Attenuation of canonical transient receptor potential-like channel 6 expression specifically reduces the diacylglycerol-mediated increase in intracellular calcium in human myometrial cells.
An increase in intracellular Ca(2+) ([Ca(2+)](i)) as a result of release of Ca(2+) from intracellular stores or influx of extracellular Ca(2+) contributes to the regulation of smooth muscle contractile activity. Human uterine smooth muscle cells exhibit receptor-, store-, and diacylglycerol (OAG)-mediated extracellular Ca(2+)-dependent increases in [Ca(2+)](i) (SRCE) and express canonical transient receptor potential-like channels (TRPC) mRNAs (predominantly TRPC1, -4, and -6) that have been implicated in SRCE. To determine the role of TRPC6 in human myometrial SRCE, short hairpin RNA constructs were designed that effectively targeted a TRPC6 mRNA reporter for degradation. One sequence was used to produce an adenovirus construct (TC6sh1). TC6sh1 reduced TRPC6 mRNA but not TRPC1, -3, -4, -5, or -7 mRNAs in PHM1-41 myometrial cells. Compared with uninfected cells or cells infected with empty vector, the increase in [Ca(2+)](i) in response to OAG was specifically inhibited by TC6sh1, whereas SRCE responses elicited by either oxytocin or thapsigargin were not changed. Similar findings were observed in primary pregnant human myometrial cells. When PHM1-41 cells were activated by OAG in the absence of extracellular Na(+), the increase in [Ca(2+)](i) was partially reduced. Furthermore, pretreatment with nifedipine, an L-type calcium channel blocker, also partially reduced the OAG-induced [Ca(2+)](i) increase. Similar effects were observed in primary human myometrial cells. These findings suggest that OAG activates channels containing TRPC6 in myometrial cells and that these channels act via both enhanced Na(+) entry coupled to activation of voltage-dependent Ca(2+) entry channels and a nifedipine-independent Ca(2+) entry mechanism to promote elevation of intracellular Ca(2+).
Chung
Daesuk
D
Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA.
Kim
Yoon-Sun
YS
Phillips
Jennifer N
JN
Ulloa
Aida
A
Ku
Chun-Ying
CY
Galan
Henry L
HL
Sanborn
Barbara M
BM
D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D004075:Diglycerides / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D004526:Efficiency; D005260:Female; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D009215:Myometrium / Q000187:drug effects* / Q000378:metabolism; D011247:Pregnancy; D034741:RNA, Small Interfering / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D000074287:TRPC6 Cation Channel
C520323:1-oleoyl-2-acetoyl-sn-glycerol; D004075:Diglycerides; D034741:RNA, Small Interfering; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D012964:Sodium; D002118:Calcium