Calcium channel ligand binding to intact, concanavalin A and cyclic AMP-treated cells of the immune system.

To examine whether the cells of immune system express calcium channel-forming proteins, we studied the binding of calcium channel ligands, known to detect certain types of the above channels in excitable tissues, to murine splenic and human peripheral blood mononuclear cells. Specific (i.e., displaceable by excess cold ligand) binding of the 3H-labelled dihydropyridine drugs PN200-110 and nitrendipine, was not detected in these cells. Specific binding of a phenylalkylamine drug, [3H]verapamil, was detected, but cannot be attributed to the existence of certain specialized receptors, since multiple [3H]verapamil binding sites (about 10(6) per cell) appeared to be occupied. [3H]Verapamil binding to murine splenic mononuclear cells was inhibited following exposure to either the polyclonal T-cell activator, concanavalin A, or a cell-permeable analogue of the second messenger, cyclic AMP, suggesting that processes of lymphocyte activation and/or intracellular signalling may down-modulate at least some of calcium channel ligand binding sites.

[The effect of the binding of the antigenic determinant of tetrodotoxin-sensitive protein on the phytohemagglutinin-induced proliferative response of the mononuclear cells in mouse spleen]. / Izuchenie vliianie sviazyvaniia antigennoi determinanty tetrodotoksinchuvstvitel'nogo belka na proliferativnyi otvet mononuklearnykh kletok selezenki myshi, indutsiruemyi fitogemaggliutininom.

Mammalian brain-derived "tetrodotoxin-sensitive protein" has been shown to share an epitope with as yet unidentified structure of the human and rodent lymphocyte surface. Previously obtained observations that a monoclonal antibody to this epitope induces a proliferative response of murine splenic mononuclear cells are confirmed. However, this antibody fails to modify the phytohemagglutinin-induced response. Moreover, lectin with submitogenic concentration inhibited the antibody-induced response provided that monocytes were present in the culture. The antibody-induced proliferation appeared to be less monocyte-dependent than the lectin-induced one. Taken together, these findings argue against hypothesis that a lymphocyte structure with epitope of the "tetrodotoxin-sensitive protein" is associated with either T cell receptor for antigen or interleukin-2 receptor.

Second messenger-induced modulation of Thy-1 antigen expression: an ELISA study using murine thymic and neuroblastoma cells.

Freshly isolated cells of murine thymuses and in vitro cultured murine neuroblastoma cells were seeded into microplates, treated with either phorbol myristate acetate (PMA) or dibutyryl-cyclic adenosine monophosphate (DBcAMP), and then processed for enzyme immunoassay to analyze with G4 monoclonal antibody the expression of Thy-1.2 antigen epitope. Pretreatment of thymic cells with PMA resulted in little, if any, decrease of Thy-1 expression, while treatment of these cells with DBcAMP caused a significant down-modulation of the epitope. DBcAMP did not affect binding of another murine IgG antibody to the thymic cells. Modulation of the epitope on thymic cells caused by DBcAMP was dose-dependent with maximal effect seen at the drug concentration of 10(-4) M. However, at various doses of DBcAMP (10(-6) to 10(-3) M) we were unable to detect any significant shift of Thy-1 expression on neuroblastoma cells. Though mechanisms of the above phenomena need further elucidation, we conclude that cellular ELISA may provide a useful alternative to more commonly used cytofluorimetric studies for the analysis of immune cell-surface antigen expression and its pharmacological and physiological modulation.

Monoclonal antibody to brain cytoplasmic tetrodotoxin-sensitive protein detects an epitope associated with lymphocytes and involved in lymphocyte activation.

We have previously derived a monoclonal antibody, BIP-4, which is specific to a mammalian brain protein representing a type of sodium channel. Here we show that this antibody detects an epitope associated with lymphocytes and that it triggers a proliferative response of the cells. BIP-4 epitope can be detected on both human peripheral blood and murine splenic mononuclear cells. Surface immuno-globulin-negative (i.e. resting T) lymphocytes are neither bound by the antibody nor proliferate to it. Proliferative response exerted in 7-day cultures of murine splenic mononuclear cells by recombinant interleukin-2 was blocked by BIP-4 antibody. We conclude that the epitope shared by a type of brain sodium channel protein and lymphocyte surface is involved in some, yet unrecognized, step of immune cell activation.

Possible relationship of brain cytoplasmic tetrodotoxin-sensitive protein to voltage-gated sodium channel shown by monoclonal antibody.

Biochemical events leading to the formation of mature membrane-associated sodium channel proteins are not completely understood. We have recently purified a protein from the cytoplasm of brain cells, which is able to become incorporated into liposomes and induce neurotoxin-dependent sodium permeability. Here we report data on a monoclonal antibody derived against this protein. This antibody crossreacts with cell membrane preparations. The antibody binding to viable neuroblastoma cells is inhibited by veratrine, indicating that membrane molecules antigenically related to the cytoplasmic protein may also be related to the voltage-gated sodium channel.

[Binding of monoclonal antibodies to cerebral cytoplasmic tetrodotoxin-sensitive protein to human lymphocytes]. / Sviazyvanie monoklonal'nykh antitel k tsitoplazmaticheskomu tetrodotoksinchuvstvitel'nomu belku golovnogo mozga s limfotsitami cheloveka.

To examine whether lymphocytes express antigenic determinants of brain cytoplasmic tetrodotoxin-sensitive protein (CTSP), anti-CTSP monoclonal antibody (Bab) binding to human peripheral blood lymphocytes was studied using ELISA assay. It was shown that the Mab bound human lymphocytes in a dose-dependent fashion. Halph-maximal binding of this Mab was significantly enhanced if test-cells were pretreated with concanavalin A or with mixed lymphocyte culture supernatant. Results are discussed from the point of view of the hypothesis that CTSP are metabolic precursors of membrane sodium channels.

[Effect of helium and argon on the oxygen uptake by lymphocytes]. / Vliianie geliia i argona na potreblenie kisloroda limfotsitami.

The effect of inert helium and argon gases on the tissue respiration has been studied on lymphocyte suspensions of white rats. It is shown that normoxic helium-oxygen mixture induces almost a two-fold increase of the O2 uptake by lymphocytes as compared with the control (air). No deviations in the value of the studied parameter are revealed in case of replacement of nitrogen from air by argon. Significance of the membrane structure in realization of effects of inert gases is under discussion.

[Monoclonal antibodies to cytoplasmic tetrodotoxin-sensitive brain protein. The effect of veratrine on antibody binding to neuroblastoma cells]. / Monoklonal'nye antitela k tsitoplazmaticheskomu tetrodotoksinchuvstvitel'nomu belku golovnogo mozga. Vliianie veratrina na sviazyvanie antitel s kletkmai neiroblastomy.

Recently a glycoprotein capable to induce tetrodotoxin-sensitive sodium permeability being incorporated to liposomes was purified from the cytoplasm of the bovine brain. It is shown that a monoclonal antibody derived against this protein binds intact murine neuroblastoma cells. Veratrine, neurotoxin referred to modulate the activity of voltage-gated sodium channels, is shown to compete with the antibody for the neuroblastoma surface epitope. It is postulated that molecular moiety bound with the antibody is either identical or spatially related to veratrine (veratridine) binding site.

[Antibody adsorption to tetrodotoxin-sensitive cytoplasmic proteins by murine neuroblastoma cells]. / Adsorbtsiia antitel k tsitoplazmaticheskim tetrodotoksinchuvstvitel'nym belkam kletkami myshinoi neiroblastomy.

The study was aimed to examine properties of polyclonal antibodies elicited after immunization by cytoplasmic nerve cell glycoproteins forming sodium channels in liposomes. It was shown that intact murine neuroblastoma cells can absorb these antibodies. Absorbing cell dose-effect curves were found to have a characteristic form able to shift when the cell culture time or serum concentration in the growth medium varied.