Lawrence J. Alvarez

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.

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.

Immunolocalization of Na-K-ATPase, Na-K-Cl and Na-glucose cotransporters in the conjunctival epithelium.

Purpose. To investigate possible regional expression of transport systems in the conjunctival epithelium given distinct differences in morphological appearance between the bulbar and palpebral epithelia as well as variations found in the proportions of Na absorptive versus Cl secretory activities in electrophysiological studies. Methods. Mouse monoclonal antibodies against the a1-subunit of Na-K-ATPase and Na-K-Cl cotransporter (NKCC1) and a rabbit polyclonal antibody against the Na-glucose cotransporter (SGLT1) were used in immunoblotting and immunofluorescent labeling of frozen fixed sections isolated from either the bulbar and palpebral regions of the conjunctiva. Results. Western blot analysis clearly demonstrated the presence of Na-K-ATPase, NKCC1 and SGLT1 proteins in both bulbar and palpebral conjunctiva. Indirect immunofluorescence studies on bulbar and palpebral conjunctival portions revealed intense staining by the anti-NKCC1 and anti-a1-Na-K-ATPase antibodies exclusively at the basolateral surfaces, whereas the anti-SGLT1 antibody was used with porcine conjunctiva to elicit strong and unambiguous staining along the apical plasma membrane. Conclusions. Proteins that mediate the transport activities of the Na-K-ATPase and Na-K-Cl cotransporter are uniformly distributed throughout the palpebral and bulbar regions of the rabbit conjunctival epithelium. Although the Na-glucose cotransporter could be detected in immunoblots of the rabbit, this cotransporter appears to be uniformly distributed as well, based upon immunohistochemical sections of the pig conjunctiva. Thus, it appears likely that mechanisms for Cl secretion and Na absorption reside in both bulbar and palpebral segments of the conjunctival epithelium.

Acetylcholine modulation of the short-circuit current across the rabbit lens

Rabbit lenses were bathed within a bicameral Ussing-type chamber under short-circuit conditions. In this situation the short-circuit current (Isc) reflects, across the anterior aspect, the presence of anteriorly facing K+ conductance(s) plus the Na(+)-K+ pump current. Across the posterior surface the Isc is primarily carried by the movement of Na+ from the posterior bathing solution to the lens. Addition of acetylcholine (ACh) to the posterior hemichamber did not affect the translens electrical parameters; but, its introduction to the anterior bath at 1 microM immediately reduced the Isc from 8.91 +/- 1.47 to 5.84 +/- 1.28 microA cm-2 and increased the translens resistance from 1.50 +/- 0.08 to 1.59 +/- 0.09 K omega cm2 (+/- S.E.S; P < 0.05 as paired values, n = 25 lenses). The suppressed Isc gradually recovered and reached 75% of the control value 5 min after the introduction of the neurotransmitter. In six cases the recovery was nearly complete (> or = 95% of control) within this time. The preaddition of 0.1 microM atropine prevented an effect by 1 microM ACh. When atropine was added within 1 min of ACh, the suppressed Isc immediately recovered. The ACh-elicited Isc suppression was averted in lenses pre-exposed to either K+ channel blockers (quinidine or barium) or to the endoplasmic reticular Ca(2+)-ATPase inhibitor thapsigargin (Tg: 0.1 microM), which in itself produced Isc inhibitions similar to those seen with ACh under control conditions. Similarly comparable were the ACh-evoked Isc inhibitions garnered upon introduction of the agonist to lenses bathed in the absence of extracellular Ca2+. In these cases, however, the Isc recovered fully within 2-3 min. This condition also revealed that the anterior removal of medium Ca2+ increased the Isc by about 50%, a completely reversible phenomenon; Ca2+ restoration in the presence of the Ca2+ channel blocker, nifedipine (10 microM), blunted markedly the reversal to the control Isc. Overall, these results suggest that ACh receptor activation induces the release of intracellularly stored Ca2+, which in turn leads to the temporary deactivation of a K+ conductance(s); in addition, secondary Ca2+ inflow may further extend the observed inhibition. During this study, the Isc of about 30% of the lenses used spontaneously oscillated (common duration of 30 min, with a mean peak frequency of 0.76 +/- 0.32 cycle min-1 and mean amplitude of 4.07 +/- 2.65 microA cm-2; +/- S.D.S, n = 24). Experiments attempted to determine the sensitivity of the oscillatory activity to ACh. Tg, nifedipine, and the phorbol ester PMA.(ABSTRACT TRUNCATED AT 400 WORDS)

Contribution from a pH- and tonicity-sensitive K+ conductance to toad translens short-circuit current

Studies of toad (Bufo marinus) lenses mounted in Ussing-type chambers revealed that: (1) the translens short-circuit current (Isc) across the posterior surface is primarily carried by the movement of Na+ from the posterior bathing solution to the lens; (2) across the anterior face the majority of the Isc is mediated by Ba(2+)-sensitive channels and the remaining current is rapidly reduced by ouabain; (3) most of the anterior K+ conductance is of the tonicity-sensitive, quinidine-inhibitable type (i.e. hypotonic shifts increase Isc and hypertonic shifts decrease Isc; quinidine pretreatment eliminates such responses); (4) 86Rb+ uptake is stimulated by alkaline pH and occurs primarily across the anterior surface with quinidine the most potent inhibitor of this process; and (5) the Na(+)-K+ pump can maintain lens [Na+] and [K+] for at least 20 hr in a Ringers solution near neutral pH but not at pH 8.7 (a pH used in some studies with this lens). It is concluded that the Isc can be viewed as a representation of the current across the epithelial basolateral membrane, a surface dominated by pH- and tonicity-sensitive K+ channels. The direction of the Isc response to tonicity changes suggests a role for these channels in epithelial volume regulation.

Beta-adrenergic stimulation of Na+–K+–2Cl− cotransport activity in the rabbit lens

Experimental maneuvers known to increase cellular cAMP levels evoked a stimulation in the K(+) influx across the anterior surfaces of isolated rabbit lenses, as measured by 86Rb(+) uptake. For this, the lenses were mounted in a modified Ussing-type chamber and exposed to the radiolabel under short-circuit conditions. The enhanced, cAMP-elicited flux was attributed to the basolateral Na(+)-K(+)-2Cl(-) cotransporter given its preclusion by bumetanide, a highly selective inhibitor of this symport, and the ineffectiveness of ouabain in mitigating the stimulation. The ouabain- plus bumetanide-insensitive K(+) uptake, which is about 10% of the total influx and represents passive entry of the radiolabel, was not affected by cAMP-elevating conditions. Forskolin, an activator of adenylyl cyclase; epinephrine, a non-selective adrenergic agonist; and the beta-selective agents, isoproterenol and terbutaline, were among the drugs used to elicit the increase in bumetanide-sensitive K(+) inflow. In experiments with isoproterenol, the stimulated influx evoked by the agonist was inhibited in lenses simultaneously exposed to propranolol. Other observations included that the stimulation of bumetanide-sensitive K(+) influx with forskolin was eliminated in lenses pretreated with the protein kinase inhibitors, staurosporine or H-89. However, these drugs were ineffective in preventing the increased influx produced by calyculin A, a phosphatase inhibitor, suggesting modulation of the cotransporter by at least two independent pathways. The cAMP-generating stimuli also produced an inhibition of the short-circuit current across the lens and an increase in translens resistance. These latter effects suggest that cAMP elevation also evokes an inhibition in an epithelial conductance(s) simultaneously to the stimulation of the cotransporter. As such, this study provides the first indication for the regulation of lens transport by adrenoceptors, presumably of the beta-2 subtype.

Characterization of serotonergic receptors in rabbit, porcine and human conjunctivae

Purpose. To characterize the serotonin (5-HT) receptors linked to the modulation of adenylyl cyclase activity in rabbit, porcine and human conjunctivae. Methods. Serotonin receptor-subtype expression was examined using reverse transcription-polymerase chain reaction (RT-PCR) and receptor subtype-specific polyclonal antibodies for the immunofluorescent labeling of conjunctival cryosections. In addition, measurements of the effects of serotonergics on the short-circuit current (I sc) across rabbit and porcine conjunctivae were contrasted. Results. RT-PCR assays indicated the expression of 5-HT 1B and 5-HT 1D receptors, subtypes negatively coupled to adenylyl cyclase, in the rabbit conjunctiva. This approach also suggested the co-expression of 5-HT 1B, 5-HT 1D, 5-HT 1F, 5-HT 4 and 5-HT 7 mRNA’s in the porcine conjunctiva, and 5-HT 1D, 5-HT 1F and 5-HT 7 in the human conjunctiva. Since the 5-HT 4 and 5-HT 7 receptors are positively linked to adenylyl cyclase, these results implied that the porcine and human tissues exhibited subtypes both positively and negatively linked to the enzyme. However, immunohistochemical observations, using currently available antibodies solely localized the 5-HT 7 moiety in the porcine and human epithelia, suggested that the 1B/1D forms may be minor elements. Consistent with this prospect, 5-HT was a stimulant of the transepithelial I sc across the porcine conjunctiva, an opposite response from earlier findings that demonstrated inhibitory effects by 5-HT on the rabbit I sc, which are now explained by the localization of the 1B/1D receptors in the rabbit stratified epithelium. Conclusions. The 5-HT receptors expressed by mammalian conjunctivae are not identical. In terms of 5-HT receptor expression, the porcine tissue may be a more appropriate model for human, than is the rabbit, in that 5-HT may serve as a secretagogue in the human epithelium.

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.

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.

Effects of Ca2+ on rabbit translens short-circuit current: evidence for a Ca2+ inhibitable K+ conductance

PURPOSE To characterize the effects of medium Ca2+ levels on rabbit lens electrical properties. Early studies with wholly submerged lenses had shown that Ca2+ removal from the bath resulted in an increased Rb+ efflux, a consequence of an increased Na+ Permeability and lens depolarization. METHODS Lenses were bathed with Ussing-type chambers under short-circuited conditions, an arrangement in which the translens short-circuit current (Isc) is carried out across the posterior lens surface mainly by an influx of Na+, and across the anterior face largely by a K+ efflux. RESULTS Under the present conditions in which the effects of Ca2+ were characterized unilaterally, the above established effects could only be ascribed to the posterior surface. When Ca2+ removal was limited to the anterior face, the Isc increased from 11.87 +/- 1.17 to 17.04 +/- 1.52 microA/cm2 (means +/- SEs, n = 18; an accompanying translens resistance (Rt) decrease of 0.23 +/- 0.049 K omega.cm2 was also recorded). Conversely, increasing the control, anterior-bath [Ca2+] from 1.8 to 3.6 mM reduced the K+ efflux-dependent Isc from 10.54 +/- 1.09 to 8.93 +/- 1.02 (n = 10, with an Rt increase of 0.11 +/- 0.013). These changes were reversible Na(+)-independent, and fully inhibited by the presence of K+ channel blockers (quinidine or Ba2+). Inhibitions of the Ca2+ effects were also obtained with strontium, a Ca2+ surrogate. The Isc was less responsive to changes in the Ca2+ content of the posterior bath. Removal of the cation caused a gradual 1.65 +/- 0.72 microA/cm2 increase (n = 9, with an Rt decrease of 0.090 +/- 0.021 K omega.cm2). In the absence of posterior Na+, Ca2+ withdrawal resulted in highly variable responses, with some specimens exhibiting salient current increases, suggesting that an outwardly directed, posterior efflux of an anion could also have been affected. During the course of this study it was consistently observed that the removal of Na+ from the anterior bath led to an Isc decrease of 2.62 +/- 0.22 microA/cm2 (n = 32, with an Rt increase of 0.35 +/- 0.029 k omega.cm2). This change occurred in both the presence of ouabain and the absence of Ca2+, suggesting that it did not result from an inhibition of the Na(+)-K+ pump current nor from a reversal in putative Na+/Ca2+ exchange activity. Small Isc increases upon anterior Na+ withdrawal (1.68 +/- 0.17, n = 7), consistent with Na+ efflux from the lens, could only be observed with K+ channels inhibited with Ba2+. Also congruent with the observations of a relatively limited anterior Na+ permeability, was the finding that the induction of nonspecific cation channels with amphotericin B reduced the Isc by following Na+ from the anterior bath to enter the lens. Thus, changes in lens Isc can differentiate changes in K+ permeability across the native anterior epithelium from changes in Na+ permeability. CONCLUSIONS Overall, these results suggest that lens Ca2(+)-mobilizing agents (e.g. acetylcholine) could trigger the inhibition of epithelial K+ conductance(s) by the direct action of Ca2+ on K+ channels.