Modulation of Intracellular Ca2+ Concentration by Vitamin B12 in Rat Thymocytes

Abstract

ABSTRACTWe have studied several novel effects of vitamin B12 (cyanocobalamin) on cellular Ca2+ homeostasis in rat thymocytes. We determined the effect of various concentrations of vitamin B12 on intracellular Ca2+ concentration ([Ca2+]i) and parameters of Ca2+in signaling using the fluorescent dye Fura-2. The basal [Ca2+]i in Ca2+-containing media was 115 ± 5 nM but in vitamin B12 (10 nM)-treated thymocytes [Ca2+]i was decreased to 60 ± 15 nM (mean ± SEM) during the first 5 min. The decline in [Ca2+]i was accompanied by an increase in the endoplasmic reticulum Ca2+ store, presumably as a result of Ca-ATPase activation. At the same time 100 nM–10 mM B12 induced the accumulation of Ca2+ in mitochondria. Somewhat higher concentrations of B12 (1–10 μM) had no effect on [Ca2+]i. A further increase in B12 concentration with range from 50 μM to 1 mM caused a dose-dependent elevation of [Ca2+]i from the basal level (115 ± 5 nM) up to 200 ± 50 nM in thymocytes, and this elevation was partially blocked in Ca2+-free media. This high concentration of vitamin B12 caused a gradual decrease of endoplasmic reticulum Ca2+ stores by means of Ca-ATPase inhibition. The B12-induced increase in [Ca2+]i was not observed after depletion of intracellular Ca2+ stores, induced by addition of 2′,5′-di(tert-butyl)-1,4-benzohydroquinone (BHQ), an inhibitor of endoplasmic reticulum Ca 2+-ATPase, concanavalin A, or arachidonic acid. These studies show that vitamin B12 regulates [Ca2+]i via several different mechanisms at different B12 concentrations. Participation of G proteins and calmodulin activity in B12-mediated [Ca2+]i increase is discussed.