Subcellular and multicelluar organization of calcium signaling in liver

Cytosolic Ca2+ ([Ca2+]i) is a ubiquitous intracellular messenger in mammalian cells, and imaging studies of single living cells loaded with fluorescent Ca2+ indicators have demonstrated that the [Ca2+]i changes induced by extracellular agonists are often organized in complex temporal and spatial patterns. A number of hormones that regulate hepatic metabolism, including vasopressin and α1adrenergic agonists, bring about their effects through a rise in [Ca2+]i mediated by the Ca2+-mobilizing second messenger IP3. Imaging studies of isolated hepatocytes have demonstrated that the [Ca+]i responses during continuous exposure to these hormones consist of a series of discrete [Ca+]i spikes, the frequency of which is determined by the dose of agonist. By contrast, the amplitude and kinetics of the individual [Ca+]i spikes are not affected by changing the agonist dose. In addition to the temporal organization in the form of [Ca2+]i oscillations, the [Ca2+]i changes are also spatially organized into regenerative [Ca2+]i waves that propagate throughout the cytoplasm and nucleoplasm of the cell from a discrete plasma membrane locus. These oscillatory [Ca2+]i waves are generated by the complex interplay of [Ca2+]i and IP3 in regulating the gating properties of the IP3- receptor Ca2+ channel, which is located in the endoplasmic reticulum Ca2+ store.</jats:p

Apoptosis and its suppression in hepatocytes culture

In order to achieve the goal of developing extracorporeal liver support devices, it is necessary to optimise bioprocess environment such that viability and function are maximised. Optimising culture medium composition and controlling the constitution of the cellular microenvironment within the bioreactor have for many years been considered vital to achieving these aims. Coupled to this is the need to understand apoptosis, the prime suspect in the demise of animal cultures, including those of hepatocytes. Results presented here show that absent nutrients including glucose and amino acids play a substantial part in the induction of apoptosis. The use of chemical apoptosis inhibitors was utilised to investigate key components of hepatic apoptosis where caspases, predominantly caspase 8, were implicated in staurosporine (STS)-induced HepZ apoptosis. Caspase 9 and 3 activation although recorded was of less significance. Interestingly, these results were not consistent with those of mitochondrial membrane depolarisation where inhibition of caspase activation appeared to drive depolarisation. Inhibition of mitochondrial permeability transition and use of anti-oxidants was unsuccessful in reducing apoptosis, caspase activation and mitochondrial membrane depolarisation. In further studies, the anti-apoptotic gene bcl-2 was over-expressed in HepZ, resulting in a cell line that was more robust and resistant to death induced by glucose and cystine deprivation and treatment with STS. Bcl-2 did not however show significant cytoprotectivity where apoptosis was stimulated by deprivation of glutamine and serum. Overall, results indicated that although apoptosis can be curbed by use of chemical inhibitors and genetic manipulation, their success is dependent on apoptotic stimuli