Presence of functional type B natriuretic peptide receptor in human ocular cells.

PURPOSE: To study the effects of natriuretic peptides on cyclic guanosine monophosphate (cGMP) production and calcium mobilization in cultured human ocular cells. METHODS: Cultured simian virus 40-transformed (HTM-3) and nontransformed (HTM-16) human trabecular meshwork (TM) cells and nontransformed human ciliary muscle (CM) cells were used. Accumulation of cGMP in cells lysate was measured by radioimmunoassay. Intracellular calcium concentration was measured by microscope-based ratiofluorometry. RESULTS: Both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) increased the accumulation of cGMP in HTM-3, HTM-16, and CM cells. In the nontransformed TM cells, CNP was five times more efficacious (maximal effect of CNP was 497% +/- 44% that of ANP) and 10 times more potent than ANP (ANP, log [EC50] = -6.99 +/- 0.08; CNP, log [EC50] = -7.96 +/- 0.20). Similar results were seen in HTM-3 and CM cells. Under the assay conditions used, the peptides increased only the production of cGMP without changing its degradation rate. The peptide-induced increase of cGMP in the TM and CM cells correlated with suppression of carbachol-induced calcium mobilization in the cell. CONCLUSIONS: It is known that CNP, but not ANP, selectively activates the guanylyl cyclase associated with the type B natriuretic peptide receptor (NPR-B). Thus, the data suggest that NPR-B is the primary functional NPR in the TM and CM cells. The effects on cGMP and calcium produced by the activation of this receptor are expected to alter TM and CM contractility and may affect aqueous humor hydrodynamics and intraocular pressure.

Effects of muscarinic agents on cultured human trabecular meshwork cells.

Intracellular calcium measurements were performed in cultured human trabecular meshwork cells preloaded with the cell permeant dye fura 2-AM. Fluctuations in calcium levels were then monitored with microscope-based ratio fluorometry. Carbachol increased intracellular calcium in a dose-dependent manner; as did oxotremorine-M, aceclidine, and pilocarpine. Carbachol's effect was blocked by the non-selective muscarinic antagonist atropine, as well as by muscarinic receptor subtype-selective antagonists such as pirenzepine (M1-selective), p-fHHSiD (M3-selective), and 4-DAMP (M1, M3 subtypes). Rank order of potencies for the antagonists' effects was atropine = 4-DAMP > p-fHHSiD > pirenzepine, a profile suggesting that the M3 receptor subtype is essential in the carbachol effect. Phospholipase C activity was estimated via measurement of total production of inositol phosphates in cultured human trabecular meshwork cells pre-exposed to 3H-myoinositol. In these cells, carbachol also stimulated phosphoinositide production in a dose-dependent manner, and an antagonist profile similar to that seen for calcium response was obtained when carbachol was used as the effector. The data indicate that muscarinic effects on cultured human trabecular meshwork calcium mobilization and phospholipase C activity are mediated by an M3-like receptor subtype. Therefore, the muscarinic M3 receptor may play a role in trabecular meshwork cell function(s).

Preliminary characterization of a transformed cell strain derived from human trabecular meshwork.

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.

Single-cell contraction assay for human ciliary muscle cells. Effect of carbachol.

PURPOSE: The authors developed an assay to observe the contraction of a single human ciliary muscle cell. METHODS: Cultured human ciliary muscle cells were partially detached from the culture dish by incubation with a nonenzymatic dissociation buffer and treated with carbachol or pilocarpine. Contraction was quantified by measuring the cross-sectional surface areas of the cells. RESULTS: Carbachol decreased the cell surface area in a time-dependent manner. Contraction was observed within 1 min after the addition of carbachol and completed in less than 15 min. The effect of carbachol was dose dependent. For example, at 10 min after treatment with 10 mumol/l carbachol, the relative surface areas of cells decreased to 47% +/- 4% (mean +/- standard error of the mean, n = 7, with surface area at 0 min defined as 100%). The relative surface areas were 74% +/- 4% (n = 7) after 1 mumol/l and 100% +/- 9% (n = 7) after 0.1 mumol/l carbachol treatment. This contractile effect was antagonized by pretreatment with atropine, a specific muscarinic antagonist. CONCLUSIONS: A simple method was established to study the functional changes of human ciliary muscle cells.

Insulin-dependent alterations of phorbol ester binding to adipocyte subcellular constituents. Evidence for the involvement of protein kinase C in insulin action.

The binding of tritiated phorbol-12,13-dibutyrate (3H-PBu2) was employed to estimate the mass of protein kinase C associated with plasma membranes and cytosol isolated from untreated and insulin-treated adipocytes. Binding of 3H-PBu2 to both plasma membranes and cytosol was rapid, achieving a steady state within minutes. Treatment of cells with physiological concentration of insulin (0.67 nM) caused a 42% increase (from 0.92 +/- 0.08 to 1.30 +/- 0.12 pmol 3H-PBu2/mg protein, p less than 0.0001) and a 27% decrease (from 0.41 +/- 0.07 to 0.30 +/- 0.05 pmol 3H-PBu2/mg protein, p less than 0.020) in phorbol ester bound to cytosol and plasma membranes, respectively. The half-maximal concentrations of unlabelled PBu2 needed to displace 3H-PBu2 bound to cytosol from control and insulin-treated cells were 54 and 13 pM, respectively. These data indicate that insulin modifies protein kinase C in adipocytes.

Chelation of intracellular calcium blocks insulin action in the adipocyte.

The hypothesis that intracellular Ca2+ 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 microM 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 CaCl2 and the Ca2+ ionophore A23187. These conditions had no effect on basal activity and omission of CaCl2 from the buffer prevented the restoration of insulin-stimulated glucose uptake by A23187. Quin-2 loading also inhibited insulin-stimulated glucose oxidation (IC50, 11 microM quin-2 AM) and the ability of insulin to inhibit cAMP-stimulated lipolysis (IC50, 78 microM quin-2 AM), without affecting their basal activities. Incubation of cells with 100 microM 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.

Evidence that protein kinase C is involved in regulating glucose transport in the adipocyte.

The role of protein kinase C in the mechanism of stimulation of glucose transport in rat adipocytes was investigated. Glucose transport was stimulated by dioleoylglycerol (DOG), tetradecanoyl phorbol acetate (TPA) and phospholipase C (PLC). Agents that inhibit protein kinase C (polymyxin B, gossypol and quercitin) also inhibited glucose transport that had been stimulated by DOG, TPA, PLC and insulin.