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Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua (P.G.A., G.M., G.G.N.), I-35–121 Padua, Italy; Department of Histology and Embryology, Poznan School of Medicine (A.M., L.K.M.), PL-60781, Poznan, Poland; and Department of Pharmacology, Tulane University Medical Center (H.C.C.), New Orleans, Louisiana 70112-2699
Address all correspondence and requests for reprints to: Prof. G. G. Nussdorfer, Department of Human Anatomy and Physiology, Section of Anatomy, Via Gabelli 65, I-35121 Padova, Italy. E-mail: ggnanat{at}ipdunidx.unipd.it
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Introduction |
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There is general agreement that ADM induces vasodilation through the activation of adenylate cyclase (AC)- coupled receptors of the CGRP1 subtype (4, 6). Several investigations have also found that ADM acts as growth modulator of many cell systems cultured in vitro, but the results are conflicting. To summarize, an antiproliferogenic effect has been reported in rat vascular smooth muscle cells (VSMC) (7) and mesangial cells (8, 9). In contrast, other investigators observed a clear-cut growth-promoting effect of ADM in rat VSMC (10) and Swiss 3T3 fibroblasts (11, 12).
Binding sites for ADM have been demonstrated in human and rat adrenal zona glomerulosa (ZG), and the bulk of the evidence indicates that they are CGRP1 receptors (for review, see Ref. 13). ADM, like PAMP (14), was found to inhibit angiotensin II (Ang-II)- and K+-stimulated aldosterone secretion through a mechanism probably involving the impairment of agonist-enhanced Ca2+ influx (15, 16, 17, 18, 19, 20, 21). Despite the large mass of data concerning the acute effect of ADM on adrenal cortex, in vivo studies dealing with the possible modulatory action of this peptide on adrenal growth are not yet available.
Hence, we decided to address this issue using the technique of in situ perfusion of the isolated rat adrenal gland (22), because it allows the delivery of ADM and other chemicals directly to the gland and study of their effects in vivo without any possible interference with other systemic mechanisms involved in the regulation of adrenal growth, e.g. the kidney renin-angiotensin system and hypothalamo-pituitary-adrenal axis (for review, see Ref. 23), which are likely to be affected by ADM (5, 6, 13).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP and
[3H]thymidine were obtained from Amersham Pharmacia Biotech (Aylesbury, UK), and antimitogen-activated
protein kinase (anti-MAPK) p42/p44 polyclonal antibody was purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Adult male Sprague Dawley rats (260 ± 30 g BW) were purchased from Charles River Laboratories, Inc. (Como, Italy). They were housed four per cage, kept under a 12-h light, 12-h dark cycle (illumination onset at 0800 h) at 23 C, and maintained on a standard diet and tap water ad libitum. The protocol of the experiments described below was approved by the local ethical committee for animal studies.
In situ adrenal perfusion
Rat were anesthetized with Nembutal, and the left adrenal gland
was perfused in situ, as previously detailed (24). Briefly,
perfusion medium was introduced, via a cannula inserted in the coeliac
artery, into an isolated segment of aorta from which the adrenal
arteries arise; after flowing through the adrenal gland, medium was
collected by a cannula inserted into the renal vein. Perfusion medium
[medium 199 and Krebs-Ringer bicarbonate buffer (2:1, vol/vol)
containing 0.2% glucose and 5 mg/ml human serum albumin] was gassed
with 95% air and 5% CO2, maintained at 37 C,
and delivered at a constant rate of 2 ml/10 min for 180 min.
The following chemicals were added to the perfusion medium: 1) ADM (from 10-10–10-6 M); 2) 10-8 M ADM in the presence of CGRP-(8–37) or ADM-(22–52) (from 10-9–10-5 M); and 3) 10-8 M ADM in the presence of H-89, AACOCF3, indomethacin, phenidone, U-73122, or calphostin C (10-5 M), tyrphostin (10-6 or 10-5 M), SQ-22536 or InsP3S3 (10-4 M), PD-98059 or U0216 (10-5 or 10-4 M), and Rp-cAMP-S (10-3 M). The concentrations of the chemicals used in the third experiment, according to the current literature (see Discussion), were approximately the maximal effective ones. In the case of the first perfusion experiment, in some instances 0.1 mg colchicine dissolved in 200 µl medium was injected into the perfusion cannula at 120 min (24). The following experiments were also carried out: 1) 10-9 M ACTH in the presence of H-89 (10-5 M), SQ-22536 (10-4 M), or Rp-cAMP-S (10-3 M); and 2) 10-9 M Ang-II in the presence of U-73122 (10-5 M), calphostin C (10-5 M), or InsP3S3 (10-4 M).
Measurement of the mitotic index
Adrenal glands of colchicine-injected rats were removed, fixed
in Bouin’s solution, and embedded in paraffin. Adrenals were sectioned
at 6 µm, and sections were stained with hematoxylin-eosin. The
mitotic index (percentage of metaphase-arrested cells) was calculated
at x400 by counting 5000 cells in the ZG and zona
fasciculata-reticularis (ZF/R) of each adrenal gland.
Measurement of DNA synthesis
Perfused adrenals were immediately collected under sterile
conditions, gently decapsulated to separate capsule-ZG, hemisected,
demedullated under the dissecting microscope, and then quartered.
Adrenal capsule-ZG and ZF/R quarters were put in the perfusion medium
containing 200 U/ml penicillin, 10 µg streptomycin, and 2 µCi/ml
[3H]thymidine. The incubation was carried out
for 180 min in a shaking bath at 37 C in an atmosphere of 95% air-5%
CO2. At the end of the incubation, the medium was
removed, and the samples were washed twice with ice-cold Krebs-Ringer
bicarbonate buffer and frozen at -20 C. DNA was recovered from each
specimen without phenol extraction and ethanol precipitation (24),
using the Nuclei Clean Kit (Sigma), and its radioactivity
was measured in a liquid scintillation counter (model 1211,
LKB, Stockholm, Sweden). Results were expressed as counts
per min/100 mg tissue. Due to the very peculiar vascularization of the
rat adrenals, where arterioles have an almost exclusive extracapsular
location (25), VSMCs represent no more than 0.05% of the entire
cortical cell population. Consequently, they cannot significantly bias
the effect of ADM on parenchymal cell DNA synthesis.
Dispersed adrenocortical cells
Dispersed ZG (capsular) and ZF/R (inner) cells were obtained
from the adrenals of nonperfused rats by collagenase digestion and
mechanical disaggregation (14). The viability of dispersed cells was
checked by the trypan blue exclusion test and was greater than 92%.
ZF/R cell contamination in capsular cell preparations, as evaluated by
phase microscopy, was always less than 7%, and ZG or medullary
chromaffin cell contaminations in inner cell preparations were less
than 0.5% and virtually absent, respectively.
Dispersed cells were put in medium 199 and Krebs-Ringer bicarbonate buffer with 0.2% glucose containing 5 mg/ml BSA. They were incubated with 10-8 M ADM alone or in the presence of CGRP-(8–37) or ADM-(22–52) (10-6 M). Other ZG cell preparations were preincubated for 30 min with 10-5 M tyrphostin-23, and then exposed to ADM (10-8 M). The incubation was carried out in a shaking bath at 37 C for 15 min and was stopped by two quick washes with ice-cold PBS.
Cell extract preparation
Dispersed cells were rapidly lysed by the addition of ice-cold
extraction buffer containing 12.5 mM Tris-HCl (pH 7.4), 2
mM EDTA, 2 mM EGTA, 25 mM
ß-glycerophosphate, 2 mM sodium vanadate, 10
µM phenylmethylsulfonylfluoride, 1 µg/ml leupeptin, and
5 µg/ml apoprotin. Cells were Dounce homogenized (20 strokes; Kontes
Co., Vineland, NJ) on ice for 1 min, and homogenates were centrifuged
(at 4 C) at 800 x g for 10 min and then at 12,000
x g for 15 min (26). Supernatants were removed, the protein
concentration was determined by the Lowry method using BSA as a
standard, and supernatants were stored at -80 C.
Measurement of tyrosine kinase (TK) activity
The assay procedure followed with few modifications that
detailed by Nichols and Morimoto (27). Briefly, the cell extract (25
µg) was incubated with 1 mg/ml of the TK substrate
poly(Glu4,Tyr1) in the
presence of 20 mM HEPES (pH 7.4), 5 mM
MnCl2, 10 mM
MgCl2, 10 µM ATP, and 5 µCi
[-32P]ATP in a final volume of 50 µl for
30 min at 30 C. The reaction mixture (40 µl) was pipetted onto
2.5 x 2.5-cm squares of Whatman 3MM Chr filter paper
(Clifton, NJ), which were washed four times with 10% trichloroacetic
acid and then dried. 32P incorporation was
measured in a liquid scintillation counter (1900 TR, Packard Instrument
Co., Meriden, CT).
Measurement of MAPK activity
MAPK activity was assayed by immune complex kinase assay as
described previously (26) with slight modifications. The cell lysate
was incubated with 2 µg anti-MAPK antibody (1:3000 dilution) for 120
min at 4 C. The immunoprecipitate was recovered by incubation with 30
µl protein A-Sepharose (Pharmacia Biotech, Uppsala,
Sweden) overnight at 4 C by centrifuging and washing three times with
cell lysis buffer and once with a kinase buffer containing 12.5
mM Tris-HCl (pH 7.4), 2 mM EGTA, 10
mM MgCl2, 1 mM
dithiothreitol, and 50 µg/ml PKA inhibitor. Immunoprecipitates were
incubated with 0.4 mg/ml myelinic basic protein substrate in a final
volume of 40 µl kinase buffer containing 50 µM ATP and
3 µCi [-32P]ATP for 30 min at 30 C. The
reaction was stopped by the addition of 10 µl 25% trichloroacetic
acid. The reaction mixture (25 µl) was spotted onto 2.5 x
2.5-cm squares of Whatman P81 photocellulose paper, which
were washed four times with 75 mM phosphoric acid, and then
dried. 32P incorporation was measured by liquid
scintillation.
Statistics
Data were expressed as the mean ± SEM, and
statistical comparison was performed using ANOVA, followed by Duncan’s
multiple range test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The
ADM (10-8 M)-induced increase in ZG
DNA synthesis was inhibited by both CGRP-(8–37) and ADM-(22–52) in a
concentration-dependent manner; the maximal effective concentration was
10-7 M (Fig. 2). The two antagonists were equipotent
(-log10 IC50 ±
SD, 9.4 ± 1.2 vs. 9.9 ± 1.4); the
efficacy of ADM-(22–52) was slightly higher than that of CGRP-(8–37),
but the difference was not significant (percent decrease ±
SD elicited by the maximal effective
concentration, 92 ± 15% vs. 79 ± 11%;
P > 0.05; n = 5).
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, upper panels), which,
in contrast, effectively counteracted the ACTH
(10-9 M)-induced
increase in DNA synthesis (Fig. 3, lower panels); nor 2) by
10-5 M U-73122,
10-4 M
InsP3S3, and
10-5 M calphostin C (Fig. 4, upper panels), which
conversely evoked a sizeable decrease in the Ang-II
(10-9 M)-evoked rise in
DNA synthesis (Fig. 4, lower panels). A concentration of
10-5 M AACOCF3,
indomethacin, or phenidone did not alter the ADM
(10-8 M)-induced increase
in the [3H]thymidine incorporation into
capsule-ZG DNA (Fig. 5). In contrast, the
effect of 10-8 M ADM was
concentration dependently blocked by either PD-98059 and U0216 or
tyrphostin-23 (Fig. 6).
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2-fold) and MAPK activity (70%) in ZG, but not ZF/R,
cells, and the effect was abolished by both 10-6
M CGRP-(8–37) and ADM-(22–52) (Fig. 7). The preincubation of cells with
10-5 M tyrphostin-23 abolished the
ADM effect on TK and MAPK activities (Fig.
8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our study also provides insight into the mechanisms underlying the ZG growth-promoting action of ADM. Our strategy was to employ selective and potent inhibitors of the main signaling pathways currently known to mediate the effect of the growth promoters and to study whether they block the proliferogenic action of the maximal effective concentration of ADM on rat ZG.
Neither the AC inhibitor SQ-22536 (29), the cAMP blocker Rp-cAMP-S (30), nor the selective PKA antagonist H-89 (24) affected ADM-enhanced DNA synthesis in rat ZG. The possibility that insufficient concentrations of the inhibitors were used is ruled out by the finding that these concentrations are able to efficaciously counteract the ZG proliferogenic effect of ACTH, which is mainly, if not exclusively, mediated by activation of the AC/PKA cascade (23, 31). These observations apparently conflict with the reported involvement of cAMP signaling in the proliferogenic effect of ADM on cultured fibroblasts (11, 12) and human tumor cell lines (32). However, they agree with findings obtained in rat VSMC cultures (10).
Other main transduction pathways involved in the ZG proliferogenic effect of Ang-II and endothelin-1 are the PLC-dependent (24, 33, 34) and PLA2-dependent (35) cascades. Our results indicate that neither of these pathways underlies the proliferogenic effect of ADM. In fact, the PLC inhibitor U-73122 (24), the PKC inhibitor calphostin C (21), and the IP3 blocker InsP3S3 (21), at concentrations that significantly impair the proliferogenic effect of Ang-II, did not affect the ADM-induced rise in ZG DNA synthesis. Likewise, a complete ineffectiveness was observed for the PLA2 inhibitor AACOCF3 (36), the cyclooxygenase (COX) inhibitor indomethacin (24), and the mixed COX/lipoxygenase inhibitor phenidone (24).
p42/p44 MAPKs are ubiquitous members of a family of serine/threonine kinases that are known to play a crucial role in cellular proliferation (for review, see Refs. 37, 38). MAPK cascade involves a series of cytoplasmic phosphorylations, where p21-activated kinase (PAK or MAPK kinase kinase kinase) activates Raf (MAPK kinase kinase), which, in turn, phosphorylates MEK1/2 (MAPK kinase), which eventually activates extracellular signal-regulated kinase-1/2. Activated extracellular signal-regulated kinases translocate to the nucleus, where they phosphorylate transcriptional factors that induce expression of the growth-associated nuclear protooncogene c-fos, leading to G0 to G1 and G2 to M transition of the cell cycle. Receptor TK plays a pivotal role in the MAPK cascade, because by binding to its agonists it activates Ras, a peptide belonging to a family of low mol wt GTP-binding proteins, which activates PAK and Raf.
Our present study provides strong evidence that ADM elicits rat ZG cell proliferation by activating the above-summarized TK-MAPK signaling pathway. This contention is based on the following findings: 1) the TK inhibitor tyrphostin-23 (24), the MEK1 inhibitor PD-98059 (39), and the MEK1/MEK2 noncompetitive antagonist U0126 (40) block the ADM-induced rise in ZG DNA synthesis; 2) none of these antagonists per se evokes any apparent effect on the basal rate of DNA synthesis, thereby making unlikely a possible nonspecific toxic effect on ZG cells; and 3) ADM selectively activates TK and MAPK activities in ZG cells, and the effect is blocked by either CGRP-(8–37) and ADM-(22–52) or tyrphostin-23.
Compelling evidence indicates that, in addition to TK, G protein-coupled receptors can activate MAPK cascade (for review, see Refs. 41, 42). PKC activates MAPK through a Ras-independent mechanism (43, 44), and PKA seems to exert an analogous action (45). Accordingly, ET-1 and Ang-II stimulate mitogenesis and MAPK activity of ZG cells through PLC/PKC-independent and -dependent pathways (24, 33), and cAMP induces MAPK activation and cell proliferation in many cell systems, including ZG cells (31), although the ability of ACTH to stimulates MAPK in rat ZG cells has been recently denied (46). There is also proof that cross-talk among TK-, PKC-, and PKA-dependent signaling pathways occurs in the regulation of MAPK activity in bovine adrenocortical cells (47, 48). However, our findings seem to rule out the possibility that PKA or PKC cascades play a relevant role in the ZG proliferogenic effect of ADM.
This contention agrees with the findings obtained by Iwasaki et al. (10) in cultured rat VSMC, thereby suggesting a specificity in the proliferogenic action of ADM compared with that of Ang-II, ET-1, and other growth-promoting agents, e.g. GnRH (42). At present, it is possible to provide only a tentative explanation for this finding. The Ang-II-induced Ras-dependent Raf-1 activation in bovine ZG cells has been recently reported to be negatively modulated by Ca2+ influx (49). However, the involvement of Ca2+ in GnRH-evoked MAPK activation has been clearly demonstrated (50, 51). Moreover, the rise in intracellular Ca2+ is known to play a role in the activation, not only of PKC, but also of PKA (for review, see Ref. 52). Hence, it could be hypothesized that the inhibitory effect of ADM on Ca2+ channels of ZG cells (15, 16, 17, 18, 19, 20, 21) may impair the activation of PKC and PKA, thereby making the growth-promoting action of ADM exclusively dependent on the TK-activated MAPK cascade.
According to the cell migration theory (for review, see Ref. 23), ZG in mammals is the cambium layer involved in adrenocortical cell renewal, which suggests that ADM may enhance and maintain the growth of the entire gland. The physiological relevance of the present findings remains to be assessed. However, in light of the well recognized inhibitory action of ADM on Ca2+-dependent agonist-stimulated aldosterone secretion, they stress the complex role played by this peptide in the regulation of adrenocortical physiology. The possibility that ADM may act as an aldosterone secretion regulator in adult growth-quiescent adrenals and, as previously suggested (32, 53), as a growth promoter in immature or tumorous glands merits study.
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Footnotes |
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Received October 18, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunits
activate mitogen-activated protein kinase via a novel protein kinase
C-dependent mechanism. J Biol Chem 271:1266–1269-melanocyte-stimulating hormone and corticotropin in
the rat adrenal cortex. Biochem J 305:433–438
T3–1). Mol Cell Endocrinol 122:33–50[CrossRef][Medline]
-T3–1 cell line:
differential roles of calcium and protein kinase C. Endocrinology 138:1673–1682This article has been cited by other articles:
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