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Phylogenetic aspects of cardiac hormones as revealed by immunocytochemistry, electronmicroscopy, and bioassay
Peptides. Vol. 6. Suppl. 3. pp. 321-331. 1985. e AnkhoInternationalInc. Printedin the U.S.A.
0196-9781/85 $3.00 + .00
Phylogenetic Aspects of Cardiac Hormones as Revealed by Immunocytochemistry, Electronmicroscopy, and Bioassay M. R E I N E C K E , 1 M. N E H L S A N D W. G. F O R S S M A N N
D e p a r t m e n t o f A n a t o m y III, University o f Heidelberg, Heidelberg, F . R . G .
REINECKE, M., M. NEHLS AND W. G. FORSSMANN. Phylogenetic aspects of cardiac hormones as revealed by immunocytochemistr3", electronmicroscopy, and bioassay. PEPTIDES 6: Suppl. 3,321-331, 1985.--A hormone family of cardiac peptides has recently been isolated and biochemically and pharmacologically characterized by the relaxation of vascular smooth muscle, diuretic and natriuretic activities. The cardiac hormones are stored in specific granules of the atrial myoendocrine cells. Since data is available only from mammals (rat, pig, man) we started a phylogenetic study by investigating representatives of the higher vertebrate classes (birds, reptiles, amphibians, bony fish) as well as an invertebrate species, the gastropod mollusc Helix pomatia. Homologous cardiac hormones of the cardiodilatin (CDD) family which exerted a dose-dependent relaxant effect on the rabbit aorta were extracted from the atria of all species studied and from the ventricles of amphibians and teleosts. The storage sites of cardiac hormones were localized by electronmicroscopy and immunocytochemistry using antisera against several sequences of pig CDD and applying the peroxidaseantiperoxidase technique. CDD-immunoreactivity (CDD-IR) was observed in myoendocrine cells in the atria of all vertebrate species studied, and in amphibians and teleosts also in the ventricles. In the snail, however, CDD-IR was present in nerve endings of the atrium and in perikarya of the subesophageal ganglion as well as in fibers of the intestinal nerve, while no CDD-IR was detected in heart muscle cells. In correlation, no "specific" granules were observed in myocardiocytes of the snail and vascular smooth muscle relaxant bioactivity was present in extracts of the subesophageal ganglion. The findings indicate that in the vertebrates studied the cardiodilatin-immunoreactive substances seem to constitute an endocrine system in the heart. In the snail, in contrast, they are present in a neuro-cardiac axis. This seems to represent a model unique in phylogeny. Cardiac hormones Cardiodilatin Mammals Birds Reptiles Amphibians Gastropods Immunocytochemistry Electronmicroscopy Bioassay
E N D O C R I N E - L I K E myocardial cells characterized by specific granules have first been described by Kisch [19], Bompiani et al. [I] and Jamieson and Palade [17]. The majority of these myoendocrine cells occur in the atrial appendages of the mammalian heart. More than a decade later functional studies were carried out on the myoendocrine cells [20] which demonstrated a relation between the water electrolyte balance and the content of specific granules in the atrial myoendocrine cells. Atrial extracts of those cardiac regions containing myoendocrine cells were later shown to exert two main biological effects: (1) Diuresis and natriuresis [5] and (2) relaxation of vascular smooth muscle [3, 7, 12]. Using these biological actions as bioassays from atrial extracts of some mammalian species several peptide hormones were then isolated and different names, like, e.g., cardionatfin, atrial natriuretic factor, atriopeptin, were given to these peptides [4, 8, 18, 27, 29]. Our working group was the first to demonstrate the occurrence of a larger 126 amino acid containing cardiac
Bony fish
hormonal peptide which we called cardiodilatin (CDD) [12, 13, 14]. The C-terminal of the CDD-molecule contains the amino acid sequences of the cardiac peptides described by the various groups, cDNA and genom analysis have confined that all cardiac hormonal peptides belong to one family, the members of which exhibit different parts of the primary structure of a hormone precursor [16,30]. This precursor is extended by a signal peptide of 25 amino acids at the N-terminal portion of cardiodilatin. The actual knowledge about the occurrence of cardiac hormones of the cardiodilatin family is almost restricted to a few mammalian species. Some recent investigations, however, indicate that the principle of cardiodilatin-like hormones is also existent in nonmammalian species: De Bold and Salerno [6] found biological activity in extracts of frog heart, Reinecke et al. [24] presented preliminary results demonstrating the presence of cardiac hormones in several submammalian species, and Nehls et al. [22] showed the existence of cardiodilatin-like material in the snail Helix
'Requests for reprints should be addressed to Prof. Dr. M. Reinecke, Anatomisches Institut III, Universitiit Heidelberg, Im Neuenheimer Feld 307, D-6900 Heidelberg, F.R.G.
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FIG. 1. (a.b) Localization of CDD-IR in the human (a) and porcine (b) right atrium. CDD-IR is concentrated at the nuclear poles of myoendocrine cells which occur intermingled between the atrial myocardiocytes. (a) x640. lb) x 590. (c) Myoendocrine cell from the right auricle of Tttpaia behtngeri showing an accumulation of specific atrial granules at the nuclear pole. x 22600. Id~ Dose-dependent vasorelaxant effect of pig atrial extract on the rabbit aorta.
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PHYLOGENETIC ASPECTS OF CARDIAC HORMONES
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TABLE 1 AMOUNTOF WET WEIGHT(rag)OF EXTRACTEDCARDIACTISSUE CAUSINGA VASODILATIONOF 50% OF THE PRECONTRACTED RABBIT AORTA
positive controls [11]. As positive controls sections of porcine cardiac atria were subjected to parallel incubation. Since all controls indicated the specificity of the obtained immunoreactions the stained structures are called cardiodilatin-immunoreactive (CDD-IR).
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Electronmicroscopy Small tissue specimens of the atria and ventricles of all species investigated were fixed by immersion in a solution containing 1.25% formaldehyde and 1.25% glutardialdehyde in a 0.2 M phosphate buffer [9]. After rinsing in the buffer the specimens were postfixed in 1% OsO~ diluted in 0.2 M phosphate buffer, dehydrated in graded series of ethanol and routinely embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate. A Zeiss EM 10 was used for analysis and photography.
Extraction and Bioassay
pomatia. Since phylogenetic studies are thought to be of great importance for a variety of not only phylogenetic problems but also for pharmacological and physiological questions [23], we investigated the possible occurrence of cardiac hormones of the CDD family in the higher submammalian classes as well as in an invertebrate species. The study was carried out by correlating immunocytochemical and electronmicroscopical results with those obtained by bioassays. METHOD As representatives of their classes the following vertebrate species were used for immunohistochemical and electronmicroscopical analysis and in part also for bioassay: Mammals: Man, Tupaia belangeri, pig, dog, cat, rat, guinea pig; birds: Gallus g. domesticus. Coturnix c.japonicus; reptiles: Anolis carolinensis; amphibians: Rana esculenta; teleost bony fish: Salmo gairdneri. In addition, the gastropod mollusc Helix pomatia was investigated.
Cardiac atria and ventricles of the following species (number of individuals in parentheses) were extracted: Pig (50), chicken (12), Anolis carolinensis (15), Rana esculenta (30), Salmo gairdneri (30), Helix pomatia (30). As for the snail also cerebral (30) and subesophageal ganglia (30) were extracted. After animals were sacrificed, the tissue for extraction was dissected rapidly and immediately deep frozen. After defrosting, tissue was weighed and boiled in fivefold (weight/volume) volumes of distilled water for 10 min. After boiling an equal amount of ice-cold 0.4 M acetic acid was added and the mixture was stirred for 2.5 hr at 4°C. Thereafter, the crude extract was centrifuged at 50,000 g for 30 min. Supernatant was collected and prepurified using Sep-Pak capsules with 80% acetonitrile in 0.1% trifluoro acetic acid as solvent. The vasorelaxant effect exerted on the rabbit aorta as described for mammalian cardiac hormones [12] was used as bioassay. After an equilibration period of 2 hr in a standard physiological salt solution, helical strips of rabbit aorta were precontracted by 10-7 M norepinephrine. After the contraction had reached a plateau, increasing amounts of the prepurified extracts were added cumulatively.
lmmunocytochemistry All mammalian and avian species were anesthetized by intraperitoneai injection of Nembutal ®, the reptiles and amphibians and the snails by using ether-chloroform vapour and the bony fish by adding MS 222® to the water. Human atrial tissue which is excised to introduce the tube for extracorporal circulation during coronary by-pass operation was also investigated in our study (approved by the ethic committee of the University of Heidelberg). The hearts and in case of the snails also the cerebral and subesophageal ganglia were rapidly dissected and fixed by immersion in Bouin's fluid without acetic acid. After fixation the specimens were dehydrated in ascending concentrations of ethanol and routinely embedded in paraplast. Sections were cut at 7 tzm and processed for a modification [11] of the peroxidaseantiperoxidase (PAP) technique [28]. All antisera applied in this study were raised in rabbit against different portions of cardiodilatin (CDD) and are described in detail elsewhere [15]. Throughout the submammalian species studied only C-terminal region-specific antisera were reactive. A detailed immunocytochemical study related to phylogeny will be published elsewhere [26]. The specificity of the immunohistochemical reactions was tested by a variety of negative and
RESULTS VERTEBRATES
Immunocytochemistry and Electronmicroscopy Mammals. In the atria of all mammalian species investigated CDD-IR was localized in myoendocrine cells of both atrial appendages. In the remaining atrial myocardium CDD-IR cells occurred only infrequently. The distribution patterns of the CDD-IR cells in the different species showed a high degree of correspondence. In the myoendocrine cells CDD-IR was accumulated adjacent to the nuclear poles (Fig. la,b) and occurred only rarely in other cytoplasmic regions. By means of electronmicroscopy the myoendocrine cells of the species studied were characterized by specific moderate electron-dense granules (Fig. lc). These were present mainly in the perinuclear region adjacent to the Golgi apparatus. This location is identical to that of the CDD-IR as observed in the light microscope. In the ventricles neither CDD-IR nor myoendocrine cells were to be found. Birds. In contrast to the mammalian hearts, in the hearts of the avian species investigated, i.e., chicken and quail,
328
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P H Y L O G E N E T I C ASPECTS OF CARDIAC HORMONES CDD-IR cells were observed only very infrequently. They were faintly stained (Fig. 2a,b) and located in both atrial appendages, while no CDD-IR cells seemed to occur in ventricles. In the electronmicroscope, myoendocrine cells were found in corresponding location. Like in mammals, the secretory granules were concentrated in the perinuclear regions (Fig. 2c). The density of the granules, however, was extremely low. Reptiles. In the heart of the lizard species Anolis carolinensis which was studied as representative of the reptiles CDD-IR cells occurred throughout the atria (Fig. 3a,b) with the highest density in the appendages. In the ventricles no CDD-IR cells were observed. In correlation, in all regions of the atria myoendocrine cells exhibiting heteromorphous secretory granules (Fig. 3c) were identified. Amphibians. In the atria of the frog Rana esculenta CDD-IR cells were present in large numbers (Fig. 4a). They were distributed throughout the atrial myocardium without showing any preferential location. Furthermore, in contrast to the vertebrate classes described above CDD-IR cells occurred in moderate numbers also in the ventricle. They were restricted mostly to the outer ventricle wall (Fig. 4b). Myoendocrine cells (Fig. 4c) were found in the same locations, i.e., they occurred both in the atria and in the ventricle. Bony fish. A fresh water teleost, the rainbow trout, was investigated as representative of the bony fish. Throughout the atrium of the trout CDD-IR cells (Fig. 5a) occurred in high densities. Like in the frog heart, in the ventricle of the trout also CDD-IR cells were found, however, in by far lower numbers (Fig. 5b). In the electronmicroscope myoendocrine cells were identified in corresponding locations. In contrast to the other vertebrate species studied, the secretory granules of the trout myoendocrine cells were smaller and less heteromorphous (Fig. 5c).
Bioassay Prepurified atrial extracts from pig (Fig. ld), chicken (Fig. 2d), lizard (Fig. 3d), frog (Fig. 4d) and trout (Fig. 5d) as well as ventricular extracts from frog and trout exerted potent vasorelaxant effects on the rabbit aorta precontracted by norepinephrine. The vascular smooth muscle relaxation achieved was clearly dose-dependent. Additional information is presented in Table I which gives the amount of extracted cardiac tissue which is necessary to cause a relaxation of 50%. The values obtained differ widely between the representatives of the different classes. The lowest amounts of atrial extracts were necessary with the mammals, intermediate ones with lizard, frog and trout, while high amounts of chicken extract had to be applied to cause an aortic relaxation of 50%. While no unequivocal dilation of rabbit aorta could be yielded applying extracts of pig, chicken and lizard ventricles, ventricular extracts of frog and trout caused vasodilations. However fairly high amounts were necessary to cause an aortic relaxation of 50% (Table 1). GASTROPODS
hnmunohistochenlistry and Electromnicroscopy The immunohistochemical investigation of the snail heart revealed the occurrence of CDD-IR structures. These were found exclusively in the atrium whereas no CDD-IR occurred in the ventricle. The CDD-IR was not present in the atrial myocardiocytes but was confined to round or ovoid
329 structures (Fig. 6a). These were located intermingled between the myocardiocytes as well as free in the atrial lumen. Their average diameter surpassed that of the myocardiocytes. Electronmicroscopical analysis of the corresponding areas of the snail heart revealed the existence of nerve varicosities characterized by heteromorphous electron-dense granules (Fig. 6b). The varicosities occurred in the same overall distribution as the light microscopically identified CDD-IR structures. No secretory granules were observed within the atrial myocardiocytes. In the visceral ganglion of the subesophageal complex CDD-IR perikarya were found either singularly or in small groups (Fig. 6c). Furthermore, CDD-IR was present in nerve fibers of the neuropile as well as in fibers of the intestinal nerve which projects to the cardiac region. In the cerebral ganglia CDD-IR perikarya occurred only very infrequently.
Bioassay Atrial and subesophageal extracts prepurified by the use of Sep-Pak capsules exerted smooth, muscle relaxation of rabbit aortic strips which was clearly dose-dependent (Figs. 6d,e). With extracts of the ventricle and the cerebral ganglia, however, no vascular smooth muscle relaxation was found. DISCUSSION The results presented here indicate that the occurrence of cardiac hormones of the CDD-family is not restricted to mammals. They seem to be present also in the hearts of avian, reptilian, amphibian and bony fish species as has been demonstrated here by immunocytochemistry, electronmicroscopy and bioassay. While a variety of investigations were carried out on the localization [2, 13, 15, 21] and physiology (for review see [10]) of cardiac hormones in mammals there is except for a short communication of our group [24] only one study dealing with the possible existence of cardiac hormones in submammalian vertebrates [6]. In contrast to our results the authors did not succeed to detect biological activity of cardiac hormones in the chicken heart. This discrepancy may be due to the different extraction and/or bioassay methods applied as well as to the paucity of myoendocrine and CDD-IR cells, respectively, in the chicken, While so far no results are available on the occurrence of cardiac hormones in reptiles and bony fish, in agreement with our results myoendocrine cells, as well as cardiac hormone bioactivity, were found in the frog heart |6|. Since for each vertebrate class investigated only one or two representatives were studied to date one should be careful not to overdue the interpretations. However, some conclusions may be drawn. In the phylogenetic line leading to the birds the cardiac hormones may have lost their physiological importance since in the avian heart CDD-IR cells or myoendocrine cells, respectively, occur very infrequently. These results are supported by the bioassay: The low density of CDD-IR cells in the chicken atrium is reflected by the high amount of tissue extract necessary to cause a relaxation of 50% of the rabbit aorta. However, pharmacological studies in avian species must be carried out to clarify this hypothesis. The morphological result that the occurrence of CDD-IR cells or myoendocrine cells, respectively, in the hearts of the mammals, birds and reptiles studied was restricted to the atria while in the amphibians and bony fish they were additionally in the ventricles also correlates with the bioassay: No vascular smooth muscle relaxation was obtained from
330
REINECKE, NEHLS AND FORSSMANN
ventricular extracts of pig, chicken and lizard whereas ventricular extracts of frog and trout caused relaxation of rabbit aortic strips. Thus, there may be a phylogenetic trend of concentrating the cardiac hormone producing cells in the atria. A particular interesting finding [22] of our phylogenetic studies is also included in this investigation, i.e., that in the snail Helix pomatia, a gastropod mollusc, a CDD-like hormonal substance is present in atrial nerves and not in myocardial cells as was found in all vertebrate species studied so far. Thus, at least in the snail, a CDD-IR neurosecretory axis occurs which projects from the subesophageal ganglion to the cardiac atrium. This result stimulated our efforts to investigate whether CDD-IR also exists in the mammalian nervous system. In fact, an immunocytochemical study o f hypothalamic regions of Tupaia belangeri showed the presence of CDD-IR neurones which may be involved in cardiovascular and water balance regulation [15]. We have further indication that CDD can be postu-
lated to be a neurotransmitter or neuromodulator in other regions of the nervous system as well as in other species [25]. Our present interest is focused on the problem of whether the CDD-like neuroendocrine system observed in the snail is typical for gastropods or if it constitutes a general phenomenon of invertebrates. Furthermore, the functional significance of the neuro-cardiac CDD-IR axis in the snail is presently under investigation. Interesting phylogenetic questions concern the points of how this neuroendocrine system has been transformed into a pure cardiac endocrine system and where along the phylogenetic tree this step has been taken place.
ACKNOWLEDGEMENTS For skillful technical assistance we thank Mrs. Barbara Briihl and Mr. Richard Hertel. The study was supported by the German Research Foundation (Re 520/1-2).
REFERENCES I. Bompiani, G. D., C. Rouiller and P. Y. Hatt. Le tissu de conduction du coeur chez le rat. Etude au microscope ~lectronique. Arch Mal Coeur 52: 1257-1274, 1959. 2. Cantin, M., J. Gutkowska, G. Thibault, R. E. Milne, S. Ledoux, S. MinLi and C. Chapeau. Immunocytochemical localization of atrial natriuretic factor in the heart and salivary glands. Histochemistry 80:113-127, 1984. 3. Currie, M. G., D. M. Geller, B. R. Cole, J. G. Boylan, W. YuSheng, S. W. Holmberg and P. Needleman. Bioactive cardiac substances: potent vasorelaxant activity in mammalian atria. Science 221: 71-73, 1983. 4. Currie, M. G., D. M. Cieller, B. R. Cole, N. R. Siegel, K. F. Fok, S.P. Adams, S.R. Eubanks, G.R. Gallupi and P. Needleman. Purification and sequence analysis of bioactive atrial peptides (atriopeptins). Science 223: 67-69, 1984. 5. De Bold, A. J., H. B. Borenstein, A. T. Veress and H. Sonnenberg. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci 28: 89-94, 1981. 6. De Bold, A. J. and T. A. Salerno. Natriuretic activity of extracts obtained from hearts of different species and from various rat tissues. Can J Physiol Pharmacol 61: 127-130, 1983. 7. Deth, R. C., K. Wong, S. Fukozawa, R. Rocco, J. L. Smart, J. Lynch and R. Awad. Inhibition of rat aorta contractile response by natriuresis-inducing extract of rat atrium. Fed Proc 41: 983, 1982. 8. Flynn, T. G., M. L, De Bold and A. J. De Bold. The amino acid sequence of an atrial peptide with potent diuretic and natriuretic properties. Biochem Biophys Res Commun 117: 859-865, 1983. 9. Forssmann, W. G. General methods in transmission electron microscopy of the nervous system. In: Techniques in Neuroanatomical Research, edited by Ch. Heym and W. G. Forssmann. Berlin: Springer, 1981, pp. 21-40. 10. Forssmann, W. G. Cardiac hormones with renal effects: potential role in health and disease. Contrib Nephrol, in press, 1985. 11. Forssmann, W. G., V. Pickel, M. Reinecke, D. Hock and J. Metz. Immunohistochemistry and immunocytochemistry of nervous tissue. In: Techniques in Neuroanatomical Research, edited by Ch. Heym and W. G. Forssmann. Berlin: Springer, 1981, pp. 171-205. 12. Forssmann, W. G., D. Hock, F. Lottspeich, A. Henschen, V. Kreye, M. Christmann, M. Reinecke, J. Metz and V. Mutt. The right auricle of the heart is an endocrine organ. Cardiodilatin as a peptide hormone candidate. Anat Embryol (Berlin) 168: 307-313, 1983.
13. Forssmann, W. G., C. Birr, M. Cadquist, M. Christmann, R. Finke, A. Henschen, D. Hock, H. Kirchheim, V. Kreye, F. Lottspeich, J. Metz, V. Mutt and M. Reinecke. The auricular myocardiocytes of the heart constitute an endocrine organ. Characterization of a porcine cardiac peptide hormone, cardiodilatin-126. Cell Tissue Res 238: 425--430, 1984. 14. Forssmann, W. G., D. Hock, F. Kirchheim, J. Metz, V. Mutt and M. Reinecke. Cardiac hormones: Morphological and functional aspects. Clin Exp Hyperten Theor3" Pract A6: 1873-1878, 1984. 15. Forssmann, W. G. and V. Mutt. Cardiodilatin-immunoreactive neurons in the hypothalamus ofTupaia. Anat Embryol 172: i-5, 1985. 16. Greenberg, B. D., G. H. Bencen, J. J. Seilhamer, J. A. Lewicki and J. C. Fiddes. Nucleotide sequence of the gene encoding human atrial natriuretic factor precursor. Nature 312: 656-658, 1984. 17. Jamieson, J. D. and G. E. Paiade. Specific granules in atrial muscle cells. J Cell Biol 23: 151-172, 1964. 18. Kangawa, K. and H. Matsuo. Purification and complete amino acid sequence of a-human atrial natriuretic polypeptide (ahANP). Biochem Biophys Res Commun 118: 131-139, 1984. 19. Kisch, B. Electron microscopy of the atrium of the heart. I. Guinea pig. Exp Med Sarg 14: 99-112, 1956. 20. Marie, J. P., H. Guillemot and P. Y. Hatt. Le degr~ de granulation des cardiocytes auriculaires, l~tude planim~trique au cours de diff~rents apports d'eau et de sodium chez le rat. Pathol Biol 24: 549-554, 1976. 21. Metz, J., V. Mutt and W. G. Forssmann. Immunohistochemical localization of cardiodilatin in myoendocrine cells of the cardiac atria. Anat Embryol 170: 123-127, 1984. 22. Nehls, M., M. Reinecke, R. E. Lang and W. G. Forssmann. Biochemical and immunological evidence for a cardiodilatin-like substance in the snail neuro-cardiac axis. Proc Natl Acad Sei USA, accepted for publication, 1985. 23. Reinecke, M. and W. G. Forssmann. Why do we study the phylogeny of neuropeptide hormones? In: Evolution and Tumour Pathology of the Neuroendocrine System. edited by S. Falkmer, R. H~tkanson and F. Sundler. Amsterdam: Elsevier Biomedical Press, 1984, pp. 1-5. 24. Reinecke, M., M. Nehls, R. E. Lang and W. G. Forssmann. Phylogenetic aspects of cardiac hormones as revealed by electronmicroscopy, immunohistochemistry, radioimmunoassay, bioassay and biochemistry. Neural and Endocrine Peptides and Receptors '85, Fifth Int. Wash. Spring Symp., (Abstract) 33: 1985.
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25. Reinecke, M. and W. G. Forssmann. The cardiac hormone cardiodilatin: also a neurotransmitter? Neurosci Lett. in preparation. 1985. 26. Reinecke, M. and W. G. Forssmann. Immunocytochemical localization of cardiac hormones in the hearts of representatives of all vertebrate classes. Cell Tissue Res, in preparation, 1985. 27. Seidah. N. G., C. Lazure, M. Chr¢tien G. Thibault, R. Garcia, M. Cantin, J. Genest, R. F. Nutt, S. F. Brady, T. A. Lyle, W. J. Paleveda, C. D. Colton and T. M. Ciccarone. Amino acid sequence of homologous rat at/fal peptides: natriuretic activity of native and synthetic forms. Proc Natl Acad Sci USA 81: 2640-2644, 1984.
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0196-9781/85 $3.00 + .00
Phylogenetic Aspects of Cardiac Hormones as Revealed by Immunocytochemistry, Electronmicroscopy, and Bioassay M. R E I N E C K E , 1 M. N E H L S A N D W. G. F O R S S M A N N
D e p a r t m e n t o f A n a t o m y III, University o f Heidelberg, Heidelberg, F . R . G .
REINECKE, M., M. NEHLS AND W. G. FORSSMANN. Phylogenetic aspects of cardiac hormones as revealed by immunocytochemistr3", electronmicroscopy, and bioassay. PEPTIDES 6: Suppl. 3,321-331, 1985.--A hormone family of cardiac peptides has recently been isolated and biochemically and pharmacologically characterized by the relaxation of vascular smooth muscle, diuretic and natriuretic activities. The cardiac hormones are stored in specific granules of the atrial myoendocrine cells. Since data is available only from mammals (rat, pig, man) we started a phylogenetic study by investigating representatives of the higher vertebrate classes (birds, reptiles, amphibians, bony fish) as well as an invertebrate species, the gastropod mollusc Helix pomatia. Homologous cardiac hormones of the cardiodilatin (CDD) family which exerted a dose-dependent relaxant effect on the rabbit aorta were extracted from the atria of all species studied and from the ventricles of amphibians and teleosts. The storage sites of cardiac hormones were localized by electronmicroscopy and immunocytochemistry using antisera against several sequences of pig CDD and applying the peroxidaseantiperoxidase technique. CDD-immunoreactivity (CDD-IR) was observed in myoendocrine cells in the atria of all vertebrate species studied, and in amphibians and teleosts also in the ventricles. In the snail, however, CDD-IR was present in nerve endings of the atrium and in perikarya of the subesophageal ganglion as well as in fibers of the intestinal nerve, while no CDD-IR was detected in heart muscle cells. In correlation, no "specific" granules were observed in myocardiocytes of the snail and vascular smooth muscle relaxant bioactivity was present in extracts of the subesophageal ganglion. The findings indicate that in the vertebrates studied the cardiodilatin-immunoreactive substances seem to constitute an endocrine system in the heart. In the snail, in contrast, they are present in a neuro-cardiac axis. This seems to represent a model unique in phylogeny. Cardiac hormones Cardiodilatin Mammals Birds Reptiles Amphibians Gastropods Immunocytochemistry Electronmicroscopy Bioassay
E N D O C R I N E - L I K E myocardial cells characterized by specific granules have first been described by Kisch [19], Bompiani et al. [I] and Jamieson and Palade [17]. The majority of these myoendocrine cells occur in the atrial appendages of the mammalian heart. More than a decade later functional studies were carried out on the myoendocrine cells [20] which demonstrated a relation between the water electrolyte balance and the content of specific granules in the atrial myoendocrine cells. Atrial extracts of those cardiac regions containing myoendocrine cells were later shown to exert two main biological effects: (1) Diuresis and natriuresis [5] and (2) relaxation of vascular smooth muscle [3, 7, 12]. Using these biological actions as bioassays from atrial extracts of some mammalian species several peptide hormones were then isolated and different names, like, e.g., cardionatfin, atrial natriuretic factor, atriopeptin, were given to these peptides [4, 8, 18, 27, 29]. Our working group was the first to demonstrate the occurrence of a larger 126 amino acid containing cardiac
Bony fish
hormonal peptide which we called cardiodilatin (CDD) [12, 13, 14]. The C-terminal of the CDD-molecule contains the amino acid sequences of the cardiac peptides described by the various groups, cDNA and genom analysis have confined that all cardiac hormonal peptides belong to one family, the members of which exhibit different parts of the primary structure of a hormone precursor [16,30]. This precursor is extended by a signal peptide of 25 amino acids at the N-terminal portion of cardiodilatin. The actual knowledge about the occurrence of cardiac hormones of the cardiodilatin family is almost restricted to a few mammalian species. Some recent investigations, however, indicate that the principle of cardiodilatin-like hormones is also existent in nonmammalian species: De Bold and Salerno [6] found biological activity in extracts of frog heart, Reinecke et al. [24] presented preliminary results demonstrating the presence of cardiac hormones in several submammalian species, and Nehls et al. [22] showed the existence of cardiodilatin-like material in the snail Helix
'Requests for reprints should be addressed to Prof. Dr. M. Reinecke, Anatomisches Institut III, Universitiit Heidelberg, Im Neuenheimer Feld 307, D-6900 Heidelberg, F.R.G.
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324
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PHYLOGENETIC ASPECTS OF CARDIAC HORMONES
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TABLE 1 AMOUNTOF WET WEIGHT(rag)OF EXTRACTEDCARDIACTISSUE CAUSINGA VASODILATIONOF 50% OF THE PRECONTRACTED RABBIT AORTA
positive controls [11]. As positive controls sections of porcine cardiac atria were subjected to parallel incubation. Since all controls indicated the specificity of the obtained immunoreactions the stained structures are called cardiodilatin-immunoreactive (CDD-IR).
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Electronmicroscopy Small tissue specimens of the atria and ventricles of all species investigated were fixed by immersion in a solution containing 1.25% formaldehyde and 1.25% glutardialdehyde in a 0.2 M phosphate buffer [9]. After rinsing in the buffer the specimens were postfixed in 1% OsO~ diluted in 0.2 M phosphate buffer, dehydrated in graded series of ethanol and routinely embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate. A Zeiss EM 10 was used for analysis and photography.
Extraction and Bioassay
pomatia. Since phylogenetic studies are thought to be of great importance for a variety of not only phylogenetic problems but also for pharmacological and physiological questions [23], we investigated the possible occurrence of cardiac hormones of the CDD family in the higher submammalian classes as well as in an invertebrate species. The study was carried out by correlating immunocytochemical and electronmicroscopical results with those obtained by bioassays. METHOD As representatives of their classes the following vertebrate species were used for immunohistochemical and electronmicroscopical analysis and in part also for bioassay: Mammals: Man, Tupaia belangeri, pig, dog, cat, rat, guinea pig; birds: Gallus g. domesticus. Coturnix c.japonicus; reptiles: Anolis carolinensis; amphibians: Rana esculenta; teleost bony fish: Salmo gairdneri. In addition, the gastropod mollusc Helix pomatia was investigated.
Cardiac atria and ventricles of the following species (number of individuals in parentheses) were extracted: Pig (50), chicken (12), Anolis carolinensis (15), Rana esculenta (30), Salmo gairdneri (30), Helix pomatia (30). As for the snail also cerebral (30) and subesophageal ganglia (30) were extracted. After animals were sacrificed, the tissue for extraction was dissected rapidly and immediately deep frozen. After defrosting, tissue was weighed and boiled in fivefold (weight/volume) volumes of distilled water for 10 min. After boiling an equal amount of ice-cold 0.4 M acetic acid was added and the mixture was stirred for 2.5 hr at 4°C. Thereafter, the crude extract was centrifuged at 50,000 g for 30 min. Supernatant was collected and prepurified using Sep-Pak capsules with 80% acetonitrile in 0.1% trifluoro acetic acid as solvent. The vasorelaxant effect exerted on the rabbit aorta as described for mammalian cardiac hormones [12] was used as bioassay. After an equilibration period of 2 hr in a standard physiological salt solution, helical strips of rabbit aorta were precontracted by 10-7 M norepinephrine. After the contraction had reached a plateau, increasing amounts of the prepurified extracts were added cumulatively.
lmmunocytochemistry All mammalian and avian species were anesthetized by intraperitoneai injection of Nembutal ®, the reptiles and amphibians and the snails by using ether-chloroform vapour and the bony fish by adding MS 222® to the water. Human atrial tissue which is excised to introduce the tube for extracorporal circulation during coronary by-pass operation was also investigated in our study (approved by the ethic committee of the University of Heidelberg). The hearts and in case of the snails also the cerebral and subesophageal ganglia were rapidly dissected and fixed by immersion in Bouin's fluid without acetic acid. After fixation the specimens were dehydrated in ascending concentrations of ethanol and routinely embedded in paraplast. Sections were cut at 7 tzm and processed for a modification [11] of the peroxidaseantiperoxidase (PAP) technique [28]. All antisera applied in this study were raised in rabbit against different portions of cardiodilatin (CDD) and are described in detail elsewhere [15]. Throughout the submammalian species studied only C-terminal region-specific antisera were reactive. A detailed immunocytochemical study related to phylogeny will be published elsewhere [26]. The specificity of the immunohistochemical reactions was tested by a variety of negative and
RESULTS VERTEBRATES
Immunocytochemistry and Electronmicroscopy Mammals. In the atria of all mammalian species investigated CDD-IR was localized in myoendocrine cells of both atrial appendages. In the remaining atrial myocardium CDD-IR cells occurred only infrequently. The distribution patterns of the CDD-IR cells in the different species showed a high degree of correspondence. In the myoendocrine cells CDD-IR was accumulated adjacent to the nuclear poles (Fig. la,b) and occurred only rarely in other cytoplasmic regions. By means of electronmicroscopy the myoendocrine cells of the species studied were characterized by specific moderate electron-dense granules (Fig. lc). These were present mainly in the perinuclear region adjacent to the Golgi apparatus. This location is identical to that of the CDD-IR as observed in the light microscope. In the ventricles neither CDD-IR nor myoendocrine cells were to be found. Birds. In contrast to the mammalian hearts, in the hearts of the avian species investigated, i.e., chicken and quail,
328
REINECKE, NEHLS AND FORSSMANN
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P H Y L O G E N E T I C ASPECTS OF CARDIAC HORMONES CDD-IR cells were observed only very infrequently. They were faintly stained (Fig. 2a,b) and located in both atrial appendages, while no CDD-IR cells seemed to occur in ventricles. In the electronmicroscope, myoendocrine cells were found in corresponding location. Like in mammals, the secretory granules were concentrated in the perinuclear regions (Fig. 2c). The density of the granules, however, was extremely low. Reptiles. In the heart of the lizard species Anolis carolinensis which was studied as representative of the reptiles CDD-IR cells occurred throughout the atria (Fig. 3a,b) with the highest density in the appendages. In the ventricles no CDD-IR cells were observed. In correlation, in all regions of the atria myoendocrine cells exhibiting heteromorphous secretory granules (Fig. 3c) were identified. Amphibians. In the atria of the frog Rana esculenta CDD-IR cells were present in large numbers (Fig. 4a). They were distributed throughout the atrial myocardium without showing any preferential location. Furthermore, in contrast to the vertebrate classes described above CDD-IR cells occurred in moderate numbers also in the ventricle. They were restricted mostly to the outer ventricle wall (Fig. 4b). Myoendocrine cells (Fig. 4c) were found in the same locations, i.e., they occurred both in the atria and in the ventricle. Bony fish. A fresh water teleost, the rainbow trout, was investigated as representative of the bony fish. Throughout the atrium of the trout CDD-IR cells (Fig. 5a) occurred in high densities. Like in the frog heart, in the ventricle of the trout also CDD-IR cells were found, however, in by far lower numbers (Fig. 5b). In the electronmicroscope myoendocrine cells were identified in corresponding locations. In contrast to the other vertebrate species studied, the secretory granules of the trout myoendocrine cells were smaller and less heteromorphous (Fig. 5c).
Bioassay Prepurified atrial extracts from pig (Fig. ld), chicken (Fig. 2d), lizard (Fig. 3d), frog (Fig. 4d) and trout (Fig. 5d) as well as ventricular extracts from frog and trout exerted potent vasorelaxant effects on the rabbit aorta precontracted by norepinephrine. The vascular smooth muscle relaxation achieved was clearly dose-dependent. Additional information is presented in Table I which gives the amount of extracted cardiac tissue which is necessary to cause a relaxation of 50%. The values obtained differ widely between the representatives of the different classes. The lowest amounts of atrial extracts were necessary with the mammals, intermediate ones with lizard, frog and trout, while high amounts of chicken extract had to be applied to cause an aortic relaxation of 50%. While no unequivocal dilation of rabbit aorta could be yielded applying extracts of pig, chicken and lizard ventricles, ventricular extracts of frog and trout caused vasodilations. However fairly high amounts were necessary to cause an aortic relaxation of 50% (Table 1). GASTROPODS
hnmunohistochenlistry and Electromnicroscopy The immunohistochemical investigation of the snail heart revealed the occurrence of CDD-IR structures. These were found exclusively in the atrium whereas no CDD-IR occurred in the ventricle. The CDD-IR was not present in the atrial myocardiocytes but was confined to round or ovoid
329 structures (Fig. 6a). These were located intermingled between the myocardiocytes as well as free in the atrial lumen. Their average diameter surpassed that of the myocardiocytes. Electronmicroscopical analysis of the corresponding areas of the snail heart revealed the existence of nerve varicosities characterized by heteromorphous electron-dense granules (Fig. 6b). The varicosities occurred in the same overall distribution as the light microscopically identified CDD-IR structures. No secretory granules were observed within the atrial myocardiocytes. In the visceral ganglion of the subesophageal complex CDD-IR perikarya were found either singularly or in small groups (Fig. 6c). Furthermore, CDD-IR was present in nerve fibers of the neuropile as well as in fibers of the intestinal nerve which projects to the cardiac region. In the cerebral ganglia CDD-IR perikarya occurred only very infrequently.
Bioassay Atrial and subesophageal extracts prepurified by the use of Sep-Pak capsules exerted smooth, muscle relaxation of rabbit aortic strips which was clearly dose-dependent (Figs. 6d,e). With extracts of the ventricle and the cerebral ganglia, however, no vascular smooth muscle relaxation was found. DISCUSSION The results presented here indicate that the occurrence of cardiac hormones of the CDD-family is not restricted to mammals. They seem to be present also in the hearts of avian, reptilian, amphibian and bony fish species as has been demonstrated here by immunocytochemistry, electronmicroscopy and bioassay. While a variety of investigations were carried out on the localization [2, 13, 15, 21] and physiology (for review see [10]) of cardiac hormones in mammals there is except for a short communication of our group [24] only one study dealing with the possible existence of cardiac hormones in submammalian vertebrates [6]. In contrast to our results the authors did not succeed to detect biological activity of cardiac hormones in the chicken heart. This discrepancy may be due to the different extraction and/or bioassay methods applied as well as to the paucity of myoendocrine and CDD-IR cells, respectively, in the chicken, While so far no results are available on the occurrence of cardiac hormones in reptiles and bony fish, in agreement with our results myoendocrine cells, as well as cardiac hormone bioactivity, were found in the frog heart |6|. Since for each vertebrate class investigated only one or two representatives were studied to date one should be careful not to overdue the interpretations. However, some conclusions may be drawn. In the phylogenetic line leading to the birds the cardiac hormones may have lost their physiological importance since in the avian heart CDD-IR cells or myoendocrine cells, respectively, occur very infrequently. These results are supported by the bioassay: The low density of CDD-IR cells in the chicken atrium is reflected by the high amount of tissue extract necessary to cause a relaxation of 50% of the rabbit aorta. However, pharmacological studies in avian species must be carried out to clarify this hypothesis. The morphological result that the occurrence of CDD-IR cells or myoendocrine cells, respectively, in the hearts of the mammals, birds and reptiles studied was restricted to the atria while in the amphibians and bony fish they were additionally in the ventricles also correlates with the bioassay: No vascular smooth muscle relaxation was obtained from
330
REINECKE, NEHLS AND FORSSMANN
ventricular extracts of pig, chicken and lizard whereas ventricular extracts of frog and trout caused relaxation of rabbit aortic strips. Thus, there may be a phylogenetic trend of concentrating the cardiac hormone producing cells in the atria. A particular interesting finding [22] of our phylogenetic studies is also included in this investigation, i.e., that in the snail Helix pomatia, a gastropod mollusc, a CDD-like hormonal substance is present in atrial nerves and not in myocardial cells as was found in all vertebrate species studied so far. Thus, at least in the snail, a CDD-IR neurosecretory axis occurs which projects from the subesophageal ganglion to the cardiac atrium. This result stimulated our efforts to investigate whether CDD-IR also exists in the mammalian nervous system. In fact, an immunocytochemical study o f hypothalamic regions of Tupaia belangeri showed the presence of CDD-IR neurones which may be involved in cardiovascular and water balance regulation [15]. We have further indication that CDD can be postu-
lated to be a neurotransmitter or neuromodulator in other regions of the nervous system as well as in other species [25]. Our present interest is focused on the problem of whether the CDD-like neuroendocrine system observed in the snail is typical for gastropods or if it constitutes a general phenomenon of invertebrates. Furthermore, the functional significance of the neuro-cardiac CDD-IR axis in the snail is presently under investigation. Interesting phylogenetic questions concern the points of how this neuroendocrine system has been transformed into a pure cardiac endocrine system and where along the phylogenetic tree this step has been taken place.
ACKNOWLEDGEMENTS For skillful technical assistance we thank Mrs. Barbara Briihl and Mr. Richard Hertel. The study was supported by the German Research Foundation (Re 520/1-2).
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