Comparative sialomics between hard and soft ticks: implications for the evolution of blood-feeding behavior

Ticks evolved various mechanisms to modulate their host's hemostatic and immune defenses. Differences in the anti-hemostatic repertoires suggest that hard and soft ticks evolved anti-hemostatic mechanisms independently, but raise questions on the conservation of salivary gland proteins in the ancestral tick lineage. To address this issue, the sialome (salivary gland secretory proteome) from the soft tick, Argas monolakensis, was determined by proteomic analysis and cDNA library construction of salivary glands from fed and unfed adult female ticks. The sialome is composed of approximately 130 secretory proteins of which the most abundant protein folds are the lipocalin, BTSP, BPTI and metalloprotease families which also comprise the most abundant proteins found in the salivary glands. Comparative analysis indicates that the major protein families are conserved in hard and soft ticks. Phylogenetic analysis shows, however, that most gene duplications are lineage specific, indicating that the protein families analyzed possibly evolved most of their functions after divergence of the two major tick families. In conclusion, the ancestral tick may have possessed a simple (few members for each family), but diverse (many different protein families) salivary gland protein domain repertoire.     

Characterization of anti-hemostatic factors in the argasid, Argas monolakensis: implications for the evolution of blood-feeding in the soft tick family

To date, the only anti-hemostatic factors characterized for softs ticks are for Ornithodoros moubata and Ornithodoros savignyi, ticks that feeds mainly on mammals. This includes thrombin (ornithodorin and savignin), fXa (TAP and fXaI) and platelet aggregation (disagegin and savignygrin) inhibitors that belong to the BPTI-Kunitz protein family. This raises the question on how well anti-hemostatic factors will be conserved in other soft tick genera that feeds on other vertebrates such as birds. We characterized the anti-hemostatic factors from Argas monolakensis, a soft tick that feeds mainly on Californian gulls. The main anti-clotting factor (monobin) is an ortholog of ornithodorin and savignin and shows similar slow tight-binding kinetics. The main anti-platelet activities are apyrase and fibrinogen receptor antagonists (monogrins). The monogrins are orthologs of disagregin and savignygrin and like savignygrin presents the RGD integrin-recognition motif on the BPTI substrate-binding presenting loop. This implies that the anti-hemostatic factors evolved in the ancestral soft tick lineage and has been maintained in soft tick species from two distinct genera with different host preferences. The Argas derived anti-hemostatic factors bind to mammalian targets with affinities similar to that observed for their orthologs in the Ornithodoros genus. This cross-reactivity could have facilitated the switching of soft ticks from avian to mammalian hosts and can explain in part the ability of Argas ticks, to feed on humans, thereby remaining a possible health risk.     

Nuttalliella namaqua: a living fossil and closest relative to the ancestral tick lineage: implications for the evolution of blood-feeding in ticks

Ticks are monophyletic and composed of the hard (Ixodidae) and soft (Argasidae) tick families, as well as the Nuttalliellidae, a family with a single species, Nuttalliella namaqua. Significant biological differences in lifestyle strategies for hard and soft ticks suggest that various blood-feeding adaptations occurred after their divergence. The phylogenetic relationships between the tick families have not yet been resolved due to the lack of molecular data for N. namaqua. This tick possesses a pseudo-scutum and apical gnathostoma as observed for ixodids, has a leathery cuticle similar to argasids and has been considered the evolutionary missing link between the two families. Little knowledge exists with regard to its feeding biology or host preferences. Data on its biology and systematic relationship to the other tick families could therefore be crucial in understanding the evolution of blood-feeding behaviour in ticks. Live specimens were collected and blood meal analysis showed the presence of DNA for girdled lizards from the Cordylid family. Feeding of ticks on lizards showed that engorgement occurred rapidly, similar to argasids, but that blood meal concentration occurs via malpighian excretion of water. Phylogenetic analysis of the 18S nuclear and 16S mitochondrial genes indicate that N. namaqua grouped basal to the main tick families. The data supports the monophyly of all tick families and suggests the evolution of argasid-like blood-feeding behaviour in the ancestral tick lineage. Based on the data and considerations from literature we propose an origin for ticks in the Karoo basin of Gondwanaland during the late Permian. The nuttalliellid family almost became extinct during the End Permian event, leaving N. namaqua as the closest living relative to the ancestral tick lineage and the evolutionary missing link between the tick families.     

A leukotriene receptor antagonist modulates iNOS in the lung and in a leukotriene-free cell model.

Nitric oxide (NO), an important cell signaling molecule, is considered a marker of inflammatory response and is elevated in asthmatics. This study investigated the effects of montelukast (a leukotriene receptor antagonist) on iNOS expression and activity in a Brown Norway (BN) rat allergic inflammation model and in L2 lung epithelial cells. Allergic inflammation was induced by ovalbumin (OVA) injection in BN rats followed by treatment with either montelukast or dexamethasone (DX). Allergen inhalation was performed, and post-allergen Penh was measured 5 min after the challenge. Cysteinyl leukotriene levels were measured in bronchoalveolar lavage (BAL) fluid and lung iNOS expression and activity determined. These parameters were also measured in cytokine stimulated L2 lung epithelial cells. iNOS expression was significantly higher in OVA challenged rats compared to the naive, DX, and montelukast treated groups, as confirmed by immunohistochemistry and Western blot analysis. However, no significant differences in NOS activity were found. Cysteinyl leukotriene measured in BAL was significantly higher in all OVA challenged rats compared to naive controls. Incubation of L2 cells with a mixture of interferon gamma (IFNgamma), lipopolysaccharide (LPS), and tumor necrosis factor (TNFalpha) resulted in high levels of nitrite formation resulting from iNOS induction. Treatment of cytokine stimulated cells with DX or montelukast significantly decreased iNOS expression and activity. No detectable cysteinyl leukotrienes were found in the supernatant fluid of L2 cells. This study confirms the ability of montelukast to modulate iNOS function and raises the possibility that changes in iNOS expression and activity may occur via pathways independent of cysteinyl leukotrienes.     

Adaptation of ticks to a blood-feeding environment: evolution from a functional perspective

Ticks had to adapt to an existing and complex vertebrate hemostatic system from being free-living scavengers. A large array of anti-hemostatic mechanisms evolved during this process and includes blood coagulation as well as platelet aggregation inhibitors. Several questions regarding tick evolution exist. What was the nature of the ancestral tick? When did ticks evolve blood-feeding capabilities? How did these capabilities evolve? Did host specificity influence the adaptation of ticks to a blood-feeding environment? What are the implications of tick evolution for future research into tick biology and vaccine development? We investigate these questions in the light of recent research into protein superfamilies from tick saliva. Our conclusions are that the main tick families adapted independently to a blood-feeding environment. This is supported by major differences observed in all processes involved with blood-feeding for hard and soft ticks. Gene duplication events played a major role in the evolution of novel protein functions involved in tick-host interactions. This occurred during the late Cretaceous and was stimulated by the radiation of birds and placental mammals, which provided numerous new niches for ticks to adapt to a new lifestyle. Independent adaptation of the main tick families to a blood-feeding environment has several implications for future tick research in terms of tick genome projects and vaccine development.     

Metabolism and analysis of cysteinyl leukotrienes in the monkey

Predominant hepatobiliary elimination from blood and subsequent enterohepatic circulation of cysteinyl leukotrienes is demonstrated in the monkey Macaca fascicularis. From intravenous [3H]leukotriene C4, about 40% were recovered as metabolites in bile and about 20% in urine within 5 h. [3H]Leukotriene E4 was a predominant metabolite of defined structure in blood plasma, bile, and urine. From intraduodenal [3H]leukotriene C4, about 5% were recovered as metabolites in bile and about 8% in urine within 8 h. Endogenous cysteinyl leukotrienes generated in vivo were measured after implantation of a subcutaneously looped biliary bypass. Tapping of the loop allowed access to bile and prevented interference by leukotrienes produced by surgical trauma (Denzlinger, C., Rapp, S., Hagmann, W., and Keppler, D. (1985) Science 230, 330-332). Endogenous cysteinyl leukotrienes were analyzed in bile, urine, and blood plasma by the sequential use of high-performance liquid chromatography and a radioimmunoassay that was optimized for leukotriene E4 as a predominant metabolite detected in the tracer studies. Biliary leukotriene E4 rose from less than 0.2 to 9 nmol/liter, when leukotriene synthesis was elicited in anesthesized monkeys by staphylococcal enterotoxin B administered intragastrically. This study provides an approach to the analysis of cysteinyl leukotrienes in primates and serves to define the role of these mediators under pathophysiological as well as physiological conditions in vivo.     

The Mitochondrial Genomes of Nuttalliella namaqua (Ixodoidea: Nuttalliellidae) and Argas africolumbae (Ixodoidae: Argasidae): Estimation of Divergence Dates for the Major Tick Lineages and Reconstruction of Ancestral Blood-Feeding Characters

Ixodida are composed of hard (Ixodidae), soft (Argasidae) and the monotypic Nuttalliellidae (Nuttalliella namaqua) tick families. Nuclear 18S rRNA analysis suggested that N. namaqua was the closest extant relative to the last common ancestral tick lineage. The mitochondrial genomes of N. namaqua and Argas africolumbae were determined using next generation sequencing and de novo assembly to investigate this further. The latter was included since previous estimates on the divergence times of argasids lacked data for this major genus. Mitochondrial gene order for both was identical to that of the Argasidae and Prostriata. Bayesian analysis of the COI, Cytb, ND1, ND2 and ND4 genes confirmed the monophyly of ticks, the basal position of N. namaqua to the other tick families and the accepted systematic relationships of the other tick genera. Molecular clock estimates were derived for the divergence of the major tick lineages and supported previous estimates on the origins of ticks in the Carboniferous. N. namaqua larvae fed successfully on lizards and mice in a prolonged manner similar to many argasids and all ixodids. Excess blood meal-derived water was secreted via the salivary glands, similar to ixodids. We propose that this prolonged larval feeding style eventually gave rise to the long feeding periods that typify the single larval, nymphal and adult stages of ixodid ticks and the associated secretion of water via the salivary glands. Ancestral reconstruction of characters involved in blood-feeding indicates that most of the characteristics unique to either hard or soft tick families were present in the ancestral tick lineage.     

Leukotriene-induced contraction is mediated by cysteinyl leukotriene receptor CysLT1 in guinea pig fundus but by CysLT1 and CysLT2 in antrum

AimsLeukotriene D₄ (LTD₄) causes contraction of the stomach through unclear receptors. The aim of the present study is to characterize the cysteinyl leukotriene receptor (CysLT) mediating leukotriene-induced muscle contraction in the stomach.Main methodsWe measured contraction of gastric muscle strips isolated from the guinea pig fundus and antrum caused by cysteinyl leukotrienes, including LTC₄, LTD₄ and LTE₄, as well as the dihydroxy leukotriene LTB₄ in vitro.Key findingsIn both fundic and antral muscle strips, LTC₄ and LTD₄ caused marked whereas LTE₄ caused moderate, concentration-dependent contractions. In contrast, LTB₄ caused only small contraction. The relative potencies for cysteinyl leukotrienes to cause contraction in both fundus and antrum were LTC₄ = LTD₄ > LTE₄. The LTD₄-induced contraction was not affected by tetrodotoxin or atropine, suggesting that the action is not neurally mediated. The LTD₄-induced contraction in the fundus was almost abolished by the CysLT₁ selective antagonist montelukast. In contrast, the LTD₄-induced contraction in the antrum was only partially inhibited by montelukast or the dual CysLT₁ and CysLT₂ antagonist BAY u9773. This antral contraction was almost abolished by the combination of montelukast and BAY u9773, indicating enhancement of inhibition.SignificanceThe results of the present study demonstrate that cysteinyl leukotrienes LTC₄, LTD₄ and LTE₄ cause moderate to marked whereas the dihydroxy leukotriene LTB₄ causes small muscle contraction in the stomach in vitro. The leukotriene-induced contraction is mediated by CysLT₁ in fundus but by CysLT₁ and CysLT₂ in antrum.     

Function, mechanism and evolution of the moubatin-clade of soft tick lipocalins

The "moubatin-clade" of soft tick lipocalins, although monophyletic, shows clear signs of paralogy as indicated by the various functions associated with this protein family. This includes anti-platelet (moubatin), anti-complement (OMCI) and toxic (TSGP2) activities in the vertebrate host. In order to understand the evolution of function and how it relates to the various paralogs in this clade, we characterized a number of different proteins in regard to undefined function and mechanism. By utilizing gain-of-function for TSGP2 and loss-of-function for TSGP3, we show that inhibition of collagen-induced platelet aggregation by moubatin and TSGP3 is due to scavenging of thromboxane A(2). Moubatin, TSGP2 and TSGP3 are also able to bind leukotriene B(4) with high affinity. TSGP2 and TSGP3, but not moubatin, binds complement C5, with kinetics that indicates that conformation change occurs during interaction. A conserved loop and histidine residue in the sequences of OMCI, TSGP2 and TSGP3 are implicated in the interaction with complement C5. The data presented suggest that the ancestral function evolved in this clade was aimed at inhibition of vasoconstriction, platelet aggregation and neutrophil aggregation, primarily by scavenging of thromboxane A(2) and leukotriene B(4). C5 complement targeting activity evolved within this clade, probably within the Old World Ornithodorinae. The moubatin-clade itself most probably derived from the related histamine and serotonin-binding lipocalin sub-family that is conserved within the Argasidae.     

Regulation of the eicosanoid pathway by tumour necrosis factor alpha and leukotriene D4 in intestinal epithelial cells

In this study the mRNA and protein levels of the key enzymes involved in eicosanoid biosynthesis and the cysteinyl leukotriene receptors (CysLT₁R and CysLT₂R) have been analysed in non-transformed intestinal epithelial and colon cancer cell lines. Our results revealed that tumour necrosis factor alpha (TNF-α), and leukotriene D₄ (LTD₄), which are inflammatory mediators implicated in carcinogenesis, stimulated an increase of cyclooxygenase-2 (COX-2), in non-transformed epithelial cells, and 5-lipoxygenase (5-LO) in both non-transformed and cancer cell lines. Furthermore, these mediators also stimulated an up-regulation of LTC₄ synthase in cancer cells as well as non-transformed cells. We also observed an endogenous production of CysLTs in these cells. TNF-α and LTD₄, to a lesser extent, up-regulate the CysLT₁R levels. Interestingly, TNF-α also reduced CysLT₂R expression in cancer cells. Our results demonstrate that inflammatory mediators can cause intestinal epithelial cells to up-regulate the expression of enzymes needed for the biosynthesis of eicosanoids, including the cysteinyl leukotrienes, as well as the signal transducing proteins, the CysLT receptors, thus providing important mechanisms for both maintaining inflammation and for tumour progression.     

Human endothelial cells stimulate leukotriene synthesis and convert granulocyte released leukotriene A4 into leukotrienes B4, C4, D4 and E4

Incubation of human endothelial cells with leukotriene A4 resulted in the formation of leukotrienes B4, C4, D4 and E4. Endothelial cells did not produce leukotrienes after stimulation with the ionophore A23187 and/or exogenously added arachidonic acid. However, incubation of polymorphonuclear leukocytes with ionophore A23187 together with endothelial cells led to an increased synthesis of cysteinyl-containing leukotrienes (364%, mean, n = 11) and leukotriene B4 (52%) as compared to leukocytes alone. Thus, the major part of leukotriene C4 recovered in mixed cultures was attributable to the presence of endothelial cells. Similar incubations of leukocytes with fibroblasts or smooth muscle cells did not cause an increased formation of leukotriene C4 or leukotriene B4. The increased biosynthesis of cysteinyl-containing leukotrienes and leukotriene B4 in coincubation of leukocytes and endothelial cells appeared to be caused by two independent mechanisms. First, cell interactions resulted in an increased production of the total amount of leukotrienes, suggesting a stimulation of the leukocyte 5-lipoxygenase pathway, induced by a factor contributed by endothelial cells. Secondly, when endothelial cells prelabeled with [35S]cysteine were incubated with either polymorphonuclear leukocytes and A23187, or synthetic leukotriene A4, the specific activity of the isolated cysteinyl-containing leukotrienes were similar. Thus, transfer of leukotriene A4 from stimulated leukocytes to endothelial cells appeared to be an important mechanism causing an increased formation of cysteinyl-containing leukotrienes in mixed cultures of leukocytes and endothelial cells. In conclusion, the present study indicates that the vascular endothelium, when interacting with activated leukocytes, modulates both the quantity and profile of liberated leukotrienes.     

The role of leukotrienes in the pathogenesis of systemic sclerosis

Systemic sclerosis (SSc, scleroderma) is an autoimmune disease characterized by widespread vascular injury and progressive fibrosis of the skin and internal organs. SSc-related involvement of the lungs, heart, kidneys and/or the gastrointestinal system accounts for the increased mortality of scleroderma patients. Despite the progress which has recently been made in this field, the treatment of SSc is still unsatisfactory due to the low efficacy and/or high toxicity of available therapies. Leukotrienes are a family of lipid mediators synthesized from arachidonic acid in a process mediated by 5-lipoxygenase; they include leukotriene B4 and a group of cysteinyl leukotrienes: C4, D4, and E4. Leukotrienes play an important role in the regulation of all the processes vital to the pathogenesis of SSc, namely inflammation, vascular function and connective tissue remodeling. The available data suggests that an excessive synthesis of leukotrienes may contribute to the development and progression of SSc. Accordingly, blockade of leukotriene pathways appears to be a new, promising target for the treatment of SSc.     

Regulation of the eicosanoid pathway by tumour necrosis factor alpha and leukotriene D4 in intestinal epithelial cells

In this study the mRNA and protein levels of the key enzymes involved in eicosanoid biosynthesis and the cysteinyl leukotriene receptors (CysLT₁R and CysLT₂R) have been analysed in non-transformed intestinal epithelial and colon cancer cell lines. Our results revealed that tumour necrosis factor alpha (TNF-α), and leukotriene D₄ (LTD₄), which are inflammatory mediators implicated in carcinogenesis, stimulated an increase of cyclooxygenase-2 (COX-2), in non-transformed epithelial cells, and 5-lipoxygenase (5-LO) in both non-transformed and cancer cell lines. Furthermore, these mediators also stimulated an up-regulation of LTC₄ synthase in cancer cells as well as non-transformed cells. We also observed an endogenous production of CysLTs in these cells. TNF-α and LTD₄, to a lesser extent, up-regulate the CysLT₁R levels. Interestingly, TNF-α also reduced CysLT₂R expression in cancer cells. Our results demonstrate that inflammatory mediators can cause intestinal epithelial cells to up-regulate the expression of enzymes needed for the biosynthesis of eicosanoids, including the cysteinyl leukotrienes, as well as the signal transducing proteins, the CysLT receptors, thus providing important mechanisms for both maintaining inflammation and for tumour progression.     

Cysteinyl leukotrienes mediate histamine hypersensitivity ex vivo by increasing histamine receptor numbers

BACKGROUND: Hyperresponsiveness to histamine is a key feature of a variety of pathological conditions, including bronchial asthma, food allergy, colitis ulcerosa, and topical allergic disorders. Cells isolated from hyperresponsive individuals do not display exaggerated histamine responses ex vivo and thus the molecular mechanisms underlying histamine responsiveness remain obscure. Importantly, several in vivo observations implicate cysteinyl leukotrienes as possible mediators of increased histamine responses. We decided to investigate whether cysteinyl leukotrienes enhance the cellular reaction to histamine in cell types involved in pathological and immunological histamine hyperresponsiveness, as this might provide an in vitro system for studying histamine responsiveness and could shed light on the underlying molecular mechanisms. MATERIALS AND METHODS: Histamine responsiveness was determined by measuring histamine-induced prostaglandin E(2) production. Scatchard analysis was performed to determine the number of histamine H(1) receptors. Mouse macrophages, primary isolated human peripheral blood monocytes, and human umbilical smooth muscle cells were investigated before and after cysteinyl leukotriene stimulation. Results: In all three cell types tested, cysteinyl leukotrienes instantaneously enhanced histamine-induced prostaglandin E(2) production. This increase in prostaglandin E(2) production coincided with the immediate and transient appearance of additional H(1) receptors on the plasma membrane. CONCLUSIONS: Cysteinyl leukotrienes prime histamine responses by recruiting additional histamine receptors in immunologically relevant cell types in vitro.     

Evolution of hematophagy in ticks: common origins for blood coagulation and platelet aggregation inhibitors from soft ticks of the genus Ornithodoros

Identification and characterization of antihemostatic components from hematophagous organisms are useful for the elucidation of the evolutionary mechanisms involved in adaptation to a highly complex host hemostatic system. Although many bioactive components involved in the regulation of the host's hemostatic system have been described, the evolutionary mechanisms of how arthropods adapted to a blood-feeding environment have not been elucidated. This study describes common origins of both blood coagulation inhibitors and platelet aggregation inhibitors (PAIs) from soft ticks of the genus Ornithodoros. Neighbor-joining analysis indicates that fXa, thrombin, and PAIs share a common ancestor. Maximum parsimony analysis and a phylogeny based on root mean square deviation values of alpha-carbon backbone structures suggest a novel evolutionary pathway by which different antihemostatic functions have evolved through a series of paralogous gene duplication events. In this scenario, the thrombin inhibitors preceded the fXa and PAIs. This evolutionary model explains why the tick serine protease inhibitors have inhibition mechanisms that differ from that of the canonical bovine pancreatic trypsin inhibitor (BPTI)-like inhibitors. Higher nonsynonymous-to-synonymous substitution rates indicate positive Darwinian selection for the fXa and PAIs. Comparison with hemostatic inhibitors of hard ticks suggests that the two main tick families have independently evolved novel antihemostatic mechanisms. Independent evolution of these mechanisms in ticks points to a rapid divergence between tick families that could be dated between 120 and 92 MYA. This coincides with current molecular phylogeny views on the early divergence of modern birds and placental mammals in the Late Cretaceous, which suggests that this event might have been a driving force in the evolution of hematophagy in ticks.     

Inhibition of leukotriene A4 hydrolase/aminopeptidase by captopril

Captopril ((2S)-1-(3-mercapto-2-methyl-propionyl)-L-proline) inhibited the bifunctional, Zn(2+)-containing enzyme leukotriene A4 hydrolase/aminopeptidase reversibly and competitively with Ki = 6.0 microM for leukotriene B4 formation and Ki = 60 nM for L-lysine-p-nitroanilide hydrolysis at pH 8. Inhibition was independent of pH between pH 7 and 8, the optimum range for each catalytic activity. Half-maximal inhibition of leukotriene B4 formation by intact erythrocytes and neutrophils required 50 and 88 microM captopril, respectively. In neutrophils and platelets neither 5(S)-hydroxyeicosatetraenoic acid, 12(S)-hydroxyeicosatetraenoic acid, nor leukotriene C4 formation were reduced, indicating selective inhibition of leukotriene A4 hydrolase/aminopeptidase, not 5-lipoxygenase, 12-lipoxygenase, or leukotriene C4 synthase. In whole blood, captopril inhibited leukotriene B4 formation with an accompanying redistribution of substrate toward formation of cysteinyl leukotrienes. The decrease in leukotriene B4 was more substantial than the corresponding increase in cysteinyl leukotrienes suggesting that nonenzymatic hydration predominates over transcellular metabolism of leukotriene A4 by platelets during selective inhibition of leukotriene A4 hydrolase. Enalapril dicarboxylic acid and Glu-Trp-Pro-Arg-ProGln-Ile-Pro-Pro which inhibit angiotensin-converting enzyme: angiotensin I, bradykinin, and N-[3-(2-furyl)acryloyl]Phe-Gly-Gly which are substrates; and chloride ions which activate angiotensin-converting enzyme did not modulate leukotriene A4 hydrolase/aminopeptidase activity. The results indicate that: (i) the sulfhydryl group of captopril is an important determinant for inhibition of leukotriene A4 hydrolase/aminopeptidase, probably by binding to an active site Zn2+; (ii) aminopeptidase and leukotriene A4 hydrolase display differential susceptibility to inhibition; (iii) there is minimal functional similarity between angiotensin-converting enzyme (peptidyl dipeptidase) and leukotriene A4 hydrolase/aminopeptidase; (iv) captopril may be a useful prototype to identify more potent and selective leukotriene A4 hydrolase inhibitors.     

Eosinophils and cysteinyl leukotrienes

Eosinophils are the main source of the cysteinyl leukotrienes, LTC(4)/D(4)/E(4), which are lipid mediators that play major roles in the pathogenesis of asthma and other forms of allergic inflammation. Here, we review the mechanisms governing eosinophil LTC(4) synthesis, focusing on the distinct intracellular domains that regulate eicosanoid formation and function within eosinophils. Cysteinyl leukotrienes exert their actions by engaging specific receptors. As recently shown, eosinophils express CysLT1 and CysLT2, the only cloned receptors for cysteinyl leukotrienes. Therefore, here we also present some of the new findings regarding the paracrine/autocrine activation of these CysLT receptors on eosinophils, and discuss some data on novel intracrine effects of LTC(4) triggered by a putative third CysLT receptor expressed intracellularly within eosinophils.     

Leukotriene C4 metabolism by hepatoma cells deficient in the uptake of cysteinyl leukotrienes

Uptake and metabolism of the cysteinyl leukotrienes C4 and E4 (LTC4 and LTE4) were studied in AS-30D hepatoma cell suspensions and compared with rat hepatocytes. The hepatoma cells were deficient in the uptake of [3H]LTC4 and [3H]LTE4 but took up, in control experiments, L-[14C]glutamine and [14C]adenosine in a time-dependent manner. By contrast, isolated hepatocyte suspensions incubated under the same conditions took up [3H]LTC4 and [3H]LTE4 as well as L-[14C]glutamine and [14C]adenosine. The hepatoma cells deficient in the uptake of cysteinyl leukotrienes metabolized extracellular [3H]LTC4 to [3H]LTD4 and to [3H]LTE4. Addition of acivicin, an inhibitor of gamma-glutamyltransferase, largely prevented metabolism of [3H]LTC4 by the hepatoma cells. Sonication of the cells did not enhance the formation of [3H]LTD4 and [3H]LTE4 from [3H]LTC4. We conclude that ectoenzymes of AS-30D hepatoma cells catalyze the conversion of LTC4 to LTE4 via LTD4. As compared to hepatocytes, these neoplastic cells have lost the uptake system for cysteinyl leukotrienes and may serve in studies on leukotriene metabolism by cell-surface enzymes.     

Leukotriene uptake by hepatocytes and hepatoma cells.

The uptake of tritiated cysteinyl leukotrienes (LTC4, LTD4, LTE4) and LTB4 was investigated in freshly isolated rat hepatocytes and different hepatoma cell lines under initial-rate conditions. Leukotriene uptake by hepatocytes was independent of an Na+ gradient and a K+ diffusion potential across the hepatocyte membranes as established in experiments with isolated hepatocytes and plasma membrane vesicles. Kinetic experiments with isolated hepatocytes indicated a low-Km system and a non-saturable system for the uptake of cysteinyl leukotrienes as well as LTB4 under the conditions used. AS-30D hepatoma cells and human Hep G2 hepatoma cells were deficient in the uptake of cysteinyl leukotrienes, but showed significant accumulation of LTB4. Moreover, only LTB4 was metabolized in Hep G2 hepatoma cells. Competition studies on the uptake of LTE4 and LTB4 (10 nM each) indicated inhibition by the organic anions bromosulfophthalein, S-decyl glutathione, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate, probenecid, docosanedioate, and hexadecanedioate (100 microM each), but not by taurocholate, the amphiphilic cations verapamil and N-propyl ajmaline, and the neutral glycoside ouabain. Cholate and the glycoside digitoxin were inhibitors of LTB4 uptake only. Bromosulfophthalein, the strongest inhibitor of leukotriene uptake by hepatocytes, did not inhibit LTB4 uptake by Hep G2 hepatoma cells under the same experimental conditions. Leukotriene-binding proteins were analyzed by comparative photoaffinity labeling of human hepatocytes and Hep G2 hepatoma cells using [3H]LTE4 and [3H]LTB4 as the photolabile ligands. Predominant leukotriene-binding proteins with apparent molecular masses in the ranges of 48-58 kDa and 38-40 kDa were labeled by both leukotrienes in the particulate and in the cytosolic fraction of hepatocytes, respectively. In contrast, no labeling was obtained with [3H]LTE4 in Hep G2 cells. With [3H]LTB4 a protein with a molecular mass of about 48 kDa was predominantly labeled in the particulate fraction of the hepatoma cells, whereas in the cytosolic fraction a labeled protein in the range of 40 kDa was detected. Our results provide evidence for the existence of distinct uptake systems for cysteinyl leukotrienes and LTB4 at the sinusoidal membrane of hepatocytes; however, some of the inhibitors tested interfere with both transport systems. Only LTB4, but not cysteinyl leukotrienes, is taken up and metabolized by the transformed hepatoma cells.