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Biomedical subjects

M J Karnovsky

Publications and source records attributed to M J Karnovsky.

At least 109 records · Page 6Linked to original sources
PubMed1986

Dietary cholesterol-induced changes in macrophage characteristics. Relationship to atherosclerosis.

In diet-induced hypercholesterolemia, circulating monocytes adhere to the endothelium of the vessel wall and emigrate into the intima. Atherosclerotic lesions may develop, characterized by the presence of lipid-laden macrophages and proliferating smooth muscle cells recruited from the media. Using rat peritoneal macrophages, the authors examined the influence of diet-induced hypercholesterolemia on several variables of macrophage function that may contribute to lesion formation, including adhesion to bovine aortic endothelial cells (BAECs) and vascular smooth muscle cells (VSMCs), the production of chemoattractants and mitogens for VSMCs, and the release of the reactive oxygen species, superoxide. In general, a hypercholesterolemia-induced augmentation of macrophage function was observed. In comparison with macrophages from normal animals (N M phi s), macrophages from hypercholesterolemic animals (H M phi s) were 50-80% more adhesive to BAECs and VSMCs. H M phi-secreted products increased VSMC migration 6 to 7-fold, whereas N M0s only stimulated motility 2.5-fold. In addition, H M phi-conditioned media produced increased VSMC growth 5-fold, compared with a 2.5-fold increase produced by N M phi-conditioned media. Although the production of superoxide was found to be the same for both N M phi s and H M phi s, the release of superoxide by macrophages found in the intima of hypercholesterolemic animals may contribute to the necrosis of cells in the developing lesion. These results suggest that dietary cholesterol may accelerate atherosclerotic lesion formation by inducing specific changes in the properties of circulating monocytes and intimal macrophages.

Animals↗
PubMed1986

Glomerular endothelial cells secrete a heparinlike inhibitor and a peptide stimulator of mesangial cell proliferation.

The regulation of cell growth in the kidney glomerulus plays a key role in many physiologic and pathologic processes. In this communication the authors have examined the possible role of glomerular endothelial cells as potential regulators of mesangial cell proliferation. Conditioned medium was collected from confluent cultures of glomerular endothelial cells and tested for its effects on glomerular mesangial cell and vascular smooth muscle cell growth. When glomerular endothelial cell-conditioned medium was mixed 1:1 with normal growth medium, the growth of these two closely related cell types was inhibited by 60-70%. If the conditioned medium was diluted to 1:9, a stimulation of mesangial and smooth muscle cells growth was seen. Approximately 70% of the antiproliferative activity was destroyed by a highly purified heparinase; the other 30% was sensitive to trypsin. Approximately 90% of the mitogenic activity was protease-sensitive. These results suggest that glomerular endothelial cells may participate in part in mesangial cell growth regulation via a heparin-mediated mechanism.

Animals↗
PubMed1986

Focal and segmental glomerulosclerosis following a single intravenous dose of puromycin aminonucleoside.

Focal and segmental glomerulosclerosis (FSGS) represents a final pathologic pattern of a number of human renal disorders. Among laboratory models, repeated intraperitoneal injections of the aminonucleoside of puromycin (PA) produces a histologic pattern not unlike the human process. A single intravenous dose of this drug usually results in glomerular morphologic changes in rats resembling those in human nephrotic syndrome with minimal changes. This report describes acute and chronic glomerular injury that begins as early as 8 days after a single central administration of PA and progresses to FSGS within an 18-week period. It seems likely that minimal change disease and FSGS are two pathologic processes in the same continuum of disease. In this model, the severity and persistence of the glomerular lesion may represent irreversible glomerular epithelial cell (GEC) injury secondary to the toxic effects of PA.

Animals↗
PubMed1985

Binding and internalization of heparin by vascular smooth muscle cells.

Previous work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H-heparin to these cells occurs via specific, high-affinity binding sites (Kd = 10(-9) M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1-2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4 degrees C, a diffuse surface staining pattern was observed. After warming the cells to 37 degrees C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37 degrees C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high-affinity binding sites and the initial rapid uptake of 3H-heparin, suggests that heparin enters smooth muscle cells by both receptor-mediated and other endocytic pathways.

Animals↗
PubMed1985

Effect of heparin on vascular smooth muscle cells. I. Cell metabolism.

Previous work from our laboratory has shown that heparin inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. The mechanism of action of this glycosaminoglycan is unknown. In this communication, we have examined the antiproliferative effect of heparin on smooth muscle and other cell types, and have investigated several aspects of heparin on smooth muscle cell metabolism. Smooth muscle and closely related cell types from several species, including human, were much more sensitive to heparin than any other cell type tested, including primary and established cell lines, normal and transformed cell pairs, fibroblasts, epithelial, and endothelial cells. Flow microfluorimetric analysis of cell cycle distribution indicated that heparin blocked either the G0----S transition or a very early S-phase event in smooth muscle cells. Heparin rapidly inhibited DNA and RNA synthesis, but did not affect the rate of protein synthesis. The decrease in nucleic acid synthesis could be accounted for by an inhibition of thymidine and uridine uptake. Interestingly, heparin did not block amino acid or glucose transport. Although no change in the overall rate of protein synthesis was observed in the presence of heparin, we noted at least two changes in the synthesis of specific proteins by smooth muscle cells: two 35,000-dalton proteins which appeared in the culture medium of heparin-treated cells, and the transient disappearance of a 48,000-dalton protein in the substrate attached material of smooth muscle cells exposed to heparin. The role of the observed changes in smooth muscle cell metabolism is yet to be determined, but they may provide valuable clues to the molecular mechanisms controlling the antiproliferative activity of heparin.

Animals↗
PubMed1985

Effect of heparin on vascular smooth muscle cells. II. Specific protein synthesis.

Heparin suppresses the proliferation of vascular smooth muscle cells both in vivo and in vitro. The mechanism of action of the antiproliferative activity of heparin is not known. We have detected differences in the synthesis of specific proteins when vascular smooth muscle cells are exposed to heparin and report here that many characteristics of these protein alterations parallel the properties of the antiproliferative activity. The induction into the culture medium of a pair of proteins of approximately 35,000 dalton mw in heparin-treated smooth muscle cell cultures and the antiproliferative effect of heparin share the following characteristics: 1) the effect is reversible, 2) the effect is specific for smooth muscle cells, 3) anticoagulant and non-anticoagulant heparin are equally effective, 4) the effect is lost with time in culture and, 5) heparin is the most potent glycosaminoglycan in producing the effect. Furthermore, heparin causes a transient suppression of a 48,000 dalton substrate-attached protein, whereas chondroitin sulfate A and C and dermatan sulfate had much less effect. Dextran sulfate was almost as effective as heparin in suppressing the synthesis of the substrate-attached protein. These proteins appear to be noncollagenous and the induced synthesis of the 35,000 dalton proteins is inhibited by actinomycin D. Although a direct relationship between these specific protein changes and the antiproliferative effect of heparin has not been proven, these protein alterations may play a crucial role in the effect of heparin on smooth muscle cell growth.

Carbohydrate Metabolism↗
PubMed1985

Inhibition of rat cervical epithelial cell growth by heparin and its reversal by EGF.

The effects of heparin on the in vitro growth of rat cervical epithelial cells were examined. Heparin was found to inhibit in a dose dependent fashion the log-phase growth of rat cervical epithelial cells (RCEC) grown in the absence of medium supplements. An inhibition of growth is observed at concentrations as low as 500 ng/ml and 50% inhibition of growth occurs at a concentration of 5 micrograms/ml. The growth inhibitory activity of heparin is independent of anticoagulant activity since three separate non-anticoagulant preparations of heparin all inhibit growth. Other glycosaminoglycans including chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, hyaluronic acid, and keratin sulfate do not inhibit the growth of rat cervical epithelial cells. The ability of heparin to inhibit the log-phase growth of rat cervical epithelial cells is dependent on the composition of the medium in which the cells are grown. The addition of greater than or equal to 7.5 ng/ml epidermal growth factor to epithelial cultures blocks the growth inhibitory activity of heparin. These results suggest that components of the extracellular matrix modulate the growth responses of epithelial cells and may be important in regulating cellular proliferation in normal and pathological states.

Animals↗
PubMed1985

Release of superoxide and change in morphology by neutrophils in response to phorbol esters: antagonism by inhibitors of calcium-binding proteins.

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.

Animals↗
PubMed1985

Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli.

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.

Amino Acid Sequence↗
PubMed1985

Heparin inhibits mesangial cell proliferation in habu-venom-induced glomerular injury.

The authors have investigated the ability of anticoagulant heparin and nonanticoagulant heparin to inhibit mesangial-cell proliferation after the administration of habu (Trimeresurus flavorivids) snake venom to rats. Rats given injected habu venom exhibited glomerular capillary cystic lesions 6 to 24 hours later, and marked mesangial proliferation was noted within the cyst after 3 days. At 7 days 87% of these lesions (nodules) contained primarily mesangial cells embedded in a dense matrix and fibrin. A decrease in the frequency of nodules and the persistence of cysts indicate effective antiproliferative treatment. When anticoagulant heparin treatment extended from 18 hours after venom administration until sacrifice at 7 days, the percentage of nodules was reduced to 40%. Nonanticoagulant heparins resulted in some, but inconsistent, inhibition of mesangial-cell proliferation. The mechanism of the antiproliferative action of heparin on mesangial cells is not known but may be similar to that for vascular smooth muscle growth regulation. The authors suggest that endogenous heparin in the glomerular basement membrane and mesangial matrix may exert an antiproliferative effect under normal conditions. Loss of this inhibition due to glomerular damage might be reversed by the addition of exogenous heparin.

Animals↗
PubMed1985

Effects of the aminonucleoside of puromycin on glomerular epithelial cells in vitro.

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.

Adenosine↗
PubMed1985

Heparin and glomerular epithelial cell-secreted heparin-like species inhibit mesangial-cell proliferation.

The regulation of cell growth in the kidney glomerulus plays a key role in many physiologic and pathologic processes. In this communication, the authors examine the possible role of heparin-like species as inhibitors of mesangial-cell proliferation. Heparin profoundly inhibited the growth of cultured mesangial cells in a dose-dependent manner, with an ED50 = 5-10 micrograms/ml. The antiproliferative activity of heparin was reversible and specific for mesangial cells as the target cell in the glomerulus. Heparin was much more effective than other glycosaminoglycans. Cultured glomerular epithelial cells were found to secrete both stimulators and inhibitors of mesangial-cell growth. Approximately half of the inhibitory activity was destroyed by a highly purified heparinase; the other half was sensitive to trypsin. Approximately 80% of the mitogenic activity was protease-sensitive. These results suggest that heparin and glomerular epithelial cells may participate in mesangial-cell growth regulation.

Animals↗
PubMed1984

Studies on the mechanism of superoxide release from human neutrophils stimulated with arachidonate.

cis-Unsaturated fatty acids stimulate release of superoxide (O-2) by human neutrophils (Badwey, J. A., Curnutte, J. T., Robinson, J. M., Berde, C. B., Karnovsky, M. J., and Karnovsky, M. L. (1984) J. Biol. Chem. 259, 7870-7877). The rate of O-2 release due to arachidonate (105 +/- 24 S.D., nmol of O-2/min/10(7) cells) was comparable to optimal values obtained with other stimuli. Antagonists of calcium-binding proteins (i.e. phenothiazines, naphthalene sulfonamides) inhibited the release of O-2 in a fashion compatible with the involvement of calmodulin in these phenomena. Synthetic substrates for and an inhibitor of chymotrypsin-like proteases (e.g. N-benzoyl-L-tyrosine ethyl ester, L-1-tosylamido-2-phenylethyl chloromethyl ketone) also blocked O-2 release. Antagonists of calcium-binding proteins and of proteases were effective in this context with neutrophils stimulated with a variety of agents. The implications of these data for recent reports concerning the mechanism of action of cis-unsaturated fatty acids on phagocytes is discussed.

Arachidonic Acid↗
PubMed1984

Superoxide release by neutrophils: synergistic effects of a phorbol ester and a calcium ionophore.

Exposure to combined suboptimal concentrations of 4 beta-phorbol 12-myristate 13-acetate and the calcium ionophore A23187 stimulates superoxide release from guinea pig neutrophils to rates ca. 3.5-fold greater than the sum of the rates elicited by each of the agents added separately. This effect was largely dependent upon the presence of calcium in the extracellular medium. The data are discussed in relation to recent reports concerning the interactions of phorbol-esters with cells and the mechanism of activation of superoxide release by neutrophils.

Animals↗
PubMed1984

Effects of free fatty acids on release of superoxide and on change of shape by human neutrophils. Reversibility by albumin.

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.

Arachidonic Acid↗
PubMed1984

Partition of fatty acids and fluorescent fatty acids into membranes.

We have measured the membrane/water partition of free fatty acids, of a fluorescent analogue, and of chlorpromazine into plasma membranes from lymphoma, platelets, red cells, and liposomes of egg phosphatidylcholine. Three different methods were used: hygroscopic desorption [ Conrad , M.J., & Singer, S.J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5202-5206; Conrad , M.J., & Singer, S.J. (1981) Biochemistry 20, 808-818], centrifugation, and fluorescence titration. The partition coefficients for chlorpromazine and all fatty acid/membrane combinations were in the range 10(2)-10(5). No significant partition difference was observed between liposomes and plasma membranes. These results suggest, therefore, that fatty acids and chlorpromazine can readily partition into artificial and cell plasma membranes.

Animals↗
PubMed1984

Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells.

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.

Animals↗