Calcium ions protect cell-substratum adhesion receptors against proteolysis : Evidence from immunoabsorption and electroblotting studies

An antibody preparation against BHK cell wheat germ agglutinin receptors (anti-WGA-R), which inhibits fibronectin-mediated cell adhesion, was characterized by immunoabsorption and electroblotting experiments. The results indicated that a 48 kD cell surface WGA-R receptor is protected from proteolysis by Ca²⁺ ions and is important in fibronectin-mediated cell adhesion.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

Qualitative and quantitative interactions of lectins with untreated and neuraminidase-treated normal, wild-type, and temperature-sensitive polyoma-transformed fibroblasts.

The lectin receptors of confluently grown hamster BHK, wild type polyoma virus transformed PyBHK, and temperature-sensitive polyoma transformed ts3-PyBHK fibroblasts were investigated using cell agglutination, quantitative (125I)lectin binding, and ferritin-lectin labeling. PyBHK and permissively grown ts3-PyBHK cells agglutinated more strongly with Ricinus communis I agglutinin (RCA-I)compared to BHK and nonpermissively grown ts3-PyBHK, although saturation binding of (125I)RCA-I to these cells at 4 degrees resulted in a twofold difference in lectin-binding sites on BHK and nonpermissively grown ts3-PyBHK cells (1.0-1.3 x 10 7 sites/cell) compared to PyBHK and permissively grown ts3-PyBHK (0.4-0.6 x 10 7 sites/cell). These cells bound equivalent amounts of (125I)concanavalin A (0.8-1 x 10 7 sites/cell) and (125I)wheat germ agglutinin (1-2.2 x 10 7 sites/cell). Under these binding conditions little endocytosis occurred, as judged by the subsequent release of greater than 90% cell-bound (125I)RCA-I by the RCA-I inhibitor lactose and localization of ferritin-RCA-I exclusively to the extracellular plasma membrane surface. However, if the binding is performed at 22 degrees, only 50% of the bound lectin can be removed by lactose, and ferritin-RCA-I is localized inside the cell within endocytotic vesicles. The relative mobility of RCA-I receptors was examined on ts3-PyBHK cells by the ability of ferritin-RCA-I to induce clustering of its receptors at 22 degrees. RCA-I receptors on permissively grown ts3-PyBHK cells appeared to be more mobile than on nonpermissively grown cells. BHK and PyBHK cells were treated with neuraminidase, and the resulting enzyme-treated cells were assayed for lectin agglutinability and quantitative binding of RCA-I, concanavalin A, and wheat germ agglutinin. Neuraminidase treatment resulted in decreased concanavalin A and wheat germ agglutinability and a slight increase in RCA-I agglutinability. The enzyme-treated BHK and PyBHK cells bound less (125I)wheat germ agglutinin (2.8 x 10 6 and 2.2 x 10 6 sites/cell, respectively) and 2.5 and 6.2 times more (125I)RCA-I (2.5-3 x 10 7) and 3.5-4 x 10 7 sites/per cell, respectively). There was no change in the number of concanavalin A binding sites after neuraminidase treatment. The increase in RCA-I binding sites approximated the decrease in wheat germ agglutinin binding sites indicating that the predominant penultimate oligosaccharide residue to sialic acid on these cells is D-Gal.     

Induction of cell spreading by substratum-adsorbed ligands directed against the cell surface

Studies were carried out to compare the spreading of baby hamster kidney (BHK) cells, which occurs by an interaction between the cells and a specific serum glycoprotein (ASF) adsorbed onto the substratum surface, with the spreading of BHK cells that occurs by an interaction between the cells and substrata coated with ligands directed at various cell surface determinants. The ligands tested were polycationic ferritin, concanavalin A (ConA) and antibody directed against BHK plasma membranes. Cell spreading onto ASF and ligand-coated substrata were similar even though different cell surface components were apparently involved. The similarities were:

1. 1. The shape of the spread cells.

2. 2. The inhibition of cell spreading by conditions that interfere with metabolic activity, block free sulfhydryl groups, or interfere with microtubules and microfilaments.

3. 3. The similar reorganization of certain cell surface antigenic determinants during cell spreading onto any of the substrata.

The results indicate that cell spreading is a general cellular response to specific cell-substratum interactions but does not depend upon binding between a unique cell surface receptor and the substratum.     

Fibroblast spreading and phagocytosis: similar cell responses to different-sized substrata

Experiments were carried out to test the hypothesis that cell spreading and phagocytosis are similar cell responses to different-sized substrata. The following morphological and biochemical studies provided evidence for this supposition. Cells phagocytosed 1.09-micron and 5.7-micron latex beads, but were unable to completely ingest 15.8-micron or 25.7-micron beads. With the larger beads, the cells spread around the bead surfaces with an appearance typical of cells spread on culture dishes. Biochemical studies with cytochalasin D, azide, and iodoacetate, as well as temperature-dependence studies, demonstrated similar responses of cell spreading and phagocytosis to these treatments. Similar cell surface receptors were involved in cell spreading and phagocytosis based upon experiments using antibodies to baby hamster kidney (BHK) cell wheat germ agglutinin receptors. And finally, BHK cell variants with defective plasma fibronectin (pFN) receptors were unable to spread on pFN-coated dishes or ingest pFN-coated beads. Evidence also is presented concerning the "contact" process in cell adhesion. It was found that azide and low temperature inhibited cell attachment per se but did not block fibronectin-receptor interactions based upon cell binding of pFN-coated beads. A possible explanation for the contact process is presented based upon the resistance of cells and beads to shear forces.     

Double label freeze-etch study of the relative topography of concanavalin A and wheat germ agglutinin receptors at the myoblast surface

This report records for the first time double-label freeze-etch electron microscopy of cells in culture. On L6 rat myoblasts, receptors for wheat germ agglutinin and concanavalin A were found distributed together in irregular granular microclusters of cell surface material up to 60 nm in diameter. Simultaneous localization of two different receptor families to such small regions using colloidal gold and ferritin to differentiate between lectin markers proved difficult in our hands. We were able to achieve the desired result using native concanavalin A and ferritin-conjugated wheat germ agglutinin, whose shadowed diameters are measurably different.     

Adhesive specificity of developing cerebellar cells on lectin substrata

Ten lectins, each with a different carbohydrate-binding specificity, have been coupled to tissue culture substrata with carbodiimide [1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene sulfonate] and assayed for their efficacy as substrates for the carbohydrate-specific adhesion of cells dissociated from mouse cerebellum at embryonic Day 13 and postnatal Days 0 and 7. On surfaces treated with concanavalin A, succinyl-concanavalin A, Lens culinaris agglutinin, and wheat germ agglutinin, both embryonic and early postnatal cerebellar cells formed a monolayer. On surfaces coupled with Ricinus communis_I agglutinin (120,000 daltons) both embryonic and postnatal cells formed cellular aggregates with extensive fiber outgrowth. On surfaces treated with peanut agglutinin, Dolichos bifloris agglutinin, Wistaria floribunda agglutinin, soybean agglutinin, or Ulex europaeus_I agglutinin, embryonic cerebellar cells formed cellular aggregates with a cell viability of 25–35% and little or no fiber outgrowth. Postnatal cerebellar cells, in contrast, formed cellular aggregates with a cell viability of 60–70% and extensive fiber outgrowth. On surfaces treated with Ulex europaeus_I agglutinin, cells from postnatal Day 7 formed limited areas of monolayer in addition to cellular aggregates. After 12 hr in vitro the specific attachment of cerebellar cells to lectin-derivatized substrata was inhibited 60–80% by the inclusion of free hapten carbohydrate (50–100 mM) in the growth medium. The addition of soluble concanavalin A or Ricinus communis_I agglutinin (100 μg/ml) was toxic. These studies suggest the presence of glycoconjugate-binding sites for concanavalin A, Lens culinaris agglutinin, and wheat germ agglutinin which promote cerebellar cellular adhesion.     

Role of integrin receptors in manganese-dependent BHK cell spreading on albumin-coated substrata.

In manganese-containing medium, tissue cells can spread on albumin and other substrata typically nonadhesive for cells in calcium/magnesium-containing medium. To learn whether integrin receptors play a role in Mn-dependent adhesion, we tested the effects of RGD peptides and polyclonal anti-fibronectin receptor antibodies on BHK cell spreading on fibronectin and albumin-coated substrata. In Ca/Mg-containing medium on fibronectin substrata, the RGD-related peptides GRG-DSP and GRGDS but not RGDS inhibited cell spreading. In Mn-containing medium, spreading on albumin was inhibited by GRGDSP and GRGDS and also by RGDS. GRGESP, on the other hand, did not inhibit cell spreading under any condition tested. Antibodies directed against fibronectin receptors also inhibited Mn-dependent cell spreading on albumin substrata, but higher levels of antibody were required than were necessary to inhibit Ca/Mg-dependent spreading on fibronectin. On the basis of these results, we suggest that integrin receptors, but probably not fibronectin receptors, mediate Mn-dependent BHK cell spreading on albumin.     

Adhesion sites of murine fibroblasts on cold insoluble globulin-adsorbed substrata

The attachment and detachment behavior of three mouse fibroblast cell lines adhering to plastic tissue culture substrata coated with the serum protein cold-insoluble globulin (CIg) resembles that seen on the usual serumcoated substrata. The transformed cell line SVT2 spreads more extensively on the CIg-coated than on the serum-coated substratum, while the nontransformed Balb/c 3T3 line and concanavalin A-selected “revertant” of SVT2 are equally well spread on both substrata. In all three cases, immunofluorescence microscopy using antibodies to CIg suggests that the cells are more tightly apposed to the CIg-coated substratum than to the serum-coated substratum. Substrate-attached material (SAM), which contains cell-substratum adhesion sites and which is left after EGTA-mediated detachment of cells, is enriched for cell surface fibronectin and glycosaminoglycans (GAG). When cells are seeded onto CIg-coated substrata rather than serum-coated substrata, there is an increased deposition of GAG but a comparable deposition of cellular proteins. The protein distribution of the two types of SAM are identical as analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, including fibronectin content. This indicates that substratum-bound CIg cannot functionally substitute for cell surface fibronectin in these adhesion sites. Analysis of the GAG deposited on CIg-coated substrata reveals that hyaluronate and the chondroitins are increased to a much greater extent than heparan sulfate; however, the ratio of hyaluronate to the various chondroitin species is invariant. These data provide further evidence that hyaluronate and the chondroitins are deposited in adhesion sites in well-defined stoichiometric proportions, possibly as supramolecular complexes, and that CIg may mediate adhesion of cells in the serum layer by binding to GAG-containing proteoglycans.     

BHK cell variant with defective fibronectin receptor function

Baby hamster kidney cells were mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine and selected to obtain a population of non-attaching cells. The cell variant FN-1 was cloned from the non-attaching cell population, recloned, and tested for cell adhesive interactions using four different assays of fibronectin (pFN) receptor function: cell attachment and spreading on culture dishes and cell binding and phagocytosis of latex beads. On pFN-coated culture dishes, FN-1 cells had decreased attachment compared to parental cells and were unable to spread. With pFN-coated beads, only one third as many pFN-bead binding sites could be detected on FN-1 cells as on the parental cells, and the FN-1 cells were unable to phagocytose the pFN-coated beads. In other studies, the variant cells were able to attach normally and spread partially on substrata coated with polycationic ferritin, concanavalin A, or anti-BHK cell surface antibody. The results suggest that the pFN-receptor function of FN-1 cells is defective.     

Stimulation of the membrane-bound, magnesium-dependent adenosine triphosphatase of mouse neuroblastoma by concanavalin A and wheat germ agglutinin.

The effect of the plant lectins concanavalin A and wheat germ agglutinin on the membrane-bound Mg⁺²-dependent ATPase of an adrenergic clone of mouse neuroblastoma was examines. When cell membranes were treated with concanavalin A or wheat germ agglutinin, a dose-related increase in ATPase-specific activity was observed. Maximal stimulation was greater with wheat germ agglutinin than with concanavalin A; half-maximal and maximal stimulation occurred at similar lectin concentrations. Concanavalin A-dependent stimulation was blocked by α-methylmannoside but not by N-acetylglucosammine. Conversely, stimulation with wheat germ agglutinin was prevented by N-acetylglucosamine but not by α-methylmannoside. The combined effects of concanavalin A and wheat germ agglutinin were greater than the individual effects of either, but were not additive. The results suggest that these lectins interact specifically with membrane glycoproteins or glycolipids, resulting in enhancement of Mg⁺²-dependent ATPase activity.     

Regulation of integrin-mediated adhesion at focal contacts by cyclic AMP

Cyclic AMP (cAMP) elevation causes diverse types of cultured cells to round partially and develop arborized cell processes. Renal glomerular mesangial cells are smooth, muscle-like cells and in culture contain abundant actin microfilament cables that insert into substratum focal contacts. cAMP elevation causes adhesion loss, microfilament cable fragmentation, and shape change in cultured mesangial cells. We investigated the roles of the classical vitronectin (αVβ3 integrin) and fibronectin (α5β1 integrin) receptors in these changes. Mesangial cells on vitronectin-rich substrata contained microfilament cables that terminated in focal contacts that stained with antibodies to vitronectin receptor. cAMP elevation caused loss of focal contact and associated vitronectin receptor. Both fibronectin and its receptor stained in a fibrillary pattern at the cell surface under control conditions but appeared aggregated along the cell processes after cAMP elevation. This suggested that cAMP elevation caused loss of adhesion mediated by vitronectin receptor but not by fibronectin receptor. We plated cells onto fibronectin-coated slides to test the effect of ligand immobilization on the cellular response to cAMP. On fibronectin-coated slides fibronectin receptor was observed in peripheral focal contacts where actin filaments terminated, as seen with vitronectin receptor on vitronectin-coated substrata, and in abundant linear arrays distributed along microfilaments as well. Substratum contacts mediated by fibronectin receptor along the length of actin filaments have been termed fibronexus contacts. After cAMP elevation, microfilaments fragmented and fibronectin receptor disappeared from peripheral focal contacts, but the more central contacts along residual microfilament fragments appeared intact. Also, substratum adhesion was maintained after cAMP elevation on fibronectin—but not on vitronectincoated surfaces. Although other types of extracellular matrix receptors may also be involved, our observations suggest that cAMP regulates adhesion at focal contacts but not at fibronexus-type extracellular matrix contacts. © 1993 Wiley-Liss, Inc.     

Modulation of WGA binding sites on marrow sinus endothelium in state of stimulated erythropoiesis: a possible mechanism regulating the rate of cell egress

To study the regulation of cellular and molecular traffic across the marrow-blood barrier, rat marrow endothelial surface was incubated with ferritin-conjugated concanavalin A, wheat germ agglutinin (WGA), recinus communis agglutinin I, and phytohemagglutinin. Normal animals were compared with those after erythropoietic stimulation (phenylhydrazine-induced hemolysis, phlebotomy). A selective and significant reduction in the density of WGA receptors, but not other lectins was noted congruent to the degree of reticulocytosis. Neuraminidase treatment also reduced WGA binding sites and the surface negative charge as detected by polycationic ferritin (PCF). Thus, the reduction in WGA binding sites, may reflect a decrease in the density of membrane sialic acid, rendering the endothelial surface charge less negative and providing an electrostatic attraction for the negatively charged surface of reticulocytes. The findings may also be explained by an increase in the frequency of WGA-excluding fenestrae in the endothelium. These areas, lacking sialic acid, may provide unstable areas in the membrane suitable for the passage of cells and molecules in both directions. It is concluded that, by modulating the density of sialic acid residues, the endothelium may regulate the traffic of cells and molecules across the marrow-blood barrier.     

Collagen can modulate cell interactions with fibronectin

We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed.     

Identification of a novel glycoprotein (AGp110) involved in interactions of rat liver parenchymal cells with fibronectin

We have identified an integral membrane glycoprotein in rat liver that mediates adhesion of cultured hepatocytes on fibronectin substrata. The protein was isolated by affinity chromatography of detergent extracts on wheat germ lectin-Agarose followed by chromatography of the WGA binding fraction on fibronectin-Sepharose. The glycoprotein (AGp110), eluted at high salt concentrations from the fibronectin column, has a molecular mass of 110 kD and a pI of 4.2. Binding of immobilized AGp110 to soluble rat plasma fibronectin required Ca2+ ions but was not inhibited by RGD peptides. Fab' fragments of immunoglobulins raised in rabbits against AGp110 reversed the spreading of primary hepatocytes attached onto fibronectin-coated substrata, but had no effect on cells spread on type IV collagen or laminin substrata. The effect of the antiserum on cell spreading was reversible. AGp110 was detected by immunofluorescence around the periphery of the ventral surface of substratum attached hepatocytes, and scattered on the dorsal surface. Immunohistochemical evidence and Western blotting of fractionated liver plasma membranes indicated a bile canalicular (apical) localization of AGp110 in the liver parenchyma. Expression of AGp110 is tissue specific: it was found mainly in liver, kidney, pancreas, and small intestine but was not detected in stomach, skeletal muscle, heart, and large intestine. AGp110 could be labeled by lactoperoxidase-catalyzed surface iodination of intact liver cells and, after phase partitioning of liver plasma membranes with the detergent Triton X-114, it was preferentially distributed in the hydrophobic phase. Treatment with glycosidases indicated extensive sialic acid substitution in at least 10 O-linked carbohydrate chains and 1-2 N-linked glycans. Immunological comparisons suggest that AGp110, the integrin fibronectin receptor and dipeptidyl peptidase IV, an enzyme involved in fibronectin-mediated adhesion of hepatocytes on collagen, are distinct proteins.     

A method for the fine-structural demonstration of lectin receptors

A new two-step method using an Fc-fragment/ferritin conjugate as a marker for the visualization of lectin-binding sites on neuronal and other cell membranes is described. In this study of rat synaptosomes, three lectins were tested: concanavalin A, mistletoe lectin I and wheat germ agglutinin. The specificity of the method was proved by control experiments.     

Concanavalin A and wheat germ agglutinin receptor activity of sialoglycopeptides isolated from the surface of Novikoff hepatoma cells

Novikoff ascites hepatoma cells were highly agglutinable by the plant lectins concanavalin A and wheat germ agglutinin. Treatment of the intact cells with papain released from the cell surface a glycopeptide fraction which possessed concanavalin A and wheat germ agglutinin receptor activity, as judged by its ability to inhibit lectin-induced hemagglutination. A component of the cell-surface glycopeptide fraction, excluded from Sephadex G-50, possessed lectin receptor activities reflecting the cytoagglutination properties of the intact cells from which it was derived. Further resolution of this component by pronase digestion, gel filtration, and ion-exchange chromatography resulted in the isolation of sialoglycopeptides which exhibited potent and specific concanavalin A receptor activity.     

Pinocytosis and locomotion in amoebae XIV. Demonstration of two different receptor sites on the cell surface ofAmoeba proteus

Summary Two different receptor sites, located on the cell surface ofAmoeba proteus were detected by using fluorescent analog cytochemistry (FAC) and electron microscopy (EM). Bovine serum albumin labeled with fluoresceine-isothiocyanate (FITC-BSA) and unlabeled ferritin bind, in a pH-dependent manner, as cations at the outer filaments of the mucous layer. The anionic receptor sites show a high affinity for Ca-ions which suppress the binding capacity of FITC-BSA and ferritin at low pH-values. The cation receptors obviously play an important role in the initiation of pinocytosis as demonstrated by the internalization, intracellular translocation and sequestration of the FITC-BSA. FITC- or ferritin-labeled concanavalin A (FITC-Con A, ferritin-Con A) bind predominantly in a pH-independent manner at the tips of the outer filaments and the basal zone of the mucous layer. The binding capacity of FITC-Con A is not influenced by external Ca-ions. Other lectins such asDolichos bifloris agglutinin (DBA), peanut agglutinin (PNA),Ricinus communis agglutinin I (RCA I), soybean agglutinin (SBA),Ulex europaeus agglutinin I (UEA I) and wheat germ agglutinin (WGA) are not specifically bound to the cell surface. So far, no experimental evidence has been gathered for the definitive function of a Con-A receptor in the mucos layer ofAmoeba proteus.Abbreviations BSA bovine serum albumin - Con A concanavalin A - CTC chlorotetracycline - DBA Dolichos bifloris agglutinin - DTE dithioeritritol - FITC fluorosceine-isothiocyanate - IEP iso electric point - PIPES 1-4-piperazine-diethane sulfonic acid - PNA peanut agglutinin - RCA I Ricinus communis agglutinin I - SBA soybean agglutinin - Uac uranylacetat - UEA I Ulex europaeus agglutinin I - WGA wheat germ agglutinin     

Regulation of surfactant phospholipid secretion from isolated rat alveolar type II cells by lectins

The major surfactant-associated protein is a potent inhibitor of surfactant phospholipid secretion from isolated type II cells. Since the major surfactant-associated protein contains a carboxy terminal polypeptide domain which is homologous to the lectin-like liver mannose-binding protein, we tested whether lectins inhibit surfactant phospholipid secretion from rat alveolar type II cells. Concanavalin A, wheat germ agglutinin and Maclura pomifera agglutinin were potent inhibitors of surfactant phospholipid secretion. When adenosine 5'-triphosphate (ATP) was utilized as a secretagogue, the IC50 values for inhibition of surfactant phospholipid secretion were 5.10(-7) (wheat germ agglutinin), 1.10(-6) (concanavalin A) and 2.5.10(-5) M (M. pomifera agglutinin). Similar results were obtained when 12-O-tetradecanoylphorbol 13-acetate was utilized as a secretagogue: IC50 values of 1.10(-6) M for concanavalin A and wheat germ agglutinin and 2.5.10(-5) M for M. pomifera agglutinin. Hapten sugars were utilized to antagonize the inhibitory effect of the lectins. N-Acetyl-D-glucosamine significantly reversed inhibition of phospholipid secretion by wheat germ agglutinin in a dose-dependent fashion and methyl alpha-D-mannoside significantly reversed inhibition of phospholipid secretion by concanavalin A. N-Acetyl-D-galactosamine had no significant effect on inhibition of secretion produced by any of the lectins. The inhibitory effect of the lectins did not appear to be due to cytotoxicity since lactate dehydrogenase was not released above control levels and the inhibition of the surfactant phospholipid secretion by wheat germ agglutinin could be reversed after treatment of cells with wheat germ agglutinin by washing the lectin from the cells followed by treatment of the cells with ATP. These studies demonstrate a direct inhibitory effect of plant lectins on phospholipid secretion from type II cells in vitro.