[Ion channels in B lymphocytes].
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B-Lymphocytes↗
Biomedical subjects
D Choquet
Publications and source records attributed to D Choquet.
Defective transmembrane calcium influx demonstrated in a primary immunodeficiency by video-imaging.
A rise of intracellular calcium concentration triggered by the engagement of various membrane receptors is a key event in the control of cell growth. This increase involves both a release of calcium from intracellular stores and the opening of a transmembrane calcium conductive pathway. Using video imaging to measure intracellular calcium concentration in individual fura-2-loaded cells, we detected a defect in calcium influx in lymphocytes and fibroblasts collected from patients affected by a rare and new form of primary immunodeficiency. In these cells, pharmacological agents such as thapsigargin or ionomycin, and the physiological activator bradykinin, only induced transient increases in cytoplasmic calcium level, due to the emptying of internal stores, while in control cells, this initial step is followed by an additional and sustained transmembrane calcium influx. The fact that calcium influx is absent in patient's fibroblasts indicates that the related deficiency, which is clinically associated with a lack of proliferation of T lymphocytes, also affects cells of the non-hematopoietic lineages. This study emphasizes the adequacy of single cell imaging for determining whether some forms of pathologies are associated with a disregulation of ionic fluxes, and for identifying them accurately.
B-Lymphocytes↗
The (YXXL/I)2 signalling motif found in the cytoplasmic segments of the bovine leukaemia virus envelope protein and Epstein-Barr virus latent membrane protein 2A can elicit early and late lymphocyte activation events.
The cytoplasmic domains of the transducing subunits associated with B and T cell antigen receptors contain a common amino acid motif consisting of two precisely spaced Tyr-X-X-Leu/Ile sequences (where X corresponds to a variable residue). Expression of a single copy of this motif suffices to initiate B or T cell activation. The bovine leukaemia virus (BLV) is a B cell lymphotropic retrovirus which causes a non-neoplasic proliferation of B cells. The cytoplasmic domain of the BLV transmembrane envelope glycoprotein, gp30, possesses two overlapping copies of the Tyr-X-X-Leu/Ile-containing motif which could participate in the induction of B cell activation. Similarly, the N-terminal cytoplasmic domain of the latent membrane protein 2A (LMP2A) of the Epstein-Barr virus (EBV) contains a single copy of the Tyr-X-X-Leu/Ile-containing motif which could play a critical role in B cell transformation. To determine whether these two virus-encoded cytoplasmic domains are endowed with signalling functions, we constructed chimeric proteins by replacing the cytoplasmic tail of CD8-alpha with that of either BLV gp30 or EBV LMP2A. We show here that, once separately expressed in B or T cell lines, these chimeras are capable of triggering both calcium responses and cytokine production when cross-linked with an antibody to CD8-alpha. Furthermore, using site-directed mutagenesis, we demonstrated unequivocally that this signalling function may be accounted for by the Tyr-X-X-Leu/Ile motifs they contain.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Pattern of potassium channel expression in proliferating B lymphocytes depends upon the mode of activation.
Ionic channel expression is highly regulated during mitogenesis. But it is not clear whether these regulations only follow intrinsic programs during the course of the cell cycle or if they also depend upon the external factors used to promote cell activation. B lymphocytes express two classes of potassium channels and can be stimulated to enter the cell cycle by distinct pathways. Thus, we have analyzed, with the patch-clamp technique, if the expression of channels varies when the cells are activated by different signals that lead to cell proliferation. We found that stimulation through Ag receptors increases the expression of calcium- and voltage-activated potassium channels, whereas a bacterial mitogen, LPS, only enhances the expression of the latter. Moreover, channel expression can still be modified in proliferating cells because stimulation of LPS-activated cells through Ag receptors induces rapid expression of calcium-activated channels. The use of inhibitors of mRNA synthesis revealed that this process depends upon gene transcription. Thus, differential induction of the expression of potassium channels is not only linked to the entry into the cell cycle but depends also on pathways of stimulation.
Animals↗
Cross-linking of IgG receptors inhibits membrane immunoglobulin-stimulated calcium influx in B lymphocytes.
By cross-linking membrane immunoglobulins (mIg), the antigenic stimulation of B lymphocytes induces an increase in intracellular free calcium levels ([Ca2+]i) because of a combination of release from intracellular stores and transmembrane influx. It has been suggested that both events are linked, as in a number of other cases of receptor-induced increase in [Ca2+]i. Conversely, in B lymphocytes, type II receptors for the Fc fragment of IgG (Fc gamma RII) inhibit mIg-mediated signaling. Thus, we have investigated at the level of single cells if these receptors could act on specific phases of mIg Ca2+ signaling. Lipopolysaccharide-activated murine B splenocytes and B lymphoma cells transfected with intact or truncated Fc gamma RII-cDNA were used to determine the domains of Fc gamma RII implicated in the inhibition of the Ca2+ signal. [Ca2+]i was measured in single fura-2-loaded cells by microfluorometry. The phases of release from intracellular stores and of transmembrane influx were discriminated by using manganese, which quenches fura-2, in the external medium as a tracer for bivalent cation entry. The role of membrane potential was studied by recording [Ca2+]i in cells voltage-clamped using the perforated patch-clamp method. Cross-linking of mIgM or mIgG with F(ab')2 fragments of anti-Ig antibodies induced a sustained rise in [Ca2+]i due to an extremely fast and transitory release of Ca2+ from intracellular stores and a long lasting transmembrane Ca2+ influx. The phase of influx, but not that of release, was inhibited by membrane depolarization. The increase in [Ca2+]i occurred after a delay inversely related to the dose of ligand. Co-cross-linking mIgs and Fc gamma RII with intact anti-Ig antibodies only triggered transitory release of Ca2+ from intracellular stores but no Ca2+ influx, even when the cell was voltage-clamped at negative membrane potentials. These transitory Ca2+ rises had similar amplitudes and delays to those induced by cross-linking mIgs alone. Thus, our data show that Fc gamma RII does not mediate an overall inhibition of mIg signaling but specifically affects transmembrane Ca2+ influx without affecting the release of Ca2+ from intracellular stores. Furthermore, this inhibition is not mediated by cell depolarization. Thus, Fc gamma RII represents a tool to dissociate physiologically the phases of release and transmembrane influx of Ca2+ triggered through antigen receptors.
Animals↗
Doppler echocardiographic evaluation of valve regurgitation in healthy volunteers.
OBJECTIVE: To study the prevalence and the characteristics of physiological valve regurgitation. DESIGN: Pulsed wave Doppler echocardiography, continuous wave Doppler echocardiography and Doppler colour flow mapping were performed prospectively in healthy volunteers. SETTING: Echocardiography laboratory in a city hospital. PATIENTS: 32 consecutive healthy volunteers (age 21-49 years, mean age 29.4). MAIN OUTCOME MEASURES: Identification of regurgitation with colour Doppler flow mapping and measurement of the jet area, jet length, and maximal velocity of the regurgitation. RESULTS: Regurgitation was recorded at the pulmonary (100%), tricuspid (100%), mitral (56%), and aortic valves (6%). The velocity of pulmonary and tricuspid regurgitation was similar to that predicted from the pressure gradient calculated from the Bernoulli equation. The jet area and jet length were generally small. CONCLUSION: Trivial regurgitation from the pulmonary, tricuspid, and mitral valves is common in healthy people. It is important to take such regurgitation into account when valve disease is diagnosed.
Adult↗
[Contribution of transesophageal echocardiography in the diagnosis of intra- and para-cardiac masses].
The authors detected 59 thrombi and 7 intra- or paracardiac tumors in 58 patients in a series of 1,100 transesophageal echocardiography. Twenty-six of the 51 patients with a thrombus were in sinus rhythm; 25 had atrial fibrillation. In 44 cases, the thrombus was single and in 7 cases there were multiple thrombi. A phenomenon of spontaneous contrast in the left atrium was observed in 24 patients (47%). In 31 cases (53%) the thrombi were located in the left auricle, in 21 cases (36%) in the left atrium, in 4 cases in the left ventricle and in 3 cases in the right atrium. Transthoracic echocardiography only detected 25% of these thrombi. The superiority of transesophageal echocardiography was particularly evident for the detection of thrombi in the left auricle (31 by transesophageal echocardiography versus 2 by transthoracic echocardiography) and in the left atrium (13 by transesophageal echocardiography versus 7 by transthoracic echocardiography). Five myxomas were diagnosed by transesophageal echocardiography and 4 of them were identified by transthoracic echocardiography. The site of implantation of the tumor was located in all 5 cases by transesophageal echocardiography. Two right paracardiac tumours were only visible by transesophageal echocardiography. Transesophageal echocardiography is therefore very useful in the diagnosis of thrombi in the left atrium and auricle, of rare hypodense myxomas and paracardiac tumors. In addition, it enables precise localisation of the site of implantation of nearly all intracardiac tumors.
Aged↗
[Aneurysm of the interatrial septum. Contribution of transesophageal echography. Relation with systemic embolic complications].
The aim of this study was to evaluate the contribution of transesophageal echocardiography to the diagnosis of aneurysms of the interatrial septum and to identify the role played by this condition in unexplained systemic embolic accidents. Thirty two aneurysms of the interatrial septum were discovered in a consecutive series of 751 transesophageal echocardiograms, i.e. an incidence of 4.2 per cent. The diagnosis was possible by transthoracic echocardiography in only 9 cases (28%). Search for a patent foramen ovale by a contrast test was positive in 87 per cent of cases of aneurysm of the interatrial septum as compared with 45 per cent for patients without an aneurysm (p < 0.01). In patients in whom transesophageal echocardiography was requested for etiological evaluation of an ischemic vascular accident of embolic origin (n = 191), an aneurysm of the interatrial septum was found in 8.3 per cent of cases. In patients with no history of a systemic embolic accident (n = 560), the incidence of septums of the interatrial septum was 2.8 per cent (p < 0.01). Furthermore, 50 per cent of patients with an aneurysm of the interatrial septum had a history of systemic embolism. Transesophageal echocardiography thus appears to be superior to transthoracic echocardiography in the positive diagnosis of aneurysms of the interatrial septum and our study is suggestive of their emboligenic nature.
Adult↗
Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes.
B lymphocytes and macrophages express closely related immunoglobulin G (IgG) Fc receptors (Fc gamma RII) that differ only in the structures of their cytoplasmic domains. Because of cell type-specific alternative messenger RNA splicing, B-cell Fc gamma RII contains an insertion of 47 amino acids that participates in determining receptor function in these cells. Transfection of an Fc gamma RII-negative B-cell line with complementary DNA's encoding the two splice products and various receptor mutants indicated that the insertion was responsible for preventing both Fc gamma RII-mediated endocytosis and Fc gamma RII-mediated antigen presentation. The insertion was not required for Fc gamma RII to modulate surface immunoglobulin-triggered B-cell activation. Instead, regulation of activation involved a region of the cytoplasmic domain common to both the lymphocyte and macrophage receptor isoforms. In contrast, the insertion did contribute to the formation of caps in response to receptor cross-linking, consistent with suggestions that the lymphocyte but not macrophage form of the receptor can associate with the detergent-insoluble cytoskeleton.
Amino Acid Sequence↗
Differential regulation of voltage- and calcium-activated potassium channels in human B lymphocytes.
The expression and characteristics of K+ channels of human B lymphocytes were studied by using single and whole-cell patch-clamp recordings. They were gated by depolarization (voltage-gated potassium current, IKv, 11-20 pS) and by an increase in intracellular Ca2+ concentration (calcium-activated potassium current, IKCa, 26 pS), respectively. The level of expression of these channels was correlated with the activational status of the cell. Both conductances are blocked by tetraethylammonium, verapamil, and charybdotoxin, and are insensitive to apamin; 4-aminopyridine blocks IK, preferentially. We used a protein kinase C activator (PMA) or antibodies to membrane Ig (anti-mu) to activate resting splenocytes in culture. Although IKv was recorded in the majority of the resting lymphocytic population, less than 20% of the activated cells expressed this conductance. However, in this subset the magnitude of IKv was 20-fold larger than in resting cells. On the other hand, IKCa was detected in nearly one half of the resting cells, whereas all activated cells expressed this current. The magnitude of IKCa was, on average, 30 times larger in activated than in nonactivated cells. These results probably reflect that during the course of activation 1) the number of voltage-dependent K+ channels per cell decreases and increases in a small subset and 2) the number of Ca(2+)-dependent K+ channels per cell increases in all cells. We suggest that the expression of functional Ca(2+)- and voltage-activated K+ channels are under the control of different regulatory signals.
B-Lymphocytes↗
Role of associated gamma-chain in tyrosine kinase activation via murine Fc gamma RIII.
Type III receptors for the Fc portion of IgG (Fc gamma RIII), initially characterized on macrophages and NK cells, are also expressed on several pre-B cell lines. Surface expression of Fc gamma RIII requires the association of the ligand binding alpha-chain with homodimeric gamma-chains. Type II Fc gamma R is homologous to Fc gamma RIII alpha-chain in the extracellular portion and differs in the transmembrane and cytoplasmic domains. The role of Fc gamma R in cell activation was investigated by expressing Fc gamma RIII and the lymphocyte-specific b1 isoform of Fc gamma RII (Fc gamma RIIb1) in an Fc gamma R-negative, sIgG-positive B-cell line. We found that, in contrast to Fc gamma RIIb1, Fc gamma RIII triggers the same events of cell activation as sIG i.e. Ca2+ mobilization, tyrosine phosphorylation and IL-2 secretion. By expressing cytoplasmic domain-lacking Fc gamma RIII alpha-chain in the absence or in the presence of gamma-chains, we demonstrated that cell activation via Fc gamma RIII requires the co-expression of gamma-chains, and is independent of the cytoplasmic portion of the alpha-chain. Furthermore, the cytoplasmic portion of the gamma-chain, fused to the extracellular and transmembrane domains of Fc gamma RII confers on the chimeric receptor the ability to trigger cell activation. Mutation of one tyrosine residue in the cytoplasmic domain of the gamma-chain prevented triggering of cytoplasmic signals. We therefore demonstrate that a tyrosine-containing motif, present in the cytoplasmic domain of the associated gamma-chain, is necessary and sufficient to trigger cell activation via Fc gamma RIII.
Animals↗
Calcium influx through nicotinic receptor in rat central neurons: its relevance to cellular regulation.
The Ca2+ permeability of a nicotinic acetylcholine receptor (nAChR) in the rat CNS was determined using both current and fluorescence measurements on medial habenula neurons. The elementary slope conductance of the nAChR channel was 11 pS in pure external Ca2+ (100 mM) and 42 pS in standard solution. Ca2+ influx through nAChRs resulted in the rise of cytosolic Ca2+ concentration ([Ca2+]i) to the micromolar range. This increase was maximal under voltage conditions (below -50 mV) in which Ca2+ influx through voltage-activated channels was minimal. Ca2+ influx through nAChRs directly activated a Ca(2+)-dependent Cl- conductance. In addition, it caused a decrease in the GABAA response that outlasted the rise in [Ca2+]i. These results underscore the physiological significance of Ca2+ influx through nAChR channel in the CNS.
Animals↗
Mechanism of 4-aminopyridine action on voltage-gated potassium channels in lymphocytes.
The mechanism by which 4-aminopyridine (4-AP) blocks the delayed rectifier type potassium (K+) channels present on lipopolysaccharide-activated murine B lymphocytes was investigated using whole-cell and single channel patch-clamp recordings. 4-AP (1 microM-5 mM) was superfused for 3-4 min before applying depolarizing pulses to activate the channel. During the first pulse after application of 4-AP above 50 microM, the current inactivated faster, as compared with the control, but its peak was only reduced at high concentrations of 4-AP (Kd = 3.1 mM). During subsequent pulses, the peak current was decreased (Kd = 120 microM), but the inactivation rate was slower than in the control, a feature that could be explained by a slow unblocking process. After washing out the drug, the current elicited by the first voltage step was still markedly reduced, as compared with the control one, and displayed very slow activation and inactivation kinetics; this suggests that the K+ channels move from a blocked to an unblocked state slowly during the depolarizing pulse. These results show that 4-AP blocks K+ channels in their open state and that the drug remains trapped in the channel once it is closed. On the basis of the analysis of the current kinetics during unblocking, we suggest that two pathways lead from the blocked to the unblocked states. Computer simulations were used to investigate the mechanism of action of 4-AP. The simulations suggest that 4-AP must bind to both an open and a nonconducting state of the channel. It is postulated that the latter is either the inactivated channel or a site on closed channels only accessible to the drug once the cell has been depolarized. Using inside- and outside-out patch recordings, we found that 4-AP only blocks channels from the intracellular side of the membrane and acts by reducing the mean burst time. 4-AP is a weak base (pK = 9), and thus exists in ionized or nonionized form. Since the Kd of channel block depends on both internal and external pH, we suggest that 4-AP crosses the membrane in its nonionized form and acts from inside the cell in its ionized form.
4-Aminopyridine↗
Assessment of severity of mitral regurgitation by measuring regurgitant jet width at its origin with transesophageal Doppler color flow imaging.
BACKGROUND: The ability of transesophageal color Doppler echocardiography to provide high-resolution images of both cardiac structure and blood flow in real time is advantageous for many clinical purposes. This study was performed to determine the utility of the regurgitant jet width at its origin measured by transesophageal Doppler color flow imaging in the assessment of severity of mitral regurgitation. METHODS AND RESULTS: Sixty-three consecutive patients with mitral regurgitation underwent transesophageal color Doppler examination, and the diameter of regurgitant jet at its origin was measured. Both right and left cardiac catheterizations were performed within 24 hours of Doppler studies, and angiographic grading of mitral regurgitation and regurgitant stroke volume were evaluated. There was a close relation between the jet diameter at its origin measured by transesophageal Doppler color flow imaging and the angiographic grade of mitral regurgitation (r = 0.86, p less than 0.001). A jet diameter of 5.5 mm or more identified severe mitral regurgitation (grade III or IV) with a sensitivity of 92%, specificity of 92%, and positive and negative predictive values of 88% and 95%, respectively. In 31 patients with isolated mitral regurgitation, the jet diameter correlated well with the regurgitant stroke volume determined by a combined hemodynamic-angiographic method (r = 0.85, p less than 0.001). A jet diameter of 5.5 mm or more identified a regurgitant stroke volume of 60 ml or more with a sensitivity of 88%, specificity of 93%, and positive and negative predictive values of 94% and 87%, respectively. CONCLUSIONS: The regurgitant jet width at its origin measured by transesophageal Doppler color flow imaging provides a simple and useful method of measuring the severity of mitral regurgitation, and it may allow differentiation between mild and severe mitral regurgitation.
Angiocardiography↗
Determination of left ventricular inflow by pulsed Doppler echocardiography: influence of mitral orifice area and blood velocity measurements.
Pulsed Doppler echocardiography was performed in 30 patients to assess the influence of mitral orifice area and velocity on the determination of mitral stroke volume and inflow. Aortic forward stroke volume and outflow were measured at the centre of the aortic annulus, and compared with mitral flow measurements calculated by three methods. Both mitral stroke volume and inflow derived from an instantaneous velocity-area method showed an excellent correlation with aortic flow measurements. The other two methods, which determined mitral stroke volume and inflow based on a mean mitral valve area and diastolic velocity integral, significantly underestimated mitral flow measurements. This study indicates that the instantaneous velocity-area method offers a reliable means for quantitating left ventricular inflow.
Adult↗
Assessment of severity of aortic regurgitation by M-mode colour Doppler flow imaging.
To assess the value of measuring the aortic regurgitant jet diameter at its origin by M-mode colour Doppler imaging, 82 patients with aortic regurgitation underwent, within 72 h of each other, colour Doppler examination and angiography. After excluding one patient without colour Doppler aortic regurgitation and five with a highly eccentric regurgitant jet, we found a close relationship between the jet diameter at its origin measured by M-mode colour Doppler and the angiographic grade of aortic regurgitation (r = 0.88). A jet diameter greater than or equal to 12 mm identified severe aortic regurgitation (grade III or IV) with a sensitivity of 86.4% and a specificity of 94.4%. In 38 patients, the jet diameter correlated well with the regurgitant fraction measured by a combined haemodynamic-angiographic method (r = 0.88). A jet diameter greater than or equal to 12 mm identified a regurgitant fraction greater than or equal to 40% with a sensitivity of 88.2% and a specificity of 95.2%. This study indicates that the size of the regurgitant jet diameter at its origin measured by M-mode colour Doppler provides a simple and useful measure of the severity of aortic regurgitation. It may allow differentiation between mild or moderate and severe aortic regurgitation and evaluation of regurgitant fraction.
Angiography↗
Non-invasive measurement of the regurgitant fraction by pulsed Doppler echocardiography in isolated pure mitral regurgitation.
OBJECTIVE: To assess the usefulness of pulsed Doppler echocardiography as a method of measuring the regurgitant fraction in patients with mitral regurgitation. PATIENTS AND METHODS: Twenty controls and 27 patients with isolated mitral regurgitation underwent Doppler studies. In the patients the study was performed within 48 hours of cardiac catheterisation. Aortic outflow was measured in the centre of the aortic annulus, and mitral inflow was derived from the flow velocity at the tip of the leaflets and the area of the elliptical mitral opening. The regurgitant fraction was calculated as the difference between the two flows divided by the mtiral inflow. RESULTS: In the 20 controls the two flows were almost identical (mitral inflow, 4.44 (SD 0.88) l/min; aortic outflow, 4.58 (SD 0.84) l/min), with a mean regurgitant fraction of 4.2 (SD 8.4)%. In patients with mitral regurgitation, the mitral inflow was significantly higher than the aortic outflow (8.8 (3.6) v 4.3 (1.1) l/min). In most patients the Doppler-derived regurgitant fraction (45.8 (19.2)%) accorded closely with the regurgitant fraction (41.3 (SD 17.8)%) determined by the haemodynamic technique. CONCLUSION: Pulsed Doppler echocardiography, with an instantaneous velocity-valve area method for calculating mitral inflow, reliably measured the severity of regurgitation in patients with mitral regurgitation.
Adult↗
[Determination of transmitral blood flow by pulsed echodoppler. Correlation with aortic blood flow in 30 patients].
The aim of this study was to assess the validity of mitral valve blood flow measured by pulsed Doppler echocardiography (PDE) with the sample volume positioned at the tips of the mitral leaflets. Thirty patients with a mean age of 38.4 years underwent calculation of transmitral blood flow: by Touche's method (A) in which the mitral orifice is assumed to be an ellipse with a constant long axis equal to the diameter of the mitral annulus and a variable short axis equal to the distance between the mitral leaflets measured on the M mode recording. The velocities are recorded by PDE with the sample volume at the tips of the mitral leaflets. The instantaneous cardiac output is equal to the surface multiplied by the instantaneous velocity. The integration of the instantaneous outputs throughout the whole of diastole by a computer programme provides the stroke volume; by a simplification of this method (B) which considers the short axis of the mitral ellipse to be constant and equal to the mean mitral valve leaflet separation measured from the M mode recording, and; by Hoit's method (C) which calculates mitral valve surface area from the M mode recording alone. The transmitral blood flow was calculated by these three methods and compared to the classical PDE aortic cardiac output measurement during the same examination, the accuracy of which has been previously demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗