Austin K. Mircheff

The pathology of dry eye: the interaction between the ocular surface and lacrimal glands.

BACKGROUND Most dry-eye symptoms result from an abnormal, nonlubricative ocular surface that increases shear forces under the eyelids and diminishes the ability of the ocular surface to respond to environmental challenges. This ocular-surface dysfunction may result from immunocompromise due to systemic autoimmune disease or may occur locally from a decrease in systemic androgen support to the lacrimal gland as seen in aging, most frequently in the menopausal female. HYPOTHESIS Components of the ocular surface (cornea, conjunctiva, accessory lacrimal glands, and meibomian glands), the main lacrimal gland, and interconnecting innervation act as a functional unit. When one portion is compromised, normal lacrimal support of the ocular surface is impaired. Resulting immune-based inflammation can lead to lacrimal gland and neural dysfunction. This progression yields the OS symptoms associated with dry eye. THERAPY Restoration of lacrimal function involves resolution of lymphocytic activation and inflammation. This has been demonstrated in the MRL/lpr mouse using systemic androgens or cyclosporine and in the dry-eye dog using topical cyclosporine. The efficacy of cyclosporine may be due to its immunomodulatory and antiinflammatory (phosphatase inhibitory capability) functions on the ocular surface, resulting in a normalization of nerve traffic. CONCLUSION Although the etiologies of dry eye are varied, common to all ocular-surface disease is an underlying cytokine/receptor-mediated inflammatory process. By treating this process, it may be possible to normalize the ocular surface/lacrimal neural reflex and facilitate ocular surface healing.

Calcium-activated endoplasmic reticulum stress as a major component of tumor cell death induced by 2,5-dimethyl-celecoxib, a non-coxib analogue of celecoxib

A drawback of extensive coxib use for antitumor purposes is the risk of life-threatening side effects that are thought to be a class effect and probably due to the resulting imbalance of eicosanoid levels. 2,5-Dimethyl-celecoxib (DMC) is a close structural analogue of the selective cyclooxygenase-2 inhibitor celecoxib that lacks cyclooxygenase-2–inhibitory function but that nonetheless is able to potently mimic the antitumor effects of celecoxib in vitro and in vivo. To further establish the potential usefulness of DMC as an anticancer agent, we compared DMC and various coxibs and nonsteroidal anti-inflammatory drugs with regard to their ability to stimulate the endoplasmic reticulum (ER) stress response (ESR) and subsequent apoptotic cell death. We show that DMC increases intracellular free calcium levels and potently triggers the ESR in various tumor cell lines, as indicated by transient inhibition of protein synthesis, activation of ER stress–associated proteins GRP78/BiP, CHOP/GADD153, and caspase-4, and subsequent tumor cell death. Small interfering RNA–mediated knockdown of the protective chaperone GRP78 further sensitizes tumor cells to killing by DMC, whereas inhibition of caspase-4 prevents drug-induced apoptosis. In comparison, celecoxib less potently replicates these effects of DMC, whereas none of the other tested coxibs (rofecoxib and valdecoxib) or traditional nonsteroidal anti-inflammatory drugs (flurbiprofen, indomethacin, and sulindac) trigger the ESR or cause apoptosis at comparable concentrations. The effects of DMC are not restricted to in vitro conditions, as this drug also generates ER stress in xenografted tumor cells in vivo, concomitant with increased apoptosis and reduced tumor growth. We propose that it might be worthwhile to further evaluate the potential of DMC as a non-coxib alternative to celecoxib for anticancer purposes. [Mol Cancer Ther 2007;6(4):1262–75]

A Unified Theory of the Role of the Ocular Surface in Dry Eye

It is our belief that the pathology of dry eye occurs when systemic androgen levels fall below the threshold necessary for support of secretory function and generation of an anti-inflammatory environment (Fig. 3). When this occurs, both the lacrimal gland and the ocular surface become irritated and inflamed, and they secrete cytokines that interfere with the normal neural connections that drive the tearing reflex. This leaves the lacrimal gland in an isolated condition, perhaps exacerbating atrophic alterations of the glandular tissue. These changes allow for antigen presentation at the surface of the lacrimal acinar cells and increase lymphocytic infiltration of the gland. A similar series of events may be occurring on the ocular surface. From this hypothesis we conclude: 1. The ocular surface, lacrimal gland, and interconnecting innervation act as an integrated servo-mechanism. 2. Once the lacrimal gland loses its androgen support, it is subject to immune/neurally mediated dysfunction. 3. The ocular surface is an appropriate target for dry eye therapeutics.

Androgen support of lacrimal gland function

The effects of dihydrotestosterone (DHT) (1 mg/kg) on biochemical parameters related to lacrimal secretion, basal tear flow rate, and pilocarpine-stimulated lacrimal gland fluid secretion, in mature ovariectomized rabbits were studied. The effects of the synthetic estrogen, diethylstilbestrol (DES) (100 μg/kg), on lacrimal gland biochemical parameters in normal mature female rabbits was also studied. Ovariectomy decreased the total serum levels of testosterone (T) by 88.5% and androstenedione by 35.9%, without changing the levels of dehydroepiandrosterone (DHEA) or its sulfate. Ovariectomy caused a significant regression of the lacrimal glands, decreasing total DNA by 35%, and total protein by 22%. DHT treatment of ovariectomized animals prevented lacrimal gland regression, increasing total gland DNA (31%) and total protein (18%). DHT treatment also increases Na+,K+-ATPase activity (29%) and ß-adrenergic receptor binding sites (23%) compared to the ovariectomized group. DHT increased pilocarpine stimulated lacrimal gland fluid secretion (13.26±1.47 μL/min) compared to the ovariectomized group (7.72±0.41 μL/min), but DHT treatment paradoxically decreased basal tear flow rate (1.02±0.04 μL/min) as compared to the ovariectomized rabbits (1.96±0.12 μL/min). DES decreased the total serum T from 59.33±10.54 pg/mL to 21.5±6.06 pg/mL. DES decreased total Na+,K+-ATPase by 12% and increased ß-adrenergic receptor binding sites by 83.3%. These results suggest that androgens play a major role in supporting lacrimal gland secretory function. Additionally, they suggest that estrogens may influence certain aspects of lacrimal functions, although it is not clear to what extent those actions are elicited directly or indirectly.

In vivo PTH provokes apical NHE3 and NaPi2 redistribution and Na-K-ATPase inhibition

The aim of this study was to test the hypothesis that in vivo administration of parathyroid hormone (PTH) provokes diuresis/natriuresis through redistribution of proximal tubule apical sodium cotransporters (NHE3 and NaPi2) to internal stores and inhibition of basolateral Na-K-ATPase activity and to determine whether the same cellular signals drive the changes in apical and basolateral transporters. PTH-(1-34) (20 U), which couples to adenylate cyclase (AC), phospholipase C (PLC), and phospholipase A2 (PLA2), or [Nle8,18,Tyr34]PTH-(3-34) (10 U), which couples to PLC and PLA2 but not AC, were given to anesthetized rats as an intravenous bolus followed by low-dose infusion (1 U. kg-1. min-1 for 1 h). Renal cortex membranes were fractionated on sorbitol density gradients. PTH-(1-34) increased urinary cAMP excretion 3-fold, urine output (V) 2.0 +/- 0.1-fold, and lithium clearance (CLi) 2.8 +/- 0.3-fold. With this diuresis/natriuresis, 25% of NHE3 and 18% of NaPi2 immunoreactivity redistributed from apical membranes to higher density fractions containing intracellular membrane markers, and basolateral Na-K-ATPase activity decreased 25%. [Nle8,18,Tyr34]PTH-(3-34) failed to increase V or CLi or to provoke redistribution of NHE3 or NaPi2, but it did inhibit Na-K-ATPase activity 25%. We conclude that in vivo PTH stimulates natriuresis/diuresis associated with internalization of apical NHE3 and NaPi2 and inhibition of Na-K-ATPase activity, that cAMP-protein kinase A stimulation is necessary for the natriuresis/diuresis and NHE3 and NaPi2 internalization, and that Na-K-ATPase inhibition is not secondary to depressed apical Na+ transport.The aim of this study was to test the hypothesis that in vivo administration of parathyroid hormone (PTH) provokes diuresis/natriuresis through redistribution of proximal tubule apical sodium cotransporters (NHE3 and NaPi2) to internal stores and inhibition of basolateral Na-K-ATPase activity and to determine whether the same cellular signals drive the changes in apical and basolateral transporters. PTH-(1-34) (20 U), which couples to adenylate cyclase (AC), phospholipase C (PLC), and phospholipase A2(PLA2), or [Nle8,18,Tyr34]PTH-(3-34) (10 U), which couples to PLC and PLA2 but not AC, were given to anesthetized rats as an intravenous bolus followed by low-dose infusion (1 U ⋅ kg-1 ⋅ min-1for 1 h). Renal cortex membranes were fractionated on sorbitol density gradients. PTH-(1-34) increased urinary cAMP excretion 3-fold, urine output (V) 2.0 ± 0.1-fold, and lithium clearance (CLi) 2.8 ± 0.3-fold. With this diuresis/natriuresis, 25% of NHE3 and 18% of NaPi2 immunoreactivity redistributed from apical membranes to higher density fractions containing intracellular membrane markers, and basolateral Na-K-ATPase activity decreased 25%. [Nle8,18,Tyr34]PTH-(3-34) failed to increase V or CLi or to provoke redistribution of NHE3 or NaPi2, but it did inhibit Na-K-ATPase activity 25%. We conclude that in vivo PTH stimulates natriuresis/diuresis associated with internalization of apical NHE3 and NaPi2 and inhibition of Na-K-ATPase activity, that cAMP-protein kinase A stimulation is necessary for the natriuresis/diuresis and NHE3 and NaPi2 internalization, and that Na-K-ATPase inhibition is not secondary to depressed apical Na+ transport.

Reversible effects of acute hypertension on proximal tubule sodium transporters

Acute hypertension provokes a rapid decrease in proximal tubule sodium reabsorption with a decrease in basolateral membrane sodium-potassium-ATPase activity and an increase in the density of membranes containing apical membrane sodium/hydrogen exchangers (NHE3) [Y. Zhang, A. K. Mircheff, C. B. Hensley, C. E. Magyar, D. G. Warnock, R. Chambrey, K.-P. Yip, D. J. Marsh, N.-H. Holstein-Rathlou, and A. A. McDonough. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F1004-F1014, 1996]. To determine the reversibility and specificity of these responses, rats were subjected to 1) elevation of blood pressure (BP) of 50 mmHg for 5 min, 2) restoration of normotension after the first protocol, or 3) sham operation. Systolic hypertension increased urine output and endogenous lithium clearance three- to fivefold within 5 min, but these returned to basal levels only 15 min after BP was restored. Renal cortex lysate was fractionated on sorbitol gradients. Basolateral membrane sodium-potassium-ATPase activity (but not subunit immunoreactivity) decreased one-third to one-half after BP was elevated and recovered after BP was normalized. After BP was elevated, 55% of the apical NHE3 immunoreactivity, smaller fractions of sodium-phosphate cotransporter immunoreactivity, and apical alkaline phosphatase and dipeptidyl-peptidase redistributed to membranes of higher density enriched in markers of the intermicrovillar cleft (megalin) and endosomes (Rab 4 and Rab 5), whereas density distributions of the apical cytoskeleton protein villin were unaltered. After 20 min of normalized BP, all the NHE3 and smaller fractions of the other apical membrane proteins returned to their original distributions. These findings suggest that the dynamic regulation of proximal tubule sodium transport by acute changes in BP may be mediated by rapid reversible regulation of sodium pump activity and relocation of apical sodium transporters.

Delineation of sodium-stimulated amino acid transport pathways in rabbit kidney brush border vesicles

SummaryWe have confirmed previous demonstrations of sodium gradient-stimulated transport ofl-alanine, phenylalanine, proline, and β-alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these14C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability ofd-glucose to inhibit tracer uptake.l-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the14C-l-alanine uptake could be inhibited by proline and β-alanine, while lysine and MeAIB were no more effective thand-glucose. Roughly 50% of the14C-phenylalanine uptake could be inhibited by proline and β-alanine; lysine was as effective as proline and β-alanine, and the effects of pairs of these amino acids at 50mm each were not cumulative. MeAIB was no more effective thand-glucose. We conclude that three pathways mediate the uptake of neutral,l, α-amino acids. One system is inaccessible to lysine, proline, and β-alanine. The second system carries a major fraction of thel-alanine flux; it is sensitive to proline and β-alanine, but not to lysine. The third system carries half the14C-phenylalanine flux, and it is sensitive to proline, lysine, and β-alanine. Since the neutral,l, α-amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classicalA system, and since all of thel-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classicalASC system.l-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective thand-glucose in inhibiting lysine uptake, while proline and β-alanine appear to inhibit a component of the lysine flux. We conclude that the14C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, β-alanine, andl-alanine, and one which is inhibited byl-alanine and phenylalanine but inaccessible to proline, β-alanine, and MeAIB. Fluxes of14C-proline and14C-MeAIB are completely inhibited byl-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit14C-β-alanine uptake. However, β-alanine inhibits only 38% of the14C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports β-alanine.

Lacrimal fluid and electrolyte secretion: A review

Lacrimal gland fluid is an important component of the precorneal tear film. The rate of lacrimal gland fluid secretion is controlled primarily by parasympathetic innervation, and it is, apparently, modulated by sympathetic innervation. Lacrimal gland fluid is produced in two stages, secretion of a primary fluid which resembles an isotonic ultrafiltrate of plasma in the acinus-early intercalated duct region, and secretion of a KCl-rich fluid in subsequent ductal elements. Little is known about the electrolyte transport mechanisms of the ductal epithelia. Recent work using a variety of techniques, including tracer flux measurements, intracellular electrical recording, intracellular ion activity measurements, patch clamping, and analytical subcellular fractionation, supports a model for transcellular Cl-secretion in the acinus which involves Cl--selective channels in the apical plasma membrane and an array of Na+/H+ antiporters, Cl-/HCO3-antiporters, K+ channels, and Na,K-ATPase in the basal-lateral plasma membrane.

Lacrimal histopathology and ocular surface disease in a rabbit model of autoimmune dacryoadenitis.

Purpose. To study the effects of induced autoimmune dacryoadenitis on lacrimal gland function, histopathology, and ocular surface disease in a rabbit model. Methods. One lacrimal gland was surgically excised from each experimental rabbit, and epithelial cells were purified, cultured, irradiated, and then cocultured with autologous peripheral blood lymphocytes (PBLs) for 5 days. Autoimmune dacryoadenitis was induced by injecting the autologous mixed cell reactions (AMCRs) into the rabbits remaining lacrimal gland. Normal rabbits and rabbits with both lacrimal glands injected with nonstimulated PBLs were examined as controls. Eyes were evaluated biweekly for 8 weeks by slit-lamp biomicroscopy, Schirmer testing, tear break-up time measurement, and rose bengal examination. Sections of lacrimal glands removed at 8 weeks post-operation were immunostained using antibodies against rabbit class II major histocompatibility complex molecule (MHC-II), CD4, CD8, CD18, and rabbit thymic lymphocyte antigen (RTLA). Relative numbers of positively stained cells were quantified with a ChromaVision image analysis system. Results. During an 8-week period, a continuous decrease in tear production and stability, accompanied by a continuous increase in rose bengal staining, occurred in eyes in which AMCR-PBL had been injected into the ipsilateral lacrimal glands. Similar, though generally less severe, changes occurred in eyes contralateral to the AMCR-PBL-injected eyes. No obvious changes by 8 weeks in these parameters were found in eyes in which the lacrimal glands had been injected with nonstimulated PBLs or in the lacrimal gland-excised eyes contralateral to normal eyes. Interstitial cells in normal lacrimal glands expressed CD18 and RTLA antigens, but few expressed CD4, CD8, or MHC-II. Focal mononuclear cell infiltrates were only found in lacrimal glands from animals with induced autoimmune dacryoadenitis. These cells were predominantly positive for CD4 (7.3-fold increase), RTLA (7.8-fold increase), or CD18 (42-fold increase). MHC-II expression in interstitial and ductal epithelial cells was also significantly greater in these animals than in control animals. The mononuclear cell infiltrates were frequently found enveloping venules, some of which appeared to be high endothelial cell venules. The ductal epithelium also contained CD4 and CD8 immunopositivity, within the epithelium, at the lumenal surface, or surrounding the ducts. Occasionally CD4 and CD8 immunopositive cells could be identified within the acinar lumens. Conclusions. Injection of activated PBLs (i.e., AMCR-PBLs) in the lacrimal gland induces autoimmune dacryoadenitis with immunopathologic features similar to those of Sjögrens syndrome. The lacrimal immunopathology is accompanied by typical clinical manifestations of dry eye syndrome. The persistent significant dry eye does not appear to result just from failure of the diseased gland but from a more general dysfunction of the surface secretory tissues.

Cytoplasmic dynein participates in apically targeted stimulated secretory traffic in primary rabbit lacrimal acinar epithelial cells

A major function of the acinar cells of the lacrimal gland is the production and stimulated release of tear proteins into ocular surface fluid. We investigate the participation of cytoplasmic dynein in carbachol-stimulated traffic to the apical plasma membrane in primary rabbit lacrimal acinar epithelial cells. Confocal fluorescence microscopy revealed a major carbachol-induced, microtubule-dependent recruitment of cytoplasmic dynein and the dynactin complex into the subapical region. Colocalization studies, sorbitol density gradient/phase partitioning analysis and microtubule-affinity purification of membranes showed that some dynein and dynactin complex were associated with VAMP2-enriched membranes. Adenovirus-mediated overexpression of p50/dynamitin inhibited the recruitment and colocalization of dynein, the dynactin complex and VAMP2 in the subapical region. Nocodazole treatment and p50/dynamitin overexpression also depleted subapical stores of rab3D in resting acini, suggesting that dynein activity was also involved in maintenance of rab3D-enriched secretory vesicles. These data implicate cytoplasmic dynein in stimulated traffic to the apical plasma membrane in these secretory epithelial cells.