The Role of Carbonic Anhydrase in Blood Ion and Acid-Base Regulation 1

Transcription

1 AMER. ZOOL., 24: (1984) The Role of Carbonic Anhydrase in Blood Ion and Acid-Base Regulation 1 RAYMOND P. HENRY 2 Department of Physiology, G4, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania SYNOPSIS. The role of carbonic anhydrase (CA) in ion transport processes of aquatic and terrestrial arthropod species is reviewed. In both insects and crustaceans CA is found in a variety of ion transporting tissues. The bulk of CA activity in crustaceans is concentrated in the posterior gills, which are morphologically and biochemically adapted for ion transport. The enzyme can be specifically localized to gill lamellae which contain large populations of salt transporting chloride cells. Enzyme activity in the posterior gills of species having the ability to regulate blood ion concentrations increases when these organisms are acclimated to environmental salinities in which they ion regulate. In stenohaline, ion conforming species branchial CA activity is uniformly low, being only 5 10% that in regulating species. Studies on the blue crab, Calhnectes sapidus, using the specific CA inhibitor acetazolamide have shown that the enzyme is indeed important in blood ion regulation. Blood Na + and Cl~ concentrations are both severely lowered in drug-treated animals acclimated to low salinity, while they remain virtually unaffected in animals acclimated to high salinity, in which the animal is an ion conformer. High salinity acclimated crabs treated with acetazolamide do not survive transfer to low salinity, and mortality is related to a breakdown in the ion regulatory mechanism. Branchial CA most likely functions in the hydration of respiratory CO 2 to H + and HCO,~, which serve as counterions for the active uptake of Na + and Cl~, respectively. In terrestrial species the role of CA is unclear and merits further investigation. INTRODUCTION Since its discovery in 1932 by Meldrum and Roughton, the enzyme carbonic anhydrase (CA) (E.C ) has been the subject of intense study. Most of the work, however, has been concentrated on the mammalian erythrocyte enzyme. As a result, much is known about the physiological, biochemical and physical-chemical properties of red cell CA (for reviews see Maren, 1967; Lindskog etai, 1971;Nahas and Schaefer, 1974; Wyeth and Prince, 1977; Bauer et ai, 1980). Carbonic anhydrase catalyzes the reversible hydration/ dehydration reaction of CO 2 and water, as shown below (for a detailed discussion of the reaction mechanism see Coleman, 1980). CA CO 2 + H 2 O ~ H + + HCO 3 - ^^ H 2 CO 3 *^ In the red cell the enzyme functions primarily in CO 2 transport and excretion, as 1 From the Symposium on Cellular Mechanisms of Ion Regulation in Arthropods presented at the Annual Meeting of the American Society of Zoologists, December 1982, at Louisville, Kentucky. 2 Present address: Department of Zoology, 101 Cary Hall, Auburn University, Auburn, Alabama shown in Figure 1. CO 2 from respiring tissue enters the plasma and diffuses into the red cell where it is rapidly hydrated to H + and HCO 3 ". The H + ion is buffered by intracellular hemoglobin while HCO 3 " is transported back to the plasma in exchange for Cl~. Thus the bulk of CO 2 transported by the blood is in the form of plasma HCO 3 ". At the site of gas exchange, the lung, the series of reactions takes place in reverse and CO 2 is excreted in the gas form. Erythrocyte CA thus allows the large pool of otherwise slow reacting plasma HCO 3 " to be utilized in CO 2 excretion. This well-known scheme is inapplicable to the arthropods, however, for the reason that with one known exception (the larval form of Chironomous is unique by having both CA and a respiratory pigment in its blood) (Roeder, 1953), these organisms (and most other invertebrates as well) lack both circulating erythrocytes and blood CA activity (Levenbrook and Clark, 1950; Buck and Friedman, 1958; Waterman, 1960; Burnett et ai, 1981; Henry and Cameron, 1982a). But because the products of the hydration reaction are charged ionic species it has long been recognized that carbonic anhydrase could play an important role in various ion transport processes: that of

2 242 RAYMOND P. HENRY HMHCO; ;co 9 3 (fast) * FIG. 1. Carbonic anhydrase-catalyzed CO 2 reactions in the vertebrate erythrocyte. generating counterions (i.e., H + and HCO 3 ~) for the ionic species being transported. Ion transport mechanisms in the arthropods have been studied in relation to primarily two physiological processes. Aquatic species (crustaceans) which can invade estuarine waters of low salinity possess the ability to regulate blood ion concentrations significantly above those in the ambient medium (reviewed by Kirschner, 1979). Na + and Cl~, which make up over 90% of the total blood ions, are actively transported from the medium against an electrochemical gradient to the blood by the gills. There is a large body of evidence supporting the hypothesis of Na + /H + (or NH + 4 ) and C1VHCO 3 ~ exchange across the branchial, and thus there is good reason to suspect that gill CA may be involved. The second process occurs in terrestrial arthropods which are faced with an entirely different problem, that of combatting water loss. Ion transport in relation to water reabsorption has been more thoroughly studied in the insect species (reviewed by Phillips, 1981) although terrestrial crustaceans appear to have equally efficient mechanisms (Bliss, 1968). The reabsorption of water in the insect hindgut and rectum is accomplished by the active transport of Na +, K + and Cl" from the urine. Again, H + and HCO 3 " generated from the catalyzed hydration of CO 2 and water could function as counterions. This review will deal with the potential contribution of carbonic anhydrase to ion transport processes in arthropods, specifically the crustaceans and insects. The distribution of the enzyme and its biochemical properties will be discussed in relation to its function in ion regulation. Also, more direct evidence from physiological studies using CA inhibitors will be presented. DISTRIBUTION OF CARBONIC ANHYDRASE Carbonic anhydrase was initially discovered in arthropods almost 50 yr ago (Ferguson et at., 1937) and since that time the enzyme has been found in a wide variety of aquatic and terrestrial species. A summary of much of the purely documentary work, including the species, the tissue where the enzyme was detected and the assay method used, is presented in Table 1. Carbonic anhydrase appears to be fairly ubiquitous, being found in a number of different tissue types. Among the insect species CA activity has been found in ion transporting epithelia as well as in other tissues. The presence of the enzyme has also been documented histochemically in Malpighian tubules of two species of praying mantis (Polya and Wirtz, 1965). Despite the relatively large number of species examined, information on the distribution of CA in the insects remains rather superficial. There are a number of reasons for this. First, although many species have been examined, the work has been concentrated on only a few tissues. Some of the earlier research was done on general body parts such as head and abdomen, with no attempt made to separate individual tissues. Second, a good deal of the data on enzyme activity has been obtained using the manometric "boat" assay; this technique was abandoned quite some time ago because of lack of sensitivity resulting from the reaction being limited by diffusion of CO 2 and not by the catalyzed production of CO 2 (see Davis [1963] for a detailed discussion of CA assay methods). Furthermore, most of the data on insects in Table 1 was originally reported in a semi-quantitative manner (as the ratio of the catalyzed to the uncatalyzed

3 CARBONIC ANHYDRASE FUNCTION IN ARTHROPODS 243 reaction rates) making it difficult to compare levels of enzyme activity either among different species or among different tissues within a single species. Given these facts, it would be useful for the distribution of carbonic anhydrase in insects to be reexamined in a systematic, quantitative manner under standard conditions of temperature, ph, ionic strength and buffer capacity, all of which affect the CO 2 reaction. The distribution of CA in the crustaceans is somewhat similar to that in insects insofar as enzyme activity appears to be found in a variety of tissues, but seems to always be present in high levels in those tissues responsible for ion transport (i.e., gills, Table 1). Recent work by Henry and Cameron (1982a) has shown that not only do the gills possess the bulk of CA activity in the animal, but also that enzyme activity is distributed heterogenously among the individual gill pairs, and it is dependent upon environmental salinity (Table 2). In crabs typically having from 7 to 9 gill pairs, the posterior 2 to 3 gill pairs have substantially more CA activity than do the anterior gills. Among the species examined this difference occurs only in those organisms capable of regulating blood ion concentrations. The gills in such species (e.g., C. sapidus) are morphologically distinct; the posterior gills possess dense populations of salt transporting "chloride cells" which are absent in the anterior gills (Copeland and Fitzjarrell, 1968; Aldridge and Cameron, 1979). The anterior gills are believed to function strictly in gas exchange while the posterior gills have a mixed respiratory and ion regulating function. Pequeux and Gilles (1981), using isolated perfused gills from the crab Enocheir sinensis, demonstrated this functional difference by showing that Na + fluxes in the anterior gills were entirely passive, while those in the posterior gills were responsible for net Na + influx. The anterior-posterior difference in branchial CA activity is greatest when C. sapidus is osmo- and ion regulating. It is of interest to note that this difference is not found in a stenohaline osmo- and ion conforming species (L. emarginata), and the values for branchial CA activity in this species are only about 5% of the maximum values in C. sapidus (Table 2). The heterogenous distribution of branchial CA is also seen in a terrestrial crab, G. lateraiis. The smaller anterior-posterior difference, which is due to the anterior gills having higher enzyme activity than those in C. sapidus, is correlated with the presence of chloride cell patches in all of the gills; the posterior gills having larger patches (Copeland, 1968). Branchial carbonic anhydrase activity can be further localized in C. sapidus as a result of a unique feature of the fifth gill pair. In low salinity acclimated animals the posterior lamellae of the fifth gill possess a large patch of chloride cells which is absent from the anterior lamellae (Aldridge, 1977; Henry and Cameron, 1982a). From a crude dissection along the midline of the fifth gill, separating the anterior and posterior lamellae, Henry and Cameron (i982a) showed that the posterior lamella contains about 75% of the total CA activity in the gill (Table 3). The pattern of distribution of branchial CA is remarkably similar to that of another gill enzyme which is known to be important in ion transport: the Na + /K + -ATPase (Towle, 1981). In the blue crab ATPase activity is also found to be concentrated in the posterior gills, and activity increases proportionally more in these gills during the animal's transition from ion conformity to regulation. Furthermore, this enzyme has also been localized to the area of the lamellae containing dense patches of chloride cells (Neufeld et ai, 1980). The distribution of CA in arthropod tissues suggests that the enzyme is indeed involved in ion transport process. Its presence in ion transporting tissues such as insect midgut and Malpighian tubules, and crustacean gills is a strong indication that the enzyme plays a role in blood ion regulation. In crustaceans carbonic anhydrase has been found in greatest abundance in the specific gills believed to be responsible for ion transport, and CA activity has been correlated with the presence of salt transporting chloride cells in the lamellae. Enzyme activity increases to a greater

4 K3 Spei les atimiis mnenas aih\giapsit\ aas.sipes dlttnu\ wiprm'isu.s inmirih ameriianu.s imiilus pohphemus.ihiiua rmarginata.nrdimima mrnifex.tndisnma guaiihitmi.illline/tes snpidus t'kircnius late rails TABLE 1. Tissue Cuticle Whole body Mantle Muscle Muscle Muscle Branchial Muscle Heart Antennal gland Branchial Stomach mucosa Muscle Heart Antennal gland Branchial Muscle Heart Branchial Carbonic anhydrase activity in various tissues of selected arthropod species. CA Activity 1.5 mmol COj/min mg Pro. 8.1 E.U./mg Pro.' 3.6 E.U. 2.3 E.U. 65 E.U./g 20 E.U./g 325 E.U./g 150 E.U./g 8 E.U./g 45 E.U./g 0.05 mmol CO 2 /min mg Pro E.U./mg Pro. 0 E.U./mg Pro. 0.8 mmol CO 2 /min mg Pro mmol CO 2 /min mg Pro mmol CO 2 /min mg Pro mmol CO 2 /min mg Pro mmol CO 2 /min mg Pro. 54 E.U./g 0 E.U./g 0.9 mmol CO 2 /min mg Pro mmol CO 2 /min mg Pro mmol COj/min mg Pro mmol CO 2 /min mg Pro mmol CO 2 /min mg Pro. 0.7 mmol COj/min mg Pro. 0.2 mmol CO 2 /min mg Pro. 0.3 mmol CO 2 /min mg Pro mmol CO 2 /min mg Pro. Method Colorimetric ApH Colorimetric ApH Reference Giraud, 1981 Burnett el al., 1981 Costlow, 1959 Ferguson et al., 1937 Ferguson et al., 1937 Ferguson et al., 1937 Henry and Cameron, 1982o Randall and Wood, 1981 Henry and Cameron, 1982o Sobotka and Kami, 1941 Henry and Cameron, 1982n Henry and Cameron, 1982o 50 < o z D y x

5 TABLE 1. Continued Species Tissue CA Activity Method Reference dlo/ilioin rernpia'' glnis urticae'' ietis bmssicae*' anessa u> h haleta bitcrphnln*' osmotnche patona 1 ' auduca ie.xla'' usca miisca usfu (lomeslmi apilla japonira enplanetn amerirana colopendm sp. Fat body Integument Head Abdomen Total body Abdomen Abdominal contents Head Fat body Gut Coxal muscle Body segments Poison claw 2.1 E.U./mg Pro. 0.2 E.U./mg Pro. 0.7 E.U./mg Pro. 0.5 E.U./mg Pro. 0.4 E.U./mg Pro. 0.5 E.U./mg Pro. 2 W.A. units 2 W.A. units 2 W.A. units 4.6 E.U./g 18 E.U./g 2.1 E.U. 5.5 E.U./g 18 E.U./g 2.1 E.U. 1.3 E.U. 1.8 E.U. 0.9 E.U E.U./g 1.25 E.U./g 28 E.U./g Turbeck and Foder, 1970 Turbeck and Foder, 1970 Turbeck and Foder, 1970 Turbeck and Foder, 1970 Turbeck and Foder, 1970 Turbeck and Foder, 1970 Johnston and Jungreis, 1979 Sobotka and Kann, 1941 Anderson and March, 1956 Sobotka and Kann, 1941 Anderson and March, 1956 Sobotka and Kann, 1941 ' E.U. (enzyme unit) usually expressed as (Vc Vu)/Vu where Vc = the catalyzed reaction rate and Vu = the uncatalyzed rate. For the specific assay onditions of ph, temperature and assay media see the original references. h Larval stage of development. > CS O Z n z i < D 73 C Z n 0 z H X O "0 0 D

6 246 RAYMOND P. HENRY TABLE 2. Carbonic anhydrase activity in three species of crustaceans from various habitats.* Species Environment mm mosm Na* C. sapidus (anterior) (posterior) Backfin muscle Antennal gland C. sapidus (anterior) (posterior) Backfin muscle Antennal gland L. emarginata (anterior) (posterior) C. laterahs (anterior) (posterior) Cheliped muscle Terrestrial Data summarized from Figures 4 8 in Henry and Cameron, 1982a. extent in the ion regulating gills under environmental conditions in which blood ion concentrations are regulated. In contrast, branchial CA activity is very low and distributed uniformly among the gills of a stenohaline, osmo- and ion conforming species. PROPERTIES OF ARTHROPOD CA Very little work has been done on the biochemical characteristics of arthropod CA even though such information would undoubtedly shed more light on the enzyme's physiological importance. A review of the few studies on arthropod CA indicates that not only may there be fundamental differences between it and the well-known vertebrate erythrocyte enzyme, TABLE 3. Localization of carbonic anhydrase activity m anterior (respiratory) and posterior (ion- and osmoregulatory) lamellae of gill number 5 of low salinity (250 mosm) adapted C. sapidus.* Specific activity (percent of total) Crab Anterior Posterior Mean ± SF ± 3 * Data from Henn and Cameron, 1982« ± 3 mosm Blood mm Na* Cl- ci CA Activity (pmol COj/ mm mg Pro.) 150 ± ± ± ± ± ± ± ± but there may also be differences between insect and crustacean CA as well. Branchial CA from two species of crustaceans (C. sapidus and G. lateralis) is inhibited by increasing concentrations of NaCl and activity of the dehydration reaction is inversely related to ph. The enzymes show a temperature optimum of about 25 C, approximately the temperature at which the animals are most active (Henry and Cameron, 1982). In contrast, carbonic anhydrase from the midgut of the larvae of the insect Hyalophora cecropia is stimulated by low concentrations of K 2 SO 4, KC1, KNO 3, KI and KBr (Turbeck and Foder, 1970). Also, CA from the midgut of Manduca sexta (clarva) appears to be stimulated by choline chloride and KC1, while the enzyme from fat body tissue and the integument seems to be insensitive (Johnston and Jungreis, 1979). Finally, a substantial amount of carbonic anhydrase from the midgut of H. cecropia appears to be particulate in nature (Turbeck and Foder, 1970). A complete biochemical characterization of crustacean gill CA is currently in progress in my laboratory. CARBONIC ANHYDRASE FLNCTION Crustacean gill CA was originally thought to be important in CO 2 excretion by virtue

7 CARBONIC ANHYDRASE FUNCTION IN ARTHROPODS 247 of its being found in high concentrations in the respiratory organ (Ferguson et al., 1937), and this idea still persists. The crustacean gill, in addition to being the organ of O 2 and CO 2 exchange, is also the site of blood osmo- and ion regulation and blood acid-base balance (Smith and Linton, 1971; Mangum and Towle, 1977; Cameron, 1978a, b; Truchot, 1978, 1979). Carbonic anhydrase has been studied in relation to all three physiological functions, the most common approach taken involving the use of one of the highly specific sulfonamide inhibitors (e.g., acetazolamide, or Diamox). Data from such studies appear to support a role for branchial CA in the processes of blood ion and acid-base regulation. In C. sapidus acclimated to 250 mosm salinity inhibition of branchial carbonic anhydrase by acetazolamide disrupts the animal's ability to maintain blood Na + and Cl~ concentrations at normal values above those in the ambient medium (Fig. 2). The reduction in blood ion concentrations (and therefore total blood osmolality) is dependent upon the dose of inhibitor used; the maximum effect was seen after 24 hr of exposure to the inhibitor at which time blood Na + and Cl" concentrations were depressed by 115 and 192 mm, respectively. The time course of blood ion reduction and recovery corresponds very nicely to that of branchial CA inhibition; following an injection of acetazolamide maximal inhibition is achieved between 12 and 24 hr, with 100% of enzyme activity being recovered by 96 hr (Henry and Cameron, 1983). By 96 hr blood ion concentrations are also restored to pre-injection control values. In contrast, acetazolamide has virtually no effect on blood osmolality and ion concentrations in blue crabs acclimated to high salinity (865 mosm) at which the animal is a conformer. Blood Na + and Cl" concentrations are lowered by less than 5% of control values, and total osmolality is not significantly altered (Fig. 3). The same results were also observed in another euryhaline crab, Pachygrapsus crassipes treated with Diamox. Blood Cl" concentrations were lowered between 40 and 75 maf in low salinity acclimated animals, but were '. 700 ^ _ s o ZOO ft T I M E I hr) FIG. 2. Prebranchial blood osmolality, Na + and Cl" concentrations in C. sapidus acclimated to 250 mosm prior to and after an injection of acetazolamide. Open circles/dashed lines represent saline-injected controls. Closed circles represent drug-treated animals, with concentrations of acetazolamide shown in the figure. Mean ± SE, n = 6. T = 22 C. From Henry and Cameron, unaffected by the drug in animals acclimated to full strength seawater (Burnett et al., 1981). When C. sapidus acclimated to 865 mosm are transferred directly to low salinity (250 mosm) blood ion concentrations reach new steady-state values by about 24 hr (Fig. 4). High salinity acclimated animals treated with acetazolamide (10" 4 M) fail to survive the transfer to low salinity, with about 80% mortality occurring by 48 hr, and 100% by 96 hr (Henry and Cameron, 19826). The failure to survive the transfer can be related to a breakdown in the ion regulatory process, as blood Na + and Cl" concentrations decline steadily in low salinity. By 48 hr blood Na + is 147 mm below control values and blood Cl" is 162 mm lower (Fig. 4). There is no effect of acetazolamide on either O 2 uptake or CO 2 production in C. sapidus (Henry and Cameron, 1983) which

8 248 RAYMOND P. HENRY X Q J 480 ut j 490 E 480 ^ o 800 E R ' TIME ( hr) FIG. 3. Prebranchial blood ionic and acid-base parameters for C. sapidus acclimated to 865 mosm prior to and after an injection of acetazolamide (10~ 5 M in blood). Open circles/dashed lines represent controls. Mean ± SE, n = 6. T = 22 C. From Henry and Cameron, reinforces the idea that the mortality resulted from the animal's inability to ion regulate. An interesting aspect of the effects of CA inhibition on blood ion regulation is that Cl~ concentrations appear to be lowered to a greater degree than Na + in both the acute stages of low salinity acclimation and in fully acclimated blue crabs. In acclimated animals the maximum difference (Na + -Cl~) is about 29 mm (Henry and Cameron, 1983). The increase in the Na + -Cl" difference results in a relative increase in positive charge in the blood, and probably reflects an increase in the overall strong ion difference (S.I.D.) (Stewart, 1978, 1981). This increase in positive charge is partially offset by an increase in blood HCO 3 ~ of 8 mm which results in a corresponding increase in blood ph of 0.25 units (Henry and Cameron, 1983). Thus, inhibition of branchial CA in the blue crab results in a breakdown of the ion regulatory mechanism accompanied by a blood alkalosis which is of nonrespiratory origin (blood Pco 2 remains constant during CA inhibition) ZOO 12 TIME FIG. 4. Prebranchial blood osmolality and ion concentrations for C. sapidus acclimated to 865 mosm and then transferred to 250 mosm. Open circles represent controls; solid circles represent animals given an injection of acetazolamide (10~ 4 M) prior to transfer. Mean ± SE, n = 6. T = 25 C. From Henry and Cameron, 1982ft. From these data a model of branchial CA function in ion transport and acid-base regulation can be constructed (Fig. 5). The enzyme is shown as functioning primarily in the hydration of CO 2 to H + and HCO 3 ", which serve as counterions in the uptake of Na + and Cl~, respectively. The products of the hydration reaction are continuously being drawn away and the enzyme is seen as scavenging respiratory CO 2 as it diffuses from the blood to the medium. The movement of Na + into the blood is believed to occur via a basolateral Na + /K + -ATPase, while Cl" is believed to move down an electrical gradient across the basal membrane of the gill (discussed in detail by Kirschner, 1979). According to this scheme inhibition of gill CA would inhibit the ion uptake mechanisms through the depletion of counterions; passive diffusion of Na + and Cl" from the blood to the medium would be unaffected. Studies on ion movements in crustaceans (hr)

9 CARBONIC ANHYDRASE FUNCTION IN ARTHROPODS 249 Fie. 5. A model of branchial carbonic anhydrase function in the osmo-regulating blue crab. Dashed lines represent movement by diffusion; solid lines represent some form of coupled transport. From Henry and Cameron, have shown that acetazolamide affects Na + and Cl~ fluxes differently. Net uptake of both ions is dramatically reversed by acetazolamide in the blue crab and the crayfish, Astacus leptodactylus (Ehrenfeld, 1974; Cameron, 1979). In both species Na + influx is inhibited by about 70% while passive Na + efflux is unaffected. The loss of blood Na + most likely results from simple "leaking" of Na + from the animal to the medium when the rate of Na + influx is reduced to below that for passive efflux. Net loss of blood Cl" is, however, a result of over a doubling of Cl" efflux which swamps the relatively slight (30-40%) increase in Cl" influx in acetazolamide-treated animals. It has been suggested that when HCO 3 ~ becomes limiting C1~/C1~ exchange substitutes for C1"/HCO 3 ", resulting in both the higher influx and efflux rates (Ehrenfeld, 1974). Certainly more work is necessary before the exact mechanisms of Na + and Cl~ transport are elucidated, but it appears that acetazolamide affects Na + and Cl" transport differently, resulting in a proportionally greater loss of blood Cl". Very little work has been done on the Na + and Cl" transport mechanisms in aquatic insects, but Stobbart (1971) suggested that the anal papillae of the larva of Aides aegypti are capable of active Na + and Cl" uptake, in part by Na + /H + and C1~/HCO 3 " exchange. The role of carbonic anhydrase has not been investigated in any detail in aquatic insects, however, and must at this time remain speculative. As mentioned above, terrestrial species of arthropods utilize ion transport as a means of conserving water in a dry habitat. In a fully terrestrial species of crab, G. lateralis, acetazolamide caused a dramatic increase in the concentrations of the major blood ions. Na + and Cl" increased by 150 mm and 95 mm, respectively, while total osmolality increased by over 300 mosm after 96 hr (Henry and Cameron, 1983). Mortality in these animals was high, 40% by 96 hr and 100% by 7 days, and although the conclusions are preliminary, it is possible that both the increase in blood ion concentrations and mortality resulted from the loss of tissue and hemolymph water. The bulk of the research done on ion transport in the terrestrial species has been performed on insects with the aim of explaining the mechanisms of water reabsorption used to prevent desiccation. It is beyond the scope of this discussion to review in detail all the existing data on insect ion regulation; rather this section will focus on data pertinent to carbonic anhydrase function while relying heavily on a recent review by Phillips (1981) for general background information on ion transport. Blood ion concentrations are controlled by selective reabsorption from the urine. Primary urine is formed by secretion in the Malpighian tubules, and although some ion reabsorption can occur in the lower sections of the tubules and the anterior hindgut, the rectum is the principal site of ion transport (Goh and Phillips, 1978; Phillips, 1981). Between 80 and 95% of Na + K + and water is recovered by the rectum and hindgut (Phillips, 1981). Water reabsorption occurs via osmosis which results from the transport of one or more of the following ions: K +, Na + and Cl" from the urine into the rectal (Goh and Phillips, 1978; Phillips, 1981). Haskell et al. (1965) have presented evidence that CA is involved in active K + transport by the midgut of the silkworm. High concentrations of the CA inhibitor cardrase (10" 3 M) inhibited the potassium current by 23% and 36% depending on whether the drug was applied to the lumen or the blood side of the isolated midgut.

10 250 RAYMOND P. HENRY Another inhibitor, hygroton, was without effect even at 10" 3 M; however, 10~ 4 M sodium sulfide (a competitive inhibitor of CA) caused a reversible 31% inhibition of the K + current, and 10~ 3 M resulted in an 87% inhibition. The authors suggest that H + transport may play a role in the generation of the short circuit current by the midgut. These results are not conclusive; the concentration of cardrase needed for even a small degree of inhibition was 10 5 times the Ki for the drug, and the observed inhibition may have been an artifact of the non-specific action of high concentrations of sulfonamides (Maren, 1977). Other studies appear to confirm that H + and/or HCO 3 ~ transport is involved in generating the short circuit current. Williams et al. (1978) demonstrated that Na +, K + and Cl" transport could not account for the short circuit current in the isolated locust rectum, and they proposed either the transport of H + to the lumen or of HCO 3 ~ to the hemolymph as being responsible. Carbonic anhydrase has been detected in the locust rectum, and acetazolamide (5 x 10~ 4 M) inhibits the short circuit current by 25-40% (Williams et al, 1978). CA in the rectal could be involved in generating H + for secretion, or in removing HCO 3 " that had been transported from the lumen, but the enzyme could not be involved in both processes simultaneously unless H + and HCO 3 ~ transport occurred separately in distinct areas of the rectum. Regardless, it appears that the role of CA is not analogous to its function in branchial epithelia since Cl" transport in the insect rectum does not occur via C1~/HCO 3 " exchange (Phillips, 1981). The precise role of CA in the overall process of ion and water reabsorption in insects will only become more clearly denned through further research. REFERENCES Aldridge, J. B Structure and respiratory function in the gills of the blue crab, Callinectes sapidus (Rathbun). M.A. Thesis, The University of Texas at Austin. Aldridge, J. B. and J. N. Cameron CO 2 exchange in the blue crab, Cnlhnecte\ wpidus (Rathbun). J. Exp. Zool. 207: Anderson, A D. and R. B. March Inhibitors of carbonic anhydrase in the American cockroach, Periplaneta americana (L.). Can.J. Zool. 34: Bauer, C, G. Gros, and H. Bartels. (eds.) Biophysics and physiology of carbon dioxide. Springer- Verlag, New York. Bliss, D. E Transition from water to land in decapod crustaceans. Amer. Zool. 8: Buck, J. and S. Friedman Cyclic CO 2 release in diapausing pupae. III. CO 2 capacity of the blood: Carbonic anhydrase. J. Ins. Physiol. 2: Burnett, L. E., P. J. Woodson, M. G. Rietow, and V. C. Vilicich Crab gill intra-epithelial carbonic anhydrase plays a major role in hemolymph CO 2 and chloride ion regulation. J. Exp. Biol. 92: Cameron, J. N. 1978a. NaCl balance in blue crabs, Callinectes sapidus, in fresh water. J. Comp. Physiol. 123B: Cameron, J. N Effects of hypercapnia on blood acid-base status, NaCl fluxes and trans-gill potential in freshwater blue crabs, Callinectes sapidus. J. Comp. Physiol. 123B: Cameron, J. N Effects of inhibitors on ion fluxes, trans-gill potential and ph regulation in freshwater blue crabs, Callinectes sapidus (Rathbun). J. Comp. Physiol. 133B: Coleman, J. E Current concepts of the mechanism of action of carbonic anhydrase. In C. Bauer, G. Gros, and H. Bartels (eds.), Biophysics and physiology of carbon dioxide, pp Springer- Verlag, New York. Copeland, D. E Fine structure of salt and water uptake in the land crab, Cecarcinus lateralis. Amer. Zool. 8: Copeland, D. E. and A. T. Fitzjarrell The salt absorbing cells in the gills of the blue crab, Callinectes sapidus Rathbun with notes on modified mitochondria. Z. Zellforsch. 92:1-22. Costlow, J. D Effects of carbonic anhydrase inhibitors on shell development and growth of Balanus improvisus Darwin. Physiol. Zool. 32: Davis, R. P The measurement of carbonic anhydrase activity. Methods of Biochemical Analysis 11: Ehrenfeld,J Aspects of ionic transport mechanisms in crayfish, Astacus leptodactylus. J. Exp. Biol. 61: Ferguson, J. K. W., L. Lewis, and J. Smith The distribution of carbonic anhydrase in certain marine invertebrates. J. Cell. Comp. Physiol. 10: Giraud, M Carbonic anhydrase activity in the integument of the crab, Carcinus maenas during the intermolt cycle. Comp. Biochem. Physiol. 69A: Goh, S. and J. E. Phillips Dependence of prolonged water absorption by in vitro locust rectum on ion transport. J. Exp. Biol. 72: Haskell, J. A., R. D. demons, and W. R. Harvey Active transport by the Cecropia midgut. I. Inhibitors, stimulants, and postassium transport. J. Cell. Comp. Physiol. 65:45-56.

11 CARBONIC ANHYDRASE FUNCTION IN ARTHROPODS 251 Henry, R. P. and J. N. Cameron. 1982a. The distribution and partial characterization of carbonic anhydrase in selected aquatic and terrestrial decapod crustaceans. J. Exp. Zool. 221: Henry, R. P. and J. N. Cameron Acid-base balance in the euryhaline blue crab, Callinectes sapidus, during acclimation from high to low salinity. J. Exp. Biol. 101: Henry, R. P. and J. N. Cameron The role of branchial carbonic anhydrase in respiration, ion regulation and acid-base balance in the aquatic crab, Callinectes sapidus and the terrestrial crab, Gecarcinus lateralis. J. Exp. Biol. 103: Johnston, J. W. and A. M. Jungreis Comparative properties of mammalian and insect carbonic anhydrases: Effects of potassium and chloride on the rate of CO 2 hydration. Comp. Biochem. Physiol. 62B Kirschner, L. B Control mechanisms in crustaceans and fishes. In R. Gilles (ed.), Mechanisms of osmoregulatwn in animals: Maintenance of cell volume, pp J. Wiley and Sons, New York. Levenbrook, L. and A. M. Clark The physiology of carbon dioxide transport in insect blood. II. The effect of insect blood on the rate of hydration of CO 2. J. Exp. Biol. 27: Lindskog, S., L. E. Henderson, K. K. Kannan, A. Liljas, P. O. Nyman, and B. Strandberg Carbonic anhydrase. In P. D. Boyer (ed.), The enzymes, Vol. V, pp Academic Press, New York. Mangum.C. P. and D. W. Towle Physiological adaptations to unstable environments. Am. Sci. 65: Maren, T. H Carbonic anhydrase: Chemistry, physiology and inhibition. Physiol. Rev. 47: Maren, T. H Use of inhibitors in physiological studies of carbonic anhydrase. Am. J. Physiol. 232:F Meldrum, N. U. and F. J. W. Roughton The CO 2 catalyst present in blood. J. Physiol. (London) 75:15. Nahas, G. and K. E. Schaefer. (eds.) Carbon dioxide and metabolic regulations. Springer-Verlag, New York. Neufeld, G. J., C. W. Holiday, and J. B. Pritchard Salinity adaptations of gill Na,K-ATPase in the blue crab, Callinectes sapidus. J. Exp. Zool. 211: Pequeux, A. and R. Gilles Na + fluxes across isolated perfused gills of the Chinese crab, Enocheir sinensis. J. Exp. Biol. 92: Phillips, J. E Comparative physiology of in sect rectal function. Am. J. Physiol. 241:R Polya.J. B.and A.J. Wirtz Studies on carbonic anhydrase. I. A review of recent investigations. Enzymol. 28: Randall, D.J. and C. M. Wood Carbon dioxide excretion in the land crab, Cardisoma carmfex. J. Exp. Zool. 218: Roeder, K. D Insect physiology. John Wiley and Sons, New York. Smith, D. S. and J. R. Linton Potentiometric evidence for the active transport of sodium and chloride across excised gills of Callinectes sapidus. Comp. Biochem. Physiol. 39A: Sobotka, H. and S. Kann Carbonic anhydrase in fishes and invertebrates. J. Cell. Comp. Phys. 17: Stewart, P. A Independent and dependent variables of acid-base control. Respir. Physiol. 33: Stewart, P. A How to understand acid-base. Elsevier, New York. Stobbart.R.H Evidence for Na + /H + and Cl"/ HCO S " exchanges during independent sodium and chloride uptake by the larva of the mosquito, Aedes aegypti (L.). J. Exp. Biol. 54: Towle.D. W Role of Na + + K + -ATPase in ion regulation by marine and estuarine animals. Mar. Biol. Lett. 2: Truchot, J. P Mechanisms of extracellular acid-base regulation as temperature changes in decapod crustaceans. Respir. Physiol. 33: Truchot, J. P Mechanisms of the compensation of blood respiratory acid-base disturbances in the shore crab, Carcinus maenas. J. Exp. Zool. 210: Turbeck, B. O. and B. Foder Studies on a carbonic anhydrase from the midgut of larvae of Lepidoptera. Biochem. Biophys. Acta 212: Waterman, T. H Physiology of Crustacea, Vol I. Academic Press, New York. Williams, D., J. E. Phillips, W. T. Prince, and J. Meredith The source of short-circuit current across locust rectum. J. Exp. Biol. 77: Wyeth, P. and R. Prince Carbonic anhydrase. Inorg. Perspect. Biol. Med. 1:37-71.

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