GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
87, 171-177 (1992)
Frog Lymph Heart Synthesizes and Stores Immunoreactive Natriuretic Peptide
Atrial
HOON RYU,* KYUNG Woo CHO, SUHN HEE KIM, SUNG Zoo KIM, SEON HEE OH, YUN HA HWANG, AND GEUM YEONG LEE* Department
of Physiology, Jeonbug
Medical National
School, and *Department of Biology, University, Jeonju 560-180, Republic
College of Natural of Korea
Sciences,
Accepted October 12, 1991 The presence of immunoreactive atria1 natriuretic peptide (irANP) and ANP gene expression in the frog lymph heart was examined by a radioimmunoassay (RIA) combined with HPLC and by Northern blot hybridization of total RNA. Serial dilution curve of the lymph heart extract was paralleled with the RIA standard curve. The lymph heart contained 153.32 t 35.80 pg of irANP/mg of wet tissue. The major form of irANP in the frog lymph heart was high molecular weight on reverse-phase and gel permeation high performance liquid chromatography as in the frog atria and ventricles. The frog lymph heart, as well as frog atria and ventricles, was shown to express mRNA coding for ANP. Dense core secretory granules similar to those observed in the mammalian atria were also found in the frog lymph heart. The presence of irANP and the expression of ANP gene in the frog lymph heart suggest that the lymph heart may participate in the regulation of homeostasis of lymph circulation and blood volume change through the synthesis and release of ANP. 6 1992 Academic press, IW.
Atria1 natriuretic peptide (ANP), now well known as a hormone stored in and released from mammalian atria1 cardiocytes, possesses variable biological effects: induction of diuresis and natriuresis (De Bold et al., 1981; Garcia et al., 1982), vasodilation (Garcia et al., 1984), and inhibition of aldosterone and renin release (De Lean et al., 1984; Obana et al., 1985). ANP has been found and characterized in a wide range of mammalian (Chapeau et al., 1985; De Bold and Salerno, 1983) and submammalian (Netchitailo et al., 1986a, 1988; Kim et al., 1989a) animals. It has previously been found by immunohistochemistry, bioassay, or radioimmunoassay that the frog atria and ventricles synthesize and store ANP (Chapeau et al., 1985; Netchitailo et al., 1986a, 1988). Frog ANP has also been known to be closely related to mammalian ANP in structure and pharmacological activities (De Bold and Salerno, 1983; Lazure et al., 1988; Sakada et al., 1988).
Interestingly, electron microscopic studies have revealed that the developing and adult frog lymph heart is very rich in dense core granules similar to atria1 specific granules in the blood heart of the adult frog (Markozashvili and Rumyantsev, 1984; Rumyantsev and Krylova, 1990). Therefore, we investigated the possible occurrence of irANP in the adult frog lymph heart by radioimmunoassay and characterized the molecular profile of irANP by reverse-phase and gel permeation high-performance liquid chromatography. We also examined ANP gene expression in the frog lymph heart by Northern blot hybridization for human ANP cDNA. MATERIALS Preparation
AND METHODS
of Samples
Adult frogs, Rana dybowskii, weighing 23-28 g were used. Frogs were captured during winter season and 171 0016-6480192 $4.00 Copyright 6 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
172
RYU ET AL.
were adapted at temperature 9” before experimentation for about 2 weeks. Frogs were pithed and the lymph hearts located at the end of the vertebral column were rapidly removed under a stereomicroscope. Both atria and ventricles were also obtained. Tissues were placed into prechilled tubes containing 0.1 N acetic acid with aprotinin (200 kallikrein inhibitor units/ml (KIU/ml)), phenylmethylsulfonyl fluoride (PMSF, 0.4%), ethylenediamine tetraacetic acid (EDTA, 10 n&), and soybean trypsin inhibitor (SBTI, 50 benzoyl arginine ethyl ester units/ml (BAEE/ml)). After weighing, tissues were boiled for 10 min and homogenized using a Polytron homogenizer. Tissue extracts were centrifuged at 10,OOOg for 15 min at 4”. The supematant was collected and applied on the Sep-Pak Cl8 cartridges (Waters Associates, Milford, MA) and irANP was extracted as described previously (Cho et al., 1988a,b). In brief, samples were applied on a SepPak Cl8 cartridge previously activated with 4 ml of acetonitrile and 0.1% trifluoroacetic acid (TFA). The cartridge was rewashed with 4 ml of 0.1% TFA and the adsorbed peptide was eluted with 3 ml of 60% acetonitrile in 0.1% TFA. The eluent was subsequently dried using a Speed-Vat concentrator (Savant, Hicksville, NY).
Radioimmunoassay (RIA) for irANP The content of irANP in the frog lymph heart was determined by RIA as previously described (Cho et al., 1988a,b). Briefly, anti-ANP antibody was raised against a synthetic atriopeptin III (AP III, NovaBiothem, Laeufelfingen, Switzerland) and AP III (Peninsula Laboratories, Belmont, CA) was iodinated using the chloramine-T method (Kim et al., 198913). The tissue extracts were reconstituted with Tris-acetate buffer (0.1 M, pH 7.4, containing 0.2% neomycin, 10 mM EDTA, 50 BAEE/ml SBTI, 200 KIUiml aprotinin, 0.4 mg% PMSF, 0.02% sodium azide, and 1% bovine serum albumin). After incubation with anti-ANP antibody for 24 hr at 4”, “-?-ANP was added and incubated for 24 hr at 4” again. The separation of free from bound forms was achieved by the double antibody technique.
Reverse-Phase (RP) and Gel Permeation (GPC) High-Performance Liquid Chromatography (HPLC) To elucidate the molecular profile of irANP in the frog lymph heart, 10-12 frogs were sacrificed and tissues were immediately put into liquid nitrogen. After boiling in 0.1 N acetic acid for 10 min, samples were processed as described above. The eluates were dried
using a Speed-Vat concentrator, reconstituted with 200 p,l of 0.1% TFA, and centrifuged at 10,OOOgfor 10 min at 4”. One hundred microliters of aliquots was subjected to the RP-HPLC on a p,Bondapak (Waters Associates, Milford, MA). Elution was performed with the linear gradient of 20 to 60% acetonitrile in 0.1% TFA for 40 min at a flow rate of 1 ml/min. Gel permeation HPLC was also performed on a TSK-GEL G 2000 SW column (7.5 x 300 mm, Toyo Soda, Tokyo, Japan) and eluted with 30% acetonitrile in 0.1% TFA. The flow rate was 0.3 ml/min and the fraction volume was 0.3 ml. The fractionated samples were dried using a Speed-Vat concentrator and their irANP content was determined by RIA.
Northern Blot Analysis Total RNA was isolated from frozen (-70”) frog lymph hearts, atria, and ventricles by the guanidinium monothiocyanate technique (Chirgwin et al., 1979). The lymph hearts from 40 frogs were removed and immediately put into liquid nitrogen. Frog atria and ventricles were used as control tissue for ANP mRNA. After quantification by absorption at 260 nm, 21 p,g of total RNA from lymph hearts as well as 58 pg of atria1 and 43 +g of total ventricular RNA were denatured, electrophoresed on 1.1% agarose gel containing 5% formaldehyde, and transferred to nylon membrane (Bio-Rad, Richmond, CA). A 580-bp Sac-PsfL restricted ANP cDNA cloned in a phAF48 (Nakayama et al., 1984) was a kind gift from Dr. S. Nakanish (Kyoto, Japan) through Dr. S. J. Kim (Bethesda, MD). The cDNA was labeled with [3ZP]dCTP (50 l&i) (Amersham) by random priming method (Amersham, U.S.A.) and denatured at 100” for 10 min. Membranes were prehybridized at 65” in 1% BSA, 7% sodium dodecyl sulfate (SDS), 1 mA4 EDTA, and 0.5 M sodium phosphate buffer (pH 7.4). Hybridization was performed by adding 1 x lo6 cpm/ml of the ANP probe and incubating for 16-20 hr at 65”. The membrane was washed twice in 1 M sodium chloridesodium citrate buffer and 0.1% SDS at 42” for 30 min. The membranes were exposed to an X-ray film with intensifying screens at -70” for 2 or 3 days.
Electron Microscopy To investigate the presence of secretory granules in the frog lymph heart, the posterior lymph hearts were excised and immediately fixed with 2.5% glutaraldehyde for 2 hr. All the samples were posffrxed in 1% osmium tetroxide (0~0,) and embedded in Epon 812. Thin sections were cut from Epon-embedded portions of the lymph heart with a diamond knife. These samples were stained with uranyl acetate and lead citrate,
irANP IN THE FROG LYMPH and the fine structure was examined with a JEOL 12OOEX electron microscope.
RESULTS Immunoreactive Atria1 Natriuretic in Frog Lymph Heart
Peptide
The serial dilution of extracts of the frog (R. dybowskii) lymph heart, atria, and ventricles inhibited the binding of [12jI]AP III to anti-AP III antiserum and the dilution curve for the lymph heart was relatively parallel with the standard curve of AP III, as shown in Fig. 1. This shows that the extract of the frog lymph heart possesses ir.ANP . The contents of irANP in the frog lymph heart, atria, and ventricles are summarized in Table 1. The levels of irANP in the frog lymph heart are 30 to 40 times lower than detected in the atrium.
I
10
I
I
102
173
HEART
Molecular Profiles Lymph Heart
of irANP
in the Frog
Elution profiles of the extracts of the frog lymph heart, atria, and ventricles on RPHPLC are shown in Fig. 2. The retention time of the synthetic peptide AP III and the purified rat pro-ANP were 15 and 30 min, respectively. The major peak of the frog lymph heart exhibited the same r~t~~~~~~ time as rat pro-ANP. Gel permeative HPLC showed that most irANP in the frog lymph heart coeluted with rat pro-A suggesting that this peptide corresponds to a high molecular weight form (Fig. 3). Northern
Blot Hybridization
A~~~y~~~
To determine whether the identified irANP in the frog lymph heart arises from endogenous synthesis of the pro-ANP molecule, we isolated total RNA from the frog
I
I
I
*--a
APill
O---O A-A A--).
lymph heart atria veritricles
standard
1
103
AP III ( pg/tube)
1. A representative standard curve of atriopeptin III (AP III), and serial dilution curves of the extracts of lymph heart, atria, and ventricles of the frog, Rann dybowskii. The absolute amounts of tissues used in dilution curves were 80 mg of lymph heart, I8 mg of atrium, and 42 mg of ventricle. FIG.
174
RYU ET AL. TABLE
THE LEVELS OF irANP dybowskii) LYMPH HEART,
Sample
1 IN THE FROG (Rana ATRIA, AND VENTRICLES
Levels of irANP
Lymph heart Atrium Ventricle
153.32 ? 5290.00 524.50
35.80 pg/mg pg/mg pg/mg
L 1210.00 + 116.62
Values are the mean f SEM of six experiments.
posterior lymph heart, atria, and ventricles and analyzed it for the presence of frog ANP mRNA transcripts. As shown in Fig. 4, Northern blot hybridization analysis revealed that the frog lymph heart contains ANP transcripts with size comparable to that of frog atria1 and ventricular transcripts.
ANP. Indeed, the pharmacological effect of frog ANP was found to be similar to that of mammalian ANP (Lihrmann et al., 1988), and was found to be highly homologous to rat (II-ANP (Sakada et al., 1988). The finding of ANP transcripts in the frog lymph heart is of particular interest, in that no groups have reported the presence of ANP mRNA. Since the description of extraatrial expression of ANP gene by Gardner et al. (1986), it has been thought that some tissue-specific factor, present predominantly in the atria, is required for efficient expression of this gene. Recently, extracardiac sources of ANP such as hypothalamus (Tanaka et al., 1984; Netchitailo
Electron
Microscopy
In the striated muscle cell of the frog lymph heart numerous secretory granules with electron dense core were observed (Fig. 5). We also found the secretory granules in the myoblast-like cell of the frog lymph heart (data not shown).
F’m ANP
AP Ill
T
T
A 1.2
r c -* --
-0.8
405
0
E 206
.
___---
6OE
--
a4 /fJLJ
oAi
k...
B
DISCUSSION
6
The present study demonstrates the presence of irANP and ANP gene expression in the frog lymph heart. IrANP in the frog lymph heart seems to exclusively consist of a high molecular weight form, which probably represents pro-ANP as in the frog atria and ventricles. In addition, we found that the frog lymph heart locally transcribes ANP mRNA. This finding suggests that the frog lymph heart synthesizes and stores ANP. Earlier studies have reported the presence of ANP in the frog atria and ventricles (De Bold and Salreno, 1983; Chapeau et al., 1985; Netchitailo et al., 1986a, 1988; Lazure et al., 1988; Sakada et al., 1988; Kim et al., 1989a). They suggested that frog ANP is structurally related to mammalian
4
2 0
2 1 0 0
10 Retention
20
30 time(min
40 )
FIG. 2. Reverse-phase HPLC profiles of irANP in the frog lymph heart (A), atria (B), and ventricles (C) of the frog. Arrowheads indicate the retention times of AP III and rat pro-ANP.
irANP IN THE FROG LYMPH
et al., 1986b), central nervous system (Netchitailo et al,, 1987), thymocytes (Vollmar et dl., 199Oa), spleen (Vollmar et al., 1989), gastrointestinal tract (Vollmar et al., 1988), interrenal gland (Lihrmann et al., 1988), ovary (Kim et al., 1989b), and various lymph nodes (Vollmar and Schulz, 1990b) have been described. It has been quite evident that the synthesis of ANP is by no means restricted to the cardiocytes. But the role of ANP in extracardiac tissues remains unknown. It was suggested that ANP in extracardiac tissues is synthesized not for circulation as a hormone but to serve some local paracrine, autocrine, or neurotrans-
Retention
time(min)
FIG. 3. Gel permeation HPLC profiles of irANP in lymph heart (A), atria (B), and ventricles (C) of the frog. Arrowheads indicate the retention times of blue dextran (Vo), bovine serum albumin (BSA), cytochrome C (CC), and AP III.
HEART
AT
175
LH
VT
FIG. 4. Blot hybridization analysis of total RNA from the frog lymph heart (LH), atria (AZ’), and ventricles (VT). RNA from the frog lymph heart (21 pg), atria (58 kg), and ventricles (43 pg) was anilyzed by blot hybridization using a 32P-labded hANP cDNA probe as described in the text.
mitter function (Gardner et al., 1986). Alternatively, the presence of ANP in ce1l.sof immune organs raises the possibility of a physiological function for ANP in the immune system (Gutkowska and Nemer, 1989). It was reported that the frog lymph heart wall is distinguished as three layers: an ,inner layer formed by endothelial cells lining the organ cavity, a middle layer made up of muscle fibers, and an outer one composed of fibrous connective tissue (Rumyantsev and Krylova, 1990). Ultrastructural studies of the frog lymph heart have shown the electron-dense core granules accumulated in peripheral region of sarsop’lasm of lymph heart muscles. In the present study, we have also found numerous electron-dense granules in the striated muscle cells and in the myoblast-like cells of the adult frog lymph heart (Fig. 5). In summary, the frog lymph heart was found to contain pro-ANP and to express its gene. Our findings raise the possibility that ANP in the frog lymph heart ,could be another extracardiac source of AMP secretion to the circulation and may serve a role
176
RYU ET AL.
FIG. 5. Electron micrograph of the frog lymph heart. Note a number of secretory granules (G). The frog lymph heart was fixed with 2.5% glutaraldehyde and embedded in Epon 812. Nucleus (N), mitochondria (m), myofilaments (M). Original magnification x4000. Bar = 2 urn.
as a biological regulator in lymph circulation and in body fluid homeostasis (Baldwin et al., 1990). ACKNOWLEDGMENTS We thank Dr. S. Nakanish and Dr. S. J. Kim for providing ANP cDNA. The authors thank especially Prof. Moo Sam Lee, Dept. of Anatomy, for providing facilities for the electron microscopy. We also thank Prof. Won Koo Lee, Dept. of Biology, and Mr. Byung Mun Ko, Dept. of Anatomy, for their critical suggestions on the experiments, and Miss Kyung Hwa Nam, Miss Hea Kyung Cho, and Miss Yeo Lae Eun for their fine technical assistance. This study was supported by a Graduate Fellowships and a research grant from the Korea Science and Engineering Foundation, and by a grant-in-aid from the Ministry of Education, Korea.
REFERENCES Baldwin, A. L., Rozum, J. S., and Gore, R. W. (1990). Routes of water and small solute transport between blood and lymph in frog. Physiol. Zool. 63, 1141-l 156.
Chapeau, C., Gutkowska, J., Schiller, P. W., Miline, R. W., Thibault, G., Garcia, R., Genest, J., and Cantin, M. (1985). Localization of immunoreactive synthetic atrial natriuretic factor (ANF) in the heart of various animal species. J. H&o&em. Cytochem. 33, 541-550. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. Cho, K. W., Seul, K. H., Kim, S. H., Ryu, H., Seul, K. M., and Koh, G. Y. (1988a). Epicardial release of immunoreactive atria1 natriuretic peptides in inside-out perfused rabbit atria. Biochem. Biophys. Res. Commun. 153, 811-817. Cho, K. W., Seul, K. H., Ryu, H., Kim, S. H., and Koh, G. Y. (1988b). Characteristics of distentioninduced release of immunoreactive atria1 natriuretic peptide in isolated perfused rabbit atria. Regul. Pept. 22, 333-345. De Bold, A. J., Borenstein, H. B., Veress, A. T., and Sonnenberg, H. (1981). A rapid and potent natriuretic response to intravenous injection of atria1 myocardial extract in rats. Life Sci. 28, 89-94. De Bold, A. J., and Salerno, T. A. (1983). Natriuretic
irANP IN THE FROG LYMPH activity of extracts obtained from heart of different species and from various rat tissues. Can. J. Physiol. Pharmacol. 61, 127-130. De Lean, A., Racz, K., Gutkowska, J., Nguyen, T., Cantin, M., and Genest, J. (1984). Specific receptor-mediated inhibition by synthetic atria1 natriuretic factor of hormone-stimulated steroidogenesis in cultured bovine adrenal cells. Endocrinology 115, 1636-1638. Garcia, R., Cantin, M., Thibault, G., Ong, H., and Genest, J. (1982). Relationship of specific granules to the natriuretic and diuretic activity of rat atria. Experientia 38, 1071-1073. Garcia, R., Thibault, G., Cantin, M., and Genest, J. (1984). Effect of a purified atrial natriuretic factor on rat and rabbit vascular strips and vascular beds. Am. J. Physiol. 247, R34--R39. Gardner, D. G., Deschepper, C. F., Ganong, W. F., Hane, S., Fiddes, J., Baxter, J. D., and Lewicki, J. (1986). Extra-atria1 expression of the gene for atria1 natriuretic factor. Proc. Natl. Acad. Sci. USA 83, 6697-6701. Gutkowska, J., and Nemer, M. (1989). Structure, expression, and function of atrial natriuretic factor in extraatrial tissues. Endocrine Rev. 10,519-536. Kim, S. H., Cho, K. W., Koh, G. Y., Seul, K. H., So, J. N., and Ryu, H. (1989a). Phylogenetic study on the immunoreactive atria1 natriuretic peptide in the heart. Gen. Comp. Endocrinol. 14, 127-135. Kim, S. H,, Cho, K. W., Seul, K. H., Ryu, H., and Koh, G. Y. (1989b). Presence of immunoreactive atria1 natriuretic peptide in follicular fluid, ovary and oVarim perfusates. Life Sci. 45, 1581-1589. Lazure, C., Gng, H.,’ McNicoll, N., Netchitailo, P., Chsetien, M., De Lean, A., and Vaudry, H. (I%%). The amino sequences of frog heart atrial natriuretic-like peptide and mammalian ANF are closely related. FE&S Lett. 238, 300-306. Lihrmann, I., Net&i&lo, P., Feuilloley, M., Cantin, M., D&rue, C., Leboulenger, F., De Lean, A., and Vat&y, II. (1988) Effect of atrial natriuretic fac&or on @rticosteroid production by perifused frog interrenal slices. Gen. Comp. Endocrinol. 71, 55-62. MarKozashvili, M. I., and Rumyantsev, P. P. (1984). Dltrastructure of muscle fibers and cells synthesizing D,NA in lymph hearts of developing frogs and chick embryos. Cell Tissue Res. 238,369-379. Nakayama,, K., Ohkubo, H., Hirose, T., Inayama, S., and Nakanishi, S. (1984). mRNA sequence for human cardio#ttin~atrial natriuretic factor precursor &td &n.tlation of precursor mRNA in rat atria. Ndruri 31D, 699-701~
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Netchitailo, P., Feuilloley, L. M., Pelletier, G., Cantin, M., De Lean, A., Leboulenger, F., and Vaudry, H. (1986a). Localization and characterization of atrial natriuretic factor (ANF)-like peptide in the frog atrium. Peptides 7, 573-579. Netchitailo, P., Feuilloley, M., Pelletier, G., Cantin, M. ) Leboulenger, F., Andersen, A., and Van&y, H. (1986b). Localization of atria1 natriuretic factor (ANF)-immunoreactive material in the hypothalamo-pituitary complex of the frog. Newossi. Lett. 72, 141-146. Netchitailo, P., Feuilloley, M., Pelletier, G., Leboulenger, F., Cantin, M., Gutkowska, J., and Vaudry, II. (1987). Atrial natriuretic factor like immunoreactivity in the central nervous system of the frog. Neuroscience 22, 341-359. Netchitailo, P., Feuilloley, L. M., Pelletier, a?., De Lean, A., Ong, II., Cantin, M., Gutkowska, J., Leboulenger, F., and Vat&y, A. (1988). Localization and identification of immunoreactive atrial natriuretic factor (ANF) in the frog ventricle. Peptides 9, l-6. Obana, K., Naruse, M., Naruse, K., Sakurai, II., Demura, II., Inagami, T., and Shizume, K. (1985). Synthetic rat atrial natriuretic factor inhibits in vitro and in vivo renin secretion in rats. Endocrinology 117, 1282-1284. Rumyantsev, P. P., and Krylova, M. I. (1990). Ultrastructure of myofibers and cells synthesizing DNA in’ the developing and regenerating lymphheart muscles. Int. Rev. CyroE. 120, l-52. Sakada, J., Kangawa, K., and Matsuo. H. (1988). Identification of new atria1 natriuretic peptides in frog heart. Biochem. Biophys, Res. Commun. 155, 1338+1345. Tanakaj. I., Misono, K. S., and Inagami, T. (1984). At&l natriuretic factor in rat hypothalamus, atria and plasma: Determination by specific radioimmunoassay. Biochem. Biophys. Res. Commun. 124, 663668. Vollmar, A. M., Friedrich, A., Sinowatz, F., and Schulz, R. (1988). Presence of atria1 natriuretic peptide-like material in guinea pig intestine. Peptides 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., Hanze, J., and Schulz, R. t199Oa). The rat thymus-A site of atrial natriuretic peptide synthesis. Peptides 11, 33-37. Vollmar, A. M., and Schulz, R. (1990b). Atria1 natriuretit peptide in lymphoid organs of various species. Comp. Biochem. Physiol. 96,459-463.
AND
COMPARATIVE
ENDOCRINOLOGY
87, 171-177 (1992)
Frog Lymph Heart Synthesizes and Stores Immunoreactive Natriuretic Peptide
Atrial
HOON RYU,* KYUNG Woo CHO, SUHN HEE KIM, SUNG Zoo KIM, SEON HEE OH, YUN HA HWANG, AND GEUM YEONG LEE* Department
of Physiology, Jeonbug
Medical National
School, and *Department of Biology, University, Jeonju 560-180, Republic
College of Natural of Korea
Sciences,
Accepted October 12, 1991 The presence of immunoreactive atria1 natriuretic peptide (irANP) and ANP gene expression in the frog lymph heart was examined by a radioimmunoassay (RIA) combined with HPLC and by Northern blot hybridization of total RNA. Serial dilution curve of the lymph heart extract was paralleled with the RIA standard curve. The lymph heart contained 153.32 t 35.80 pg of irANP/mg of wet tissue. The major form of irANP in the frog lymph heart was high molecular weight on reverse-phase and gel permeation high performance liquid chromatography as in the frog atria and ventricles. The frog lymph heart, as well as frog atria and ventricles, was shown to express mRNA coding for ANP. Dense core secretory granules similar to those observed in the mammalian atria were also found in the frog lymph heart. The presence of irANP and the expression of ANP gene in the frog lymph heart suggest that the lymph heart may participate in the regulation of homeostasis of lymph circulation and blood volume change through the synthesis and release of ANP. 6 1992 Academic press, IW.
Atria1 natriuretic peptide (ANP), now well known as a hormone stored in and released from mammalian atria1 cardiocytes, possesses variable biological effects: induction of diuresis and natriuresis (De Bold et al., 1981; Garcia et al., 1982), vasodilation (Garcia et al., 1984), and inhibition of aldosterone and renin release (De Lean et al., 1984; Obana et al., 1985). ANP has been found and characterized in a wide range of mammalian (Chapeau et al., 1985; De Bold and Salerno, 1983) and submammalian (Netchitailo et al., 1986a, 1988; Kim et al., 1989a) animals. It has previously been found by immunohistochemistry, bioassay, or radioimmunoassay that the frog atria and ventricles synthesize and store ANP (Chapeau et al., 1985; Netchitailo et al., 1986a, 1988). Frog ANP has also been known to be closely related to mammalian ANP in structure and pharmacological activities (De Bold and Salerno, 1983; Lazure et al., 1988; Sakada et al., 1988).
Interestingly, electron microscopic studies have revealed that the developing and adult frog lymph heart is very rich in dense core granules similar to atria1 specific granules in the blood heart of the adult frog (Markozashvili and Rumyantsev, 1984; Rumyantsev and Krylova, 1990). Therefore, we investigated the possible occurrence of irANP in the adult frog lymph heart by radioimmunoassay and characterized the molecular profile of irANP by reverse-phase and gel permeation high-performance liquid chromatography. We also examined ANP gene expression in the frog lymph heart by Northern blot hybridization for human ANP cDNA. MATERIALS Preparation
AND METHODS
of Samples
Adult frogs, Rana dybowskii, weighing 23-28 g were used. Frogs were captured during winter season and 171 0016-6480192 $4.00 Copyright 6 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
172
RYU ET AL.
were adapted at temperature 9” before experimentation for about 2 weeks. Frogs were pithed and the lymph hearts located at the end of the vertebral column were rapidly removed under a stereomicroscope. Both atria and ventricles were also obtained. Tissues were placed into prechilled tubes containing 0.1 N acetic acid with aprotinin (200 kallikrein inhibitor units/ml (KIU/ml)), phenylmethylsulfonyl fluoride (PMSF, 0.4%), ethylenediamine tetraacetic acid (EDTA, 10 n&), and soybean trypsin inhibitor (SBTI, 50 benzoyl arginine ethyl ester units/ml (BAEE/ml)). After weighing, tissues were boiled for 10 min and homogenized using a Polytron homogenizer. Tissue extracts were centrifuged at 10,OOOg for 15 min at 4”. The supematant was collected and applied on the Sep-Pak Cl8 cartridges (Waters Associates, Milford, MA) and irANP was extracted as described previously (Cho et al., 1988a,b). In brief, samples were applied on a SepPak Cl8 cartridge previously activated with 4 ml of acetonitrile and 0.1% trifluoroacetic acid (TFA). The cartridge was rewashed with 4 ml of 0.1% TFA and the adsorbed peptide was eluted with 3 ml of 60% acetonitrile in 0.1% TFA. The eluent was subsequently dried using a Speed-Vat concentrator (Savant, Hicksville, NY).
Radioimmunoassay (RIA) for irANP The content of irANP in the frog lymph heart was determined by RIA as previously described (Cho et al., 1988a,b). Briefly, anti-ANP antibody was raised against a synthetic atriopeptin III (AP III, NovaBiothem, Laeufelfingen, Switzerland) and AP III (Peninsula Laboratories, Belmont, CA) was iodinated using the chloramine-T method (Kim et al., 198913). The tissue extracts were reconstituted with Tris-acetate buffer (0.1 M, pH 7.4, containing 0.2% neomycin, 10 mM EDTA, 50 BAEE/ml SBTI, 200 KIUiml aprotinin, 0.4 mg% PMSF, 0.02% sodium azide, and 1% bovine serum albumin). After incubation with anti-ANP antibody for 24 hr at 4”, “-?-ANP was added and incubated for 24 hr at 4” again. The separation of free from bound forms was achieved by the double antibody technique.
Reverse-Phase (RP) and Gel Permeation (GPC) High-Performance Liquid Chromatography (HPLC) To elucidate the molecular profile of irANP in the frog lymph heart, 10-12 frogs were sacrificed and tissues were immediately put into liquid nitrogen. After boiling in 0.1 N acetic acid for 10 min, samples were processed as described above. The eluates were dried
using a Speed-Vat concentrator, reconstituted with 200 p,l of 0.1% TFA, and centrifuged at 10,OOOgfor 10 min at 4”. One hundred microliters of aliquots was subjected to the RP-HPLC on a p,Bondapak (Waters Associates, Milford, MA). Elution was performed with the linear gradient of 20 to 60% acetonitrile in 0.1% TFA for 40 min at a flow rate of 1 ml/min. Gel permeation HPLC was also performed on a TSK-GEL G 2000 SW column (7.5 x 300 mm, Toyo Soda, Tokyo, Japan) and eluted with 30% acetonitrile in 0.1% TFA. The flow rate was 0.3 ml/min and the fraction volume was 0.3 ml. The fractionated samples were dried using a Speed-Vat concentrator and their irANP content was determined by RIA.
Northern Blot Analysis Total RNA was isolated from frozen (-70”) frog lymph hearts, atria, and ventricles by the guanidinium monothiocyanate technique (Chirgwin et al., 1979). The lymph hearts from 40 frogs were removed and immediately put into liquid nitrogen. Frog atria and ventricles were used as control tissue for ANP mRNA. After quantification by absorption at 260 nm, 21 p,g of total RNA from lymph hearts as well as 58 pg of atria1 and 43 +g of total ventricular RNA were denatured, electrophoresed on 1.1% agarose gel containing 5% formaldehyde, and transferred to nylon membrane (Bio-Rad, Richmond, CA). A 580-bp Sac-PsfL restricted ANP cDNA cloned in a phAF48 (Nakayama et al., 1984) was a kind gift from Dr. S. Nakanish (Kyoto, Japan) through Dr. S. J. Kim (Bethesda, MD). The cDNA was labeled with [3ZP]dCTP (50 l&i) (Amersham) by random priming method (Amersham, U.S.A.) and denatured at 100” for 10 min. Membranes were prehybridized at 65” in 1% BSA, 7% sodium dodecyl sulfate (SDS), 1 mA4 EDTA, and 0.5 M sodium phosphate buffer (pH 7.4). Hybridization was performed by adding 1 x lo6 cpm/ml of the ANP probe and incubating for 16-20 hr at 65”. The membrane was washed twice in 1 M sodium chloridesodium citrate buffer and 0.1% SDS at 42” for 30 min. The membranes were exposed to an X-ray film with intensifying screens at -70” for 2 or 3 days.
Electron Microscopy To investigate the presence of secretory granules in the frog lymph heart, the posterior lymph hearts were excised and immediately fixed with 2.5% glutaraldehyde for 2 hr. All the samples were posffrxed in 1% osmium tetroxide (0~0,) and embedded in Epon 812. Thin sections were cut from Epon-embedded portions of the lymph heart with a diamond knife. These samples were stained with uranyl acetate and lead citrate,
irANP IN THE FROG LYMPH and the fine structure was examined with a JEOL 12OOEX electron microscope.
RESULTS Immunoreactive Atria1 Natriuretic in Frog Lymph Heart
Peptide
The serial dilution of extracts of the frog (R. dybowskii) lymph heart, atria, and ventricles inhibited the binding of [12jI]AP III to anti-AP III antiserum and the dilution curve for the lymph heart was relatively parallel with the standard curve of AP III, as shown in Fig. 1. This shows that the extract of the frog lymph heart possesses ir.ANP . The contents of irANP in the frog lymph heart, atria, and ventricles are summarized in Table 1. The levels of irANP in the frog lymph heart are 30 to 40 times lower than detected in the atrium.
I
10
I
I
102
173
HEART
Molecular Profiles Lymph Heart
of irANP
in the Frog
Elution profiles of the extracts of the frog lymph heart, atria, and ventricles on RPHPLC are shown in Fig. 2. The retention time of the synthetic peptide AP III and the purified rat pro-ANP were 15 and 30 min, respectively. The major peak of the frog lymph heart exhibited the same r~t~~~~~~ time as rat pro-ANP. Gel permeative HPLC showed that most irANP in the frog lymph heart coeluted with rat pro-A suggesting that this peptide corresponds to a high molecular weight form (Fig. 3). Northern
Blot Hybridization
A~~~y~~~
To determine whether the identified irANP in the frog lymph heart arises from endogenous synthesis of the pro-ANP molecule, we isolated total RNA from the frog
I
I
I
*--a
APill
O---O A-A A--).
lymph heart atria veritricles
standard
1
103
AP III ( pg/tube)
1. A representative standard curve of atriopeptin III (AP III), and serial dilution curves of the extracts of lymph heart, atria, and ventricles of the frog, Rann dybowskii. The absolute amounts of tissues used in dilution curves were 80 mg of lymph heart, I8 mg of atrium, and 42 mg of ventricle. FIG.
174
RYU ET AL. TABLE
THE LEVELS OF irANP dybowskii) LYMPH HEART,
Sample
1 IN THE FROG (Rana ATRIA, AND VENTRICLES
Levels of irANP
Lymph heart Atrium Ventricle
153.32 ? 5290.00 524.50
35.80 pg/mg pg/mg pg/mg
L 1210.00 + 116.62
Values are the mean f SEM of six experiments.
posterior lymph heart, atria, and ventricles and analyzed it for the presence of frog ANP mRNA transcripts. As shown in Fig. 4, Northern blot hybridization analysis revealed that the frog lymph heart contains ANP transcripts with size comparable to that of frog atria1 and ventricular transcripts.
ANP. Indeed, the pharmacological effect of frog ANP was found to be similar to that of mammalian ANP (Lihrmann et al., 1988), and was found to be highly homologous to rat (II-ANP (Sakada et al., 1988). The finding of ANP transcripts in the frog lymph heart is of particular interest, in that no groups have reported the presence of ANP mRNA. Since the description of extraatrial expression of ANP gene by Gardner et al. (1986), it has been thought that some tissue-specific factor, present predominantly in the atria, is required for efficient expression of this gene. Recently, extracardiac sources of ANP such as hypothalamus (Tanaka et al., 1984; Netchitailo
Electron
Microscopy
In the striated muscle cell of the frog lymph heart numerous secretory granules with electron dense core were observed (Fig. 5). We also found the secretory granules in the myoblast-like cell of the frog lymph heart (data not shown).
F’m ANP
AP Ill
T
T
A 1.2
r c -* --
-0.8
405
0
E 206
.
___---
6OE
--
a4 /fJLJ
oAi
k...
B
DISCUSSION
6
The present study demonstrates the presence of irANP and ANP gene expression in the frog lymph heart. IrANP in the frog lymph heart seems to exclusively consist of a high molecular weight form, which probably represents pro-ANP as in the frog atria and ventricles. In addition, we found that the frog lymph heart locally transcribes ANP mRNA. This finding suggests that the frog lymph heart synthesizes and stores ANP. Earlier studies have reported the presence of ANP in the frog atria and ventricles (De Bold and Salreno, 1983; Chapeau et al., 1985; Netchitailo et al., 1986a, 1988; Lazure et al., 1988; Sakada et al., 1988; Kim et al., 1989a). They suggested that frog ANP is structurally related to mammalian
4
2 0
2 1 0 0
10 Retention
20
30 time(min
40 )
FIG. 2. Reverse-phase HPLC profiles of irANP in the frog lymph heart (A), atria (B), and ventricles (C) of the frog. Arrowheads indicate the retention times of AP III and rat pro-ANP.
irANP IN THE FROG LYMPH
et al., 1986b), central nervous system (Netchitailo et al,, 1987), thymocytes (Vollmar et dl., 199Oa), spleen (Vollmar et al., 1989), gastrointestinal tract (Vollmar et al., 1988), interrenal gland (Lihrmann et al., 1988), ovary (Kim et al., 1989b), and various lymph nodes (Vollmar and Schulz, 1990b) have been described. It has been quite evident that the synthesis of ANP is by no means restricted to the cardiocytes. But the role of ANP in extracardiac tissues remains unknown. It was suggested that ANP in extracardiac tissues is synthesized not for circulation as a hormone but to serve some local paracrine, autocrine, or neurotrans-
Retention
time(min)
FIG. 3. Gel permeation HPLC profiles of irANP in lymph heart (A), atria (B), and ventricles (C) of the frog. Arrowheads indicate the retention times of blue dextran (Vo), bovine serum albumin (BSA), cytochrome C (CC), and AP III.
HEART
AT
175
LH
VT
FIG. 4. Blot hybridization analysis of total RNA from the frog lymph heart (LH), atria (AZ’), and ventricles (VT). RNA from the frog lymph heart (21 pg), atria (58 kg), and ventricles (43 pg) was anilyzed by blot hybridization using a 32P-labded hANP cDNA probe as described in the text.
mitter function (Gardner et al., 1986). Alternatively, the presence of ANP in ce1l.sof immune organs raises the possibility of a physiological function for ANP in the immune system (Gutkowska and Nemer, 1989). It was reported that the frog lymph heart wall is distinguished as three layers: an ,inner layer formed by endothelial cells lining the organ cavity, a middle layer made up of muscle fibers, and an outer one composed of fibrous connective tissue (Rumyantsev and Krylova, 1990). Ultrastructural studies of the frog lymph heart have shown the electron-dense core granules accumulated in peripheral region of sarsop’lasm of lymph heart muscles. In the present study, we have also found numerous electron-dense granules in the striated muscle cells and in the myoblast-like cells of the adult frog lymph heart (Fig. 5). In summary, the frog lymph heart was found to contain pro-ANP and to express its gene. Our findings raise the possibility that ANP in the frog lymph heart ,could be another extracardiac source of AMP secretion to the circulation and may serve a role
176
RYU ET AL.
FIG. 5. Electron micrograph of the frog lymph heart. Note a number of secretory granules (G). The frog lymph heart was fixed with 2.5% glutaraldehyde and embedded in Epon 812. Nucleus (N), mitochondria (m), myofilaments (M). Original magnification x4000. Bar = 2 urn.
as a biological regulator in lymph circulation and in body fluid homeostasis (Baldwin et al., 1990). ACKNOWLEDGMENTS We thank Dr. S. Nakanish and Dr. S. J. Kim for providing ANP cDNA. The authors thank especially Prof. Moo Sam Lee, Dept. of Anatomy, for providing facilities for the electron microscopy. We also thank Prof. Won Koo Lee, Dept. of Biology, and Mr. Byung Mun Ko, Dept. of Anatomy, for their critical suggestions on the experiments, and Miss Kyung Hwa Nam, Miss Hea Kyung Cho, and Miss Yeo Lae Eun for their fine technical assistance. This study was supported by a Graduate Fellowships and a research grant from the Korea Science and Engineering Foundation, and by a grant-in-aid from the Ministry of Education, Korea.
REFERENCES Baldwin, A. L., Rozum, J. S., and Gore, R. W. (1990). Routes of water and small solute transport between blood and lymph in frog. Physiol. Zool. 63, 1141-l 156.
Chapeau, C., Gutkowska, J., Schiller, P. W., Miline, R. W., Thibault, G., Garcia, R., Genest, J., and Cantin, M. (1985). Localization of immunoreactive synthetic atrial natriuretic factor (ANF) in the heart of various animal species. J. H&o&em. Cytochem. 33, 541-550. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. Cho, K. W., Seul, K. H., Kim, S. H., Ryu, H., Seul, K. M., and Koh, G. Y. (1988a). Epicardial release of immunoreactive atria1 natriuretic peptides in inside-out perfused rabbit atria. Biochem. Biophys. Res. Commun. 153, 811-817. Cho, K. W., Seul, K. H., Ryu, H., Kim, S. H., and Koh, G. Y. (1988b). Characteristics of distentioninduced release of immunoreactive atria1 natriuretic peptide in isolated perfused rabbit atria. Regul. Pept. 22, 333-345. De Bold, A. J., Borenstein, H. B., Veress, A. T., and Sonnenberg, H. (1981). A rapid and potent natriuretic response to intravenous injection of atria1 myocardial extract in rats. Life Sci. 28, 89-94. De Bold, A. J., and Salerno, T. A. (1983). Natriuretic
irANP IN THE FROG LYMPH activity of extracts obtained from heart of different species and from various rat tissues. Can. J. Physiol. Pharmacol. 61, 127-130. De Lean, A., Racz, K., Gutkowska, J., Nguyen, T., Cantin, M., and Genest, J. (1984). Specific receptor-mediated inhibition by synthetic atria1 natriuretic factor of hormone-stimulated steroidogenesis in cultured bovine adrenal cells. Endocrinology 115, 1636-1638. Garcia, R., Cantin, M., Thibault, G., Ong, H., and Genest, J. (1982). Relationship of specific granules to the natriuretic and diuretic activity of rat atria. Experientia 38, 1071-1073. Garcia, R., Thibault, G., Cantin, M., and Genest, J. (1984). Effect of a purified atrial natriuretic factor on rat and rabbit vascular strips and vascular beds. Am. J. Physiol. 247, R34--R39. Gardner, D. G., Deschepper, C. F., Ganong, W. F., Hane, S., Fiddes, J., Baxter, J. D., and Lewicki, J. (1986). Extra-atria1 expression of the gene for atria1 natriuretic factor. Proc. Natl. Acad. Sci. USA 83, 6697-6701. Gutkowska, J., and Nemer, M. (1989). Structure, expression, and function of atrial natriuretic factor in extraatrial tissues. Endocrine Rev. 10,519-536. Kim, S. H., Cho, K. W., Koh, G. Y., Seul, K. H., So, J. N., and Ryu, H. (1989a). Phylogenetic study on the immunoreactive atria1 natriuretic peptide in the heart. Gen. Comp. Endocrinol. 14, 127-135. Kim, S. H,, Cho, K. W., Seul, K. H., Ryu, H., and Koh, G. Y. (1989b). Presence of immunoreactive atria1 natriuretic peptide in follicular fluid, ovary and oVarim perfusates. Life Sci. 45, 1581-1589. Lazure, C., Gng, H.,’ McNicoll, N., Netchitailo, P., Chsetien, M., De Lean, A., and Vaudry, H. (I%%). The amino sequences of frog heart atrial natriuretic-like peptide and mammalian ANF are closely related. FE&S Lett. 238, 300-306. Lihrmann, I., Net&i&lo, P., Feuilloley, M., Cantin, M., D&rue, C., Leboulenger, F., De Lean, A., and Vat&y, II. (1988) Effect of atrial natriuretic fac&or on @rticosteroid production by perifused frog interrenal slices. Gen. Comp. Endocrinol. 71, 55-62. MarKozashvili, M. I., and Rumyantsev, P. P. (1984). Dltrastructure of muscle fibers and cells synthesizing D,NA in lymph hearts of developing frogs and chick embryos. Cell Tissue Res. 238,369-379. Nakayama,, K., Ohkubo, H., Hirose, T., Inayama, S., and Nakanishi, S. (1984). mRNA sequence for human cardio#ttin~atrial natriuretic factor precursor &td &n.tlation of precursor mRNA in rat atria. Ndruri 31D, 699-701~
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Netchitailo, P., Feuilloley, L. M., Pelletier, G., Cantin, M., De Lean, A., Leboulenger, F., and Vaudry, H. (1986a). Localization and characterization of atrial natriuretic factor (ANF)-like peptide in the frog atrium. Peptides 7, 573-579. Netchitailo, P., Feuilloley, M., Pelletier, G., Cantin, M. ) Leboulenger, F., Andersen, A., and Van&y, H. (1986b). Localization of atria1 natriuretic factor (ANF)-immunoreactive material in the hypothalamo-pituitary complex of the frog. Newossi. Lett. 72, 141-146. Netchitailo, P., Feuilloley, M., Pelletier, G., Leboulenger, F., Cantin, M., Gutkowska, J., and Vaudry, II. (1987). Atrial natriuretic factor like immunoreactivity in the central nervous system of the frog. Neuroscience 22, 341-359. Netchitailo, P., Feuilloley, L. M., Pelletier, a?., De Lean, A., Ong, II., Cantin, M., Gutkowska, J., Leboulenger, F., and Vat&y, A. (1988). Localization and identification of immunoreactive atrial natriuretic factor (ANF) in the frog ventricle. Peptides 9, l-6. Obana, K., Naruse, M., Naruse, K., Sakurai, II., Demura, II., Inagami, T., and Shizume, K. (1985). Synthetic rat atrial natriuretic factor inhibits in vitro and in vivo renin secretion in rats. Endocrinology 117, 1282-1284. Rumyantsev, P. P., and Krylova, M. I. (1990). Ultrastructure of myofibers and cells synthesizing DNA in’ the developing and regenerating lymphheart muscles. Int. Rev. CyroE. 120, l-52. Sakada, J., Kangawa, K., and Matsuo. H. (1988). Identification of new atria1 natriuretic peptides in frog heart. Biochem. Biophys, Res. Commun. 155, 1338+1345. Tanakaj. I., Misono, K. S., and Inagami, T. (1984). At&l natriuretic factor in rat hypothalamus, atria and plasma: Determination by specific radioimmunoassay. Biochem. Biophys. Res. Commun. 124, 663668. Vollmar, A. M., Friedrich, A., Sinowatz, F., and Schulz, R. (1988). Presence of atria1 natriuretic peptide-like material in guinea pig intestine. Peptides 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., Hanze, J., and Schulz, R. t199Oa). The rat thymus-A site of atrial natriuretic peptide synthesis. Peptides 11, 33-37. Vollmar, A. M., and Schulz, R. (1990b). Atria1 natriuretit peptide in lymphoid organs of various species. Comp. Biochem. Physiol. 96,459-463.
