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Luke B. Potts, Lih Kuo, Wenjuan Xu, Travis W. Hein; Signaling Pathway for Porcine Retinal Arteriolar Constriction to PKC Activation: Roles of L-type Voltage-operated Calcium Channels, Myosin Light Chain Kinase and Myosin Light Chain Phosphatase. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6845. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Increased protein kinase C (PKC) activity has been implicated in several ischemic retinal diseases, possibly due to increased retinal arteriolar constriction. Opening of plasmalemmal calcium channels and subsequent calcium/calmodulin-mediated activation of myosin light chain (MLC) kinase, as well as inhibition of myosin light chain phosphatase (MLCP), contribute to smooth muscle contraction by regulating MLC phosphorylation. However, involvement of these signaling entities in PKC-elicited retinal vasoconstriction has not been ascertained. Hence, the purpose of this study was to determine the roles of L-type voltage-operated calcium channels (L-VOCCs) and MLC kinase in PKC activation-induced vasoconstriction. Whether the activation of PKC or L-VOCCs leads to MLCP phosphorylation in retinal arterioles was also assessed.
We utilized an isolated vessel approach and pharmacological tools to assess porcine retinal arteriolar constriction to activators of PKC (PDBu, 0.1 μM) and L-VOCC (Bay K 8644, 1 μM). Immunoblotting was used to determine phospho-MLCP levels in retinal arterioles.
Isolated and pressurized (55 cmH2O) porcine retinal arterioles (~80 µm maximum diameter) developed stable basal tone (~42% of maximum diameter), and constricted by 22±6% and 16±5% in response to PDBu and Bay K 8644, respectively. The broad-spectrum PKC inhibitor Gö-6983 (3 µM) and L-VOCC blocker nifedipine (1 µM) prevented, as well as reversed, the vasoconstriction elicited by PDBu. Inhibition of MLC kinase with ML-9 did not prevent the development of vasoconstrictions to PDBu or Bay K 8644, but reversed vasoconstrictions to both agonists. Both PDBu and Bay K 8644 elicited an increase in phospho-MLCP levels relative to control.
PKC activation stimulates calcium influx through L-VOCCs, which leads to retinal arteriolar constriction. MLC kinase is activated by calcium from L-VOCCs for maintenance, but not for development, of PDBu-induced retinal vasoconstriction. Increased inhibitory phosphorylation of MLCP with PDBu and Bay K 8644 treatments suggests that this signaling mechanism may enhance MLC phosphorylation and subsequently contribute to retinal vasoconstriction in response to PKC activation.
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