CC14 and CBrC13 cause a rapid decline in the capacity of the ER to sequester calcium. This discovery has led to a new hypothesis which holds that loss of calcium from the ER causes a physiologically unacceptable rise in cytosolic free calcium, which, through unknown mechanisms, causes disruption in intracellular traffic of VLDL (resulting in fatty liver), and other pathological changes. This hypothesis views elevated cytosolic calcium as a kind of toxicological second messenger. We planto test the """"""""calcium hypothesis"""""""" directly by measuring cytosolic free calcium in normal and toxicant-treated hepatocytes by use of the calcium binding fluorescent probe, quin-2, which we have synthesized, and by use of aequorin. Concomitantly, loss of calcium from the ER will be monitored by use of chlorotetracycline. Further, we plan to test the """"""""calcium hypothesis"""""""" by investigating whether and to what extent the pathological consequences of halogenated hydrocarbon action on isolated hepatocytes can be mimiked by controlled elevations of hepatocyte cytosolic calcium achieved by use of calcium ionophores such as A23187. We plan to determine whether the toxic action of halogenated hydrocarbons can be exacerbated by use of mitochondrial poisons. Conversely, we plan to investigate whether the action of agents such as CC14, CBrC13, and other hepatotoxins can be blocked by calcium antagonists such as tetracaine and TMB-8. On the assumption that pathological action of excess cytosolic calcium may be mediated through calmodulin, we plan to test whether calmodulin-blocking agents can ameliorate the action of CC14, CBrC13, etc. Along another line, we plan to carry out experiments designed to determine whether an exaggerated deregulation of hepatocyte calcium homeostasis may play a role in the potentiatin of halogenated hydrocarbon toxicity by certain alcohols and ketones. Similarly, we plan to determine whether the increased susceptibility of induced rats to halogenated hydrocarbons may involve an exaggerated dumping of calcium from the ER to the hepatocyte cytosol. Finally, calcium sequestration by isolated microsomal vesicles of hepatic origin may involve 2 calcium sequestering systems. Resolution of the system complexity, which we plan to carry out, is a necessary first step before it will be possible to determine whether any observed action of xenobiotics on microsomal vesicle calcium sequestration is due to direct action on transmembrane calcium pumping, or to increased back-diffusion of intravesicular calcium.
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