The enzyme carbonic anhydrase (CA), which is concentrated mainly in the osmoregulatory tissue of the gills, appears to be required for ion regulation but not for CO2 excretion. An injection of the CA inhibitor acetazolamide produced an inhibition of between 90 and 100 %, which took 6 h to be fully effective, and 48 –96 h to wear off. During the period of inhibition in Callinectes sapidus there was no change in either O2 uptake or CO2 excretion, nor was there any increase in blood In blue crabs acclimated to 250mosM salinity, at which the animals are ion regulators, inhibition of CA caused both Na+ and Cl− concentrations in the blood to be lowered, with Cl− being lowered to a greater degree. As a result of an increase in the Na+–Cl− difference the animal experienced a ‘metabolic’ alkalosis: elevated blood pH and HCO3− at constant The data are consistent with the hypothesis that branchial CA functions in providing H+ and HCO3− as counterions for Na+ and Cl− transport through the hydration of respiratory CO2.
In the terrestrial Gecarcinus lateralis, inhibition of CA caused an increase in blood but did not alter O2 uptake or CO2 excretion. After an initial acidosis, blood pH and HCO2− increased and remained elevated. Blood osmolality, Na+, Cl− and Ca2+ concentrations all increased, and the animals experienced a high rate of mortality. These data suggest that CA in the land crab is also important in blood ion regulation, probably to combat desiccation.
The physiological functions of carbonic anhydrase in the vertebrates are well known. The erythrocyte enzyme is critical in the transport and excretion of metabolically produced CO2, and carbonic anhydrase in the kidney is active in the resorption/ excretion of bicarbonate ions and thus is involved in controlling the long-term acid base status of the blood (Woodbury, 1965; Davenport, 1974).
The overwhelming majority of the invertebrates which have been examined, including decapod crustaceans, however, lack red blood cells, and also lack any CA activity in the blood (Aldridge, 1977; Burnett, Woodson, Rietow & Vilicich, 1981; see also Henry & Cameron, 1982a for a more detailed treatment). Also the antennal gland of the crustaceans, which does not appear to play a role in blood acid-base regulation (Cameron & Batterton, 1978), does not have a high concentration of CA (Henry & Cameron, 1982a). Instead, the major concentration of CA is found in the gills (Aldridge, 1977; Burnett et al. 1981; Henry & Cameron, 1982a).
Crustacean gills perform a number of physiological functions. Primarily respiratory organs of O2 and CO2 exchange in both air-and water-breathing animals, the gills of the water breathers also function as organs of osmoregulation, ion regulation, and acid-base balance (Smith & Linton, 1971 ; Towle, Palmer & Harris, 1976; Mangum & Towle, 1977; Cameron, 1978a,b,Truchot, 1978, 1979). Gill carbonic anhydrase has been implicated in all of the above functions, but much of the evidence is circumstantial and some researchers have reported conflicting results.
Ever since it was first found in the gill tissue of invertebrates, carbonic anhydrase has been suggested as having an important role in CO2 excretion (Ferguson, Lewis & Smith, 1937), but closer examination has revealed that the enzyme is primarily associated with the individual gills and the patches of salt transporting cells within those gills which are involved in osmo-and ion regulation (Henry & Cameron, 1982a). Very little enzyme activity is found in the respiratory lamellae.
Aldridge & Cameron (1979), and Cameron (1979a) reported no changes in either blood pH or total CO2 (CT) in freshwater-adapted Callinectes sapidus after an injection of the CA inhibitor acetazolamide (Diamox). They concluded on the basis of that experiment that the gill enzyme was not important in CO2 excretion. They did not, however, confirm the assumption that the enzyme was indeed fully inhibited.
In contrast to this, Burnett et al. (1981) reported a significant disruption of blood pH, CT and Cl− ion concentration in the euryhaline marine crab, Pachygrapsus crassipes, after treatment with acetazolamide, with the most dramatic effect being on animals acclimated to salinities in which they regulated their haemolymph chloride ion concentrations significantly above those of the medium. The authors concluded that gill CA is important in both blood Cl− regulation and CO2 excretion. The latter conclusion is rather tenuous and does not appear to be supported by their data as they reported an increase in blood pH, rather than the expected respiratory acidosis.
A respiratory acidosis was observed in the semi-terrestrial crab, Cardisoma camifex, after an injection of acetazolamide (Randall & Wood, 1981), but CO2 excretion was not significantly lowered. Also, only two animals were used in the pH and determinations, and one of those died during the experiment. So despite the efforts of a number of workers, we are left without a clear picture of the physiological role of crustacean gill carbonic anhydrase. This report presents a study of gill CA function in respiration, ion regulation and acid-base balance in the aquatic crab, Callinectes sapidus, and the terrestrial crab, Gecarcinus lateralis. The two species used in this study are interesting because C. sapidus is an osmo-and ion conformer at high salinity (800 –1000mosM) but the animal regulates blood salts in low salinity; G. lateralis, which is fully terrestrial, maintains its blood hypo-osmotic and ionic to full-strength sea water (see Henry & Cameron, 1982a,b for details).
MATERIALS AND METHODS
Experimental handling of animals
Callinectes sapidus and Gecarcinus lateralis were collected and maintained as described previously (Henry & Cameron, 1982a). For experimentation, blue crabs were placed in large, flow-through aquaria of either 250 or 865 mosM salinity (details given by Henry & Cameron, 1982b). G. lateralis were put in individual Plexiglas chambers of approximately 400 ml volume. A layer of moist sand was placed in the bottom of each chamber and humidified air was pumped through an opening in the top. Each chamber was covered with black plastic to minimize disturbances to the animal.
Post-branchial (arterial) blood was sampled from the heart through rubber septa glued over small holes in the carapace using a 1 ml syringe and 23 ga needle (Henry & Cameron, 1982b). Injections of substances into the animal were performed through these septa. Prebranchial (venous) blood was sampled from the infrabranchial sinuses at the bases of the walking legs.
In vivo inhibition of gill CA by acetazolamide
A 0·1 M solution of the soluble salt sodium acetazolamide was made in Cortland saline (Wolf, 1963) which had been adjusted to pH 9·0 by the addition of NaOH. The solution was then titrated down to pH8·3 with 0·1N-HCL without any loss of acetazolamide from precipitation. This was done in order to bring the acetazolamide solution as close to the pH of the crabs’ blood (7·5–8·0) as possible while maintaining solubility of the drug.
The blood volume of the crab was estimated as roughly one third of the total fresh weight (Gleeson & Zubkoff, 1977) and the appropriate volume of inhibitor was injected to give a 10−4M concentration in the blood.
Previous to and at various times after injection, animals were killed, the 7th gill pair in C. sapidus and the 9th in G. lateralis were dissected out, and the pH-stat assay procedure was carried out as described in detail elsewhere (Henry & Cameron, 1982a). Briefly, the assay involves measuring the catalysed rate of dehydration in a buffered solution of bicarbonate by holding the pH constant through the addition of HCL from an auto-titration apparatus (Radiometer-Copenhagen). The amount of titrant added per unit time is a reliable measure of the velocity of the reaction. Activity per g fresh weight of tissue and specific activity were reported as a percentage of the normal, pre-injection level in that gill pair.
Blood osmotic, ionic and acid-base parameters
Pre-and/or post-branchial blood samples (˜200μl) were taken before and after an acetazolamide injection (10−3-10−5 M final concentration in the blood), bracketing the time period in which the gill enzyme was found to be inhibited. Blood pH and total CO2 (CT) were measured immediately; pH was determined on a 50μl sample using an acid-base analyser (Radiometer-Copenhagen PHM 71) and a water-jacketed capillary pH electrode which was thermostatted to the experimental temperature (20–22°C). CT was determined on 20μl of blood using a conductometric method which depends on conversion of all combined forms (bicarbonates, etc.) to dissolves CO2 gas by acidification, removal in a carrier gas stream, followed by absorption in alkali and detection by differential conductivity (Maffly, 1968; J. N. Cameron, unpublished).
The remaining blood was allowed to clot in the syringe; the clot was disrupted by expulsion into a centrifuge tube and separated from plasma via centrifugation (Eppen-dorf Model 5412). Blood osmolality, Na+ and Cl− concentrations were measured by flame photometry and Ag-titration as described in detail by Henry & Cameron (1982b). Calcium was determined by atomic absorption (Perkin-Elmer 303).
A second set of animals was used to obtain blood values, since in C. sapidus the blood clotted before all measurements could be made on one sample, and G. lateralis were too small to allow serial sampling of large enough volumes of blood. A water-jacketed electrode and acid-base analyser (Radiometer-Copenhagen PHM 71), with the scale expanded four times, were used in the determinations (Henry & Cameron, 1982b). Control animals were injected with Cortland saline and sampled as above.
Whole-animal respiratory parameters
Oxygen uptake and carbon dioxide excretion were measured in both species previous to and after an acetazolamide injection (10−3M in blood) or an injection of Cortland saline (controls). Individual blue crabs were placed in sealed Plexiglas chambers through which air-equilibrated water flowed. Oxygen uptake was determined from the difference between the inflowing and outflowing water multiplied by the flow rate and expressed on a fresh-weight-specific basis for each crab. No auxiliary stirring in the chamber was used. Water was measured using a water-jacketed electrode (Radiometer-Copenhagen) and acid-base analyser (PHM 71). The electrode was calibrated using a sodium sulphite/sodium borate zero solution and air-equilibrated water. was determined in the same fashion, with total inspired and expired CO2 being determined on 150μl of water using the same procedure described above for CT in blood.
Individuals of G. lateralis were placed in sealed Plexiglas chambers through which humidified air was pumped. and total CO2 in the open chambers were measured as described above, and then the chambers were sealed with a rubber stopper. At 1 h after the chambers had been sealed, the drop in (and increase in total CO2) was measured on air samples that were withdrawn through a rubber septum using a 22 ga needle and 1 ml syringe. At the end of the experiment the animals were weighed and and were expressed on a fresh-weight-specific basis.
Inhibition of gill carbonic anhydrase
The time course of gill CA inhibition by a 10−4M-blood acetazolamide concentration in both species is shown in Fig. 1. At very low levels of activity it is difficult to measure the difference between the catalysed and uncatalysed rates of dehydration accurately. The natural variation in the uncatalysed and catalysed rates under idea conditions (using pure bovine CA which causes no foaming) is 2 and 5 %, respectively (Henry & Cameron, 1982a), and this increases when foaming occurs through the use of a crude tissue homogenate. Therefore, for the maximum degree of inhibition, 95 ± 5 % at 6–24 h, it can only be said that between 90 and 100% of the enzyme activity was inhibited. The difference may be due to actual residual activity or it may be due to the inability of the assay to distinguish accurately between 0 and 5 % activity. A higher concentration of the drug (10−3M), therefore, was also used in the physiological studies.
Blood acid-base parameters: Callinectes acclimated to low salinity
In blue crabs acclimated to 250mosM water for at least 2 weeks, there was a profound effect of CA inhibition on the blood acid-base status, which was directly related to the dose of acetazolamide given to the animal (Fig. 2). The transient depression in pH at 2 h for the lower concentrations of acetazolamide was probably related to stress brought about from handling the animals twice during that time period and not an effect of the inhibition. At 2h the dose given did not completely inhibit the gill enzyme (Fig. 1) and blood pH was restored to near control values by 12–24 h, during which time gill CA is still inhibited by the drug. A 10−3 M concentration of the inhibitor, which according to Maren (1977) is enough to give a 10−4M concentration intracellularly and inhibit 99–99% of the enzyme activity, caused angificant elevation of pH (0·25 units) between 4 and 48 h (P< 0·01, Student’s i test). The gradual return to the resting value of 7·90 by 96 h (Fig. 2) paralleled the time course for gill CA inhibition and recovery (Fig. 1).
All concentrations of acetazolamide used were effective in altering blood bicarbonate concentrations, but the degree of change was dose-dependent (Fig. 2). The lower concentrations, 10−5 and 10−4M, caused an elevation of only about 2 mm, be this was significant (P < 0·05, t test). A 10−3 M concentration of acetazolamide in the blood almost doubled the resting HCO3− concentration, but pre-injection levels were also restored by 96 h (Fig. 2).
In contrast to pH and CT, blood was virtually unaffected by 10−3M acetazolamide. in control animals was elevated slightly, probably being attributable to handling stress, and the in the drug-treated crabs were also raised only a small degree (1 Torr) (Fig. 2). In both cases there was no significant difference among the means (P> 0·05, F test).
Blood osmotic and ionic parameters: Callinectes acclimated to low salinity
Inhibition of carbonic anhydrase clearly disrupted the blue crab’s ability to maintain its blood osmolality above that of the ambient medium (250 mosM) (Fig. 3). Also, the effect appeared to be dose-dependent and more prolonged at the higher drug concentrations, with blood osmolality being depressed between about 5 and 35 %. In all cases, however, blood osmolality was restored to normal, resting values by 96 h.
The response of the two major ions in the blood, Na+ and Cl−, which make up over 90 % of the blood osmolality, was also dose-dependent and followed the same pattern (Fig. 3). At any particular time during the course of CA inhibition, however, blood Cl− concentrations were lowered more than those of Na+ (Fig. 3). For example, for the 10−3 M-acetazolamide treatment, blood Na+ was lowered by 70 ± 9, 100 ± 12, and 115 ± 15 mm at 6, 12 and 24 h, respectively, while blood Cl− was depressed by 81 ± 8, 126 ± 12, and 152 ± 14 mm respectively for those same times (mean ± S.E., n = 6). The differences at 12 and 24h were significant (P<0·05, t test), and corresponded to the largest changes in blood acid-base status (Figs 2, 3).
Blood acid-base, osmotic and ionic parameters: Callinectes acclimated to high salinity
In C. sapidus acclimated to 865 mosM, blood osmolality and ion concentrations conform to those of the ambient medium, and 10−3 M-acetazolamide had only slight effects on the blood acid-base status and blood ion concentrations (Fig. 4). There was no significant difference among mean osmolality values over the time course (P> 0·40, F test). Sodium and chloride concentrations were depressed by less than 5 % of resting values, but again, the concentrations of the two ions were affected different At 6 and 12 h, blood Cl− was lowered 5 ± 1 and 20 ± 4mm, while the differences for Na+ were only 2 ± 0·5 and 12 ± 3 mm.
Blood pH in the acetazolamide-injected animals did not differ from that in the controls over the initial 6 h, but it was slightly below control values at 12 and 24 h (Fig. 4). Total CO2 increased significantly (P < 0·05, t test) by almost 3 mm over 12 h, but then returned to control values between 24 and 48 h. The perturbation in blood acid-base status occurred concomitantly with the disruption in the pre-injection Na+–Cl−difference, as was the case in the low salinity acclimated animals, but the effects of an acetazolamide injection in the seawater-acclimated crabs were slight compared to those in blue crabs acclimated to low salinity (Figs 2, 3, 4).
Blood acid-base parameters: Gecarcinus
A more typical respiratory acidosis took place in G. lateralis during the inito period after a 10−4 or 10−3 M-acetazolamide injection, with blood increasing 2–7Torr, respectively, over the first 6h (Fig. 5). Blood pH fell approximately OT units and CT rose about 3 mm over the first 3 h. By 12 h, dropped about 2 Torr, but CT had increased another 2mm and blood pH was restored to resting values regardless of drug dosage (Fig. 5). By 96 h, CT had more than doubled, and blood pH also increased to greater than resting values despite the fact that also remained elevated.
Mortality was a factor in this series of experiments, since with 10−4 or 10−3 M-etazolamide 40 % of the crabs had died by 96 h, and 100 % by 7 days. Those animals used in the determination for 10−3 M-acetazolamide all died by 48 h. The high rate of mortality was not seen in the animals treated with 10−6 M acetazolamide, but neither were the effects on pH and CT
Blood osmotic and ionic parameters: Gecarcinus
The effect of 10−4 M-acetazolamide on blood Na+, Cl− and Ca2+ was to increase their concentrations and therefore also increase blood osmolality (Fig. 6). The increase in blood Na+ from 515 ± 17 to 655 ± 14 mm was greater than that for Cl− (490 ± 12 to 585 ± 18mm)between24and 96h, while blood Ca2+ rose from20±0 ·5to 27 ±0 ·8mm (mean ± S.E. n = 6). The increase in the blood concentrations of the major ionic Secies in G. lateralis corresponded to the increase in the HCO3− concentration and the rise in pH (Fig. 5). Both the ionic and acid-base parameters failed to return to restint values by 96 h, and the high rate of mortality made it impossible to follow the expeiment beyond that time.
For blue crabs acclimated to 250mosM, the inhibition of CA by 10 −4M acetazolamide had no effect on either oxygen uptake or carbon dioxide ex cretion Neither an injection of Cortland saline nor acetazolamide caused any significant change in the resting values of or (P > 0 · 05, t test ; Table 1 ). The values for RQ were high, being around 2 (Table 1), but they are typical of aquatic crustaceans (Bosworth, O’Brien & Amberson, 1940). The RQ did not change sig nificantly over the time course of the experiment (P> 0 ·05 t test), with the exception of one high value of 2 ·62 at 48 h.
A similar pattern was seen in G. lateralis. An injection of 10 −4 M-acetazolamide did not significantly alter and also did not significantly lower (P> 0 ·05, t test; Table 1). RQ values of slightly less than 1 were typical of air-breathing animals in general, and with the exception of one high value at 24 h, they did not vary significant ly throughout the experiment (P> 0 ·05, t test; Table 1).
Inhibition of gill CA by acetazolamide
There is a lag of about 4 h between the time of an injection of acetazolamide and the time of 95 % inhibition of the gill enzyme (Fig. 1), which is probably due to the time needed for the drug to cross the basal membrane of the gill and effect inhibition. The delay in action of acetazolamide has been until now undocumented. In humans an injection of acetazolamide takes only minutes to block the activity of the red cell enzyme (Maren, 1967). This difference probably explains the negative results repor ted in previous studies of the effects of acetazolamide. Cameron (1979a) found no change in blood pH, CT and Cl− ion flux in C. sapidus after a Diamox injection, but he only waited 1-2 h before taking the measurements. Similarly, Aldridge & Cameron (1979) reported no change in blood pH or CT at 3 and 6h after injection; in neither study was CA activity monitored, thus making it impossible to confirm whether or not there was a significant degree of inhibition. In contrast, after 12 h of exposure to acetazolamide Burnett et al. (1981) did see an effect on pH, CT and Cl− ion concentration in the blood of P. crassipes.
The drug is also cleared rather slowly from the two decapod species, compared to mammals. The inhibition does not begin to wear off until between 24 and 48 h in the crabs, whereas the half time of the drug in rabbit plasma is only 100 min (Maren, 1967), with over 90% being recovered in the animal’s urine. The mechanism of clearance was not investigated, but it probably involves diffusive loss, excretion, and metabolic breakdown.
For the blue crab the correlation between the time courses of CA inhibition (Fig. 1) and those for the changes in blood pH, CT, Na+ and Cl− concentrations (Figs 2, 3) is very good, showing that when the enzyme is indeed inhibited there are specifiy effects. This emphasizes the importance of monitoring enzyme activity in order M ensure that the inhibitor is effective. It should be mentioned that caution should be used in comparing enzymological data obtained in vitro with in vivo blood measurements, but in this case the two appear to be compatible.
That a dose-dependent response of the physiological parameters to the drug was seen indicates that not all of the enzyme is inhibited by a 10 ·5 or 10 −4 M concentration in blood (Figs 2, 3, 4). It is possible that in C. sapidus, regardless of the amount of drug used, some of the branchial enzyme (5 –10 %, Fig. 1) remained functional. That would explain why, although blood ion concentrations were severely depressed, they always remained above those in the ambient medium. Carbonic anhydrase is one of the most efficient enzymes known and, at least in the vertebrates, as little as 0 ·1 % of normal activity is enough to maintain certain physiological functions (i.e., CO2 excretion) (Maren, 1967). This may also be the case in the blue crab because, although ion-and osmoregulatory functions were severely cut back, they were never completely eliminated.
CO2excretion in C. sapidus
The inhibition of branchial CA by acetazolamide had no significant effect on either blood (Fig. 2) or whole-animal CO2 excretion (Table 1 ). These results contradict the suggestion of Woodson, Burnett, Cullinan & Alberico (1980) and Burnett et al. (1981) that gill CA is important in CO2 excretion in another aquatic crab, P. crassipes. The rise in blood pH is also opposite from what one would expect (i.e., a respiratory acidosis) if an inhibition of CA were retarding CO2 excretion (Fig. 2).
Interestingly, Burnett et al. (1981) also saw a rise in pH along with elevated total CO2 (CT) and lowered blood Cl−, as is the case in the blue crab. Without the data for and CO2 excretion, however, they interpreted the rise in CT ( = HCO3− at physiological pH) as resulting from the inability of the animal to excrete its CO2. The same results can just as easily occur in a metabolic alkalosis (see below).
If CA is not required for CO2 excretion, then the process must be a passive one, depending on the diffusion of CO2 from blood to water along its gradient. This is possible even though blood is relatively low in aquatic crabs (2 –3 Torr) because of the high solubility of CO2 in water (Dejours, 1975). It has also been suggested that a disequilibrium in the blood, resulting from the absence of blood CA, could increase the gradient at the site of excretion, the gills (Cameron, 1979b; R. P. Henry & J. N. Cameron, unpublished observations).
Also, it is unlikely that blood HCO3− is the major species of CO2 which is excreted, as is the case in the vertebrates, since the basal membrane of the gill is generally considered to be impermeable to HCO3−, and in the hypothesis of Randall & Wood (1981), the transport of HCO3− from blood into the gill is the critical step in the process. Crustacean blood contains no CA activity (Henry & Cameron, 1982a) thus limiting the conversion of plasma HCO3− to CO2 to the slow, uncatalysed rate which is much slower than the transit time of blood in the gill (Cameron, 1979b). It is possible that membrane-bound CA exposed to plasma would facilitate CO2 excretion via the catalysed dehydration of plasma HCO3− to CO2, but this is unlikely since Cl−is a strong competitive inhibitor of CA (Maren, 1967 ; Henry & Cameron, 1982a), and blue crab blood has 250mm-Cl− vs 10mm-HCO3−.
Carbonic anhydrase function in crabs 219
The intracellular location of the enzyme also presents problems in CO2 excretion, Gutknecht, Bisson & Tosteson (1977), using an in vitro system, have shown that HCO3− diffusion across a membrane is limited by the diffusion of HCO3− through the unstirred layer at the surface of the membrane, a process which is very slow. CA on the blood side of the membrane (the typical vertebrate condition) would facilitate HCO3− diffusion by catalysing the dehydration reaction, thus keeping a high Pcoi in the unstirred layer; diffusion of HCO3− would actually take place as CO2. CA on the intracellular side of the membrane (the invertebrate condition) would have no effect on HCO3− diffusion from the blood.
Acid-base balance and ion regulation in C. sapidus
The acid-base disturbance that is caused by the inhibition of branchial CA by acetazolamide is typical of a metabolic alkalosis characterized by elevated blood pH and HCO3− at constant (Fig. 2). At the same time that acid-base status is disrupted, blood Na+ and Cl− concentrations are significantly lowered (Fig. 3); Cl− is lowered proportionally more than Na+ in every case, with the greatest difference between the concentrations of these two ions occurring together with the largest increase in pH and HCO3−. It appears that acid-base balance in the low salinity acclimated blue crab is linked to the ion regulatory process and, more specifically, to the difference in Na+-Cl−.
There is a large body of evidence supporting the hypothesis that Na+ and Cl− regula tion in both fish and crabs is accomplished by Na+/H+ (or NH4+) and Cl−/OH− (or HCO3−) exchanges (see reviews by Kirschner, 1979; Heisler, 1980). The exchanges have been considered to be independent of each other (Garcia Romieu & Maetz, 1964), and the relative rates of Na+/H+ and C1−/HCO3− exchanges which control the con centrations of the two major ions in the blood have been shown to be sensitive to blood acid-base disturbances (Truchot, 1975, 1979; Cameron, 1976, 1978b).
The inhibition of CA by acetazolamide has also been shown to depress both Na+ and Cl− uptake in various animals (Payan & Maetz, 1973; Dietz, 1974; Garcia Romieu & Ehrenfeld, 1975; Alvarado, Dietz & Mullen, 1975), so it is not unreasonable to suggest that the depression in blood Na+ and Cl− concentrations by acetazolamide in C. sapidus is a result of the inhibition of the Na+/H+ and Cl−/ HCO3− exchanges. Payan & Maetz (1973) reported that, although Na+ uptake was depressed by acetazolamide, Na+ efflux remained unchanged. With only the uptake mechanisms impaired by the drug, it appears that the animal ‘leaks’ ions into the medium thus reducing their concentrations in the blood. Cameron (1978a) has shown that passive Cl− efflux in the blue crab is about 30 % greater than that for Na+, so if the uptake of both ions is affected equally by CA inhibition the higher rate of passive Cl− efflux could explain why blood Cl− was lowered more than Na+.
Regardless of the cause, the effect of acetazolamide is to increase the Na+–Cl−difference in the blood. This may result in an increase in the strong ion difference (SID) in blood (Stewart, 1978). The observed change in the Na+-Cl− difference is in the direction that would be expected to produce an alkalosis resulting from an increase in HCO3− concentrations which would offset the relative increase in positive charge. The largest increase in blood HCO3− occurs at the same time as the largest lange in the Na+-Cl− difference, but only makes up about 25 % of the change. The complete SID was not measured; in fact, to do so is impossible. Since other strong ions were not measured, the change in the Na+-Cl− difference may hare overestimated the change in the total SID, and the rise in HCO3− may have actually accounted for more of the charge difference. Presumably changes in other strong ions (Ca2+, or K+, e.g.), total weak acid (mostly protein), or ion activity account for the rest of the apparent charge difference.
The same general patterns of blood ion and acid-base disturbance in response to 10−3 M-acetazolamide are seen in the high salinity acclimated blue crab, but to a much lesser degree. At that salinity (865 mosM) C. sapidus is an osmoconformer, its blood ion concentrations, for the most part, passively reflecting those in the ambient medium. The very small change in the Na+-Cl− difference (˜8 mm) and the equally small rise in HCO3− (˜3 mm) most probably represent baseline levels of the active ion exchange processes which, although not needed for ion regulation, are used in controlling the acid-base status of the blood. Such a mechanism would be employed to restore normal acid-base status of the blood following a disturbance such as an acidosis resulting from strenuous exercise (e.g., by swimming) (Cameron, 1978b).
The role of branchial carbonic anhydrase
It is unlikely that branchial CA plays a direct role in ion uptake in the blue crab, since the actual transport process is believed to be handled by membrane-bound Na+/ K+ (NH4+) and CK/HCOs− ATPases (Kerstetter & Kirschner, 1974; Towle et al. 1976; Magnum & Towle, 1977; DePew & Towle, 1979). Na+ and Cl− transport are considered to be independent of each other (reviewed by Kirschner, 1979) but both are affected by the CA inhibitor acetazolamide. The most plausible way for CA to play a role in ion regulation without being directly responsible for ion transport is for it to be involved in providing the counter-ions (HCO3− and H+) for the exchanges via the intracellular hydration of respiratory CO2 (Fig. 7), an hypothesis, that is similar to the model of Kirschner (1979) for fish. This model shows the proposed mechanisms of Na+ and Cl− uptake and movement into the blood and the involvement of branchial CA (for a detailed discussion of the transport mechanisms see Kirschner, 1979). CO2 is shown as diffusing from blood to water down its gradient as discussed above.
Since branchial CA is involved in supplying counter ions for the ion transporting enzymes which maintain Na+ and Cl− concentrations in the blood, and since a change in the concentrations of these ions appears to change the SID and thus to alter the acid-base status of the blood, gill CA is also involved in blood acid-base regulation, although in an indirect manner. It appears, then, that the role of branchial CA is primarily non-respiratory, in the sense that the enzyme has little or no function in the respiratory process of CO2 excretion.
Respiration and acid-base balance in G. lateralis
The role of branchial CA in the terrestrial crab, G. lateralis, is less clear. Although there is a clear respiratory acidosis during the early stages of inhibition of the gill enzyme (Fig. 5), there is no significant lowering of CO2 excretion (Table 1). MCOJ is actually elevated at 24 h after an injection of the inhibitor. This is the same pattern as seen in the semi-terrestrial crab, Cardisoma carnifex by Randall & Wood (1981). Thus, although inhibition of CA causes an increase in the actual process CO2 excretion appears to be occurring via diffusion down the gradient from blood to air. The increase in blood at 24 h might explain the increased CO2 excretion value at that time, but this is highly speculative since was also elevated at 6 and 12 h while was not. The elevation of blood may indicate a respiratory function for the branchial enzyme, but its activity does not seem to be critical to normal CO2 excretion.
The apparent respiratory acidosis was virtually fully compensated after 12 h by a rise in blood HCO3− (Fig. 5), but HCO3− concentrations continued to rise through the duration of the experiment. By 96 h HCO3− concentrations were almost three times resting values, and blood pH was also elevated for the 10−4 M-acetazolamide injection.
Concomitant with the rise in blood HCO3− was an elevation in blood Na+, Cl− and Ca2+ concentrations (Fig. 6). The steepest rise, 24–96 h, coincided with the onset of mortality in these animals. Na+ increased to a greater degree than did Cl−, thus altering the resting Na+-Cl− difference and presumably also the SID. The continued increase in blood HCO3− may also involve the increased Na+-Cl− difference, but even so, it did not entirely make up the charge difference.
The increase in the concentrations of the blood ions indicates that branchial CA is involved in ion regulation, in this case probably to combat desiccation. Without the functioning enzyme the crab appears to dry out: blood osmolality increases by over 300mosM and the animal dies. If this is so, then the direction of ion transport in G. lateralis is outward, the opposite direction to that in C. sapidus. Very little is known about the mechanism of outward ion transport (reviewed by Kirschner, 1979), and there is not enough information either to propose a mechanism or to propose a role of branchial CA in the process.
The increase in blood Ca2+, which parallels that of HCO3−, is indicative of yet third potential role of Ca in the terrestrial crab. Integument CA in crabs is believed of function in laying down and calcifying the exoskeleton (Giraud, 1981), and with the enzyme inhibited, exoskeletal CaCO3 could dissolve, elevating blood Ca2+ and HCO3” concentrations. This is plausible since the exoskeleton has been suggested to be an important source of blood buffers in crabs (deFur, Wilkes & McMahon, 1980; Henry, Kormanik, Smatresk & Cameron, 1981).
The function of CA in the terrestrial G. lateralis appears to be complex, in the respect that there is evidence that the gill enzyme is involved in the regulation of both blood and ion concentrations and that CA in the integument may be needed for CaCO3 deposition in the shell. An injection of acetazolamide inhibits CA throughout the entire animal, and the responses of these three areas are superimposed upon each other. More research is needed to clarify the function of the gill enzyme and to separate the responses of CA in other tissues in this species.