The sensitized liver represents a rich source of endogenous leukotrienes

The ability of livers to produce endogenous leukotrienes after immunological stimulation was tested with organs from rats and guinea pigs. Passive sensitization of rats in vivo with monoclonal murine antidinitrophenol-IgE before antigen challenge in the isolated perfused liver system elicited a rapid hepatic production and biliary excretion of leukotrienes as judged by radioimmunoassay after separation of individual leukotrienes by high-performance liquid chromatography. Within 10 min after antigen infusion, mainly leukotriene C4, but also leukotriene D4 and N-acetyl-leukotriene E4, appeared in the bile. The biliary excretion rate of antigen-induced cysteinyl leukotrienes rose from less than 2 pmol.min-1.(kg body mass)-1 before challenge to about 30 pmol.min-1.(kg body mass)-1 for 20 min before it declined toward prechallenge level. Quantitatively similar hepatic production of cysteinyl leukotrienes was elicited in isolated perfused guinea pig livers challenged with ovalbumin after active sensitization of the animals with ovalbumin plus Al(OH)3. To exclude extrahepatic contributions to the observed leukotriene production, both passive sensitization with anti-dinitrophenol-IgE and subsequent antigen challenge were performed on isolated rat livers perfused with blood-free medium. Such exclusively hepatic sensitization and challenge also resulted in massive production of leukotrienes. The biliary excretion rate of cysteinyl leukotrienes amounted to approximately 20 pmol.min-1.(kg body mass)-1 during the 10 to 20 min period after antigen challenge as compared with less than 1 pmol.min-1.(kg body mass)-1 before challenge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bronchodilatory and mast cell stabilising activity of Cressa cretica L.: evaluation through in vivo and in vitro experimental models

ObjectiveTo evaluate the effect of ethylacetate fraction (Fr-Et) and methanolic fraction (Fr-Me) obtained from Cressa cretica L.(C. cretica) L. on experimental models for bronchodilatory activity and mast cell stabilising activity.MethodsThe effect of Fr-Et and Fr-Me were studied on acetylcholine and histamine aerosol-induced broncospasm using guinea pigs as experimental animals. Also, the effects of these fractions were evaluated on the isolated guinea pig tracheal preparations. Besides this mast cell degranulation effect was assessed using egg albumin and compound 48/80 on rat peritoneal mast cells.ResultsSignificant increase in preconvulsion time was observed due to pretreatment with the fractions when guinea pigs were exposed to histamine and acetylcholine aerosol. Fr-Et and Fr-Me significantly increased the preconvulsion in a dose depended manner that suggestive of bronchodilating activity. Fr-Et and Fr-Me exhibited a significant concentration dependant relaxant effect on guinea pig trachea pre-contracted with CCh, K⁺ and histamine. The results revealed that Fr-Et to be more potent than Fr-Me in relaxing histamine and K⁺ and calcium induced contraction than CCh induced contractions. Studies on the fractions in protecting mast cell degranulation, which were elicited by the egg albumin as well as synthetic compound 48/80 revealed both the fractions significantly protect the mast cell degranulation, which release mediators such as histamine and proinflammatory cytokines through various stimuli in a dose depended manner.ConclusionsThus our study established the bronchodilator activity, and mast cell stabilizing activity which are important mediators that provoke or sustain in asthma.     

Immunohistochemical localization of histamine-stimulated increases in cyclic GMP in guinea pig lung

A significant number of asthmatic subjects are provoked by allergic reactions. The underlying pathophysiologic event is mast cell degranulation with the release and generation of the mediators of anaphylaxis. Histamine, one of the major mast cell mediators, causes 10- to 50-fold increases in guinea pig lung cyclic 3',5'-guanosine monophosphate (cyclic GMP) through H1 receptor stimulation. Employing monoclonal antibodies directed at cyclic GMP, immunocytochemical techniques were used to identify those specific cells in lung responding to histamine stimulation with increases in cyclic GMP. The most responsive cells were alveolar and parenchymal macrophages, pleural lining cells, and endothelial and epithelial cells. Little or no increases in bronchial or vascular smooth muscle cyclic GMP was noted. At the height of the reaction, a generalized increase in cyclic GMP staining of all alveolar cells was observed. These findings suggest that the lining cells of the lung including macrophages, mesothelial, endothelial, and epithelial cells may be the most responsive cells to histamine released during allergic responses. The absence of muscular staining suggests that cyclic GMP does not participate in histamine-stimulated muscle contraction.     

Mast cell renin and a local renin-angiotensin system in the airway: role in bronchoconstriction

We previously reported that mast cells express renin, the rate-limiting enzyme in the renin-angiotensin cascade. We have now assessed whether mast cell renin release triggers angiotensin formation in the airway. In isolated rat bronchial rings, mast cell degranulation released enzyme with angiotensin I-forming activity blocked by the selective renin inhibitor BILA2157. Local generation of angiotensin (ANG II) from mast cell renin elicited bronchial smooth muscle contraction mediated by ANG II type 1 receptors (AT(1)R). In a guinea pig model of immediate type hypersensitivity, anaphylactic mast cell degranulation in bronchial rings resulted in ANG II-mediated constriction. As in rat bronchial rings, bronchoconstriction (BC) was inhibited by a renin inhibitor, an AT(1)R blocker, and a mast cell stabilizer. Anaphylactic release of renin, histamine, and beta-hexosaminidase from mast cells was confirmed in the effluent from isolated, perfused guinea pig lung. To relate the significance of this finding to humans, mast cells were isolated from macroscopically normal human lung waste tissue specimens. Sequence analysis of human lung mast cell RNA showed 100% homology between human lung mast cell renin and kidney renin between exons 1 and 10. Furthermore, the renin protein expressed in lung mast cells was enzymatically active. Our results demonstrate the existence of an airway renin-angiotensin system triggered by release of mast-cell renin. The data show that locally produced ANG II is a critical factor governing BC, opening the possibility for novel therapeutic targets in the management of airway disease.     

5-lipoxygenase inhibition reduces intrahepatic vascular resistance of cirrhotic rat livers: a possible role of cysteinyl-leukotrienes

BACKGROUND & AIMS: Cysteinyl-leukotrienes (Cys-LTs) increase intrahepatic vascular resistance in normal rat livers. CCl4 cirrhotic rat livers have increased Cys-LT production and 5-lipoxygenase messenger RNA (mRNA) expression. The aim of this study was to investigate the role of 5-lipoxygenase-derived eicosanoids regulating intrahepatic vascular tone in control and CCl4-induced cirrhotic rat livers. METHODS: In different groups of portally perfused control and cirrhotic rat livers, the following were analyzed: a portal perfusion pressure (PP) dose-response curve to LTD4; the effects on PP caused by either vehicle, the selective 5-lipoxygenase inhibitor AA-861, the selective Cys-LT1 receptor antagonist MK-571, or the dual Cys-LT1 and Cys-LT2 receptor antagonist BAY u9773; and immunohistochemistry for 5-lipoxygenase in liver sections of cirrhotic and control livers. RESULTS: Cirrhotic livers have a hyperesponse to LTD4. In control livers, AA-861 and MK-571 produced a moderate and similar reduction in PP. In cirrhotic livers, 5-lipoxygenase inhibition produced a marked and significantly greater reduction in PP than in controls. However, no effect on PP was observed after MK-571 or BAY u9773. 5-Lipoxygenase-positive cells were markedly increased in cirrhotic livers. CONCLUSIONS: Our results suggest that 5-lipoxygenase-derived eicosanoids may contribute to the increased intrahepatic vascular resistance of cirrhotic rat livers and therefore the pathogenesis of portal hypertension.     

Comparative studies of mediator release from guinea pig lung mast cells and basophils

Guinea pig lung mast cells and blood basophils were isolated and purified and their mediator release characteristics were compared. Upon stimulation with the antigen ovalbumin (OA) of cells passively sensitized with antiovalbumin (anti-OA) antibody, both cell types released histamine. The sensitivity and maximal response (20 to 25% histamine release) to OA was similar for both cells and was unaffected by cell purification. Antigen-induced histamine release (HR) was dependent upon added calcium to a similar extent (1 mM Ca++ maximal release) in both cell types; OA stimulation of passively sensitized mast cells also released leukotriene bioactivity (maximal release, 52 +/- 7 units/10(6) mast cells). There was no correlation between OA-induced leukotriene release and mast cell purity. No leukotriene bioactivity was detected in actively (sheep blood sensitization) or passively (anti-OA) sensitized basophils. Both lung mast cells and blood basophils released histamine in response to the secretagogues calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate (TPA); TPA-induced HR from mast cells was independent of added calcium. In basophils, TPA-induced HR was only partially independent of added calcium. While both cell types were poorly responsive to the secretagogue 48/80, only the lung mast cell demonstrated inconsistent HR to concanavalin A (Con-A). Phosphatidylserine had no effect on HR provoked by antigen, Con-A, or compound 48/80. These observations demonstrate similarities and differences in mediator release characteristics between guinea pig lung mast cells and blood basophils that are similar to those observed with human lung mast cells and basophils. These observations also suggest a lack of influence on mediator release by other cell types present in dispersed lung cell and mixed leukocyte preparations.     

Slow reacting substances of anaphylaxis: Identification of leukotrienes C-1 and D from human and rat sources

Slow reacting substance(s) of anaphylaxis (SRS-A) was isolated from both human (lung) and rat sources and compared with three synthetic SRS-As of known structure-leukotrienes (LTs) C-1, C-2, and D. Reversed-phase liquid chromatography was used both as a final purification step and a means of comparison of biologically derived and synthetic substances. Two major peaks of SRS-A activity of both rat and human origin corresponded chromatographically with LTC-1 and LTD, respectively, and had equivalent specific activities on the guinea pig ileum. With guinea pig ileum, the specific activities (units/pmol) for synthetic leukotrienes and anaphylactic peaks were (mean +/- SEM): synthetic LTC-1, 1.93 +/- 0.13; SRS-A(rat) peak I, 1.69 +/- 0.43; synthetic LTD, 6.10 +/- 1.15; SRS-A(rat) peak II, 7.14 +/- 0.51; and SRS-A(hu) peak II, 1.90. Both synthetic LTC-1 and LTD and their SRS-A natural counterparts had a preferential contractile activity on guinea pig peripheral airway compared to central airways and were at least 200 times more active than histamine on peripheral airways on a molar basis. Leukotriene D is the major SRS-A of human lung and accounts for almost all of the biological activity. It likely is formed from leukotriene C-1 in vivo by an enzymic process of the well-known gamma-glutamyltransferase type.     

Trichomonas vaginalis-secreted cysteinyl leukotrienes promote migration,degranulation and MCP-1 production in mast cells

Trichomonas vaginalis, a flagellated extracellular protozoan parasite that infects the human genitourinary tract, is usually transmitted by sexual contact. Our previous study showed that the leukotriene B₄ (LTB₄), a T vaginalis-secreted lipid mediator, induces interleukin (IL)-8 production and promotes mast cell degranulation and migration via BLT1 in human. In this study, we investigated whether T vaginalis produces another leukotrienes and whether it causes increased MCP-1 production, mast cell migration and degranulation by activating mast cells. We found that cysteinyl leukotrienes (CysLTs) were contained in T vaginalis-derived secretory product (TvSP) by ELISA. The TvSP-stimulated human mast cell line (HMC-1) exhibited significantly increased monocyte chemoattractant protein-1 (MCP-1) secretion compared to the unstimulated cells. Inhibition of NOX2 activation of cells by treatment of NOX inhibitor or NOX2 siRNA reduced TvSP-stimulated MCP-1 production in HMC-1 cells. It was also confirmed that the receptor for CysLTs is expressed in mast cells. The CysLT receptor (CysLTR) antagonist inhibited TvSP-stimulated MCP-1 production of mast cells, as well as ROS production, migration and degranulation of mast cells, and reduced phospho-NF-kB expression. These results suggest that T vaginalis-secreted CysLTs promote migration, degranulation and MCP-1 production in human mast cells through CysLT receptor-mediated NOX2 activation.     

Antigen-induced release of airway epithelium-derived inhibitory factor

The ability of guinea pig trachea to produce a vasoactive epithelium-derived inhibitory factor (EpDIF) in response to mast cell-derived mediators was assessed in a coaxial bioassay system. The mast cell degranulating agent compound 48/80 (10 micrograms/ml) and histamine caused reductions in phenylephrine-induced tone in endothelium-denuded rat aorta preparations mounted coaxially within epithelium-intact guinea pig tracheal tube tissue. Relaxation responses to histamine and to compound 48/80 (10 micrograms/ml) were markedly reduced in the presence of mepyramine (50 microM) or when the epithelium was removed from coaxially mounted guinea pig trachea, indicating that they were mediated via the release of EpDIF. Coaxial bioassay assemblies were also prepared using EpDIF donor tracheal tissue obtained from guinea pigs actively sensitized to ovalbumin. Subsequent challenge with ovalbumin (10(-7) to 10(-1) mg/ml) produced concentration-dependent relaxation mediated by EpDIF. Ovalbumin-induced relaxation responses were not inhibited in the presence of either mepyramine (20 and 100 microM) or SKF 104353-Z2 (10 microM) alone but were significantly reduced when both mepyramine (20 microM) and SKF 104353-Z2 (10 microM) were present. Antigen-induced relaxation was apparently mediated by EpDIF in response to mast cell-derived histamine and leukotrienes. Rat tracheal airway smooth muscle did not relax in response to EpDIF, suggesting selectivity of action on vascular smooth muscle. Vasoactive EpDIF may play a role in protecting against antigen-induced bronchoconstriction by regulating bronchial circulation flow.     

Smooth muscle contraction and release of histamine and slow-reacting substance of anaphylaxis in pulmonary tissues isolated from guinea pigs passively sensitized with IgG1 or IgE antibodies

In previous studies, we have provided evidence that different Fc receptors mediate antigen-induced pulmonary smooth muscle contractile responses after passive sensitization of guinea pigs with IgG1 or IgE antibodies. In this study, we examined the relationship between contraction and release of histamine and slow-reacting substance of anaphylaxis (leukotrienes) in superfused trachea and parenchymal strips as well as mediator release from minced lung fragments after passive sensitization of guinea pigs with IgG1 or IgE antibodies. Guinea pigs were immunized to produce either IgG1 or IgG1 and IgE using oxazolone-guinea-pig albumin or oxazolone-Ascaris plus cyclophosphamide, respectively. The contaminating IgG1 in the IgE-rich serum was removed by passage over a protein A-Sepharose column. Normal guinea pigs were passively sensitized intraperitoneally or intravenously with injections of either IgG1 or IgE 1 or 2 days before in vitro studies. Superfused tissues were challenged with 10(-1) mg/ml antigen (oxazolone-human serum albumin conjugate), and contractions and histamine and leukotriene release were monitored at discrete time intervals thereafter. At equivalent levels of contraction, substantially more histamine and leukotrienes were released from tissues taken from IgG1-sensitized animals. The amounts of histamine released from lung parenchymal strips and trachea in the IgE-sensitized state were approximately 5 and 38%, respectively, of those released from corresponding tissues in the IgG1-sensitized state. The leukotriene release from tissues isolated from IgE-sensitized animals was less than 4% of that released from tissues in the IgG1-sensitized state. Similar differences in mediator release were seen in comparable studies on minced lung fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contractile activities of structural analogs of leukotrienes C and D: role of the polar substituents.

Twenty-three structural analogs of the leukotriene components of slow reacting substance of anaphylaxis (SRS-A), in which the polar regions of the leukotriene were systematically modified, were tested for their contractile activities on guinea pig pulmonary parenchymal strips and guinea pig ileum. The structural modifications allowed evaluation of the separate contributions of the four polar units in the C-1 to C-6 region of the SRS-A leukotrienes to smooth muscle spasmogenic activity. The free NH2-terminal amino group of the S-linked peptide was necessary for full activity, and its deletion or substitution reduced activity by more than one but less than two orders of magnitude. A similar level of importance was apparent for the free glycine carboxyl group. In contrast, a free eicosanoid carboxyl at C-1 is not required for full activity on the airway and for substantial activity on the ileum. A role for the C-5 hydroxyl is indicated by the inactivity of the one available 5-desoxy analog. Nucleophilic, divalent sulfur is not critical to leukotriene D (LTD) activity, in that one sulfoxide had substantial function. The conformational relationship between the eicosanoid and peptide moieties of LTD is of considerable importance in that epimers at the C-5 or C-6 position were less active than LTD by more than two orders of magnitude. Several lines of evidence suggest that the relative geometrical arrangement of the C20 chain and the peptide unit is important to activity, consistent with the existence of a true receptor for LTD.     

Intestinal mucosal injury is associated with mast cell activation and leukotriene generation duringNippostrongylus-induced inflammation in the rat

We examined mucosal injury in the jejunum of the rat during infection with the nematode parasite, Nippostrongylus brasiliensis (Nb). Injury was documented morphologically (increase in crypt length with or without villus atrophy) and biochemically (activities of digestive or proliferative enzymes) and related to mast cell activation and leukotriene generation. At day 4 crypt length and thymidine kinase activity were increased; no changes in villus parameters were recorded. No evidence of mast cell activation was found and leukotriene levels in the mucosa were normal. At day 7, the gut was acutely inflamed and edema was present at the tips of the villi. This progressed to enterocyte detachment, resulting in villus atrophy with decreased activities of brush border enzymes. At this stage mucosal histamine was decreased and rat mast cell protease II (RMCP II) was increased in serum, indicating mast cell activation. In addition, mucosal leukotrienes (LTB4, LTC4, LTE4) were present in significant quantities. Following worm expulsion, the villus abnormalities resolved and serum RMCP II returned to normal. However, the crypt hyperplasia persisted. Our results suggest that during Nb infection at least two components of injury can be identified. One component, epithelial injury at the villus tips, may be related to activation of mucosal mast cells.     

Platelet activating factor stimulates secretion of mucin by explants of rodent airways in organ culture

Platelet activating factor (PAF; 1-o-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine-3-phosphocholine) a potential mediator of anaphylaxis, stimulates secretion of mucin by explants of trachea from four separate rodent species (guinea pig, rat, rabbit, ferret) in organ culture. Enhanced secretion is not a result of cell damage or release of histamine by cells within the explants (e.g., platelets). It is inhibited by equimolar concentrations of the potent PAF-receptor antagonist, Ro 19-3704. PAF provokes production of immunoreactive peptidyl leukotrienes (ir-LTC4, LTD4, LTE4) within the explants. The stimulatory effect of PAF on mucin secretion is blocked by equimolar concentrations of nordihydroguiaretic acid (NDGA) a "mixed" inhibitor of both cyclo- and lipoxygenase pathways of arachidonic acid metabolism. Leukotrienes are localized within tracheobronchial epithelium by immunohistochemical staining, and physical removal of epithelium from explants inhibits production of leukotrienes in vitro under nonstimulated conditions and after exposure to PAF. In addition, the stimulatory effect of PAF on mucin secretion is not altered by FPL-55712, a receptor antagonist of LTD4. These results are consistent with the hypothesis that PAF stimulates secretion of mucin by activating biosynthesis of lipoxygenase products (e.g., peptidyl leukotrienes) within epithelial cells of the respiratory mucosa.     

Leukotriene E4-induced airway hyperresponsiveness of guinea pig tracheal smooth muscle to histamine and evidence for three separate sulfidopeptide leukotriene receptors.

Bronchial hyperresponsiveness to contractile agonists and nonspecific irritants is a characteristic feature of bronchial asthma. The mechanisms causing this hyperirritability are unknown. The existence of separate receptors for leukotrienes C4 and D4 (LTC4 and LTD4) has been demonstrated previously by physiologic and radioligand binding studies. The rank order of potency of the sulfidopeptide leukotrienes for contracting tracheal spirals [leukotriene E4 (LTE4) greater than LTD4 = LTC4] is different from that for contracting parenchymal strips (LTD4 greater than LTE4 greater than LTC4), thereby suggesting the existence of a separate receptor for LTE4. We now report that LTE4, the most stable of the leukotrienes comprising slow reacting substance of anaphylaxis, enhances the contractile response of guinea pig tracheal spirals but not of parenchymal strips to histamine in a time- and dose-dependent fashion. The ability of LTE4 to increase histamine responsiveness occurred after removal of the free agonist and recovery of the tissues to baseline tensions and was not produced by leukotrienes C4 and D4, which elicited the same magnitude of contraction of tracheal smooth muscle as LTE4. These findings suggest that LTE4-induced airway hyperirritability is not mediated by the contractile response per se and may be mediated through a receptor distinct from those for leukotrienes C4 and D4.     

Increased 5-lipoxygenase activity in massive hepatic cell necrosis in the rat correlates with neutrophil infiltration.

Rats were treated with heat-killed Propionibacterium acnes and subsequent injection of a small amount of lipopolysaccharide after 7 days. After 24 hr most of the rats died of massive liver cell necrosis. Nonparenchymal liver cells were isolated from this liver injury model and incubated with arachidonic acid. Reverse-phase high-pressure liquid chromatography detected the 5-lipoxygenase metabolites (leukotriene B4 and 5-hydroxy-arachidonic acid), whereas these compounds were produced in negligible amounts when the rats were treated with P. acnes only. Immunohistochemical studies with 5-lipoxygenase antiserum revealed that the injured livers contained a large number of positively stained round cells with segmented nuclei, which were rarely found in the livers treated with P. acnes only. These positively stained cells were histologically identified as neutrophils. The results suggested that the increased 5-lipoxygenase activity in the injured rat liver is attributable to the infiltrating neutrophils rather than to nonparenchymal hepatic cells.     

Generation of leukotriene C4 from a subclass of mast cells differentiated in vitro from mouse bone marrow.

Bone marrow-derived mast cells, differentiated in vitro, demonstrate surface IgE, receptors and contain histamine in metachromatic granules, which are composed of chondroitin sulfate E proteoglycan rather than heparin proteoglycan. Activation of this subclass of mast cells with calcium ionophore A23187 resulted in generation of immunoreactive C-6-sulfidopeptide leukotriene in a dose- and time-dependent fashion. Isolation of immunoreactive C-6-sulfldopeptide leukotriene by reverse-phase high-performance liquid chromatography (RP-HPLC) revealed a retention time and a specific biologic activity identical to those of synthetic leukotriene C4 (LTC4). Neither radiolabeled nor immunoreactive conversion products of [3H]LTC4/LTC4 were recognized during RP-HPLC resolution of the supernatants. The failure of fresh bone marrow cultures to generate C-6-sulfidopeptide leukotriene in response to ionophore indicates that leukotriene generation is dependent upon cellular differentiation into a mast cell population. The amount of LTC4 generated during optimal ionophore stimulation, 90.9 +/- 7.5 ng per 10(6) cells, contrasts with the relatively low amounts of C-6-sulfidopeptide leukotriene generated by the conventional heparin-containing rat mast cells or mouse mastocytoma cells.     

Hepatic mucosal mast cell hyperplasia in rats with secondary biliary cirrhosis

Mast cells have been shown to play a role in many chronic inflammatory and fibrotic disorders. However, their possible contribution to the pathological changes that occur in liver cirrhosis is unknown. To explore this, we examined whether changes in hepatic mast cell number and mediator content were associated with fibrotic changes in experimental biliary cirrhosis. Rats were studied 7, 14, or 21 days after bile duct resection (BDR). Hepatic mast cells were identified by histochemical and immunohistochemical stains. Rat mast cell protease II (RMCP-II), a marker of mast cell degranulation, was measured in liver by enzyme-linked immunosorbent assay. Hepatic collagen deposition was assessed by Sirius Red F3BA staining. In day 21 BDR rats, there was a one- to twofold increase (P < .001) in the number of hepatic mast cells, but this was not observed in day 7 or 14 BDR rats. Mild fibrotic changes were noted in BDR rat livers as early as 7 days after induction of cholestasis. Significant expansion and organization of fibrous tissue had occurred in day 14 BDR rats which progressed to bridging fibrosis by day 21. Liver RMCP-II levels were decreased by 50 percent (P < .05) and mast cell degranulation was apparent as shown by histamine immunostaining. These results suggest that hepatic mast cell hyperplasia and degranulation occur during prolonged cholestasis in the rat. Although these changes do not correlate with the onset of hepatic fibrosis, they do occur at a time during which there is significant deposition and organization extracellular matrix elements. Hepatic mast cells, by releasing profibrogenic mediators, may contribute to fibrotic changes in biliary cirrhosis. (Hepatology 1996 Apr;23(4):888-95)     

Slow reacting substances (leukotrienes): enzymes involved in their biosynthesis.

Slow reacting substances (leukotrienes C4, D4, E4) are synthesized in vivo by a combination of two previously unrelated pathways: lipoxygenase oxygenation of arachidonic acid and the glutathione detoxification pathway. Enzymes involved in the latter pathway (glutathione transferase [RX: glutathione R-transferase, EC 2.5.1.18]; gamma-glutamyltransferase [(5-glutamyl)-peptide: amino acid 5-glutamyltransferase, EC 2.3.2.2] ) have been investigated in guinea pig lung and rat basophilic leukemia (RBL-1) cells. We report data on levels of enzymic activity both before and during the release of slow reacting substances. Both glutathione transferase and gamma-glutamyltransferase are present in significant quantities in guinea pig lung and RBL-1 cells. A model for the changes in gamma-glutamyltransferase during leukotriene release is proposed for the cell line, and differences from the guinea pig lung system are reported. Leukotriene C4 is converted to the more potent leukotriene D4 by the action of gamma-glutamyltransferase on guinea pig ileum during bioassay. gamma-Glutamyltransferase may represent a control feature in the biosynthesis of leukotriene D4, and thus be involved in leukotriene-induced bronchoconstriction in the lung.     

Histamine-containing neurons in the rat hypothalamus.

A specific antiserum against histamine was produced in rabbits, and an immunohistochemical study of histamine-containing cells was carried out in rat brain. The antiserum bound histamine in a standard radioimmunoassay and stained mast cells located in various rat and guinea pig tissues. Enterochromaffin-like cells in the stomach and neurons in the posterior hypothalamic area could be detected with this antiserum. The staining was highly specific and was not abolished by preabsorption with histidine, histidine-containing peptides, serotonin, or catecholamines, whereas preabsorption with histamine completely abolished the staining. Immunoglobulins of this antiserum purified by affinity chromatography stained the same cells as did the crude antiserum, whereas the serum fraction, which was not absorbed by histamine-affinity ligand, failed to stain any neuron. Histamine-immunoreactive neuronal cell bodies were found only in the hypothalamic and premammillary areas of colchicine-treated rats. The largest group of cells was seen in the caudal magnocellular nucleus and medially on the dorsal and ventral aspects of the ventral premammillary nucleus. Immunoreactive nerve fibers, but no cell bodies, were detected in other parts of the brain. Histamine-immunoreactive mast cells were found in the median eminence and pituitary gland. The results suggest that histamine-containing neurons are located only in a small area of the posterior hypothalamus, and these cells are probably the source of ascending and descending fibers detected in other brain areas.