The effects of hypercapnia on force and rate of contraction and intracellular pH of perfused ventricles from the land snail Helix lucorum (L.)

The effect of hypercapnic acidosis on heart performance has been studied extensively in vertebrates, and it is known that an increase in the extracellular partial pressure of carbon dioxide reduces the force and rate of heart contraction (Orchard and Kentish, 1990; Driedzic and Gesser, 1994). An increase in in extracellular fluids causes a decrease in the intracellular pH (pH_i) of myocytes, which is thought to reduce contractile force through H⁺ competing for intracellular Ca²⁺ binding sites (Williamson et al., 1976). On the other hand, it has been shown by Langer (1985) and Langer et al. (1989) that low extracellular pH (pH_e) reduces the rate of Ca²⁺ entry into heart cells. The mechanisms by which H⁺ might reduce Ca²⁺ entry into heart cells are not well known. It is believed that Ca²⁺ and H⁺ act in a competitive manner for the same binding sites on sarcolemma and indeed, several studies have shown that, when increased extracellularly, Ca²⁺ improves cardiac performance during hypercapnic acidosis (Gesser and Poupa, 1979; Lagerstrand and Poupa, 1980; Williamson et al., 1976). However, some data indicate that low extracellular pH causes conformational changes in Ca²⁺ channels or transporters, resulting in decreased Ca²⁺ entry into myocytes (Iijima and Hagiwara, 1986a); Krafte and Kass, 1988; Klockner and Isenberg, 1994).

Although hypercapnia is a common response of many molluscs to various environmental conditions, little is known regarding the effects of hypercapnic acidosis on the pH_i of molluscan hearts and on cardiac activity. Hypercapnia and respiratory acidosis are greatly developed in land pulmonate snails during periods of estivation (Barnhart, 1986; Barnhart and McMahon, 1987; Rees and Hand, 1990) and the elevation of in the haemolymph of estivating snails results in a decrease in pH_e and pH_i (Barnhart and McMahon, 1988; Rees et al., 1991). On the other hand, mobilization of CaCO₃ stores caused by hypercapnia causes an increase in Ca²⁺ levels in the haemolymph of estivating snails (Burton, 1976; Barnhart, 1986). Increases in Ca²⁺ levels in the haemolymph of estivating land snails play an important role in the acid–base balance (Burton, 1976); however, the exact role of Ca²⁺ ions on heart activity in snails during estivation remains unknown. In the present work, we studied the effect of hypercapnic salines, containing different concentrations of Ca²⁺, on the force and rate of contraction of isolated ventricles from the land snail Helix lucorum. This was done in order to elucidate how heart activity is influenced by hypercapnia in land pulmonates during estivation. In addition, the pH_i of the perfused ventricles was determined in order to examine whether it plays any key role in the regulation of heart activity. Moreover, we examined whether low extracellular pH affects entry of Ca²⁺ into the heart cells. To examine this, using fura-2 we determined the intracellular concentration of Ca²⁺ in slices of heart muscle superfused under normal and hypercapnic conditions.

It is known that, as well as Ca²⁺, the levels of some other solutes (e.g. Mg²⁺, K⁺) increase in the haemolymph of estivating snails (Barnhart, 1986). We measured the concentrations of Na⁺, Mg²⁺, K⁺ and HCO₃⁻ in the haemolymph of snails estivating for 3 months and then we created a saline simulating the composition of the corresponding haemolymph. Afterwards hearts were perfused with this saline in order to obtain some data concerning the effect of hypercapnia in relation to the changes of solutes in haemolymph on the heart activity.

Adult specimens of Helix lucorum (L.) were collected in the vicinity of Edessa, in northern Greece. The snails were kept in an active state at a temperature of 25±0.5 °C and subjected to a 10.00 h:14.00 h L:D photoperiod in large glass boxes, with a daily supply of lettuce leaves and water. High humidity (85±1 %) was maintained by sprinkling the interior of the boxes with water every day. To induce estivation, active snails were removed and transferred to glass boxes without food and water, but with ample aeration. The snails were kept for 3 months in a dormant state at the same conditions of temperature and photoperiod as described above.

Haemolymph samples from active snails and those estivating for 1, 2 and 3 months were collected as described by Pedler et al. (1996). The concentrations of monovalent (Na⁺, K⁺) and divalent (Ca²⁺, Mg²⁺) cations in the haemolymph of active and estivating snails were measured by atomic absorption spectrophotometry as described by Wieser (1981).

Perfusion of isolated ventricles was performed as described by Wernham and Lukowiak (1983). After removing the shell, the heart was dissected out and a cannula was placed in the ventricle through the auricle. The auricle–ventricle junction was ligated so that the auricle attached to the ventricle was not filled with the perfusion buffer. A small hook, connected by a thread to a force-displacement transducer, was placed on the tip of the ventricle. The perfusion saline was delivered into the ventricle through a three-way valve at a pressure head of 8 cm H₂O. The normal saline used was composed according to measured concentrations of monovalent and divalent cations and the [HCO₃⁻]_ecalculated in the haemolymph of normal snails (Table 1). The composition of the normal saline (saline A) was: 46 mmol l⁻¹NaCl, 3.2 mmol l⁻¹KCl, 1.25 mmol l⁻¹mgCl₂, 6.85 mmol l⁻¹CaCl₂and 21 mmol l⁻¹NaHCO₃(see also Table 2). Saline A was equilibrated with air and its pH was adjusted to pH 7.75 prior to the experiments.

Four experimental protocols (all performed at 25 °C) were applied to the isolated perfused ventricles. The purpose of the first experiment was to examine the effect of hypercapnic salines, in the presence of normal levels (6.85 mmol l⁻¹) of extracellular Ca²⁺, on the force and rate of heart contraction as well as on pH_i. The first hypercapnic saline had the same composition as saline A except that it was equilibrated with 10 % CO₂in air, pH 7.2 (saline B) (Table 2). Two other hypercapnic salines used had the same composition as saline A except that they were equilibrated with 10 % of CO₂in air and contained either 15 mmol l⁻¹NaHCO₃(pH 7.0) (saline C) or 5 mmol l⁻¹NaHCO₃(pH 6.6) (saline D; Table 2). Gas mixtures were obtained using a Woesthoff (Bochum, Germany) gas-mixing pump. Before perfusion of ventricles with any of the above hypercapnic salines, ventricles were preincubated with saline A until a stable heart frequency was obtained. Ventricles were perfused with the above hypercapnic salines for 1 h and recordings of ventricle beats were monitored continuously on a chart recorder. To determine the pH_iof ventricles perfused under the conditions described above, we repeated the experiment and ventricles were removed from the perfusion system at intervals of 10, 30 and 60 min after perfusion with all of the above hypercapnic salines. Hearts were frozen in liquid nitrogen and held thus until pH_iwas determined. Ventricles perfused with the saline A were used as controls (0 min).

The purpose of second experiment was to examine whether changes in extracellular level of Ca²⁺affect the heart activity as well as pH_iunder hypercapnic acidosis. We therefore perfused ventricles in the presence of extracellular concentrations of Ca²⁺lower or higher than 6.85 mmol l⁻¹. Specifically, ventricles were perfused initially with saline A containing 6.85 mmol l⁻¹Ca²⁺and then with the saline B containing one of the following concentrations of Ca²⁺: 3 mmol l⁻¹, 11 mmol l⁻¹, 15 mmol l⁻¹or 30 mmol l⁻¹. The pH_iof ventricles was determined after perfusing them with the above hypercapnic salines for 1 h.

The importance of extracellular Ca²⁺on pH_iand heart activity under hypercapnic acidosis was examined further in the third experiment. Specifically, we examined the effect of an organic and inorganic blockers of Ca²⁺entry into cells on the force and rate of ventricle contraction and on the pH_iof ventricles perfused under hypercapnic conditions. Ventricles were perfused initially with saline A containing 6.85 mmol l⁻¹Ca²⁺and then the perfusate was changed to saline B containing the same concentration of Ca²⁺as saline A plus 10⁻⁴mol l⁻¹verapamil or 10⁻⁴mol l⁻¹Co²⁺, both of which are known to affect Ca²⁺entry into molluscan heart cells (Devlin, 1993a,b). Ventricles were perfused under the above conditions for 1 h and recordings were taken as described in the first experiment. To determine the pH_iof ventricles perfused under the above conditions, we repeated the experiment and ventricles were removed from the perfusion system at intervals of 10, 30 and 60 min and kept frozen in liquid nitrogen until pH_iwas determined. Ventricles perfused with saline A were used as controls (0 min).

In the fourth experiment, we examined the effect of a saline that simulated the composition of haemolymph of snails after estivating for 3 months on the force and rate of ventricle contraction and on pH_i. This saline consisted of measured concentrations of divalent and monovalent cations and the [HCO₃⁻]_ecalculated in the haemolymph of estivating snails (Table 1): 49 mmol l⁻¹NaCl, 4.8 mmol l⁻¹KCl, 12.6 mmol l⁻¹mgCl₂, 27 mmol l⁻¹CaCl₂and 20 mmol l⁻¹NaHCO₃. The pH of this saline was adjusted to a value of 7.4. P_CO2of the saline was determined using a P_CO2electrode after pH adjustment and was found to be about 27 mmHg. Before applying the above saline, ventricles were perfused initially with saline A in the presence of normal levels of Ca²⁺(6.85 mmol l⁻¹). Ventricles were perfused with this saline for 1 h and recordings of ventricle beats were taken as described previously. To determine the pH_iof ventricles perfused under the above conditions we repeated the experiment and the ventricles were removed from the perfusion system at intervals of 10, 30 and 60 min. They were frozen in liquid nitrogen and thus stored until pH_iwas determined. Ventricles perfused with saline A were used as controls.

The pH_iwas determined by the homogenate method developed by Pörtner et al. (1990). In brief, ventricles were ground under liquid nitrogen and then 100 mg of tissue powder were put into an Eppendorf vial (600 μl) containing 200 μl ice-cold medium (160 mmol l⁻¹KF, 1 mmol l⁻¹nitrilotriacetic acid, pH 7.4). After completely filling the vial with the medium, the mixture was first stirred with a needle in order to release air bubbles, mixed in a Vortex mixer, and then centrifuged for 30 s. Within 3 min after thawing of the tissue powder in the medium, the pH of the supernatant was measured at 25 °C using a capillary pH electrode G299A, as described previously for the determination of pH_e. In addition to determining pH_iin the perfused ventricles, we also determined pH_iof hearts from active snails and those estivating for 1, 2 or 3 months.

The results are presented as means ± S.E.M. Significance of differences was tested using Bonferonni’s test, which permits multiple comparisons to be taken into consideration. The limit of significance was set at various levels, as indicated in the corresponding Tables and Figures.

The effect of estivation on acid–base variables and the concentrations of divalent and monovalent cations are given in Table 1, which also shows the pH of haemolymph (pH_e) and the intracellular pH (pH_i) of hearts from both active and estivating snails for 1, 2 and 3 months. pH_e, although variable, declined progressively during estivation and was determined to be 7.44±0.03 after 3 months. increased significantly within the first 2 months and afterwards it decreased slightly. Similarly, the concentration of [HCO₃⁻]_eincreased within the first 2 months, but afterwards it decreased to control levels. From the determined divalent and monovalent cations, Ca²⁺and Mg²⁺showed the most pronounced changes in the haemolymph of estivating snails. Both Ca²⁺and Mg²⁺increased during estivation up to about 27.25±1.89 mmol l⁻¹and 12.63±1.43 mmol l⁻¹after 3 months, respectively. In contrast, the pH_iof hearts remained at control levels during estivation.

Under normal conditions of perfusion, ventricles beat at a stable rate (29±2 beats min⁻¹, N=10) and only a slight reduction (6 %) in force of contraction was observed after 1 h of perfusion (Fig. 1A). However, there was a gradual reduction of force of contraction as a function of pH_e(Fig. 1A), while rate of contraction (beats min⁻¹) declined between pH 7.75 and 7.2, without further changes at lower pH values (Fig. 1B). Specifically, the contractile force of ventricles decreased by about 25 %, 30 % and 47 % after 60 min of perfusion with salines B, C and D, respectively, in the presence of 6.85 mmol l⁻¹Ca²⁺. The pH_i(7.179±0.025) remained stable in the ventricles perfused for 60 min with saline A (Fig. 1C). Also, perfusion of ventricles with the hypercapnic salines B and C did not cause any significant reduction in pH_i. Perfusion of ventricles with saline D, however, caused a significant reduction in pH_icompared to control values (P<0.001) within the first 30 min of perfusion. After 30 min of perfusion, however, pH_iwas recovering slowly towards the control level (Fig. 1C).

Perfusion of ventricles with the hypercapnic saline B in the presence of 3 mmol l⁻¹Ca²⁺caused greater decreases in the contractile force (66 %) compared to ventricles perfused with the corresponding hypercapnic saline, but containing 6.85 mmol l⁻¹Ca²⁺(25 %) (Fig. 2). Increases in the concentration of Ca²⁺in the hypercapnic saline B to >6.85 mmol l⁻¹did not further improve the contractility and rate of contraction of ventricles and had nearly the same effects as perfusion in the presence of 6.85 mmol l⁻¹Ca²⁺. pHi was determined to be 7.174±0.03, 7.21±0.035, 7.20±0.025 and 7.185±0.036 after 60 min of perfusion with saline B containing 3 mmol l⁻¹, 11 mmol l⁻¹, 15 mmol l⁻¹or 30 mmol l⁻¹of Ca²⁺, respectively.

The effects of the combination of hypercapnia and veparamil or hypercapnia and Co²⁺on the contractile force, rate of contraction and pH_iof ventricles are shown in Fig. 3. Perfusion of ventricles with the saline B in the presence of verapamil (10⁻⁴mol l⁻¹) caused a marked reduction in the force and rate of ventricle contraction (Fig. 3A). Similar results were obtained in the presence of Co²⁺(10⁻⁴mol l⁻¹) (Fig. 3B). In both cases, however, the pH_iof ventricles, after an initial increase, remained stable during perfusion (Fig. 3C).

The effects of the saline simulating the composition of haemolymph of snails estivating for 3 months on the perfused ventricles are shown in Fig. 4. The contractile force of ventricles was reduced by about 18 % (Fig. 4A) and the rate of ventricle beating by about 60 % (Fig. 4B) after perfusion with the above saline. The pH_iremained stable in the ventricles perfused under the same conditions (Fig. 4C).

To examine whether low extracellular pH affects the influx of Ca²⁺into the heart cells, we determined the intracellular concentration of Ca²⁺in ventricles perfused with salines A and B, containing either 3 mmol l⁻¹Ca²⁺or 27 mmol l⁻¹Ca²⁺(Fig. 5). There was no effect on the level of intracellular Ca²⁺in ventricles perfused with the hypercapnic saline B containing 27 mmol l⁻¹Ca²⁺, while perfusion of ventricles in the presence of 3 mmol l⁻¹Ca²⁺caused a significant decrease in the level of intracellular Ca²⁺.

Similar to other land snails (Barnhart, 1986; Rees and Hand, 1990), Helix lucorumexperiences hypercapnia and respiratory acidosis during estivation (Table 1). The results from in vitro experiments on isolated perfused ventricles indicate that hypercapnia may negatively affect the heart activity in estivating snails. Specifically, it seems that hypercapnia causes a marked decrease in the rate and, to a lesser extent, in the ability of heart muscle to generate force. However, these changes in heart activity seem to be correlated with a reduction in extracellular pH rather than with that of intracellular pH. As shown in Fig. 1, a reduction of pH in the perfusates, by changing the concentration of bicarbonates, resulted in a significant reduction in the contractility (Fig. 1A) and rate of ventricle beating (Fig. 1B). However, there does not seem to be any correlation between heart activity and changes in pH of intracellular fluids, since perfusion of ventricles with hypercapnic salines does not appear to cause a significant reduction of pH_i(Fig. 1C). Only when ventricles were perfused with hypercapnic saline containing 5 mmol l⁻¹ NaHCO₃, did pH_ifall significantly within the first 30 min; thereafter it recovered at a slow rate (Fig. 1C).

The reduction in force was more significant when ventricles were treated with hypercapnic salines containing 3 mmol l⁻¹Ca²⁺(Fig. 2). Nevertheless, even in this case, the changes in ventricle activity did not appear to correlate with changes in the pH_i, since pH_idid not change after 60 min of perfusion in the presence of low levels of Ca²⁺. The above results indicate that high extracellular concentrations of Ca²⁺may counteract the acidic effect of hypercapnia on heart contractility in land snails during estivation. On the contrary, increased extracellular levels of Ca²⁺do not seem to restore the rate of heart contraction during hypercapnia. However, concentrations of Ca²⁺higher than 6.85 mmol l⁻¹did not completely restore the contractility of the heart during perfusion with hypercapnic salines (Fig. 2). This response of ventricles’ contractility to increasing extracellular Ca²⁺under hypercapnic acidosis seems to be similar to that observed under normal conditions of perfusion. As reported, the contractile tension of ventricles rises to a plateau level with increasing concentrations of Ca²⁺(Burton and Mackay, 1970).

The data obtained using verapamil and Co²⁺(Fig. 3) show a high dependence of heart activity on the rate of Ca²⁺entry into the cells. These results are similar to those obtained from other land snails and they show that extracellular Ca²⁺plays an important role in the snail heart, both in excitation–contraction and in the generation of the action potentials (Elekes et al., 1973; Kiss and S.-Roza, 1973; Paul, 1961). Our results indicate that H⁺ions might negatively influence Ca²⁺entry into the heart cells of Helix lucorumand that increases in extracellular Ca²⁺might counteract this negative effect (Fig. 5). The intracellular concentration of Ca²⁺was about 135±16 nmol l⁻¹in the ventricles of normal snails, a value which is similar to that reported for muscles from other invertebrates (Ishii et al., 1989), vertebrates (Batle et al., 1993) and also for the brain of the turtle Trachemys scripta(Bickler, 1992). Superfusion of ventricle slices under hypercapnia in the presence of 27 mmol l⁻¹Ca²⁺did not cause any change in the intracellular concentration of Ca²⁺. In contrast, the latter decreased significantly when slices of ventricles were superfused under the same hypercapnic conditions but in the presence of 3 mmol l⁻¹Ca²⁺(Fig. 5). The above results are similar to those reported for vertebrates, where hypercapnic acidosis depresses cardiac activity. The mechanisms by which low extracellular pH modulates heart activity in vertebrates are not well understood. It has been reported that low pH reduces contractile force through competition by H⁺for Ca²⁺-binding sites intracellularly and, possibly, extracellularly (Williamson et al., 1976). Competition of H⁺for the Ca²⁺binding sites is supported by studies demonstrating a reversal of acidosis depression by increasing extracellular Ca²⁺(Williamson et al., 1976; Yee and Jackson, 1984; Lagerstrand and Poupa, 1980; Gesser and Poupa, 1979). On the other hand, it has been shown that the strength of cardiac muscle contraction is determined by the magnitude of Ca²⁺bound to sarcolemmal surface receptors (Langer, 1985; Philipson et al., 1980; Bers et al., 1981) which, in turn, corresponds to the concentration of extracellular Ca²⁺(Philipson et al., 1980). Moreover, it has been reported that conformational changes in Ca²⁺channels or transporters and changes in the voltage dependence of channel gating caused by low pH might decrease Ca²⁺entry into heart cells (Iijima and Hagiwara, 1986; Ohmori and Yoshii, 1977). Besides, H⁺can block the channel, possibly in the channel pore (Krafte and Kass, 1988; Klockner and Isenberg, 1994).

Although it becomes obvious from the results presented that low extracellular pH has a negative effect on heart activity, it is unclear what is the exact role of extracellular pH on the modulation of heart activity in Helix lucorumduring estivation. This difficulty is due to the fact that the levels of acid–base parameters vary in the haemolymph of Helix lucorumduring estivation (Table 1) and, as has been reported previously, these changes may reflect periodic bursts of ventilation in land snails (Barnhart and McMahon, 1987; Rees and Hand, 1990). Moreover, the levels of several solutes change in the haemolymph of estivating snails and they may also be involved in the modulation of heart activity. Specifically, the levels of of Ca²⁺and Mg²⁺, which have opposite effects on heart activity (Burton and Mackay, 1970; Burton and Loudon, 1972) increase significantly in the haemolymph of estivating snails compared to controls (Table 1). Perfusion of ventricles with the saline simulating the haemolymph of estivating snails caused a reduction in contractile force of about 18 % (Fig. 4A) and a reduction in the rate of contraction of about 59 % (Fig. 4B). However, the above conditions of perfusion did not cause any change in pH_iof ventricles (Fig. 4C). Taking into consideration all the above data, it could be concluded that it is not only the extracellular pH but the combination of the acid–base status and the various solutes which may affect heart activity in land snails during estivation. Moreover, recent data indicate that the biogenic amines serotonin and dopamine are involved in the modulation of heart activity in estivating snails (Rofalikou et al., 1999).

According to the results presented, the heart of Helix lucorumseems to defend itself against a drop in extracellular pH and maintains a stable pH_iduring estivation (Table 1). The maintenance of pH_iat stable levels seems to be in conflict with the effect of artificial hypercapnia on the pH_iof whole body of other land snail species. Determination of pH_iof the whole body either by DMO (Barnhart and McMahon, 1988) or by NMR (Rees et al., 1991) has shown that artificial hypercapnia causes decreases in the pH_i. The acid–base variables in the haemolymph of Helix lucorum(Table 1) are in accordance with those reported for the haemolymph from other land snails (Barnhart, 1986; Rees and Hand, 1991) and the pH_iin the heart of Helix lucorumis similar to that reported for the hearts of other molluscs (Ellington, 1993; Kinsey and Ellington, 1995). Perhaps pH_ifluctuations in tissues as small as the heart cannot be recorded when pH_iis determined by DMO and NMR in the whole body. The recovery of pH_iin the perfused ventricles of Helix lucorumunder hypercapnia indicates that mechanisms of ion exchange of acid–base equivalents between intracellular and extracellular compartments may exist in cardiac muscles. This suggestion is in accordance with data which indicate that regulation of pH_iin the nervous and muscular system of land snails may involve tightly linked Cl^––HCO₃⁻and Na⁺–H⁺exchange (Thomas, 1977; Ellington, 1993; Zange et al., 1990). The physiological importance of pH_imaintainance at stable levels in the heart of land snails during estivation is not clear. However, recent data indicate that the circulatory system of snails is involved in gas exchange during estivation (Rofalikou et al., 1999). Consequently, maintainance of heart pH_iat stable levels may be of great physiological importance since it might preserve heart activity.

The authors wish to thank D. Haas for his technical assistance with the determination of intracellular pH.