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M P Henkart

Publications and source records attributed to M P Henkart.

12 recordsLinked to original sources
PubMed1984

Cytolytic activity of purified cytoplasmic granules from cytotoxic rat large granular lymphocyte tumors.

Purified cytoplasmic granules from cytotoxic rat large granular lymphocytes (LGL) tumors were cytolytic to erythrocytes, splenocytes, and a number of different lymphoid tumor cells. Granule concentrations of approximately 1 microgram/ml granule protein were adequate to lyse 100% of the erythrocytes, while the nucleated cells required up to 100 micrograms/ml granule protein to achieve complete lysis. Cytoplasmic granules purified from noncytotoxic lymphoid cells did not contain detectable cytolytic activity; purified granules from rat mast cells and rat liver lysosomes likewise failed to display cytolytic activity. However, granules prepared from normal rat peripheral blood LGL were cytolytic. Granule-mediated lysis of erythrocytes and nucleated cells was complete within 3 min at room temperature. The lytic activity required calcium at concentrations of 10(-4)-10(-2) M; magnesium or barium failed to replace calcium, while strontium could replace calcium at 10(-3)-10(-2) M when nucleated cells were the target. Exposure of LGL tumor granules to calcium before the addition of target cells resulted in an inactivation of granule cytolytic activity over the course of 20 min at room temperature. Granule cytolytic activity was heat and Pronase sensitive, and could be solubilized by 2 M salt. Examination of granules exposed to calcium in the electron microscope using negative staining showed that calcium treatment of granules results in the formation of ring-shaped structures previously described to be associated with LGL-mediated cytotoxicity. These results provide support for the hypothesis that the cytotoxic processes mediated by LGL are a secretory event characterized by the release of cytolytic material from the cytoplasmic granules after triggering by a surface receptor. The results further suggest that the ring structures visible in the electron microscope are associated with the lytic event.

Animals↗
PubMed1984

Liposomes as targets for granule cytolysin from cytotoxic large granular lymphocyte tumors.

Purified cytoplasmic granules from rat large granular lymphocyte tumors having natural killer activity and/or antibody-dependent cell-mediated cytotoxicity induced a rapid, dose-dependent release of the water-soluble marker carboxyfluorescein from liposomes made of phosphatidylcholine. A solubilized, partially purified cytolytic preparation termed "cytolysin" from these granules showed identical properties. Marker release induced by granules or the cytolysin was strongly dependent on the presence of Ca2+ at a concentration of 0.1 mM or higher in the medium; Ca2+ could be replaced by higher concentration of Sr2+ but not by Ba2+ or by Mg2+. These properties strikingly parallel the lytic effects that granules and granule cytolysin exert on cells. Marker release from liposomes was stopped instantaneously when an excess of EGTA was added to the medium. The remaining carboxyfluorescein inside the liposomes was present at the original internal concentration, indicating that marker release was all-or-none from individual liposomes. Liposomes comprised of lipid in the solid phase released marker more slowly than did comparable liposomes containing fluid-phase lipids. Variation of the lipid headgroup had only minor effects on the cytolysin-induced marker release. Electron microscopy of liposomes exposed to cytolysin in the presence of Ca2+ showed cylindrical structures of 15-nm diameter inserted into the membrane concomitant with the penetration of negative stain into the liposome. These properties of large granular lymphocyte granule cytolysin strongly suggest that it operates through a mechanism similar to the membrane attack of complement.

Animals↗
PubMed1984

Purification and properties of cytoplasmic granules from cytotoxic rat LGL tumors.

To evaluate the role of NK cell granules in the lytic activity of NK cells, cytoplasmic granules of rat NK tumors were purified by centrifugation of the cell homogenates in a Percoll gradient. Analysis of such gradients showed a band of light-scattering material near the bottom of the tube; assay of gradient fractions for lytic activity against SRBC showed a potent lytic activity giving a sharp peak in this region. Complete lysis of SRBC was achieved with less than 1 microgram/ml protein of the most active fractions. Examination in the electron microscope showed that a pool of fractions containing lytic activity consisted of pure cytoplasmic granules showing similar morphology to those found in the LGL tumors. The lytic band was associated with a peak in the activity of four different lysosomal enzymes. Analysis of Percoll gradient fractions showed that marker enzymes for mitochondria, plasma membrane, and cytosol were well separated from this activity peak. Analysis of the Percoll gradient fractions by SDS gel electrophoresis showed that this granule fraction was free of contamination of proteins from other parts of the gradient. The granules contained major protein bands of 62, 58, 30, 29, and 28 kilodaltons. In addition to protein, the purified granule fractions contain hexose and uronic acid, but no nucleic acids or phospholipids were detected in chemical assays. Major amounts of chymotryptic, tryptic, and elastase activities were not present, nor were peroxidase or lysozyme activities detectable in substantial amounts. These data show that NK tumor cell cytoplasmic granules contain a potent lytic activity and have biochemical properties that distinguish them from granules present in granulocytes and mast cells.

Animals↗
PubMed1979

Evidence for an intracellular calcium store releasable by surface stimuli ifibroblasts (L cells).

A spontaneously occurring or electrically elicited hyperpolarizing activation (HA) in L cells was previously shown to be due to a specific increase in the membrane K+ permeability (Nelson et at. 1972. J. Gen. Physiol. 60:58--71). Intracellular injection of Ca++ elicits an identical hyperpolarizing response which suggests that the increased K+ permeability associated with the HA is mediated by an increase in cytoplasmic Ca++. In zero-Ca, EGTA-containing saline the proportion of cells in which HA's can be evoked decreases, but the amplitude of those HA's that are produced is comparable to that of HA's in normal Ca saline. Co++ does block the HA but only after a period of 2 h or longer; D-600 does not affect the HA. The observations, with others, suggest that the primary source of the Ca mediating the HA response is intracellular. In L cells the endoplasmic reticulum forms morphologically specialized appositions with the surface membrane which resemble structures at the triads of muscle that are thought to mediate coupling between surface membrane electrical activity and contraction via Ca release from the sarcoplasmic reticulum. The similar structures in L cells may mediate coupling between surface membrane electrical, mechanical, or chemical stimuli and the HA response via release of Ca from the endoplasmic reticulum. Surface-coupled release of Ca from intracellular stores might also regulate a number of other intracellular functions in nonmuscle cells.

Calcimycin↗
PubMed1979

Oscillatory membrane potential changes in cells of mesenchymal origin: the role of an intracellular calcium regulating system.

A number of mesenchymal cells (fibroblasts, macrophages and megakaryocytes) respond to a variety of stimuli with large hyperpolarizations lasting several seconds (the H.A. response). The H.A. responses can occur as repetitive trains or oscillations. These hyperpolarizations are due to an increase of the surface membrane permeability to potassium ions which is probably mediated by an increase in the cytoplasmic free calcium ion concentration. Evidence is discussed which suggests that the source of this increased calcium, is least in part, an intracellular sequestering system, probably the endoplasmic reticulum. A model capable of producing oscillatory changes in membrane potential is proposed based on such an intracellular calcium sequestering and releasing system.

Calcium↗
PubMed1978

Endoplasmic reticulum sequesters calcium in the squid giant axon.

Axons were loaded with calcium, rapidly frozen, and freeze-substituted. The endoplasmic reticulum, in addition to mitochondria, contained calcium deposits, as indicated by electron probe x-ray microanalysis. Oxalate injected into living axons helped to preserve calcium-containing deposits during preparation for microscopy. It is concluded that the endoplasmic reticulum is a calcium-sequestering compartment in the squid giant axon.

Animals↗
PubMed1973

Presynaptic inhibition at inhibitory nerve terminals. A new synapse in the crayfish stretch receptor.

Previous physiological evidence has shown that the receptor neuron of the slowly adapting stretch receptor organ of crayfish receives synapses from three inhibitory axons, while the receptor muscle is innervated by both excitatory and inhibitory axons. Fine structural studies have indicated that after certain preparative procedures synaptic vesicles in the inhibitory terminals on dendrites of the receptor neuron appear small and elongate, while those in the excitatory terminals on the receptor muscle are round and larger. This study describes a new synapse between two inhibitory nerve endings on the receptor neuron. One axon, containing small elongate vesicles, forms a presynaptic chemical contact with another morphologically similar axon that, itself, presumably releases inhibitory transmitter onto the receptor neuron. A second type of presynaptic axo-axonic synapse, analogous to one previously described in another crustacean muscle, was also found between presumed inhibitory and excitatory nerve terminals on the receptor muscle. Thus, the stretch receptor has a relatively complex organization with a morphological basis for two types of presynaptic inhibition: one on excitatory terminals and the other on inhibitory terminals.

Animals↗
PubMed1971

Excitation-contraction coupling in amphioxus muscle cells.

1. Excitation-contraction coupling was studied in myotomal muscles of amphioxus, Branchiostoma californiense.2. The action potential of a muscle cell produces a twitch with a rise time of 30-40 msec at 11 degrees C and its Q(10) is about 2.2.3. The twitch increases in amplitude with increasing external Ca concentration and is abolished in Ca-free saline (1 mM-EGTA and 55.7 mM-MgCl(2)); the twitch amplitude is suppressed by Co or La ions.4. Caffeine at concentrations above 1 mM in the external saline causes a prolongation of the action potential and a contracture which lasts several minutes.5. After exposure to caffeine the responsiveness of the muscle to subsequent applications of caffeine recovers in normal saline in 20-30 minutes but not in Ca-free saline.6. The amplitude of the caffeine contracture is independent of the external Ca concentration and is unaltered after the twitch is eliminated in Ca-free saline.7. After exposure to caffeine a full-sized twitch can be obtained before the responsiveness to caffeine shows any significant recovery.8. It is concluded that the twitch is produced by the Ca influx resulting from the increased permeability of the muscle cell membrane to Ca during the action potential and that the Ca mobilized by caffeine is not necessary to the initiation of the twitch.9. Electronmicroscopy shows the existence of sarcoplasmic reticulum.

Action Potentials↗