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Biomedical subjects

M J Karnovsky

Publications and source records attributed to M J Karnovsky.

At least 163 records · Page 9Linked to original sources
PubMed1980

Specializations in filopodial membranes at points of attachment to the substrate.

A mouse cell line (LM), which grows predominantly as spindle-shaped cells with numerous filopodia, was employed in this study. These filopodial projections appear to be important as sites of attachment to the substratum in LM cells. Morphologically the filopodia are slender projections from the cell body which usually attach to the substrate at their distal ends (filopodial footpads). Freeze-fracture of monolayer cultures in situ preserves the spatial relationship of filopodial processes to that of the cell body. Examination of these freeze-fracture preparations reveals a striking difference in the density of intramembrane particles (IMP) in the filopodial-footpad plasmalemma compared with the plasmalemma of the cell body (number of IMP in footpad > cell body). Additionally, there is a marked difference in the number of filipin-sterol complexes on the cell body, compared with the filopodial footpad, implying a difference in the cholesterol content in these regions (filipin-sterol complexes in footpad < cell body). These data suggest a structural and functional specialization of the filopodial-footpad plasma membrane which may be related to cell adhesion.

Animals↗
PubMed1980

Inhibition of rat arterial smooth muscle cell proliferation by heparin. II. In vitro studies.

We studied in vitro the effects heparin on the growth of rat aortic smooth muscle cells. Measurements of growth were monitored by [3H]thymidine uptake and changes in cell number over a period of 3 days. Our results show that heparin-highly anticoagulant or nonanticoagulant-significantly inhibits growth of smooth muscle cells. We also show that this is a highly specfic interaction with regard to molecule and cell type: i.e., other polyanions, except for a low molecular weight dextran sulfate, do not have the same effect on growth, and not all cells are inhibited by heparin; e.g., endothelial cell growth actually is enhanced. After removing antithrombin from our media, we carried out experiments which show that heparin is effective even though thrombin, a potent mitogenic agent, is still present and active. We also found that passing the platelet extract over a heparin column did not remove all of the motogenic activity of the platelet preparation. Both experiments indicate an inhibitory role for the heparin molecule, per se. Our results support the findings of a recent paper (Guyton et al., 1980) showing that heparin can limit the size of myointimal plaques in rats after carotid injuries by inhibiting smooth muscle cell proliferation.

Animals↗
PubMed1980

Contraction of cultured rat glomerular cells of apparent mesangial origin after stimulation with angiotensin II and arginine vasopressin.

Studies to identify the physiological role of glomerular mesangial cells were undertaken using homogeneous cultures of rat glomerular cells of apparent mesangial origin (MS). Cultured MS cells were treated with arginine vasopressin (AVP), angiotensin II (AGII), prostaglandin E(2), and parathyroid hormone. AVP (0.1 nM) and AGII (1 nM) stimulated contraction of MS cells in vitro that was complete by 2 min at 37 degrees C or 10 min at 23 degrees C as observed by phase contrast and electron microscopy. Relaxation recurred 15 min after hormonal addition at 23 degrees C. Similar experiments in cloned rat glomerular epithelial cells or "renin"-producing cells did not demonstrate a contractile response. The contraction of MS cells was independent of cyclic AMP (cAMP) and cyclic 3',5'-guanosine monophosphate (cGMP) production, even when cyclic nucleotides were measured as early as 30 s after hormonal stimulation. To demonstrate that contraction was a function of hormone-receptor interaction, binding of [(3)H](8-lysine)vasopressin was studied. Specific binding for 1.6 and 5 nM hormone was both time- and dose-dependent. The estimated apparent affinity was 10 nM. In late MS cell passages (>16th) that no longer demonstrated hormone-stimulated contraction, no specific binding of [(3)H](8-lysine)vasopressin was observed. Incubations were modified to optimize the conditions for detecting the effect of hormones on cell cyclic nucleotide content. A supramaximal concentration of AVP (200 nM) increased the cAMP content of MS cells twofold in the presence of a phosphodiesterase inhibitor. Similar experiments with prostaglandin E(2) (1 mug/ml) led to a 1.5-6-fold increase in MS cell cAMP content, but no effect on contraction was observed. Neither hormone altered cGMP content. These data are further support for the independence of contraction and cyclic nucleotide production. Our studies suggest that MS cells are the equivalent of smooth muscle cells in the glomerulus and that their contraction may be important in control of glomerular filtration.

1-Methyl-3-isobutylxanthine↗
PubMed1980

Evaluation of the polyene antibiotic filipin as a cytochemical probe for membrane cholesterol.

Distribution of the filipin-cholesterol complexes has been studied by freeze-fractures techniques. While filipin-cholesterol complexes are formed in fixed as well as unfixed cells, the distribution of the complexes varies with fixation. Furthermore, filipin induces dramatic rearrangement of intramembrane particles in unfixed, but not in fixed, cells. Examination of thin sections of filipin-treated cells shows that filipin alone induces substantial cellular damage, while filipin treatment simultaneous to fixation or following fixation does not cause noticeable damage. For filipin to be an effective cytochemical probe of membrane structure, cellular damage must be minimized; this is best achieved by treating cells with filipin following glutaraldehyde fixation.

Animals↗
PubMed1980

Adhesion of leukocytes to endothelium: roles of divalent cations, surface charge, chemotactic agents and substrate.

The attachment of polymorphonuclear leukocytes (PMN) to the endothelial lining of blood vessels is an important initial event in the acute inflammatory reaction. Experiments were carried out to examine some of the parameters of this adhesive interaction and the extent to which the substrate (endothelium) plays a role in controlling the process. Using a monolayer collection assay for adhesion, we found a requirement for divalent cations. Mn2+ produces maximal adhesion followed by Zn2+ greater than Ni2+ greater than Mg2+ greater than Ba2+ greater than Ca2+. Pretreatment of either the endothelium of PMN with chemotactic agents (zymosan-activated serum, a bacterial filtrate, C5a and formyl-methionyl-leucyl-phenylalanine) causes an increase in the attachment of PMN to endothelial monolayers. Modulations of net surface charge, as measured by laser Doppler electrophoresis, are not correlated to alterations in adhesion. Mn2+, which produces maximal adhesion, reduces the net surface charge to the same extent as Ca2+, which has the smallest effect on adhesion. Similarly, reductions in net surface charge are not responsible for the increases in adhesion due to the chemotactic agents because there are no differences in electrophoretic mobilities between cells treated with either chemotactic agents or their appropriate controls. Scatchard analysis of the binding of the chemotactic tripeptide, formyl-methionyl-leucyl-phenylalanine, indicates the presence of high-affinity binding sites on the surface of the endothelium. The results suggest a role for the endothelium in mediating the adhesive interaction with the PMN.

Animals↗
PubMed1980

The role of negative charge in spontaneous aggregation of transformed and untransformed cell lines.

No correlation between net negative surface charge as determined by electrophoretic light-scattering techniques and the rates of spontaneous aggregation of 3T3MIT and SVPy 3T3MIT cells has been found. Neuraminidase treatment of both 3T3MIT cells and SVPy 3T3MIT cells causes a significant decrease in electrophoretic mobility but only the 3T3MIT cells show an increase in spontaneous aggregation. An increase in spontaneous aggregation of 3T3MIT cells is seen after growth in 200 mM urea for 18 h but no change in net surface charge occurs. The distribution of anionic sites on the membranes of cells was determined using the ultrastructural marker polycationized ferritin. The distribution of polycationized ferritin-binding sites was essentially identical for both cell lines under all conditions when they were labelled at 4 degrees C. When the cells were labelled with polycationized ferritin at 37 degrees C it was found that cells which have a high net rate of spontaneous aggregation also show rearrangement of anionic sites on their surface membrane. Clustering and rearrangement of anionic sites at 37 degrees C correlate with high rates of spontaneous aggregation.

Animals↗
PubMed1979

Ionophore A23187 disrupts membrane structure by modifying protein-lipid interactions.

The physiological function of membrane-boudn protein clearly depends on the nature of its neighbouring lipid. However, the question of how critical the protein is to the structure and function of membrane lipid has received much less attention. There is some evidence that lipid surrounding membrane protein, 'boundary lipid', may be more ordered than continuum lipid. Protein can also alter the enthalpy and temperature of pure lipid-phase transitions. However, controversy surrounds the question of whether membrane proteins determine long-range aspects of lipid structure or merly perturb their local environments. Here, we demonstrate that a lipophilic substance, the calcium ionophore, A23187 (Lilly), dramatically disorders the lipid structure of the membrane and that this phenomenon depends on the presence of proteins.

Animals↗
PubMed1979

Regional membrane specialization in the thin limbs of Henle's loops as seen by freeze-fracture electron microscopy.

Rat thin limbs of Henle were studied by freeze-fracture electron microscopy. Thin limb segments in both short- and long-looped nephrons were identified by previously developed ultrastructural criteria, continuity with known thick segments, and architectural relationships in the outer medulla. Intramembrane particle (IMP) density and the number of intramembrane fibrils comprising the zonula occludens were determined for each morphologically identifiable thin limb segment. The IMP density on the protoplasmic faces of both the luminal and abluminal membranes of the upper portion of the descending thin limb (DTL) of the long-looped nephron is quantitatively greater than in the short-looped thin limb, lower portion of the long-looped DTL, and in the ascending thin limb. The zonulae occludens in the long-looped upper DTL consists of a single fibril; the long-looped lower DTL contains 3.13 +/- 0.14 fibrils; the ascending thin limb contains 1.31 +/- 0.09 fibrils; and the short-looped DTL contains 3.75 +/- 0.19 fibrils. These studies further support the contention that there is anatomic heterogeneity among the thin limb segments. Because direct physiologic studies in the thin limbs are incomplete and conflicting, the need for correlative physiologic studies on anatomically characterized structures is indicated.

Animals↗
PubMed1979

Lymphocyte surface and cytoplasmic changes associated with translational motility and spontaneous capping of Ig.

Murine B-lymphocytes during translatory motion undergo a series of changes with respect to their morphology and distribution of surface immunoglobulins (Ig). The sequence of events comprising these changes was followed by fluorescence microscopy and a correlated detection of surface features by scanning microscopy on exactly the same cell. A round, presumably non-motile lymphocyte exhibited a random distribution of Ig and its surface displayed evenly distributed microvilli. Formation of a ruffled edge at one pole, accompanied by a decreased fluorescence at this pole marked the initial events of lymphocyte motility. In the subsequent stages, the ruffled edge became progressively prominent and displayed a constriction at its base, while the microvilli were displaced to the opposite pole. Ig in such lymphocytes was localized at the trailing, microvilli-rich pole. Thin sections of motile lymphocytes revealed Ig, microtubules, microfilaments and coated vesicles as the characteristic features of the trailing end. These observations may have bearing on the mechanism of lymphocyte motility and spontaneous capping of Ig.

Animals↗
PubMed1979

Membrane alterations and other morphological features associated with polyethylene glycol-induced cell fusion.

Polyethylene glycol (PEG) induces rapid fusion of LM cells. Membrane fusion, as detected by formation of pentalaminar membrane arrays, occurs as early as 1 min after PEG treatment. The entire cell surface arrears to be capable of fusion since fusion occurs in regions where pseudopodia make contact with each other or with a neighbouring cell body and also in areas where cells are in contact along their entire periphery. Cytoskeletal components showed no apparent deleterious effect from PEG treatment or subsequent cell fusion as determined by thin-section EM. Freeze-fracture of monolayer cultures reveals a thermotropic rearrangement of intramembranous particles following PEG treatment.

Animals↗
PubMed1979

Smooth muscle cell proliferation in the occluded rat carotid artery: lack of requirement for luminal platelets.

The relationship of intimal smooth muscle cell proliferation in the permanently occluded rat carotid artery to the presence or absence of luminal platelets was examined. Blood was rinsed from the arterial lumen immediately after occlusion and was replaced by autologous, citrated platelet-rich plasma (PRP, 6 to 20 X 10(5) platelets/microliter) or filtered platelet-poor plasma (PPP, less than 100 platelets/microliter). Occluded arteries were studied after 1 to 28 days by light and electron microscopy. Events occurring within the first 2 days included fibrin clot formation, endothelial degeneration and denudation, transmural migration of polymorphonucelar leukocytes and monocytes, and, in PRP-filled arteries, degranulation and disappearance of platelets. By 7 days a neointima was formed by macrophages and undifferentiated cells. The latter cells had some features of vascular smooth muscle cells and were apparently derived from medial cells which traversed the internal elastic lamina. After 14 days, identifiable smooth muscle cells emerged as the predominant cell type in a rapidly growing intimal plaque. No differences could be discerned between arteries originally filled with PRP or PPP. This experimental model is similar to atherosclerosis in dimensions of avascular area and in coexistence of degenerative, inflammatory, and proliferative processes. Cell proliferation deep within an atherosclerotic plaque could be initiated by factors other than platelets, perhaps by products of inflammatory cells.

Animals↗