Dataset Information

Pharmacological profile of store-operated channels in cerebral arteriolar smooth muscle cells.

ABSTRACT: 1. In this study, we determined a pharmacological profile of store-operated channels (SOCs) in smooth muscle cells of rabbit pial arterioles. Ca(2+)-indicator dyes, fura-PE3 or fluo-4, were used to track [Ca(2+)](i) and 10 micro M methoxyverapamil (D600) was present in all experiments on SOCs to prevent voltage-dependent Ca(2+) entry. Store depletion was induced using thapsigargin or cyclopiazonic acid. 2. SOC-mediated Ca(2+) entry was inhibited concentration dependently by Gd(3+) (IC(50) 101 nM). It was also inhibited by 10 micro M La(3+) (70% inhibition, N=5), 100 micro M Ni(2+) (57% inhibition, N=5), 75 micro M 2-aminoethoxydiphenylborate (66% inhibition, N=4), 100 micro M capsaicin (12% inhibition, N=3) or preincubation with 10 micro M wortmannin (76% inhibition, N=4). It was completely resistant to 1 micro M nifedipine (N=5), 10 micro M SKF96365 (N=6), 10 micro M LOE908 (N=14), 10-100 micro M ruthenium red (N=1+2), 100 micro M sulindac (N=4), 0.5 mM streptomycin (N=3) or 1 : 10,000 dilution Grammostolla spatulata venom (N=4). 3. RT-PCR experiments on isolated arteriolar fragments showed expression of mRNA species for TRPC1, 3, 4, 5 and 6. 4. The pharmacological profile of SOC-mediated Ca(2+) entry in arterioles supports the hypothesis that these SOCs are distinct from tonically active background channels and several store-operated and other nonselective cation channels described in other cells. Similarities with the pharmacology of TRPC1 support the hypothesis that TRPC1 is a subunit of the arteriolar smooth muscle SOC.

SUBMITTER: Flemming R

PROVIDER: S-EPMC1573921 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Pharmacological profile of store-operated channels in cerebral arteriolar smooth muscle cells.

Flemming R R Xu S Z SZ Beech D J DJ

British journal of pharmacology 20030701 5

1. In this study, we determined a pharmacological profile of store-operated channels (SOCs) in smooth muscle cells of rabbit pial arterioles. Ca(2+)-indicator dyes, fura-PE3 or fluo-4, were used to track [Ca(2+)](i) and 10 micro M methoxyverapamil (D600) was present in all experiments on SOCs to prevent voltage-dependent Ca(2+) entry. Store depletion was induced using thapsigargin or cyclopiazonic acid. 2. SOC-mediated Ca(2+) entry was inhibited concentration dependently by Gd(3+) (IC(50) 101 nM ...[more]

PMID: 12839869

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Project description:Key pointsIn vascular smooth muscle cells (VSMCs), activation of Ca2+ -permeable store-operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediates Ca2+ entry pathways that regulate contraction, proliferation and migration, which are processes associated with vascular disease. Activation of TRPC1-based SOCs requires protein kinase C (PKC) activity, which is proposed to phosphorylate TRPC1 proteins to promote channel opening by phosphatidylinositol 4,5-bisphosphate (PIP2 ). We investigated the identity of the PKC isoform involved in activating TRPC1-based SOCs in rat mesenteric artery VSMCs. TRPC1-based SOCs were reduced by PKCδ inhibitors and knockdown of PKCδ expression. Store depletion induced interactions between TRPC1 and PKCδ and PKCδ-dependent phosphorylation of TRPC1. Furthermore, generation of store-operated interactions between PIP2 and TRPC1 and activation of TRPC1-based SOCs by PIP2 required PKCδ. These findings reveal that PKCδ activity has an obligatory role in activating TRPC1-based SOCs, through regulating PIP2 -mediated channel opening.AbstractIn vascular smooth muscle cells (VMSCs), stimulation of Ca2+ -permeable canonical transient receptor potential channel 1 (TRPC1)-based store-operated channels (SOCs) mediates Ca2+ entry pathways that regulate cell contraction, proliferation and migration, which are processes associated with vascular disease. It is therefore important to understand how TRPC1-based SOCs are activated. Stimulation of TRPC1-based SOCs requires protein kinase C (PKC) activity, with store-operated PKC-dependent phosphorylation of TRPC1 essential for channel opening by phosphatidylinositol 4,5-bisphosphate (PIP2 ). Experimental protocols used to activate TRPC1-based SOCs suggest that the PKC isoform involved requires diacylglycerol (DAG) but is Ca2+ -insensitive, which are characteristics of the novel group of PKC isoforms (δ, ε, η, θ). Hence, the present study examined whether a novel PKC isoform(s) is involved in activating TRPC1-based SOCs in contractile rat mesenteric artery VSMCs. Store-operated whole-cell cation currents were blocked by Pico145, a highly selective and potent TRPC1/4/5 channel blocker and T1E3, a TRPC1 blocking antibody. PKCδ was expressed in VSMCs, and selective PKCδ inhibitory peptides and knockdown of PKCδ expression with morpholinos oligomers inhibited TRPC1-based SOCs. TRPC1 and PKCδ interactions and phosphorylation of TRPC1 induced by store depletion were both reduced by pharmacological inhibition and PKCδ knockdown. In addition, store-operated PIP2 and TRPC1 interactions were blocked by PKCδ inhibition, and PKCδ was required for PIP2 -mediated activation of TRPC1 currents. These results identify the involvement of PKCδ in stimulation of TRPC1-based SOCs and highlight that store-operated PKCδ activity is obligatory for channel opening by PIP2 , the probable activating ligand.

Dataset Information

Pharmacological profile of store-operated channels in cerebral arteriolar smooth muscle cells.

Publications

Pharmacological profile of store-operated channels in cerebral arteriolar smooth muscle cells.

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