Effects of a specific inhibitor of the 5-lipoxygenase pathway on mediator release from human basophils and mast cells

We have characterized the effect of a specific inhibitor of the 5-lipoxygenase pathway on mediator release from human basophils and lung mast cells. In four experiments with purified basophils the phenothiazine derivative, L651-392, proved to be an effective inhibitor of leukotriene (LT) C4 release in the range 1-10 microM (P less than .005) although the release of histamine was unaffected by the highest doses of the drug. The release of 5-hydroxyeico-satetraenoic acid (5-HETE) from purified basophils (n = 2) was also reduced from 8.6 ng/10(6) basophils to below the limit of detection (1.2 ng/10(6) basophils). Intermediate concentrations of L651-392 (0.5 microM) reduced both the rate at which LTC4 was produced and the final concentration. The release of LTC4 was complete within 20-30 min and was unaffected by increased incubation periods (up to 90 min). HPLC analysis revealed that the drug was not promoting the metabolism of LTC4 to LTD4 or LTE4. Basophils (average purity = 78 +/- 3, n = 3) labeled with 3H-arachidonic acid (AA) were challenged with anti-IgE (0.1 microgram/ml) and the lipid mediators analyzed by HPLC. Control cells released 3H-LTC4, 3H-HETE, unmetabolized 3H-AA, and an unidentified metabolite, whereas those pretreated with L651-392 released only 3H-AA and the unknown metabolite with no detectable 3H-LTC4 or 3H-HETE. The specificity of the drug for the 5-lipoxygenase pathway was confirmed in three experiments with human lung mast cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of the complement peptides, C5a and C5a des Arg on human basophil and lung mast cell histamine release.

The ability of purified anaphylatoxins to induce human lung mast cell mediator release was investigated. In eight anti-IgE responsive (histamine release = 22 +/- 5%, mean +/- SEM) mast cell preparations of 1-96% purity, C5a and C5a des Arg (0.55 pg/ml to 55 micrograms/ml), failed to elicit or potentiate histamine release; lung fragments were similarly unresponsive. The related peptide C3a was also inactive. All anaphylatoxins failed to induce mast cell leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) release. LTC4 release was also negligible from basophils where C5a was a potent histamine release stimulus. Supernatants from C5a-challenged mast cells remained fully active on basophils, excluding carboxypeptidase inactivation of C5a as an explanation for the lung mast cell results. In contrast to lung, skin mast cells were C5a-responsive (histamine release = 8 +/- 1%, at 55 micrograms/ml, n = 2). We conclude that C5a, though devoid of activity on the human lung mast cell, is a human basophil and skin mast cell secretagogue. These findings demonstrate significant organ-specific heterogeneity in mast cell responsiveness.     

Interleukin 5 modifies histamine release and leukotriene generation by human basophils in response to diverse agonists

Human interleukin 5 (IL-5), known as a selective colony-stimulating factor of the eosinophil lineage and activator of mature eosinophils, also profoundly influences the mediator release profile of human basophils. IL-5 by itself triggers neither granule release nor de novo synthesis of lipid mediators. However, at low concentrations (0.1-10 ng/ml), IL-5 rapidly primes basophils for enhanced histamine release and leukotriene C4 (LTC4) generation in response to all established basophil agonists. LTC4 generation is more strongly affected by IL-5 than histamine release. In particular, IL-5 renders basophils capable of producing large quantities of LTC4 in response to C5a, which, without the cytokine, induces histamine release only. Finally, IL-5 renders basophils responsive to agonists (neutrophil-activating peptide 1 and C3a), which are otherwise inefficient triggers for basophil mediator release. The effects are similar to the recently established bioactivity of IL-3 on basophils, with the exception of its influence on IgE-dependent basophil activation, which is less pronounced. Thus, IL-5 strongly modulates the function not only of eosinophils but also of basophils, the two major effector leukocyte types involved in allergic inflammatory processes, e.g., in asthma.     

Mechanism of inhibitory effects of azelastine on smooth muscle contraction.

The mechanism of inhibitory effects of azelastine, an antiallergic and antiasthmatic agent, on depolarization- and alpha-1 adrenergic agonist-induced contractions of intact smooth muscle was studied. The effects of azelastine on membrane currents were determined in isolated guinea pig ileum smooth muscle cells with the whole-cell clamp technique; the effects on contraction were evaluated in receptor- and G-protein-coupled, alpha-toxin-permeabilized rabbit femoral artery and portal vein smooth muscle strips. Azelastine (1-20 microM), like dihydropyridines, inhibited spontaneous rhythmic and high K(+)-induced contractions, mainly through inhibition of the voltage-dependent (L-type) Ca++ current. The tonic component of high K+ contractions was inhibited more than the phasic component, correlating to voltage-dependent inhibition of Ca++ current by the drug. Azelastine (IC50 of 0.25 microM), a known histamine blocker, also reversibly inhibited alpha-1 agonist-induced contractions in the presence and absence of extracellular Ca++. Both major pathways of pharmacomechanical coupling, agonist-induced Ca++ release from the sarcoplasmic reticulum and Ca++ sensitization of the regulatory/contractile apparatus were blocked by the same concentration of drug in permeabilized as in intact muscle. Inositol 1,4,5-trisphosphate-induced Ca++ release and guanosine 5'-O-(tau-thiotriphosphate)-induced Ca++ sensitization, however, were not inhibited. Azelastine at high (greater than 10 microM) concentrations reversibly inhibited Ca(++)-activated contraction, more potently at lower Ca++ concentration and in phasic smooth muscle, but inhibited neither adenosine 5'-O-(tau-thiotriphosphate)-induced, Ca(++)-independent nor phorbol ester-induced contractions. These results indicate that azelastine is a genuine Ca++ antagonist that inhibits voltage-gated Ca++ inward current and agonist-induced Ca++ release and Ca++ sensitization.     

Doxorubicin blocks the increase in intracellular Ca++, part of a second messenger system in N1E-115 murine neuroblastoma cells

Exposure of differentiated N1E-115 murine neuroblastoma cells, microinjected with the Ca(++)-sensitive photoprotein aequorin, to doxorubicin for 1 hr, but not for 2 min, produced a reversible block of the rise in intracellular free Ca++ [( Ca++]i) produced by histamine. The resting level of [Ca++]i was increased from 0.23 to 1.22 microM (P less than 0.05) by 10(-4) M histamine. After exposure to 10(-6) M doxorubicin for 1 hr, histamine increased [Ca++]i to only 0.34 microM (P less than 0.05 compared to the histamine alone value). Doxorubicin exposure for 1 hr completely blocked the increase in inositol trisphosphate caused by histamine. There was no block by doxorubicin of the release of intracellular Ca++ after microinjection of the cells with inositol 1,4,5-trisphosphate. Based on the results from studies with differentiated N1E-115 neuroblastoma cells doxorubicin may: 1) block the histamine-induced rise in [Ca++]i by decreasing synthesis of inositol polyphosphates, 2) block plasma membrane Ca++ channels that allow entry of extracellular Ca++ in response to histamine and/or 3) prevent recovery of histamine receptors after desensitization.     

Stereoselective modulation of ryanodine-sensitive calcium channels by the delta isomer of hexachlorocyclohexane (delta-HCH).

delta-Hexachlorocyclohexane (delta-HCH) is shown to be 30-fold more potent as a positive inotropic agent with rat atrial strips compared with lindane (gamma-HCH). Threshold and ED50 values for enhanced contractile force at a pacing frequency of 0.5 Hz are less than 1 microM and 2.2 microM for delta-HCH and 40 microM and 63 microM for gamma-HCH, respectively. Contracture developed in atria exposed to greater than 4 microM delta-HCH (ED50 = 11 microM) but not in atria exposed to gamma-HCH. Uptake and release of Ca++ measured from actively loaded cardiac sarcoplasmic reticulum (SR) vesicles is measured with antipyrylazo III. Although delta-HCH (30 microM) decreases Ca(++)-dependent ATPase by 20%, it does not significantly alter Ca++ loading in the presence of ruthenium red. Addition of delta-HCH (5-50 microM) after loading is complete causes rapid, dose-dependent release of Ca++ from SR. Ca++ release induced by delta-HCH is markedly stereoselective. Compared with gamma-HCH (50 microM), delta-HCH (50 microM) induces a nearly 20-fold higher initial rate of Ca++ release (4.3 nmol of Ca++/mg/sec). Studies with [3H]ryanodine demonstrate that delta-HCH sharply inhibits Ca(++)- or daunorubicin-activated radioligand binding (IC50 = 37 and 25 microM, respectively, logit slope = 2). Inhibition of [3H]ryanodine-binding by delta-HCH is stereoselective inasmuch as IC50 values for alpha, beta and gamma isomers are greater than 100 microM. The delta-HCH modified Ca++ channel appears to proceed by a noncompetitive mechanism (reducing Bmax in equilibrium experiments) with respect to the conformationally sensitive binding site for [3H]ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contrast media are incomplete secretagogues acting on human basophils and mast cells isolated from heart and lung,but not skin tissue

To investigate the mechanisms of anaphylactoid reactions to radiocontrast media, in vitro mediator release induced by three iodinated contrast agents was examined using peripheral blood basophils and mast cells purified from human lung parenchyma, heart, and skin tissues. Three iodinated contrast agents, sodium and meglumine salts of ioxaglic acid, sodium and meglumine salts of ioxithalamic acid, and ioversol, were incubated with basophils purified from peripheral blood and human mast cells isolated and purified from different anatomical sites. Release of preformed (histamine and tryptase) and de novo synthesized mediators (prostaglandin D₂ and leukotriene C₄) into the supernatans was determined at various contrast medium concentrations after incubation for 60 min. Ioxaglate (0.2–0.3 M), ioxithalamate (0.3–0.5 M), and to a lesser extent ioversol (0.3–0.5 M) induced histamine release from basophils in a concentration-dependent manner. All three induced the release of preformed mediators (histamine and tryptase) from human lung, but not from skin mast cells. They also induced histamine and tryptase release from human heart mast cells. However, they did not induce the de novo synthesis of leukotriene C₄ or prostaglandin D₂ from human basophils or any type of mast cell examined. Cross-linking of IgE by anti-IgE induced the release of leukotriene C₄ or prostaglandin D₂ from human basophils or mast cells. Mannitol, an osmotic stimulus, induced the release of histamine from human basophils, but to a lesser extent from mast cells. These results show that different contrast media can differ in their ability to release mediators from enriched preparations of human basophils and mast cells. The three contrast agents examined act on basophils and mast cells as incomplete secretagogues, causing the release of preformed mediators, but not the de novo synthesis of chemical mediators. It may be useful to measure plasma tryptase levels to detect adverse reactions caused by iodinated radiographic contrast materials.     

Alpha-2 adrenergic inhibition of Ca(++)-evoked [3H]norepinephrine release from synaptosomes is blocked by depolarization

The Ca(++)-evoked release of [3H]norepinephrine was used in these studies to investigate presynaptic regulation of norepinephrine release. In hippocampal synaptosomes, previously unexposed to Ca++ during isolation and superfusion, 1.25 mM Ca++ evoked a modest (4 to 7% of total stores) release of [3H]norepinephrine with 4.5 mM [K+] present. The alpha-2 adrenergic agonist clonidine inhibited 60% of the Ca(++)-evoked [3H]norepinephrine release. The alpha-2 adrenergic antagonists idazoxan and yohimbine reversed clonidine inhibition of release whereas the alpha-1 antagonist prazosin did not. Increasing the [K+] before Ca++ exposure increased [3H]norepinephrine release, and at 20 [K+] the release increased to over 20% of total stores. However, at [K+] above 9 mM, inhibition of Ca(++)-evoked release by clonidine decreased, and by 20 mM [K+] clonidine no longer inhibited release. Release was unaffected by 5 microM idazoxan or the opiate antagonist naloxone at 15 or 20 mM [K+]. The K+ channel blockers tetraethylammonium (5 mM) and 4-aminopyridine (0.1 mM) increased Ca(++)-evoked release almost 4-fold above control (4.5 mM [K+] present). Neither clonidine nor idazoxan affected Ca(++)-evoked release with the K+ channel blockers present. Therefore, even though K+ channel blockers and 20 mM [K+] increase neurotransmitter release, it is not autoreceptor activation by released endogenous norepinephrine that is responsible for blocking alpha-2 inhibition, but the depolarization produced by these treatments. The 20 mM [K+] blockade of alpha-2 inhibition was decreased by lowering the [Ca++] in the superfusion buffer. Therefore, synaptosomal accumulation of Ca++ may partially explain the loss of alpha-2 inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

9-methyl-7-bromoeudistomin D, a powerful radio-labelable Ca++ releaser having caffeine-like properties, acts on Ca(++)-induced Ca++ release channels of sarcoplasmic reticulum.

9-Methyl-7-bromoeudistomin D (MBED), a derivative of eudistomin D isolated from a marine tunicate, induced Ca++ release from the heavy fraction of fragmented sarcoplasmic reticulum (HSR) in the same way as that of caffeine, followed by spontaneous Ca++ reuptake in the Ca++ electrode experiment. The rate of 45Ca++ efflux from HSR vesicles was accelerated markedly by MBED or caffeine in a concentration-dependent manner. The 50% effective concentrations of MBED and caffeine were approximately 1 microM and 1 mM, respectively, indicating that MBED is 1000 times more potent than caffeine in HSR. Procaine, ruthenium red or Mg++ caused concentration-dependent inhibition of MBED-triggered Ca++ release from HSR. The bell-shaped profile of Ca++ dependence for MBED is very similar to that of caffeine. The caffeine-produced maximum response of 45Ca++ efflux was increased further by adenosine-5'-(beta, gamma-methyl-ene)triphosphate, whereas that was not changed by MBED. MBED also caused Ca++ release from sarcoplasmic reticulum (SR) of chemically skinned fibers. These stimulatory effects of MBED on the Ca++ release from skeletal muscle SR were almost indistinguishable from those of caffeine except the difference in potencies. The [3H]ryanodine binding to junctional terminal cisternae membranes was not inhibited by MBED or caffeine. MBED did not cause Ca++ release from the light fraction of fragmented SR and turbidity change of mitochondrial suspension. These observations suggest a most likely idea that MBED binds to the caffeine-binding site in the Ca channel protein and thus produces the potentiation of Ca(++)-induced Ca++ release from SR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of antigen and Ca++-ionophore-induced peptidoleukotriene release from guinea pig lung preparations using high-performance liquid chromatography

This study was undertaken to investigate antigen-induced peptidoleukotriene release from lungs of sensitized guinea pigs using a recently developed solid-phase extraction and high-performance liquid chromatography assay system. This release was compared to the response due to Ca++-ionophore (A23187) challenge. Incubation of lung fragments (0.6 g) from actively sensitized guinea pigs with ovalbumin (3 micrograms/ml) for 20 min at 37 degrees C resulted in the release of 40 to 60 ng of leukotriene (LT)D4 detected in the extracted filtrate (40-50% recovery of LTD4). The amount of LTD4 determined using the high-performance liquid chromatography assay correlated well with the quantity determined by a LTC4 radioimmunoassay. LTD4 release was saturable and was optimal at a tissue concentration of 0.6 g/2.5 ml of buffer. Kinetic analysis of LT generation showed that after antigen challenge, LTC4 levels peaked at 3 min and declined rapidly with time; LTD4 levels then increased significantly, reaching a maximum at 15 min and decreased slightly at 60 min. LTE4 was not detected until 30 min after antigen challenge after which it increased slowly. The kinetic results permit an estimation of the rate of LTD4 and LTE4 formation to be 5 and 0.17 ng/min/g of lung, respectively. In contrast to antigen challenge, LTD4 release from Ca++-ionophore-stimulated lung fragments was not saturable and was biphasic with increasing amounts of tissues. Moreover, LTD4 produced by Ca++-ionophore stimulation could not be detected during the first 10 min but thereafter increased linearly with incubation time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Macrophage inflammatory protein-1 alpha activates basophils and mast cells

Macrophage inflammatory protein-1 (MIP) is a recently cloned cytokine that causes neutrophilic infiltration and induces an inflammatory response. We studied the effect of MIP-1 alpha on histamine secretion from basophils and mast cells. Leukocytes from allergic and normal subjects were studied. MIP-1 alpha caused dose-dependent release of histamine from basophils of 14 of 20 allergic donors at concentrations of 10(-9)-10(-7) M, and the mean release was 13.50 +/- 2.9% at the highest concentration. In the same experiments, the mean histamine release by anti-immunoglobulin E and monocyte chemotactic and activating factor (MCAF) (10(-7) M) was 32 +/- 7% and 31 +/- 3%, respectively. The cells from only 2 of 10 normal subjects released histamine in response to MIP-1 alpha. Histamine release by MIP-1 alpha was rapid, and almost complete within the first 3 min. MIP-1 alpha-induced degranulation was a calcium-dependent noncytotoxic process. MIP-1 alpha showed chemotactic activity for purified basophils that was comparable to MCAF. Both MIP-1 alpha and MCAF at 10(-7) M concentration elicited a chemotactic response that was 40% of the maximal response to C5a (1 microgram/ml). Murine MIP-1 alpha induced histamine release from mouse peritoneal mast cells in a dose-dependent manner. Thus, we have established that MIP-1 alpha is a novel activator of basophils and mast cells.     

Quantitative, phenotypic, and functional evaluation of basophils in myelodysplastic syndromes

BACKGROUND: The myelodysplastic syndromes (MDS) are a group of clonal haematological disorders characterized by cytopenia(s), reduced differentiation-capacity of myeloid cells, and impaired leukocyte function. However, little is known so far about basophil granulocytes in MDS. DESIGN: We have compared the numbers, phenotype and function of basophils in MDS patients with those in healthy subjects. A total numer of 23 patients with MDS (refractory anaemia, n = 8; refractory anaemia with ringsideroblasts, n = 7; refractory anaemia with excess of blasts/refractory anaemia with excess of blasts in transformation, n = 8) and 20 healthy donors were included. RESULTS: The numbers of blood basophils in MDS patients (34.6 +/- 62.9 microL-1) was lower compared to healthy controls (58.6 +/- 64.9 microL-1). Correspondingly, whole blood histamine levels were lower in MDS patients (MDS 34.1 +/- 29.1 ng mL-1 vs. normal donors 72.0 +/- 36.9 ng mL-1). Like "normal" basophils, basophils in MDS expressed interleukin-3 receptor alpha (CD123), E-NPP3 (CD203c), CR1 (CD35), CR3 (CD11b), CR4 (CD11c), membrane co-factor protein (CD46), decay-accelerating factor (CD55) and membrane attack complex inhibitory factor (CD59), as well as receptors for C3a, C5a (CD88), and IgE. Recombinant human (rh) C5a and anti-IgE induced significant release of histamine from basophils in both groups of donors without significant differences between MDS and healthy controls. CONCLUSIONS: The absolute numbers of basophils in MDS patients are lower than in normal donors. However, basophils in MDS do not differ from their "normal counterparts" in terms of complement receptor expression, IgE-receptor expression, or functional responses to respective ligands.     

Inhibition of immunoglobulin, but not polypeptide base-stimulated release of histamine and arachidonic acid by anti-inflammatory steroids

Incubation overnight of purified rat mast cells with glucocorticoids inhibited the release of histamine and [1-14C]arachidonic acid (and its metabolites) stimulated by three immunoglobulin (Ig) E-like secretagogues, anti-IgE, the antigen-ovalbumin and concanavalin A. In contrast, pretreatment with glucocorticoids did not affect either histamine or [1-14C]arachidonic acid release stimulated by somatostatin, compound 48/80 or the calcium ionophore A23187. Glucocorticoids inhibited IgE-like arachidonic acid and histamine release with an order of potency similar to their in vivo anti-inflammatory potencies (i.e., fluocinolone greater than dexamethasone greater than hydrocortisone greater than cortisone). This inhibition required several hours and was temperature-dependent, suggesting a specific glucocorticoid receptor mechanism. IgE-stimulated Ca++ influx was decreased by hydrocortisone pretreatment. These results suggest that glucocorticoids specifically uncouple IgE-mediated calcium flux with subsequent inhibition of histamine and arachidonic acid release.     

Studies on maitotoxin-induced intracellular Ca(2+) elevation in chinese hamster ovary cells stably transfected with cDNAs encoding for L-type Ca(2+) channel subunits.

The aim of the present study was to characterize the role played by different L-type Ca(2+) channel subunits in [Ca(2+)](i) increase induced by maitotoxin (MTX). In the presence of 5 mM extracellular K(+), MTX (0.01-0.5 ng/ml) induced a significant concentration-dependent increase in Fura-2-monitored [Ca(2+)](i) in single Chinese hamster ovary (CHO) cells expressing the alpha(1c) (CHOCalpha9 cells) or the alpha(1c)beta(3)alpha(2)delta (CHOCalpha9beta3alpha2/delta4 cells) subunits of voltage-gated Ca(2+) channels (VGCCs), whereas the effect was much reduced in wild-type CHO cells lacking VGCCs. In addition, MTX effect on CHOCalpha9, CHOCalpha9beta3alpha2/delta4, and GH(3) cells (0.01-0.1 ng/ml) was inhibited by the selective L-type Ca(2+) channel entry-blocker nimodipine (10 microM); a nimodipine-insensitive component was still present, particularly at high (>1 ng/ml) toxin concentrations. In CHOCalpha9beta3alpha2/delta4 cells, depolarizing concentrations of extracellular K(+) (55 mM) reinforced the [Ca(2+)](i) increase induced by MTX (0.1 ng/ml), and this effect was prevented by nimodipine (10 microM). Finally, patch-clamp experiments in CHOCalpha9beta3alpha2/delta4 cells showed that low MTX concentrations (0.03 ng/ml) induced the occurrence of an inward current at -60 mV, which was completely prevented by Cd(2+) (100 microM) and by nimodipine (10 microM), whereas the same dihydropyridine concentration (10 microM) failed to prevent the electrophysiological effects of a higher toxin concentration (3 ng/ml). In conclusion, the results of the present study showed that MTX-induced [Ca(2+)](i) elevation involves two components: 1) an action on L-type VGCCs at the pore-forming alpha(1c) subunit level, which is responsible for the greatest rise of [Ca(2+)](i); and 2) a VGCC-independent mechanism that is present both in excitable and in nonexcitable cells and is responsible for a lower elevation of [Ca(2+)](i).     

Defective histamine release in chronic urticaria.

Histamine release from peripheral blood leukocytes challenged with anti-human IgE was studied in patients with chronic urticaria and nonatopic controls. 19 of 23 controls, but only 6 of 20 patients, released over 20% of the total available leukocyte histamine. The response to anti-IgE concentrations of 1.66, 0.33, 0.066, and 0.013 mug antibody N/ml was significantly lower in patients than in controls. Serum IgE levels were significantly higher in the patients but total histamine content of about 10(7) leukocytes was not. Deuterium oxide (D2O) greatly increased histamine release (in both groups), indicating that the anti-IgE interacted with the basophils of urticaria patients. Passive sensitization of leukocytes with biologically active IgE was achieved in both patients and control subjects whose cells responded to anti-IgE, but was not achieved in either patients or control subjects whose cells were nonresponsive to anti-IgE challenge. 125I-anti-IgE autoradiographic studies revealed no obvious quantitative abnormality in the amount of basophil-bound IgE in chronic urticaria patients. Ionophore stimulation of aliquots of the same leukocytes used for anti-IgE challenge demonstrated that the urticaria patients'' basophils were capable of releasing normal amounts of histamine. Leukocyte cyclic AMP levels in the two groups were not significantly different either in base-line levels or in responsiveness to stimulation with isoproterenol. These data indicate that chronic urticaria patients have a (acquired?) defect in leukocyte histamine release that occurs after the anti-IgE-IgE interaction, but before the actual (second-stage) release process, and that is comparable to the phenomenon of desensitization.     

Inhibitory action of peripheral-type benzodiazepines on dopamine release from PC12 pheochromocytoma cells.

Characteristics of the benzodiazepine inhibition of dopamine (DA) release in PC12 cells were investigated. Diazepam inhibited DA release evoked by high concentrations of extracellular K+ in a dose-dependent manner (IC50, 10 microM). Ro 5-4864 [7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepine- 2-one], a peripheral-type benzodiazepine, also inhibited DA release effectively. PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline carboxamide], a benzodiazepine generally considered a peripheral-type benzodiazepine receptor antagonist, did not antagonize the inhibition induced by diazepam, but rather inhibited DA release itself. On the other hand, the central-type benzodiazepines, clonazepam and Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a] [1,4]benzodiazepine-3-carboxylate) did not affect the DA release. Diazepam, Ro 5-4864 and PK 11195 also inhibited a Ba(++)-current carried by voltage-gated Ca++ channels, and diazepam suppressed an increase in intracellular Ca++ evoked by 80 mM extracellular K+ as measured by the fura-2 method. These results suggest that the inhibitory action of diazepam and other benzodiazepines on DA release from PC12 cells may be mediated through one type of peripheral-type benzodiazepine receptors which are coupled to voltage-gated Ca++ channels and that these receptors may not necessarily be the same as those in other tissues.     

Cutaneous late-phase response to allergen. Mediator release and inflammatory cell infiltration.

To better define the inflammatory infiltrates and kinetics of mediator release during the cutaneous late-phase reaction (LPR), we examined skin biopsies at 8 h, and skin chamber cell counts and mediator release for 12 h after antigen challenge. Compared with the control sites, the antigen-stimulated biopsy sites contained 14 times as many basophils (P less than 0.01) and six times as many eosinophils (P less than 0.001) with one to two fold more mononuclear cells (P less than 0.03) and neutrophils (P less than or equal to 0.01). Similar changes were found in the skin chambers. Although there were neutrophils in the control chamber, they were only twice as numerous in the antigen challenged site (P less than 0.01). Eosinophils were 35-fold (P less than or equal to 0.03) more prevalent in the antigen chamber than the control chamber for hours 8-12 and basophils were noted starting in the eighth hour and were 20-fold (P less than or equal to 0.03) more concentrated in the antigen chamber during the next 4 h. The mononuclear cells were not significantly different between antigen and control blisters. With respect to inflammatory mediators, there was an initial peak of histamine (13.2 +/- 2.9 ng/ml) in the blister fluid at 1 h. The level then fell to approximately 2 ng/ml, followed by a secondary rise starting at the eighth hour and increasing to 9.8 +/- 2.8 ng/ml by the twelfth hour. This secondary increase in histamine correlated significantly (r = 0.81, P less than 0.05) with the observed influx of basophils. PGD2 in the blister fluid rose to 371+/-25 pg/ml during the first 4 h and then slowly decreased to half this level during the last 4 h. Thus, the cutaneous LPR has been shown to manifest a secondary increase in histamine levels and a markedly specific increase in eosinophils and basophils with mediator release apparently being derived from the latter cells.     

Norepinephrine and adenosine triphosphate release in different ratio from guinea pig vas deferens by high potassium chloride, ouabain and monensin

Release of norepinephrine (NE) and ATP from the guinea pig vas deferens evoked by ouabain in combination with monensin or by high KCl was measured by a high-pressure liquid chromatography-ECD and luciferin-luciferase assay, respectively. Ouabain (10-100 microM) induced a concentration-dependent liberation of NE, which was enhanced by 10 microM monensin, a Na+-ionophore. The marked NE release elicited by the combined administration of both the drugs was unaffected by Ca++-removal but was reduced by lowering Na+ from the medium. This NE release in the Ca++-free medium was diminished markedly after treatment with 6-hydroxydopamine or reserpine and in low-temperature (25 degrees C) medium. This release was also decreased by ruthenium red (10-30 microM), an uptake inhibitor of Ca++ to mitochondria, and carbonyl cyanide-m-chlorophenyl hydrazone (10 microM), a metabolic inhibitor. On the other hand, 100 mM KCl caused a moderate, extracellular Ca++-dependent release of NE. ATP-outflow from the tissue evoked by 100 microM ouabain plus 10 microM monensin was almost unaltered by Ca++-removal but was inhibited by 6-hydroxydopamine or prazosin (0.3 microM), whereas release induced by high KCl was reduced by 6-hydroxydopamine and Ca++-free medium but was unaffected by prazosin. ATP/NE ratios at respective maximum effluxes evoked by 100 mM KCl and ouabain plus monensin were 6.59 and 0.22, respectively. These findings suggest that there may be more than one site of corelease for NE and ATP. Ouabain plus monensin seems to produce an extracellular Ca++-independent neuronal release of NE and ATP from the cytoplasmic and vesicular storage sites which predominantly release NE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-independent phosphorylation of smooth muscle myosin light chain by okadaic acid isolated from black sponge (Halichondria okadai)

Previously, we have shown that okadaic acid (OA), isolated from black sponge (Halichondria okadai) causes contraction even in the absence of Ca++ in the saponin-permealized taenia isolated from guinea pig cecum. In the present study, mechanism of action of OA was examined using native actomyosin extracted from chicken gizzard smooth muscle. In the absence of Ca++, OA (0.1-1 microM) induced superprecipitation and increased the Mg++-adenosine triphosphatase activity. The OA-induced superprecipitation was enhanced by Ca++ at a concentration (greater than 0.1 microM) which did not activate the calmodulin-dependent myosin light chain (MLC) kinase. The effect of OA was not affected by the calmodulin inhibitor, trifluoperazine, at a concentration (100 microM) needed to inhibit the Ca++-induced response, but was inhibited markedly by the nonselective kinase inhibitors, amiloride (1 mM) and K-252a (5 microM). The OA-induced superprecipitation in the absence of Ca++ was accompanied by phosphorylation of the 20 K dalton MLC, which also was enhanced by low concentration of Ca++ (greater than 0.1 microM). OA did not change the phosphatase activity which dephosphorylates the phosphorylated MLC. An activator of Ca++- and phospholipid-dependent protein kinase, 12-O-tetradecanoylphorbol 13-acetate (1 microM), did not modulate superprecipitation or phosphorylation of MLC in the presence and absence of OA. Furthermore, inhibitors of Ca++ and phospholipid-dependent protein kinase, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride (400 microM) and polymyxin B (100 micrograms/ml), affected neither superprecipitation nor phosphorylation of MLC induced by OA. With a reconstituted system containing purified myosin and MLC kinase, OA induced only slight phosphorylation of MLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of histamine release from human basophils by human platelet factor 4.

Human basophils were stimulated to release histamine noncytotoxically by purified human platelet factor 4 (PF4) and the synthetic substituent peptide PF4(59-70). Histamine release was augmented significantly by 10(-7) M PF4 and 10(-5) M PF4(59-70), increased in a concentration-dependent manner, and attained a maximum at 3 X 10(-5) M PF4 and 3 X 10(-4) M PF4(59-70) similar to that achieved by goat anti-human myeloma IgE. PF4 (1-60) failed to initiate the release of histamine, which confirmed that the critical determinant of activity is in the carboxy-terminal sequence. Histamine release from basophils by optimally effective concentrations of PF4 and PF4(59-70) reached a plateau by 1-3 min, as contrasted with 10 min or longer for anti-IgE. The elimination of calcium and magnesium from the buffer suppressed the release of histamine by anti-IgE by 79-83%, but had no effect on that elicited by PF4(59-70). The rate of uptake of [125I]PF4 by purified basophils was similar on a molar basis to the rate of release of histamine by the same concentrations of PF4. The noncytotoxic release of histamine from human basophils by PF4 thus is temporally and biochemically distinct from that mediated by IgE and may be similar to that evoked by other polycationic stimuli.