Influence of yeasts and of their constituents on nucleoside uptake in peritoneal murine macrophages

A marked reduction of [3H]-uridine uptake was observed when mouse peritoneal macrophages (pM phi) were exposed to heat-killed Candida albicans or Saccharomyces cerevisiae. By contrast, an increased nucleoside uptake was promoted by yeast products such as zymosan, laminarin, or yeast cell-wall extracts, which are mainly composed of beta-glucans and alpha-mannans. In a search for the active fungal component(s), the uptake process was shown to be differently affected by monosaccharides and polysaccharides. These findings support the view that a specific recognition of a pM phi membrane receptor is mediating the effect of the various substances.     

Expression of interferon-beta during the triggering phase of macrophage cytocidal activation. Evidence for an autocrine/paracrine role in the regulation of this state.

The expression of cytocidal activity is induced by the sequential interaction of macrophages with a priming stimulus, such as interferon (IFN)alpha, -beta, or -gamma, and a triggering stimulus, such as poly(I.C) or lipopolysaccharide. However, most triggering stimuli are also capable of inducing IFN expression. This suggested to us the possibility that in addition to its role in initially priming macrophages for cytocidal activity, IFN may also be expressed during the triggering stage where it may potentially contribute to the regulation of cytocidal activity. We have explored this question by (i) attempting to dissociate IFN-inducing activity from triggering activity with a variety of structurally related and charge-related polyanions; (ii) determining if macrophages express IFN during the triggering stage; and (iii) questioning if IFN produced during the triggering stage contributes to the regulation of cytocidal activation. Exposure of unprimed macrophages to a triggering concentration of poly(I.C) alone failed to induce IFN beta expression. However, exposure of IFN beta-primed cells to poly(I.C) dramatically increased the expression of IFN beta mRNA. Priming with IFN gamma was likewise found to increase the expression of IFN beta mRNA in response to a triggering concentration of polyribonucleotides. Three approaches were adopted to ascertain if the increased expression of IFN beta contributed to cytocidal activation. First, macrophages derived from strains of mice which differ in their susceptibility to IFN induction by poly(I.C) were primed with IFN beta, washed, and triggered with poly(I.C). Under these conditions, macrophages derived from stain B10.A(2R), which are hyporesponsive to poly(I.C) in terms of IFN induction, also showed a diminished capacity to express Bf, a marker of cytocidal activation. Second, exposure of IFN-primed macrophages to poly(I.C) in the presence of anti-IFN alpha/beta antibody was found to reduce substantially the synthesis of NO2/NO3, an alternative marker of macrophage cytocidal activation. Third, exposure of IFN-primed macrophages to the calcium ionophores ionomycin or A23187, which do not induce the production of IFN beta during triggering, led to an abbreviated expression of Bf compared with stimuli that induce IFN beta expression such as poly(I.C). However, the capacity to synthesize Bf in response to A23187 was partially reconstituted when macrophages were triggered with the ionophore in the continuous presence of IFN beta. Collectively, these data show that IFN beta is expressed during the triggering stage of macrophage cytocidal activation and suggest that it plays an important and previously unsuspected role in the expression of this state.     

Long term endocrine regulation of nucleoside transporters in rat intestinal epithelial cells

We studied the regulation of nucleoside transporters in intestinal epithelial cells upon exposure to either differentiating or proliferative agents. Rat intestinal epithelial cells (line IEC-6) were incubated in the presence of differentiating (glucocorticoids) or proliferative (EGF and TGF-alpha) agents. Nucleoside uptake rates and nucleoside transporter protein and mRNA levels were assessed. The signal transduction pathways used by the proliferative stimuli were analyzed. We found that glucocorticoids induce an increase in sodium-dependent, concentrative nucleoside transport rates and in protein and mRNA levels of both rCNT2 and rCNT1, with negligible effects on the equilibrative transporters. EGF and TGF-alpha induce an increase in the equilibrative transport rate, mostly accounted for by an increase in rENT1 activity and mRNA levels, rENT2 mRNA levels remaining unaltered. This effect is mimicked by another proliferative stimulus that functions as an in vitro model of epithelial wounding. Here, rENT1 activity and mRNA levels are also increased, although the signal transduction pathways used by the two stimuli are different. We concluded that differentiation of rat intestinal epithelial cells is accompanied by increased mature enterocyte features, such as concentrative nucleoside transport (located at the brush border membrane of the enterocyte), thus preparing the cell for its ultimate absorptive function. A proliferative stimulus induces the equilibrative nucleoside activities (mostly through ENT1) known to be located at the basolateral membrane, allowing the uptake of nucleosides from the bloodstream for the increased demands of the proliferating cell.     

Cells of the J774 macrophage cell line are primed for antibody-dependent cell-mediated cytotoxicity following exposure to gamma-irradiation.

Activation of macrophages (M phi) for host defense against tumor cells follows a sequence of priming events followed by an initiating stimulus that results in production and release of cytotoxic molecules that mediate target cell killing. We have developed a model to study specific macrophage cytotoxicity in vitro utilizing a cultured murine M phi cell line, J774. Specific cytotoxicity of cultured human gastrointestinal tumor cells is achieved in the presence of murine IgG2a monoclonal antibody (mAb) 17-1-A. The ability of these cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) is greatly enhanced following gamma-irradiation. ADCC can be demonstrated at mAb 17-1-A concentrations greater than or equal to 1 microgram/ml and effector/target cell ratios greater than or equal to 2. Exposure to doses greater than or equal to 10 Gy of gamma-irradiation increases ADCC threefold. Varying the duration from J774 M phi exposure to gamma-irradiation until addition of antibody-coated target cells showed that the primed state for ADCC is stable for at least 8 days but approximately 24 hr is required for complete development of the primed state. mAb-dependent target cell death begins 8 hr after addition of mAb and labeled target cells to primed effector cells and is complete by 24 hr. Incubation of unirradiated J774 M phi effector cells with recombinant murine interferon-gamma (rmIFN-gamma) also results in enhanced ADCC, but the extent of target cell killing achieved is less than that following priming by gamma-irradiation. Concomitant priming of gamma-irradiated J774 M phi with rmIFN-gamma increases the extent of ADCC. Further study of irradiated J774 cells may elucidate the molecular pathways utilized by M phi for achieving and maintaining the primed state for ADCC. Irradiated J774 cells will also provide a homogenous, stably primed cell type in which to examine the mechanism(s) of cytotoxicity employed by tumoricidal M phi.     

Human granulocyte-macrophage colony-stimulating factor and other cytokines prime human neutrophils for enhanced arachidonic acid release and leukotriene B4 synthesis

Human recombinant granulocyte-macrophage CSF (GM-CSF) "primes" neutrophils for enhanced biologic responses to a number of secondary stimuli. Here, we examined the properties of neutrophil priming by GM-CSF and other growth factors such as human rTNF and granulocyte CSF. Although GM-CSF has a negligible direct effect on [3H]arachidonic acid release, it enhances or "primes" neutrophils for three- to fivefold increased release of [3H]arachidonic acid, induced by 1.0 microM A23187 and the chemotactants FMLP, platelet-activating factor, and leukotriene B4 (LTB4) (all 0.1 microM). The priming effects of GM-CSF were concentration- and time-dependent (maximum 100 pM, 1 h at 23 degrees C), and consistent with the determined dissociation constant of the human GM-CSF receptor. Indomethacin (10(-8) M), cycloheximide (100 micrograms/ml), and pertussis toxin (200 ng/ml, 2 h at 37 degrees C) had no effect on GM-CSF-, A23187, or platelet-activating factor-induced [3H]arachidonic acid release. The lipoxygenase inhibitor, nordihydroguaiaretic acid, however, totally abolished A23187-induced [3H]arachidonic acid release from both diluent- and GM-CSF-treated neutrophils. Consistent with this observation, we found that GM-CSF-pretreated neutrophils synthesize increased levels of LTB4 after stimulation with A23187 and chemotactic factors. GM-CSF enhances neutrophil arachidonic acid release and LTB4 synthesis, and thereby may amplify the inflammatory response to chemotactic factors and other physiologically relevant stimuli.     

Modulation of macrophage function by gamma-irradiation. Acquisition of the primed cell intermediate stage of the macrophage tumoricidal activation pathway

Macrophage activation for tumor cell killing is a multistep pathway in which responsive macrophages interact sequentially with priming and triggering stimuli in the acquisition of full tumoricidal activity. Although this synergistic response of normal macrophages to sequential incubation with activation signals has been well established, characterization of the intermediate stages in this pathway has been difficult, due in large measure to the instability of the intermediate cell phenotypes. We have developed a model system for examination of macrophage-mediated tumor cell lysis, with the use of the murine macrophage tumor cell line RAW 264.7. These cells, like normal macrophages, exhibit a strict requirement for interaction with both interferon-gamma (IFN-gamma, the priming signal) and bacterial lipopolysaccharide (LPS, the triggering signal) in the development of tumor cytolytic activity. In this system, the priming effects of IFN-gamma decay rapidly after withdrawal of this mediator and the cells become unresponsive to LPS triggering. We have recently observed that gamma-irradiation of the RAW 264.7 cells also results in development of a primed activation state for tumor cell killing. The effects of gamma-radiation on the RAW 264.7 cell line are strikingly similar to those resulting from incubation with IFN-gamma, with the exception that the irradiation-induced primed cell intermediate is stable and responsive to LPS triggering for at least 24 hr. Treatment with gamma-radiation also results in increased cell surface expression of major histocompatibility complex-encoded class I antigens; however, class II antigen expression is not induced. Irradiation-induced development of the primed phenotype is not solely the result of cytostatic effects as treatment of the cells with a radiomimetic drug, mitomycin C, results in decreases in [3H]thymidine incorporation that are similar to those observed after irradiation, without concomitant development of cytolytic potential. In addition, priming by gamma-radiation does not appear to be mediated by the release of soluble autoregulatory factors. This alternate pathway for induction of the primed macrophage activation state should serve as a useful tool for identification of molecules important to the functional potential of primed cells, and for elucidation of the biochemical mechanisms of the priming event in tumoricidal activation.     

Role of protein synthesis in the activation of cytotoxic mouse macrophages by lymphokines

The role of protein synthesis during the activation of macrophages (M phi) by lymphokines (LK) was studied. Peritoneal murine macrophages elicited by proteose-peptone (pM phi) were activated with LK (supernatants from normal mouse spleen cells pulsed with concanavalin A) and tested for cytotoxicity in an 18 hr assay against 111In-labeled L5178Y lymphoma target cells. Reversible (cycloheximide and puromycin) or poorly reversible (emetine and pactamycin) inhibitors of protein synthesis were added during activation, and their effects on pM phi-mediated cytotoxicity and pM phi protein synthesis were measured. Minimal concentrations of inhibitors, reducing the rate of protein synthesis by more than 90% without toxic effects on macrophages, were chosen. Exposure of pM phi to LK for 2 to 18 hr in the presence of reversible inhibitors of protein synthesis did not affect the induction of cytolytic activity, indicating that protein synthesis was not required during the activation period. In contrast, activation of macrophages for 2 hr in the presence of poorly reversible inhibitors of protein synthesis resulted in a considerable reduction of cytolytic activity. The impairment of cytotoxic activity was also evident when pM phi were treated with such drugs during the first 2 hr of an 18 hr exposure to LK or when LK-activated macrophages were treated for 2 hr with the drugs before the addition of the targets. These results demonstrate that active protein synthesis is not required during the exposure of pM phi to LK, but that new proteins have to be synthesized to allow the expression of the cytotoxic activity in LK-activated pM phi.     

Extracellular inosine is modulated by H2O2 and protects sertoli cells against lipoperoxidation and cellular injury

Extracellular purines are involved in the regulation of a wide range of physiological processes, including cytoprotection, ischemic preconditioning, and cell death. These actions are usually mediated via triggering of membrane purinergic receptors, which may activate antioxidant enzymes, conferring cytoprotection. Recently, it was demonstrated that the oxidative stress induced by cisplatin up-regulated A1 receptor expression in rat testes, suggesting an involvement of purinergic signaling in the response of testicular cells to oxidant injury. In this article, we report the effect of hydrogen peroxide on purinergic agonist release by cultured Sertoli cells. Extracellular inosine levels are strongly increased in the presence of H2O2, suggesting an involvement of this nucleoside on Sertoli cells response to oxidant treatment. Inosine was observed to decrease H2O2-induced lipoperoxidaton and cellular injury, and it also preserved cellular ATP content during H2O2 exposure. These effects were abolished in the presence of nucleoside uptake inhibitors, indicating that nucleoside internalisation is essential for its action in preventing cell damage.     

Picolinic acid, a catabolite of tryptophan, as the second signal in the activation of IFN-gamma-primed macrophages.

We have studied the effects of picolinic acid, a product of tryptophan degradation, on the activation of mouse peritoneal macrophages (M phi). Picolinic acid acts synergistically with IFN-gamma in activating M phi from C57BL/6 mice. Moreover, M phi from C3H/HeJ mice and C3H/HeN that do not become cytotoxic in response to IFN-gamma alone could be fully activated by exposure to picolinate plus IFN-gamma. These results indicate that picolinic acid is a potent costimulator of M phi activation that functions as a second signal. Inasmuch as we have previously demonstrated that the activation of cytotoxic M phi correlates with specific changes in ribosomal RNA (rRNA), we investigated whether picolinic acid could modify M phi RNA metabolism. Picolinic acid inhibited the synthesis of total M phi RNA, the accumulation of newly synthesized 28S rRNA, and augmented the steady state levels of rRNA precursors (pre-rRNA). These changes in RNA metabolism were similar to those previously described in murine M phi activated in vitro or in vivo to express tumoricidal activity. These results demonstrate that picolinic acid is a potent, biologic M phi second signal, suggest that the changes in rRNA are causally connected with the expression of tumoricidal activity, and suggest the existance of an autocrine effect mediated by picolinic acid.     

The NADPH oxidase of human polymorphonuclear leukocytes. Evidence for regulation by multiple signals

Activation of the membrane-bound NADPH oxidase in human polymorphonuclear leukocytes can be triggered by chemoattractants, the tumor promoter phorbol myristate acetate or the calcium ionophore A23187. We have shown previously that these stimuli have markedly different temporal patterns of oxidase activation (McPhail, L. C., and Snyderman, R. (1983) J. Clin. Invest. 72, 192-200), suggesting that each follows, at least in part, a unique transductional pathway. We now report that if leukocytes were sequentially exposed to any of several combinations of heterologous stimuli, the pattern of activation by the second stimulus was strikingly altered, resulting in a more rapid rate and enhanced level of oxidase activation by the second stimulus. This suggests that exposure of cells to the first stimulus (priming) had influenced an intermediate also used by the second stimulus. The signal for priming could be clearly distinguished from the signal causing oxidase activation by the dose-response curves for each, as well as by the use of several pharmacologic agents. In addition, if leukocytes were given sequential doses of homologous stimuli, either partial (phorbol myristate acetate) or full (N-formyl-methionyl-leucyl -phenylalanine and A23187) desensitization of oxidase activation was observed. These results demonstrate that these stimuli share a common intermediate in the pathway of oxidase activation. Moreover, the data indicate that NADPH oxidase activation is regulated by at least three distinct signals: signal 1 (priming), signal 2 (activation), and signal 3 (inactivation). It is likely that more than one intracellular messenger exerts a modulating influence on NADPH oxidase activity and that its regulation involves the interplay between several cellular control proteins.     

Activation of lymphocytes by a thiol-derivatized nucleoside: characterization of cellular parameters and responsive subpopulations

Lymphocyte activation, whether specific or nonspecific, is generally conceptualized as initiated by the binding of an activating ligand to a surface membrane receptor, followed by transduction of the signal across the cell membrane. In many situations several qualitatively distinct signals are required. We have recently described a new class of lymphocyte activator, the C8 bromine substituted guanine ribonucleosides, that traverse the cell membrane, bypassing classical triggering mechanism(s), apparently activating the lymphocyte at an intracellular site. However, the identity of the lymphocyte population(s) activated, as well as the nature of any cellular interactions involved in activation, has not been studied heretofore. The present experiments describe the cellular parameters of lymphocyte activation by a thiol substituted member of this class of activators, 8-mercaptoguanosine (8MGuo). Upon addition of this nucleoside derivative to cultures of murine spleen cells, a marked increase in [3H]TdR uptake and blast transformation ensues. Normal splenic B cells and spleen cells from congenitally athymic (nu/nu) mice are responsive to 8MGuo, whereas thymocytes and splenic T cells are not. Two subpopulations of B cells appear to be involved in the response to this nucleoside. The predominant one is a mature population that bears surface delta-chains, la antigens, C receptors, and (by indirect evidence) the Lyb3, 5, and 7 antigens. These cells also bear mu-chain and Fc receptors. In addition, a second, minor subpopulation of less mature cells that bear only mu-chain and Fc receptors also appears to be reactive to 8MGuo. The existence of this immature, reactive B cell subset was confirmed by observation of 8MGuo responsiveness in lymphocytes from 4-day-old mice whose cells do not yet exhibit these later-appearing markers. Accessory cells appear to play a minimal, if any, role in the 8MGuo response. These results establish two distinct B cell subpopulations as the major and minor cellular targets of C8-derivatized nucleosides, and suggest that the activation process results from a direct interaction between the nucleoside and target cell.     

Stable expression of a recombinant sodium-dependent, pyrimidine-selective nucleoside transporter (CNT1) in a transport-deficient mouse leukemia cell line.

Previous studies of nucleoside transport in mammalian cells have identified two types of activities: the equilibrative nucleoside transporters and concentrative, Na+-nucleoside cotransporters. Characterization of the concentrative nucleoside transporters has been hampered by the presence in most cells and tissues of multiple transporters with overlapping permeant specificities. With the recent cloning of cDNAs encoding rat and human members of the concentrative nucleoside transporter (CNT) family, it is now possible to study the concentrative transporters in isolation by use of functional expression systems. We report here the isolation of a nucleoside transport-deficient subline of L1210 mouse leukemia (L1210/DNC3) that is a suitable recipient for stable expression of cloned nucleoside transporter cDNAs. We have used L1210/DNC3 as the recipient in gene transfer studies to develop a stable cell line (L1210/DU5) that produces the recombinant concentrative nucleoside transporter with selectivity for pyrimidine nucleosides (CNT1) that was initially identified in rat intestine (Q.Q. Huang, S.Y. Yao, M.W. Ritzel, A.R.P. Paterson, C.E. Cass, and J.D. Young. 1994. J. Biol. Chem. 269: 17,757-17,760). L1210/DU5 was used to examine the permeant selectivity of recombinant rat CNT1 by comparing a series of nucleoside analogs with respect to (i) inhibition of inward fluxes of [3H]thymidine, (ii) initial rates of transport of 3H-analog, and (iii) cytotoxicity to L1210/DU5 versus the parental transport-deficient cell line. By all three criteria, recombinant CNT1 transported 5-fluoro-2'-deoxyuridine and 5-fluorouridine well and cytosine arabinoside poorly. Although some purine nucleosides (2'-deoxyadenosinedeoxyadeno-2'-deoxyadenosine, 7-deazaadenosine) were potent inhibitors of CNT1, they were poor permeants when uptake was measured directly by analysis of isotopic fluxes or indirectly by comparison of cytotoxicity ratios. We conclude that comparison of analog cytotoxicity to L1210/DU5 versus L1210/DNC3 is a reliable indirect predictor of transportability, suggesting that cytotoxicity assays with a panel of such cell lines, each with a different recombinant nucleoside transporter, would be a valuable tool in the development of antiviral and antitumor nucleoside analogs.     

Identification of a novel point mutation in ENT1 that confers resistance to Ara-C in human T cell leukemia CCRF-CEM cells

The genetic basis for the Ara-C resistance of CCRF-CEM Ara-C/8C leukemia cells was investigated. DNA sequencing revealed that these cells expressed an equilibrative nucleoside transporter 1 (ENT1) with a single missense mutation resulting in glycine to arginine replacement (G24R). To test the importance of this residue, additional G24 mutants were created and examined for [3H]-uridine and [3H]-Ara-C uptake. Both a G24E and G24A mutant showed reduced ENT1-dependent activity. An EGFP-tagged G24R ENT1 displayed plasma membrane localization even though it was unable to bind [3H]-NBMPR, an ENT1-specific inhibitor. These results define G24 as critical amino acid for ENT1 nucleoside uptake and suggest that mutations in TM1 may provide a mechanism for Ara-C resistance in CCRF-CEM Ara-C/8C cells.     

Comparative effects of various classes of mouse interferons on macrophage activation for tumor cell killing

The effects of mouse interferon-alpha (MuIFN-alpha), -beta (MuIFN-beta), and -gamma (MuIFN-gamma) on macrophage activation for tumor cell killing were determined by using proteose peptone-elicited peritoneal macrophages from C3H/HeN and C3H/HeJ mice under conditions that either included or were free of detectable endotoxin. Alone, under the conditions used, none of the interferons was able to activate macrophages directly for tumor cell killing. However, with a second signal provided to responsive macrophages by contaminating endotoxin, added bacterial lipopolysaccharide (LPS), or heat-killed Listeria monocytogenes (HKLM), all three types of interferon induced cytolytic activity, with MuIFN-gamma approximately 500 to 1000-fold more active than either MuIFN-alpha or -beta. Thus, all three interferons were able to prime macrophages for killing but required a second signal before cytolytic activity could be expressed. When MuIFN-gamma was mixed with either MuIFN-alpha or -beta and placed on macrophages, little or no killing developed. Mixtures of MuIFN-gamma with either MuIFN-alpha or -beta did increase the sensitivity of macrophages to triggering by LPS, however, compared with macrophages treated with MuIFN-gamma alone. The results are collectively important because they i) confirm that significant quantitative differences exist between the various interferons with regard to their capacity to prime macrophages for tumor cell killing; ii) indicate that to be an efficient activator each type of interferon must be combined with a second stimulus, such as LPS or HKLM; iii) show that neither MuIFN-alpha nor -beta can provide an efficient second triggering signal for macrophages that are primed by MuIFN-gamma; and iv) document that mixtures of MuIFN-gamma with either MuIFN-alpha or -beta are most efficient at inducing priming, compared with any one of the interferons used alone.     

Purine uptake and release in rat C6 glioma cells: nucleoside transport and purine metabolism under ATP-depleting conditions

Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.     

Biosynthetic human GM-CSF modulates the number and affinity of neutrophil f-Met-Leu-Phe receptors

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) modulates the function of mature neutrophils by priming for enhanced chemotaxis and oxidative metabolism in response to N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). Our studies establish a relationship between f-Met-Leu-Phe receptor number and affinity and neutrophil chemotaxis and oxidative metabolism. A brief (5- to 15-min) exposure to physiologic concentrations of GM-CSF (10 pM to 100 pM) enhances f-Met-Leu-Phe-induced neutrophil chemotaxis by 85%, correlating with a rapid threefold increase (46,000/cell to 150,000/cell) in high-affinity neutrophil f-Met-Leu-Phe receptors. More prolonged incubation (1 to 2 hr) of neutrophils with GM-CSF is accompanied by a change to low-affinity f-Met-Leu-Phe receptors (Kd = 29 nM to Kd = 99 nM) concomitant with priming for enhanced neutrophil oxidative metabolism. Moreover, enhanced chemotactic responses to f-Met-Leu-Phe are no longer evident after more prolonged incubation of neutrophils with GM-CSF. These results show that a single lymphokine (GM-CSF) induces sequential changes in neutrophil f-Met-Leu-Phe receptor number and affinity that may enhance different physiologic responses.     

Functional expression and characterization of a sodium-dependent nucleoside transporter hCNT2 cloned from human duodenum

We have cloned and functionally expressed a sodium-dependent human nucleoside transporter, hCNT2, from a CNS cancer cell line U251. Our cDNA clone of hCNT2 had the same predicted amino acid sequence as the previously cloned hCNT2 transporter. Of the several cell lines studied, the best hCNT2 transport function was obtained when transiently expressed in U251 cells. Na(+)-dependent uptake of [3H]inosine in U251 cells transiently expressing hCNT2 was 50-fold greater than that in non-transfected cells, and uptake in Na(+)-containing medium was approximately 30-fold higher than that at Na(+)-free condition. The hCNT2 displayed saturable uptake of [3H]inosine with K(m) of 12.8 microM and V(max) of 6.66 pmol/mg protein/5 min. Uptake of [3H]inosine was significantly inhibited by the purine nucleoside drugs dideoxyinosine and cladribine, but not by acyclic nucleosides including acyclovir, ganciclovir, and their prodrugs valacyclovir and valganciclovir. This indicates that the closed ribose ring is important for binding of nucleoside drugs to hCNT2. Among several pyrimidine nucleosides, hCNT2 favorably interacted with the uridine analogue floxuridine. Interestingly, we found that benzimidazole analogues, including maribavir, 5,6-dichloro-2-bromo-1-beta-D-ribofuranosylbenzimidazole (BDCRB), and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), were strong inhibitors of inosine transport, even though they have a significantly different heterocycle structure compared to a typical purine ring. As measured by GeneChip arrays, mRNA expression of hCNT2 in human duodenum was 15-fold greater than that of hCNT1 or hENT2. Further, the rCNT2 expression in rat duodenum was 20-fold higher than rCNT1, rENT1 or rENT2. This suggests that hCNT2 (and rCNT2) may have a significant role in uptake of nucleoside drugs from the intestine and is a potential transporter target for the development of nucleoside and nucleoside-mimetic drugs.     

Cognitive processes and dynamic time structures. Introduction of a measuring instrument for studying the Micro-Vigilance-Shift-(MVS) hypothesis]

The psychophysiological measuring method for the determination of the auditory order threshold (OT) is steadily gaining in importance, both for the diagnosis and treatment of speech disorders. Observed intraindividual variability of (macro-) vigilance led to the hypothesis of discontinuous cognitive processing in the central nervous system. The base for the variability of microvigilance is hypothesized to be in the phase difference of the alpha rhythm. To test for this hypothesis, we developed an EEG-comparator, which allows for a phase-dependent triggering of external stimuli. In direct comparison with stochastic (i.e. non-phase-dependent) stimulus presentation, the threshold in phase-dependent OT-testing is distinctly lower. Optimal results occurred at phase angels of phi = 90 degrees and phi = 270 degrees. Our findings support the hypothesis of a correlation between alpha rhythm and vigilance processes. Furthermore, there seems to be evidence that memory processes go with changes in vigilance, and in this context the alpha phase correlation seems to be important.     

Uptake properties of lamivudine (3TC) by a continuous renal epithelial cell line

The purpose of this study was to characterize the renal uptake properties of the cytidine analog and antiretroviral agent 3TC. The uptake of radiolabelled 3TC was measured at 37 degrees C in a continuous porcine renal epithelial cell line (i.e., LLC-PK1 cells) grown as a monolayer on an impermeable support. 3TC (5 microM) uptake (37 degrees C) by the monolayer cells was saturable (Km = 1.2 +/- 0.2 mM) but not significantly altered by various dideoxynucleoside analog drugs, nucleosides, and nucleoside transport inhibitors, suggesting that a nucleoside transporter is not involved in 3TC uptake. A number of endogenous organic cation probes and inhibitors significantly reduced 3TC uptake by the monolayer cells. Quinine, trimethoprim (TMP), and tetraethylammonium (TEA) inhibited 3TC uptake in a dose dependent manner with IC50 values of 0.6 mM, 0.63 mM, and 1.9 mM, respectively. In turn, the uptake of the typical organic cation substrate TEA was inhibited by high concentrations of 3TC. An outwardly directed proton gradient significantly increased the uptake of 3TC by the monolayer cells, suggesting the involvement of a proton exchange process. Conversely, in the presence of monensin, a Na+/H+ ionophore, the uptake of 3TC was significantly reduced. These results suggest that the uptake of 3TC by a cultured renal epithelium may be mediated by an organic cation-proton exchanger. The observed clinical interaction between 3TC and trimethoprim may be explained by competition for a common renal organic cation tubular transporter.     

Mechanism and regulation of neutrophil priming by platelet-activating factor

Although a weak direct stimulus of superoxide anion (O₂^?) production, platelet-activating factor (PAF) markedly enhances responses to chemotactic peptides (such as n-formyl-met-leu-phe, FMLP) and phorbol esters (such as phorbol myristate acetate, PMA) in human neutrophils. The mechanism of priming was explored first through inhibition of steps in the signal transduction pathway at and following PAF receptor occupation. Priming was not altered by pertussis toxin or intracellular calcium chelation, but the PAF receptor antagonist WEB 2086 and the protein kinase C (PKC) inhibitors sphinganine and staurosporine significantly inhibited the primed response. In order to study the regulation of PAF priming, the effect of PAF alone was desensitized by exposure to escalating doses of PAF prior to exposure to the secondary stimuli. The priming effect of PAF was not desensitized under these conditions. The role of PKC in desensitization was also studied. Prior exposure to PAF also desensitized the increase in membrane PKC activity evoked by a single concentration of PAF. However, when the PAF response was desensitized, PKC priming of the response to FMLP or PMA still occurred, suggesting that PKC activity may play a role in the maintenance of the primed state despite PAF desensitization. These data suggest that: (1) PAF priming is receptor- and PKC-mediated but is independent of pertussis toxin-inhibitable G-proteins or intracellular calcium, (2) during migration in vivo, neutrophils may be desensitized to the direct effects of PAF but maintain the capacity for enhanced responses to other stimuli, (3) desensitization of PAF-induced particulate PKC activity also occurs, but PAF primes PKC activity despite PAF desensitization, and (4) distinct mechanisms govern the direct and priming effects of PAF on oxidative metabolism. © 1993 Wiley-Liss, Inc.