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Is an increase in duodenal bicarbonate concentration after STa really enhanced bicarbonate ion secretion?

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