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I Pecht

Publications and source records attributed to I Pecht.

At least 109 records · Page 6Linked to original sources
PubMed1990

A glycolipid-specific monoclonal antibody modulates Fc epsilon receptor stimulation of mast cells.

In an effort to identify membrane components participating in coupling stimulus to secretion in mast cells, monoclonal antibodies were produced from spleen cells of mice immunized with plasma membranes isolated from rat mast cells of the RBL-2H3 line. The resultant mAbs were screened by their capacity to modulate the secretory response of these cells to crosslinking of their type 1 Fc epsilon receptor (Fc epsilon RI). Following this scheme, we obtained a hybridoma designated B17, which secretes an IgM-class mAb (B17) that binds to and modulates secretion from RBL-2H3 cells. By immunoblotting, B17 was shown to bind to a membrane component of low molecular weight, later identified as a glycolipid. While B17 partially inhibits IgE binding to RBL-2H3 cells, no noticeable inhibition of B17 binding by IgE was observed. mAb B17 does not cause any secretory response on its own, and its modulatory effect on Fc epsilon RI-mediated secretion is bimodal: it either enhances or inhibits secretion, depending on the B17 dose and also on the nature and dose of the agent used for crosslinking the Fc epsilon RI. When secretion was induced by IgE and suboptimal or optimal doses of multivalent antigen, B17 (2-80 nM) caused an increase in secretion. However, higher doses of B17 (greater than 150 nM) inhibited secretion. Secretion induced by supraoptimal doses of antigen, or by the Fc epsilon RI-specific mAb F4 was inhibited by B17 at all the dose range tested (2-200 nM). In contrast, B17 had no effect on secretion induced by Ca2+ ionophores. These results demonstrate that Fc epsilon RI function is modulated by a mAb binding to a membrane glycolipid.

Animals↗
PubMed1990

Phosphorylation of type II Fc gamma receptor on activated human B lymphocytes.

Activation of resting human B lymphocytes either by cross-linking their membranal IgM or by phorbol esters has been previously demonstrated to modulate the type II receptor for Fc gamma domains (Fc gamma RII): shortly after stimulation a decrease in IgG binding capacity and an enhancement of Fc gamma RII expression were observed. These were followed by the release of Fc gamma RII fragments from the cell membrane. Since protein phosphorylation is well-established signal transduction element, we examined whether Fc gamma RII may be a target of such activation induced phosphorylation. Resting (high density) and activated (low density) human tonsil B lymphocytes were stimulated either by cross-linking their surface IgM (sIgM) or by the phorbol ester TPA. This treatment induced specific phosphorylation of a 36 kd membrane protein. This polypeptide was shown to specifically bind to IgG-coated Sepharose beads or to monoclonal Fc gamma RII-specific antibody-coated Affi-Gel 10 beads; thus, it most probably corresponds to the Fc gamma RII of these cells. In addition, phosphorylation of a 20 kd protein with similar binding characteristics was also observed in several experiments. Both serine and tyrosine were the amino acids that underwent phosphorylation in the 36 kd Fc gamma RII. The extent of Fc gamma RII phosphorylation correlated with the increase in receptor expression as monitored by specific mAb binding and, at the same time, with the decrease in the capacity to bind IgG-sensitized erythrocytes. These results suggest that stimulation-induced phosphorylation of Fc gamma RII on B cells is an early signal transduction element involved in controlling B cell response.

Amino Acids↗
PubMed1989

Direct binding of a myasthenia gravis related epitope to MHC class II molecules on living murine antigen-presenting cells.

MHC gene products present antigenic epitopes to the antigen receptor on T cells. Nevertheless, direct binding of such epitopes to MHC class II proteins on normal living antigen-presenting cells (APCs) has not yet been demonstrated. We have previously shown a significant difference in the ability of T cells of myasthenia gravis (MG) patients to proliferate in response to the synthetic peptide p195-212 of the human acetylcholine receptor (AChR) alpha-subunit in comparison to healthy controls. The observed proliferative responses correlated significantly with HLA-DR5. Moreover, lymph node cells of various mouse strains that were primed with the T cell epitope, p195-212, were found to proliferate to different extents. To investigate these observations further, we designed an assay for direct binding of p195-212 to MHC class II proteins on the surface of freshly prepared splenic adherent cells. Binding of a biotinylated p195-212 was monitored using phycoerythrin-avidin by flow cytometry. Fifteen to sixty per cent of the cells were labeled following incubation with the biotinylated peptide. Binding was observed only to splenic adherent cells derived from mouse strains of which T cells were capable of proliferating in response to p195-212. The binding specificity, in terms of epitope structure and its site of interaction on the cells, was shown by its inhibition with an excess of the unlabeled peptide or with the relevant monoclonal anti-I-A antibodies. These results constitute the first direct evidence for the specific binding of a T cell epitope to live APC.

Amino Acid Sequence↗
PubMed1989

Mast cell stimulation by monoclonal antibodies specific for the Fc epsilon receptor yields distinct responses of arachidonic acid and leukotriene C4 secretion.

The release of arachidonic acid ([3H]AA) and leukotriene C4 (LTC4) from the rat mucosal mast cells of the RBL-2H3 line stimulated by Fc epsilon receptor-specific monoclonal antibodies (mAb), by IgE and multivalent antigen, or by Ca2+ ionophores, was investigated. In parallel, secretion of the granular enzyme beta-hexosaminidase was also assayed. The release of [3H]AA and LTC4 in response to stimulation by three Fc epsilon RI-specific mAb shows similar quantitative differences to those observed for the secretion of granule-stored mediators. The mAb F4 induced a substantial release of both [3H]AA and LTC4, which is as high as that induced by IgE and multivalent antigen. mAb J17 and H10 were found to induce an insignificant release of [3H]AA, but while J17 did induce release of LTC4, H10 failed to induce it, even though both J17 and H10 caused substantial release of beta-hexosaminidase. Ca2+ ionophores were found to be relatively more effective in inducing release of [3H]AA and LTC4 than in causing the secretion of granular mediators, as compared to cell stimulation by Fc epsilon RI aggregation. These results illustrate that the cell responses of degranulation and de novo synthesis and release of mediators have different sensitivities to stimulation by (a) configurationally distinct Fc epsilon RI dimers, (b) Fc epsilon RI aggregates induced by IgE and multivalent antigen, and (c) Ca2+ ionophores.

Animals↗
PubMed1989

Long-range intramolecular electron transfer in azurins.

The Cu(II) sites of azurins, the blue single copper proteins, isolated from Pseudomonas aeruginosa and Alcaligenes spp. (Iwasaki) are reduced by CO2- radicals, produced by pulse radiolysis, in two distinct reaction steps: (i) a fast bimolecular phase, at the rates (5.0 +/- 0.8) x 10(8) M-1.s-1 (P. aeruginosa) and (6.0 +/- 1.0) x 10(8) M-1.s-1 (Alcaligenes); (ii) a slow unimolecular phase with specific rates of 44 +/- 7 s-1 in the former and 8.5 +/- 1.5 s-1 for the latter (all at 298 K, 0.1 M ionic strength). Concomitant with the fast reduction of Cu(II), the single disulfide bridge linking cysteine-3 to -26 in these proteins is reduced to the RSSR- radical ion as evidenced by its characteristic absorption band centered at 410 nm. This radical ion decays in a unimolecular process with a rate identical to that of the slow Cu(II) reduction phase in the respective protein, thus clearly suggesting that a long-range intramolecular electron transfer occurs between the RSSR- radicals and the Cu(II) site. The temperature dependence of the internal electron transfer process in both proteins was measured over the 4 degrees C to 42 degrees C range. The activation parameters derived are delta H* = 47.5 +/- 4.0 and 16.7 +/- 1.5 kJ.mol-1; and delta S not equal to = -56.5 +/- 7.0 and -171 +/- 18 J.K-1.mol-1, respectively. Using the Marcus theory, we found that the intramolecular electron transfer rates and their activation parameters observed for the two azurins correlate well with the distances between the reactive sites, their redox potential, and the nature of the separating medium. Thus, azurins with distinct structural and reactivity characteristics isolated from different bacteria or modified by site-directed mutagenesis can be used in comparing long-range electron transfer process between their conserved disulfide bridge and the Cu(II) sites.

Alcaligenes↗
PubMed1988

Possible orientational constraints determine secretory signals induced by aggregation of IgE receptors on mast cells.

Three biologically active monoclonal antibodies (mAbs) specific for the monovalent, high-affinity membrane receptor for IgE (Fc epsilon R) were employed in analysing the secretory response of mast cells of the RBL-2H3 line to crosslinking of their Fc epsilon R. All three mAbs (designated F4, H10 and J17) compete with each other and with IgE for binding to the Fc epsilon R. Their stoichiometry of binding is 1 Fab:1 Fc epsilon R, hence, the intact mAbs can aggregate the Fc epsilon Rs to dimers only. Since all three mAbs induce secretion, we conclude that Fc epsilon R dimers constitute a sufficient 'signal element' for secretion of mediators for RBL-2H3 cells. The secretory dose-response of the cells to these three mAbs are, however, markedly different: F4 caused rather high secretion, reaching almost 80% of the cells' content, while J17 and H10 induced release of only 30-40% mediators content. Both the intrinsic affinities and equilibrium constants for the receptor dimerization were derived from analysis of binding data of the Fab fragments and intact mAbs. These parameters were used to compute the extent of Fc epsilon R dimerization caused by each of the antibodies. However, the different secretory responses to the three mAbs could not be rationalized simply in terms of the extent of Fc epsilon R dimerization which they produce. This suggests that it is not only the number of crosslinked Fc epsilon Rs which determines the magnitude of secretion-causing signal, but rather other constraints imposed by each individual mAb are also important.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

A monoclonal antibody that inhibits secretion from rat basophilic leukemia cells and binds to a novel membrane component.

Rat basophilic leukemia (RBL-2H3) cells, like mast cells and basophils, carry monovalent membrane receptors with high affinity for IgE (Fc epsilon R). Cross-linking of these receptors provides the immunologic stimulus which initiates a series of biochemical events, culminating in secretion of inflammatory mediators. In an attempt to identify membrane components involved in the stimulus-secretion coupling of these cells, hybridomas were produced from splenocytes of mice immunized with intact RBL-2H3 cells. Here we report the production of a mAb (designated G63) that inhibits the Fc epsilon R-mediated secretion from RBL cells. At low degrees of Fc epsilon R aggregation, the mAb G63-induced inhibition may be complete, whereas at the maximum of secretion the inhibition is in the range of 30 to 40%. The relative degree of inhibition of secretion is dependent on the dose of mAb G63. Furthermore, inhibition requires the bivalency of G63, as the Fab fragments are inactive. The number of antigenic epitopes recognized by G63 per RBL-2H3 cell is 1.8 x 10(4) epitopes/cell, as determined by direct binding studies of 125I-labeled Fab fragments of G63. This number is 20 to 30 times smaller than that of Fc epsilon R on the same cells. The membrane component to which G63 binds has been identified by immunoprecipitation as a glycoprotein with an apparent Mr of 58 to 70 kDa. All of these results, and the fact that no competition for binding to RBL cells between mAb G63 and IgE can be resolved, indicate that mAb G63 binds to a membrane component which is distinct from the Fc epsilon R. mAb G63 suppresses the Fc epsilon R-mediated rise in cytoplasmic concentration of free Ca2+ ions, known to be one of the biochemical signals involved in the stimulus-secretion coupling in RBL-2H3 cells. G63 does not affect, however, the degranulation induced by the Ca2+ ionophore A23187. Therefore, mAb G63 probably exerts its inhibitory effect on a step preceding the rise in cytoplasmic free Ca2+. Thus, mAb G63 defines a previously unidentified membrane component that is involved in one of the early steps of the RBL-2H3 activation mediated by their Fc epsilon R.

Animals↗
PubMed1988

Three-dimensional model of stellacyanin and its implications for electron transfer reactivity.

Experimental data were combined with computational methods in constructing a hypothetical three-dimensional model for the blue single copper protein Rhus stellacyanin (St). The known sequence of stellacyanin and its homology with plastocyanin (Pc) were used together with the results of spectroscopic studies of the protein that yielded the current assignment of two histidines, one cysteine and a disulfide sulfur as copper ligands in stellacyanin. By computer graphics and energy minimization the folding of the protein was predicted. The model structure is somewhat less regular than Pc as judged by surface area and energy comparisons, but it is a stable structure. Besides rotation of one imidazole ring the copper site undergoes no change even in the absence of the copper ion and the model shows that the site can be constructed with the four assumed copper ligands without forming a strained system. The structure also indicates that a carbonyl oxygen atom is near the copper, thus the site may have analogy to the Alcaligenes denitrificans azurin (Az) site, although the amino acid sequence is more homologous to that of Pc. The model indicates that aspartate 49, reductively labeled by Cr(III), is near the copper center and homologous to the site labeled by Cr(III) on Pc. Also homologous to Pc is a tyrosine residue adjacent to the aspartate. This tyrosine has been implicated in Pc electron transfer and thus is probably involved in electron transfer reactivity of St as well. The higher reactivity of St with small-molecule redox reagents compared to Az and Pc, may be due to the proximity of the above-mentioned aspartate 49 to the Cu, or the greater exposure of one of the Cu cysteine ligands, in the predicted structure as compared to that in the known Pc and Az structures.

Amino Acid Sequence↗
PubMed1988

Isolation and purification of an Fc epsilon receptor activated ion channel from the rat mast cell line RBL-2H3.

Derivatives of the antiallergic drug cromolyn [disodium 5,5'-[(2-hydroxy-1,3-propanediyl)-bis(oxy)]bis [4-oxo-(4H-1-benzopyran)-2- carboxylate]], which can be conjugated covalently at the propane 2-position to macromolecules and to insoluble matrices, were synthesized. Conjugates of these derivatives with macromolecules were examined for their binding to cells of the rat basophilic leukemia line RBL-2H3, which is widely employed as a model for immunologically induced mast cell degranulation. Only those drug-protein conjugates in which the cromolyn analogue with an amino group at the propane 2-carbon instead of the hydroxyl was linked to the carrier by glutaraldehyde were found to exhibit specific and saturable binding to these cells. Analysis of the binding data for these conjugates yielded an apparent binding constant of 3.8 +/- 0.2 X 10(8) M-1 and an apparent number of binding sites for the probe of 4000-8000 per cell. The conjugates found to bind specifically to the cells were also immobilized on agarose matrices and employed in an affinity-based isolation of the membrane component responsible for the observed binding. A single labeled polypeptide was eluted from these columns, onto which either whole cell lysates or solubilized purified plasma membranes of surface-radioiodinated RBL-2H3 cells had been adsorbed. This membrane protein appears on autoradiograms of nonreducing SDS-PAGE as a single broad band of approximately 110,000 daltons (Da) apparent molecular mass. On autoradiograms of reducing gels, the only band detected has an apparent mass of approximately 50,000 Da and appears narrower. Elution of the columns with the drug and disulfide-reducing agents or with the latter alone resulted in significantly higher yields of the 50-kDa polypeptide. Both the intact and reduced proteins bind strongly to immobilized concanavalin A and less so to immobilized wheat germ agglutinin, suggesting that the isolated intact protein is probably a dimer of two glycosylated subunits of similar molecular mass. Treatment of the reduced protein with endoglycosidase F leads to a decrease in its apparent molecular mass by approximately 12 kDa, suggesting that the extent of glycosylation of this polypeptide is approximately 25%. As shown in the following paper, the intact protein constitutes a Ca2+ channel that is activated upon IgE-Fc epsilon receptor aggregation.

Affinity Labels↗
PubMed1988

Calcium specificity of the antigen-induced channels in rat basophilic leukemia cells.

Ion channels, activated upon IgE-Fc epsilon receptor aggregation by specific antigen, were studied in micropipet-supported lipid bilayers. These bilayers were reconstituted with purified IgE-Fc epsilon receptor complex and the intact 110-kDa channel-forming protein, both isolated from plasma membranes of rat basophilic leukemia cells (line RBL-2H3). In order to identify the current carrier through these ion channels and to determine their ion selectivity, we investigated the currents flowing through the IgE-Fc epsilon receptor gated channels in the presence of a gradient of Ca2+ ions. Thus, the solution in which the micropipet-supported bilayer was immersed contained 1.8 mM CaCl2, while the interior of the micropipet contained 0.1 microM Ca2+ (buffered with EGTA). Both solutions also contained 150 mM of a monovalent cation chloride salt (either K+ or Na+). The currents induced upon specific aggregation of the IgE (by either antigen or anti-IgE antibodies) were examined over a range of potentials imposed on the bilayer. The type of conductance event most frequently observed under the employed experimental conditions was a channel that has a slope conductance of 3 pS and a reversal potential practically identical with the calculated value for the reversal potential of calcium (134 +/- 11 mV in the presence of sodium, 125 +/- 13 mV in the presence of potassium). These results indicate that this channel is highly selective for calcium against the monovalent cations sodium and potassium. This same channel has a conductance of 4-5 pS in the presence of symmetrical solutions containing only 100 mM CaCl2 and 8 pS in the presence of 0.5 M NaCl with no calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Thermodynamics of oligosaccharides binding to a dextran-specific monoclonal IgM.

The binding thermodynamics of seven different oligosaccharide haptens to the dextran-specific IgM secreted by the murine plasmacytoma MOPC-104E were studied by direct calorimetric measurements. The enthalpy change values observed for the binding process range between -5 and -16 kcal/mol depending on the hapten and the temp of measurement. The antibody-hapten interactions were characterized by a positive heat capacity change [delta Cp approximately 300 cal/(mol.degree)] and a resultant process of enthalpy-entropy compensation. The calculated change in unitary entropy of the reaction, delta Su, ranged between -20 and -30 eu (4 degrees C), corresponding to an expected entropy loss due to immobilization of the hapten molecules. The entropy of binding increased with rising temp, thus compensating for the decreasing enthalpy contribution to the free energy of binding. The data are consistent with a hapten binding induced conformational transition to a more relaxed state in the immunoglobulin molecule.

Animals↗
PubMed1988

Ionic signalling in mast cells; antigen and ionophore induced changes in cytosolic pH.

Stimulation of cells of the rat basophilic leukemia line RBL-2H3, which are used as a model in biochemical studies of mast cells, by antigen or by the calcium ionophore ionomycin, are known to cause secretion of mediators of inflammation. These stimuli have now been found to cause a decrease in the cells' cytosolic pH. This acidification process was monitored by the fluorescent indicator 2',7'-bis (carboxyethyl)-5(6)-carboxyfluorescein (BCECF) introduced into these cells. The antigen induced acidification was the result of specific aggregation of membrane residing IgE, reached values up to 0.03 pH units and required the presence of sodium and calcium ions in the incubation medium. It was amiloride resistant but was blocked by the metabolic inhibitor deoxyglucose. Ionomycin caused a dose dependent decrease in cytosolic pH which was also sensitive to the pH of the extracellular medium. The acidification reached more than 0.1 pH units at optimal, non-cytotoxic, doses of ionomycin (1 microM) and decreased markedly as the medium pH increased from 7.0 to 8.0. The antigen and ionophore induced cytosolic acidification processes are interpreted as being the result of the increased concns of free cytosolic calcium ions rather than the effect of direct activation of a sodium-proton exchanger. Further investigation of this process is in progress.

Animals↗
PubMed1988

Isotype restriction of murine antibodies towards the loop region of hen's egg white lysozyme.

Monoclonal antibodies specific for the loop determinant (residues 64-80) of hen's egg white lysozyme demonstrated an immunoglobulin class restriction. Only IgM response against the loop could be evoked in mice, irrespective of whether the immunogen was the intact native lysozyme as such, or the loop peptide covalently conjugated to a synthetic carrier, poly-DL-alanyl-poly-L-lysine (A-L). Despite the fact that in polyclonal antisera from mice immunized with lysozyme, the ELISA-titre of anti-loop reactivity was very low, 26% of the total anti-lysozyme response could be accounted for by the loop when expressed as the percentage of anti-lysozyme hybridoma colonies producing monoclonal antibodies reactive with the loop. The results can be interpreted either as determinant controlled isotype restriction, or alternatively, as an affinity restriction leading to the phenomenon that antibodies of isotypes other than IgM are formed, but are of too low avidity to be detected by the ELISA method.

Animals↗
PubMed1988

Preferred sites and pathways for electron transfer in blue copper proteins.

Long-range electron transfer reactions proceed within and between metalloproteins at relatively fast rates and with marked specificities. The blue single copper proteins are well known electron carriers with their redox center being of limited accessibility to solvent and solutes. The question of where and how electrons are transferred to and from the copper-ion have been investigated. One experimental approach developed in order to pursue these problems is that of reductively labeling several representative, yet structurally distinct blue single copper proteins; azurin, plastocyanin, and stellacyanin with chromium ions. In all three cases, a substitution inert Cr(III)-adduct is formed when the oxidized protein is reduced by Cr(II)ag ions. In azurin, Cr(III) binds to the Glu-91 carboxylate approximately 10 A from the copper center. In both plastocyanin and stellacyanin the Cr(III) label is most probably also coordinated to carboxylate groups, present in plastocyanin, and in stellacyanin 12 A and 6 A, respectively, from the copper center. The salient feature emerging from examination of the three copper proteins is that a pi-facilitated electron transfer (E.T.) pathway may be operative; in azurin, E.T. proceeds via an extended imidazole ring system, and in plastocyanin and stellacyanin via a weakly coupled pi-system. Therefore, a case emerges for suggesting that this is the common feature of the long-distance intramolecular E.T. in this class of metalloproteins. These pathways are most probably a regulatory alternative to the E.T. site recognized at the exposed, "Northern" imidazole coordinated to copper in all these proteins.

Azurin↗
PubMed1987

Oligomerization and ring closure of immunoglobulin E class antibodies by divalent haptens.

Cross-linking of antibodies constitutes a widespread initiation signal for their respective effector functions. Cross-linking IgE-class antibodies provide the triggering signal to mast cells for their degranulation process. To obtain a quantitative insight into these cross-linking processes, the interactions between a DNP-specific monoclonal antibody of the IgE class and a series of divalent DNP haptens with spacers of different length and flexibility have been studied by fluorescence titration experiments. These were analyzed by employing the theoretical model developed by Dembo and Goldstein [Dembo, M., & Goldstein, B. (1978) J. Immunol. 121, 345-353] in a fitting procedure. Equilibrium constants that describe the aggregation and ring-closure processes caused by divalent hapten binding have been used as free parameters. The intrinsic binding constants were determined by fluorescence titrations with corresponding monovalent haptens. The main results are the following: (1) The divalent haptens with a short and flexible spacer [i.e., N alpha, N epsilon-di-(DNP)-L-lysine,meso-bis[(DNP-beta-Ala)amino]succinate, and bis[(DNP-tri-D-Ala)amino]heptane, having a maximal DNP-DNP distance of gamma = 14, 21, and 45 A, respectively] effect aggregation of the antibodies mainly into closed dimers. (2) The divalent hapten family with long and rigid oligoproline spacers di(DNP)-Ahx-Asp-(Pro)n-Lys with n = 24, 27, and 33 (i.e., gamma = 100, 110, and 130 A) causes aggregation of the antibodies predominantly into closed dimers and trimers. The corresponding equilibrium constants of the respective ring-closure processes decrease significantly with longer spacer length. (3) Evidence was found that intramolecularly monomeric ring closure of the IgE antibodies is caused by haptens containing oligoproline spacers with n = 37 or 42 (gamma = 130-150 A). The equilibrium constant of the ring-closure process increases with spacer length. This increase in stability indicates a difference in the imposed strain. Furthermore, the latter results imply that the distance between the two binding sites of the IgE molecule lies in the range dictated by the rigid oligoproline part of the respective hapten's spacer, i.e., 115-130 A. (4) Nearly all oligomeric ring-closure processes proceed relatively slowly with an approximate lower limit of a half-life of 5-10 s. This slowing down of the aggregation and ring-closure processes most probably reflects steric factors.

Calorimetry↗
PubMed1987

Stimulus-secretion coupling mechanisms in mast cells.

We have investigated several aspects of the complex sequence of events, transmitting the antigen-induced signal of cross-linking immunoglobulin E (IgE) resident in the membrane surface of mast cells into the signals yielding the final process of mediator release. Already the initial phase of this cascade still requires a better understanding. Namely, we are still missing a clear physical description of the effective stimulus-producing antigen-IgE complex in terms of size and spatial requirements. We are investigating this problem on a well-defined cell line (rat basophilic leukemia-RBL-2H3) using synthetic divalent haptens and a monoclonal IgE. A subsequent phase following IgE aggregation is a transient increase in the free calcium concentration in these cells' cytosol. The source of the Ca2+ and the way by which it enters the cytosol are studied predominantly by examining antigen-induced channel activity in the cells' membrane allowing Ca2+ influx from the exterior medium. Finally, we have observed that under certain experimental conditions, antigen-induced degranulation can be achieved even without a rise in cytosolic free calcium. In our search for alternative second messengers, we examine the potential candidacy of the cytosolic Na+/H+ balance. So far, we have found that antigen-stimulated secretion does require extracellular sodium and involves changes in its cytosolic pH. However, further studies are required to clarify its possible role as a coupling element.

Animals↗
PubMed1987

Ion channels activated by specific Ti or T3 antibodies in plasma membranes of human T cells.

T lymphocytes are activated to proliferate via a surface membrane receptor recognizing the antigen/major histocompatibility complex. This membrane component is comprised of at least five polypeptide subunits, collectively termed the Ti-T3 receptor complex. A transient increase in cytosolic free calcium occurs as an early event in the T-cell activation process and is necessary for induction of the endogenous IL-2 and certain other genes. Monoclonal antibodies specific to epitopes of either the Ti or the T3 components were shown to be effective agonists, also leading to such transient rises in cytosolic free calcium and activating the lymphocytes. Here we show, using micropipette-supported bilayers formed from membranes of the human T-cell line REX, that Ti- or T3-specific antibodies cause opening of ligand gated ion channels. Both types of specific antibodies yielded similar histograms of conductance amplitudes which show a channel with a conductance of 2-3 pS in symmetrical 100 mM CaCl2 solutions. These channels allow the passage of calcium and barium ions and are blocked by lanthanum ions, suggesting that they are specific for calcium. We propose that these channels, by allowing the entry of external calcium, may account for a large fraction of the rise in intracellular calcium observed upon triggering of the Ti-T3 receptor.

Antibodies, Monoclonal↗