J. Peter Gierow

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)

Sjögren's autoimmunity: how perturbation of recognition in endomembrane traffic may provoke pathological recognition at the cell surface.

CD4 T cell antigen recognition requires presentation by major histocompatibility complex Class II molecules (MHC II). B cell surface immunoglobulins recognize antigens independently of MHC II, but activation typically requires CD4 cell cytokines as accessory signals. Plasma membrane–endomembrane traffic in lacrimal gland acinar cells, targets of autoimmune activity in Sjögrens syndrome, may satisfy both requirements. The Golgi protein galactosyltransferase and the lysosomal proteins cathepsin B and cathepsin D appear at the plasma membranes during sustained secretomotor stimulation. The RNA transcription termination factor La, a frequent target of Sjögrens autoantibodies, appears in the acinar cell cytoplasm and plasma membranes during viral infection and during in vitro exposure to cytokines. MHC II cycle through endomembrane compartments which contain La, galactosyltransferase, cathepsin B and cathepsin D and which are sites of proteolysis. This traffic may permit trilateral interactions in which B cells recognize autoantigens at the surface membranes, CD4 T cells recognize peptides presented by MHC II, B cells provide accessory signals to CD4 T cells, and CD4 T cells provide cytokines that activate B cells. Acinar cells stimulate lymphocyte proliferation in autologous mixed cell reactions, confirming that they are capable of provoking autoimmune responses. Copyright

Immunogold Localization of Prolactin in Acinar Cells of Lacrimal Gland

Prolactin (PRL) is known to be involved in osmoregulation and control of cellular differentiation in lower vertebrates (Horobbin, 1980). In mammals, PRL influences sodium and water transport in kidney (Loretz and Bern, 1982) and sweat glands (Robertson et al., 1989), and stimulates the secretion of both fluid and protein in the mammary gland (Olivier-Bousquet, 1978; Houdebine and Dijane, 1989). Although PRL is primarily a product of mammotrophes in the anterior pituitary, it is known to be elaborated by other cell types in the body, including lymphocytes (Buskila, et al., 1991; Lavelle, 1992). PRL receptors also have been found on a number of cells besides mammary secretory cells, again including lymphocytes, and these facts implicate the hormone in immunomodulation, as well as in its better known function in lactogenesis. In fact, Buskila and co-workers (1991) have proposed that PRL may serve as a master hormone controlling immune system function.

Presence of α-and β-Integrin Subunits in Rabbit Lacrimal Gland Acinar Cells Cultured on a Laminin-Rich Matrix

Integrins play critical roles in the interactions between the extracellular matrix and the cell by directing a number of functions, e.g. cell adhesion, cytoskeletal organization, and signal transduction. It is our hypothesis that they are also involved in modulating the regulation of secretion by lacrimal gland acinar cells, either by direct interaction with intracellular signaling cascades, or through modulation of cytoskeletal organization. Relatively little is known about the integrins in the lacrimal gland, but our laboratory is presently surveying for potential candidates capable of modulating secretion by lacrimal acinar cell. We have previously reported the presence of α2 αv, β1 and β4 integrin subunits in cells obtained by a cytospin technique.1, 2 The present study is an expanded survey for integrin subunits using rabbit lacrimal gland acinar cells in primary culture on laminin coated multi-well slides.

Role of Protein Kinases in Regulation of Apical Secretion and Basal-Lateral Membrane Recycling Traffic in Reconstituted Rabbit Lacrimal Gland Acini

Cholinergic stimulation activates at least two different membrane traffic functions in lacrimal gland acinar cells: (i) exocytic release of stored secretory proteins across the apical plasma membranes, which is followed by retrieval and recycling of secretory vesicle constituents, and (ii) recycling traffic between basal-lateral plasma membranes and endomembrane compartments, e.g., membranes involved in translocation of neurotransmitter and neuropeptide receptors and of Na,K-ATPase and other ion transport proteins. The signal transduction and effector mechanisms involved in regulated protein secretion have been studied most extensively in rat extraorbital lacrimal glands.1–4 Work with this preparation has produced considerable evidence implicating both branches of the phosphoinositide cascade, including Ca2+-mediated activation of Ca2+/calmodulin-dependent protein kinase and diacylglycerol-mediated activation of protein kinase C.5,6 A role for phospholipase D has also been suggested.7 In contrast, relatively little is known about regulation of the basal-lateral plasma membrane-endomembrane recycling traffic. Therefore, the goals of the present study were to assess the effects of protein kinase activators and inhibitors on secretion of β-hexosaminidase, a marker for function of the apical secretory pathway,8 and on endocytosis of Lucifer Yellow, a marker for function of the basal-lateral plasma membrane-endomembrane recycling pathway, in rabbit lacrimal gland acinar cells.9,10 Accordingly, we employed the phorbol ester, phorbol-12-myristate-13-acetate (PMA); the Ca2+-ionophore, A23187; the protein kinase C inhibitor, calphostin c; and the Ca2+/calmodulin-dependent protein kinase (CaM kinase II) inhibitor, KN62.

Subcellular Organization of Ion Transporters in Lacrimal Acinar Cells: Secretagogue-Induced Dynamics

The electrolyte-driven secretion of water is one of the major functions of the lacrimal gland, and impairment of this function is an obvious cause of dry eyes. As reviewed elsewhere (Mircheff, 1986, 1989), Alexander et al. showed in 1972 that the lacrimal gland produces fluid in two stages, secretion of a NaCl-rich solution in the acini, and secretion of a KCl-rich solution in the ducts. Studies of ductal transport have begun only recently (Saito, this volume). In contrast, modern concepts of acinar secretory mechanisms began to emerge more than ten years ago, when Dartt and coworkers (1981) presented the first evidence that the lacrimal glands conform to the principles of epithelial electrolyte secretion formulated by Silva and coworkers (1977),

Adenosine A2 receptor presence and synergy with cholinergic stimulation in rabbit lacrimal gland.

Purpose: Secretion from the lacrimal gland is an important part of the well-being of the eye, and a central part in the search for treatment of dry eye syndrome. Adenosine has stimulatory effects on the lacrimal gland, and can potentiate the effect of the cholinergic agonist carbachol (Cch). The aim of the present study is to investigate the presence of the adenosine A2 receptor subtypes A2A and A2B in the rabbit lacrimal gland, and to characterize their role in regulated acinar cell secretion. Methods: Expression of the receptors was investigated using reverse transcriptase-PCR (RT-PCR) and immunofluorescence, and secretion effects were studied using a secretion assay in isolated lacrimal gland acinar cells. Results: Presence of both receptors was detected by RT-PCR and immunofluorescence. The secretion assay revealed a minor effect of stimulation of the A2 receptors, and a strong synergistic effect with the cholinergic agonist Cch. The synergistic effect was significantly reduced by the A2B antagonist PSB 1115, but not by the A2A antagonist SCH 58261, indicating that A2B is the receptor responsible for this potentiation. Conclusions: The study reveals the presence of the adenosine A2 receptor subtypes as well as a role for them in lacrimal gland secretion, and especially in the synergy with purinergic and cholinergic stimulation.

Acinar Cell Basal-Lateral Membrane—Endomembrane Traffic May Mediate Interactions with Both T Cells and B Cells

The lacrimal glands are normally populated by significant numbers of IgA-producing plasma cells and T lymphocytes, with CD8 cells in twofold excess over CD4 cells. The numbers of these cells increase during normal aging, and there has been speculation that they may exacerbate the lacrimal dysfunction of primary lacrimal deficiency. Lymphocytic infiltration increases much more dramatically in Sjogren’s syndrome, an immune system disorder in which CD4 cells dominate the T lymphocyte population, and the antibody-producing population includes large numbers of B cells. The B cell infiltrates are polyclonal and often produce IgG and IgM against intracellular antigens, such as the ribonuclear proteins Ro and La and a variety of Golgi proteins. Because of the severity of the ocular surface disease and the debilitating and potentially life-threatening nature of the systemic manifestations of Sjogren’s syndrome, it is important to identify the mechanisms that lead to CD4 cell and B cell proliferation in the lacrimal glands.

Endocytosis and Exocytosis in Rabbit Lacrimal Gland Acinar Cells

The main function of the lacrimal gland is to maintain the surface of the eye moist, well lubricated, and free of irritants. It achieves this function by secreting ions, proteins, and water. Recent studies have shown that stimulation of lacrimal cells by carbachol, an acetylcholine analogue, not only triggers release of secretory products across the apical plasma membrane and activates various ion transporters involved in the trans-epithelial secretion of Na+ and Cl−, but it also causes Na+, K+-ATPase pump units to be mobilized from a cytoplasmic pool and inserted into the baso-lateral plasma membrane (Yiu et al., 1988). Recent studies suggest that carbachol also increases the rate of endocytosis at the baso-lateral surface (Lambert et al., 1993). We have analyzed the internalization and release of a fluorescent fluid phase marker, Lucifer Yellow, in order to learn more about the control of membrane trafficking in acinar cells from rabbit lacrimal glands and to discern the roles of distinct intracellular compartments. In an attempt to evaluate the extent to which fluid phase marker internalization reflects the retrieval of secretory vesicle membranes, we have also measured protein secretion.

Hypothesis for autoantigen presentation and T cell activation.

Investigators have appreciated for many years that autoimmune processes lead to the periductal fibrosis and acinar atrophy characteristic of Sjogren’s Syndrome. Some observations can be interpreted as suggesting that chronic, low-grade autoimmune phenomena account for qualitatively similar, but quantitatively less severe, changes regarded as age-related lacrimal gland atrophy (Damato et al., 1984). We wish here to review several lines of clinical and basic research which have produced new insights into cellular and molecular processes that might play critical roles in the initiation of such autoimmune phenomena.