PPAR? regulates resistance vessel tone through a mechanism involving RGS5-mediated control of protein kinase C and BKCa channel activity
Ketsawatsomkron P, Lorca RA, Keen HL, Weatherford ET, Liu X, Pelham CJ, Grobe JL, Faraci FM, England SK, Sigmund CD
Cells used in publication:
arteries mesenteric (MA)
Tissue Origin: aortic
4D Nucleofector Y-Unit Transfection of intact mesenteric arteries: Blood vessels were isolated in chilled Krebs buffer and transferred to a mixture of Nucleofector solution (AD2, 4D-Nucleofector Y unit) and 100 nM siRGS5 or negative control (NC) non-targeting siRNA sequences (IDT, Coralville, IA). After 25 min incubation at room temperature, blood vessels were electroporated with program CM138. Following a subsequent 5 min incubation, one mL of warm DMEM/F12 (serum free) was gently added to the mixture and the plate was incubated for 30 hr before samples were collected for qRT-PCR or vascular reactivity assay. Confocal microscopy (Zeiss S10) was used to visualize the fluorescence signal (red) from vessels transfected with dye-labeled oligo control (IDT, IA).
RATIONALE: Activation of peroxisome proliferator-activated receptor-? (PPAR?) by thiazolidinediones lowers blood pressure, whereas PPAR? mutations cause hypertension. Previous studies suggest these effects may be mediated through the vasculature, but the underlying mechanisms remain unclear. OBJECTIVE: To identify PPAR? mechanisms and transcriptional targets in vascular smooth muscle and their role in regulating resistance artery tone. METHODS AND RESULTS: We studied mesenteric artery (MA) from transgenic mice expressing dominant-negative (DN) mutant PPAR? driven by a smooth muscle cell-specific promoter. MA from transgenic mice exhibited a robust increase in myogenic tone. Patch clamp analysis revealed a reduced large conductance Ca(2+)-activated K(+) (BKCa) current in freshly dissociated smooth muscle cell from transgenic MA. Inhibition of protein kinase C corrected both enhanced myogenic constriction and impaired the large conductance Ca(2+)-activated K(+) channel function. Gene expression profiling revealed a marked loss of the regulator of G protein signaling 5 (RGS5) mRNA in transgenic MA, which was accompanied by a substantial increase in angiotensin II-induced constriction in MA. Small interfering RNA targeting RGS5 caused augmented myogenic tone in intact mesenteric arteries and increased activation of protein kinase C in smooth muscle cell cultures. PPAR? and PPAR? each bind to a PPAR response element close to the RGS5 promoter. RGS5 expression in nontransgenic MA was induced after activation of either PPAR? or PPAR?, an effect that was markedly blunted by DN PPAR?. CONCLUSIONS: We conclude that RGS5 in smooth muscle is a PPAR? and PPAR? target, which when activated blunts angiotensin II-mediated activation of protein kinase C, and preserves the large conductance Ca(2+)-activated K(+) channel activity, thus providing tight control of myogenic tone in the microcirculation.
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