autocrine function of adrenomedullin in peripheral nerves of rats

Brain Research 955 (2002) 64–71 www.elsevier.com / locate / brainres

Research report

Paracrine / autocrine function of adrenomedullin in peripheral nerves of rats b ¨ Charles E. Dumont a , *, Roman Muff a , Beat Fluhmann , Jan A. Fischer a , Walter Born a a

Research Laboratory for Calcium Metabolism and Departments of Orthopedic Surgery and Medicine, University of Zurich, Klinik Balgrist, Forchstrasse 340, 8008 Zurich, Switzerland b Human Nutrition and Health, Roche Vitamins Ltd, 4070 Basel, Switzerland Accepted 26 June 2002

Abstract The presence of adrenomedullin (AM) and of an AM receptor were investigated in highly enriched primary cultures of Schwann cells and perineural fibroblasts of newborn and adult rats. AM was released into the conditioned medium of adult perineural fibroblasts (17496629 pgeq / 10 5 cells per 24 h). mRNA encoding AM was also predominantly expressed in adult perineural fibroblasts. mRNA encoding the calcitonin receptor-like receptor (CRLR) and the receptor-activity-modifying proteins (RAMP) 1, -2 and -3 were demonstrated in all the primary cells, but the levels of RAMP1 mRNA relative to 18s rRNA were 10-fold lower than those of CRLR and RAMP2 and -3 encoding mRNA. The results are consistent with the expression of CRLR / RAMP2 and CRLR / RAMP3 heterodimeric AM receptors in all the primary cells examined. AM stimulated cAMP accumulation in newborn (EC 50 0.6260.29 nM) and adult (EC 50 0.4560.03 nM) rat Schwann cells and in newborn (EC 50 0.7960.50 nM) and adult (EC 50 1.0660.72 nM) rat perineural fibroblasts. The EC 50 of calcitonin gene-related peptide stimulated cAMP production was 93- to 100-fold higher than those of AM in the four types of primary cells studied. The co-expression of AM and its receptor in perineural fibroblasts and the expression of an AM receptor in Schwann cells suggest autocrine and / or paracrine modes of action of AM in peripheral nerves.  2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Glia and other non-neuronal cells Keywords: Adrenomedullin; Calcitonin receptor-like receptor; Receptor-activity-modifying protein; Schwann cell; Fibroblast; Cell interaction

1. Introduction Human and rat adrenomedullin (AM) are 52- and 50amino acid polypeptides belonging to the calcitonin (CT) family of peptides that also includes CT, a- and b-CT gene-related peptide (CGRP) and amylin [31]. AM, CGRP and amylin share six amino acid ring structures, linked by disulfide bonds between cysteine residues, and amidated C-termini, both required for the biological activity of the peptides. AM and CGRP are potent vasodilatory and

* Corresponding author. Present address: Universitatsklinik ¨ Balgrist, ¨ Forchstrasse 340, 8008 Zurich, Switzerland. Tel.: 141-1-386-3075; fax: 141-1-386-1609. E-mail address: [email protected] (C.E. Dumont).

hypotensive peptides that interact with receptors on endothelial and vascular smooth muscle cells [22]. Adrenomedullin was first isolated from a pheochromocytoma, but subsequently found in the majority of the tissues examined, consistent with a wide range of biological actions [11,14]. In the brain, immunoreactive AM is widely distributed. It was recognized in neurons, astrocytes and glial cell tumors [23,25–27,29], and AM encoding mRNA was increased in interferon-g treated rat astrocytes [15]. AM stimulated the proliferation of several tumor cell lines including those of neuroblastoma or glioblastoma origin [18,21]. Auto- and / or paracrine modes of action, neutralized with antibodies to AM, have been proposed [21]. Mitogenic activity of AM was also observed in AM producing retinal pigment epithelial cells [28]. AM-

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03365-6

C.E. Dumont et al. / Brain Research 955 (2002) 64–71

provoked proliferation of Swiss 3T3 fibroblasts is mediated by cAMP/ PKA dependent signal transduction [32]. In rat renal mesangial cells, on the other hand, AM inhibited [ 3 H]thymidine incorporation and suppressed proliferation of quiescent and PDGF stimulated cells [6,24]. Adrenomedullin receptors linked to cAMP formation have been revealed in mouse and rat fibroblast cell lines, in rat astrocytes in primary culture and in the human oligodendroglial cell line KG1C [7,13,30,33]. An initially orphan CT receptor-like receptor (CRLR) of the B family of G protein-coupled receptors with seven transmembrane domains requires co-expression of associated receptor-activity-modifying proteins (RAMP) for functional expression [17]. A CRLR / RAMP1 complex is a CGRP receptor. However, with RAMP2 or -3 the CRLR forms AM receptors [3,8,12]. Along these lines, RT-PCR analysis revealed expression of mRNA encoding the CRLR and RAMP1, -2 and -3 in the human oligodendroglial KG1C cell line [30]. Both the CLRL / RAMP1 and -2 defined CGRP and AM receptors are coupled to cAMP production [3,17]. Perineural fibroblasts and Schwann cells are non-neuronal resident cells in peripheral nerves required for the formation of the extracellular matrix, and the basal lamina and myelin sheath, respectively (for review see Ref. [9]). Both Schwann cells and fibroblasts proliferate after nerve injury, and Schwann cells remain the predominant cell type in vacated endoneural tubes. Local interactions among neurons, Schwann cells and perineural fibroblasts are critical for proper development and regeneration of the peripheral nervous system. Here, we have demonstrated the release of AM from perineural fibroblasts, and have identified AM receptors in Schwann cells and perineural fibroblasts. Taken together the results imply local signaling of AM in perineural fibroblasts and Schwann cells of peripheral nerves.

2. Materials and methods

2.1. Materials Rat (r) AM and rCGRP were supplied by Phoenix Pharmaceuticals (Belmont, CA, USA). Recombinant heregulin-b1 was donated by M. Sliwkowski (Genentech, South San Francisco, CA, USA). Plastic ware for tissue culture was purchased from Becton Dickinson (Basel, Switzerland) and culture media, fetal calf serum (FCS) and B27 supplemented mixture from Life Technologies (Basel, Switzerland). Trypsin was purchased from Worthington Biochemicals (Lakewood, NJ, USA) and collagenase and dispase from Roche Diagnostics (Rotkreuz, Switzerland). Other chemicals and reagents were purchased from Fluka (Buchs, Switzerland) at the highest grade available.

65

2.2. Tissue culture Newborn rat Schwann cells and perineural fibroblasts were prepared according to Brockes et al. [2]. Briefly, 3-day-old Wistar rats were decapitated and both sciatic nerves and dorsal root ganglions were aseptically dissected and kept in Leibovitz’s L15 medium. The sciatic nerves were minced and incubated together with dorsal root ganglion cells in L15 medium containing 1.5 mg / ml collagenase and 0.25 mg / ml trypsin. Subsequently, the tissue suspension was triturated with a Pasteur pipette and 18- and 24-gauge needles and passed through a 70-mm mesh nylon filter. The cells were preplated for 30 min in non-coated tissue culture flasks and adherent cells were newborn rat perineural fibroblasts. They were maintained in culture in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 100 U / ml penicillin, 100 mg / ml streptomycin and 2 mM L-glutamine (seeding medium). The supernatant containing enriched Schwann cells was transferred to another non-coated flask and the remaining fibroblasts were eliminated by repetitive additions of 10 25 M cytosine-b-D-arabinofuranoside on days 1, 2 and 3, and subsequent complement lysis was carried out in Leibovitz’s L15 medium containing 0.5% bovine serum albumin (L15–BSA). The cells were treated for 30 min at 4 8C with rabbit Thy 1.1 antiserum (Cedarlane, Ont., Canada) at 1:32 final dilution. They were then washed with L15–BSA and incubated for 30 min at 37 8C with a 1:10 dilution of rabbit complement (Cedarlane) in L15–BSA. Surviving Schwann cells were seeded in polyL-lysine coated flasks in seeding medium containing 10% heat-inactivated FCS, 8 nM heregulin and 2 mM forskolin. Adult rat Schwann cells and perineural fibroblasts were prepared according to a protocol of Casella et al. [4]. Adult female rats (250–280 g) were killed by CO 2 asphyxiation. The two sciatic nerves were aseptically dissected and kept in Leibovitz’s L15 medium. Then 2–5-mm long nerve fascicle segments were prepared under 2.53 magnification and ten explants were pooled in 35-mm petri dishes and cultured in seeding medium containing 8 nM heregulin and 2 mM forskolin. The medium was changed every 3 days. Adherent cells, predominantly perineural fibroblasts, were passaged two to three times without growth factors to eliminate contaminating Schwann cells before use. On day 14, the nerve explants, containing Schwann cells, were transferred to a new culture dish and incubated overnight in DMEM containing 15% FCS, 0.25% dispase, 0.05% collagenase and 25 mM HEPES. On the following day the nerve fragments were triturated with a Pasteur pipette and a 24-gauge needle. The cell suspensions containing the adult rat Schwann cells were passed through a 70-mm mesh nylon filter and cultured on laminin-coated 35-mm petri dishes in seeding medium containing 8 nM heregulin and 2 mM forskolin. The Rat-2 cell line (CRL 1764) of fibroblast origin was

66

C.E. Dumont et al. / Brain Research 955 (2002) 64–71

obtained from the European Collection of Animal Cell Culture (Porton Down, Salisbury, UK) and grown in tissue culture dishes under the conditions used for the perineural fibroblasts. All the cells were cultured at 37 8C in a humidified atmosphere of 95% air and 5% CO 2 .

2.3. Immunofluorescence staining Monoclonal antibodies to low affinity nerve growth factor receptor (anti-NGFR) or to Thy 1.1 (anti-Thy 1.1) (Chemicon, Temecula, CA, USA) were used for the immunocytochemical characterization of Schwann cells or fibroblasts. First 100 cells / ml were seeded into slide flasks and grown under the culture conditions described for the individual cell types. Then after 3 days, the Schwann cells and the fibroblasts were fixed with 4 and 2% freshly prepared paraformaldehyde, respectively. Blocking was carried out for 30 min at room temperature in phosphatebuffered saline (PBS), pH 7.4 containing 0.05% sodium azide, 2% BSA and 2% goat serum (blocking buffer). Next 0.1% Triton X-100 was added to the blocking buffer for Schwann cell preparations. After blocking, the cells were incubated overnight at 4 8C with mouse anti-NGFR (1:15) or mouse anti-Thy 1.1 (1:1500) in blocking buffer. Subsequently, the cells were washed three times in PBS and incubated for 1 h at room temperature with a 1:75 final dilution of tetramethyl rhodamine isothiocyanate (TRITC)conjugated rabbit anti-mouse IgG (Dako, Zug, Switzerland) in PBS, containing 0.05% sodium azide, 0.01% Triton X-100, and 1% rat serum. Negative control staining of cells was carried out with TRITC-conjugated rabbit anti-mouse IgG in the absence of the first antibodies. After washing in PBS, the cells were mounted in Immu-Mount (Shandon, Pittsburgh, PA, USA) and viewed with a Nikon E600 microscope equipped with a mercury lamp, a Y-Fl epifluorescence attachment and a G-2A filter block (Nikon, ¨ Kusnacht, Switzerland).

2.4. Fluorescent activated cell sorting ( FACS) Immunostaining of living Schwann cells and of fibroblasts was performed with the NGFR and Thy 1.1 antibodies, respectively, at the dilution described for immunostaining. The cells were resuspended in 10% FCS in PBS (blocking solution), and counted. 10 6 cells were incubated for 1 h at 4 8C with the first antibodies. The cells were subsequently washed twice and then incubated in the blocking solution for 1 h at 4 8C with a 1:100 dilution of an FITC-conjugated polyclonal anti-mouse IgG (Pharmingen, Basel, Switzerland). The cells were washed twice in blocking buffer and fixed with 1% formaldehyde at 4 8C and analyzed with a CellCalibour FACS emitting an argon laser beam at 495 nm (Becton Dickinson, Basel, Switzerland). 10 5 cells were analyzed per tube, and the data were processed using the CellQuest version 1.2 software (Bec-

ton Dickinson, Immunocytometry Systems, Basel, Switzerland). A combination of forward scatter (FSC) and sideways light scatter (SSC) was used to gate cells. The fluorescence distribution was displayed as single histogram for fluorescence measured at 530 nm (FL-height). The signals were acquired in a linear mode for FSC and in a logarithmic mode for SSC and FL-height. The threshold levels were set with negative control cells.

2.5. Adrenomedullin measurement The cells were seeded in 25-cm 2 flasks and cultured until they reached confluence. The medium was then removed and the cells incubated in serum-free DMEM supplemented with B27 supplement mixture, 100 U / ml penicillin, 100 mg / ml streptomycin and 2 mM Lglutamine. After 2 days the medium was collected, boiled for 5 min and kept at 220 8C until analyzed. For peptide extraction, the media were equilibrated with 0.1% trifluoroacetic acid (TFA) overnight at 4 8C and subsequently applied to Sep-Pak C 18 cartridges (Millipore, Milford, MA, USA). The absorbed peptides were eluted with 80% acetonitrile in 0.1% TFA. A 50-ml aliquot of radioimmunoassay buffer (0.1 M sodium phosphate, pH 7.4, 0.05 M NaCl, 0.1% Triton X-100, 1 ng / ml BSA) was added and the extracts evaporated in a Speedvac concentrator (Savant Instruments, Hicksville, NY, USA). They were redissolved in distilled H 2 O. Overall recoveries of [ 125 I]rAM added to individual medium samples before extraction were 80.268.5% (mean6S.D.) [33]. Concentrations of AM in medium extracts were measured by radioimmunoassay with a rAM antiserum (Phoenix Pharmaceuticals, Belmont, CA, USA; 1:2000 final dilution). Different dilutions of the samples were incubated with the antiserum for 3 days at 4 8C in the absence of radioligand followed by 3 days with 7.5310 5 Bq of [ 125 I]rAM (743 10 12 Bq / mmol). Separation of antibody-bound from free AM was carried out with dextran-coated charcoal. [ 125 I]rAM in supernatants and charcoal sediments was measured in a g-counter (Kontron, Zurich, Switzerland). The ratio of antibody-bound and free [ 125 I]rAM was calculated by correcting for non-antibody-bound radioactivity in the supernatant in the absence of rAM antiserum. The limit of detection of synthetic rAM used as a standard was 40 pg / tube and up to 0.5 mg / tube of synthetic rCGRP or CT inhibited [ 125 I]rAM binding by less than 5% of maximal binding.

2.6. Quantitative RT-PCR Total RNA of 5310 6 to 10 7 cells was isolated using the RNeasy kit from Qiagen (Basel, Switzerland). The first strand cDNA was synthesized from total RNA by reverse transcription with the SuperScript II姠 Reverse Transcriptase Kit (Life Technologies, Basel, Switzerland) and

C.E. Dumont et al. / Brain Research 955 (2002) 64–71

random hexamers. The cDNA was amplified using the multiplex real time quantitative TaqMan姠 technology in a 7700 Sequence Detector with the Universal Master Mix (PE Biosystems, Foster City, CA, USA) as described [10]. PCR-primers (Life Technologies, Basel, Switzerland) and TaqMan姠-probes (Integrated DNA Technologies, Coralville, IA or PE Biosystems) were: CRLR (59-CAGGATCCCATTCAACAAGGA-39, 59-TTCCAGCATAGCCATCCGT-39 and 59-FAM-AAGGCCTTTACTGCAACAGAACCTGG-Tamra-39), RAMP1 (59-GGCTCTGCTTGCCATGG-39, 59-CGGCAGGCAGTGACCA-39 and 59-FAM-CCTCTGGCTGCTGCTGGCTCAT-Tamra-39), RAMP2 (59-CCCTCCGCTGTTACTGCTG-39, 59-GATTGATTCAGGGACTCCGG-39 and 59-FAM-TGCTGCTGGGCGCTGTCTCA-Tamra-39), RAMP3 (59-GCTGCACCTTCTCCCTCTG-39, 59-CATTGCAACCGCATACTTGG-39 and 59-FAM-TGCTGCTGCTTTGTGGTGAGTGTG-Tamra-39), AM (59-TTGGGCTCCAGGACAAGC-39, 59-TGGGCTGTGCTCTGAGTGC-39 and 59-FAM-AGCACGTCTAGCACCCCACAACC-Tamra-39), with 18s rRNA as reference gene (59-CGGCTACCACATCCAAGGAA-39, 59-GCTGGAATTACCGCGGCT-39 and 59-VICTGCTGGCACCAGACTTGCCCTC-Tamra-39). mRNA levels were measured in triplicates and normalized to 18s rRNA levels. Gene expression was calculated using the DCT method according to the manufacturer’s protocol [16].

67

flat with a polygonal shape and stained for the Thy 1.1 marker protein. Both newborn and adult rat perineural fibroblasts were enriched after three passages to between 95 and 100%. Newborn Schwann cells were 98% pure after complement lysis, and adult rat Schwann cells were enriched after two passages to between 95 and 97%. FACS analysis revealed homogeneous cell populations of both Schwann cells (Fig. 2) and fibroblasts (not shown).

3.2. Adrenomedullin release from perineural fibroblasts Continuous treatment of fibroblasts with dexamethasone has been shown to increase immunoreactive rAM in the incubation medium [19]. Here, the release of AM from control Rat-2 fibroblasts was 91629 pgeq / 10 5 cells per 24 h (n55); the amounts were increased 5.660.4-fold in the presence of 10 26 M dexamethasone (P,0.01; n54). Also 17506630 pgeq / 10 5 cells per 24 h immunoreactive AM were released from adult rat perineural fibroblasts (n53). A small increase to 24306780 pgeq / 10 5 cells per 24 h was observed with 10 26 M dexamethasone (P,0.2). AM was below 20 pgeq / 10 5 cells per 24 h in conditioned medium of newborn and adult rat Schwann cells and of newborn perineural fibroblasts in the absence and presence of 10 26 M dexamethasone (n53).

3.3. Expression of mRNA encoding AM, CRLR, and RAMP1, -2 and -3

2.7. Cyclic AMP formation Schwann cells and perineural fibroblasts were grown to confluence in 24-well plates. Growth factors were withdrawn 24 h before stimulation. cAMP was measured by radioimmunoassay as described [20].

2.8. Statistical analysis Results are means6standard error of the mean (S.E.M.). Differences between means were determined by one-way analysis of variance (ANOVA). A value of P,0.05 was considered as statistically significant. The half-maximal effective dose (EC 50 ) was calculated by non-linear regression analysis using Fig.P 6.0 software (Biosoft, Cambridge, UK).

3. Results

3.1. Cell characterization Potential cross-contamination of fibroblasts and Schwann cells kept in primary culture was analyzed by immunocytochemistry and FACS. Adherent Schwann cells displayed characteristic bipolar spindle-like shapes and stained for the Schwann cell specific immunoreactive low affinity NGF-receptor (Fig. 1). Perineural fibroblasts were

Messenger RNA encoding AM was predominantly expressed in the Rat-2 fibroblast cell line and the adult perineural fibroblasts (Fig. 3). In the two cell types the cellular content of mRNA encoding AM corresponded to the amounts of immunoreactive AM released into the medium. Rat-2 cells with AM receptors predominantly expressed CRLR and RAMP2 encoding mRNA [7]. The levels of mRNA encoding RAMP3 were 100-fold lower and those of RAMP1 were undetectable under the experimental conditions used. CRLR and RAMP2 and -3 encoding mRNA were expressed in primary newborn and adult rat Schwann cells and in perineural fibroblasts. RAMP1 encoding mRNA was also present in all primary cells, but the levels relative to 18s rRNA were at least ten times lower than those of CRLR and RAMP2 and -3 encoding mRNA. The levels of CRLR, RAMP2 and -3 encoding mRNA relative to 18s rRNA were comparable in newborn and adult Schwann cells, and higher in newborn and adult perineural fibroblasts. Taken together, the results indicate the expression of CRLR / RAMP2 and CRLR / RAMP3 AM receptors in all the cell types investigated.

3.4. Stimulation of cAMP formation by AM and CGRP The functional expression of AM receptors in primary Schwann cells and perineural fibroblasts was revealed by AM evoked stimulation of cAMP production (Fig. 4). In

68

C.E. Dumont et al. / Brain Research 955 (2002) 64–71

Fig. 1. Immunocytochemical identification of newborn (A, B) and adult (E, F) rat Schwann cells and of newborn (C, D) and adult (G, H) perineural fibroblasts. The cells were stained with antibodies to the Schwann cell-specific low affinity NGFR (A, C, E, G) and the fibroblast-specific Thy 1.1 (B, D, F, H). Scale bar represents 40 mm.

newborn and adult Schwann cells a maximal 8-fold stimulation by AM of cAMP formation was obtained with EC 50 of 0.6260.29 nM (n55) and 0.4560.03 nM (n53), respectively. In newborn and adult perineural fibroblasts rAM revealed a maximal 10–20-fold stimulation of cAMP formation with EC 50 of 0.7960.50 nM (n53) and 1.0660.72 nM (n53), respectively. rCGRP, on the other hand, stimulated cAMP production with a 93-fold higher EC 50 of 42.1613.5 nM than AM in adult rat Schwann cells. AM was also over 100-fold more potent than CGRP in Schwann cells of newborn rats and in perineural fibroblasts of both newborn and adult animals.

4. Discussion An understanding of mechanisms involved in the development and regeneration of peripheral nerves is a prerequisite for successful reconstruction of their lesions. Here, AM was released from adult perineural fibroblasts. The expression of AM receptors in the same cells and in

newborn and adult Schwann cells suggests local regulatory roles of AM in peripheral nerves. Endothelial and smooth muscle cells of blood capillaries surrounding peripheral nerves are potential additional sources of AM interacting locally with AM receptors revealed here in Schwann cells and perineural fibroblasts. In the central nervous system direct dose-dependent excitation of neurons by AM has been observed in the area prostrema [1]. In peripheral nerves direct actions of AM on neurons, e.g. in nerve injury, remain to be demonstrated. Rat-2 cells of fibroblast origin release AM and express AM receptor [7]. The release of AM is increased with dexamethasone (this study; [7]). However, in the present study, AM was undetectable in the medium of newborn perineural fibroblasts. Interestingly, the amounts released into the medium were 19 times higher in adult perineural fibroblasts than in the Rat-2 fibroblast cell line. Quantitative RT-PCR analysis of AM encoding mRNA revealed comparable results. The results indicate developmental regulation of AM expression in perineural fibroblasts. The presence of specific AM receptors, not recognizing

C.E. Dumont et al. / Brain Research 955 (2002) 64–71

69

Fig. 2. FACS analysis of newborn (top panels) and adult (bottom panels) rat Schwann cells. Histograms combining FSC and SSC (left panels) and for fluorescence distribution of control cells stained with the second rabbit anti-mouse antibodies alone (middle panels) and of Schwann cells stained with mouse anti-NGFR and rabbit anti-mouse IgG (right panels).

Fig. 3. Expression of mRNA encoding AM (j), and RAMP1 (l), -2 (o), -3 (9), the CRLR (h) normalized to 18s rRNA in Rat-2 fibroblasts (1), newborn rat Schwann cells (2) and perineural fibroblasts (3), and adult rat Schwann cells (4) and perineural fibroblasts (5). Results are means6S.E.

70

C.E. Dumont et al. / Brain Research 955 (2002) 64–71

Fig. 4. cAMP formation in rat Schwann cells and perineural fibroblasts. Newborn (a, b) and adult (c, d) Schwann cells (a, c) and perineural fibroblasts (b, d) were treated for 15 min at 37 8C with rAM (d) or raCGRP (s) in the presence of 1 mM IBMX. Results are means6S.E.M.

CGRP to any great extent, was revealed in primary Schwann cells and in perineural fibroblasts of newborn and adult rats. The EC 50 of cAMP formation of AM was 1 nM and that of CGRP a 100-fold higher. This is in agreement with the stimulation of cAMP production by over 10 nM CGRP in newborn rat Schwann cells and fibroblasts from sciatic nerves [5]. Related AM receptors were described in cultured cells co-expressing the CRLR and RAMP2 [3,12]. Quantitative RT-PCR analysis of total RNA isolated from control Rat-2 cells and from the four types of primary cells revealed comparable levels of RAMP2 and CRLR encoding mRNA when normalized to 18s rRNA. Interestingly, the relative expression levels of RAMP3 mRNA exceeded those of RAMP2 mRNA in all primary cells, but they were 100-fold lower in Rat-2 cells. Along these lines, coexpression of the human CRLR and human RAMP3 in HEK 293T cells and of the rat CRLR and mouse RAMP3 in COS-7 cells revealed AM- and mixed-type AM / CGRP receptors, respectively [8,12]. The levels of RAMP1 mRNA normalized to 18s rRNA were 10-fold lower than those of CRLR, RAMP2 and -3 encoding mRNA in all primary cell types. RAMP1 encoding mRNA was undetectable in Rat-2 cells. Taken together the results indicate predominant expression of CRLR / RAMP2 and CRLR / RAMP3 AM receptors in Rat-2 cells and in primary Schwann cells and perineural fibroblasts of newborn and adult rats. The expressed AM receptor proteins remain to be identified once well characterized antibodies to the cloned CRLR and RAMP of the rat become available.

In conclusion, perineural fibroblasts of adult rats in primary culture release AM into the pericellular medium. High affinity AM receptors linked to cAMP production have been identified in perineural fibroblast and in Schwann cells of newborn and adult rats. They presumably correspond to the CRLR / RAMP2 and CRLR / RAMP3 complexes encoded by the corresponding mRNA found to be co-expressed at comparable levels in the individual cell types. The co-existence of AM and its receptors in nonneuronal cells enriched from peripheral nerves suggests local regulatory actions of this peptide likely important for nerve regeneration.

Acknowledgements The authors thank Dr M. Sliwkowski (Genentech, South San Francisco, CA, USA) for the heregulin. This work was supported by the Swiss National Science Foundation, the University of Zurich and the Schweizerische Verein Balgrist.

References [1] M.A. Allen, A.V. Ferguson, In vitro recordings from area postrema neurons demonstrate responsiveness to adrenomedullin, Am. J. Physiol. 270 (1996) R920–R925. [2] J.P. Brockes, K.L. Fields, M.C. Raff, Studies on cultured rat

C.E. Dumont et al. / Brain Research 955 (2002) 64–71

[3]

[4]

[5]

[6]

[7]

[8]

[9] [10]

[11] [12]

[13]

[14]

[15]

[16]

[17]

[18]

Schwann cells. I. Establishment of purified populations from cultures of peripheral nerve, Brain Res. 165 (1979) 105–118. ¨ ¨ N. Buhlmann, K. Leuthauser, R. Muff, J.A. Fischer, W. Born, A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1, Endocrinology 140 (1999) 2883– 2890. G.T. Casella, R.P. Bunge, P.M. Wood, Improved method for harvesting human Schwann cells from mature peripheral nerve and expansion in vitro, Glia 17 (1996) 327–338. L. Cheng, M. Khan, A.W. Mudge, Calcitonin gene-related peptide promotes Schwann cell proliferation, J. Cell Biol. 129 (1995) 789–796. E.N. Chini, E. Choi, J.P. Grande, J.C. Burnett, T.P. Dousa, Adrenomedullin suppresses mitogenesis in rat mesangial cells via cAMP pathway, Biochem. Biophys. Res. Commun. 215 (1995) 868–873. H.A. Coppock, A.A. Owji, C. Austin, P.D. Upton, M.L. Jackson, J.V. Gardiner, M.A. Ghatei, S.R. Bloom, D.M. Smith, Rat-2 fibroblasts express specific adrenomedullin receptors, but not calcitonin-generelated-peptide receptors, which mediate increased intracellular cAMP and inhibit mitogen-activated protein kinase activity, Biochem. J. 338 (1999) 15–22. N.J. Fraser, A. Wise, J. Brown, L.M. McLatchie, M.J. Main, S.M. Foord, The amino terminus of receptor activity modifying proteins is a critical determinant of glycosylation state and ligand binding of calcitonin receptor-like receptor, Mol. Pharmacol. 55 (1999) 1054– 1059. S.Y. Fu, T. Gordon, The cellular and molecular basis of peripheral nerve regeneration, Mol. Neurobiol. 14 (1997) 67–116. M. Heim, J. Johnson, F. Boess, I. Bendik, P. Weber, W. Hunziker, B. ¨ Fluhmann, Phytanic acid, a natural peroxisome proliferator-activated receptor (PPAR) agonist, regulates glucose metabolism in rat primary hepatocytes, FASEB J. 16 (2002) 718–722. J.P. Hinson, S. Kapas, D.M. Smith, Adrenomedullin, a multifunctional regulatory peptide, Endocr. Rev. 21 (2000) 138–167. K. Husmann, P.M. Sexton, J.A. Fischer, W. Born, Mouse receptoractivity-modifying-proteins-1, -2, -3: amino acid sequence, expression and function, Mol. Cell. Endocrinol. 162 (2000) 35–43. Y. Isumi, N. Minamino, T. Katafuchi, M. Yoshioka, T. Tsuji, K. Kangawa, H. Matsuo, Adrenomedullin production in fibroblasts: its possible function as growth regulator of Swiss 3T3 cells, Endocrinology 139 (1998) 2552–2563. K. Kitamura, K. Kangawa, M. Kawamoto, Y. Ichiki, S. Nakamura, H. Matsuo, T. Eto, Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma, Biochem. Biophys. Res. Commun. 192 (1993) 553–560. W. Kuchinke, R.P. Hart, G.M. Jonakait, Identification of mRNAs regulated by interferon-gamma in cultured rat astrocytes by PCR differential display, Neuroimmunomodulation 2 (1995) 347–355. K.J. Livak, S.J. Flood, J. Marmaro, W. Giusti, K. Deetz, Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization, PCR Methods Appl. 4 (1995) 357–362. L.M. McLatchie, N.J. Fraser, M.J. Main, A. Wise, J. Brown, N. Thompson, R. Solari, M.G. Lee, S.M. Foord, RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor, Nature 393 (1998) 333–339. M.J. Miller, A. Martinez, E.J. Unsworth, C.J. Thiele, T.W. Moody, T. Elsasser, F. Cuttitta, Adrenomedullin expression in human tumor

[19] [20]

[21]

[22] [23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

71

cell lines. Its potential role as an autocrine growth factor, J. Biol. Chem. 271 (1996) 23345–23351. N. Minamino, K. Kikumoto, Y. Isumi, Regulation of adrenomedullin expression and release, Microsc. Res. Tech. 57 (2002) 28–39. J. Moran, W. Hunziker, J.A. Fischer, Calcitonin and calcium ionophores: cyclic AMP responses in cells of a human lymphoid line, Proc. Natl. Acad. Sci. USA 75 (1978) 3984–3988. L. Ouafik, S. Sauze, F. Boudouresque, O. Chinot, C. Delfino, F. Fina, V. Vuaroqueaux, C. Dussert, J. Palmari, H. Dufour, F. Grisoli, P. Casellas, N. Brunner, P.M. Martin, Neutralization of adrenomedullin inhibits the growth of human glioblastoma cell lines in vitro and suppresses tumor xenograft growth in vivo, Am. J. Pathol. 160 (2002) 1279–1292. W.K. Samson, Adrenomedullin and the control of fluid and electrolyte homeostasis, Annu. Rev. Physiol. 61 (1999) 363–389. F. Satoh, K. Takahashi, O. Murakami, K. Totsune, M. Sone, M. Ohneda, H. Sasano, T. Mouri, Immunocytochemical localization of adrenomedullin-like immunoreactivity in the human hypothalamus and the adrenal gland, Neurosci. Lett. 203 (1996) 207–210. K. Segawa, K. Minami, T. Sata, A. Kuroiwa, A. Shigematsu, Inhibitory effect of adrenomedullin on rat mesangial cell mitogenesis, Nephron 74 (1996) 577–579. J. Serrano, L.O. Uttenthal, A. Martinez, A.P. Fernandez, J. Martinez de Velasco, D. Alonso, M.L. Bentura, M. Santacana, J.R. Gallardo, R. Martinez-Murillo, F. Cuttitta, J. Rodrigo, Distribution of adrenomedullin-like immunoreactivity in the rat central nervous system by light and electron microscopy, Brain Res. 853 (2000) 245–268. K. Takahashi, F. Satoh, E. Hara, M. Sone, O. Murakami, T. Kayama, T. Yoshimoto, S. Shibahara, Production and secretion of adrenomedullin from glial cell tumors and its effects on cAMP production, Peptides 18 (1997) 1117–1124. K. Takahashi, M. Nakayama, K. Totsune, O. Murakami, M. Sone, T. Kitamuro, A. Yoshinoya, S. Shibahara, Increased secretion of adrenomedullin from cultured human astrocytes by cytokines, J. Neurochem. 74 (2000) 99–103. T. Udono, K. Takahashi, M. Nakayama, O. Murakami, Y.K. Durlu, M. Tamai, S. Shibahara, Adrenomedullin in cultured human retinal pigment epithelial cells, Invest. Ophthalmol. Vis. Sci. 41 (2000) 1962–1970. Y. Ueta, K. Kitamura, T. Isse, I. Shibuya, N. Kabashima, S. Yamamoto, K. Kangawa, H. Matsuo, T. Eto, H. Yamashita, Adrenomedullin-immunoreactive neurons in the paraventricular and supraoptic nuclei of the rat, Neurosci. Lett. 202 (1995) 37–40. Y. Uezono, E. Nakamura, Y. Ueda, I. Shibuya, Y. Ueta, H. Yokoo, T. Yanagita, Y. Toyohira, H. Kobayashi, N. Yanagihara, A. Wada, Production of cAMP by adrenomedullin in human oligodendroglial cell line KG1C: comparison with calcitonin gene-related peptide and amylin, Mol. Brain Res. 97 (2001) 59–69. S.J. Wimalawansa, Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: a peptide superfamily, Crit. Rev. Neurobiol. 11 (1997) 167–239. D.J. Withers, H.A. Coppock, T. Seufferlein, D.M. Smith, S.R. Bloom, E. Rozengurt, Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells, FEBS Lett. 378 (1996) 83–87. U. Zimmermannn, J.A. Fischer, K. Frei, A.H. Fischer, R.K. Reinscheid, R. Muff, Identification of adrenomedullin receptors in cultured rat astrocytes and in neuroblastoma3glioma hybrid cells (NG108-15), Brain Res. 724 (1996) 238–245.