Canonical
transient receptor potential (
TRPC) proteins may play a role in regulating changes in
intracellular calcium ([Ca(2+)](i)). Human
myometrium expresses
TRPC4,
TRPC1 and
TRPC6 mRNAs in greatest relative abundance. Contributions of
TRPC4 to increases in [Ca(2+)](i) were assessed in PHM1-41 and primary human uterine
smooth muscle (UtSMC) cells using short hairpin
RNAs (
shRNAs). Based on a reporter
assay screen, one
shRNA was
selected to construct an
adenoviral expression vector (TC4sh1). TC4sh1 induced both
mRNA and protein
TRPC4 knockdown in PHM1-41 cells without affecting expression of other
TRPCs. Signal-regulated Ca(2+) entry (SRCE), defined as a stimulus- and
extracellular Ca(2+)-dependent increase in [Ca(2+)](i), was measured in PHM1-41 cells treated with
oxytocin (
G-protein coupled receptor (
GPCR)-stimulated),
thapsigargin (store depletion-stimulated), and OAG (diacylglycerol-stimulated), using
Fura-2. Cells infected with TC4sh1 exhibited
attenuated oxytocin-, ATP- and PGF2alpha-mediated SRCE, but no change in thapsigargin- or OAG-stimulated SRCE. Similar results were obtained in primary uterine
smooth muscle cells. Additionally, cells expressing TC4sh1 exhibited a significantly smaller increase in channel activity in response to
oxytocin administration than did cells infected with empty
virus. These data show that, in human myometrial cells,
knockdown of endogenous
TRPC4 specifically
attenuates GPCR-stimulated, but not thapsigargin- or OAG-stimulated
extracellular calcium-dependent increases in [Ca(2+)](i). These data imply that, in this cellular context, the mechanisms regulating
extracellular Ca(2+)-dependent increases in [Ca(2+)](i) are differentially affected by different
signaling pathways.
