Structure-activity relationships of neurokinin A (4-10) at the human tachykinin NK(2) receptor: the role of natural residues and their chirality.

A structure-activity study of neurokinin A (NKA) (4-10) was performed to investigate the importance of residue and chirality for affinity and efficacy at the NK(2) receptor in human colon circular muscle. Two series of NKA(4-10) analogues were produced with either L-alanine or the D-enantiomer substituted. Their activities were determined in vitro by means of radioligand binding and isolated smooth muscle pharmacology. NKA was more potent than NKA(4-10) at the human, unlike the rabbit, NK(2) receptor. The contractile response of NKA(4-10) was unaffected by N-terminal acetylation. L-Ala substitution of Asp(4), Val(7), Leu(9), and Met(10) caused an 8- to 80-fold decrease, and substitution of Phe(6) caused a 5000-fold decrease in binding affinity (P < 0.01). Positions Ser(5) and Gly(8) were not significantly affected. In functional studies, a similar pattern was observed. The replacement of residues with their respective D-enantiomer drastically reduced binding affinity and functional potency, particularly at positions 6 and 7 (P < 0.05). NKA(4-10) analogues L-Ala(6), L-Ala(8), D-Phe(6), D-Val(7), and D-Met(10) were partial agonists. An excellent correlation was observed between binding and functional data (r = 0.95). A retro-inverso analogue of NKA(4-10) was inactive. In conclusion, the side chains of Asp(4), Phe(6), Val(7), Leu(9), and Met(10) are structurally important features of NKA(4-10) for agonist activity, and changes in amino acid chirality are detrimental to binding affinity and functional activity. Overall, our data are broadly similar to those of previous studies in the rat. However, at the human NK(2) receptor, unlike the rat, [Ala(8)]NKA(4-10) was an antagonist.

Bufokinin: actions and distribution in the toad cardiovascular system.

1. Bufokinin is a substance P-like neuropeptide and potent spasmogen isolated from the intestine of the cane toad Bufo marinus. In the present study, we investigated the effects of bufokinin on systemic blood pressure and heart rate in the anaesthetized toad and the distribution of bufokinin-like immunoreactivity in the toad vasculature. 2. Intravenous bufokinin caused a dose-dependent fall in systemic blood pressure (maximum fall 20 mmHg) with an ED50 of 2.9 pmol. At higher doses, the effect was prolonged and blood pressure did not return to baseline within 60 min. There was no significant change in heart rate associated with hypotension. 3. Bufokinin-like immunoreactivity was mapped in whole mounts of toad blood vessels and organs using a mouse polyclonal antibody BK3 (at 1:5000) and the avidin-biotin method. Bufokinin-immunoreactive fibres were associated with most blood vessels examined: a moderately dense perivascular network of varicose fibres was present around renal arteries, with sparser immunoreactive fibres in the ventral aorta, sciatic artery, anterior abdominal vein and hepatic portal vein. 4. Bufokinin-immunoreactive fibres, mainly following blood vessels, were seen in whole mounts of the urinary/bladder and tongue, but not in the air sac. In the heart ventricle, varicose fibres were found in the valve cusps, intracardiac ganglia, epicardium and myocardium close to the endocardium, but not in the rest of the myocardium. 5. The vasodepressor action of bufokinin and the presence of bufokinin-like immunoreactivity in varicose fibres in various vessels suggest a role for bufokinin in haemodynamic regulation and/or sensory nerve function in the toad. The lack of any reflex tachycardia in response to the falls in blood pressure was of note.

Bufokinin: immunoreactivity, receptor localization and actions in toad intestine and mesenteric circulation.

In this study, we have mapped the immunoreactivity and the binding sites for bufokinin, a tachykinin peptide from the toad intestine. Dense bufokinin-immunoreactive fibers were present at the myenteric plexus, but no cell bodies were stained, suggesting an extrinsic origin. Bufokinin nerve fibers were also associated with submucosal blood vessels and mesenteric arteries. Autoradiographic binding sites for [(125)I]Bolton-Hunter-bufokinin were densely localized over the intestinal circular and longitudinal muscle, submucosal blood vessels and the endothelium of mesenteric arteries. Mesenteric veins had minimal immunoreactivity and binding sites. In the anesthetized toad, topical application of bufokinin onto the mesentery caused a 2.7-fold increase in arterial blood flow, observed using intravital microscopy. This study supports a role for bufokinin as an endogenous spasmogen and hemodynamic regulator in the toad intestine.

Characterization of the [125I]-neurokinin A binding site in the circular muscle of human colon.

Neurokinin A (NKA) is a potent contractile agonist of human colon circular muscle. These responses are mediated predominantly through tachykinin NK2 receptors. In the present study, the NK2 receptor radioligand [125I]-NKA has been used to characterize binding sites in this tissue, using tachykinin agonists and antagonists. 125INKA labelled a single, high affinity binding site. Specific binding (95% of total binding) of [125I]-NKA was saturable (K(D) 0.47+/-0.05 nM), of high capacity (Bmax 2.1+/-0.1 fmol mg(-1) wet weight tissue) and reversible (kinetically derived K(D) 0.36+/-0.07 nM). The rank order of agonists competing for the [125I]-NKA binding site was neuropeptide gamma (NPgamma) > or = NKA > or = [Lys5, MeLeu9,Nle10]NKA (4-10) (NK2 agonist) >> substance P (SP) > neurokinin B (NKB) > or = [Pro9]SP (NK1 agonist) >> senktide (NK3 agonist), indicating binding to an NK2 site. The nonpeptide selective NK2 antagonist SR48968 showed higher affinity for the [125I]-NKA site than selective peptide NK2 antagonists. The rank order of potency for NK2 antagonists was SR48968 > or = MEN11420 > GR94800 > or = MEN10627 > MEN10376 > or = R396. The NK1 antagonist SR140333 was a weak competitor. The competition curve for SP could be resolved into two sites. When experiments were repeated in the presence of SR140333 (0.1 microM), the curve for SP became monophasic and showed a significant shift to the right, whereas curves to NKA and NKB were unaffected. In conclusion, binding of the radioligand [125I]-NKA to membranes from circular muscle is predominantly to the NK2 receptor. There may be a small component of binding to the NK1 receptor. The NK2 receptor mediates circular muscle contraction, whereas the role of the NK1 receptor in circular muscle is unclear.

Structure-activity studies at the rat tachykinin NK2 receptor: effect of substitution at position 5 of neurokinin A.

A series of analogues of neurokinin A(4-10) was synthesized using solid phase techniques with Chiron pins, and purified by HPLC. The potencies of 10 peptides with substitution at Ser5 were assessed at rat fundus NK2 receptors. In membrane binding studies with [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10), all compounds except [Asp5]NKA(4-10) showed reasonable affinity, and analogues with Lys and Arg substitutions were five-fold more potent than NKA(4-10). In functional studies, all peptides were able to contract the rat isolated fundus strips. Analogues with Phe, His and Asn substitutions were substantially weaker in functional than in binding studies, whereas there was an excellent correlation (r = 0.95) between binding and functional potency for the remaining seven peptides. [Phe5]NKA(4-10) is in fact neurokinin B(4-10) and this residue may be critical in determining selectivity between NK2 and NK3 receptors. Analogues with a basic residue (Lys, Arg) at position 5 showed both increased affinity and functional potency, whereas the neutral [Asn5]NKA(4-10) was equally as weak in contractile studies as the acidic [Asp5]NKA(4-10). However, [Glu5]NKA(4-10) and [Gln5]NKA(4-10) were no different from NKA(4-10). Our results could indicate the presence of a negative charge on the NK2 receptor, close to position 5 of NKA. This would facilitate interaction with positively charged side chains and impede interaction with negatively charged side chains, particularly the inflexible side chain of aspartic acid. Thus, not only the charge, but also the length of the side chain of the residue at position 5, seems to be important for interaction with the rat NK2 receptor.

Binding and functional potency of neurokinin A analogues in the rat fundus: A structure-activity study.

Structure-activity relationships of neurokinin A (NKA) and the two analogues NKA(4-10) and [Nle10]NKA(4-10) were investigated at the rat fundus NK-2 receptor, using selected amino acid substitutions. Both radioligand binding with [125I][Lys5,Tyr(I2)7,MeLeu9, Nle10] NKA(4-10) and functional studies were performed and correlated. In membrane binding experiments loss of His1 and Lys2, or replacement of Lys2 with Ala did not substantially alter binding affinity of NKA. NKA(4-10) free acid was unable to compete with the radioligand. [Nle10]NKA(4-10) binding affinity to rat fundus membrane preparations was decreased when substituting Asp4 with Gln or Asn, or Val7 with either Tyr or Ile. Replacement of Ser5 with the negatively charged Glu also decreased the binding affinity, but substitution with the positively charged Lys substantially increased the affinity of [Nle10] NKA(4-10) for the NK-2 receptor. Lengthening NKA(4-10) or [Nle10]NKA(4-10) with Ala11 or Nle11, respectively, decreased the binding affinity of the peptide. In both binding and functional studies, replacement of any of the residues of NKA(4-10), except for Ser5, with alanine decreased the affinity of the peptide for the NK-2 receptor. Ala substitutions at positions 4, 6, and very obviously at 8, 9 and 10 of NKA(4-10) yielded peptides unable to achieve a maximum contractile response, although they did not demonstrate antagonist activity. These data confirm the importance of the NKA carboxyl terminus, and the requirement for Phe6, Val7, Gly8, Leu9 and Met10 integrity for interaction with the NK-2 receptor. They also suggest that Ser5 is a good site to target modifications leading to the design of new potential drugs.

Effect of pattern of preceding inspiration on FEV1 in asthmatic children.

In adults, both peak expiratory flow (PEF) and forced expiratory volume in one second (FEV1) are significantly influenced by the time course of the inspiration preceding the forced expiration. The aim of this study was to evaluate the effects of three different inspiratory manoeuvres on PEF, FEV1, and forced vital capacity (FVC) in asthmatic children. Twenty five symptomless asthmatic children performed forced expiration preceded by three different inspiratory manoeuvres, which consisted of: a rapid inspiration with a 2 s end-inspiratory breathhold (Manoeuvre No. 1); a rapid inspiration without an end-inspiratory breathhold (Manoeuvre No. 2); and a slow inspiration lasting about 5 s with an end-inspiratory breathhold of at least 4 s (Manoeuvre No. 3). All manoeuvres were performed in a randomly assigned sequence each morning for three consecutive days. In each session, the manoeuvres were repeated three times and the highest value was chosen. Both FVC and FEV1 obtained with Manoeuvre No. 3 were significantly lower than the corresponding values obtained with Manoeuvre Nos. 1 and 2. The mean (SD) FVC values were 2.76 (0.66) L with Manoeuvre No. 1, 2.67 (0.58) L with Manoeuvre No. 2 and 2.52 (0.52) L with Manoeuvre No. 3. The corresponding values of FEV1 were 2.25 (0.53), 2.22 (0.53) and 2.07 (0.44) L, respectively. By contrast, the values of PEF, obtained with a portable peak flow meter, were similar with the three different inspiratory manoeuvres. The results of this study show that in symptomless asthmatic children the preceding inspiratory manoeuvre may influence forced vital capacity and forced expiratory volume in one second. Hence, in order to reduce variability due to interference by physiological factors and so improve reproducibility of pulmonary function tests, the inspiratory manoeuvres must be accurately standardized.

Bronchial reactivity in asthmatic children at high and low altitude. Effect of budesonide.

Inhaled steroids may control bronchial inflammation in asthmatics exposed to allergens. In this study we evaluated whether prophylactic budesonide would prevent relapse of asthma in children re-exposed to offending allergens at sea level, after a period of antigen avoidance at high altitude. Thirty children received either budesonide (200 micrograms b.i.d.) or placebo (double-blind). Following a 4-wk baseline period and 2 wk of treatment at high altitude, children were treated for 3 mo at sea level. Methacholine challenge and pulmonary function studies were performed before and after baseline period, after the 2 wk of treatment in the mountain environment, and at the end of treatment. ECP serum levels were evaluated after the baseline period and at the end of treatment. PEFR and symptoms were recorded in a diary card during the study. The increase in methacholine provocative dosage was greater, although not significant (p = 0.096), in the budesonide than in the placebo group after the treatment at high altitude and remained higher at the end of the treatment (p = 0.04). ECP levels increased in both the groups with no significant difference. Our results confirm that budesonide, in addition to its efficacy in treating pre-existent airway inflammation, is effective in preventing the increase of reactivity in asthmatic children re-exposed to allergens.

Peak expiratory flow variation and bronchial hyperresponsiveness in asthmatic children during periods of antigen avoidance and reexposure.

Changes of diurnal variation of peak expiratory flow rate (%PEF variation) and their relationship with bronchial hyperresponsiveness (BHR) to methacholine (PC20) were evaluated in 12 children with mild-to-moderate asthma and house-dust mite allergy, during successive periods of stay in a mite-free environment at high altitude (1756 m) and at their home at sea level. The children remained at the high altitude from October until the end of December; then they spent a 3-week period at home and returned to high altitude residence in January. PEF was measured daily, in the morning and in the evening, during the 3 months' stay at high altitude and them for 10 days after the return in January. PC20 was assessed in 8/12 children, once a month from October to December, and at the return in January. Mean absolute PEF values did not change significantly throughout the study. From October to December, patients showed a significant decrease of mean %PEF variation (P = 0.04), while PC20 showed an increase (P = 0.05). After the 3 weeks at home, both %PEF variation (P = 0.03) and PC20 (P = 0.05) significantly worsened. The correlation between PC20 values and mean %PEF variation in the 2 days before and after each methacholine test was r = -0.63 (P = 0.001). Our data suggest that there is a beneficial effect of a prolonged stay in a mite-free environment, on both PEF variability and BHR, also in asthmatic children with good pulmonary function. PEF variability and bronchial responsiveness to methacholine were significantly correlated also for small changes of the two variables.

Evidence for a high molecular weight pre-robustoxin molecule in the venom of the male Sydney funnel-web spider (Atrax robustus).

Robustoxin is the lethal polypeptide toxin in Atrax robustus venom. A monoclonal antibody was produced using synthetic, unfolded robustoxin conjugated to keyhole limpet haemocyanin as the immunogen. This monoclonal antibody did not protect newborn mice against challenge with the crude venom of the male Sydney funnel-web spider, but did slightly prolong their survival time. Western blotted crude venom of the male Sydney funnel-web spider showed two monoclonal antibody binding bands. One band at low M(r) corresponded to robustoxin (M(r) 4854), while the other higher M(r) band (approximately 37,000) may be due to a pre-robustoxin molecule.

Benzyl-benzoate foam: effects on mite allergens in mattress, serum and nasal secretory IgE to Dermatophagoides pteronyssinus, and bronchial hyperreactivity in children with allergic asthma.

Home mattresses of 24 asthmatic children with house dust mite allergy were sprayed with either benzyl-benzoate foam or placebo in a double blind fashion, 10 days before the children left the residential house for asthmatic children Istituto Pio XII (located in the Italian Alps in an environment free of mites) and went back to their own home for the Christmas and Easter holidays. A further group of 8 children, whose mattresses received no treatment, was kept as an absolute control. Two days after spraying, benzyl-benzoate or placebo were vacuumed from the mattresses. Acarex test was performed immediately before spraying and at the end of each holiday period of 20 and 10 days, respectively. Bronchial hyperreactivity as well as serum and nasal secretory specific IgE for Dermatophagoides pteronyssinus were assessed in all children immediately before leaving and within 48 hr after returning to the residential house. The results of the study show that sprayed benzyl-benzoate foam was no more effective than placebo in reducing the level of house dust mite recovered from patients' mattresses, or in reducing bronchial hyperreactivity and IgE concentration in serum and nasal secretions.

Antigen avoidance in a mountain environment: influence on basophil releasability in children with allergic asthma.

BACKGROUND: The influence of natural antigen avoidance in an environment free of relevant allergens (Istituto Pio XII, Misurina, Italian Alps, 1756 m) and of antigen exposure (sea level) on basophil releasability, as well as on bronchial hyperreactivity (BHR) and specific IgE serum level, were investigated in a group of children with asthma who were allergic to Dermatophagoides pteronyssinus. METHODS: Twenty allergic children with asthma participated in the study. Spontaneous and antigen-induced histamine release, BHR, and serum IgE were investigated at the time of admission, after 40 and 80 days of antigen avoidance, and after 15 days of exposure at sea level. RESULTS: Significant drops in antigen-induced basophil histamine release, BHR, and specific IgE serum level but not in spontaneous basophil histamine release were observed after 40 days of antigen avoidance and were confirmed at a further evaluation after 40 more days. After 15 days of antigen exposure at sea level, specific antigen-induced basophil histamine release, BHR, and serum IgE but not spontaneous basophil histamine release increased promptly, even if not significantly. CONCLUSION: In children with allergic asthma, antigen-induced basophil releasability, BHR, and specific IgE serum levels appear to be modifiable by periods of antigen avoidance or exposure.

Monoclonal antibodies neutralizing the haemolytic activity of box jellyfish (Chironex fleckeri) tentacle extracts.

1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri). 2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots. 3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions. 4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions.

Comparison of nedocromil sodium and sodium cromoglycate administered by pressurized aerosol, with and without a spacer device in exercise-induced asthma in children.

To compare the effectiveness of nedocromil sodium (NS) and sodium cromoglycate (SCG) administered by metered dose inhaler (MDI) in preventing exercise-induced asthma (EIA), 12 asthmatic children with EIA were studied in a randomized, double-blind, cross-over, placebo-controlled study. NS and SCG were given by MDI alone, and by MDI with a 700 ml spacer device (Fisonair, Fisons, UK), in order to assess the benefit of using such a device. Following a baseline exercise challenge, the protective effect of NS, SCG or placebo was evaluated in each subject. The percentage fall in forced expiratory volume in one second, and percentage protection were measured. NS and SCG provided a significant and comparable protection from EIA, and both were better than placebo. No further improvement was observed after drug administration via the spacer. Both NS and SCG are effective in preventing EIA in children, when administered at the recommended clinical dose, and the use of a spacer for administering the drug provides no advantage if the technique of inhalation is good.

Preliminary study on the effect of broxaterol on bronchial hyperresponsiveness in asthmatic children: influence of allergen exposure.

Increased bronchial hyperresponsiveness (BHR) has been reported in adult asthmatic patients after regular treatment with beta 2-agonists. In this study we evaluated the effect of a 4-weeks treatment period with broxaterol, a new beta 2-agonist, on BHR in asthmatic children living in two different environmental conditions. Two groups of patients, 24 domiciled at sea level, allergen exposed (group 1), and 24 resident at high altitude (Misurina, 1,756 m) in an allergen-free environment (group 2), have been tested. Children were randomly treated with broxaterol 400 micrograms q.i.d. or sodium chromoglycate 10 mg q.i.d. (as control treatment) by the metered dose inhaler for 4 weeks. Pulmonary function tests and methacholine challenge were performed at the beginning and at the end of the study. Throughout the study period a diary card was completed and peak expiratory flow rate (PEFR) was measured three times daily. Forty-two of the patients admitted concluded the study. No significant change was observed in the methacholine PC20 throughout the study period regardless of the type of treatment and/or environment. The amplitude percentage mean of diurnal changes in PEFR during the study period showed no statistically significant differences between treatments or centres, or in the interaction of treatment with centre, time with treatment, time with centre, time with centre and treatment (p > 0.05). Therefore in this pilot study regular treatment with broxaterol for 4 weeks did not cause an increase in BHR in asthmatic children both in conditions of allergen exposure and in an environment free of offending allergens.

An endogenous antitoxin to the lethal venom of the funnel web spider, Atrax robustus, in rabbit sera.

1. An endogenous antitoxin fraction was isolated from non-immune rabbit sera by affinity chromatography with robustoxin bound to the solid support. 2. Robustoxin is the sole lethal toxin in the venom of the male funnel web spider, Atrax robustus. 3. The fraction was found to contain IgG and IgM immunoglobulins. 4. This fraction prevented or reversed the lethal actions of the crude venom in newborn mice, in mouse phrenic nerve-hemidiaphragm preparations, and in anaesthetized monkeys. 5. The antitoxin fraction is of potential value in the therapy of human envenomation by A. robustus.

Protection of monkeys against the lethal effects of male funnel-web spider (Atrax robustus) venom by immunization with a toxoid.

A stable toxoid was prepared from robustoxin (the lethal polypeptide neurotoxin in the venom of the male funnel-web spider, Atrax robustus) by polymerization with glutaraldehyde. This material was non-toxic in new-born mice. Administration of the toxoid to three Macaca fascicularis monkeys (50-80 micrograms/kg s.c. at 14-day intervals for 8-12 weeks) produced no toxic effects; anti-robustoxin antibodies were detected in serum samples by immunodiffusion tests within 13-27 days. In vivo evidence of successful protection with the toxoid was obtained by challenging the monkeys with male A. robustus venom (50 micrograms/kg i.v.) under anaesthesia with pentobarbitone (one monkey), or with ketamine, halothane and nitrous oxide, 1-26 weeks after the last injection of the toxoid. Only minor respiratory, cardiovascular and skeletal motor disturbances were produced, and all monkeys recovered fully and uneventfully. Challenge with the same dose of venom in non-immunized or robustoxin N-terminal decapeptide ovalbumin conjugate-treated monkeys resulted in typical lethal neurotoxic effects, culminating in severe hypotension or death from circulatory and respiratory failure within 280 min.

Direct immunization with synthetic peptidyl-polyamide resin. Comparison with antibody production from free peptide and conjugates with carrier proteins.

An 11-amino acid residue peptidyl-linkage agent-polyamide resin complex was synthesized by the fluorenylmethyloxycarbonyl (Fmoc)-polyamide solid-phase system. Mice were immunized with the free peptide, peptidyl-resin and peptide coupled to the carrier proteins ovalbumin (Ova) and keyhole limpet haemocyanin (KLH). The immunogenicity of these materials was assessed by measurement of the capacity of the various antisera to bind the peptide in an enzyme-linked immunosorbent assay (ELISA). The peptidyl-resin exhibited enhanced immunogenicity compared to the free peptide. It is suggested that the time needed for screening for immunogenicity of large numbers of synthetic peptides thus be greatly shortened by using peptidyl-resins for immunization. This method eliminates laborious cleavage of peptide from resin, purification, coupling to carrier and the difficulties of handling peptides of low solubility.

Stabilization of lethal and hemolytic activities of box jellyfish (Chironex fleckeri) venom.

The stability of both the lethal and hemolytic activities of box jellyfish (Chironex fleckeri) tentacle extract was assessed after various extraction procedures. Both activities were higher when no buffers or water were used during the initial extraction. Also, when the extract was first filtered through a Sep-pak C18 cartridge, the residual lethal titre, after incubation for 24 hr at room temperature, was increased 16-fold and hemolysis was increased 2.6-fold. Evidence for proteolytic activity in the extract was also obtained and monitored by size exclusion HPLC.

Oxygen carriage and carbonic anhydrase activity in the blood of a marsupial, the Tammar wallaby (Macropus eugenii), during early development.

Blood O2 transport and Hb type have been studied in pouch young and adult of a marsupial, the Tammar Wallaby. The O2-Hb equilibrium curves (at 35.5 degrees C and PCO2 = 34 Torr) had a high P50 in the first few days of life, up to 49 Torr. This fell to 32 Torr by 2 weeks of age. Also (delta log P50/delta PCO2) was low but it rose to adult levels by 2 weeks of age. The curves in these early pouch young showed a change in Hill coefficient (nH) at between 32 and 62% saturation, nH rising to more than 4.0 at higher O2 saturations. This indicates interaction between more than 4 Hb subunits. Model calculations showed that such curves could be produced by a mixture of 2 Hb components; one with a low P50 and low nH, and one with a high P50 and high nH. In this model the nH values were different from the nH values of either component. The temperature effect on P50 in early pouch young was higher than in adult Tammars and similar to that reported for adult eutherians. In the first 4 days all red cells were nucleated and four Hb types were present. Carbonic anhydrase activity in the blood before birth was about 30% of the adult levels. These levels remained until 2 days after birth, when a rapid rise in activity began, near-adult levels being reached at 5 days despite the animals being still very immature.