The role of protein kinase C in modulation of aqueous humor outflow facility Rahul N. Khurana a , Pei-Feng Deng a , David L. Epstein a , P. Vasantha Rao a,b, * a Department of Ophthalmology, Duke University Medical Center, Durham, NC, USA b Department of Pharmacology and Cancer, Duke University Medical Center, Durham, NC, USA Received 11 March 2002; accepted in revised form 15 September 2002 Abstract The elevated intraocular pressure that is commonly associated with glaucoma is believed to arise due to impairment of trabecular meshwork (TM) function. Although the TM and Schlemm’s canal (SC) comprise the major route for aqueous humor outflow, little is known about the potential signaling mechanisms involved in the regulation of aqueous outflow. Based on knowledge regarding the role of protein kinase C (PKC) in vascular biology, we sought to understand the contribution of the PKC pathway towards outflow function by studying the modulation of contractile and morphological characteristics of TM and SC cells. We investigated the involvement of PKC in regulation of myosin light chain (MLC) phosphorylation, formation of actin stress fibers and integrin – ECM adhesions (focal adhesions) in human TM and SC cells and correlated these changes with aqueous outflow facility measured in an enucleated porcine whole eye perfusion model. Expression and distribution of PKC isoforms (a and 1) in TM and SC cells and tissues was confirmed by Western blot and immunohistochemical analysis, respectively. Both, pharmacological activators (phorbol-12-myristate 13-acetate (PMA) and phorbol-12,13- dibutyrate (PDBu)) and inhibitors (staurosporine and GF109203X) of PKC were found to induce changes in cell shape (retraction and rounding up) and cytoskeletal organization in human TM and SC cells. While PMA and PDBu produced an increase in formation of actin stress fibers and focal adhesions and in MLC phosphorylation, PKC inhibitors were observed to induce contrasting effects in these cells. Intriguingly, both PDBU and GF109203X caused increases in aqueous outflow facility in the perfusion model. The PKC inhibitor (GF109203X) increased outflow by 46% while the PKC activator (PDBu) only increased outflow by 27%. These results suggest that PKC might play an important role in modulation of aqueous outflow facility by regulating MLC phosphorylation and thereby, the morphological and cytoskeletal characteristics of TM and SC cells. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: cytoskeleton; myosin light chain phosphorylation; protein kinase C; trabecular meshwork; Schlemm’s canal; contraction; perfusion; aqueous humor outflow 1. Introduction Understanding the potential regulatory mechanisms influencing aqueous humor outflow facility has important implications for unraveling the etiology of glaucoma and developing better therapy. The elevated intraocular pressure that is commonly associated with glaucoma is believed to arise mainly due to impairment of trabecular meshwork (TM) function (Epstein, 1987a; Lutjen-Drecoll, 1998; Johnson and Erickson, 2000). The TM has smooth muscle like properties and is actively involved in aqueous humor dynamics through contractile mechanisms (de Kater, Shahsafaei, and Epstein, 1992; Wiederholt, Thieme, and Stumpff, 2000). The contractile and relaxation properties of TM and SC cells are believed to influence morphological and cell adhesion characteristics and thereby through these mechanisms could alter paracellular permeability and the geometry of the aqueous outflow pathway (Kaufman and Erickson, 1982; Epstein et al., 1987b; Erickson-Lamy, Schroeder, and Epstein, 1992; Epstein, Roberts, and Skinner, 1997; O’Brien et al., 1997; Tian et al., 1998; Epstein, Rowlette, and Roberts, 1999; Kaufman et al., 1999; Peterson et al., 2000; Tian et al., 2000a; Wiederholt et al., 2000). Interestingly, agents such as cytochalasin, latruncu- lin, H-7, ethacrynic acid, BDM, M-7, staurosporine, vinblastin and endothelin, which influence cellular acto- myosin and microtubule dynamics and cell adhesion, have 0014-4835/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S0014-4835(02)00255-5 Experimental Eye Research 76 (2003) 39–47 www.elsevier.com/locate/yexer * Author for correspondence: Dr P. Vasantha Rao, Department of Ophthalmology, Duke University Medical Center, Box 3802, Durham, NC 27710, USA. E-mail address: rao00011@mc.duke.edu (P. Vasantha Rao).