14C Fixation by Leaves and Leaf Cell Protoplasts of the Submerged Aquatic Angiosperm Potamogeton lucens: Carbon Dioxide or Bicarbonate?

Carbon dioxide elimination by cardiomyocytes: a tale of high carbonic anhydrase activity and membrane permeability.

Carbonic anhydrase: chemistry, physiology, and inhibition.

Injection of carbonic anhydrase isoform II (CA) into Xenopus frog oocytes increased the rate of H+ flux via the rat monocarboxylate transporter isoform 1 (MCT1) expressed in the oocytes. MCT1 activity was assessed by changes of intracellular H+ concentration measured by pH-selective microelectrodes during application of lactate. CA-induced augmentation of the rate of H+ flux mediated by MCT1 was not inhibited by ethoxyzolamide (10 microM) and did not depend on the presence of added CO2/HCO3- but was suppressed by injection of an antibody against CA. Deleting the C terminus of the MCT1 greatly reduced its transport rate and removed transport facilitation by CA. Injected CA accelerated the CO2/HCO3(-)-induced acidification severalfold, which was blocked by ethoxyzolamide and was independent of MCT1 expression. Mass spectrometry confirmed activity of CA as injected into the frog oocytes. With pulldown assays we demonstrated a specific binding of CA to MCT1 that was not attributed to the C terminus of MCT1. Our results suggest that CA enhances MCT1 transport activity, independent of its enzymatic reaction center, presumably by binding to MCT1.

Photosynthetic bicarbonate utilization in Porphyra haitanen-sis (Bangiales, Rhodophyta)

The occurrence of an active CO2 transport system and of carbonic anhydrase (CA) has been investigated by mass spectrometry in the marine, unicellular rhodophyte Porphyridium cruentum (S.F. Gray) Naegeli and two marine chlorophytes Nannochloris atomus Butcher and Nannochloris maculata Butcher. Illumination of darkened cells incubated with 100 μM H13CO3− caused a rapid initial drop, followed by a slower decline in the extracellular CO2 concentration. Addition of bovine CA to the medium raised the CO2 concentration by restoring the HCO3−–CO2 equilibrium, indicating that cells were taking up CO2 and were maintaining the CO2 concentration in the medium below its equilibrium value during photosynthesis. Darkening the cell suspensions caused a rapid increase in the extracellular CO2 concentration in all three species, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. CA activity was detected by monitoring the rate of exchange of 18O from 13C18O2 into water. Exchange of 18O was rapid in darkened cell suspensions, but was not inhibited by 500 μM acetazolamide, a membrane‐impermeable inhibitor of CA, indicating that external CA activity was not present in any of these species. In all three species, the rate of exchange was completely inhibited by 500 μM ethoxyzolamide, a membrane‐permeable CA‐inhibitor, showing that an intracellular CA was present. These results demonstrate that the three species are capable of CO2 uptake by active transport for use as a carbon source for photosynthesis.

since the 1970s, over 200 studies with acetazolamide have shown it safe and 60–80% effective in acute mountain sickness (AMS). Despite much investigation, our understanding of its action in AMS remains incomplete and more complicated than generally taught. This should come as no surprise

By the process of pH polarity, several submersed angiosperms can use bicarbonate as carbon source for photosynthesis. Under conditions of relatively high light intensity and low CO2 availability, the pH of the apoplast and unstirred layer becomes acid at one side of the leaf and alkaline at the other. In the acid region, bicarbonate is converted into CO2, which diffuses into the leaf where it is fixed. Previous experiments on the light-dependent reduction of extracellular electron acceptors led to the hypothesis of redox regulation. Under conditions of high light and low CO2, excess reducing power in the chloroplast was supposed to be shuttled to the cytoplasm where it can upregulate the plasma membrane proton pump, leading to activation of polarity. Chlorophyll a fluorescence is an indicator for photosynthetic electron transport, the energization of thylakoids, and the reoxidation of chloroplast NADPH. It was used therefore to test redox regulation in vivo in Potamogeton lucens L. leaves. The fluoresence parameter, qP, an indicator for photochemical quenching and NADPH reoxidation, appeared to be rather insensitive to the inorganic carbon concentration and to the presence or absence of polarity. In contrast, qN, an indicator for non-photochemical quenching related to thylakoid energization, photoinhibition, and state transitions, increased under conditions of low CO2 - high light and polarity. Taken together the data show polarity to be an effective mechanism to make bicarbonate accessible as carbon source and seem to agree with the idea of redox regulation of pH polarity.Key words: bicarbonate utilization, chlorophyll a fluoresence, pH polarity, redox regulation, Potamogeton lucens, submerged aquatic macrophyte.

I was interested in the paper of Sellers et al. reporting on the ability of heat stable enterotoxin from E. coli allegedly to stimulate duodenal bicarbonate anion secretion (1). However, I believe the main conclusion is not supported by the data because the chosen physiological techniques do not prove that enhanced bicarbonate anion secretion occurred after the duodenum was exposed to the enterotoxin. Enhanced appearance of bicarbonate anion might arise by enhanced bicarbonate ion secretion but the more likely cause is cessation of hydrogen ion secretion, a known effect of STa that is likely to be the only effect of that enterotoxin (2, 3). The chosen physiological technique was in vivo perfusion followed by measurement of bicarbonate appearance in the lumen of the perfused anaesthetised mouse. Samples were taken and the bicarbonate ion concentration assayed indirectly by titration. The technique involves adding an amount of hydrochloric acid to the sample that will consume the bicarbonate that was present. Back titration with sodium hydroxide to the original sample pH, or even beyond it, estimates the amount of titratable buffer that was initially present but that was consumed by the hydrochloric acid by conversion into carbon dioxide. Any difference between the amount of HCl added and NaOH needed for the back titration allows calculation of the total amount of bicarbonate that was present. In order for this to be assayed directly and with even greater accuracy, any bicarbonate in the buffer that was converted into carbon dioxide could be measured as carbon dioxide. This was measured by the authors and there is no doubt that bicarbonate was converted into carbon dioxide, as sensed by the CO2 gas-sensing electrode. This means that the reader can be confident that bicarbonate in solution was accurately measured. However, the claim that titratable bicarbonate secretion was true bicarbonate secretion and not altered hydrogen ion secretion (Materials and Methods, “Measurement of HCO3 secretion in vivo,” paragraph 3, line 13) is a non-sequitur. The conversion of bicarbonate into carbon dioxide and the establishing that carbon dioxide was indeed formed does not by itself rule out changes in luminal bicarbonate concentration arising because of reduced hydrogen ion secretion. The authors perfused isotonic saline through the duodenum and over time, because of the concentration gradient between the lumen and the interstitial fluid or perhaps because of putative cellular bicarbonate secretion, the bicarbonate concentration increased from zero to the values recorded. In other experiments, the inclusion of STa enterotoxin in the luminal perfusate caused the bicarbonate concentration to increase still further. However, it is known that Na /H exchange occurs in the duodenum, where NHE:3 is present. Bicarbonate that enters the duodenum might be expected to react in part with secreted hydrogen ion so that the eventual bicarbonate concentration that is achieved is the result of appearance of bicarbonate anion and its removal by chemical reaction with secreted hydrogen ion. If the amount of secreted hydrogen ion is reduced by STa, a likely occurrence because it does stop luminal acidification, then it is to be expected that the bicarbonate ion concentration will be higher than normal in the perfused duodenum, as the authors probably showed. This is likely to be the explanation for the higher rates of bicarbonate appearance, given the known effects of STa. It is still possible that bicarbonate secretion is enhanced after STa exposure through the mechanism that the authors invoke but this is not something they have shown in their paper. I believe they have shown that STa reduces hydrogen ion secretion into the lumen and this is manifested by higher concentrations of bicarbonate ion. What they have not shown is that STa stimulates duodenal bicarbonate secretion, certainly not by simply verifying that bicarbonate is convertible into carbon dioxide, as their methods section states. This is a conclusion that goes beyond what the data can reasonably support. A further pharmacological argument in favour of hydrogen ion secretion not being involved is that some of experiments were done in the presence of amiloride. With amiloride inhibiting any hydrogen ion secretion, it might safely be concluded that any subsequent action of guanylin and STa might be restricted to enhanced secretion of bicarbonate ion. It is the case that amiloride might inhibit duodenal NHE:3, but evidence suggests that amiloride and its derivatives are only effective in moderate to low sodium ion containing perfusates (4). Amiloride at 1 mM concentration fails to affect the mucosal surface pH in rat proximal jejunum (5) and 100 uM ethyl-iso-propyl-amiloride (EIPA) fails to inhibit water absorption, whilst STa does inhibit water absorption in high sodium ion containing perfusates (6). Only when the sodium ion concentration is low can

The mechanism of action of sodium–glucose co‐transporter 2 inhibitors is similar to carbonic anhydrase inhibitors

Studies were performed to examine the possible effects of carbonic anhydrase (CA) inhibition on the glomerulotubular balance for bicarbonate in anesthetized dogs. Maximal CA inhibition was achieved by acetazolamide infusion and glomerular filtration rate (GFR) was reduced in a stepwise fashion by progressive clamping of the left renal artery. A close relationship (R equals 0.973) was maintained between the amount of filtered and reabsorbed bicarbonate in normal dogs with CA inhibition. A similar relationship was observed between GFR and bicarbonate reabsorption during CA inhibition in normal dogs (R equals 0.957) as well as in sodium bicarbonate loaded dogs (R equals 0.867). In these two groups, GFR in the clamped kidney was reduced to values ranging respectively from 99 to 5% and from 96 to 3%. Distal tubular blockade with ethacrynic acid and chlorothiazide, performed in normal dogs and in sodium bicarbonate loaded dogs, did not abolish glomerulotubular balance for bicarbonate during CA inhibition. This study demonstrates that the glomerulotubular balance for bicarbonate is maintained during CA inhibition whether or not distal tubular blockade is superimposed. A proportionate decrease in both fractions of bicarbonate reabsorption, either CA dependent or not mediated by CA, or an adaptive increase in the fraction of bicarbonate reabsorption not mediated by CA can explain the maintenance of glomerulotubular balance for bicarbonate.

Paradoxical effect of bicarbonate on cytoplasmic pH

Department of Clinical Pharmacology, United Medical and Dental Schools, Guy's Hospital, London, England

Calcification and ocean acidification: new insights from the coccolithophore <i>Emiliania huxleyi</i>

Carbonic anhydrase (CA) is a metalloenzyme that catalyzes the interconversion between carbon dioxide and water and dissociated ions of carbonic acid. In addition, CA performs various other functions in animals and plants, depending on the part of the living being. CAs have been found in almost all organisms. Besides, CAs are associated with several diseases, such as glaucoma, obesity, epilepsy, cancer, and so on. CAs are also involved in tumor cell growth and angiogenesis. Thus, inhibition of CA may be an attractive way of control of such diseases. Hence, CA inhibitors have been designed and developed to cure CA-associated diseases. Some examples of approved CA inhibitors are dorzolamide, methazolamide, brinzolamide, and dichlorphenamide. Furthermore, various heterocyclic scaffolds were utilized for the design of CA inhibitors. Among those, pyrazole/pyrazoline derivatives have exhibited greater potency toward CA inhibition. Hence, research that took place in the field of drug design and discovery of CA inhibition has been systematically reviewed and collated. Alongside, the structure-activity relationship has been described, followed by a description of the most potent molecules and their structural features.

The most troublesome factors for the biologist to measure accurately, or even to estimate, are carbon dioxide tension and free carbon dioxide, bicarbonates and carbonates in solution in natural waters. In order to obtain accurate knowledge of the conditions of natural waters these factors must be determined in the field, unless all necessary precautions are taken during the collection and preservation of samples to avoid any change in the factors from those in the field. This is extremely difficult and verges upon impossibility, as will be shown in later discussion. The importance of determining these factors as they exist in the field should not be minimized. One needs only to consult any good text-book on physiology to be convinced of the importance of the carbon dioxide partial pressure of the alveolar air of lung-breathing animals in maintaining a constant alkalinity of the blood: in short, the importance, in the respiratory function, of the carbon dioxide tension of the blood of animals possessing a hemoglobin system of transporting oxygen from the respiratory organs to the tissues of the body. The study of the effect of the carbon dioxide partial pressure of alveolar air on the physiology of respiration of lung-breathing animals has been an important field of investigation. On the other hand, the investigation of the effect of a rapid variation of the carbon dioxide tension of the medium surrounding gill-breathing animals has been much neglected. Doubtless the reason for this neglect is the great difficulty in determining accurately the carbon dioxide tension of water, and the lack of a simple method by which it can be determined rapidly. When the literature is examined it is found that the methods in vogue for the determination of carbon dioxide tension, free carbon dioxide, bicarbonates and carbonates, must be employed with caution. Johnston ('i6) has given an excellent criticism of the standard methods employed in water analysis. The law of mass action has been applied, and formulae have been developed, for the purpose of calculating the values of one or more of the factors from the known values of other factors, that is, we are here dealing