Endothelin-Induced Changes of Secondary Messengers in Cultured Corneal Endothelial Cells

Abstract

The effect of endothelins on corneal endothelial cells is not well understood. We have investigated the effects of endothelin-1 (ET-1), endothelin-2 (ET-2) and endothelin-3 (ET-3) on bovine corneal endothelial cellular proliferation and the secondary messenger changes in cells in the presence of ET-1. It was found that the 3H-thymidine uptake was enhanced by ET-1 significantly, whereas ET-2 and ET-3 had no effect. ET-1 remarkably affects the increase of corneal endothelial cells on 3H-thymidine, 3H-leucine, and 3H-uridine uptakes in a dose-dependent manner. The 50% effective concentrations (EC50) for ET-1, as measured by 3H-thymidine uptake, 3H-uridine uptake, and 3H-leucine uptake were 10(-8.78) M, 10(-8.53) M and 10(-8.04) M, respectively. It was found that endothelin-1 increased intracellular calcium concentration by using the method of preloading with Fura-2-AM and assaying with spectrophotometry. The cellular IP1, IP2, and IP3 were also stimulated in the presence of ET-1. Moreover, ET-1 enhanced the basal cellular cAMP and cGMP concentrations in corneal endothelial cells in a dose-dependent manner. Immunofluorescent staining revealed that ET-1 increased the fibronectin protein concentration and changed protein distribution in corneal endothelial cells. These findings indicate that endothelin-1 increases in cell proliferation and biological changes may be involved in changing intracellular calcium mobility, increasing intracellular phosphoinositides, enhancing intracellular cGMP and cAMP accumulation, and fibronectin protein synthesis.

Hyperglycaemia Inhibits Thymidine Incorporation and Cell Growth via Protein Kinase C, Mitogen‐Activated Protein Kinases and Nitric Oxide in Human Umbilical Vein Endothelium

“…Incorporation of thymidine into DNA has been used as an index of cell proliferation in HUVECs (Sobrevia et al 1996;Lau & Ma, 1996;Zanetti et al 2001;Bussolati et al 2001) and other endothelial cell types (Parenti et al 1998;Heller et al 1999;Grant et al 1999;Wu et al 2001;Kim et al 2001;Wang et al 2002;Spyridopoulus et al 2002). Thymidine incorporation was reduced in HUVECs incubated with 25 mM D-glucose for 24 h (Sobrevia et al 1995).…”

“…cGMP, a second messenger for NO (Shaul, 2002), and cAMP are increased by high levels of D-glucose (Sobrevia et al 1996), and induce inhibition (Zanetti et al 2001;Wu et al 2001;Kim et al 2001) or stimulation (Grant et al 1999;Wu et al 2001) of endothelial cell proliferation. D-Glucose also activates protein kinase C, which is associated with longer term increases in NO production in HUVECs (Pan et al 1995;Montecinos et al 2000) and inhibition of endothelial cell proliferation (Wang et al 2002;Spyridopoulus et al 2002).…”

Effect of Latanoprost on Cultured Porcine Corneal Stromal Cells

“…17,18 The cultured corneal stromal cells were plated into 12-well plates for at least 24 hr to ensure attachment. Cultured cells were incubated with serumfree RPMI 1640 medium with various concentrations of latanoprost ranging from 10 −5 , 10 −6 , 10 −7 to 10 −8 M for 1.5 hr.…”

Injury-elicited differential transcriptional regulation of phospholipid growth factor receptors in the cornea

“…Previous work has found that corneal endothelium intracellular Ca 2ϩ can be increased through receptor-mediated pathways by histamine (5, 6), purinergic agonists (5, 30), bradykinin (5,40), and endothelin-1 (5,38). Adrenergic agonists decreased [Ca 2ϩ ] i (34).…”