Secretagogue‐evoked changes in intracellular free magnesium concentrations in rat pancreatic acinar cells.

Abstract

SUMMARY1. This study employs the fluorescent dye Mag-Fura-2 acetoxymethyl ester (AM) to measure intracellular free magnesium concentration [Mg2+]i in isolated rat pancreatic acinar cells. Initially a number of protocols were investigated to develop optimal loading conditions for the dye Mag-Fura-2 AM. The procedure yielding cells which showed minimal dye loss and no adverse compartmentalization was adopted for subsequent experiments.

Intracellular magnesium transport and regulation in epithelial secretory cells

“…The decrease revealed a dosedependency with secretagogues, and atropine blocked the ACh-evoked response. Control experiments indicated that the secretagogues-induced decrease can not be attributed to a bleaching effect caused by the excitation beam, which together with the dose-dependency and the effect of atropine confirms that the observed response is not artefactual (40). Different doses of adrenaline, noradrenaline and histamine were also examined, but no changes in [Mg 2+ ] i were seen (40,42 -mobilising agents such as ACh and CCK-8, it was decided to make this point clearer by co-loading either Fura-2 or Magfura-2 loaded cells with BAPTA-AM, a Ca 2+ chelator (46).…”

“…Control experiments indicated that the secretagogues-induced decrease can not be attributed to a bleaching effect caused by the excitation beam, which together with the dose-dependency and the effect of atropine confirms that the observed response is not artefactual (40). Different doses of adrenaline, noradrenaline and histamine were also examined, but no changes in [Mg 2+ ] i were seen (40,42 -mobilising agents such as ACh and CCK-8, it was decided to make this point clearer by co-loading either Fura-2 or Magfura-2 loaded cells with BAPTA-AM, a Ca 2+ chelator (46). At a BAPTA concentration equal to 10 µM, which enabled a small rise in [Ca 2+ ] i , not enough to interfere with Magfura, the typical decrease in [Mg 2+ ] i was found after the addition of CCK-8 but the initial rise was no longer present, indicating that it was an artifact.…”

“…The affinity of Magfura-2 for Ca 2+ is not very different from that for Mg 2+ (29) and several authors have indicated the existence of a clear interference from this source (54,55 (40,41) or single cell studies (46,51). In addition, different approaches can be undertaken, including perfusion of the tissue (28,46) or the employment of static-volume cuvettes (42,43).…”

“…A suitable method for loading pancreatic acinar cells was developed in our laboratory. The protocol adopted, which is described in detail elsewhere (40), enables sufficient dye to be loaded into cells to give fluorescence readings. Loading times are short and no detectable compartmentalization is evident.…”

“…Our experiments using either a suspension of rat pancreatic acinar cells or single mouse acinar cells loaded with Magfura-2 AM showed that acetylcholine (ACh), carbamylcholine cholecystokinin-octapeptide (CCK-8), thapsigargin and ionomycin, all well known Ca 2+ -mobilising agents, induce marked changes in [Mg 2+ ] i , consisting of a sharp and transient rise, immediately followed by a gradual decline to reach a steady-state significantly lower than the resting value, which is maintained throughout the experiment (40)(41)(42)(43)45,46,102,127,130). The decrease revealed a dosedependency with secretagogues, and atropine blocked the ACh-evoked response.…”