Histamine H3 receptor-mediated inhibition of sympathetically evoked mydriasis in rats

This study was designed to determine if the histamine H3 receptor agonist R-alpha-methylhistamine would play a role in modulation of sympathetically evoked mydriasis in anesthetized rats, and if so, to ascertain the specific receptor subtype(s) involved. Reproducible frequency-response curves of pupillary dilation were generated by stimulation of the cervical preganglionic sympathetic nerve (1-32 Hz). Systemic administration of R-alpha-methylhistamine (0.3-3.0 mg kg(-1)) produced a dose-related inhibition of the evoked mydriasis. The greatest inhibition was seen at lower frequency levels, with about 43% depression observed at 2 Hz. The specific histamine H3 receptor antagonist, clobenpropit (3.0 mg kg(-1), i.v.), blocked the inhibitory effect of R-alpha-methylhistamine, whereas neither the histamine H2 receptor antagonist, cimetidine (5.0 mg kg(-1), i.v.), nor the histamine H1 receptor antagonist, chlorpheniramine (0.5 mg kg(-1), i.v.), was effective. The histamine H2 receptor agonist, dimaprit (10 mg kg(-1), i.v.), was also without effect on the evoked mydriasis. R-alpha-methylhistamine (3.0 mg kg(-1)) did not inhibit phenylephrine-induced mydriasis. These results support the conclusion that R-alpha-methylhistamine produces inhibition of sympathetically evoked mydriasis via histamine H3 receptor stimulation, presumably by an action on presynaptic histamine H3 receptors.

The histamine H4 receptor is a potent inhibitor of adhesion-dependent degranulation in human neutrophils

The histamine H4 receptor regulates the inflammatory response. However, it is not known whether this receptor has a functional role in human neutrophils. We found that fMLP (1 μM), but not histamine (0.1-1 μM), induced Mac-1-dependent adhesion, polarization, and degranulation (release of lactoferrin). A pretreatment of neutrophils with histamine (0.001-1 μM) or JNJ 28610244 (0.1-10 μM), a specific H4 receptor agonist, led to inhibition of degranulation. Total inhibition of degranulation was obtained with 0.1 μM histamine and 10 μM JNJ 28610244. Furthermore, such inhibition by histamine of degranulation was reversed by JNJ 7777120 and JNJ 28307474, two selective H4 receptor antagonists. However, neither histamine nor the H4 receptor agonist JNJ 28610244 prevented fMLP-induced, Mac-1-dependent adhesion, indicating that the H4 receptor may block signals emanating from Mac-1-controlling degranulation. Likewise, engagement of the H4 receptor by the selective agonist JNJ 28610244 blocked Mac-1-dependent activation of p38 MAPK, the kinase that controls neutrophil degranulation. We also show expression of the H4 receptor at the mRNA level in ultrapure human neutrophils and myeloid leukemia PLB-985 cells. We concluded that engagement of this receptor by selective H4 receptor agonists may represent a good, therapeutic approach to accelerate resolution of inflammation.

A selective antagonist of histamine H₄ receptors prevents antigen-induced airway inflammation and bronchoconstriction in guinea pigs:involvement of lipocortin-1

BACKGROUND AND PURPOSE: Among the pathogenic mechanisms of asthma, a role for oxidative/nitrosative stress has been well documented. Recent evidence suggests that histamine H₄ receptors play a modulatory role in allergic inflammation. Here we report the effects of compound JNJ 7777120 (JNJ), a selective H4 receptor antagonist, on antigen-induced airway inflammation, paying special attention to its effects on lipocortin-1 (LC-1/annexin-A1), a 37 kDA anti-inflammatory protein that plays a key role in the production of inflammatory mediators.

EXPERIMENTAL APPROACH: Ovalbumin (OA)-sensitized guinea pigs placed in a respiratory chamber were challenged with antigen. JNJ (5, 7.5 and 10 mg.kg⁻¹) was given i.p. for 4 days before antigen challenge. Respiratory parameters were recorded. Bronchoalveolar lavage (BAL) fluid was collected and lung specimens taken for further analyses 1 h after antigen challenge. In BAL fluid, levels of LC-1, PGD2 , LTB4 and TNF-α were measured. In lung tissue samples, myeloperoxidase, caspase-3 and Mn-superoxide dismutase activities and 8-hydroxy-2-deoxyguanosine levels were measured.

KEY RESULTS: OA challenge decreased LC-1 levels in BAL fluid, induced cough, dyspnoea and bronchoconstriction and increased PGD2 , LTB4 and TNF-α levels in lung tissue. Treatment with JNJ dose-dependently increased levels of LC-1, reduced respiratory abnormalities and lowered levels of PGD2 , LTB4 and TNF-α in BAL fluid.

CONCLUSIONS AND IMPLICATIONS: Antigen-induced asthma-like reactions in guinea pigs decreased levels of LC-1 and increased TNF-α and eicosanoid production. JNJ pretreatment reduced allergic asthmatic responses and airway inflammation, an effect associated with LC-1 up-regulation.

Peptide leucine arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin of the Northern Leopard frog, Rana pipiens

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens), This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions, pLR was synthesized and elicited rapid, noncytolytic histamine release that had a a-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin, pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action, pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i,e. mature neutrophils, pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.

Ca2+- and volume-sensitive chloride currents are differentially regulated by agonists and store-operated Ca2+ entry.

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca2+]i in a Ca2+-free medium followed by a secondary [Ca2+]i rise upon Ca2+ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca2+-activated (ICl(Ca)) and volume-regulated (ICl, swell) chloride currents. NPPB and DIDS more efficiently inhibited ICl(Ca) and ICl, swell, respectively. Cell swelling caused by hypotonic solution invariably activated ICl, swell while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling–induced ICl, swell, while its inactive analogue U-73343 had no effect. ICl(Ca) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca2+]i in the pipette solution at 500 nM. In contrast, ICl, swell could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced ICl, swell in a nonadditive manner, suggesting their convergence on a common pathway. ICl, swell and ICl(Ca) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated ICl(Ca), but abolished ICl, swell, thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that ICl, swell can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a "selection" switch between closely localized channels.

Liver fluke (Fasciola hepatica)-derived peptides activate rat peritoneal mast cells

Short peptides with sequences derived from those found in the tegumental antigen of Fasciola hepatica have been synthesised. Incubation of some of these peptides with rat peritoneal mast cells resulted in the degranulation of the cells as measured by a histamine release assay. This activity was shown to be associated with the proline-lysine-proline motif, which is responsible for the induction of mast cell degranulation by the mammalian bioactive peptide substance P. Studies on the mode of action of the fluke-derived peptide indicated that it was operating through the same biochemical pathways as substance P. The implications of these findings for the development of immune responses during parasite infections are discussed. (C) 2003 Elsevier Science B.V. All rights reserved.

Adenosine, inflammation and asthma - a review

Adenosine is a ubiquitous molecule present in every cell of the human body. It has a wide range of physiological functions mediated predominantly through specific cell surface adenosine receptors. Adenosine has both pro- and anti-inflammatory effects and acts on inflammatory and resident immune cells and antioxidant enzymes. The elevation of adenosine in the bronchoalveolar lavage (BAL) fluid of asthmatics combined with its bronchoconstrictor effect on the airways in asthmatics has led to increased research into the contribution of adenosine in the pathophysiology of inflammation and asthma. This review looks at the airway response to adenosine and at the interaction of adenosine with mast cells and basophils.

Mast cell mediator release in nonasthmatic subjects after endobronchial adenosine challenge

Background: Adenosine 5′-monophosphate (AMP) has been shown to cause bronchoconstriction in atopic subjects but to have no effect on nonatopic nonasthmatic subjects. Endobronchial AMP challenge has previously been shown to cause mast cell mediator release in asthmatic subjects, but it is unknown whether a similar response occurs in atopic nonasthmatic and nonatopic nonasthmatic control subjects who have no response to inhalation AMP challenge.

Objective: This study examined the change in mast cell–derived products after endobronchial saline challenge and AMP challenge in subjects with and without a positive inhalation response to AMP.

Methods: Inhalation challenge with AMP challenge was performed in normal, atopic nonasthmatic, and atopic asthmatic subjects. Levels of mast cell mediators were measured after endobronchial adenosine challenge and after placebo endobronchial saline challenge.

Results: There were significant increases in histamine, tryptase, protein, and prostaglandin D2 levels (P = .02, P = .02, P = .01, and P = .01, respectively) after AMP challenge compared with after saline challenge in nonatopic nonasthmatic subjects. There was no significant increase in any mediator in either of the other 2 groups.

Conclusion: This study suggests dissociation between mediator release and bronchoconstriction in response to AMP.

Inflammatory mediators in bronchoalveolar lavage samples from children with and without asthma

Background: We investigated whether eosinophils and mast cells, found in the airways of children with wheeze, were activated during relatively asymptomatic periods.
Methods: A nonbronchoscopic bronchoalveolar lavage (BAL) procedure was performed on children presenting for an elective surgical procedure. Eosinophil-derived (eosinophil cationic protein, ECP) and mast cell-derived (histamine/tryptase) mediator concentrations were measured in the BAL fluid. A detailed history and serum immunoglobulin E were used to classify the children into four groups: atopic with and without asthma, viral-associated wheeze and normal controls.
Results: The ECP concentrations in BAL from atopic asthmatic subjects were significantly higher than those measured in BAL from normal controls (P < 0.01), no other groups differed significantly. Histamine concentrations were elevated in both the atopic asthmatic and viral-associated wheeze groups compared with controls (P < 0.02) and additionally higher concentrations were obtained in atopics with asthma compared with atopics without asthma (P < 0.03). Tryptase concentrations did not differ between groups, although the tryptase and histamine concentrations correlated significantly (r = 0.78, P < 0.0001).
Conclusions: Elevated histamine concentrations were found in children with wheeze regardless of the aetiology, whereas ECP was only elevated in those asthmatics with atopy. This suggests that even in relatively quiescent periods, there is some on going activation of airway eosinophils in children with atopic asthma.

Evaluation and outcome of patients with chronic non-productive cough using a comprehensive diagnostic protocol

Background-Asthma, post-nasal drip syndrome (PNDS), and gastrooesophageal reflux (GOR) account for many cases of chronic non-productive cough (CNPC). Each may simultaneously contribute to cough even when clinically silent, and failure to recognise their contribution may lead to unsuccessful treatment.

Methods—Patients (all lifetime non-smokers with normal chest radiographs and spirometric measurements) referred with CNPC persisting for more than three weeks as their sole respiratory symptom underwent histamine challenge, home peak flow measurements, ear, nose and throat (ENT) examination, sinus CT scanning, and 24 hour oesophageal pH monitoring. Treatment was prescribed on the basis of diagnoses informed by investigation results.

RESULTS—Forty three patients (29 women) of mean age 47.5 years (range 18-77) and mean cough duration 67 months (range 2-240) were evaluated. On the basis of a successful response to treatment, a cause for the cough was identified in 35 patients (82%) as follows: cough variant asthma (CVA) (10 cases), PNDS (9 cases), GOR (8cases), and dual aetiologies (8 cases). Histamine challenge correctly predicted CVA in 15 of 17 (88%) positive tests. ENT examination and sinus CT scans each had low positive predictive values for PNDS (10 of 16 (63%) and 12 of 18 (67%) positive cases, respectively), suggesting that upper airways disease frequently co-exists but does not always contribute to cough. When negative, histamine challenge and 24 hour oesophageal pH monitoring effectively ruled out CVA and GOR, respectively, as a cause for cough.

CONCLUSION—This comprehensive approach aids the accurate direction of treatment and, while CVA, PNDS and GOR remain the most important causes of CNPC to consider, a group with no identifiable aetiology remains.

Antimicrobial peptides in the venom of the European hornet, Vespa crabro, identified as mastoparan C and crabrolin

Amphibian skin secretions are rich sources of cationic amphipathic peptides which often possess potent and broad-spectrum antimicrobial activity. However, the venoms of other animals such as hymenopteran insects, also contain peptides with these characteristics and the literature is unclear as to their antimicrobial potential. Here we subjected the venom of the European hornet, Vespa crabro, to reverse phase HPLC fractionation followed by screening of aliquots of individual fractions in bacterial zonal inhibition assays. Two major peptides possessing activity in these assays were further purified by HPLC and subjected to MALDI-TOF MS analysis and MS/MS fragmentation using an ESI mass spectrometer. The peptides were identified as mastoparan C (LNLKALLAVAKKILamide) and crabrolin (FLPLILRKIVTALamide). Replicates of both peptides were synthesised by solid-phase methodology and mean inhibitory concentrations (MICs) established against Staphylococcus aureus and Escherichia coli. Mastoparan C was found to be a potent antimicrobial with MIC values of 2 µM and 4 µM against S. aureus and E. coli, respectively. Crabrolin was found to be less potent with MIC values of > 160 µM and 40 µM for S. aureus and E. coli. Hornet venom thus contains a potent antimicrobial peptide that has been unambiguously identified as mastoparan C, a peptide that is known to affect profound histamine release from mast cells and to generally activate membrane G protein-linked receptors. It is thus highly probable that its antimicrobial effects, like those previously documented, are a result of a generalized membrane interactive and disruptive function — perhaps reflective of the authentic role of amphibian skin antimicrobials.