Hovhannes J. Gukasyan

Pharmacological modulation of fluid secretion in the pigmented rabbit conjunctiva.

We determined net fluid secretion rate across the pigmented rabbit conjunctiva in the presence and absence of pharmacological agents known to affect active Cl- secretion and Na+ absorption. Fluid flow across a freshly excised pigmented rabbit conjunctiva mounted between two Lucite half chambers was measured by a pair of capacitance probes in an enclosed cabinet maintained at 37 degrees C and a relative humidity of 70%. Fluid transport was also measured in the presence of compounds known to affect active Cl- secretion (cAMP, UTP, and ouabain), Na+ absorption (D-glucose), or under the Cl--free condition on both sides of the tissue. Net fluid secretion rate across the pigmented rabbit conjunctiva in the serosal-to-mucosal direction at baseline was 4.3+/-0.2 microl/hr/cm2 (mean +/- s.e.m.). Net fluid secretion rate was increased approximately two-fold by mucosally applied 1 mM 8-Br cAMP (8.4+/-0.4 microl/hr/cm2) and 10 microM UTP (9.8+/-0.6 microl/hr/cm2), but was abolished by either serosally applied 0.5 mM ouabain (0.3+/-0.1 microl/hr/cm2) or under the Cl--free conditions (0.06+/-0.04 microl/hr/cm2). Mucosal addition of 20 mM D-glucose decreased net fluid secretion rate to 1.0+/-0.5 microl/hr/cm2. In conclusion, the pigmented rabbit conjunctiva appears to secrete fluid secondary to active Cl- secretion. This net fluid secretion is subject to modulation by changes in active Cl- secretion rate and in mucosal fluid composition such as glucose concentration.

Correlation of In Vitro and In Vivo Kinetics of Nitric Oxide Donors in Ocular Tissues

In the eye, nitric oxide (NO) is involved in the regulation of intraocular pressure (IOP) and ocular blood flow. The main purpose of this study was to measure the kinetics of NO release from NO donors in ocular cells and tissues using in vivo and in vitro models and demonstrate the link between the kinetics of NO release with the functional effect, IOP. Nitric oxide release was measured in human ocular cells using a fluorescent dye, diaminofluorescein (DAF), following treatment with short-acting sodium nitroprusside (SNP) and longer-acting S-nitroso-N-acetylpenicillamine (SNAP) NO donors. Both SNP and SNAP were also administered topically to rabbits; IOP was measured and levels of NO and cGMP were assessed as biomarkers over a time course in the aqueous humor (AH) and iris/ciliary body (ICB). Time- and concentration-dependent increases in NO level were produced by SNP and SNAP in human ocular cells. Both NO and cGMP levels appeared to be elevated following treatment with the aforementioned NO donors in rabbit ocular tissues. Transient IOP lowering was accompanied with these biochemical estimations in rabbits, with time of maximal effect being shifted to the right for longer-acting SNAP as compared with short-acting SNP. In vitro and in vivo NO/cGMP assay results displayed a correlation between short- and longer-acting NO donors, discriminating their respective temporal actions in the eye. Due to their translatability, the in vitro DAF assay and in vivo NO fluorometric assay can therefore be potentially useful in screening novel NO donors with different temporal/kinetic profiles.

Metabolism and transport of purinergic receptor agonists in rabbit conjunctival epithelial cells.

The role of extracellular nucleotides as signaling molecules is well established in a number of biological systems. Their function in the regulation of ion1, 2 and fluid3 transport has been investigated in the pigmented rabbit conjunctiva where active Cl− secretion has been implicated as the driving force for fluid secretion.3 These agonists interact with two classes of receptors located on the cell membrane: P2X ligand-gated ion channels and P2Y G-protein-coupled receptors.4 Receptor mapping techniques have revealed the presence of P2Y type receptors in ocular epithelial cells.5

Glutathione and Its Transporters in Ocular Surface Defense

Glutathione (GSH) is an abundant antioxidant ubiquitous in nearly all cell types. Deficiency of GSH has been linked to ocular disease and viral infection. Other established vital roles of GSH include detoxification and immunoprotection. Endogenous GSH plays a protagonists role in safeguarding active transport processes compartmentalized at the interface between conjunctival mucosa and the tear film. Optimal electrokinetic transport across the conjunctival epithelium requires the mucosal presence of GSH. Glutathione is the most abundant known endogenous antioxidant molecule in tear fluid, mainly derived from conjunctival secretion. Conjunctival GSH transport, a major kinetic component of GSH turnover, occurs through multiple functionally distinct mechanisms. Cell membrane potential regulates conjunctival GSH efflux, while conjunctival GSH uptake requires extracellular Na(+). Significant modulation of GSH, its constituent amino acids, and functions of associated transporters occurs in the conjunctival epithelium with viral inflammatory disease. Topical conjunctival delivery of GSH, its metabolic precursors, or pharmacologic stimulation of endogenous conjunctival GSH secretion carry potential in alleviating viral-inflammatory conjunctivitis.

An Assessment of the Ocular Safety of Inactive Excipients Following Sub-Tenon Injection in Rabbits

PURPOSE This work characterized the safety and toleration of inactive excipients following sub-Tenon (ST) administration. METHODS Rabbits were anesthetized and eyes received an ST injection of the following test excipients: carboxy methylcellulose (CMC; low [90 kDa], mid [250 kDa], and high [700 kDa] molecular weight [MW], 0.25%-1.0% w/v), polysorbate 80 (0.02 and 0.2% w/v), polyethylene glycol 3350 (PEG; 0.2 and 1.0% w/v), poloxamer 188 (0.01 and 0.25% w/v), poloxamer 182 (2% w/v), benzyl alcohol (BA; 4% w/v), benzalkonium chloride (BAC; 0.02%, 0.04%, and 0.05% w/v), and methylcellulose (MC; 0.25% w/v). After a 1-week observation period for clinical signs of ocular tolerability, the animals were euthanized and eyes were collected for histologic examination. RESULTS The ocular tolerability of the tested excipients were ranked as follows from the innocuous to most deleterious: saline approximately PEG (1% w/v) approximately polysorbate 80 (0.2% w/v) > CMC (0.25% w/v, 90 kDa) > MC (0.25% w/v) approximately poloxomer 188 (0.25% w/v) approximately sodium citrate (pH 9) BAC (0.05% w/v) > CMC (0.5% w/v, 700 kDa) > poloxomer 182 (2% w/v) > BA (4% w/v). Clinical signs of ocular irritation were limited to redness and chemosis observed with most test excipients. The BA excipient also produced corneal opacity. Microscopic findings included histiocytic infiltration (BAC, BA, CMC, MC, and poloxamer 188), heterophilic inflammation (BA, CMC, and poloxamer 182), and edema (BAC, BA, CMC, and poloxamer 182) in episcleral tissue. The severity of the clinical and hisopathologic effects increased with the concentration of the test excipients administered. CONCLUSIONS This research has evaluated the safety profile of inactive excipients that may be used to formulate new chemical entities for the treatment of ocular disease following a ST injection.

Functional characterization and cloning of amino acid transporter B0,+ (ATB0,+) in primary cultured rat pneumocytes

Cationic amino acid transport in primary cultured rat pneumocytes exhibiting characteristics of alveolar epithelial type I‐like cells are described. Asymmetry and activator ion dependency of 3H‐L‐arginine uptake were characterized from the apical or basolateral fluid of pneumocytes grown on permeable support. Substrate specificity of transport was evaluated as a function of 3H‐L‐arginine uptake inhibition in the presence of other amino acids. Transepithelial transport studies estimated 3H‐L‐arginine flux in the apical‐to‐basolateral and basolateral‐to‐apical directions. Full length cDNA of rat amino acid transporter B0,+ (rATB0,+) was cloned and its relative expression level studied. Results indicate that uptake of 3H‐L‐arginine from apical fluid is dependent on Na+ and Cl−. Zwitterionic and cationic amino acids (excluding L‐proline and anionic amino acids) inhibited uptake of 3H‐L‐arginine from apical, but not basolateral incubation fluid. Apical‐to‐basolateral transepithelial flux of 3H‐L‐arginine was 20× higher than basolateral‐to‐apical transport. Kinetic studies of 3H‐L‐arginine uptake from apical fluid revealed maximal velocity (Vmax) and Michaelis–Menten constants (Kt) of 33.32 ± 2.12 pmol/mg protein/15 min and 0.50 ± 0.11 mM, respectively, in a cooperative process having a coupling ratio of 1.18 ± 0.16 with Na+ and 1.11 ± 0.13 with Cl−. Expression of rATB0,+ mRNA was identified by RT‐PCR and Northern analysis. Corresponding cloned 3.2 kb rATB0,+ cDNA sequence exhibits pronounced homology in deduced amino acid sequence to mouse (95% identity and 97% similarity) and human (89% identity and 95% similarity) ATB0,+ homologues. We conclude that rat pneumocytes express ATB0,+, which may partly contribute towards recovering cationic and neutral amino acids from alveolar luminal fluid. J. Cell. Physiol. 214: 645–654, 2008.

In Vivo Evaluation of 11β-Hydroxysteroid Dehydrogenase Activity in the Rabbit Eye

PURPOSE Steroids are used in a diverse range of conditions in clinical ophthalmology and one of the most significant complications is corticosteroid-induced glaucoma, which is characterized by an increase in intraocular pressure (IOP). 11beta-Hydroxysteroid dehydrogenase-1 (11beta-HSD1) is known to catalyze the interconversion of hormonally inactive cortisone to hormonally active cortisol and is widely expressed in the eye, particularly ciliary epithelium. Carbenoxolone (CBX), an 11beta-HSD1 inhibitor, has been shown to reduce IOP in healthy volunteers and patients with ocular hypertension (OHT). The purpose of this study was to: (1) develop an in vivo model for the assessment of cortisone to cortisol conversion in the eye, that is, 11beta-HSD1 activity and (2) assess the pharmacokinetic/pharmacodynamic relationship following topical treatment with 11beta-HSD1 inhibitors using an in vivo rabbit model. METHODS Potent and selective 11beta-HSD1 inhibitors were topically administered to the rabbit eye and exogenous cortisone to endogenous cortisol conversion in the eye was assessed in rabbits. Tissues were then evaluated for cortisone, cortisol, and 11beta-HSD1 inhibitor levels by LC/MS/MS. Concomitantly cortisol activity in ocular tissue samples was determined using a secondary mechanistic pLuc-GRE assay. RESULTS Topical treatment with potent and selective 11beta-HSD1 inhibitors resulted in complete inhibition in the conversion of cortisone to cortisol in the rabbit eye as well as decreased pLuc-GRE luciferase activity. The reduction of cortisone conversion was time- and dose-dependent as well as dependent on dosing volume (suggestive of increased spillover and washout with greater dosing volume). CONCLUSIONS In conclusion, topical delivery of 11beta-HSD1 inhibitors can reduce or inhibit the conversion of cortisone to cortisol in the eye, indicating that the rabbit eye possesses an active enzyme for glucocorticoid synthesis. Dosing concentration and volume play an important role in the pharmacokinetic and pharmacodynamic effects of topically delivering an 11beta-HSD1 inhibitor. The rabbit model is useful for mechanistically assessing the conversion of cortisone to cortisol in the eye.

Evaluation of miR-216a and miR-217 as Potential Biomarkers of Acute Exocrine Pancreatic Toxicity in Rats:

Detecting and monitoring exocrine pancreatic damage during nonclinical and clinical testing is challenging because classical biomarkers amylase and lipase have limited sensitivity and specificity. Novel biomarkers for drug-induced pancreatic injury are needed to improve safety assessment and reduce late-stage attrition rates. In a series of studies, miR-216a and miR-217 were evaluated as potential biomarkers of acute exocrine pancreatic toxicity in rats. Our results revealed that miR-216a and miR-217 were almost exclusively expressed in rat pancreas and that circulating miR-216a and miR-217 were significantly increased in rats following administration of established exocrine pancreatic toxicants caerulein (CL) and 1-cyano-2-hydroxy-3-butene (CHB) as well as in rats administered a proprietary molecule known to primarily affect the exocrine pancreas. Conversely, neither microRNA was increased in rats administered a proprietary molecule known to cause a lesion at the pancreatic endocrine–exocrine interface (EEI) or in rats administered an established renal toxicant. Compared with amylase and lipase, increases in miR-216a and miR-217 were of greater magnitude, persisted longer, and/or correlated better with microscopic findings within the exocrine pancreas. Our findings demonstrate that in rats, miR-216a and miR-217 are sensitive and specific biomarkers of acute exocrine pancreatic toxicity that may add value to the measurement of classical pancreatic biomarkers.

Oligopeptide Transport in Rat Lung Alveolar Epithelial Cells is Mediated by Pept2

ABSTRACTPurposeStudies were conducted in primary cultured rat alveolar epithelial cell monolayers to characterize peptide transporter expression and function.MethodsFreshly isolated rat lung alveolar epithelial cells were purified and cultured on permeable support with and without keratinocyte growth factor (KGF). Messenger RNA and protein expression of Pept1 and Pept2 in alveolar epithelial type I- and type II-like cell monolayers (±KGF, resp.) were examined by RT-PCR and Western blotting. 3H–Glycyl-sarcosine (3H–gly-sar) transmonolayer flux and intracellular accumulation were evaluated in both cell types.ResultsRT-PCR showed expression of Pept2, but not Pept1, mRNA in both cell types. Western blot analysis revealed presence of Pept2 protein in type II-like cells, and less in type I-like cells. Bi-directional transmonolayer 3H–gly-sar flux lacked asymmetry in transport in both types of cells. Uptake of 3H–gly-sar from apical fluid of type II-like cells was 7-fold greater than that from basolateral fluid, while no significant differences were observed from apical vs. basolateral fluid of type I-like cells.ConclusionsThis study confirms the absence of Pept1 from rat lung alveolar epithelium in vitro. Functional Pept2 expression in type II-like cell monolayers suggests its involvement in oligopeptide lung disposition, and offers rationale for therapeutic development of di/tripeptides, peptidomimetics employing pulmonary drug delivery.