Question: 

The transient receptor potential channel C3 (TRPC3) is the most upstream protein of endothelial stretch-induced signaling characterized so far. We here analyze possible mechanisms of stretch-induced TRPC3 activation.

Methods: 

Primary human umbilical vein endothelial cells (EC) were cultured on glass cover slips or on stretchable Flexercell membranes. Intracellular calcium ions were detected by Fura-2 fluorescence ratio using a real time CCD camera system. Analytical methods such as real time RT-PCR, Western blotting and immunofluorescence analysis were performed according to standard protocols.

Results: 

TRPC3 activates the stretch-sensitive transcription factor zyxin.Inhibition of TRPC3 by its specific inhibitor Pyr3 (10 mmol/L) efficiently blunted zyxin-induced gene expression in EC. Hypothesizing that TRPC3, similar to other TRP-channels may be activated by diacylglycerol (DAG), we exposed EC to the DAG analogue OAG (100 mmol/L) to activate TRPC3. Indeed, nuclear translocation of zyxin and zyxin-induced gene expression similar to the stretch-response was observed in response to OAG treatment. This response was inhibitable by Pyr3. Intracellular calcium measurements revealed that in EC, after protein kinase C (PKC) inhibition, OAG induces a strong calcium signal that was mostly blunted by parallel exposure of the cells to Pyr3.

Conclusion: 

TRPC3 may be activated by stretch via a release of DAG that seems to set off stretch-induced zyxin activation via an increase of cytosolic calcium into EC. The stretch-induced activation of PKC seems to inhibit OAG-induced TRPC3 activation providing a potential strategy to prevent stretch-induced endothelial gene expression.