Cornelia Geers

Carbonic anhydrase III is not required in the mouse for normal growth, development, and life span.

ABSTRACT Carbonic anhydrase III is a cytosolic protein which is particularly abundant in skeletal muscle, adipocytes, and liver. The specific activity of this isozyme is quite low, suggesting that its physiological function is not that of hydrating carbon dioxide. To understand the cellular roles of carbonic anhydrase III, we inactivated the Car3 gene. Mice lacking carbonic anhydrase III were viable and fertile and had normal life spans. Carbonic anhydrase III has also been implicated in the response to oxidative stress. We found that mice lacking the protein had the same response to a hyperoxic challenge as did their wild-type siblings. No anatomic alterations were noted in the mice lacking carbonic anhydrase III. They had normal amounts and distribution of fat, despite the fact that carbonic anhydrase III constitutes about 30% of the soluble protein in adipocytes. We conclude that carbonic anhydrase III is dispensable for mice living under standard laboratory husbandry conditions.

Effects of carbonic anhydrase inhibitors on contraction, intracellular pH and energy-rich phosphates of rat skeletal muscle.

1. The effects of carbonic anhydrase inhibitors on contractile parameters, intracellular pH (pHi) and energy‐rich phosphates were studied in isolated rat soleus and extensor digitorum longus (EDL) muscles. 2. The muscles were incubated either in Ringer solutions (95% O2/5% CO2 = control) or in solutions to which one of the inhibitors, 5 X 10(‐4) M‐chlorzolamide or 10(‐2) M‐NaCNO, had been added. Muscles were stimulated directly and contracted under isometric conditions. 3. Compared with control muscles, both inhibitor‐treated muscles showed a significantly decreased tetanic force and an increased half‐relaxation time of twitches and tetani. Chlorzolamide increased time‐to‐peak in both muscles. Cyanate decreased isometric twitch force in both muscles. 4. Both inhibitors decreased pHi in both muscles; chlorzolamide by 0.1 unit, cyanate by 0.4 unit in soleus and by 0.8 unit in EDL. 5. Chlorzolamide increased the concentrations of creatine and inorganic phosphate (Pi) in soleus (the effect of chlorzolamide was not studied in EDL). Cyanate caused these same changes in soleus as well as EDL and in addition decreased the concentrations of ATP and phosphocreatine in soleus and EDL. 6. In muscles acidified by either low external HCO3‐ (2 mM) or by elevated PCO2 (30% CO2 in the gas phase) in the bath, decreases in isometric force and increases in half‐relaxation time of tetani were observed. In addition there were increases in muscle Pi. These effects were more pronounced with 30% CO2 than with 2 mM‐HCO3‐. 7. Neither acidifying solutions prolonged either half‐relaxation time or time‐to‐peak of twitches. 8. We conclude that carbonic anhydrase inhibition exerts its effect (a) on isometric tension at least partly via an elevated Pi (perhaps in combination with lowered pHi); (b) on the half‐relaxation time of tetani by means of lowered pHi and elevated concentration of Pi; (c) on relaxation and time‐to‐peak of twitches by some unknown mechanism, neither directly by a change in pHi nor in Pi.

Carbonic anhydrase inhibition affects contraction of directly stimulated rat soleus.

We have studied the contractile parameters of directly stimulated isolated rat soleus muscles incubated in media containing the carbonic anhydrase inhibitors chlorzolamide (5.10(-4)M) or cyanate (10(-2)M). Both inhibitors caused a decrease in isometric twitch and tetanic (5s) tensions and an increase in muscle relaxation time. It is speculated that among the three types of skeletal muscle carbonic anhydrase it may be the enzyme associated with the sarcoplasmatic reticulum whose inhibition caused the observed changes in contractile parameters.

Inhibition properties and inhibition kinetics of an extracellular carbonic anhydrase in perfused skeletal muscle

We have investigated the properties of the carbonic anhydrase which is functionally available to CO2 and HCO3- in the capillary bed of skeletal muscle. We used essentially the indicator-dilution technique of Effros and Weissman (J. Appl. Physiol. 47, 1090-1098, 1979). Into hindlimbs of rabbits perfused with dextran solution we injected boli containing H14CO3- or 36Cl-, and 3H-dextran (MW 80 000) as an intravasal indicator, and observed the washout of these indicators by fractionated collection and analysis of the venous effluent. In agreement with previous studies we found that addition of 10(-4) M of the carbonic anhydrase inhibitor acetazolamide to the perfusate considerably speeds up the washout of 14C, reducing the extraction of 14C from 0.72 to 0.45. A half-maximal effect on 14C extraction was achieved with 1 x 10(-6) M acetazolamide (IC50). The carbonic anhydrase inhibitors methazolamide and benzolamide both yielded IC50 values of 10(-5) M. This pattern of inhibitory potency of the three sulfonamides is incompatible with their inhibitory effects on the two known cytosolic isoenzymes of skeletal muscle, CAII and CAIII. While cells take up acetazolamide and benzolamide extremely slowly, with half-times of several minutes to hours, the effect of both sulfonamides on 14C washout occurred very rapidly: less than 1 min, probably not more than a few seconds, were necessary to achieve inhibitory effects. We conclude that (1) a tissue carbonic anhydrase converts the injected H14CO3- quickly into 14CO2 which then diffuses into the intracellular space thus causing a washout of 14C that is much slower than that of the intravasal indicator or that of 36Cl-, (2) this carbonic anhydrase is not intra- but extracellular and presumably membrane-bound, and (3) its properties suggest that it is distinct from the well-known cytosolic carbonic anhydrases and represents a different isoenzyme.

Contractile function of papillary muscles with carbonic anhydrase inhibitors

Contractile parameters of directly stimulated rabbit papillary muscles were studied during incubation in baths containing the carbonic anhydrase inhibitors chlorzolamide (1*10(-3) M) or ethoxzolamide (1*10(-4) M). Both inhibitors caused an at least partly reversible decrease in isometric force as it has been observed in skeletal muscle, and--in contrast to the results in skeletal muscles--a decrease in time-to-peak and half-relaxation time. It is postulated that inhibition of the membrane-bound carbonic anhydrase of heart muscle might induce an intracellular acidosis and that this acidosis causes the observed effects on contractile parameters.

Effects of carbonic anhydrase inhibitors on oxygen consumption and lactate accumulation in skeletal muscle

In isolated rat soleus and extensor digitorum longus (EDL) muscles, the effects of carbonic anhydrase inhibitors were studied on oxygen consumption as well as lactate release and accumulation after incubation in inhibitors lasting long enough to produce marked changes in contractile parameters and in the concentrations of energy-rich phosphates. The inhibitors used were chlorzolamide (10(-3) M) and NaCNO (10(-2) M). Compared with control muscles, muscles treated with either of the two inhibitors showed a decrease in force, and an increase in time-to-peak as well as in relaxation time. Lactate content and release in soleus and in EDL were increased by factors of 2-3 with both inhibitors. With both inhibitors, oxygen consumption in the red soleus increased by approximately 27%, whereas in EDL, no significant change could be observed. The increase in aerobic metabolic rate in the red soleus only might indicate that the isozyme CA III, which is present only in this type of muscle, is in some way involved in keeping the oxygen consumption low. The increase in anaerobic metabolic rate occurring in both muscles can possibly be explained by increases in Pi and ADP.

Muscle Carbonic Anhydrases

In striated muscles, at least three types of carbonic anhydrase (CA) have been demonstrated: (1) a sulfonamide-resistant isozyme, CA III, which appears in the cytosol of red skeletal muscles,7,33,38 (2) a sulfonamide-sensitive cytosolic isozyme, most likely CA II,51 and (3) a membrane-bound form present in the sarcolemma16,23,57 and the sarcoplasmic reticulum (SR).5 Depending on the muscle type, these CA isozymes are variably present at these different sites in the muscle cell.