Activation of connexin-43 hemichannels can elevate [Ca2+]i and [Na+]i in rabbit ventricular myocytes during metabolic inhibition

ATP depletion due to ischemia or metabolic inhibition (MI) causes Na+ and Ca2+ accumulation in myocytes, which may be in part due to opening of connexin-43 hemichannels. Halothane (H) has been shown to reduce conductance of connexin-43 hemichannels and to protect the heart against ischemic injury. We therefore investigated the effect of halothane on [Ca2+](i) and [Na+](i) in myocytes during MI. Isolated rabbit left ventricular myocytes were loaded with 4 muM fluo-3 AM for 30 min, or with 5 mum sodium green AM for 60 min at 37degreesC. After washing, the myocytes were exposed to: (1) Normal HEPES solution; (2) MI solution (2 mM NaCN, 20 mM 2-deoxy-D-glucose and 0-glucose); or (3) MI + H (0.95 mm, 4.7 mm) for 60 min. Propidium iodide (PI, 25 muM) was added to all samples before data acquisition. The fluorescence intensity was measured by flow cytometry with 488 nm excitation and 530 nm emission for fluo-3 or sodium green, and 670 nm for PI, ;The [Ca2+](i) and [Na+](i) were then calculated by calibration. In some experiments, the effect of 10 mum tetrodotoxin (TTX) and 20 muM nifedipine (NIF) were studied. Metabolic inhibition for 60 min caused a significant increase in [Ca2+](i) and [Na+](i) in myocytes when compared to controls, which was significantly reduced by halothane in a dose-dependent fashion. In the presence of TTX and NIF, halothane also significantly reduced the rise in the [Ca2+](i) and [Na+](i) in myocytes subjected to MI. 1-heptanol, another gap junction blocker, had similar effects. Thus, halothane reduced [Ca2+](i) and [Na+](i) overload produced by MI in myocytes. This effect is not solely due to block of voltage-gated Na+ and Ca2+ channels, and is likely mediated by inhibiting the opening of connexin-43 hemichannels. (C) 2001 Academic Press.