Biomedical subjects

A K Mircheff

Publications and source records attributed to A K Mircheff.

At least 37 records · Page 2Linked to original sources

PubMed1998

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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Acute Disease↗

PubMed1997

Cholinergic stimulation of lacrimal acinar cells promotes redistribution of membrane-associated kinesin and the secretory protein, beta-hexosaminidase, and increases kinesin motor activity.

The role of the microtubule-based motor, kinesin, in membrane trafficking has been investigated in resting and stimulated acinar cells from rabbit lacrimal gland, a cholinergically controlled secretory tissue. Microtubule-dependent motors from extracts of control and carbachol-treated acini were isolated by microtubule-affinity purification and their activity was determined using a video-enhanced differential interference contrast microscopy assay for microtubule gliding. The observation that carbachol treatment resulted in a 2.2-fold stimulation of the frequency of GTP-dependent microtubule gliding in fractions isolated by microtubule-affinity purification and GTP release suggested that kinesin was a target of carbachol-induced stimulation. Resolution of membranes from resting cells by fractionation on a sorbitol density gradient followed by partitioning analysis in a dextran-polyethyleneglycol two-phase system revealed that membrane-associated kinesin codistributed with Golgi-derived membranes, a post-Golgi secretory compartment designated Hex1, membranes from a trans Golgi network-like compartment, endoplasmic reticulum and a group of putative lysosomal membranes containing cathepsin B. Comparable fractionation of carbachol-treated acini showed that stimulation caused redistributions of membrane-associated kinesin, the secretory enzyme beta-hexosaminidase, and galactosyltransferase that appeared to reflect both a reorganization within the Golgi complex and a return of material to the Golgi complex from the secretory pathway. Our findings that carbachol promotes activation of lacrimal acinar kinesin as well as major shifts in kinesin-membrane association within the secretory pathway suggests that kinesin plays a major role in secretory vesicle assembly, apical secretion, and/or secretory vesicle membrane recycling in the lacrimal gland.

Animals↗

PubMed1997

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 micrograms/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) of 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 beta-adrenergic receptor binding sites (23%) compared to the ovariectomized group. DHT increased pilocarpine stimulated lacrimal gland fluid secretion (13.26 +/- 1.47 microL/min) compared to the ovariectomized group (7.72 +/- 0.41 microL/min), but DHT treatment paradoxically decreased basal tear flow rate (1.02 +/- 0.04 microL/min) as compared to the ovariectomized rabbits (1.96 +/- 0.12 microL/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 beta-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.

Acid Phosphatase↗

PubMed1997

Adenovirus infection of the cornea causes histopathologic changes in the lacrimal gland.

PURPOSE: To explore the effects on the lacrimal gland of adenovirus infection of the cornea. METHODS: Rabbit corneas were inoculated with human adenoviruses Ad5, Ad14, or a rabbit adapted form of Ad 5, and in some instances booster inoculations were given. Sections of lacrimal glands removed 21-59 days post-inoculation were immunostained using antibodies against rabbit Class I and Class II MHC molecules, CD4, CD8, CD18, and rabbit thymic lymphocyte antigen (RTLA). Relative numbers of positively stained cells were quantified with a Metamorph image analysis system. RESULTS: RTLA and CD18 antigens were expressed on many interstitial cells in the normal lacrimal gland, but few expressed CD4 or CD8. The number of RTLA+ cells increased by 60-100% after inoculation of Ad5 and after boosting, and CD18+ cells increased from 33-100% after inoculation of Ad5 and after boosting. Booster inoculations also caused focal lymphocytic infiltration. MHC Class I was expressed on interstitial cells and duct epithelium, but not acinar cells, and there was no detectable difference after viral infection. In controls, MHC Class II was localized to a population of interstitial cells and a few acinar cells. A single inoculation of the Ad5 virus did not result in an increase in the total number of MHC Class II-positive cells at 21 days, but inoculation with the rabbit-adapted Ad 5 and booster inoculations caused a 30% increase. CONCLUSIONS: Ad5 and rabbit-adapted Ad5 infection of the cornea induce lymphocytic infiltration in the lacrimal gland, and the effect is enhanced by boosting. There is also an increase in expression of MHC Class II after inoculation with rabbit-adapted Ad5 and with booster inoculations.

Adenoviridae↗

PubMed1996

Evidence of sodium-dependent glucose transport in human erythroleukemia cells.

Sodium-dependent transport of D-glucose has been reported only in epithelial cells of small intestine and kidney, and well-differentiated tumors thereof. We observed a two-fold decrease (p < 0.05) in the intracellular distribution volume (Vic, defined as steady-state intracellular uptake divided by extracellular concentration) of the non-metabolized D-glucose analog 3-O-methylglucose (3-O-MG) when logarithmically growing K562 cells (an anaplastic human erythroleukemia) were incubated 3 h in choline-substituted, phosphate buffered saline (PBS) rather than Na+ PBS, each containing a glucose concentration ([Glu]) of 5.6 mM. Electromechanically measured cellular volume Vc differed < 10% between the different media. In Na+ PBS, Vic (3-O-MG) was approximately twice Vc and declined progressively when [Glu] was reduced to 2.8 and 0.1 mM. We conclude that, in a balanced salt medium containing glucose as the only energy source, K562 cells express a concentrative mechanism having characteristics consistent with Na(+)-dependent transport of glucose.

3-O-Methylglucose↗

PubMed1996

Na-K-ATPase in lacrimal gland acinar cell endosomal system: correcting a case of mistaken identity.

Na-K-ATPase is associated with a variety of membrane populations in lacrimal acinar cells. Acinus-like structures formed by rabbit acinar cells in primary culture were incubated with horseradish peroxidase (HRP) to label basolateral and endosomal membranes and then analyzed by electron microscopy cytochemistry with the 3-3'-diaminobenzidine reaction or by fractionation and measurement of marker catalytic activities or immunoreactivities. HRP adsorbed to basolateral membranes at 4 degrees C. Fractionation showed it associated with low-density membranes enriched in acid phosphatase and TGN38 but containing only minor amounts of Na-K-ATPase. Cells internalized HRP to cytoplasmic vesicles, Golgi structures, and lysosomes at 37 degrees C. The major endosomal compartment revealed by fractionation coincided with major peaks of Na-K-ATPase and Rab6 and secondary peaks of galactosyltransferase and gamma-adaptin. Carbachol (10 microM) increased lysosomal and Golgi labeling. Thus most of the Na-K-ATPase is located in the basolateral membrane-oriented endosomal system, concentrated in a compartment possibly related to the trans-Golgi network. Constitutive and stimulation-accelerated traffic to and from this compartment may serve several exocrine cell functions.

Animals↗

PubMed1996

Rapid redistribution and inhibition of renal sodium transporters during acute pressure natriuresis.

Acute arterial hypertension provokes a rapid decrease in proximal tubule (PT) Na+ reabsorption, increasing flow to the macula densa, the signal for tubuloglomerular feedback. We tested the hypothesis, in rats, that Na+ transport is decreased due to rapid redistribution of apical Na+/H+ exchangers and basolateral Na+ pumps to internal membranes. Arterial pressure was increased 50 mmHg by constricting various arteries. We also tested whether transporter internalization occurred when PT Na+ reabsorption was inhibited with the carbonic anhydrase inhibitor benzolamide. Five minutes after initiating either natriuretic stimuli, cortex was removed, and membranes were fractionated by density gradient centrifugation. Urine output and endogenous lithium clearance increased threefold in response to either stimuli. Acute hypertension provoked a redistribution of apical Na+/H+ exchanger NHE3, alkaline phosphatase, and dipeptidyl peptidase IV to higher density membranes enriched in the intracellular membrane markers. Basolateral membrane Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity decreased 50%, 25-30% of the alpha 1-and beta 1-subunits redistributed to higher density membranes, and the remainder is attributed to decreased activity of the transporters. Benzolamide did not alter Na+ transporter activity or distribution, implying that decreasing apical Na+ uptake does not initiate redistribution or inhibition of basolateral Na(+)-K(+)-ATPase. We conclude that PT natriuresis provoked by acute arterial pressure is mediated by both endocytic removal of apical Na+/H+ exchangers and basolateral Na+ pumps as well as decreased total Na+ pump activity.

Acute Disease↗

PubMed1996

Protein phosphorylation in Golgi, endosomal, and endoplasmic reticulum membrane fractions of lacrimal gland.

Ca2+/calmodulin- and cAMP-dependent protein kinase activities were characterized in two subcellular membrane samples. Membranes from rat lacrimal gland were isolated by differential and density gradient centrifugation into six density windows. The present study focused on membranes from density windows III and V which contain mixtures of apical, Golgi, endosomal, and endoplasmic reticulum membranes in different proportions. Phosphorylation of membrane proteins was measured by incubating the samples in [g-32P]ATP and separating the proteins by discontinuous SDS-PAGE followed by autoradiography. The amount of phosphate incorporated into specific peptide bands was quantified by densitometry. Ca2+/calmodulin-dependent protein kinase phosphorylated a 52,000 MW peptide in membranes from both density windows with a maximal increase from 0.3 to 66 microM free Ca2+. Trifluoperazine and promethazine, two inhibitors of Ca2+/calmodulin-dependent protein kinases, inhibited this phosphorylation. cAMP-dependent protein kinase phosphorylated a 22,000 MW peptide and a 91,000 MW peptide which were present in membranes from density window III only. We conclude that a Ca2+/calmodulin-dependent protein kinase activity is present in membranes from both density window III and V whereas a cAMP-dependent protein kinase activity is present only in membranes from density window III.

Animals↗

PubMed1996

Endothelin protein expression in tear glands of the rabbit.

PURPOSE: Endothelin-1 (ET-1) has been found to accelerate rabbit corneal epithelial wound healing in vivo and proliferation of rabbit and bovine epithelial cells in vitro. We have now determined that endothelin-like immunoreactivity (ET-LI) is present in the tear fluid of New Zealand white rabbits, and we have addressed the question of whether it is produced by the tear glands (lacrimal gland and Harderian gland). METHODS: Tears were obtained with capillary pipettes and lacrimal gland fluid was collected by cannulation. ET-LI was determined with a sandwich-enzyme immunoassay. The reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to determine if there are gene transcripts for prepro ET-1 in tear glands. ET-LI in the tear glands was evaluated using immunohistochemistry. RESULTS: The tears contained 13.85 +/- 3.94 pg/ml (mean +/- SEM, n = 6) ET-LI and the lacrimal gland fluid had 24.90 +/- 6.00 pg/ml (n = 6). The plasma contained 0.89 +/- 0.20 pg/ml (n = 6). Gene transcripts were identified for prepro ET-1 in the lacrimal and Harderian glands. Specific staining for ET-LI was found in the epithelial cells of the ducts of the lacrimal gland and the acinar cells of the Harderian gland. CONCLUSIONS: The higher concentrations of ET-LI in the tears and the lacrimal gland fluid than in plasma along with prepro ET-1 gene transcripts in the tear glands suggest that they secrete ET-1.

Animals↗

PubMed1995

Fluid phase endocytosis by isolated rabbit lacrimal gland acinar cells.

It is well known that lacrimal gland acinar cells retrieve secretory vesicle membrane constituents from their apical plasma membranes after stimulated exocytosis of secretory proteins. There have also been indications of a recycling traffic involving the basal-lateral plasma membranes. In an effort to document this traffic, determine how it is regulated, and discern whether it involves more than one intracellular compartment, we studied internalization of the fluid phase marker, Lucifer Yellow, and its relationship to protein release in acinar cells isolated from rabbit lacrimal glands. Loading of intracellular vesicles was apparent with fluoresence microscopy. Stimulation with carbachol increased both the rate of internalization and the intracellular volume equilibrating with extracellular fluid, suggesting the loading of two compartments. A carbachol concentration of 10 microM increased uptake by 80% during 20-min incubations at 37 degrees C. Increasing the carbachol concentration to 1 mM reduced the response by 50%, and it appeared to do so by decreasing the intracellular volume accessible to extracellular fluid, rather than the rate of endocytosis. Carbachol affected protein release differently, increasing it by 50% at 10 microM and 80% at 1 mM. Acceleration of endocytosis by 10 microM carbachol was transient, becoming negligible after 60 min. Vasoactive intestinal peptide (VIP) and isoproterenol increased internalization 35% and 25% respectively; neither reduced uptake at the highest concentrations tested; and only isoproterenol significantly affected protein secretion. Combinations of VIP and carbachol exerted synergistic effects on both fluid phase internalization and protein release. Steady-state uptake at 18 degrees C in the presence of 10 microM carbachol was equal to uptake at 37 degrees C in the absence of carbachol, suggesting a temperature block in the pathway to at least one endocytic compartment. Decreasing the temperature to 18 degrees C eliminated the inhibitory action of excessive carbachol, suggesting that the compartment whose loading was impaired by excessive carbachol was positioned distal to the temperature block. Carbachol accelerated release of marker from preloaded cells, indicating that it stimulated recycling between the plasma membranes and endocytic compartments. This effect was maximal at a concentration of 10 microM and unchanged with increasing concentrations. In accord with the hypothesis that traffic into and out of a certain compartment was particularly dependent on stimulation, a fraction of the marker taken up by optimally stimulated cells at 37 degrees C was retained unless carbachol or VIP was present in the efflux medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Hypophysectomy-induced regression of female rat lacrimal glands: partial restoration and maintenance by dihydrotestosterone and prolactin.

PURPOSE: Previous studies have implicated androgens and one or more as yet unknown pituitary or pituitary-dependent factors in the regulation of certain lacrimal gland functions. Many observations suggest that prolactin (PRL) might well be one of these factors. This study was designed to determine the effect of hypophysectomy on biochemical markers of exorbital lacrimal gland secretory capacity and to determine the extent to which dihydrotestosterone (DHT) and prolactin reverse these changes. METHODS: Female rats were hypophysectomized and, 5 days later, were treated for 2 days with DHT (0.25 or 1 mg/kg), PRL (1 or 5 mg/kg), combinations of the low or high doses of DHT and PRL, or vehicle only. The animals were killed, and crude membrane fractions were isolated from their lacrimal glands. An untreated group served as control. RESULTS: Lacrimal glands atrophied rapidly after hypophysectomy, losing 40% of their total and membrane-associated protein and 50% of their total DNA within 5 days. Total Na+,K(+)-ATPase and acid phosphatase activities and beta-adrenergic receptor number were decreased by half, whereas alkaline phosphatase activity and muscarinic cholinergic receptor number were reduced by 25% to 30%. DHT treatment increased total DNA above control values; it partially restored the amount of protein in the gland, the Na+,K(+)-ATPase and acid phosphatase activities, and the beta-adrenergic receptor number; and it fully restored the alkaline phosphatase activity. Prolactin treatment partially restored the amount of protein in the gland and the Na+,K(+)-ATPase activity; it fully restored the alkaline phosphatase activity and cholinergic receptor number; but it had no effect on the acid phosphatase activity or the beta-adrenergic receptor number. The high dose of DHT reduced the increase in cholinergic receptor number elicited by PRL. The high dose of PRL reduced the increases of total Na+,K(+)-ATPase and acid phosphatase elicited by DHT. CONCLUSIONS: These findings suggest that DHT and PRL exert general trophic actions on the lacrimal gland and specifically on lacrimal Na+,K(+)-ATPase, acid phosphatase, and neurotransmitter receptors. They also suggest that excessive levels of either hormone may be deleterious to secretory function. Because sex hormone levels are prone to wide fluctuations in women, our results also suggest a plausible hypothesis to account for the greater incidence in women of lacrimal insufficiency.

Acid Phosphatase↗

PubMed1994