ACUTE EFFECTS OF CELL ISOLATION ON INSP PROFILES IN ADULT-RAT CARDIOMYOCYTES

The isolation and culture of adult rat. cardiomyocytes was shown to ca use major changes in the contents of [H-3]-labeled inositol phosphates and inositol phospholipids. Undigested heart tissue contained high le vels of [H-3]Ins(1,4,5)P, (5364 +/- 800 ct/min/g tissue, 80+/-12 ct/mi n/mg protein) and mass content averaged 13.8 nmol/g tissue or 208 +/- mol/mg protein (mean+/-S.E.M., n=4). After collagenase digestion, [H-3 ]Ins(1,4,5)P-3 was undetectable and the mass content of Ins(1,4,5)P-3 had decreased to 0.8+/-0.2 pmol/mg protein (mean+/-S.F.M., n=4, P<0.01 ). [H-3]Ins(1,4)P-2 was reduced by 80% and [H-3]PtdIns(4,5)P-2 by 90%. These profiles remained essentially unchanged when the isolated cells were maintained in culture for up to 24 h, even though the inositol p hosphate response remained sensitive to norepinephrine. Similar to fin dings in intact tissue, the inositol phosphate response to norepinephr ine in these cells was inhibited by neither U-73122 (5 mu M) nor by ne omycin (5 mM). By 48 h in culture, the relative levels of [H-3]Ins(1,4 ,5)P-3 and [H-3]Ins(1,4)P-2 had increased in relation to the total ino sitol phosphate content and responses appeared to better reflect intac t tissue. However, while retaining insensitivity to neomycin, cells at 48 h were fully sensitive to U-73122 (5 mu M). These data demonstrate that altered inositol phosphate responses are observed in adult cardi omyocytes from the time of isolation and that while the profiles chang e over time in culture, a pattern similar to that in intact heart is n ot re-established. (C) 1997 Academic Press Limited.