3-Methylcholanthrene increases phorbol 12-myristate 13-acetate-induced respiratory burst activity and intracellular calcium levels in common carp (Cyprinus carpio L) macrophages

Abstract

Phagocytic cells play a key role in the fish immune system. They secrete reactive oxygen species (ROS) involved in their bactericidal activity. These cells are highly sensitive to pollution by polycyclic aromatic hydrocarbons and other organic pollutants. We have investigated the intracellular mechanisms by which 3-methylcholanthrene (3-MC) increased bactericidal activity of carp phagocytes. Macrophages isolated from head kidney (pronephros) and incubated 1 h with 3-MC enhanced their production of ROS when they were stimulated 1.25 h with phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C (PKC). 3-MC also produced a rapid and a sustained increase in [Ca(2+)](i) (2 h minimum). However, the cytochrome P450 1A and Ah receptor inhibitor, alpha-naphtoflavone (alpha-NF), inhibited the potentiation of PMA-induced ROS production, suggesting 3-MC metabolic activation. Moreover, alpha-NF increased [Ca(2+)](i) without macrophage ROS production, suggesting that some mechanism other than calcium release is playing a role in the stimulation of the macrophages by 3-MC. The rise in [Ca(2+)](i) induced by 3-MC was potentiated by the inhibitor of the endoplasmic reticulum calcium ATPases, thapsigargin. And treating the cells with 3-MC decreased the calcium mobilization caused by thapsigargin. These results suggest that 3-MC acts on the endoplasmic reticulum, perhaps directly on calcium ATPases, to increase intracellular calcium levels in carp phagocytes.