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Atrial natriuretic peptide in lymphoid organs of various species
Camp. Biochem. Physiol. Vol. 96A. No. 4, pp. 459463, Printed in Great Britain
ATRIAL
1990
0300-9629/90 $3.00 + 0.00 0 1990 Pergamon Press plc
NATRIURETIC PEPTIDE IN LYMPHOID OF VARIOUS SPECIES
ORGANS
ANGELIKA M. VOLLMAR and R~~DIGERSCHULZ Institute of Pharmacology, Miinchen 22, Federal
Toxicology and Pharmacy, University of Munich, Kiiniginstr. 16, D-8000 Republic of Germany. Telephone (089) 2180-2663. Fax (089) 34 23 16 (Receitied 5 January 1990)
Evidence for the occurrence of atrial natriuretic peptide (ANP) in various lymphoid organs of different species (rat, mouse, pig, chicken) is provided. 2. ANP precursor material (I-126) as well the ohvsioloaicallv active ANP (99-126). were identified bv chromatog;aphic analysis and‘RIA in extracts of’thymus,spleeh and lymph nodes of’rat, mouse and pig. 3. mRNA coding for ANP was demonstrated both in the thymus and in isolated thymocytes of these species. Furthermore, mRNA for ANP was detected in spleen and lymph nodes (rat and pig). 4. The bursa of Fabricius, thymus glands and spleen of chickens were also shown to express mRNA coding for ANP. 5. These findings provide a firm basis for a link of ANP to the immune system, a novel aspect of possible biological functions of this peptide. Abstract-l.
INTRODUCTION
weeks) and chickens (3-4 day old, white leghorn, strain A). All animals were killed by decapitation with the exception of pigs. which were iniected with T-61” euthanasia solution (Bay&, FRG). The thymus glands, spleen and lymph nodes (i.e. axial nodes, posterior and anterior cervical nodes, intestinal nodes) as well as the bursa of Fabricius of the chicken were excised and maintained at 0” or -70°C as appropriate, until biochemical analysis.
Atria1 natriuretic peptide (ANP) has been shown to be a potent diuretic, natriuretic and vasorelaxant hormone, and the heart myocytes represent the major, although not exclusive, site of synthesis and secretion of this peptide (for review, see Cantin and Genest, 1985). A high degree of conservation is seen in the structural organization of the ANP gene, as well as the amino acid sequences of ANP in various mammalian species (Nemer et al., 1984; Seidman et al., 1984). More recently, the presence of ANP has been demonstrated in birds (Miyata et al., 1988) and other submammalian vertebrates (Netchitailo et al., 1986; Sakato et al., 1988). The demonstration of ANP-like peptides in non-atria1 tissues, such as the brain and adrenals (McKenzie et al., 198.5; Zamir et al., 1986) has begun to draw attention to other possible biological roles of ANP, in addition to those already documented, e.g. the regulation of blood volume (see Needleman and Greenwald, 1986). Our own laboratory has shown the presence and synthesis of ANP-like material in lymphoid organs, like the thymus (Vollmar and Schulz, 1988) the spleen (Vollmar et al., 1989) and the intestine (Vollmar et al., 1988). We therefore suggested that ANP may be involved in immunological functions. We report here the identification of ANP-like material, and mRNA coding for ANP in a number organs of the immune system of various mammalian species and that of the domestic chicken. These new findings strengthen the concept of a link between ANP and the lymphoid system. MATERIALS
Tissue extraction Freshly removed tissue was immediately transfered to 0.1 N HCI (4°C) (1 g wet tissue/5 ml), homogenized with a polytron (I min, setting 8) and boiled for 1min in a microwave oven. After centrifugation (2O,OOOg, 20 min, 4°C) the clear supematant from which an aliquot was taken for protein determination (Bradford method) was run through a column filled with activated Amberlite XAD-4 adsorbent resin (500 mg/column, Serva, Heidelberg, FRG). ANP-like material was eluted with 80% acetonitrile in 0. I % trifluoroacetic acid (TFA), and subsequently lyophilized. Characterization and quantification of extracted ANP-like material IR-ANP was characterized as previously described (Vollmar et al., 1988). Briefly, an aliquot of the lyophilized tissue extract was dissolved in 200~1 0.1 M acetic acid, centrifuged and the clear supematant subjected to gel chromatography (Sephadex G-SO, Pharmacia LKB, Freiburg, FRG, column 9 x 1000 mm, flowrate 10 ml 0.1 M acetic acid per hour). Calibration of the column was carried out with bovine serum albumin (V,), vitamin B12 (V,), rat-pro-ANP (2-126) (Hoechst, Frankfurt, FRG) and ANP (99-126) (Novabiochem, Laufelfingen, Switzerland). For RP-HPLC analysis an aliquot of the extracts was redissolved in 100~1 0.1% TFA, centrifuged and loaded onto a HPLC C8 Ultrapore TM column (5pm, 4.6 x 750 mm, Beckman Instruments, FRG). Calibration was carried out with rat ANP fragments (99-123), (99-125). (99-126) (Novabiochem, Switzerland) and ANP (2-126) (Hoechst, Frankfurt, FRG). The peptides were eluted with a linear gradient of acetonitrile (20-55%. in 55 min) in 0. I % TFA (flowrate: 0.5 ml/min). Fractions were assayed for ANP by RIA.
AND METHODS
Animals Rats (Sprague-Dawley, male, 6 weeks), mice (BALB/c, male, 6-8 weeks), pigs (German country race, male, 1-3 459
A. M. VOLLMARand R. SCHULZ
460
IR-ANP was quantified by RIA after Sephadex G-50 gel filtration of crude extracts containing approximately equal amounts of protein. Values were corrected for recovery rates and crossreactivity of the antibody used (see below). Antisera and radioimmunoassay
Characteristics of the antiserum ‘Toni III’ have been recently described in detail (Vollmar and Schulz, 1988). Briefly, it was raised against human-ANP (99-126) and recognizes the carboxy-terminus of ANP (99-126). Antiserum ‘Toni III’ cross-reacts with ANP (2-126) and ratANP (99-l 26) to a degree of 30 and IOO%, respectively. The radioimmunoassay using ‘Toni III’ antiserum was performed as previously described (Arendt ei al., 1985) (999126) (Amersham, FRG). using ?-rat-ANP Measurement of extraction efficiency
A quantity (1 pmol) of synthetic rat-pro-ANP was added as internal standard to the various tissues and the extraction and chromatographic procedures were performed as described above. Concentration of IR-ANP in control tissue and tissue with exogenous ANP (22126) were determined by RIA (antiserum ‘Toni III’). The calculated recoveries amounted between 22 and 38%. Northern blot h~~r~dizutiffn
Total RNA was prepared from frozen (- 7OC) tissue by the LiCl-urea technique (Auffray and Rougeon, 1980). In some cases, mRNA was enriched by applying total RNA (I mg) to oligo dT celiulose columns (Boehringer, Mannheim, FRG). After quantification by absorption at 260 nm, various amounts of mRNA (up to SO kg) were denatured in 1 M glyoxal (pH 5), containing 50% DMSO and 10 mM sodium phosphate buffer (pH 6.8) and electrophoresed on a 1.2% agarose gel. The mRNA was then blotted onto Nytran l3N membranes (Schleicher & Schuell, FRG) and the transfer checked by staining the gel with ethidium bromide. A UV source was used for visualization. For preparation of an ANP-specific cRNA hybridization probe, a 580 base Pst I-restricted ANP cDNA fragment (gift from K. Bloch and 3. Seidman, Boston, MA) was subcloned into a pSP64 p&mid (Seidman ef al., 1984). The Eco RI linearized plasmid served as a template for ANP cRNA transcription using 100 IlCi ‘2P-UTP (200 Ci/mmol, Amersham, FRG) and the SP6 polymerase-promoter system (Melton et al., 1978). Prehybridization (3 hr. WC) of the filters was carried out in 50mM Na-phosphate buffer (pH 6.8) containing 1 M NaCl, 50% formamide, 200 pg/ml sheared salmon sperm DNA, 2001(g/ml yeast RNA, 5 x Denhardt’s solution. 0.2% SDS and IOmM EDTA. Hybridization was performed by incubating with 2 x lo* cpm/ml of the labeled ANP probe overnight at 65°C. The membranes were then washed twice in 0.1 x SSC containing 0.1% SDS; each wash lasted 15 min at room temperature; a further wash in the same solution was performed at 7WC for 1hr. Subsequently, the membranes were exposed to an X-ray film (Hype~lm MP, Amersham, FRG) for 3-5 days at -70’C. RESULTS Tissue levels
of ANP
immunoreactitGty
Acidic extracts of thymus glands of rat, mouse and pig were found to contain immunoreactive ANP (IR-ANP). This material was further analyzed by either Sephadex G-50 gelfiltration (Fig. la) or RPHPLC (data not shown). The IR-ANP consisted of the ANP-like precursor molecule ANP (l-126), as well as ANP (999126). These data were extended by extraction of isolated rat and mouse thymocytes, which revealed the presence of ANP-like precursor by
RP-HPLC and RIA (Fig. lb). Similar IR-ANP profiles were obtained by assaying RP-HPLC runs of acidic extracts of rat and mouse spleen. The IR-ANP was found to mainly consist of ANP (l- 126) (Fig. lc). Figure l(d) represents ANP-like material extracted from lymph nodes of rat and mouse, as well as from intestinal lymph nodes of pigs after Sephadex G-50 gel filtration. The thymus glands, spleen and bursa of Fabricius of hatched chickens were also examined for IR-ANP. However, due to the very low crossreactivity of the antiserum with chicken ANP (assayed by measuring extracts of chicken atria) only traces of IR-ANP could be detected (Table 1). Table 1 also lists the range of extracted IR-ANP (fmol/mg protein) from various lymphoid tissues of different species. These values were corrected for the tissue specific recovery (22-38%) and for the crossreactivity of antiserum ‘Toni III’ with ANP (l-126). Detection
of nzRNA coding for ANP
The presence of mRNA was examined by Northern blot hybridization of total RNA as well as of purified mRNA from various lymphoid tissues. RNA extracted from rat atria was used as a control of mRNA coding for ANP. A single hybridization band, comigrating with the rat atria1 transcript was recently demonstrated in rat thymus tissue (Vollmar et al., 1990) (Fig. 2a, lane 1). Figure 2(a), lanes 2-4 extend these observations to mouse thymus and to rat and mouse thymocytes (Fig. 2, lanes 3 and 4). mRNA coding for ANP was demonstrated in the spleen of the rat (Fig. 2a, lane 5), although not consistently. No signal was observed in the mouse spleen. These inconsistencies in detecting ANP mRNA in spleen may reflect either a very low abundance and/or a high degradation rate of the ANP mRNA and tacks correlation with the peptide levels found in this tissue (Table 1). Examination of rat anterior and posterior cervical lymph nodes and intestinal lymph nodes of the pig also revealed mRNA coding for ANP (Fig. 2, lanes 6 and 7). The results of Northern blot hybridization of ANP mRNA in chicken lymphoid tissue are shown in Fig. 2, panel b. Blotted total RNA from chicken thymus glands (lane l), spleen (lane 2) and bursa of fabricius (lane 3), show hybridization bands corresponding to rat atrial ANP transcript (lane 4), as well as to ANP transcript of chicken heart (lane 5).
DISCUSSION
The major findings reported here are the demonstration of ANP-like precursor as well as the detection of the corresponding mRNA in lymphoid organs of five vertebrates, including one avian species. These data strongly support the recently advanced concept of a link between ANP and the immune system based on earlier discoveries of the ANP precursor in the rat thymus and guinea pig spleen (Vollmar and Schulz, 1988; Vollmar et al., 1989). Moreover, this material was shown to be confined to thymocytes. The shorter IR-ANP fragments detected most iikely reflects degradation of the prohormone, a mechanism well known to occur during tissue extraction (Voilmar et al., 1988).
461
ANP in lymphoid organs
(a)
f 80 < 3
f fi
a I 5
60
40
20
IO
30
20 Fraction
Fraction
number
number a N
(d)
(cl
IO
20 Fraction
IO
40
30
20 Fraction
number
30
number
Fig. 1.Chromatographic analysis of ANP immunoreactivity extracted from rat, mouse and pig lymphoid organs. (a) Sephadex G-50 gel filtration of acidic extracts of whole rat (a), mouse ( x ) and pig (0) thymus tissues. (b) RP-HPLC profile of ANP-immunoreactivity present in isolated rat (0) and mouse (x ) thymocytes. (c) RP-HPLC analysis of acidic extract of mouse ( x) and rat spleen (0). (d) Sephadex G-50 separation of various lymph node extracts: mouse cervical anterior and posterior lymph nodes ( x ). rat axial lymph nodes (0) and pig intestinal (0) lymph nodes. Calibration of the chromatographic columns was carried out as described under “Materials and Methods”. Arrows indicate positions at which ANP fragments elute.
Table I.
IR-ANP (fmol/mg
Tissue Thymus Spleen Lymph nodes Anterior and posterior cervical Axial Intestinal Quantification of IR-ANP n.d. = not determined.
protein) in lymphoid organs
Rat
MOW.!
Pig
Chicken
72-218 (n = 8) 56-109 (n =R)
265-400 (n = 7) 134-219 (n =X)
54-190 (n = 4) n.d.
< IO
143.-225 (n =5) 86-168 (n = 6) n.d.
96-334 (n = 6) n.d.
n.d.
n.d.
ad.
n.d.
n.d.
50-405 (n = 4)
n.d.
c5
has been performed as described under “Methods”
462
A.M. VOLLMAR and R. SCHULZ
a
b
for ANP in lymphoid tissue af mouse, rat, pig (panel af (20 pgf from rat and mouse thymus tissue. Lanes 3 and 4: total RNA (SO&75 pg) extracted from isolated rat and mouse thymocytes, respectively. Lane 5: mRNA (5Opg) from rat spleen. Lanes 6 and 7: total RNA (SO-75 pg) from rat cervical lymph node and pig intestinal lymph nodes, respectively. Lane 8: total RNA (0.5 fig) from rat atria used as control for mRNA coding for ANP. (b) total RNA (3Opg) from chicken bursa of Fabricius (lane l), spleen (lane 2) and thymus (lane 3). As control, lane 4 shows RNA extracted from chicken heart (20 pg) and lane 5, the ANP transcript from rat atria (0.5 pg). RNA was isolated, electrophoresed and hybridized as described in *‘Materials and Methods”. Fig. 3, Northern
blot anagysis of mRNA
coding
and chicken (panel bf (a) lanes 1 and 2: mRNA
The presence of IR-ANP prohormone without exception in all mammalian tissues investigated suggests its synthesis therein. Verification of this was attempted by screening these tissues for the corresponding mRNA by Northern blot hybridization. Although mRNA coding for ANP was not unequivocally detected in all tissues containing the IR-ANP, its presence in isolated thymocytes from the rat and mouse stresses the view that the lymphoid cells express ANP. Again, these experiments extend previous wark showing ANP transcripts in whole thymus extracts of rats (Vollmar et al., 1990). The inconsistent detection of ANP-mRNA in the spleen may be due to specific turnover rates and to high nuclease activities. Our attempts to detect II?-ANP in the chicken were hampered by the fow cross-reactivity of the antiserum employed with chicken ANP. Therefore, demonstration of mRNA coding for ANP in tissues from this species would circumvent this problem and provides an indication of avian lymphatic tissues synthesize ANP. These experiments present evidence for mRNA in the chicken thymus, the bursa of Fabricius and the spleen showing that this might be the case. These observations are interesting in that they demonstrate that ANP is also found in birds, which have an immune system that is organized differently from that of the mammalian immune system (White, 1981). in summary, the present experiments with primary and secondary lymphatic tissues demonstrates the synthesis of ANP in immune tissues of a variety of
mammalian, and one avian, species. Future characterization of the ANP producing ceil types in these various organs should contribute to our understanding of how ANP and the immune system communicate. Acknoit,led~ements-‘.-We would like to thank Dr K. Heinritzi (Munich, FRG) for providing the pig tissues. Dr R. M. Arendt (Munich. FRG) for donating the antiserum ‘Toni III’, as well as Drs’ Seipcke, Preibisch and Schiine for the synthetic ANP (2-126). We are grateful to Dr R. E. Lang (Heidelberg, FRG) for his help in performing the Northern blot hybridization and to Dr K. Bloch and Dr J. Seidman (Boston, MA) for providing the ANP cDNA. The valuable technical assistance of MS G. Hach and U. Riiberg and the secretarial support of Ms B. Feriings are gratefully a~know~~d~~. Supported by the DFG.
REFERENCES Arendt R. M., Stan@ E., Zlhringer J., Liebisch D. C. and Herz A. (1985) Demonstration and characterization of alpha-human atria1 natriuretic factor in human plasma. FEBS Lrtr. 189, 57”.61. Auffray C. and Rougeon F. (1980) Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor K’NA. Eur. J. Biochem. 107, 303-314. Cantin M. and Genest J. (1985) The heart and the atria1 natriuretic factor. En&c&. lieu. 6, 107-127. McKenzie J., Tannaka T.. Misono N. and Inagami T. (1985) ~mmunocytochem~ca~ localization of atria1natriuretic factor in the kidney, adrenai medulla, pituitary and atrium of rat. J. Hisrochem. Cyrochem 338, 828-832.
ANP in iymphoid organs Melton D. A,, Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K. and Green M. R. (1978) Effmient ia a&o synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promotor. Nucieie Acids Res. 12, 7035-7056. Miyata A.. Minamino N., Kangawa K. and Matsuo H. (1988) Identification of a 29 amino acid natriuretic peptide in chicken heart. Biochem. Biophys. Res. Commun. 155, 1330-1337.
Needleman P. J, and Greenwald J. E. (1986) Atriopeptin: a cardiac hormone intimately involved in fluid, electrolyte. and blood pressure homeostasis. Are%* Engl. J. Med. 314, 828-834,
Nemer M., Chamberland M., Sirois D., Argentin S., Drouin J,, Dixon R. A., Zivin R. A. and Condra J. (1984) Gene structure of human cardiac hormone precursor, pronat~o-d~iatin~ Nature 3t2, 654-656. Netchitailo P., Feuilloley M., Pelletier G., Cantin M., De Lean A., Leboulenger-F. and Vaudry H. (1986) Locahzation and characterization of atrial natriuretic factor (ANF)-like peptide in the frog atrium. Peptides 7, 5733579. Sakato I., Kangawa K. and Matsuo II. (1988) Identification of new atrial natriuretic peptides in frog heart. Biochem. Biophys. Rex. Commun. 155, 1338-1345.
463
Seidman C., Duby A. D., Choi E., Graham R. M., Haber E.. Homcv C.. Smith J. A. and Seidman J. C. (1984) The structure of rat preproatrial natriuretic factor as defined by complementary DNA clone. Science 225, 324-326. Vollmar A. M., Friedrich A., Sinowatz F. and Schulz R. (1988) Presence of atria1 natriuretic peptide-like material in the guinea pig intestine. Peprides 9, 965-971. Vollmar A. M., Friedrich A. and Schulz R. (1989) Immunoreactive atria1 natriuretic peptide in the guinea pig spleen. Life Sci. 45, 1293-1297. Vollmar A. M., Lang R. E., Hatme J. and Schulz R. (1990) The rat thymus-a site of atrial natriuretic peptide synthesis. Peptides It, 33-37. Voltmar A. M. and Schulz R. (1988) Evidence for the presence of ANP precursor materiaf in the rat thymus. Biuchem. Biophys. Rex C5mmn. 155, 700-708. White R. G. (1981) The structural organization of avian lymphoid tissues. In Aoiun Immunoio& (Edited by Rose M. E., Payne L. N. and Freeman B. M.), pp. 21.-42. British Poultry Science, Edinburgh. Zamir N., Skofitsch G., Eskay R. L. and Jakobowitz D. M (1986) Distribution of immunoreactive atria1 natriuretic peptides in the central nervous system of the rat. Brain Res. 365, IO5-ill.
ATRIAL
1990
0300-9629/90 $3.00 + 0.00 0 1990 Pergamon Press plc
NATRIURETIC PEPTIDE IN LYMPHOID OF VARIOUS SPECIES
ORGANS
ANGELIKA M. VOLLMAR and R~~DIGERSCHULZ Institute of Pharmacology, Miinchen 22, Federal
Toxicology and Pharmacy, University of Munich, Kiiniginstr. 16, D-8000 Republic of Germany. Telephone (089) 2180-2663. Fax (089) 34 23 16 (Receitied 5 January 1990)
Evidence for the occurrence of atrial natriuretic peptide (ANP) in various lymphoid organs of different species (rat, mouse, pig, chicken) is provided. 2. ANP precursor material (I-126) as well the ohvsioloaicallv active ANP (99-126). were identified bv chromatog;aphic analysis and‘RIA in extracts of’thymus,spleeh and lymph nodes of’rat, mouse and pig. 3. mRNA coding for ANP was demonstrated both in the thymus and in isolated thymocytes of these species. Furthermore, mRNA for ANP was detected in spleen and lymph nodes (rat and pig). 4. The bursa of Fabricius, thymus glands and spleen of chickens were also shown to express mRNA coding for ANP. 5. These findings provide a firm basis for a link of ANP to the immune system, a novel aspect of possible biological functions of this peptide. Abstract-l.
INTRODUCTION
weeks) and chickens (3-4 day old, white leghorn, strain A). All animals were killed by decapitation with the exception of pigs. which were iniected with T-61” euthanasia solution (Bay&, FRG). The thymus glands, spleen and lymph nodes (i.e. axial nodes, posterior and anterior cervical nodes, intestinal nodes) as well as the bursa of Fabricius of the chicken were excised and maintained at 0” or -70°C as appropriate, until biochemical analysis.
Atria1 natriuretic peptide (ANP) has been shown to be a potent diuretic, natriuretic and vasorelaxant hormone, and the heart myocytes represent the major, although not exclusive, site of synthesis and secretion of this peptide (for review, see Cantin and Genest, 1985). A high degree of conservation is seen in the structural organization of the ANP gene, as well as the amino acid sequences of ANP in various mammalian species (Nemer et al., 1984; Seidman et al., 1984). More recently, the presence of ANP has been demonstrated in birds (Miyata et al., 1988) and other submammalian vertebrates (Netchitailo et al., 1986; Sakato et al., 1988). The demonstration of ANP-like peptides in non-atria1 tissues, such as the brain and adrenals (McKenzie et al., 198.5; Zamir et al., 1986) has begun to draw attention to other possible biological roles of ANP, in addition to those already documented, e.g. the regulation of blood volume (see Needleman and Greenwald, 1986). Our own laboratory has shown the presence and synthesis of ANP-like material in lymphoid organs, like the thymus (Vollmar and Schulz, 1988) the spleen (Vollmar et al., 1989) and the intestine (Vollmar et al., 1988). We therefore suggested that ANP may be involved in immunological functions. We report here the identification of ANP-like material, and mRNA coding for ANP in a number organs of the immune system of various mammalian species and that of the domestic chicken. These new findings strengthen the concept of a link between ANP and the lymphoid system. MATERIALS
Tissue extraction Freshly removed tissue was immediately transfered to 0.1 N HCI (4°C) (1 g wet tissue/5 ml), homogenized with a polytron (I min, setting 8) and boiled for 1min in a microwave oven. After centrifugation (2O,OOOg, 20 min, 4°C) the clear supematant from which an aliquot was taken for protein determination (Bradford method) was run through a column filled with activated Amberlite XAD-4 adsorbent resin (500 mg/column, Serva, Heidelberg, FRG). ANP-like material was eluted with 80% acetonitrile in 0. I % trifluoroacetic acid (TFA), and subsequently lyophilized. Characterization and quantification of extracted ANP-like material IR-ANP was characterized as previously described (Vollmar et al., 1988). Briefly, an aliquot of the lyophilized tissue extract was dissolved in 200~1 0.1 M acetic acid, centrifuged and the clear supematant subjected to gel chromatography (Sephadex G-SO, Pharmacia LKB, Freiburg, FRG, column 9 x 1000 mm, flowrate 10 ml 0.1 M acetic acid per hour). Calibration of the column was carried out with bovine serum albumin (V,), vitamin B12 (V,), rat-pro-ANP (2-126) (Hoechst, Frankfurt, FRG) and ANP (99-126) (Novabiochem, Laufelfingen, Switzerland). For RP-HPLC analysis an aliquot of the extracts was redissolved in 100~1 0.1% TFA, centrifuged and loaded onto a HPLC C8 Ultrapore TM column (5pm, 4.6 x 750 mm, Beckman Instruments, FRG). Calibration was carried out with rat ANP fragments (99-123), (99-125). (99-126) (Novabiochem, Switzerland) and ANP (2-126) (Hoechst, Frankfurt, FRG). The peptides were eluted with a linear gradient of acetonitrile (20-55%. in 55 min) in 0. I % TFA (flowrate: 0.5 ml/min). Fractions were assayed for ANP by RIA.
AND METHODS
Animals Rats (Sprague-Dawley, male, 6 weeks), mice (BALB/c, male, 6-8 weeks), pigs (German country race, male, 1-3 459
A. M. VOLLMARand R. SCHULZ
460
IR-ANP was quantified by RIA after Sephadex G-50 gel filtration of crude extracts containing approximately equal amounts of protein. Values were corrected for recovery rates and crossreactivity of the antibody used (see below). Antisera and radioimmunoassay
Characteristics of the antiserum ‘Toni III’ have been recently described in detail (Vollmar and Schulz, 1988). Briefly, it was raised against human-ANP (99-126) and recognizes the carboxy-terminus of ANP (99-126). Antiserum ‘Toni III’ cross-reacts with ANP (2-126) and ratANP (99-l 26) to a degree of 30 and IOO%, respectively. The radioimmunoassay using ‘Toni III’ antiserum was performed as previously described (Arendt ei al., 1985) (999126) (Amersham, FRG). using ?-rat-ANP Measurement of extraction efficiency
A quantity (1 pmol) of synthetic rat-pro-ANP was added as internal standard to the various tissues and the extraction and chromatographic procedures were performed as described above. Concentration of IR-ANP in control tissue and tissue with exogenous ANP (22126) were determined by RIA (antiserum ‘Toni III’). The calculated recoveries amounted between 22 and 38%. Northern blot h~~r~dizutiffn
Total RNA was prepared from frozen (- 7OC) tissue by the LiCl-urea technique (Auffray and Rougeon, 1980). In some cases, mRNA was enriched by applying total RNA (I mg) to oligo dT celiulose columns (Boehringer, Mannheim, FRG). After quantification by absorption at 260 nm, various amounts of mRNA (up to SO kg) were denatured in 1 M glyoxal (pH 5), containing 50% DMSO and 10 mM sodium phosphate buffer (pH 6.8) and electrophoresed on a 1.2% agarose gel. The mRNA was then blotted onto Nytran l3N membranes (Schleicher & Schuell, FRG) and the transfer checked by staining the gel with ethidium bromide. A UV source was used for visualization. For preparation of an ANP-specific cRNA hybridization probe, a 580 base Pst I-restricted ANP cDNA fragment (gift from K. Bloch and 3. Seidman, Boston, MA) was subcloned into a pSP64 p&mid (Seidman ef al., 1984). The Eco RI linearized plasmid served as a template for ANP cRNA transcription using 100 IlCi ‘2P-UTP (200 Ci/mmol, Amersham, FRG) and the SP6 polymerase-promoter system (Melton et al., 1978). Prehybridization (3 hr. WC) of the filters was carried out in 50mM Na-phosphate buffer (pH 6.8) containing 1 M NaCl, 50% formamide, 200 pg/ml sheared salmon sperm DNA, 2001(g/ml yeast RNA, 5 x Denhardt’s solution. 0.2% SDS and IOmM EDTA. Hybridization was performed by incubating with 2 x lo* cpm/ml of the labeled ANP probe overnight at 65°C. The membranes were then washed twice in 0.1 x SSC containing 0.1% SDS; each wash lasted 15 min at room temperature; a further wash in the same solution was performed at 7WC for 1hr. Subsequently, the membranes were exposed to an X-ray film (Hype~lm MP, Amersham, FRG) for 3-5 days at -70’C. RESULTS Tissue levels
of ANP
immunoreactitGty
Acidic extracts of thymus glands of rat, mouse and pig were found to contain immunoreactive ANP (IR-ANP). This material was further analyzed by either Sephadex G-50 gelfiltration (Fig. la) or RPHPLC (data not shown). The IR-ANP consisted of the ANP-like precursor molecule ANP (l-126), as well as ANP (999126). These data were extended by extraction of isolated rat and mouse thymocytes, which revealed the presence of ANP-like precursor by
RP-HPLC and RIA (Fig. lb). Similar IR-ANP profiles were obtained by assaying RP-HPLC runs of acidic extracts of rat and mouse spleen. The IR-ANP was found to mainly consist of ANP (l- 126) (Fig. lc). Figure l(d) represents ANP-like material extracted from lymph nodes of rat and mouse, as well as from intestinal lymph nodes of pigs after Sephadex G-50 gel filtration. The thymus glands, spleen and bursa of Fabricius of hatched chickens were also examined for IR-ANP. However, due to the very low crossreactivity of the antiserum with chicken ANP (assayed by measuring extracts of chicken atria) only traces of IR-ANP could be detected (Table 1). Table 1 also lists the range of extracted IR-ANP (fmol/mg protein) from various lymphoid tissues of different species. These values were corrected for the tissue specific recovery (22-38%) and for the crossreactivity of antiserum ‘Toni III’ with ANP (l-126). Detection
of nzRNA coding for ANP
The presence of mRNA was examined by Northern blot hybridization of total RNA as well as of purified mRNA from various lymphoid tissues. RNA extracted from rat atria was used as a control of mRNA coding for ANP. A single hybridization band, comigrating with the rat atria1 transcript was recently demonstrated in rat thymus tissue (Vollmar et al., 1990) (Fig. 2a, lane 1). Figure 2(a), lanes 2-4 extend these observations to mouse thymus and to rat and mouse thymocytes (Fig. 2, lanes 3 and 4). mRNA coding for ANP was demonstrated in the spleen of the rat (Fig. 2a, lane 5), although not consistently. No signal was observed in the mouse spleen. These inconsistencies in detecting ANP mRNA in spleen may reflect either a very low abundance and/or a high degradation rate of the ANP mRNA and tacks correlation with the peptide levels found in this tissue (Table 1). Examination of rat anterior and posterior cervical lymph nodes and intestinal lymph nodes of the pig also revealed mRNA coding for ANP (Fig. 2, lanes 6 and 7). The results of Northern blot hybridization of ANP mRNA in chicken lymphoid tissue are shown in Fig. 2, panel b. Blotted total RNA from chicken thymus glands (lane l), spleen (lane 2) and bursa of fabricius (lane 3), show hybridization bands corresponding to rat atrial ANP transcript (lane 4), as well as to ANP transcript of chicken heart (lane 5).
DISCUSSION
The major findings reported here are the demonstration of ANP-like precursor as well as the detection of the corresponding mRNA in lymphoid organs of five vertebrates, including one avian species. These data strongly support the recently advanced concept of a link between ANP and the immune system based on earlier discoveries of the ANP precursor in the rat thymus and guinea pig spleen (Vollmar and Schulz, 1988; Vollmar et al., 1989). Moreover, this material was shown to be confined to thymocytes. The shorter IR-ANP fragments detected most iikely reflects degradation of the prohormone, a mechanism well known to occur during tissue extraction (Voilmar et al., 1988).
461
ANP in lymphoid organs
(a)
f 80 < 3
f fi
a I 5
60
40
20
IO
30
20 Fraction
Fraction
number
number a N
(d)
(cl
IO
20 Fraction
IO
40
30
20 Fraction
number
30
number
Fig. 1.Chromatographic analysis of ANP immunoreactivity extracted from rat, mouse and pig lymphoid organs. (a) Sephadex G-50 gel filtration of acidic extracts of whole rat (a), mouse ( x ) and pig (0) thymus tissues. (b) RP-HPLC profile of ANP-immunoreactivity present in isolated rat (0) and mouse (x ) thymocytes. (c) RP-HPLC analysis of acidic extract of mouse ( x) and rat spleen (0). (d) Sephadex G-50 separation of various lymph node extracts: mouse cervical anterior and posterior lymph nodes ( x ). rat axial lymph nodes (0) and pig intestinal (0) lymph nodes. Calibration of the chromatographic columns was carried out as described under “Materials and Methods”. Arrows indicate positions at which ANP fragments elute.
Table I.
IR-ANP (fmol/mg
Tissue Thymus Spleen Lymph nodes Anterior and posterior cervical Axial Intestinal Quantification of IR-ANP n.d. = not determined.
protein) in lymphoid organs
Rat
MOW.!
Pig
Chicken
72-218 (n = 8) 56-109 (n =R)
265-400 (n = 7) 134-219 (n =X)
54-190 (n = 4) n.d.
< IO
143.-225 (n =5) 86-168 (n = 6) n.d.
96-334 (n = 6) n.d.
n.d.
n.d.
ad.
n.d.
n.d.
50-405 (n = 4)
n.d.
c5
has been performed as described under “Methods”
462
A.M. VOLLMAR and R. SCHULZ
a
b
for ANP in lymphoid tissue af mouse, rat, pig (panel af (20 pgf from rat and mouse thymus tissue. Lanes 3 and 4: total RNA (SO&75 pg) extracted from isolated rat and mouse thymocytes, respectively. Lane 5: mRNA (5Opg) from rat spleen. Lanes 6 and 7: total RNA (SO-75 pg) from rat cervical lymph node and pig intestinal lymph nodes, respectively. Lane 8: total RNA (0.5 fig) from rat atria used as control for mRNA coding for ANP. (b) total RNA (3Opg) from chicken bursa of Fabricius (lane l), spleen (lane 2) and thymus (lane 3). As control, lane 4 shows RNA extracted from chicken heart (20 pg) and lane 5, the ANP transcript from rat atria (0.5 pg). RNA was isolated, electrophoresed and hybridized as described in *‘Materials and Methods”. Fig. 3, Northern
blot anagysis of mRNA
coding
and chicken (panel bf (a) lanes 1 and 2: mRNA
The presence of IR-ANP prohormone without exception in all mammalian tissues investigated suggests its synthesis therein. Verification of this was attempted by screening these tissues for the corresponding mRNA by Northern blot hybridization. Although mRNA coding for ANP was not unequivocally detected in all tissues containing the IR-ANP, its presence in isolated thymocytes from the rat and mouse stresses the view that the lymphoid cells express ANP. Again, these experiments extend previous wark showing ANP transcripts in whole thymus extracts of rats (Vollmar et al., 1990). The inconsistent detection of ANP-mRNA in the spleen may be due to specific turnover rates and to high nuclease activities. Our attempts to detect II?-ANP in the chicken were hampered by the fow cross-reactivity of the antiserum employed with chicken ANP. Therefore, demonstration of mRNA coding for ANP in tissues from this species would circumvent this problem and provides an indication of avian lymphatic tissues synthesize ANP. These experiments present evidence for mRNA in the chicken thymus, the bursa of Fabricius and the spleen showing that this might be the case. These observations are interesting in that they demonstrate that ANP is also found in birds, which have an immune system that is organized differently from that of the mammalian immune system (White, 1981). in summary, the present experiments with primary and secondary lymphatic tissues demonstrates the synthesis of ANP in immune tissues of a variety of
mammalian, and one avian, species. Future characterization of the ANP producing ceil types in these various organs should contribute to our understanding of how ANP and the immune system communicate. Acknoit,led~ements-‘.-We would like to thank Dr K. Heinritzi (Munich, FRG) for providing the pig tissues. Dr R. M. Arendt (Munich. FRG) for donating the antiserum ‘Toni III’, as well as Drs’ Seipcke, Preibisch and Schiine for the synthetic ANP (2-126). We are grateful to Dr R. E. Lang (Heidelberg, FRG) for his help in performing the Northern blot hybridization and to Dr K. Bloch and Dr J. Seidman (Boston, MA) for providing the ANP cDNA. The valuable technical assistance of MS G. Hach and U. Riiberg and the secretarial support of Ms B. Feriings are gratefully a~know~~d~~. Supported by the DFG.
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