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Voltage-gated [Ca.sup.2+] channels ([Ca.sub.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.sub.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.sub.V] blockers on cell proliferation and cell cycle were also investigated. Whole cell recordings of 3T3-L1 preadipocytes showed low-threshold [Ca.sub.V], which could be inhibited by mibefradil, [Ni.sup.2+] ([IC.sub.50] of 200 [micro]M), and NNC55-0396. Dominant expression of [[alpha].sub.1G] mRNA was detected among [Ca.sub.V] transcripts ([[alpha].sub.1A]-[[alpha].sub.1I]), supported by expression of [Ca.sub.V]3.1 protein encoded by [[alpha].sub.1G] gene, with immunohistochemical studies and Western blot analysis, siRNA targeted for [[alpha].sub.1G] markedly inhibited [Ca.sub.V]. Dominant expression of [[alpha].sub.1G] mRNA and expression of [Ca.sub.V]3.1 protein were also observed in mouse primary cultured preadipocytes. Expression level of [[alpha].sub.1G] mRNA and [Ca.sub.V]3.1 protein significantly decreased in differentiated adipocytes. Mibefradil, NNC55-0396, a selective T-type [Ca.sub.V] blocker, but not diltiazem, inhibited cell proliferation in response to serum. NNC55-0396 and siRNA targeted for am also prevented cell cycle entry/progression. The present study demonstrates that the [Ca.sub.V]3.1 T-type [Ca.sup.2+] channel encoded by [[alpha].sub.1G] subtype is the dominant [Ca.sub.V] in mouse preadipocytes and may play a role in regulating preadipocyte proliferation, a key step in adipose tissue development. 3T3-L1; adipose; [[alpha].sub.1G]; cell cycle doi: 10.1152/ajpcell.00488.2009.

Abstract: Voltage-gated Na+ channel (I Na) encoded by SCN9A mRNA is expressed in cultured human bronchial smooth muscle cells. We investigated the effects of dexamethasone on I Na, by using whole-cell voltage clamp techniques, reverse transcriptase/polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR. Acute application of dexamethasone (10−6 M) did not affect I Na. However, the percentage of the cells with I Na was significantly less in cells pretreated with dexamethasone for 48 h, and the current-density of I Na adjusted by cell capacitance in cells with I Na was also decreased in cells treated with dexamethasone. RT-PCR analysis showed that α and β subunits mRNA of I Na mainly consisted of SCN9A and SCN1β, respectively. Treatment with dexamethasone for 24–48 h inhibited the expression of SCN9A mRNA. The inhibitory effect of dexamethasone was concentration-dependent, and was observed at a concentration higher than 0.1 nM. The effect of dexamethasone on SCN9A mRNA was not blocked by spironolactone, but inhibited by mifepristone. The inhibitory effects of dexamethasone on SCN9A mRNA could not be explained by the changes of the stabilization of mRNA measured by using actinomycin D. These results suggest that dexamethasone inhibited I Na encoded by SCN9A mRNA in cultured human bronchial smooth muscle cells by inhibiting the transcription via the glucocorticoid receptor. [Copyright &y& Elsevier]