Constriction of Retinal Venules to Endothelin-1: Obligatory Roles of ETA Receptors, Extracellular Calcium Entry, and Rho Kinase

Abstract

Purpose: Endothelin-1 (ET-1) is a potent vasoconstrictor peptide implicated in retinal venous pathologies such as diabetic retinopathy and retinal vein occlusion. However, underlying mechanisms contributing to venular constriction remain unknown. Thus, we examined the roles of ET-1 receptors, extracellular calcium (Ca2+), L-type voltage-operated calcium channels (L-VOCCs), Rho kinase (ROCK), and protein kinase C (PKC) in ET-1-induced constriction of retinal venules.

Methods: Porcine retinal venules were isolated and pressurized for vasoreactivity study using videomicroscopic techniques. Protein and mRNA were analyzed using molecular tools.

Results: Retinal venules developed basal tone and constricted concentration-dependently to ET-1. The ETA receptor (ETAR) antagonist BQ123 abolished venular constriction to ET-1, but ETB receptor (ETBR) antagonist BQ788 had no effect on vasoconstriction. The ETBR agonist sarafotoxin S6c did not elicit vasomotor activity. In the absence of extracellular Ca2+, venules lost basal tone and ET-1-induced constriction was nearly abolished. Although L-VOCC inhibitor nifedipine also reduced basal tone and blocked vasoconstriction to L-VOCC activator Bay K8644, constriction of venules to ET-1 remained. The ROCK inhibitor H-1152 but not PKC inhibitor Gö 6983 prevented ET-1-induced vasoconstriction. Protein and mRNA expressions of ETARs and ETBRs, along with ROCK1 and ROCK2 isoforms, were detected in retinal venules.

Conclusions: Extracellular Ca2+ entry via L-VOCCs is essential for developing and maintaining basal tone of porcine retinal venules. ET-1 causes significant constriction of retinal venules by activating ETARs and extracellular Ca2+ entry independent of L-VOCCs. Activation of ROCK signaling, without involvement of PKC, appears to mediate venular constriction to ET-1 in the porcine retina.

Figure 1

Vasomotor response of isolated and pressurized porcine retinal venules to ET-1. (A) Venular diameters were recorded before (R: resting diameter) and after 30-minute treatment (T) with an ET-1 receptor antagonist. In the absence of receptor antagonist, retinal venules constricted to ET-1 in a concentration-dependent manner (Control; n = 9). ET_AR antagonist BQ123 (1 μM; n = 5), but not ET_BR antagonist BQ788 (0.1 μM; n = 5), inhibited venular constriction to ET-1. Both antagonists did not alter resting basal tone. *P < 0.05 versus Control. (B) Representative images of a porcine retinal venule at resting diameter and after constriction in response to ET-1 (0.1 nM). (C) The response of retinal venules to ET_BR agonist sarafotoxin S6c was examined (n = 6).

Figure 2

Molecular analyses of ET-1 receptors in porcine retinal venules. (A) Equal amounts of total RNA isolated from porcine retinal venules (RV) and neural retina tissue (RT) were reverse transcribed and then analyzed by real-time PCR for detection of ET_AR, ET_BR, and GAPDH mRNAs. The ET_AR and ET_BR transcripts were normalized to GAPDH expression and presented as relative mRNA expression. n = number of pigs studied. (B) Equal amount of protein was loaded for Western blot analyses of ET_AR and ET_BR in RVs and neural RT from pigs. Data represent four independent experiments.

Figure 3

Roles of extracellular calcium and L-VOCCs in vasoconstriction of isolated retinal venules to ET-1. (A) Venular diameters were recorded before (R: resting diameter) and after 30-minute treatment (T) of the vessel with Ca²⁺-free solution (n = 10) or L-VOCC inhibitor nifedipine (1 μM; n = 7). In the absence of treatment (Control; n = 12), retinal venules constricted to ET-1 in a concentration-dependent manner. Both Ca²⁺-free solution and nifedipine abolished resting tone, and the venular constriction to ET-1 was attenuated in the Ca²⁺-free solution. The vasoconstrictor response to ET-1 remained intact in the presence of nifedipine. #P < 0.05 versus R; *P < 0.05 versus Control. (B) The effect of L-VOCC agonist Bay K8644 (6 μM) on resting diameter (R) was examined in the absence (n = 3) or presence of nifedipine (1 μM; n = 3). *P < 0.05 versus R.

Figure 4

Roles of ROCK and PKC in vasoconstriction of isolated retinal venules to ET-1. (A) Venular diameters were recorded before (R: resting diameter) and after 30-minute treatment (T) with ROCK inhibitor H-1152 (3 μM; n = 6) or PKC inhibitor Gö 6983 (10 μM; n = 7). In the absence of treatment (Control; n = 12), retinal venules constricted to ET-1 in a concentration-dependent manner. The resting diameter of retinal venules was not altered by either drug treatment. The ET-1–induced constriction was abolished by H-1152 but not Gö 6983. *P < 0.05 versus Control. (B) Venular diameters were recorded under resting conditions (R) and at different concentrations of PKC activator PDBu (n = 6). (C) Equal amount of protein was loaded for Western blot analyses of ROCK isoforms (ROCK1 and ROCK2) and p38 in retinal venules (RV) and neural retina tissue (RT). Data represent three independent experiments.