Early stimulation of potassium uptake in lymphocytes treated with PHA.
TL;DR: The results thus suggest that the categorization of lymphoid cells into the “short-lived” and the "long-lived" categories is a gross oversimplification, and are consistent with the interpretation that there are a multitude of subclasses of lymphoids cells, having discretely different life expectancies in the range from a few days or less to well over 1 month.
About: This article is published in Experimental Cell Research.The article was published on 1970-11-01. It has received 182 citations till now. The article focuses on the topics: Lymph node & Spleen.
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TL;DR: This chapter discusses the interactions of lectins with animal cell surfaces, which have proven to be quite useful for clinical blood typing and structural studies of blood group substances, in analysis of the surface structure of normal and tumor cells, and so on.
Abstract: Publisher Summary This chapter discusses the interactions of lectins with animal cell surfaces. Lectin interactions with cell surfaces can be put into a proper framework for understanding complex phenomena such as lectin-induced mitogenic stimulation, cell agglutination, and lectin-mediated cell toxicity. Lectins have been isolated from a wide variety of plants and animals, from legumes to horseshoe crabs. Lectins generally have the property of agglutinating cells through their cell surface oligosaccharide determinants, and in some cases these determinants have been elucidated by determining the sequences or sugars responsible for saccharide inhibition of agglutination. Because of the carbohydrate-binding specificities of lectins, some of these agglutinins have proven to be quite useful for clinical blood typing and structural studies of blood group substances, in analysis of the surface structure of normal and tumor cells, for specific isolation of glycoproteins and oligo- and mucopolysaccharides, in studies on mitogenesis, as antigen-antibody models, and so on. They have also proven to be invaluable as specific molecular probes for studying membrane, cell, and tissue structure and organization.
974 citations
TL;DR: 3H-thymidine incorporation by T lymphocytes following PHA stimulation is inhibited by the ‘classical’ K+ channel blockers tetraethylammonium and 4-aminopyridine, and also by quinine, suggesting that K+ channels may play a part in mitogenesis.
Abstract: Membrane receptors and ion transport mechanisms probably have an important role in lymphocyte activation leading to T-lymphocyte proliferation in the immune response. Here we have applied a gigaohm-seal patch clamp technique1 to reveal the identity and properties of ion channels in human T lymphocytes. A voltage-dependent potassium channel bearing a resemblance to the delayed rectifier of nerve and muscle cells was found to be the predominant ion channel in these cells. In the whole cell recording conformation, the channels open with sigmoid kinetics during depolarizing voltage steps, reaching a maximum K+ conductance of 3–5 nS. The current subsequently becomes almost completely inactivated during a long-lasting depolarization. Currents through single K+ channels recorded in whole cell and outside-out patch recording conformations reveal a unitary channel conductance of about 16 pS in normal Ringer solution. Thus, the peak current corresponds to approximately 200–300 conducting K+ channels per cell. Phytohaemag-glutinin (PHA), at concentrations that produce mitogenesis, alters K+ channel gating within 1 min of addition to the bathing solution, causing channels to open more rapidly and at more negative membrane potentials. 3H-thymidine incorporation by T lymphocytes following PHA stimulation is inhibited by the ‘classical’ K+ channel blockers tetraethylammonium and 4-aminopyridine, and also by quinine, at doses found to block the K+ channel in voltage-clamped T lymphocytes, suggesting that K+ channels may play a part in mitogenesis.
730 citations
TL;DR: Increased turnover of phospholipid fatty acids, following lymphocyte stimulation, requires a transferase to incorporate new fatty acids and a means of generating lysophosphatides and free fatty acids as substrates for this enzyme.
Abstract: Publisher Summary This chapter discusses fatty acids and immunity. Like all mammalian cells, lymphocytes contain a variety of fatty acids, some of which, the essential fatty acids, must be supplied to the cells because they are unable to synthesize them. Of the essential fatty acids, linoleic acid and the derivative arachidonic acid are major components of the phospholipids of lymphocyte membranes, while α-linolenic and its derivatives are only minor components. A wide variety of fatty acids produce effects on both the lymphoid and reticuloendothelial systems; the actual effects produced depend on the method of administration and the chemical nature of the fatty acid. Arachidonic acid is an immediate prostaglandin precursor and plays a central role in lymphocyte activation. Membrane phospholipid fatty-acid composition alters when lymphocytes are stimulated, either by antigen or by mitogens, such as phytohaemagglutinin or concanavalin A. Increased turnover of phospholipid fatty acids, following lymphocyte stimulation, requires a transferase to incorporate new fatty acids and a means of generating lysophosphatides and free fatty acids as substrates for this enzyme.
221 citations
TL;DR: The finding that quiescent non-dividing small lymphocytes could be triggered into a derepressed state of active growth and proliferation in vitro by phytohaemagglutinin (PHA) was a major advance in the realisation of this potential.
Abstract: One of the motivating forces in lymphocyte biology is the belief that the behaviour of lymphocytes, and more particularly their response to antigen, provides a unique opportunity to analyse the molecular and cellular events involved in the induction and expression of 'differentiation'. A major advance in the realisation of this potential was the finding (Nowell 1960) that quiescent non-dividing small lymphocytes could be triggered into a derepressed state of active growth and proliferation in vitro by phytohaemagglutinin (PHA). The response observed involves parameters common to many systems in which cell growth and proliferation are occurring, e.g. tissue regeneration and embryogenesis, and includes enhanced glycolysis, lysosomal enzyme activity, enhanced nuclear template activity, histone acetylation, DNA polymerase enzyme activity, etc. More specific changes indicative of 'differentiated' activities of lymphocytes, e.g. immunoglobulin synthesis, may also occur, as will be discussed in this review. Although precise quantitation of the number of responding cells has yet to be reported it is clear that the number of cells reacting to PHA and similar stimulants Is considerably greater than one would expect to observe with a classical 'immunological' response in which antigen recognition by relatively small numbers of cells occurs (i.e. 'cional' selection). The response to PHA and other mitogens (see below) is therefore generally considered to be non-specific, i.e. it is a polyclonal lymphocyte response initiated by
216 citations
TL;DR: Lysolecithin acyltransferase showed specific activities between 10 to 12 nmoles·(mg protein)−1·min−1 in the plasma membrane compared to 3–4 nmoles–(mgprotein)− 1·min+1 in endoplasmic reticulum and thus appears to be a plasma membrane component in lymphocytes.
211 citations
References
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TL;DR: The present study arose from the observation that a more intense colour was sometimes produced if, instead of being heated at 1000 for 10 min., the reaction mixture was allowed to stand overnight at room temperature.
Abstract: Of the colour reactions available for the determination and identification of deoxyribonucleic acid (DNA), the reaction with diphenylamine in a mixture of acetic and sulphuric acids at 1000 (Dische, 1930) has been perhaps the most widely used. The present study arose from the observation that a more intense colour was sometimes produced if, instead of being heated at 1000 for 10 min., the reaction mixture was allowed to stand overnight at room temperature. As a result of this observation the procedure has been modified, principally by adding acetaldehyde to the reagents and by allowing the solution to stand for about 17 hr. at 30° instead of heating it at 1000. The modified method is 3-5 times as sensitive as Dische's original procedure, and several substances which interfere in the original method do not do so in the modified procedure. Some observations on the mechanism of the reaction have been made; in particular it was discovered that there is a liberation of inorganic orthophosphate from DNA during the early stages of the reaction. This finding has a bearing on the structure of DNA. The modified method has already been used in an investigation of nucleic acid metabolism during bacteriophage multiplication (Burton, 1955).
13,649 citations
TL;DR: The viability of column-separated cells was shown by their non-staining with trypan blue, motility, phagocytic ability, oxygen consumption, and survival or development in tissue culture, whereas Nowell’s blast-like, dividing, phytohemagglutinin cells were produced only in cultures containing lymphocytes.
437 citations
TL;DR: In vitro ouabain, at relatively low concentrations, inhibits the blastogenic action of PHA as well as the incorporation of tritiated nucleosides into nucleic acid; excess potassium in the culture medium diminishes or prevents this effect of ouABain.
Abstract: DURING a study of the blastogenic action of phyto-haemagglutinin (PHA) on small lymphocytes in vitro1, we found cause to speculate that interference with membrane ion transport might selectively inhibit part of the sequence of events2–7 required for cell transformation. We therefore investigated8 the effect of the cardiac glycoside, ouabain (‘G-Strophanthin’), which is known selectively to inhibit coupled sodium and potassium transport9–11. We now report that in vitro ouabain, at relatively low concentrations, inhibits the blastogenic action of PHA as well as the incorporation of tritiated nucleosides into nucleic acid; excess potassium in the culture medium diminishes or prevents this effect of ouabain.
140 citations
TL;DR: The results are consistent with a postulated working hypothesis that lymphocyte stimulation involves an early and protracted activation of membrane transport Na-K-ATPase, and is critically dependent on a subsequent increase of intracellular potassium which is suppressed by ouabain.
109 citations
TL;DR: It is suggested that PHA induces early changes in the surface of lymphocytes and the consequent redistribution of acid hydrolases may play a role in remodeling processes of the stimulated cells.
Abstract: Subcellular fractions were isolated by differential centrifugation from pure suspensions of human blood lymphocytes incubated with and without phytohemagglutinin (PHA). Between 30 and 120 min after addition of PHA to intact cells, redistribution of acid hydrolases (beta glucuronidase, acid phosphatase), from a 20,000 g x 20 min granular fraction into the corresponding supernatant, was observed. No increase in total acid hydrolase activity was found at these times. The mitochondrial marker enzyme, malate dehydrogenase, did not undergo redistribution. Granules derived from PHA-treated cells became more fragile upon subsequent incubation with membrane-disruptive agents in vitro (streptolysin S, filipin). These changes were associated with an increase in the over-all permeability of the stimulated cell to substances in the surrounding medium, such as neutral red. Augmentation of dye entry into lymphocytes required intact metabolism as judged by response to temperature and inhibitors (cyanide, antimycin A, 2,4-dinitrophenol). PHA, however, did not release enzyme activity from hydrolase-rich granules in vitro or render them more susceptible to subsequent challenge with membrane-disruptive agents. These studies suggest that PHA induces early changes in the surface of lymphocytes. The consequent redistribution of acid hydrolases may play a role in remodeling processes of the stimulated cells.
103 citations