Acute Effects of Cell Isolation on InsP Profiles in Adult Rat Cardiomyocytes

The isolation and culture of adult rat cardiomyocytes was shown to cause major changes in the contents of [3H]-labeled inositol phosphates and inositol phospholipids. Undigested heart tissue contained high levels of [3H]Ins(1,4,5)P3(5364±800 ct/min/g tissue, 80±12 ct/min/mg protein) and mass content averaged 13.8 nmol/g tissue or 208±36 pmol/mg protein (mean± ,n=4). After collagenase digestion, [3H]Ins(1,4,5)P3was undetectable and the mass content of Ins(1,4,5)P3had decreased to 0.8±0.2 pmol/mg protein (mean± ,n=4,P<0.01). [3H]Ins(1,4)P2was reduced by 80% and [3H]PtdIns(4,5)P2by 90%. These profiles remained essentially unchanged when the isolated cells were maintained in culture for up to 24 h, even though the inositol phosphate response remained sensitive to norepinephrine. Similar to findings in intact tissue, the inositol phosphate response to norepinephrine in these cells was inhibited by neither U-73122 (5μ ) nor by neomycin (5 m ). By 48 h in culture, the relative levels of [3H]Ins(1,4,5)P3and [3H]Ins(1,4)P2had increased in relation to the total inositol phosphate content and responses appeared to better reflect intact tissue. However, while retaining insensitivity to neomycin, cells at 48 h were fully sensitive to U-73122 (5μ ). These data demonstrate that altered inositol phosphate responses are observed in adult cardiomyocytes from the time of isolation and that while the profiles change over time in culture, a pattern similar to that in intact heart is not re-established.