Influence of cell isolation and incubation procedures on Ca2+ dependence of glucose transport in isolated cardiac myocytes.
Insulin stimulates hexose transport in isolated myocytes of adult rats but data are conflicting regarding the Ca2+-dependence of this effect, demonstrated previously in intact heart muscle. 3-O-methyl-D-glucose and 2-deoxy-D-glucose transport was compared in cells prepared and incubated by different published procedures. Ca2+-dependence of hexose transport stimulation by insulin was found only with incubation in a modified Joklik tissue culture medium buff red with bicarbonate at pH 7.4. At pH 7.0 or when bicarbonate was replaced by N-2-hydroxyethylpiperazine-N'-2 ethane sulfonic acid (HEPES) stimulation by insulin was not Ca2+-dependent, nor was there a Ca2+-dependent increment in insulin effect in physiological saline media regardless of pH or buffer (HEPES, 3-[N-morpholino] propanesulfonic acid (MOPS) or bicarbonate). The ATP content of cells incubated in HEPES or MOPS media was reduced. Ca2+-dependence seems to require the presence of bicarbonate and another factor(s) in tissue culture medium. The response is also influenced by the isolation procedure and may be related in part to preservation of an intact glycocalix. Thus, details in cell isolation and incubation procedures may strongly influence the behaviour of the cells. Isolated cardiac myocytes are acceptable as a valid model only if it can be demonstrated in each specific case that the characteristics present in intact tissue are maintained unaltered in the isolated cells.
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000502:physiology*; D002469:Cell Separation / Q000379:methods*; D005947:Glucose / Q000378:metabolism*; D006601:Hexoses / Q000378:metabolism; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D008297:Male; D003012:Microbial Collagenase / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains; D012508:Sarcolemma / Q000378:metabolism
D006601:Hexoses; D007328:Insulin; D003012:Microbial Collagenase; D005947:Glucose; D002118:Calcium