Simultaneous measurement and imaging of intracellular Ca2+ and H+ transport in isolated rabbit gastric glands

Abstract

1. Activation of H(+) secretion in the intracellular canaliculi of parietal cells occurs on an unknown time scale with ill-defined kinetics for the coupling of H(+) secretion and the elevation of intracellular calcium ([Ca(2+)](i)) stimulated by secretagogues. 2. We developed a method to determine H(+) secretion in isolated rabbit gastric glands with spatio-temporal resolution, using the fluorescent indicator Lysosensor Yellow-Blue (LYB). Glands accumulated the dye exclusively in the intracellular canaliculi of parietal cells and the gland lumen. Dye fluorescence in the acid spaces of the glands increased upon stimulation of acid secretion by carbachol, histamine and forskolin. Simultaneous fluorescence measurements of acid secretion and [Ca(2+)](i) at 1 s resolution were made by joint loading of LYB and Fluo-3. 3. Carbachol-stimulated H(+) secretion was detected in the gland lumen as early as 3 s after the onset of the [Ca(2+)](i) spike. H(+) accumulation appeared to be transient and paralleled the release component of the [Ca(2+)](i) spike. Short and repetitive stimulations with carbachol elicited repetitive responses in [Ca(2+)](i) and H(+) secretion. 4. Histamine or forskolin stimulated H(+) secretion with a delayed onset (around 2 min) and a sustained response. Acid secretion was temporally unrelated to the oscillatory Ca(2+) responses. 5. The striking difference in the kinetics of activation of H(+) secretion by cholinergic and cAMP-dependent secretagogues indicates that two distinct mechanisms are operating in the final stimulation of the pump, in spite of both eliciting a [Ca(2+)](i) response.

Secretory state regulates Zn2+ transport in gastric parietal cell of the rabbit

“…As shown in Fig. 5, fluorescence of the reporter LysoSensor DND-160 at different emission wavelengths (460 nm or 528 nm) behaved in accordance with the recognized responses (3,31) and suggested the feasibility of emission ratiometric reporting. In control studies, no fluorescence was observed when TVs were not present, indicating that the measurements were not attributable to residual dye.…”

“…To perform these studies, the fluorescent reporter LysoSensor DND-160 was used to monitor acidity within isolated TVs. Initially, we evaluated sequestration of this reporter and its ability to monitor pH ratiometrically in isolated TVs (excitation 360 nm; emission at 460 nm and 528 nm) (3,6,19,31). In these protocols, the ratio of emission intensities (460 nm/ 528 nm) was measured in TVs (8-to 10-mg aliquots) loaded with the reporter in 96-well plates and then exposed to ICB containing the protonophores FCCP (20 M) and nigericin (7 M), adjusted to clamp pH levels over the range 3.4 -7.4.…”

Heterogeneity of acid secretion induced by carbachol and histamine along the gastric gland axis and its relationship to [Ca2+]i

“…In our previous work we characterized the temporal relationship in real time between acid secretion and Ca 2ϩ changes stimulated by carbachol and histamine observing a striking difference in the kinetics of activation of H ϩ secretion. The acid response to a pulse of carbachol was fast, small, transient, and synchronized with the Ca 2ϩ spike, whereas histamine induced a slow, large, and sustained H ϩ secretion with a long lag time (140 s), which was unrelated to the Ca 2ϩ signal (36). Since the activation of HCl secretion by both types of secretagogues involves the translocation of H ϩ /K ϩ pumps contained in tubulovesicular structures to the plasma membrane and the activation of K ϩ and Cl Ϫ channels (41,46), this raises the question as to which are the coupling mechanisms between the elevation of different second messengers and the activation of secretion.…”

“…However, measurements of gastric secretion were determined in separate experiments using a radiolabel method with low temporal resolution. To improve the measurement of the kinetics of acid secretion and its relationship to [Ca 2ϩ ] I , we previously developed a methodology using fluorescent dyes to simultaneously measure [Ca 2ϩ ] i and secretion (36). In our previous work we characterized the temporal relationship in real time between acid secretion and Ca 2ϩ changes stimulated by carbachol and histamine observing a striking difference in the kinetics of activation of H ϩ secretion.…”

“…Acetylcholine binds to M 3 -muscarinic receptors leading to increases in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) (1,7). Histamine binds to H 2 receptors resulting in the elevation of both [Ca 2ϩ ] i and cyclic AMP (cAMP) (8,10,27,36).…”