Carbon Dioxide Transport and Carbonic Anhydrase in Blood and Muscle

Viktig studie med alt om hvordan CO2 går fra celle til vev til blod og forholdet mellom bikarbonat og melkesyre.

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.

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 CO2in skeletal muscle has to take into account this contribution of lactic acid and its involvement in kinetics and equilibria of CO2reactions.

Table 1.

CO2 transport in blood at rest and exercise

Arterial Rest Exercise
Venous v-a diff, mmol/l blood Venous v-a diff, mmol/l blood
mM mmol/l blood mM mmol/l blood mM mmol/l blood
pH† 7.40 7.37 7.145*
PCO 2 40 46 78*
Dissolved 1.23 0.68 1.42 0.78 0.10 2.40 1.32 0.64
Bicarbonate 24.58 13.52 26.38 14.51 0.99 26.65* 14.66 1.14
Carbamate 0.54 0.30 0.55 0.30 0.01 0.44 0.24 −0.06
Sum plasma 26.35 14.49 28.35 15.59 1.10 29.49 16.22 1.72
Red blood cell
pH† 7.20 7.175 6.996
Hb, g/l 333
Hct† 0.45
HbO2, fract† 0.97 0.75 0.25
Dissolved 1.23 0.4 1.42 0.46 0.06 2.40 0.78 0.38
Bicarbonate 15.47 5.01 16.84 5.46 0.44 18.91 6.13 1.11
Carbamate 1.66 0.75 1.86 0.84 0.09 2.12 0.95 0.21
Sum RBC 18.37 6.16 20.12 6.75 0.59 23.43 7.86 1.70
Total CO2 20.65 22.34 1.69 24.08 3.42

1.  Dissolved CO2

Only a small portion, ∼5% of total arterial content, is present in the form of dissolved CO2.

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−.

During a heavy work load of the muscle, high levels of lactic acid are present in addition to CO2, aggravating the decrease in pH. With this low pH, the fraction of HCO3− 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.

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 CO2bound 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.

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 PCO 2in venous plasma is counteracted or overruled by a decrease in carbamate caused by the fall in pH.

Fig. 2.

Diffusion constants of CO2 (in cm2·min−1·atm−1) at 22°C in different tissues as a function of the protein concentration (points) and in hemoglobin solutions of different hemoglobin concentrations (solid line). [Redrawn from Gros and Moll (64).]

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.

Therefore, the HCO3− concentration gradient per CO2concentration gradient is higher at low PCO 2, and vice versa. This implies that the relative contribution of facilitated diffusion is highest at lowest PCO 2values and decreases consistently with increasing PCO 2 (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.

This very much higher concentration difference of the bound H+ compensates for the lower diffusion coefficients of mobile buffers.

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.

Fig. 3.

Calculated CO2 fluxes across a layer of buffer solution as a function of the average pH value in this layer. The boundary CO2partial pressures are constant with 6.65 and 5.32 kPa (50 and 40 mmHg), respectively. The solution is 66 mM phosphate with varying contents of base. Thickness of the layer is 180 μm. Carbonic anhydrase is assumed to be present in excess. Solid curve represents the total flux of CO2, and dashed curve represents the flux by free diffusion only. [Redrawn from Gros et al. (67).]

1.  Dissolved CO2

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).

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.

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).

3.  H+

Proton permeability of phospholipid vesicles is five times higher than HCO3− permeability, 1.8 × 10−5cm/s (127). However, because the H+concentration gradient across the cell membrane is very small (intracellular pH 7.2, extracellular pH 7.4, ΔpH 0.2), the product permeability × concentration gradient, is also very small:P H+ × 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.

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).

Thus, in the presence of lactate, the above H+ flux estimate would have to be raised to ∼4 × 10−9mmol·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.

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.

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.

1.  Catalysis by CA in blood

Carbonic anhydrase is found in the blood of all vertebrates.

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).

membrane-bound CA IV was found to be associated with capillary endothelium, sarcolemma, and sarcoplasmic reticulum (SR) (24).

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 PCO 2 and decreases in arterial pH by producing an increase in CO2excretion 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.

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 PCO 2 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 (VO 2max), 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.

In heavily exercising muscle, in addition to CO2, lactic acid is produced and the additional H+ shift the equilibrium of the hydration/dehydration reaction toward CO2 and have to be buffered and eliminated from the cell. Intracellular pH of skeletal muscle can become very low and can decrease from ∼7.2 at rest to a value as low as 6.6–6.7 (6, 110) or to even lower values of 6.2–6.4 (119,152, 183) during maximal exercise. Accordingly, during maximal work, HCO3 −concentration is only two times that of dissolved CO2, whereas during rest, the ratio of HCO3 −/CO2 is ∼13. As a result, less facilitation of CO2 diffusion can be expected to take place during heavy exercise. At the same time, the “CO2store” in the muscle, HCO3 −, will be mobilized by the intracellular metabolic acidosis producing high PCO 2 values in muscle tissue and in the venous blood leaving the exercising muscle.

The intracellular H+ transport capacity, which suffices to transport H+ at a rate equal to the rate of HCO3− transport as it results from a HCO3 − concentration difference in the millimolar range (facilitated CO2 diffusion), will also suffice to transport H+ at a rate equaling the lactate flux that results from a lactate concentration difference in the millimolar range (lactic acid transport). Thus lactic acid, which is almost completely dissociated at physiological pH values, can be efficiently transported through the cell interior utilizing this facilitated H+ transport system. It may be noted that this H+ transport system under conditions of exclusively aerobic metabolism is used by the cell to maintain a facilitation of CO2 diffusion, whereas under conditions of dominating anaerobic glycolysis and low intracellular pH, it is mainly used to transport H+ along with the lactate anion through the intracellular space, a prerequisite for the elimination of lactic acid from the cell.

Fig. 4.

Schematic representation of proposed role of sarcoplasmic reticulum (SR) carbonic anhydrase (CA) in Ca2+ transport across the SR membrane. Catalyzed CO2 hydration within the SR provides protons that are exchanged for Ca2+ across SR membrane. Ca2+ uptake requires rapid H+ production within SR, as shown; Ca2+ release requires rapid H+buffering. Other counterions of Ca2+ appear to be Mg2+ and K+. As indicated at right, scheme on left is projected into a cross section through SR or L system. [From Geers et al. (55).]

During the capillary transit, the blood takes up CO2, H+, and lactate from the muscle cell via the interstitial space. Chemical and transport events that occur during gas and lactic acid exchange, which we have included in our calculations, are with few exceptions shown in Figure 6. With steady-state conditions, it is assumed that within each part of the interstitial space along the capillary wall concentrations are constant. Thus the sum of influx and efflux into this compartment and the rate of change of chemical rection has to be zero for CO2, HCO3 −, H+, and lactate.

The reactions and transport events included in the analysis are described as follows.

For CO2,

1) hydration/dehydration reaction catalyzed by CA or uncatalyzed;

2) diffusion between skeletal muscle cell, interstitial space, and erythrocytes; and

3) binding of CO2 to hemoglobin within erythrocytes (not shown in Fig. 6).

For HCO3 −, hydration/dehydration reaction catalyzed by CA or not (see point 1);

4) diffusion from interstitial space into plasma, and vice versa; and

5) movement between plasma and erythrocytes via anion exchanger.

For H+, hydration/dehydration reaction catalyzed by CA or not (see point 1);

6) buffered by proteins inside erythrocytes and plasma (not shown in Fig. 6);

7) cotransport of H+ and lactate ions across the sarcolemmal and red cell membrane;

8) release of H+by carbamate reaction (not shown in Fig. 6);

9) uptake of H+due to deoxygenation of hemoglobin (not shown in Fig. 6); and

10) diffusion across the capillary wall.

For lactate, cotransport of H+ and lactate ions across the sarcolemma and erythrocytes (see point 7);

11) movement of lactate ions via anion exchanger between plasma and erythrocytes.

An increase of the intramuscular partial pressure of CO2 to values as high as 13.3 kPa (100 mmHg) does not require any higher CA activity inside erythrocytes.

The same holds for a higher HCO3 − permeability of the erythrocyte membrane: when the HCO3 − permeability is assumed to be three times higher than the standard value used, the arteriovenous differences are unaltered compared with those seen in Figure 8. This implies that no additional CA would be necessary if the permeability of the erythrocyte membrane for HCO3 − were higher. (It may be noted that massive reduction of HCO3 −permeability per se decreases CO2 excretion in the lung, Ref. 23).

In other words, neither does the HCO3 −permeability of the red cell membrane set a limit to CO2uptake, nor does red cell CA activity ever become limiting at increased levels of CO2production. A possible situation where intraerythrocytic CA may become more critical, which has to our knowledge never been investigated, is a severe lactic acidosis with low intraerythrocytic pH values. At a pH of 6.4, the activity of CA II decreases to ∼30% of its value at pH 7.2 (99), and in this situation, more enzyme will be necessary to maintain the required activity.

More H+ arrive in the blood, and pH in plasma and in red blood cells decreases. This implies that, at a given PCO 2(here 55 mmHg), the total CO2 bound in the blood is reduced, because both HCO3 − and carbamate decrease with decreasing pH. Thus lactic acidosis decreases the total CO2release from muscle into blood at a given PCO 2. Because CO2 production continues, the consequence is a rise in tissue and venous blood PCO 2 values. For the whole body, heavy exercise therefore is associated with rather high muscle venous PCO 2 values and, as is well known, part of this CO2 is mobilized by lactic acid from the CO2 stores in muscle and blood.

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