2--7--bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl-ester and fura-2-am

Intestinal nutrient transport and sensing are of emerging interest in research on obesity and diabetes and as drug targets. Appropriate in vitro models are lacking that allow both, studies on transport processes as well as sensing and subsequent incretin hormone secretion including intracellular signaling. We here demonstrate that murine small-intestinal organoids are the first in vitro model system enabling concurrent investigations of nutrient and drug transport, sensing and incretin hormone secretion as well as fluorescent live-cell imaging of intracellular signaling processes. By generating organoid cultures from wild type mice and animals lacking different nutrient transporters, we show that organoids preserve the main phenotypic features and functional characteristics of the intestine. This turns them into the best in vitro model currently available and opens new avenues for basic as well as medical research.

Topics: Acids; Animals; Biological Transport; Calcium; Cell Survival; Fluoresceins; Fluorescence; Food; Fura-2; Incretins; Intestinal Mucosa; Intestine, Small; Intracellular Space; Mice, Inbred C57BL; Molecular Imaging; Organoids; Pharmaceutical Preparations; Reproducibility of Results

Changes in intracellular pH (pH(i)) and cytosolic calcium concentration ([Ca(2+)](c)) caused by the glutamate agonist domoate (DOM) were studied in single cultured mouse cerebellar granule cells (CGC) by using the fluorescent probes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and simultaneous evaluation of cytosolic calcium concentration with the fluorescent dye Fura-2 acetoxymethyl ester (Fura-2 AM). DOM caused a concentration-dependent increase in [Ca(2+)](c) and a concentration-dependent intracellular acidification of CGC. DOM-induced intracellular acidification was completely abolished by the use of Ca(2+)-free medium, suggesting that it was due mostly to an influx of extracellular calcium. The pH(i) decrease caused by DOM was also completely blocked in the presence of the AMPA/kainate receptor antagonist CNQX, indicating that the DOM-induced intracellular acidification was caused by DOM activation of the AMPA/kainate subtype of glutamate receptors. Different mechanisms that could be involved in DOM-induced pH(i) decrease, such as displacement of H(+) by Ca(2+) from a common intracellular binding site, DOM-induced alteration of pH(i) regulation mechanisms, and a possible acidification caused by DOM-induced increase of mitochondrial Ca(2+) uptake, were excluded. DOM-induced intracellular acidification was completely prevented by inhibitors of the plasma membrane calcium adenosine triphosphatase (ATPase) (PMCA), including orthovanadate, lanthanum extracellular pH of 8.5, and the specific PMCA inhibitor caloxin 2A1. Our results therefore indicate that PMCA is involved in DOM-induced intracellular acidification in primary cultures of CGC. Simultaneous recording of [Ca(2+)](c) and pH(i) indicates that the increase in intracellular calcium evoked by DOM will activate the calcium extrusion mechanisms through the calcium pump, which, in turn, will decrease intracellular pH by countertransport of H(+) ions.

Topics: Acid-Base Equilibrium; Animals; Calcium; Calcium-Transporting ATPases; Cell Membrane; Cells, Cultured; Cerebellum; Cytosol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Fluoresceins; Fluorescent Dyes; Fura-2; Glutamic Acid; Image Processing, Computer-Assisted; Kainic Acid; Mice; Neuromuscular Depolarizing Agents; Neurons; Receptors, AMPA

The effect of different neurotransmitters on the intracellular pH (pHi) and intracellular calcium (Ca2+i) was studied in cultured astrocytes from neonatal rat cerebellum, using the fluorescent dyes 2,7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) and Fura-2. Application of glutamate or kainate (100 microM) in a HEPES-buffered, CO2/HCO3(-) -free saline induced a decrease in pHi and an increase in Ca2+i. Amplitude and time course of the pHi and Ca2+i transients were different. Glutamate and kainate evoked a mean acidification of 0.22 +/- 0.05 (n = 29) and 0.20 +/- 0.09 (n = 12) pH units, respectively. The changes in pHi and Ca2+i induced by kainate, but not by glutamate, were inhibited by 6-cyano-7-dinitroquinozalin-2,3-dion (CNQX; 50 microM). In order to elucidate the mechanism of the agonist-induced acidification, whether the pHi changes were secondary to the Ca2+ rises was tested. In the absence of extracellular Ca2+, the kainate-induced Ca2+i transient was suppressed, while the intracellular acidification was only reduced by 13%. Removal of extracellular Ca2+ reduced the glutamate-induced pHi change by 8%, while the second component of the Ca2+i transient was abolished. Application of trans-( +/- )-1-amino-(1S,3R)-cyclopentadicarboxylic acid (t-ACPD, 100 microM), a metabotropic glutamate receptor agonist, and of noradrenaline (20 microM) evoked a Ca2+i increase, but no change of pHi. D-aspartate, which has a low affinity to glutamate receptors, but is known to be transported by the glutamate uptake system in some astrocytes, evoked an intracellular acidification, similar to that induced by glutamate, but no Ca2+i transient. The results suggest that the kainate-induced acidification is only partly due to the concomitant Ca2+i rise, while the glutamate/aspartate-induced acidification is mainly due to the activation of the glutamate uptake system.

Topics: Animals; Animals, Newborn; Aspartic Acid; Astrocytes; Calcium; Cells, Cultured; Cerebellum; Cycloleucine; Excitatory Amino Acid Agonists; Fluoresceins; Fluorescent Dyes; Fura-2; Glutamic Acid; Hydrogen-Ion Concentration; Kainic Acid; Neurotoxins; Norepinephrine; Rats; Receptors, Metabotropic Glutamate

This study reports on the changes in intracellular calcium concentration ([Ca2+]in) and intracellular pH ([pH]in) that occur in K562 target cells during interaction with human Natural Killer (NK) cells. The data were obtained using a quantitative fluorescence microscope and fluorescent ratio probes specific for [Ca2+]in (Fura-2-AM) and [pH]in (BCECF-AM). Results demonstrate that two types of target cell response to the attack by an NK cell can be distinguished. The target cell either dies immediately, due to the complete breakdown of the membrane impermeability, or the initial membrane damage (i.e., increased membrane permeability) is repaired and the cell "escapes" immediate death. During both responses an increase of [Ca2+]in takes place in the target cells. In the cells that die immediately, however, [Ca2+]in reaches higher levels (approximately 1,400 nM) than in the cells that restore the initial damage (approximately 700 nM). Changes in target cell [pH]in are also detected during both responses. The direction of the change (acidification or alkalinization) as well as the level of the change depend on extracellular pH ([pH]ex). Also, [pH]in remains changed during the time the cells were followed (10 min). The programming time (i.e., the time from the initiation of the cytotoxic process to the time that a change in the physiological parameter was detected) of the killing process that leads to an immediate target cell death appears to be shortest at [pH]ex 7.3-7.6 (approximately 3 min).

Topics: Calcium; Calibration; Cell Membrane Permeability; Cytotoxicity, Immunologic; Fluoresceins; Fluorescent Dyes; Fura-2; Homeostasis; Humans; Hydrogen-Ion Concentration; Intracellular Fluid; Killer Cells, Natural; Leukemia, Erythroblastic, Acute; Membrane Glycoproteins; Microscopy, Fluorescence; Neoplastic Stem Cells; Perforin; Pore Forming Cytotoxic Proteins; Tumor Cells, Cultured