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Physiological Reviews, Vol. 80, No. 2, April 2000, pp. 681-715
Copyright ©2000 by the American Physiological Society
Zentrum Physiologie, Medizinische Hochschule, Hannover, Germany
I. INTRODUCTION
II. CARBON DIOXIDE TRANSPORT IN BLOOD
A. Transport Forms of CO2 in Blood
B. Transport Within the Intracellular Compartment
C. Transport Across the Erythrocyte Membrane
E. Interconversion Between CO2 and HCO3
E. Interconversion Between CO2 and Carbamate
III. CARBON DIOXIDE TRANSPORT IN MUSCLE
A. CO2 Production in Muscle
B. Localization of CA in Skeletal Muscle
C. Transport in the Intracellular Compartment
D. Transport Across Sarcolemma
E. Transport Across Capillary Walls
IV. KINETIC REQUIREMENTS OF THE PROCESSES INVOLVED IN ELIMINATION OF CARBON DIOXIDE AND LACTIC ACID FROM MUSCLE AND UPTAKE INTO BLOOD
A. Theoretical Model of CO2 and Lactic Acid Exchange in Muscle
B. Reactions Included in the Model and Their Mathematical Form
C. Permeability of the Capillary Wall to Lactate
D. Effect of CA at Different Localizations on Equilibration of Intravascular pH, CO2 Excretion, and Excretion of Lactic Acid
V. APPENDIX
A. Interstitial Concentrations of CO2, HCO3, H+, and Lactate
B. Concentration Changes Within Plasma and Erythrocytes
C. Volumes
D. Solutions
E. Indexes Used
F. Abbreviations and Values of Parameters Used
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ABSTRACT |
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Geers, Cornelia and
Gerolf Gros.
Carbon Dioxide Transport and Carbonic Anhydrase in Blood and
Muscle. Physiol. Rev. 80: 681-715, 2000.
CO2
produced within skeletal muscle has to leave the body finally via
ventilation by the lung. To get there, CO2 diffuses from
the intracellular space into the convective transport medium blood with
the two compartments, plasma and erythrocytes. Within the body,
CO2 is transported in three different forms: physically dissolved, as HCO3
, or as carbamate. The relative
contribution of these three forms to overall transport is changing
along this elimination pathway. Thus the kinetics of the interchange
have to be considered. Carbonic anhydrase accelerates the
hydration/dehydration reaction between CO2,
HCO3
, and H+. In skeletal muscle, various
isozymes of carbonic anhydrase are localized within erythocytes but are
also bound to the capillary wall, thus accessible to plasma; bound to
the sarcolemma, thus producing catalytic activity within the
interstitial space; and associated with the sarcoplasmic reticulum. In
some fiber types, carbonic anhydrase is also present in the sarcoplasm.
In exercising skeletal muscle, lactic acid contributes huge amounts of
H+ and by these affects the relative contribution of the
three forms of CO2. With a theoretical model, the complex
interdependence of reactions and transport processes involved in
CO2 exchange was analyzed.
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I. INTRODUCTION |
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One of the major requirements of the body is to eliminate CO2. The large, but highly variable, amount of CO2 that is produced within muscle cells has to leave the body finally via ventilation of the alveolar space. To get there, diffusion of CO2 has to occur from the intracellular space of muscles into the convective transport medium blood, and diffusion out of the blood has to take place into the lung gas space across the alveolocapillary barrier.
Carbon dioxide in the body is present in three different forms:
dissolved, bound as bicarbonate, or bound as carbamate. The relative
contribution of these different forms to overall CO2 transport changes markedly along this elimination pathway, because for
diffusion across membrane barriers, another form is more appropriate than for transport within intra- or extracellular compartments. Thus
the kinetics of the interchange between forms become critically important. In addition, the products of one such interchange, the
hydration reaction of CO2, HCO3
, and
H+, are required for a great variety of other cellular
functions such as secretion of acid or base and some reactions of
intermediary metabolism. In exercising skeletal muscle, the other
"end product" of metabolism, lactic acid, contributes huge amounts
of H+ and by these affects the predominance of the three
forms of CO2, because HCO3
as well as
carbamate are critically dependent on the concentration of
H+. Discussion of the overall transport of CO2
in skeletal muscle has to take into account this contribution of lactic
acid and its involvement in kinetics and equilibria of CO2
reactions. This interdependence of CO2 and lactic acid
elimination is one major aspect of this review, which as far as we know
has not before been reviewed in detail.
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II. CARBON DIOXIDE TRANSPORT IN BLOOD |
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A. Transport Forms of CO2 in Blood
Carbon dioxide transport forms in blood have been thoroughly reviewed by Klocke (105). We only briefly summarize their respective contribution to overall CO2 exchange. Table 1 sums up the contribution of the various forms in the two compartments plasma and erythrocytes: in whole arterial and venous blood during rest and during heavy exercise.
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1. Dissolved CO2
Only a small portion, ~5% of total arterial content, is present
in the form of dissolved CO2. Using a solubility
coefficient SCO2 of 3.21 × 10
5
M/Torr (35) for plasma at 37°C, this gives 1.28 mM
dissolved CO2, or, using SCO2 of
3.08 × 10
5 M/Torr (7), 1.23 mM
dissolved CO2 at a PCO2 of 5.32 kPa
(40 Torr). At rest, the contribution of dissolved CO2 to
the total arteriovenous CO2 concentration difference is
only ~10%. However, during heavy exercise, the contribution of
dissolved CO2 can increase sevenfold and then makes up
almost one-third of the total CO2 exchange.
2. CO2 bound as HCO3
The majority of CO2 in all compartments is bound as
HCO3
. The ratio of HCO3
over
dissolved CO2 is given by the Henderson-Hasselbalch
equation
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in total CO2 is
diminished. Although at pH 7.4 HCO3
is 20-fold
compared with dissolved CO2, it is only 13-fold at the
normal intraerythrocytic pH of 7.2, and the ratio may fall to much
lower values at plasma pH values of considerably below 7 during maximal
exercise. Therefore, although the absolute arteriovenous difference is
higher during exercise than during rest, the relative contribution of
HCO3
to overall exchange is less. For the
example of heavy exercise given in Table 1, HCO3
contributes only two-thirds of total CO2 exchange,
whereas at rest this figure is ~85%.
3. CO2 bound as carbamate
The amount of CO2 bound as carbamate to hemoglobin in erythrocytes or to plasma proteins depends on O2 saturation of hemoglobin and 2,3-diphosphoglycerate (2,3-DPG) concentration in the case of erythrocytes, and on H+ concentration in the case of both red blood cells and plasma (61, 68, 134, 135). During passage of blood through muscle, O2 saturation and H+ concentration change considerably, in particular during exercise. However, the increase in hemoglobin desaturation and the increase in H+ concentration experienced by red blood cells in the capillary during exercise affect the amount of CO2 bound to hemoglobin in opposite directions. Whereas deoxygenation of hemoglobin increases the amount of CO2 bound to hemoglobin, acidification decreases the amount of carbamate formed by hemoglobin.
To calculate carbamate concentrations within erythrocytes, we use a
single set of constants for the
- and
-chains in the oxy state of
hemoglobin and separate constants for the
- and
-chains in the
deoxy state. Because the calculation of carbamate is dependent on the
values of the carbamate equilibrium constant (pKc) and the ionization equilibrium
constant of the amino group (pKz) employed,
errors in the determination of these constants in different studies can
lead to changes in the calculation of the carbamate. Therefore, we use
two different sets of constants to give an estimate of the variability
of the calculated carbamate concentrations.
For oxyhemoglobin, we use the binding constants of Gros et al.
(68) (number of CO2 binding sites per
hemoglobin tetramer n = 2, pKc = 4.73, pKz = 7.16), and for deoxyhemoglobin, their constants for the
-chain
-amino groups of n = 2, pKc = 5.19, pKz = 7.05. However, because their measurements were done in the absence of
2,3-DPG, their carbamate equilibrium constant
(Kc) for the
-amino groups of the
-chains of deoxyhemoglobin was not used. On the other hand,
intraerythrocytic concentration of 2,3-DPG has effectively no influence
on the binding of CO2 to oxyhemoglobin and to the
-chain
-amino groups of deoxyhemoglobin (132). For the
-chain
-amino group of deoxyhemoglobin, the ionization constant
(Kz; pKz = 6.13) estimated by Gros et al. (68) was used in
conjunction with the CO2 binding constant
for this same
amino group given by Perella et al. (135) for the presence
of 2,3-DPG. Perella et al. (135) determined this figure by
measuring CO2 binding of hemoglobin whose
-amino groups
were differentially blocked by cyanate. Using their value of
, one obtains together with the above value of
pKz a carbamate equilibrium constant
pKc = 5.06 for these groups. Thus we
describe CO2 binding by the
-amino groups of the
-chains of deoxyhemoglobin in the presence of 2,3-DPG with
n = 2, pKz = 6.13, and
pKc = 5.06. With these constants we
estimate a contribution of only ~5% (0.09 mM) of carbamate to
overall CO2 exchange during rest (Table 1). When the
pKz and pKc
values reported by Perella et al. (134) and the data of
Perella et al. (135) are combined in an analogous fashion to estimate binding constants in the presence of 2,3-DPG, a
contribution of 9% (0.16 mM) of carbamate to CO2 exchange
during rest is calculated. The former estimate of 0.09 mM or 5% agrees
nicely with measurements of Böning et al. (13). From
their data, an arteriovenous difference for carbamate of 0.09 mM is
calculated for the blood gas values of Table 1. It should be noted that
all these estimates of the contribution of carbamate to overall
CO2 exchange are lower than the value of 12.6% calculated
by Klocke (105). However, Klocke's use of the data of
Perella et al. (135), which are valid for pH 7.4 rather
than the normal intraerythrocytic pH 7.2, may have led to a substantial
overestimate of the role of carbamate because carbamate formation
increases drastically with increasing pH. Thus it appears that a
contribution of 5% by carbamate is a reasonable estimate although
markedly lower than previously believed.
During heavy exercise as defined in Table 1, ~6% (10.8% with the data of Perella and co-workers, Refs. 134, 135) of the arteriovenous concentration difference of total CO2 is calculated to be due to a change in carbamate. Böning et al. (13) have measured an ~10% contribution to overall exchange during aerobic exercise, but during heavy exercise with considerable anaerobic metabolism they found that carbamate does not contribute to CO2 exchange at all; arterial blood contained a carbamate concentration that was higher by 0.06-0.13 mol/mol hemoglobin than that of venous blood in the presence of lactic acid. The data of Table 1 thus represent an intermediate position between these extreme types of exercise.
Carbamate concentration in plasma does not contribute to overall CO2 exchange according to Table 1, which is in agreement with Klocke's conclusion (105). During heavy exercise, arterial plasma contains an even higher concentration of carbamate than venous plasma. The physicochemical reason for this is that, in the absence of an oxylabile carbamate fraction as exhibited by hemoglobin, the increase in carbamate by the elevated PCO2 in venous plasma is counteracted or overruled by a decrease in carbamate caused by the fall in pH.
B. Transport Within the Intracellular Compartment
Figure 1 shows that overall
CO2 transport is the sum of the diffusion of 1)
dissolved CO2 and 2) CO2 bound
as HCO3
. The contribution of HCO3
to CO2 transport is called "facilitated CO2
diffusion" and was first described by Longmuir et al.
(111). Gros and Moll (66) and Gros et al.
(67) have shown that facilitated CO2 diffusion involves a flux of H+ equivalent to that of
HCO3
, a fact which matches the other fact that
hydration of CO2 produces equal amounts of H+
and HCO3
. Facilitated CO2 diffusion by
HCO3
diffusion under steady-state conditions then
requires 1) rapid conversion of CO2 into
HCO3
and H+, which at the short diffusion
distances as they occur in cells (<1 mm) implies that the presence of
carbonic anhydrase (CA) catalyzing CO2 hydration is
essential for facilitation to occur, and 2) equal fluxes of
H+ and HCO3
, where 3)
significant fluxes of H+ can only be achieved when they
occur by facilitated H+ diffusion, i.e., by the diffusion
of mobile buffers carrying H+ and present at concentrations
comparable to that of HCO3
. This leads to the
following scheme of facilitated CO2 diffusion shown in
Figure 1.
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1. Diffusion of dissolved CO2
Diffusion coefficients (=diffusion constant/SCO2) have been measured under conditions where only little facilitated diffusion is present. In a 33g% hemoglobin solution, the hemoglobin concentration that prevails inside the red blood cell, the CO2 diffusion coefficient, is reduced to less than one-half of its value in water (Table 2). It is not clear why the figure of Uchida et al. (173) is three times lower than the figure of Gros and Moll (64) for this condition. Compared with diffusion in water, diffusion is hindered by the presence of hemoglobin as it is by the presence of other intracellular proteins. It appears that proteins are virtually impermeable to CO2 and represent the major obstacles to CO2 diffusion within cells (64). Accordingly, CO2 diffusion constants decrease in a defined manner with increasing hemoglobin concentration (curve in Fig. 2) that can be explained quantitatively on the basis of the geometry of the water space in a hemoglobin solution (64). Similarly, the CO2 diffusion constant in various tissues varies systematically with the protein concentration of these tissues (points in Fig. 2). It is obvious that, for a given protein concentration, the CO2 diffusion constants in these different cells or tissues agree very nicely.
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2. Diffusion of HCO3
The diffusion coefficients for HCO3
are about
one-half as great as those for CO2, and in the presence
of proteins, its diffusion can be expected to be hindered to an extent
comparable to that observed for CO2 diffusion. The
HCO3
diffusion coefficient is calculated from the
equivalent conductivity of HCO3
(67,
108), 11.7 × 10
6 cm2/s in
pure water at 25°C, and is reduced to 8.7 × 10
6
cm2/s at physiological ionic strength and 25°C
(67). Again, Uchida et al. (173) report a
surprisingly low value of 1.4 × 10
6 cm2/s
for HCO3
diffusion in "100% hemolysate" at
37°C. The relative contribution of HCO3
to total
CO2 diffusion (=facilitated diffusion) can be evaluated on
the basis of these diffusion coefficients. The contribution of
HCO3
diffusion, and thus of facilitated diffusion, to
total CO2 diffusion depends greatly on the partial pressure
range of CO2 in which the transport process takes place.
This can be predicted from the general shape of CO2 binding
curves, which are steep at low PCO2 values and
become flatter with increasing PCO2. Therefore, the HCO3
concentration gradient per CO2
concentration gradient is higher at low PCO2,
and vice versa. This implies that the relative contribution of
facilitated diffusion is highest at lowest PCO2
values and decreases consistently with increasing
PCO2 (66, 67).
3. Diffusion of H+
The diffusion coefficient of free H+ in aqueous
solutions at 25°C is 9.3 × 10
5 cm2/s
(123), i.e., H+ possess a more than five times
greater diffusivity in water than CO2. Nevertheless, free
diffusion of H+ is a rather ineffective mechanism of
H+ transport, because at physiological values of pH, the
H+ concentration gradients within cells cannot exceed the
order of 10
7 to 10
8 M. In the presence of
buffering substances at physiological concentrations of
10
3 to 10
2 M, such differences of free
H+ concentrations are accompanied by concentration
differences of buffered H+ of at least of 10
3
to 10
2 M or more. This very much higher concentration
difference of the bound H+ compensates for the lower
diffusion coefficients of mobile buffers. The diffusion coefficient for
a mobile buffer such as phosphate is of the same order of magnitude as
that of HCO3
, 7.0 × 10
6
cm2/s (67). Consider as an example an
intracellular pH difference of 0.1 between absolute pH values of 7.1 and 7.2; then, the expected flux of H+ by free
H+ diffusion, estimated as diffusion coefficient
(D) × concentration difference (
c), gives 9.3 × 10
5 × 1.62 × 10
8
mmol·cm
1·s
1 = 1.5 × 10
12 mmol·cm
1·s
1.
Estimating facilitated H+ flux by diffusion of buffered
H+ with the assumption of a buffer capacity of 40 mM/
pH
and the above value of D for phosphate in an analogous
fashion yields 7.0 × 10
6 × 0.1 × 40 × 10
3 mmol·cm
1·s
1 = 2.8 × 10
8
mmol·cm
1·s
1. Thus facilitated
H+ diffusion by buffer diffusion in this example is more
than 10,000 times more effective than free diffusion of H+.
It has been shown that not only the diffusion of low-molecular-weight buffers such as phosphate (67) but also the diffusion of
protein buffers (66) is a highly effective means of
H+ transport. In the case of very large protein molecules,
it has even been shown that facilitated H+ transport occurs
very efficiently not only by translational but in addition by
rotational protein diffusion (62, 63). Thus facilitated CO2 diffusion essentially occurs by diffusion
of HCO3
and simultaneous buffer-facilitated
H+ diffusion. That buffer mobility is indispensable for
this process to take place has been shown by Gros et al.
(67) by demonstrating that immobilized phosphate buffer
cannot entertain facilitated CO2 diffusion.
Al-Baldawi and Abercrombie (3) have reported measurements
of H+ diffusion in cytoplasm extracted from giant neurons
of a marine invertebrate. An apparent diffusion coefficient for
H+ of only 1.4 × 10
6 cm2/s
was determined, which was 5 times lower than the estimated diffusion
coefficient of the mobile buffers and 70 times lower than the diffusion
coefficient of free H+. This appears to be in contradiction
to the above considerations. However, because the authors performed
their measurements under non-steady-state conditions by observing the
relaxation of pH after a sudden pH change at one surface of the
cytoplasm sample, it appears likely that this value represents a
substantial underestimate of the apparent H+ diffusivity
that one would observe under steady-state conditions. A pH
transient will be greatly slowed down by the presence of buffers whose
buffering capacity is so overwhelming compared with free H+
concentration. This problem was aggravated in the experiments of
Al-Baldawi and Abercrombie (3) by the presence in the
cytoplasm of a substantial fraction of immobile buffers.
The contribution of facilitated diffusion to overall diffusion depends
on the actual concentration differences for HCO3
and
for buffered H+. These in turn are dependent on the actual
pH gradient and the pK value(s) of the mobile buffer(s)
present. As an example, calculated CO2 fluxes are shown in
Figure 3 as a function of the average pH
with boundary PCO2 values of 5.32 and 6.65 kPa
(40 and 50 mmHg) in a 66 mM phosphate solution (pK 6.84, 25°C). Total CO2 diffusion is more than twice as high as
free diffusion of dissolved CO2 in a pH range of
~6.9-7.8. Thus more than one-half of the CO2 transport in this model system occurs by facilitated diffusion, which
means that at a physiological pH of the intracellular or extracellular
spaces more HCO3
than CO2 molecules
contribute to total CO2 flux within the compartment. Facilitated diffusion does not reach its maximum exactly at the pK value of the phosphate, i.e., at the maximal buffer
capacity of the solution. The reason for this is that whereas above pH 6.84 the buffer capacity decreases, the pH difference across the layer
increases markedly. The sum of these two effects leads to an increase
with increasing pH in the concentration differences of
HCO3
and of the H+-carrying
H2PO4
beyond the pK of the
phosphate buffer. Under physiological conditions, the course of this
curve may be different, since buffering is accomplished by different
sets of buffers with more than one pK value. Although
proteins, which are important buffers in intact cells, possess a lower
diffusivity than inorganic phosphate, their large buffer capacity for
H+ results in a facilitation of CO2 diffusion
in intact cells that is of a similar order of magnitude as shown in
Figure 3 for phosphate. For the condition within red blood cells, in a
hemoglobin solution of 30 g% Hb at 38°C and at roughly physiological
pH and PCO2, Gros and Moll (65)
have measured a contribution of facilitated CO2 diffusion
of ~85% to total intraerythrocytic CO2 transport.
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C. Transport Across the Erythrocyte Membrane
Although total CO2 flux across the membrane can again
be considered as the sum of diffusion of dissolved CO2 and
of HCO3
with accompanying H+, the
relative contributions of dissolved and bound CO2 to
overall CO2 flux across the erythrocyte membrane are quite
different compared with diffusion within the intracellular compartment.
1. Dissolved CO2
Cell membranes are generally considered to be highly permeable to
gases such as CO2 or O2, with one of the few
exceptions being the apical membrane of parietal and chief cells of
gastric glands, which have been described to possess "no detectable
permeability to NH3, NH4+, CO2,
and HCO3
" and whose surface area times permeability
product was found to be about three orders of magnitude lower than that
of the basolateral membranes of the same cells (15,
176). Erythrocyte membranes, though, are highly permeable
to CO2, the absolute permeability values cited being in the
range of 0.35-3 cm/s (Table 3), as has
been thoroughly discussed by Klocke (105). More recently, Forster et al. (47) measured the rate of depletion of
C16O18O in erythrocyte suspensions by mass
spectroscopy. They evaluated their measurements to give the
CO2 permeability of the human red cell membrane and
obtained a value of ~1 cm/s. If we simplistically equate
physiological CO2 concentration with CO2
concentration difference across the membrane
(cCO2), we can estimate the possible order of
magnitude of physiologically occurring CO2 fluxes from PCO2 × cCO2 = 1 cm/s × 1 mM = 1 × 10
3
mmol·cm
2·s
1.
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2. HCO3
Permeability for HCO3
of artificial phospholipid
vesicles, which are devoid of any anion exchanger, is six orders of
magnitude lower (Table 3; Ref. 127) than it is for dissolved
CO2. However, erythrocyte membranes of all vertebrates
with the exception of agnathans (hagfishes and lampreys; see reviews,
Refs. 80, 126, 136) do have a rapid anion
(HCO3
/Cl
) exchange protein, capnophorin
or band 3 (see review by Jennings, Ref. 90), which exchanges
HCO3
for Cl
at a ratio of 1:1. An
overview of the distribution of this transporter in red blood cells is
given in Table 4. Thus the permeability of the erythrocyte membrane to HCO3
is considerably
increased over that of lipid bilayers but still about three to four
orders of magnitude lower than the permeability for dissolved
CO2 (Table 3). Net driving force for
HCO3
/Cl
exchange is proportional to the
difference in the electrochemical potential for both ions. The turnover
number of this transporter is 4-5 × 104
Cl
·s
1·transporter
molecule
1 at 37°C (16, 56).
With this figure and the number of 1.0-1.2 × 106
copies of capnophorin per red blood cell (90), a maximal
flux across the membrane of ~6 × 10
5 mmol
Cl
·cm
2·s
1 can be
calculated using an erythrocytic volume of 80 × 10
12 cm3 and a red cell surface of 1.6 × 10
6 cm2. Maximal
HCO3
flux is expected to be in the same range as that
of Cl
. Flux measurements at 0°C have shown a maximal
value for HCO3
flux of 1% of this theoretical value,
~5 × 10
7 mmol
HCO3
·cm
2·s
1
(48). When the large temperature dependence of the anion
transporter with a turnover number at 0°C of only ~200
Cl
·s
1·molecule
1
(56) is taken into account, this agrees well with the
value calculated above for maximal flux at 37°C. Values of
HCO3
permeability of erythrocyte membranes are
similar for humans and birds (as well as in several other species). In
red blood cells of adult humans and of chicken HCO3
permeability
(PHCO3
) was
measured to be 5.6 × 10
4 and 7 × 10
4 cm/s, respectively (160). These latter
authors showed that a functionally active band 3 protein is present in
the erythrocyte membrane of the chicken at very early stages of
development. In the dogfish (Mustelus canis), flux of
HCO3
across red cell membrane was reported to equal
flux across human red cell membranes (128). It should be
mentioned that a very large number of measurements have been reported
for the HCO3
permeability of the human red blood cell
(e.g., Refs. 19, 30, 88, 103), most of which yielded numbers very
similar to that of Sieger et al. (160), as cited in Table
3. Estimating the physiologically possible HCO3
flux
across the red cell membrane in a fashion analogous to that used above
for CO2, one obtains the following:
PHCO3
× cHCO3
= 5 × 10
4 cm/s × 10 mM = 5 × 10
6
mmol·cm
2·s
1, which is ~10 times less
than the maximal flux of 6 × 10
5
mmol·cm
2·s
1 estimated above from
turnover number and number of copies of capnophorin per red blood cell
but more than two orders of magnitude smaller than the above
CO2 flux.
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3. H+
Proton permeability of phospholipid vesicles is five times higher
than HCO3
permeability, 1.8 × 10
5
cm/s (127). However, because the H+
concentration gradient across the cell membrane is very small (intracellular pH 7.2, extracellular pH 7.4,
pH 0.2), the product permeability × concentration gradient, is also very small:
PH+ × cH+ = 1.8 × 10
5 cm/s × 2.3 × 10
8 M = 4 × 10
13 mmol
H+·cm
2·s
1. Thus diffusion
of free H+ across the membrane is so small that it cannot
support any facilitated CO2 diffusion. However, in addition
to free H+ diffusion, there are several other more
efficient mechanisms of H+ transport across the red cell
membrane. The significance of these pathways for overall H+
flux across the erythrocytic membrane depends on the specific conditions. Proton fluxes (or reverse OH+ fluxes) in
erythrocyte membranes can be achieved via 1) the
Jacobs-Stewart cycle, which includes
HCO3
/Cl
exchange via capnophorin;
2) H+ flux via HCl cotransport (or
OH+-Cl
countertransport); and 3)
H+ flux via H+-lactate cotransport and nonionic
lactic acid diffusion.
Bisognano et al. (12) measured H+ fluxes
across red cell membranes by a pH stat method. They induced these
fluxes by establishing an outward Cl concentration gradient through
increasing the osmotic strength of the extracellular solution with
sucrose. Without lactate/lactic acid present, two-thirds of the
measured apparent H+ flux was mediated via the
Jacobs-Stewart cycle (89), and one-third was due
to a DIDS-sensitive HCl cotransport. The Jacobs-Stewart cycle
brings about a H+ efflux from the red blood cell by the
following sequence of processes: 1) H+ in the
cell reacts with HCO3
to give CO2,
2) CO2 leaves the cell and 3) is
hydrated extracellularly to give H+ and
HCO3
, where 4) the H+ remains
outside while the HCO3
enters the cell in exchange
for Cl
via the Cl
/HCO3
exchanger. Bisognano et al. (12) base their study on the
assumption that the Jacobs-Stewart cycle-mediated H+
flux is inhibitable by the CA inhibitor ethoxzolamide, whereas HCl
cotransport is not affected by this drug but suppressed by DIDS. Under
their conditions, an initial flux via HCl cotransport was observed at
0.5 × 10
9 mmol
H+·cm
2·s
1, compared with 1 × 10
9 mmol
H+·cm
2·s
1 mediated by the
Jacobs-Stewart cycle, giving a total H+ flux by these
two mechanisms of 1.5 × 10
9
mmol·cm
2·s
1.
Compared with free H+ diffusion, this value is very high;
it is considerably lower than the HCO3
flux estimated
above and substantially lower than the estimated CO2 flux.
A third mechanism of H+ transport across the red cell
membrane is by the H+/lactate carrier and by nonionic
diffusion of lactic acid, both of which require the presence of lactate
(27, 138). Proton fluxes via this mechanism
were calculated from flux measurements of Fishbein et al.
(42) and of Poole and Halestrap (138) in human red blood cells to be 2-2.5 × 10
9 mmol
H+·cm
2·s
1. Thus, in the
presence of lactate, the above H+ flux estimate would have
to be raised to ~4 × 10
9
mmol·cm
2· s
1, which is much lower than
the flux estimate for HCO3
. The fluxes of both ions,
however, are more than two orders of magnitude smaller than a
physiological CO2 flux. Thus a significant facilitation of
CO2 diffusion across the red cell membrane does not occur.
It may be noted that the activity of the H+-lactate
cotransport can be increased considerably in Plasmodium falciparum-infected human erythrocytes (96).
In conclusion, the permeability of dissolved CO2 is much
greater than the effective permeability of HCO3
and
H+. At the same time, more than two-thirds of the
CO2 transported in either red blood cells or plasma is
transported in the form of HCO3
. This makes it appear
essential that CO2 and HCO3
can be
converted into each other quite rapidly at the boundary between the two
compartments: intraerythrocytic space and plasma. A high velocity of
this interconversion is achieved by the enzyme CA.
Although HCO3
and H+ are produced
in equal amounts by the hydration of CO2, the distribution
of the two products among the two compartments, intraerythrocytic space
and plasma, is quite different at electrochemical equilibrium.
Bicarbonate is transported to a larger fraction within plasma than
within erythrocytes because the equilibrium pH of the plasma is more
alkaline than the intraerythrocytic pH (Table 1). In contrast,
H+ are transported to a larger fraction within erythrocytes
than in plasma because the nonbicarbonate buffer capacity of
erythrocytes exceeds that of plasma by a factor of ~10.
The CO2 entering the blood during capillary passage through the peripheral tissue can encounter two different situations: 1) a situation where CA is present in red blood cells only, or 2) a situation where CA is available in red blood cells and in plasma,
A) RAPID CATALYSIS OCCURS ONLY WITHIN ERYTHROCYTES. Carbon
dioxide enters the red blood cells, and there is rapidly converted to
HCO3
and H+. When the red blood cell has
reached the end of the capillary, electrochemical equilibrium across
erythrocyte membrane is not yet established, because H+
concentration and even more so HCO3
concentration are
too high within red blood cells compared with plasma concentrations. A
significant fraction of the intraerythrocytic HCO3
has left the cell via HCO3
/Cl
exchange
already during capillary transit. After blood has left the capillary,
part of HCO3
and H+ that has been
produced within the red blood cell is dehydrated back to give
CO2; CO2 then leaves the cell and enters the
plasma, where the slow uncatalyzed reaction hydrates CO2 to
establish final equilibrium. During this postcapillary process, the
plasma pH shifts slowly in the acidic direction.
B) RAPID CATALYSIS OCCURS IN ERYTHROCYTES AND IN PLASMA.
Carbon dioxide enters red blood cells and is rapidly converted there, but also within plasma, to HCO3
and H+.
Equilibration between the two compartments is no longer rate limited by
the slow CO2 hydration reaction in plasma. The pH of the
blood leaving the capillary can be expected to be constant and in
equilibrium. However, as is shown in section
IVD2, in the presence of lactic acid
production in the tissue, this must not necessarily be so. At the end
of capillary transit, it may even be possible that H+
concentration in plasma is too high compared with intraerythrocytic concentration for equilibrium to be established across the erythrocytic wall, since equilibration of lactic acid across this wall is a slow process.
For CO2 excretion, it is essential that there is a rapid chemical reaction of CO2. However, how and how fast acid-base equilibrium is established in the blood depends on the sites of CA localization and its activity. This and the velocity of other CO2 reactions will therefore be considered in the following sections.
E. Interconversion Between CO2 and
HCO3
The interconversion between CO2 and
HCO3
without a catalyst is rather slow and may
require more than 1 min to approach completion, a time much too long
compared with the capillary transit time, which is ~1 s. Carbon
dioxide hydration/dehydration reactions are accelerated between 13- and
25,000-fold by intraerythrocytic CA activities (46,
47). With such a CA activity, the interconversion between
CO2 and HCO3
inside erythrocytes requires
only 2 ms for 95% completion.
The participants of this reaction, HCO3
and, in
particular, the H+ (see sects.
IIIC1, IIID, and
IV) are involved in other reactions, so the time
course of their concentration is also dependent on the kinetics of
other reactions. For a complete understanding of the overall time
course of CO2 exchange, these reactions have also to be considered.
1. Catalysis by CA in blood
Carbonic anhydrase is found in the blood of all vertebrates. With respect to location, isozyme types, and coexpression of the anion exchanger (capnophorin), there are very interesting variations between different species from the lower vertebrates to mammals (Table 4) that may shed light on the increasing efficiency of CO2 excretion in the course of evolution.
A) CYTOPLASM OF ERYTHROCYTES. Carbonic anhydrase was first detected by Meldrum and Roughton (118) [for review, see Maren (113) and Klocke (105)]. Briefly, isozyme CA I with a relatively low specific activity occurs in the red blood cells of all vertebrate groups with the exception of the cat family and a few other species; it is absent in the red blood cells of the cat, lion, jaguar, tiger, leopard, ox, chicken, and frog (18, 115). In primitive agnathans (lamprey, hagfish) and elasmobranchs (dogfish), CA I is the only isozyme present (114). It is somewhat less inhibitable by sulfonamides and considerably more susceptible to anions than CA II. Isozyme CA II with a specific activity that is ~10 times higher than that of CA I is probably the most widespread form and occurs in the red blood cells of all vertebrates except agnathans and elasmobranchs.
A mechanism for rapid HCO3
/Cl
exchange
across the erythrocyte membrane is present in almost all vertebrate red
blood cells and is missing only in the most primitive vertebrate group,
the agnathans (lamprey, hagfish; Refs. 39, 131, 171, 172). The consequence of this has been discussed by Nikinmaa (126).
In these fish this precludes the utilization of plasma
HCO3
in CO2 excretion on a physiological
time scale. Together with the rather flat CO2 binding
curve, CO2 transport in these animals appears rather
inefficient, yet in the lamprey, CO2 transport potential is
as great as in the highly active teleosts despite the missing anion
exchanger. In this species, the disadvantage is overcome by a high
intraerythrocytic pH, resulting in a high intracellular binding
capacity for CO2 and a marked Haldane effect. However, the
intracellular buffering capacity is separated from the plasma
compartment in lampreys due to the low membrane permeability to
HCO3
. As a consequence, extracellular metabolic acid
loads cause marked fluctuations in plasma pH. Thus the major advantage
gained by the rapid anion exchanger appears to consist of an improved
effective extracellular buffering (with access to the intracellular
buffering power) rather than in a major improvement of gas transport
(126).
The acceleration of the hydration-dehydration velocity by CA within
erythrocytes is considerable. An activity (factor by which the rate of
CO2 hydration is accelerated) of 13-14,000 was reported by
Forster and Itada (46), and figures of 23,000 and 25,000 have been obtained by Wistrand (184) and by Forster et al.
(47). The cytosolic CA enzymes may be not uniformly
distributed within the red blood cells. Some indications have been
proposed to suggest that their concentration may be increased near the
cellular border. Interactions of CA are reported with the plasmalemmal
anion exchanger (capnophorin = band 3; Ref. 98), and it would seem
a most efficient place to catalyze
CO2-HCO3
hydration-dehydration
reaction in close neighborhood to
HCO3
/Cl
exchange. Parkes and Coleman
(133) reported an enhancement of CA activity by
erythrocytic membranes; CA II and CA I activity were increased 3.5- and
1.6-fold, respectively, by the presence of red cell membranes. Whether
in their erythrocytic membrane preparation the effective structure was
the band 3 protein can only be speculated. Although these observations
have not been confirmed yet by other investigators, there are several
studies of the problem whether part of red cell CA is firmly bound to the erythrocyte membrane. Whereas Enns (40) had found CA
in red cell ghosts, later studies by Tappan (170),
Rosenberg and Guidotti (145), and Randall and Maren
(139) came to the conclusion that red cell CA is a truly
cytosolic enzyme and not membrane bound.
B) PLASMA. No CA activity aside from that attributable to lysed erythrocytes was ever found in plasma, with the sole exception of dogfish (Scyliorhinus canicula; Ref. 186). Also, there is no CA activity on the outside surface of erythrocytes or available to plasma, as was confirmed by Effros et al. (37).
However, in tissues where especially large amounts of CO2
leave or enter the blood, there is extracellular catalysis available to
the capillary plasma in some species. This is achieved by an extracellular CA bound to membranes, the membrane-bound isozyme CA
IV, which provides catalytic activity to the plasma. Such a membrane-bound CA was found in the lung (36,
104, 150) and gill (28). In
skeletal muscle, the first evidence for the presence of such a CA in
dog, cat, and rabbit was provided by measurements of the distribution
space of labeled HCO3
(37,
50, 51, 189) and by measurements
of the postcapillary pH kinetics (128).
With the histochemical method of Hanson, staining of endothelial
membranes as well as sarcolemma was observed in skeletal muscle
(140, 141). Immunohistochemical studies
employing anti-CA IV antibodies at the light microscopic level
revealed staining of capillary walls, which would indicate that CA IV
is associated with endothelial cell membranes (157). With
the use of semithin sections and a more sensitive immunocytochemical
technique, however, capillaries and sarcolemma were found to be
stained, and in antibody-treated ultrathin sections of skeletal
muscle studied by electron microscopy, membrane-bound CA IV was
found to be associated with capillary endothelium, sarcolemma, and
sarcoplasmic reticulum (SR) (24). That sarcolemmal and SR
CA IV were visible in ultrathin sections but not in cryosections was
attributed to a poor accessibility of CA IV at these locations in the
7-µm-thick untreated cryosections. Another approach has been used by
Geers et al. (51). Measuring the space of distribution of
labeled HCO3
and its reduction by CA inhibitors,
Geers et al. (51) observed that the effectiveness of
macromolecular CA inhibitors of different molecular size
(Prontosil-dextrans of mol wt 5,000 vs. 100,000) indicates the presence
of CA in the interstitial space. Indirect although strong evidence for
this CA to be associated with the sarcolemma was obtained by
intracellular and cell surface pH microelectrode measurements on
skeletal muscle fibers by deHemptinne et al. (25). They
observed a transient alkaline pH shift on the surface of these fibers
upon exposure to propionate, whose magnitude was greatly increased in
the presence of the CA inhibitor acetazolamide in the extracellular
space. This was interpreted to show that in the presence of
extracellular sarcolemmal CA CO2-HCO3
acts as a rapid and efficient source of H+ that enter the
cells together with the propionate ion. Analysis of isolated
sarcolemmal vesicles demonstrated the presence of high activities of CA
associated with the sarcolemma (179), and it was shown by
Western blotting with anti-CA IV antibody that this sarcolemmal CA
is isozyme CA IV (175). In conclusion then, the present
state of evidence indicates that in skeletal muscle, membrane-bound
CA IV is associated with endothelial as well as with sarcolemmal
membranes, in addition to a CA bound to the sarcoplasmic reticulum membrane.
In the case of lung tissue, it was estimated by Bidani et al. (11) that the CA of rat pulmonary vasculature catalyzes the extracellular hydration-dehydration reaction by a factor of 130-150, a figure that may appear rather small compared with the intraerythrocytic activity of >10,000. However, the calculations described below indicate that an activity of 100 in the plasma within the capillary bed of skeletal muscle should be sufficient physiologically (see sect. IVD2). No estimates of intracapillary CA activity in muscle capillaries are available.
The localization pattern of CA in blood is further complicated by the presence of a CA inhibitor in the plasma of some species, which ensures the absence of any activity of soluble CA in plasma. This was first described for dog and fish (14) and later for pig, sheep, rabbit, and various fish species (74, 75, 77, 83, 109, 137, 149). The molecular size of this endogenous CA inhibitor was determined to be 10-30 kDa for the eel (74) and 79 kDa for the pig (148).
In view of the presence of an extracellular membrane-bound CA activity available to plasma in lung and muscle, the existence of a plasma CA inhibitor seems astonishing. However, some experiments indicate that the plasma inhibitor inhibits erythrocytic cytosolic CA rather well, but CA from lung tissue homogenate (membrane-bound CA; dog) only incompletely (83). Similarly, plasma inhibitor from pigs showed a less than complete inhibition of vascular CA activity in lungs of rats (78), where the vascular activity of CA is known to be CA IV located on the extracellular luminal surface of capillary endothelial cells.
It may be hypothesized that, due to its molecular size, the plasma inhibitor has no access to the interstitial space. Thus a CA associated with the sarcolemma may be left uninhibited by the plasma inhibitor, because a macromolecule of this size may not enter the interstitial space to a great extent. Even the endothelial membrane-bound CA IV in the lung appears to be only partly inhibited, and this should also be true for capillary CA IV of muscle (and, for example, of the brain).
We conclude that the plasma inhibitor will inhibit erythocytic CA released from any hemolyzed red blood cells throughout the blood vessels rather completely, thus reducing the dehydration/hydration reaction in the plasma to the uncatalyzed velocity, whereas in vascular regions with a membrane-bound CA, e.g., muscle, heart, lungs, and others, a marked catalysis of the dehydration/hydration reaction can take place even in the presence of a plasma inhibitor. Thus CA activity available to plasma appears to be confined to precise localizations within the circulation that are equipped with a capillary CA.
The effect of presence of CA in the plasma has been studied by Wood and Munger (186) for the rainbow trout. They found that CA attenuated postexercise increases in PCO2 and decreases in arterial pH by producing an increase in CO2 excretion during exercise. However, the normal postexercise hyperventilation was also greatly attenuated when CA was present in the plasma, as was the normal increase in the plasma levels of epinephrine and norepinephrine. They concluded that CO2 is an important secondary drive to ventilation in fish, and by increasing CO2 excretion by the presence of CA in the plasma this drive is diminished. The plasma CA inhibitor will ensure that no CA activity of hemolysed erythrocytes is present and thus will contribute to maintain a high level of ventilation in certain situations, which will be favorable for O2 supply.
E. Interconversion Between CO2 and Carbamate
The kinetics of oxylabile carbamate was thoroughly reviewed by Klocke (105). We only briefly summarize the relevant facts in these sections.
1. In plasma
The kinetics of this interconversion do not seem to be important for overall CO2 kinetics, since venous and arterial plasma carbamate concentrations are almost identical. The kinetics of plasma carbamate formation were characterized by Gros et al. (61) by a half-time of this reaction of 0.047 s.
2. In erythrocytes
The binding of CO2 to hemoglobin in solution has long
been known to be quite rapid, requiring a time to reach completion of 0.1-0.2 s (44), which corresponds to a half-time of
~0.04 s. Gros et al. (68) have studied the kinetics of
hemoglobin carbamate formation in a wide range of pH and
PCO2 values using a pH stopped-flow technique. They determined kinetic constants for the forward reaction of amino groups and CO2, ka,
for the
-chain and the
-chain
-NH2 groups in
addition to the e-NH2 groups of human oxy- and
deoxyhemoglobin. Table 5 shows the
complete set of constants reported by Gros et al. (68)
with ka, pKz,
and pKc, which describe their kinetic measurements. With these constants, overall carbamate kinetics under
physiological conditions of pH and PCO2 were
estimated to possess a half-time of 200 ms and to require ~1 s to
reach equilibrium by 95%. Their measurements were performed in the
absence of 2,3-DPG, and these times may become slightly shorter when
2,3-DPG is present. Nevertheless, their estimate of the half-time
of carbamate formation is considerably greater than that of Forster et
al. (44) but is similar to Klocke's estimate
(102) of the half-time of mobilization of oxylabile
carbamate of 0.12 s, which was obtained in the presence of
2,3-DPG.
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III. CARBON DIOXIDE TRANSPORT IN MUSCLE |
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A. CO2 Production in Muscle
Unlike most other tissues, muscle exhibits a vast range of aerobic
(and anaerobic) metabolic rates. In humans, O2 consumption of muscle tissue can rise 15- to 20-fold from resting values of ~10
µmol·min
1·100 g
1, and even higher
increases have been reported from 6.3 mmol·min
1·100
g
1 at rest to 200 µmol·min
1·100
g
1 at maximal exercise of a small muscle group (forearm;
Ref. 73). Carbon dioxide production rates can be calculated from these
O2 consumption rates using a RQ of ~0.85. The
PCO2 values in the venous blood leaving the
skeletal muscle have also been measured and are ~5.32-5.99 kPa
(40-45 mmHg) at rest and can rise to as much as ~13.3 kPa (100 mmHg)
during exercise (for example, Ref. 95).
Although different muscle types and different mammalian species have
vastly different maximal specific O2 consumption rates, maximal specific mitochondrial O2 consumption differs
considerably less. At maximum O2 consumption
(VO2max), mitochondria of different species
consumed 4.56 ± 0.61 ml
O2·min
1·ml
1
(87). This indicates that it is essentially mitochondrial
density in muscle fibers that determines maximal specific
O2 consumption of these fibers.
Carbon dioxide produced within muscle mitochondria has to diffuse
through the intracellular compartment and cross the sarcolemmal membrane and the capillary wall to reach the convective medium blood.
Because all the membranes crossed by CO2 along this
diffusion pathway are considered highly permeable to CO2,
their surface area is of no relevance for CO2 transport,
but this area may be important for the permeation of ions associated
with gaseous exchange. Among these membrane barriers, the capillary
wall has by far the smallest surface area, only ~1/5 of the entire
area of the sarcolemma (185) and only ~1/200 of the
total area of the inner mitochondrial membranes (87). The
following fluxes of respiratory gases occur across the surface of the
capillary wall. Maximal flow of O2 per area of capillary
wall is 1.3-1.9 µl
O2·min
1·cm
2 as calculated
from Conley et al. (20), where the higher values have been
measured in more athletic species (dog, pony) and the lower values in
less athletic ones (goat, calf). Corresponding maximal total
CO2 flux across capillary wall can be expected to be
~15% lower than the respective O2 fluxes (~60 nmol
CO2·min
1·cm
2). The area of
inner mitochondrial membranes being larger, CO2 flux across
this membrane is expected to be ~0.3 nmol
CO2·min
1·cm
2. This may
indicate that the capillary wall could be a significant barrier to ion
fluxes associated with CO2 transport.
B. Localization of CA in Skeletal Muscle
As discussed for erythrocytes, dissolved CO2 gas with
few exceptions is highly permeable across biological membranes, but the
more abundant species, HCO3
, is not. This
permeability difference requires conversion of HCO3
to CO2 at a sufficient speed in red blood cells as well as
in muscle cells before permeation of the membrane occurs, and only CA
can ensure this rapid conversion.
Several types of CA are present in various parts of skeletal muscle as has been reviewed by Gros and Dogson (60) and recently by Henry (79). Table 6 gives a summary of CA localizations in skeletal muscle.
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1. Capillary endothelium
As discussed in section IID, there is clear evidence that skeletal muscle capillaries possess an endothelial membrane-bound CA. The evidence that this enzyme is CA IV appears convincing.
2. Sarcolemma
Carbonic anhydrase associated with this membrane was found with a
variety of methods. The first evidence for the presence of an
extracellular catalysis of the hydration/dehydration reaction in the
capillary bed of skeletal muscle arose from functional studies. From
observation of the distribution space of
H14CO3
and its reduction in the presence
of CA inhibitors, it was concluded that an extracellular CA in
mammalian skeletal muscle exists (38). Its inhibition
properties were different from those of the cytosolic enzymes, and it
was concluded that this isozyme is present in the interstitial space
and probably bound to the sarcolemma (50, 51). Other functional studies have indicated that an
extracellular CA available to plasma is involved in fast pH
equilibration of the venous effluent and the interstitial space. With
the use of a stop-flow technique, it was shown that an electrolyte
perfusate had access to a CA while it passed through the hindlimb
capillary bed. By inhibiting this extracellular CA, a pH disequilibrium developed in the venous saline perfusate, and the pH of the effluent slowly became more acidic after the saline had left the capillary bed,
i.e., a so-called postcapillary acid pH shift developed
(129). It appears likely now that these effects, at least
partly, are due to capillary endothelial CA IV.
Transient changes of surface pH, induced by sudden addition and
withdrawal of propionic acid, were magnified when CA was inhibited in
isolated soleus muscle from mouse and rat and in cardiac muscle from
sheep, rabbit, and cat (25). Transient changes of surface pH induced by a sudden increase and decrease of
PCO2 were blocked by CA inhibitors in a
nonvertebrate muscle, the crayfish muscle (151). In the
former case, surface pH change is due to a net movement of
H+ across the membrane together with propionate. When
sarcolemmal CA is blocked, the efficacy of the
CO2/HCO3
buffer is diminished, producing
an increased pH transient. In the latter experiment, inhibition of the
sarcolemmal CA activity leads to a suppression of surface pH transients
that are caused by net fluxes of CO2, which go along with
rapid changes in surface PCO2 that are followed
by H+ production or consumption when CA is present on the
cell surface, but are not (or very slowly) when CA is inhibited. These
effects are clearly due to a CA bound to the external surface of muscle fibers.
In sarcolemmal vesicles, a CA was found in preparations from red and from white muscles of the rabbit. The inhibition constants (Ki) of this sarcolemmal CA toward acetazolamide, chlorzolamide, and cyanate were shown to be different from those of CA II or CA III (179). With CA IV antibodies this sarcolemmal CA was identified to be CA IV (175). In semithin and ultrathin sections of rat soleus muscle, CA IV was found immunocytochemically to be associated with the sarcolemma in addition to its association with the capillary endothelium and the SR (24). In mammalian heart, CA IV was also found to be associated with the sarcolemma in addition to the capillaries (156). Thus the present state of information indicates that sarcolemmal CA is the membrane-bound, glycosyl-phosphatidylinositol (GPI)-anchored isoform CA IV.
3. Cytoplasm
It is well known that the sulfonamide-resistant isozyme CA III is present in high concentration in the cytoplasm of mammalian skeletal muscle type I (slow-oxidative fibers) (review, Ref. 60). A sulfonamide-sensitive CA, probably CA II, was found in the cytosol of white muscles of the rabbit (161) and in the mouse soleus (21). In the latter case, however, no measurements of the erythrocytic contamination of the muscle homogenates were done; thus CA II of erythrocytes could be present in these experiments. In contrast, in the former case, red cell contamination was carefully controlled. In the cytoplasm of white as well as red hindlimb muscles of the rat, no sulfonamide-sensitive CA was present (54). For other animal groups, studies are scarce; from functional studies, the presence of a CA in the cytosol of crayfish muscle was deduced (151), and there is one report indicating that frog white muscle has a CA II-type isozyme (155).
Mammalian heart muscle appears to contain no cytosolic enzyme but high activities of the membrane-bound form (17, 54).
4. SR
A membrane-bound CA was detected in preparations of SR vesicles from red as well as white skeletal muscle of the rabbit (17). This finding was confirmed by histochemical results employing the fluorescent CA inhibitor dansylsulfonamide for CA staining (17, 26). Although it has not been possible to visualize SR-CA immunocytochemically at the light microscopic level (157), membrane-bound CA IV was found to be associated with SR by electron microscopy and use of CA IV antibodies (24). This latter finding corresponds with a reaction of SR membranes with anti-CA IV in Western blots (175). Wetzel and Gros (180) have found that nevertheless the inhibitory properties of the CA of SR toward sulfonamides are significantly different from those of the CA of the sarcolemma, which may indicate either that SR-CA is an isoform somewhat different from CA IV or has different properties because of a different membrane environment. In the mammalian heart, immunocytochemical evidence at the electron microscopic level indicates that CA IV is also associated with the SR (156).
5. Mitochondria
Mitochondrial CA, CA V, is present in high activities in liver and kidney mitochondria (see review, Ref. 60). In skeletal muscle, mitochondrial CA was found only in muscles of the guinea pig but was not detectable in skeletal muscles of the rabbit or the rat or the mouse (Table 6). Carbonic anhydrase V has been detected in immunocytochemical studies in the mitochondria of rat skeletal muscle in addition to those in several other rat <