Download The Role of Carbonic Anhydrase in Blood Ion and Acid

AMER. ZOOL., 24:241-251 (1984)
The Role of Carbonic Anhydrase in Blood Ion and
Acid-Base Regulation1
RAYMOND P. HENRY 2
Department of Physiology, G4, University of Pennsylvania,
School of Medicine, Philadelphia, Pennsylvania 19104
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 CO2 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. 4-2-1-1) 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 CO2 and water, as
shown below (for a detailed discussion of
the reaction mechanism see Coleman,
1980).
CA
CO2 + H2O ~ H+ + HCO 3 ^ ^ H2CO3*^
In the red cell the enzyme functions primarily in CO2 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, 2730 December 1982, at Louisville, Kentucky.
2
Present address: Department of Zoology, 101 Cary
Hall, Auburn University, Auburn, Alabama 36849.
shown in Figure 1. CO2 from respiring tissue enters the plasma and diffuses into the
red cell where it is rapidly hydrated to H +
and HCO3". 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 CO2 transported
by the blood is in the form of plasma
HCO3". At the site of gas exchange, the
lung, the series of reactions takes place in
reverse and CO2 is excreted in the gas form.
Erythrocyte CA thus allows the large pool
of otherwise slow reacting plasma HCO3"
to be utilized in CO2 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
241
242
RAYMOND P. HENRY
HMHCO; •
3
(fast)
;co9
*
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
FIG. 1. Carbonic anhydrase-catalyzed CO2 reactions
in the vertebrate erythrocyte.
generating counterions (i.e., H + and
HCO3~) 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
NH4+) and C1VHCO3~ exchange across the
branchial epithelium, 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 CO2 and water could
function as counterions.
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 CO2 and
not by the catalyzed production of CO2 (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
CARBONIC ANHYDRASE FUNCTION IN ARTHROPODS
243
reaction rates) making it difficult to com- species are only about 5% of the maximum
pare levels of enzyme activity either among values in C. sapidus (Table 2). The heterdifferent species or among different tissues ogenous distribution of branchial CA is also
within a single species. Given these facts, seen in a terrestrial crab, G. lateraiis. The
it would be useful for the distribution of smaller anterior-posterior difference,
carbonic anhydrase in insects to be reex- which is due to the anterior gills having
amined in a systematic, quantitative man- higher enzyme activity than those in C.
ner under standard conditions of temper- sapidus, is correlated with the presence of
ature, pH, ionic strength and buffer chloride cell patches in all of the gills; the
capacity, all of which affect the CO2 reac- posterior gills having larger patches (Copetion.
land, 1968).
Branchial carbonic anhydrase activity can
The distribution of CA in the crustaceans is somewhat similar to that in insects be further localized in C. sapidus as a result
insofar as enzyme activity appears to be of a unique feature of the fifth gill pair. In
found in a variety of tissues, but seems to low salinity acclimated animals the postealways be present in high levels in those rior lamellae of the fifth gill possess a large
tissues responsible for ion transport (i.e., patch of chloride cells which is absent from
gills, Table 1). Recent work by Henry and the anterior lamellae (Aldridge, 1977;
Cameron (1982a) has shown that not only Henry and Cameron, 1982a). From a crude
do the gills possess the bulk of CA activity dissection along the midline of the fifth
in the animal, but also that enzyme activity gill, separating the anterior and posterior
is distributed heterogenously among the lamellae, Henry and Cameron (i982a)
individual gill pairs, and it is dependent showed that the posterior lamella contains
upon environmental salinity (Table 2). In about 75% of the total CA activity in the
crabs typically having from 7 to 9 gill pairs, gill (Table 3).
the posterior 2 to 3 gill pairs have substanThe pattern of distribution of branchial
tially more CA activity than do the anterior CA is remarkably similar to that of another
gills. Among the species examined this dif- gill enzyme which is known to be important
ference occurs only in those organisms in ion transport: the Na + /K + -ATPase
capable of regulating blood ion concentra- (Towle, 1981). In the blue crab ATPase
tions. The gills in such species (e.g., C. sapi- activity is also found to be concentrated in
dus) are morphologically distinct; the pos- the posterior gills, and activity increases
terior gills possess dense populations of salt proportionally more in these gills during
transporting "chloride cells" which are the animal's transition from ion conforabsent in the anterior gills (Copeland and mity to regulation. Furthermore, this
Fitzjarrell, 1968; Aldridge and Cameron, enzyme has also been localized to the area
1979). The anterior gills are believed to of the lamellae containing dense patches
function strictly in gas exchange while the of chloride cells (Neufeld et ai, 1980).
posterior gills have a mixed respiratory and
The distribution of CA in arthropod tision regulating function. Pequeux and Gilles sues suggests that the enzyme is indeed
(1981), using isolated perfused gills from involved in ion transport process. Its presthe crab Enocheir sinensis, demonstrated ence in ion transporting tissues such as
this functional difference by showing that insect midgut epithelium and Malpighian
Na+ fluxes in the anterior gills were entirely tubules, and crustacean gills is a strong
passive, while those in the posterior gills indication that the enzyme plays a role in
were responsible for net Na+ influx.
blood ion regulation.
In crustaceans carbonic anhydrase has
The anterior-posterior difference in
branchial CA activity is greatest when C. been found in greatest abundance in the
sapidus is osmo- and ion regulating. It is of specific gills believed to be responsible for
interest to note that this difference is not ion transport, and CA activity has been
found in a stenohaline osmo- and ion con- correlated with the presence of salt transforming species (L. emarginata), and the porting chloride cells in the lamellae.
values for branchial CA activity in this Enzyme activity increases to a greater
K3
TABLE 1.
Spei les
Tissue
Catimiis mnenas
Paih\giapsit\ aas.sipes
Cuticle
Gills
Whole body
Mantle
Gills
Muscle
Gills
Muscle
Muscle
Gills
Gills
Gills
Branchial
epithelium
Gills
Muscle
Heart
Antennal
gland
Branchial
epithelium
Stomach
mucosa
Gills
Gills
Muscle
Heart
Antennal
gland
Branchial
epithelium
Gills
Muscle
Heart
Branchial
epithelium
Bdlttnu\ wiprm'isu.s
Hinmirih ameriianu.s
Limiilus pohphemus
l.ihiiua rmarginata
C.nrdiMima mrnifex
C.tndisnma guaiihitmi
(.illline/tes snpidus
Ct'Kircnius late rails
Carbonic anhydrase activity in various tissues of selected arthropod species.
CA Activity
Method
Reference
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 CO2/min mg Pro.
0.27 E.U./mg Pro.
0 E.U./mg Pro.
Manometric boat
Electrometric ApH
Colorimetric ApH
Colorimetric ApH
Manometric boat
Manometric boat
Manometric boat
Manometric boat
Manometric boat
Manometric boat
pH Stat
Manometric boat
Manometric boat
Giraud, 1981
Burnett el al., 1981
Costlow, 1959
0.8 mmol CO2/min mg Pro.
0.02 mmol CO 2 /min mg Pro.
0.04 mmol CO2/min mg Pro.
0.06 mmol CO2/min mg Pro.
pH
pH
pH
pH
Henry and Cameron, 1982o
0.05 mmol CO2/min mg Pro.
pH Stat
54 E.U./g
Manometric boat
0 E.U./g
0.9 mmol CO2/min mg Pro.
0.15 mmol CO2/min mg Pro.
0.12 mmol COj/min mg Pro.
0.09 mmol CO2/min mg Pro.
Manometric boat
pH Stat
pH Stat
pH Stat
pH Stat
0.03 mmol CO2/min mg Pro.
pH Stat
0.7 mmol COj/min mg Pro.
0.2 mmol CO2/min mg Pro.
0.3 mmol CO2/min mg Pro.
0.15 mmol CO2/min mg Pro.
pH
pH
pH
pH
Ferguson et al., 1937
Ferguson et al., 1937
Ferguson et al., 1937
Henry and Cameron, 1982o
Randall and Wood, 1981
50
•<
Stat
Stat
Stat
Stat
Stat
Stat
Stat
Stat
o
z
D
y
x
Sobotka and Kami, 1941
Henry and Cameron, 1982n
Henry and Cameron, 1982o
TABLE 1.
Species
Hdlo/ilioin rernpia''
Aglnis urticae''
Pietis bmssicae*'
Vanessa u>
h
Phaleta bitcrphnln*'
Cosmotnche patona1'
Mauduca ie.xla''
Musca miisca
Musfu (lomeslmi
Papilla japonira
Penplanetn amerirana
Scolopendm sp.
Tissue
Midgut
epithelium
Midgut
epithelium
Midgut
epithelium
Midgut
epithelium
Midgut
epithelium
Midgut
epithelium
Fat body
Midgut
Integument
epithelium
Head
Abdomen
Total body
Abdomen
Abdominal
contents
Head
Fat body
Gut
Coxal muscle
Body segments
Poison claw
Midgut
CA Activity
Continued
Method
Reference
2.1 E.U./mg Pro.
pH Stat
Turbeck and Foder, 1970
0.2 E.U./mg Pro.
pH Stat
Turbeck and Foder, 1970
0.7 E.U./mg Pro.
pH Stat
Turbeck and Foder, 1970
0.5 E.U./mg Pro.
pH Stat
Turbeck and Foder, 1970
0.4 E.U./mg Pro.
pH Stat
Turbeck and Foder, 1970
0.5 E.U./mg Pro.
pH Stat
Turbeck and Foder, 1970
2 W.A. units
2 W.A. units
2 W.A. units
Electrometric ApH
Electrometric ApH
Electrometric ApH
Johnston and Jungreis, 1979
4.6 E.U./g
18 E.U./g
2.1 E.U.
5.5 E.U./g
18 E.U./g
Manometric boat
Manometric boat
Electrometric ApH
Manometric boat
Manometric boat
Sobotka and Kann, 1941
2.1 E.U.
1.3 E.U.
1.8 E.U.
0.9 E.U.
0.28 E.U./g
1.25 E.U./g
28 E.U./g
Electrometric ApH
Electrometric ApH
Electrometric ApH
Electrometric ApH
Manometric boat
Manometric boat
Manometric boat
Anderson and March, 1956
>
CS
O
Z
n
z
i
•<
D
Anderson and March, 1956
Sobotka and Kann, 1941
73
C
Z
n
0
z
H
X
O
"0
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
conditions of pH, temperature and assay media see the original references.
h
Larval stage of development.
0
D
246
RAYMOND P. HENRY
TABLE 2.
Carbonic anhydrase activity in three species of crustaceans from various habitats.*
Environment mM
Species
C. sapidus
Gills (anterior)
Gills (posterior)
Backfin muscle
Antennal gland
C. sapidus
Gills (anterior)
Gills (posterior)
Backfin muscle
Antennal gland
L. emarginata
Gills (anterior)
Gills (posterior)
C. laterahs
Gills (anterior)
Gills (posterior)
Cheliped muscle
Blood mM
mOsm
Na*
Cl-
mOsm
Na*
ci-
865
426
489
882
443
449
CA Activity
(pmol COj/
mm mg Pro.)
150 ± 45
230 ± 13
85
75
250
116
131
665
353
338
255 ± 15
838 ± 82
120
80
950
—
551
978
—
556
48 ± 10
59 ± 10
Terrestrial
769
381
378 ± 22
613 ± 37
200
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,
T A B L E 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
1
23.8
18.5
33.0
26.2
25.3 ± 3
76.5
81.5
67.0
73.8
74.7 ± 3
2
3
4
Mean ± SF
* Data from Henn and Cameron, 1982«.
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 epithelium of
the larvae of the insect Hyalophora cecropia
is stimulated by low concentrations of
K2SO4, KC1, KNO3, 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 epithelium 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 CO2 excretion by virtue
CARBONIC ANHYDRASE FUNCTION IN ARTHROPODS
247
of its being found in high concentrations
700
in the respiratory organ (Ferguson et al.,
1937), and this idea still persists. The crus'. ^50
tacean gill, in addition to being the organ
600
of O 2 and CO2 exchange, is also the site of
blood osmo- and ion regulation and blood
°
550
acid-base balance (Smith and Linton, 1971;
500
Mangum and Towle, 1977; Cameron,
1978a, b; Truchot, 1978, 1979). Carbonic
anhydrase has been studied in relation to _- 35 0
all three physiological functions, the most
300
6
common approach taken involving the use
o
250
of one of the highly specific sulfonamide
inhibitors (e.g., acetazolamide, or Diamox). 2
Data from such studies appear to support
a role for branchial CA in the processes of
350
blood ion and acid-base regulation.
-• 300
In C. sapidus acclimated to 250 mOsm
salinity inhibition of branchial carbonic
6
250
anhydrase by acetazolamide disrupts the
° ZOO
animal's ability to maintain blood Na+ and
ft 2 4 6
12
24
48
96
Cl~ concentrations at normal values above
TIME
I hr)
those in the ambient medium (Fig. 2). The
reduction in blood ion concentrations (and FIG. 2. Prebranchial blood osmolality, Na+ and Cl"
therefore total blood osmolality) is depen- concentrations in C. sapidus acclimated to 250 mOsm
to and after an injection of acetazolamide. Open
dent upon the dose of inhibitor used; the prior
circles/dashed lines represent saline-injected conmaximum effect was seen after 24 hr of trols. Closed circles represent drug-treated animals,
exposure to the inhibitor at which time with concentrations of acetazolamide shown in the
blood Na+ and Cl" concentrations were figure. Mean ± SE, n = 6. T = 22°C. From Henry
depressed by 115 and 192 mM, respec- and Cameron, 1983.
tively. The time course of blood ion reduction and recovery corresponds very nicely
to that of branchial CA inhibition; follow- unaffected by the drug in animals accliing an injection of acetazolamide maximal mated to full strength seawater (Burnett et
inhibition is achieved between 12 and 24 al., 1981).
hr, with 100% of enzyme activity being
When C. sapidus acclimated to 865 mOsm
recovered by 96 hr (Henry and Cameron, are transferred directly to low salinity (250
1983). By 96 hr blood ion concentrations mOsm) blood ion concentrations reach new
are also restored to pre-injection control steady-state values by about 24 hr (Fig. 4).
values.
High salinity acclimated animals treated
4
In contrast, acetazolamide has virtually with acetazolamide (10" M) fail to survive
no effect on blood osmolality and ion con- the transfer to low salinity, with about 80%
centrations in blue crabs acclimated to high mortality occurring by 48 hr, and 100% by
salinity (865 mOsm) at which the animal is 96 hr (Henry and Cameron, 19826). The
a conformer. Blood Na+ and Cl" concen- failure to survive the transfer can be related
trations are lowered by less than 5% of to a breakdown in+ the ion regulatory procontrol values, and total osmolality is not cess, as blood Na and Cl" concentrations
in low salinity. By 48 hr
significantly altered (Fig. 3). The same decline steadily
+
results were also observed in another eury- blood Na is 147 mM below control values
haline crab, Pachygrapsus crassipes treated and blood Cl" is 162 mM lower (Fig. 4).
with Diamox. Blood Cl" concentrations There is no effect of acetazolamide on
were lowered between 40 and 75 mAf in either O2 uptake or CO2 production in C.
low salinity acclimated animals, but were sapidus (Henry and Cameron, 1983) which
6
s
248
RAYMOND P. HENRY
900
9
800
700
7.80
600
7
500
X
Q.
-J
70
480
uT 4 70
5 460
400
j
E
^
490
480
470
300
460
200
400
o
800
E
R
2
4
6
'2
TIME
24
48
96
( 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, 1983.
300
ZOO
12
TIME
(hr)
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.
reinforces the idea that the mortality
resulted from the animal's inability to ion
regulate.
An interesting aspect of the effects of CA
From these data a model of branchial
inhibition on blood ion regulation is that CA function in ion transport and acid-base
Cl~ concentrations appear to be lowered to regulation can be constructed (Fig. 5). The
a greater degree than Na+ in both the acute enzyme is shown as functioning primarily
stages of low salinity acclimation and in in the hydration of CO2 to H+ and HCO 3 ",
fully acclimated blue crabs. In acclimated which serve as counterions in the uptake
animals the maximum difference (Na+-Cl~) of Na+ and Cl~, respectively. The products
is about 29 mM (Henry and Cameron, of the hydration reaction are continuously
1983). The increase in the Na+-Cl" differ- being drawn away and the enzyme is seen
ence results in a relative increase in positive as scavenging respiratory CO2 as it diffuses
charge in the blood, and probably reflects from the blood to the medium. The movean increase in the overall strong ion dif- ment of Na+ into the blood is believed to
ference (S.I.D.) (Stewart, 1978, 1981). This occur via a basolateral Na + /K + -ATPase,
increase in positive charge is partially offset while Cl" is believed to move down an elecby an increase in blood HCO3~ of 8 mM trical gradient across the basal membrane
which results in a corresponding increase of the gill (discussed in detail by Kirschner,
in blood pH of 0.25 units (Henry and Cam- 1979). According to this scheme inhibition
eron, 1983). Thus, inhibition of branchial of gill CA would inhibit the ion uptake
CA in the blue crab results in a breakdown mechanisms through the depletion of
of the ion regulatory mechanism accom- counterions; passive diffusion of Na+ and
panied by a blood alkalosis which is of non- Cl" from the blood to the medium would
respiratory origin (blood Pco 2 remains be unaffected.
constant during CA inhibition).
Studies on ion movements in crustaceans
CARBONIC ANHYDRASE FUNCTION IN ARTHROPODS
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, 1983.
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 HCO3~
becomes limiting C1~/C1~ exchange substitutes for C1"/HCO3", 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~/HCO3" exchange. The role of carbonic anhydrase has not been investigated
in any detail in aquatic insects, however,
and must at this time remain speculative.
249
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 epithelium (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.
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 105
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 HCO3~ 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
HCO3~ 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 epithelium 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 HCO3~
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~/HCO3" 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.
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