Involvement of CaV3.1 T-type calcium channels in cell proliferation in mouse preadipocytes.

Voltage-gated Ca(2+) channels (Ca(V)) are ubiquitously expressed in various cell types and play vital roles in regulation of cellular functions including proliferation. However, the molecular identities and function of Ca(V) remained unexplored in preadipocytes. Therefore, whole cell voltage-clamp technique, conventional/quantitative real-time RT-PCR, Western blot, small interfering RNA (siRNA) experiments, and immunohistochemical analysis were applied in mouse primary cultured preadipocytes as well as mouse 3T3-L1 preadipocytes. The effects of Ca(V) blockers on cell proliferation and cell cycle were also investigated. Whole cell recordings of 3T3-L1 preadipocytes showed low-threshold Ca(V), which could be inhibited by mibefradil, Ni(2+) (IC(50) of 200 muM), and NNC55-0396. Dominant expression of alpha(1G) mRNA was detected among Ca(V) transcripts (alpha(1A)-alpha(1I)), supported by expression of Ca(V)3.1 protein encoded by alpha(1G) gene, with immunohistochemical studies and Western blot analysis. siRNA targeted for alpha(1G) markedly inhibited Ca(V). Dominant expression of alpha(1G) mRNA and expression of Ca(V)3.1 protein were also observed in mouse primary cultured preadipocytes. Expression level of alpha(1G) mRNA and Ca(V)3.1 protein significantly decreased in differentiated adipocytes. Mibefradil, NNC55-0396, a selective T-type Ca(V) blocker, but not diltiazem, inhibited cell proliferation in response to serum. NNC55-0396 and siRNA targeted for alpha(1G) also prevented cell cycle entry/progression. The present study demonstrates that the Ca(V)3.1 T-type Ca(2+) channel encoded by alpha(1G) subtype is the dominant Ca(V) in mouse preadipocytes and may play a role in regulating preadipocyte proliferation, a key step in adipose tissue development.

Comparative effects of azelnidipine and other Ca2+-channel blockers on the induction of inducible nitric oxide synthase in vascular smooth muscle cells.

Overproduction of nitric oxide by inducible nitric oxide synthase contributes to the progression of cardiovascular disease. We investigated the effects of azelnidipine and other Ca2+-channel blockers on nitric oxide production by cultured aortic smooth muscle cells isolated from Wistar rats and human umbilical vein endothelial cells (HUVECs), using the Griess reaction and oxyhemoglobin method. Release of lactic dehydrogenase (LDH) was measured to evaluate cell damage, and immunohistochemistry was performed to examine the expression of inducible nitric oxide synthase and nitrotyrosine protein. Azelnidipine and other Ca2+-channel blockers inhibited the release of nitric oxide induced by lipopolysaccharide plus interferon-gamma. Azelnidipine inhibited it most potently among the Ca2+-channel blockers tested (azelnidipine, amlodipine, nifedipine, diltiazem, verapamil, and nicardipine) at a concentration of 10 microM. Longer stimulation with these agents induced the expression of inducible nitric oxide synthase and nitrotyrosine, with an increase of lactic dehydrogenase release, whereas azelnidipine suppressed these changes. In human umbilical vein endothelial cells, azelnidipine enhanced basal nitric oxide production by endothelial nitric oxide synthase. In conclusion, azelnidipine potently inhibited the induction of inducible nitric oxide synthase and then nitric oxide production in vascular smooth muscle cells, while enhancing constitutive nitric oxide production by endothelial cells. Azelnidipine may inhibit nitrotyrosine expression and cell damage caused by overproduction of nitric oxide, suggesting a mechanism for its cardiovascular protective effect.