Non-mitogenic binding of phytohemagglutinin to lymphocytes

³H-PHA is bound to lymphocytes both at 37°C and at 0°C. However, the lectin is rapidly shed from cells which have been pulsed at 0°C and no DNA-synthesis takes place even if the pulsed cells are kept at 37°C for 64 hours. Cells pulsed at 37°C and subsequently at 0°C do not shed the bound lectin. They are induced to synthesize DNA when subsequently incubated at 37°C for 64 hours.     

Ouabain binding to preimplantation rabbit blastocysts

Ciliary ganglia (CG) from 8-day-old chick embryos were cultured as explants on a highly adhesive collagen substratum in the presence of the ciliary neuronotrophic factor (CNTF). A remarkable correlation was found between the formation of an outgrowth of ganglionic nonneuronal cells and the timing and extent of neuritic development outside the ganglion. Neurites were not seen to emerge from the ganglion before the onset (24 hr after explantation) of a nonneuronal cell outgrowth. After nonneurons began to migrate over the collagen substratum, neurites could be seen to extend up to, but not beyond the distal limit of the nonneuronal outgrowth. Time-lapse analysis showed that neuritic growth cones could move in synchrony with a nonneuron with which they were in contact as well as over the nonneuronal cell surface, but not on the collagen located distally to the external edge of the nonneuronal outgrowth.Freshly dissected CGs were also grown as secondary explants on preformed host monolayers of ganglionic nonneurons. These secondary explants showed considerable neuritic development within 24 hr, while control ganglia explanted on collagen had not produced neurites. Autoradiographic experiments indicated that this neuritic outgrowth occurred on nonneuronal cells emerging precociously from the secondary explant, rather than on the preexisting host nonneurons. Electron microscopy of 24-hr explants demonstrated that, inside the ganglion, neurites were also very closely associated with the surface of nonneuronal cells.Neuritic behavior in this nonneuron/collagen terrain is compared with previously described observations of CG explants on polyornithine (PORN) or dissociated CG neurons on PORN or collagen. These observations led to the identification of a PORN-bindable neurite promoting factor (PNPF) which does not bind to, and is not active on, collagen. The hypothesis is discussed that PNPF molecules are present on the surface of nonneuronal cells and that the cells owe to those molecules their competence as a suitable terrain for the elongation of neuritic processes.     

Phytohemagglutinin-lymphocyte interaction : Characterization of binding sites on pig lymphocytes for I-labelled phytohemagglutinin

A small fraction only (0.33%) of the added ¹²⁵I-labelled PHA was bound by pig lymphocytes under culture conditions giving optimal stimulation of transformation. The amount bound corresponded to about 30 000 binding-sites/cell. Pig lymphocytes can, however, bind very much more more PHA than this, corresponding to about 600 000 sites/cell. The PHA-membrane receptor complex formed under conditions giving optimal stimulation was solubilized in 1% sodium deoxycholate. It had a molecular weight of about 300 000 and a sedimentation coefficient of about 9S. In contrast, the majority of the complexes formed in the presence of excess PHA were probably dissociated by sodium deoxycholate. These binding sites had a much lower affinity for PHA than those that mediate transformation.     

Temperature-dependent binding of hydrophilic beta-adrenergic receptor ligands to intact human lymphocytes

Important differences in binding characteristics between agonists and antagonists of the beta-adrenergic receptor have been described. However, these observations have been complicated since most available antagonists are much more lipophilic than agonists. In order to separate out those binding characteristics of agonist vs. antagonist from those characteristics of lipophilic vs. hydrophilic ligands, we have studied competition of the hydrophilic ligands isoproterenol (agonist) and CGP-12177 (antagonist) with [125I]iodopindolol binding in intact human lymphocytes. Analyzing competition curves from assays performed at 13 degrees C, 25 degrees C and 37 degrees C we demonstrated that at lower temperatures there was a decrease in IC50 for isoproterenol but not for CGP-12177. Using cells preincubated with isoproterenol then extensively washed, competition curves with both isoproterenol and CGP-12177 were biphasic, and characterized by the appearance of a population of receptors with a low affinity for both hydrophilic ligands. Furthermore, at lower temperatures the biphasic nature of these curves was accentuated and was characterized by a 6-fold and 40-fold increase in the apparent KD of a population of low affinity sites for isoproterenol and CGP-12177, respectively.     

Muscarinic cholinergic binding sites on intact human lymphocytes

Using the muscarinic chalinergic ligand [³H]-quinuclidinyl benzilate, we have demonstrated that intact, viable human lymphocytes possess specific muscarinic binding sites. The binding is saturable, proportional to cell number, and is displaceable by atropine, benztropine, trihexyphenidyl and scopolamine. The apparent kd is 67 nM and the number of binding sites per cell is on the order of 5 × 10⁴. Not only do these findings provide a pharmacological basis for the observed effects of muscarinic agents on lymphocyte function, they also demonstrate the utility of human peripheral blood lymphocytes for investigation of abnormalities of the muscarinic cholinergic system.     

Ouabain binding to phospholipid-dependent adenosine triphosphatase.

The role of phospholipid in the binding of ouabain to the (Na+ + K+)-dependent adenosine triphosphatase was studied. Enzyme preparations obtained from rabbit kidney were treated with Lubrol WX to remove the phospholipid component essential for ATPase activity. Reconstituted enzyme samples were prepared by the addition of phosphatidylserine and sedimentation of an enzymically active lipid-protein complex. The binding of ouabain to both kinds of preparations was measured under equilibrium conditions with the use of 3H-labelled ouabain and initial ouabain concentrations in the range 0.01-1 micrometer. The main findings were: (i) (Mg2+ + Pi) promoted binding of significant quantities of ouabain only to the reconstituted enzyme; (ii) the absence of added Na+, (Mg2+ + ATP) similarly promoted binding only to the reconstituted samples; (iii) the addition of Na+ in the presence of (Mg2+ + ATP) increased the amount of ouabain bound to the reconstituted enzyme when the ouabain concentration was below about 0.1 micrometer, but it had no effect when the ouabain concentration was about 1 micrometer; (iv) (Mg2+ + ATP) induced ouabain binding to the depleted enzyme only when Na+ was also added; (v) the amount of ouabain bound to both depleted and reconstituted enzymes was the same in the presence of (Mg2+ + ATP + Na+); (vi) the reconstituted enzyme appeared to have a greater affinity for Na+ than did the depleted enzyme.     

Ouabain binding to renal tubules of the rabbit

It is well known that ouabain, a specific inhibitor of Na-K ATPase-dependent transport, interferes with renal tubular salt reabsorption. In this study, we employed radiochemical methods to measure the kinetics of [3H]ouabain binding to slices of rabbit renal medulla and high resolution quantitative autoradiography to determine the location and number of cellular binding sites. The kinetics obeyed a simple bimolecular reaction with an association constant of 2.86 +/- 0.63 SD x 10(3) M-1 min-1 and a dissociation constant of 1.46 x 10(-3) min-1, yielding an equilibrium binding constant of 0.51 x 10(-6) M. Binding was highly dependent upon temperature. At a concentration of 10(-6) M, the rate of accumulation between 25 degrees C and 35 degrees C exhibited a Q10 of 1.8. At 0 degree C the rate of ouabain dissociation was negligible. The specificity of binding was demonstrated with increasing potassium concentrations. At a concentration of 1 microM, 6 mM, and 50 mM K+ produced a 2.5- and 7-fold decrease, respectively, in the rate of ouabain accumulation observed at zero K+. Binding was completely inhibited by 1 mM strophanthin K. The major site of ouabain binding was the thick ascending limb; little or no binding was observed in thin limbs and collecting ducts. Moreover, binding was confined to the basolateral membranes. From autoradiographic grain density measurements, it was estimated that each cell contains over 4 x 10(6) ouabain binding sites or Na-K ATPase molecules. These results taken together with physiological and biochemical observations suggest that Na-K ATPase plays a key role in salt reabsorption by this segment.     

A muscarinic receptor type in human lymphocytes: a comparison of 3H-QNB binding to intact lymphocytes and lysed lymphocyte membranes

Human blood lymphocytes from normal blood donors exhibited specific binding of the muscarinic antagonist 3H-quinuclidinyl benzilate (3H-QNB). The 3H-QNB binding to intact viable lymphocytes as well as to lysed lymphocyte membranes "P2" was saturable and displaceable by both muscarinic agonists and antagonists. For the lysed lymphocyte membranes "P2" a single binding site with a Bmax of 109 pmol/g protein and a Kd of 15 nM was obtained. Intact viable lymphocytes also showed one binding site with a Kd of 24 nM and a Bmax of 1556 pmol/g protein. The higher Bmax value might be explained in terms of uptake of the ligand when using intact cells or through loss of binding sites when using lysed lymphocyte membranes "P2". IC50 values were lower by a factor of 10(2) for atropine and scopolamine and by 10(4) for pirenzepine when lysed lymphocyte membranes "P2" were used instead of intact viable lymphocytes.     

Response of baboon peripheral blood lymphocytes to phytohemagglutinin: time-course and dose-response relationships.

Optimal conditions for studying phytohemagglutinin(PHA)-induced transformation of baboon peripheral blood lymphocytes were determined. Maximal stimulation, as determined by uptake and incorporation of tritiated thymidine (total cell and trichloroacetic-acid-precipitable radioactivity), occurred at a PHA level of 12.5 microgram in a culture volume of 0.25 ml containing 2 x 10(5) lymphocytes. Optimal stimulation occurred after a total incubation period of 114 h, the last 18 h of which was in the presence of 1muCi of the labeled DNA precursor per culture. While there was considerable variation in the extent of responsiveness of lymphocytes from individual animals, the shape of the dose-response and time-course curves for most mitogen concentrations was generally similar.     

Kinetics of proliferative processes induced by phytohemagglutinin in irradiated lymphocytes

The kinetics of the induced process of normal and exposed human lymphocytes to DNA synthesis and mitosis has been analyzed in detail. It was shown that the capacity of the exposed lymphocytes of repeated entering the phase of DNA synthesis was sharply decreased and the average length of the prereplicative period increased.     

Membrane viscosity of lymphocytes and influence of phytohemagglutinin

The membrane viscosity of peripheral blood lymphocytes (PBLs) of equine, bovine and canine was measured by the use of time-resolved fluorescence depolarization technique with 1, 6-diphenyl-1,3,5-hexatriene (DPH). The viscosity values were 0.55, 0.59 and 0.50 poise for equine, bovine and canine PBLs, respectively. These values were compared with steady-state anisotropies and order parameters measured from electron spin resonance (ESR) of 5-doxyl stearic acid. Both values were increased with increase of viscosity. The fluid property of the membranes stimulated with phytohemagglutinin-P (PHA) was measured with steady-state fluorescence anisotropy and ESR. Little change of membrane fluidity was recognized with both methods during the stimulation with PHA. It appears that PHA activation process for these lymphocytes does not included large increase of the membrane fluidity which significantly accelerate the diffusion velocity of receptors in the plasma membrane.     

Cell kinetics and radiosensitivity of Cercopithecus aethiops sabaceus lymphocytes stimulated by phytohemagglutinin

The use of the BudR-Giemsa technique has shown that the peripheral blood lymphocytes of Cercopithecus aethiops sabaceus display a very fast answer to PHA-stimulation. At 32 h culture time numerous mitoses are already present and at 48 h more than 30% of the dividing cells are in second mitosis. Exposure to X-irradiation results in a higher incidence of unstable aberrations than in human lymphocytes.     

Methylation of transfer RNA during transformation of human lymphocytes by phytohemagglutinin

Methylation of transfer RNA during phytohemagglutinin induced transformation of human lymphocytes was studied by labeling the tRNA $\text{in}\text{vivo}$ with (methyl-H³)-methionine and measuring the distribution of tritium in the methylated nucleotides. An alteration in the pattern of methylation occurred within hours after PHA-stimulation and this change was maintained through several cell generations. There was a 50 to 94% increase in the relative amount of methylated N²-methyl-guanine (1 to 3 hr) and a 40 to 59% decrease in the relative amount of 1-methyladenine (1 to 12 hr). Treatment of the stimulated cells with Actinomycin D prevented the subsequent methylation not tRNA. However, inhibition of protein synthesis by puromycin did not effect the early changes observed in the methylation of tRNA.