title: Involvement of CaV3.1 T-type calcium channels in cell proliferation in mouse preadipocytes.
abstract: 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.
authors.affiliation: Department of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Oguri
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Tanaka
authors.forename: Haruko
authors.initials: H
authors.lastname: Iida
authors.forename: Kentarou
authors.initials: K
authors.lastname: Meguro
authors.forename: Haruhito
authors.initials: H
authors.lastname: Takano
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Oonuma
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nishimura
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Morita
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Yamasoba
authors.forename: Ryozo
authors.initials: R
authors.lastname: Nagai
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Nakajima
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D050156:Adipogenesis; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020747:Calcium Channels, T-Type / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D049109:Cell Proliferation / Q000187:drug effects; D007150:Immunohistochemistry; D008564:Membrane Potentials; D051379:Mice; D008813:Mice, Inbred ICR; D018408:Patch-Clamp Techniques; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C498197:Cacna1g protein, mouse; D002121:Calcium Channel Blockers; D020747:Calcium Channels, T-Type; D012333:RNA, Messenger; D002118:Calcium