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M J Karnovsky

Publications and source records attributed to M J Karnovsky.

At least 127 records · Page 7Linked to original sources
PubMed1984

Leukotriene B4 action on endothelium mediates augmented neutrophil/endothelial adhesion.

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.

Animals↗
PubMed1984

Localization of calmodulin in differentiating pulmonary type II epithelial cells.

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.

Animals↗
PubMed1984

Inhibition of platelet function by cis-unsaturated fatty acids.

The uptake of free fatty acids has previously been shown to affect the capping of lymphocytes, and there is evidence that different types of fatty acids may partition into separate lipid domains in cell surface membranes. In studies of gel-filtered human platelets, we found that cis-unsaturated fatty acids (1-35 microM) inhibited platelet shape change, aggregation, and secretion of 5-hydroxytryptamine induced by thrombin, adenosine diphosphate (ADP), collagen, U46619 (a thromboxane A2 analog), or plant lectins, but not that induced by A23187, a calcium ionophore. Trans-unsaturated and saturated fatty acids had little or no inhibitory effect. The inhibitory effects of cis-unsaturated fatty acids were not affected by inhibition of adenylate cyclase or cyclooxygenase. 14C-labeled fatty acids were taken up into platelet lipids. The maximum platelet-inhibitory effect of cis-unsaturated fatty acids was seen when over 90% of the platelet label was still in the form of free fatty acids. Platelet inhibition could be reversed by washing the platelets by gel filtration. Binding of platelet agonists to the platelet was not inhibited by the fatty acids. Cis-unsaturated fatty acids, but not trans-unsaturated or saturated fatty acids, decreased fluorescence polarization of platelets or isolated platelet membranes monitored with 1,6-diphenyl- 1,3,5-hexatriene. The potency of the fatty acids as inhibitors of platelet aggregation was inversely correlated with their melting points. These data suggest that inhibition of receptor-mediated platelet responses by cis-unsaturated fatty acids results from perturbation of the platelet membrane in specific lipid domains.

Blood Platelets↗
PubMed1984

Heparin-like molecules as regulators of atherogenesis.

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.

Animals↗
PubMed1983

Comparative biochemical and cytochemical studies on superoxide and peroxide in mouse macrophages.

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.

Animals↗
PubMed1983

Control of erythrocyte shape by calmodulin.

Erythrocytes are deformable cells whose shapes can be altered by treatments with a variety of drugs. The forms the erythrocyte may assume vary continuously from the spiny "echinocytes" or crenated cells at one extreme to highly folded and dented "cupped" cells at the other extreme. Examination of 39 compounds for cup-forming activity revealed a remarkable correlation between their ability to form cupped cells and their inhibitory activity against the calcium regulatory protein, calmodulin. Calmodulin is known to interact with several erythrocyte proteins including spectrin, spectrin kinase, and the Ca++ ATPase calcium pump of the membrane. These proteins regulate the form of the cytoskeleton as well as intracellular calcium and ATP levels. It is proposed that calmodulin is required to maintain normal erythrocyte morphology and that in the presence of calmodulin inhibitors, the cell assumes a cupped shape.

Anesthetics, Local↗
PubMed1983

Role of cholesterol in the capping of surface immunoglobulin receptors on murine lymphocytes.

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.

Actinin↗
PubMed1983

Ultrastructural localization of 5'-nucleotidase in guinea pig neutrophils based upon the use of cerium as capturing agent.

A cytochemical method for 5'-nucleotidase localization in which cerium serves as the capture agent in order to enzymatically detect liberated inorganic phosphate has been developed. The method has been established in cell-free model systems and in guinea pig neutrophils where 5'-nucleotidase is restricted to the plasmalemma as an ectoenzyme. This cerium-based method gives better results for ultrastructural localization of 5'-nucleotidase than conventional lead-based methods.

5'-Nucleotidase↗
PubMed1983

Ultrastructural localization of several phosphatases with cerium.

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.

Acid Phosphatase↗
PubMed1982

Inhibition of vascular smooth muscle cell growth by endothelial cell-derived heparin. Possible role of a platelet endoglycosidase.

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 described in the preceding communication 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 to 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 is presented.

Animals↗
PubMed1982

Inhibition of cap formation on lymphocytes by free fatty acids is not mediated by a depletion of ATP.

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.

Adenosine Triphosphate↗
PubMed1982

Effects of urea treatment on the divalent cation-independent cell aggregation of 3T3MIT fibroblasts.

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.

Animals↗
PubMed1982

Prostaglandin production by homogeneous cultures of rat glomerular epithelial and mesangial cells.

Prostaglandin production was measured in homogeneous cultures that had been characterized as glomerular epithelial and mesangial cells. Glomerular epithelial cells produced ten times more cyclooxygenase products than mesangial cells; 88 to 95% of its products were prostacyclin. Mesangial cells produced predominantly prostaglandin E2 (57 to 66%); only 14 to 15% of the prostaglandins produced by mesangial cells was PGI2. Cultured glomerular epithelial and mesangial cells produced little prostaglandin D2, thromboxane, 12-L-hydroxyeicosatetraenoic acid, or 6-sulfido-peptide-containing leukotrienes.

Angiotensin II↗
PubMed1982

Differentiation-dependent stimulation of neovascularization and endothelial cell chemotaxis by 3T3 adipocytes.

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.

Adipose Tissue↗