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Cells isolated from the trabecular meshwork (TM) of a male glaucoma patient were transformed by transfection with an origin defective mutant of SV40 virus. Transformation dramatically increased the growth rate of these cells (designated HTM-3 cells), allowing biochemical and pharmacological characterization. The HTM-3 cells had cytoskeletal components that were reported to be present in TM tissue and non-transformed TM cells. Vimentin, tubulin and smooth muscle specific alpha-actin, but not desmin, were localized in these cells by immunocytochemistry. The extracellular matrix components collagen types I, III and IV, fibronectin and laminin were found in HTM-3 cells as well as their non-transformed parental cells. As predicted, the protein profile of the HTM-3 cells revealed by two-dimensional gel electrophoresis was different from that of the non-transformed cells, probably due to the enhanced growth characteristics of these cells. Furthermore, HTM-3 cells had various intracellular second messenger systems that responded to pharmacological agents. Forskolin, prostaglandin E2, beta-adrenergic and adenosine A2 agonists stimulated the adenylyl cyclase in these cells, whereas muscarinic, serotonergic, dopaminergic and other agonists were ineffective. Sodium nitroprusside increased the intracellular concentration of cGMP, demonstrating the presence of a functional guanylyl cyclase. Phospholipase C activity in these cells was also detected. Muscarinic agonists, histamine and bradykinin, but not adrenergic, serotonergic agonists or prostaglandins, increased phosphoinositide turnover. These drug responses of HTM-3 cells agree with published data on primary TM cells and TM tissues, suggesting that the transformed cells may be a valid substitute for certain pharmacological studies of TM.

The hypothesis that intracellular Ca21 is an essential component of the intracellular mechanism of insulin action in the adipocyte was evaluated. Cells were loaded with the Ca2+ chelator quin-2, by preincubating them with quin-2 AM, the tetrakis(acetoxymethyl) ester of quin-2. Quin-2 loading inhibited insulin-stimulated glucose transport (IC50, 26 ,AM quin-2 AM) without affecting basal activity. The ability of insulin to stimulate glucose uptake in quin-2-loaded cells could be partially restored by preincubating cells with buffer supplemented with 1.2 mM CaC12 and the Ca21 ionophore A23187.These conditions had no effect on basal activity and omission of CaC12 from the buffer prevented the restoration of insulinstimulated glucose uptake by A23187. Quin-2 loading also inhibited insulin-stimulated glucose oxidation (IC50, 11 ,IM quin-2 AM) and the ability of insulin to inhibit cAMPstimulated lipolysis (IC50, 78 ,uM quin-2 AM), without affecting their basal activities. Incubation of cells with 100 ,AM quin-2 or quin-2 AM had no effect on intracellular ATP concentration or the specific binding of 125I-labeled insulin to adipocytes. These findings suggest that intracellular Ca2+ is an essential component in the coupling of the insulin-activated receptor complex to cellular physiological/metabolic machinery. Furthermore, differing quin-2 AM dose-response profiles suggest the presence of dual Ca2+-dependent pathways in the adipocyte. One involves insulin stimulation of glucose transport and oxidation, whereas the other involves the antilipolytic action of insulin.The possible involvement of Ca2+ in the cellular mechanism of insulin action has been the subject of considerable study (1-3). Clausen et al. (4)

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