Simultaneous measurements of cytosolic pH and calcium interactions in bovine lactotrophs using optical probes and four-wavelength quantitative video microscopy.
The properties of pH and calcium homeostasis have been investigated in single bovine lactotrophs by the use of the fluorescent indicators 2',7'-bis(carboxyethyl)-carboxyfluorescein (BCECF) and fura-2 respectively. A method of simultaneous recording from both dyes loaded in the same cell was used. Despite slight crosstalk between the two dyes, physiologically relevant information about the interrelationship between pH and calcium homeostasis was obtained. Three types of interactions were recorded. First, an increase in calcium due to the discharge of intracellular stores by thyrotropin-releasing hormone resulted in no change in cytosolic pH. Secondly, alkalinization by the addition of a weak base, NH4Cl, induced a large transient (around 1000 nM) and a small (a few tens of nanomoles per liter) sustained increase in cytosolic calcium. The former is partly due to release from agonist-sensitive stores. Thirdly, upon the removal of NH4Cl the cytoplasm became acidic, which induced a release of calcium from intracellular stores in some cells. In addition we demonstrate that the recovery from acid load is sensitive to extracellular Na+, suggesting the presence of Na+/H+ exchange mechanisms in bovine lactotrophs. Interestingly we have also found that, at rest, removal of Na+ from the bathing medium results in a decrease in resting [Ca2+]i, paralleled by a reduction in pHi. This suggests a role for Na+/H+ exchange in determining resting [Ca2+]i.
Zorec
R
R
Department of Neurobiology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, UK.
Hoyland
J
J
Mason
W T
WT
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D003600:Cytosol / Q000378:metabolism; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016257:Fura-2; D006863:Hydrogen-Ion Concentration; D008297:Male; D008853:Microscopy; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D014741:Video Recording
D005452:Fluoresceins; D005456:Fluorescent Dyes; D000643:Ammonium Chloride; D013973:Thyrotropin-Releasing Hormone; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D002118:Calcium; D016257:Fura-2