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To determine if singlet oxygen (O2(1 delta g)) is produced by neutrophils (PMNs) during the process of phagocytosis, glass beads were coated with a specific chemical trap for O2(1 delta g), 9,10-diphenylanthracene (DPA). Singlet oxygen, but not other reactive oxygen species, reacts rapidly with DPA at a rate of kr = 1.3 x 10(6) M-1 s-1 to form a stable product, DPA-endoperoxide (Corey, E. J., and Taylor, W. C. (1964) J. Am. Chem. Soc. 86, 3881-3882; Wasserman, H. H., Scheffer, J. R., and Cooper, J. L. (1972) J. Am. Chem. Soc. 94, 4991-4996; Turro, N. J., Chow, M.-F., and Rigaudy, J. (1981) J. Am. Chem. Soc. 103, 7218-7224). The production of DPA-endoperoxide was determined by ultraviolet spectroscopy as a decrease in DPA absorbance at 355 nm. The absorbance of DPA was normalized to the absorbance of perylene, which was included in the coating on the beads as a nonreactive, internal standard. In the present study, DPA- and perylene-coated beads were initially allowed to adhere to fibronectin-coated coverslips. PMNs were then added to the bead-coated coverslips and allowed to adhere and phagocytose the beads for 1 h at 37 degrees C. In some experiments, 4B-phorbol-12-myristate-13-acetate (PMA) (1 ng/2.5 x 10(7) cells/ml), a known activator of the PMN NADPH-oxidase, was added as a co-stimulant. The amount of O2(1 delta g) produced by phagocytically stimulated PMNs was calculated to be 11.3 +/- 4.9 nmol of O2(1 delta g)/1.25 x 10(6) cells. Low dose PMA co-stimulation increased the production of O2(1 delta g) to 14.1 +/- 4.1 nmol/1.25 x 10(6) cells. Averaged together these amounts represent approximately 19 +/- 5.0% of the total oxygen consumed by PMNs in response to DPA- and perylene-coated beads. The specificity of the DPA reaction with O2(1 delta g) was confirmed by warming to 120 degrees C, which releases O2(1 delta g) from the DPA-endoperoxide, regenerating the parent DPA compound (Wasserman et al., 1972; Turro et al., 1981) and the absorbance at 355 nm. In addition, beta-carotene, an avid quencher of O2(1 delta g), was included in the coating of some bead preparations; assays in which these beads were used showed no change in the absorbance at 355 nm. Singlet oxygen production by myeloperoxidase was also measured using the coated bead assay and the results suggest that this is a major pathway by which singlet oxygen is generated in phagocytically stimulated PMNs.(ABSTRACT TRUNCATED AT 400 WORDS)

Progressive graft arteriosclerosis is responsible for the majority of late deaths occurring in cardiac transplant recipients. In order to define a model of this disease in the rat, we exchanged heterotopic cardiac allografts between MHC-compatible inbred strains. Lewis rats served as donors and F-344 rats as recipients. Twenty allografts were followed by daily palpation and removed at the time of terminal rejection or on the 120th postoperative day for pathologic study. Sixteen allografts (80%) survived at least three weeks, and five allografts (25%) survived indefinitely. The majority of arteries (greater than 90%) examined demonstrated significant intimal disease; histologic findings in lesions in allografts rejecting at early time points included intense mononuclear cell infiltration of the intima, while lesions in long-term-surviving allografts demonstrated fibrous intimal thickening, which is characteristic of graft arteriosclerosis seen clinically. A limited course of cyclosporine therapy in F-344 recipients increased the incidence of indefinite allograft survival from 25% to 86%, and was associated with a modest reduction in the amount of intimal disease observed. These results suggest that this model should be useful in future studies regarding the pathogenesis and therapy of cardiac graft arteriosclerosis.

We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.

Basic fibroblast growth factor (bFGF) is mitogenic for smooth muscle cells (SMC) and angiogenic. We examined the in vivo effects of bFGF in balloon denuded carotid arteries of laboratory rats. bFGF was administered continuously from polymer-based devices at 34 ng/d into the periadventitial space of rat carotid arteries for 2 wk. Intimal hyperplasia was not observed in the absence of injury or with lipopolysaccharide induced endothelial dysfunction. Different degrees of vascular injury produced proportionally more intimal hyperplasia. bFGF increased the intimal hyperplastic response 1.3-fold with severe vascular injury, and 2.4-fold with more mild injury. Increased cell proliferation, not extracellular matrix production, accounted for these effects. Cell density was unchanged for the control and bFGF-treated groups, and the number of proliferating intimal cells at 2 wk rose to an amount equivalent to the increase in mass; 1.9- and 4.0-fold for severe and lesser injury, respectively. The relative ability of heparin to reduce SMC proliferation was not altered by the presence of bFGF.bFGF also induced profound angiogenesis within and surrounding the polymeric releasing device, and in the vasa vasorum immediately around the injured arteries. bFGF's effect on vasa was linearly related to the amount of SMC proliferation within the blood vessel. Thus, the in vivo mitogenic and angiogenic potential of bFGF are coupled, and may be similarly modulated by the products of local injury and/or factors in the vessel wall.

Recently, much experimental evidence has accumulated contending that hypercholesterolemia is an aggravating factor in the progression of initial glomerular injury to glomerulosclerosis. Many of the features of progressive glomerular disease share biological properties with those of atherosclerosis, and data suggesting a noxious role for oxidized low-density lipoprotein in the glomerulosclerosis process are emerging. This review discusses these issues and examines the pathobiology of cholesterol-induced glomerular injury.

The proliferation of vascular smooth muscle cells (VSMC) is strongly inhibited by whole heparin both in vitro and in vivo. To identify and characterize antiproliferative, but nonanticoagulant heparin derivatives, heparin fragments made by periodate treatment were produced and acylated with 2-, 4-, or 6-carbon chain lengths. In culture, the 4- and 6-carbon acylated compounds were more effective than whole heparin in inhibiting serum stimulated VSMC growth at equal mass or approximately equal mean molar concentrations. Further testing was performed in the rat carotid balloon injury model. Myointimal VSMC proliferation produced by balloon catheterization of rat carotid arteries was inhibited by the 4-carbon acylated compound as effectively as heparin at the same mass dose. Importantly, unlike heparin, the 4-carbon acylated compound had no anticoagulant effect in vivo. These experiments suggest nonanticoagulant, acylated heparin derivatives may have a pharmacologic role in preventing myointimal proliferative lesions that are responsible for failures of vascular surgeries and angioplasties.

Arachidonate activation of the NADPH-oxidase in intact neutrophils and in a cell-free O2- generation system was compared to synergistic activation in response to arachidonate and agents that effect protein phosphorylation. In intact neutrophils, suboptimal doses of retinal which increase protein phosphorylation, or 4B-phorbol 12-myristate 13-acetate (PMA) an activator of protein kinase C, induced minimal O2- release, but primed neutrophils to release enhanced amounts of O2- in response to 2.5 microM arachidonate. In contrast to retinal or PMA, okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, did not induce any release of O2-, but significantly increased the maximal rate and duration of O2- release in response to arachidonate. In the cell-free system, only arachidonate induced O2- generation. Consistent with previous findings, activation of the cell-free system was dependent of the presence of light membranes, cytosol, NADPH, Mg2+, and 82 microM arachidonate. Pretreatment of neutrophils with suboptimal doses of PMA or retinal had little effect on the arachidonate-stimulated release of O2- in cell-free preparations of these cells. However, cytosol (but not light membranes) from PMA or retinal-primed neutrophils was more effective in completing resting membrane NADPH-oxidase activity when compared to cytosol from resting cells. The addition of protein kinase C inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine decreased the effectiveness of PMA-primed cytosol to complete the cell-free system, but had little effect on cytosol obtained from cells primed with retinal. The addition of protein phosphatase inhibitors, p-nitrophenyl phosphate or okadaic acid to neutrophil cavitates increased 3-fold the release of O2- in cell-free preparations of these cells. Okadaic acid and p-nitrophenyl phosphate also increased the effectiveness of both cytosol and light membranes to complete the cell-free system when combined with cytosol or light membranes from resting neutrophils, respectively, indicating that both fractions are affected by the inhibition of protein phosphatase activity. These data indicate that increases in protein phosphorylation alone do not lead to the activation of the NADPH-oxidase, but in addition to the requirement of an anionic amphiphile, the release of O2- from intact neutrophils or in the cell-free system is increased by stimulus activation of protein kinase C or more impressively by inhibition of protein phosphatase activity.

The authors investigated the effect of heparin on the development of myointimal proliferative lesions in a rat vein graft model. Intimal thickening in this model was most pronounced in the anastomotic regions, and was composed principally of vascular smooth muscle cells, as identified by immunocytochemistry with anti-muscle actin antibody, HHF-35. Medial thickening was less cellular, and evenly distributed throughout the grafts. Continuous, intravenous infusion of whole heparin at 0.3 mg/kg/hr effectively inhibited the development of myointimal proliferative lesions, although with no effect on medial thickening. The authors suggest that heparin, through its antiproliferative activity for vascular smooth muscle cells, may have a potentially important pharmacologic role in preventing vein graft failure, which most commonly results from the development of myointimal proliferative lesions.

We show that tubular structures present in phorbol ester-stimulated macrophages are sensitive to commonly used chemical fixatives (i.e., they usually become fragmented during fixation). These structures are well preserved in macrophages that are physically fixed by rapid-freezing and subsequent freeze-substitution in osmium-acetone. We have developed methods that combine rapid-freezing, freeze-substitution, and enzyme cytochemistry for preservation of these tubular structures and for detection of endocytosed material (i.e., horseradish peroxidase). This method of rapid-freeze cytochemistry may be useful in other situations where chemical fixation does not adequately preserve cell structures, particularly of membrane compartments.

To investigate NADPH-oxidase activation, we studied the effects of free fatty acid (FFA), their uncharged derivatives, and calcium on membrane lipid structure and superoxide anion (O2-) release from intact neutrophils and in cell-free O2(-)-generating systems. This study determined that in calcium-free phosphate-buffered saline (PBS), cis-unsaturated FFA (cis FFA), trans-unsaturated FFA (trans FFA), and to a limited extent saturated FFA decreased the polarization of the membrane lipid structure probe 1,6-diphenyl-1,3,5-hexatriene (DPH), and these decreases correlated with partitioning of the FFA into the plasmalemma and the release of O2- from intact neutrophils and in cell-free preparations. Although a decrease in DPH polarization was always observed under conditions that resulted in the release of O2-, there was not a direct correlation between the amount of decrease in DPH polarization and the release of O2-. Trans FFA did not induce a dose-dependent decrease or as dramatic a decrease in DPH polarization compared with cis FFA, yet the trans FFA stimulated a greater release of O2- at 2.5, 5, and 10 microM concentrations. In addition, responses of the neutrophil to trans FFA, but not cis FFA, were differentially affected by the presence of calcium. When 0.1 mM calcium was added to the PBS decreases in DPH polarization in response to trans FFA were reduced by greater than 60%, whereas O2- was reduced by only 25-36%. The addition of 0.1 mM calcium 3 min after the trans FFA had partitioned into the membrane also reversed by 50-65% decreases in DPH polarization but did not affect the release of O2-. In the presence of 0.9 mM calcium, only the cis FFA decreased DPH polarization or stimulated the release of O2-. Calcium is known to interact more readily with the ionized carboxyl group of trans FFA, neutralizing the anionic charge through an electrostatic interaction. In support of the requirement for the ionized carboxyl group, structurally similar uncharged cis, trans, and saturated fatty alcohols; methyl esters; and aldehydes decreased DPH polarization in the absence and presence of 0.9 mM calcium; however, none of these compounds stimulated the release of O2-. These results indicate that, in addition to the partitioning of FFA into the plasmalemma, which results in calcium-modulatable decreases in DPH polarization, activation or assembly of the NADPH-oxidase requires the ionized carboxyl group.

Recent studies have shown that nonrenal extracellular matrices are composed of collagenous proteins with properties that are different from those of fibrillar collagens and type IV collagen. The structures of these newly described collagens suggest that they may provide connections between specific matrix molecules and, in so doing, partially determine the three-dimensional structure of the matrix. The molecular composition and organization of normal and diseased mesangial matrix are incompletely understood. As a means to further understand the structure of the mesangial matrix, we have further defined the collagenous proteins synthesized by rat mesangial cells. By using monospecific antibodies, we have shown that these cells secrete types III and IV collagen. Metabolic labeling and electrophoretic analysis of proteins isolated from the culture medium revealed that mesangial cells also synthesize and secrete short-chain collagenous proteins of 80, 75, and 69 kDa which differ in their binding to concanavalin A. Cell-free translation of mesangial mRNA demonstrated the presence of two additional collagenase sensitive proteins, 40 and 35 kDa in size, which were not detected in the culture medium. These results demonstrate that mesangial cells synthesize several short-chain collagenous proteins which may be similar to short-chain collagens isolated from nonrenal tissues. Further definition of these proteins will lead to a greater understanding of the extracellular matrix elaborated by mesangial cells.

Continuous intravenous infusion of heparin suppresses smooth muscle cell proliferation in rats after endothelial injury but may lead to hemorrhage and other complications. The anticoagulant property has been removed from chemically modified heparin without loss of antiproliferative effect but use of such compounds is still limited. In this study ethylene-vinyl acetate copolymer matrices containing standard and modified heparin were placed adjacent to rat carotid arteries at the time of balloon dendothelialization. After 14 days arterial occlusion by smooth muscle cell proliferation was defined. Matrix delivery of both heparin compounds effectively diminished this proliferation in comparison to controls without producing systemic anticoagulation or side effects. In addition, this mode of therapy appeared more effective than the administration of the same agents by either intravenous pumps or heparin/polymer matrices placed in a subcutaneous site distant from the injured carotid artery. Thus, heparin's inhibition of smooth muscle cell proliferation after vascular injury might be most effective within the microenvironment of the injured vessel wall, and the accelerated atherosclerosis or restenosis that often follows angioplasty and other vascular interventions might best be treated with site-specific therapy.

Heparin is a potent inhibitor of vascular smooth muscle cell (VSMC) growth. In this paper we show that heparin suppressed the induction of c-fos and c-myc mRNA in rat and calf VSMC. This effect of heparin is closely associated with its growth-inhibitory activity, as shown by isolating and characterizing a strain of rat VSMC that was resistant to heparin's antiproliferative effect; heparin did not suppress c-fos mRNA induction in these cells. Moreover, neither a nonantiproliferative heparin fragment or other glycosaminoglycans that lack growth-inhibitory activity repressed c-fos or c-myc mRNA levels. The effect of heparin on c-fos mRNA induction was selective for specific mitogens, as heparin inhibited c-fos mRNA induction in phorbol 12-myristate 13-acetate (TPA) stimulated but not epidermal growth factor (EGF) stimulated VSMC. The effect of heparin on gene expression is independent of ongoing protein synthesis, and inhibition of c-fos mRNA is at the transcriptional level. These results suggest that heparin may selectively inhibit a protein kinase C-dependent pathway for protooncogene induction and that this may be one mechanism used by heparin to inhibit cell proliferation.

Polymorphonuclear leukocytes can produce active oxygen species such as hydrogen peroxide and superoxide under various conditions. Because these substances can be toxic to cells, it is possible that the interaction between the circulating leukocytes and the blood vessel wall, either in normal circulation or during the acute inflammatory response, could damage the endothelial lining. Using an in vitro system of cultured endothelial cells and isolated polymorphonuclear leukocytes, we have measured the levels of detectable superoxide when neutrophils are attached to either endothelial monolayers or to plastic. Our results show that the levels of superoxide, on a per-cell basis, are lower when the neutrophils are attached to endothelium than when attached to plastic, even if the neutrophils are stimulated with phorbol myristate acetate. This is also reflected in data showing that no injury occurs to the endothelial cells, as measured by 51Cr release, under these same conditions. When endothelial cells are pretreated with an inhibitor of superoxide dismutase, diethyldithiocarbamate, the levels of superoxide detected are the same for neutrophils stimulated on plastic and those on the endothelial monolayer, suggesting that endothelial superoxide dismutase may remove a portion of the neutrophil-generated superoxide from the detection system. Further evidence for the role of endothelium in destroying superoxide is suggested by results that show that the level of detectable superoxide released from neutrophils attached to formalin-fixed endothelial monolayers is the same as that for neutrophils attached to plastic. It is important to note that with the inhibitor of superoxide dismutase present, the endothelial monolayers do not display enhanced 51Cr release under the conditions employed. When both endothelial catalase and glutathione reductase are inhibited, we detect increased 51Cr release from endothelial cells in response to stimulated neutrophils. Our results show that the endothelial cells are important in affecting the apparent reduction of toxic oxygen products derived from polymorphonuclear leukocytes attached to their surface.

We have examined the nature of focal glomerular sclerosis (FGS) in fawn-hooded (FH) rats. The fawn-hooded rat develops pathologic features similar to those observed in steroid-resistant focal glomerular sclerosis, ie, by light microscopy some of the glomeruli appear normal but others show areas of solidification confined to one or two lobules of the tuft. The pathogenesis of this disease is not well known and there is a great need for an animal model. In the FH animal, a marked difference in the development of the lesion was noted between male and female rats. Fifty percent of 4-month-old males had proteinuria in excess of 10 mg/day (none of the females had significant proteinuria), while all 12-month-old males had proteinuria in excess of 45 mg/day (female 12-month-old FH rats had mean proteinuria of 7 mg/day). At 6 months of age continuing through 12 months of age, male FH rats had mesangial deposits of IgG, IgM, and, occasionally, C3, demonstrable by immunofluorescence, whether or not FGS was present. Subepithelial electron-dense deposits were never seen by electron microscopy either at 6 of 12 months. Six-month-old animals frequently did not exhibit FGS. Instead, the glomerular epithelial cells, exhibited fusion of foot processes, vacuolization, and, in some areas, focal loss of the epithelial covering on the glomerular basement membrane (GBM). Six-month-old males with proteinuria exhibited focal loss of negative charge from all layers of the filtration barrier. The GBM from sclerotic glomeruli of 12-month-old rats was commonly denuded of epithelium. None of the animals in this study was uremic. FH rats demonstrated FGS associated with progressive glomerular epithelial cell injury.

3T3 cells that have undergone adipose differentiation in vitro secrete factor(s) that stimulate angiogenesis (neovascularization) in vivo. When medium containing 0.5% fetal calf serum was conditioned by 3T3-F442A adipocytes, it stimulated angiogenesis when placed on the chicken chorioallantoic membrane. Control medium or medium conditioned by preadipocytes did not stimulate angiogenesis, even at much higher doses. Thus, the production of the angiogenic activity is strongly dependent upon differentiation of the adipocytes. The degree of the angiogenic response to adipocyte-conditioned medium was potentiated by heparin; heparin added to unconditioned medium or to preadipocyte-conditioned medium was not angiogenic. The adipocyte-conditioned medium also strongly stimulated, in a differentiation-dependent fashion, the motility of aortic and capillary endothelial cells in a modified Boyden chamber assay. Checkerboard analysis cells indicated that 75% of the motility-stimulating activity was chemotactic in nature. The chemotactic activity has an apparent specificity for endothelial cells, in that chemotaxis of smooth muscle cells and fibroblasts was stimulated to a much lesser extent. These results, in conjunction with our previous demonstration of an endothelial cell mitogen produced by 3T3 adipocytes [Castellot, J. J., Jr., Karnovsky, M. J. & Spiegelman, B. M. (1980) Proc. Natl. Acad. Sci. USA 77, 6007-6011] indicate that the differentiation of these cells is closely linked to the production of factors that stimulate angiogenesis in vivo and growth and chemotaxis of endothelial cells in vitro.

Brush borders isolated from epithelial cells from the small intestine of neonatal rats are able to contract in the presence of ATP and Mg2+; Ca2+ is not required. Contraction is characterized by a pinching-in of the plasma membrane in the region of the zonula adherens and a subsequent rounding of the brush borders. No movement or consistent shortening of the microvilli is observed. The contraction appears to involve the 5- to 7-nm diameter microfilaments in the terminal web which associate with the zonula adherens. These filaments bind heavy meromyosin as do the actin core filaments of the microvilli. A model for contraction is presented in which, in the intact cell, terminal web filaments and core filaments interact to produce shortening of the microvilli.

We have previously reported that cis-unsaturated free fatty acids, e.g. linoleic acid, introduced as free fatty acids into lymphocyte membranes inhibited surface immunoglobulin capping. Trans-unsaturated and saturated free fatty acids had no effect. These results were interpreted as being due to perturbation of specific membrane lipid domains. Corps, A. N., Pozzan, T., Hesketh, T. R., and Metcalfe, J. C. (1980) J. Biol. Chem. 255, 10566-10568) in a recent paper argued, however, that linoleic acid causes depletion of ATP, as measured by the luciferin/luciferase assay, and respiratory uncoupling. We now present data showing that under the same conditions as the capping inhibition, there is neither ATP depletion, as found using a hexokinase/glucose-6-phosphate dehydrogenase assay, nor respiratory uncoupling. Linoleic acid produces artifacts in ATP measurements made using the luciferin/luciferase assay which leads to an erroneous conclusion regarding the ATP dependence of the capping inhibition.

An aldehyde oxidase extracted from guinea pig granulocytes with isotonic KCl catalyzes the oxidation of a variety of aliphatic aldehydes and 2-OH-pyrimidine. The stoichiometry of the oxidation of 2-OH-pyrimidine is consistent with the reaction 2-OH-pyrimidine + OH- + O2 leads to uracil + H2O2. Between 75 and 90% of the peroxide produced results from dismutation of superoxide formed as an intermediate. The Km for 2-OH-pyrimidine is approximately 1.65 mM and the maximum velocity is 22.9 +/- 5.5 S.D. nmol of superoxide/min/10(7) cells. This same maximum velocity is observed for the substrates isobutyraldehyde, propionaldehyde, and acetaldehyde. Unlike other aldehyde oxidases, this enzyme is inactive with purines as substrates and is insensitive to antimycin A, menadione, and Triton X-100. The enzyme is inhibited by cyanide, methanol, and arsenite. The apparent molecular weight is approximately 340,000 +/- 25,000 and the pH optimum is in the range of 7.5 to 9.0. Electron cytochemistry reveals an association of this oxidase with the phagosome membrane. The potential significance of this oxidase is discussed in relation to microbicidal mechanisms.

Capping of surface Ig by anti-Ig antibodies involves a membrane perturbation requiring an energy-dependent step. Lymphocytes treated with anti-Ig are stimulated to move. Previously, we had shown that movement was not essential for capping, although it influenced the localization of the cap. We have investigated the role of cell movement and of microtubular proteins in this phenomenon. Treatment of B lymphocytes with colchicine does not affect capping of Ig nor does it affect the increase in translational movement produced by anti-Ig antibodies. Treatment of lymphocytes with cytochalasin B stops translational movement and may affect capping to some degree under appropriate circumstances. Lymphocytes treated with both drugs are impaired in capping. We surmise that there may be two cytoplasmic events regulating directly or indirectly capping: one associated with the process of translational movement, the other associated with the activity of microtubules. Lymphocytes treated with concanavalin A do not cap Ig. Colchicine reverses this inhibition. Certain experimental procedures antagonize the colchicine effect, the most striking of which is the use of cytochalasin B. Colchicine appears to increase movement of the Con A-treated lymphocyte, and this increased movement appears responsible for the accumulation of complexes to the posterior part of the cell. Con A inhibits patching of Ig by anti-Ig, and this is not reversed by colchicine.

Growth of nontransformed 3T3MIT fibroblasts in media containing 200 mM urea leads to the rapid acquisition of the transformed adhesive phenotype as evidenced by an increased rate of divalent cation-independent cell aggregation. The increased rate of divalent cation-independent cell aggregation of urea treated 3T3MIT cells shares many properties with the high rate of aggregation of transformed cells including a sensitivity to treatment with trypsin or hyaluronidase and a reduction in the presence of exogenously added hyaluronic acid. Reversal of the urea-induced increase in aggregation occurs within 24 hours in the absence of urea and can be blocked by 0.2 micrograms/ml cycloheximide. In the presence of cycloheximide, low rates of aggregation can be restored by the addition of urea-conditioned supernatents. The results of these experiments suggest that the loss of an aggregation-inhibitory activity during growth in media containing 200 mM urea is responsible for the increased rate of divalent cation-independent cell aggregation. After removal of this aggregation-inhibitory activity, the normally lowly adhesive 3T3MIT cells become phenotypically transformed with regards to the rate of divalent cation-independent cell aggregation.

Release of superoxide from human neutrophils was stimulated by cis-unsaturated fatty acids (e.g. arachidonate, linoleate). All the saturated and trans-unsaturated fatty acids tested were ineffective in this context. Binding of linoleate to neutrophils was biphasic and could be resolved into a linear and a saturable component. The extent of linoleate binding to the saturable component correlated strongly with the amount of O-2 released (r = 0.96). Palmitate, a saturated fatty acid, exhibited only linear binding to neutrophils and the binding was similar to the linear component of linoleate binding. All cis-unsaturated fatty acids tested decreased the fluorescence polarization of cis- and trans-parinaric acid used as membrane probes in suspensions of neutrophils, whereas the saturated fatty acid, myristate, increased the polarization. Fatty acids which stimulated O-2 release induced morphological changes (i.e. evaginations) in neutrophils, whereas the inactive fatty acids did not affect the cellular morphology. Effects on both the cell morphology and superoxide release were reversed by treatment of the cells with delipidated albumin. These data are discussed in relation to recent reports concerning the effects of free fatty acids on various biochemical systems.

During the initial 48 hr of incubation in immunized Mishell-Dutton spleen cell cultures, most lymphocytes exist as single cells or an occasional pair of cells (doublets). Ultrastructural examination of the area between those cells forming doublets revealed a septate-like junctional complex occurring over large portions of the plasmalemma. This junctional zone persisted in areas where extensive cytoplasmic interdigitation among cell processes was evident. Approximately 1% of the lymphocytes isolated from the top fraction of a discontinuous fetal calf serum gradient were involved in formation of doublets. The majority of these doublets showed evidence of junctional interaction. Moreover, this septate-like zone of adhesion was seen using several different fixatives and stains and remained intact after osmotic cell disruption. The junction was not demonstrable by using freeze-fracture techniques and therefore must be limited to interactions within the surface of the cells.

All-trans-retinal stimulated the release of superoxide by human and guinea pig neutrophils 63 +/- 14 SD and 53 +/- 5 SD nmol of O2-/min/10(7) cells, respectively. Superoxide release by unstimulated cells was negligible. All-trans-retinal also induced morphological changes (i.e., evaginations) in these cells. Other retinoids were effective in instigating these phenomena. The similarities of these effects to those instigated by cis-unsaturated fatty acids (Badwey, J.A., et al., 1984, J. Biol. Chem., 259:7870-7877) are discussed in light of possible mechanisms.

Using the rat model of atherosclerosis, the influence of dietary fish oil on early stages of atherosclerotic lesion formation was studied. Normocholesterolemic rats (serum cholesterol less than 100 mg/dl), moderately hypercholesterolemic rats fed cholesterol and cholic acid (serum cholesterol less than 400 mg/dl), and severely hypercholesterolemic rats fed cholesterol, cholic acid, and 2-thiouracil (serum cholesterol greater than 900 mg/dl) had their diets supplemented with 5% (w/w) "MaxEPA" fish oil for a period of 2 weeks. In each diet group safflower oil was used as a control for fish oil. Monocyte adhesion to the thoracic aorta and intimal foam cell formation were used to measure the extent of atherosclerotic lesion formation in each rat. Cholesterol and triglyceride levels were measured in both plasma and lipoprotein fractions. In normocholesterolemic rats, fish oil did not influence the morphology of the vessel wall. In moderately hypercholesterolemic rats, monocyte adhesion was the same irrespective of dietary oil, however, intimal foam cell formation was 2-fold higher in the fish oil-fed animals despite a reduction in serum cholesterol levels when compared to the safflower oil-fed animals. In severely hypercholesterolemic rats, monocyte adhesion to the vessel wall and intimal foam cell formation were both 4-fold higher in the fish oil compared with the safflower oil fed animals. These observations could not be attributed to differences in the plasma or lipoprotein profiles of safflower oil vs. fish oil fed rats. The results of this study suggest that dietary fish oil, when fed to hypercholesterolemic rats for a period of 2 weeks, enhances the rate of monocyte adhesion and fatty streak formation in the thoracic aorta.

A simple method has been devised for correlating fluorescence microscopy and scanning electron microscopy, whereby the identical cell observed by the former can be observed by the latter. With this methodology, we have studied the sequence of cell surface changes which occur when mouse B lymphocytes, bearing immunoglobulin (Ig) on their surfaces, interact with fluoresceinated anti-Ig antibodies. Initially, the pattern of staining is diffuse; then, rapidly, patching occurs, followed by the sweeping of the patches into a cap. Accompanying these events are: the disappearance of microvilli, the formation of ruffles and lamellipodia at the pole of the cell opposite the capped pole, the presence of a constriction ring beneath the cap, ameboid shapes, and the translocation of the cell in a direction opposite the cap. Whereas the cell body is smooth, the capped pole frequently shows the presence of numerous microvilli. However, not all capping cells show the changes, and cells capped in the presence of cytochalasin B usually show none of them, except that their surfaces are smooth. Furthermore, T-cells undergoing translocation show the same cell surface changes as do B-cells. Whereas the contractile apparatus of the cell is considered to underlie all these phenomena, it is concluded that the cell surface changes do not result from patching and capping per se, but rather are an expression of the cell translocation also induced by the anti-Ig antibody.

Pulmonary surfactant is synthesized by alveolar type II pneumonocytes and stored in inclusions called lamellar bodies. In the present study we have investigated the role of the calcium-binding protein, calmodulin, in regulating surfactant secretion in differentiating rat fetal type II pneumonocytes. Lamellar body secretion is stimulated in differentiating type II cells in vitro by the calcium ionophore, A23187. A23187-induced secretion is blocked by the phenothiazine drugs, trifluoperazine and chlorpromazine, but is unaffected by the inactive analogs, trifluoperazine sulfoxide and chlorpromazine sulfoxide. Immunofluorescence studies on cultured type II pneumonocytes show that the percentage of Nomarski-dense intracellular granules, which stain positively with anticalmodulin antibody, increases when the cells are stimulated with the calcium ionophore, A23187. Since these Nomarski-dense granules are positively stained by phosphine-3R, these results indicate that increased amounts of immunoreactive calmodulin appear associated with lamellar body surfaces when the cells are stimulated for secretion. In addition, ultrastructural localization of calmodulin on isolated lamellar bodies using protein A-colloidal gold indicates that calmodulin is present on their outer surfaces. The endogenous calmodulin present on the isolated lamellar bodies is not removed by treatment of the lamellar bodies with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Using the same localization technique, we found that isolated lamellar bodies are capable of binding exogenously added calmodulin in the presence of calcium. Taken together, these results implicate calmodulin in pulmonary surfactant secretion.

We have compared the subcellular sites of H2O2 and presumably also superoxide-(O2-) production, and certain aspects of metabolic responses (O2 consumption, O2- production) of stimulated neutrophils from human blood and those elicited into guinea pig peritonea. Stimulation was accomplished with either opsonized zymosan or phorbol-12-myristate-13-acetate (PMA). Striking quantitative differences were observed between these cell types with regard to the increased respiration and O2- production observed during stimulation. These differences were most apparent when opsonized zymosan served as the stimulating agent. They were minimized when the soluble stimulating agent, PMA, was used. With either stimulus, the subcellular sites of H2O2 production were the same for both types of neutrophils, i.e., the plasmalemma and phagosomal membranes. No H2O2 production could be detected cytochemically in the absence of stimulation. Treatment of both unstimulated human blood and elicited guinea pig peritoneal neutrophils with the nonpenetrating, covalently linking reagent, p-diazobenzenesulfonic acid, failed to diminish O2- production upon subsequent stimulation, in contrast to a previous report. These data are discussed in terms of the possible cytological arrangements of the respiratory enzyme(s), and the different modes of stimulation of neutrophil metabolism by various agents. Ancillary data on elicited mouse peritoneal neutrophils are presented.

Cerium ions have been used as the capture agent for inorganic phosphate released during the enzymatic hydrolysis of phosphate-containing substrates by a variety of phosphatases. Cerium phosphate reaction product accumulation is proportional to the amount of enzyme present in a cell-free model system. Ultrastructurally, cerium phosphate reaction product appears as a very fine electron-dense precipitate. Cerium appears to be a better capture agent for inorganic phosphate than lead in that reaction product is usually more uniform and more consistently reproducible when cerium is used. Furthermore, nonspecific deposits of reaction product that are commonly encountered in lead-based phosphatase reactions are virtually nonexistent when cerium is the capture agent.

The ability of phorbol derivatives to function as stimulating agents for superoxide (O2-) release by guinea pig neutrophils has been evaluated and compared to the known ability of each compound to activate protein kinase C. Those that activate the kinase also stimulate O2- release, while those that are inactive with respect to the kinase have no effect on O2- release. The same correlation was observed with respect to the ability of phorbol esters to induce morphological changes in neutrophils, i.e., vesiculation and reduction in granule content. Certain phenothiazines and naphthalene sulfonamides that are known antagonists of calcium-binding proteins blocked both phorbol ester-induced O2- release and morphological changes in these cells.

Glomerular epithelial cells (GECs) in vitro provide a useful model for the study of the mechanism(s) underlying the nephrotic syndrome of rats induced by the aminonucleoside of puromycin (PAN). Some of the toxicities of PAN are nonspecific, in that the constituent molecules of PAN (adenosine and puromycin) cause similar effects in vitro. These include GEC blebbing and rounding, reduced uptake of precursors of protein (leucine) and glycoprotein (glucosamine) synthesis, and increased permeability of the GEC membrane to adenosine. Some of the effects of PAN are not reproduced by adenosine or puromycin and are inhibited by the simultaneous presence of N6-monomethyl adenosine (MMA), a PAN analog and an in vivo blocker of nephrosis due to PAN. These processes may be related to the nephrotic syndrome and include the loss of adhesion to plastic; a reduction in the incorporation of 14C-glucosamine and 35S-sulfate both into molecules removable from the GEC surface by neuraminidase and into those moieties precipitated from the culture media by TCA; a marked reduction in the "ordering" of the lipids of the rigid GEC membrane, which is possibly dependent upon cell-surface proteins. These morphologic alterations in GECs and in the distribution of negatively charged molecules, which are either secreted or on the cell surface, correlate with observations made in PAN-induced nephrosis in rats in vivo. These include changes in the turnover and the array of sialic acid and heparan sulfate glycoprotein on the GECs and the glomerular basement membrane. The in vitro sensitivity of GECs to PAN and the effects of MMA suggest a role for these cells in in vivo aminonucleoside nephrotoxicity, where alterations in both the morphology and the anionic topology of GECs participate in the development of proteinuria.

Isolated rat renal glomeruli and cultured glomerular mesangial and epithelial cells were examined for atrial natriuretic peptide (ANP) receptors, and for ANP-stimulated cyclic guanosine monophosphate (cGMP) generation. In glomeruli from normal rats, human (1-28) 125I-ANP bound to a single population of high affinity receptors with a mean equilibrium dissociation constant of 0.46 nM. Human (1-28) ANP markedly stimulated cGMP generation, but not cAMP generation in normal rat glomeruli. Analogues of ANP that bound to the glomerular ANP receptor with high affinity stimulated cGMP accumulation, whereas the (13-28) ANP fragment, which failed to bind to the receptor, was devoid of functional activity. Cell surface receptors for ANP were expressed on cultured glomerular mesangial but not epithelial cells, and appreciable ANP-stimulated cGMP accumulation was elicited only in mesangial cells. Approximately 12,000 ANP receptor sites were present per mesangial cell, with an average value for the equilibrium dissociation constant of 0.22 nM. Feeding of a low-salt diet to rats for 2 wk resulted in marked up regulation of the glomerular ANP receptor density to a mean of 426 fmol/mg protein, compared with 116 fmol/mg in rats given a high-salt diet. A modest reduction in the affinity of glomerular ANP receptors was also observed in rats fed the low-salt diet. ANP-stimulated cGMP generation in glomeruli did not change with alterations in salt intake. We conclude that high salt feeding in the rat results in reduced glomerular ANP receptor density relative to values in salt restricted rats. Furthermore, the mesangial cell is a principal target for ANP binding in the glomerulus.

Previous work from our laboratory has demonstrated that both anticoagulant and nonanticoagulant heparin species can inhibit the proliferation of vascular smooth muscle cells in vivo and in vitro. In this communication, we report studies on the structure-function relationships of heparin to its antiproliferative effect on vascular smooth muscle cells. These structure-function studies were carried out by preparing discrete sizes of heparin fragments and by chemically modifying heparin. The compounds were tested for their ability to inhibit rat and calf aortic smooth muscle cell growth. The minimum fragment size which retains some growth inhibitory activity is a hexasaccharide; maximal antiproliferative activity was obtained with dodecasaccharide and larger fragments. Both O-sulfation and N-substitution were found to be important for the growth inhibitory effect. Comparison of the antiproliferative and anticoagulant activities of the different heparin species has allowed us to identify several heparin molecules which have lost their anticoagulant properties, but retain antiproliferative activity.

The distribution of Ia antigens was studied on murine spleen lymphocytes by an ultrastructural technique employing deep freeze-etched replicas. Ia antigens were labeled on cells from appropriate congenic and recombinant strains of mice by incubating the cells with FITC-conjugated anti-Iak antibody, followed by ferritin-coupled Fab anti-FITC. Ia antigens were detected predominantly on immunoglobulin (Ig)-bearing B lymphocytes. Antigens coded for by the entire Ik region were present on the surfaces of 95% of the positive cells (from B10.BR mice) in densely packed microclusters. Ia specificities coded for by the I-A and I-C subregions (on 4R and B10.HTT mice) exhibited a more variable pattern, with 30 to 35% of the labeled cells having sparsely distributed Ia antigens in relatively discrete microclusters. Binding of anti-Iak antibody at 37 degrees C led to patch formation but not to capping. Modulation of surface Ig left Ia antigens diffusely distributed on the cell surface, indicating that these two membrane proteins are independent molecules.

Previously, we have shown that the capping of surface immunoglobulins on murine lymphocytes can be affected by modulating the lipid environment of the surface membrane with free fatty acids. In the present study, murine lymphocytes were depleted of cholesterol by incubation with phospholipid vesicles. As the cellular cholesterol:phospholipid ratio decreased, the capping of the surface immunoglobulin was seen to decrease. This inhibition of capping could not be reversed by calcium and is not accompanied by changes in either the cytoskeletal element alpha-actinin or cellular ATP levels. Incubation of the cholesterol-depleted cells with cholesterol-containing phospholipid vesicles raised both the cholesterol:phospholipid ratio and capping levels to values close to those of untreated control cells. Remarkably, stearic acid, a saturated fatty acid, could also restore the capping levels in the cholesterol-depleted cells. On the basis of the present data and measurements of the fluorescence polarization of the probe diphenyl hexatriene, we propose a model in which the protein(s) involved in capping is located in a gel-like lipid domain, and that removal of cholesterol makes this domain less gel-like and inhibits capping. Restoration of the gel-like nature of this domain by the addition of either cholesterol or stearic acid enables the protein(s) to function normally.

Abnormalities in lipid metabolism resulting from nephrotic syndrome may play a role in the progression of initial glomerular injury to focal and segmental glomerulosclerosis (FSGS). In order to more specifically assess this, we fed male Sprague-Dawley rats, made nephrotic with a single intravenous injection of puromycin aminonucleoside (PA), either normal rodent chow (Group 1) or the same formulation supplemented with 4% cholesterol/1% cholic acid (Group 2). This 4% cholesterol/1% cholic acid-added diet was utilized because, in normal, non-nephrotic rats, this alimentary supplement produces, for the most part, only a significant rise in fasting serum cholesterol and not fasting serum triglycerides. FSGS developed 18 weeks after PA delivery, and both groups of rats were studied functionally and morphologically. Group 2 rats had significantly higher daily urine protein excretion, lower inulin clearance, and greater blood urea nitrogen concentrations than Group 1 animals. Histologically, Group 2 animals demonstrated a significantly greater percentage of glomeruli examined with segmental areas of glomerulosclerosis/hyalinosis, mesangial cell proliferation, and mesangial "foam" cells. At 2, 4, 12, and 18 weeks after PA delivery, the fasting serum cholesterol was always significantly greater in Group 2 rats, whereas in regards to fasting serum triglycerides it was only significantly elevated in Group 2 rats at 4 and 12 weeks after PA administration.

Examination of the toad urinary bladder by freeze-fracture electron-microscopy demonstrates structural specialization of the granular cell's luminal membrane compared to its basal membrane. Although both membranes appear to possess about 1,700 intramembranous particles per mum2, those of the luminal membrane tend to be significantly larger in size. In addition, the fracturing properties of the two membranes are markedly different: the majority of particles are found on fracture face B (outer membrane face), in the case of the luminal membrane, and the majority are found on fracture face A (inner membrane face), in the case of the basal membrane. While the two fracture faces of the basal membrane possess a similar distribution of particle sizes, in the case of the luminal membrane the B face was found to possess particles generally larger than those found on the A face. It was established that the probability of luminal membrane particles adhering to face B instead of face A is closely correlated with the size of the particle. The structural specialization of the granular cell's luminal membrane may have an important relationship to the characteristic permeability properties of this membrane and the capacity of this cell type to respond physiologically to the hormone vasopressin.

We have studied the interaction of free fatty acids (FFAs) with cell membranes and lipid bilayers by monitoring changes in the emission polarization of the fluorescent probes diphenylhexatriene (DPH) and anilino-naphthalene sulfonate (ANS). We found that the FFAs readily intercalate into membranes and produce significant changes in the packing of the lipid molecules. The membrane alterations could be divided into two patterns: the cis-unsaturated FFAs (designated Group A) disorder the membranes' interior (as reported by DPH) and order the head group region (as reported by ANS); the trans-unsaturated or saturated FFAs (Group B) do not alter the bilayer interior but also order the head group region. Using solution theory, the shift in transition midpoint temperatures as a function of fatty acid type was used to infer that the Group A FFAs partition into fluid domains, while Group B FFAs partition preferentially into gel-like domains. These results are explained in terms of a domain model of membrane lipid structure. Low concentrations of Group A FFAs inhibit the capping of surface immunoglobulin (Ig), whereas no effect was seen with Group B FFAs. The capping inhibition caused by Group A FFAs was reversible with increasing doses of extracellular calcium. Fluorescence photobleaching recovery showed that the Group A FFAs do not inhibit receptor immobilization associated with patch formation but rather inhibit the final energy-dependent movement of the patched receptors into a cap. We have also shown that the Group A FFAs cause a shift in membrane-bound calcium to the lipid phase from probably protein calcium-binding sites. The data have generated a model of receptor mobility invoking a trans-membrane, calcium-binding, receptor-anchoring protein, linked to the cytoskeleton. Inhibition of capping by Group A FFAs is postulated to be due to perturbation of specific lipid domains associated with this protein, such perturbation leading to conformational changes in the protein, and consequent intramembraneous calcium sequestration in the lipid phase, rendering the calcium unavailable for activation of the cytoskeleton.

Bovine aortic endothelial cells release a heparin-like substance in the presence of 0.4% fetal calf serum. This substance inhibited the growth of smooth muscle cells in vitro by about 70%. Substitution of platelet-poor plasma for serum resulted in minimal liberation of inhibitory activity from the cells unless at least 10-fold higher concentrations of platelet-poor plasma were utilized. This suggested that a platelet product was involved in the release process. Therefore, we examined the ability of the platelet heparitinase recently isolated in our laboratory to release heparin-like species from cultured endothelial cells. Our results show that when endothelial cells were exposed to serum-free medium containing 1 ng/ml of the purified platelet endoglycosidase, at least as much inhibitory activity was released as was obtained with 0.4% serum. Dose response experiments indicated that only 10 pg/ml of the enzyme were necessary to liberate 50% of the inhibitory activity from endothelial cells. The heparin-like nature of the inhibitory substance was demonstrated by its sensitivity of Flavobacterium heparinase. Utilizing appropriate controls, the release of heparin-like material by the endoglycosidase was shown to be enzyme-specific and was not due to artifacts of experimental manipulations. In addition, this enzyme did not convert prereleased material to an active component, but directly liberated the active heparin-like species from endothelial cells. A simple model describing the possible role of heparin-like components and the endoglycosidase in the normal and injured wall will be presented.

The attachment of human polymorphonuclear leukocytes (PMN) to bovine endothelial monolayers is increased by pretreatment of the endothelial cells with leukotriene B4 (LTB4). The response is dose dependent with a maximum at 10(-6) M, requires no more than 1 min of preincubation for the maximal effect, and decays linearly over 30 min once the LTB4 is removed. Preincubation of PMN with LTB4 does not augment subsequent adhesion to a plastic substratum or an untreated monolayer of endothelial cells and eliminates the augmented attachment to LTB4-pretreated endothelial cells. The simultaneous exposure of both cell types during the adhesion interaction gives a biphasic dose-related increment, suggesting that the inhibition of the neutrophil adherence is less marked than with a pretreatment interval. Thus, the LTB4-dependent adherence of PMN to endothelial cells is selectively mediated by endothelial cells, is dose related, is reversible upon removal of the agonist from the endothelial cells, and is opposed by the direct action of LTB4 on the PMN.

Maximal rates of superoxide (O-2) release, and the cytochemical locales of peroxide staining in resident, elicited, and activated macrophages have been determined. Macrophages elicited into the peritoneum with either casein (1.2% w/v) or proteose-peptone (10.0% w/v) release about twice as much O-2 as macrophages activated by infection of the animals with either Listeria monocytogenes, or Bacille Calmette-Guerin (BCG) followed by immune boosting with Purified Protein Derivative (PPD) (i.e., about 35 vs. 14-18 nmol O-2/min/10(7) cells). Macrophages elicited with thioglycollate (3.0% w/v) and resident macrophages produce negligible amounts of O-2 upon stimulation with PMA. These data are compared with those reported by other investigators who used different procedures. A cytochemical procedure for localizing peroxide has been modified for use with murine macrophages. No production of H2O2 by macrophages is detected cytochemically in the absence of stimulation. Upon exposure to PMA, resident macrophages are still largely unresponsive. Approximately 20% of the casein elicited macrophages and BCG-PPD activated macrophages exhibit H2O2 staining, which is largely restricted to the cytoplasmic vesicles and channels induced by PMA in these cells. The only exception to this staining pattern is a small population (about 2%) of activated macrophages which exhibits H2O2 staining in the cytoplasmic vesicles and channels and on the plasmalemma as well.

Irreversibly sickled cells (ISC's) are circulating erythrocytes in patients with sickle cell disease that retain a sickled shape even when oxygenated. Evidence points to a membrane defect that prevents the return of these cells to the normal biconcave shape. The erythrocyte membrane protein spectrin is believed to help control erythrocyte shape and deformability. Recent studies suggest that normally spectrin and an erythrocyte actin form a self-supporting, fibrillar, lattice-like network on the cytoplasmic membrane surface. When normal erythrocyte ghosts are extracted with Triton X-100 all the integral membrane proteins and most of the membrane lipids are removed, leaving a ghost-shaped residue composed principally of spectrin and actin. We concentrated ISC's from patients with sickle cell anemia and compared the morphology and protein composition of ghosts and Triton-extracted ghost residues prepared from these ISC's with similar preparations of reversibly sickable cells and normal cells. (a) Many ISC's formed ISC-shaped ghosts. (b) All ISC-shaped ghosts formed ISC-shaped Triton residues. (c) Spectrin, erythrocyte actin (Band 5), an unidentified Band 3 component, and Band 4.1 were the major protein components of the Triton residues. All membrane-associated sickle hemoglobin was removed by the Triton treatment. (d) No ISC-shaped ghosts or ISC-shaped Triton residues were formed when deoxygenated, sickled RSC's were lysed or Triton-extracted. ISC-shaped ghosts and Triton residues were never formed from normal cells. These observations suggest that a defect of the "spectrin-actin lattice" may be the primary abnormality of the ISC membrane. Since ISC's are rigid cells, the data support the postulate that spectrin is a major determinant of membrane deformability. Finally, they provide direct evidence that spectrin is important in determining erythrocyte shape.

The glycosaminoglycan heparin inhibits the growth of a number of different cell types in vitro including smooth muscle cells, mesangial cells, fibroblasts, and rat cervical epithelial cells (RCEC). Studies investigating the antiproliferative effects of heparin on smooth muscle cells have demonstrated the site of the cell cycle block and revealed several metabolic alterations that could be causally associated with growth inhibition. We have investigated these metabolic parameters in RCEC to determine whether they are also associated with the antiproliferative effects of heparin in epithelial cells. Heparin acts rapidly to inhibit RCEC growth with inhibition detectable by autoradiography 7 h after the addition of heparin. Heparin treated RCEC begin to enter S-phase 12 h after the removal of heparin. These findings suggest that heparin blocks RCEC in the early-to-mid G1 phase of the cell cycle rather than late in G1 or early in S-phase as has previously been demonstrated for smooth muscle cells. Unlike smooth muscle cells, the uptake of thymidine and uridine is not inhibited by heparin in RCEC. Treatment of medium with heparin-Sepharose does not reduce the subsequent growth of RCEC; heparin inhibits the growth of RCEC in heparin-Sepharose treated medium in a manner identical to that in nontreated medium. Therefore the growth inhibitory effects of heparin cannot be explained by the inactivation of mitogens present in serum. In contrast to its effects on smooth muscle cells, heparin treatment of RCEC does not result in a reduction in the binding of epidermal growth factor (EGF) to the cells. These results indicate that although heparin inhibits the growth of a variety of cell types, significant differences exist in the responses of the different cells to heparin.