In order to evaluate the contributions of physico-chemical buffering to the adjustment of intracellular pH in response to changes of temperature in tissues of dogfish (Scyliorhinus stellaris), the CO2 equilibration method for the determination of intracellular buffer values was modified to yield data for the mathematical simulation of the intracellular compartments as closed buffer systems, and for the calculation of transmembrane bicarbonate transfer in vivo.
The respective buffer values of imidazole-like and phosphate-like buffer substances were estimated to be about 39 and 11 in white muscle, 21 and 18 in red muscle and 27 and 10 mequiv/(pH.l cell water) in heart muscle. In white muscle, the observed changes of intracellular pH can be explained by physico-chemical buffering and changes of . In red muscle and heart muscle considerable amounts of bicarbonate have additionally to be transferred across the cell membrane to achieve the temperature-dependent variations of pH observed in vivo.
The regulation of intracellular pH (pHi) in response to changes of temperature is still not entirely understood. Several mechanisms are possible (Heisler, Neumann & Holeton, 1980). Of these, only change of , and changes of intracellular buffer pK values are favoured by Reeves & Malan (1976) as being exclusively responsible for the pHi adjustment in frog and turtle. In dogfish, however, it has been shown that net bicarbonate transfer between intracellular and extracellular body compartments and the environmental sea water is also important (Heisler, 1978). It has been further shown that even moderate hypercapnia at low acclimation temperature is compensated by appropriate bicarbonate concentration changes, to attain the same ΔpH/Δt relationship as in normocapnic animals (Heisler et al. 1980).
The present study assesses the relative contributions of the three mechanisms which are involved in the intracellular pH regulation in dogfish when temperature is changed. This was achieved by determining intracellular buffer values for dogfish, and then modelling the intracellular compartment as a physico-chemical buffer system, as for frog and turtle (Reeves & Malan, 1976).
Physico-chemical buffering properties can only be determined in a closed system, where bicarbonate, H+ or OH− exchange with other compartments can be excluded (Heisler & Piiper, 1971). Such conditions can be achieved by application of the CO2-equilibration technique of homogenates, which has previously been used for the determination of total intracellular buffer values at fixed temperature (Heisler & Piiper, 1971). The methodology, however, has to be modified by introduction of temperature, as well as , as a variable. The resultant pH and bicarbonate changes can then be utilized for a mathematical simulation of either the intracellular compartment modelled as a closed buffer system, or of the transmembrane bicarbonate exchanges in vivo.
Two different approaches can be used to evaluate the obtained data sets of Δt, ΔpH and Δ [HCO3] values :
(A) This first approach describes the behaviour of the intracellular buffer system via the determination of the buffer values of the predominant intracellular non-bicarbonate buffers. These are the imidazole-like buffers, with a fairly high temperature coefficient of their pK values (ΔpK/Δt ∼ –0·02 U/°C), and the phosphate-like buffers, with a low temperature coefficient (ΔpK/Δt ∼ –0·002 U/°C). The intra-cellular compartment can be modelled as if it was a closed buffer system with no transfer of non-volatile substances across the cell membrane, or the bicarbonate transfer due to changes of temperature in vivo can be calculated.
[HCO3−]a = concentration of intracellular bicarbonate determined in vivo at temperature a,
Δ [HCO3−]NB = concentration of bicarbonate produced by CO2 buffering of the intracellular non-bicarbonate (NB) buffers during the transition from temperature a to b and from in vivo at a to in vivo at b,
= concentration of bicarbonate in the closed buffer system at temperature b.
= pH of closed buffer system at temperature b,
a and pK = respective constants of the Henderson-Hasselbalch equation of the bicarbonate buffer system at b,
ΔpH’ = difference between intracellular pH at a, and ,
= determined in vivo at b.
Series A : Varied temperature
Samples of white muscle, red muscle, and heart muscle of Scyliorhinus stellaris (body weight: 0 ·3 –3 kg) were frozen in liquid nitrogen, powdered with mortar and pestle and diluted with dogfish Ringer solution (Table 1) by a factor of about 3 (for details of the procedure see Heisler & Piiper, 1971). This homogenate was equilibrated in an intermittently rotating cuvette (Fig. 1) at 10 and 20 °C ( ± 0 ·1 °C) with gases of known values (consisting of only CO2 and O2) according to the following protocol :
Equilibration at 10 °C with a known . The pH of the homogenate was adjusted by addition of either HC1 or NaHCO3 to a value falling into the range of pHi values determined in vivo for the respective tissue (Heisler, Weitz & Weitz, 1976b ; Heisler et al. 1980).
Subsequent equilibration at 20 °C with a different which was sufficient to produce a change in pH (ΔpH) of – 0 · 04 to – 0 ·08 in comparison with the value measured at the end of equilibration period a.
Re-equilibration with the used during equilibration period a.
This equilibration sequence (procedure 1) was repeated twice with different values in order to produce ΔpH’s of –0 ·15 to –0 ·22 (procedure 2) and of –0·27 to – 0·33 (procedure 3). The pH of the homogenate was recorded continuously during all equilibrations with one of two single-unit double electrolyte bridge electrodes. Each electrode was used for measurements at only one temperature with temperature-controlled precision phosphate buffers adjusted to the same ionic strength as the Ringer solution used for dilution of the muscle powder. Equilibration with each gas mixture was continued (20 –40 min) until the pH was either constant or varied only slightly at a constant rate due to metabolic release of H+ ions. Complete equilibration was checked by frequent measurements of , in the homogenate.
Series B: Constant temperature
In a separate series of experiments, equilibration procedures similar to those described above, but at constant temperature, were performed in order to determine the total buffer value of the non-bicarbonate buffers (for details of the method see Heisler & Piiper, 1971).
The behaviour of pH in the intracellular compartments as closed buffer systems, and the bicarbonate transfer between the intracellular compartments as open buffer systems and the extracellular space were calculated on the basis of equations (10), (12), (14) and (15) using , pHi and [HCO3] values reported by Heisler et al. (1980).
The buffer values determined for juvenile (0 ·3 –1 kg) and adult specimens (1 –3 kg) did not show any trend to be different from each other so the mean values of all studied specimens were calculated and are presented in Table 2. The total buffer values, both those determined as the sum of imidazole-like and phosphate-like buffer values according to the method described in this paper and those determined at constant temperature (method adopted from Heisler & Piiper, 1971) are fairly similar and somewhat smaller than values determined in mammalian and bird species using in principle the same technique (Heisler & Piiper, 1971 ; Lai, Attebery & Brown, 1973 ; Lykkeboe & Johansen, 1975).
The contribution of imidazole-like and phosphate-like buffering to the total buffer value is different in the three muscle types. In white muscle, imidazole-like buffering contributes about 80% to the total buffer value. The observed ratio for βim/βph of about 3 ·7 is very similar to the concentration ratio for imidazole and phosphate compounds of 4, calculated for frog intracellular muscle compartments by Reeves & Malan (1976). While in white muscle the contribution of phosphate-like buffering is relatively small, it is larger in the other two muscle types, especially in red muscle, where about half (47%) of the total buffer value has to be attributed to phosphate-like buffering.
The intracellular compartments have been modelled as closed buffer systems according to (10) and (14). The changes of intracellular pH that would occur under these conditions when temperature is changed are summarized in Table 3. The ΔpHi/ Δt values calculated for red muscle and for heart muscle are significantly different from the corresponding values determined in vivo with both normal and with increased at low acclimation temperature (reversed ), but for white muscle they are different only with hypercapnia at low temperature.
The net bicarbonate transfer between intracellular and extracellular compartments of the studied tissues occurring in vivo after temperature is changed by 10 °C are listed in Table 4. The transferred amounts are considerable in comparison with the normal intracellular concentration of about 1 mm, and are significantly different from zero for heart muscle and for red muscle. In white muscle the difference is only significant with increased at low temperature.
The mathematical treatment of the obtained data according to both approaches ‘A’ and ‘B’ is based on a linear correlation between the amount of buffered H+ ions and the change in pH. The linear relationship was chosen as the best approximation to the obtained correlation of Δ [HCO3] and ΔpH values determined in the individual experiments. The non-bicarbonate buffer curve in the muscle tissues of dogfish is similarly linear, as in the intracellular compartments of human erythrocytes (Dill, Edwards & Consolazio, 1937). This quasi-linearity can be explained by the overlapping sigmoid buffer curves of several buffer groups in different stereometric positions in larger molecules, which have different pK values according to their position and chemical modifications.
Both approach ‘A’ and approach ‘B’ (see Theoretical Considerations) are equivalent in their final description of the closed intracellular buffer systems and of the bicarbonate transfer in vivo. The main advantage of approach ‘A’ is that the obtained data allow the simulation of the intracellular compartment for every chosen temperature change, whereas approach ‘B’ is limited to application with the temperature step imposed during the experimental procedure. Additionally, approach ‘A’ yields buffer values for the imidazole-like and phosphate-like buffers. The calculations of these values, however, are fairly sensitive to mis-estimates of the chosen mean ΔpK values.
The error introduced by such mis-estimates is dependent on the relative buffer value of the respective buffer group. Considering the range covered by the ΔpK values of different imidazole-like and phosphate-like buffer substances (Edsall & Wyman, 1958) and the determined relative buffer values, mis-estimates of about ±20% have to be considered possible, even when the similarity between the total buffer values determined at constant temperature and the values of (βim/βph) suggests that the obtained values fit quite well the actual conditions.
While mis-estimates of the Δ pK values have considerable influence on the calculated buffer values, the calculations of ΔpHi/Δt for the closed buffer systems (Table 3) and of the bicarbonate transfers (Table 4) are completely independent of the ΔpK values according to the applied mathematical treatment. They are only influenced by the and pK values used as constants for the bicarbonate calculations by means of the Henderson-Hasselbalch equation. Application of the values for dogfish plasma to the non-diluted intracellular compartments may result in a maximal error of 5%, a value that does not influence the significance of the data presented in Tables 3 and 4.
While all sources of error discussed above cannot significantly affect the results presented in this paper, autolysis of ATP and creatine phosphate (CP) in the muscle sample and later in the homogenate would affect the quantitative conclusions of this paper considerably. Decomposition of ATP and CP would release fairly large amounts of inorganic phosphate, which is a good buffer in the physiological pH range. As CP, and ATP in the presence of Mg2+, hardly buffer in the physiological pH range, this mechanism would result in an overestimate of the buffer value for phosphate-like substances. But even the assumption of a maximal overestimate would not change the qualitative conclusions of this paper: even with a phosphate buffer value of zero the ΔpH/Δt values for the closed buffer system in red muscle and heart muscle would still be significantly different in the same direction from the in vivo values as those given in Table 3. Also the bicarbonate transfers for these tissues would be significantly different from zero in the same direction as those reported in Table 4.
It is, however, unlikely that significant autolysis can take place during the applied procedure. The muscle samples were deep frozen precipitously in liquid nitrogen within 20 s from the start of the excision and then kept frozen until the muscle powder was diluted with Ringer solution. During the equilibration procedure any decomposition of CP or ATP would have been recorded as a considerable rise in homogenate pH measured at the end of equilibration C in comparison with pHa (see Procedure), resulting from the release of OH− ions (for CP: 0· 5 mmol OH− /mmol CP decomposed, Meyerhoff & Lohmann, 1928). As we have not observed such alkaline pH shifts in our experiments and as the hypoxic time of the tissues can be considered to be only a very few seconds, we believe that no significant decomposition of ATP or CP has affected the reported results, especially as our phosphate-like buffer values appear rather low with respect to inorganic phosphate concentrations measured in fish muscle tissues (i.e. muscle of resting trout: 44· 5 mmol/kg fresh tissue, Tomlinson, Geiger & Kay, 1965).
White muscle appears to be the only one of the studied tissues in which the intracellular pH is adjusted after changes of temperature more or less without the necessity of bicarbonate being transferred across the cell membrane. The Δ pH/Δt value in the closed intracellular buffer system is at normal environmental almost the same as observed in vivo (see Table 3). When the value is reversed by hypercapnia at low acclimation temperature, however, the closed buffer system Δ pHi/Δ t is significantly smaller than in vivo and a significant amount of bicarbonate has to be transferred across the cell membrane to achieve the in vivo steady state value (Table 4). In the other two studied tissues, red muscle and heart muscle, the closed buffer system Δ pHi/Δ t values differ by a factor of 2 from the in vivo conditions. The amount of bicarbonate that has to be transferred to attain the in vivo equilibrium is in heart muscle about 3 times as large as the normal intracellular content, and in red muscle about 7 times as large. It is quite evident on the basis of the data summarized in Tables 3 and 4 that net bicarbonate transfer processes play an important role for the pHi adjustment in dogfish after temperature changes. Bicarbonate transfer, however, appears to be not a special mechanism for acid-base regulation after temperature changes, since it has been shown to be significantly involved in acid-base regulation under various conditions: in hypercapnia at constant temperature in mammalian tissues (Heisler & Piiper, 1972 ; Strome, Clancy & Gonzalez, 1976), dogfish tissues (Heisler et al. 1976a) and teleost fish tissues (Heisler et al. 1978), after exhausting exercise in mammalian and amphibian tissues (Benadé & Heisler, 1978) and in dogfish muscle tissues (Holeton & Heisler, 1978). These accumulated data suggest that net bicarbonate (or H+ or OH-) transfer is similarly widespread among species and tissues as the transport system for H+, OH− or bicarbonate across the cell membrane serving the maintenance of the steady state intracellular/extracellular bicarbonate distribution. It may even be that both transfers are mediated by the same regulator mechanism which is modulated with changing setpoint values under various conditions. The extent, however, to which net bicarbonate movements are actually involved in acid-base regulation may be quite variable according to the contributions of other mechanisms.
Apparently the importance of this mechanism is smaller in the two air-breathing species frog and turtle. When temperature is changed in these animals, the intracellular pH is adjusted by changes of buffer pK values and changes in (Reeves & Malan, 1976). As the pK values change almost immediately with temperature and as appears to be adjusted by changes of ventilation, at least in turtles, in less than 1 h (Jackson & Kagen, 1976), the deviation of the intracellular pH from the steady state values for the new temperature can be expected to be small and of only short duration (Reeves & Malan, 1976). Therefore, this type of regulation appears to be rather advantageous. But water breathing animals have no choice of mechanism, for they are fairly limited in the adjustment of , as a result of the low oxygen content of their breathing medium. Bicarbonate transfer is a relatively slow method of acid-base adjustment in response to temperature change (Heisler, 1978) but this disadvantage is mitigated by the fact that the extent and the rate of temperature changes most fish normally encounter are small in comparison with those faced by air breathing ectotherms.
The present results have shown that in dogfish the changes of intracellular pH in response to changes of temperature can be explained on the basis of physico-chemical buffering and changes of only in white muscle under normal conditions. In red muscle and heart muscle, and also in white muscle when is reversed by hypercapnia at low acclimation temperature, the pHi is predominantly and probably ultimately regulated by means of intracellular/extracellular bicarbonate transfer processes.
The authors gratefully acknowledge the support by the Stazione Zoologies at Naples.