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Biomedical subjects

M L Karnovsky

Publications and source records attributed to M L Karnovsky.

At least 37 records · Page 2Linked to original sources
PubMed1988

Synergistic stimulation of neutrophils. Possible involvement of 5-hydroxy-6,8,11,14-eicosatetraenoate in superoxide release.

Neutrophils stimulated with optimal amounts of tumor-promoters that activate protein kinase C (e.g. mezerein, phorbol 12,13-dibutyrate) are known to release large quantities of superoxide: approximately 40-50 nmol O2-/min/10(7) cells. Previous studies have shown that treatment of neutrophils with the calcium ionophore A23187, or with 5-hydroxy-6,8,11,14-eicosatetraenonate (5-HETE), dramatically increased the ability of these cells to release O2- in response to suboptimal concentrations of the stimulants mentioned. In this manuscript, we provide data relevant to the basis of this augmentation of O2- release. The synergy with ionophore A23187 exhibited a partial requirement for extracellular Ca2+, whereas that with 5-HETE exhibited a near absolute requirement for that cation. Neutrophils stimulated with optimal amounts of tumor-promoters are known to exhibit a redistribution of protein kinase C activity from the soluble to a particulate fraction. A redistribution of kinase activity was not observed in cells stimulated synergistically. On the other hand, ionophore A23187 and 5-HETE increased the binding of a suboptimal amount of [3H] phorbol 12,13-dibutyrate to intact neutrophils by approximately 25 and 50%, respectively. Inhibitors of protein kinase C (i.e. sphingosine, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) substantially blocked O-2 release from neutrophils stimulated either synergistically or with optimal levels of tumor-promoters. These data suggest a role for 5-HETE in modulating O-2 release by neutrophils and are discussed in relation to models of the interactions of protein kinase C with membranes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1987

Cell surface dynamics of neutrophils stimulated with phorbol esters or retinoids.

Neutrophils undergo rapid morphological changes as well as metabolic perturbations when stimulated with certain phorbol esters. Stimulated cells initially exhibit pronounced projections emanating from the cell bodies, followed by rounding of the cells, reduction in granule number, and the appearance of intracellular vesicles. We show these vesicles to be derived, at least in part, from the plasmalemma. The experimental approach involved labeling stimulated and unstimulated cells with native ferritin and cationized ferritin, along with the cytochemical localization of ecto-5'-nucleotidase. The labeling patterns of the vesicles indicate that these structures are involved in both phorbol ester-stimulated adsorptive and fluid-phase endocytosis. Neutrophils stimulated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) exhibit two distinct rates of superoxide release in which the second, prolonged level is approximately 50% of the initial rate. All-trans-retinal, which we have recently shown to stimulate O2- release but not granule exocytosis or cell vesiculation, induces a single prolonged rate of maximal O2- release. Neutrophils treated with both all-trans-retinal and TPA exhibit only a single sustained rate of maximal O2- release similar to that observed with all-trans-retinal alone. Moreover, treatment of cells with all-trans-retinal blocks the vesiculation of neutrophils induced by TPA in a dose-dependent manner. This observation provides a possible explanation for the differences in the kinetics of superoxide release.

Animals↗
PubMed1986

Features of the translocation of protein kinase C in neutrophils stimulated with the chemotactic peptide f-Met-Leu-Phe.

The effects of f-Met-Leu-Phe (fMLP) on neutrophils, i.e. elevation of the levels of cytoplasmic Ca2+ and intramembranous diacylglycerol, would be expected to be accompanied by translocation of protein kinase C (PKC) to the plasmalemma. However, fMLP-induced PKC translocation could hitherto be demonstrated only when cells were additionally treated with cytochalasin B. We show here that treatment of guinea pig neutrophils with fMLP alone does lead to a significant PKC translocation which can be inhibited by pertussis toxin. The translocation can be detected only if the incubation is terminated within 30 sec after addition of fMLP, the termination is rapid, e.g. by application of a freeze clamp-technique, and the concentration of Ca2+ chelators in the buffer used for lysing the cells is low.

Animals↗
PubMed1986

Specific binding sites for muramyl peptides on murine macrophages.

Two radiolabeled (125I) muramyl peptide derivatives of high specific activity were prepared: a tripeptide with an iodinated C-terminal tyrosine methyl ester (Ligand I), and a muramyl tripeptide with a C-terminal lysine derivatized with Bolton-Hunter reagent (Ligand II). These were used to characterize binding of muramyl peptides to monolayers of murine macrophages. Saturable high-affinity binding to resident, caseinate-elicited, and Listeria-activated peritoneal cells was observed with both radioligands. Binding affinities varied with the state of activation of the macrophages, and KD values ranged from 48 +/- 33 pM (for resident macrophages, Ligand I) to 1020 +/- 90 pM (for activated macrophages, Ligand II). Specific binding sites were also found on a macrophage-derived cell line. The ability of several unlabeled muramyl peptides to compete with Ligands I and II for their binding sites was tested. Competition was stereospecific and correlated with known biological activities of these compounds (i.e., immunoadjuvanticity, pyrogenicity, and somnogenicity). The sites identified here for Ligands I and II may mediate some of the effects that muramyl peptides have previously been demonstrated to have on macrophages.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1986

Retinoids stimulate the release of superoxide by neutrophils and change their morphology.

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.

Animals↗
PubMed1986

Superoxide production by polymorphonuclear leukocytes. A cytochemical approach.

Phagocytosis by polymorphonuclear leukocytes triggers a burst of oxidative metabolism resulting in hydrogen peroxide and superoxide production, and these active oxygen species function in the killing of microorganisms. A new cytochemical technique, based on a manganese dependent diaminobenzidine oxidation, has been developed to detect superoxide in these cells. It has been shown that superoxide generation is associated with the plasma membrane in cells activated by particulate (zymosan) and non-particulate (phorbol myristate acetate) stimuli. This membrane activity is maintained during invagination such that reduced oxygen is generated within the endocytic vacuoles. Reaction product is absent from unstimulated cells; additionally, formation of precipitate is blocked by omission of Mn++, low temperature, glutaraldehyde prefixation, and the presence of superoxide dismutase in the incubation medium.

3,3'-Diaminobenzidine↗
PubMed1986

all-trans-Retinal stimulates superoxide release and phospholipase C activity in neutrophils without significantly blocking protein kinase C.

all-trans-Retinal was previously shown to stimulate high levels of superoxide release by guinea pig neutrophils. When the cells, previously labeled with [3H]inositol, are treated with all-trans-retinal, they exhibit a decrease in the levels of [3H]inositol phospholipids and an increase in the accumulation of [3H]inositol phosphates. The maximal accumulation of inositol phosphates and the optimal rate of superoxide release occurred together at approximately 7 min after stimulation. The levels of [3H]inositol phosphates accumulated were comparable to those observed when the cells were stimulated with a chemotactic peptide. In direct measurements, using concentrations that stimulate intact cells maximally, all-trans-retinal was found not to inhibit protein kinase C from the cytosol of neutrophils significantly. This contrasts with the situation with this kinase obtained from other sources. These observations represent additional effects of vitamin A on cells.

Free Radicals↗
PubMed1986

Apparent absence of a translocase in the cerebral glucose-6-phosphatase system.

In the hepatocyte endoplasmic reticulum, a substrate transporter could provide a means of regulating hydrolysis of glucose-6-phosphate by specifically modulating access of the substrate to the hydrolase. Several characteristics of the cerebral microsomal enzyme suggest that such an hypothesis is untenable in the brain. These are: (a) the inability of the enzyme in either untreated or detergent-disrupted brain microsomes to distinguish between glucose-6-phosphate and mannose-6-phosphate; (b) the close agreement of the apparent Km values for either substrate in intact or disrupted microsomal preparations; (c) the constancy of the latency toward both substrates over a wide concentration range; (d) the inability of nonpenetrating, covalently-linking reagents [e.g., 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)] to affect the accessibility of the hydrolase to its substrate; (e) the absence of a putative transporter polypeptide, such as that of the liver, in experiments where tritiated H2DIDS, polyacrylamide gel electrophoresis, and radioautography are applied to brain microsomes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Comparative aspects of the production of oxygen radicals by phagocytic cells, and aspects of other effector substances.

The author reviews the information gleaned from a great variety of sources on the factors influencing the production of oxygen radicals by phagocytic cells, with reference to inflammation. He emphasizes the value of comparative studies, mentioning particularly the effect of leukocyte perturbants, fatty acids and somnogenic leukocyte modulators.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1986

Muramyl peptides in mammalian tissues and their effects at the cellular level.

Muramyl peptides (MPs), presumably breakdown products of bacterial cell walls, have been found in the brain, liver, and kidney of the rat. They exert multiple physiological effects on higher animals as immunoadjuvants, activators of macrophages, pyrogens, antitumor agents, inducers of contractility of smooth muscle, and promoters of slow-wave sleep, as well as nonspecific protectors of animals against infection. Structure-function relationships of these substances have been extensively studied, especially with respect to somnogenicity. In the role an intact muramyl ring is required, and the 1,6-anhydro form is active. The presence of free carboxyls or amides on the glutamyl and diaminopimelyl entities have important effects. The stereochemistry is crucial: the alanine adjacent to the N-acetylmuramyl entity must be L, and the glutamate must be D. Studies were carried out with murine macrophages to establish mechanisms of action of these glycopeptides. There are two populations of binding sites for MPs on those cells. When compounds of different structure are compared, binding ability correlates with pyrogenic and somnogenic activity. Serotonin competes with these agents for binding sites. Binding of that substance induces at least one macrophage response characteristic of the binding of MP.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1985

Muramyl peptides and serotonin interact at specific binding sites on macrophages and enhance superoxide release.

Serotonin at low micromolar concentrations inhibited binding of two [125I]-labeled muramyl peptides to resident mouse peritoneal cells and to a macrophage-derived cell line, PU5-1.8-F7. Binding of [3H]serotonin was inhibited in parallel fashion. Overnight incubation with serotonin or muramyl peptide enhanced the release of superoxide by both types of cells when later stimulated with phorbol myristate acetate. Serotonin antagonists decreased binding of muramyl peptide and serotonin and diminished the subsequent enhancement of superoxide release. A cell line variant lacking detectable binding sites for muramyl peptide was far less responsive (superoxide release) than the parent line, to either drug. The data are consistent with sharing of a common set of receptors on the macrophage by muramyl peptide and serotonin and with involvement of these receptors in enhancing superoxide release.

Acetylmuramyl-Alanyl-Isoglutamine↗