Interest in ERbeta vasorelaxation was raised by the observation that ERbeta-deficient mice develop sustained hypertension as they age. Therefore we decided to investigate acute ERbeta-signalling mechanism in isolated aortic smooth muscle from male rats. DPN was therefore tested in this preparation, for its effects on membrane potential, calcium signal.

Methods: 

Membrane potential in isolated aortic smooth muscle cells was measured by flow cytometry, using a previously published method. Briefly, cells were loaded for 15 minutes with 1 mM bis-oxonol sensitive probe DIBAC2(3) (Invitrogen, Carlsbad, Ca). Fluorescence quantification was performed by using flow cytometry (Epics Elite, Coulter Hielary, Fl) with a 70 mM flow cell at 37 °C. Cells were excited with an argon laser at 488 nm and the emitted fluorescence was 52525 nm. Fluorescence readings were expressed in arbitrary units. Free cytosolic calcium in isolated aortic smooth muscle cells was measured by flow cytometry, using a previously published method. Briefly, cells were loaded for 15 minutes with Fluo 3-AM (Invitrogen, Carlsbad, Ca). Fluorescence quantification was performed by using flow cytometry (Epics Elite, Coulter Hielary, Fl) with a 70 mM flow cell at 37 °C. Cells were excited with an argon laser at 488 nm and the emitted fluorescence was 52525 nm. Fluorescence readings were expressed in arbitrary units.

Results: 

In isolated rat aortic smooth muscle cells, 2 mM phenylephrine induced a 33 10% (n=7) increase in the fluorescence signal of bis-oxonol sensitive probe DIBAC2(3), thus indicating membrane depolarization. DPN fully abolished phenylephrine depolarization, and even reversed it at high concentrations . The concentration-response curve for DPN antagonism of membrane depolarization superposed with those for cAMP increase and vasorelaxant action . Free cytosolic calcium in isolated aortic smooth muscle cells was measured as described in the Methods Section. 2 mM Phenylephrine induced a 20.3 2.5% increase in the Fluo 3-AM fluorescence signal in cells treated with vehicle, and of 2.2 1.8% in the presence of 100 mM DPN.

Conclusion: 

The relaxation induced by DPN implies the repolarization of the membrane and the decrease of the intracellular ionic calcium.