Cytosolic free magnesium concentration [Mg2+]; and its regulation were studied in cultured embryonic chicken heart cells by use of the fluorescent indicator 2-[2-(5-carboxy)oxazole]-5-hydroxy-6-aminobenzofuran-N,N,Otriacetic acid (Furaptra). The intracellular location ofFuraptra was confirmed by its complete release from cells upon addition of saponin. The basal [Mg2+J], which averaged 0.48 ± 0.03 mM (n = 31), increased 3-fold on perfusion with sodium-free solution. This increase could not simply be attributed to intracellular sodium-extracellular magnesium exchange be- Indeed, even the basal [Mg2+] level is controversial (7,8). Page and Polimeni (9) showed that the rate of 28Mg uptake in perfused rat heart is increased by increasing extracellular Mg2+ ([Mg2+]0) and that 28Mg efflux is also stimulated by increasing [Mg2+]0 (Mg2+-Mg2' exchange). In frog skeletal muscle, 28Mg uptake is increased in alkaline solution and depressed in acid solution (10). The properties of sodiummagnesium exchange have been studied in squid axon (11,12) and barnacle muscle fibers (13 tTo whom reprint requests should be addressed. 2981The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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To assess the role of pH in cellular Mg homeostasis, cytosolic pH (pHi) was manipulated by the NH4Cl prepulse technique; pHi, cytosolic Mg2+ (Mgi), and cytosolic Ca2+ (Cai) were measured fluorometrically in single cultured embryonic chicken heart cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), FURAPTRA, and fura-2, respectively. The basal values obtained were as follows: pHi = 7.21 +/- 0.10 (n = 7), [Mg]i = 0.51 +/- 0.08 mM (n = 9), [Ca]i = 126 +/- 15 nM (n = 7). When cells were perfused with 10 mM NH4Cl solution for 5 min, a transient alkalinization (0.53 U) of the cytosol was accompanied by a transient decrease (0.12 mM) in [Mg]i and a transient increase (59 nM) in [Ca]i; these changes approached control levels within 5 min. Upon removal of NH4Cl, a transient acidification (0.89 U) of the cytosol was accompanied by a transient increase (0.10 mM) in [Mg]i and a transient increase (125 nM) in [Ca]i; again, these changes returned toward control levels within 5 min. No significant changes in total cell Mg or Ca were observed during these manipulations. NH4Cl-evoked changes in [Mg]i were not altered significantly by either Mg-free or Ca-free conditions. Changes in [Mg]i were inversely correlated with changes in pHi and were not secondary to changes in [Ca]i. The results suggest that pHi modulates Mgi, probably by affecting cytosolic Mg binding and/or the transport of Mg across subcellular organelles.

A simple method for incorporating aequorin into mammalian cells to measure cytosolic ionized Ca2+ is described and compared with scrape loading and hypoosmotic treatment (HOST). The procedure consists of incubating the cells for 10 min and centrifuging them at 200 g for 30 s in the presence of aequorin. This method incorporates the same amount of photoprotein as scrape loading but 70% less than HOST. Cytosolic ionized Ca2+ has been measured in hepatocytes, kidney cells and tubules, macrophages, and cardiac myocytes loaded with aequorin by this new procedure.