Functional characterization of organic cation drug transport in the pigmented rabbit conjunctiva (original) (raw)
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Carrier-mediated transport of monocarboxylate drugs in the pigmented rabbit conjunctiva
Investigative ophthalmology & visual science, 1998
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. 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 (Isc) and 14C-L.-lactate transport. When added to the mucosal side at 37 degrees C and at pH 7.4, applications of as much as 40 mM L- and D-lactate increased Isc in a saturable manner. By contrast, no change in Isc was observed at 4 degrees C or under the mucosal Na+-free condition. 14C-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 nons...
Arginine vasopressin transport and metabolism in the pigmented rabbit conjunctiva
European Journal of Pharmaceutical Sciences, 1998
The purpose of this study was to evaluate the transepithelial transport and metabolism of arginine vasopressin (AVP) in the pigmented rabbit conjunctiva, both in the absence and presence of protease inhibitors. The apparent permeability coefficient, Papp, for 'H-AVP was determined in the modified Ussing chamber, and AVP metabolites were monitored by reversed phase HPLC using a C,, column. At 50 nM donor 'H-AVP, the P,,, in the mucosal-to-serosal (ms) direction was about five times higher than that in the opposite direction. Excess (0.1 mM) AVP decreased the Pap,, for labelled AVP in the mucosal-to-serosal (ms) direction by about 50%. However, intact AVP transport showed neither concentration nor direction dependence. HPLC analysis revealed two subspecies of 'H-AVP in the receiver fluid and virtually no degradation products in the donor fluid following 3 h flux experiments. 'H-AVP transported in the ms direction underwent extensive hydrolysis (73%), which was decreased by 33% with mucosal application of 2 mM camostat mesylate (an aminopeptidase inhibitor) or by 27% with 0.5 mM leupeptin (a serine protease inhibitor). By contrast, 'H-AVP transported in the serosal-to-mucosal (sm) direction resulted in only 37% hydrolysis, and mucosal application of either inhibitor did not significantly affect the P,,, for intact AVP. These data suggest that intact AVP transport in the conjunctiva may be mediated mostly by passive diffusion and enzymatic degradation of AVP may be mediated by proteolytic enzymes present on the mucosal side of the conjunctiva. 0 1998 Elsevier Science B.V.
Characterization of Brimonidine Transport in Retinal Pigment Epithelium
Investigative Opthalmology & Visual Science, 2006
PURPOSE. To investigate the involvement of carrier-mediated transport mechanisms in brimonidine transport in retinal pigment epithelium (RPE). METHODS. The transport of [ 3 H]-brimonidine in bovine RPEchoroid explants was evaluated in a modified Ussing chamber. The uptake of [ 3 H]brimonidine was evaluated in differentiated ARPE-19 cells cultured on permeable transwell filters. RESULTS. The transport of brimonidine into (choroid-to-retina transport [inward]) and out of (retina-to-choroid transport [outward]) the eye in bovine RPE-choroid explants was temperature dependent. Both inward and outward brimonidine transport decreased at 5 M compared with 10 nM. The melanin pigmentation of RPE did not significantly affect tissue permeability at either brimonidine dose. A saturable component was identified for the inward transport with the apparent Michaelis-Menten constant and a maximum transport rate of 51 M and 148 pmol/(cm 2 ⅐h), respectively. Both apical (representing retina-to-choroid transport) and basolateral (representing choroidto-retina transport) brimonidine uptake in ARPE-19 cells showed temperature dependence. Apical uptake was higher than basolateral uptake at 37°C and was decreased to 70% in the presence of NaN 3 or in the absence of extracellular Na ϩ. Besides ␣2-agonists, apical uptake was inhibited by verapamil, desipramine, and quinidine, but not by MPP ϩ (1-methyl-4phenylpyridinium), TEA (tetraethylammonium), decynium-22, carnitine, PHA (p-aminohippurate), alanine, or inosine. Basolateral brimonidine uptake increased by 35% at extracellular pH of 6 and decreased by 50% under cell-depolarized conditions of high medium K ϩ and 1 M valinomycin. Temperaturedependent components of basolateral uptake were not saturated at doses up to 2 mM. CONCLUSIONS. A carrier-mediated transport process for brimonidine in RPE was demonstrated in bovine RPE-choroid explants and polarized ARPE-19 cells. This transport system may play a significant role in modulating the movement of brimonidine into and out of the eye.
Identification and Localization of Acid-base Transporters in the Conjunctival Epithelium
Experimental Eye Research, 2001
Acid-base transporters of rabbit and porcine conjunctival epithelia were identi®ed and localized with immunoblotting and immunohistochemical techniques using speci®c antibodies against carriers commonly found in epithelia, i.e. the Cl À /HCO À 3 exchanger (AE2), Na /H exchanger (NHE-1, -2, -3) and the electrogenic Na -(n)HCO À 3 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 identi®ed 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 speci®c, 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-speci®c. For con®rmation 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 À -dependent pH i effects consistent with the existence of Cl À /HCO À 3 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 À 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 À -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 À secretion is variable and dependent upon individual rates of metabolic CO 2 production. Overall, the present study provides an initial identi®cation 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 ®lm but may complement the Na -2Cl À -K cotransporter in effectuating Cl À secretion.
Utility of transporter/receptor(s) in drug delivery to the eye
World Journal of Pharmacology, 2013
The eye is a highly protected organ, and designing an effective therapy is often considered a challenging task. The anatomical and physiological barriers result in low ocular bioavailability of drugs. Due to these constraints, less than 5% of the administered dose is absorbed from the conventional ophthalmic dosage forms. Further, physicochemical properties such as lipophilicity, molecular weight and charge modulate the permeability of drug molecules. Vision-threatening diseases such as glaucoma, diabetic macular edema, cataract, wet and dry age-related macular degeneration, proliferative vitreoretinopathy, uveitis, and cytomegalovirus retinitis alter the pathophysiological and molecular mechanisms. Understanding these mechanisms may result in the development of novel treatment modalities. Recently, transporter/receptor targeted prodrug approach has generated significant interest in ocular drug delivery. These transporters and receptors are involved in the transport of essential nutrients, vitamins, and xenobiotics across biological membranes. Several influx transporters (peptides, amino acids, glucose, lac-tate and nucleosides/nucleobases) and receptors (folate and biotin) have been identified on conjunctiva, cornea, and retina. Structural and functional delineation of these transporters will enable more drugs targeting the posterior segment to be successfully delivered topically. Prodrug derivatization targeting transporters and receptors expressed on ocular tissues has been the subject of intense research. Several prodrugs have been designed to target these transporters and enhance the absorption of poorly permeating parent drug. Moreover, this approach might be used in gene delivery to modify cellular function and membrane receptors. This review provides comprehensive information on ocular drug delivery, with special emphasis on the use of transporters and receptors to improve drug bioavailability.
Experimental Eye Research, 2004
The purposes of this study were to provide functional evidence for the presence of a peptide transporter on blood-ocular barriers and to elucidate the mechanism of a dipeptide transport across these barriers following systemic administration. Glycylsarcosine was chosen as a model dipeptide and [ 3 H] glycylsarcosine was administered through the marginal ear vein of New Zealand white rabbits. At the end of an experimental period, vitreous humor, retina and aqueous humor were collected. Time dependent uptake of glycylsarcosine into ocular tissues was studied at 5, 10, 15 and 30 min. Competitive inhibition studies were performed by intravenous administration of [ 3 H] glycylsarcosine with and without various inhibitors. Concentration-dependent ocular uptake of glycylsarcosine was carried out by administration of various concentrations of unlabelled glycylsarcosine spiked with a fixed amount of [ 3 H] glycylsarcosine. Time-dependent uptake of glycylsarcosine into vitreous humor, retina and aqueous humor for a period of 30 min following systemic administration was linear. Ocular uptake of glycylsarcosine was inhibited by peptide transporter substrates such as dipeptides (glycylproline and carnosine) and captopril but not by non-substrates such as amino acids. Concentration-dependent selfinhibition of glycylsarcosine ocular uptake was also observed. The results indicate that model dipeptide is transported across bloodocular barriers via a carrier-mediated process. In conclusion, an oligopeptide transport system is involved in the transport of glycylsarcosine across blood-ocular barriers. This information may be utilized to design transporter/receptor targeted drug delivery systems for efficient ocular uptake from systemic administration.
Phorbol ester modulation of active ion transport across the rabbit conjunctival epithelium
Experimental eye …, 1999
Protein kinase C (PKC) activation elicits diverse cell-type specific effects on key epithelial transporters. The present work examined the influence of phorbol esters, which are known activators of PKC isoenzymes, on the short-circuit current (I sc), a direct measure of net transcellular electrolyte transport, of the rabbit conjunctiva. In this preparation, the I sc measures a Na +-dependent, bumetanide-inhibitable Cl − transport in the basolateral-to-apical direction plus an amiloride-resistant Na + absorptive process in the opposite direction. Additions of phorbol 12-myristate-13-acetate (PMA) to the basolateral bathing media did not affect the transepithelial electrical parameters ; but its introduction to the apical bath at 1 and 10 µ elicited a transient ($ 2 min duration) I sc spike followed by a sustained reduction relative to the control level. Such PMA-elicited I sc reductions were from 14n0p2n0 to 3n1p0n8 µA cm −# (p...'s, n l 3) at 1 µ and from 16n5p1n9 to 4n6p0n7 µA cm −# (n l 22) at 10 µ. The former concentration failed to produce extensive I sc reductions in 3 other experiments. Similar results were obtained with phorbol 12,13-dibutyrate (PDBu). Its apical administration at 0n1 µ reduced the I sc from 18n5p4n1 to 7n8p2n0 (n l 3), and from 16n5p2n9 to 6n9p1n2 (n l 7) when introduced at 1 µ. The phorbol-evoked I sc reductions occurred without a simultaneous change in transepithelial resistance (R t). However, after about 15-20 min, R t gradually declined by about 25 %. In contrast to these results, treatment with a phorbol ester known not to activate PKC (4-α-PMA) did not affect the electrical parameters when added at 10 µ. PMA-and PDBu-evoked I sc reductions could be obtained with conjunctiva that were (1) pretreated with bumetanide, (2) bathed in Cl −-free media, and (3) pretreated with amphotericin B, changes consistent with a likely inhibition of the basolateral Na + \K + pump. Such I sc inhibitions were attenuated with conjunctiva pre-exposed to 1 µ staurosporine, a nonselective kinase inhibitor known to suppress PKC activity. Staurosporine, in itself, produced a rapid 26 % I sc inhibition (n l 15) along with a 17% R t increase upon its apical introduction. These electrical responses were less extensive in Cl −-free media and absent in amphotericin B-treated conjunctiva, suggesting the presence of a kinase-mediated regulation of apical channels for both Na + and Cl −. Overall, these results imply that in addition to previously demonstrated epinephrine-elicited, up-regulation of Cl − secretion, mechanisms may also exist, via PKC activation, to suppress Na + \K + pumping and consequently reduce transepithelial transport rates.