Species dependence for binding of small molecule agonist and antagonists to the C5a receptor on polymorphonuclear leukocytes

This study investigated the receptor binding affinities of a C5a agonist and cyclic antagonists for polymorphonuclear leukocytes (PMNs) isolated from human, sheep, pig, dog, rabbit, guinea pig, rat and mouse. The affinities of the two small molecule antagonists, F-[OPdChaWR] and AcF-[OPdChaWR], and the agonist, YSFKPMPLaR, revealed large differences in C5a receptor (C5aR) affinities between species. The antagonists bound to human, rat and dog PMNs with similar high affinities, but with lower affinities to PMNs from all other species. The C5a agonist also bound with varying affinities between species, but showed a different affinity profile to the antagonists. In contrast, recombinant human C5a had similar affinity for PMNs of all species investigated. The low correlation between the affinities of the antagonists and the agonist between species either suggests that different receptor residues are important for distinguishing between agonist/antagonist binding, or that the agonist and antagonist peptides bind to two distinct sites within the C5aR.

Characterization of a glycine receptor domain that controls the binding and gating mechanisms of the beta-amino acid agonist, taurine

The beta -amino acid, taurine, is a full agonist of the human glycine receptor al subunit when recombinantly expressed in a mammalian (HEK293) cell line, but a partial agonist of the same receptor when expressed in Xenopus oocytes. Several residues in the Ala101-Thr112 domain have previously been identified as determinants of beta -amino acid binding and gating mechanisms in Xenopus oocyte-expressed receptors. The present study used the substituted cysteine accessibility method to investigate the role of this domain in controlling taurine-specific binding and gating mechanisms of glycine receptors recombinantly expressed in mammalian cells. Asn102 and Glu103 are identified as taurine and glycine binding sites, whereas Ala101 is eliminated as a possible binding site. The N102C mutation also abolished the antagonistic actions of taurine, indicating that this site does not discriminate between the putative agonist- and antagonist-bound conformations of beta -amino acids. The effects of mutations from Lys104-Thr112 indicate that the mechanism by which this domain controls beta -amino acid-specific binding and gating processes differs substantially depending on whether the receptor is expressed in mammalian cells or Xenopus oocytes. Thr112 is the only domain element in mammalian cell-expressed GlyRs which was demonstrated to discriminate between glycine and taurine.

A single beta subunit M2 domain residue controls the picrotoxin sensitivity of alpha beta heteromeric glycine receptor chloride channels

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the alpha beta heteromeric glycine receptor relative to the alpha homomeric receptor. By analogy with structurally related receptors, the beta subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the alpha subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the alpha homomeric receptor. Furthermore, the converse F282T mutation in the beta subunit increased the picrotoxin sensitivity of the alpha beta heteromeric receptor. The P278G mutation in the beta subunit did not affect the picrotoxin sensitivity of the alpha beta heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to alpha subunit T258 is specifically required for picrotoxin sensitivity. Mutations to alpha subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin las an allosterically acting 'competitive' antagonist) binds to this residue.

Two new classes of conopeptides inhibit the alpha 1-adrenoceptor and noradrenaline transporter

Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets alpha (1)-adrenoceptors (rho -TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (chi -MrIA and chi -MrIB from the mollusk-hunting C. marmoreus). rho -TIA and chi -MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs.

Antagonism of the two-needle pine stem rust fungi Cronartium flaccidum and Peridermium pini by Cladosporium tenuissimum in vitro and in planta

Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 x 10(7) conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4 degreesC. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wail and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host tell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known beta -1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.

The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-LI adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA/AM.. While the ionophores A23187 or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degreesC, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/cahnodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.

Clinical trials with glycoprotein IIB/IIIA antagonists - No benefit without bleeding?

As the glycoprotein GPIIb/IIIa receptor is the final common pathway in platelet aggregation, antagonists of this receptor cause a profound inhibition of aggregation induced by any agonist. The short-term efficacy and safety of GPIIb/IIIa antagonists in patients undergoing coronary angioplasty was demonstrated with murine 7E3 Fab, but this antibody was immunogenic. Abciximab is a chimeric human-mouse monoclonal antibody that is less immunogenic. The first major trial with a GPIIb/IIIa antagonist was the EPIC trial with abciximab, which showed that abciximab reduced the ischemic complications of coronary balloon angioplasty and atherectomy in high-risk patients, but increased the risk of bleeding. Subsequent studies showed that using less concurrent heparin reduced bleeding. Abciximab also reduced the rate of revascularization. Further studies have shown that the benefits of abciximab extended to all patients undergoing angioplasty (EPILOG), including patients with unstable angina (CAPTURE) and acute myocardial infarction (RAPPORT). Clinical trials with eptifibatide and tirofiban have failed to demonstrate benefit, at the doses used, in angioplasty. Abciximab and eptifibatide, but not oral xemilofiban, improve the safety of the coronary stenting procedure. Shortterm intravenous treatment with lamifiban, eptifibatide or tirofiban is beneficial in acute coronary syndromes (unstable angina, non-Q wave myocardial infarction). Orally active GPIIb/IIIa antagonists are being developed for use in acute coronary syndromes and myocardial infarction. However, no benefit has been shown with lefradafiban in acute coronary syndromes and sibrafiban and orbofiban are harmful. Eptifibatide, lamifiban and abciximab improve coronary patency in myocardial infarction, and long-term trials of GPIIb/IIIa antagonists are being conducted in acute myocardial infarction. Abciximab can cause thrombocytopenia, and all the GPIIb/IIIa antagonists increase the incidence of bleeding, but there is no excess of intracranial hemorrhage. (C) 2001 Prous Science. All rights reserved.

Pharmacotherapy of intermittent claudication

Intermittent claudication (IC) is leg muscle pain, cramping and fatigue brought on by exercise and is the primary symptom of peripheral arterial disease. The goals of pharmacotherapy for IC are to increase the walking capacity/quality of life and to decrease rates of amputation. In 1988, pentoxifylline was the only drug that had reasonable supportive clinical trial evidence for being beneficial in IC. Since then a number of drugs have shown benefit or potential in IC. Cilostazol, a specific inhibitor of phosphodiesterase 3 and activator of lipoprotein lipase, clearly increases pain-free and absolute walking distances in claudicants. However, cilostazol does cause minor side effects including headache, diarrhoea, loose stools and flatulence. Naftidrofuryl, a serotonin (5-HT2) receptor antagonist and antiplatelet drug, is beneficial in claudicants. Inhibitors of platelet aggregation (including nitric oxide from L-arginine or glyceryl trinitrate) and anticoagulants (low molecular weight heparin, defibrotide) probably have both short and long-term benefits in IC. In addition, intravenous infusions of prostaglandins (PGs) PGE1 and PGI2 have an established role in severe peripheral arterial disease and the recent introduction of longer lasting and/or oral forms of the PGs makes them more likely to be useful in the IC associated with less severe forms of the disease. There are some exciting new approaches to the treatment of IC, including propionyl-L-carnitine and basic fibroblast growth factor (bFGF).

ReoPro rules: results from the 'DoTirofiban and ReoPro give similar efficacy trial' (TARGET) - A Key Paper Evaluation (Topol, Ej et al)

In the treatment of atherosclerotic disease, stenting in the presence of a glycoprotein (GP) IIb/IIIa antagonist is becoming an increasingly common procedure. The ‘Do Tirofiban and ReoPro Give Similar Efficacy Trial’ (TARGET) was designed to determine whether the cheaper tirofiban was as effective and safe as abciximab in the prevention of ischaemic events with stenting. Unexpectedly, abciximab was shown to be superior to tirofiban. Tirofiban is a selective GP IIb/IIIa antagonist whereas abciximab has additional anti-inflammatory actions, which may contribute to its superiority.

Long-lasting synaptic modification in the rat hippocampus resulting from NMDA receptor blockade during development

Recent reports have suggested that proper maturation of synapses in the hippocampus requires activation of NMDA receptors. We previously demonstrated that neonatal ethanol exposure results in a lasting reduction in synaptic strength in the hippocampus. To determine if this reduction was due to ethanol's effects on NMDA receptors, we investigated long-term changes in synaptic properties resulting from administration of NMDA receptor antagonists to neonatal animals. Rats were injected daily from PND 4-9 with either the noncompetitive NMDA receptor antagonist MK-801, the competitive NMDA receptor antagonist CPP, or the AMPA receptor antagonist NBQX. Control rats were either injected daily with physiological saline during the same period or left to develop normally. Hippocampal slices were prepared from nembutal-anesthetized animals between PND 35 and PND 40. The maximum pEPSP and PS values were not significantly different between controls and NMDA antagonist-treated animals. However, slices from animals injected with NMDA receptor antagonists required higher stimulus currents to attain comparable pEPSPs. The ratio of the slope of the pEPSP to the amplitude of the presynaptic volley was also reduced, as were pEPSP responses to specific stimulus currents. None of these effects were observed in slices prepared from animals treated with the AMPA receptor antagonist NBQX. Glutamate receptor antagonism did not produce lasting changes in long-term potentiation or paired-pulse facilitation. These results indicate activation of NMDA receptors during development is necessary for proper development of synapses. (C) 2001 Wiley-Liss, Inc.

Endogenous endothelin-1 limits exercise-induced vasodilation in hypertensive humans

Essential hypertension is a common disorder, associated with increased endothelin-l-mediated vasoconstrictor tone at rest. We hypothesized that increased vasoconstrictor activity of endothelin-1 might explain why the normal decrease in peripheral vascular resistance in response to exercise is attenuated in hypertensive patients. Therefore, we investigated the effect of endothelin A (ETA) receptor blockade on the vasodilator response to handgrip exercise. Forearm blood flow responses to handgrip exercise (15%, 30%, and 45% of maximum voluntary contraction) were assessed in hypertensive patients and matched normotensive subjects, before and after intra-arterial infusions of the ETA receptor antagonist BQ-123; a control dilator, hydralazine; and placebo (saline). Preinfusion (baseline) vasodilation in response to exercise was significantly attenuated at each workload in hypertensive patients compared with normotensive subjects. Intra-arterial infusions of hydralazine and saline did not increase the vasodilator response to exercise in either hypertensives or normotensives at any workload. The vasodilator response to exercise was markedly enhanced after BQ-123 at the 2 higher workloads in hypertensives (157 +/- 48%, P < 0.01; 203 &PLUSMN; 58%, P < 0.01) but not in normotensives. This suggests that the impaired vasodilator response to exercise in hypertensive patients is, at least in part, a functional limitation caused by endogenous ETA receptor-mediated vasoconstriction. Treatment with endothelin receptor antagonists may, therefore, increase exercise capacity in essential hypertension.

Oral administration of a 12% sucrose solution did not decrease behavioural indicators of distress in piglets undergoing tail docking, teeth clipping and ear notching

Sucrose has been shown to attenuate the behavioural response to painful procedures in human infants undergoing circumcision or blood collection via heelstick. Sucrose has also been found to have a behaviour-modifying effect in neonatal rats exposed to a hot plate. The effect was abolished in neonatal rats by injection of the opioid antagonist naltrexone, suggesting that it was mediated by endogenous opioids. In this experiment, the behaviour of 571 newborn Large White x Landrace hybrid piglets in a specific-pathogen-free piggery of the University of Queensland was recorded during and after the routine management practices of tail docking, ear notching and teeth clipping. Piglets were randomly assigned to receive 1.0 ml of a 12% sucrose solution (treatment group) or a placebo (1.0 ml of air) administered via syringe in the mouth, 60 s before commencement of one of the management procedures. Behaviours were recorded at the time of the procedure, and then 2 min after completion of the procedure. Piglets that received the sucrose solution did not behave significantly differently from piglets receiving the placebo. Regardless of whether sucrose or placebo was administered, piglets undergoing the routine management procedures showed significantly greater behavioural responses than piglets undergoing no procedure. It was concluded that under commercial conditions, a 12% sucrose solution administered I min prior to surgery was not effective in decreasing the behavioural indicators of distress in piglets undergoing routine management procedures, Further research into methods of minimising distress caused to piglets by these procedures is recommended.

Antagonism by NKP608 of substance P-induced plasma protein exudation in a novel preparation of perfused trachea in rats and guinea-pigs in vivo

Objective: To examine whether NKP608, a novel 1-benzoyl-2-benzyl-4-aminopiperidine NK1 receptor antagonist, inhibits substance P (SP)-induced airway plasma protein exudation in vivo. Material: Anaesthetised English shorthair guinea-pigs and Wistar rats. Treatment: Tachykinin peptides were applied topically onto the trachea and antagonists administered intravenously. Methods: Tracheal segments isolated in situ were perfused with saline and plasma-derived protein assayed in the perfusate. Results: SP (1 muM) caused plasma protein exudation, which was abolished by an NK1 antagonist (RP 67580, 1.75 mumol/kg) but unaffected by an NK2 antagonist (SR 48968, 1.75 mumol/kg) indicating the response is NK1-receptor-mediated. This was confirmed with a response to an NK1 agonist ([Sar(9), Met(O-2)(11)]-SP, 1 muM) but none to an NK2 agonist ([betaAla(8)]-neurokinin A(4-10), 1 muM). NKP608 inhibited SP responses with estimated ID50 values (mumol/kg) of 0.0044 (guinea-pigs) and 0.19 (rats). Conclusions: NKP608 is an antagonist in vivo of NK1 receptor-induced tracheal plasma protein exudation and is more potent in guinea-pigs than rats.

A novel bioassay for human somatogenic activity in serum samples supports the clinical reliability of immunoassays

OBJECTIVE Because there is discordance between different immunoassay values for serum hGH, and because clinical state may not correlate with immunoreactive hGH, we have developed an assay to accurately measure serum hGH somatogenic bioactivity. The results of this assay were compared with the Elegance two-site ELISA assay across 135 patient samples in a variety of clinical states. DESIGN The somatogenic assay was based on stable expression of hGH receptor in the murine BaF line, allowing these cells to proliferate in response to hGH. To eliminate interference by other growth factors in serum, we created a specific antagonist of the hGH receptor (similar to Trovert or Pegvisomant) which allowed us to obtain a true measure of hGH somatogenic activity by subtraction of the activity in the presence of the antagonist. The assay was carried out in microtiter plates over 24 h, with oxidation of a chromogenic tetrazolium salt (MTT) as the endpoint. PATIENTS These encompassed a number of different clinical conditions related to short stature, including idiopathic short stature, neurosecretory dysfunction and renal failure, as well as obese patients on dietary restriction and normal volunteers. MEASUREMENTS In addition to the colourimetric (MTT) response to hGH, we measured free hGH by stripping out GHBP-bound hGH using beads coupled to a monoclonal antibody to the GHBP (GH binding protein). All samples were measured in both bioassay and ELISA assay. RESULTS This bioassay was sensitive (5 mU/l or 2 mug/l) and precise, and not subject to interference by the GHBP. There was a good correlation (r = 0.95) between bioactivity and immunoactivity across clinical states. There was, however, an increased bioactivity during secretory peaks (over 25 mU/l), which has been reported previously for the Nb2 bioassay. Free hGH did not correlate with clinical state. CONCLUSIONS Because the results of the Elegance ELISA and the bioassay correlate well, even though there is greater bioactivity at higher hormone concentrations, it is evident that an appropriate immunoassay is able to act as a reliable indicator for clinical assessment. In those rare cases where bio-inactive GH exists, our bioassay should provide an appropriate means to demonstrate this.