D-Galactosamine Induced Hepatocyte Apoptosis is Inhibited in vivo and in Cell Culture by a Calcium Calmodulin Antagonist, Chlorpromazine, and a Calcium Channel Blocker, Verapamil

Abstract

Studies were conducted in C57BL/6N Crj male mice and in cultured hepatocytes to clarify the relationship between galactosamine (GalN) induced apoptosis and [Ca²⁺]i kinetics. Chlorpromazine (CPZ), a Ca²⁺-calmodulin antagonist, and verapamil (VR), a Ca²⁺-channel blocker each inhibited GalN-induced DNA fragmentation and the appearance of apoptotic bodies. The kinetics of calcium uptake were evaluated using a calcium analyzer with the acetoxymethyl ester of fura-PE3 (fura-PE3/AM, 2.5 M) as the calcium reporter. An increase in [Ca²⁺]i was detected in the cultured hepatocytes within 3 hours after treatment with 20 mM GalN; this increase was inhibited by pretreatment with either 20 M CPZ or 30 M VR. Ca²⁺ imaging by confocal laser scanning microscopy showed that increase in [Ca²⁺]i after treatment with GalN was initially localized around nuclei, while [Ca²⁺]i signals were later diffuse and observed throughout the cytoplasm. The activities of lactate dehydrogenase (LDH) and serum glutamate-pyruvate transaminase (sGPT), used as indicators of plasma membrane damage and leakage, however, were not reduced by pretreatment with CPZ or VR. From these findings, we infer that the DNA fragmentation in GalN-induced hepatocyte apoptosis is associated with an elevation in the perinuclear concentration of Ca²⁺, but GalN-induced necrotic cell death is triggered through pathway(s) that are insensitive to blockage of Ca ²⁺ influx and therefore appear to occur independently of elevation in [Ca²⁺]i. These results help to clarify the role of calcium flux in hepatocyte apoptosis and necrosis induced by exposure to hepatotoxins in vivo and in vitro.