Voltage-dependent modulation of single N-Type Ca2+ channel kinetics by receptor agonists in IMR32 cells.

The voltage-dependent inhibition of single N-type Ca(2+) channels by noradrenaline (NA) and the delta-opioid agonist D-Pen(2)-D-Pen (5)-enkephalin (DPDPE) was investigated in cell-attached patches of human neuroblastoma IMR32 cells with 100 mM Ba(2+) and 5 microM nifedipine to block L-type channels. In 70% of patches, addition of 20 microM NA + 1 microM DPDPE delayed markedly the first channel openings, causing a four- to fivefold increase of the first latency at +20 mV. The two agonists or NA alone decreased also by 35% the open probability (P(o)), prolonged partially the mean closed time, and increased the number of null sweeps. In contrast, NA + DPDPE had little action on the single-channel conductance (19 versus 19.2 pS) and minor effects on the mean open time. Similarly to macroscopic Ba(2+) currents, the ensemble currents were fast activating at control but slowly activating and depressed with the two agonists. Inhibition of single N-type channels was effectively removed (facilitated) by short and large depolarizations. Facilitatory pre-pulses increased P(o) significantly and decreased fourfold the first latency. Ensemble currents were small and slowly activating before pre-pulses and became threefold larger and fast decaying after facilitation. Our data suggest that slowdown of Ca(2+) channel activation by transmitters is mostly due to delayed transitions from a modified to a normal (facilitated) gating mode. This single-channel gating modulation could be well simulated by a Monte Carlo method using previously proposed kinetic models predicting marked prolongation of first channel openings.     

Ca2+ pools and cell growth. Evidence for sarcoendoplasmic Ca2+-ATPases 2B involvement in human prostate cancer cell growth control

The present study demonstrates for the first time that intracellular calcium-ATPases and calcium pool content are closely associated with prostate cancer LNCaP cell growth. Cell growth was modulated by changing the amount of epidermal growth factor, serum, and androgene in culture media. Using the microspectrofluorimetric method with Fura-2 and Mag Fura-2 as probes, we show that in these cells, the growth rate is correlated with intracellular calcium pool content. Indeed, an increased growth rate is correlated with an increase in the calcium pool filling state, whereas growth-inhibited cells show a reduced calcium pool load. Using Western blotting and immunocytochemistry, we show that endoplasmic reticulum calcium pump expression is closely linked to LNCaP cell growth, and are a common target of physiological stimuli that control cell growth. Moreover, we clearly demonstrate that inhibition of these pumps, using thapsigargin, inhibits LNCaP cell growth and prevents growth factor from stimulating cell proliferation. Our results thus provide evidence for the essential role of functional endoplasmic reticulum calcium pumps and calcium pool in control of prostate cancer LNCaP cell growth, raising the prospect of new targets for the treatment of prostate cancer.     

Opioid agonist modulation of cytoplasmic free Ca2+ level in concanavalin A-stimulated mouse lymphocytes.

In this study the influence of mu-, delta-, and kappa-selective opioid agonists (DAMGO, DSLET, and dynorphin A (1-13)) on cytoplasmic free Ca2+ ([Ca2+]i) level in normal and concanavalin-A (Con A)-activated mouse lymphocytes was investigated. [Ca2+]i was measured using the fluorescent dye FURA-2AM. The opioid peptides at 10-12-10-7 M induced some increase in [Ca2+]i in non-activated lymphocytes. However, DAMGO and DSLET (10-13-10-7 M) considerably inhibited a Con A-induced increase in [Ca2+]i. The inhibiting effect of both peptides was higher after 20-min preincubation compare to 2-h preincubation. The effect of the kappa-agonist dynorphin A (1-13) was significantly different depending on the duration of cell pretreatment and the concentration of the peptide used. After preincubation for 20 min at low concentrations (10-12-10-11 M) it slightly stimulated, while at higher (10-10-10-7 M) concentrations it inhibited lymphocyte response to Con A. After preincubation for 2 h, pronounced stimulation of mitogen-induced Ca2+ flux was observed at peptide concentration 10-9 M. The effects of opioids were antagonized by naloxone. These data indicate that functionally active opioid receptors expressed on lymphocytes could be involved in early stages of mitogen activation.     

Negative control of store-operated Ca2+ influx by B cell receptor cross-linking

An increase in the intracellular Ca(2+) concentration by B cell receptor (BCR) cross-linking plays important roles in the regulation of B cell functions. [Ca(2+)](i) is regulated by Ca(2+) release from the Ca(2+) store as well as store-operated Ca(2+) influx (SOC). Protein tyrosine kinases downstream of BCR cross-linking were shown to regulate the mechanism for Ca(2+) release. However, it remains elusive whether BCR cross-linking regulates SOC or not. In this study, we examined the effect of BCR cross-linking on thapsigargin-induced SOC in the DT40 B cells. We found that the SOC-mediated increase in intracellular Ca(2+) concentration was inhibited by BCR cross-linking. Using a membrane-potential-sensitive dye, we found that BCR cross-linking induced depolarization, which is expected to decrease the driving force of Ca(2+) influx and SOC channel conductance. When membrane potential was held constant by the transmembrane K(+) concentration gradient in the presence of valinomycin, the BCR-mediated inhibition of SOC was still observed. Thus, the BCR-mediated inhibition of SOC involves both depolarization-dependent and depolarization-independent mechanisms of SOC inhibition. The depolarization-independent inhibition of the SOC was abolished in Lyn-deficient, but not in Bruton's tyrosine kinase-, Syk- or SHIP (Src homology 2 domain containing phosphatidylinositol 5'-phosphatase)-deficient cells, indicating that Lyn is involved in the inhibition. These results show novel pathways of BCR-mediated SOC regulations.     

Rapid modulation of Ca2+ uptake in human jejunal enterocytes

The active metabolite of D vitamin, 1,25(OH)2D3, has been suggested to promote acute uptake of calcium through the intestinal lining in cell lines and murine models. In this study, the effects of D vitamin on the cytoplasmic Ca2+ of single human jejunal enterocytes, obtained with LOC-I-GUT technique, was analyzed in vivo in a fluorometric system using fura-2 as the Ca2+-sensing probe. Vitamin-promoted acute Ca2+ influx exhibited dual kinetics, indicating initial release from intracellular Ca2+ pools and fast entry from the extracellular space. Furthermore, providing a chemical clamp of membrane potential close to 0 mV did not activate voltage-sensitive calcium channels in the cellular membrane, neither was the hormone-induced Ca2+ influx affected by verapamil. This advocates that voltage-operated channels like L-type Ca2+ channels do not participate in the process of Ca2+ uptake. In fact, the existence of calcium-release-activated-calcium channels (I(CRAC)) was implied by the findings that irreversible depletion of intracellular Ca2+ stores by thapsigargin promoted Ca2+ entry. In the thapsigargin-treated enterocytes, D vitamin lost its ability to promote calcium entry indicating an important role for intracellular store-operated Ca2+ stores in the acute effects of 1,25(OH)2D3.     

Ethanol inhibits ligand-activated Ca2+ channels in human B lymphocytes.

Ethanol reportedly is immunosuppressive, interfering with lymphocyte proliferation. To investigate the basis for this immunosuppression, the effects of acute treatment with ethanol were studied on Ca2+ mobilization in tonsillar B lymphocytes and the human lymphoblastoid B-cell line, Ramos. The level of intracellular Ca2+ was monitored in cells loaded with the fluorescent dye indo-1 following stimulation with either anti-IgM antibody or platelet activating factor. The effect of ethanol was also examined on the induction of the early proto-oncogene c-fos in these cells. Ethanol inhibited ligand-activated Ca2+ mobilization due to transmembrane influx but not intracellular store release, in a dose- and time-dependent manner. This inhibition was not due to the inability of anti-IgM to bind to its surface receptor nor to membrane depolarization induced by ethanol. Ethanol also inhibited the Ca2(+)-dependent induction by anti-IgM of c-fos in these cells. The inhibitory effects of ethanol on ligand-activated Ca2+ channels and subsequent induction of c-fos may provide the basis for its immunosuppressive action.     

Modeling Ca2+ feedback on a single inositol 1,4,5-trisphosphate receptor and its modulation by Ca2+ buffers

The inositol 1,4,5-trisphosphate receptor/channel (IP₃R) is a major regulator of intracellular Ca²⁺ signaling, and liberates Ca²⁺ ions from the endoplasmic reticulum in response to binding at cytosolic sites for both IP₃ and Ca²⁺. Although the steady-state gating properties of the IP₃R have been extensively studied and modeled under conditions of fixed [IP₃] and [Ca²⁺], little is known about how Ca²⁺ flux through a channel may modulate the gating of that same channel by feedback onto activating and inhibitory Ca²⁺ binding sites. We thus simulated the dynamics of Ca²⁺ self-feedback on monomeric and tetrameric IP₃R models. A major conclusion is that self-activation depends crucially on stationary cytosolic Ca²⁺ buffers that slow the collapse of the local [Ca²⁺] microdomain after closure. This promotes burst-like reopenings by the rebinding of Ca²⁺ to the activating site; whereas inhibitory actions are substantially independent of stationary buffers but are strongly dependent on the location of the inhibitory Ca²⁺ binding site on the IP₃R in relation to the channel pore.     

Proliferation-associated changes of Ca2+ transport in myeloid leukemic cell lines

Proliferation-associated changes in calcium metabolism were investigated employing the promyelocytic HL-60 and monoblastic U-937 cell lines. The cells were stimulated to proliferate employing mitogenic factors as follows. 1) Transferrin or insulin: HL-60 cells were adjusted for growth in serum-free medium, and 24 h prior to the experiment, the cells were deprived of transferrin or insulin. The re-addition of either one of them stimulated cell proliferation as was evident by increased [3H]-tymidine incorporation activity. Cell proliferation was associated with an enhanced Ca2+ influx rate, measured by 45Ca2+ uptake activity. 2) Granulocyte-monocyte colony-stimulating factor (GM-CSF): addition of GM-CSF to proliferating or quiescent HL-60 cells resulted in increased cell proliferation, which was also accompanied by increased rate of Ca2+ influx. 3) Serum: HL-60 and U-937 were grown for 24 h in serum-depleted medium. Re-addition of serum to the cells was not associated with immediate or delayed change in calcium influx rate but rather with an immediate increase in the cytosolic free calcium concentration, measured employing the fluorescent probe, fura-2AM. This increase was independent of extracellular calcium, unaffected by verapamil, diltiazem, and lanthanum, and associated with enhanced 45Ca2+ efflux. Thus, in all three cases evoked cell proliferation was accompanied by quantitative changes in Ca2+ metabolism. While the transferrin-, insulin-, and GM-CSF-stimulated cell proliferation was accompanied by delayed increases in 45Ca2+ influx, the serum-stimulated cell proliferation was accompanied by an immediate elevation of free cytosolic Ca2+.     

Ryanodine receptor adaptation and Ca2+(-)induced Ca2+ release-dependent Ca2+ oscillations.

A simplified mechanism that mimics "adaptation" of the ryanodine receptor (RyR) has been developed and its significance for Ca2+(-)induced Ca2+ release and Ca2+ oscillations investigated. For parameters that reproduce experimental data for the RyR from cardiac cells, adaptation of the RyR in combination with sarco/endoplasmic reticulum Ca2+ ATPase Ca2+ pumps in the internal stores can give rise to either low [Cai2+] steady states or Ca2+ oscillations coexisting with unphysiologically high [Cai2+] steady states. In this closed-cell-type model rapid, adaptation-dependent Ca2+ oscillations occur only in limited ranges of parameters. In the presence of Ca2+ influx and efflux from outside the cell (open-cell model) Ca2+ oscillations occur for a wide range of physiological parameter values and have a period that is determined by the rate of Ca2+ refilling of the stores. Although the rate of adaptation of the RyR has a role in determining the shape and the period of the Ca2+ spike, it is not essential for their existence. This is in marked contrast with what is observed for the inositol 1,4,5-trisphosphate receptor for which the biphasic activation and inhibition of its activity by Ca2+ are sufficient to produce oscillations. Results for this model are compared with those based on Ca2+(-)induced Ca2+ release alone in the bullfrog sympathetic neuron. This kinetic model should be suitable for analyzing phenomena associated with "Ca2+ sparks," including their merger into Ca2+ waves in cardiac myocytes.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

Increased intracellular Ca2+ induces Ca2+ influx in human T lymphocytes.

One current hypothesis for the initiation of Ca2+ entry into nonelectrically excitable cells proposes that Ca2+ entry is linked to the state of filling of intracellular Ca2+ stores. In the human T lymphocyte cell line Jurkat, stimulation of the antigen receptor leads to release of Ca2+ from internal stores and influx of extracellular Ca2+. Similarly, treatment of Jurkat cells with the tumor promoter thapsigargin induced release of Ca2+ from internal stores and also resulted in influx of extracellular Ca2+. Initiation of Ca2+ entry by thapsigargin was blocked by chelation of Ca2+ released from the internal storage pool. The Ca2+ entry pathway also could be initiated by an increase in the intracellular concentration of Ca2+ after photolysis of the Ca(2+)-cage, nitr-5. Thus, three separate treatments that caused an increase in the intracellular concentration of Ca2+ initiated Ca2+ influx in Jurkat cells. In all cases, Ca(2+)-initiated Ca2+ influx was blocked by treatment with any of three phenothiazines or W-7, suggesting that it is mediated by calmodulin. These data suggest that release of Ca2+ from internal stores is not linked capacitatively to Ca2+ entry but that initiation is linked instead by Ca2+ itself, perhaps via calmodulin.     

Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts.

The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg(2+)-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable Kd of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 microM free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.     

Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+ removal: aberrations in human complex I deficiency

Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]_M) and the rate of cytosolic Ca²⁺ removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial Ca²⁺ indicator rhod-2 can be used to selectively buffer the Bk-induced increase in mitochondrial Ca²⁺ concentration ([Ca²⁺]_M) and, consequently, the Ca²⁺-stimulated increase in [ATP]_M, thus allowing studies of how the increase in [ATP]_M and the cytosolic Ca²⁺ removal rate are related. Luminometry of healthy fibroblasts expressing either aequorin or luciferase in the mitochondrial matrix showed that rhod-2 dose dependently decreased the Bk-induced increase in [Ca²⁺]_M and [ATP]_M by maximally 80 and 90%, respectively. Digital imaging microscopy of cells coloaded with the cytosolic Ca²⁺ indicator fura-2 revealed that, in parallel, rhod-2 maximally decreased the cytosolic Ca²⁺ removal rate by 20%. These findings demonstrate that increased mitochondrial ATP production is required for accelerating cytosolic Ca²⁺ removal during stimulation with a Ca²⁺-mobilizing agonist. In contrast, complex I-deficient patient fibroblasts displayed a cytosolic Ca²⁺ removal rate that was already decreased by 40% compared with healthy fibroblasts. Rhod-2 did not further decrease this rate, indicating the absence of mitochondrial ATP supply to the cytosolic Ca²⁺ pumps. This work reveals the usefulness of rhodamine-based Ca²⁺ indicators in examining the role of intramitochondrial Ca²⁺ in mitochondrial (patho) physiology. human skin fibroblast; OXPHOS disease; calcium ion extrusion; rhod-2; CGP-37157     

Spontaneous human B2 bradykinin receptor activity determines the action of partial agonists as agonists or inverse agonists. Effect of basal desensitization.

In this report, we show that desensitization regulates ligand-independent, spontaneous activity of the human B2 bradykinin (BK) receptor, and the level of spontaneous receptor activity determines the action of the BK antagonists and partial receptor agonists NPC17731 and HOE140 as agonists or inverse agonists. Spontaneous receptor activity was monitored by measuring basal cellular phosphoinositide (PI) hydrolysis as a function of the density of the receptor in transiently transfected HEK293 cells. Minimal spontaneous activity of the wild-type B2 receptor was detected in these cells. Mutating a cluster of serines and threonines within the fourth intracellular domain of the receptor, which is critical for agonist-promoted desensitization, significantly increased the spontaneous receptor activity. BK, the natural B2 receptor ligand and, consequently, a full agonist, stimulated PI hydrolysis at high and low levels of spontaneous receptor activity. On the other hand, the partial agonists NPC17731 and HOE140 were stimulatory, or agonists, at the lower level of receptor activity but inhibitory, or inverse agonists, at the higher level of activity. These results show that receptors are desensitized in response to their spontaneous activity. Furthermore, these results, which refute traditional theories, show that the capacity of a drug to modulate a receptor response is not intrinsic to the drug but is also dependent on the cellular environment in which the drug acts.     

Amplitude modulation of nuclear Ca2+ signals in human skeletal myotubes: a possible role for nuclear Ca2+ buffering

Video-rate confocal microscopy of Indo-1-loaded human skeletal myotubes was used to assess the relationship between the changes in sarcoplasmic ([Ca(2+)](S)) and nuclear ([Ca(2+)](N)) Ca(2+) concentration during low- and high-frequency electrostimulation. A single stimulus of 10 ms duration transiently increased [Ca(2+)] in both compartments with the same time of onset. Rate and amplitude of the [Ca(2+)] rise were significantly lower in the nucleus (4.0- and 2.5-fold, respectively). Similarly, [Ca(2+)](N) decayed more slowly than [Ca(2+)](S) (mono-exponential time constants of 6.1 and 2.5 s, respectively). After return of [Ca(2+)] to the prestimulatory level, a train of 10 stimuli was applied at a frequency of 1 Hz. The amplitude of the first [Ca(2+)](S) transient was 25% lower than that of the preceding single transient. Thereafter, [Ca(2+)](S) increased stepwise to a maximum that equalled that of the single transient. Similarly, the amplitude of the first [Ca(2+)](N) transient was 20% lower than that of the preceding single transient. In contrast to [Ca(2+)](S), [Ca(2+)](N) then increased to a maximum that was 2.3-fold higher than that of the single transient and equalled that of [Ca(2+)](S). In the nucleus, and to a lesser extent in the sarcoplasm, [Ca(2+)] decreased faster at the end of the stimulus train than after the preceding single stimulus (time constants of 3.3 and 2.1 s, respectively). To gain insight into the molecular principles underlying the shaping of the nuclear Ca(2+) signal, a 3-D mathematical model was constructed. Intriguingly, quantitative modelling required the inclusion of a satiable nuclear Ca(2+) buffer. Alterations in the concentration of this putative buffer had dramatic effects on the kinetics of the nuclear Ca(2+) signal. This finding unveils a possible mechanism by which the skeletal muscle can adapt to changes in physiological demand.     

Glutamate receptor agonists evoked Ca2+-dependent and Ca2+-independent release of [3H]d-Aspartate from cultured chick retina cells

We studied the release of [³H]d-aspartate evoked by glutamate receptor agonists from monolayer cultures of chick retina cells, and found that activation of the glutamate receptors can evoke both Ca²⁺-dependent and Ca²⁺-independent release of [³H]d-aspartate. In Ca²⁺-free (no added Ca²⁺) Na⁺ medium, the agonists of the glutamate receptors induced the release of [³H]d-aspartate with the following rank order of potency: kainate>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)∼N-methyl-d-aspartate (NMDA). In media containing 1 mM CaCl₂ the release of [³H]d-aspartate evoked by NMDA, kainate and AMPA was increased by about 112%, 20% and 39%, respectively, as compared to the release evoked by the same agonists in Ca²⁺-free medium. NMDA was the most potent agonist in stimulating the Ca²⁺-dependent release of [³H]d-aspartate, possibly by exocytosis, and AMPA was as potent as kainate. The Ca²⁺-dependent release of [³H]d-aspartate evoked by kainate was dependent on the influx of Ca²⁺ through the receptor associated channel, as well as through the N- (ω-Conotoxin GVIA-sensitive) and L- (nitrendipine-sensitive)type voltage-sensitive Ca²⁺ channels (VSCC). The exocytotic release of [³H]d-aspartate evoked by AMPA relied exclusively on Ca²⁺ entry through the L-type VSCC, whereas the effect of NMDA was partially mediated by the influx of Ca²⁺ through the receptor-associated channel, but not through L- or N-type VSCC. Thus, activation of these different glutamate receptors under physiological conditions is expected to cause the release of cytosolic and vesicular glutamate, and the routes of Ca²⁺ entry modulating vesicular release may be selectively recruited.     

A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+

Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.     

Isolation and functional analysis of human B cell populations. I. Characterization of the B1+B2+ and B1+B2- subsets

Distinct populations of human B lymphocytes can be identified by their expression and/or co-expression of the B cell-restricted antigens B1 and B2. Dual fluorochrome staining and flow cytometric cell sorting permitted the isolation of the B1+B2+ and B1+B2- cells to homogeneity. In contrast, very few B1-B2+ cells were obtainable from normal lymphoid organs. Virtually all B1+B2+ cells expressed IgM and IgD, but lacked IgG and the plasma cell antigens PCA-1 and PC-1, whereas the B1+B2- cells more frequently expressed IgG, PCA-1 and PC-1. Both populations were noncycling and were composed of similar percentages of small and large cells. The B1+B2+ cells proliferate to anti-mu or to anti-mu + PHA-LCM, but not to PHA-LCM alone. They require both T cells and PWM to produce Ig. In contrast, B1+B2-cells do not significantly proliferate to anti-mu, PHA-LCM, or anti-mu and PHA-LCM. They produce Ig in response to T cells alone without PWM. These phenotypic and functional observations provide preliminary evidence that these populations are distinct and that the B1+B2+ cell may be a "resting" B cell, whereas the B1+B2- cell appears to be more "differentiated." The present studies further suggest that they will also be helpful in characterizing B cells in some human disease states. We believe that the identification and isolation of these and similar subsets of B cells defined by differing cell surface phenotype should aid our understanding both of normal B cell differentiation and of B cell disease states.