Role of laminin-1, collagen IV, and an autocrine factor(s) in regulated secretion by lacrimal acinar cells

Lanlin Chen, J. Douglas Glass, Staci C. Walton, and Gordon W. Laurie

Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Adhesion to novel basement membrane component BM180 in the presence of laminin-1 promotes stimulus-secretion coupling in lacrimal acinar cells [G. W. Laurie, J. D. Glass, R. A. Ogle, C. M. Stone, J. R. Sluss, and L. Chen. Am. J. Physiol. 270 (Cell Physiol. 39): C1743-C1750, 1996]. The identity of the active laminin-1 site and the possibility that other promoters of coupling are present in the acinar cell microenvironment were probed by use of different substrates, media, neutralizing antibodies and cell numbers. Regulated peroxidase secretion was unaffected by basement membrane coat concentration and was detectable at reduced levels in serum-free medium. Anti-laminin-1 antibodies, particularly against sites in the beta 1 and gamma 1 chains, but not alpha 1 chains, partially suppressed regulated secretion, as did an anti-collagen IV antibody. Without effect were RGD peptide and antibodies against entactin, the beta 1-integrin subunit, and several growth factors. Increasing cell number in serum-free medium revealed an unknown, serum-maskable, secretion-enhancing activity with a remarkable specificity for regulated secretion. Stimulus-secretion coupling, therefore, appears to be modulated by several extracellular factors whose relative contributions remain to be determined.

coupling; tear; exocytosis; signaling; integrin

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

FROM ITS EARLIEST FORMATIVE stages as a cordlike ectodermal invagination of conjunctival origin, the lacrimal gland (20, 33) has some form of basement membrane investment. For many years, basement membrane was viewed simply as a supportive structure. Indeed, enzymatic dissolution of basement membrane is a prerequisite for acinar cell dispersion (9). Molecular studies now reveal this thin, resilient extracellular membrane to be a complex, adhesive gel of laminins, collagen IV, perlecan, growth factors, and other components both known and unknown (28).

Particular attention has been paid to mechanisms and consequences of cellular adhesion to basement membrane, an interaction linked in part to differentiation of epithelial and other cell types (8). Differentiation of mammary epithelial cells, as manifested by the quantity and nature of constitutive casein secretion, is basement membrane dependent (25). Could the same be true of the multistep regulated secretory pathway in excitable cells, such as lacrimal acinar cells? Extrapolation from descriptive studies in pancreas (4) suggest that development of regulated secretion is a complex process. Cell polarization coincides with the appearance of protein synthetic and signal transduction machinery, surface agonist receptors, and expression of secretory proteins and, as a consequence, the capacity to release nascent secretory proteins by the regulated secretory pathway.

Previously, we searched for differentiation factors that may be residents of lacrimal acinar basement membranes (14). Advantage was taken of a novel basement membrane substrate (referred to as BMS; Ref. 17). Adhesion to BMS was sufficient to reverse the characteristic propensity of isolated lacrimal acinar cells to lose secretagogue responsiveness in culture. Combined use of fractionated BMS and anti-BMS monoclonal antibodies led to the identification of BM180, a novel lacrimal and parotid gland basement membrane protein with cell adhesion activity that appeared to act in the presence of laminin-1 to promote stimulus-secretion coupling (14). Here we explore which laminin-1 sites may be active and whether other extracellular molecules have a modulatory role in regulated secretion by lacrimal acinar cells.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Preparation of substrate. A 10 mM EDTA extract of Engelbreth-Holm-Swarm (EHS) tumor matrix (BMS) was prepared as previously described (17). BMS contains numerous components including laminin-1, BM180 (14), and collagen IV. Purified laminin-1 was kindly provided by Dr. Roy Ogle (University of Virginia). BMS was diluted with DMEM and added at 0.044-0.06, 0.177, 0.354, 0.531, or 0.708 mg/cm2 to 96- or 48-well culture plates kept chilled on ice. Laminin-1 and also collagen I and Matrigel (both purchased from Collaborative Research, Bedford, MA) were similarly coated at 0.531 mg/cm2. For ELISA examination of basement membrane synthesis by collagen I-adherent cells, a 0.06 mg/cm2 collagen coating was used. Concentrations of 0.044-0.06 mg/cm2 gave rise to a monolayer. Concentrations >0.044-0.06 mg/cm2 formed a gel when incubated for 1 h at 37°C.

Antibodies and synthetic peptides. Antibody and peptide reagents utilized are summarized in Table 1. All antibodies, except those directed against entactin, were purified on protein A Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ) or G protein agarose (Pierce, Rockford, IL) or, alternatively, were purchased in purified form. Rabbit antibody concentration was determined using the extinction coefficient for rabbit IgG (13.5 [A1%1cm280]). Chain-specific anti-laminin-1 antibodies were provided by Dr. Y. Yamada (National Institute of Dental Research, Bethesda, MD). Chain specificity was independently verified (17). Anti-entactin antisera (6), including a negative control anti-glutathione S-transferase antiserum, were supplied by Dr. A. Chung (University of Pittsburgh, Pittsburgh, PA). A polyclonal anti-rat beta 1-integrin antiserum (2) was provided by Dr. T. Borg (University of South Carolina, Columbia, SC). ELISA detection of BM180 was performed with monoclonal antibody (MAb) 3E12 (14).

                              
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Table 1.   Summary of antibody and peptide reagents

Isolation of lacrimal acinar cells. Lacrimal acinar cells were isolated from 4- to 6-wk-old male Sprague-Dawley rats, as previously described (14). Briefly, an intracardiac wash of the vasculature with DMEM was followed by mincing and incubation in alternating solutions of EDTA and enzyme (9, 23). Released single acinar cells were collected by passage through sequential 160-µm and 25-µm Nitex filters followed by centrifugation on a discontinuous 10%, 30%, 60% Percoll gradient. Acinar cells sediment at the interface between 30 and 60%. Cell viability was 77-95%, as assessed by trypan blue exclusion. Cells were plated at 0.5-2.8 × 105 cells/cm2 on BMS, laminin-1, collagen I, or Matrigel in serum-containing medium (MOM; Ref. 9). Cells were also plated at 0.3-3.5 × 105 cells/cm2 on BMS in serum-free MOM (SFMOM; Ref. 9) medium. MOM consisted of high-glucose DMEM containing dexamethasone (1 ng/ml), putrescine (1 mM), epidermal growth factor (EGF; 50 ng/ml), L-ascorbic acid (25 µg/ml), insulin (6 µg/ml), transferrin (6 µg/ml), selenous acid (6 ng/ml), reduced glutathione (10 µg/ml), HEPES (15 mM), gentamicin (50 µg/ml), and heat-inactivated fetal calf serum (10%). SFMOM was identical to MOM, except for the omission of calf serum and addition of basic fibroblast growth factor (bFGF; 100 ng/ml). In inhibition studies, gelled BMS was incubated with DMEM-diluted anti-laminin-1, anti-entactin or anti-growth factor antibodies (10-100 µg/100 µl) for 1-2 h at 37°C and washed two times with DMEM before addition of freshly isolated cells. In other experiments, freshly isolated cells were preincubated with anti-beta 1-integrin antibodies (10-100 µg/100 µl) or synthetic peptide (100 µM) for 1 h at 37°C. Cells in the presence of antibody or peptide were then plated on BMS.

Secretion assay. The tear protein peroxidase was used as a marker of constitutive and regulated secretion. Cultures were gently washed twice overnight (18-24 h), and fresh medium was added. Constitutively secreted peroxidase was then collected for 100 min. Peroxidase secreted by the regulated secretory pathway was collected in the same manner following replacement with fresh medium to which had been added carbachol (10-4 M) and vasoactive intestinal peptide (VIP; 10-8 M). Cultures were terminated by digestion of BMS with dispase (40 U/ml; Collaborative Research) in the presence of 0.1% trypsin and 1.1 mM EDTA (GIBCO BRL, Grand Island, NY). Released cells were collected and retained for determination of total cellular peroxidase and DNA (14). Quantitation of secreted peroxidase was determined using the method of Herzog et al. (10). All secretion values were normalized to cellular DNA.

ELISA. Presence of laminin-1 and BM180 in overnight collagen-adherent cultures of mouse lacrimal acinar cells (2 × 105 cells/cm2) were detected by ELISA. Isolation of mouse lacrimal cells was similar to that used for rats, with the omission of the Percoll gradient and Nitex filtering. Cultures, or collagen-coated (0.06 mg/cm2) wells without cells, were PBS washed, fixed with 4% formaldehyde, and then PBS washed and blocked with PBS containing 1% BSA and 0.01% Tween 20. Subsequent steps, including incubation with antibodies against intact mouse laminin-1 or with anti-BM180 MAb 3E12 (14), detection, and analysis, were all carried out using a standard ELISA format, as previously described (14). Mouse lacrimal acinar cells were used to avoid background staining between the anti-rat secondary antibody (required to detect 3E12) and rat cells.

Statistical analysis. All values are expressed as means ± SE. Student's t-test was used where noted to assess statistical significance.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Lacrimal acinar cells freshly released from their native basement membranes adhere to BMS, a 10 mM EDTA extract of mouse EHS tumor basement membrane (Fig. 1), and after 24 h in culture remain capable of secreting tear proteins in a physiological manner (Fig. 2; Ref. 14). In contrast, cells on plastic are poorly adherent and generally lose much of their capacity to respond to carbachol and VIP (Fig. 2A). Regulated secretion remains constant at 40-50 mU tear peroxidase/µg cellular DNA, irrespective of the amount of underlying BMS (Fig. 2A; 41 ± 1 mU/µg, n = 62, for 0.531 mg/cm2), suggesting that key components are saturating at the lowest levels applied, even in the absence of the gelled configuration (< 0.177 mg/cm2) common to native basement membranes. Acinar cells on gelled collagen I were equally responsive (Fig. 2B). This result is a manifestation of the commonly observed propensity of epithelial cells to elaborate native basement membranes soon after adhesion to collagen I (Fig. 3; Ref. 25). Indeed, synthesis of both laminin-1 and BM180 could be readily detected (Fig. 3) by ELISA. Lower secretory levels were observed for cells cultured on gelled Matrigel (Fig. 2B) or purified laminin-1 (Fig. 2B; Ref. 14). Both constitutive and regulated tear secretion were serum enhanced but not serum dependent (constitutive secretion 4.4 ± 0.8 and 2 ± 0.7 mU/µg; regulated secretion 41 ± 1 and 11.2 ± 1.7 mU/µg, for serum-containing and serum-free media respectively; n = 40-62). Carbachol-stimulated secretion was dose dependent and atropine inhibitable (Table 2).


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Fig. 1.   Appearance of rat lacrimal acinar cells on basement membrane substrate (BMS). Freshly isolated cells just after plating (A and B) or 24 h later (C and D). Cells cultured for 24 h are adherent and display numerous secretory granules (small raised bumps) concentrated in apical cytoplasm. Secretory granules contain a number of tear proteins, including peroxidase. Cells were cultured in serum-containing medium (MOM) on BMS (0.531 mg/cm2). Similar culture conditions were used in experiments represented in Figs. 2-7, except as noted. Bars, 100 µm (for A and C) and 20 µm (for B and D).


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Fig. 2.   Regulated secretory pathway is particularly active in BMS- and collagen I-adherent lacrimal acinar cells. A: adhesion to BMS enhances capacity for regulated peroxidase secretion in a dose-independent manner between 0.044 and 0.708 mg/cm2. Plastic-adherent cells (0 mg/cm2) display lower secretion, an activity attributable to presence of serum. At concentrations >0.044 mg/cm2, BMS is in form of a gel. Values represent 7 (0-0.354 and 0.708 mg/cm2) or 62 (0.531 mg/cm2) independent measurements. B: use of gelled collagen I (Coll; 0.531 mg/cm2) or Matrigel (Mg; 0.531 mg/cm2) as adhesion substrate is equally (collagen I; P = 0.8), or less (Matrigel; P < 0.001) effective. Gelled laminin-1 (Ln; 0.531 mg/cm2)-adherent cells display less capacity for regulated secretion (P < 0.001). Occasional batches (not shown) of possibly BM180- and/or collagen IV-contaminated laminin-1 are equally active. Values represent 7 (collagen I), 10 (laminin-1), or 19 (Matrigel) independent measurements. Here and in Figs. 4-6, except as noted, regulated peroxidase secretion was assessed after stimulation with 10-4 M carbachol and 10-8 M vasoactive intestinal peptide; cumulative secretion over 100 min was normalized to µg of cellular DNA.


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Fig. 3.   Elaboration of laminin-1 and BM180 by collagen I-adherent lacrimal acinar cells (2 × 105 cells/cm2). Overnight cultures of mouse lacrimal acinar cells (solid bars), or cell-free collagen I-coated wells incubated overnight with serum-containing medium (open bars), were formaldehyde fixed, blocked, and then immunostained with antibodies against intact mouse laminin-1 (Ab LN) or mouse BM180 (Ab BM180) using an ELISA approach. Ctrl, result when primary antibody was omitted. Wells were coated with 0.06 mg/cm2 of collagen I. Use of rat lacrimal cells gave rise to background staining with anti-rat secondary antibody required to detect Ab BM180. This problem did not exist for detection of rabbit Ab LN, for which an identical level of basement membrane synthesis could be detected. OD, optical density.

                              
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Table 2.   Dose response effect of carbachol with or without VIP and atropine on peroxidase secretion by BMS-adherent lacrimal acinar cells

Anti-laminin-1 and anti-collagen IV antibodies suppress regulated secretion. Previous studies suggested that BM180 and laminin-1 together played key roles in promoting stimulus-secretion coupling in BMS-adherent lacrimal acinar cells (14). To determine which laminin-1 site(s) was involved and whether other basement membrane-associated molecules contributed, an antibody blocking approach was used (Fig. 4; Tables 3 and 4). Antibodies against intact laminin-1 partially suppressed regulated secretion by 26% (Fig. 4A), an effect that was reproduced with anti-beta 1 (35%) and anti-gamma 1 (33%) but not with two different anti-alpha 1, laminin chain-specific antibodies (Fig. 4B). Anti-collagen IV antibodies tested in parallel were also partially inhibitory (37%; Fig. 4A). Constitutive secretion was unaffected (Fig. 4A, inset). These values are similar to the level of inhibition (30-40%) commonly observed in the presence of anti-BM180 MAb 3E12 (14). In contrast, domain-specific anti-entactin antibodies (Table 3) and several different anti-growth factor antibodies (Table 4) had no effect.


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Fig. 4.   Anti-laminin-1 and anti-collagen IV antibodies partially inhibit BMS-enhanced regulated secretion. A: preincubation of BMS with antibodies against intact laminin-1 (Ab LN) or collagen (Ab IV) suppresses capacity for regulated secretion (solid symbols) in a dose-dependent manner. Values are expressed as % of no-antibody control and are means ± SE of 5 independent measurements; t-test comparing BMS vs. BMS + Ab LN or BMS + Ab IV at 50 µg/well revealed a P < 0.001. Inset: there was little or no effect of Ab LN and Ab IV on constitutive secretion (Unstim; open symbols); t-test with same format as above indicated no significant difference (P = 0.24-0.27). Constitutive secretion values were 116 ± 16 and 114 ± 18% for Ab LN and Ab IV, respectively, at 100 µg/well. Values are means ± SE of 5 independent measurements. B: association of laminin-1 activity with 2 distinct domains. Preincubation of BMS with antibodies against beta 1 chain YIGSR site (Ref. 7; amino acids 925-933; Ab beta 1) or carboxy-terminal gamma 1 chain site (amino acids 1420-1439; Ab gamma 1) partially inhibits regulated secretion. Not inhibitory were neutralizing antibodies against carboxy-terminal SN-peptide site (Ref. 17; alpha 1 chain amino acids 2179-2198; Ab alpha 1[SN]) or IKVAV site (Ref. 27; alpha 1 chain amino acids 2097-2108; Ab alpha 1[IK]). Values are means ± SE of 5 independent measurements; t-test comparing BMS vs. BMS + Ab beta 1 or BMS + Ab gamma 1 revealed a P < 0.003; t-test comparing BMS vs. BMS + Ab alpha 1[SN] or BMS + Ab alpha 1[IK] showed no significant difference (P = 0.95 and 0.21, respectively).

                              
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Table 3.   Lack of effect of anti-entactin antibodies on regulated secretion

                              
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Table 4.   Anti-growth factor antibodies have no apparent effect on regulated secretion when preincubated with BMS

Lack of effect of anti-beta 1-integrin and soluble RGD peptide on regulated secretion. Although information is lacking on lacrimal acinar cells, cellular adhesion to laminin-1 and collagen IV is most commonly mediated by beta 1-integrins. Four notable exceptions include alpha 6beta 4, alpha Vbeta 3, alpha -dystroglycan, and the putative 67/32-kDa laminin-1 receptor (19). The BM180 receptor(s) is unknown. To examine whether blocking cell surface beta 1-integrins would in turn affect stimulus-secretion coupling, acinar cells were cultured overnight in the presence of anti-beta 1-integrin antibody (Fig. 5). Regulated and constitutive secretion, assessed 24 h later, were unaffected (Fig. 5A). An alternative strategy is to interfere with RGD-dependent adhesion mechanisms [i.e., alpha Vbeta 3-laminin-1 (13), alpha 5beta 1-fibronectin (11), and alpha IIbbeta 3-fibrinogen (12)] by plating cells in the presence of soluble GRGDS synthetic peptide. Use of GRGDS peptide had no effect. Curiously, the control peptide GRGESP was slightly inhibitory (Fig. 5B).


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Fig. 5.   Anti-beta 1-integrin antibody and RGD peptide have no apparent effect on either regulated (solid bars) or constitutive (open bars) secretory pathways. A: preincubation of anti-beta 1-integrin antibody with acinar cells, followed by 24-h culture on BMS in presence of antibody, does not interfere with secretory capacity. Similar results were obtained in serum-free cultures (not shown). Values represent 3 (50 µg) or 6 (10, 100 µg) independent measurements. B: incubation with RGD peptide (100 µM) has no effect on regulated secretion, whereas some slight suppression appears to occur with control RGE peptide (P = 0.037). Values are means ± SE of 4 independent measurements.

Acinar cells produce a secretion-enhancing factor(s). In addition to tear proteins, lacrimal acinar cells may produce insoluble (i.e., basement membrane) and soluble materials that could help establish a suitable microenvironment for regulated secretory function. To examine this possibility, freshly isolated cells were plated at increasing density on BMS, and secretion was assessed after normalization to cellular DNA (Fig. 6). Normalized regulated secretion increased with plating number in serum-free cultures, whereas constitutive secretion remained constant (Fig. 6A). Addition of serum (Fig. 6B) masked this effect. Cellular aggregation in serum-free cultures was minimal (Fig. 7), suggesting that reformation of native acinar cell-like structures could not serve as a mechanism for enhanced secretion. These results are interpreted as tentatively revealing the presence of an autocrine secretion-enhancing factor(s) that specifically acts on the regulated secretory pathway to augment stimulus-secretion coupling.


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Fig. 6.   Presence of a putative autocrine enhancing activity (or activities) is revealed by culturing BMS-adherent acinar cells at increasing density without serum. A: in serum-free medium (SFMOM) capacity for regulated (Stim; solid symbols), but not constitutive (Unstim; open symbols), secretion is cell number dependent. Similar results were obtained whether cells were adherent to 0.044 (squares) or 0.531 (circles) mg/cm2 BMS. Values were normalized to µg cellular DNA and are means ± SE of 3 (1.1, 1.8, and 2.1 × 105 cells/cm2), 6 (2.8 × 105 cells/cm2), or 12 (1.4 × 105 cells/cm2) independent measurements. B: inclusion of serum in medium (MOM) masked or eliminated this effect. Values are means ± SE of 2 (1.1 and 2.1 × 105 cells/cm2) or 3 (all others) independent measurements.


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Fig. 7.   Appearance of overnight serum-free cultures plated at increasing density on BMS. A: 0.35 × 105 cells/cm2. B: 0.7 × 105 cells/cm2. C: 1.4 × 105 cells/cm2. D: 2.1 × 105 cells/cm2. E: 2.8 × 105 cells/cm2. F: 3.5 × 105 cells/cm2. Cells were adherent to 0.06 mg/cm2 BMS. Cells remain, for the most part, dispersed. In contrast, serum-containing cultures tend to display density-dependent aggregation (for example, Fig. 1C), a phenomenon that is not linked to enhanced secretion (Fig. 6B). Bar, 200 µm.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Development of the regulated secretory pathway is an intricate example of cellular differentiation possibly activated by adhesive signaling between the exocrine acinar cell and component(s) of the acinar cell basement membrane. Use of antibodies and serum-free or serum-containing lacrimal acinar cell cultures has here resulted in the identification of basement membrane collagen IV, two sites in laminin-1, and an unidentified autocrine factor(s) as likely playing contributory roles in the maintenance of the regulated secretory pathway in vitro.

Advantage was taken of the observation that gelled BMS supported stimulus-secretion coupling by overnight cultures of lacrimal acinar cells (14). Dissection of BMS by gel filtration previously identified a novel lower-molecular-weight activity defined by a monoclonal antibody as BM180, whose function appeared to be dependent on laminin-1 (14). The nature of laminin-1 active site(s) and the possible presence of accessory activators of stimulus-secretion coupling were investigated by plating lacrimal acinar cells overnight on BMS in the presence of anti-laminin beta 1 and gamma 1 chain or anti-collagen IV antibodies. Each reduced regulated secretion by 33-40% but left secretion by the constitutive pathway untouched. Not inhibitory were a diverse panel of antibodies with specificities for two laminin alpha 1 chain sites; three entactin globules and a bridging domain; the growth factors bFGF, tumor necrosis factor-alpha , EGF, insulin-like growth factor (IGF) II, and transforming growth factor (TGF)-beta ; and beta 1-integrins.

These results are in keeping with the concept that stimulus-secretion coupling is dependent on a highly selective group of basement membrane molecules that, through receptor ligation, may upregulate one or several elements in the coupling pathway(s). Noteworthy was the association of apparent coupling activity with laminin beta 1 and gamma 1 chains, rather than the strongly adhesive alpha 1 chain G domain through which most cellular attachment to laminin-1 occurs (19, 26). The beta 1 chain epitope, located near the laminin-1 cross region (amino acids 929-933), curiously includes the YIGSR sequence that was previously thought to be a major cell adhesion site via its apparent interaction with a 32/67-kDa nonintegrin receptor (7). More recent studies attribute cell adhesion activity of the cross region to a cryptic RGDS site in the alpha 1 chain, which serves as ligand for the alpha Vbeta 3 integrin receptor (13). The inability of soluble RGDS (and of an anti-beta 1-integrin antibody) to inhibit regulated secretion underlines the apparent complexity of this region and offers an opportunity to reexplore the possible functional significance of the YIGSR site or adjacent sites that might have been partially blocked by anti-YIGSR peptide antibody. How the gamma 1 chain epitope (amino acids 1420-1439), with differing location (carboxy-terminal E8 region) and sequence, contributes to the role of laminin-1 in regulated secretion and whether it and/or the beta 1 chain are capable of synergy with BM180 remains to be determined. When the lack of beta 1-integrin or RGD involvement and the apparent requirement for cellular contact with BM180 and two uncommon sites in laminin-1 are taken together, it appears that developmental activation of stimulus-secretion coupling may be the main responsibility of supplemental, rather than primary, adhesion mechanisms. Such an arrangement could subtly modulate acinar cell function in the absence of detachment-associated apoptosis.

A striking observation was the augmentation of normalized regulated, but not constitutive, secretion as lacrimal acinar cell plating density was increased under serum-free conditions. Microscopic examination of cultures suggested that enhancement was not due to the formation of higher order cellular aggregates, such as acini, the appearance of which (29) may depend on the presence of contaminating cellular aggregates at the time of plating. Curiously, serum seemed to promote density-dependent aggregation, yet secretion remained constant. What is the identity of this previously undescribed secretion-enhancing activity, which is presumed to be of lacrimal acinar cell origin (although contribution from a contaminating mesenchymal or ductal cell cannot be excluded)? From its earliest stages of development, the lacrimal gland is influenced by a complex and dynamic array of soluble and matrix signals. Thought to be destined mainly for the corneal surface are a number of lacrimal gland-derived growth factors expressed by acinar cells [EGF, TGF-alpha , and TGF-beta (30, 32)], ductal cells [EGF (22)], or periacinar connective tissue cells [hepatocyte growth factor (HGF) (15)]. bFGF and receptor mRNA for bFGF and HGF (15) have also been detected but with unknown cellular origin. In addition, both the growth factor-rich serum transudate common to all tissues and BM180 in the lacrimal acinar cell basement membrane are potential or demonstrated (BM180) modulators of tear protein secretion. Lacrimal EGF expression is first detected 2 wk after birth, in keeping with its apparent role in eyelid opening. In nonocular tissues, TGF-alpha stimulates epithelial, endothelial, and fibroblast proliferation, and HGF is involved in aggregation morphogenesis (1). bFGF could have multiple influences, including epithelial differentiation and promotion of blood vessel growth (31).

Many of these factors tentatively appear to play little or no role in the development of stimulus-secretion coupling by lacrimal acinar cells. Although exogenous bFGF, but not EGF or IGF-I, has been reported to stimulate regulated amylase secretion by cultured pancreatic acini using an atropine-insensitive pathway (3), inclusion of bFGF or EGF in our serum-free lacrimal acinar cell cultures is insufficient to support regulated secretion at low plating densities. TGF-beta and platelet-derived growth factor have been respectively detected at 8.5 ng/ml and 64 pg/ml in BMS (18), among the few growth factors surveyed. The compositionally similar Matrigel contains, in addition, bFGF (1 ng/ml) and IGF-I (7 ng/ml) (16). Yet, increasing BMS 12-fold or preincubating BMS with neutralizing anti-TGF-beta , -EGF, -bFGF, or -IGF-II antibodies had no effect on regulated secretion. Lack of a BMS dose response also argues against the candidacy of BM180, laminin-1, or collagen IV, although one might suggest that the cellular surface of gelled vs. coated BMS may not differ in terms of molar levels of available matrix-bound components. Other candidate growth factors remain that could possibly support regulated secretion. Because serum masked the effect, it may be an alternative source of the same active factor(s). Experiments testing whether the activity (or activities) is soluble (via use of conditioned media) or, alternatively, deposited onto the BMS coat are needed. When all the data are taken together, it is apparent that the regulated secretory pathway is modulated by multiple environmental influences. At the level of the acinar cell-basement membrane interface, there exists BM180, laminin-1, and collagen IV, which appear to act in a beta 1-integrin-independent manner. Also acting on the cells is a previously undescribed secretion-enhancing activity (or activities), presumably derived from lacrimal acinar cells either as a soluble factor or as a matrix factor, which could possibly synergize with basement membrane to promote lacrimal acinar cell differentiation and regulated secretory function.

    ACKNOWLEDGEMENTS

We gratefully acknowledge Drs. Albert Chung, Tom Borg, Roy Ogle, and Yoshi Yamada for protein and antibodies.

    FOOTNOTES

This work was supported by National Eye Institute Grant EY-09747 (to G. W. Laurie). J. D. Glass was supported in part by an institutional grant from the Juvenile Diabetes Foundation.

Present addresses: L. Chen, Div. of Nephrology, Dept. of Internal Medicine, University of Virginia, Charlottesville, VA 22908; J. D. Glass, Dept. of Anesthesiology, University of Virginia, Charlottesville, VA 22908.

Address for reprint requests: G. W. Laurie, Department of Cell Biology, Box 439, Health Sciences Center, University of Virginia, Charlottesville, VA 22908.

Received 5 February 1997; accepted in final form 15 April 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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