In situ measurement of pH in the secreting canaliculus of the gastric parietal cell and adjacent structures.

The gastric H(+)/K(+)-ATPase is located within an infolding (secretory canaliculus) of the apical plasma membrane of gastric parietal cells. Our aim was to measure the pH values in the cytosol and canaliculus of the acid-secreting parietal cell and the adjacent gland lumen in situ. We used ultrafine double-barreled tip-sealed microelectrodes at high acceleration rates for intracellular and canalicular measurements. Immunohistochemical staining of the parietal cells was used to identify the track of the electrode and to estimate the position of the electrode tip at the time of the last intracellular measurement. En route to the deepest regions of the mucosa, where the average gland lumen pH was approximately 3, and on advancing in steps of 2 mum, the electrode entered the cytosol of the parietal cells, where the pH value was 7.4. Advancing the electrode further resulted, in several instances, in a sharp decrease in pH to an average value of 1.7 +/- 0.2, which we interpreted as the measurement within the canaliculus. When the electrode was advanced even further, the pH reading returned to the cytosolic value. From the difference in pH between the secreting canaliculus and the adjacent gland lumen, we concluded that the released acid was immediately buffered. Thus, the only cellular structure directly exposed to the highly acidic canalicular content is the apical membrane forming the canaliculus in the parietal cell.

Gastric antibacterial efficiency is different for pepsin A and C.

The gastric lumen represents a bactericidal barrier, whose major components are an acidic pH and a family of isoenzymes of the gastric aspartate protease, pepsin. To evaluate whether specific pepsins are specialized in antibacterial protection, we tested their effects on the gastric pathogen Helicobacter pylori. In a recent study we found pepsin to affect the motility of the bacteria, one of its most important virulence factors. We were able to show that the antibacterial effect of pepsin occurs in two phases: rapid loss of motility and subsequent destruction. In the present study we used the rapid pepsin-induced bacterial immobilization as a marker of antibacterial efficiency. The proteolytic activity of different pepsins was normalized to values between 2 and 200 U/ml in the hemoglobin degradation test of Anson, performed at pH 2 and 5. We found that pepsin C completely inactivates H. pylori at proteolytic activities of 2 (pH 5) and 20 (pH 2) U/ml. In contrast, the activities of pepsin A and chymosin required to affect Helicobacter motility were ten times higher.

Significance of extracellular potassium in central respiratory control studied in the isolated brainstem-spinal cord preparation of the neonatal rat.

The significance of extracellular potassium in central respiratory control was investigated using the isolated brainstem-spinal cord preparation of the neonatal rat. Depth profiles of extracellular potassium activity ([K+])ECF in the medulla were measured with ion-sensitive microelectrodes. Although [K+]ECF increased with depth in medullary tissue during control (4 mM) and low (1 mM) potassium concentration ([K+])CSF superfusion, this gradient disappeared with higher [K+]CSF. With low [K+]CSF (1 mM), respiratory CO2 responsiveness was abolished, and increased with high [K+]CSF (8 mM). Respiratory frequency (fR) was diminished at low [K+]CSF (1 mM), and increased with elevated [K+]CSF (8 and 16 mM); with yet higher [K+]CSF (32 mM) apnea occurred after a transient increase in fR. Perforated patch recording revealed that high [K+]ECF decreased membrane resistance, depolarized membrane potential, and increased firing frequency in most of the recorded medullary neurons. High [K+]ECF also increased excitatory and inhibitory post-synaptic potentials of medullary neurons and augmented the functional connectivity among neurons. It is concluded that [K+]ECF is of importance in the maintenance of respiratory rhythm and central chemosensitivity.

Rapid loss of motility of Helicobacter pylori in the gastric lumen in vivo.

The human pathogen Helicobacter pylori has infected more than half of the world's population. Nevertheless, the first step of infection, the acute colonization of the gastric mucus, is poorly understood. For successful colonization, H. pylori must retain active motility in the gastric lumen until it reaches the safety of the mucus layer. To identify the factors determining the acute colonization, we inserted bacteria into the stomach of anesthetized Mongolian gerbils. We adjusted the gastric juice to defined pH values of between 2.0 and 6.0 by using an autotitrator. Despite the fact that Helicobacter spp. are known to survive low pH values for a certain time in vitro, the length of time that H. pylori persisted under the assay conditions within the gastric juice in vivo was remarkably shorter. In the anesthetized animal we found H. pylori to be irreversibly immotile in less than 1 min at lumen pH values of 2 and 3. At pH 4 motility was lost after 2 min. However, the period of motility increased to more than 15 min at pH 6. Blocking pepsins in the gastric lumen in vivo by using pepstatin significantly increased the period of motility. It was possible to simulate the rapid in vivo immotilization in vitro by adding pepsins. We conclude that pepsin limits the persistence of H. pylori in the gastric chymus to only a few minutes by rapidly inhibiting active motility. It is therefore likely that this short period of resistance in the gastric lumen is one of the most critical phases of Helicobacter infection.

The spatial orientation of Helicobacter pylori in the gastric mucus.

The highly motile human pathogen Helicobacter pylori lives deep in the gastric mucus layer. To identify which chemical gradient guides the bacteria within the mucus layer, combinations of luminal perfusion, dialysis, and ventilation were used to modify or invert transmucus gradients in anaesthetized Helicobacter-infected mice and Mongolian gerbils. Neither changes in lumen or arterial pH nor inversion of bicarbonate/CO2 or urea/ammonium gradients disturbed Helicobacter orientation. However, elimination of the mucus pH gradient by simultaneous reduction of arterial pH and bicarbonate concentration perturbed orientation, causing the bacteria to spread over the entire mucus layer. H. pylori thus uses the gastric mucus pH gradient for chemotactic orientation.