Inhibition of ionic transport and ATPase activities by serotonin analogues in the isolated toad lens.

The effects of serotonin and five other indoles were tested on the electrical parameters and ionic transport in the isolated toad lens. Serotonin, tryptophan and 5-hydroxy-L-tryptophan did not affect the electrical parameters of the lens at concentrations as high as 1 mM. Tryptamine, 5-methyltryptamine and 5-methoxytryptamine had dual effects: 1 mM in the posterior bathing solution depressed the potential difference of the posterior face of the lens, which resulted in an increase in the translenticular potential difference and short-circuit current; 1 mM in the anterior solution (in contact with the lens epithelium) produced a quick and pronounced reduction of the potential difference of the anterior face. This resulted in a 90-100% decline of the translenticular short-circuit current. Serotonin and tryptamine were then tested for their effect on the ATPases of lens epithelium. Both amines inhibited the enzymes with tryptamine at 5 mM completely inhibiting all ATPase activity. Since tryptophan is transported from the aqueous humor into the lens and may be converted by lens enzymes to serotonin and tryptamine, these findings may have physiological implications in cataractogenesis.

The isobutylene-isobutane alkylation process in liquid HF revisited.

Details on the mechanism of HF catalyzed isobutylene-isobutane alkylation were investigated. On the basis of available experimental data and high-level quantum chemical calculations, a detailed reaction mechanism is proposed taking into account solvation effects of the medium. On the basis of our computational results, we explain why the density of the liquid media and stirring rates are the most important parameters to achieve maximum yield of alkylate, in agreement with experimental findings. The ab initio Car-Parrinello molecular dynamics calculations show that isobutylene is irreversibly protonated in the liquid HF medium at higher densities, leading to the ion pair formation, which is shown to be a minimum on the potential energy surface after optimization using periodic boundary conditions. The HF medium solvates preferentially the fluoride anion, which is found as solvated [FHF](-) or solvated F(-.)(HF)(3). On the other hand, the tert-butyl cation is weakly solvated, where the closest HF molecules appear at a distance of about 2.9 Angstrom with the fluorine termination of an HF chain.

Reduction of water permeability by anisotonic solutions in frog corneal epithelium.

PURPOSE: To study the effects of bathing solution osmolarity and Cl- secretagogues on the diffusional water permeability (Pdw) of the isolated frog corneal epithelium. METHODS: Isolated frog corneas, with the endothelium scraped off, were mounted as a partition between Ussing-type hemichambers. Unidirectional diffusional water fluxes (Jdw) were measured by adding 3H2O to one hemichamber and sampling from the other. Electrical parameters were measured simultaneously. Jdw was determined in control isosmotic conditions and after either changes in osmolarity of the bathing solutions or the additions of amphotericin B, epinephrine, Ca2+ ionophore, and other agents. RESULTS: Apical addition of 0.5 mM HgCl2 elicited an 11-fold increase in paracellular conductance and inhibited Jdw by 36%, suggesting that Jdw was predominantly transcellular and that there was a negligible contribution of the paracellular pathway. Pretreatment of corneas with 2-mercaptoethanol prevented the effects of Hg2+ on the paracellular conductance and Jdw. A hypotonic medium on the basolateral side reversibly reduced Jdw proportionately to the reduction in osmolarity, with 40 mOsm exerting a 29% decrease. Results from an Arrhenius plot suggest that water channels closed under this condition. Apical hypertonicity (350 mOsm) reduced Jdw by approximately 12%. Basolateral hypertonicity (450 mOsm), after permeabilization of the apical membrane with amphotericin B, reduced Jdw by 15%. Epinephrine was the only Cl- secretagogue that reduced Jdw, on average by 12%. This effect, which was also observed with amphotericin B-treated corneas, was not mediated by classical beta-receptors based on the results obtained with isoproterenol and propranolol. CONCLUSIONS: Changes in basolateral osmolarity or the presence of an apical hypertonic solution decreased the diffusional water permeability (Pdw) of the corneal epithelium. Epinephrine also decreased Pdw, and this effect was localized to the basolateral membrane. The similarities, of a sequence motif found in potassium channels and beta-adrenergic receptor kinases that are regulated by the beta gamma subunit of G proteins with that found in aquaporins 2 and 5, could explain the link with epinephrine. Regardless of the mechanism, these results indicate that corneal epithelial water permeability can be regulated, presumably to protect cell volume from changes in solution osmolarity.

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 +/- SE's, 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.

Na+-K+ ATPase distribution in frog and bovine lenses.

Na+-K+ ATPase activity was measured in the capsule-epithelium and decapsulated frog and bovine lens. The decapsulated lens contained approximately 20% of the whole lens activity in the frog and 30% in the bovine. These values were measured from the aqueous homogenate of the entire decapsulated lens, an approach which may have underestimated the activity by diluting the ouabain-sensitive component in the preparation. Subsequent determinations were done on separate portions of superficial (2 to 3 mm) anterior-equatorial, and posterior bovine cortex. The activities per gram of tissue were enriched with respect to the values for the entire decapsulated bovine lens. These activities were further enriched by a sucrose density gradient centrifugation. The anterior-equatorial cortical segment contained 1.6 times the activity found in the capsule-epithelium. The posterior cortex had a much smaller but statistically significant level of Na+-K+ ATPase. It is unlikely that the observed asymmetry of the anterior-equatorial segment with the posterior cortex is exclusively due to epithelial contamination for the result would require the adherence of 62% of the epithelium. Scanning electron micrographs of 6 decapsulated bovine lenses indicated an average contamination of about 9%. This asymmetry may have a physiological role in assisting the pump mechanism of the epithelium.

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 alpha1-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-alpha1-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.

Na(+)-Cl(-)-K+ cotransport activity in cultured bovine lens epithelial cells and its absence in intact bovine lenses.

Using 86RbCl, experiments were directed to identify the pathways for K+ entry in bovine lens cells. Experiments were performed on cultured bovine lens epithelial cells, which are known to transform spontaneously in vitro, and on whole bovine lenses. In cultured epithelial monolayers, K+ inflow occurred nearly equally via the Na(+)-K+ pump and a bumetanide-sensitive, Na(+)- and Cl(-)-dependent process. In the simultaneous presence of ouabain and bumetanide, uptake was inhibited by 98%. Significant passive movement of label through K+ channels could not be detected as indicated by the lack of an effect by 5 mM Ba2+. In the presence of ouabain, the concentration of bumetanide producing half-maximal inhibition of K+ uptake was 1.2 x 10(-7) M, a value similar to those of other cell systems in which Na(+)-Cl(-)-K+ cotransport has been demonstrated by various methods. The present system was not affected by attempts to independently perturb cell Ca2+ and cAMP, but K+ uptake was inhibited upon exposure to phorbol esters and to a cell permeable analog of the protein kinase C (PKC) activator diacylglycerol. From experiments with phorbol 12-myristate-13-acetate (PMA), the reduced inflow was attributed to an inhibition of the bumetanide-sensitive pathway. In contrast, treatment with a phorbol ester known not to activate PKC, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), was ineffective; in toto suggesting that the inositol signaling system regulates K+ fluxes in these cells primarily by affecting the rate via a Na(+)-Cl(-)-K+ cotransport mechanism. These results contrasted with those garnered with the intact lens. Lenses were isolated in a bicameral Ussing-type arrangement so that their anterior and posterior aspects could be bathed independently. K+ inflow across the epithelial surface was predominantly mediated by the Na(+)-K+ pump, i.e. ouabain inhibited uptake by 87%. Addition of bumetanide to the anterior bath resulted in an uptake rate not different from that found under control conditions. The present results are consistent with findings in other cell systems which indicate that the Na(+)-Cl(-)-K+ cotransport activity is associated with highly proliferative cells, such as the cultured cells used. It remains to be determined whether activation, or expression, of the cotransporter has a role in the intact lens.

Cellular pH and Na+-H+ exchange activity in lens epithelium of Bufo marinus toad.

Incubation of lenses with the acetoxymethyl ester of 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF-AM) led to selective, prolonged entrapment of the deesterified, pH-sensitive, membrane-impermeable form of the dye (BCECF) in the lens epithelial cells allowing, thereby, for the continuous fluorometric monitoring of epithelial intracellular pH (pHi) in intact lenses. Under anaerobic conditions in physiological solution, the epithelium maintained a pHi of 7.36 +/- 0.14 (mean +/- SD, n = 6 lenses). In the absence of CO2 and HCO3-, the pHi increased to 7.83 +/- 0.38 (n = 55). In this condition, either the removal of Na+ or the addition of amiloride to the medium resulted in a decrease of pHi. This acidification was increased in amiloride-inhibitable fashion when lenses were loaded with Na+ before extracellular Na+ removal. The extent of artificially generated cellular acidosis (induced by the "NH4+ loading" technique) was markedly decreased by the presence of extracellular Na+ or reverted on its addition when the ion was initially absent; the latter reversal was inhibited by amiloride. This inhibition was by-passed by monensin addition. The existence of a Na+-H+ antiport in the epithelial cells is concluded.

HCO3- transport in the toad lens epithelium is mediated by an electronegative Na(+)-dependent symport.

The pH-sensitive cell-entrapable dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to continuously monitor epithelial intracellular pH (pHi) of intact toad lenses, enabling a description of a HCO3- transport mechanism that contributes to pHi homeostasis of this organ. In physiological medium, pH 7.40, the steady-state pHi was 7.48 +/- 0.03 (SE; n = 93). Induction of cell depolarization by either elevation of [K+] to 50 mM, addition of 0.2 mM quinidine, a K(+)-channel blocker, or addition of 0.1 mM Li+ ionophore that equalizes Na+ and K+ permeabilities elicited pHi increases (delta pHi = 0.18 +/- 0.02; P less than 0.0005; n = 13, for K+). These increases could be blocked or reverted by DIDS and were not affected by amiloride. Removal of Na+ induced an amiloride-insensitive acidification. pHi recovery seen upon Na+ reintroduction in the presence of amiloride was inhibited by DIDS. Despite the effects of DIDS on induced pHi changes, the agent did not affect control pHi. Elevation of medium HCO3- (pH to 7.7) produced a pHi increase followed by a spontaneous reversal. This increase was both DIDS and Na+ sensitive. pHi was not affected in any condition by removal (or addition) of Cl-, unless the lens was pretreated with the artificial Cl(-)-HCO3- exchanger tributyltin. Collectively, these results suggest that the primary mechanism for HCO3- movement across the lens epithelial membrane is an electronegative Na+ cotransporter and that this system is near equilibrium under normal physiological conditions.

Evidence for parallel Na(+)-H+ and Na(+)-dependent Cl(-)-HCO3- exchangers in cultured bovine lens cells.

BCECF, a cell-entrapable dye with a pH-sensitive fluorescence spectrum, was used to identify transport mechanisms contributing to pH homeostasis of cultured bovine lens epithelial cells. Cells from a spontaneously established lineage were grown on glass coverslips that fit diagonally in a standard curvette and intracellular pH (pHi) was measured. Under perfusion with a CO2-HCO3(-)-free medium (pH 7.45), pHi was 7.19 +/- 0.21 (mean +/- S.D., n = 94 cell preparations). Cell acidifications (pHi to 6.65, n = 8) induced by the 'NH(4+)-loading' method were rapidly followed by a Na(+)-dependent, amiloride-inhibitable pHi recovery. Introduction of a CO2-HCO3(-)-rich medium (pH 7.45) resulted in a small acidification (0.18 +/- 0.04 U, n = 16; P < 0.002) due to rapid CO2 entry and an ensuing slow alkalinization to a pHi near the control CO2-HCO3(-)-free value. Subsequent removal of Cl- resulted in a further alkalinization of 0.18 +/- 0.02 U (n = 13; P < 0.001). This Cl- effect was completely inhibited by the absence of Na+, but was insensitive to amiloride, suggesting the presence of a Na(+)-dependent Cl(-)-HCO3- exchanger. Consistent with this posit, the reintroduction of Na+ to cells perfused in the absence of the cation with a HCO3(-)-containing, amiloride-complemented solution resulted in a gradual recovery from the acidic pHi induced by the baseline conditions (n = 6). The amiloride-insensitive, Na(+)- and HCO3(-)-dependent recovery was completely inhibited in cells pre-incubated with DIDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of toad lens epithelial Na+/H+ exchange activity by hypertonicity.

Incubation of toad lenses with the acetoxymethyl ester of 2',7'-biscarboxyethyl-5(6)-carboxy-fluorescein led to a highly selective accumulation of the de-esterified, pH-sensitive form of the dye in the epithelial cells, enabling the continuous fluorometric monitoring of epithelial intracellular pH (pHi) in intact lenses. The effects of changes in extralenticular [Na+] and of amiloride-addition indicated that the epithelium contains an amiloride-sensitive Na+/H+ antiport. Exposure of lenses to hypertonic conditions (by the addition of sucrose to the medium) resulted in a biphasic change in pHi; a rapid initial, 'spike-like' decrease was immediately followed by a persistent reversal that raised pHi in CO2/HCO3- -rich and -free media by 0.13 and 0.18 units, respectively. Under CO2/HCO3- -free conditions, the hypertonic exposure raised pHi to a value near the calculated equilibrium position for a lens Na+/H+ exchanger. At this point, monensin addition did not affect pHi, suggesting that the tonicity shift had induced a rapid endogenous Na+/H+ exchange activity. In contrast, in the presence of 1 mM amiloride or in the absence of extralenticular Na+, sucrose addition induced only a persistent pHi decrease, which could be reversed (in the 'amiloride' case) by monensin addition. These results demonstrate that the hypertonic exposure induced an epithelial cell acidification as well as a stimulation of the Na+/H+ exchange activity which reverted the acidification. The hypertonic exposure also elicited pHi increases in lenses that had been preacidified by the 'NH4+ loading' or 'pCO2 raise' methods, indicating that the onset of the stimulation could not be attributed to a pHi decrease.(ABSTRACT TRUNCATED AT 250 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 Ringer's 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.

Identification and localization of acid-base transporters in the conjunctival epithelium.

Acid-base transporters of rabbit and porcine conjunctival epithelia were identified and localized with immunoblotting and immunohistochemical techniques using specific antibodies against carriers commonly found in epithelia, i.e. the Cl(-)/HCO3(-)exchanger (AE2), Na(+)/H(+) exchanger (NHE-1, -2, -3) and the electrogenic Na(+)-(n)HCO3(-) cotransporter (NBC). Western blot analysis demonstrated that anti-AE2 reacted with an approximate 170 kDa protein in both rabbit and pig cell membranes prepared from separately isolated bulbar and palpebral conjunctivae. NHE1 was similarly identified in these distinct conjunctival regions but results with anti-NBC were ambiguous. Histochemical examinations indicated that the AE2 and NHE1 proteins reside on the basolateral surfaces of the plasma membrane throughout the multilayered tissue. The immunostaining of porcine cryosections for AE2 and rabbit sections for NHE1 was specific, because of its abolishment following either pre-absorption with the corresponding peptide or omission of the primary antibody. Screening with anti-NBC produced weak staining of the sections that appeared to be non-specific. For confirmation of these results, the acid-base transporters present in rabbit cell cultures of conjunctival epithelia were ascertained from the changes in intracellular pH (pH(i)) evoked upon sequential superfusion with media of altered composition. This approach readily obtained Na(+)- and Cl(-)-dependent pH(i)effects consistent with the existence of Cl(-)/HCO3(-) and Na(+)/H(+)exchange activities. Evidence for the presence of NBC could not be acquired, thereby substantiating the observations from the immunodetection techniques. The identity and location of the antiporters that were found suggested that these elements could contribute to transcellular Cl(-)transport in the basolateral-to-apical direction. To test this possibility, the effects of AE and/or NHE inhibition were determined on the bumetanide-insensitive Cl(-)-dependent short-circuit current across rabbit conjunctivae freshly isolated in Ussing-type chambers. These experiments revealed that such current is indeed sustained by the antiporters. Results with acetazolamide further suggested that the contribution of the acid-base transporters towards transepithelial Cl(-)secretion is variable and dependent upon individual rates of metabolic CO(2)production. Overall, the present study provides an initial identification of the acid-base transporters present in the conjunctiva. Besides their likely role in intracellular pH regulation, the parallel, basolateral expression of AE2 and NHE1 indicates that these elements do not directly contribute to the pH of the tear film but may complement the Na(+)-2Cl(-)-K(+)cotransporter in effectuating Cl(-)secretion.

Potassium current oscillations across the rabbit lens epithelium.

Rabbit lenses expressing spontaneous oscillations in translens short-circuit current (Isc) are obtained somewhat frequently, with this phenomenon observed in approximately 30% of isolated lenses as described earlier (Exp. Eye Res. 61, 129-140, 1995). Since pharmacological protocols to consistently elicit Isc oscillations were not found, characterizations of the underlying transport processes have been limited to the application of various inhibitors on the spontaneous phenomenon. The present report extends the initial observations by confirming that oscillations are immediately inhibited upon the anterior addition of the Ca2+ channel blocker nifedipine (10 microM), and by demonstrating that other treatments which should affect epithelial Ca2+ homeostasis are also inhibitory (e.g., Bay K 8644 (10 microM), diltiazem (10 microM), EGTA (2 mm), and Ca2+-free media). Furthermore, Isc oscillations are immediately inhibited by the K+ channel blocker, Ba2+, but not by the Na+-K+ pump inhibitor, ouabain. The intracellular Ca2+ mobilizing agents thapsigargin (0.1 microM) or acetylcholine (1 microM) modified but did not permanently inhibit the oscillations, confirming earlier observations. At 50 microM, however, acetylcholine addition was inhibitory, but reversible, for oscillations restarted upon its subsequent removal. In addition, lens oscillations were also characterized under open-circuit conditions with microelectrodes inserted in the superficial cells near the equator of lenses isolated in a divided chamber. The potential difference (PD) across each lens face was recorded, as was the translens PD (PDt), which equals the difference between the PDs across each lens surface. Oscillations in PDt were obtained in 7 of 26 lenses. The oscillations arose only from an oscillation in the PD across the anterior face (PDa). While PDa and PDt oscillated with the same amplitude (approximately 12 mV) and period (approximately 70 sec), the PD across the posterior surface remained stable. During these oscillations the conductance of the anterior surface was maximal at the most positive voltage of the anterior bath with respect to the lens interior (46 mV), whereas, minimal conductance occurred at the least positive PDa (34 mV). Overall, these observations are consistent with the likely presence of voltage-operated Ca2+ channels in parallel with various Ca2+-sensitive K+ channels in the epithelial basolateral membrane. A model to explain the oscillatory pattern across the anterior face while the PD across the posterior face remains unaltered is presented.

Cyclic AMP-dependent stimulation of basolateral K(+)conductance in the rabbit conjunctival epithelium.

The regulation of Na(+)transport by cAMP in freshly isolated rabbit conjunctival epithelium, a tissue exhibiting both Cl(-)secretion and Na(+)absorption, was examined. Bulbar-palpebral segments of conjunctiva were mounted between Ussing-type hemichambers under short-circuit conditions in Cl(-)free media. In this situation, the short-circuit current (I(sc)) measures an amiloride-resistant Na(+)absorptive process in the apical-to-basolateral direction. Apical additions (each at 10 microm) of cAMP-elevating compounds, forskolin, rolipram, IBMX and epinephrine all stimulated the Na(+)-dependent I(sc)by approximately 3.5-4.5 microA cm(-2)(minimal 40% increase) and reduced transepithelial resistance (R(t)) by at least 7% (P<0.05). Pre-exposure (1 hr) to the protein kinase A (PKA) inhibitor H-89 (10 microm), which in itself inhibited the I(sc)by 0. 5 microA cm(-2), attenuated the I(sc)responses of the cAMP-elevating agents (P< 0.05, unpaired data). In reverse, H-89 promptly decreased the I(sc)by 1.5-2.5 microA cm(-2)and increased R(t)by 5% (P<0.05) in tissues pre-stimulated with either forskolin or an epinephrine plus IBMX combination. Additions of epinephrine or rolipram to apically permeabilized preparations using amphotericin B, increased the I(sc)by 12 and 22% respectively over baseline and reduced R(t)by 6% (P<0.05). Similarly, in the presence of a transepithelial K(+)gradient (apical to basolateral) and amphotericin B, cAMP elevation stimulated K diffusion across the preparation by at least 1.8 microA cm(-2)and decreased the R(t)by 4% (P<0.05), changes that were reversed by subsequent H-89 addition. Under Cl(-)rich conditions, pretreatment with 5 m m Ba(2+)reduced the basal I(sc)by 59% and blocked the cAMP-induced I(sc)stimulations typically seen in the presence of the anion. The results provide evidence for a PKA-regulated, Ba(2+)-inhibitable (voltage insensitive) basolateral K(+)conductance in rabbit conjunctival epithelial cells. The action of Cl(-)secretogogues acting via cAMP on basolateral K(+)channel activity indicates that endogenous levels of cAMP may play a role in the regulation of Cl(-)secretion and Na(+)absorption in the conjunctiva.

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)

Reduction in water permeability of the rabbit conjunctival epithelium by hypotonicity.

The effects of unilateral exposure to hypotonic media on the diffusional water permeability of the isolated rabbit conjunctiva were determined. For these experiments, a segment of the bulbar-palpebral conjunctiva was mounted between Ussing-type hemichambers under short-circuited conditions. Unidirectional diffusional water fluxes (Jdw) were measured in either direction by adding 3H2O to one hemichamber and sampling from the other. Electrical parameters were measured simultaneously. Jdw were determined in control isosmotic conditions and after dilution of one of the bathing solutions from 290 to 108 mOsMolar. This hypotonic condition reduced Jdw by 25-30% (n = 17) when applied basolaterally and by 25% (n = 6) apically. The effects were reversible and were also obtained when the opposite bathing solution contained amphotericin B, selectively permeabilizing the contralateral cell surface. From concomitant changes in transepithelial electrical resistance as well as 14C-mannitol fluxes completed under identical conditions, arguments are presented that the above effect is best explained as a cell regulated reduction in membrane water permeability. Presumably both apical and basolateral membranes can down-regulate their water permeabilities. This response, suggesting a protective mechanism to help maintain cell volume from hypotonicity, was also seen in other studies using the amphibian bladder and the frog cornea, in which the effect was only obtained basolaterally. Thus, regulation of epithelial water permeability appears to be a basic trait common to both amphibians and mammals, although tissue differences exist.

5'-Nucleotidase levels in normal and virus-transformed cells: implications for cellular aging in vitro.

5'-Nucleotidase activity in the postnuclear supernatant of normal and SV-40 virus-transformed human embryonic lung, chick embryo, and mouse embryo fibroblasts, WI-38, IMR-90, VA-13, CEF, 3T3, and SV3T3 cells was measured. In IMR-90 and CEF cells, both of which have a limited lifespan in vitro, 5'-nucleotidase activity increased 6- and 20-fold, respectively, with increasing population doublings. However, VA-13 cells, a permanent cell line, showed no increase in this enzyme activity with progressive population doublings. 5'-Nucleotidase activity was not detectable in two other permanent cell lines, 3T3 and SV3T3 cells. Of the six cell lines tested, the conspicuous enhancement in 5'-nucleotidase activity with increasing population doublings was observed only in normal cell lines and was absent in transformed cell lines. These observations suggest a molecular mechanism which may play a role in aging of normal cells in vitro and which may involve catabolism of nucleic acids.