M. J. Barahona1, J. Ollodart1, T. M. Gisinger1,3, V. M. Baratta1, Y. W. Stroehl1,2, D. Mulligan1, J. P. Geibel1,4 1Yale University, School of Medicine,Department Surgery,New Haven, CT, USA 2Charité University Medicine Berlin,Faculty Of Medicine,Berlin, BERLIN, Germany 3Paracelsus Medical University,Department Of Medicine,Salzburg, SALZBURG, Austria 4Yale University School Of Medicine,Department Of Cellular And Molecular Physiology,New Haven, CT, USA
Introduction: Gastroesophageal reflux disease is a prevalent chronic disorder, yet symptom management is often difficult to achieve or maintain. Traditional treatments involve proton pump inhibitors and H2-receptor antagonists, though many patients have persistent symptomatology. Recently, an unaccounted apical vacuolar H+-ATPase was identified and found to be activated in the absence of H,KATPase activity. This H+-ATPase is activated by the calcium-sensing receptor (CaSR), which is in turn modulated by the calcium-activated chloride channel (CaCC). Production of gastric acid via this mechanism can be inhibited by blocking gastric CaCC. Tannic acid has been shown to block the CaCC in other organ systems. Here, we demonstrate that modulation of CaSR via the calcimimetic R568 and CaCC, via tannic acid can indirectly influence acid secretion through the vacuolar H+-ATPase.
Methods: Gastric glands from rats were isolated via a hand dissection technique. The individual glands were then perfused in vitro with a K+-free HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) to suppress H,KATPase activity. The control group was perfused with 400 nM R568 hydrochloride, a positive allosteric modulator and agonist of CaSR, while the experimental group was perfused with 400 nM R568 hydrochloride and 200 µM tannic acid. Next, the extracellular calcium concentration was elevated to 5.0 mM in the presence and absence of R568 and tannic acid. The H+-ATPase activity was selectively monitored with the rate of proton extrusion (ΔpH/min) on individual parietal cells by observing a change and recovery rate of intracellular pH after acid loading the cells with various solutions, leaving only the H+-ATPase as an active proton secretory pathway.
Results: After exposure to R568 in glands from fasted animals there was increased vacuolar H+-ATPase activity with normal extracellular (1.0 mM) Ca2+ with a rate of proton extrusion increase of 0.01255 ± 0.00120 ΔpH/min. Elevations in Ca2+ concentration (1.0 mM to 5.0 mM) caused a further increase in K+-independent H+ secretion 0.01621 ± 0.00087 ΔpH/min. When tannic acid, an inhibitor of CaCC was added to the perfusates with 1.0 mM Ca2+ and 5.0 mM Ca2+, there was a significant inhibition of proton secretion under all conditions with a lower proton extrusion relative to the control 0.00296 ± 0.00027 ΔpH/min (p<0.0001) and 0.00409 ± 0.00053 ΔpH/min (p<0.0001), respectively.
Conclusion: An increase in extracellular calcium concentration leads to CaSR stimulation and elevated H+-ATPase activity. In this study, we demonstrate tannic acid-induced inhibition of the H+-ATPase through inactivation of the CaCC. The inhibition of H+-ATPase activity along with H,KATPase activity theoretically may lead to complete blockade of gastric acid production under resting and stimulated conditions. The CaSR along with CaCC can be important new pharmacologic targets to suppress acid secretion.