PGI 2 opens potassium channels in retinal pericytes by cyclic AMP-stimulated, cross-activation of PKG

Abstract

Pericytes exert an important influence on the control of retinal blood flow; however, little is known regarding the molecular basis of retinal pericyte excitability. The purpose of this study was to elucidate the signaling pathway of how prostacyclin (PGI 2), an important endogenous regulator of retinal blood flow, stimulates potassium channel activity in retinal pericytes. Retinal pericytes were isolated from porcine eyeballs and plated on glass coverslips. Immunocytochemistry was performed to verify expression of the pericyte-specific ganglioside marker, 3G5 and smooth muscle α-actin. Activity of the large-conductance, voltage- and calcium-activated potassium (BK Ca) channel was measured in retinal pericytes via single-channel patch-clamp, and channel identification was confirmed via biophysical and pharmacological characterization. PGI 2 (10 μM) or beraprost (30 μM; more stable prostacyclin analog) dramatically stimulated the activity of BK Ca channels isolated in cell-attached patches. These experiments strongly suggested that PGI 2 stimulated BK Ca channel activity via a diffusible second messenger. Similarly, chlorophenylthio (CPT)-cAMP (100 μM; membrane permeable cAMP derivative) induced a significant increase in BK Ca channel activity; however, inhibition of the cAMP-dependent protein kinase (PKA) with 300 nM KT5720 could not reverse the stimulatory effect of either PGI 2 or CPT-cAMP. In contrast, activation of BK Ca channels with either CPT-cAMP or PGI 2 was abolished by 300 nM KT5823 ( n = 5, p < 0.01), an inhibitor of the cGMP-dependent protein kinase (PKG). In addition, PGI 2-stimulated channel activity was also attenuated by Rp-8-CPT-cGMPS, which inhibits PKG activity via a different mechanism. These findings demonstrate that prostacyclin, the most abundant prostanoid in the retinal circulation, is a potent stimulator of BK Ca channel activity in retinal pericytes. Interestingly, this response appears to involve cAMP-stimulated cross-activation of PKG, and not PKA. Taken together, these findings could explain, at least in part, the cellular/molecular basis for PGI 2-induced pericyte relaxation and augmentation of blood flow in the retina. Further, we propose PKG-dependent stimulation of BK Ca channel activity as a new potential therapeutic target to combat decreased retinal blood flow seen in some disease states (e.g., diabetic retinopathy).