Nordihydroguaiaretic acid elevates osteoblastic intracellular Ca²⁺

Nordihydroguaiaretic acid (NDGA) is widely used as a pharmacological tool to inhibit lipoxygenases; however, recent evidence suggests that it increases renal intracellular [Ca²⁺]_i via novel mechanisms. Here the effect of NDGA on Ca²⁺ signaling in MG63 osteoblastic cells was explored using fura-2 as a Ca²⁺ indicator. NDGA (2-50 μM) increased [Ca²⁺]_i in a concentration-dependent manner. The signal comprised an initial rise and an elevated phase over a time period of 4 min. Removing extracellular Ca²⁺ reduced 2-50 μM NDGA-induced signals by 62±2%. After incubation with 50 μM NDGA in Ca²⁺-free medium for several minutes, addition of 3 mM CaCl₂ induced an increase in [Ca²⁺]_i. NDGA (50 μM)-induced [Ca²⁺]_i increases were not changed by pretreatment with 10 μM of verapamil, diltiazem, nifedipine, nimodipine and nicardipine. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (1 μM) inhibited 50 μM NDGA-induced [Ca²⁺]_i increases by 69± 3%. Inhibition of phospholipase C with 2 μM U73122 had little effect on 50 μM NDGA-induced Ca²⁺ release. Several other lipoxygenase inhibitors had no effect on basal [Ca²⁺]_i. At a concentration that did not increase basal [Ca²⁺]_i, NDGA (1 μM) did not alter 10 μM ATP- or 1 μM thapsigargin-induced [Ca²⁺]_i increases. Alteration of protein kinase C activity with 1 nM phorbol 12-myristate 13-acetate or 2 μM GF 109203X did not affect 50 μM NDGA-induced [Ca²⁺]_i increases. Together, the results show that NDGA increased [Ca²⁺]_i in osteoblasts in a lipoxygenase-independent manner, by releasing stored Ca²⁺ in a fashion independent of phospholipase C activity, and by causing Ca²⁺ influx.