Regulation of L-cystine transport and intracellular GSH level by a nitric oxide donor in primary cultured rabbit conjunctival epithelial cell layers

PURPOSE: Metabolism and transport of cysteine are critical for maintenance of the intracellular glutathione (GSH) level. In this study, transport mechanisms of L-cystine and regulation of GSH biosynthesis in the absence or presence of NO-induced oxidant stress were investigated in primary cultured rabbit conjunctival epithelial cells (RCECs). METHODS: RCECs were grown in membrane filters to exhibit tight barrier properties. Uptake and transepithelial transport of L-cystine were determined in the presence or absence of extracellular Na(+). Uptake was determined at 10 minutes after (14)C-L-cystine instillation into apical (a) or basolateral (b) bathing fluid. The effect of nitric oxide (NO) on L-cystine uptake, cellular GSH level, and expression level of two subunits of the rate-limiting enzyme gamma-glutamylcysteine synthetase (GCS) was examined after a 24-hour incubation of primary cultured RCECs with an NO donor, S-nitroso-N-acetylpenicillamine (SNAP; N-acetyl-3-(nitrosothio)-D-valine. RESULTS: Cellular uptake of L-cystine by RCECs occurred through both Na(+)-dependent and -independent mechanisms. Uptake from apical fluid was higher than that from basolateral fluid, except for the highest concentration of L-cystine tested (200 microM). Transepithelial permeability (P(app)) of L-cystine (at 2.5 microM) was three times higher in the a-to-b direction than in the b-to-a direction in the presence of Na(+), whereas the reverse was true in the absence of Na(+). Na(+)-dependent L-cystine uptake from apical fluid was significantly elevated in primary cultured RCECs treated for 24 hours with various concentrations (0.1-2.0 mM) of SNAP, with maximum uptake observed at 1 mM. A similar pattern of SNAP-induced increase of Na(+)-independent L-cystine uptake from apical fluid was observed, whereas no significant difference was observed for basolateral uptake. Concomitantly, a significant elevation of intracellular GSH (up to fivefold versus the control) was recorded, with the highest increase occurring at 0.1 to 0.25 mM SNAP. A parallel increase in the expression levels of both catalytic and regulatory subunits of GCS was observed by Western blot analysis of lysates from RCECs treated with 0.25 mM SNAP for 24 hours. CONCLUSIONS: L-Cystine is transported by both Na(+)-dependent and -independent amino acid transport systems in RCECs. At low substrate concentrations, L-cystine uptake was higher from apical than basolateral fluid. Permeability studies indicated net absorption of L-cystine across RCECs. SNAP caused significant increases in both L-cystine uptake and intracellular GSH level, which occurred concomitantly with elevation of both catalytic and regulatory subunits of GCS. Understanding sulfur amino acid precursor-dependent cellular mechanisms of GSH homeostasis would be of value in devising GSH-based treatment for conjunctival or other ocular disorders.     

2nd Ophthalmic Drug Development and Delivery Summit

The Second Annual Ophthalmic Drug Development and Delivery Summit was held on 19 - 20 September 2006 in San Diego, CA, US. The 2-day symposium, having a highly focused theme, was packed with cutting-edge science, insightful overviews and networking opportunities. With a total of 11 recognized specialists presenting reviews and recent results in the advancement of ocular drug development and delivery, the invited expert speaking faculty presented the latest preclinical and clinical developments in novel ophthalmic therapies and drug delivery technology. The talks included various case studies from primary investigators and pharmaceutical companies touching upon key topics: updates on current clinical trials, study design issues, sustained delivery to the eye, views of the vitreous space as a drug reservoir, new developments in dry and wet age-related macular degeneration and diabetic retinopathy, formulation for optimal drug delivery, differences and similarities in developing drugs for the eye compared with other targets, pharmacokinetics, novel ocular delivery methods and devices, delivery of proteins and peptides, focal drug delivery, non-invasive drug delivery to the eye, neuroprotection challenges, in vitro and in vivo models for glaucoma and angiogenesis for early efficacy estimation, and toxicology. Overall, the 2-day annual symposium continues to grow as an efficient platform for fostering discussion on a range of scientific topics and challenges and avenues for building collaborative partnerships in ophthalmic drug development.     

A Phase II Study of Pembrolizumab in EGFR-Mutant,PD-L1+, Tyrosine Kinase Inhibitor Naïve Patients With Advanced NSCLC

Background

Despite the significant antitumor activity of pembrolizumab in NSCLC, clinical benefit has been less frequently observed in patients whose tumors harbor EGFR mutations compared to EGFR wild-type patients. Our single-center experience on the KEYNOTE-001 trial suggested that pembrolizumab-treated EGFR-mutant patients, who were tyrosine kinase inhibitor (TKI) naïve, had superior clinical outcomes to those previously treated with a TKI. As TKI naïve EGFR-mutants have generally been excluded from pembrolizumab studies, data to guide treatment decisions in this patient population is lacking, particularly in patients with programmed death ligand 1 (PD-L1) expression ≥50%.

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

Purpose

Studies were conducted in primary cultured rat alveolar epithelial cell monolayers to characterize peptide transporter expression and function.

Methods

Freshly 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.

Results

RT-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.

Conclusions

This 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.

     

l‐Carnosine: multifunctional dipeptide buffer for sustained‐duration topical ophthalmic formulations

Objectives The use of L‐carnosine as an excipient in topical ophthalmic formulations containing gellan gum, a carbohydrate polymer with in‐situ gelling properties upon mixing with mammalian tear fluid, was developed as a novel platform to extend precorneal duration. Specific utilisation of L‐carnosine as a buffer in gellan gum carrying vehicles was characterised. Methods Buffer capacity was evaluated using 7.5, 13.3, and 44.2 mM L‐carnosine in a pH range of 5.5–7.5. Accelerated chemical stability was determined by HPLC at L‐carnosine concentrations of 5–100 mM. Combinations of 7.5 mM L‐carnosine with 0.06–0.6% (w/v) gellan gum were characterised rheologically. L‐Carnosine‐buffered solutions of gellan gum were tested for acute topical ocular tolerance in vivo in pigmented rabbits. A unique formulation combining timolol (which lowers intraocular pressure) in L‐carnosine‐buffered gellan gum was compared with Timoptic‐XE in normotensive dogs. Key findings L‐Carnosine exhibited optimal pharmaceutical characteristics for use as a buffer in chronically administered topical ocular formulations. Enhancement trends were observed in solution‐to‐gel transition of L‐carnosine‐buffered vehicles containing gellan gum vs comparators. Topical tolerability of L‐carnosine‐buffered gellan gum formulations and lowering of intraocular pressure were equivalent with timolol and Timoptic‐XE. Conclusions Functional synergy between excipients in gellan gum formulations buffered with L‐carnosine has potential for topical ocular dosage forms with sustained precorneal residence.     

Effect of adriamycin and its complexes with transition metals on induction of immune response of human lymphocytes in culture

Effect of adriamycin on effector cells of humoral and cell immune response can be enhanced or supressed by its complexation with Fe³⁺, Cu²⁺ and Co²⁺. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 7, pp. 60–62, July, 1999     

Specialized protective role of mucosal glutathione in pigmented rabbit conjunctiva

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.     

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 protagonist's 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.