Tissue plasminogen activator reduces the elevated intraocular pressure induced by prednisolone in sheep.

We have previously shown that tissue plasminogen activator (tPA) injected in the vitreous of sheep, reduced or prevented the elevation of the intraocular pressure (IOP) normally produced by the instillation of 1% prednisolone. We now report the effect of tPA when injected into the anterior chamber (AC) in amounts of 0.01, 0.001 and 0.0001 µg diluted in a volume of 50 µL. Lyophilized tPA, obtained as Actilyse 50 mg from Boehringer Ingelheim containing arginine was utilized. The Actilyse was diluted in balanced salt solution to obtain the desired amount of tPA in 50 µL. An identical solution containing only arginine was prepared to inject into the contralateral eye as a control. Six sheep of the Corriedale breed were selected. At the beginning of the study all eyes received instillation of 1% prednisolone 3 times/day for 10 days to elevate their IOP from 10 mm Hg to about 23 mm Hg. Then, 0.0001 µg was injected into one of the eyes and its effect was followed for up to 55:00 h while the instillation of prednisolone continued in both eyes. The same protocol was implemented for the 0.001 and 0.01 µg amounts after extended washout and IOP was over 22 mm Hg. The injection of 0.0001 µg into the AC had no effect on an IOP of 23.0 mm Hg at 6:00 and 30:00 h after injection. 0.001 µg of tPA reduced IOP from 23.1 to 18.6 mm Hg at 6:00 h but IOP recovered to 22.3 mm Hg at 30:00 h. Injection of 0.01 µg produced a marked and prolonged reduction of IOP. From a baseline of 23.0, IOP was reduced to 14.0, 14.7, 21.2, and 20.9 mm Hg at 5.0, 23.0, 27.0 and 45.5 h, respectively. The 0.423 µg of arginine, which is associated with 0.01 µg tPA, was injected alone and had no effect. Recombinant human tPA injected in the AC is effective in reversing steroid-induced IOP elevation in sheep. The reduction of IOP elevation may be the result of an effect on extra-cellular matrix turnover in the TM. These findings suggest that tPA may by useful as a therapeutic agent in steroid-induced glaucomas.

Sildenafil stimulates aqueous humor turnover in rabbits.

Sildenafil citrate increases ocular blood flow and accelerates the rate of anterior chamber refilling after paracentesis. The latter effect could have resulted from a reduction in outflow facility or from an increase in aqueous humor (AH) production. In this study, we used scanning ocular fluorophotometry to examine the effects of sildenafil on AH turnover, and thus, AH production in eyes of live normal rabbits. For this, the rate of aqueous humor flow (AHF) was quantified with a commercially available fluorophotometer that measured the rate of fluorescein clearance from the anterior segment, which predominantly occurs via the trabecular meshwork. After ≈2 h of control scans to determine the baseline rate of AHF, the rabbits were fed 33 mg of sildenafil and allowed ≈45 min for the drug to enter the systemic circulation. Thereafter, fluorescence scans were retaken for an additional 90-120 min. Sildenafil ingestion increased AHF by about 36%, from 2.31 µL/min to 3.14 µL/min (P < 0.001, as two-tailed paired data, n = 20 eyes). This observation indicates that sildenafil citrate, which is a phosphodiesterase type-5 inhibitor currently marketed as a vasodilator (e.g., Viagra, Revatio), stimulates AHF in rabbits. Our results seem consistent with reports indicating that the drug dilates intraocular arteries and augments intraocular vascular flow. These physiological responses to the agent apparently led to increased fluid entry into the anterior chamber. As such, the drug might have utility in patients with ocular hypotony resulting from insufficient AH formation.

Fluid circulation determined in the isolated bovine lens.

PURPOSE: In 1997, a theoretical model was developed that predicted the existence of an internal, Na(+)-driven fluid circulation from the poles to the equator of the lens. In the present work, we demonstrate with a novel system that fluid movement can be measured across the polar and equatorial surface areas of isolated cow lenses. We have also determined the effects of ouabain and reduced bath [Na(+)]. METHODS: Lenses were isolated in a chamber with three compartments separated by two thin O-rings. Each compartment, anterior (A), equatorial (E), and posterior (P), was connected to a vertical capillary graduated in 0.25 µL. Capillary levels were read every 15 minutes. The protocols consisted of 2 hours in either open circuit or short circuit. The effects of ouabain and low-Na(+) solutions were determined under open circuit. RESULTS: In 21 experiments, the E capillary increased at a mean rate of 0.060 µL/min while the A and P levels decreased at rates of 0.044 and 0.037 µL/min, respectively, closely accounting for the increase in E. The first-hour flows under short circuit were approximately 40% larger than those in open-circuit conditions. The first-hour flows were always larger than those during the second hour. Preincubation of lenses with either ouabain or low-[Na(+)] solutions resulted in reduced rates of fluid transport. When KCl was used to replace NaCl, a transitory stimulation of fluid transport occurred. CONCLUSIONS: These experiments support that a fluid circulation consistent with the 1997 model is physiologically active.

Sildenafil accelerates anterior chamber refilling after paracentesis in sheep and rabbits.

PURPOSE: Sildenafil increases ocular blood flow. Thus, the authors investigated if it also increases anterior chamber (AC) refilling after paracentesis. METHODS: Corriedale sheep and albino rabbits were used as animal models. Intraocular pressure (IOP) was measured, paracentesis performed on one eye, and AC refilling followed by observation using oblique illumination. IOP measurements continued as the AC formed. After IOP stabilization, sildenafil (100 mg) was orally administered. Forty to 60 minutes later, AH was withdrawn from the contralateral eye. The point at which IOP recovered was used to determine refilling time. Paracentesis volumes were either 60, 120, or 300 µL in sheep, and 50 or 100 µL in rabbits. RESULTS: IOP recovered in approximately 49, 56, and 50 minutes after the 60, 120, and 300 µL withdrawals in sheep. The refilling times of the contralateral eye after sildenafil ingestion were approximately 19, 26, and 37 minutes for the respective AH withdrawals. With rabbits, IOP recovered in approximately 13 minutes after the 50 and 100 µL AH withdrawals. After sildenafil, the IOP recovery times of the fellow eye were approximately 6 minutes. AH refilling rates were estimated by dividing the paracentesis volume by IOP recovery time. After sildenafil, such rates were larger than the AH formation rate attributed to secretion by the ciliary epithelium. CONCLUSIONS: Sildenafil accelerates the rate of AC refilling and might have beneficial utility as an agent enhancing fluid entry into the AC of patients who experienced AH loss during eye surgery, as well as in some cases of ocular hypotony.

Interaction between mechanical and osmotic forces in the isolated rabbit lens.

Based on our previous work showing that cow and rabbit lenses isolated with their accommodation anatomical components intact change volume during simulated accommodation in vitro, and that hyposmolality and hyperosmolality also produce volume changes, we tested the idea that exerting these forces simultaneously may add or counteract each other. Further, we attempted to find a point at which osmotic and mechanical forces may cancel each other. Using previously described methodology, we found that combined stretching and anisotonic conditions applied to a lens always produced less of a volume change than that observed on its paired lens from the fellow eye that was only subjected to anisotonic conditions. Our results suggest that a stretching force that increases the equatorial diameter by 0.4% and reduces the lens volume by 1.8% could be canceled by a hyposmotic force of about -20 to -30 mOsM. Counter-intuitively, lenses that were subjected to stretching and hyperosmolality had less volume decrease than their paired lenses only exposed to hypertonicity. This latter observation is likely due to the prevention by the mechanical stretching forces of the shortening of the equatorial diameter, which normally occurs in hypertonic media.

Gene expression changes in steroid-induced IOP elevation in bovine trabecular meshwork.

PURPOSE: To determine whether gene expression changes occur in the trabecular meshwork (TM) of cow eyes with steroid-induced intraocular pressure (IOP) elevation. METHODS: Adult female Braford cows (n = 4) were subjected to uniocular prednisolone acetate treatment for 6 weeks. IOP was monitored with an applanation tonometer. At the conclusion of the experiment, animals were euthanized, eyes were enucleated, and the TM was dissected and stored in an aqueous nontoxic tissue storage reagent. RNA was extracted and subjected to microarray analysis using commercial oligonucleotide bovine arrays. Some of the genes differentially expressed between control and experimental eyes were confirmed by quantitative RT-PCR and some of the respective proteins were studied by immunoblotting. RESULTS: IOP began to increase after 3 weeks of treatment, reaching a peak 2 weeks later. IOP differences between corticosteroid-treated and fellow control eyes were 6 ± 1 mm Hg (mean ± SD) at the conclusion of the study. Microarray analysis revealed that expression of 258 genes was upregulated, whereas expression of 187 genes was downregulated in the TM of eyes with steroid-induced IOP elevation. Genes identified to be differentially expressed include genes coding for cytoskeletal proteins, enzymes, growth and transcription factors, as well as extracellular matrix proteins and immune response proteins. A number of relevant gene networks were detected by bioinformatic analysis. CONCLUSIONS: Steroid-induced IOP elevation alters gene expression in the bovine TM. Identification of genes with changing expression in this model of open-angle glaucoma may help elucidate the primary changes occurring at the molecular level in this condition.

An hypothesis on pressure transmission from anterior chamber to optic nerve.

Few studies have characterized how pressure in the anterior chamber (AC) of the eye is transmitted via the vitreous to the vitreous-ganglion cell interface. We are aware of only one study that simultaneously measured the pressures in the AC and vitreous humor; and of only one study that simultaneously measured the pressures in the AC and the suprachoroidal space (SCS). The pressure in the AC is defined as the intraocular pressure (IOP), which when elevated beyond statistically normal limits is a recognized risk factor for glaucoma, a malady best described as an optic neuropathy with degeneration and eventual death of the retinal ganglion cells (GC's) and highly characteristic changes in the optic nerve head (ONH). Most investigators currently believe that the prevalent risk factor for GC apoptosis is ocular hypertension, but no one has demonstrated how an increase in IOP in the AC is transmitted to the GC's. In patients with primary open angle glaucoma, the pressure in the AC increases due to an increase in the resistance of the trabecular meshwork (TM) outflow pathway. We questioned how such increased pressure in the AC would be transmitted to the GC to produce the changes in the ONH seen in glaucoma. Based on our preliminary data and purview of the literature, we hypothesize that a pressure increase originating in the AC is likely transmitted via both the SCS and the vitreous, with transmission via the former pathway probably most efficient in affecting the GC. Independently of the mechanism that produces GC apoptosis, the ones that are first affected, as repeatedly shown by visual field tests, are the most peripheral ones; i.e., those whose axons are the most external as they form the ONH and enter the lamina cribrosa. There are no published reports explaining this peculiarity. The dogma is that the pressure transmitted via the vitreous is higher at the periphery because it is transmitted across a shorter distance, since the vitreous acts as a buffer that absorbs part of the pressure being transmitted. We propose that IOP is not only transmitted via the vitreous but also via the SCS. Increases in IOP could be efficiently applied via the SCS to the most external axons of the ONH as they leave the eye. Our hypothesis can also explain low-tension glaucoma in which the most peripheral GC's are also affected first, because pressure is transmitted without decay due to a reduced uveoscleral (UVS) flow.

Effect of sildenafil citrate on intraocular pressure and blood pressure in human volunteers.

Anecdotal reports have suggested that the vasodilator, sildenafil citrate, which evokes its effect via a select inhibition of PDE5, has the potential to increase intraocular pressure (IOP) in some individuals. An ocular hypertensive effect by sildenafil was also recently described in a sheep animal model. In contrast, clinical studies have not found a direct association between sildenafil ingestion (commonly consumed as Viagra) and changes in IOP. However, some such studies also reported no effects of sildenafil on systemic blood pressure (BP) at the time of the IOP determination. Given this surprising result, our purpose was to repeat a study in human volunteers in the city of Corrientes, Argentina to corroborate the effects of sildenafil on human IOP and systemic BP. For the present study, 9 healthy volunteers (male and female, 18-74 years old) were selected as subjects after ophthalmic and cardiovascular evaluation indicated that they exhibited normal parameters for their age. In a masked, placebo-controlled study, the subjects ingested 100 mg sildenafil citrate (provided as Vorst from Laboratorios Bernabo, Argentina) in one session, and a placebo on a second separate occasion. IOP was measured with a Goldman applanation tonometer by an ophthalmologist, and BP by a second physician, neither of whom witnessed the tablet ingestion by the volunteers, nor provided with information on the nature of the test compounds. A third individual administered the tablets. The average baseline IOP of this group of 9 was 13.1 ± 0.6 mm Hg. Subsequent to sildenafil ingestion, IOP increased by 26% to 16.5 ± 0.8 mm Hg 60 min later (P < 0.005, as paired data), and returned to control values within 2 h. Both systolic and diastolic BP were significantly reduced by sildenafil ingestion. At the point of maximal systemic hypotension (90 min), the systolic and diastolic pressures declined by 15% and 13%, respectively. No significant changes in IOP or BP were recorded after ingestion of the placebo. Our results suggest that sildenafil can elicit a transient IOP increase that may be of importance to patients chronically treated with PDE5 inhibitors for various vascular diseases (e.g., pulmonary hypertension). We discuss possible mechanisms by which PDE5 inhibition might lead to a rise in IOP.

Suppression of corticosteroid-induced ocular hypertension in sheep by anecortave.

OBJECTIVE: To confirm the ocular hypotensive effects of anecortave acetate on an ovine model for steroid-induced ocular hypertension. Eyes of normal sheep exhibit a robust steroid-induced ocular hypertensive response. Recent observations in an uncontrolled, interventional case series indicated that anecortave elicited hypotensive effects when administered as a sub-Tenon depot in the eyes of a small sample of patients with glaucoma. METHODS: Intraocular pressure (IOP) was monitored by Perkins applanation tonometry in 16 normal sheep receiving topically administered prednisolone acetate, 0.5%, in both eyes, 3 times daily, a protocol that doubled IOP within 12 days. Half of the sheep had received a unilateral sub-Tenon injection of anecortave in 1 eye prior to the initiation of the bilateral prednisolone instillations, while the 8 remaining sheep received the unilateral anecortave sub-Tenon depot after the IOP was maximally elevated by the prednisolone instillations. RESULTS: In these 2 sets of experiments, the presence of the anecortave depot suppressed the steroid-induced IOP elevation and reverted the elevated IOP to baseline levels. Measurements of aqueous outflow facility indicated that eyes treated with prednisolone plus anecortave exhibited a 5.8-fold higher outflow facility than the fellow eyes solely exposed to prednisolone, indicating that anecortave prevented the increase in outflow resistance produced by the corticosteroid. CONCLUSION: Elucidation of the mechanisms of action of anecortave in animal models may prove relevant to the design of novel interventions for the management of primary open-angle glaucoma.

Treatment of sheep steroid-induced ocular hypertension with a glucocorticoid-inducible MMP1 gene therapy virus.

PURPOSE: To investigate whether intracameral injection of the adenovirus vector AdhGRE.MMP1 would reduce or prevent elevated intraocular pressure (IOP) induced by corticosteroids in living animals. METHODS: Glucocorticoid-inducible adenovirus vectors carrying wild-type or mutant forms of human metalloproteinase 1 (MMP1 and mutMMP1) cDNAs were generated. An adenovirus carrying no gene (Ad5.CMV.Null) was used as an additional control. Sheep were injected intracamerally with 30 microL of each vector, either previously or after the induction of increased IOP with topical prednisolone or sub-Tenon triamcinolone under various protocols. IOP was measured with a Perkins tonometer. Inflammation was monitored by visual inspection. RESULTS: In eyes in which IOP was already elevated to 24 to 30 mm Hg, injection of AdhGRE.MMP1 reduced IOP by 70% in 24 hours and to 10 to 13 mm Hg in 48 hours. In eyes with normal IOP (9-11 mm Hg), preinjection of the virus protected against the increase in IOP normally produced by the corticosteroid. IOP remained at a level of approximately 12 mm Hg for 5 days despite the continuous application of the corticosteroid. Injections of the control viruses had no hypotensive effects. There were no signs of ocular inflammation or discomfort to the animals. CONCLUSIONS: A single dose of a gene therapy vector carrying an inducible metalloproteinase human gene can both protect against the IOP increase produced by corticosteroid instillation in the sheep model and quickly reverse the IOP increase previously elicited by the corticosteroid. These results are a first step toward a treatment of steroid-glaucoma with inducible overexpression of extracellular matrix modulator genes.

Effects of sildenafil and tadalafil on intraocular pressure in sheep: implications for aqueous humor dynamics.

PURPOSE: To determine the effects of vasodilators on intraocular pressure (IOP) and the protein content of sheep aqueous humor (AH), because the vasodilators may increase fluid leakage from the fenestrated capillaries of the ciliary body to the extracellular tissue and directly to the anterior chamber (AC) via the iris, and some senior patients (older than 70) treated with sildenafil have exhibited elevated IOP. METHODS: Experiments were performed on domestic sheep residing on a ranch in Argentina. These docile and compliant animals readily swallowed tablets of sildenafil (50 and 100 mg) and tadalafil (20 mg). IOP was monitored by Perkins applanation tonometry in 21 normal sheep receiving orally administered drugs. In addition, paracentesis was performed on six sheep to quantify changes in AH protein levels. RESULTS: Ingestion of both sildenafil and tadalafil increased sheep IOP from normal levels of approximately 9 to 11 mm Hg within 1 hour. The IOP elevation was approximately 1.6-fold with both doses of sildenafil. IOP returned to control values within 4 hours. With the longer-lasting vasodilator tadalafil, IOP remained 1.6- to 1.9-fold higher than normal for at least 48 hours and returned to control levels within 4 days. The AH protein content was approximately 39% higher in sheep given 100 mg sildenafil. CONCLUSIONS: These data are consistent with a vasodilator-evoked increase in plasma-like fluid in the AC, which likely accounts for the IOP elevation. The results are discussed with a model for AH dynamics that may be of importance to senior individuals treated for vascular diseases with these compounds.

Changes in rabbit and cow lens shape and volume upon imposition of anisotonic conditions.

In vivo, mammalian lenses have the capacity to effect fully reversible changes in shape, and possibly volume, during the accommodation process. Isolated lenses also change shape by readily swelling or shrinking when placed in anisotonic media. However, the manner by which the lens changes its shape when its volume is changed osmotically is not firmly established. Putatively, the lens could swell or shrink evenly in all directions, or manifest distinctive swelling and/or shrinking patterns when exposed to anisotonic media. The present study measured physical changes in lenses consistent with the latter alternative using methods we developed for determining rapid changes in lens shape and volume. It was found in isolated rabbit and cow lenses that the length of the axis between the anterior and posterior poles (A-P length) primarily increases under hypotonic conditions (-40 to -100 mOsM), with smaller, or no changes, in equatorial diameter (ED). Hypertonic conditions (+50 to +100 mOsM) on rabbit lenses elicited a predominant reduction in ED, while the A-P length was only marginally reduced. Hypertonic solutions of +150 mOsM were required to obtain similar changes in cow lens shape. The ratio of the A-P length to the ED was taken as a measure of "circularity". This ratio increased gradually in rabbit and cow lenses bathed in hypotonic solutions because of the increase in the A-P length. The calculated lens volume increased in tandem with the increase in "circularity". Lens circularity also increased under hypertonic conditions due to the decrease in ED, but this increase in circularity during shrinkage was not as pronounced as that which occurred during swelling. As such, the lens has a tendency upon swelling to change its shape by approaching the structure of a globular spheroid (as occurs during accommodation for near focusing), but lens shrinkage does not result in a flatter lens with a reduced A-P length as occurs during dis-accommodation for distance focusing. Moreover, osmotically evoked shape changes appear irreversible, in contrast to the mechanically elicited shape changes of accommodation.

Steroid-induced ocular hypertension in normal sheep.

PURPOSE: To determine whether the ovine eye develops elevation of intraocular pressure (IOP) in response to corticosteroid applied topically. METHODS: IOP was monitored by Perkins applanation tonometry in a group of 18 sheep receiving topically administered 0.5% prednisolone acetate in one eye (experimental), three times daily, for a period of 3 or four 4 weeks after the establishment of baseline IOP values. Perkins readings were converted to actual mm Hg using a calibration curve derived from in vivo manometric measurements. IOP was monitored for an additional 1 to 3 weeks after discontinuation of corticosteroid treatment. RESULTS: Baseline IOP in normal sheep was 10.6+/-1.4 mm Hg (mean +/- SD; n=36 eyes). The IOP of the experimental eyes began to increase after 1 week of prednisolone treatment in all sheep and reached a peak 1 week later (27.5 mm Hg experimental vs. 11.7 mm Hg fellow, control eye; P<0.001). After the discontinuation of corticosteroid instillation, the IOP of the treated eyes declined to the baseline values over the course of 1 to 3 weeks. CONCLUSIONS: Ovine eyes exhibit a robust steroid-induced ocular hypertensive response, with 100% occurrence in this trial. The mechanisms of steroid-induced glaucoma may be related to those involved in primary open-angle glaucoma and could provide insight into primary open-angle and clues to its treatment.

Inhibitions of chloride transport and gap junction reduce fluid flow across the whole porcine ciliary epithelium.

PURPOSE: To study the effects of chloride transport and gap junction inhibitors on fluid formation across the porcine ciliary epithelium. METHODS: A complete annulus of porcine iris-ciliary body preparation was mounted onto a modified Ussing type chamber to measure the fluid flow (FF) rate. The potential difference (PD) across the preparation was monitored simultaneously. The effects of several inhibitors on chloride transport and gap junction were studied. These included 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 5-(N,N-dimethyl)amiloride hydrochloride (DMA), bumetanide, niflumic acid, and heptanol. RESULTS: The average baseline FF rate was 2.56 +/- 0.07 microL/h per preparation (n = 33). DIDS (0.1 mM) or DMA (0.1 mM) showed no effect on both FF and PD when added to the blood side of the preparation. Bumetanide (0.1 mM), on the blood side, inhibited the FF by 46% and caused a slight depolarization of PD. Heptanol (3.5 mM) depolarized the PD and reduced FF by 45% and 78% through the blood and aqueous sides, respectively. Niflumic acid (1 mM at the aqueous side) also depolarized the PD and significantly inhibited the FF (62%). CONCLUSIONS: The effects of the chloride transport inhibitors on fluid formation across the porcine iris-ciliary body were comparable to that in previous chloride transport studies. The results indicated that fluid secretion by the isolated porcine ciliary epithelium is mainly driven by chloride transport. However, there may be other unidentified ion movements that drive residual FF after chloride transport is inhibited.

Surface change of the mammalian lens during accommodation.

Classical theories suggest that the surface area of the crystalline lens changes during accommodation while the lens volume remains constant. Our recent work challenged this view by showing that the lens volume decreases as the lens flattens during unaccommodation. In this paper we investigate 1) the magnitude of changes in the surface of the in vitro isolated cow lens during simulated accommodation, as well as that of human lens models, determined from lateral photographs and the application of the first theorem of Pappus; and 2) the velocity of the equatorial diameter recovery of prestretched cow and rabbit lenses by using a custom-built software-controlled stretching apparatus synchronized to a digital camera. Our results showed that the in vitro cow lens surface changed in an unexpected manner during accommodation depending on how much tension was applied to flatten the lens. In this case, the anterior surface initially collapsed with a reduction in surface followed by an increase in surface, when the stretching was applied. In the human lens model, the surface increased when the lens unaccommodated. The lens volume always decreases as the lens flattens. An explanation for the unexpected surface change is presented and discussed. Furthermore, we determined that the changes in lens volume, as reflected by the speed of the equatorial diameter recovery in in vitro cow and rabbit lenses during simulated accommodation, occurred within a physiologically relevant time frame (200 ms), implying a rapid movement of fluid to and from the lens during accommodation.

Fluid transport phenomena in ocular epithelia.

This article discusses three largely unrecognized aspects related to fluid movement in ocular tissues; namely, (a) the dynamic changes in water permeability observed in corneal and conjunctival epithelia under anisotonic conditions, (b) the indications that the fluid transport rate exhibited by the ciliary epithelium is insufficient to explain aqueous humor production, and (c) the evidence for fluid movement into and out of the lens during accommodation. We have studied each of these subjects in recent years and present an evaluation of our data within the context of the results of others who have also worked on electrolyte and fluid transport in ocular tissues. We propose that (1) the corneal and conjunctival epithelia, with apical aspects naturally exposed to variable tonicities, are capable of regulating their water permeabilities as part of the cell-volume regulatory process, (2) fluid may directly enter the anterior chamber of the eye across the anterior surface of the iris, thereby representing an additional entry pathway for aqueous humor production, and (3) changes in lens volume occur during accommodation, and such changes are best explained by a net influx and efflux of fluid.

IBMX-elicited inhibition of water permeability in the isolated rabbit conjunctival epithelium.

Agents expected to increase intracellular cAMP levels were tested on the diffusional water permeability (P(dw)) of isolated rabbit conjunctival epithelia given recent indications of the apical expression of AQP5, a water channel homologue regulated by cAMP in other cell systems. For these experiments, segments of conjunctivae were mounted between Ussing-type hemichambers under short-circuit conditions. Unidirectional water fluxes (J(dw)) were measured by adding (3)H(2)O to one hemichamber and sampling from the other, while the electrical parameters (I(sc) and R(t)) were recorded simultaneously. J(dw) were determined under control conditions and after the introduction of forskolin, dibutyryl-cAMP, rolipram and IBMX. All agents reduced J(dw), with rolipram and IBMX the most effective inhibitors (~28% reduction), while simultaneously evoking stimulations of the I(sc); suggesting that cAMP regulates ionic transport and P(dw) independently. This observation was consistent with the elimination of the IBMX-elicited I(sc) stimulations by the PKA inhibitor, H89, and the ineffectiveness of the sulfonamide in preventing the J(dw) reductions produced by the xanthine. Data from mannitol fluxes and Arrhenius plots indicated that the IBMX-elicited P(dw) reduction occurred at the level of water-transporting channels, but the specific moiety was not identified. Instead it was observed that lipophiles commonly used in other systems to uncouple cellular communication precluded the effects of IBMX on J(dw), but the mechanism for these results was not directly linked to gap-junction blockade in the conjunctiva, as assessed by the transepithelial electrical parameters. Putatively, agents such as heptanol, by also fluidizing the bilayer, may have changed the conformation of a water channel in a manner preventing down-regulation by IBMX. Nevertheless, this study uncovered an apparently unique response to cAMP elevation exhibited by the conjunctiva, namely that P(dw) declines via an H89-insensitive pathway under conditions whereby PKA-dependent electrolyte transport might be over stimulated due to excessive cAMP levels (e.g., PDE inhibition).

Fluid transport across the isolated porcine ciliary epithelium.

PURPOSE: To quantify spontaneous fluid transport across the isolated porcine ciliary epithelium and determine its sensitivity to the electrolyte transport inhibitors ouabain and bumetanide, as well as bath Cl(-) and HCO(3)(-) levels. METHODS: A complete annulus of ciliary body was mounted in a custom-designed chamber appropriate for quantifying net fluid movement, as well as the transepithelial potential difference (PD) across the in vitro ciliary epithelium. RESULTS: A spontaneous and stable fluid flow (FF) in the blood-to-aqueous direction was measured over a 4-hour period. This flux solely reflected the secretory activity of the isolated ciliary epithelium (CE), given the absence of externally applied osmotic or pressure gradients. In contrast to FF, the PD declined during the 4 hours in vitro, suggesting that the integrity of the tight junctions may have been compromised during this time so that an increased movement of counter ions via the paracellular pathway could have shunted the PD, while at the same time transcellular fluid transport remained unaffected. The FF in the blood-to-aqueous direction (2.3 +/- 0.2 muL/hr; n = 7) was eliminated by a unilateral reduction in the bath Cl(-) levels on the blood side of the preparation and restored on reintroducing the anion to the bathing medium. This linkage between FF and blood side [Cl(-)] is consistent with the existence of a net Cl(-) flux across the porcine CE in the same direction as the fluid transport. Addition of bumetanide to the blood-side bath inhibited FF by approximately 40%, whereas the removal of CO(2)/HCO(3)(-) from the blood-side bathing solution elicited a approximately 50% reduction in FF. Ouabain inhibited the FF from either side of the preparation, although the effects were more rapid when the glycoside was applied to the blood side of the tissue. Overall, these findings indicate the dependence of FF on active ionic transport by the isolated CE. CONCLUSIONS: Isolated porcine ciliary epithelial preparations transport fluid in the blood-to-aqueous direction, indicating that measurements of volumetric fluid flow across this preparation may serve as a suitable model for future studies directed toward the pharmacological control of secretion.