Intracellular Ca²⁺ regulation in hepatocytes under experimental transplantation conditions

Abstract

Under transplant conditions excessive accumulation of intracellular calcium ([Ca²⁺]_i) is considered to be a mediator of cell injury during ischaemia and re-oxygenation. To clarify this consideration as well as the necessity of calcium-free preservation solutions, we used a well-known in-vitro model. Furthermore, a new application to mimic clinical situation was established, and we evaluated the correlation between [Ca²⁺]_i change and cell survival in monolayers of isolated rat hepatocytes undergoing cold hypoxia in defined solutions and during re-oxygenation. [Ca²⁺]_i was measured in single cells by ratio imaging of Fura-2 fluorescence after various periods of cold hypoxia (ischaemia phase) in different preservation solutions [UW, HTK, EC and Krebs–Henseleit buffer (KH)] and following warm normoxic reperfusion (re-oxygenation phase) with KH. Cell survival was measured simultaneously by trypan-blue exclusion. Cell survival decreased, depending on preservation solution and preservation time. The partially tremendous [Ca²⁺]_i change under cold hypoxia did not correlate with the change in cell survival. For example, UW-stored cells showed a [Ca²⁺]_i loss from 280 nM to 56 nM, compared with KH-stored cells with a [Ca²⁺]_i increase of up to 445 nM. Our results indicate that [Ca²⁺]_i plays only a minor role in the pathomechanisms of hypoxic and re-oxygenation hepatocellular injury.