The effect of calmodulin antagonists on hyperthermic cell killing and the development of thermotolerance

Abstract

The role of calmodulin (CaM) in hyperthermic cell killing, and the development of thermotolerance in rat 13762NF mammary adenocarcinoma cells, was investigated by using the CaM antagonists W-7 [N-(6-aminohexyl)-5-chloro-l-naphthalene sulphon-amide] and W-13 [iV-(4-aminobutyl)-2-chloro-naphthalenesulphonamide] and their less active analogues W-5 [N-(6-aminohexyl)-l-naphthalenesulphonamide] and W-12 [N-(4-aminobutyl)-2-naphthalenesulphonamide]. The CaM antagonists W-7 and W-13 potentiated 43 °C cell killing (and the less active analogues did not) at a concentration compatible with CaM inhibition, thus hyperthermic perturbation of CaM-regulated processes may contribute to cellular lethality. The potentiation of hyperthermic killing by antagonists appeared to be temperature-dependent, sensitizing much more effectively to 43 °C than to 42°C killing. The effect may be related to differing primary mechanisms of hyperthermic killing activated at the two temperatures, or simply to differences in incorporation or localization of the antagonists. The presence of the CaM antagonists throughout fractionated 42°C or 43°C heating, or during continuous 42°C heating, did not significantly inhibit or potentiate me triggering and development of thermotolerance or alter the rates of heat stress protein (HSP) synthesis. Studies using CaM-agarose isolation of CaM-binding proteins indicated that binding of some HSP to CaM-agarose occurred and was Ca²⁺-dependent. The specificity and physiological relevance of these HSP binding to CaM was not clear, since their affinity was not high in these cells. Presumably W-7 would perturb any physiologically relevant CaM-protein interactions in cells but W-7 concentrations that reduced HSP and other protein binding to CaM-agarose columns by 50 per cent or more, had no effect on thermotolerance development in cells. These observations, combined with the studies that showed little effect of CaM antagonists on HSP synthesis at concentrations which potentiated cell killing, suggested that events leading to triggering or developing mermotolerance were not strongly dependent on any putative HSP binding to CaM. These studies also suggest some targets of hyperthermic cell killing at 43 °C are different from those that lead to the triggering and development of thermotolerance.

Key Words:

• calmodulin
• heat stress (shock) proteins
• hyperthermia
• thermotolerance.