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The electrocardiogram of Sepia officinalis L. (cephalopoda: coleoida) and its modulation by neuropeptides of the FMRFamide group
Camp. Eiochem.
Physiol.
Vol. 103C.
No. 2, pp. 399402,
0306~4492/92
1992
$5.00 + 0.00
0 1992PergamonPressLtd
Printedin Great Britain
THE ELECTROCARDIOGRAM OF SEPIA OFFICINALZS L. (CEPHALOPODA: COLEOIDA) AND ITS MODULATION BY NEUROPEPTIDES OF THE FMRFamide GROUP P. M. JAKOBSand R. SCHIPP Institut fiir Allgemeine und Spezielle Zoologie der Justus-Liebig-Universitiit,
D-6300 Giessen, F.R.G. (Tel. 0641-702-5837, Fax. 0641-702-2099); and Institut de Biologie Marine (F-33120) Arcachon, France (Received 30 September 1991) Abstract-l. Electrocardiogram (ECG) was recorded of the systemic heart of Sepia ojficinalisL. from both isolated auricle-ventricle preparations. 2. The peptides FMRFamide and FLRFamide evoked a concentration-dependent increase of the bioelectrical signals of the auricle (‘Y-wave) and ventricle (“RI’-spike) as well as a positive dromotropic effect, i.e. a shortening of the AV-interval.
INTRODUCTION Numerous functional studies on the isolated systemic heart of coleoid cephalopods revealed a myogenic autorhythm that is under neurocontrol by inhibitoric cholinergic and excitatoric catecholaminergic mechanisms similar to those in the vertebrate heart (survey in Kling and Jakobs, 1987). Furthermore, there is much evidence for interfering peptidergic influences in extrinsic heart control. On the one hand physiopharmacological results revealed strong and long lasting positive inotropic effects of FMRFamide on the systemic heart of Octopus vulgaris and Sepia oficinalis (Voigt et al., 1983; Wells, 1983a, b; Kling and Jakobs, 1987; Jakobs, 1991a); on the other hand there are immunhisto- and cytochemical findings to demonstrate that neuropeptides of the FMRFamidefamily occur in terminal nerve fibres of the heart wall as well as neuroendocrine axon terminals within the Vena cephalica (NSV-system) of cephalopods (Martin and Voigt, 1987; Le Gall et al., 1988; Jakobs, 1991b). However, no results have been attained thus far concerning either the action of these peptides on the somewhat neglected ECG of cephalopods, or concerning comparative analyses of simultaneously recorded acto- and electrograms of auricle-ventricle preparations of cephalopods which could help to attribute the ECG-signals to the different heart phases. The present paper will demonstrate the influence of FMRFamide and FLRFamide on the excitation pattern of the auricle and ventricle of Sepia oficinalis by comparative in situ and in vitro recordings. They will be compared with earlier in vivo recordings of free-moving Sepiu (Mislin, 1966/67) and Octopus (Smith,
1981a).
All the animals were anaesthetized by 1% ethanol/seawater before surgical procedures and dissections were carried out. The experiments were performed at temperatures of 18-20°C. For the in situ registration of the electrocardiogram (ECG) the animals were dissected ventrally and placed in a flat tube-bath. To support the circulatory functions the gills were continuously perfused by oxygenated seawater with 0.5% ethanol in order to preserve the anaesthesia. For the anterograde perfusion of the isolated systemic heart, two input tubes were placed inside the two auricles and one output cannula in the cepahlic aorta; the AV-valves as well as the aortic valve were kept intact and functioned in perfusion as well as under in situ conditions (Foti et al., 1985; Jakobs, 1991b). The genital artery as well as the renal artery were tied up by ligatures. The preload pressure was adjusted at 50 mm H,O (=490.5 Pa), the afterload pressure at 20mm H,O (= 196.2Pa). To register the actograms (AG) the aortic cannula was connected with a pressuretransducer: (HSE WlOl), an amplifier (Sachs HSE 300) and a recorder (Watanabe Mark V). For the bipolar recording of the ECG, the preparations were enclosed in a Faraday-cage. Two chlorided silver electrodes (0.1 mm in diameter) were inserted into the surface of the left auricle behind the entrance of the efferent branchial vessel and the second one into the cephalic aorta distal of the valve. The signals were amplified and recorded using an ECG-preamplifier (HSE-EKG-Koppler Type 556A) and an xt-recorder (Mark V, Watanabe). FMRFamide was purchased from Sigma Chemicals and FLRFamide from Bachem.
RESULTSAND
According to earlier studies in octopods (Smith, 1981b; Wells and Smith, 1987; Agnisola et al., 1989a, b) in both in situ as well as in vitro preparations using > =t
METHODS
H
Juvenile animals (mantle-length: 7-10.5 cm, body weight: 60-l 10 g) of both sexes of Sepia oficinalis (L.) from the Bassin d’Arcachon (Atlantic Ocean) were used in this study. Dedicated to Prof. Dr R. Bgssler’s 65th birthday.
DISCUSSION
10 set
Fig. 1. In situ recording of the electrocardiogram of Sepiu oficinalis; explanations to P and R see Fig. 3 and in text. 399
400
P. M.
JAKOBS and R. SCHIPP
ECG > 3
H
10 ssc.
Fig. 2. Simultaneously recorded gram (AG/ECG)
acto- and electrocardiofrom the isolated heart of Sepia oJk5nalis in seawater-perfusion.
the bifurcative perfusion regular and synchronous contraction waves could be observed running from the auricles to the aortic pole, that indicated a likewise directed spreading out of the excitation waves. The electrocardiograms recorded bipolarly left auricle/aorta cephalica from both isolated and in situ preparations showed similar signals with a regular rhythm and without any deformations (Figs 1, 2, 3). The single excitation period (duration: 2-3 set) was characterized by a steeply increasing spike of 1O&400 p V and a duration of 120-400 msec, immediately preceding the ventricle contraction. Accordingly, this spike can be viewed primarily as an expression of the ventricle excitation (phase of de- and repolarization) (Mislin, 1966/67; Smith, 198la). Analogous to the ECG of vertebrates it is called “R”, as distinct differences are known to exist in the anatomy and mechanisms of excitation between the hearts of both organisms. Prior to the “R”-signal, a variable wave potential “P” of 50-280 PV and 800-1700 msec occurred, which has been attributed to the auricle excitation. This is because it was absent in recordings of single ventricle preparations without auricles of the same species on the Straub cannula (Kling, 1985) or perfused isolated ventricles of Eledone cirrhosa (Smith, 198la). In in situ recordings the “P”-wave was distinctly separated from the “R”-spike, but in the ECG of perfused isolated organs it appeared, however, considerably closer to “R”, and an accentuated maximum of “P” was not always visible. The interval between the beginning of the “P”-wave until the beginning of the “R”-spike amounts to 130 msec in in vitro and 240 msec in in situ preparations. For the present, and with certain reservations, this period can be apostrophied as the “AV-Interval”, as studies on cephalopod hearts still allow no complete clarity concerning the possible
tossc
Fig. 4. ECG of Sepia-heart in-situ before and after an injection of FMRFamide (1.5 ml of 10m6 M) into the V. cephalica.
occurrence of nodal myogenic pacemaker areas within the auricles. Smith (198la, Efedone cirrhosa, his Fig. la) observed a small initial wave a in few recordings of free moving animals but was not sure if a definite “P”wave equivalent is present. On the other hand he detected spontaneous contractions within the auricleventricular junctions of unperfused heart preparations of octopods; thereby also confirming the observation about a “particular sensitivity” of this area in Octopus heart by v. Skramlik (1941). Liicke (1990) demonstrated regular autorhythmic contractions of isolated ring preparations of auricles of Sepia oficinalis, even in not stretched segments, whereas ring-shaped segmental preparations of the ventricle showed no or only infrequent spontaneous activities. In addition, structual analyses of longitudinal sections of the AV-transitional area in the same species, stained by Masson’s coloration or Bodian’s silver impregnation, suggested that although the walls of the two compartments are sharply bordered against each other, single muscle fibres from the auricles enter the ventricle myocard, indicating a probable conductive coupling of both organ areas (Kling and Schipp, 1987a). The appearance of gap junctions between auricular and ventricular muscle cells, revealed by TEM studies of the same authors, also point to a functional syncytium such as the one in the vertebrate heart. FMRFamide evoked a concentration-dependent increase of the “P”-wave and the “R”-spike in in situ
AV-IV 10 set
Fig. 3. Single ECG of the isolated Sepia-heart in higher amplification; auricular (P) and ventricular (R) signal; further explanations see in text.
Fig. 5. The action of increasing concentrations of FMRFamide on the AG and ECG of an isolated Sepia-heart in perfusion.
Neuropeptides modulate Sepia ECG AG
ECG
10-'M
-
FLRFamide
lo-‘M
-
FLRFamide
10 sec.
Fig. 6. The action of increasing FLRFamide concentrations on AG and ECG of an isolated Sepia heart in perfusion.
preparations of the systemic heart (Figs 4, 5). After injections of l-l.5 ml of 10e6 M FMRFamide into the vena cephalica an increased depolarisation amplitude of 31% for the auricle (‘Y-wave) and about 14% for the ventricle (“R”-spike) was recorded. The distance from the beginning of the auricle excitation (“P”) to the beginning of the ventricle depolarisation (“AV-interval”) (Rushmer, 1972) appeared about 13.8% shorter. As mentioned above, the ECG of non-treated isolated organs already showed a much shortened AV-interval in comparison to the in situ preparations. After applying low concentrations of FMRFamide (lo-’ M) the “P’‘-wave was only visible as a preceding shoulder of the “R”-complex. A FMRFamide-concentration of 10m6M induced a total fusion of both signals. In that case, only the amplitude of the “R”-spike could be precisely determined; after a heart perfusion with FMRFamide at a concentration of lo-’ M it (“R”) increased by 21% and at a concentration of 10e6 M by 35%. The duration of the total excitation period (beginning of ‘Y-wave until end of “R’‘-spike) did not alter significantly. After applying FLRFamide the ECG of the isolated perfused systemic heart of Sepia also showed a concentration-dependent rise of the “P”-wave (lo-’ M FLRFamide+ 19%; 10m6M FLRFamide-r25%) and “R”-spike (lo-’ M FLRFamide+57%; lo6 M FLRFamide-+86%; Fig. 6), with a simultaneous increase of a negative spike prior
401
to the ventricle depolarisation. A double peak of “R” sometimes observed in recordings during the SWperfusion (Fig. 6) altered to one steeply increased “R”-peak under the action of this peptide. Furthermore, the AV-interval not clearly measurable under SW-perfusion but more clearly marked off after FLRFamide applications was shortened in the step from lo-’ to 10m6M FLRFamide by 33.5%. The findings concerning the action of the both tetrapeptides on the AG confirm earlier studies about their synergic and primarily positive inotropic effect on the cephalopod heart (Voigt et al., 1983; Wells 1983 a, b; Jakobs, 1991a). But as mentioned above, there are no comparable earlier results either about actions of these peptides or other neurotransmitters on the ECG of cephalopods. Concerning the action mechanisms of FMRFamide at the cellular level in molluscs, there are primarily results on gastropod neurons, which revealed opposite bioelectrical effects, indicating that this peptide acts via multiple receptors (Cottrell and Davies, 1987; Kobayashi and Muneoka, 1989). In some neurons of Helix uspersu FMRFamide induced depolarization, in others, hyperpolarisation (Shelvelkin and Nikitin, 1988). For the Aplysia neuron R14 an FMRFamide-induced slow inward current is described that is carried by Na+ and depends inversely on external Ca2+ (Brezina et al., 1987; Ichinose and McAdoo, 1988). In the heart excitatory neuron, PON, of Achatina this peptide increased the background K + -conductance and decreased inward Ca2+-current (Kobayashi and Muneoka, 1989). A point of a special interest for understanding the increase of auricular and ventricular depolarisation as well as the inotropic effects observed here in Sepia heart is represented by the recent discovery of a receptor (or second messenger) operated FMRFamide Ca2+-channel in Lymnueu heart muscle cells whose activation seems to generate a CaP mediated inward current (preliminary reported in Brezden and Gardner, 1990). Acknowledgements-The authors would like to thank Prof. C. Cazaux, who provided the facilities for work in the “Institut de Biologie Marine d’Arcachon”, G. Balser, S. Beckermann, B. Fronk, M. Kopsch, R. M. Lawson and H. Schmidt for their valuable technical assistance. The investigations were supported by grants of the “Deutsche Forschungsgemeinschaft”.
REFERENCES
Agnisola C., Cariello L., Desnatis A., Miralto A. and Tota B. (1989a) Chronotropic and inotropic effects of atria1 peptides on the isolated systemic heart of Octopus vulnaris. J. camp. Phvsiol. 158B. 637441. Agniiola C., Conic M:, Acierno k. and Tota B. (1989b) Effects of cardiodilatin I-16 and ANF on the coronary flow in the isolated systemic heart of Ocfopus vulgaris. Comp. Biochem. Physiol. 942, 731-733. Brezden B. L. and Gardner D. R. (1990) Non-voltage-gated calcium channels in snail heart ventricle cells. J. exp. Biol. 150,187-203. Brezina V., Eckert R. and Erxleben C. (1987) Suppression of calcium current by an endogenous neuropeptide in neurones of Aplysia californica. J. Physiol. 388, 565-595.
402
P. M. JAKOB~ and R. SCHIPP
Cottrell G. A. and Davies N. W. (1987) Multiple receptor sites for a molluscan peptide (FMRFamide) and related peptides of Helix. J. Physiol. 382, 5148. Foti L., Genoino I. T. and Agnisola G. (1985) In vitro cardiac performance in Ocfopus uulguris (Lam.). Come. Biochem. Physiol. 82C, 483488. . _ Ichinose M. and McAdoo D. J. (1988) The voltaaedependent, slow inward current unduced by the neuropeptide FMRFamide in Apiysiu neuron R 14. J. Neurosci. 8, 3891-3900. Jakobs P. M. (1991a) Effects of FMRFamide and some analogues on the isolated systemic heart of Sepia oficinalis L. in orthograde perfusion. 1st Int. Symposium on the Cuttlefish Sepia, Caen, June 1-3, 1989, Centre Publ. University of Caen. Jakobs P. M. (1991b) Pharmakologische immunhistound cytochemische Untersuchungen zur Bestimmung und Lokahsation peptiderger Rezeptoren im Zentralherzen von Sepia ojicinalis L. (Cephalopoda: Coleoidea). Inaug. Diss. JLU-Giessen. Kling G. (1985) Vergleichende histochemisch-cytologische und pharmakologische Untersuchungen zur Innervation des Zentralherzens von Sepia ojicinalis L. (Cephalopoda: Coleoidea) Inaug. Diss. JLU-Giessen. Kling G. and Jakobs P. M. (1987) Cephalopod myocardial receptors: Pharmacological studies bn the isolated heart of Seaia officinalis (L.). Exoerientia 43. 51 l-525. Kling G. &d Schipp’ R. ‘(1987a) Comparative ultrastructural and cytochemical analysis of the cephalopod systemic heart and its innervation. Experientiu 43, 502-S Il. Kling G. and Schipp R. (1987b) Effects of biogenic amines and related agonists and antagonists on the isolated heart of the common cuttlefish Sepia oflcinalis (L.). Cornp. Biochem. Physiol. 87C, 25 I-285. Kobayashi M. and Muneoka Y. (1989) Functions, receptors, and mechanisms of the FMRFamide-related peptides. Biol. Bull. 177, 206-209. LeGall S., Feral C., van Minnen J. and Marchand C. R. (1988) Evidence for peptidergic innervation of the endocrine optic gland in Sepia by neurons showing FMRFamide-like immunoreactivity. Brain Res. 462, 83-88. Liicke J. (1990) Vergleichende physiologisch-pharmakologische und cytochemische Untersuchungen zu Auto-
matie und Ca*+-Stoffwechsel des Zentralherzens von Sepia oficinalis L. (Cephalopoda; Coleoidea). Unpublished. Martin R. and Voigt K. H. (1987) The neurosecretory system of the 0ctonu.r vena cava: a neurohemal organ. Experientia 43, 537-543. Mislin. H. (1966) Uber die Beziehune zwischen Atmune und Kreislauf bei ‘Cephalopoden (Se& offcinalis L.).-Synchronregistrierung von Elektrocardiogramm (EKG) und Atembcwegung am schwimmenden. Tier. Verh. Dlsch. Zool. Ges. 175-181. Mislin H. (1967) Experimentelle Untersuchungen des Blutkreislaufs bei Tintenfischen. Umsch. Wiss. Tech. 289-290. Rushmer R. F. (1972) Structure and function of the cardiovascular system. In Organ Physiology, pp. l-296. W. B. Saunders, ~Philadelphia~ . __ _. Shelvelkin A. V. and Nikitin V. P. 11988) Effect of FMRFamide on activity of devensive behavior command neurons of fed and hungry snails (Helix pomatiu). Bull. exp. Biol. Med. 105, 618-620. Skramlik E. v. (1941) Uber den Kreislauf bei den Weichtieren. Ergeb. Biol. 18, 88-286. Smith P. J. S. (1981a) The octonod ventricular cardiogram. Camp. Biochem. ihysiol. 70.& 103-105. Smith P. J. S. (1981b) The role of venous pressure in regulation of output from the heart of the Octopus, Eledone cirrhosa (Lam.). J. exp. Biol. 93, 243-255. Voigt K. H., Kiehling C., Friisch D., Bickel U., Geis R. and Martin R. (1983) Identity and function of neuropeptides in the vena cava neuropil of Octopus. In Molluscan Neuroendocrinology (edited by Lever J. and Boer H. H.) pp. 228-234. North Holland Publishing Company, Amsterdam. Wells, M. J. (1983a) Circulation in cephalopods. In The Mollusca Vol. 5 (edited by Saleuddin A. S. M. and Wilbur K. M.), pp. 239-290. Academic Press, New York. Wells M. J. (1983b) Hormones and the Circularion in Octopus (edited by Lever J. and Boer H. H.), pp. 221-228. North Holland Publishing Company, Amsterdam. Wells M. J. and Smith P. J. S. (1987) The performance of the Octopus circulatory system: a triumph of engineering over design. Experientia 43, 487499.
Physiol.
Vol. 103C.
No. 2, pp. 399402,
0306~4492/92
1992
$5.00 + 0.00
0 1992PergamonPressLtd
Printedin Great Britain
THE ELECTROCARDIOGRAM OF SEPIA OFFICINALZS L. (CEPHALOPODA: COLEOIDA) AND ITS MODULATION BY NEUROPEPTIDES OF THE FMRFamide GROUP P. M. JAKOBSand R. SCHIPP Institut fiir Allgemeine und Spezielle Zoologie der Justus-Liebig-Universitiit,
D-6300 Giessen, F.R.G. (Tel. 0641-702-5837, Fax. 0641-702-2099); and Institut de Biologie Marine (F-33120) Arcachon, France (Received 30 September 1991) Abstract-l. Electrocardiogram (ECG) was recorded of the systemic heart of Sepia ojficinalisL. from both isolated auricle-ventricle preparations. 2. The peptides FMRFamide and FLRFamide evoked a concentration-dependent increase of the bioelectrical signals of the auricle (‘Y-wave) and ventricle (“RI’-spike) as well as a positive dromotropic effect, i.e. a shortening of the AV-interval.
INTRODUCTION Numerous functional studies on the isolated systemic heart of coleoid cephalopods revealed a myogenic autorhythm that is under neurocontrol by inhibitoric cholinergic and excitatoric catecholaminergic mechanisms similar to those in the vertebrate heart (survey in Kling and Jakobs, 1987). Furthermore, there is much evidence for interfering peptidergic influences in extrinsic heart control. On the one hand physiopharmacological results revealed strong and long lasting positive inotropic effects of FMRFamide on the systemic heart of Octopus vulgaris and Sepia oficinalis (Voigt et al., 1983; Wells, 1983a, b; Kling and Jakobs, 1987; Jakobs, 1991a); on the other hand there are immunhisto- and cytochemical findings to demonstrate that neuropeptides of the FMRFamidefamily occur in terminal nerve fibres of the heart wall as well as neuroendocrine axon terminals within the Vena cephalica (NSV-system) of cephalopods (Martin and Voigt, 1987; Le Gall et al., 1988; Jakobs, 1991b). However, no results have been attained thus far concerning either the action of these peptides on the somewhat neglected ECG of cephalopods, or concerning comparative analyses of simultaneously recorded acto- and electrograms of auricle-ventricle preparations of cephalopods which could help to attribute the ECG-signals to the different heart phases. The present paper will demonstrate the influence of FMRFamide and FLRFamide on the excitation pattern of the auricle and ventricle of Sepia oficinalis by comparative in situ and in vitro recordings. They will be compared with earlier in vivo recordings of free-moving Sepiu (Mislin, 1966/67) and Octopus (Smith,
1981a).
All the animals were anaesthetized by 1% ethanol/seawater before surgical procedures and dissections were carried out. The experiments were performed at temperatures of 18-20°C. For the in situ registration of the electrocardiogram (ECG) the animals were dissected ventrally and placed in a flat tube-bath. To support the circulatory functions the gills were continuously perfused by oxygenated seawater with 0.5% ethanol in order to preserve the anaesthesia. For the anterograde perfusion of the isolated systemic heart, two input tubes were placed inside the two auricles and one output cannula in the cepahlic aorta; the AV-valves as well as the aortic valve were kept intact and functioned in perfusion as well as under in situ conditions (Foti et al., 1985; Jakobs, 1991b). The genital artery as well as the renal artery were tied up by ligatures. The preload pressure was adjusted at 50 mm H,O (=490.5 Pa), the afterload pressure at 20mm H,O (= 196.2Pa). To register the actograms (AG) the aortic cannula was connected with a pressuretransducer: (HSE WlOl), an amplifier (Sachs HSE 300) and a recorder (Watanabe Mark V). For the bipolar recording of the ECG, the preparations were enclosed in a Faraday-cage. Two chlorided silver electrodes (0.1 mm in diameter) were inserted into the surface of the left auricle behind the entrance of the efferent branchial vessel and the second one into the cephalic aorta distal of the valve. The signals were amplified and recorded using an ECG-preamplifier (HSE-EKG-Koppler Type 556A) and an xt-recorder (Mark V, Watanabe). FMRFamide was purchased from Sigma Chemicals and FLRFamide from Bachem.
RESULTSAND
According to earlier studies in octopods (Smith, 1981b; Wells and Smith, 1987; Agnisola et al., 1989a, b) in both in situ as well as in vitro preparations using > =t
METHODS
H
Juvenile animals (mantle-length: 7-10.5 cm, body weight: 60-l 10 g) of both sexes of Sepia oficinalis (L.) from the Bassin d’Arcachon (Atlantic Ocean) were used in this study. Dedicated to Prof. Dr R. Bgssler’s 65th birthday.
DISCUSSION
10 set
Fig. 1. In situ recording of the electrocardiogram of Sepiu oficinalis; explanations to P and R see Fig. 3 and in text. 399
400
P. M.
JAKOBS and R. SCHIPP
ECG > 3
H
10 ssc.
Fig. 2. Simultaneously recorded gram (AG/ECG)
acto- and electrocardiofrom the isolated heart of Sepia oJk5nalis in seawater-perfusion.
the bifurcative perfusion regular and synchronous contraction waves could be observed running from the auricles to the aortic pole, that indicated a likewise directed spreading out of the excitation waves. The electrocardiograms recorded bipolarly left auricle/aorta cephalica from both isolated and in situ preparations showed similar signals with a regular rhythm and without any deformations (Figs 1, 2, 3). The single excitation period (duration: 2-3 set) was characterized by a steeply increasing spike of 1O&400 p V and a duration of 120-400 msec, immediately preceding the ventricle contraction. Accordingly, this spike can be viewed primarily as an expression of the ventricle excitation (phase of de- and repolarization) (Mislin, 1966/67; Smith, 198la). Analogous to the ECG of vertebrates it is called “R”, as distinct differences are known to exist in the anatomy and mechanisms of excitation between the hearts of both organisms. Prior to the “R”-signal, a variable wave potential “P” of 50-280 PV and 800-1700 msec occurred, which has been attributed to the auricle excitation. This is because it was absent in recordings of single ventricle preparations without auricles of the same species on the Straub cannula (Kling, 1985) or perfused isolated ventricles of Eledone cirrhosa (Smith, 198la). In in situ recordings the “P”-wave was distinctly separated from the “R”-spike, but in the ECG of perfused isolated organs it appeared, however, considerably closer to “R”, and an accentuated maximum of “P” was not always visible. The interval between the beginning of the “P”-wave until the beginning of the “R”-spike amounts to 130 msec in in vitro and 240 msec in in situ preparations. For the present, and with certain reservations, this period can be apostrophied as the “AV-Interval”, as studies on cephalopod hearts still allow no complete clarity concerning the possible
tossc
Fig. 4. ECG of Sepia-heart in-situ before and after an injection of FMRFamide (1.5 ml of 10m6 M) into the V. cephalica.
occurrence of nodal myogenic pacemaker areas within the auricles. Smith (198la, Efedone cirrhosa, his Fig. la) observed a small initial wave a in few recordings of free moving animals but was not sure if a definite “P”wave equivalent is present. On the other hand he detected spontaneous contractions within the auricleventricular junctions of unperfused heart preparations of octopods; thereby also confirming the observation about a “particular sensitivity” of this area in Octopus heart by v. Skramlik (1941). Liicke (1990) demonstrated regular autorhythmic contractions of isolated ring preparations of auricles of Sepia oficinalis, even in not stretched segments, whereas ring-shaped segmental preparations of the ventricle showed no or only infrequent spontaneous activities. In addition, structual analyses of longitudinal sections of the AV-transitional area in the same species, stained by Masson’s coloration or Bodian’s silver impregnation, suggested that although the walls of the two compartments are sharply bordered against each other, single muscle fibres from the auricles enter the ventricle myocard, indicating a probable conductive coupling of both organ areas (Kling and Schipp, 1987a). The appearance of gap junctions between auricular and ventricular muscle cells, revealed by TEM studies of the same authors, also point to a functional syncytium such as the one in the vertebrate heart. FMRFamide evoked a concentration-dependent increase of the “P”-wave and the “R”-spike in in situ
AV-IV 10 set
Fig. 3. Single ECG of the isolated Sepia-heart in higher amplification; auricular (P) and ventricular (R) signal; further explanations see in text.
Fig. 5. The action of increasing concentrations of FMRFamide on the AG and ECG of an isolated Sepia-heart in perfusion.
Neuropeptides modulate Sepia ECG AG
ECG
10-'M
-
FLRFamide
lo-‘M
-
FLRFamide
10 sec.
Fig. 6. The action of increasing FLRFamide concentrations on AG and ECG of an isolated Sepia heart in perfusion.
preparations of the systemic heart (Figs 4, 5). After injections of l-l.5 ml of 10e6 M FMRFamide into the vena cephalica an increased depolarisation amplitude of 31% for the auricle (‘Y-wave) and about 14% for the ventricle (“R”-spike) was recorded. The distance from the beginning of the auricle excitation (“P”) to the beginning of the ventricle depolarisation (“AV-interval”) (Rushmer, 1972) appeared about 13.8% shorter. As mentioned above, the ECG of non-treated isolated organs already showed a much shortened AV-interval in comparison to the in situ preparations. After applying low concentrations of FMRFamide (lo-’ M) the “P’‘-wave was only visible as a preceding shoulder of the “R”-complex. A FMRFamide-concentration of 10m6M induced a total fusion of both signals. In that case, only the amplitude of the “R”-spike could be precisely determined; after a heart perfusion with FMRFamide at a concentration of lo-’ M it (“R”) increased by 21% and at a concentration of 10e6 M by 35%. The duration of the total excitation period (beginning of ‘Y-wave until end of “R’‘-spike) did not alter significantly. After applying FLRFamide the ECG of the isolated perfused systemic heart of Sepia also showed a concentration-dependent rise of the “P”-wave (lo-’ M FLRFamide+ 19%; 10m6M FLRFamide-r25%) and “R”-spike (lo-’ M FLRFamide+57%; lo6 M FLRFamide-+86%; Fig. 6), with a simultaneous increase of a negative spike prior
401
to the ventricle depolarisation. A double peak of “R” sometimes observed in recordings during the SWperfusion (Fig. 6) altered to one steeply increased “R”-peak under the action of this peptide. Furthermore, the AV-interval not clearly measurable under SW-perfusion but more clearly marked off after FLRFamide applications was shortened in the step from lo-’ to 10m6M FLRFamide by 33.5%. The findings concerning the action of the both tetrapeptides on the AG confirm earlier studies about their synergic and primarily positive inotropic effect on the cephalopod heart (Voigt et al., 1983; Wells 1983 a, b; Jakobs, 1991a). But as mentioned above, there are no comparable earlier results either about actions of these peptides or other neurotransmitters on the ECG of cephalopods. Concerning the action mechanisms of FMRFamide at the cellular level in molluscs, there are primarily results on gastropod neurons, which revealed opposite bioelectrical effects, indicating that this peptide acts via multiple receptors (Cottrell and Davies, 1987; Kobayashi and Muneoka, 1989). In some neurons of Helix uspersu FMRFamide induced depolarization, in others, hyperpolarisation (Shelvelkin and Nikitin, 1988). For the Aplysia neuron R14 an FMRFamide-induced slow inward current is described that is carried by Na+ and depends inversely on external Ca2+ (Brezina et al., 1987; Ichinose and McAdoo, 1988). In the heart excitatory neuron, PON, of Achatina this peptide increased the background K + -conductance and decreased inward Ca2+-current (Kobayashi and Muneoka, 1989). A point of a special interest for understanding the increase of auricular and ventricular depolarisation as well as the inotropic effects observed here in Sepia heart is represented by the recent discovery of a receptor (or second messenger) operated FMRFamide Ca2+-channel in Lymnueu heart muscle cells whose activation seems to generate a CaP mediated inward current (preliminary reported in Brezden and Gardner, 1990). Acknowledgements-The authors would like to thank Prof. C. Cazaux, who provided the facilities for work in the “Institut de Biologie Marine d’Arcachon”, G. Balser, S. Beckermann, B. Fronk, M. Kopsch, R. M. Lawson and H. Schmidt for their valuable technical assistance. The investigations were supported by grants of the “Deutsche Forschungsgemeinschaft”.
REFERENCES
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