Extramedullary hematopoiesis in a child with hereditary spherocytosis: an uncommon cause of an adrenal mass.

We report a case of extramedullary hematopoiesis presenting as an adrenal mass in a young male with hereditary spherocytosis. The unilateral adrenal mass was discovered during an abdominal ultrasound performed for jaundice. CT and MR imaging were subsequently performed, followed by an excisional biopsy at the time of splenectomy and cholecystectomy. Although extramedullary hematopoiesis is a rare cause of an adrenal mass, the diagnosis must be considered in any patient with a history of a congenital hemolytic disorder such as hereditary spherocytosis. In this regard, the morbidity of an unnecessary procedure may be avoided.

The lung tumor promoter, butylated hydroxytoluene (BHT), causes chronic inflammation in promotion-sensitive BALB/cByJ mice but not in promotion-resistant CXB4 mice.

An inflammatory response accompanies the reversible pneumotoxicity caused by butylated hydroxytoluene (BHT) administration to mice. Lung tumor formation is promoted by BHT administration following an initiating agent in BALB/cByJ mice, but not in CXB4 mice. To assess the contribution of inflammation to this differential susceptibility, we quantitatively characterized inflammation after one 150 mg/kg body weight, followed by three weekly 200 mg/kg ip injections of BHT into male mice of both strains. This examination included inflammatory cell infiltrate and protein contents in bronchoalveolar lavage (BAL) fluid, cyclooxygenase (COX)-1 and COX-2 expression in lung extracts, and PGE(2) and PGI(2) production by isolated bronchiolar Clara cells. BAL macrophage and lymphocyte numbers increased in BALB mice (P<0.0007 and 0.02, respectively), as did BAL protein content (P<0.05), COX-1 and COX-2 expression (P<0.05 for each), and PGI(2) production (P<0.05); conversely, these indices were not perturbed by BHT in CXB4 mice. BALB mice fed aspirin (400 mg/kg of chow) for two weeks prior to BHT treatment had reduced inflammatory cell infiltration. Our results support a hypothesis that resistance to BHT-induced inflammation in CXB4 mice accounts, at least in part, for the lack of effect of BHT on lung tumor multiplicity in this strain.

Electrospray ionization and tandem mass spectrometry of cysteinyl eicosanoids: leukotriene C4 and FOG7.

The cysteinyl leukotrienes, LTC4, LTD4 and LTE4, and the recently described cysteinyl eicosanoid, 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG7) have been analyzed by tandem mass spectrometry. Both [M-H]- and [M+H]+ ions were produced by electrospray ionization and collision-induced dissociation of these molecular ion species were studied using both an ion trap and a triple quadrupole instrument. Product ion spectra obtained were characteristic of the structure of the cysteinyl leukotrienes and mechanisms of ion formation were investigated by using deuterium-labeled analogs. The product ion spectrum obtained following collision-induced dissociation of the [M-H]- anion from FOG7 was devoid of significant structural information and further studies of collision activation of the [M+H]+ spectrum were therefore examined. Positive ion MS3 spectra obtained in the ion trap from the gamma-glutamate cleavage products of FOG7 and its derivative (d7-FOG7) afforded an abundant ion not observed in spectra generated from the cysteinyl leukotrienes. Formation of this fragment ion likely occurred via a McLafferty-type rearrangement to afford cleavage of the C6-C7 bond adjacent to the sulfur atom and was valuable for the identification of the structure of FOG7 and defining the biosynthetic pathway as a 1,4-Michael addition of glutathione to 5-oxo-eicosatetraenoic acid (5-oxo-ETE).

Overexpression of Bcl-2 suppresses the calcium activation of a mitochondrial megachannel.

The molecular mechanism(s) by which Bcl-2 regulates apoptosis is poorly understood. Bcl-2 suppresses apoptosis by inhibiting calcium activation of the permeability transition of mitochondria. In this patch-clamp study, overexpression of Bcl-2 in mitochondria of cultured cells suppressed calcium activation of a high conductance channel that may underlie the permeability transition. All other single channel parameters were identical when multiple conductance channel activities of mitochondria from control and Bcl-2 overexpressing cells were compared. Bcl-2 forms channels in artificial membranes; however, no novel channel activities could be linked to Bcl-2 overexpression, suggesting Bcl-2 does not form channels in native inner membranes of mitochondria.

Synthesis of 5-oxo-6,8,11,14-eicosatetraenoic acid and identification of novel omega-oxidized metabolites in the mouse macrophage.

The metabolism of arachidonic acid by the 5-lipoxygenase pathway not only leads to the formation of leukotrienes but also to the biologically active eicosanoid 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). The synthesis of 5-oxo-ETE was investigated in the elicited peritoneal macrophage and the formation of 5-hydroxyeicosatetraenoic acid (5-HETE) as well as 5-oxo-ETE was quantitated using stable isotope dilution tandem mass spectrometry. The metabolism of 5-oxo-ETE in these same cells led to the formation of a series of novel less lipophilic metabolites oxidized near the methyl terminus that were structurally characterized using electrospray LC/MS and LC/MS/MS. Five novel metabolites of 5-oxo-ETE were identified including 5,18-diHETE, 5,19-diHETE, 5-oxo-19-HETrE, 5-oxo-18-HETrE, and 5,19-diHETrE. These metabolites corresponded to omega-1 and omega-2 oxidation of 5-oxo-ETE presumably formed by a specific cytochrome P450. There was no evidence for the formation of omega-oxidation (20-hydroxy metabolites), which are known products of metabolism of 5-oxo-ETE in other cell types. None of the metabolites were found to elevate intracellular calcium release, suggesting that this metabolic pathway may result in inactivation of 5-oxo-ETE. This is the first report of the biosynthesis of 5-oxo-ETE by tissue resident cell outside of the blood and the formation of novel omega-1 and omega-2 oxidation of this eicosanoid.

A novel glutathione containing eicosanoid (FOG7) chemotactic for human granulocytes.

A biologically active glutathione adduct of the eicosanoid 5-oxo-eicosatetraenoic acid has been observed as a product formed within the murine peritoneal macrophage. This five-oxo glutathione adduct (FOG(7)) was structurally characterized using electrospray tandem mass spectrometry as a 1,4 Michael addition product 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid. FOG(7) was found to be highly potent in stimulating eosinophil as well as neutrophil chemotaxis, also capable of initiating actin polymerization, without elevating intracellular free calcium ion concentration within either the eosinophil or polymorphonuclear leukocyte. These biological responses suggest that either FOG(7) activates a subset of receptors mediating the broader biological activity of the parent eicosanoid 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) or that a receptor not activated by 5-oxo-ETE participates in the chemotactic activity of FOG(7). The only other known biologically active glutathione adduct has been leukotriene C(4) (LTC(4)), another eicosanoid that exerts potent effects through the Cys-LT receptor. The biochemical parallel between the formation of LTC(4) and FOG(7) suggests an interesting mechanism by which biologically active eicosanoids derived from electrophilic intermediates may have unique distribution and prolonged efficacy in vivo.

Covalent binding of LTA(4) to nucleosides and nucleotides.

Leukotriene A(4) (LTA(4)) is a chemically reactive conjugated triene epoxide that is formed by 5-lipoxygenase and is an intermediate in the formation of the biologically active eicosanoids leukotriene B(4) and leukotriene C(4). The present study was undertaken to determine whether or not LTA(4) could serve as an electrophilic species that nucleosides and nucleotides could attack, ultimately resulting in a covalent adduct. Electrospray ionization mass spectrometry and tandem mass spectrometry were used to study the covalent binding of LTA(4) with uridine, cytidine, adenosine, and guanosine. The reaction with guanosine was found to yield five major and at least six minor adduct species. Reversed phase HPLC and mass spectrometric data suggested that the guanosine attacked LTA(4) either at carbon-12 or carbon-6 with opening the epoxide at carbon-5 to yield a series of adducts characterized by the molecular anion [M-H](-) at m/z 600.3. Reactions of LTA(4) with mixtures of nucleosides and nucleotides revealed that guanine-containing nucleosides were the most reactive toward LTA(4). The facility of the reaction of guanine with LTA(4) raises the possibility that this intermediate of leukotriene biosynthesis formed on or near the cellular nuclear envelope may react with nucleosides and nucleotides present in RNA or DNA.

Differential metabolism of exogenous and endogenous arachidonic acid in human neutrophils.

Leukotrienes can be produced by cooperative interactions between cells in which, for example, arachidonate derived from one cell is oxidized to leukotriene A(4) (LTA(4)) by another and this can then be exported for conversion to LTB(4) or cysteinyl leukotrienes (cys-LTs) by yet another. Neutrophils do not contain LTC(4) synthase but are known to cooperate with endothelial cells or platelets (which do have this enzyme) to generate cys-LTs. Stimulation of human neutrophils perfusing isolated rabbit hearts resulted in production of cys-LTs, whereas these were not seen with perfused hearts alone or isolated neutrophils. In addition, the stimulated, neutrophil-perfused hearts generated much greater amounts of total LTA(4) products, suggesting that the hearts were supplying arachidonate to the neutrophils and, in addition, that this externally derived arachidonate was preferentially used for exported LTA(4) that could be metabolized to cys-LTs by the coronary endothelium. Stable isotope-labeled arachidonate and electrospray tandem mass spectrometry were used to differentially follow metabolism of exogenous and endogenous arachidonate. Isolated, adherent neutrophils at low concentrations (to minimize transcellular metabolism between them) were shown to generate higher proportions of nonenzymatic LTA(4) products from exogenous arachidonate (deuterium-labeled) than from endogenous (unlabeled) sources. The endogenous arachidonate, on the other hand, was preferentially used for conversion to LTB(4) by the LTA(4) hydrolase. This result was not because of saturation of the LTA(4) hydrolase, because it occurred at widely differing concentrations of exogenous arachidonate. Finally, in the presence of platelets (which contain LTC(4) synthase), the LTA(4) synthesized from exogenous deuterium-labeled arachidonate was converted to cys-LTs to a greater degree than that from endogenous sources. These experiments suggest that exogenous arachidonate is preferentially converted to LTA(4) for export (not intracellular conversion) and raises the likelihood that there are different intracellular pathways for arachidonate metabolism.

Malignancies in pediatric patients with ataxia telangiectasia.

BACKGROUND: Patients with ataxia telangiectasia (AT), known to have an inherent increased susceptibility to the development of cancer, may present with malignancies that are unusual for the patient's age, are often difficult to diagnose clinically and radiographically and respond poorly to conventional therapy. MATERIALS AND METHODS: We reviewed the clinical presentation and imaging studies of 12 AT patients who developed malignancies. RESULTS: Eight of the twelve patients developed non-Hodgkin's lymphoma (CNS, thorax, bone), two developed Hodgkin's disease, and two were diagnosed with gastrointestinal mucinous adenocarcinoma. CONCLUSION: The lymphomas were commonly extra nodal, and infiltrative rather than mass-like. The recognition of the tumors was often delayed due to confusion with the known infectious complications in AT patients.

Activation of the epidermal platelet-activating factor receptor results in cytokine and cyclooxygenase-2 biosynthesis.

Recent studies suggest that the lipid mediator platelet-activating factor (PAF) is involved in keratinocyte function and skin inflammation. Indeed, PAF is found in association with inflammatory skin diseases, intradermal injections of PAF induce inflammation, and keratinocytes express functional PAF receptors (PAF-R). One mechanism by which the keratinocyte PAF-R could contribute to epidermal functions and inflammatory states would be through the synthesis of inflammatory regulators, such as PAF, PGs, and cytokines. The ability of the epidermal PAF-R to induce the synthesis of these immunomodulators was tested using a model system created by transduction of the PAF-R-negative human epidermal cell line KB with the PAF-R. Activation of this epidermal PAF-R resulted in arachidonic acid release, and the biosynthesis of PAF and PGE2. In addition, the KB PAF-R triggered increased levels of mRNA and protein for the inducible isozyme of cyclooxygenase (COX-2) as well as IL-6 and IL-8, both of which have been implicated in skin inflammatory processes. Studies with the human keratinocyte-derived epidermal cell line HaCaT revealed that activation of the endogenous PAF-R led to the increased accumulation of COX-2, IL-6, and IL-8 mRNA similar to that seen with the KB PAF-R model system. Finally, treatment of HaCaT keratinocytes with IL-8 resulted in PAF biosynthesis, indicating the existence of a positive feedback loop between IL-8 and PAF in epidermal cells. These studies suggest involvement of PAF and the PAF-R in the epidermal cytokine network.

Expression of the platelet-activating factor receptor results in enhanced ultraviolet B radiation-induced apoptosis in a human epidermal cell line.

Recent studies have demonstrated that ultraviolet B radiation (UVB) damages human keratinocytes in part by inducing oxidative stress and cytokine production. Severe UVB damage to the keratinocyte can also result in apoptosis or programmed cell death. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to epidermal cell damage and epidermal cells express PAF receptors, it is not known whether PAF is involved in UVB-induced epidermal cell apoptosis. These studies examined the role of the PAF system in UVB-induced epidermal cell apoptosis using a novel model system created by retroviral-mediated transduction of the PAF receptor-negative human epidermal cell line KB with the human PAF receptor (PAF-R). Expression of the PAF-R in KB cells did not affect base-line growth or apoptosis, yet resulted in a decrease in the lag time between treatment of the cells and the induction of apoptosis following irradiation with 400 J/m2 UVB. This effect was inhibited by pretreatment with the PAF-R antagonists WEB 2086 and A-85783, confirming involvement of the PAF-R in this process. At lower doses (100-200 J/m2) of UVB, only KB cells that expressed the PAF-R became apoptotic. Treatment of PAF-R-expressing KB clones with the metabolically stable PAF-R agonist 1-hexadexyl-2-N-methylcarbamoyl-3-glycerophosphocholine (CPAF) alone did not induce apoptosis but augmented the degree of apoptosis observed if CPAF was used in combination with lower doses (200 J/m2) of UVB irradiation. Interestingly, UVB irradiation was found to stimulate PAF synthesis only in PAF-R-expressing KB cell clones. The antioxidants N-acetyl cysteine, 1,1,3,3-tetramethyl-2-thiourea, and vitamin E inhibited both UVB-induced PAF biosynthesis as well as the augmentation of UVB-induced apoptosis in PAF-R-expressing KB clones, suggesting the possibility that UVB stimulates the production of oxidized lipid species with PAF-R agonistic activity in this model system. Thus, these studies indicate that a component of UVB-induced epidermal cell cytotoxicity can be modulated by PAF-R activation through the production of PAF and PAF-like species.

Structural characterization of the covalent attachment of leukotriene A3 to leukotriene A4 hydrolase.

Leukotriene A4 (LTA4) hydrolase catalyzes the conversion of the unstable epoxide LTA4 [5(S)-trans-5,6-oxido-11,14-cis-eicosatetraenoic acid] into proinflammatory LTB4. During the process of catalyzing this reaction, the enzyme is suicide inactivated by its substrate. In addition, LTA3, and analogue of LTA4 that lacks the C14-C15 double bond, is a potent suicide inhibitor of LTA4 hydrolase. We have synthesized [3H]LTA3 and used this ligand to demonstrate that LTA3 can covalently label LTA4 hydrolase and that this labeling is specifically competed for by bestatin and LTA4. Incubation of recombinant human LTA4 hydrolase with LTA3 followed by proteolysis (endoproteinase Lys-C) resulted in a peptide map with a single modified peptide defining the location of the LTA3 covalent attachment region. This modified 21-amino-acid peptide had a UV absorption spectrum corresponding to a conjugated triene chromophore which established conservation of this structural unit after covalent interaction of LTA3 with LTA4 hydrolase. MALDI-TOF mass spectrometric analysis of the 21-amino-acid peptide adduct revealed an abundant MH+ at m/z 2658, consistent with the predicted nominal mass of the sequenced peptide with the addition of a single LTA3 moiety. Proteolysis of LTA4 hydrolase modified with LTA3 was performed sequentially with endo-Asp-N and endo-Lys-C. The resulting peptide isolated by reverse-phase high-performance liquid chromatography was analyzed by mass spectroscopy revealing two related peptides, D371-K385 (m/z 2018.0) and D375-K385 (m/z 1577.8), both of which retained the elements of LTA3. Postsource decay of m/z 1577.8 resulted in an abundant ion at m/z 536 and an ion of lesser abundance at m/z 856 consistent with cleavage between V381 and P382 that supported assignment of the modified tyrosine residue at Y383. These results suggest nucleophilic attack of a tyrosine residue (Y383) at the conjugated triene epoxide of LTA3 resulting in a triene ether carbinol covalent adduct.

Two high conductance channels of the mitochondrial inner membrane are independent of the human mitochondrial genome.

Patch-clamp techniques were used to characterize the channel activity of mitochondrial inner membranes of two human osteosarcoma cell lines: a mitochondrial genome-deficient (rho0) line and its corresponding parental (rho+) line. Previously, two high conductance channels, mitochondrial Centum picoSiemen (mCS) and multiple conductance channels (MCC), were detected in murine mitochondria. While MCC was assigned to the protein import in yeast mitochondria, the role of mCS is unknown. This study demonstrates that mCs and MCC activities from mouse mitochondria are indistinguishable from those of human mitochondria. The channel activities and their functional expression levels are not altered in cells lacking mtDNA. Hence, rho0 cells may provide a model system for elucidating the role of mitochondrial channels in disease processes and apoptosis.

Identification and pharmacological characterization of platelet-activating factor and related 1-palmitoyl species in human inflammatory blistering diseases.

Through its pro-inflammatory effects on leukocytes, endothelial cells, and keratinocytes, the lipid mediator platelet-activating factor (PAF) has been implicated in cutaneous inflammation. Although the 1-alkyl PAF species has been considered historically the most abundant and important ligand for the PAF receptor (PAF-R), other putative ligands for this receptor have been described including 1-acyl analogs of sn-2 acetyl glycerophosphocholines. Previous bioassays have demonstrated a PAF-like activity in lesions of the autoimmune blistering disease bullous pemphigoid. To assess the actual sn-2 acetyl glycerophosphocholine species that result in this PAF agonistic activity, we measured PAF and related sn-2 acetyl GPCs in fresh blister fluid samples from bullous pemphigoid and noninflammatory (suction-induced) bullae by mass spectrometry. We report the presence of 1-hexadecyl as well as the 1-acyl PAF analog 1-palmitoyl-2-acetyl glycerophosphocholine (PAPC) in inflammatory blister fluid samples. Because PAPC is the most abundant sn-2 acetyl glycerophosphocholine species found in all samples examined, the pharmacological effects of this species with respect to the PAF-R were determined using a model system created by transduction of a PAF-R-negative epidermoid cell line with the PAF-R. Radioligand binding and intracellular calcium mobilization studies indicated that PAPC is approximately 100x less potent than PAF. Though a weak agonist, PAPC could induce PAF biosynthesis and PAF-R desensitization. Finally, intradermal injections of PAF and PAPC into the ventral ears of rats demonstrated that PAPC was 100x less potent in vivo. These studies suggest possible involvement of PAF and related species in inflammatory bullous diseases.

Oxidative metabolism of a rexinoid and rapid phase II metabolite identification by mass spectrometry.

LGD1069 (Targretin), a retinoid "X" receptor-selective ligand, or rexinoid, is in clinical trials for treating cancer. Biologically-active oxidized LGD1069 metabolites have been observed in patient plasma samples, making corresponding structural characterizations necessary. Formation of multiple metabolite isomers in vivo has created technical challenges in metabolite structural analysis; however, mass spectrometry (MS) was able to pinpoint two sites of Phase I metabolism. A carbon-13 trideuterated analog was used as an isotopic marker to probe Phase II metabolism of LGD1069. Rats were orally gavaged with an equimolar mixture of LGD1069 and [13C2H3]LGD1069, then anesthetized prior to bile-duct cannulation. Bile was collected for 7 hr, extracted, and concentrated. Recovered metabolites were analyzed by narrow-bore, gradient liquid chromatography (LC) with negative ion, electrospray ionization MS detection. When resultant total ion chromatograms were interrogated for mass spectra exhibiting isotope clusters separated by 4 daltons, 13 such clusters corresponding to Phase II LGD1069 metabolites of nine different molecular weights were detected. Acyl-glucuronide and taurine conjugates of both parent compound and hydroxy-LGD1069 were observed. The sulfate and taurine conjugates of oxo-LGD1069 were also identified, as were 6,7-dihydroxy-LGD1069 taurine, LGD1069 ether glucuronide, and a secondary conjugate (taurine) of the latter. Identities of selected conjugates were confirmed by MS/MS. The results of this study demonstrate that when combined with traditional GC/MS and MS/MS data, the isotope cluster technique can provide powerful selectivity in identifying numerous Phase II drug metabolites during a single LC/MS analysis.

Gas chromatographic/mass spectrometric analysis of oxo and chain-shortened leukotriene B4 metabolites. Leukotriene B4 metabolism in Ito cells.

Analysis by gas chromatography/mass spectrometry (GC/MS) of derivatized metabolites formed following incubation of leukotriene B4 (LTB4) incubation with Ito cells extends previous knowledge concerning fragmentation mechanisms for derivatized hydroxy-substituted unsaturated fatty acids. LTB4 was metabolized by rat Ito cells, a hepatic perisinusoidal stellate cell, by the delta 10- and delta 14-reductase pathways, resulting in the formation of 10,11-dihydro-LTB4 and 10,11,14,15-tetrahydro-LTB4. Formation of the intermediate metabolites, 12-oxo-10,11-dihydro-LTB4 and 12-oxo-10,11,14,15-tetrahydro-LTB4, was also observed. GC/electron impact (EI) MS analysis of the 12-oxo metabolites, derivatized as the pentafluorobenzyl ester/ trimethylsilyl ether compounds, resulted in unique fragmentations indicative of the oxo substituent and double bond positions. Further metabolism of 10,11-dihydro-LTB4 and 10,11,14,15-tetrahydro-LTB4 by carboxy terminus beta-oxidation resulted in chain-shortened monohydroxy metabolites. Possible intermediates in this metabolism, which resulted in loss of the original C-5 hydroxy substituent from LTB4, were identified as 2,4,6-conjugated triene-containing C-18 metabolites. The absence of a double bond allylic to the trimethylsiloxy ether in derivatized 10,11,14,15-tetrahydro LTB4 metabolites strikingly reduced the abundance of alpha-cleavage ions observed in the EI mass spectra of these compounds, thus suggesting the importance of formation of an allylic stabilized radical in such alpha-cleavage reactions. Lacking a favorable alpha-cleavage reaction, GC/EIMS analysis of 10-hydroxy-2,4,6-octadecatrienoic acid resulted in the formation of m/z 91, which may arise via cyclization of the conjugated triene moiety. In addition, GC/MS analysis of derivatized metabolites containing the 2,4,6 conjugated triene moiety resulted in a unique fragment ion in the electron capture ionization mass spectra that also may arise via cyclization of the conjugated triene with formation of m/z 121.

Activity of the mitochondrial multiple conductance channel is independent of the adenine nucleotide translocator.

The functional relationship between the adenine nucleotide translocator (ANT) and the mitochondrial multiple conductance channel (MCC) was investigated using patch-clamp techniques. MCC activity with the same conductance, ion selectivity, voltage dependence, and peptide sensitivity could be reconstituted from inner membrane fractions derived from mitochondria of ANT-deficient and wild-type Saccharomyces cerevisiae. In addition, the MCC activity of mouse kidney mitoplasts was unaffected by carboxyatractyloside, a known inhibitor of ANT and inducer of a permeability transition. These results suggest that MCC activity is independent of ANT.

Identification of functional platelet-activating factor receptors on human keratinocytes.

Platelet-activating factor (PAF) is a potent inflammatory mediator that has been shown to be produced by human keratinocytes and is thought to play a role in cutaneous inflammation. Immunofluorescence and radioligand binding studies were used to characterize PAF receptors (PAF-R) on human keratinocytes and the human epidermoid cell lines A-431 and HaCaT. Indirect immunofluorescence studies demonstrated anti-PAF-R staining of primary cultures of human keratinocytes, A-431 cells, and HaCaT cells. Primary cultures of human fibroblasts and the melanoma cell line SK-30 failed to show immunostaining above that seen with control antiserum. With indirect immunofluorescence studies of sections of normal human skin, a granular anti-PAF-R staining pattern was noted on the keratinocyte cell membranes. A-431 cells readily metabolized PAF by deacetylation-reacylation at 37 degrees C, but not at 4 degrees C. Binding studies on crude membrane preparations of A-431 cells conducted at 4 degrees C demonstrated specific binding that reached saturation by 120 min. Scatchard analysis of PAF binding data revealed a single class of high-affinity (KD = 6.3 +/- 0.3 nM) PAF binding sites. The immunofluorescence and radioligand binding sites were shown to be functional PAF-Rs, as 10 pM to 1 microM PAF increased intracellular calcium in primary cultures of human keratinocytes, A-431 cells, and HaCaT cells, whereas PAF treatment of primary cultures of human fibroblasts or the melanoma cell line SK-30 did not result in changes in the intracellular calcium concentration. The structurally dissimilar PAF-R antagonists CV-6209, Ro19-3704, and alprazolam all inhibited the PAF-induced calcium changes in A-431 cells. The CV-6209 inhibition was seen at doses that competed with the PAF binding to these cells. These studies provide the first evidence for the presence of a functional PAF-R expressed on human keratinocytes, suggesting that this lipid mediator may play an important role in normal keratinocytes or in inflammatory dermatology.

Metabolism of 6-trans-isomers of leukotriene B4 in cultured hepatoma cells and in human polymorphonuclear leukocytes. Identification of a delta 6-reductase metabolic pathway.

The intermediate metabolic events which degrade hydroxy polyunsaturated fatty acids is largely unknown. Such molecules are common products of lipid peroxidation and lipoxygenase catalyzed oxidation of arachidonic acid. Metabolism of two 5,12-dihydroxyeicosatetraenoic acids, 6-trans-LTB4 (leukotriene B4), and 6-trans-12-epi-LTB4 was studied in HepG2 cells (a human-derived hepatoma cell line). Extensive metabolism was observed with a major metabolite identified as 4-hydroxy-6-dodecenoic acid for both epimers. Incubation of 6-trans-LTB4 epimers at shorter times revealed the formation of intermediate metabolites, including 6-hydroxy-4,8-tetradecadienoic acid and 8-hydroxy-4,6,10-hexadecatrienoic acid suggesting beta-oxidation as the major pathway leading to the formation of the common terminal metabolite. Two additional metabolites were structurally elucidated as 5-oxo-6,7-dihydro-LTB4 and 6,7-dihydro-LTB4 which have not been previously described. Formation of 5-oxo-6,7-dihydro-LTB4 and 6,7-dihydro-LTB4 were also observed during metabolism of 6-trans-12-epi-LTB4 in human polymorphonuclear leukocytes. Of particular interest is the metabolism of these compounds by beta-oxidation from the carboxyl terminus, a process which is not observed with leukotriene B4 or leukotriene C4. Identification of these metabolites suggested the operation of the 5-hydroxyeicosanoid dehydrogenase pathway followed by a delta 6-reductase metabolic pathway which has not been previously described. This pathway of beta-oxidation may limit the activity of various 5,12-diHETEs including nonenzymatic hydrolysis products of LTA4 and also the recently described B4-isoleukotrienes.