Morphological characteristics and stimulus-secretion coupling in bovine adrenal chromaffin cell cultures

Chromaffin cells isolated from adult bovine adrenal medullae were plated on collagen-coated dishes in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. After 8–16 h in culture, the chromaffin cells had started to develop one, two or, more rarely, three neurites. The neurites grew at a rate of7.25 ± 0.07μm/day showing a linear correlation between process length and age of the culture. The neurites displayed one or more varicosity-like structures. Some neurites made long lasting contacts with other cell bodies and processes. Cytochemical tests indicated positive catecholamine fluorescence in boti cell bodies and processes. Fluorometric determinations indicated that by day 7 the catecholamine content was75.7 ± 1% of that found on day 1 and that noradrenaline and adrenaline represented24.6 ±1% and75.3 ± 1% of the total amine content respectively. Catecholamines were released from these cultured chromaffin cells in response to stimulation by either 56 mm KCl or acetylcholine. The stimulation-induced release of amines was Ca²⁺-dependent and was blocked by increasing the extracellular c oncentration of Mg²⁺. Substitution of (a) extracellular Na²⁺ by either choline or sucrose or (b) Ca²⁺ by Ba²⁺ produced a sharp increase in catecholamine output. Acetylcholine produced a doserelated increase in catecholamine release; half maximal release was produced by3.5 × 10^?5m acetylcholine and maximal release by 10^?4m acetylcholine. The cholinergic receptor of cultured chromaffin cells appears to be nicotinic since catecholamine release was stimulated by nicotine and carbamylcholine but not by pilocarpine. Acetylcholine-evoked catecholamine release was blocked by d-tubocurarine (ID₅₀ = 5.5 × 10^?7m), and by hexamethonium (ID₅₀ = 8 × 10^?6m). However, the acetylcholine-evoked amine release was not blocked by α-bungarotoxin at a concentration (1.25 × 10^?7m) which blocked frog rectus abdominus responses to acetylcholine (10^?6–10^?3m). Tetrodotoxin (5 × 10^?6m) produced47 ± 2% inhibition of the secretory response to acetylcholine (10^?4m) but it did not modify the amine release in response to 56 mm KCl.Adult bovine chromaffin cells in culture display some of the morphological and functional characteristics of sympathetic neurones. Consequently, the bovine chromaffin cell in culture appears to be a promising model for studies on development and secretion.     

Differential effects of concanavalin a on acetylcholine and potassium-evoked release of catecholamines from cultured chromaffin cells

Exposure of cultured chromaffin cells to concanavalin A (5 × 10⁷–10^?5 m) produced a dose-related and non-competitive inhibition of the secretory responses to acetylcholine (5 × 10^?6–10^?4M). Studies with fluorescein isothiocyanate-conjugated concanavalin A indicated that after 20 min of exposure to concanavalin A, the lectin was taken up by the chromaffin cells. The uptake of concanavalin A, which was not modified by colchicine (10^?3 m) treatment, took place without apparent intermediate stage of ‘patch’ or ‘cap’ formation. The responses to acetylcholine were promptly restored by α-methyl-d-mannoside in cells exposed to concanavalin A for different periods of time (10min–8h). After 8h of exposure to concanavalin A, some lectin was still present on cell surfaces, as indicated by the experiments with fluorescein isothiocyanate conjugated immunoglobulin G anti-concanavalin A.The secretory responses to depolarizing concentrations of K⁺ remained unchanged in chromaffin cells exposed to concanavalin A for different periods of time (20 min–8 h). The results indicate that concanavalin A blocks the cholinergic responses of the chromaffin cell and that either the ‘concanavalin A-cholinergic receptor complex’ or the ‘surface concanavalin A receptor’ which modified the cholinergic response remains on the cell surface during the uptake of part of the lectin. Furthermore, the results show that the internalization of concanavalin A does not interfere with the cellular mechanism of amine extrusion.     

D2-but not D1-dopamine receptors are involved in the inhibitory control of alpha-melanocyte-stimulating hormone release from the rat hypothalamus

Summary Release of alpha-melanocyte-stimulating hormone (-MSH) from slices of rat hypothalamus superfused with artificial cerebro-spinal fluid (ACSF) was quantified by radioimmunoassay. Addition of 10^-6 M quinpirole, a D2-dopamine receptor agonist, to the superfusion medium caused a significant (P < 0.001) reduction in the amount of -MSH released upon depolarisation with 50 mM potassium from 319 ±37% to 110 ±16% of basal release in normal ACSF (mean ±S.E.M.). Basal peptide release in the presence of quinpirole was unaffected. Sulpiride, a D2-dopamine receptor antagonist, at a concentration of 10^-6 M, induced a significant (P < 0.05) increase of both basal and potassium-stimulated -MSH release to 203 ±21% and 447 ±88% of basal release in normal ACSF respectively. The latter increases were abolished when sulpiride and quinpirole were added in combination. SK&F 38393-A and SCH 23390, a D1-dopamine agonist and antagonist respectively, had no significant effect on either basal or potassiumstimulated -MSH release. It is proposed that endogenous dopamine exerts an inhibitory control on -MSH release from the rat hypothalamus via D2-dopamine receptors and that in isolated hypothalamic slices there is a tonic inhibition of peptide release due to the activity of this system.     

Selective detection of adenosine A1 receptor-dependent G-protein activity in basal and stimulated conditions of rat brain [35S]guanosine 5′-(γ-thio)triphosphate autoradiography

[³⁵S]Guanosine 5′-(γ-thio)triphosphate autoradiography is a novel technique to detect receptor-dependent activation of G-proteins in brain tissue sections. While an increasing number of reports using this approach are beginning to appear, little effort has been directed to the identification of factors responsible for the heterogeneously distributed [³⁵S]guanosine 5′-(γ-thio)triphosphate signal in basal conditions. The present study demonstrates that endogenously formed adenosine generates a widespread and prominent adenosine A₁ receptor-dependent signal in basal conditions using this technique. Treatment of rat brain tissue sections with the A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine dose-dependently (ec₅₀<10 nM) suppressed basal [³⁵S]guanosine 5′-(γ-thio)triphosphate binding in a region-specific manner, an effect fully mimicked by the adenosine-depleting enzyme adenosine deaminase, and less so by the A₁ antagonist cirsimarin and by caffeine. That adenosine was continuously formed during the incubation is supported by the constant requirements of adenosine deaminase in order to suppress basal radioligand binding and further by the fact that low micromolar concentrations of adenine nucleotides evoked only adenosine-mimicking and fully 8-cyclopentyl-1,3-dipropylxanthine-sensitive binding responses. In the presence of adenosine deaminase, all responses to adenine nucleotides were abolished, indicating that prior conversion to adenosine was required. Upon stimulation, this technique selectively detected A₁ receptor-activated G-proteins, as the non-selective agonists adenosine and 2-chloroadenosine and the A₁-selective agonist N⁶-p-sulfophenyladenosine all evoked only 8-cyclopentyl-1,3-dipropylxanthine-sensitive responses in identical gray matter areas, and also in several white matter areas such as the corpus callosum, anterior commissure, optic tract and cerebellar white matter. Dose–response studies revealed region-specific differences in the magnitude of A₁ receptor-stimulated G-protein activation, with the highest response (nine-fold over basal) detectable in the hippocampus. No response to the A_2A-selective agonist 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5′-N-ethylcarboxamidoadenosine or the A₃-selective agonist 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide was detected in any region.These data reveal that a significant amount of noise inherent to [³⁵S]guanosine 5′-(γ-thio)triphosphate autoradiography can be eliminated by removal of the adenosine signal, a step likely facilitating detection of responses to other receptors. Furthermore, the data establish [³⁵S]guanosine 5′-(γ-thio)triphosphate autoradiography as a novel and selective approach to directly assess A₁ receptor–G-protein coupling in anatomically defined regions of the central nervous system.     

Mechanisms Involved in Activation of Human Eosinophil Exocytosis by Substance P: An in Vitro Model of Sensory Neuroimmunomodulation

Substance P (SP), a tachykinin with a wide range of biological activities including a priming effect on human eosinophil chemotaxis, was investigated for its influence on eosinophil cytotoxic function measured as degranulation of eosinophil-derived neurotoxin (EDN). Peripheral blood was obtained from healthy volunteers and the degranulation assays were performed using radioimmunoassay (RIA). SP and its C-terminal elicited EDN release in a time-dependent mode at a narrow range of doses with optimal activity of 10^?6M. FK888 (NK-1 receptor antagonist) inhibited EDN release stimulated by SP in dose dependency, also a complete inhibition was observed when eosinophils were preincubated with 1000ng/ml pertussis toxin (PTX). Pre-exposure of eosinophils to staurosporine resulted in blockage of SP-induced EDN release in a dose-dependent mode. On the other hand, SP at 10^?7M and 10^?8M primed eosinophils to suboptimal dose (10^?8M) of Platelet activating factor (PAF) resulting into significant enhancement of EDN release. SP(4–11) fragment showed a similar activity while SP(1–4) fragment was not active. SP priming of eosinophils was not affected by Ca²⁺ depletion, however, it caused a change in the pattern of the intracellular calcium influx against the suboptimal dose of PAF. These results suggest that SP i) may induce human eosinophil matrix protein degranulation through a receptor mediated mechanism coupled to PTX sensitive G protein(s) with the probability of linkage to phospholipase C activation, and, ii) primes human eosinophils for an exalted inflammatory response through a Ca²⁺ independent pathway.     

Ion-induced release of LHRH,TRH and α-MSH from hypothalamic synaptosomes and its inhibition by vinblastine

Homogenates of rat hypothalamic tissue were fractionated by means of discontinuous sucrose density gradient centrifugation. Immunoreactive luteinizing hormone releasing hormone (LHRH), thyrotropin releasing hormone (TRH), and -melanocyte stimulating hormone (-MSH) were found to be concentrated in the synaptosome-enriched fraction. This fraction was suspended in 0.32 M sucrose and the release of the three peptides was investigated. After incubation, the synaptosomes were re-isolated by ultrafiltration, and the concentration of each peptide in the ultrafiltrate was determined by radioimmunoassay. When the synaptosomal fraction was incubated at 30° C in 0.32 M sucrose containing either 60 mM K⁺-2 mM Ca²⁺ or 140 mM Na⁺ alone a release of LHRH, TRH, and -MSH occurred. Of the total content 30–50% of LHRH but only about 10% of TRH and -MSH was releasable. When the synaptosome preparation was preincubated for 30 min at 30°C with 10^–4 M vinblastine. K⁺- as well as Na⁺-induced release of LHRH, THR, and -MSH was inhibited, and the stimulatory effect of each cation was almost totally blocked by preincubation with 5×10^–4 and 10^–3 M vinblastine. The inhibitory action of vinblastine (5×10^–4 M) did not affect the oxidation of glucose to CO₂ by the synaptosomes. The results of the present investigation demonstrate that synaptosome-enriched fractions of hypothalamic origin are more stable with respect to LHRH, TRH, and -MSH release during incubation in isotonic sucrose than they are in ionic solutions, and that the peptides are released by a vinblastine-sensitive mechanism.Supported by grants from the National Institute of Arthritis, Metabolism, and Digestive Diseases (AMO 1237), the National Institute of Child Health and Human Development (HDO8672), and the National Institutes of Health contract (5-P17-HL1487-06)Supported by Grant No. 512-6951 from the Danish Medical Research Council     

Modulation by 310-1310-1310-1stimulation of IgE-mediated14C-serotonin release from rat mast cells

The formaldehyde method was used to examine the effects of clonidine and methoxamine on IgE-mediated¹⁴C-serotonin release from rat mast cellsin vitro. Clonidine (10^?11?10^?8 M) caused dose-related enhancement of the mediator release 7min after the antigen challenge yohimbine (10^?6 M) blocked this enhancement by clonidine (10^?6 M), but prazosin (10^?6 M) did not. Methoxamine did not enhance this immunological release reaction at concentrations up to 10^?6 M. PGE₁ (2×10^?8?2×10^?5 M), isoproterenol (10^?10?10^?8 M), dopamine (4×10^?8?4×10^?8 M) and aminophylline (6×10^?6?6×10^?4 M) caused dose-related inhibition of this mediator release 1 min after antigen challenge. Clonidine (10^?13?10^?12 M), but not methoxamine (10^?8?10^?6 M), reversed dose-dependently this inhibition of mast cells by PGE₁ (2×10^?6 M), isoproterenol (10^?8 M), dopamine (4×10^?6 M); yohimbine (10^?8 M) antagonized this reversing action of clonidine (10^?12 M), but prazosin (10^?10 M) did not. Neither clonidine (10^?14?10^?11 M) nor methoxamine (10^?8?10^?6 M) reversed the inhibitory action of aminophylline (2×10^?4 M). These results suggest that clonidine enhances IgE-mediated¹⁴C-serotonin release from rat mast cells and also antagonizes the inhibition of mast cells by PGE₁, isoproterenol and dopamine, but not by aminophylline in this immunological reaction through α₂-adrenergic receptors, and that the inhibition of adenylate cyclase of mast cells is one of the biochemical actions of α₂-adrenergic mechanisms.     

Presence of a high affinity uptake system for catecholamines in cultured bovine adrenal chromaffin cells

The uptake of catecholamines was investigated in bovine adrenal chromaffin cells cultured for 2 and 7 days. These cells, after their attachment onto the collagen-coated plates, began to develop processes which progressively increased in length with time in culture. Process outgrowth of a few cells was apparent on day 2 in culture. However, by day 7 most of the chromaffin cells possessed processes with a mean length of52.68 ± 1.27μm(n= 202). There was found to exist in both 2-day and 7-day-old cultures an uptake mechanism for (?)noradrenaline which in many aspects simulated the neuronal uptake₁ transport system in that it was saturable, followed Michaelis-Menten kinetics, had a high affinity for (?)noradrenaline with apparentK_m values ranging from 0.35–0.48 μm (n = 4) and 0.46 ? 0.67 μm (n = 4) on day 2 and day 7 respectively. This uptake process was blocked by low concentrations of desipramine (10^?7 M), a specific inhibitor of uptake₁, and like the neuronal uptake mechanism exhibited absolute Na⁺ dependency in which Li⁺ could not adequately replace Na⁺. However, uptake by the chromaffin cells did not show any stereochemical specificity towards the (?) form of noradrenaline nor did it exhibit structural specificity for (?)noradrenaline over (?)adrenaline.Therefore, it appeared that a high affinity accumulating mechanism for noradrenaline was present in cultured chromaffin cells isolated from adult bovine adrenal medulla. The properties of this uptake system did not vary significantly from day 2 to day 7 and thus did not parallel the morphological changes seen in culture.     

The muscarinic agonist pilocarpine inhibits DNA and interferon-γ synthesis in peripheral blood mononuclear cells

Lymphocyte basal DNA synthesis and proliferative responses to phytohemagglutinin (PHA) showed a dose-dependent (5 × 10⁻⁵ − 5 × 10⁻³ M) inhibition by the muscarinic agonist pilocarpine, in contrast to the basal enhancing effect produced by the M₂ muscarinic-nicotinic agonist carbacol. The effect of pilocarpine was reversed by both atropine (1 × 10⁻⁶ M) and pirenzepine (1 × 10⁻⁷ − 1 × 10⁻⁸ M), M₁ − M₂ and M₁ muscarinic antagonists, respectively. The effect of pilocarpine may thus be specific for the M₁ muscarinic receptor. Pilocarpine also inhibited interferon-gg (IFN-γ)-PHA induced production, but was unable to reverse the pokeweed mitogen (PWM)-induced DNA synthesis. Distinct immunoregulatory activities are suggested for cholinergic muscarinic receptors M₁ and M₂.     

Attenuation of induced-anxiety in rats by chlordiazepoxide: Role of raphe dorsalis benzodiazepine binding sites and serotoninergic neurons

In chronically implanted awake rats, microinjections of chlordiazepoxide (5 × 10^?7M) into the dorsal raphésignificantly attenuated the inhibition of lever-pressing for food elicited by a signal of punishment. This effect is abolished by prior application of 5,7-dihydroxytryptamine into the dorsal raphé(3 weeks after the infusion of the neurotoxin, dorsal raphétryptophan hydroxylase activity was reduced to 25% of control values). Furthermore, the disinhibitory effect of intra raphéchlordiazepoxide can be mimicked or potentiated by intra raphédorsalis application of serotonin (10^?7 or 10^?8 M, respectively). Further evidence for a crucial interaction between benzodiazepines and serotoninergic processes are provided by in vitro experiments showing that chlordiazepoxide or diazepam (10^?5 M) are able to facilitate the K⁺ -evoked [³H]serotonin release from rat midbrain slices. Finally, a high density of [³H]flunitrazepam binding sites was found in the dorsal (and the median) raphénucleus, the Kd and B_max values being not altered by prior infusion of 5,7-dihydroxytryptamine.These in vitro data suggest possible means by which intra raphé(and perhaps peripherally administered) benzodiazepines may affect the activity of serotoninergic neurons and thereby produce their effects on experimental anxiety.     

Characterization and localization of ouabain receptors in Schistosoma mansoni

Saturable ouabain binding sites were detected in intact male schistosomes. The K_D for binding of ouabain to Schistosoma mansoni was 2.6 × 10^?7 M and to Schistosoma japonicum 2.9 × 10^?7 M. The binding of ouabain to the receptor demonstrated pharmacological specificity. Binding sites, obtained by differential centrifugation, were associated with fractions containing tegument (58%) and microsomal membranes (33%). Binding sites were concentrated in tegumental membranes, i.e., a 19-fold enrichment of receptors was found in membranes isolated from intact schistosomes exposed to Triton X-100. The antiparasitic drugs praziquantel (IC₅₀ = 9 × 10^?7 M) and Ro-11-3128 (IC₅₀ = 5 × 10^?6 M) inhibit binding of [³H]ouabain to intact parasites in a pharmacologic specific manner. Both praziquantel and Ro-11-3128 are without effect (IC₅₀ > 10^?4 M) on [³H]ouabain binding to homogenates of S. mansoni. These findings indicate that ouabain receptors are present in S. mansoni and that these receptors represent Na⁺-K⁺ pump sites. In addition, the characteristics and location of these receptors are consistent with previous observations on the physiological action of ouabain on the parasite.     

Affinity of the interaction between Fcgamma receptor type III (FcγRIII) and monomeric human IgG subclasses. Role of FcγRIII glycosylation

Binding of the Fc region of IgG antibodies to low affinity Fcγ receptors (FcγR) triggers important effector functions in the immune system. The type IIIb FcγR (FcγRIIIb or CD16) is a heavily glycosylated protein anchored to the membrane of neutrophils by a glycosylphosphatidylinositol link. This receptor contributes to cell activation by IgG immune complexes. To better understand the nature of the ligand-receptor association, we have studied the affinity and kinetics of the interaction between human IgG subclasses and two soluble forms of FcγRIIIb (sFcγRIIIb or sCD16) corresponding to the 188 N-terminal amino acids of the extracellular region of the receptor, a glycosylated one made in eucaryotic cells (euc.sCD16) and a non-glycosylated one (proc.sCD16) made in Escherichia coli. Experiments using a BIAcore^TM instrument, to measure protein binding in real time, showed that monomeric human IgG1 and IgG3, but not IgG2, IgG4, IgA and divalent antigen-binding fragments (F(ab′)₂) of IgG1, bound to immobilized euc.sCD16 with an affinity constant (K_A) of 1.3 ± 0.6 × 10⁶ M^?1 and 2.6 ± 0.4 × 10⁵ M^?1, respectively. The affinity constant of proc.sCD16 for human IgG1 was in the same range (1.1 ± 0.2 × 10⁶ M^?1), whereas that for human IgG3 was twofold higher (4.2 ± 0.4 × 10⁵ M^?1). The specificity of the non-glycosylated receptor for human IgG subclasses bound to Sepharose was IgG1 > IgG3 ? IgG4 ? IgG2. Thus, the extracellular polypeptide of FcγRIIIb dictates the interaction of the receptor with IgG subclasses although glycosylation plays an inhibitory role in the interaction with human IgG3.     

Steroid Sex Hormones and Macrophage Function: Modulation of Reactive Oxygen Intermediates and Nitrite Release

PROBLEM: In general, females have a more active immune response than do males. The effects of female sex hormones on lymphocytes have been studied extensively but their effects on macrophages are poorly understood. METHOD: In this study, peritoneal macrophages (M0) obtained from male rats were treated in vitro with estradiol (E₂), progesterone (P), testosterone (TS), or hydrocortisone (HC) and their effects on superoxide, hydrogen peroxide, and nitrite release determined. RESULTS: At concentrations between 10^?10 and 10^?9 M, female and male sex hormones had no significant effect on superoxide release but, at concentrations above or below that range, these hormones stimulated the release of these reactive oxygen intermediates (ROI). In contrast, M0 treated with HC generally exhibited either unaltered or reduced ROI release. CONCLUSIONS: These findings suggest that female sex hormones regulate ROI release by Mø in a manner not entirely shared by other steroid hormones. At most concentrations used, E₂, P, TS, and HC significantly inhibited nitrite release by Mø. However, with 10^?10 M of E₂ or 10^?9M of P, nitrite release by Mø was not affected. In the presence of anti-TNF antibody, the amounts of superoxide and hydrogen peroxide release were moderately reduced but nitrite release was dramatically inhibited. The sensitivity of Mø to variations in the concentrations of female sex hormones may contribute to gender-related differences in the immune response.     

Regulation of cell cyclic AMP in medullary thick ascending limb of Henle in a rat model of chronic renal failure

Chronic renal failure (CRF) is accompanied by adaptive changes in electrolyte reabsorption in the thick ascending limb of Henle of surviving nephrons. To study the cellular mechanism of this adaptation, we measured intracellular cAMP in micro-dissected medullary thick ascending limb (mTAL) segments in rats with CRF. mTAL exhibited in CRF an increase of basal cAMP from 25.6 ± 10.0 in controls to 65.8 ± 11.3 fmol mm^?1 tubule in CRF (P < 0.05). Vasopressin and calcitonin stimulated mTAL adenylate-cyclase in a dose-dependent manner in controls but failed to stimulate in CRF. Likewise, maximal stimulation with 10^?3 M 3-isobutyl-1-methylxanthine (IBMX) plus 10^?5 M forskolin increased cAMP in controls to 63.0 ± 16.0 but not in CRF, where maximal stimulated values remained at 63.1 ± 18.8 fmol mm^?1 tubule (P NS). Alpha₂-adrenoreceptor activation with clonidine at concentrations ranging from 10^?8 to 10^?6 M diminished cAMP production by 37% in CRF (P < 0.05), whereas no differences were found in controls. Thus, the basal intracellular cAMP is increased in rat mTAL in CRF. The finding that neither forskolin nor vasopressin were able to further augment intracellular cAMP would suggest that stimulatory pathways of the adenylate-cyclase system are activated in the basal state. However, mTAL cells in CRF seem to retain the response of normal epithelium to inhibitory pathways such as the one mediated by alpha₂-adrenoreceptors.     

Effects of "systemic" budesonide concentrations on in vitro allergen-induced activation of blood mononuclear cells isolated from asthmatic patients

Blood levels of inhaled corticosteroids are significantly lower than those measured in the lung, but their concentration could still have anti-inflammatory effects. To determine whether budesonide, at concentrations similar to those obtained in blood after drug inhalation (10 ^?9 M), could downregulate the allergen-induced activation of mononuclear cells, we studied 21 atopic patients, sensitized to Dermatophagoides pteronyssinus (Der p). On blood mononuclear cells, isolated from these patients, incubated with Der p allergen extract and with or without budesonide, we evaluated: 1) the proliferative response of T cells; 2) the expression of two surface activation markers, the HLA-DR antigens and the interleukin (IL)-2 receptors; and 3) the release of cytokines known to modulate the allergic processes. Allergen-induced T-cell proliferation was associated with increased HLA-DR antigen and IL-2 receptor expression (P < 0.001), and with increased release of IL-2, interferon-gamma (IFN-γ), IL-1β, tumor necrosis factor-alpha (TNF-α), and granulocyte/macrophage colony-stimulating factor (GM-CSF). The addition of budesonide at the beginning of the cell cultures induced a dose-dependent inhibition of T-cell proliferation, still significant (P < 0.05) at the lowest concentrations tested (10 ^?9 and 10^?10 M). A significant inhibitory effect on T-cell proliferation was also present when budesonide (10 ^?9 M) was added to the cell cultures 3 or 5 days after the beginning of the cell cultures. In addition, budesonide 10^?9 M significantly decreased the expression of IL-2 receptors (P < 0.05), but not of HLA-DR antigens, and significantly reduced the release of IL-1β and GM-CSF (JP < 0.05), but not of IL-2, IFN-γ, and TNF-α. Thus, the blood concentrations of budesonide, after drug inhalation, may exert some anti-inflammatory effect, downregulating both “local” (through the bronchial circulation) and “systemic” allergen-specific immune reactions.     

Altered ventriculo-arterial coupling during exercise in athletes releasing biomarkers after endurance running

Exercise can lead to release of biomarkers such as cardiac troponin T (cTnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP), a poorly understood phenomenon proposed to especially occur with high-intensity exercise in less trained subjects. We hypothesised that haemodynamic perturbations during exercise are larger in athletes with cTnT release, and studied athletes with detectable cTnT levels after an endurance event (HIGH; n?=?16; 46?±?9?years) against matched controls whose levels were undetectable (LOW; n?=?11; 44?±?7?years). Echocardiography was performed at rest and at peak supine bicycle exercise stress. Left ventricular (LV) end-systolic elastance (E _LV a load-independent measure of LV contractility), effective arterial elastance (E _A a lumped index of arterial load) and end-systolic meridional wall stress were calculated from cardiac dimensions and brachial blood pressure. Efficiency of cardiac work was judged from the ventriculo-arterial coupling ratio (E _A/E _LV: optimal range 0.5–1.0). While subgroups had similar values at rest, we found ventriculo-arterial mismatch during exercise in HIGH subjects [0.47 (0.39–0.58) vs. LOW: 0.73 (0.62–0.83); p?<?0.01] due to unopposed increase in E _LV (p?<?0.05). In LOW subjects, a greater increase occurred in E _A during exercise (+81?±?67?% vs. HIGH: +39?±?32?%; p?=?0.02) which contributed to a maintained coupling ratio. Subjects with higher baseline NT-proBNP had greater systolic wall stress during exercise (R ²?=?0.39; p?<?0.01) despite no correlation at rest (p?=?ns). In conclusion, athletes with exercise-induced biomarker release exhibit ventriculo-arterial mismatch during exercise, suggesting non-optimal cardiac work may contribute to this phenomenon.     

Inhibition by acetylcholine of the stimulation-evoked release of [3H]acetylcholine from the guinea-pig myenteric plexus

The longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum was incubated with [³H]choline and then superfused with Tyrode's solution. Exposure to [³H]choline resulted in the formation of [³H]acetylcholine which was released upon electrical field stimulation. The effects of exogenous acetylcholine, physostigmine and scopolamine on the stimulation-evoked release of [³H]acetylcholine were studied.In the absence of a cholinesterase inhibitor exogenous acetylcholine (10^?5 M) only slightly inhibited (by 26%) the evoked release of [³H]acetylcholine. If the cholinesterase activity of the preparation was reduced by about 50% in the presence of 10^?7M physostigmine, exogenous acetylcholine caused a concentration-dependent depression of the release evoked at 1 Hz. At a concentration of 10^?5 M acetylcholine the release was reduced by 76%. Scopolamine (10^?9 M) shifted the concentration-response curve for the inhibitory action of acetylcholine in a parallel manner to the right. From the dose ratio a pA₂ value of 9.8 for scopolamine against the release-inhibitory effect of acetylcholine was calculated. Physostigmine also inhibited the stimulation-evoked release of [³H]acetylcholine in a concentration-dependent manner, the maximal effect measured being an 85% reduction by 10^?5 M physostigmine. In the absence of a cholinesterase inhibitor scopolamine enhanced the evoked release of [³H]acetylcholine. The facilitatory effect was more marked at a stimulation frequency of 3 Hz (2-fold increase) than at 1 Hz (1.4-fold increase).It is concluded that extracellular acetylcholine decreases the stimulation-evoked release of neuronal acetylcholine. This inhibition is specifically mediated by a stimulation of presynaptic muscarinic receptors. The increase by scopolamine of the evoked release of [³H]acetylcholine suggests that previously liberated acetylcholine may trigger the negative feedback mechanism of acetylcholine release even if the cholinesterase activity is not inhibited, and that the presynaptic muscarinic receptors of the myenteric plexus have a physiological role in regulating the release of acetylcholine.     

Presynaptic effect of L-glutamic acid on the release of dopamine in rat striatal slices

The release of [³H] dopamine ([³H] DA) was estimated in serial superfusate fractions of rat striatal slices continuously superfused with L-[3,5-³H]-tyrosine. L-glutamic acid (5 · 10^?5 M), but not the D-stereoisomer, increased the spontaneous release of [³H] DA (60%). The stimulating effect of L-glutamic acid was still observed in the presence of tetrodotoxin (5 · 10^?7 M), suggesting that the amino-acid acts at a presynaptic site. Moreover, atropine (10^?6 M) or pempidine (10^?5 M) which blocks the acetylcholine (ACh) evoked release of [³H] DA did not reduce the stimulatory effect of L-glutamic acid on [³H] DA release, thus excluding the possible intervention of striatal cholinergic neurons. The data obtained support the hypothesis of a direct control of DA release from nerve terminals by glutamatergic neurons.