Cells Acutely Harvested from Inner-Wall Cells and Explant-Derived Cells from Inner- and Outer-Wall of Schlemm's Canal Display Functional Adenosine Receptors

Abstract

Abstract: : Purpose: Adenosine receptor (AR) agonists alter aqueous humor outflow, but at unknown sites. We developed a novel technique to isolate cells from Schlemm’s canal (SC) inner wall by enzymatic treatment, and tested whether acutely harvested and explant-derived cells from SC respond to selective AR agonists. Methods: Inner wall SC tissue partially separated from trabecular meshwork was dissected from human donor corneal rims. SC cells were dissociated in clumps by 14-16 hr incubation with collagenase IV, elastase, hyaluronidase and DNAase. Single-cell suspensions were obtained after a further 5-min trypsin-DNAase step. Cells were incubated in laminin-coated culture plates with F99 medium and 10% FCS, 20 ng/ml bFGF and 10 ng/ml EGF without subsequent growth factor supplementation. Mixed inner and outer wall SC cells were isolated by a previously described explant-like technique involving 3-weeks incubation after placing a suture in the SC. Effects of AR agonists on intracellular Ca²⁺ of subconfluent cells were measured by fura-2 fluorescence. Acutely harvested cells were studied at passage P0, and explant-derived cells at P3-5. Results: Confluent primary cultures were initially polygonal with cobblestone, sheet-like, monolayered morphology and strong contact inhibition. At later passage, cells became fusiform and were linearly arranged in monolayers, as with explant-derived cells. All A₁ (30 nM ADAC, 100 nM CPA), A₂A (100 nM CGS-21680) and A₃ (100 nM Cl-IB-MECA) selective AR agonists tested increased Ca²⁺. ADAC and CGS-21680 increased Ca²⁺ of acutely harvested cells by 35% and 76%, respectively. CPA, CGS-21680 and Cl-IB-MECA increased Ca²⁺ of explant-derived cells by 38%, 100% and 153%, respectively. Conclusions: Cells can now be cultured both selectively from human inner wall SC and more quickly than the explant-derived culture of mixed inner and outer wall cells. Cells obtained by both techniques respond to AR agonists, consistent with a role of adenosine in modulating aqueous humor outflow.

Keywords: outflow: trabecular meshwork • second messengers: pharmacology/physiology • intraocular pressure