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Vincent H.l. Lee - One of the best experts on this subject based on the ideXlab platform.
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Carrier-Mediated Transport of NG-Nitro-L-Arginine, a Nitric Oxide Synthase Inhibitor, in the Pigmented Rabbit Conjunctiva1
2016Co-Authors: Ken-ichi Hosoya, Yoshihide Horibe, Kwang-jin Kim, Vincent H.l. LeeAbstract:In this study, the transport mechanism of NG-nitro-L-arginine (L-NA), a nitric oxide synthase inhibitor that may be useful for alleviating intraocular inflammation, was characterized in the Pigmented Rabbit conjunctiva. L-NA, when applied to the mu-cosal side of the conjunctiva, led to dose-dependent increases in the short-circuit current (Isc) at 37°C but not at 4°C or under the Na1-free condition. Serosally added 1 mM L-NA did not elicit any change in the Isc. Mucosally added 1 mM L-NA elicited a net absorptive Na1 flux of 0.09 mEq/(cm2zhr), compa-rable with the Isc change. L-NA transport at 0.1 mM in the mucosal-to-serosal (ms) direction was 22 times greater than that in the serosal-to-mucosal direction. There was a good correlation between the ms flux of L-NA and the Isc changes elicited by L-NA under the same experimental conditions. L-N
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Carrier-mediated transport of monocarboxylate drugs in the Pigmented Rabbit conjunctiva. Invest Ophthalmol Vis Sci
2013Co-Authors: Yoshihide Horibe, Ken-ichi Hosoya, Kwang-jin Kim, Vincent H.l. LeeAbstract:PURPOSE. TO determine whether an Na +-dependent monocarboxylate transport process exists on the mucosal side of the Pigmented Rabbit conjunctiva and to evaluate how it may contribute to the absorption of ophthalmic monocarboxylate drugs. METHODS. L-Lactate was used as a model substrate. The excised Pigmented Rabbit conjunctiva was mounted in a modified Ussing chamber for the measurement of short-circuit current (/sc) and 14 C-L-lactate transport. RESULTS. When added to the mucosal side at 37°C and at pH 7.4, applications of as much as 40 mM L- and D-lactate increased /sc in a saturable manner. By contrast, no change in /sc was observed at 4°C or under the mucosal Na +-free condition. l4 C-L-Lactate transport in the mucosal-to-serosal (m-s) direction at 0.01 mM revealed directionality, temperature dependency, Na + dependency, and ouabain sensitivity, but not pH dependency. L-Lactate transport in the m-s direction consisted of a saturable Na +-dependent process by the transcellular pathway and a nonsaturable process by the paracellular pathway. For the saturable process, the apparent Michaelis-Menten constant was 1.
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Cidofovir transport in the Pigmented Rabbit conjunctiva
Current Eye Research, 2009Co-Authors: Ken-ichi Hosoya, Vincent H.l. LeeAbstract:PURPOSE. To characterize cidofovir ((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine) transport in the Pigmented Rabbit conjunctiva and to evaluate the formulation influence on its transport. METHODS. The excised Pigmented Rabbit conjunctiva was mounted in the modified Ussing chamber. Cidofovir transport was initiated by applying 3 H-cidofovir to the donor compartment and assessed by measuring the radioactivity accumulated in the receiver fluid over 180 min. RESULTS. Cidofovir flux in the mucosal-to-serosal direction increased proportionally with drug concentration over the 0.01 to 1 mM range. Cidofovir transport (0.01 mM) at 37°C in the mucosal-to-serosal direction was not significantly different from that in the opposite direction or from that at 4°C. Hypotonicity (80 mOsm/kg), 0.5% EDTA, and 0.0125% benzalkonium chloride increased the apparent permeability coefficient of cidofovir 3, 21, and 49 times, respectively. This was accompanied by a corresponding 43%, 86%, and 96% reduction in the transconj...
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Contribution of Na+-glucose cotransport to the short-circuit current in the Pigmented Rabbit conjunctiva
Current Eye Research, 2009Co-Authors: Ken-ichi Hosoya, Kwang-jin Kim, Udaya Bhaskar Kompella, Vincent H.l. LeeAbstract:The objective of this study was to determine whether Na+-glucose cotransport contributed to the residual short-circuit current (Isc) in the Pigmented Rabbit conjunctiva not accounted for by CI- secretion. The Isc of the Pigmented Rabbit conjunctiva was measured following exposure of its mucosal or serosal side to varying concentrations of D-glucose or phloridzin in a glutathione-bicarbonated Ringer's solution (GBR). Addition of D-glucose to the mucosal side of the conjunctiva bathed in glucose-free GBR elevated Isc up to 26 ± 6% in a dose-dependent manner (K0.5 = 1.2 mM), while mucosal (but not serosal) addition of phloridzin reduced the Isc (IC50 = 0.05 mM) of the conjunctiva bathed in regular GBR. In a mucosal Na+-free medium, neither 20 mM D-glucose nor 0.5 mM phloridzin was effective. In a mucosal glucose-containing medium, deletion of Naf reduced Isc up to 27 ± 4%. It was, however, restored to its initial value upon the addition of 17.5–141 mM Na+ to a medium containing 5 mM D-glucose. Hill plot anal...
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Ocular esterase composition in albino and Pigmented Rabbits: Possible implications in ocular prodrug design and evaluation
Current Eye Research, 2009Co-Authors: Vincent H.l. Lee, Shih-chieh Chang, Clarence M. Oshiro, Ronald E. SmithAbstract:A methodology was developed to determine the proportion of acetyl-(AChE) and butyrylcholinesterase (BuChE) in the albino and Pigmented Rabbit eye. It was found that BuChE contributed over 75% of the cholinesterase activity in all the ocular tissues but the corneal epithelium of the albino Rabbit. This esterase was principally responsible for the parabolic chain length dependence of ocular hydrolysis of model naphthyl ester prodrugs reported previously. In contrast, when incubated with AChE, the rate of hydrolysis of these esters decreased monotonically with increasing ester chain length. Together these findings suggest that esters whose chain length exceeds 4 carbons will be hydrolyzed primarily by BuChE. It is suggested that the dominance of BuChE in ocular tissues is another factor which merits consideration in the design and evaluation of ocular ester prodrugs.
Kwang-jin Kim - One of the best experts on this subject based on the ideXlab platform.
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Carrier-Mediated Transport of NG-Nitro-L-Arginine, a Nitric Oxide Synthase Inhibitor, in the Pigmented Rabbit Conjunctiva1
2016Co-Authors: Ken-ichi Hosoya, Yoshihide Horibe, Kwang-jin Kim, Vincent H.l. LeeAbstract:In this study, the transport mechanism of NG-nitro-L-arginine (L-NA), a nitric oxide synthase inhibitor that may be useful for alleviating intraocular inflammation, was characterized in the Pigmented Rabbit conjunctiva. L-NA, when applied to the mu-cosal side of the conjunctiva, led to dose-dependent increases in the short-circuit current (Isc) at 37°C but not at 4°C or under the Na1-free condition. Serosally added 1 mM L-NA did not elicit any change in the Isc. Mucosally added 1 mM L-NA elicited a net absorptive Na1 flux of 0.09 mEq/(cm2zhr), compa-rable with the Isc change. L-NA transport at 0.1 mM in the mucosal-to-serosal (ms) direction was 22 times greater than that in the serosal-to-mucosal direction. There was a good correlation between the ms flux of L-NA and the Isc changes elicited by L-NA under the same experimental conditions. L-N
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Carrier-mediated transport of monocarboxylate drugs in the Pigmented Rabbit conjunctiva. Invest Ophthalmol Vis Sci
2013Co-Authors: Yoshihide Horibe, Ken-ichi Hosoya, Kwang-jin Kim, Vincent H.l. LeeAbstract:PURPOSE. TO determine whether an Na +-dependent monocarboxylate transport process exists on the mucosal side of the Pigmented Rabbit conjunctiva and to evaluate how it may contribute to the absorption of ophthalmic monocarboxylate drugs. METHODS. L-Lactate was used as a model substrate. The excised Pigmented Rabbit conjunctiva was mounted in a modified Ussing chamber for the measurement of short-circuit current (/sc) and 14 C-L-lactate transport. RESULTS. When added to the mucosal side at 37°C and at pH 7.4, applications of as much as 40 mM L- and D-lactate increased /sc in a saturable manner. By contrast, no change in /sc was observed at 4°C or under the mucosal Na +-free condition. l4 C-L-Lactate transport in the mucosal-to-serosal (m-s) direction at 0.01 mM revealed directionality, temperature dependency, Na + dependency, and ouabain sensitivity, but not pH dependency. L-Lactate transport in the m-s direction consisted of a saturable Na +-dependent process by the transcellular pathway and a nonsaturable process by the paracellular pathway. For the saturable process, the apparent Michaelis-Menten constant was 1.
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Contribution of Na+-glucose cotransport to the short-circuit current in the Pigmented Rabbit conjunctiva
Current Eye Research, 2009Co-Authors: Ken-ichi Hosoya, Kwang-jin Kim, Udaya Bhaskar Kompella, Vincent H.l. LeeAbstract:The objective of this study was to determine whether Na+-glucose cotransport contributed to the residual short-circuit current (Isc) in the Pigmented Rabbit conjunctiva not accounted for by CI- secretion. The Isc of the Pigmented Rabbit conjunctiva was measured following exposure of its mucosal or serosal side to varying concentrations of D-glucose or phloridzin in a glutathione-bicarbonated Ringer's solution (GBR). Addition of D-glucose to the mucosal side of the conjunctiva bathed in glucose-free GBR elevated Isc up to 26 ± 6% in a dose-dependent manner (K0.5 = 1.2 mM), while mucosal (but not serosal) addition of phloridzin reduced the Isc (IC50 = 0.05 mM) of the conjunctiva bathed in regular GBR. In a mucosal Na+-free medium, neither 20 mM D-glucose nor 0.5 mM phloridzin was effective. In a mucosal glucose-containing medium, deletion of Naf reduced Isc up to 27 ± 4%. It was, however, restored to its initial value upon the addition of 17.5–141 mM Na+ to a medium containing 5 mM D-glucose. Hill plot anal...
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clathrin and caveolin 1 expression in primary Pigmented Rabbit conjunctival epithelial cells role in plga nanoparticle endocytosis
Molecular Vision, 2003Co-Authors: Mohamed G Qaddoumi, Hovhannes J. Gukasyan, Kwang-jin Kim, Jasmine Davda, Vinod Labhasetwar, Vincent H.l. LeeAbstract:Purpose: The internalization of poly (dl-lactide-co-glycolide, PLGA) nanoparticles in Rabbit conjunctival epithelial cells (RCEC) was previously shown to occur by an endocytic process, as evidenced its energy-dependence, inhibition by the vesicle formation blocker cytochalasin D, and by the characteristic display of punctate distribution under confocal microscopy. In addition, clathrin protein was implicated in the endocytosis of these nanoparticles in vascular smooth muscle cells. We sought to examine the expression of clathrin and caveolin-1 in RCECs and to determine whether they play a role in PLGA nanoparticle endocytosis. Methods: PLGA (50:50) nanoparticles (100 nm in diameter) containing 6-coumarin (fluorescent marker, 0.05% w/v) were used in this study. The effect of pharmacological treatments aimed at disrupting formation of clathrin-coated vesicles (hypertonic challenge and intracellular K + depletion) and caveolae (nystatin and filipin) on apical uptake of nanoparticles in primary cultured RCEC was investigated. Transferrin was chosen as a marker for clathrin-dependent endocytosis from the basolateral aspect, whereas cholera toxin B subunit was chosen as a marker for caveolae-mediated endocytosis. The staining pattern of nanoparticles in RCECs was compared with that of clathrin heavy chain (HC) and caveolin-1 under fluorescent confocal microscopy to examine possible colocalization using clathrin HC and caveolin-1 mouse monoclonal antibodies (mAb). Two pairs of primers were designed (based on conserved regions of clathrin and caveolin-1 gene in different species) to amplify a 744-bp and 152-bp fragment of clathrin HC and caveolin-1 gene, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) to detect the message for clathrin HC and caveolin-1 was performed using total RNA prepared from freshly isolated RCECs. HEK293 cells were used as positive control for clathrin gene expression, whereas Rabbit heart muscle and HEK cells were used as positive control for caveolin-1 gene expression. The RT-PCR products were separated using 2% agarose gel electrophoresis. Western blot analysis was performed to detect the expression of both clathrin and caveolin-1 proteins in RCECs using mouse mAbs. HeLa cells and A431 epidermoid cells were used as positive controls. The effect of transfection of RCECs (using Lipofectamine 2000™ reagent) with specific antisense oligonucleotides designed against the Rabbit clathrin isoform on clathrin protein expression and PLGA nanoparticle uptake was investigated. Results: Apical uptake of nanoparticles in primary cultured RCECs was decreased by 45% and 35%, respectively, as a result of K + depletion and hypertonic media treatments. Likewise, the same treatments significantly decreased the basolateral uptake of FITC-transferrin by 50%. In contrast, nystatin and filipin had no effect on apical uptake of nanoparticles and cholera toxin B subunit in RCECs, suggesting a lack of the involvement of caveolae in the internalization of these two agents. Confocal microscopy showed fluorescent staining of cell membrane in the presence clathrin mAb, but not in the presence of caveolin-1 mAb, with partial overlap with a nanoparticle staining pattern. RT-PCR confirmed the presence of the clathrin HC gene, but not the caveolin-1 gene, in RCECs as indicated by a 744-bp fragment of the gene. However, caveolin-1 gene was detected in other Rabbit tissues such as the epithelium of the cornea and trachea, and heart muscle, as indicated by a 152-bp fragment of the gene. Western blot analysis revealed a clathrin HC band (180 kDa) in RCEC culture and HeLa cells. However, caveolin-1 protein (22 kDa) was not detected in RCEC culture, but was detected in A431 cells. Transfection of RCECs with antisense oligonuceotide directed against clathrin HC resulted in knockdown of the clathrin HC protein in a concentration dependent manner. However, clathrin HC protein knockdown had no effect on apical uptake of nanoparticles in RCECs. Conclusions: Our findings indicate that endocytosis of nanoparticles in primary cultured RCECs occurs mostly independently of clathrin- and caveolin-1-mediated pathways. In addition, the gene and protein expression of clathrin HC, but not caveolin-1, was identified in Rabbit conjunctival epithelial cells.
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specialized protective role of mucosal glutathione in Pigmented Rabbit conjunctiva
Investigative Ophthalmology & Visual Science, 2003Co-Authors: Hovhannes J. Gukasyan, Kwang-jin Kim, Ram Kannan, Robert A Farley, Vincent H.l. LeeAbstract:PURPOSE To investigate mechanisms of H(2)O(2)-induced reduction in rates of active ion transport (I(sc)) across the Pigmented Rabbit conjunctival tissue and the protective role afforded by mucosal glutathione (GSH). METHODS Changes in I(sc) and specific binding properties of ouabain were evaluated in a modified Ussing chamber setup, using conjunctival tissues freshly excised from Pigmented Rabbits. Effective concentrations of H(2)O(2) at which 50% of I(sc) was inhibited (IC(50)) were determined for the mucosal and serosal instillation of the agent. The rate of exogenous H(2)O(2) consumption in the mucosal and serosal bathing fluids was estimated. Mucosal 8-Br cAMP at 3 mM, serosal bumetanide at 0.5 mM, and both mucosal and serosal bathing of the conjunctiva with Na(+)-free bicarbonated Ringer's solution (BRS) were used to estimate contributions of conjunctival ion transport mechanisms in I(sc) changes elicited by mucosal H(2)O(2) at IC(50). Specific binding of (3)H-ouabain to the serosal side of the conjunctiva was estimated in the presence of mucosal or serosal H(2)O(2) to assess the role of functional Na(+)/K(+)-ATPase pumps in H(2)O(2) injury. The effect of mucosally instilled GSH and other reductive and nonreductive agents on possible restoration of oxidant-induced decrease in conjunctival I(sc) was also determined. RESULTS Mucosal and serosal H(2)O(2) decreased conjunctival I(sc) gradually in a dose-dependent manner. The mucosal IC(50) of H(2)O(2)was 1.49 +/- 0.20 mM, whereas the serosal IC(50) was estimated at 10.6 +/- 2.0 micro M. The rate of H(2)O(2) consumption from mucosal fluid was six times faster than that from serosal fluid. Conjunctival tissues pretreated with mucosal H(2)O(2) at IC(50) retained approximately 50% of their maximum 8-Br cAMP-dependent increases in I(sc). Serosal bumetanide did not further reduce the I(sc) beyond the initial 70% decrease caused by mucosal H(2)O(2). When conjunctiva was bathed with Na(+)-free BRS on both the mucosal and serosal sides, before or after addition of mucosal H(2)O(2), the combined effects were additive, decreasing I(sc) by up to 95% to 99%. Mucosal, but not serosal, GSH or reduced L-glutathione mono-ethyl ester (GSH-MEE) superfusion of conjunctival tissues pre-exposed to mucosal H(2)O(2) at IC(50) recovered to 60% to 80% of the initial pre-H(2)O(2) I(sc) after approximately 100 minutes. The specific binding of (3)H-ouabain to the serosal side of the tissue was inhibited by 85% in the presence of mucosal or serosal treatment with H(2)O(2) at their respective IC(50) values. Pretreatment for 60 minutes with either 5 mM GSH, 2 mM GSH-MEE, or 0.1 mM ebselen, when instilled into the mucosal fluid, resulted in 30%, 45%, or 55% reductions, respectively, in ouabain binding after exposure to mucosal H(2)O(2) at IC(50). Furthermore, mucosal posttreatment with 10 mM GSH or 5 mM GSH-MEE of conjunctival tissues pre-exposed to mucosal H(2)O(2) resulted in a 30% recovery of the ouabain-binding level above that observed in tissues exposed to 1.5 mM H(2)O(2) alone on the mucosal side. By contrast, the decrease in conjunctival I(sc) or in the ouabain-binding level elicited by serosal H(2)O(2) at IC(50) was irreversible. CONCLUSIONS A higher mucosal IC(50) of [H(2)O(2)] on conjunctival I(sc) corresponds to the faster consumption of exogenous H(2)O(2) from mucosal bathing fluid. In addition, actively secreted GSH by conjunctival epithelial cells may help reduce the injury by mucosally applied H(2)O(2). Injury by H(2)O(2) may directly affect vital membrane components (e.g., Na(+),K(+)-ATPase) involved in active ion transport across conjunctiva. Mucosal protection by GSH (or its analogues) of active conjunctival ion transport may be useful in maintaining the physiological functions of conjunctiva under oxidative stress.
Ken-ichi Hosoya - One of the best experts on this subject based on the ideXlab platform.
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Carrier-Mediated Transport of NG-Nitro-L-Arginine, a Nitric Oxide Synthase Inhibitor, in the Pigmented Rabbit Conjunctiva1
2016Co-Authors: Ken-ichi Hosoya, Yoshihide Horibe, Kwang-jin Kim, Vincent H.l. LeeAbstract:In this study, the transport mechanism of NG-nitro-L-arginine (L-NA), a nitric oxide synthase inhibitor that may be useful for alleviating intraocular inflammation, was characterized in the Pigmented Rabbit conjunctiva. L-NA, when applied to the mu-cosal side of the conjunctiva, led to dose-dependent increases in the short-circuit current (Isc) at 37°C but not at 4°C or under the Na1-free condition. Serosally added 1 mM L-NA did not elicit any change in the Isc. Mucosally added 1 mM L-NA elicited a net absorptive Na1 flux of 0.09 mEq/(cm2zhr), compa-rable with the Isc change. L-NA transport at 0.1 mM in the mucosal-to-serosal (ms) direction was 22 times greater than that in the serosal-to-mucosal direction. There was a good correlation between the ms flux of L-NA and the Isc changes elicited by L-NA under the same experimental conditions. L-N
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Carrier-mediated transport of monocarboxylate drugs in the Pigmented Rabbit conjunctiva. Invest Ophthalmol Vis Sci
2013Co-Authors: Yoshihide Horibe, Ken-ichi Hosoya, Kwang-jin Kim, Vincent H.l. LeeAbstract:PURPOSE. TO determine whether an Na +-dependent monocarboxylate transport process exists on the mucosal side of the Pigmented Rabbit conjunctiva and to evaluate how it may contribute to the absorption of ophthalmic monocarboxylate drugs. METHODS. L-Lactate was used as a model substrate. The excised Pigmented Rabbit conjunctiva was mounted in a modified Ussing chamber for the measurement of short-circuit current (/sc) and 14 C-L-lactate transport. RESULTS. When added to the mucosal side at 37°C and at pH 7.4, applications of as much as 40 mM L- and D-lactate increased /sc in a saturable manner. By contrast, no change in /sc was observed at 4°C or under the mucosal Na +-free condition. l4 C-L-Lactate transport in the mucosal-to-serosal (m-s) direction at 0.01 mM revealed directionality, temperature dependency, Na + dependency, and ouabain sensitivity, but not pH dependency. L-Lactate transport in the m-s direction consisted of a saturable Na +-dependent process by the transcellular pathway and a nonsaturable process by the paracellular pathway. For the saturable process, the apparent Michaelis-Menten constant was 1.
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Cidofovir transport in the Pigmented Rabbit conjunctiva
Current Eye Research, 2009Co-Authors: Ken-ichi Hosoya, Vincent H.l. LeeAbstract:PURPOSE. To characterize cidofovir ((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine) transport in the Pigmented Rabbit conjunctiva and to evaluate the formulation influence on its transport. METHODS. The excised Pigmented Rabbit conjunctiva was mounted in the modified Ussing chamber. Cidofovir transport was initiated by applying 3 H-cidofovir to the donor compartment and assessed by measuring the radioactivity accumulated in the receiver fluid over 180 min. RESULTS. Cidofovir flux in the mucosal-to-serosal direction increased proportionally with drug concentration over the 0.01 to 1 mM range. Cidofovir transport (0.01 mM) at 37°C in the mucosal-to-serosal direction was not significantly different from that in the opposite direction or from that at 4°C. Hypotonicity (80 mOsm/kg), 0.5% EDTA, and 0.0125% benzalkonium chloride increased the apparent permeability coefficient of cidofovir 3, 21, and 49 times, respectively. This was accompanied by a corresponding 43%, 86%, and 96% reduction in the transconj...
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Contribution of Na+-glucose cotransport to the short-circuit current in the Pigmented Rabbit conjunctiva
Current Eye Research, 2009Co-Authors: Ken-ichi Hosoya, Kwang-jin Kim, Udaya Bhaskar Kompella, Vincent H.l. LeeAbstract:The objective of this study was to determine whether Na+-glucose cotransport contributed to the residual short-circuit current (Isc) in the Pigmented Rabbit conjunctiva not accounted for by CI- secretion. The Isc of the Pigmented Rabbit conjunctiva was measured following exposure of its mucosal or serosal side to varying concentrations of D-glucose or phloridzin in a glutathione-bicarbonated Ringer's solution (GBR). Addition of D-glucose to the mucosal side of the conjunctiva bathed in glucose-free GBR elevated Isc up to 26 ± 6% in a dose-dependent manner (K0.5 = 1.2 mM), while mucosal (but not serosal) addition of phloridzin reduced the Isc (IC50 = 0.05 mM) of the conjunctiva bathed in regular GBR. In a mucosal Na+-free medium, neither 20 mM D-glucose nor 0.5 mM phloridzin was effective. In a mucosal glucose-containing medium, deletion of Naf reduced Isc up to 27 ± 4%. It was, however, restored to its initial value upon the addition of 17.5–141 mM Na+ to a medium containing 5 mM D-glucose. Hill plot anal...
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Nucleotide Stimulation of Cl− Secretion in the Pigmented Rabbit Conjunctiva
Journal of Pharmacology and Experimental Therapeutics, 1999Co-Authors: Ken-ichi Hosoya, Kwang-jin Kim, Hideo Ueda, Vincent H.l. LeeAbstract:We evaluated the role of extracellular UTP and other nucleotides in the regulation of active ion transport across the Pigmented Rabbit conjunctiva. When added to the mucosal side of the conjunctiva, UTP (0.01–1000 μM), increased the short-circuit current by up to 14.6 ± 2.1 μA/cm 2 . The half-maximal concentration was 11.4 ± 2.3 μM. The serosal absence of Cl − , serosal presence of 10 μM bumetanide, and mucosal presence of 0.3 mM N- phenylanthranilic acid significantly reduced the change in the short-circuit current (ΔIsc) induced by 10 μM UTP by 78, 77, and 42%, respectively. Mucosal 10 μM UTP significantly increased 36 Cl flux in the serosal-to-mucosal direction by 0.17 μEq/cm 2 /h, while not affecting mucosal-to-serosal 36 Cl flux. By contrast, 22 Na transport in either direction was unaffected. The rank order of ΔIsc elicited by adenosine and nucleotides was consistent with the predominant involvement of P2Y purinergic receptors in the UTP effect on conjunctival ion transport. Moreover, the ΔIsc elicited by UTP was inhibited by 0.05 and 1 mM suramin (a P2-purinergic receptor antagonist), resulting in a rightward shift of the half-maximal concentration to 106.7 ± 1.3 μM. In conclusion, the primary effect of UTP on ion transport in the Pigmented Rabbit conjunctiva is stimulation of Cl − secretion, possibly at the P2Y 2 and/or the P2Y 4 receptor on the mucosal side of the tissue. Because of the coupling of fluid flow with Cl − secretion, UTP or its analogs may be considered for stimulating transconjunctival fluid flow in the dry-eye state.
Udaya Bhaskar Kompella - One of the best experts on this subject based on the ideXlab platform.
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Contribution of Na+-glucose cotransport to the short-circuit current in the Pigmented Rabbit conjunctiva
Current Eye Research, 2009Co-Authors: Ken-ichi Hosoya, Kwang-jin Kim, Udaya Bhaskar Kompella, Vincent H.l. LeeAbstract:The objective of this study was to determine whether Na+-glucose cotransport contributed to the residual short-circuit current (Isc) in the Pigmented Rabbit conjunctiva not accounted for by CI- secretion. The Isc of the Pigmented Rabbit conjunctiva was measured following exposure of its mucosal or serosal side to varying concentrations of D-glucose or phloridzin in a glutathione-bicarbonated Ringer's solution (GBR). Addition of D-glucose to the mucosal side of the conjunctiva bathed in glucose-free GBR elevated Isc up to 26 ± 6% in a dose-dependent manner (K0.5 = 1.2 mM), while mucosal (but not serosal) addition of phloridzin reduced the Isc (IC50 = 0.05 mM) of the conjunctiva bathed in regular GBR. In a mucosal Na+-free medium, neither 20 mM D-glucose nor 0.5 mM phloridzin was effective. In a mucosal glucose-containing medium, deletion of Naf reduced Isc up to 27 ± 4%. It was, however, restored to its initial value upon the addition of 17.5–141 mM Na+ to a medium containing 5 mM D-glucose. Hill plot anal...
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contribution of na glucose cotransport to the short circuit current in the Pigmented Rabbit conjunctiva
Current Eye Research, 1996Co-Authors: Ken-ichi Hosoya, Kwang-jin Kim, Udaya Bhaskar Kompella, Vincent H.l. LeeAbstract:The objective of this study was to determine whether Na+-glucose cotransport contributed to the residual short-circuit current (Isc) in the Pigmented Rabbit conjunctiva not accounted for by CI- secretion. The Isc of the Pigmented Rabbit conjunctiva was measured following exposure of its mucosal or serosal side to varying concentrations of D-glucose or phloridzin in a glutathione-bicarbonated Ringer's solution (GBR). Addition of D-glucose to the mucosal side of the conjunctiva bathed in glucose-free GBR elevated Isc up to 26 ± 6% in a dose-dependent manner (K0.5 = 1.2 mM), while mucosal (but not serosal) addition of phloridzin reduced the Isc (IC50 = 0.05 mM) of the conjunctiva bathed in regular GBR. In a mucosal Na+-free medium, neither 20 mM D-glucose nor 0.5 mM phloridzin was effective. In a mucosal glucose-containing medium, deletion of Naf reduced Isc up to 27 ± 4%. It was, however, restored to its initial value upon the addition of 17.5–141 mM Na+ to a medium containing 5 mM D-glucose. Hill plot anal...
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Cyclic AMP modulation of active ion transport in the Pigmented Rabbit conjunctiva.
Journal of Ocular Pharmacology and Therapeutics, 1996Co-Authors: Udaya Bhaskar Kompella, Kwang-jin Kim, Michael H.i. Shiue, Vincent H.l. LeeAbstract:ABSTRACT This study was conducted to determine whether active ion transport in the Pigmented Rabbit conjunctiva was subjected to cyclic AMP modulation. 8-BrcAMP and compounds that may affect intracellular cAMP levels were tested for their effects on the conjunctival short-circuit current (Isc) following mucosal application. Conjunctival Isc was increased by 1 mM 8-BrcAMP, 0.001-20 μM forskolin, 0.01-2 mM theophylline, 2 μM l-epinephrine, and 20 μM terbutaline in a Cl−-containing but not in a Cl−-free medium. The increase in Isc induced by 1 mM 8-BrcAMP was reversed by 1 mM N-phenylanthranilic acid, suggesting enhancement of Cl− secretion by cAMP. Similarly, the increase in Isc induced by 20 μM terbutaline was blunted by 1 μM timolol, suggesting a role for β2 adrenergic modulation of Cl− secretion. Collectively, these results are consistent with the notion of cAMP modulation of conjunctival active ion transport.
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Possible existence of Na(+)-coupled amino acid transport in the Pigmented Rabbit conjunctiva.
Life Sciences, 1995Co-Authors: Udaya Bhaskar Kompella, Kwang-jin Kim, Michael H.i. Shiue, Vincent H.l. LeeAbstract:The objective of the present study was to determine whether Na+-amino acid cotransport contributed to the short-circuit current (Isc) in the isolated Pigmented Rabbit conjunctiva. Glycine, L-arginine, D-arginine, and L-glutamic acid were the amino acids tested, and bioelectric measurements were made in the Using chamber. The Isc was increased from 4% (L-glutamic acid) to 44% (L-arginine). The EC50 was 0.35 mM for glycine, 0.06 mM for L-arginine, 0.16 mM for D-arginine, and 1 mM for L-glutamic acid. No elevation in Isc was seen in the absence of Na+ in the mucosal bathing fluid. The above findings are consistent with the possible existence of a Na+-amino acid cotransport process on the apical side of the Pigmented Rabbit conjunctiva.
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possible existence of na coupled amino acid transport in the Pigmented Rabbit conjunctiva
Life Sciences, 1995Co-Authors: Udaya Bhaskar Kompella, Kwang-jin Kim, Michael H.i. Shiue, Vincent H.l. LeeAbstract:The objective of the present study was to determine whether Na+-amino acid cotransport contributed to the short-circuit current (Isc) in the isolated Pigmented Rabbit conjunctiva. Glycine, L-arginine, D-arginine, and L-glutamic acid were the amino acids tested, and bioelectric measurements were made in the Using chamber. The Isc was increased from 4% (L-glutamic acid) to 44% (L-arginine). The EC50 was 0.35 mM for glycine, 0.06 mM for L-arginine, 0.16 mM for D-arginine, and 1 mM for L-glutamic acid. No elevation in Isc was seen in the absence of Na+ in the mucosal bathing fluid. The above findings are consistent with the possible existence of a Na+-amino acid cotransport process on the apical side of the Pigmented Rabbit conjunctiva.
Yoshihide Horibe - One of the best experts on this subject based on the ideXlab platform.
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Carrier-Mediated Transport of NG-Nitro-L-Arginine, a Nitric Oxide Synthase Inhibitor, in the Pigmented Rabbit Conjunctiva1
2016Co-Authors: Ken-ichi Hosoya, Yoshihide Horibe, Kwang-jin Kim, Vincent H.l. LeeAbstract:In this study, the transport mechanism of NG-nitro-L-arginine (L-NA), a nitric oxide synthase inhibitor that may be useful for alleviating intraocular inflammation, was characterized in the Pigmented Rabbit conjunctiva. L-NA, when applied to the mu-cosal side of the conjunctiva, led to dose-dependent increases in the short-circuit current (Isc) at 37°C but not at 4°C or under the Na1-free condition. Serosally added 1 mM L-NA did not elicit any change in the Isc. Mucosally added 1 mM L-NA elicited a net absorptive Na1 flux of 0.09 mEq/(cm2zhr), compa-rable with the Isc change. L-NA transport at 0.1 mM in the mucosal-to-serosal (ms) direction was 22 times greater than that in the serosal-to-mucosal direction. There was a good correlation between the ms flux of L-NA and the Isc changes elicited by L-NA under the same experimental conditions. L-N
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Carrier-mediated transport of monocarboxylate drugs in the Pigmented Rabbit conjunctiva. Invest Ophthalmol Vis Sci
2013Co-Authors: Yoshihide Horibe, Ken-ichi Hosoya, Kwang-jin Kim, Vincent H.l. LeeAbstract:PURPOSE. TO determine whether an Na +-dependent monocarboxylate transport process exists on the mucosal side of the Pigmented Rabbit conjunctiva and to evaluate how it may contribute to the absorption of ophthalmic monocarboxylate drugs. METHODS. L-Lactate was used as a model substrate. The excised Pigmented Rabbit conjunctiva was mounted in a modified Ussing chamber for the measurement of short-circuit current (/sc) and 14 C-L-lactate transport. RESULTS. When added to the mucosal side at 37°C and at pH 7.4, applications of as much as 40 mM L- and D-lactate increased /sc in a saturable manner. By contrast, no change in /sc was observed at 4°C or under the mucosal Na +-free condition. l4 C-L-Lactate transport in the mucosal-to-serosal (m-s) direction at 0.01 mM revealed directionality, temperature dependency, Na + dependency, and ouabain sensitivity, but not pH dependency. L-Lactate transport in the m-s direction consisted of a saturable Na +-dependent process by the transcellular pathway and a nonsaturable process by the paracellular pathway. For the saturable process, the apparent Michaelis-Menten constant was 1.
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functional characterization of organic cation drug transport in the Pigmented Rabbit conjunctiva
Investigative Ophthalmology & Visual Science, 2000Co-Authors: Hideo Ueda, Yoshihide Horibe, Kwang-jin Kim, Vincent H.l. LeeAbstract:PURPOSE. To characterize carrier-mediated organic cation drug transport in the Rabbit conjunctiva. METHODS. The transport of [ 14 C]guanidine, the model substrate, in the excised Pigmented Rabbit conjunctiva was evaluated in the modified Ussing chamber. Tetraethylammonium (TEA) transport also was investigated to determine substrate specificity. RESULTS. The apparent permeability coefficient for guanidine and TEA in the mucosal-to-serosal (ms) direction was 5.4 and 49.6 times greater than that in the serosal-to-mucosal (sm) direction, respectively. Guanidine transport in the ms (but not sm) direction revealed temperature and concentration dependency over 0.02 to 10 mM with an apparent Michaelis-Menten constant of 3.1 mM and a maximal flux of 11.4 nmol/(cm 2 h). Net guanidine transport measured at 0.1 mM across the conjunctiva was decreased by 71% or 82%, respectively, on the addition of 1 μM valinomycin (a K + ionophore) in both bathing fluids or in a high K + buffer in the mucosal fluid. Interestingly, net guanidine transport was reduced, rather than enhanced, by 63% upon acidifying the mucosal bathing fluid. By contrast, net guanidine transport was not affected by the serosal presence of 0.5 mM ouabain (a Na + ,K + -ATPase inhibitor), by the mucosal and serosal presence of 0.1 μM monensin (a Na + ionophore) or 0.3 μM carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP, a H + ionophore). Guanidine transport in the ms direction was polyspecific, as indicated by the 48% to 82% inhibition by structurally diverse amines. In particular, guanidine ms transport was inhibited by the antiglaucoma drugs dipivefrine (72%), brimonidine (70%), and carbachol (78%). CONCLUSIONS. A carrier-mediated organic cation transport process appears to exist in the conjunctiva, mediating the absorption of organic amines, including certain amine-type ophthalmic drugs. This process may be driven by an inside-negative apical membrane potential difference.
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Carrier-mediated transport of monocarboxylate drugs in the Pigmented Rabbit conjunctiva.
Investigative Ophthalmology & Visual Science, 1998Co-Authors: Yoshihide Horibe, Ken-ichi Hosoya, Kwang-jin Kim, Vincent H.l. LeeAbstract:PURPOSE. To determine whether an Na + -dependent monocarboxylate transport process exists on the mucosal side of the Pigmented Rabbit conjunctiva and to evaluate how it may contribute to the absorption of ophthalmic monocarboxylate drugs. METHODS. L-Lactate was used as a model substrate. The excised Pigmented Rabbit conjunctiva was mounted in a modified Ussing chamber for the measurement of short-circuit current (I sc ) and 14 C-L-lactate transport. RESULTS. When added to the mucosal side at 37°C and at pH 7.4, applications of as much as 40 mM L- and D-lactate increased I sc in a saturable manner. By contrast, no change in I sc was observed at 4°C or under the mucosal Na + -free condition. 14 C-L-Lactate transport in the mucosal-to-serosal (m-s) direction at 0.01 mM revealed directionality, temperature dependency, Na + dependency, and ouabain sensitivity, but not pH dependency. L-Lactate transport in the m-s direction consisted of a saturable Na + -dependent process by the transcellular pathway and a nonsaturable process by the paracellular pathway. For the saturable process, the apparent Michaelis-Menten constant was 1.9 mM, the maximum flux was 8.9 nanomoles/cm 2 per hour. and the apparent Na + L-lactate coupling ratio was 2:1. 14 C-L-Lactate transport in the m-s direction was significantly inhibited (46% to 83%) by the mucosal presence of various monocarboxylate compounds, but not by dicarboxylate compounds, zwitterionic compound, D-glucose, amino acids, and peptidomimetic antibiotics. Monocarboxylate nonsteroidal anti-inflammatory drugs and the antibacterial fluoroquinolones inhibited 14 C-L-lactate transport by 40% to 85%, whereas prostaglandins and cromolyn had no effect. CONCLUSIONS. An Na + -dependent monocarboxylate transport process that may be used by nonsteroidal anti-inflammatory and fluoroquinolone antibacterial drugs for transport appears to be present on the mucosal side of the Pigmented Rabbit conjunctiva. A possible physiologic role for the Na + -dependent monocarboxylate transport process may be to salvage tear lactate.
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carrier mediated transport of ng nitro l arginine a nitric oxide synthase inhibitor in the Pigmented Rabbit conjunctiva
Journal of Pharmacology and Experimental Therapeutics, 1998Co-Authors: Ken-ichi Hosoya, Yoshihide Horibe, Kwang-jin Kim, Vincent H.l. LeeAbstract:In this study, the transport mechanism of N G -nitro-l-arginine (L-NA), a nitric oxide synthase inhibitor that may be useful for alleviating intraocular inflammation, was characterized in the Pigmented Rabbit conjunctiva. L-NA, when applied to the mucosal side of the conjunctiva, led to dose-dependent increases in the short-circuit current (I sc ) at 37°C but not at 4°C or under the Na + -free condition. Serosally added 1 mM L-NA did not elicit any change in the I sc . Mucosally added 1 mM L-NA elicited a net absorptive Na + flux of 0.09 μEq/(cm 2 ·hr), comparable with the I sc change. L-NA transport at 0.1 mM in the mucosal-to-serosal (ms) direction was 22 times greater than that in the serosal-to-mucosal direction. There was a good correlation between the ms flux of L-NA and the I sc changes elicited by L-NA under the same experimental conditions. L-NA transport was saturable, with a K m of 0.35 mM and a maximal flux of 290 pmol/(cm 2 ·min). Hill analysis of L-NA flux observed at 0.1 mM L-NA in response to varying Na + concentrations in the mucosal bathing fluid yielded a Hill coefficient of 0.98, suggesting a 1:1 coupling between Na + and L-NA. Moreover, ms 3 H-L-NA transport was inhibited by basic amino acids (L-Arg and L-Lys) and a neutral amino acid (L-Leu), but not by an acidic amino acid (L-Glu) and the d-stereoisomer of L-NA. In the case of L-Arg, inhibition was competitive with a K i of 0.034 mM. Taken together, the above findings are consistent with the involvement of the L-Arg transport system B 0,+ in the conjunctival transport of L-NA.