Cytosolic calcium after carbon tetrachloride, 1,1-dichloroethylene, and phenylephrine exposure: Studies in rat hepatocytes with phosphorylase a and quin2

Abstract

Carbon tetrachloride (CCl 4) and 1,1-dichloroethylene (DCE), both hepatotoxins, inhibit sequestration of Ca 2+ by rat liver endoplasmic reticulum (ER) both in vivo and in vitro. It is possible that, as a result, cytosolic Ca 2+ concentrations rise in liver cells. In experiments presented here, isolated hepatocytes were exposed to CCl 4, DCE, and phenylephrine (PE), a non-hepatotoxic alpha 1-adrenergic agent that mobilizes Ca 2+. Cytoplasmic Ca 2+ concentrations were evaluated by two methods: (1) indirectly by assaying the activity of glycogen phosphorylase a, and (2) directly by monitoring the fluorescence of quin2. In primary hepatocyte cultures, CCl 4, DCE, and PE exposure increased the activity of phosphorylase a at 5 min from 39 ± 2 to 130 ± 12, 80 ± 13, and 97 ± 10 nmoles PO 4 3−/ mg protein/min respectively. In rat hepatocyte suspensions loaded with quin2 and exposed to CCl 4, DCE, or PE, cytosolic Ca 2+ concentrations were elevated within 20 sec to 0.83 ± 0.13, 0.59 ± 0.06 and 0.99 ± 0.14 μM Ca 2+ respectively. Basal Ca 2+ levels in these cells averaged 0.25 ± 0.03 μM. Thus, CCl 4 and PE apparently increased cytosolic Ca 2+ levels to approximately the same extent, whereas DCE was somewhat less effective. The durations of the effects of CCl 4 and PE were examined further by determining their time courses of elevated phosphorylase a activity. In hepatocyte cultures, increased phosphorylase a activity persisted through at least 60 min following CCl 4 exposure. In contrast, phosphorylase a activity returned to basal levels by 20 min after PE. Increases in cytoplasmic Ca 2+ levels that are sustained rather than transient may distinguish these hepatotoxic chlorinated aliphatic hydrocarbons from non-toxic hormonal agents.