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M L Karnovsky

Publications and source records attributed to M L Karnovsky.

At least 19 recordsLinked to original sources
PubMed1995

Muramyl dipeptide mimicry in the regulation of murine macrophage activation by serotonin.

Muramyl peptides (MPs) are regulators of macrophage function. That the activities of MPs may be mediated by serotonin (5-HT) is supported by earlier work that demonstrated specific binding sites for MPs on macrophages that competitively bind 5-HT. Both mediators were also shown to enhance the production of superoxide anion (an antibacterial agent) by these cells. We now report on two additional macrophage activation phenomena affected by 5-HT: phagocytosis and induction of tumor necrosis factor alpha (TNF) mRNA. Serotonin acts as a muramyl peptide-like agonist by increasing phagocytosis of tubercle bacilli by murine peritoneal macrophages, and as a partial agonist/antagonist in the induction of mRNA for tumor necrosis factor. These observations provide further evidence for a serotonergic involvement in some of the physiological responses to MPs.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1994

Intestinal absorption and excretion of octapeptides composed of D amino acids.

Octapeptides synthesized from D amino acids were absorbed from the intestine and excreted in urine of normal rats drinking 5% glucose/1% creatinine containing the 125I-labeled peptides at 0.1-25 mg/dl. The rats ingested fluid at the rate of about 20 ml/hr and produced urine at 15 ml/hr for several hours during the nocturnal feeding period. Sixty-one +/- 4% of the ingested creatinine and 50 +/- 3% of a lipid-insoluble D octapeptide (EASASYSA, 784 Da) were excreted intact in the urine. The steady-state molar rate of absorption-excretion of creatinine equaled or exceeded the maximum rate of carrier-mediated intestinal transport of glucose, suggesting that both the creatinine and the D octapeptide were transported paracellularly by solvent drag through absorptive cell junctions that were dilated by the glucose. More than 70% of the ingested glucose was also absorbed paracellularly. The results demonstrate that intact oligopeptides can be absorbed efficiently from the intestine when they are not hydrolyzed by membrane-bound peptidases of the brush border. The results also provide support for recent theories proposing that coupling of membrane digestion with paracellular solvent drag accounts for a major fraction of normal intestinal absorption of nutrients.

Amino Acid Sequence↗
PubMed1994

Methoxatin (PQQ) in guinea-pig neutrophils.

PQQ, also called methoxatin, has been isolated from guinea-pig neutrophils. The organic cations diphenyleneiodonium (DPI) and diphenyliodonium (BPI) and the aromatic o-diamine 4,5-dimethylphenylenediamine (DIMPDA) sequester synthetic PQQ and inhibit its redox-cycling activity in a model system. Standards were made of adducts of tritiated PQQ with unlabeled DIMPDA and of unlabeled PQQ with tritiated DPI or DIMPDA. PQQ adducts were isolated from guinea-pig neutrophils with each of the tritiated inhibitors. They were separated and defined by high-performance liquid chromatography (HPLC). Tiron, a disodium benzene disulphonic acid, broke the DPI-PQQ adduct isolated from neutrophils and released free PQQ. Both DPI and DIMPDA, as well as BPI, blocked O2.- release by stimulated neutrophils. The blockade exerted by these inhibitors was released by the addition of PQQ to the cell suspensions. The data demonstrate the presence of PQQ in guinea-pig neutrophils and suggest that it has a possible role, direct or indirect, in the O2.(-)-producing respiratory burst.

Animals↗
PubMed1994

Aspects of the release of superoxide by leukocytes, and a means by which this is switched off.

Although great progress has been made in understanding the respiratory burst of leukocytes that produce superoxide (O2-), it is possible that a component or components, might have been overlooked. Furthermore, O2- production and its sequels, though cardinal in bactericidal action, might ultimately be damaging to the host's own cells. It is important, therefore, that a biologic mechanism exist to turn off O2- production by stimulated leukocytes. This article offers evidence that methoxatin (PQQ), a redox-cycling orthoquinone, might be involved in O2- production by leukocytes. This is based on the fact that inhibitors of O2- production, such as diphenylene iodonium (DPI) and 4,5-dimethylphenylene diamine (DIMPDA), were shown to sequester PQQ in leukocytes, i.e., to form adducts with that substance. Addition of PQQ to cells blocked with the inhibitors partially restored O2- release. With respect to turning off cellular O2- release, a factor was observed to be released to the medium by old macrophages (14 days old, but not by those less than 7 days old). Such conditioned medium, when added to stimulated neutrophils or macrophages, blocked O2- release. This factor was sensitive to proteases, exhibited molecular sizes of 3 and 11 kDa, and its action was independent of the nature of the stimulus applied to the leukocytes. It was partially purified by column (sizing) chromatography and HPLC. It seems to be a general modulator of the release of reactive oxygen species by phagocytes and is irrespective of phagocytic cellular type, or species from which the cells were derived.

Chromatography, High Pressure Liquid↗
PubMed1992

Serotonergic aspects of the response of human platelets to immune-adjuvant muramyl dipeptide.

The human platelet serotonergic responses of aggregation and uptake were shown to be modulated by muramyl peptides. Muramyl dipeptide inhibited serotonin uptake in a temperature dependent and stereospecific manner. It also blocked the binding to platelets of [3H]imipramine, a well-known inhibitor of serotonin uptake. Muramyl dipeptide decreased serotonin2 (5-HT2) mediated platelet aggregation, and blocked the binding of a 5-HT2-specific ligand: lysergic acid diethylamide. Since muramyl peptides are released upon degradation of bacteria, the findings offer a possible mechanism for neuro-immune modulation by emphasizing the interaction between 5-HT (a neurotransmitter) and muramyl peptides (immuno-adjuvants).

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1991

Role of biological membranes in slow-wave sleep.

Two involvements of cellular membranes in slow-wave sleep (SWS) are discussed. In the first the endoplasmic reticulum (ER) is focussed upon, and in the second, the plasmalemma, where specific binding sites (receptors?) for promoters of slow-wave sleep are believed to be located. The study concerning the ER focuses on an enzyme in the brain, glucose-6-phosphatase, which, although present at low levels, manifests greatly increased activity during SWS compared to the waking state. The work on the plasmalemma has to do with the specific binding of muramyl peptides, inducers of slow-wave sleep, to various cells, and membrane preparations of various sorts, including those from brain tissue. Such cells as macrophages from mice, B-lymphocytes from human blood, and cells from a cell line (C-6 glioma) have been examined in this context.

Animals↗
PubMed1990

A sensitive and specific assay for superoxide anion released by neutrophils or macrophages based on bioluminescence of polynoidin.

Using the luminescent protein polynoidin, present in the bioluminescent system isolated from the marine annelid Harmothoe lunulata, we have developed a new method to measure, specifically, superoxide anion (O2-) released by macrophages or neutrophils. A small quantity of an aqueous crude extract of polynoidin is used to detect O2- released by stimulated cells. Light emission is linearly dependent on the number of cells over a wide range (10(3) to 10(7) cells), and the assay is thus more sensitive than either luminol or ferricytochrome c reduction. Luminescence is enhanced 20% by mannitol, 80% by catalase, and is totally quenched by superoxide dismutase. For the same number of cells, neutrophils showed a threefold higher release of O2- and a twofold faster first-order light decay than stimulated macrophages, in accordance with data obtained by other methods.

Animals↗
PubMed1990

Epithelial macrophages secrete a deactivating factor for superoxide release.

The release of superoxide anion (O2-) by inflammatory macrophages, multinucleated giant cells, and epithelioid cells, obtained by the insertion of round glass coverslips into the subcutaneous tissue of mice, was investigated. O2- was shown to be spontaneously released by cells on the surface of glass coverslips implanted up to 7 days, but not by cells obtained 14 or 21 days after coverslip implantation. The former showed increased O2- release when stimulated by phorbol myristate acetate, whereas cells harvested after 14 or 21 days implantation did not. The induction of delayed type hypersensitivity around coverslips implanted for 5 days increased spontaneous O2- release by these cells by 40%. On the other hand, when the same protocol was used with coverslips implanted for 14 days, O2- release was not detected. These results were viewed in regard to the composition of the cell population at each time point. When coverslips were removed after 14 days of implantation and the cells incubated for 30 minutes in vitro, the medium so conditioned inhibited O2- release by cells of 5 day old preparations. This indicates the release by cells on the longer term coverslips of a substance that inhibits O2- production by cells of coverslips implanted for 5 days only. This inhibitory activity could be suppressed by treating the conditioned medium with proteases. The factor was, however, heat stable and exerted its effects even when the test cells were exposed to phorbol myristate acetate.

Animals↗
PubMed1990

HLA class II-restricted binding of muramyl peptides to B lymphocytes of normal and narcoleptic subjects.

The propensity for narcolepsy, a clinical sleep disorder of unknown etiology, is virtually totally included within the HLA-DR2,DQw1 (DRw15,DQw6) phenotype. The disorder is characterized by decreased sleep latency, early onset of rapid eye movement sleep, and a paucity of nocturnal slow-wave sleep. Muramyl peptides, naturally occurring bacterial cell wall peptidoglycans, potently enhance the duration and amplitude of slow-wave sleep in animals, bind to murine mononuclear cells, and exhibit a major histocompatibility complex-restricted immunoadjuvant effect in mice. We examined the binding of muramyl peptides to peripheral blood mononuclear leukocytes of HLA-typed normal (n = 13) and narcoleptic (n = 10) subjects. Muramyl peptides bound specifically and with high affinity to normal B- but not T-lymphocyte-enriched preparations. There was no significant specific binding to B-cell-enriched preparations from narcoleptic patients. Furthermore, B-lymphocyte-enriched preparations of normal individuals who had the HLA-DR2,DQw1 phenotype (n = 8) exhibited a lower level of specific binding than those of normals who did not have this phenotype (n = 5, p less than 0.001). These observations are an additional indication of the relevance of muramyl peptides to slow-wave sleep and provide a basis for a better understanding of the relation between narcolepsy and the MHC at the biochemical level.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1989

Protein phosphorylation associated with synergistic stimulation of neutrophils.

Neutrophils treated with optimal amounts of tumor promoters that activate protein kinase C (e.g. mezerein) release large quantities of superoxide (O2-) and exhibit an intense phosphorylation of two proteins with molecular masses of approximately 47 and 49 kDa. These cells can also be stimulated synergistically to release a comparable amount of O2-. This involves treatment with a suboptimal amount of a tumor promoter and an agent capable of elevating cellular Ca2+. Neutrophils treated in the former fashion exhibit a redistribution of the activity of protein kinase C from a soluble to a particulate fraction that is stable in the presence of Ca2+ chelators, whereas cells stimulated synergistically do not do so to an appreciable extent (Badwey, J. A., Robinson, J. M., Horn, W., Soberman, R. J., Karnovsky, M. J., and Karnovsky, M. L. (1988) J. Biol. Chem. 263, 2779-2786). In this paper, we report that neutrophils stimulated synergistically do exhibit a significant incorporation of 32P into the 47-kDa protein, but with little labeling of the 49-kDa species. This labeling of the 47-kDa protein was greater than the sum of those observed with each agent added separately but was less than that observed in cells stimulated with optimal amounts of tumor promoters alone. An inhibitor of protein kinase C (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) blocked O2- release and the phosphorylation of the 47-kDa protein under all conditions of stimulation mentioned, whereas an inhibitor of cyclic nucleotide-dependent kinases had no effect on these phenomena. Thus, labeling of the 47-kDa protein can occur in the absence of a "tight" translocation of protein kinase C to membrane and was always observed during synergy. The data support a role for protein kinase C and the 47-kDa phosphoprotein in the synergistic stimulation of neutrophils.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Paradoxical effects of retinal in neutrophil stimulation.

Retinal stimulates the activity of phospholipase C and superoxide (O2-) release in neutrophils. The latter response is comparable in magnitude to that observed when phorbol 12-myristate 13-acetate (PMA) is the stimulating agent. Cells treated with retinal, however, do not undergo degranulation, nor do they exhibit the formation of intracellular vesicles, as is commonly observed with other agents (e.g. Lochner, J. E., Badwey, J. A., Horn, W., and Karnovsky, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7673-7677). Retinal promotes redistribution of the activity of protein kinase C from a soluble to a particulate fraction in neutrophils, and this redistribution precedes O2- release. Superoxide release stimulated with retinal, however, is largely insensitive to inhibitors of protein kinase C (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7); staurosporine). These compounds substantially block both O2- release and the phosphorylation of two proteins with molecular masses of about 47 and 49 kDa when the stimulus is PMA. The data indicate that retinal and PMA elicit the formation of active protein kinase C complexes of different natures, or that the mechanism of stimulation of O2- release by retinal does not involve this kinase. The significance of these observations to the common use of retinoids as inhibitors of protein kinase C is discussed.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Phosphorylation of both 47 and 49kDa proteins accompanies superoxide release by neutrophils.

Neutrophils stimulated with activators of protein kinase C (i.e., 4 beta-phorbol 12-myristate 13-acetate; sn-1,2-dioctanoylglycerol) exhibit a dramatic, dose-dependent incorporation of 32P[Pi] into two proteins with molecular weights of ca. 47 and 49kDa. Proteins of the same molecular weights are also labelled when the cells are stimulated with a chemotactic peptide. However, with the latter stimulus, labelling of the 47kDa species is transient whereas that of the 49kDa entity persists. Labelling of both proteins always accompanied the release of O2-stimulated by these agents. The kinetics of labelling are compatible with the involvement of both phosphoproteins in the stimulation of these cells.

Animals↗
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↗