Mediation of calcium-independent contraction in trabecular meshwork through protein kinase C and rho-A.

PURPOSE: Inhibition of protein kinase C (PKC) and rho-kinase (ROCK) may represent a new way of influencing outflow facility through isolated relaxation of the trabecular meshwork (TM). This work was performed to investigate the existence of calcium-independent contraction in this smooth-muscle-like tissue and its modulation by targeting the rho-guanosine triphosphatase (GTPase)-mediated pathway. METHODS: Isometric tension measurements of bovine TM and ciliary muscle (CM) were performed. Intra- and extracellular calcium buffering was accomplished with EGTA and 1, 2-bis(2-aminophenoxy)-ethane-N,N:,N:,N:',N:'-tetra-acetic acid tetrakis/acetoxymethhyl ester (BAPTA-AM) followed by stimulation of PKC with phorbolester (PMA) or 4alpha-phorbol. Calcium-independent contraction was blocked using the highly specific ROCK inhibitor Y-27632. Western blot analysis and immunoprecipitation was performed using human TM cells. RESULTS: In TM, carbachol induced partial contraction under conditions of extracellular calcium depletion (22. 1% +/- 2.3% versus 100%, n = 9). The membrane-permeable calcium chelator BAPTA-AM completely blocked this response (1.1% +/- 1.4% versus 100%, n = 9). When calcium was completely blocked, PMA induced contraction in TM (16.7% +/- 5.9% versus 100%, n = 9) but not in CM (1.8% +/- 2.5% versus 100%, n = 6). The inactive PMA analogue 4alpha-phorbol did not induce contraction, indicating that activation of PKC is involved in this contractile response. The ROCK inhibitor Y-27632 completely blocked the calcium-independent PMA-induced contraction in TM. Western blot analysis and immunoprecipitation revealed the expression of the rho-A protein in human TM cells. CONCLUSIONS: The data indicate that contrary to CM, the TM features calcium-independent contractile mechanisms linked to rho-A and PKC isoforms that do not require calcium for activation. ROCK inhibitors may allow specific modulation of the TM to enhance outflow facility, thus lowering intraocular pressure.

The effects of protein kinase C on trabecular meshwork and ciliary muscle contractility.

PURPOSE: The possible role of protein kinase C (PKC) inhibitors in novel pressure-lowering drugs is currently under investigation. To gain further insight into regulation of contractility by PKC in trabecular meshwork (TM) and ciliary muscle (CM), the effects of various PKC inhibitors and activators were tested. METHODS: Isometric tension measurements of bovine TM and CM strips were performed. PKC was stimulated by phorbol ester and by the diacylglycerol analogue diC8. PKC blockade was accomplished using H7 and myristoilated PKC substrate (mPKC). Western blot analysis was used to identify specific PKC isoforms in human trabecular meshwork (HTM), human ciliary muscle (HCM), and bovine TM and CM. RESULTS: In tissues precontracted by carbachol PKC antagonist H7 led to a relaxation of TM (25+/-7.2 versus 100%; n = 8) with no effect on CM. mPKC substrate selectively blocks PKC. This substance led to relaxation of TM (32.8+/-7.4 versus 100%, n = 7), whereas CM was not affected. PMA at concentrations of 10(-6) M led to a slow contraction of both tissues that was more marked in TM. DiC8 and 4alpha-phorbol had no effect on contractility. Western blot analysis revealed expression of calcium-dependent PKC-alpha and calcium-independent PKC-epsilon isoforms in HTM and HCM. PKC-epsilon expression was more pronounced in HTM than in HCM. Similar PKC isoform expression was found in native bovine tissue. CONCLUSIONS: PKC isoforms show different tissue distributions in human and bovine TM and CM. Contractility differences exist in both tissues in response to PKC antagonists and agonists. The data indicate that PKC may be involved in regulation of aqueous humor outflow by the TM. Thus, inhibition of PKC may represent a new way of influencing outflow facility through isolated relaxation of TM.