Vasoconstriction resulting from dynamic membrane trafficking of TRPM4 in vascular smooth muscle cells.

The melastatin (M) transient receptor potential (TRP) channel TRPM4 mediates pressure and protein kinase C (PKC)-induced smooth muscle cell depolarization and vasoconstriction of cerebral arteries. We hypothesized that PKC causes vasoconstriction by stimulating translocation of TRPM4 to the plasma membrane. Live-cell confocal imaging and fluorescence recovery after photobleaching (FRAP) analysis was performed using a green fluorescent protein (GFP)-tagged TRPM4 (TRPM4-GFP) construct expressed in A7r5 cells. The surface channel was mobile, demonstrating a FRAP time constant of 168 +/- 19 s. In addition, mobile intracellular trafficking vesicles were readily detected. Using a cell surface biotinylation assay, we showed that PKC activation with phorbol 12-myristate 13-acetate (PMA) increased (approximately 3-fold) cell surface levels of TRPM4-GFP protein in <10 min. Similarly, total internal reflection fluorescence microscopy demonstrated that stimulation of PKC activity increased (approximately 3-fold) the surface fluorescence of TRPM4-GFP in A7r5 cells and primary cerebral artery smooth muscle cells. PMA also caused an elevation of cell surface TRPM4 protein levels in intact arteries. PMA-induced translocation of TRPM4 to the plasma membrane was independent of PKCalpha and PKCbeta activity but was inhibited by blockade of PKCdelta with rottlerin. Pressure-myograph studies of intact, small interfering RNA (siRNA)-treated cerebral arteries demonstrate that PKC-induced constriction of cerebral arteries requires expression of both TRPM4 and PKCdelta. In addition, pressure-induced arterial myocyte depolarization and vasoconstriction was attenuated in arteries treated with siRNA against PKCdelta. We conclude that PKCdelta activity causes smooth muscle depolarization and vasoconstriction by increasing the number of TRPM4 channels in the sarcolemma.

Endothelium-dependent cerebral artery dilation mediated by TRPA1 and Ca2+-Activated K+ channels.

Although it is well established that changes in endothelial intracellular [Ca(2+)] regulate endothelium-dependent vasodilatory pathways, the molecular identities of the ion channels responsible for Ca(2+) influx in these cells are not clearly defined. The sole member of the ankyrin (A) transient receptor potential (TRP) subfamily, TRPA1, is a Ca(2+)-permeable nonselective cation channel activated by electrophilic compounds such as acrolein (tear gas), allicin (garlic), and allyl isothiocyanate (AITC) (mustard oil). The present study examines the hypothesis that Ca(2+) influx via TRPA1 causes endothelium-dependent vasodilation. The effects of TRPA1 activity on vascular tone were examined using isolated, pressurized cerebral arteries. AITC induced concentration-dependent dilation of pressurized vessels with myogenic tone that was accompanied by a corresponding decrease in smooth muscle intracellular [Ca(2+)]. AITC-induced dilation was attenuated by disruption of the endothelium and when the TRPA1 channel blocker HC-030031 was present in the arterial lumen. TRPA1 channels were found to be present in native endothelial cells, localized to endothelial cell membrane projections proximal to vascular smooth muscle cells. AITC-induced dilation was insensitive to nitric oxide synthase or cyclooxygenase inhibition but was blocked by luminal administration of the small and intermediate conductance Ca(2+)-activated K(+) channel blockers apamin and TRAM34. BaCl(2), a blocker of inwardly rectifying K(+) channels, also inhibited AITC-induced dilation. AITC-induced smooth muscle cell hyperpolarization was blocked by apamin and TRAM34. We conclude that Ca(2+) influx via endothelial TRPA1 channels elicits vasodilation of cerebral arteries by a mechanism involving endothelial cell Ca(2+)-activated K(+) channels and inwardly rectifying K(+) channels in arterial myocytes.