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Biomedical subjects

R W Lambert

Publications and source records attributed to R W Lambert.

At least 19 recordsLinked to original sources
PubMed1996

Excimer laser effects on human corneal endothelium. Modulation by serum factor(s).

OBJECTIVE: To determine the possibility of endothelial cell damage after excimer laser ablation. METHODS: Endothelial cell densities and morphology of human corneas after photoablations or mechanical keratectomy were compared with those of the untreated mates after 1 week of culture with or without serum. RESULTS: Corneas cultured in serum-free medium after ablation to a depth of 150 microns showed endothelial cell densities reduced to 60% of untreated, mate corneas; ultrastructural analysis showed endothelial cell damage not seen in untreated mates. Corneas ablated to the same depth and cultured in serum-enriched medium showed no endothelial cell density loss, nor did corneas cultured in serum-free medium after an ablation to a depth of 50 microns or mechanical keratectomies averaging 95 microns. CONCLUSIONS: Endothelial cell loss in deep laser resections may be prevented by factor(s) in fetal bovine serum. The apparent lack of cell loss in clinical studies may be related to the protective action of similar factors in aqueous humor.

Aged↗
PubMed1996

Comparison of the UniversalKeratome and the Automated Corneal Shaper.

PURPOSE: To compare the morphologic appearance and measurements of in situ keratomileusis performed with the UniversalKeratome (UK) with those done with the Automated Corneal Shaper (ACS). SETTING: Surgical suite within private practice. METHODS: Procedures were performed the same day on mate eye-bank eyes. In situ keratomileusis was done using existing nomograms for each instrument to resect a cap thickness of 160 microns and a myopic resection of 100 microns. Intraocular pressures were increased by inflating the globes with balanced salt solution and were measured with the suction fixation rings in place. The excised caps and stromal resections were measured twice independently after surgery, again after tissue fixation, and then evaluated with light and scanning electron microscopy. RESULTS: No complications were encountered. Compared with the ACS, the UK was easy to set up, use, clean, and take down. Its excised tissue dimensions were greater and more predictable, it resected a concave shaped lenticule (edges imperceptibly blending with the host stroma), and it created a smoother power resection surface and primary resection base. CONCLUSIONS: Smoother, predictable tissue resection, and simple assembly/disassembly and use give the UK an apparent advantage over the ACS. The UK corrects astigmatism and hyperopia by changing the shape of the poly(methyl methacrylate) optical insert.

Aged↗
PubMed1996

Analysis of cytomegalovirus infection and replication in acinar epithelial cells of the rat lacrimal gland.

PURPOSE: The objectives of this investigation were threefold: to advance understanding of the nature and impact of herpesvirus infection in the lacrimal gland; to determine the influence of gender and sex hormones on viral infectivity and replication capacity in lacrimal tissue; and to compare the susceptibilities of lacrimal, submandibular, and parotid cells to viral invasion. METHODS: Acinar epithelial cells were isolated from lacrimal or salivary glands from intact, orchiectomized, ovariectomized, or sham-operated rats, cultured on Matrigel in serum-free media, and briefly exposed to rat cytomegalovirus (RCMV). Cells were then incubated for varying time intervals, and RCMV titers and secretory component (SC) levels in media or cell extracts were measured by plaque assay or radioimmunoassay. Exocrine glands also were obtained from rats after RCMV inoculation in vivo and were analyzed for viral infection. RESULTS: These findings demonstrated that RCMV invades the rat lacrimal gland after intravenous or intraperitoneal viral inoculation; RCMV infects and undergoes a time-, dose-, strain-, and gender-dependent replication in acinar epithelial cells from rat lacrimal tissue; the magnitude of RCMV replication in acinar epithelial cells in vitro may, in fact, induce an acute increase in the cellular production of SC; and the extent of viral replication in lacrimal and salivary gland epithelial cells displays distinct, tissue-specific variations. CONCLUSIONS: The herpesvirus RCMV invades and replicates in acinar epithelial cells from the rat lacrimal gland. The magnitude of the viral infection may be significantly influenced by gender and alterations in the hormonal environment.

Animals↗
PubMed1995

Androgen control of secretory component mRNA levels in the rat lacrimal gland.

The purpose of this investigation was to determine whether the known gender-related differences in, and the endocrine control of, the production of secretory component (SC) by the rat lacrimal gland are associated with alterations in SC mRNA content. Levels of SC mRNA were measured in lacrimal tissues of intact, sham-operated, castrated, hypophysectomized, and testosterone-treated male and female adult rats by Northern blot procedures, which utilized a specific, [alpha-32P]-labelled rat SC cDNA probe. For control purposes, SC mRNA amounts were standardized to the beta-actin content in experimental blots. The location of SC mRNA in lacrimal glands was evaluated by in situ hybridization techniques, which involved exposure of tissue sections to sense or anti-sense [35S]-labelled SC RNA probes. Our results demonstrate that: (1) lacrimal glands of male rats contain a significantly greater amount of SC mRNA than those of female rats, and that this difference co-exists with distinct, gender-associated variations in the distribution of SC mRNA in lacrimal tissue; (2) orchiectomy or hypophysectomy, but not ovariectomy or sham surgery, leads to a marked decline in the lacrimal SC mRNA content; and (3) testosterone, but not placebo, administration to castrated male and female rats induces a significant increase in the SC mRNA levels in lacrimal tissue. Overall, these findings show that gender, androgens and the hypothalamic-pituitary axis exert a considerable influence on the SC mRNA content in the rat lacrimal gland.

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↗
PubMed1994

Rabbit lacrimal acinar cells in primary culture: morphology and acute responses to cholinergic stimulation.

PURPOSE: The rabbit lacrimal gland yields large numbers of viable acinar cells that, when exposed to carbachol, respond with accelerated protein release, fluid phase endocytosis (Lucifer yellow uptake), and Na/H antiport activation. The current study was undertaken to determine whether such cells exhibit similar responses after having been maintained in primary culture. METHODS: Cells were isolated from 2-kg, juvenile male New Zealand White rabbits and maintained in a supplemented DMEM/Ham's F-12 medium for up to 72 hours. RESULTS: Electron microscopy showed the organization of freshly isolated cells to be highly polarized, with secretory vesicles at one pole and nucleus at the other; vesicles were heterogeneous in size and in the electron density of their contents. The cells remained polarized after overnight culture, but the secretory vesicle population was more homogeneous in size and content, and the cells tended to aggregate. After 72 hours, roughly half the cells retained good morphology and cytoplasmic polarization, but the vesicles were enlarged and their contents less electron dense. Cells that had been maintained overnight responded to the addition of 10 microM carbachol with a 32.2% +/- 15.5% (n = 12, P < 0.04) increase in the total amount protein released during a standard 20-minute incubation. This represented a mean 125% increase in the temperature-dependent component of protein release. The protein secretory response was decreased to 14.6% +/- 6.1% (n = 3, P < 0.07) for cells that had been maintained for 72 hours. In the same samples, carbachol increased fluid phase endocytosis by 38.3% +/- 8.1% (P < 0.01) and 70.9% +/- 13.4% (P < 0.025), respectively. The protein secretory response was partially, and the endocytic response fully blocked by 1 mM atropine. CONCLUSIONS: This model could be useful as a simplified system in which to study regulation of acinar cell function over days, rather than hours, as is required in fresh tissue models.

Alkalies↗
PubMed1994

Neuroendocrinimmune modulation of secretory component production by rat lacrimal, salivary, and intestinal epithelial cells.

PURPOSE: To evaluate the kinetics, receptor specificity, molecular basis, and site selectivity of the endocrine and neural regulation of secretory component (SC) synthesis by rat lacrimal gland acinar cells. METHODS: Acinar cells from the rat lacrimal and submandibular glands, as well as epithelial cells (IEC-6) from the rat small intestine, were cultured in supplemented, serum-free media and treated with dihydrotestosterone, cholera toxin, carbachol, vehicle, or other agents for varying time periods. Media SC levels were measured by radioimmunoassay. RESULTS: The authors' findings with lacrimal gland acinar cells demonstrate that: a significant, temporal delay exists between the initiation of stimulatory or inhibitory signals and the eventual cellular SC response to regulatory compounds; the parasympathetic analogue, carbachol, exerts a dual effect on SC output, i.e., an early stimulation (hours) followed by an extended suppression (days); the androgen and cholinergic control of SC is receptor-mediated; and the androgen modulation of SC may involve the induction of gene expression. In addition, the authors' results show that distinct, tissue-specific variations occur in the nature of SC regulation: Compounds that control SC output by lacrimal acinar cells do not necessarily alter SC production by epithelial cells from the rat submandibular gland or small intestine. CONCLUSIONS: These findings advance the authors' understanding of the neuroendocrine regulation of SC synthesis in acinar cells from the lacrimal gland. Moreover, the authors' results indicate that the nature of the neural, endocrine, and immune control of lacrimal SC may be unique.

Animals↗
PubMed1993

Influence of gender and the endocrine environment on the distribution of androgen receptors in the lacrimal gland.

Androgens are known to regulate both the structure and function of lacrimal tissue in a variety of species. To explore the endocrine basis for this hormone action, the following study was designed to: (1) determine the cellular distribution of androgen receptors in the lacrimal gland; and (2) examine the influence of gender and the endocrine environment on the glandular content of these binding sites. Lacrimal glands were obtained from intact, castrated, hypophysectomized, diabetic or sham-operated male or female adult rats, mice or hamsters, as well as from orchiectomized rats exposed to placebo compounds or physiological levels of testosterone. The cellular location of androgen receptors was evaluated by utilizing an immunoperoxidase protocol, in which a purified rabbit polyclonal antibody to the rat androgen receptor was used as the first antibody. Our findings with lacrimal glands showed that: (1) androgen receptors are located almost exclusively in nuclei of epithelial cells; (2) the cellular distribution or intranuclear density of these binding sites is far more extensive in glands of males, as compared to females; (3) orchiectomy or hypophysectomy, but not sham-surgery or diabetes, lead to a dramatic reduction in the immunocytochemical expression of androgen receptors; and (4) testosterone administration to orchiectomized rats induces a marked increase in androgen receptor content, relative to that in placebo-exposed glands. Our results also reveal that a 10 kb androgen receptor mRNA exists in the rat lacrimal gland. Overall, these findings demonstrate that gender and the endocrine system may significantly influence the distribution of androgen binding sites in rat lacrimal tissue. Moreover, our results show that androgens up-regulate their own lacrimal gland receptors.

Animals↗
PubMed1993

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini.

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.

Amiloride↗
PubMed1993

Plasma membrane internalization and recycling in rabbit lacrimal acinar cells.

PURPOSE: The purpose of this study was to examine internalization and recycling of plasma membrane constituents in lacrimal gland acinar cells. METHODS: Acinar cells were isolated from rabbit lacrimal glands. Surface-expressed reactive groups were biotinylated at 4 degrees C with sulfo-N-hydroxysuccinimidyl-biotin. Incorporated biotin was then labeled with avidin-horseradish peroxidase complex for light microscopy, with avidin-lucifer yellow conjugate for fluorescence microscopy, and quantitative fluorometry, and with avidin-ferritin conjugate for electron microscopy. RESULTS: At 4 degrees C labels remained at the surfaces of intact cells. Surface avidin-lucifer yellow decreased markedly, giving way to punctate cytoplasmic labeling, on warming to 37 degrees C. Electron microscopy of cells warmed after labeling with avidin-ferritin revealed ferritin in smooth vesicles underlying the plasma membranes, in vesicles adjacent to Golgi membranes, and in multivesicular bodies. Incubation at 37 degrees C before chilling and labeling with avidin-lucifer yellow decreased the cells' capacity to bind avidin-lucifer yellow by 95%, with t0.5 < 0.5 min. If cells were then incubated with avidin-lucifer yellow at 37 degrees C, they took up the marker with a time course that indicated that 60% of the initial biotin either recycled back to the plasma membrane or remained in intracellular compartments that could be reached by endocytosed extracellular fluid. Internalized biotin communicated with extracellular avidin-lucifer yellow with a t0.5 of 2 min, and this process was accelerated by carbachol at concentrations of 10 mumol/l and 1 mmol/l. CONCLUSIONS: Acinar cell plasma membrane constituents participate in an ongoing, secretagogue-modulated recycling traffic between small surface-expressed pools and 10- to 20-fold larger intracellular pools.

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