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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.

Voltage-gated [Na.sup.+]] channel currents ([I.sub.Na]) are expressed in several types of smooth muscle cells. The purpose of this study was to evaluate the expression of [I.sub.Na], its functional role, pathophysiology in cultured human (hASMCs) and rabbit aortic smooth muscle cells (rASMCs), and its association with vascular intimal hyperplasia. In whole cell voltage clamp, [I.sub.Na] was observed at potential positive to -40 mV, was blocked by tetrodotoxin (TTX), and replacing extracellular [Na.sub.+] with N-methyl-D-glucamine in cultured hASMCs. In contrast to native aorta, cultured hASMCs strongly expressed SCN9A encoding [Na.sub.v]1.7, as determined by quantitative RT-PCR. [I.sub.Na] was abolished by the treatment with SCN9A small-interfering (si)RNA (P < 0.01). TTX and SCN9A siRNA significantly inhibited cell migration (P < 0.01, respectively) and horseradish peroxidase uptake (P < 0.01, respectively). TTX also significantly reduced the secretion of matrix metalloproteinase-2 6 and 12 h after the treatment (P < 0.01 and P < 0.05, respectively). However, neither TTX nor siRNA had any effect on cell proliferation. L-type [Ca.sup.2+] channel current was recorded, and [I.sub.Na] was not observed in freshly isolated rASMCs, whereas TTX-sensitive [I.sub.Na] was recorded in cultured rASMCs. Quantitative RT-PCR and immunostaining for [Na.sub.v]1.7 revealed the prominent expression of SCN9A in cultured rASMCs and aorta 48 h after balloon injury but not in native aorta. In conclusion, these studies show that [I.sub.Na] is expressed in cultured and diseased conditions but not in normal aorta. The [Na.sub.v]l.7 plays an important role in cell migration, endocytosis, and secretion. [Na.sub.v]1.7 is also expressed in aorta after balloon injury, suggesting a potential role for [Na.sub.v]1.7 in the progression of intimal hyperplasia. vascular smooth muscle; sodium channel; SCN9A; balloon injury

The A-type voltage-dependent K+ current (IA) has been identified in several types of smooth muscle cells including the pulmonary artery (PA), but little is known about the pharmacological and molecular characteristics of IA in human pulmonary arterial smooth muscle cells (hPASMCs). We investigated IA expressed in cultured PASMCs isolated from the human main pulmonary artery, using patch-clamp techniques, reverse transcriptase–polymerase chain reaction (RT–PCR), quantitative real-time RT–PCR and immunocytochemical studies.With high EGTA and ATP in the pipette, the outward currents were dominated by a transient K+ current (IA), followed by a relatively small sustained outward current (IK).IA was inhibited by 4-aminopyridine (4-AP) concentration-dependently, and could be separated pharmacologically into two components by tetraethylammonium (TEA) sensitivity. A component was sensitive to TEA, and the second component was insensitive to TEA.IA was inhibited by blood depressing substrate (BDS)-II, a specific blocker of KV3.4 subunit, and phrixotoxin-II, a specific blocker of KV4.2 and 4.3.Flecainide inhibited IA concentration-dependently, but it inhibited it preferentially in the presence of TEA (TEA-insensitive IA).Systematic screening of expression of KV genes using RT–PCR showed the definite presence of transcripts of the IA-encoding genes for KV3.4, KV4.1, KV4.2 and KV4.3 as well as the IK-encoding genes for KV1.1, KV1.5 and KV2.1. The real-time RT–PCR analysis showed that the relative abundance of the encoding genes of IA α-subunit and KV channel-interacting proteins (KChIPs) was KV4.2>KV3.4>KV4.3 (long)>KV4.1, and KChIP3≫KChIP2, respectively.The presence of KV3.4, KV4.2 and KV4.3 proteins was also demonstrated by immunocytochemical studies, and confirmed by immunohistochemical staining using intact human PA sections.These results suggest that IA in cultured hPASMCs consists of two kinetically and pharmacologically distinct components, probably KV3.4 and KV4 channels.British Journal of Pharmacology (2005) 146, 49–59. doi:10.1038/sj.bjp.0706285; published online 6 June 2005 [ABSTRACT FROM AUTHOR]

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]