Nitric oxide-induced mobilization of intracellular calcium via the cyclic ADP-ribose signaling pathway

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from beta-NAD+ by ADP-ribosyl cyclases in sea urchin eggs and in several mammalian cells (Galione, A., and White, A. (1994) Trends Cell Biol. 4, 431 436). Pharmacological studies suggest that cADPR is an endogenous modulator of Ca2+-induced Ca2+ release mediated by ryanodine-sensitive Ca2+ release channels. An unresolved question is whether cADPR can act as a Ca2+-mobilizing intracellular messenger. We show that exogenous application of nitric oxide (NO) mobilizes Ca2+ from intracellular stores in intact sea urchin eggs and that it releases Ca2+ and elevates cADPR levels in egg homogenates. 8-Amino-cADPR, a selective competitive antagonist of cADPR-mediated Ca2+ release, and nicotinamide, an inhibitor of ADP-ribosyl cyclase, inhibit the Ca2+-mobilizing actions of NO, while, heparin, a competitive antagonist of the inositol 1,4,5-trisphosphate receptor, did not affect NO-induced Ca2+ release. Since the Ca2+-mobilizing effects of NO can be mimicked by cGMP, are inhibited by the cGMP-dependent-protein kinase inhibitor, Rp-8-pCPT-cGMPS, and in egg homogenates show a requirement for the guanylyl cyclase substrate, GTP, we suggest a novel action of NO in mobilizing intracellular calcium from microsomal stores via a signaling pathway involving cGMP and cADPR. These results suggest that cADPR has the capacity to act as a Ca2+-mobilizing intracellular messenger.     

Conjugation of blocked ricin to an anti-CD19 monoclonal antibody increases antibody-induced cell calcium mobilization and CD19 internalization

CD19 (B4) is a signal transduction molecule restricted to the B-cell lineage and the target of the immunotoxin anti-B4-blocked ricin (anti-B4-bR), which is composed of the monoclonal antibody (MoAb) anti-B4 and the modified plant toxin blocked ricin. To explore the influence of conjugation of blocked ricin to anti-B4 on functional activation of CD19, we investigated the effects of anti-B4-bR, and that of unconjugated anti-B4, on intracellular calcium mobilization and ligand/receptor internalization. The data showed that anti-B4-bR was more potent than anti-B4 in triggering cell calcium mobilization. Two other immunotoxins that bind to the B-cell surface, anti-CD20-bR and anti-CD38-bR, were devoid of the calcium increasing effect of anti-B4-bR. Furthermore, anti-B4 conjugated to ricin A-chain was also without effect in Namalwa cells, indicating that the ricin B-chain component was required for anti-B4-bR effect. Anti-B4-bR-induced calcium mobilization was inhibited in the presence of lactose, yet the calcium response induced by cross-linking anti-B4-bR with a second step antibody was not affected. The extent of CD19 modulation induced by anti-B4-bR was higher than that induced by anti-B4, and lactose dampened the effect of the immunotoxin down to that of the MoAb. Moreover, the number of internalized immunotoxin molecules was higher than that of unconjugated MoAb. Although a mechanism involving dimerization of the immunotoxin cannot be excluded, our findings suggest that the residual binding activity of the blocked ricin B-chain to cell surface molecules plays an important role in the greater calcium fluxes and greater internalization rate of anti-B4-bR, and is of functional significance in the mechanism of intoxication of cells by the immunotoxin.     

Protection by L-2-oxothiazolidine-4-carboxylic acid of hydrogen peroxide-induced CD3zeta and CD16zeta chain down-regulation in human peripheral blood lymphocytes and lymphokine-activated killer cells

The cell lethality and permeability induced in Escherichia coli B/r and Escherichia coli BS-1 by 60Co gamma-ray irradiation, ultrasound and hyperthermia in media containing different concentrations of NaCl have been investigated. It was shown that independently from the nature of damaging factors hypotonic media increase while hypertonic media in certain range of osmolyte concentration decrease sensitivity of cells to action of this factors. It was proposed that discovered phenomenology was caused by salt modification of status of the cell osmotic homeostasis destabilizing by ionizing radiation, ultrasound or hyperthermia and was not related with the system of dark repair of DNA.     

Activation of phospholipase C-beta1 via Galphaq/11 during calcium mobilization by calcitonin gene-related peptide

Interaction of calcitonin gene-related peptide (CGRP) with its receptors leads to stimulation of adenylyl cyclase and/or phospholipase C (PLC). While regulation of adenylyl cyclase is thought to involve the G-protein Gs, it is not known whether activation of PLC results from coupling the receptor to Gq family proteins or whether beta gamma subunits released from receptor-activated Gs activate PLC. We used human bone cells OHS-4 bearing CGRP receptors in which CGRP activates only the PLC signaling pathway to determine how CGRP acts. CGRP increased the concentration of intracellular calcium ([Ca2+]i) within 5 s via a Ca2+ influx through voltage-gated calcium channels and by mobilizing calcium from the endoplasmic reticulum. The activation of effectors, like PLC coupled to G-proteins, is the early event in the pathway leading to inositol 1,4,5-trisphosphate formation, which is responsible for Ca2+ mobilization. Western blotting demonstrated a range of PLC-beta isoforms (beta1, beta3, beta4, but not beta2) and G-proteins (Galphaq/11 and Galphas). Only phospholipase C-beta1 is involved in the mobilization of Ca2+ from the endoplasmic reticulum of Fura-2-loaded confluent OHS-4 cells and the formation of inositol 1,4,5-trisphosphate by CGRP; PLC-gamma have no effect. Activation of PLC-beta1 by CGRP involves the Galphaq/11 subunit, which is insensitive to pertussis toxin, but not Gbeta gamma subunits. We therefore believe that CGRP causes the activation of two separate G-proteins.     

T cell receptor zeta allows stable expression of receptors containing the CD3gamma leucine-based receptor-sorting motif

The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently of phosphorylation and leads to rapid internalization and sorting of these chimeras to lysosomal degradation. Because the TCRzeta chain rescues incomplete TCR complexes from lysosomal degradation and allows stable surface expression of fully assembled TCR, we addressed the question whether TCRzeta has the potential to mask the CD3gamma leucine-based motif. By studying CD4/CD3gamma and CD16/CD3gamma chimeras, we found that CD16/CD3gamma chimeras associated with TCRzeta. The CD16/CD3gamma-TCRzeta complexes were stably expressed at the cell surface and had a low spontaneous internalization rate, indicating that the leucine-based motif in these complexes was inactive. In contrast, the CD4/CD3gamma chimeras did not associate with TCRzeta, and the leucine-based motif in these chimeras was constitutively active resulting in a high spontaneous internalization rate and low expression of the chimeras at the cell surface. Thus, our data demonstrate that TCRzeta allows stable cell surface expression of receptors containing CD3gamma leucine-based motifs by its potential to mask such motifs.     

The site of estradiol sensitization of the gonadotrope is prior to calcium mobilization

In primary pituitary cell cultures prepared from ovariectomized rats, estradiol-17B (E2) sensitizes gonadotropes to stimulation of luteinizing hormone (LH) release by gonadotropin releasing hormone (GnRH). The calcium ionophore A23187, which stimulates LH release from the cells by Ca2+ mobilization at a post-receptor locus, and veratridine, which stimulates LH release by activation of endogenous ion channels, were used to localize the site of E2 action. Cells cultured in medium which was charcoal stripped (to remove steroids) or which contained 10(-8) M added E2 responded equally well to the ionophore and equally well to veratridine, indicating that the molecular locus of E2 action precedes Ca2+ mobilization. This type of analysis can be used to locate the site of action of compounds which alter the responsiveness of the pituitary to GnRH.     

Role of calciotrophic hormones in calcium mobilization of lactation

In two consecutive studies (Study A and Study B), we evaluated the effects of increasing doses of HBOC-201, a bovine hemoglobin-based oxygen carrier, on hemodynamics and oxygen transport in patients undergoing preoperative hemodilution for elective abdominal aortic surgery. After the induction of anesthesia and the exchange of 1 L of blood for 1 L of lactated Ringer's solution, 24 patients (12 in each study) were randomly assigned to receive, within 30 min, a predetermined volume of either HBOC-201 or 6% hydroxyethyl starch (Study A 6.9 mL/kg; Study B 9.2 mL/kg). Monitored variables included systemic and pulmonary arterial pressures, arterial and mixed venous blood gases, and calculations of cardiac index (CI), systemic (SVRI) and pulmonary (PVRI) vascular resistance indices, oxygen delivery index (DO2I), oxygen consumption index (VO2I), and oxygen extraction ratio (O2ER). In both studies, the infusion of HBOC-201 was associated with increases in SVRI (Study A 121%; Study B 71%) and PVRI (Study A 70%; Study B 53%) and with a decrease in CI (29% both studies). Hemodilution with HBOC-201 maintained the arterial oxygen content at levels higher than hemodilution with hydroxyethyl starch, but the advantage of a greater oxygen-carrying capacity was offset by the increase in SVRI, with a resulting net decrease in both CI and DO2I (Study A 30%; Study B 28%); VO2I was maintained by increased O2ER. In terms of hemodynamics and oxygen transport, hemodilution with bovine hemoglobin in these doses provided no apparent benefit over hemodilution with hydroxyethyl starch. Implications: Bovine hemoglobin in doses ranging between 55 and 97 g of hemoglobin increased vascular resistance and decreased cardiac output in anesthetized surgical patients. In terms of hemodynamics and oxygen transport, hemodilution with bovine hemoglobin in these doses provided no apparent benefit over hemodilution with hydroxyethyl starch.     

Reconstitution of a voltage and calcium dependent potassium channel from rat cerebellum

Membrane vesicles from rat cerebellum were reconstituted into lipid bilayers. The activity of two different potassium channels was recorded: (1) a small conducting voltage dependent potassium channel insensitive to [Ca2+]i, (2) a calcium and voltage dependent potassium channel (KCa). KCa channels had a conductance of (302+/-15) pS (n=5) and were activated by [Ca2+]i and membrane depolarizations. They were blocked by tetraethylamonium (TEA) and charybdotoxin (CTX) but insensitive to noxiustoxin (NTX). Finally, we showed the blocking effect of Androctonus australis Hector (AaH) scorpion venom on KCa channels from rat cerebellum.     

T cells from patients with Hodgkin's disease have a defective T-cell receptor zeta chain expression that is reversible by T-cell stimulation with CD3 and CD28

To investigate the mechanisms underlying the deficiency of T lymphocytes from patients with Hodgkin's disease, we investigated the expression of the T-cell receptor (TCR) zeta chain in patients with Hodgkin's disease. By flow cytometry using an anti-zeta chain monoclonal antibody, peripheral blood T lymphocytes from patients with untreated Hodgkin's disease were shown to express decreased levels of the TCR zeta chain. After stimulation by combined CD3 and CD28 cross-linking, T cells from Hodgkin's disease patients upregulated zeta chain protein expression to normal values within 48 hours and achieved a cytolytic potential and levels of interleukin (IL)-2 secretion that were not different from T cells obtained from healthy controls. These results show that downregulation of the TCR zeta chain in Hodgkin's T lymphocytes is a reversible event. Costimulation of CD3 and CD28 is a novel approach for overcoming the T-cell deficiency in Hodgkin's disease and might be exploited clinically. As upregulation of the zeta chain can also be achieved using bispecific monoclonal antibodies (BI-MoAbs) with specificity for tumor antigens and CD3 and CD28, respectively, an immunotherapy with CD3/CD30 and CD28/CD30 Bi-MoAbs may overcome and should therefore, not be jeopardized by the inherent T-cell deficiency in patients with Hodgkin's disease.     

Phenotype-genotype relationships in complementation group 3 of the peroxisome-biogenesis disorders

The peroxisome-biogenesis disorders (PBDs) are a set of often lethal genetic diseases characterized by mental retardation and defective peroxisomal matrix protein import. Mutations in PEX12 are known to underlie the disease in two patients from complementation group 3 of the PBDs. Here we show that all patients from this group carry mutations on both alleles of PEX12. A comparison between PEX12 genotypes and the clinical and cellular phenotypes of the corresponding PBD patients suggests a relatively straightforward relationship between genotype and phenotype in this group of the PBDs, such that the loss of PEX12 function leads to more-severe cellular and clinical phenotypes. However, one patient who presented relatively mild clinical and cellular phenotypes was a compound heterozygote for two seemingly severe mutations on each PEX12 allele. PEX12 mRNA present in the patient's cells was derived from only one allele, the one that carried a 2-bp deletion early in the PEX12 coding region, c.26,27Delta. The deduced protein product of this mRNA would contain only the first eight amino acids of the protein, and yet this mutant PEX12 cDNA displayed significant PEX12 activity in a functional complementation assay. Surprisingly, the PEX12/c.26, 27Delta cDNA directed the synthesis of a 29-kD PEX12 protein in vitro, a result that is consistent with translation initiation at a downstream AUG codon. Transfection studies confirmed the expression of similarly sized PEX12 proteins from the PEX12/c.26,27Delta allele. Thus, it appears that translation initiation at internal AUG codons may modulate disease phenotypes and should be considered whenever unexpectedly mild phenotypes result from severe mutations early in the coding region.     

Phosphorylation and coupling of zeta-chains to activated T-cell receptor (TCR)/CD3 complexes from peripheral blood T-cells of elderly humans

Aging is often accompanied by altered T-cell signaling and functions. Signals mediated through the T-cell receptor (TCR)/CD3 complex are associated with tyrosine phosphorylations of zeta-chains by the regulated activities of protein tyrosine kinases p56(lck) and p59(fyn) as well as protein tyrosine phosphatases. In the present investigation, the coupling and phosphorylation of zeta-chains to TCR/CD3 immunocomplexes were examined in peripheral blood T-cells from 13 elderly and young humans stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3epsilon monoclonal antibody OKT3. Western blots analyzing the non-covalent coupling of zeta-chains to TCR/CD3 immunocomplexes from Brij-96 detergent lysates of anti-CD3 ligated T-cells showed that the levels of zeta-chains within TCR/CD3 immunocomplexes from T-cells of elderly and young subjects did not significantly differ. By contrast, the levels of phosphorylated zeta-chains generated during in vitro phosphorylations of TCR/CD3 immunocomplexes from elderly subjects were significantly reduced and averaged 44% of those observed for anti-CD3epsilon ligated T-cells from young subjects. Analyses of the levels of zeta-chain coupling and phosphorylations in T-cells from each of the 13 elderly individuals also showed that the reductions in zeta-chain phosphorylations were heterogeneous and unrelated to modest reductions in coupling. Furthermore, the age-related decreases in zeta-chain phosphorylations were not due to diminished frequencies of CD3epsilon+ cells or densities of CD3epsilon surface receptors and could be observed without reductions in epsilon-chain phosphorylations. These results suggest that aberrancies of zeta-chain phosphorylations can occur in T-cells of elderly humans independent from any uncoupling of zeta-chains to activated TCR/CD3 complexes.     

Development and function of T cells in T cell antigen receptor/CD3 zeta knockout mice reconstituted with Fc epsilon RI gamma

Engagement of alpha-beta T cell receptors (TCRs) induces many events in the T cells bearing them. The proteins that transduce these signals to the inside of cells are the TCR-associated CD3 polypeptides and zeta-zeta or zeta-eta dimers. Previous experiments using knockout (KO) mice that lacked zeta (zeta KO) showed that zeta is required for good surface expression of TCRs on almost all T cells and for normal T cell development. Surprisingly, however, in zeta KO mice, a subset of T cells in the gut of both zeta KO and normal mice bore nearly normal levels of TCR on its surface. This was because zeta was replaced by the Fc epsilon RI gamma (FcR gamma). These cells were relatively nonreactive to stimuli via their TCRs. In addition, a previous report showed that zeta replacement by the FcR gamma chain also might occur on T cells in mice bearing tumors long term. Again, these T cells were nonreactive. To understand the consequences of zeta substitution by FcR gamma for T cell development and function in vivo, we produced zeta KO mice expressing FcR gamma in all of their T cells (FcR gamma TG zeta KO mice). In these mice, TCR expression on immature thymocytes was only slightly reduced compared with controls, and thymocyte selection occurred normally and gave rise to functional, mature T cells. Therefore, the nonreactivity of the FcR gamma + lymphocytes in the gut or in tumor-bearing mice must be caused by some other phenomenon. Unexpectedly, the TCR levels of mature T cells in FcR gamma TG zeta KO mice were lower than those of controls. This was particularly true for the CD4+ T cells. We conclude that FcR gamma can replace the functions of zeta in T cell development in vivo but that TCR/CD3 complexes associated with FcR gamma rather than zeta are less well expressed on cells. Also, these results revealed a difference in the regulation of expression of the TCR/CD3 complex on CD4+ and CD8+ T cells.     

Expression of CD38 increases intracellular calcium concentration and reduces doubling time in HeLa and 3T3 cells

CD38 is a bifunctional ectoenzyme, predominantly expressed on hematopoietic cells during differentiation, that catalyzes the synthesis (cyclase) and the degradation (hydrolase) of cyclic ADP-ribose (cADPR), a powerful calcium mobilizer from intracellular stores. Due to the well established role of calcium levels in the regulation of apoptosis, proliferation, and differentiation, the CD38/cADPR system seems to be a likely candidate involved in the control of these fundamental processes. The ectocellular localization of the cyclase activity, however, contrasts with the intracellular site of action of cADPR. Here we demonstrate that ectocellular expression of human CD38 in CD38(-) HeLa and 3T3 cells results in intracellular CD38 substrate (NAD+ + NADH) consumption and product (cADPR) accumulation. Furthermore, a causal relationship is established between presence of intracellular cADPR, partial depletion of thapsigargin-sensitive calcium stores, increase in basal free cytoplasmic calcium concentration, and decrease of cell doubling time. The significant shortening of the S phase in CD38(+) HeLa cells, as compared with controls, demonstrates an effect of intracellular cADPR on the mammalian cell cycle.     

A gene variation of 14-3-3 zeta isoform in rat hippocampus

A variant form of 14-3-3 zeta was isolated from the rat hippocampal cDNA library. The cloned cDNA is 1687 bp in length and it contains an entire ORF (nt = 63-797) with 245 amino acids that is characteristic to 14-3-3 zeta subtype. By comparing with reported sequences of 14-3-3 zeta, we found three nucleotide substitutions within the coding sequence in our clone; C<-->T transition at nt = 325 and G<-->C transversions at nt = 387 and 388. Both are missense mutations, leading ACG (Thr) to ATG (Met) and CGT (Arg) to GCT (Ala) conversions at residue 88 and 109, respectively. Our results show that at least three different genetic variants of 14-3-3 zeta are present in rat species which results in protein variations. Such mutation in the amino acid sequence is an important indication of the diverse functions of this protein and may also contribute to the recent contradictory observations regarding the role of the 14-3-3 zeta subtype.     

A genetic study of aggregation in the cellular slime mould Dictyostelium discoideum using complementation analysis

A series of aggregation-deficient (aggregateless) mutants were isolated in genetically marked haploid strains of the cellular slime mould Dictyostelium discoideum. Diploids were produced from pairs of such haploid mutants by a fusion system based on this organism's parasexual cycle. The diploids were isolated from the haploids by using complementation of non-allelic growth-temperature-sensitive mutations and selection at the restrictive temperature. Complementation between the aggregateless mutations uas then assessed in 419 diploids so formed. The non-complementing aggregateless mutations fell into five complementation groups (ago A, B, C, D, and E) and a dominant aggregation class that allowed little or no aggregation when present in a diploid with any of the other mutations tested or the parental wild type. Complicating factors, including partial dominance, multiple mutations, and possible interallelic conplementation, are discussed. Data on the linkage of the aggregateless mutations was obtained by using recessive drug resistance mutations on three linkage groups to segregate haploids from the diploids. Calculations from our results suggest a genetic complexity of about 50 genes that are specific and essential for aggregation.     

Activation-induced apoptosis of mature T cells is dependent upon the level of surface TCR but not on the presence of the CD3 zeta ITAM

Activation-induced cell death (AICD) occurs primarily in recently activated T cells after a second TCR triggering. Since a threshold in the activation status may be critical for AICD, it is likely that the CD3 ITAM, docking sites for tyrosine kinases, regulate AICD. A 'threshold model' for AICD was tested by using two targeted mutant mouse strains lacking either the zeta chain (CD3zeta-/-) or the ITAM of the zeta chain (CD3zeta-/-:Tgzetadelta67-150). Although the T cells from the CD3zeta-/- mice express extremely low levels of surface TCR, a subpopulation (approximately 18%) of activated T cells could be induced to express TCR/FcepsilonRI gamma by using a powerful polyclonal activation protocol. These activated TCR/FcRI gamma T cells were capable of undergoing AICD, but its induction required 10 times as much anti-CD3epsilon mAb as that required for AICD of wild-type T cells. Thus, the intensity of AICD correlated with the level of CD3 expression and was less efficient with activated, CD3zeta(-/-)-derived T cells. By contrast, AICD of T cells from the CD3zeta-/-:Tgzetadelta67-150 mice could be induced with low doses of anti-CD3epsilon mAb and the extent of AICD was comparable to T cells from wild-type mice. The AICD induced in T cells from CD3-/-, CD3zeta-/-:Tgzetadelta67-150 and normal controls was specifically inhibited by Fas-Ig fusion proteins. Our data support the 'threshold model' of AICD by demonstrating that AICD is controlled by the strength of T cell activation.     

Intermediates of prothrombin activation induce intracellular calcium mobilization in rat aortic smooth muscle cells

OBJECTIVES: To determine the diagnostic utility and net cost of magnetic resonance imaging (MRI) in the management of clinically and sonographically inconclusive scrotal lesions. METHODS: A multicenter retrospective review identified 34 patients diagnosed with scrotal MRI following inconclusive clinical and ultrasound (US) evaluation. Final diagnoses were based on surgery (n = 18) or clinical and US follow-up (n = 16). Final diagnoses of 29 testicular lesions were as follows: orchitis (n = 11), infarct (n = 6), neoplasm (n = 6), rupture (n = 3), torsion (n = 2), and radiation fibrosis (n = 1). Final diagnoses of five extratesticular lesions were as follows: epididymitis (n = 2), epididymal abscess (n = 2), and neoplasm (n = 1). Management plans prior to and following MRI findings were formulated by a general urologist and a urologic oncologist. The costs of the pre-MRI and post-MRI management plans were estimated using the Medicare reimbursement schedule. RESULTS: The leading US diagnosis was correct for 10 of 34 patients (29%) and the leading MRI diagnosis was correct for 31 of 34 patients (91%). MRI improved the management plan of the general urologist and urologic oncologist in 19 patients (56%) and 17 patients (50%), respectively. MRI worsened the management plan of both clinicians in 1 patient. Management was unchanged in all other patients. The overall net cost savings were $543 to $730 per patient for the urologic oncologist and the general urologist, respectively, and $3833 per patient originally scheduled for surgery. CONCLUSIONS: Use of MRI after inconclusive clinical and US evaluation of scrotal lesions may improve management, decrease the number of surgical procedures, and result in net cost savings.