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.

Animals↗

PubMed1992

Isolation and subcellular fractionation analysis of acini from rabbit lacrimal glands.

The rabbit has been a useful model for in vivo studies of the pharmacologic control of lacrimal gland fluid secretion. However, by contrast with rodent exorbital lacrimal glands, the rabbit lacrimal gland has not been subjected to detailed cellular, subcellular, or biochemical analyses. Procedures were developed to isolate rabbit lacrimal acini by collagenase digestion and mechanical dispersion. The preparations exhibited good morphology, and trypan blue exclusion rates generally exceeded 90%. The isolated acini responded to carbachol by releasing protein and increasing Na+ unidirectional influx rates. The presence of muscarinic cholinergic and beta-adrenergic receptors was indicated by specific binding of the muscarinic cholinergic antagonist, 3H-N-methylscopolamine (3H-NMS; dissociation constant, Kd, 0.55 nmol/l), and the beta-adrenergic antagonist, 3H-CGP12177 (Kd, 0.34 nmol/l). The maximal binding values measured in crude membrane preparations were 79 fmol/mg for 3H-NMS and 40 fmol/mg for 3H-CGP12177. Subcellular fractionation analyses showed various membrane populations, including a series of Golgi-derived populations admixed with a major endoplasmic reticulum-derived population, a population that may represent the basal-lateral plasma membranes, and a series of populations with characteristics suggesting they are involved in the assembly or recycling of basal-lateral membrane constituents. The authors believe the ability to isolate and analyze acinar preparations from the rabbit lacrimal gland will facilitate various studies of acinar cell biochemistry and physiology that would be impractical with the relatively smaller amounts of material that can be obtained from rat or mouse exorbital lacrimal glands.

Animals↗

PubMed1991

pH-sensitive anion exchanger in rat lacrimal acinar cells.

Basolateral membranes from rat lacrimal acinar cells contain Na(+)-H+ and Cl(-)-HCO3- antiport activities [Invest. Ophthalmol. Visual Sci. 28: 1726-1729, 1989; Am. J. Physiol. 255 (Gastrointest. Liver Physiol. 18): G367-G373, 1988]. This study evaluated factors involved in coupling ion fluxes through these antiporters. 22Na+ flux into acini isolated from rat exorbital glands was 94 +/- 6 nmol.mg-1.min-1, and it was accelerated threefold by 10(-5) M carbachol; neither resting nor stimulated influx was affected by bumetanide. It is, therefore, likely that a portion of the carbachol-dependent Na+ influx is mediated by Na(+)-H+ antiporters. 36Cl- flux into Cl(-)-loaded, unstimulated acini was 275 +/- 21 nmol.mg-1.min-1; Cl- flux into HCO3(-)-loaded acini was 204 +/- 2; Cl- flux into acini loaded with both Cl- and HCO3- was 253 +/- 32; and influx in the absence of exchangeable intracellular anions was 176 +/- 13. Therefore, Cl(-)-Cl- self-exchange represented the major component of anion exchanger-mediated Cl- flux into resting cells. As pHi was increased above 7.2 by potassium-nigericin pH clamping, Cl- fluxes into Cl(-)- and HCO3(-)-containing acini, but not into Cl(-)-depleted acini, were significantly accelerated. SITS completely abolished the pHi-activated increment of Cl(-)-Cl- exchange. Carbachol increased Cl- unidirectional flux into Cl(-)-loaded cells by 25% (P less than 0.1), apparently as a result of Na(+)-H+ antiporter-mediated cytoplasmic alkalinization.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1991

Secretagogue-induced redistributions of Na,K-ATPase in rat lacrimal acini.

Lacrimal acinar cells secrete macromolecular products in an approximately isotonic, sodium chloride (NaCl)-rich fluid. The mechanisms of macromolecular product secretion depend in part on a recycling traffic of membrane constituents between the Golgi complex and the apical plasma membrane. In contrast, the acinar cell's mechanisms for secreting Na+ and Cl- depend largely on the fluxes of these ions through transporters expressed in the apical and basal-lateral membranes. In addition to accelerating the recycling of secretory vesicle membrane constituents, the cholinergic agonist carbachol also triggers a net redistribution of sodium potassium adenosine triphosphatase (Na,K-ATPase) ion pumps between Golgi-associated pools and the basal-lateral plasma membranes (Yiu SC, et al: J Membrane Biol 102:185, 1988). In the present study, acinar preparations from rat lacrimal glands were stimulated with either carbachol, epinephrine, or isoproterenol. All three agonist stimulated release of the secretory protein lactoperoxidase, but only carbachol significantly accelerated Na+ undirectional influx. Subcellular fractionation analyses of resting and stimulated preparations indicated that carbachol caused a significant translocation of Na,K-ATPase activity from a Golgi-associated compartment to the basal-lateral plasma membranes. Neither adrenergic agonist significantly increased the basal-lateral membrane Na,K-ATPase activity, but each triggered a distinct pattern of redistributions of Na,K-ATPase and the Golgi membrane marker, galactosyltransferase. The carbachol-induced augmentation of basal-lateral membrane Na,K-ATPase activity represents a mechanism by which the cell might compensate for increased Na+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Class II antigen expression by lacrimal epithelial cells. An updated working hypothesis for antigen presentation by epithelial cells.

It has been suggested that aberrant expression of Class II histocompatibility antigens (HLA) is involved in T cell activation and leads to autoimmunity. Although Class II antigen expression was found in various nonlymphoid tissues, including salivary glands, its expression on lacrimal epithelial cells has not been reported. In this study, 12 cadaver lacrimal glands were analyzed for HLA-DR and for the numbers and distributions of T suppressor cells (Ts), T helper cells (Th), B cells, and macrophages. None of these cases exhibited the high numbers of inflammatory cells, tissue damage, and fibrosis characteristic of Sjogren's syndrome. The HLA-DR-positive epithelial cells were detected in ten cases; they represented from less than 1% to more than 70% of the epithelial cells. In these ten positive cases, there were greater numbers of T cells per millimeter squared (229 +/- 94 [mean +/- the standard error of the mean]) than in the two HLA-DR-negative cases (37 +/- 1 [mean +/- range]). Three lacrimal gland specimens tested were negative for immunoglobulin (Ig) G-bearing B cells, and two of the three specimens tested had IgA-bearing cells. Acinar cells were isolated from rat and rabbit lacrimal glands and cultured overnight in serum-free media supplemented with several potential mediators of Class II antigen expression: interferon-gamma, carbachol, or isoproterenol. Freshly isolated cells did not express Class II antigens at detectable levels, but in most experiments, they began to express the antigen even in the absence of putative mediators.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

PubMed1990

Subcellular distribution of muscarinic acetylcholine receptors in rat exorbital lacrimal gland.

The muscarinic acetylcholine receptor (MAChR) is an important mediator of parasympathetic regulation of secretion by the rat exorbital lacrimal gland. In order to survey the subcellular distribution of MAChR in lacrimal acinar cells, we have measured the binding of the specific muscarinic cholinergic antagonist [3H]-quinuclidinyl benzilate ([3H]-QNB) to membrane samples isolated from rat exorbital lacrimal glands by differential and equilibrium density gradient centrifugation. Binding of [3H]-QNB in all membrane fractions was consistent with the presence of a single class of receptor which was muscarinic in nature on the basis of its Kd for [3H]-QNB (0.30-0.35 nM) and its ability to interact with the muscarinic agonists carbachol and methachol and the antagonist atropine. MAChR were present at the highest specific activity in acinar cell basal-lateral plasma membrane-derived populations, where Bmax was as high as 1960 fmole/mg protein. However, the density distributions of MAChR and of other membrane markers indicated that the receptors were present also in membranes derived from cytoplasmic structures, where Bmax values ranged from 50.4 to 152.8 fmole/mg protein. Stimulation with 10 microM carbachol for 30 min led to a 20% (P less than 0.05) increase in the relative MAChR content of a population of membranes derived from the acinar cell basal-lateral membrane; an apparent tendency for MAChR activity to decrease in other membrane populations suggests that stimulation might cause a redistribution of MAChR between cytoplasmic pools and the cell surface membranes.

Animals↗

PubMed1989

Evaluation of skin stapling for wound closure in the emergency department.

Automatic skin staplers have been commonly used for surgical wound closure for many years. The efficiency and ease of placement of skin staplers make them an attractive alternative to suture repair of selected lacerations in the emergency department. Emergency physicians, however, have been reluctant to use staplers in the ED. We evaluated skin staples in 76 patients presenting with 87 lacerations to the scalp, trunk, or extremities, excluding hands and feet. Patients returned to the ED in two and seven to ten day for wound check and staple removal. Skin stapling was assessed for efficiency, cosmetic results, complications, and cost-effectiveness. Only one significant complication was noted in our study group - a dehiscence of a scalp laceration secondary to hematoma collection. There was also a minor dehiscence of a superficial laceration of the leg due to inadequate primary closure, which did not result in any cosmetic deformity. No infectious complications, delayed wound healing, or cosmetic problems were seen. Skin stapling was easier and quicker than suture repair at a lower overall cost in most circumstances. Our study shows skin stapling to be an efficient and cost-effective alternative method to suture wound closure for selected lacerations in patients presenting to the ED, without compromising wound healing or cosmetic results.

Adolescent↗

PubMed1989

Synthesis and antiviral activity of phosphonoacetic and phosphonoformic acid esters of 5-bromo-2'-deoxyuridine and related pyrimidine nucleosides and acyclonucleosides.

Phosphonoacetic acid (PAA, 1) was coupled with various acyclonucleosides, 2'-deoxyuridines, cytidines, and arabinosyluracils, with 2,4,6-triisopropylbenzenesulfonyl chloride (TPS) or dicyclohexylcarbodiimide (DCCI) as condensing agents, to give a range of phosphonate esters. The carboxylic ester linkage of PAA to the 5'-position of 5-bromo-2'-deoxyuridine (BUdR, 3) was achieved via the mixed anhydride formed from (diethylphosphono)acetic acid and trifluoroacetic anhydride. Phosphonoformic acid (PFA, 2) was coupled with BUdR by using the DCCI method to give the phosphonate ester. Of these compounds only phosphonate esters in the 2'-deoxyuridine series showed significant activity against herpes simplex virus types 1 and 2. The BUdR-PAA derivative and the BUdR-PFA derivative were highly active, especially the latter, which was more active than the parent nucleoside BUdR against the type 2 virus. The active compounds may exert their effects by extracellular or intracellular hydrolysis to the corresponding antiviral agents, but an intrinsic component of antiviral activity may also be involved.

Antiviral Agents↗

PubMed1989

Effects of 13-cis-retinoic acid on the hamster meibomian gland.

Systemic administration of 13-cis-retinoic acid caused a reduction of acinar tissue in the hamster meibomian gland. Histologic examination of treated meibomian gland tissue revealed a thickening of gland ductal epithelium and a decrease in the numbers of mature lipid-laden acinar cells. Morphometric analysis showed a reduction of up to 75% in mean volume of meibomian acinar tissue from animals fed a high dose of 13-cis-retinoic acid. Clinical observations in these animals included alopecia and weight loss. Ocular complications included crusting of the eyelid margin and the external surface of the lid and erythema of the conjunctiva. The model supports previous observations by the authors that systemic 13-cis-retinoic acid affects meibomian gland structure in a laboratory animal. The ocular side effects described in this model suggest that future functional studies may yield important insights into the complex relationship between meibomian gland morphology and function, and events on the ocular surface.

Animals↗

PubMed1988

Stimulation-associated redistribution of Na,K-ATPase in rat lacrimal gland.

To test the possibility that stimulation of secretion leads Na,K-ATPase to be recruited from cytoplasmic pools and inserted into basal-lateral plasma membranes, we surveyed the subcellular distributions of Na,K-ATPase in resting and stimulated fragments of rat exorbital lacrimal gland. After a two-dimensional separation procedure based on differential sedimentation and density gradient centrifugation, we defined six density windows, which differ from one another in their contents of biochemical markers. The membranes equilibrating in window I could be identified as a sample of basal-lateral membranes; in resting preparations these membranes contained Na,K-ATPase enriched 16.6-fold with respect to the initial homogenates. Windows II through VI contained various cytoplasmic membrane populations; these accounted for roughly 80% of the total recovered Na,K-ATPase activity. Thirty-minute stimulation with 10 microM carbachol caused a 1.4-fold increase (P less than 0.05) in the total Na,K-ATPase content of window I; this increase could be largely accounted for by a 1.7-fold decrease in the total Na,K-ATPase content of density window V. Acid phosphatase activity also redistributed following stimulation, but it was recruited from a different source, and it was inserted into membranes equilibrating in windows II and III as well as into the membranes of window I.

Acid Phosphatase↗