Monitoring of intracellular ammonium in perfused rat salivary gland by nitrogen-14 nuclear magnetic resonance spectroscopy.
We have observed the changes in the intracellular ammonium (NH4+) content and the intracellular pH during administration of 20 mM NH4Cl (the ammonium pulse experiment) using nitrogen-14 and phosphorus-31 nuclear magnetic resonance spectroscopy (14N and 31P NMR) at 8.45 T. In the isolated perfused rat mandibular salivary gland, resonances of trimethylamines (-328 p.p.m.) and betaine (-329 p.p.m. from the resonance of NO3-) were detected. A chemical shift reagent, 10 mM of dysprosium triethylenetetramine-N,N,N',N",N"',N"'-hexaacetic acid (Dy(TTHA], was used to discriminate between the resonances from the extracellular NH4+ (-352 p.p.m.) and the intracellular NH4+ (-355 p.p.m.). During the NH4Cl application, the intracellular NH4+ content [( NH4+]i) increased quickly to ca. 50 mmol per litre intracellular fluid (ICF), then increased gradually to ca. 70 mmol per litre ICF. The intracellular pH (pHi), calculated from the 31P chemical shift of inorganic phosphate, increased transiently by 0.5 pH units and then decreased gradually in spite of the high level of [NH4+]i. The initial increase of [NH4+]i, which was observed by 14N NMR, was larger than that calculated from the intracellular pH on an assumption of a non-ionic diffusion process for ammonia. These results suggest a possibility of influx of NH4+, and also suggest an activation of cellular buffering mechanism that extrudes the excess bases from the cells.
Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Japan.
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Murakami
D000641:Ammonia / Q000378:metabolism*; D000643:Ammonium Chloride / Q000378:metabolism*; D000818:Animals; D004492:Edetic Acid / Q000031:analogs & derivatives; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008345:Manganese / Q000494:pharmacology; D009584:Nitrogen; D010477:Perfusion; D010758:Phosphorus; D051381:Rats; D011919:Rats, Inbred Strains; D012044:Regression Analysis; D013363:Submandibular Gland / Q000378:metabolism*; D014783:Viscosity
D000643:Ammonium Chloride; D010758:Phosphorus; D008345:Manganese; D000641:Ammonia; C026059:triethylenetetraminehexaacetic acid; D004492:Edetic Acid; D009584:Nitrogen