Measurement of intracellular ionized magnesium concentration in myocytes isolated from the septomarginal band of sheep hearts.
The preparation by collagenase dispersion is described of isolated, calcium-tolerant myocytes from the septomarginal (moderator) band dissected from the sheep heart. Cells obtained were small rods (85 x 9 microns), with pronounced striations characteristic of cardiac myocytes. Isolated cells were loaded with the fluorescent ion-sensitive probes Mag-fura-2 or fura-2, for use in microspectrofluorimetry experiments to measure cytosolic free magnesium ([Mg2+]i) and calcium ([Ca2+]i) respectively; cells remained usable for up to 8 h after isolation. Glycolysis and electron transport were inhibited by a short exposure (4 min) to deoxyglucose (15 mM) and cyanide (2 mM), added simultaneously. This appeared to produce a small, but not statistically significant (P = 0.056) rise in [Mg2+]i, presumably resulting from Mg2+ liberated following consumption of MgATP. Inhibition of the Na pump by strophanthidin (20 microM), followed by removal of external Na in the presence of strophanthidin, caused an increase in both [Mg2+]i and [Ca2+]i. The time course of changes in the two ions were dissimilar, so it seems unlikely that the observed rise in the [Mg2+]i was due solely to a direct effect of [Ca2+]i on Mag-fura-2 or due to the displacement of [Mg2+]i by Ca from binding sites. Evidence is presented that suggests sodium-magnesium exchange plays a role in the regulation of myocyte [Mg2+]i.
Gow
I F
IF
Department of Physiology, University Medical School, Edinburgh, UK.
Latham
T
T
Ellis
D
D
Flatman
P W
PW
D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D003486:Cyanides / Q000494:pharmacology; D003847:Deoxyglucose / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D016257:Fura-2; D006019:Glycolysis / Q000187:drug effects; D006329:Heart Conduction System / Q000187:drug effects; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D011690:Purkinje Fibers / Q000187:drug effects / Q000378:metabolism; D012756:Sheep; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D013050:Spectrometry, Fluorescence
D000963:Antimetabolites; D003486:Cyanides; D003847:Deoxyglucose; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D016257:Fura-2