 3.3- (P<0.05) and 10-fold (P<0.005) that in normal whole adrenals. The greatest expression of ADM mRNA was found in some pheochromocytomas, but the variation was again considerable within this tumor group (Fig. 1, Table 1).
In primary cultures of Cushing’s adenoma cells, accu- mulation of ADM mRNA was reduced by approximately 40, 50 and 70% after 24 h of treatment with ACTH (30 nm), dibutyryl cAMP ((Bu)2cAMP; 1 mM) and
| n | ADM | P450scc | |
|---|---|---|---|
| Tissue | |||
| Normal adrenal | 9 | 100 (47-148) | 100 (58-142) |
| Adrenal medulla | 5 | 326 (135-667)* | 55 (11-80) |
| Adrenocortical adenoma | |||
| (Cushing's) | 5 | 85 (50-131) | 71 (10-114) |
| Adjacent gland | 3 | 51 (39-63) | 16 (7-22) |
| Adrenocortical adenoma | |||
| (Conn's) | 3 | 87 (39-150) | 92 (54-145) |
| Adjacent gland | 3 | 97 (52-150) | 129 (76-172) |
| Adrenocortical adenoma | |||
| (Virilizing) | 2 | 70 (55-85) | 144 (77-211) |
| Adjacent gland | 1 | 245 | 172 |
| Adrenocortical adenoma | |||
| (Non-functional) | 3 | 302 (63-650) | 77 (52-124) |
| Adjacent gland | 2 | 236 (140-331) | 147 (131-163) |
| Adrenocortical hyperplasia | |||
| (Bilateral) | 5 | 79 (43-123) | 112 (103-125) |
| Adrenocortical hyperplasia (Nodular) | 3 | 58 (9-91) | |
| 146 (118-190) | |||
| Adrenocortical carcinoma | 4 | ||
| (Functional) | 321 (10-1155) | 64 (5-11) | |
| Adrenocortical carcinoma | 1 | ||
| (Non-functional) | 950 | 107 | |
| Pheochromocytoma | 16 | 1018 (51-2208) ** | <1 |
| Adjacent gland | 9 | 136 (15-331) | 108 (19-217) |
*P<0.05, ** P<0-005 compared with normal whole adrenals.
staurosporine (100 nM) respectively (Fig. 3) (P<0-05 for all treatments; pooled data). The inhibition by ACTH and (Bu)2CAMP was dose-dependent and detectable from concentrations of 300 PM and 10 µM upwards respectively (data not shown). 12-O-Tetradecanoyl phorbol 13-acetate (TPA; 160 nm) stimulated the expression of ADM mRNA 18-77% after 24 h of stimulation (P<0-05). The inhibitory effect of ACTH and (Bu)2CAMP on the expression of ADM mRNA was reversed by TPA treatment, but TPA did not significantly affect the inhibition caused by stau- rosporine treatment (Fig. 3). 1-(5-Isoquinolinesulfonyl)- 2-methylpiperazine dihydrochloride (H-7; 100 µM) inhibited basal (by 90%) and TPA-induced expression of ADM mRNA (Fig. 4). As in our previous report on cultured normal adrenal cells (Liu et al. 1996), the expres- sion of P450 scc mRNA in cultured Cushing’s adenoma cells was also induced by ACTH, (Bu)2cAMP and staurosporine treatments, but TPA had no effect. TPA did not significantly modify the effects of ACTH or (Bu)2CAMP on the accumulation of P450 scc mRNA (Fig. 3). As in the case of the ADM mRNA expression, H-7 treatment inhibited P450 scc mRNA accumulation
(Fig. 4). We also detected ADM mRNA expression in primary cultures prepared from a Conn’s adenoma, a non-functional adrenocortical adenoma, a Cushing’s carcinoma, and two nodular adrenocortical hyperplasias (data not shown).
In primary cultures of normal adrenal cells, ACTH (30 nM; Fig. 5) and (Bu)2CAMP (1 mM) had no significant effect on ADM mRNA accumulation, even though they increased the concentration of P450 scc mRNA more than threefold. TPA (160 nM) had no significant effect on either ADM or P450 scc mRNA concentrations. As in Cushing’s adenoma cells, staurosporine (100 nm) inhibited the expression of ADM mRNA, and increased that of P450 scc mRNA (Fig. 5). The effect of staurosporine on both ADM and P450 scc mRNA expression was dose- (from 10 nM upwards) and time-dependent. The inhibition of ADM mRNA accumulation could be seen after 6 h, with the maximal effect occurring at 24 h. The induction of P450 scc mRNA was detectable at 12 h with the maximal concentration detected at 48 h (data not shown).
In primary cultures of pheochromocytoma cells, expres- sion of ADM mRNA was maintained for at least 2 weeks.
control
(Bu)2CAMP
ACTH/(Bu)2CAMP
ACTH
ACTH/TPA
ACTH/ST
(Bu)2cAMP/TPA
(Bu)2cAMP/ST
TPA
TPA/ST
control
ST
28S
18S
ADM
28S
18S
P450scc
28S
28S
The steady-state concentration of ADM mRNA was increased two- to fourfold by (Bu)2CAMP in a concen- tration of 1 mM after 24 h of treatment (Fig. 6) (P<0-05 in pooled data). This stimulation was maintained for at least 3 days in a dose-dependent manner, with induction detectable from a concentration of 10 µM upwards. The protein kinase inhibitor, staurosporine (100 nm), had no significant effect on either the basal or the stimulated expression of ADM mRNA (Fig. 6). However, there was a slight increase in ADM mRNA concentration after treatment with TPA (160 nM; Fig. 6). Nerve growth factor (200 µg/l) and dexamethasone (500 ng/ml) had no significant effect on ADM mRNA expression after 1 and 3 days of treatment (data not shown).
Discussion
Our data show that the ADM gene is expressed in adrenocortical neoplasms in vivo and its mRNA concen-
control
H-7
TPA
H-7/TPA
H-7/ST
ST
28S
18S
ADM
28S
— 18S
P450scc
28S
28S
tration is regulated by multiple factors in adrenocortical cells in vitro. It is possible that the ADM mRNA detected in dissected normal adrenal cortex and adrenal hyperplasia samples may be derived from chromaffin cells present within all three zones of the human adrenal cortex (Bornstein et al. 1994). However, it is unlikely that the detected expression of ADM mRNA in adrenocortical tumors comes from adrenal chromaffin cells. This view is supported by our recent study which showed that the chromaffin tissue-specific marker, chromogranin A, was undetectable in adrenocortical tumors (Liu et al. 1997). The greatest expression of ADM mRNA in adrenocortical samples was in some adrenocortical carcinomas. This demonstrates a significant potential of adrenocortical cells for ADM production.
A possible physiological and pathophysiological function of ADM in normal adrenal cortex and adrenal tumors, respectively, is not clear at present. ADM might function as an autocrine/paracrine growth factor or regulator of steroidogenesis in adrenocortical cells (Miller et al. 1996, Withers et al. 1996). However, at present there is no direct evidence to support this hypothesis.
TPA/ST
ACTH/TPA/ST
control
ACTH
ACTH/TPA
ACTH/ST
TPA
ST
28S
18S
ADM
28S
— 18S
P450scc
28S
28S
Regulation of ADM and P450 scc mRNA expression was different in both normal adrenal and Cushing’s adenoma cells. In Cushing’s adenoma cells, ACTH inhib- ited the expression of ADM mRNA, whereas it increased P450 scc mRNA concentration. These effects seem to be mediated through the cAMP-dependent protein kinase A pathway, as (Bu)2cAMP had the same effect as ACTH. However, both ACTH- and (Bu)2CAMP-induced changes in ADM mRNA expression were reversed by the protein kinase C activator, TPA, indicating the possible interaction of protein kinase A- and C-dependent pathways. The tonic effect of protein kinase C on ADM mRNA expression was inhibited by the relatively specific protein kinase C inhibitor, staurosporine, and the non-specific protein kinase inhibitor, H-7. In cultured normal adrenal cells, ACTH and (Bu)2CAMP treatments did not modify the expression of ADM mRNA, but the inhibitory effect of staurosporine was similar to that in the Cushing’s adenoma cells. Whether this reflects a differ- ence of protein kinase activities in Cushing’s adenoma and normal adrenal cells is not clear.
(Bu)2CAMP
(Bu)2CAMP/ST
control
TPA
TPA/ST
ST
28S
18S
ADM
28S
28S
Although human pheochromocytomas are one of the most abundant production sites of ADM and PAMP (Kuwasako et al. 1995, Washimine et al. 1994), the regulation of ADM gene expression in chromaffin cells has not previously been reported. The present study demon- strated that the regulation of ADM mRNA expression in pheochromocytoma cells may involve the cAMP- dependent protein kinase A pathway. It has been reported that cAMP is capable of increasing extracellular cate- cholamine concentrations by enhancing their synthesis in the cytosol and inhibiting their translocation into storage vesicles (Nakanishi et al. 1995). Therefore, (Bu)2CAMP- induced expression of ADM may be involved in the feed-back inhibition of catecholamine secretion simul- taneously stimulated by (Bu)2CAMP. This situation may be similar to that in chromaffin cells treated with nicotine. Nicotine is able to induce ADM secretion in cultured bovine adrenal medullary cells (Katoh et al. 1994) and the production of catecholamines in PC12 cells through the cAMP-dependent protein kinase A pathway (Hiremagalur et al. 1993). Therefore, the increased ADM and PAMP production induced by nicotine may inhibit the co- secretion of catecholamines (Katoh et al. 1995). The regulation of ADM gene expression seems to be tissue- specific. Dexamethasone and cortisol stimulated ADM production in cultured rat vascular smooth muscle cells (Minamino et al. 1995). However, we could not find any significant effect of glucocorticoids on ADM mRNA expression in cultured pheochromocytoma cells.
In summary, our study has shown that ADM mRNA is expressed both in adrenocortical tumors and in pheo- chromocytomas, and the mechanisms regulating ADM mRNA accumulation in primary cultures of adreno- cortical cells are different from those in pheochromo- cytoma cells. ADM may be involved in the autocrine and paracrine regulation of both adrenocortical and chromaffin cell functions.
Acknowledgements
Ms Merja Haukka and Ms Eija Heiliö are thanked for their technical assistance, Dr Johanna Arola for help in preparation of the tissues, and Dr Pantelis Georgiades (Cambridge, UK) for helpful comments. This work was financially supported by the Cancer Society of Finland, the Culture Foundation of Finland, and the Jalmari and Rauha Ahokas Foundation (to J L).
References
Arnheim N 1979 Characterization of mouse ribosomal gene fragments purified by molecular cloning. Gene 7 83-96.
Bornstein SR, Gonzalez-Hernandes JA, Ehrhart-Bornstein M, Adler G & Scherbaum WA 1994 Intimate contact of chromaffin and cortical cells within the human adrenal gland forms the cellular basis for important intraadrenal interactions. Journal of Clinical Endocrinology and Metabolism 78 225-232.
Chung B, Matteson KJ, Voutilainen R, Mohandas TK & Miller WL 1986 Human cholesterol side-chain cleavage enzyme, P450 scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta. Proceedings of the National Academy of Sciences of the USA 83 8962-8966.
Hiremagalur B, Nankova B, Nitahara J, Zeman R & Sabban EL 1993 Nicotine increases expression of tyrosine hydroxylase gene: involvement of protein kinase A-mediated pathway. Journal of Biological Chemistry 268 23704-23711.
Houchi H, Yoshizumi M, Shono M, Ishimura Y, Ohuchi T & Oka M 1996 Adrenomedullin stimulates calcium efflux from adrenal chromaffin cells in culture: possible involvement of an Na+/Ca2+ exchange mechanism. Life Sciences 58 PL35-40.
Kapas S & Hinson JP 1996 Actions of adrenomedullin on the rat adrenal cortex. Endocrine Research 22 861-865.
Katoh F, Niina H, Kitamura K, Ichiki Y, Yamamoto Y, Kangawa K, Eto T & Wada A 1994 Ca2+-dependent cosecretion of adreno- medullin and catecholamines mediated by nicotinic receptors in bovine cultured adrenal medullary cells. FEBS Letters 348 61-64. Katoh F, Kitamura K, Niina H, Yamamoto R, Washimine H, Kangawa K, Yamamoto Y, Kobayashi H, Eto T & Wada A 1995 Proadrenomedullin N-terminal 20 peptide (PAMP), an endogenous anticholinergic peptide: its exocytotic secretion and inhibition of catecholamine secretion in adrenal medulla. Journal of Neurochemistry 64 459-461.
Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H & Eto T 1993a Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochemical and Biophysical Research Communications 192 553-560.
Kitamura K, Sakata J, Kangawa K, Kojima M, Matsuo H & Eto T 1993b Cloning and characterization of cDNA encoding a precursor for human adrenomedullin. Biochemical and Biophysical Research Communications 194 720-725.
Kitamura K, Kangawa K, Ishiyama Y, Washimine H, Ichiki Y, Kawamoto M, Minamino N, Matsuo H & Eto T 1994 Identification and hypotensive activity of proadrenomedullin N-terminal 20 peptide (PAMP). FEBS Letters 351 35-37.
Kuwasako K, Kitamura K, Ichiki Y, Kato J, Kangawa K, Matsuo H & Eto T 1995 Human proadrenomedullin N-terminal 20 peptide in pheochromocytoma and normal adrenal medulla. Biochemical and Biophysical Research Communications 211 694-699.
Liu J, Kahri AI, Heikkila P, Ilvesmaki V & Voutilainen R 1995 H19 and insulin-like growth factor-II gene expression in adrenal tumors and cultured adrenal cells. Journal of Clinical Endocrinology and Metabolism 80 492-496.
Liu J, Heikkila P, Kahri AI & Voutilainen R 1996 Expression of the steroidogenic acute regulatory protein mRNA in adrenal tumors and cultured adrenal cells. Journal of Endocrinology 150 43-50.
Liu J, Voutilainen R, Kahri AI & Heikkila P 1997 Expression patterns of the c-myc gene in adrenocortical tumors and pheochromo- cytomas. Journal of Endocrinology 152 175-181.
Mazzocchi G, Rebuffat P, Gottardo G & Nussdorfer GG 1996 Adrenomedullin and calcitonin gene-related peptide inhibit aldosterone secretion in rats, acting via a common receptor. Life Sciences 58 839-844.
Miller MJ, Martinez A, Unsworth EJ, Thiele CJ, Moody TW, Elsasser T & Cuttitta F 1996 Adrenomedullin expression in human tumor cell lines: its potential role as an autocrine growth factor. Journal of Biological Chemistry 271 23345-23351.
Minamino N, Shoji H, Sugo S, Kangawa K & Matsuo H 1995 Adrenocortical steroids, thyroid hormones and retinoic acid augment the production of adrenomedullin in vascular smooth muscle cells. Biochemical and Biophysical Research Communications 211 686-693.
Montuenga LM, Martinez A, Miller MJ, Unsworth EJ & Cuttitta F 1997 Expression of adrenomedullin and its receptor during embryogenesis suggests autocrine or paracrine modes of action. Endocrinology 138 440-451.
Nakanishi N, Onozawa S, Matsumoto R, Hasegawa H & Yamada S 1995 Cyclic AMP-dependent modulation of vesicular monoamine transport in pheochromocytoma cells. Journal of Neurochemistry 64 600-607.
Nishikimi T, Kitamura K, Saito Y, Shimada K, Ishimitsu T, Takamiya M, Kangawa K, Matsuo H, Eto T, Omae T & Matsuoka H 1994 Clinical studies on the sites of production and clearance of circulating adrenomedullin in human subjects. Hypertension 24 600-604.
Satoh F, Takahashi K, Murakami O, Totsune K, Sone M, Ohneda M, Abe K, Miura Y, Hayashi Y, Sasano H & Mouri T 1995 Adrenomedullin in human brain, adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma. Journal of Clinical Endocrinology and Metabolism 80 1750-1752. Satoh F, Takahashi K, Murakami O, Totsune K, Sone M, Ohneda M, Sasano H & Mouri T 1996 Immunocytochemical localization of adrenomedullin-like immunoreactivity in the human hypothalamus and the adrenal gland. Neuroscience Letters 203 207-210.
Schell DA, Vari RC & Samson WK 1996 Adrenomedullin: a newly discovered hormone controlling fluid and electrolyte homeostasis. Trends in Endocrinology and Metabolism 7 7-13.
Takano K, Yamashita N & Fujita T 1996 Proadrenomedullin NH2-terminal 20 peptide inhibits the voltage-gated Ca2+ channel current through a pertussis toxin-sensitive G protein in rat pheochromocytoma-derived PC12 cells. Journal of Clinical Investigation 98 14-17.
Washimine H, Kitamura K, Ichiki Y, Yamamoto Y, Kangawa K, Matsuo H & Eto T 1994 Immunoreactive proadrenomedullin N-terminal 20 peptide in human tissue, plasma and urine. Biochemical and Biophysical Research Communications 202 1081-1087.
Washimine H, Asada Y, Kitamura K, Ichiki Y, Hara S, Yamamoto Y, Kangawa K, Sumiyoshi A & Eto T 1995 Immunohistochemical identification of adrenomedullin in human, rat, and porcine tissue. Histochemistry and Cell Biology 103 251-254.
Withers DJ, Coppock HA, Seufferlein T, Smith DM, Bloom SR & Rozengurt E 1996 Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells. FEBS Letters 378 83-87.
Yamaguchi T, Baba K, Doi Y & Yano K 1995 Effect of adrenomedullin on aldosterone secretion by dispersed rat adrenal zona glomerulosa cells. Life Sciences 56 379-387.
Yamaguchi T, Baba K, Doi Y, Yano K, Kitamura K & Eto T 1996 Inhibition of aldosterone production by adrenomedullin, a hypotensive peptide, in the rat. Hypertension 28 308-314.
Received 24 March 1997 Accepted 9 July 1997