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O A Candia

Publications and source records attributed to O A Candia.

At least 19 recordsLinked to original sources
PubMed1992

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)

Animals↗
PubMed1992

Characterization of active and passive Na+ and K+ transport in normal rat lens by the short-circuiting technique.

An initial characterization of the lenticular ionic permeabilities of the isolated Sprague-Dawley rat lens utilizing short-circuiting techniques was carried out to provide the basis for further studies of mechanisms underlying cataractogenesis associated with salt-sensitive genetic hypertension in the rat. Both active and passive Na+ and K+ transport were evaluated by varying ionic concentrations in the bathing solutions facing the anterior and posterior sides of the lens, as well as by the addition of BaCl2 and ouabain. In general, the ionic permeabilities and transport properties of the rat lens are qualitatively similar to those previously described in other species. Ionic replacement studies showed the presence of Na+ and K+ channels at both surfaces of the lens, with the anterior side K+ conductance being larger than the posterior. In contrast, Na+ conductance was similar at both lens surfaces. The effects of ouabain confirmed the presence of the Na(+)-K(+)-ATPase at the lens epithelium, while the effects of serial addition of BaCl2 and ouabain suggested that the contribution of K+ diffusion to the short-circuit current may be considerably greater than the electrogenic component of the Na(+)-K+ pump.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Ascorbate-stimulated active Na+ transport in rabbit ciliary epithelium.

In a physiological medium (134 mM Na+ concentration), unidirectional blood-to-aqueous and aqueous-to-blood Na+ fluxes across the isolated rabbit ciliary epithelium are large, rendering the detection of a net transport difficult. At 134 mM an active component for Na+ may be obscured by diffusional fluxes and a bidirectional Na(+)-Cl- cotransport. Considering that the active transport saturates at about 30 mM, experiments were performed at this reduced Na+ concentration to minimize the influence of diffusional pathways. A net blood-to-aqueous Na+ flux that ranged from 0.25 to 0.81 mu eq/hr was obtained. Addition of ascorbic acid to the aqueous side under this condition increased the blood-to-aqueous flux with little effect on the flux in the opposite direction. Ouabain inhibited both the Na+ and ascorbate-stimulated Na+ transport. The increase in blood-to-aqueous Na+ flux by ascorbate was also observed in tissues bathed with [Na+] closer to physiological levels (100 mM). These results indicate that the rabbit ciliary epithelium transports Na+ into the posterior chamber. Since aqueous ascorbate stimulates Na+ transport, it may be implicated in both Na+ movement and aqueous humor secretion. However, the rate of Na+ transport can only account for a small fraction of total aqueous humor production.

Animals↗
PubMed1990

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

Forskolin-induced HCO3- current across apical membrane of the frog corneal epithelium.

Forskolin (and other Cl- secretagogues) does not affect the very small Na(+)-originated short-circuit current (Isc) across frog corneal epithelium bathed in Cl- free solutions. However, forskolin in combination with increased PCO2 bubbling of the solutions (5-20% CO2) stimulated Isc proportionally to PCO2 to a maximum of approximately 8 microA/cm2. This current could be eliminated and reinstated by sequentially changing the gas composition of the bubbling to 100% air and 20% CO2-80% air. The same effects were observed when PCO2 changes were limited to the apical-side solution. Stroma-to-tear HCO3- movement was deemed unlikely, since the increase in Isc was observed with a HCO3(-)-free solution on the stromal side and CO2 gassing limited to the tear side. From the effects of ouabain and tryptamine, at least 80% of the Isc across the basolateral membrane can be accounted for by the Na+ pump current plus K+ movement from cell to bath. Methazolamide also inhibited Isc. Current across the apical membrane cannot be attributed to an electronegative Na(+)-HCO3- symport given the insensitivity of Isc to a disulfonic stilbene and the fact that stroma-to-tear Na+ fluxes did not increase on stimulation of Isc. The tear-to-stroma Na+ flux also remained unaltered, negating an increased apical bath-to-cell Na+ flow. The forskolin-20% CO2 manipulation produced a depolarization of the intracellular potential, a reduction in the apical-to-basolateral resistance ratio, and a decrease in transepithelial resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

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)

Animals↗
PubMed1989

Prostaglandins and transepithelial ionic transport.

Given the complexity of the preparation and the fact that the measured potential difference and SCC result from the algebraic contribution of several apparent ionic components (as well as unidentified ones) in the various cell membranes of the preparation, the effect of PGF2 alpha on the SCC requires further elucidation, for example, by determination of the effects of this PG on H14CO3- fluxes. However, experiments already completed suggest that PGF2 alpha can effectively stimulate the SCC of the ciliary epithelium. Since stimulation of the SCC can be expected to reflect enhanced fluid secretion, the effect of PGF2 alpha on the ciliary epithelium, at least in rabbits, can be regarded as a potentially beneficial one, providing increased production of aqueous humor. It remains to be seen whether PGF2 alpha has a similar stimulatory effect on ciliary epithelium transport function in primates whose aqueous humor, in contrast to rabbits, is deficient in HCO3- as compared to blood plasma. The possibility that PGF2 alpha does stimulate aqueous humor secretion in the human eye is suggested by the fact that PGF2 alpha-1-isopropylester applied topically to the human eye in doses greater than the minimal dose required to cause ocular hypotension yields an initial increase in intraocular pressure (see Alm and Villumsen, 1989) and a small increase in rate of aqueous production was detected in the PGF2 alpha treated eyes of cynomolgus monkeys (Nilsson et al., 1989). These considerations suggest that the effects of PGs on the transport function of the mammalian ciliary epithelium deserves further attention.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

Role of alpha 1- and alpha 2-adrenergic receptors in Cl- transport across frog corneal epithelium.

Norepinephrine, 10(-6) M, reduced Cl- transport by 26% in 75% of isolated frog corneal epithelia. This inhibition was not previously reported. Since beta-adrenergic agonists are known to only stimulate Cl- transport, the action of specific alpha 1- and alpha 2-agonists on Cl- transport and electrical parameters was investigated. Phenylephrine, an alpha 1-agonist always stimulated the Cl(-)-dependent short-circuit current (Isc), but less than the beta-agonists. UK-14,304-18 (UK), a selective alpha 2-agonist, reduced both the Isc (by 31% at 10(-5) M) and the stroma-to-tear unidirectional Cl- flux. UK hyperpolarized the apical membrane potential difference and increased the transepithelial resistance and apical-to-basolateral resistance ratio. UK reduced forskolin-stimulated adenylate cyclase activity by 36%. The electrophysiological effects of UK are consistent with a reduction of the Cl- permeability at the apical membrane. Pretreatment with UK sensitized the tissue for a greater effect by forskolin. Results show that the frog corneal epithelium also possesses alpha 1- and alpha 2-receptors, the latter negatively coupled to the adenylate cyclase system. Cl- transport is thus regulated by an interaction between the positive effects of beta- and alpha 1-stimulation and the negative influence of alpha 2-stimulation.

Adenylyl Cyclases↗
PubMed1988

Active transport of ascorbate across the isolated rabbit ciliary epithelium.

The transepithelial transport of ascorbate across the isolated rabbit ciliary epithelium (CE) was investigated. Unidirectional 14C-ascorbate fluxes were measured in the presence of equal concentrations of ascorbate on both sides of the tissue within the range of 0.025 to 1 mM. The blood to aqueous (Bl----Aq) flux increased from 6 to 95 nmoles/hr and showed nonlinearity and saturation. The aqueous to blood (Aq----Bl) flux increased, for the same range, from 0.5 to 23 nmoles/hr in a linear fashion. The permeability calculated from the Aq----Bl flux was similar to the CE permeability for mannitol suggesting that the Aq----Bl flux is mainly paracellular. The flux ratio Bl----Aq/Aq----Bl was between 4 to 12. Anoxia, ouabain and low Na+ in the media inhibited the Bl----Aq flux indicating that the transport system requires energy and a Na+ gradient. 3-O-methyl-D-glucose, D-isoascorbic acid and phlorizin also inhibited the Bl----Aq flux, suggesting that ascorbate and glucose may share a common carrier mechanism. Although the isolated CE preparation was clearly capable of flux separation and active transport, the rate of ascorbate transport measured in vitro is insufficient to maintain the aqueous ascorbate concentration observed in vivo.

3-O-Methylglucose↗
PubMed1987

Cl- secretagogues increase basolateral K+ conductance of frog corneal epithelium.

The stromal-to-tear transport of Cl- by the corneal epithelium of the frog is increased by pharmacological effectors (secretagogues) that are known to raise the intracellular levels of cyclic AMP or Ca2+. It has been shown in the past that the Cl- secretagogues increase the apical membrane permeability to Cl- and thus facilitate the cell-to-tear flux of the anion. In this report, we combine transepithelial and microelectrode studies to show that three of these secretagogues, epinephrine, the Ca2+ ionophore A23187, and forskolin, also increase the K+ conductance of the basolateral membrane by two- to threefold. The increase in the K+ conductance is not dependent on membrane potential, since this increase occurred equally when the basolateral membrane potential either exceeded 60 mV, as measured with microelectrodes, or was depolarized by voltage clamping after apical permeabilization with amphotericin B. It is proposed that both Cl- and K+ conductances are under the control of intracellular mediators that act independently on each pathway. The increase in basolateral K+ conductance favors the Cl- secretory process.

Amphotericin B↗
PubMed1987

Electrically silent Na+ and Cl- fluxes across the rabbit ciliary epithelium.

Unidirectional Na+, Cl-, and mannitol fluxes were measured across the isolated and short-circuited rabbit iris-ciliary body as functions of the Cl- concentration in the bathing solution (0, 26 and 80 mM). At constant Na+ concentration, Na+ fluxes increased on the average from 8.0 to 11.9 mu eq/hr as the Cl- concentration was raised and vice versa. Cl- fluxes also increased more than expected from simple diffusion; from 2.0 (26 mM) to 8.1 mu eq/hr (80 mM). Mannitol permeability (9.0 X 10(-7) cm/sec) was independent of the Cl- concentration and similar to that measured in "tight" epithelia. In Cl(-)-free solutions, there was good agreement between the measured electrical resistance and that calculated with the partial conductance equation. In Cl(-)-rich solutions, the calculated resistance was smaller than the measured resistance, suggesting electrically silent fluxes of Na+ and Cl-. These silent fluxes were of similar magnitude and possibly coupled with each other. This mechanism may provide an additional pathway for electrolyte movement across the ciliary epithelium.

Animals↗
PubMed1987

Electrical parameters of the isolated monkey ciliary epithelium and effects of pharmacological agents.

The electrical properties of the isolated monkey ciliary epithelium (CE) were determined in an Ussing-type chamber. In a Hepes, HCO3- buffered solution, transepithelial potential difference (PD), short-circuit current (SCC) and electrical resistance (Rt) were -2.5 mV (aqueous-side negative), 8.5 microA and 246 omega, respectively. Epinephrine (0.01 mM) increased the SCC and PD across the isolated monkey CE when added to the aqueous-side bathing solution but was without effect when added to the blood-side bathing solution. Forskolin (0.01 mM) increased the SCC and PD when added to the bathing solution on either side. A disulfonic stilbene, DIDS (0.1 mM), reduced the SCC and PD when added to the aqueous-side bathing solution but was without effect when added to the blood-side bathing solution. Ouabain (0.1 mM) added to the aqueous-side produced a transient stimulation followed by a gradual inhibition of the SCC and PD. On the blood-side, ouabain directly inhibited the SCC and PD towards zero. Although the general electrical properties were similar to those of the isolated rabbit iris-ciliary epithelium, there were differences in the effects of these pharmacological agents on the electrical properties.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Forskolin effects on frog and rabbit corneal epithelium ion transport.

The effects of forskolin on the electrophysiological parameters of the isolated corneal epithelium from bullfrog (Rana catesbeiana) were investigated. Forskolin stimulated the short-circuit current (SCC) and transepithelial potential difference (PDt), while reducing the transepithelial resistance. These effects were absent in Cl- -free bathing solutions. Furosemide, added either before or after forskolin, completely blocked the effects. Epinephrine and A23187, added after forskolin, produced only a small additional stimulation of the SCC. Propranolol neither blocked nor reduced the effect of forskolin. Forskolin increased the stroma to tear 36Cl flux by 61% and the tear to stroma 36Cl flux by 64%. Intracellular recordings showed that forskolin depolarized the potential difference across the apical membrane and reduced the apical/basolateral resistance ratio. Intracellular recordings in the isolated rabbit epithelium showed the same effects by forskolin except that there was only a brief stimulation of PDt, after which it stabilized slightly below the control level. These results are consistent with an increase in apical membrane permeability similar to that produced by adenosine 3',5'-cyclic monophosphate, epinephrine, and the Ca2+ ionophore A23187.

Animals↗
PubMed1986

Na+-K+ pump stoichiometry and basolateral membrane permeability of frog corneal epithelium.

The Na+-K+ pump flux ratio and the Na+ and K+ permeability of the basolateral membrane of the isolated frog corneal epithelium were studied with the aid of microelectrodes by analyzing the effects of ouabain, Ba2+, and amphotericin B. The experiments were done in Cl(-)-free solutions, a situation that approximates that of static head. Ouabain produced a quick depolarization of the potential difference across the basolateral membrane (PDb) from -72 to -62 mV without a change in resistance. Ba2+ (3 mM) rapidly lowered PDb from -74 to -57 mV and decreased the apical-to-basolateral resistance ratio. The effects of ouabain and Ba2+ were additive. The Na+-K+ flux ratio at the pump was calculated to be 1.78, substantially less than when the tissue is in a level flow condition, suggesting a variable stoichiometry. The K+ and Na+ resistances of the basolateral membrane were 15.7 and 5.5 k omega X cm2, respectively, allowing K+ and Na+ currents that approximately matched those produced by the Na+-K+ pump. The resistance of the basolateral membrane (4.0 k omega X cm2) was double that reported in Cl(-)-rich solutions, suggesting that Cl- contributes to the conductance of this membrane.

Amphotericin B↗