A Calcium-Dependent Mechanism Responsible for Increasing the Freezing Tolerance of the Bivalve Mollusc Modiolus Demissus

The primary site of extracellular freezing injury in many cell types is the plasma membrane (Lovelock, 1957; Farrant, Walter & Armstrong, 1967; Menz, 1971; Pribor, 1971). The mechanism of injury involves the disruption of the cell membrane caused either mechanically as cells shrink beyond a minimum cell volume (Meryman, 1971), or chemically as specific salts attain toxic concentrations in the frozen state leading to protein or lipoprotein denaturation (Lovelock, 1957; Pribor, 1971). In fact, solutes such as glycerol and various amino acids and sugars reduce injury to cells resulting from extracellular ice formation by interacting directly with membranes (Heber, Tyankova & Santarius, 1971; Santarius, 1973; Mazur, Miller & Leibo, 1974). Ca²⁺ binds to cell membranes (e.g. Shaltz & Marinetti, 1972) and also increases the freezing tolerance of algal cells (Terumoto, 1967) and the bivalve mollusc M. demissus (Murphy, 1977) when present in the extracellular medium. However, the mechanism of Ca²⁺ protection during freezing remains unknown.

In order to determine the relative importance of Ca²⁺ as a solute for increasing freezing tolerance and to define the mechanism of Ca²⁺ protection during extracellular ice formation, I have examined the Ca²⁺-dependent mechanism responsible for increasing the freezing tolerance of the intertidal, marsh-dwelling mussel Modiolus demis sus following low-temperature acclimation. The results of these experiments indicate that the site of Ca²⁺ action appears to be at the cell membrane and that the cell membrane is the primary site of freezing injury. Furthermore, although Ca²⁺ is the only blood solute associated with the increased freezing tolerance of M. demissus, the increase in blood Ca²⁺ resulting from -low-temperature acclimation can account for only 40 % of the total increase in freezing tolerance resulting from low temperature acclimation. Therefore, the total freezing tolerance mechanism involves other components.

Modiolus demissus were collected and stored as previously described (Murphy, 1977). The procedures for freezing intact M. demissus and for determining freezing mortality followed the methods of Murphy & Pierce (1975). Isolated strips of foot muscle from M. demissus were frozen and changes in freezing injury expressed as changes in the minimum voltage stimulus necessary to elicit a twitch contraction (Murphy, 1977).

A time course of the changes in both blood Ca²⁺ and freezing tolerance of M. demissus was determined. A group of 200 mussels was acclimated for 2 weeks in 33·7‰ artificial sea water (ASW) (Wilkins, 1972) with a 3·0 mM-Ca²⁺ concentration at 15 °C. Subsequently, these mussels were transferred to ASW (33·7‰) containing 9·6 mM-Ca²⁺. At various times following the transfer, ten mussels were removed and either blood Ca²⁺ concentrations were measured using a murexide assay (as previously described by Murphy (1977)) or the mussels were frozen to −10 °C for 12 h to determine percent mortality. The salinity of all sea-water samples was determined with a freezing-point depression osmometer (see Murphy, 1977).

The influence of Ca²⁺ on freezing tolerance was also determined by specifically displacing Ca²⁺ from cell membranes with lanthanum ions (La³⁺) (Weiss, 1974). Isolated strips of foot muscle were first incubated in either ASW for 6 h, or ASW for 5 h followed by incubation in ASW containing 2·0 mM-La³⁺ for 1 h. Next, the tissue was frozen for 12 h at −8·0 °C, thawed for 1 h in ASW, and the responses (threshold voltages^-1) compared. The salinity (31.9‰) and temperature (25 °C) of the ASW solutions were constant throughout. Furthermore, in order to dissolve LaCl₃, the pH of the ASW was lowered from 7·8 to 6·5 using 0·1 N-HCl. Control studies showed that a pH as low as 4·1 had no significant effect on freezing tolerance (P > 0·5).

Foot tissue was isolated from M. demissus, cut into strips, and immediately frozen for 12 h to various temperatures between − 6·0 °C and − 13·1 °C. The degree of freezing injury was then determined by measuring the contractility of the muscle following a 1 h thawing period according to the following procedure. Each tissue strip was suspended vertically in air with a silk suture between a fixed metal hook and a force-displacement transducer (Grass Instrument Co., Model FT. 03 C). The suspended tissue was then immersed in a series of 31·8‰ ASW solutions containing progressively increasing concentrations of a specific chemical stimulus. The degree of contractility was expressed as the concentration of the stimulus needed to elicit a sustained contraction for 1·5 min as displayed on an oscillograph (Grass Instrument Co., Model 79C).

The total change in freezing tolerance resulting from temperature acclimation was obtained by acclimating groups of mussels in 31·8‰ Instant Ocean at either o or 23 °C for 5 weeks. Strips of foot tissue were obtained from both groups of mussels and incubated for 6 h (25 °C) in 20 ml of blood from mussels acclimated to the same conditions. Blood osmotic concentrations were 945 m-osmol/kg H₂O and 941 m-osmol/kg H_aO for the o and 23 °C acclimated groups respectively. Following incubation, the total change in freezing tolerance was determined by freezing both groups of tissue to − 10 °C for 12 h, thawing the tissue in ASW (940 m-osmol/kg H₂O) at 25 °C for 1 h, and then measuring the difference in the threshold voltages. To determine the effect of changes in total blood solutes resulting from temperature acclimation on freezing tolerance, a similar experiment was performed following incubation of foot tissue from 23 °C-acclimated mussels in blood from either o or 23 °C acclimated mussels. Finally, the influence of the changes in blood Ca²⁺ resulting from temperature acclimation on freezing tolerance was determined. Strips f foot muscle from 23 °C-acclimated mussels were incubated in ASW containing either 8·0 or 12·0 mM-Ca²⁺ for 6 h at 25 °C. Following incubation, both groups were frozen, thawed, and the difference in the threshold voltages determined.

A time course of the changes in blood Ca²⁺ and freezing tolerance showed that blood Ca²⁺ concentrations of M.demissus acclimated in 33·7‰ low Ca²⁺-ASW (3·0 mM) and transferred to isosmotic, high Ca²⁺-ASW (9·6 mM) increased from 4·2 to 6·3 mM within 2·5 h. Simultaneously, the percentage survival of M. demissus frozen at −10 °C increased from 10−20% to 40−70% following the 2·5 h exposure to high Ca²⁺-ASW (Fig. 1). Six hours after the transfer to high Ca²⁺-ASW, the blood Ca²⁺ concentration increased further to 8·5 mM and the percentage survival ranged between 60 and 70%. By 19 h, blood Ca²⁺ concentrations had stabilized between 9·0 and 10·3 mM and did not increase any further for the duration of the experiment (8·25 days). Percentage survival values also levelled off after 19 h and ranged between 70 and 90% thereafter (Fig. 1).

The displacement of Ca²⁺ from cell membranes by La³⁺ influenced the freezing tolerance of isolated foot muscle. The contractile responses of the freeze-thawed tissue incubated in 2·0 mM-LaCl₃ relative to non-frozen control tissue were significantly lower than the relative contractile responses of freeze-thawed tissue incubated without LaCl₃ present (P < 0·001) (Table 1).

The relative response of freeze-thawed foot muscle from M. demissus to CaCl₂ remained constant following exposure to temperatures between − 8·0 and −13·1 °C (P > 0·5) (Fig. 2). In contrast, although the relative responses of freeze-thawed tissue to Ach and KC1 followed the same pattern as the relative CaCl₂ responses when frozen to − 8·1 °C (P > 0·2), the responses to these stimuli dropped significantly below the CaCl₂ response when frozen to temperatures between − 8·1 and −10·2 °C (P < 0·001) (Fig. 2). Similarly, freeze-thawed foot tissue frozen to − 8·1 °C showed the same relative response to caffeine as to CaCl₂, but exposure to temperatures between − 8·1 and −10·2 °C significantly reduced the caffeine response below that of the response to CaCl₂ (P < 0·01) (Fig. 2).

A comparison of the changes in the contractile responses of freeze-thawed foot muscle from M. demissus acclimated at 23 °C and incubated in blood from either 23 or o °C acclimated animals showed that an increased freezing tolerance could be induced in isolated foot muscle by incubation in blood from o °C acclimated animals (Table 2). This increase in freezing tolerance, however, accounted for only 38% of the total increase in freezing tolerance which occurred when foot tissue from mussels acclimated at o °C was incubated in blood from o °C acclimated animals. Similarly, the freezing tolerance of foot muscle from 23 °C acclimated animals incubated in ASW with 12·0 mM-Ca²⁺ was increased above that of foot muscle incubated in ASW with 8·0 mM-Ca²⁺. This increase accounted for only 41 % of the total increas in freezing tolerance (Table 2). Thus, although the increases in blood Ca^a+ which accompany low-temperature acclimation could account for the effect of blood solutes on freezing tolerance, changes in blood solutes alone do not account for the total change in freezing tolerance resulting from low-temperature acclimation.

The results presented here indicate that the increase in the blood Ca²⁺ concentration of M. demissus following low-temperature acclimation increases freezing tolerance directly by binding to cell membranes. Transferring M. demissus from low Ca²⁺-ASW to high Ca²⁺-ASW showed that the resulting increases in blood Ca²⁺ with time were paralleled by similar increases in freezing tolerance. Furthermore, the effect of an increase in blood Ca²+ on freezing tolerance occurred rapidly (within 2·5 h). Taken together, these data indicate that Ca²+ affects freezing tolerance directly by its presence in the blood and not secondarily by altering some biochemical or physiological process. The reduced freezing tolerance of isolated foot muscle, incubated in ASW supplemented with La²⁺ prior to freezing, indicated that the Ca²⁺ effect on freezing tolerance is at the plasma membrane surface. La³⁺ does not penetrate the cell membrane and, in addition, binds specifically to Ca²⁺ binding sites at the cell membrane surface, displacing bound Ca²⁺ from such sites (Weiss, 1974). Mg²⁺ also competes for Ca²⁺ binding sites on plasma membranes (e.g. Hemminki, 1974). Therefore, since Mg²⁺ is only 20% as effective as Ca²⁺ for increasing the freezing tolerance of M. demissus (Murphy, 1977), the reduced blood Mg²⁺ concentrations observed following low-temperature acclimation (Murphy, 1977) may also serve to increase freezing tolerance by making available a greater proportion of Ca²⁺ for binding to membrane sites.

As stated previously, a mechanism for increasing the freezing tolerance of M. demissus appears to involve an increased binding of Ca²⁺ to cell membranes. This suggests that the primary site of extracellular freezing injury is the cell membrane. The cell membrane is, in fact, the primary site of freezing injury in a variety of cell types (Lovelock, 1957; Farrant et al. 1967; Menz, 1971; Pribor, 1971). Changes in the response of freeze-thawed foot muscle to chemical stimuli following exposure to a series of subfreezing temperatures suggests that the plasma membrane is also the primary site of injury in M. demissus. The threshold concentration of CaCl₂ needed to elicit a contraction of freeze-thawed foot muscle relative to non-frozen controls remained the same following exposure to temperatures as low as −13 °C. In contrast, exposure to temperatures between −8 and −10 °C resulted in an almost complete loss of response to Ach, KC1 and caffeine. The loss of response of freeze-thawed muscle to both KC1 or Ach which elicit muscle contraction by plasma membrane depolarization (for review see Prosser, 1973 a) and caffeine which causes contraction by eliciting a Ca²⁺ release from either sarcoplasmic reticulum or plasma membranes (e.g. Huddart & Syson, 1975) implies that damage to the plasma membrane resulted from exposure to temperatures between −8 and −10 °C. The persistence of the response of freeze-thawed muscle to CaCl₂, however, is probably due to the direct interaction of Ca²⁺ with the intracellular contractile proteins (for review see Prosser, 1973b).

The increase in blood Ca²⁺ which occurs during low-temperature acclimation is the only blood solute change which is associated with the increase in the freezing tolerance of M. demissus. However, this increase in blood Ca²⁺ can account for only 40 % of the total observed change in freezing tolerance resulting from low temperature acclimation. A comparison of the differences in the freezing tolerance of M. demissus acclimated either anaerobically (13 °C) or aerobically (0 °C) showed that although the blood Ca²⁺ concentration of the anaerobic mussels was 4-fold greater than that of the low-temperature acclimated mussels, the low-temperature acclimated mussels still had a higher freezing tolerance. Therefore, part of the total increase in freezing tolerance involves mechanisms other than the blood Ca²⁺ increase and, apparently, depends directly on exposure to low temperature.

In conclusion, the increased binding of Ca²⁺ to cell membranes may reduce membrane damage during freezing either by physically stabilizing the membrane against the mechanical disruption caused by cell shrinkage or, perhaps, by preventing the denaturation of membrane components as specific tissue solutes concentrate to toxic levels in the frozen state. The other possible mechanisms which exist may involve either changes in the concentrations of specific intracellular solutes such as amino acids, organic acids, or sugars; or changes in the structural components of membranes, specifically lipids. Such changes could stabilize membranes against the stresses imposed during freezing (Heber et al. 1971; Santarius, 1971, 1973; Simino-vitch, Singh & De La Roche, 1975). These possible mechanisms are being investigated.

I wish to thank Dr. S. K. Pierce Jr. for his critical reading of the manuscript.

This work was supported by NSF Grant BMS 72-02465-A01 to Dr. S. K. Pierce Jr. and a Chesapeake Bay Funds Grant, University of Maryland.

Contribution number 60 from the Tallahassee, Sopchoppy and Gulf Coast Marine Biological Association.