John R. Boot

Calcium channel subtypes contributing to acetylcholine release from normal, 4‐aminopyridine‐treated and myasthenic syndrome auto‐antibodies‐affected neuromuscular junctions

Acetylcholine release at the neuromuscular junction relies on rapid, local and transient calcium increase at presynaptic active zones, triggered by the ion influx through voltage‐dependent calcium channels (VDCCs) clustered on the presynaptic membrane. Pharmacological investigation of the role of different VDCC subtypes (L‐, N‐, P/Q‐ and R‐type) in spontaneous and evoked acetylcholine (ACh) release was carried out in adult mouse neuromuscular junctions (NMJs) under normal and pathological conditions. ω‐Agatoxin IVA (500 nM), a specific P/Q‐type VDCC blocker, abolished end plate potentials (EPPs) in normal NMJs. However, when neurotransmitter release was potentiated by the presence of the K+ channel blocker 4‐aminopyridine (4‐AP), an ω‐agatoxin IVA‐ and ω‐conotoxin MVIIC‐resistant component was detected. This resistant component was only partially sensitive to 1 μM ω‐conotoxin GVIA (N‐type VDCC blocker), but insensitive to any other known VDCC blockers. Spontaneous release was dependent only on P/Q‐type VDCC in normal NMJs. However, in the presence of 4‐AP, it relied on L‐type VDCCs too. ACh release from normal NMJs was compared with that of NMJs of mice passively injected with IgGs obtained from patients with Lambert‐Eaton myasthenic syndrome (LEMS), a disorder characterized by a compromised neurotransmitter release. Differently from normal NMJs, in LEMS IgGs‐treated NMJs an ω‐agatoxin IVA‐resistant EPP component was detected, which was only partially blocked by calciseptine (1 μM), a specific L‐type VDCC blocker. Altogether, these data demonstrate that multiple VDCC subtypes are present at the mouse NMJ and that a resistant component can be identified under ‘pharmacological’ and/or ‘pathological’ conditions.

In vitro modulation of the eosinophil-dependent enhancement of the permeability of the bronchial mucosa.

1 Basolateral to apical albumin flux has been measured in sheets of bovine bronchial and tracheal mucosa mounted in vitro. 2 Addition of guinea‐pig peritoneal eosinophils or neutrophils to the basolateral side of such tissues had no significant influence on the transmucosal flux of albumin in either the bronchial or tracheal mucosa. 3 Stimulation of eosinophils or neutrophils by the calcium ionophore A23187, or by their presentation to an opsonized airways mucosa, resulted in a significant increase in the transbronchial flux of albumin. This effect was seen after only 60 min incubation of the leucocytes with the bronchial mucosa, and was no greater when the contact time was extended to 180 min. Incubation of bronchial mucosal tissues with 1 mg ml−1polyarginine for 3 h produced a significant increase in albumin flux, but was ineffective at 0.5 mg ml−1. 4 In contrast to the bronchial mucosa, the tracheal mucosa appeared resistant to the effects of stimulated eosinophils and neutrophils. 5 The lipoxygenase inhibitor AA‐861 failed to influence the ability of eosinophils to augment the transmembrane flux of albumin. However, insertion of a Millipore filter mask between the eosinophils and the bronchial mucosa significantly inhibited the eosinophil‐dependent enhancement of mucosal permeability. 6 The broad spectrum antiproteinase α2‐macroglobulin achieved almost total ablation of the action of stimulated eosinophils in the bronchial mucosa. These results suggest that proteinases may make a significant contribution to the genesis of epithelial injury, whereas leukotrienes do not.

Differential effects of ω-conotoxin GVIA and MVIIC on nerve stimulation induced contractions of guinea-pig ileum and rat vas deferens

Abstract ω-Conotoxin GVIA and ω-conotoxin MVIIC caused similar concentration-dependent reductions of the electrically induced twitch responses of guinea-pig ileum. The inhibitory effects of ω-conotoxin GVIA were long-lasting whereas those to ω-conotoxin MVIIC were readily reversed. Preincubation with ω-conotoxin MVIIC prevented the irreversible inhibition of ω-conotoxin GVIA suggesting a common site of interaction. However, unlike ω-conotoxin GVIA which caused inhibition, ω-conotoxin MVIIC did not affect the electrically induced twitch responses of the rat vas deferens nor did it prevent the irreversible inhibition with ω-conotoxin GVIA. This study indicates the possible existence of different subtypes of the N-type voltage-dependent calcium channel.

Bicyclo[2.2-1]heptanes as novel triple re-uptake inhibitors for the treatment of depression

A series of substituted naphthyl containing chiral [2.2.1] bicycloheptanes were prepared utilizing asymmetric Diels-Alder chemistry. This paper describes structure-activity relationships in this series. The N-methyl 2-naphthyl analogue (16d) and its des-methyl analogue (17d) are active triple re-uptake inhibitors both in vivo and in vitro.

The comparative in vitro pharmacology of leukotriene D4 and its isomers

Abstract The 5R,6S and 5S,6R isomers of leukotriene D4 (LTD4), 9cis LTD4, 9cis,11trans LTD4 and 11trans LTD4 were synthesized for comparative pharmacological studies on intestinal and respiratory smooth muscle preparations. The 5S,6R isomers are biologically active, modification of the double bond stereochemistry causing only a moderate reduction in activity. The 5R,6S isomers possess approximately 1% the biological activity of their respective 5S,6R forms.

γ-Hexachlorocyclohexane stimulation of macrophage phospholipid hydrolysis and leukotriene production

gamma-Hexachlorocyclohexane stimulates arachidonic acid release from macrophage phospholipids. It is also a powerful stimulator of leukotriene C4 production, yet (by comparison with zymosan) produces only a small effect on prostaglandin production. This suggests that synthesis of leukotrienes and prostaglandins can be independently regulated. The most likely mechanism of action of gamma-hexachlorocyclohexane is through its effect on phosphatidylinositol metabolism.

Stimulated eosinophils and proteinases augment the transepithelial flux of albumin in bovine bronchial mucosa

1 The apical to basolateral transmucosal flux of albumin has been measured in isolated sheets of bovine bronchial and tracheal mucosa. Under resting conditions the net unidirectional flux in the bronchial mucosa was not significantly different from that measured previously for the basolateral to apical vector. In contrast, the apical to basolateral flux in the tracheal mucosa was significantly lower than that measured in the opposite direction. 2 Addition of guinea‐pig peritoneal eosinophils to the apical side of the tissues had no significant effect on the transmucosal flux of albumin in either the bronchial or tracheal mucosa. 3 When eosinophils were stimulated with the ionophore A23187 or by opsonic adherence to tissues treated with a guinea‐pig anti‐bovine airway epithelium antibody, the bronchial mucosal sheets that had been exposed showed a significant increase in the transmucosal flux of albumin. However, tissues from the tracheal mucosa were resistant to the effects of stimulated eosinophils. 4 Histologically, sheets of mucosa from bovine main bronchi that had been exposed to stimulated eosinophils were characterized by epithelial injury consisting of loss of columnar epithelium from the underlying basal cell layer and biomatrix. Much less evidence of cellular injury was observed in tracheal tissues. 5 Bacterial collagenases applied to the apical side of the sheets were shown to increase the permeability of the bronchial mucosa to albumin and to produce histological changes that had similarities with the pattern of damage produced by stimulated eosinophils. 6 These observations demonstrate that the ability of eosinophils to injure the bronchial mucosa is independent of the side of the tissue on which they are present. Furthermore, key aspects of the injury process may be reproduced, at least in part, by metalloproteinases.

Synthesis of 8,9-leukotriene C3 and D3

Abstract An asymmetric epoxidation provides a short convenient synthesis of 8,9-leukotrienes C 3 and D 3 .

LY393615, a novel neuronal Ca2+ and Na+ channel blocker with neuroprotective effects in models of in vitro and in vivo cerebral ischemia

In the present studies we have examined the effects of a new calcium channel blocker, LY393615 ((N-Butyl-[5,5-bis-(4-fluorophenyl)tetrahydrofuran-2-yl]methylamine hydrochloride, NCC1048) in a model of hypoxia-hypoglycaemia in vitro and in a gerbil model of global and in two rat models of focal cerebral ischaemia in vivo. Results indicated that LY393615 protected against hypoxia-hypoglycaemic insults in brain slices and also provided significant protection against ischaemia-induced hippocampal damage in gerbil global cerebral ischaemia when dosed at 10, 12.5 (P<0.05) or 15 mg/kg i.p. (P<0.01) 30 min before and 2 h 30 min after occlusion. The compound penetrated the brain well after a 15 mg/kg i.p. dose and had a half-life of 2.5 h. In further studies LY393615 was protective 1 h post-occlusion when administered at 15 mg/kg i.p. followed by 2 doses of 5 mg/kg i.p. 2 and 3 h later. LY393615 dosed at 15 mg/kg i.p. followed by 2 further doses of 5 mg/kg i.p. (2 and 3 h later) also produced a significant reduction in the infarct volume following Endothelin-1 (Et-1) middle cerebral artery occlusion in the rat when administration was initiated immediately (P<0.01) or 1 h (P<0.05) after occlusion. The compound was also evaluated in the intraluminal monofilament model of focal ischaemia. The animals had the middle cerebral artery occluded for 2 h, and 15 min after reperfusion LY393615 was administered at 15 mg/kg i.p. followed by 2 mg/kg/h i.v. infusion for 6 h. There was no reduction in infarct volume using this dosing protocol. In conclusion, in the present studies we have reported that a novel calcium channel blocker, LY393615, with good bioavailability protects against neuronal damage caused by hypoxia-hypoglycaemia in vitro and both global and focal cerebral ischaemia in vivo. The compound is neuroprotective when administered post-occlusion and may therefore be a useful anti-ischaemic agent.

Peptide neurotoxins, small-cell lung carcinoma andneurological paraneoplastic syndromes.

Peptide neurotoxins isolated from the venom of snakes, spiders and snails have represented invaluable tools for the identification and characterisation of membrane ion channels and receptors in vertebrate cells, including human neurons. We here report on the use of these toxins for the characterisation of membrane ion channels and receptors expressed by one of the most aggressive human cancers, small-cell lung carcinoma. This tumour shares many properties with other neuro-endocrine cell types, including the ability of firing action potentials and release hormones in a calcium-dependent manner. Toxins such as alpha-bungarotoxin and omega-conotoxins, among others, have been successfully used to characterise neuronal nicotinic receptors and voltage-dependent calcium channels, respectively, in human small-cell lung carcinoma cells. These receptors and ion channels are not only crucial for the growth of this specific tumour, but also represent autoantigens against which cancer patients build an autoimmune response. Although the aim of this autoimmune response is eventually the destruction of the cancer cells, the circulating antibodies cross-react with similar ion channels and receptors present in normal neurons or other cells, causing a number of different paraneoplastic diseases, the best characterised of which is the Lambert-Eaton myasthenic syndrome. Conotoxin-based radioimmunoassays have become an invaluable tool for the diagnosis and follow up of these paraneoplastic disorders and could represent a step forward in the early diagnosis of small-cell lung carcinoma itself.