Biomedical subjects

W C Hubbard

Publications and source records attributed to W C Hubbard.

At least 55 records · Page 3Linked to original sources

PubMed1994

Dry air- and hypertonic aerosol-induced bronchoconstriction and cellular responses in the canine lung periphery.

Dry air and hypertonic saline both create an osmotic stress to the airways, whilst dry air alone induces transient cooling of the airway mucosa. It is unclear whether these two stimuli lead to bronchoconstriction via the same mechanisms. We compared airflow- and hypertonic aerosol-induced bronchoconstriction (AIB and HIB, respectively) in the canine lung periphery, using a wedged bronchoscope to measure collateral system resistance (Rcs). Bronchoalveolar lavage (BAL) was used to examine changes in cells and mediators during AIB and HIB. We found that: 1) peripheral airways are not refractory to either dry air or hypertonic aerosols, and do not exhibit cross-refractoriness to these stimuli; 2) differences in strength of stimulus can alter the magnitude but not the time course of HIB; 3) within an individual, AIB and HIB are significantly correlated; 4) epithelial cells recovered in BAL fluid (BALF) are significantly elevated after AIB, and are similarly increased after HIB; 5) when compared to control, mediators recovered in BALF are significantly elevated after AIB but not HIB; 6) HIB is not altered by cyclo-oxygenase inhibition; and 7) lavage with hypertonic fluid does not affect the number of epithelial cells recovered, although the concentrations of some mediators are increased. We speculate that differences in cell and mediator profiles reflect differences in the time course of AIB and HIB that result from the modulation of temperature sensitive pathways that occurs during dry air, but not during hypertonic aerosol challenge.

Aerosols↗

PubMed1994

Release of inflammatory mediators from guinea pig trachea by electrical field stimulation: lack of neuronal involvement.

Electrical field stimulation (EFS; 5-10 V, 1 ms, 20 Hz for 1 min) of isolated guinea pig trachea resulted in a rapid increase in tone that is blocked by either atropine or tetrodotoxin (TTX). EFS of tracheal spirals also caused large increases in the release of certain prostanoids with release of prostaglandin (PG)D2, PGE2 and PGF2 alpha (16.5-, 3.0- and 4.1-fold, respectively). In contrast to the smooth muscle response, however, EFS-induced release of prostanoids was not significantly altered in the presence of TTX. Removal of the epithelium reduced the amount of prostanoids released by EFS. Thus, EFS-induced production of PGD2, PGE2 and PGF2 alpha was significantly reduced by about 30%, 70% and 80% in epithelium-denuded tissues, respectively. Direct vagal stimulation caused a rapid contraction of the trachealis but failed to elicit increases in the release of histamine or arachidonic acid metabolites. Furthermore, the selective stimulant of C-type sensory fibers capsaicin (3 microM) or exogenously applied substance P (1 microM) or neurokinin A (1 microM) failed to induce histamine, leukotriene or prostanoid release from guinea pig tracheal rings. Although, the mechanism involved in stimulation of arachidonic acid metabolism by EFS is unclear, this effect in part involves the epithelium but apparently is not mediated by airway elements sensitive to TTX, direct vagal stimulation or tachykinins.

Animals↗

PubMed1994

Effect of ethacrynic acid on aqueous outflow dynamics in monkeys.

PURPOSE: To determine the long-term effect of ethacrynic acid (ECA) on aqueous outflow dynamics in ocular normotensive monkeys. METHODS: (1) Twelve cynomolgus monkeys received 10 microliters of 2.5 mM (= 7.5 micrograms) ECA intracamerally in one eye, vehicle in the other; outflow facility (perfusion) was determined at 1 hour, 24 hours, or 1 week, and intraocular pressure (IOP; applanation) at 24 hours, 1 week, and 6 to 7 weeks later. Six other cynomolgi received 10 microliters of 0.13 or 1.3 mM phalloidin in one eye 45 minutes before receiving ECA OU; facility was measured 1 hour after ECA. (2) Groups of five rhesus monkeys underwent intracameral injection of 2.5, 5.0, or 10 micrograms ECA in one eye, vehicle in the other, with IOP measured hours to weeks thereafter. (3) Five rhesus monkeys received 540 micrograms of ECA unilaterally as a 30-microliter topical drop once daily for 4 days. On the first and fourth treatment days, baseline IOP, pupil diameter, and refraction were measured immediately before and again at 2, 4, and 7 hours after topical treatment. RESULTS: (1) ECA divided by vehicle facility averaged 1.83 +/- 0.23 (SEM) (P < 0.02, n = 6), 1.50 +/- 0.27 (P < 0.11, n = 7), and 1.05 +/- 0.10 (n = 7) at 1 hour, 24 hours, and 1 week, respectively. IOP was 1 to 2 mm Hg lower in ECA eyes 24 hours (P < 0.05, n = 5) and 6 to 7 weeks (P < 0.05, n = 7) after treatment. Phalloidin did not diminish the 1-hour ECA facility effect. (2) Five (but not 2.5 or 10) micrograms of ECA lowered IOP 1 to 3 mm Hg, starting at 2 hours and lasting up to 48 hours. The maximum ECA effect (-2.6 +/- 0.25 mm Hg; P < 0.001, n = 5) occurred at 4 hours. IOP, corneal thickness and endothelial cell count, and anterior chamber depth were not significantly different 8 weeks after 5 micrograms ECA or vehicle. (3) Once-daily unilateral topical application of 540 micrograms of ECA induced no change in IOP, refraction, or pupil diameter compared to contralateral vehicle-treated control eyes. There were no significant ECA-related ocular bio-microscopic abnormalities. CONCLUSIONS: ECA may lower IOP and increase outflow facility longer than previously thought, but not by affecting meshwork actin filaments.

Animals↗

PubMed1993

IL-3 alters free arachidonic acid generation in C5a-stimulated human basophils.

IL-3 is known to enhance the secretion of several mediators from human basophils activated by receptor-mediated stimuli. IL-3 can cause a qualitative change in the mediator release pattern for C5a-mediated stimulation; without IL-3, C5a causes no leukotriene release whereas in the presence of IL-3, significant leukotriene occurs. This study examines the influence of a 15-min pretreatment of basophils with IL-3 (10 ng/ml) on several signal transduction events. Basophils stimulated with C5a typically displayed only a transient cytosolic Ca2+ response [Ca2+]i, attributed to the release of intracellular calcium stores. IL-3 had sporadic, statistically nonsignificant, effects on the peak of this initial response as well as inconsistent effects on the generation of a second phase in the [Ca2+]i response (i.e., that which is caused by the influx of extracellular Ca2+). IL-3 also had no effect on resting [Ca2+]i levels. Challenge of basophils in the presence of EGTA had little effect on the amount of leukotrienes generated. This maneuver did not influence the initial transient elevation of [Ca2+]i. Although basophil leukotriene release is usually slow, after exposure to IL-3 it was found that leukotriene release occurred rapidly, during the brief window of time (0 to 45 s) in which the [Ca2+]i transient occurred. These results suggested that the generation of AA was accelerated by pretreatment with IL-3. Subsequent mass measurements of free AA by gas chromatography-mass spectroscopy showed: 1) IL-3 itself caused no free AA generation; 2) without IL-3, C5a stimulated little or no free AA generation; and 3) in the presence of IL-3, C5a generated substantial levels of free AA at an accelerated rate. A similar acceleration in the rate of free AA generation, without any apparent increase in the amount, occurred in IL-3-primed basophils stimulated with FMLP. Additional studies showed that IL-3 had no effects on whole cell ATP levels and that the kinase inhibitor, staurosporine, did not inhibit the ability of IL-3 to cause enhanced responses to C5a. We conclude that the primary effect of a short period of pretreatment with IL-3 is to couple the generation of free AA to C5a-mediated stimulation in particular and to accelerate its generation after other stimuli. Enhancements of the [Ca2+]i response, while sporadic and at best modest, may have some influence on enhanced mediator release but did not clearly explain the functional effects of IL-3.

Arachidonic Acid↗

PubMed1993

Influence of arachidonic acid on indices of phospholipase A2 activity in the human neutrophil.

The present studies were conducted to understand better the regulation of phospholipase A2 (PLA2)-dependent mobilization of lipid mediators by arachidonic acid (C20:4). After stimulation of human neutrophils, g.l.c./m.s. analysis of non-esterified fatty acids indicated that the quantity of C20:4 increased as a function of time after stimulation, from undetectable quantities to > 800 pmol/10(7) cells. In contrast with C20:4, the quantities of other free fatty acids such as oleic and linoleic were high in resting cells and did not change after stimulation. Some 15% of the C20:4 released from cellular lipids remained cell-associated. To examine the effect of C20:4 on its own release, neutrophils were exposed to [2H8]C20:4, to differentiate it by g.l.c./m.s. from naturally occurring C20:4. In A23187-stimulated neutrophils, low concentrations (5-10 microM) of [2H8]C20:4 added just before A23187 increased the quantity of C20:4 produced by the cell, whereas higher concentrations (30-50 microM) decreased the quantity of C20:4 released from phospholipids. As other measures of PLA2 activity, the effects of C20:4 on production of platelet-activity factor (PAF) and leukotriene B4 (LTB4) were assessed. C20:4 treatment just before stimulation of neutrophils blocked PAF and LTB4 production in a concentration-dependent manner (IC50 10-20 microM). The effect of C20:4 was not blocked by the cyclo-oxygenase inhibitor naproxine (10 microM), nor could it be mimicked by 1 microM LTB4, 5-hydroxyeicosa-6,8,11,14-tetraenoic acid (5HETE), 5-hydroperoxyeicosa-6,8,11,14-tetraenoic acid (5HPETE) or 15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15HETE). The 5-lipoxygenase (5LO) inhibitor zileuton induced a concentration-dependent decrease in PAF, with a maximal effect of a 50% decrease at 10-50 microM. The decrease in PAF by the 5LO inhibitor could not be circumvented by addition of 1 microM 5HETE, 5HPETE and LTB4, and may be attributed to the capacity of zileuton to increase the quantity of C20:4 in A23187-treated neutrophils. The inhibitory effect of C20:4 (20-40 microM) on PAF production could be antagonized by the protein kinase C inhibitor staurosporine (30 nM), but not by inhibitors of protein kinase A, tyrosine kinase or calmodulin kinase II. Taken together, these data demonstrate that C20:4 is selectively released from membrane phospholipids of A23187-stimulated neutrophils, and this C20:4 may play an important role in regulating the mobilization of C20:4 by altering PLA2 activity.

Arachidonic Acid↗

PubMed1993

Comparison of clarithromycin and penicillin VK suspensions in the treatment of children with streptococcal pharyngitis and review of currently available alternative antibiotic therapies.

In a randomized investigator-blinded study, 506 children ages 6 months to 12 years with positive rapid direct antigen tests for Group A beta-hemolytic Streptococcus (GABHS) received treatment with either clarithromycin suspension, 7.5 mg/kg twice daily, or penicillin VK suspension, 13.3 mg/kg three times per day for 10 days. Signs and symptoms of pharyngitis or tonsillitis were evaluated and throat cultures were obtained before treatment, once during treatment and 4 to 6 days and 19 to 25 days posttreatment. All GABHS isolates were susceptible in vitro to clarithromycin. Successful clinical responses at the end of treatment were demonstrated in 169 of 176 (96%) evaluable clarithromycin-treated patients and 179 of 191 (94%) evaluable penicillin-treated patients. GABHS was successfully eradicated at end of treatment in 168 of 183 (92%) evaluable clarithromycin-treated patients compared with 162 of 199 (81%) evaluable penicillin-treated patients (P = 0.004). There were no significant changes in hematologic or serum chemistry parameters in either group. Both drugs were well-tolerated. The incidence and nature of adverse events were similar in the clarithromycin and penicillin groups, except for gastrointestinal complaints reported in 35 of 250 (14%) clarithromycin recipients compared with 12 of 256 (5%) penicillin recipients (P < or = 0.001). The results indicate that twice daily clarithromycin was as safe and effective as three times daily penicillin VK in the treatment of children with streptococcal pharyngitis or tonsillitis. Clarithromycin was statistically superior to penicillin VK in the eradication of GABHS.

Child↗

PubMed1993

Endothelin-induced contraction and mediator release in human bronchus.

1. To elucidate the role of acetylcholine and various autacoids in endothelin-1 (ET-1)-induced contraction in human bronchus, the effects of various receptor antagonists were examined. In addition, the ability of ET-1 to stimulate the release of histamine, peptidoleukotrienes and prostanoids was determined. 2. ET-1 was a potent and effective contractile agonist in human bronchus, possessing similar potency and efficacy to leukotriene D4 (LTD4); EC50 (-log M): ET-1 = 7.76 +/- 0.09, n = 7; LTD4 = 8.46 +/- 0.53, n = 7; P > 0.2; maximum response (% 10 microM pre-carbachol): ET-1 = 103.8 +/- 17.4, n = 7; LTD4 = 95.5 +/- 9.3, n = 7; P > 0.6. 3. The cyclo-oxygenase inhibitor, sodium meclofenamate (1 microM) or the potent and selective thromboxane receptor antagonist, SQ 29,548 (1 microM) were without significant effect on ET-1 concentration-response curves. 4. In the presence of sodium meclofenamate (1 microM), the muscarinic receptor antagonist, atropine (1 microM), the platelet activating factor (PAF) receptor antagonist, WEB 2086 (1 microM) or the combination of the H1-histamine receptor antagonist, mepyramine (10 microM) and the leukotriene receptor antagonist, SK&F 104353 (10 microM), were without marked effect on ET-1 concentration-response curves. In addition, the combination of all four receptor antagonists did not antagonize ET-1-induced contraction. 5. ET-1 (0.3 microM) did not stimulate the release of histamine or immunoreactive leukotrienes from human bronchus. 6. ET-1 (0.3 microM) significantly stimulated the release of prostaglandin D2 (PGD2), 9alpha, 11beta PGF2 (PGD2 metabolite), PGE2, 6-keto PGF1alpha (PGI2 metabolite), PGF2alpha, and thromboxane B2 (TxB2) a lower concentration, 10 nM, was without effect on prostanoid release. The production of PGD2 was increased 7.5 fold, whereas the release of the other prostanoids was stimulated only about 1.6 to 2.7 fold.7. These data provide evidence that ET-1 elicits contraction of human isolated bronchus predominantly via a direct mechanism with no significant involvement of the release of acetylcholine, leukotrienes,histamine or PAF. Although ET-1 increased the release of several prostanoids they did not have a significant modulatory effect on the smooth muscle contraction.

Acetylcholine↗

PubMed1993

Relative contributions of direct and indirect mechanisms mediating endothelin-induced contraction of guinea-pig trachea.

1. The present study was undertaken to determine the mechanism of action of endothelin-1 (ET-1)-induced contraction of the guinea-pig isolated trachea. 2. ET-1 (1 nM-0.3 microM) produces a concentration-dependent contraction of guinea-pig trachea with an EC50 of approximately 25 nM. The combination of the peptidoleukotriene receptor antagonist, SK&F 104353 (10 microM) and the H1-histamine receptor antagonist, mepyramine (10 microM), which abolishes antigen-induced contraction in guinea-pig trachea, was without effect on ET-1 concentration-response curves. Furthermore, the platelet-activating factor (PAF) receptor antagonist, WEB 2086, (1 or 10 microM) did not inhibit ET-induced contraction. 3. ET-1 (0.3 microM) did not stimulate histamine or immunoreactive peptidoleukotriene release from guinea-pig isolated trachea. 4. The release of various prostanoids from guinea-pig trachea was increased significantly by ET-1 (0.3 microM); the profile of release was prostaglandin D2 (PGD2) = PGE2 = 6-keto PGF1 alpha (PGI2 metabolite) > thromboxane B2 = PGF2 alpha >> 9 alpha, 11 beta PGF2 (PGD2 metabolite). ET-1-induced release of prostaglandins, which was about 30% of that elicited by antigen in sensitized tissues, was not affected by epithelium removal and was observed in tissues from which the smooth muscle had been removed. Previous studies in our laboratory indicated that indomethacin potentiated contraction produced by high concentrations of ET-1, whereas a thromboxane receptor antagonist was without appreciable effect on ET-1 concentration-response curves. 5. Pretreatment of tissues for 1 h with capsaicin (10 microM), which depletes different sensory neurones, produced a small, but significant, inhibitory effect on ET-1 concentration-response curves in the presence but not the absence of the epithelium. The combination of the NK1 tachykinin receptor antagonist,CP-96,345 (0.1 microM), and the NK2 tachykinin receptor antagonist, SR 48968 (0.1 microM), was without effect on ET-l concentration-response curves but substantially antagonized capsaicin-induced contraction.6. The present data suggest that in guinea-pig isolated trachea, ET- 1 produces contraction predominantly via a direct mechanism: there is no significant contribution of the release of histamine,leukotrienes, PAF, or tachykinins (acting on NK1 or NK2 receptors). Although ET-1 evokes the release of an array of prostanoids from the trachea they do not appear to have a major influence on the contractile response.

Animals↗

PubMed1993

Endothelin receptor subtypes in human and guinea-pig pulmonary tissues.

1. In this study the endothelin (ET) receptor subtypes mediating contractions produced by ET-1 in human and guinea-pig pulmonary tissues were investigated. In addition the receptor responsible for ET-1-induced prostanoid release in human bronchus was determined. 2. In human bronchus and human pulmonary artery ET-1 (0.1 nM-0.3 microM) was a potent and effective contractile agent (pD2 = 7.58 +/- 0.15, n = 6, and 8.48 +/- 0.11, n = 7, respectively). BQ-123 (1-10 microM), a potent and selective ETA receptor antagonist, potently antagonized ET-1-induced contraction in human pulmonary artery (pKB = 6.8 with 1 microM BQ-123, n = 7) but had no effect in human bronchus (n = 6). 3. Sarafotoxin S6c (0.1 nM-0.1 microM), the ETB-selective agonist, did not contract human pulmonary artery (n = 5), but potently and effectively contracted human bronchus: pD2 = 8.41 +/- 0.17, maximum response = 74.4 +/- 3.1% of 10 microM carbachol; n = 5. BQ-123 (1-10 microM) did not antagonize sarafotoxin S6c-induced contraction in human bronchus (n = 5). 4. ET-1 potently contracted guinea-pig trachea, bronchus, pulmonary artery and aorta (pD2 = 8.15 +/- 0.14, 7.72 +/- 0.12, 8.52 +/- 0.12, and 8.18 +/- 0.12, respectively, n = 6-14). BQ-123 (0.1-10 microM)antagonized ET-1-induced contractions in guinea-pig pulmonary artery (pKB = 6.7 with 1 microM BQ-123,n = 6), aorta (pKB = 7.1 with 1 microM BQ-123, n = 6) and trachea (pKB = 6.2 with 1 microM BQ-123, n = 6) butwas without marked effect in bronchus (n = 4). In contrast, sarafotoxin S6c (0.1 nM-0.l microM) did not contract guinea-pig aorta (n = 4) or guinea-pig pulmonary artery (n = 6) but potently and effectively contracted guinea-pig bronchus: pD2= 8.55 +/- 0. 1; maximum contraction = 63.6 +/0 3.1% of 10 microM carbachol,n = 4. Sarafotoxin S6c (0.1 nM-0. 1 microM) was a much less effective agonist in guinea-pig trachea:maximum contraction = 13.9 +/- 2.5% of 10 JM carbachol, n = 4; P< 0.0001, compared to bronchus.Contractions produced by sarafotoxin S6c in guinea-pig bronchus or trachea were unaffected by BQ-123(IO microM, n=4).5. Significant differences were observed in the efficacy, relative to carbachol, but not the potency of sarafotoxin S6c in guinea-pig airways, with a much greater maximum contractile response in bronchus(69.6 +/- 2.4% of 10 microM carbachol, n = 6) or lower region of the trachea (48.5 +/- 5.9% of 10 microM carbachol,n = 6) than in the middle region of the trachea (14.4 +/- 4.0% of 10 microM carbachol, n = 6) or the upper region of the trachea (19.3 +/- 2.7% of 10 microM carbachol, n = 6). There were minimal regional differences in either ET-1-induced contraction or the potency of BQ-123 (3 microM) for inhibition of responses to ET-1 in guinea-pig airways.6. Release of various prostanoids in human bronchus induced by ET-1 (0.3 microM) was essentially abolished with 10 IM BQ-123.7. These data provide evidence that distinct ET receptors mediate ET-1-induced contraction in human pulmonary artery, guinea-pig pulmonary artery and guinea-pig aorta (ETA subtype) compared with human bronchus and guinea-pig bronchus (non-ETA, perhaps ETB subtype). Contractions to ET-1 in guinea-pig trachea appear to involve both ETA and non-ETA (ETB?) receptor subtypes. Furthermore,regional differences appear to exist in the relative distribution of ET receptor subtypes in guinea-pig airways. In human bronchus ET-1-induced prostanoid release, unlike the contractile response, appears to be mediated via ETA receptor activation.

Animals↗

PubMed1993

Importance of prostanoids in calcium chelator-induced airway constriction.

To elucidate the mechanism of calcium chelator-induced airway constriction, we examined the relationship between prostanoids and histamine in bronchoalveolar lavage (BAL) fluid and the magnitude of airway constriction in peripheral airways of anesthetized Basenji-Greyhound dogs. A wedged bronchoscope technique was used to measure collateral system resistance (Rcs) before and after aerosol challenges. Sublobar segments were challenged either with acetylcholine or with Na2EDTA in the presence or absence of meclofenamate sodium (3 mg/kg iv) or methylprednisolone (2 mg.kg-1.day-1). After measurements of Rcs, BAL was performed, and the fluid was analyzed for prostanoids with the use of gas chromatography-mass spectrometry. Sublobar segments challenged with Na2EDTA showed increased concentrations of prostaglandin (PG) D2 but no increases in PGE2, PGF2 alpha, 9 alpha,11 beta-PGF2, 6-keto-PGF1 alpha, thromboxane B2, or histamine. There was a strong relationship (r = 0.84, P = 0.005) between changes in Rcs after Na2EDTA and concentrations of PGD2 in BAL fluid. Acetylcholine, which increased Rcs to a similar degree as Na2EDTA did, produced no significant increase in prostanoid concentrations. Changes in Rcs after Na2EDTA and concentrations of PGD2 were reduced in the presence of meclofenamate or methylprednisolone. These data support the idea that the mechanism of calcium chelator-induced bronchoconstriction involves the release of bronchoconstricting prostanoids.

Acetylcholine↗

PubMed1993

Dietary n-3 fatty acid effects on neutrophil lipid composition and mediator production. Influence of duration and dosage.

Healthy volunteers supplemented their usual Western diets with Promega fish oil supplement (eicosapentaenoic acid [EPA], 0.28 g; docosahexaenoic acid [DCHA], 0.12 g; other n-3 fatty acids 0.10 g per capsule) using three protocols. Initial experiments (protocol 1 and 2) investigated the kinetics of incorporation of n-3 fatty acids into serum and neutrophil lipids after 10 capsules/d of Promega. EPA was rapidly detected in both serum and neutrophil lipids; the arachidonic acid (AA) to EPA ratio in neutrophil phospholipids showed a maximal reduction of 49:1 to 8:1 within 1 wk of beginning supplementation. EPA was preferentially incorporated into phosphatidyl-ethanolamine and phosphatidylcholine but not phosphatidylinositol. Long-term supplementation for up to 7 wk did not influence the AA/EPA ratio or the distribution of EPA among neutrophil phospholipids in a manner that was not observed after the first week. Neutrophils produced similar quantities of platelet-activating factor and slightly lower quantities of leukotriene B4 during long-term supplementation when compared with presupplementation values. Experiments examining the influence of Promega dosage indicated that the AA/EPA ratio in neutrophil lipids decreased in a dose-dependent manner. Only when the dose was increased to 15 capsules/d was there a reduction in the AA/DCHA ratio in neutrophil lipids. The quantity of AA in neutrophil lipids remained relatively constant at all supplement doses. Taken together, the current study demonstrates the capacity of n-3 fatty acids provided with a Western diet to be rapidly incorporated into neutrophil lipids. However, dietary n-3 fatty acids appear not to significantly reduce arachidonate content within neutrophil phospholipids. Constant arachidonate levels may account for the lack of large reductions in the biosynthesis of lipid mediators by neutrophils after fish-oil supplementation.

Adult↗

PubMed1992

Evidence for different mechanisms involved in the formation of lyso platelet-activating factor and the calcium-dependent release of arachidonic acid from human neutrophils.

Recent studies suggest that the first step in platelet-activating factor (PAF) biosynthesis, 1-alkyl-2-lyso-GPC (lyso PAF) formation, may be initiated by the selective transfer of arachidonate from 1-alkyl-2-arachidonoyl-GPC to an acceptor lyso phospholipid by a CoA-independent transacylase activity (CoA-IT). The present study was designed to determine whether the formation of 1-alkyl-2-lyso-GPC and the release of arachidonic acid can occur by different mechanisms. These experiments examined both the formation of 1-[3H]alkyl-2-lyso-GPC from 1-[3H]alkyl-2-acyl-GPC and the release of arachidonic acid from membrane phospholipids as determined by GC/MS in neutrophil homogenates under various conditions. The addition of unlabelled lyso phospholipids to neutrophil homogenates stimulated the time-dependent formation of 1-[3H]alkyl-2-lyso-GPC from 1-[3H]alkyl-2-acyl-GPC. Without exogenous lyso phospholipids, little 1-[3H]alkyl-2-lyso-GPC was formed in this reaction. The activity which catalyzed the formation of 1-[3H]alkyl-2-lyso-GPC had characteristics identical to CoA-IT as indicated by the fact that both reactions were: independent of Ca2+, Mg2+, CoA and CoA fatty acids, located in microsomal fractions, and stable in 10 mM dithiothreitol. In sharp contrast to the aforementioned reaction, addition of lyso phospholipids did not affect the quantity of arachidonic acid released from membrane phospholipids. Furthermore, there was a Ca(2+)-independent release of arachidonic acid from membrane phospholipid that was increased 4 to 5-fold after the addition of 5 mM Ca2+. Finally, Ca(2+)-dependent arachidonic acid release was inhibited by putative phospholipase A2 inhibitors, aristolochic acid and scalaradial, at concentrations where neither the production of 1-[3H]alkyl-2-lyso-GPC nor Ca(2+)-independent arachidonic acid release was altered. Together these data imply that there may be different mechanisms involved in the formation of 1-alkyl-2-lyso-GPC and arachidonic acid from membrane phospholipids.

Acyltransferases↗

PubMed1992

Platelet activation in the lung after antigen challenge in a model of allergic asthma.

The purpose of this study was to investigate whether platelets are activated and release their products in the human lung after antigen challenge. Using subsegmental antigen challenge as a model of asthma, bronchoalveolar lavage fluids from ragweed-allergic asthmatic subjects were assayed for the alpha granule products, platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG), prior to challenge (baseline) and at 5 min and 19 h after challenge with ragweed antigen. Airway segments challenged with normal saline were used as controls. Five minutes after antigen challenge, levels of platelet products in BAL fluid were not elevated from baseline or normal saline control levels. However, 19 h after antigen challenge, a 10-fold increase in platelet products in BAL fluids was found. The mean PF4 levels increased from baseline and saline control values of less than 1.0 to 7.2 ng/ml (p less than 0.05) 19 h after antigen challenge. beta-TG increased from baseline and control levels of less than 1.0 to 6.6 ng/ml (p less than 0.05). Elevations in PF4 and beta-TG were highly correlated with each other (r = 0.98, p less than 0.0001). Levels of platelet products during the 19-h response correlated with albumin, with kinins, with the prostaglandins 6-keto-PGF1 alpha, PGE2, and PGF2 alpha, and with the eosinophil-derived proteins, eosinophil-derived neurotoxin and eosinophil peroxidase. We conclude that platelet activation in the lung is a feature of the late inflammatory response to antigen challenge and that platelets may play an important role in allergic inflammation and asthma.

Adult↗

PubMed1992

Ozone-induced acute tracheobronchial epithelial injury: relationship to granulocyte emigration in the lung.

To investigate the relationship between granulocyte emigration and epithelial injury in specific airway generations of the tracheobronchial tree following short-term ozone exposure, we exposed rhesus monkeys for 8 h to 0.00 (controls) or 0.96 ppm ozone with post-exposure periods of 1, 12, 24, 72, and 168 h in filtered air before necropsy. There were five control and three exposed monkeys for each of the post-exposure times for a total of 20 monkeys. Neutrophils isolated from peripheral blood and labeled with 111In-tropolonate were infused in the cephalic vein in unanesthetized monkeys (except the 1-h group) 4 to 5 h before necropsy. The trachea and microdissected bronchi (fourth and ninth generations) and respiratory bronchioles (fifteenth generation) from the right upper lobe of each monkey were examined by electron microscopy. Labeled neutrophil influx into lung tissue and bronchoalveolar lavage fluid (BALF) was maximal at 12 h and returned to baseline by 24 h after exposure. This was in contrast to total neutrophils (labeled and unlabeled) in BALF, which were significantly elevated through 24 h after exposure but returned to baseline by 72 h. Lavage protein was significantly elevated at 24 h after exposure but was at control levels at all other times. Morphometric observations showed epithelial necrosis at 1 and 12 h in the trachea and bronchioles but continued to be observed in significant numbers at 24 h after exposure in bronchi. A significant increase in the labeling index of epithelial cells was observed at 12 h only in bronchi. Epithelial necrosis and repair was associated with the presence of granulocytes in the epithelium and interstitium of all airway levels. However, eosinophils were maximally increased in the epithelium and interstitium of bronchi at 24 h after exposure when epithelial necrosis was maximal in these airways and when lavage protein was significantly elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Characterization of platelet-activating factor synthesized by normal and granulocyte-macrophage colony-stimulating factor-primed human eosinophils.

Platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3- phosphocholine) is a mediator involved in the pathogenesis of inflammatory diseases associated with tissue eosinophil infiltration. Previous studies utilizing bioassay or assaying enzymes associated with PAF biosynthesis have suggested that human eosinophils produce PAF. The present study has extended these initial studies by identifying and quantifying the different PAF molecular species and analogues synthesized by human eosinophils in response to A23187 and f-Met-Leu-Phe (FMLP). Gas chromatography-mass spectrometric analysis indicated that A23187-stimulated eosinophils produce at least three molecular species of PAF. The predominant species is 1-hexadecyl-2-acetyl-GPC (16:0) followed by 1-octadecyl-2-acetyl-GPC (18:0) and 1-octadecyl-2-acetyl-GPC (18:1). Eosinophils stimulated with FMLP produce approximately 100-fold smaller quantities of PAF relative to those produced in response to A23187 and only the 16:0 molecular species could be measured. A small percentage (comprising between 2 and 5%) of the 2-acetylated phospholipids produced by eosinophils was the 1-acyl analogue of PAF. Long-term (72 hr) incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in a three- to fourfold increase in PAF synthesis from eosinophils stimulated with FMLP, without changes in the profile of PAF molecular species or in the percentage of the 1-acyl analogue of PAF. These data indicate that human eosinophils can produce various molecular species of PAF and that this process can be quantitatively enhanced by GM-CSF.

Calcimycin↗

PubMed1991

Arachidonic acid metabolism during antigen and ionophore activation of the mouse bone marrow derived mast cell.

This study has examined the metabolism of arachidonic acid in the mouse bone marrow-derived mast cell (BMMC) during immunologic and nonimmunologic activation. The predominant pools of endogenous arachidonate in the mast cells were found in ethanolamine (46%), choline (39%) and inositol (14%) containing glycerolipids. Initial studies established conditions where equilibrium labelling of these major phospholipids in the BMMC could be reached. Upon challenge, arachidonate was lost from all major phospholipid classes (phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylinositol). There was a small but significant increase in the amount of label associated with phosphatidic acid during cell activation. Arachidonate was distributed among 1-acyl, 1-alkyl and 1-alk-1-enyl-linked subclasses of PC and PE. The rank order of loss of labelled arachidonate from the major PE and PC subclasses during antigen and ionophore activation was 1-alk-enyl-2-arachidonoyl-GPE greater than 1-acyl-2-arachidonoyl-GPC greater than 1-acyl-2-arachidonoyl-GPE greater than 1-alkyl-2-arachidonoyl-GPC. Labelled products released into the supernatant fluids and free arachidonic acid within the cell accounted for the bulk of arachidonate lost from phospholipids. Labelled products in the supernatant fluids were composed of LTB4, LTC4, PGD2 and free arachidonic acid. BMMC phospholipids were also labelled for 24 hr with [3H]choline, [3H]myoinositol or [14H]ethanolamine and labelled 2-lyso phospholipids were measured after cell activation. Radioactivity in lysophospholipids from PC, PE and PI increased significantly between 30 s and 2 min after antigen activation and then declined. Taken together, these studies suggest that arachidonate is mobilized predominantly from PE and in particular 1-alk-1-enyl-2-arachidonoyl-GPE by the direct removal of arachidonate from the sn-2 position of the molecule. Most of this arachidonate is then released from cells as eicosanoids or free fatty acid.

Animals↗

PubMed1991

Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology.

As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells.

Cell Separation↗

PubMed1991

Studies on the reduction of endogenously generated prostaglandin G2 by prostaglandin H synthase.

Prostaglandin H synthase oxidizes arachidonic acid to prostaglandin G2 (PGG2) via its cyclooxygenase activity and reduces PGG2 to prostaglandin H2 by its peroxidase activity. The purpose of this study was to determine if endogenously generated PGG2 is the preferred substrate for the peroxidase compared with exogenous PGG2. Arachidonic acid and varying concentrations of exogenous PGG2 were incubated with ram seminal vesicle microsomes or purified prostaglandin H synthase in the presence of the reducing cosubstrate, aminopyrine. The formation of the aminopyrine cation free radical (AP.+) served as an index of peroxide reduction. The simultaneous addition of PGG2 with arachidonic acid did not alter cyclooxygenase activity of ram seminal vesicle microsomes or the formation of the AP.+. This suggests that the formation of AP.+, catalyzed by the peroxidase, was supported by endogenous endoperoxide formed from arachidonic acid oxidation rather than by the reduction of exogenous PGG2. In addition to the AP.+ assay, the reduction of exogenous versus endogenous PGG2 was studied by using [5,6,8,9,11,12,14,15-2H]arachidonic acid and unlabeled PGG2 as substrates, with gas chromatography-mass spectrometry techniques to measure the amount of reduction of endogenous versus exogenous PGG2. Two distinct results were observed. With ram seminal vesicle microsomes, little reduction of exogenous PGG2 was observed even under conditions in which all of the endogenous PGG2 was reduced. In contrast, studies with purified prostaglandin H synthase showed complete reduction of both exogenous and endogenous PGG2 using similar experimental conditions. Our findings indicate that PGG2 formed by the oxidation of arachidonic acid by prostaglandin H synthase in microsomal membranes is reduced preferentially by prostaglandin H synthase.

Animals↗