Nicotinic acid modulates intracellular calcium concentration and disassembles the cytoskeleton

Abstract

Nicotinic acid (NA), a member of the vitamin B family, is well known for its functions in the treatment and prevention of atherosclerosis due to decreasing plasma levels of low-density lipoprotein cholesterol. In recent years, the major side effect of NA, cutaneous flushing, has also attracted extensive attention. However, the effects of NA in other aspects of physiology or cell biology have remained elusive. The present study provided evidence that high concentrations of NA were able to first reduce and later elevate intracellular [Ca2+] in the NIH3T3 cell line. The reduction of the intracellular Ca2+ concentration was achieved within the initial 10 sec, and was preceded by a gradual elevation of intracellular [Ca2+]. Notably, marked accumulation of opaque materials in the perinuclear region was observed in NIH3T3 cells treated with 70 mM NA. Further analysis revealed that treatment with 70 mM NA for 1 h disassembled the microtubule and F‑actin cytoskeleton systems and resulted in β‑tubulin degradation in an ubiquitin‑proteasome-dependent manner. These data indicated that high concentrations of NA disrupted cytoskeleton structures, which may have contributed to minus end (nucleus region) to plus end (cell membrane region)-directed transport processes and resulted in the deposition of material in the perinuclear region. Artificially increasing [Ca2+] adding CaCl2 to the culture media effected the disassembly of F‑actin, while it had no apparent effect on microtubules. These results suggested that the disruption of the cytoskeleton systems was not entirely due to the NA-induced elevation of [Ca2+]. Finally, microinjection of NA into xenopus embryos blocked the transport of melanosomes to the peripheral cellular area. In conclusion, the present study indicated that NA disassembles F‑actin and microtubule systems, thereby blocking cytoskeleton-dependent intracellular transport.

Figure 1

Time lapse assessment of intracellular [Ca²⁺] levels upon exposure to different concentrations of NA. (A) Visualization of intracellular [Ca²⁺] upon exposure to different concentration of nicotinic acid and other drugs by staining with Fluo3 acetoxymethyl Ca²⁺ indicator; (B) time lapse statistical curve of intracellular Ca²⁺ levels upon exposure to different NA concentrations; (C) TG inhibits the elevation of [Ca²⁺] induced by NA (pink curve) and cAMP analog 8-Br-cAMP delays and alleviates NA-induced primary [Ca²⁺] decrease (yellow curve). F₀: Fluo3-labeled intensity prior to exposure to NA or other drugs; F_t: Fluo3-labeled intensity at time-point t. NA, nicotinic acid; TG, thapsigargin.

Figure 2

NA disassembles the cytoskeleton and deposits opaque materials at the peri-nuclear region. Compared with (A) the PBS-incubated control group, (B) 70 mM NA results in evident nucleolar accumulation of opaque materials at peri-nucleus regions in NIH3T3 cells. In the PBS-incubated NIH3T3 cells, (C) F-actin and (D) microtubules are filament-rich as is apparent in (E) the merged image. Exposure to 30 mM NA for 1 h partially disassembled (F) F-actin and (G) microtubules as is apparent in (H) the merged image. 70 mM NA completely disassembled (I) F-actin and (J) microtubules as apparent in (K) the merged image. Microtubule structure was disassembled when exposed to 70 mM NA for 1 h in both (L and M) 293T cells and (N and O) CHO-K1 cells. (Magnification, ×250). NA, nicotinic acid.

Figure 3

External addition of CaCl₂ to the culture media and 1 h incubation disrupted the F-actin filaments, stained with Texas Red-X phalloidin, but did not affect the microtubules, stained with fluorescein isothiocyanate-conjugated antibody. (Magnification, ×250).

Figure 4

NA caused microtubule component β-tubulin degradation in an ubiquitin-proteasome pathway. NA, nicotinic acid.

Figure 5

Microinjection of 70 ng NA into xenopus embryos inhibited the melanosome transport. NA, nicotinic acid. (Magnification, ×30).