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1.
Mucosal Immunol ; 4(1): 53-65, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20811345

ABSTRACT

The prevalence of asthma has steadily increased during the last decade, probably as the result of changes in the environment, including reduced microbial exposure during infancy. Accordingly, experimental studies have shown that deliberate infections with live pathogens prevent the development of allergic airway diseases in mice. Bacterial extracts are currently used in children suffering from repeated upper respiratory tract infections. In the present study, we have investigated whether bacterial extracts, commercially available as Broncho-Vaxom (BV), could prevent allergic airway disease in mice. Oral treatment with BV suppressed airway inflammation through interleukin-10 (IL-10)-dependent and MyD88 (myeloid differentiation primary response gene (88))-dependent mechanisms and induced the conversion of FoxP3 (forkhead box P3)(-) T cells into FoxP3(+) regulatory T cells. Furthermore, CD4(+) T cells purified from the trachea of BV-treated mice conferred protection against airway inflammation when adoptively transferred into sensitized mice. Therefore, treatment with BV could possibly be a safe and efficient strategy to prevent the development of allergic diseases in children.


Subject(s)
Asthma , Bacteria , Respiratory System , T-Lymphocytes, Regulatory , Animals , Mice , Administration, Oral , Adoptive Transfer , Asthma/immunology , Asthma/prevention & control , Bacteria/cytology , Bacteria/immunology , CD4-Positive T-Lymphocytes/immunology , Cytokines/immunology , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Interleukin-10/metabolism , Mice, Inbred BALB C , Myeloid Differentiation Factor 88/metabolism , Respiratory System/immunology , T-Lymphocytes, Regulatory/immunology
2.
Eur J Pharmacol ; 428(2): 277-86, 2001 Oct 05.
Article in English | MEDLINE | ID: mdl-11675046

ABSTRACT

Calcium dobesilate possesses antioxidant properties and protects against capillary permeability by reactive oxygen species in the rat peritoneal cavity, but whether a similar action can take place in the diabetic rat retina is unknown. We investigated the oral treatment of diabetic rats with calcium dobesilate on the prevention of free radical-mediated retinal injury induced by ischemia/reperfusion (90 min ischemia followed by 3 min and/or 24 h of reperfusion). Streptozotocin-induced diabetic rats were orally treated with 50 and 100 mg/kg of calcium dobesilate for 10 days (n=12 in each group). In the first series of studies, calcium dobesilate was found to significantly reduce the maldistribution of ion content in diabetic ischemic/reperfused rat retina. Thus, in diabetic rats treated with 100 mg/kg/day calcium dobesilate, ischemia/reperfusion provoked: (i) 27.5% increase in retinal Na(+) content compared to 51.8% in the vehicle-treated group (P<0.05), and (ii) 59.6% increase in retinal Ca(2+) content compared to 107.1% in vehicle-treated animals (P<0.05). In the second series of studies, calcium dobesilate was found to significantly protect diabetic rat retina against inhibition of Na(+)/K(+)-ATPase and Ca(2+)/Mg(2+)-ATPase activities by ischemia/reperfusion (54% and 41% reduction, respectively, with 100 mg/kg of calcium dobesilate) and also against changes in retinal ATP, reduced glutathione (GSH), and oxidized glutathione (GSSG) contents. In the third series of experiments, rats treated with 100 mg/kg of calcium dobesilate reduced the hydroxyl radical signal intensity to 41% (measured by electron paramagnetic resonance), induced by ischemia/reperfusion in diabetic rat retina. Finally, 100 mg/kg calcium dobesilate significantly reduced retinal edema (measured by the thickness of the inner plexiform layer) in diabetic rats. In conclusion, oral treatment with calcium dobesilate significantly protected diabetic rat retina against oxidative stress induced by ischemia/reperfusion. Whether the antioxidant properties of calcium dobesilate explain, at least in part, its beneficial therapeutic effects in diabetic retinopathy deserves further investigation.


Subject(s)
Antioxidants/pharmacology , Calcium Dobesilate/pharmacology , Diabetes Mellitus, Experimental/complications , Diabetic Retinopathy/prevention & control , Reperfusion Injury/complications , Adenosine Triphosphate/metabolism , Animals , Antioxidants/therapeutic use , Ca(2+) Mg(2+)-ATPase/drug effects , Ca(2+) Mg(2+)-ATPase/metabolism , Calcium/metabolism , Calcium Dobesilate/therapeutic use , Cations/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetic Retinopathy/etiology , Diabetic Retinopathy/metabolism , Free Radicals/metabolism , Glutathione/drug effects , Glutathione/metabolism , Glutathione Disulfide/drug effects , Glutathione Disulfide/metabolism , Magnesium/metabolism , Male , Potassium/metabolism , Rats , Rats, Sprague-Dawley , Retina/drug effects , Retina/metabolism , Retina/pathology , Sodium/metabolism , Sodium-Potassium-Exchanging ATPase/drug effects , Sodium-Potassium-Exchanging ATPase/metabolism
3.
J Biol Chem ; 269(41): 25570-5, 1994 Oct 14.
Article in English | MEDLINE | ID: mdl-7523388

ABSTRACT

When single rat hepatocytes were stimulated with the phospholipase C-activating hormone, vasopressin (from 300 pM to 1 microM), the [Ca2+]i signals were always "all-or-none" responses. At low concentrations of vasopressin, Ca2+ release was maximal because liberation of additional inositol 1,4,5-trisphosphate (IP3) by photolysis of its caged precursor at the top of the [Ca2+]i spike failed to increase [Ca2+]i further. However, if IP3 was generated by photolysis of caged IP3 in previously unstimulated cells, [Ca2+]i increased immediately, and the magnitude of the response was a graded function of the quantity of IP3 released. We also analyzed the kinetics of activation of intracellular IP3 receptor/Ca2+ channels by monitoring the quench of sequestered dye by the entry of cytoplasmic Mn2+ into fura-2-loaded intracellular IP3-sensitive organelles. This Mn(2+)-induced quench was precipitous and always preceded by a delay inversely related to the vasopressin concentration. In hepatocytes stimulated with 10 nM vasopressin, IP3 increased slowly, and the half-time of the IP3 rise was comparable with the latency for the release of intracellular calcium. The slow rise in IP3 would be predicted to produce accelerating Ca2+ release. This is consistent with the results of the Mn2+ quench experiments, which revealed accelerating activation of intracellular IP3-regulated calcium channels. We conclude that this accelerating release of Ca2+, which does not occur with instantaneous increases in IP3 due to flash photolysis, is likely to be important for generating the all-or-none Ca2+ mobilization that initiates the processes of intracellular [Ca2+]i oscillations.


Subject(s)
Calcium/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Liver/physiology , Signal Transduction/physiology , Vasopressins/pharmacology , Animals , Calcium Channels/metabolism , Cell Compartmentation , Dose-Response Relationship, Drug , Enzyme Activation , Fura-2/metabolism , Inositol 1,4,5-Trisphosphate Receptors , Ion Channels/metabolism , Liver/cytology , Liver/drug effects , Male , Manganese/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear/metabolism , Type C Phospholipases/metabolism
4.
J Biol Chem ; 269(40): 25158-64, 1994 Oct 07.
Article in English | MEDLINE | ID: mdl-7929204

ABSTRACT

Different elements of voltage-gated Ca2+ influx and their role for cytosolic-free Ca2+, [Ca2+]i, were studied in cells of the pituitary line GH3B6. Single cell monitoring of [Ca2+]i with the fluorescent probe indo-1, as well as fast on-line ratio [Ca2+]i imaging with fura-2, were combined with electrophysiological recordings using the perforated patch configuration of the patch clamp technique. [Ca2+]i signals are generated by Ca2+ influx both during action potentials and in between. Steady state Ca2+ influx at resting potentials (-60 to -40 mV) was voltage-gated and sensitive to the dihydropyridine antagonist PN 200-110. Ca2+ influx could be demonstrated in physiological conditions as inward Ca2+ currents of maximally 10 pA, which were triggered when stepping up the holding voltage beyond a narrow threshold around -50 mV. Inward currents were well correlated with [Ca2+]i elevations (R = -0.8; p < 0.001). It is concluded that dihydropyridine-sensitive, low threshold voltage-gated steady state Ca2+ currents, which may be tonically activated at resting potentials, provide for [Ca2+]i signaling in excitable endocrine cells in a mode that parallels the modulation of Ca2+ influx during action potentials.


Subject(s)
Calcium Channels/physiology , Calcium/metabolism , Dihydropyridines/pharmacology , Pituitary Gland/metabolism , Animals , Cell Line , Cytosol/metabolism , Isradipine/pharmacology , Membrane Potentials
6.
Eur J Pharmacol ; 227(2): 173-80, 1992 Oct 01.
Article in English | MEDLINE | ID: mdl-1330649

ABSTRACT

In pituitary cells, electrical activity generates characteristic oscillations of the cytosolic free Ca2+ concentration, [Ca2+]i. These oscillations are controlled by activators as well as by inhibitors of secretion. We studied, in single fura-2-loaded cells, the role of protein phosphorylation in modulating [Ca2+]i oscillations, using either okadaic acid, an inhibitor of protein phosphatases, or activators of protein kinases A and C. Okadaic acid always increased rapidly both the frequency and amplitude of [Ca2+]i oscillations. In contrast, activation of protein kinases A or C generated more complex kinetic [Ca2+]i patterns: phosphorylation due to both kinases resulted in a sustained activation of [Ca2+]i oscillations in about one-third of the cells, whereas two-thirds of the cells responded by an arrest of [Ca2+]i oscillations. This transient phase of arrest was followed, after a few minutes, by a recovery of [Ca2+]i oscillations, often with enhanced frequency. During the arrest, depolarizing the cells with an external microelectrode could not trigger an increase in [Ca2+]i. We conclude that: (i) the fine regulation between phosphorylation/dephosphorylation events is crucial for the modulation of [Ca2+]i oscillations, and (ii) protein kinases A and C can control Ca2+ influx bidirectionally.


Subject(s)
Calcium/metabolism , Pituitary Gland/metabolism , Protein Kinase C/metabolism , Proteins/metabolism , Action Potentials , Animals , Cytosol/metabolism , Electric Stimulation , Enzyme Activation , Ethers, Cyclic/pharmacology , Fura-2 , Okadaic Acid , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation/drug effects , Pituitary Gland/cytology , Tumor Cells, Cultured
7.
J Neuroendocrinol ; 4(4): 473-81, 1992 Aug.
Article in English | MEDLINE | ID: mdl-21554632

ABSTRACT

Cells of the pituitary tumour cell line GH(4) C(1) were exposed to epidermal growth factor, estradiol and insulin for 5 days, a treatment which resulted in 1) increased prolactin storage in secretory granules, 2) the loss of spontaneous [Ca(2+) ](1) oscillations, and 3) a selective reduction of the protein G(s) α, seen in immunoblots, cholera toxin labelling, and vasoactive intestinal peptide stimulation of adenylyl cyclase. In contrast, the glucocorticoid dexamethasone, which increases the expression of G(s) α, partially restored spontaneous [Ca(2+) ](1) oscillations and decreased prolactin storage. It is concluded that G(s) α levels in tumoral cells result in spontaneous electrical activity which may empty prolactin stores by the continuous activation of exocytosis.

8.
J Immunol ; 147(3): 980-3, 1991 Aug 01.
Article in English | MEDLINE | ID: mdl-1907309

ABSTRACT

Variations in the cytosolic free Ca2+ concentration [( Ca2+]i) upon LPS exposure were studied in single rat peritoneal macrophages loaded with fura-2 under carefully controlled conditions. Of a total of 60 cells examined, 47% responded to LPS (1 microgram/ml) with an increase in [Ca2+]i. Macrophages were heterogeneous with regard to the LPS response, with individual cells exhibiting single rapid and transient increases in [Ca2+]i, multiple transients, or slower and more sustained variations. In 62% of the responding cells, a second exposure to LPS elicited a [Ca2+]i rise, although usually to a slightly lower peak value. Thus, rapid desensitization to LPS does not occur in the majority of these macrophages. EGTA did not abolish the response of those cells that exhibited a single rapid transient in [Ca2+]i, indicating that the source of the initial [Ca2+]i rise was the intracellular stores. There was no obvious correlation between the type of response to LPS and the initial morphologic features (rounded vs polarized) of the cells. Our present work shows unequivocally that LPS induces increases in macrophage [Ca2+]i and, thereby, lends substantial support to the hypothesis that [Ca2+]i is a second messenger in LPS-mediated activation of the macrophage.


Subject(s)
Calcium/metabolism , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Animals , Calcium/pharmacokinetics , Egtazic Acid/pharmacology , In Vitro Techniques , Platelet Activating Factor/pharmacology , Rats
9.
Eur J Pharmacol ; 206(4): 271-7, 1991 Apr 25.
Article in English | MEDLINE | ID: mdl-1680718

ABSTRACT

Adenosine is a potent paracrine/autocrine feedback inhibitor of cell activation in a variety of tissues. Adenosine action was studied in pituitary cells, in which spontaneous electrical activity causes characteristic oscillations of the cytosolic free Ca2+ concentration, [Ca2+]i. Cells of the GH3B6 rat pituitary tumor line were studied by microspectrofluorimetry using the Ca2+ probes indo-1 and fura-2, in part in combination with electrophysiological tight seal whole cell recordings, obtained with the novel approach of patch perforation. It was demonstrated that adenosine receptor activation by N6-(R-phenyl-isopropyl)-adenosine (PIA) caused a block of electrical activity and abolished the ensuing alterations in [Ca2+]i. PIA mimicked the inhibitory action of somatostatin. Adenosine effects are mediated by A1 receptors in these cells and are antagonized by IBMX, an adenosine receptor blocker. PIA also suppressed action potentials that were elicited by the activation of protein kinase C with the phorbol ester PMA, or during the second phase of TRH action. In contrast, no interference was notable on TRH-induced intracellular Ca2+ mobilization. In addition to the abolition of Ca2+ transients, PIA lowers basal [Ca2+]i in some cells. It is proposed that in addition to the inhibition of adenylate cyclase, A1 receptor action on [Ca2+]i is an important element in the control of excitable pituitary cells.


Subject(s)
Calcium/physiology , Pituitary Gland/physiology , Receptors, Purinergic/physiology , Action Potentials/physiology , Adenosine/pharmacology , Animals , Calcium/metabolism , Enzyme Activation , Indoles , Phenylisopropyladenosine/pharmacology , Pituitary Gland/cytology , Protein Kinase C/metabolism , Somatostatin/pharmacology , Thyrotropin-Releasing Hormone/physiology
10.
J Hirnforsch ; 32(5): 617-25, 1991.
Article in English | MEDLINE | ID: mdl-1812176

ABSTRACT

Polyclonal anti-idiotypic antiserum raised against both rabbit and monoclonal anti-dopamine (DA) antibodies was produced in rabbits. It was characterized for its specificity and was shown to (1) inhibit the binding of both polyclonal and monoclonal idiotypic anti-DA antibodies directed to immobilized DA conjugates; (2) inhibit the binding of (3H) DA to rat brain membranes; (3) to cross-react with a peptide extracted from a neuroblastoma cell line (NCB-20), known to express functional DA receptors. Finally, immunocytochemical studies were performed on paraformaldehyde-fixed rat brain. Anti-idiotypic antibodies were used to visualize the cellular and subcellular distribution of DA receptor binding sites in the striatum, a region that contains both D1 and D2 receptors subtypes. Under the electron microscope, the immune reaction product was observed to be concentrated in postsynaptic sites belonging mainly to dendritic spines, while presynaptic structures were sparsely labeled.


Subject(s)
Antibodies, Anti-Idiotypic , Antibodies, Monoclonal , Brain/metabolism , Dopamine/immunology , Receptors, Dopamine/analysis , Animals , Brain/ultrastructure , Cell Line , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Corpus Striatum/metabolism , Dendrites/metabolism , Dendrites/ultrastructure , Dopamine/metabolism , Enzyme-Linked Immunosorbent Assay , Mice , Microscopy, Immunoelectron , Neuroblastoma , Rabbits/immunology , Rats , Rats, Inbred Strains , Receptors, Dopamine/metabolism
11.
J Neuroendocrinol ; 3(3): 253-60, 1991 Jun 01.
Article in English | MEDLINE | ID: mdl-19215461

ABSTRACT

Abstract Quinacrine, a fluorescent basic molecule, accumulates in secretory granules of pituitary cells, as was revealed by its colocalization with immunoreactive prolactin. Thus quinacrine fluorescence may be used to monitor secretory activity at the single cell level. Rat pituitary cells in primary culture were loaded with quinacrine and stimulated with physiological secretagogues, such as thyrotrophin-releasing hormone or bradykinin, which induced a multiphasic lowering of fluorescence, corresponding to the loss of quinacrine contained in exocytosed granules. Quinacrine was further used in combination with the fluorescent calcium probe fura-2, in order to monitor simultaneously exocytosis and variations in the cytosolic free calcium concentration, [Ca(2+)](i). With an appropriate selection of the excitation wavelengths, in dual excitation microfluorimetry experiments, it was possible to distinguish between fluorescence changes due to altered [Ca(2+)](i) versus quinacrine exocytosis. Transient elevations of [Ca(2+)](i) were provoked in individual pituitary cells by enhancing calcium influx through voltage gated channels. In part of the cells an initial increase in [Ca(2+)](i) coincided with stimulated quinacrine release. The approach was also applied to cells of the neuroblastoma line NCB20, where stimulation with bradykinin caused a transient rise in [Ca(2+)](i), concomitantly with enhanced exocytosis. No increase in exocytosis was ever detected without an elevation of [Ca(2+)](i), suggesting that in both cellular systems, an increase in [Ca(2+)](i), is absolutely necessary, but not sufficient to induce secretion.

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