GTPγS-induced calcium transients and exocytosis in human neutrophils

Exocytosis and intracellular free calcium ([Ca²⁺]_in) were simultaneously recorded in single human neutrophils using patch-clamp capacitance measurements and the fura-2 fluorescence ratio method. Intracellular application of guanosine-5-O(3-thiotriphosphate) (GTPS) stimulates both exocytosis and a calcium transient. The calcium transient starts to develop after a lag phase of 40s and normally appears to trigger the onset of exocytosis indicated by the beginning of the capacitance increase. After this delay [Ca²⁺]_in increases from 150 nM to 600 nM with a sigmoidal time course. The peak concentration is reached within 30 s but the main increase occurs during 3s. [Ca²⁺]_in subsequently decays within 1–2 min to a level which is close to the resting value. This calcium transient is due to calcium release from inositoltrisphosphate-sensitive intracellular stores. Exocytosis also occurs if the calcium transient is abolished by intracellular EGTA but the lag phase is markedly prolonged. The GTPS-induced calcium transient is very similar to that observed after stimulation with N-formyl-methionyl-leucyl-phenylalanine. The interplay between guanine nucleotides, [Ca²⁺]_in and exocytosis in neutrophils closely resembles previous results obtained in mast cells suggesting a similar regulation of exocytosis in both cell types.     

Sulfhydryl modification induces calcium entry through IP₃-sensitive store-operated pathway in activation-dependent human neutrophils

As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT₁MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca²⁺]_c) without external calcium, followed by a second [Ca²⁺]_c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca²⁺]_c change of Ca²⁺-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca²⁺]_c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP₃) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP₃-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner.     

Involvement of adenosine 3′:5′-cyclic monophosphate in lipid mobilization in Locusta migratoria

The rôle of cyclic AMP in hormone-induced lipid mobilization in Locusta migratoria was investigated. Injection of a corpus cardiacum extract into adult female locusts resulted in an increased level of cyclic AMP in the fat body. The cAMP concentration is maximal at about 5 min of incubation and returns to the resting level after about 10 min. The dose-response curve is linear up to about 0.01 corpus cardiacum pair equivalents.Dibutyryl-cyclic AMP mimics the lipid mobilizing effect of corpus cardiacum extract. After flight the cyclic AMP concentration in fat body increased. Injection of corpus cardiacum extract had no effect on flight muscle cyclic AMP concentration.     

Phosphatidylinositol 3′-kinase signalling supports cell height in established epithelial monolayers

Cell–cell interactions influence epithelial morphogenesis through an interplay between cell adhesion, trafficking and the cytoskeleton. These cellular processes are coordinated, often by cell signals found at cell–cell contacts. One such contact-based signal is the phosphatidylinositol 3′-kinase (PI3-kinase; PI3K) pathway. PI3-kinase is best understood for its role in mitogenic signalling, where it regulates cell survival, proliferation and differentiation. Its precise morphogenetic impacts in epithelia are, in contrast, less well-understood. Using phosphoinositide-specific biosensors we confirmed that E-cadherin-based cell–cell contacts are enriched in PIP₃, the principal product of PI3-kinase. We then used pharmacologic inhibitors to assess the morphogenetic impact of PI3-kinase in MDCK and MCF7 monolayers. We found that inhibiting PI3-kinase caused a reduction in epithelial cell height that was reversible upon removal of the drugs. This was not attributable to changes in E-cadherin expression or homophilic adhesion. Nor were there detectable changes in cell polarity. While Myosin II has been implicated in regulating keratinocyte height, we found no effect of PI3-kinase inhibition on apparent Myosin II activity; nor did direct inhibition of Myosin II alter epithelial height. Instead, in pursuing signalling pathways downstream of PI3-kinase we found that blocking Rac signalling, but not mTOR, reduced epithelial cell height, as did PI3-kinase inhibition. Overall, our findings suggest that PI3-kinase exerts a major morphogenetic impact in simple cultured epithelia through preservation of cell height. This is independent of potential effects on adhesion or polarity, but may occur through PI3-kinase-stimulated Rac signaling.     

New roles for the major human 3′-5′ exonuclease TREX1 in human disease

Aicardi-Goutières syndrome (AGS), Systemic Lupus Erythematosus (SLE), Familial Chilblain Lupus (FCL), and Retinal Vasculopathy and Cerebral Leukodystrophy (RVCL) {a new term encompassing three independently described conditions with a common etiology - Cerebroretinal Vasculopathy (CRV), Hereditary Vascular Retinopathy (HVR), and Hereditary Endotheliopathy, Retinopathy and Nephropathy (HERNS)} - have previously been regarded as distinct entities. However, recent genetic analysis has demonstrated that each of these diseases maps to chromosome 3p21 and can be caused by mutations in TREX1, the major human 3′-5′exonuclease. In this review, we discuss the putative functions of TREX1 in relationship to the clinical, genetic and functional characteristics of each of these conditions.     

Fcγ receptors exhibit different phagocytosis potential in human neutrophils

In neutrophils, two receptors for IgG antibodies, namely FcγRIIA and FcγRIIIB are constitutively expressed, and a third one, FcγRI, can be upregulated by interferon-γ. Whether FcγRIIIB is capable of triggering phagocytosis by itself is still controversial. The main role of FcγRI has not been clearly established in these cells. To address this problem, neutrophils were treated with interferon-γ, and then phagocytosis mediated by each type of Fcγ receptor was evaluated by flow cytometry. FcγRIIA was the most efficient receptor for phagocytosis. FcγRIIIB could mediate phagocytosis but much less efficiently than FcγRIIA. Both FcγRIIA- and FcγRIIIB-mediated phagocytosis were blocked by inhibitors of Src family kinases, Syk, PI 3-K, and ERK. In contrast, interferon-γ-induced FcγRI was not able to mediate phagocytosis. Also, FcγRI did not activate ERK in the nucleus, but was however able to stimulate an efficient calcium rise. These data show that different neutrophil Fcγ receptors possess different phagocytosis capabilities: FcγRIIA and FcγRIIIB, but not FcγRI, promote phagocytosis.     

Cl⁻/HCO₃⁻ exchange activity in fMLP-stimulated human neutrophils

It is well known that chemotactic agents active Na(+)/H(+) exchanger, increasing intracellular pH of neutrophils, but their effect on bicarbonate transporters have not been established yet. To study the effect of fMLP on the activity of Cl(-)/HCO(3)(-) exchange, the rate of pH recovery after acute Cl(-) readmission in cell subjected to an alkaline load by CO(2) washout in a Cl-free medium was measured. The activity of the exchanger was reduced to 72% of control when cells were pre-incubated for 5 min with 0.1 μM fMLP and reached 48% of control in steady state after acute exposure. After extracellular bicarbonate or TMA addition the rate recovery of intracellular pH was reduce at 72% and at 84%, respectively. The inhibitory effect on the intracellular pH recovery was not affected by blockers of Na(+)/H(+) exchange. We conclude from these studies that an increase of pH(i) produced for this chemotactic agent is facilitated by the simultaneous activation of Na(+)/H(+) exchange and inhibition of Cl(-)/HCO(3)(-) exchange in neutrophils.     

Structure–activity relationships in human RNA 3′-phosphate cyclase

RNA 3′-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2′,3′ cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP_i; transfer of AMP from Rtc1 to an RNA 3′-phosphate to form an RNA(3′)pp(5′)A intermediate; and attack by the terminal nucleoside O2′ on the 3′-phosphate to form an RNA 2′,3′ cyclic phosphate product and release AMP. Here we used the crystal structure of Escherichia coli RtcA to guide a mutational analysis of the human RNA cyclase Rtc1. An alanine scan defined seven conserved residues as essential for the Rtc1 RNA cyclization and autoadenylylation reactions. Structure–activity relationships were clarified by conservative substitutions. Our results are consistent with a mechanism of adenylate transfer in which attack of the Rtc1 His320 nucleophile on the ATP α phosphorus is facilitated by proper orientation of the PP_i leaving group via contacts to Arg21, Arg40, and Arg43. We invoke roles for Tyr294 in binding the adenine base and Glu14 in binding the divalent cation cofactor. We find that Rtc1 forms a stable binary complex with a 3′-phosphate terminated RNA, but not with an otherwise identical 3′-OH terminated RNA. Mutation of His320 had little impact on RNA 3′-phosphate binding, signifying that covalent adenylylation of Rtc1 is not a prerequisite for end recognition.     

α-Tocopherol increases caspase-3 up-regulation and apoptosis by β-carotene cleavage products in human neutrophils

It has been found that β-carotene cleavage products (CarCP), besides having mutagenic and toxic effects on mitochondria due to their prooxidative properties, also initiate spontaneous apoptosis of human neutrophils. Therefore, it was expected that antioxidants such as α-tocopherol would inhibit the stimulation of apoptosis and caspase-3 activity by CarCP. However, we found that α-tocopherol increases caspase-3 up-regulation and stimulation of apoptosis of human neutrophils by CarCP. Ascorbic acid does not alter this caspase-3 up-regulating and proapoptotic effect exerted by α-tocopherol. Both α-tocopherol and ascorbic acid, in the absence of CarCP, decrease intracellular caspase-3 activity and spontaneous apoptosis of neutrophils. Uric acid alone or in combination with CarCP does not exert apparent effects on caspase-3 activity and apoptosis. Up-regulating effect of α-tocopherol is not observed in the presence of retinol that markedly stimulates apoptosis by itself, whereas increase of caspase-3 activity is induced by concomitant addition of α-tocopherol and β-ionone, a cyclohexenyl degradation product of β-carotene with shorter aliphatic chain.     

Does inositol tetrakisphosphate play a role in the receptor-mediated control of calcium mobilization?

The evidence for and against an important role for inositol 1,3,4,5 tetrakisphosphate (Ins 1,3,4,5 P4) in receptor-mediated Ca2+ mobilization is reviewed. Data obtained from patch-clamp whole-cell current recording studies on internally perfused exocrine acinar cells show that the acetylcholine (ACh)-evoked sustained increase in Ca2+-dependent K+ current caused by an increase in [Ca2+]i cannot be mimicked by internal application of inositol 1,4,5-trisphosphate (Ins 1,4,5 P3), but only by a combination of Ins 1,4,5 P3 and Ins 1,3,4,5 P4. The sustained response evoked by Ins 1,4,5 P3 + Ins 1,3,4,5 P4 is dependent on the presence of external Ca2+ as is the effect of ACh. Only those inositol trisphosphates able to evoke Ca2+ release from internal stores can support the action of Ins 1,3,4,5 P4 in evoking responses that are acutely dependent on extracellular Ca2+ (Ca2+ influx). The various arguments presented against an involvement of Ins 1,3,4,5 P4 are discussed. The main point emerging is that most studies are inadequately controlled and it is concluded that there is a strong need for whole-cell current recording studies combined with pipette fluid exchange to be carried out in many more systems. The major problem in this field is that the precise site and mechanism of action of Ins 1,3,4,5 P4 are unknown and that the pathway for Ca2+ uptake during receptor activation is inadequately defined.     

Correlation between chemotactic peptide-induced changes in chlorotetracycline fluorescence and F-actin content in human neutrophils: a role for membrane-associated calcium in the regulation of actin polymerization?

Several observations indicate that the triggering event for receptor-mediated actin polymerization takes place in or close to the plasma membrane. Stimulation of human neutrophils with the chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) causes rapid and transient changes in both chlorotetracycline (CTC) fluorescence and the cellular content of filamentous actin (F-actin), thus suggesting a regulatory role for membrane-bound calcium in actin polymerization. In the present study, tetracaine, a proposed antagonist to membrane-bound calcium, totally inhibited the rebinding of the membrane calcium released by fMet-Leu-Phe. This was accompanied by a magnified and sustained increase in the cellular content of F-actin. In agreement, N-ethylmaleimide, an inhibitor of motile functions, completely abolished the fMet-Leu-Phe-triggered changes in both CTC fluorescence and F-actin content and rapidly reversed the responses when added after the peptide. The tumor promoter phorbol-12-myristate-13-acetate, caused only small changes in CTC fluorescence and F-actin content, and reduced a subsequent fMet-Leu-Phe-induced CTC response and actin polymerization. Inhibition of the breakdown of phosphatidylinositol 4,5-bisphosphate, by calcium depletion, had no significant effects on the fMet-Leu-Phe-induced CTC response and alterations in F-actin content, whereas pretreatment with pertussis toxin totally inhibited both these responses. Consequently, the strong correlation between changes in CTC fluorescence and F-actin content, found in this study, suggests a triggering or modulating role of membrane-associated calcium on actin polymerization in human neutrophils.     

Identification of elements in human long 3′ UTRs that inhibit nonsense-mediated decay

The nonsense-mediated mRNA decay (NMD) pathway serves an important role in gene expression by targeting aberrant mRNAs that have acquired premature termination codons (PTCs) as well as a subset of normally processed endogenous mRNAs. One determinant for the targeting of mRNAs by NMD is the occurrence of translation termination distal to the poly(A) tail. Yet, a large subset of naturally occurring mRNAs contain long 3′ UTRs, many of which, according to global studies, are insensitive to NMD. This raises the possibility that such mRNAs have evolved mechanisms for NMD evasion. Here, we analyzed a set of human long 3′ UTR mRNAs and found that many are indeed resistant to NMD. By dissecting the 3′ UTR of one such mRNA, TRAM1 mRNA, we identified a cis element located within the first 200 nt that inhibits NMD when positioned in downstream proximity of the translation termination codon and is sufficient for repressing NMD of a heterologous reporter mRNA. Investigation of other NMD-evading long 3′ UTR mRNAs revealed a subset that, similar to TRAM1 mRNA, contains NMD-inhibiting cis elements in the first 200 nt. A smaller subset of long 3′ UTR mRNAs evades NMD by a different mechanism that appears to be independent of a termination-proximal cis element. Our study suggests that different mechanisms have evolved to ensure NMD evasion of human mRNAs with long 3′ UTRs.     

Analysis of developmental switching in transgenic mice with 5′ and 3′ deletions in the human β globin gene

Our interest in thecis-acting elements that promote the up-regulation of the globin gene has led to a systematic deletion analysis of portions of the globin gene in the context of the HS2 and globin gene using transgenic mice. In constructs that delete the 5 region to only 265 bp, high-level erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between –122 and –265. In addition, a construct in which the entire globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3 region of the globin gene was deleted in order to examine the effect of a previously defined 3 enhancer region. With deletion of this region, the expression of the human globin gene in transgenic mice is unchanged relative to the parental constructs.     

Molecular basis of 2′,3′-dideoxycytidine-induced drug resistance in human cells

Human monoblastoid cells (U937) grown in the presence of therapeutically relevant dideoxycytidine concentrations (0.1 M) become resistant to the drug thanks to an altered deoxycytidine kinase. In this paper we show that deoxycytidine kinase mRNA is significally reduced in drug-resistant U937 cells (U937-R) although the deoxycytidine kinase promoter is normal. A number of nucleotide deletions, insertions and substitutions was found in the coding region of deoxycytidine kinase gene. Several identified mutations result in truncated forms of the enzyme or in the introduction of stop codons: in one case a complete lack of exon 4 was found. Thus, the deoxycytidine kinase gene accumulates mutations at a very high rate, as already reported for other cytidine analogues (i.e. Ara C ) suggesting that the design of new antiviral or anticancer drugs of the cytidine family should take into account the potential development of cell resistance as a critical factor in drug failure.     

Effects of adenosine 3′ : 5′-monophosphate and guanosine 3′ : 5′-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle

The effects of adenosine 3′ : 5′-monophosphate (cyclic AMP), guanosine 3′ : 5′-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P).While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10^?5 M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP.Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10^?8 M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10^?8 M, while with cyclic AMP a concentration of 10^?5 M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P.These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.     

1α,25-dihydroxy-vitamin D-3 regulates ATP-dependent calcium transport in basolateral plasma membranes of rat enterocytes

Basolateral plasma membrane vesicles of rat small intestinal epithelium accumulate calcium through an ATP-dependent pumping system. The activity of this system is highest in duodenum and decreases towards the ileum. This distribution along the intestinal tract is similar as the active calcium absorption capacity of intact intestinal epithelial segments. ATP-dependent calcium uptake in basolateral membrane vesicles from duodenum and ileum increased significantly after repletion of young vitamin D-3-deficient rats with 1α,25-dihydroxy-vitamin D-3. Ca²⁺-ATPase activity in duodenal basolateral membranes increased to the same extend as ATP-dependent calcium transport, but (Na⁺ + K⁺)-ATPase activity remained unaltered.     

Effect of cyclic 3′:5′-AMP derivatives,prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture

Summary The secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N⁶, O^2′-dibutyryl cyclic 3′:5′-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process. malignant trophoblast cultures were incubated with 3′:5′-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmuno-assayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretio of hCG, the N⁶- and O^2′-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3′:5′-GMP (cGMP), dbcBMP, 5′-AMP, adenosine, L-epinephrine and prostaglandins E₁, E₂, F_1α and F_2α were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast. This investigation was supported by Grant Nos. CA14232 and CA16539 awarded by the National Cancer Institute, DHEW.