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Lowering intraocular pressure (IOP) is currently the only strategy documented to slow the onset and progression of glaucomatous blindness. Ouabain, a cardiotonic glycoside inhibitor of Na(+), K(+)-activated ATPase, was recently reported to enhance outflow facility in porcine anterior segments at concentrations as low as 30 nM for ≥4 h, suggesting a novel approach to lowering IOP. The underlying mechanism is unknown, but associated cytoskeletal changes were observed in porcine trabecular meshwork cells. We have previously found that changes in ATP release and subsequent ectoenzymatic conversion to adenosine may play a role in linking cytoskeletal remodeling with modulation of outflow resistance. We now tested whether altered ATP release might also be a mediator of ouabain's effect on outflow facility. ATP release from transformed human TM5 and explant-derived human trabecular meshwork cells was measured by the luciferin-luciferase reaction. Matrix metalloproteinases (MMPs) were studied by zymography, cell Na(+) concentration by SBFI fluorometry, gene expression of ATP-release pathways by real-time PCR, cell volume by electronic cell sorting and cell viability by the LDH and MTT methods. Actin was examined by confocal microscopy of phalloidin-stained cells. Contrary to expectation, ouabain at concentrations ≥10 nM inhibited swelling-triggered ATP release from TM5 cells after ≥4 h of exposure. Inhibition was enhanced by increasing ouabain concentration and exposure time. Similar effects were produced by the reversible cardiac aglycone strophanthidin. Ouabain also inhibited swelling-activated ATP release from explant-derived native human TM cells. Ouabain (4 h, 30 nM and 100 nM) did not alter gene expression of the ATP-release pathways, and cell viability was unchanged by exposure to ouabain (30 nM-1 μM). Preincubation with 30 nM ouabain for 4 h did not detectably change Na(+) level, the regulatory volume decrease (RVD) or the actin cytoskeleton of TM5 cells, but did inhibit hypotonicity-elicited ATP release. Moreover, even when N-methyl-d-glucosamine replaced Na(+) in the extracellular fluid, ouabain still inhibited swelling-initiated ATP release at 100 nM. In the absence of ouabain, extracellular ATP stimulated MMP secretion, which was largely blocked by inhibiting conversion of ATP to adenosine, as expected. In contrast, ouabain reduced ATP release, but did not alter secretion of MMP-2 and MMP-9 from cells pretreated for ≤4 h. The results suggest that: (1) ouabain can trigger enhancement of outflow facility independent of its transport and actin-restructuring effects exerted at higher concentration and longer duration; (2) ouabain exerts parallel independent effects on ATP release and outflow facility; and (3) these effects likely reflect ouabain-induced changes in the scaffolding and/or signaling functions of Na(+), K(+)-activated ATPase.

Our guiding hypothesis is that ecto-enzymatic conversion of extracellular ATP to adenosine activates A(1) adenosine receptors, reducing resistance to aqueous humor outflow and intraocular pressure. The initial step in this purinergic regulation is ATP release from outflow-pathway cells by mechanisms unknown. We measured similar ATP release from human explant-derived primary trabecular meshwork (TM) cells (HTM) and a human TM cell line (TM5). Responses to 21 inhibitors indicated that pannexin-1 (PX1) and connexin (Cx) hemichannels and P2X(7) receptors (P2RX(7) ) were comparably important in modulating ATP release induced by hypotonic swelling, whereas vesicular release was insignificant. Consistent with prior studies of PX1 activity in certain other cells, ATP release was lowered by the reducing agent dithiothreitol. Overexpressing PX1 in HEK293T cells promoted, while partial knockdown (KD) in both HEK293T and TM5 cells inhibited hypotonicity-activated ATP release. Additionally, KD reduced the pharmacologically defined contribution of PX1 and enhanced those of Cx and P2RX(7) . ATP release was also triggered by raising intracellular Ca(2+) activity with ionomycin after a prolonged lag time and was unaffected by the PX1 blocker probenecid, but nearly abolished by P2RX(7) antagonists. We conclude that swelling-stimulated ATP release from human TM cells is physiologically mediated by PX1 and Cx hemichannels and P2X(7) receptors, but not by vesicular release. PX1 appears not to be stimulated by intracellular Ca(2+) in TM cells, but can be modulated by oxidation-reduction state. The P2RX(7) -dependent component of swelling-activated release may be mediated by PX1 hemichannels or reflect apoptotic magnification of ATP release, either through itself and/or hemichannels.

The only effective intervention to slow onset and progression of glaucomatous blindness is to lower intraocular pressure (IOP). Among other modulators, adenosine receptors (ARs) exert complex regulation of IOP. Agonists of A(3)ARs in the ciliary epithelium activate Cl(-) channels, favoring increased formation of aqueous humor and elevated IOP. In contrast, stimulating A(1)ARs in the trabecular outflow pathway enhances release of matrix metalloproteinases (MMPs) from trabecular meshwork (TM) cells, reducing resistance to outflow of aqueous humor to lower IOP. These opposing actions are thought to be initiated by cellular release of ATP and its ectoenzymatic conversion to adenosine. This view is now supported by our identification of six ectoATPases in trabecular meshwork (TM) cells and by our observation that external ATP enhances TM-cell secretion of MMPs through ectoenzymatic formation of adenosine. ATP release is enhanced by cell swelling and stretch. Also, enhanced ATP release and downstream MMP secretion is one mediator of the action of actin depolymerization to reduce outflow resistance. Inflow and outflow cells share pannexin-1 and connexin hemichannel pathways for ATP release. However, vesicular release and P2X(7) release pathways were functionally limited to inflow and outflow cells, respectively, suggesting that blocking exocytosis might selectively inhibit inflow, lowering IOP.

ATP release by nonpigmented (NPE) and pigmented (PE) ciliary epithelial cells is the enabling step in purinergic regulation of aqueous humor formation, but the release pathways are unknown. We measured ATP release from primary cultures of bovine mixed NPE and PE (bCE) cells and transformed bovine NPE and PE cells, using the luciferin-luciferase reaction. Hypotonicity-triggered bCE ATP release was inhibited by the relatively selective blocker of pannexin-1 (PX1) hemichannels (probenecid, 1 mM, 47 ± 2%), by a connexin inhibitor (heptanol, 1 mM, 49 ± 4%), and by an inhibitor of vesicular release (bafilomycin A1, 25 ± 2%), but not by the P2X7 receptor (P2RX7) antagonist KN-62. Bafilomycin A1 acts by reducing the driving force for uptake of ATP from the cytosol into vesicles. The reducing agent dithiothreitol reduced probenecid-blockable ATP release. Similar results were obtained with NPE and PE cell lines. Pannexins PX1–3, connexins Cx43 and Cx40, and P2RX7 were identified in native cells and cell lines by RT-PCR. PX1 mRNA expression was confirmed by Northern blots; its quantitative expression was comparable to that of Cx43 by real-time PCR. Heterologous expression of bovine PX1 in HEK293T cells enhanced swelling-activated ATP release, inhibitable by probenecid. We conclude that P2RX7-independent PX1 hemichannels, Cx hemichannels, and vesicular release contribute comparably to swelling-triggered ATP release. The relatively large response to dithiothreitol raises the possibility that the oxidation-reduction state is a substantial regulator of PX1-mediated ATP release from bovine ciliary epithelial cells.

The purpose of the study was to determine whether novel, selective antagonists of human A 3 adenosine receptors (ARs) derived from the A 3 -selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A 3 AR antagonists lowered mouse IOP measured with SNMS tonometry by 3–5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A 3 AR agonists and the dihydropyridine A 3 AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 min. Like MRS 1191, agonist-based A 3 AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A 3 ARs derived from the potent, selective A 3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A 3 AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly-selective A 3 AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A 3 antagonists for study in multiple animal models.

Intraocular pressure is directly dependent on aqueous humor flow into, and resistance to flow out of, the eye. Adenosine has complex effects on intraocular pressure. Stimulation of A1and A2Aadenosine receptors changes intraocular pressure oppositely, likely through opposing actions on the outflow of aqueous humor. While the cellular sites regulating outflow resistance are unknown, the cells lining the inner wall of Schlemm's canal (SC) are a likely regulatory site. We applied selective adenosine receptor agonists to SC cells in vitro to compare the responses to A1and A2Astimulation. Parallel studies were conducted with human inner-wall SC cells isolated by a novel enzyme-assisted technique and with cannula-derived mixed inner- and outer-wall SC cells. A1agonists increased whole cell currents of both inner-wall and cannula-derived SC cells. An A2Aagonist reduced currents most consistently in specifically inner-wall SC cells. Those currents were also increased by A2B, but not consistently affected by A3, stimulation. A1, A2A, and A3agonists all increased SC-cell intracellular Ca2+. The electrophysiological results are consistent with the possibility that inner-wall SC cells may mediate the previously reported modulatory effects of adenosine on outflow resistance. The results are also consistent with the presence of functional A2B, as well as A1, A2A, and A3adenosine receptors in SC cells.

The eye’s aqueous humor is secreted by a bilayered ciliary epithelium comprising pigmented (PE) and nonpigmented (NPE) epithelial cell layers. Stromal Cl−enters the PE cells and crosses gap junctions to the NPE cells for release into the aqueous humor. Maxi-Cl−channels are expressed in PE cells, but their physiological significance is unclear. To address this question, excised patches and whole native bovine PE cells were patch clamped, and volume was monitored by calcein fluorescence. In symmetrical 130 mM NaCl, cAMP at the cytoplasmic surface of inside-out patches produced concentration-dependent activation of maxi-Cl−channels with a unitary conductance of 272 ± 2 pS ( n = 80). Voltage steps from 0 to ±80 mV, but not to ±40 mV, produced rapid channel inactivation consistent with the typical characteristics of maxi-Cl−channels. cAMP also activated the maxi-Cl−channels in outside-out patches. In both cases, maxi-Cl−channels were reversibly inhibited by SITS and 5-nitro-2-(phenylpropylamino)benzoate (NPPB). Decreasing cytoplasmic Cl−concentration reduced both open-channel probability and unitary conductance. Similarly, the membrane-permeant 8-bromo-cAMP stimulated outward and inward whole cell currents; the stimulation was larger at higher intracellular Cl−concentration. As with unitary currents, cAMP-triggered whole cell currents displayed inactivation at ±80 but not at ±40 mV. Moreover, cAMP triggered NPPB-sensitive shrinkage of PE cells. The results suggest that cAMP directly activates maxi-Cl−channels of native PE cells that contribute to Cl−release particularly from Cl−-loaded cells. These cAMP-activated channels provide a potential mechanism for reducing and modulating net aqueous humor secretion by facilitating Cl−reabsorption into the ciliary stroma.

The volume of certain subpopulations of trabecular meshwork (TM) cells may modify outflow resistance of aqueous humor, thereby altering intraocular pressure. This study examines the contribution that Na+/H+, Cl−/HCO[Formula: see text]exchange, and K+-Cl−efflux mechanisms have on the volume of TM cells. Volume, Cl−currents, and intracellular Ca2+activity of cultured human TM cells were studied with calcein fluorescence, whole cell patch clamping, and fura 2 fluorescence, respectively. At physiological bicarbonate concentration, the selective Na+/H+antiport inhibitor dimethylamiloride reduced isotonic cell volume. Hypotonicity triggered a regulatory volume decrease (RVD), which could be inhibited by the Cl−channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the K+channel blockers Ba2+and tetraethylammonium, and the K+-Cl−symport blocker [(dihydroindenyl)oxy]alkanoic acid. The fluid uptake mechanism in isotonic conditions was dependent on bicarbonate; at physiological levels, the Na+/H+exchange inhibitor dimethylamiloride reduced cell volume, whereas at low levels the Na+-K+-2Cl−symport inhibitor bumetanide had the predominant effect. Patch-clamp measurements showed that hypotonicity activated an outwardly rectifying, NPPB-sensitive Cl−channel displaying the permeability ranking Cl−> methylsulfonate > aspartate. 2,3-Butanedione 2-monoxime antagonized actomyosin activity and both increased baseline [Ca2+] and abolished swelling-activated increase in [Ca2+], but it did not affect RVD. Results indicate that human TM cells display a Ca2+-independent RVD and that volume is regulated by swelling-activated K+and Cl−channels, Na+/H+antiports, and possibly K+-Cl−symports in addition to Na+-K+-2Cl−symports.

Purpose Trabecular meshwork (TM) cell volume is a determinant of aqueous humor outflow resistance, and thereby IOP. Regulation of TM cell volume depends on chloride ion (Cl-) release through swelling-activated channels (ICl,Swell), whose pore is formed by LRRC8 proteins. Chloride ion release through swelling-activated channels has been reported to be regulated by calcium-activated anoctamins, but this finding is controversial. Particularly uncertain has been the effect of anoctamin Ano6, reported as a Ca2+-activated Cl- (CaCC) or cation channel in other cells. The current study tested whether anoctamin activity modifies volume regulation of primary TM cell cultures and cell lines. Methods Gene expression was studied with quantitative PCR, supplemented by reverse-transcriptase PCR and Western immunoblots. Currents were measured by ruptured whole-cell patch clamping and volume by electronic cell sizing. Results Primary TM cell cultures and the TM5 and GTM3 cell lines expressed Ano6 3 to 4 orders of magnitude higher than the other anoctamin CaCCs (Ano1 and Ano2). Ionomycin increased cell Ca2+ and activated macroscopic currents conforming to CaCCs in other cells, but displayed significantly more positive mean reversal potentials (+5 to +12 mV) than those displayed by ICl,Swell (-14 to -21 mV) in the same cells. Nonselective CaCC inhibitors (tannic acid>CaCCinh-A01) and transient Ano6 knockdown strongly inhibited ionomycin-activated currents, ICl,Swell and the regulatory volume response to hyposmotic swelling. Conclusions Ionomycin activates CaCCs associated with net cation movement in TM cells. These currents, ICl,Swell, and cell volume are regulated by Ano6. The findings suggest a novel clinically-relevant approach for altering cell volume, and thereby outflow resistance, by targeting Ano6.

The β-adrenergic antagonist timolol reduces ciliary epithelial secretion in glaucomatous patients. Whether inhibition is mediated by reducing cAMP is unknown. Elemental composition of rabbit ciliary epithelium was studied by electron probe X-ray microanalysis. Volume of cultured bovine pigmented ciliary epithelial (PE) cells was measured by electronic cell sizing; Ca2+activity and pH were monitored with fura 2 and 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Timolol (10 μM) produced similar K and Cl losses from ciliary epithelia in HCO[Formula: see text]/CO2solution but had no effect in HCO[Formula: see text]/CO2-free solution or in HCO[Formula: see text]/CO2solution containing the carbonic anhydrase inhibitor acetazolamide. Inhibition of Na+/H+exchange by dimethylamiloride in HCO[Formula: see text]/CO2solution reduced Cl and K comparably to timolol. cAMP did not reverse timolol's effects. Timolol (100 nM, 10 μM) and levobunolol (10 μM) produced cAMP-independent inhibition of the regulatory volume increase (RVI) in PE cells and increased intracellular Ca2+and pH. Increasing Ca2+with ionomycin also blocked the RVI. The results document a previously unrecognized cAMP-independent transport effect of timolol. Inhibition of Cl−/HCO[Formula: see text] exchange may mediate timolol's inhibition of aqueous humor formation.

Rabbit ciliary body and cornea were mounted in Ussing-type chambers in Tyrode's under voltage clamp and the effects of heptanol, a gap junction inhibitor, on the short circuit current generated by each of the respective epithelia were determined. Studies were carried out either in control conditions or following amphotericin B permeabilization of either the basolateral membrane of the nonpigmented epithelium of the ciliary body or the apical membrane of the corneal epithelium, respectively. Previous studies have shown that, following these permeabilizations, short circuit currents are established, reflecting aqueous (or tear)-to-serosa Na+ fluxes, and that Na+ translocation through gap junctions connecting the individual layers of these tissues constitutes the major rate limiting step. Heptanol inhibited most of the short circuit current of the amphotericin B-modified ciliary body and cornea and of the unmodified ciliary body epithelium (control). In all these cases, the apparent IC50 was about 0.8 M. In the unmodified corneal epithelium, where ion translocation across the apical membrane constitutes the main rate limiting step for active secretion, 0.4 or 0.8 mM heptanol induced short circuit current increases; partial inhibition was observed only at high concentrations known to cause maximal inhibition of junctional permeability. Heptanol also enhanced the volume regulatory decrease of cultured human NPE cells, a process dependent on cell swelling-induced stimulation of Cl- and K+ permeabilities. Combined with our previous results demonstrating the lack of heptanol effects on other epithelial functions, these data suggest that the effect of heptanol on the active ciliary body transepithelial transport is primarily due to inhibition of the nonpigmented-pigmented junctional path and that this path is a potential site of rate limitation for the secretory process.

Protein kinase C (PKC) is a major regulator of a broad range of cellular functions. Activation of PKC has been reported to stimulate Na+ transport across frog skin epithelium by increasing the apical Na+ permeability. This positive natriferic response has not been observed with other epithelial preparations, and could reflect the specific experimental conditions of different laboratories, or species or organ specificity of the response to PKC. In the present study, measurements were conducted with skins and urinary bladders from the same animals of two different species. The PKC activator TPA uniformly increased the transepithelial Na+ transport (measured as amiloride-sensitive short-circuit current, ISC, across skins from Rana temporaria and Bufo marinus, and inhibited ISC across bladders from the same animals. Inhibitors of PKC (staurosporine, H-7 and chelerythrine) partially blocked the TPA-induced stimulation of ISC across frog skin. The specificity of the PKC response by amphibian skin could have reflected an induction of moulting, similar to that observed with aldosterone. However, light micrographs of paired areas of frog skin revealed no evidence of the putative moulting. Separation of stratum corneum from the underlying stratum granulosum could be detected following application of aldosterone. We conclude that the effect of PKC on epithelial Na+ channels is organ, and not species specific. The stimulation of Na+ permeability in amphibian skin does not arise from sloughing of the stratum corneum. These observations are consistent with the hypothesis that the natriferic action arises from the calcium-independent isozyme of PKC previously detected in frog skin.

Electrophysiologic and volumetric evidence link the swelling-activated Cl- channels [gCl(Vol)] of nonpigmented ciliary epithelial (NPE) cells with the Cl(-)-channel/Cl(-)-channel regulator protein pICln. However, inhibitors (verapamil and dideoxyforskolin) of another Cl- channel/regulator (MDR1) have been found to inhibit the volume-activated transport response [the regulatory volume decrease (RVD)] of bovine NPE cells. We have addressed the possible molecular basis for the NPE Cl- channels by volumetric measurements of ODM human NPE cells in hypotonic and isotonic test solutions, and by polymerase chain reaction (PCR) cloning and Northern analyses of the same cells. Verapamil and dideoxyforskolin did inhibit the RVD. However, at a concentration (100 microM) which blocks90% of the MDR1-associated Cl- currents, forskolin had no effect on the volume-activated Cl- channels or on the inhibition of those channels by protein kinase C. High concentrations of ATP (3.5 and 10 mM) and niflumic acid (IC50 approximately 200 microM) also block [gCl(Vol)]. The RVD is inhibited by 9-phenylanthranilic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), unaffected by anthracene-9-carboxylic acid (9-AC), and stimulated by ionomycin. The Cl(-)-channel blockers NPPB, niflumic acid, DPC and 9-AC, and the Ca2(+)-ionophore ionomycin had qualitatively similar effects on the rate of staurosporine-activated isotonic cell shrink-age. These results support the concept that the volume-sensitive protein pICln regulates the Cl- channels, and that the same conduits subserve volume- and staurosporine-activated Cl- release. Of the cloned and sequenced Cl- channels, ClC-3 uniquely conforms to the stationary currents and PKC sensitivity of the NPE Cl- channels. PCR amplifications of human cDNA libraries from ciliary body, NPE cells and retina with primers based on human ClC-3 and ClC-4 cDNA, and Northern analyses using the products generated indicated that ciliary epithelial cells express transcripts for ClC-3 (but not ClC-4). We suggest that ClC-3 provides the same conduit for both volume-activated and isotonically staurosporine-activated Cl- channels of human nonpigmented ciliary epithelial cells.

Cells (ODM C1-2/SV40) derived from human non-pigmented ciliary epithelial cells were studied by electronic cell sizing. The time course of the cell volume (vc) was monitored after suspending cells in paired experimental and control, isosmotic and hyposmotic solutions of identical ioniccomposition. Following anisosmotic cell swelling, the cells displayed the regulatory volume decrease (RVD) previously described. The RVD primarily reflects loss of cell KCl since: (1) the K+-channel blockers quinidine and Ba2+ both inhibit the RVD; and (2) replacement of external Cl− with gluconate or addition of the Cl− channel blocker NPPB also inhibits the RVD. Bicarbonate has previously been reported to speed the RVD. This action likely reflects pH dependence of the channels since: (1) increasing the external pH speeds the RVD, whether or not HCO3− is present; and (2) DIDS (a blocker of Cl− channels and of Cl−/HCO 3− exchange) is an effective inhibitor of the RVD, even after blocking Cl−/HCO3− exchange by removing external HCO3−. The RVD could also be inhibited by reducing the availability of Ca 2+, either by omitting Ca2+ from the external medium or by blocking mobilization of intracellular Ca2+ with TMB-8. Furthermore, the RVD was slowed and incomplete in the presence of the calcium/calmodulin blocker trifluoperazine. We conclude that anisosmotic swelling triggers a series of events, mediated at least in part by calcium/calmodulin, leading to the extrusion of KCl through parallel K+ and Cl− channels.

Activators of protein kinase C (PKC) stimulate Na+ transport (JNa) across frog skin. We have examined the effect of Ca2+ on PKC stimulation of JNa. Both the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and the diacyl-glycerol sn-1,2-dioctanoylglycerol (DiC8) were used as PKC activators. Blocking Ca2+ entry into the cytosol (either from external or internal stores) reduced the subsequent natriferic effect of the PKC activators. This negative interaction did not simply reflect saturation of activation of the apical Na+ channels, since the stimulations produced by blocking Ca2+ entry and adding cyclic AMP were simply additive. The Ca2+ dependence of the natriferic effect could have reflected either a direct action of cytosolic Ca2+ on PKC or an indirect action on the final receptor site (the Na+ channel). To distinguish between these possibilities, the TPA- and phospholipid-dependent kinase activity of broken-cell preparations was assayed. The kinase activity was not stimulated by physiological levels of Ca2+, and in fact was inhibited at millimolar concentrations of Ca2+. We conclude that the effects of Ca2+ on the natriferic response to PKC activators are indirect. Reducing cytosolic uptake of Ca2+ may have stimulated Na+ transport by a chemical modification of the apical channels observed in other tight epithelia. The usual stimulation of Na+ transport produced by PKC activators in frog skin may reflect the operation of a nonconventional form of PKC. This enzyme is Ca2+ independent and seems related to the nPKC or PKC epsilon observed in other systems.

Activation of A1 and A2A subtype adenosine receptors (AR) likely exert opposing effects on outflow of aqueous humor, and thereby, on intraocular pressure. Selective agonists of adenosine receptor (AR) subtypes have previously been applied to trabecular meshwork (TM) and Schlemm's canal (SC) cells to identify the site(s) of differential purinergic modulation. However, the apparent changes in volume monitored by previously measuring projected cell area might have partially reflected cell contraction and relaxation. In addition, whole-cell current responses of the TM cells previously described were highly variable following application of selective A1, A2A and A3 agonists. The complexity of the electrophysiologic responses may have reflected cell heterogeneity of the populations harvested from collagenase digestion of TM explants. We now report measurements of TM-cell volume using calcein fluorescence quenching, an approach independent of contractile state. Furthermore, we have applied selective AR agonists to a uniform population of human TM cells, the hTM5 cell line. A1, but not A2A or A3, AR agonists triggered TM-cell shrinkage. Both A1 and A2A AR agonists produced reproducible increases in TM-cell whole-cell currents of similar magnitude. The results suggest that previous measurements of explant-derived TM cells may have reflected a range of responses from phenotypically different cell populations, and that the opposing effects of A1 and A2A agonists on outflow resistance are not likely to be mediated by actions on a single population of TM cells. These opposing effects might reflect AR responses by two or more subpopulations of TM cells, by TM and SC cells or by inner-wall SC cells, alone.

Purpose: Antagonists to A3 adenosine receptors (ARs) lower mouse intraocular pressure (IOP), but extension to humans is limited by species variability. We tested whether the specific A3AR antagonist MRS 1292, designed to cross species, mimicks the effects of other A3AR antagonists on cultured human nonpigmented ciliary epithelial (NPE) cells and mouse IOP. Methods: NPE cell volume was monitored by electronic cell sorting. Mouse IOP was measured with the Servo-Null Micropipette System. Results: Adenosine triggered A3AR-mediated shrinkage of human NPE cells. Shrinkage was blocked by MRS 1292 (IC50 = 42 ± 11 nM, p < 0.01) and by another A3AR antagonist effective in this system, MRS 1191. Topical application of the A3AR agonist IB-MECA increased mouse IOP. MRS 1292 reduced IOP by 4.0 ± 0.8 mmHg at 25-μ M droplet concentration (n = 10, p < 0.005). Conclusions: MRS 1292 inhibits A3AR-mediated shrinkage of human NPE cells and reduces mouse IOP, consistent with its putative action as a cross-species A3 antagonist.

A(1) adenosine receptors (ARs) reduce, and A(2)ARs increase intraocular pressure, partly by differentially altering resistance to aqueous humor outflow. It is unknown whether the opposing effects of A(1)AR and A(2)AR agonists are mediated at different outflow-pathway cell targets or by opposing actions on a single cell target. We tested whether a major outflow-pathway cell, the trabecular meshwork (TM) cell might constitute the primary AR-agonist target and respond differentially to A(1), A(2A) and A(3)AR agonists. Receptor activation in human TM cells was identified by applying subtype-selective AR agonists: CPA and ADAC for A(1)ARs, CGS 21680 and DPMA for A(2A)ARs, and Cl-IB-MECA and IB-MECA for A(3)ARs. Stimulation of A(1), A(2A) and A(3)ARs elevated Ca(2+), measured with fura-2. Whole-cell patch clamping indicated that AR agonists activated ion channels non-uniformly, possibly reflecting variability in magnitude of agonist-triggered second-messenger responses. A(1), A(2A) and A(3)AR agonists all reduced volume, determined by calcein cell imaging. The endogenous source of adenosine delivery to the outflow pathway could be the TM cells since these cells were stimulated to release ATP by hypotonic perfusion. We conclude that: (1) TM cells express functional A(1), A(2A) and A(3)ARs; and (2) the reported differential effects of AR agonists on aqueous humor outflow are not mediated by differential actions on TM-cell Ca(2+) and volume, but likely by actions on separate cell targets.

Purines regulate intraocular pressure. Adenosine activates Cl−channels of nonpigmented ciliary epithelial cells facing the aqueous humor, enhancing secretion. Tamoxifen and ATP synergistically activate Cl−channels of pigmented ciliary epithelial (PE) cells facing the stroma, potentially reducing net secretion. The actions of nucleotides alone on Cl−channel activity of bovine PE cells were studied by electronic cell sorting, patch clamping, and luciferin/luciferase ATP assay. Cl−channels were activated by ATP > UTP, ADP, and UDP, but not by 2-methylthio-ATP, all at 100 μM. UTP triggered ATP release. The second messengers Ca2+, prostaglandin (PG)E2, and cAMP activated Cl−channels without enhancing effects of 100 μM ATP. Buffering intracellular Ca2+activity with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′- tetraacetic acid or blocking PGE2formation with indomethacin inhibited ATP-triggered channel activation. The Rp stereoisomer of 8-bromoadenosine 3′,5′-cyclic monophosphothioate inhibited protein kinase A activity but mimicked 8-bromoadenosine 3′,5′-cyclic monophosphate. We conclude that nucleotides can act at >1 P2Y receptor to trigger a sequential cascade involving Ca2+, PGE2, and cAMP. cAMP acts directly on Cl−channels of PE cells, increasing stromal release and potentially reducing net aqueous humor formation and intraocular pressure.

Medical therapy of glaucoma commonly aims at slowing aqueous humor formation by the ocular ciliary epithelial bilayer, but underlying mechanisms are poorly understood. The first step in secretion is NaCl uptake from the stroma into the pigmented ciliary epithelial (PE) cell layer by electroneutral transporters. After crossing gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, solute is released into the aqueous humor. Published data have indicated that both paired Na+/H+ and Cl-/HCO3- antiporters and the Na+-K+-2Cl- symporter are involved in net uptake. The molecular identities of the paired antiporters have not been elucidated. We have studied continuously cultured bovine PE cells. Acid-activated 22Na+ uptake was inhibited by cariporide, EIPA (ethyl-isopropyl-amiloride) and amiloride, at concentrations characteristic of the NHE-1 isoform. Videomicroscopy of BCECF-loaded PE cells verified the presence of an EIPA-inhibitable Na+/H+ antiporter. Removing external Cl- also triggered an alkalinization, which was Na+-independent and could be inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Application of hypotonicity followed by return to isotonicity triggered a regulatory volume increase, which was pharmacologically similar to the uptake mechanisms described for intact rabbit ciliary epithelium. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of RNA from the human ciliary body detected expression of the AE2 Cl-/HCO3- exchanger, but not of AE1, cAE3 or bAE3. Immunostaining of bovine PE cells also revealed the presence of AE2 epitope. We conclude that paired NHE-1 Na+/H+ and AE2 Cl-/HCO3- antiporters are important components in the initial step in aqueous humor formation.

Chloride release from nonpigmented ciliary epithelial (NPE) cells is a final step in forming aqueous humor, and adenosine stimulates Cl− transport by these cells. Whole cell patch clamping of cultured human NPE cells indicated that the A3-selective agonist 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)- N-methyl-β-d-ribofuranuronamide (IB-MECA) stimulated currents ( I IB-MECA) by ∼90% at +80 mV. Partial replacement of external Cl−with aspartate reduced outward currents and shifted the reversal potential ( V rev) from −23 ± 2 mV to −0.0 ± 0.7 mV. Nitrate substitution had little effect. Perfusion with the Cl− channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid inhibited the currents. Partial Cl− replacement with aspartate and NO3 −, and perfusion with NPPB, had similar effects on the swelling-activated whole cell currents ( I Swell). Partial cyclamate substitution for external Cl− inhibited inward and outward currents of both I IB-MECA and I Swell. Both sets of currents also showed outward rectification and inactivation at large depolarizing potentials. The results are consistent with the concept that A3-subtype adenosine agonists and swelling activate a common population of Cl− channels.

The aqueous humour is formed by a bilayered ciliary epithelium (Cole, 1977), whose cells intercommunicate through gap junctions (Spitznas & Reale, 1975; Raviola & Raviola, 1978; Green et al. 1985; Coca-Prados et al. 1992; Edelman et al. 1994; Oh et al. 1994; Schutte & Wolosin, 1996). Secretion is generally thought to reflect a primary transfer of solute, largely NaCl, from the stroma to the aqueous humour, providing an osmotic driving force for the secondary osmotic transfer of water (Cole, 1977), although a more direct coupling between water and solute may also proceed across epithelia (Meinild et al. 1998). The activity of Cl− channels is likely to be a rate-limiting factor in aqueous humour secretion, given the low baseline level of channel activity (Coca-Prados et al. 1995). Release of Cl− by the non-pigmented ciliary epithelial (NPE) cells into the adjacent aqueous humour would enhance secretion, and Cl− release by the pigmented ciliary epithelial (PE) cells into the neighbouring stroma would reduce net secretion (Civan, 1998). Purines affect both aqueous humour dynamics and the cellular physiology of the ciliary epithelium, although the connection between these processes is unclear. At the in vivo level, A1-adenosine receptor stimulation has been shown to decrease intraocular pressure in rabbits (Crosson, 1995) and cynomologus monkeys (Tian et al. 1997) while A2-adenosine receptor agonists lead to elevated intraocular pressure in rabbits and cats (Crosson & Gray, 1996), but the effects on aqueous humour formation are uncertain. At the isolated cell level, most of the work concerns the NPE cells. In vitro studies have suggested that the NPE cells possess A1-adenosine receptors which lower [cAMP], A2-adenosine receptors which elevate [cAMP] and P2U2 receptors which increase phosphoinositide hydrolysis when stimulated (Wax et al. 1993). Adenosine acts with acetylcholine to elevate [Ca2+]i in the NPE cells (Farahbakhsh & Cilluffo, 1997), while ATP alone elevates [Ca2+]i (Shahidullah & Wilson, 1997). NPE cells have been reported to release ATP to the extracellular surface, where ATP can be metabolized to adenosine by ecto-enzymes (Mitchell et al. 1998). Single cell studies were tied to ionic, and thus aqueous, transport when adenosine agonists were shown to reduce NPE cell volume via Cl− pathways, activate whole cell Cl− channels in these same cells, and increase the short circuit current when applied to the aqueous face of iris ciliary body under conditions which isolated Cl− movement (Carreet al. 1997). The combination of these three techniques suggested that this stimulation of Cl− transport could lead to an increase in aqueous humour production and a recent report suggests this stimulation is mediated primarily by the A3-adenosine receptor (Mitchell et al. 1999). Considerably less information is available about the effects of purines on the PE cells. The cells have been reported to possess A1, A2 and P2Y2 receptors (Wax et al. 1993) and ATP elevates [Ca2+]i (Shahidullah & Wilson, 1997). In addition, the PE cells store and release ATP, and can degrade it extracellularly (Mitchell et al. 1998). However, the functional implications of these observations on either cellular or whole tissue physiology are unclear. In the present study we have asked how ATP affects the volume of PE cells in order to provide an initial link between purines, PE cells and net aqueous humour formation.

The aqueous humor of the eye is secreted by the bilayered ciliary epithelium, consisting of the pigmented (PE) cell layer facing the stroma and the nonpigmented (NPE) cell layer facing the aqueous humor. Cells within each layer and between the two layers are linked by gap junctions, forming a ciliary epithelial syncytium. Unidirectional secretion from the stroma to the aqueous proceeds both through the cells (the transcellular pathway) and between the cells (the paracellular pathway). Net formation of aqueous humor must, however, be the algebraic sum of unidirectional secretion and unidirectional reabsorption from the aqueous humor back into the stoma. The mechanisms potentially underlying reabsorption of aqueous humor by the NPE cells have recently been addressed by studying the regulatory response (RVI) of anisosmotically shrunken NPE cells. The results indicated that epithelial Na+ channels with a high affinity to amiloride likely contribute to reabsorption of solute from the aqueous humor. We have substantiated this possibility by using Northern analysis to identify in human ciliary body RNA a 3.7-kb transcript corresponding to the α-subunit of the amiloride-sensitive, αβγ-ENaC epithelial sodium channel. We have also found that the Na+-channel inhibitor benzamil inhibits the RVI without affecting the cell volume of isotonic cell suspensions. This observation supports the hypothesis that the low conductance, highly selective epithelial Na+ channel is activated by shrinkage and contributes to unidirectional reabsorption as aqueous humor. Examples are provided of how the integrative regulation of aqueous humor formation can involve conjugate actions on both unidirectional secretion and reabsorption. J. Exp. Zool. 279:498–503, 1997. © 1997 Wiley-Liss, Inc.

Ciliary epithelial cells possess multiple purinergic receptors, and occupancy of A1 and A2 adenosine receptors is associated with opposing effects on intraocular pressure. Aqueous adenosine produced increases in short-circuit current across rabbit ciliary epithelium, blocked by removing Cl− and enhanced by aqueous Ba2+. Adenosine’s actions were further studied with nonpigmented ciliary epithelial (NPE) cells from continuous human HCE and ODM lines and freshly dissected bovine cells. With gramicidin present, adenosine (≥3 μM) triggered isosmotic shrinkage of the human NPE cells, which was inhibited by the Cl− channel blockers 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) and niflumic acid. At 10 μM, the nonmetabolizable analog 2-chloroadenosine and AMP also produced shrinkage, but not inosine, UTP, or ATP. 2-Chloroadenosine (≥1 μM) triggered increases of whole cell currents in HCE cells, which were partially reversible, Cl− dependent, and reversibly inhibited by NPPB. Adenosine (≥10 μM) also stimulated whole cell currents in bovine NPE cells. We conclude that occupancy of adenosine receptors stimulates Cl− secretion in mammalian NPE cells.

The M-1 cell line is derived from the mouse cortical collecting duct and displays the low-conductance, highly Na(+)-selective channel activity of the alpha,beta, gamma-heterotrimeric epithelial Na+ channel (ENaC). The short-circuit current (Isc) across M-1 monolayers was 89 +/- 4 microA/cm2, and the transepithelial conductance was 2.1 +/- 0.2 mS/cm2. Isc was abolished by blocking the Na+ pump with ouabain. Both Isc and transepithelial conductance (gT) were inhibited by benzamil > amiloride >> dimethylamiloride. Under our experimental conditions, vasopressin, vasopressin, forskolin, and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) had no detectable effects on Isc or gT. Increasing apical Na+ entry with nystatin increased Isc. The possible regulation of the M-1 Na+ channel by cAMP-activated protein kinase A (PKA) was further examined with excised inside-out patches. The open-time probability (Po) was not fixed, displaying substantial variance. Perfusion with ATP itself, with the catalytic subunit of PKA with ATP, or with alkaline phosphatase had no consistent effect on Po, the unitary current, or the kinetics of the M-1 Na+ channel. The data are consistent with the concept that PKA stimulates ENaCs by phosphorylating a site with access to but not within the apical membrane patch during cell-attached and excised-patch studies.

Cells (ODM/SV40) derived from human non-pigmented ciliary epithelial cells were studied by electronic cell sizing. After transiently suspending the cells in hypotonic solution, isotonicity was restored by addition of sucrose. The cell volumes (vc) initially fell below those of control isotonic suspensions, and subsequently increased towards the baseline level. This secondary increase in vc is termed the regulatory volume increase (RVI). Results obtained with ionic substitutions and transport inhibitors indicate that four ionic mechanisms can support the RVI in these cells: coupled Na+/H+ and Cl-/HCO3- antiports, a Na+/Cl- symport, a Na+/K+/2Cl- symport, and a Na+ channel in parallel with a Cl-/HCO3- antiport. Arachidonic acid metabolites regulate the RVI very differently from their effects on the regulatory volume response (RVD) of the same cells to cell swelling. Prostaglandin E2 (PGE2), leukotriene (LTD4) and the PKC-inhibitor staurosporine all inhibit the RVI. Blockade of the cyclooxygenase, lipoxygenase and epoxygenase pathways of arachidonic acid metabolism [with 5,8,11,14-eicosatetraynoic acid (ETYA)] produces a net acceleration of the RVI. In contrast, PGE2 and staurosporine stimulate, LTD4 has no effect, and ETYA inhibits the RVD. We suggest that knowledge of the ionic mechanisms and intracellular signalling underlying the RVI phenomenon may provide a basis for reducing the rate of net aqueous humor formation by increasing the rate of reabsorption of fluid from the aqueous humor into the non-pigmented ciliary epithelial cells.

Transport defects by retinal pigment epithelial (RPE) and other cells are observed in experimental models of diabetes mellitus. Recent studies have established that glucose concentration, per se, is the critical risk factor in the pathogenesis of diabetic complications. This study was designed to test whether transport alterations could be produced in the simplest model of diabetes, sustained exposure of cultured cells to a high-glucose environment. The regulatory transport responses to acute changes in cell volume were measured in order to assess the effects of glucose on a range of transport processes. Continuous lines of nontransformed human retinal pigment epithelial (hRPE) cells were grown for two weeks with either 5.6 low glucose (LG) or 26.0 high glucose (HG) mM in paired experiments. The cell volumes of suspended cells were studied in hypo-, iso- and hypertonic solutions containing the same ionic composition. Hypotonic swelling triggered a regulatory volume decrease (RVD), inhibited by reducing the chemical driving force for K+ efflux, or blocking K+ channels (with Ba2+) or Cl- channels (with NPPB). Thus, the RVD of the hRPE cells likely reflects efflux of K+ and Cl- through parallel channels. Shrinkage caused a regulatory volume increase (RVI), which was inhibited by blocking Na+/H+ (with dimethylamiloride) or Cl-/HCO3- exchange (with DIDS). Bumetanide inhibited the RVI significantly only when the K+ concentration was increased above the baseline level. Therefore, the RVI under our baseline conditions likely reflects primarily Na+/H+ and Cl-/HCO3- antiport exchange. Growth in high-glucose medium had no substantial effect on the RVD, but reduced the rate constant of the RVI by approximately 50%. The RVI was unaffected by growth in high-mannitol medium. Stimulation of protein kinase C (PKC) with DiC8 increased the RVI of HG-cells, but not of LG-cells. The DiC8-induced stimulation was bumetanide insensitive and abolished by 1 mM amiloride. Other transport effects of PKC (on the RVD) were unaltered in the HG-cells. We conclude that sustained elevation of extracellular glucose, per se, can downregulate the Na+/H+ antiport of target cells, an effect noted in streptozotocin-treated rats, and that this downregulation does not reflect interruption of the PKC-signaling pathway.

To test whether adenosine triphosphate (ATP) release links cytoskeletal remodeling with release of matrix metalloproteinases (MMPs), regulators of outflow facility and intraocular pressure.ATP release was measured by luciferin-luciferase. Ecto-ATPases from transformed human trabecular meshwork (TM) cells (TM5) and explant-derived TM cells were identified by RT-PCR. Actin was visualized by phalloidin staining. Cell viability was assayed by lactate dehydrogenase and thiazolyl blue tetrazolium bromide methods and propidium iodide exclusion, gene expression by real-time PCR, and MMP release by zymography. Cell volume was monitored by electronic cell sorting.Hypotonicity (50%) and mechanical stretch increased ATP release with similar pharmacologic profiles. TM cells expressed ecto-ATPases E-NPP1-3, E-NTPD2, E-NTPD8, and CD73. Prolonged dexamethasone (DEX) exposure (≥ 2 weeks), but not brief exposure (3 days), increased cross-linked actin networks and reduced swelling-triggered ATP release. Cytochalasin D (CCD) exerted opposite effects. Neither DEX nor CCD altered the cell viability, gene expression, or pharmacologic profile of ATP-release pathways. DEX accelerated, and CCD slowed, the regulatory volume decrease after hypotonic exposure. Activating A(1) adenosine receptors (A(1)ARs) increased total MMP-2 and MMP-9 release. DEX reduced total A(1)AR-triggered MMP release, and CCD increased the active form of MMP-2 release. The A(1)AR agonist CHA and the A(1)AR antagonist DPCPX partially reversed the effects of DEX and CCD, respectively.Cytoskeletal restructuring modulated swelling-activated ATP release, in part by changing the duration of cell swelling after hypotonic challenge. Modifying ATP release is expected to modulate MMP secretion by altering ecto-enzymatic delivery of adenosine to A(1)ARs, linking cytoskeletal remodeling and MMP-mediated modulation of outflow facility.

Insulin stimulates Na+ transport across frog skin, toad urinary bladder, and the distal renal nephron. This stimulation reflects an increase in apical membrane Na+ permeability and a stimulation of the basolateral membrane Na,K-exchange pump. Considerable indirect evidence has suggested that the apical natriferic effect of insulin is mediated by activation of protein kinase C. However, no direct information has been available documenting that insulin and protein kinase C indeed share a common pathway in stimulating Na+ transport across frog skin. In the present work, we have studied the interaction of insulin and phorbol 12-myristate 13-acetate (PMA), a documented activator of protein kinase C. Preincubation of skins with 1,2-dioctanoylglycerol, another activator of protein kinase C, increases baseline Na+ transport and reduces the subsequent natriferic response to PMA. Preincubation with PMA markedly reduces the subsequent natriferic action of insulin. This effect does not appear to primarily reflect PMA-induced internalization of insulin receptors. The insulin receptors are localized on the basolateral surface of frog skin, but the application of PMA to this surface is much less effective than mucosal treatment in reducing the response to insulin. Preincubation with D-sphingosine, an inhibitor of protein kinase C, also reduces the natriferic action of insulin. The current results provide documentation that insulin and protein kinase C share a common pathway in stimulating Na+ transport across frog skin. The data are consistent with the concept that the natriferic effect of insulin on frog skin is, at least in part, mediated by activation of protein kinase C.

Whole skins and isolated epithelia were bathed with isotonic media (congruent to 244 mOsm) containing sucrose or glucose. The serosal osmolality was intermittently reduced (congruent to 137 mOsm) by removing the nonelectrolyte. Transepithelial and intracellular electrophysiological parameters were monitored while serosal osmolality was changed. Serosal hypotonicity increased the short-circuit current (ISC) and the basolateral conductance, hyperpolarized the apical membrane (psi mc), and increased the intracellular Na+ concentration. The increases in apical conductance and apical Na+ permeability (measured from Goldman fits of the relationship between amiloride-sensitive current and psi mc) were not statistically significant. To verify that the osmotically induced changes in ISC were mediated primarily at the basolateral membrane, the basolateral membrane potential of the experimental area was clamped close to 0 mV by replacing the serosal Na+ with K+ in Cl--free media. The adjoining control area was exposed to serosal Na+. Serosal hypotonicity produced a sustained stimulation of ISC across the control, but not across the adjoining depolarized tissue area. The current results support the concept that hypotonic cell swelling increases Na+ transport across frog skin epithelium by increasing the basolateral K+ permeability, hyperpolarizing the apical membrane, and increasing the electrical driving force for apical Na+ entry.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) activates protein kinase C (PKC) and produces an early stimulation of Na+ transport across frog skin. The ionic basis for this stimulation was studied with combined transepithelial and intracellular electrical measurements. In an initial series of experiments, TPA approximately doubled the amiloride-sensitive short-circuit current (ISC), apical Na+ permeability (PapNa), and apical membrane conductance without affecting the basolateral membrane conductance. The apical effects led to a marked depolarization of the short-circuited skin and a small increase in intracellular Na+ concentration. TPAs increase of PapNa was sufficient to explain the stimulation of basolateral Na+ transport when both the voltage and substrate dependence of the pump were taken into account. After the early stimulation, TPA later depressed ISC. Added at this point (congruent to 1-2 h after TPA administration), insulin had no effect on ISC, whereas a partial response to vasopressin was still observed. Measured either early or late after TPA addition, the phorbol ester reduced insulin binding by congruent to 40%. Insofar as 60% of the specific binding is retained, the abolishment of insulin's natriferic response is unlikely to result from the TPA-induced reduction in hormonal binding. The data provide further support for the concept that activation of PKC produces an early stimulation of Na+ transport by increasing apical Na+ permeability, and that part of insulin's natriferic effect may be mediated by PKC activation.

The phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) stimulates baseline Na+ transport across frog skin epithelium and partially inhibits the natriferic response to vasopressin. The effects are produced largely or solely when TPA is added to the mucosal surface of the tissue. Although TPA activates protein kinase C, it has other effects, as well. Thus, the biochemical basis for the effects and the ionic events involved have been unclear. Furthermore, the physiologic implications have been obscure because of the sidedness of TPA's actions. We now report that two synthetic diacylglycerols (DAG) replicate the stimulatory and inhibitory effects of TPA on frog skin. DAG is the physiologic activator of PKC. In this tissue, it produces half-maximal stimulation at a concentration of less than or equal to 19 microM. In contrast to TPA, DAG is about equally effective from either tissue surface. In a series of eight experiments, DAG was found to depolarize the apical membrane. Diacylglycerol also increases the paracellular conductance of frog skins bathed with mucosal Cl- Ringer's solution. The latter effect can be minimized by replacing NO3- for Cl- in the mucosal solution. Under these conditions, combined intracellular and transepithelial measurements indicated that DAG increased both the apical Na+ permeability and intracellular Na+ concentration. These results are qualitatively similar to the effects of cyclic 3',5'-AMP on this tissue, suggesting that activation of PKC by DAG causes phosphorylation of the same or nearby gating sites phosphorylated by cAMP. We propose that apical Na+ entry is regulated in part by activation of PKC, and that insulin may be a physiologic trigger of this activation.