Sites of the barium effect on helix heart muscle cells

Comp. Biochem. PhysioL, 1975, VoL 52C. pp. 133 to 137. Per(iamon Press. Printed in Great Britain

SITES OF THE BARIUM EFFECT ON H E L I X HEART MUSCLE CELLS L. ERDfLYI, F. J o o a n d N. HAL,~SZ Department of Animal Physiology, Attila J6zsef University and Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary

(Received 17 March 1975) Abstract I. With the enhancement of the extracellular barium concentration (Ba2+)0 the amplitude maximum (F~) of the spontaneous contractions of the heart muscle initially increased, but then decreased. The time to peak force (tl) and the relaxation time 02} increased, while the mean velocity of force development (S~) and the mean velocity of relaxation ($2) decreased. 2. The plateau phase of both the auricular and the ventricular action potentials increased in proportion to the barium concentration as a function of time, and were closely correlated with the increase of t 1. 3. In barium-containing medium excitatory junction potentials (EJP) were led off from the atrial myocardial fibres; electron-microscopic examinations suggested that this can probably be explained by the enhanced spontaneous transmitter release in various types of neuromuscular junction (NMJ). INTRODUCTION THE EFFECTS of the barium ion on the function of heart muscle cells have been studied in detail on various vertebrate and invertebrate species. The effects of the b a r i u m ion on the muscle m e m b r a n e and on the generation of the action potential have been analyzed in m a m m a l s by Antoni & Oberdisse (1965); P a p p et al. (1967); P a p p a n o (1970) a n d others, a n d in embryonic chick hearts and cultured chick heart cells by Sperelakis & L e h m k u h l (1966, 1968); P a p p a n o & Sperelakis (1969) and Sperelakis & Lee (1971). Similar examinations were m a d e on frog heart by Le D o u a r i n et al. (1968) a n d Hermsmeyer & Sperelakis (1970), while Lignon & Le Douarin (1971) dealt with the frequency-enhancing effect of the barium ion in lampetra heart. As regards the invertebrates, i m p o r t a n t studies have been made by Irisawa & Kobayashi (1964) on Limulus heart. The changes in the E C G of Helix heart on the action of alkaline earth metal ions were first analyzed in the work of Lovatt Evans (1913) and Arvanitaki & C a r d o t (1933). D a t a obtained by intracellular technique and extending to the individual alkaline earth metal ions were reported by Kiss & S.-Rozsa (1971, 1973) on the generation of action potential in Helix heart. The changes in the E C G recorded with unipolar technique on the action of the barium ion, and the question of barium calcium antagonism, were touched on in our earlier publications (Erd61yi, 1968, 1971). The present paper deals with the effects of three different concentrations of the b a r i u m ion on the ventricle and atrium of Helix. A study was made of (i) the effects on the spontaneous contractility of the ventricle, (ii) the effects on the ventricular action potential generation a n d the contraction, a n d (iii) the effects on the auricular action potential generation a n d contraction. MATERIALS AND METHODS The experiments were made on the isolated ventricle and atrium of Helix pomatia L. The muscular contraction was

recorded by means of an electro-mechanical transducer with linear characteristics. One gram on the tongue of the transducer caused a deflection of less than 0.1 ram, and thus the recording of the contraction obtained was regarded as isometric. In some of the experiments the contraction of the isolated complete ventricle stretched horizontally were registered with a pen recorder from the output of a DISA Universal Indicator. The contraction curves were photographed together by phototechnical means. Parameters as follows were examined: the height of the curve (Fc = force of contraction), the time to peak force (tl), the relaxation time (t2), the mean velocity of force development ($1), and the mean velocity of relaxation ($2) (Reiter, 1972). The values of tl and t 2 were measured at the 50% height of the contraction curve, and the 50~ Fc value was taken into consideration in the calculation of S 1 and S,. Throughout the entire duration of the experiments the resting force (FR) was 1 g. In other experiments the contractions and the intracellular action potentials were recorded simultaneously from the partially immobilized, horizontally suspended isolated ventricle or atrium. The intracellular action potentials were led off by the conventional technique, with floating glass micropipette electrodes with resistances of 8 20 MfL Physiological solution with the following ionic composition was used in the experiments: 111.I mM NaCI, 1.8 mM KC1, 1.08 mM CaCl 2 and 2-39 mM NaHCO 3 (Ringer solution as recommended by Jullien). The barium solutions contained 1, 4 or 8 mM BaCI 2, the isoosmolality being maintained by proportional decrease of the amount of NaC1. Electron-microscopic examinations were carried out on Helix atrium. Fixation was performed for 2 hr at 4°C in physiological solution dissolved in 43/0 glutaraldehyde, with postfixation for 30 min in 1~o OsO4. Physiological solution was used for washing. After dehydration the material was embedded in Araldite (Durcupan, Fluka) and sections were prepared with a Porte~Blum ultramicrotome. Photographs were taken with a JEOL i00 B electron-microscope. RESULTS

1. Effect on the spontaneous contractility of the isolated ventricle The effect of the barium ion on the spontaneous contractility of the isolated ventricle was analyzed in five parallel experiments. The results of one typical

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L. ERDI~LYI, F. JOO A N D N. HALb,SZ

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Fig. 1. Effects of physiological solution containing 1 mM barium on the contractility of the isolated ventricle and on the formation of the intracellular action potential. (a) recording of typical experiment at two different speeds. The commencement of the barium effect is indicated by an arrow. 0-3 g = 1 division; 3.45 min and 5 sec - 1 division. (b) spontaneous contraction curves recorded in physiological solution containing I mM barium. (c) the same, but with a barium content of 4raM. (d) the same, but with a barium content of 8 mM. The first curve shows the control (c) recorded in physiological solution. The other curves are recordings prepared at the indicated times (min) following the beginning of the barium effect. 0.17 g = 1 division; 3 sec = 1 division. (e) spontaneous action potential and contraction curves of isolated ventricle. (f) after 2 rain in solution containing l mM barium. (g) in physiological solution after washing for 10min. Vertical calibration: 10mV, 0-6g. Horizontal calibration: (e), (g) 250mscc, (fl 500 reset.

e x p e r i m e n t are s h o w n in Fig. la for a b a r i u m dose o f 1 m M . Figure 1 (b~d) illustrate c o n t r a c t i o n curves r e c o r d e d in physiological solution c o n t a i n i n g 1, 4 a n d 8 m M BaC12, respectively, for an e x p e r i m e n tal period o f 30rain. The change in F c is s h o w n in Fig. 2(a) as a function o f time a n d as a function o f the b a r i u m concentration, expressed as a percentage o f the control. The figures clearly indicate that, c o m p a r e d to the control, the value of F¢ increases with the increase o f the b a r i u m ion concentration, a n d the time c o n s t a n t o f the increase decreases with the dose employed. The increase is steepest for a c o n c e n t r a t i o n o f 8 m M , and least for 1 raM. The decrease of F c follows later, this being p r o p o r t i o n a l to the b a r i u m ion c o n c e n t r a t i o n . T h e variations o f t l and t 2 m e a s u r e d at the 50°;, F~ value are depicted in Fig. 2 (b a n d c). It can readily be seen from the graphs that the values of t~ a n d t~ increased with time. here too the increase being p r o p o r t i o n a l to the b a r i u m ion concentration.

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Fig. 2. (a) Variation of the value of Fc, expressed as a percentage of the isolated ventricle control, as functions of time and the barium ion concentration. The various tonic levels are also shown. (b) Variation of the value of t~ measured at the 50~o Fc value for the isolated ventricle, as a function of time and expressed as a percentage of the control. (c) the same for tz. (d) the same for Sl. (e) the same for S 2. + + : in medium containing 1 mM barium; O O: in medium containing 4 m M barium; @-----O: in medium containing 8 m M barium.

The values o f S 1 and $2, which were calculated from t 1 and t 2 a n d the 50~o F~ value via the formulae F i t 1 and F i t 2, respectively, exhibited an essentially decreasing tendency. A t e m p o r a r y increase was

Sites of the barium effect

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observed only in the physiological solution with a barium content of 4 m M (cf. Fig. 2d and 2e).

2. Effect on the ventricular action potential generation and the contraction A study was made of the role of the barium effect in the connection of the action potential and the contraction. For this, the intracellular action potentials were recorded simultaneously with the related contractions. Figure l(e) shows such a recording. The main parameters of the intracellular action potentials exhibited st good agreement with the data reported by Kiss & S.-Rdzsa (1971, 1973). As regards the connection of the action potentials and the contraction, we found a similarity with the data of Nomura (1963, 1965) relating to Dolabella heart. In the case of the Helix ventricle the contraction similarly lasted up to the beginning of the repolarization, and the relaxation proceeded in parallel with the repolarization up to the appearance of the following action potential. There was a close correlation between the length of the action potential plateau and the contraction time. On the action of the barium ion the plateau phase of the action potential was progressively lengthened with the increase of t 1, and this has led to the increase of the duration of the action potential. The value of the membrane potential did not decrease perceptibly in the dose interval examined (within 60 min). In this respect a difference showed up in comparison with vertebrate hearts, where the membrane potential undergone depolarization on the action of the barium ion (Antoni & Oberdisse, 1965; Hermsmeyer & Sperelakis, 1970). In spite of this it may be said that, compared to the control, a temporary depolarization of the membrane occurs in the course of the plateau phase of action potentials of abnormally lengthened duration. Figure l(f) illustrates the effect of 1 m M barium ion after 2 rain. The recording clearly reveals the reversible lengthening of the plateau phase (Fig. lg). From this point of view the results appreciably resemble the data recorded by Irisawa & Kobayashi (1964) with an extracellular method on oyster heart, in so far as they too observed an increase in the duration of the action potentials on the action of the barium ion. 3. Effect on the auricular action potential generation and contraction Essentially similar results were obtained in the experiments with the isolated atrium. Figure 3 shows auricular action potentials and contraction curves recorded in physiological solution containing 4 mM barium ion. The resting potential of the atrial myocardial fibres is 40-50 mV, the peak potential is 2040mV, and the duration of the action potentials is 50(01400 msec, depending on the frequency (Fig. 3a). Here too the barium ion caused an increase in the duration of the action potential, while an after-potential frequently appeared in addition to the peak potential (Fig. 3b~). The change in the contraction curve was similar to that described for the ventricle. Here again the synchronous increase of t 1 and the plateau phase of the action potential was striking.

Fig. 3. Sinmltancously recorded action potentials and contraction curve of isolated atrium. (a) control in physiological solution. (b) in medium containing 4 mM barium, after 2 min. (c} after 4 min. (d) after 6 min, when junction potentials too appear in greater numbers. (e) after 8 min. (fl in physiological solution after washing for 15 min. Vertical calibration : 10 mV, 0-I g. Horizontal calibration : 600 msec. (g) various vesicle-containing neuromuscular junctions (NMJ) of atrial myocardial fibres. The inset shows heterogcneous junction potentials recorded in medium containing 4mM barium. 6mV and 500msec = 1 division. N 1 csv-containing axon terminal. N 2 = dcv-containing axon terminal. 4. E[Jbct on the twuromuscular junction In some recordings from the atrial myocardial fibres barium-induced excitatory junction potentials (EJP) were observed. Figure 3(d) shows such junction potentials. It can clearly be seen from the figure that the junction potentials were fairly heterogeneous, being 2mV, with durations of 100-300msec. On the action of the barium ion there was probably an enhancement of the spontaneous transmitter release from the nerve terminals in the heart muscle, and it was this which indirectly induced the appearance of the junction potentials. Kiss & Elekes (1972) recorded similar junction potentials in the case of the ventricular myocardial fibres in a medium with high calcium content, which is known to be capable of enhancing the spontaneous transmitter release. By means of electron-microscopic examinations neuromuscular junctions, consisting of nerve terminals of different types, could be detected in the atrium. Neuromuscular junctions occurred frequently and were generally characterized by the close connection of each cardiac fibre with two or three nerve terminals having various vesicle contents (Fig. 3g). All

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L. ERDI~LYI,F. Jo6 AND N. HAL~SZ DISCUSSION

Fig. 4. (a) axon terminal containing neurosecretory granules, in close connection with the atrial myocardial fibres, g = glial granules, M = mitochondria, Mf = myofilaments, sv = synaptic vesicles, N 3 = nsv-containing axon terminals. The arrow shows the position of the junction connection. (b) The junction connection at higher magnification. N 3 = nsv-containingaxon terminals, gran = neurosecretory granules, Mf = myofilaments, csv = clear synaptic vesicles.

three synaptic vesicle forms known in the various species of molluscs occurred in the axon terminals (Gerschenfeld, 1963; Hanneforth, 1965; Nisbet & Plummer, 1969; Barrantes, 1970; M c K e n n a & Rosenbluth, 1973, etc.). Similarly to the nerve fibres terminating in the ventricle (Kiss & Elekes, 1972), in the atrium too the neuromuscular junctions containing clear synaptic vesicles (csv) and dense-core vesicles (dcv) most often predominated. Comparatively rarely, neuromuscular junctions were also formed by axon terminals containing neurosecretory granules. A connection of such a type is shown at two different magnifications in Fig. 4. Besides the neurosecretory granules, which vary in size from 1200 to 2000 A, the occurrence of clear synaptic vesicles in the direct vicinity of the presynaptic m e m b r a n e was characteristic of these axon terminals. Similar granule-containing nerves were first described in H e l i x by Schlotte (1963), while Cottrell & Osborne (1969) succeeded in detecting a complete neurosecretory pathway in the atrium, which was found to terminate freely towards the cavity of the heart in the vicinity of the atrioventricular boundary. We were able to distinguish the neuromuscular connections of the axon terminals of this system, but further examinations are necessary to elucidate the physiological role of these connections.

Similarly as in a number of invertebrates and vertebrates, in Helix heart muscle cells the duration of the action potential gradually increased in a barium containing medium, which suggests a mechanism with a fundamentally similar site of action. It was demonstrated on Romela muscle by Werman et al. (1961), and on Helix ganglion cells by Kostyuk (1967) that the barium ion resulted in a decrease of the membrane resistance in the lengthened plateau phase of the action potentials, although otherwise, similarly to the vertebrate heart, the resistance of the resting membrane increased here too (Hermsmeyer & Sperelakis, 1970). It may be assumed that in the case of the H e l i x heart muscle the increase of the plateau phase up to a certain critical value was the factor which had a favourable effect on the functioning of the excitatory-contraction system and on the ionic mechanism leading to increase of the force of contraction. It is probable that later, with the enhanced influx of the barium ion, this effect was shifted in the direction of the depression. The cause of the decrease of $1 was probably the inhibition of the enzymes participating in the heart function. Of interest from this aspect is the result of Sperelakis & Lee (1971), who observed an almost complete inhibition by barium ion of ( N a + - K +) ATP-ase isolated from embryonic chick hearts. For a number of invertebrate and vertebrate hormone-producing organs, neuromuscularis junctions and synapses, the barium ion is known to enhance the spontaneous transmitter release (Douglas et al., 1961; Hailer et al., 1965; Elmqvist & Feldman, 1965; Katz & Miledi, 1969; Berlind & Cooke, 1971 ; Paton et al., 1971 etc.). Our investigations showed that in the H e l i x atrium too the barium ion is able to facilitate spontaneous transmitter release, which can affect NMJ-s of several types. From a morphological aspect the H e l i x atrium is of interest because of the presence of junctions containing neurosecretory granules; in their structural appearance these junctions show a considerable similarity to the terminals described in the vertebrate neurohypophysis and in the neurosecretory terminals of certain invertebrates (Nagasawa et al., 1970; Dhainaut-Courtois, 1972; etc.). REFERENCES

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