A pertussis toxin-sensitive G protein is required to induce histamine release from rat peritoneal mast cells by bradykinin

Bradykinin, kallidin (Lys-bradykinin) and [Thi^5,8,d-Phe⁷]-bradykinin a functional B2 antagonist, induce histamine release from rat peritoneal mast cells. The histamine release is dependent upon added calcium when mast cells are placed in calcium-free medium 30 min before being triggered with the kinins. Histamine release was dose-dependently inhibited by pertussis toxin (1–100 ng/ml) and by benzalkonium chloride (0.1–3 g/ml). The efficiency of ionophore A23187 on histamine release was affected neither by pertussis toxin nor by benzalkonium chloride. The parallel responses of rat peritoneal mast cells to kinins and to substance P suggest that these peptides have the same mechanisms of action i.e. activation of a pertussis toxin-sensitive G protein and of phospholipase C defining a peptidergic triggering pathway of mast cells.     

Eotaxin-2 activates chemotaxis-related events and release of reactive oxygen species via pertussis toxin-sensitive G proteins in human eosinophils

Eosinophils play an important role in allergic and autoimmune diseases. They are activated by distinct chemokines, leading to the immigration into the inflamed tissue, and mediate tissue damage by releasing reactive oxygen species. Recently, eotaxin was found to have the broadest spectrum of activities of all eosinophil-activating CC chemokines. In this study we investigated the effect of the novel CC chemokine, eotaxin-2, on eosinophil effector functions and compared its activity with eotaxin. Using nitrobenzoxadiazole-phallacidin staining and flow cytometry, we show that eotaxin-2 induced rapid and transient actin polymerization, a prerequisite for cell migration and modulation of the respiratory burst, in eosinophils in the same range of efficacy as observed for eotaxin. Eotaxin-2 induced the release of reactive oxygen species in a dose-dependent manner; half maximal and maximal release were found at 50 ng/ml and 500 ng/ml, respectively. Surprisingly, the efficacy of eotaxin-2 was comparable to that of eotaxin and C5a. Release of reactive oxygen species was inhibited by pertussis toxin, indicating the involvement of G_i proteins in the signaling of eotaxin-2. Moreover, the anti-CC chemokine receptor 3 (CCR3) monoclonal antibody, 7B11, was able to inhibit transient rise in the cytosolic Ca²⁺ concentration and the release of reactive oxygen species following stimulation with eotaxin-2. Therefore, eotaxin-2 represents a potent CC chemokine for human eosinophils activating chemotaxis-related events, such as actin polymerization, and the respiratory burst via the CCR3. Moreover, the efficacy of eotaxin-2 seems to be in the same range as that of eotaxin which might re-evaluate the recent profile of activity of CC chemokines in the activation of human eosinophils.     

Role of phospholipase A2 and split lipid products in histamine release from mast cells

Inflammation Research -     

Role of pertussis toxin-sensitive G-proteins in synaptic transmission and plasticity at corticostriatal synapses

The role of pertussis toxin (PTX)-sensitive G-proteins in corticostriatal synaptic transmission and long-term synaptic depression (LTD) was examined using extracellular field potential and whole cell voltage-clamp recordings in striatal slices. High-frequency stimulation (HFS) produced LTD, defined as long-lasting decreases both in synaptically driven population spikes (PSs) measured with field potential recording and in excitatory postsynaptic currents (EPSCs) measured with whole cell recording. Striatal LTD could not be induced in slices obtained from rats that had received a unilateral intrastriatal injection of PTX. However, LTD could be induced in slices obtained from paired control slices. Furthermore, striatal LTD was prevented by pretreatment with N-ethylmaleimide (NEM), another compound that disrupts the function of PTX-sensitive G-proteins. NEM, itself, also potentiated PS and EPSC amplitudes. In addition, NEM increased the frequency and amplitude of both spontaneous and miniature EPSCs and decreased the paired-pulse facilitation ratio, suggesting that it may act on both pre- and postsynaptic sites. The findings suggest that PTX-sensitive G-proteins have multiple roles at corticostriatal synapses, including regulation of synaptic transmission at both pre- and postsynaptic sites, and a key role in striatal LTD.     

A pertussis toxin-sensitive G protein mediates inhibition by morphine of spontaneous electrical activity of oxytocin neurones in anaesthetized rats

We investigated the effects of intracerebroventricular (i.c.v.) pertussis toxin upon the sensitivity of supraoptic oxytocin neurones to intravenous morphine (1-5000 g/kg) in urethane-anaesthetized rats. The maximal inhibitory capacity of morphine was diminished by prior administration of pertussis toxin. Some cells were tested with both morphine and with the kappa-opioid agonist U50,488 (1-5000 g/kg): U50,488-induced inhibition of firing rate was apparently unimpaired by pertussis toxin pre-treatment. The opioid inhibition of firing rate seen in the absence of and after pertussis toxin pretreatment was naloxone-reversible. Thus a pertussis toxin-sensitive G protein may mediate the inhibitory action of morphine upon supraoptic putative oxytocin neurones or inputs to them.     

Presence of a pertussis toxin-sensitive G protein alpha subunit in Sporothrix schenckii.

As an initial step in the study of the role of G proteins in signal transduction in Sporothrix schenckii, we identified a Galphai subunit using different experimental approaches. Western blots of fungal membrane preparations using anti-Galphacommon and anti-Galphai1-Galphai2 antibodies identified a band of approximately 41 kDa. Pertussis toxin-catalyzed adenosine diphosphate (ADP)-ribosylation of these membrane fractions confirmed the presence of a protein substrate of 41 kDa. A 357 bp polymerase chain reaction (PCR) product obtained using fungal DNA as template and primers targeted to conserved Galphai sequences, was used as a probe to isolate a clone from an S. schenckii genomic library. A partial sequence for a Galphai subunit was obtained from this clone. The sequence was completed using the rapid amplification of cDNA ends (RACE) technique with mycelium and yeast cDNA. The cDNA sequence revealed a 1059 bp open reading frame encoding a 353 amino acid Galphai subunit of 41 kDa, more than 90% identical to the CPG-1 of Cryphonectria parasitica, and GNA-1 of Neurospora crassa. The genomic sequence was obtained by PCR using fungal DNA, and revealed a 1250 bp sequence and the presence of three introns. These results provide evidence for the first time of the presence and expression of a Galphai homolog in a pathogenic dimorphic fungus.     

Epithelial cell-derived human beta-defensin-2 acts as a chemotaxin for mast cells through a pertussis toxin-sensitive and phospholipase C-dependent pathway

Mast cells are known to accumulate at the sites of inflammation in response to chemoattractants generated in the local milieu. Since human beta-defensin-2 (hBD-2) is generated in several epithelial tissues where mast cells are present and because we have recently reported that this human antibacterial peptide induces mast cell degranulation, we thus hypothesized that hBD-2 could be a mast cell chemotaxin. Here we report that hBD-2 directly and specifically induces mast cell migration with an optimal concentration of 3 microg/ml. Checkerboard analysis showed that the migration was more chemotactic rather than chemokinetic. Moreover, Scatchard analysis using 125I-labeled hBD-2 revealed that mast cells have at least two classes of receptors, high- and low-affinity receptors, for this peptide. Moreover, the competitive binding assay suggested that hBD-2 is unlikely to utilize CCR6, a functional receptor for hBD-2-mediated dendritic and T cell migration, on mast cells. In addition, treatment of mast cells with G protein inhibitor, pertussis toxin, and phospholipase C inhibitor, U-73122, abolished the cell chemotaxis in response to hBD-2, indicating that the G protein-phospholipase C signaling pathway is involved in hBD-2-induced mast cell activation. Thus, we suggest that hBD-2, which was originally believed to be involved in innate host defense, may participate in the recruitment of mast cells to inflammation foci.     

Opioid-induced adenylyl cyclase supersensitization in human embryonic kidney 293 cells requires pertussis toxin-sensitive G proteins other than G(i1) and G(i3)

Chronic activation of opioid receptors in cultured mammalian cells is known to induce adenylyl cyclase (AC) supersensitization via the pertussis toxin-sensitive G(i/o) proteins. To examine the role of G(i1) and G(i3) in opioid-induced AC supersensitization, pertussis toxin-resistant mutants of Galpha(i1) and Galpha(i3) (Galpha(i1)CG and Galpha(i3)CG) were stably co-expressed with different opioid receptors (mu, delta or kappa) in human embryonic kidney (HEK 293) cells. Although the opioid receptors were capable of inhibiting AC via Galpha(i1)CG and Galpha(i3)CG in pertussis toxin-treated cells, AC supersensitization induced by chronic opioid treatment remained sensitive to pertussis toxin. Our results demonstrated that despite their ability to interact with opioid receptors, the pertussis toxin-sensitive G(i1) and G(i3) proteins on their own are incapable of supporting opioid-induced AC supersensitization.     

Effect of phospholipase A2 inhibitor on substance P-induced histamine release from rat peritoneal mast cells]

Rat peritoneal mast cells purified on a Percoll gradient were challenged with substance P and effect of phospholipase A2 inhibitor ONO-RS-082 on substance P-induced histamine release from the cells were investigated. Substance P at the concentration of 10(-5) M caused a significant histamine release and the amount of histamine release reached to its submaximum at 1 min after the challenge and then slowly increased. ONO-RS-082 inhibited the substance P-induced histamine release in a concentration-dependent manner at the concentration from 10(-6) to 10(-4) M, suggesting that phospholipase A2 may play some roles in the process of substance P-induced histamine release from rat peritoneal mast cells.     

Imipramine inhibits histamine and serotonin secretion induced by various secretagogues

Inflammation Research -     

Further characterisation of substance P induced histamine release from human bronchoalveolar lavage mast cells

Inflammation Research -     

Desensitization in the process of histamine secretion induced by antigen and dextran

1. Antigen challenge of sensitized rat peritoneal mast cells in the absence of calcium failed to release histamine. The release when calcium was added subsequently declined rapidly, this desensitization being almost complete in 4 min.2. Phosphatidyl serine (10 mug/ml.) reduced the rate of desensitization so that decay of the response to added calcium was not complete after 16 min.3. Exposing normal cells to dextran resulted in a slow rate of desensitization, the response to phosphatidyl serine added with calcium having decayed by only 27% within 10 min.4. Phosphatidyl serine added with dextran prevented desensitization so that the response to subsequently added calcium did not decay even after an interval of 20 min.5. Cells activated by dextran and calcium became rapidly desensitized as shown by decay of the response to added phosphatidyl serine which was almost complete by 5 min.6. Histamine release by the calcium ionophore (A 23187) added to cells at intervals before the addition of calcium did not show significant decay.7. Desensitization of the cells to antigen did not change their response to the ionophore.     

Effect of phospholipase A2 inhibitor ONO-RS-082 on substance P-induced histamine release from rat peritoneal mast cells.

Rat peritoneal mast cells purified on a Percoll gradient were challenged with substance P (SP) and the effect of phospholipase A2 inhibitor ONO-RS-082 on SP-induced histamine release from the cells was investigated. 10(-5) mol/l SP caused a significant histamine release and the amount of histamine release reached maximum at 1 min after the challenge. ONO-RS-082 inhibited the SP-induced histamine release in a concentration-dependent manner at the concentration from 10(-6) to 10(-4) mol/l, suggesting possible involvement of phospholipase A2 in SP-induced histamine release from rat peritoneal mast cells.     

Oxidant-mediated activation of cytosolic phospholipase a(2) in pulmonary endothelium: role of protein kinase C alpha and a pertussis toxin-sensitive protein.

The authors have previously demonstrated that the oxidant t-buOOH stimulates phospholipase A(2) (PLA(2)) activity in bovine pulmonary artery endothelial cells (S. Chakraborti et al. American Journal of Physiology, 257, L430-L437, 1989). Herein, the authors sought to investigate the mechanism by which t-buOOH stimulates PLA(2) activity and the role of protein kinase C (PKC) in this scenario. Treatment of bovine pulmonary artery endothelial cells with t-buOOH stimulated an aprotinin-sensitive protease activity, PKC activity, and PLA(2) activity in the cell membrane. Pretreatment with intracellular Ca(2+) chelator (BAPTA-AM), PKCalpha inhibitor (Go6976), cPLA(2) inhibitor (AACOCF(3)), and pertussis toxin prevented t-buOOH-stimulated PLA(2) activity. Immunoblot studies with aprotinin, cPLA(2), PKCalpha, and Gialpha antibodies revealed their presence in the endothelial membrane. Immunoblot studies of the cell membrane isolated from t-buOOH-stimulated cells with cPLA(2) and PKCalpha antibodies elicited an apparent increase in their immunoreactive protein profiles along with an additional 47-kDa immunoreactive fragment in the membrane. t-buOOH caused Gialpha phosphorylation in the membrane and pretreatment with Go6976 prevented the phosphorylation. Overall, these results suggest that t-buOOH stimulates an aprotinin-sensitive protease activity that proteolytically activates PKCalpha and that subsequently phosphorylates a pertussis toxin-sensitive protein, resulting in the stimulation of cPLA(2) activity in the cell membrane.     

Thermal analgesia and substance P depletion induced by capsaicin in guinea-pigs

Treatment of adult guinea-pigs with subcutaneous doses of capsaicin of 1, 5, 50, 200, 200, 500, 500 mg/kg in daily succession increased skin flinch latency and depleted substance P from dorsal root ganglia. Similar treatment of animals with doses of 50, 100, 100, 100, 100 mg/kg significantly increased skin flinch latency and hot-plate escape latency and depleted substance P from dorsal root ganglia and dorsal spinal cord. Capsaicin had no effect on levels of substance P in other CNS regions or in any region of the gastrointestinal tract. Responses to mechanical pressure were not altered by capsaicin treatment.Depletion of primary afferent substance P in guinea-pigs appears to result in substantial thermal antinociception without producing comparable pressure antinociception.     

Role of histamine receptors in rabbit pancreatic exocrine secretion stimulated by cholecystokinin and secretin

An investigation was made of the effects of H1 and H2 receptor agonists and antagonists on rabbit pancreatic exocrine secretion stimulated by secretin and cholecystokinin (CCK). The H1 agonist 2-thiazolylethylamine elicited dose-dependent increases in the rate of secretion. Increases in pancreatic juice flow and enzyme output were also noted after H2 antagonist cimetidine. In contrast, the H1 antagonist chlorpheniramine and H2 agonist dimaprit caused reductions in flow and enzyme output. The results suggest that H1 receptors have stimulative effects and H2 receptors have inhibitory effects on exocrine rabbit pancreas.     

Compound 48/80 and substance P induced release of histamine and serotonin from rat peritoneal mast cells

The effect of substance P and compound 48/80 on histamine and serotonin release from not isolated and isolated mast cells have been compared in experimentsin vitro. The response of not isolated and isolated mast cells were virtually identical. The release of both amines, in response to 48/80 and substance P, was dose-dependent. The percentage of histamine released by 48/80 was significantly higher than the percentage of serotonin, the difference being higher at lower concentrations of compound 48/80 after 15 min of incubation. Substance P also showed a tendency to higher efficiency for histamine than for serotonin release. In contrast to 48/80, the dose-response curves for histamine and serotonin release were parallel. These results support the view that the ratio between histamine and serotonin release depends on the liberator used. They also showed that this ratio can depend on the concentration of the agent inducing secretion. The results indicate that substance P as well as 48/80 act rather selectively as histamine liberators and that there is some difference in releasing properties of 48/80 and substance P.     

Role of lymphocyte activation by substance P in rheumatoid arthritis.

Peripheral mononuclear cells taken from rheumatoid arthritis (RA) patients, which had previously been treated in vitro with the neuropeptide Substance P (SP) followed by stimulation with the mitogen PHA, showed a significantly higher percent positivity of lymphocytes with the double-markers CD4+ CD2+, CD8+ CD25+, CD4+, HLA-DR+ and CD8+ HLA-DR+ than those of the cells untreated by SP. No effect of SP was detected in normal controls, except that the cells with the double-marker CD4+ CD25+ were slightly increased. Furthermore, in RA patients the percentage of CD4+ CD45R- cells was increased, while that of CD4+ CD45R+ cells was decreased, by pre-treatment with SP and stimulation with PHA. Monocytes of patients with rheumatoid arthritis after the treatment with SP released a significantly higher amount of oxygen-intermediate than those of normal controls with or without SP. From these results, it could be considered that SP plays an important role in RA inflammation.