Recombinant human receptors and functional assays in the discovery of altinicline (SIB-1508Y), a novel acetylcholine-gated ion channel (nAChR) agonist.

Neuronal nicotinic acetylcholine receptors (nAChRs) are a class of ion channels with significant potential as molecular targets for the design of drugs to treat a variety of CNS disorders. The discovery that neuronal nAChRs are further subdivided into multiple subtypes suggests that drugs which act selectively at specific nAChR subtypes might effectively treat Parkinson's disease (PD), Alzheimer's disease (AD), schizophrenia, ADHD, depression, anxiety or pain without the accompanying adverse side effects associated with non-selective agents such as nicotine (1) and epibatidine. Altinicline (SIB-1508Y) is a novel, small molecule designed to selectively activate neuronal nAChRs and is undergoing clinical evaluation for the treatment of PD. It was selected from a series of compounds primarily on the basis of results from functional assays, including (a) measurement of Ca2+ flux in stable cell lines expressing specific recombinant human neuronal nAChR subtypes; (b) determination of in vitro and in vivo neurotransmitter release; (c) in vivo models of PD. Biological data on both altinicline and the series of compounds from which it was selected are reported.

Analgesic profile of the nicotinic acetylcholine receptor agonists, (+)-epibatidine and ABT-594 in models of persistent inflammatory and neuropathic pain.

The anti-nociceptive and locomotor effects of the nicotinic acetylcholine receptor (nAChR) agonists (+)-epibatidine and ABT-594 were compared in the rat. Acute thermal nociception was measured using the tail flick test. Mechanical hyperalgesia was measured as paw withdrawal threshold (PWT) in response to a mechanical stimulus in two animal models of persistent pain; (1) 24 h following subplantar injections of Freund's complete adjuvant (FCA) into the left hind paw or (2) 11-15 days following a partial ligation of the left sciatic nerve. Disruption of locomotor function was assessed using an accelerating rotarod device. In all tests, (+)-epibatidine was significantly more potent than ABT-594. Both (+)-epibatidine and ABT-594 dose-dependently increased tail flick latencies but only at doses that also disrupted performance in the rotarod test. On the other hand, (+)-epibatidine and ABT-594 dose-dependently reversed inflammatory and neuropathic hyperalgesia at significantly lower doses than that needed to disrupt performance in the rotarod test. In summary, ABT-594 is less potent than (+)-epibatidine in assays of acute and persistent pain and in the rotarod assay. However, ABT-594 displayed a clearer separation between its motor and anti-hyperalgesic effects. This shows that nicotinic agonists with improved selectivity between the nicotinic receptor subtypes could provide strong analgesic effects with a much improved therapeutic window.

The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents.

Neuronal nicotinic acetylcholine receptors (NAChRs) are pentameric ligand-gated ion channel receptors which exist as different functional subunit combinations which apparently subserve different physiological functions as indicated by molecular biological and pharmacological techniques. It is possible to design and synthesize novel compounds that have greater selective affinities and efficacies than nicotine for different NAChRs, which should translate into different behavioral profiles and therapeutic potentials. Examples of NAChR agonists studied are nicotine, SIB-1508Y, SIB-1553A and epibatidine. These compounds have different degrees of selectivity for human recombinant NAChRs, different neurotransmitter release profiles in vitro and in vivo and differential behavioral profiles. Preclinical studies suggest that SIB-1508Y is a candidate for the treatment of the motor and cognitive deficits of Parkinson's disease, whereas SIB-1553A appears to have potential as a candidate for the treatment of Alzheimer's disease. Epibatidine has a strong analgesic profile, however the ratio between pharmacological activity and undesirable effects is so low that it is difficult to envisage the use of this compound therapeutically. Nicotine has a broad profile of pharmacological activity, for instance demonstrating activity in models for cognition and analgesia. As for epibatidine, the adverse effects of nicotine severely limits its therapeutic use in humans. The discovery of subtype-selective NAChR agonists such as SIB-1508Y and SIB-1553A provides a new class of neuropsychopharmacological agents with better therapeutic ratios than nonspecific agents such as nicotine.

Conformationally restricted analogues of nicotine and anabasine.

A series of conformationally restricted analogues of nicotine has been synthesized and evaluated as agonists of neuronal acetylcholine receptors. Compound 2 (SIB-1663), which selectively activated human recombinant alpha 2 beta 4 and alpha 4 beta 4 nAChRs, was shown to be active in animal models of Parkinson's disease and pain.

Partial characterization of eosinophilic granule cells (EGCs) and identification of mast cells of the intestinal lamina propria in rainbow trout (Oncorhynchus mykiss). Biochemical and cytochemical study.

The role of intestinal eosinophilic granule cells (EGCs) is still a subject of discussion. The aim of this study was to obtain additional functional data for a better characterization of these cells. Biochemical studies indicated the presence of small amounts of histamine, a characteristic and consistent marker of mast cells, in the posterior gut. On the other hand, histamine is always absent from homogenates of isolated EGCs. Using colorimetric assays, we were able to show aryl sulphatase B activity (18.5 +/- 3.7 nM nitrocatechol/10(6) cells) and detected peroxidase (1.86 +/- 0.03 ng/10(6) cells) in EGC homogenates. A cytochemical study enabled us to localize peroxidase in the granules of EGCs. These cells can also phagocytose latex beads. EGCs should thus be considered as homologous with mammalian eosinophils and not with mast cells. The screening for cells in the mucosae containing chondroitin sulphate revealed sparsely represented cells in the loose connective tissue in immediate proximity to blood capillaries. These cells could be mucosal mast cells.

Endocytosis and intracellular degradation of heterologous protein by eosinophilic granulocytes isolated from rainbow trout (Oncorhynchus mykiss) posterior intestine.

Adherence capacity to tissue substrate, endocytosis capacity for heterologous and homologous proteins, and proteolytic activity were determined in intestinal granulocytes (EGCs) isolated from healthy adult rainbow trout. The percentage of cells that could adhere to a smooth plastic surface increased with increasing incubation time. Endocytosis was effective for heterologous (human immunoglobulin G, IgGh; ovine somatotropin, oST) but not homologous proteins (recombinant trout somatotropin, rtST). The activity of cathepsin D increased significantly after the endocytosis of a heterologous protein. Finally, the analysis of immunoblots of homogenates of granulocytes incubated in the presence of the two different proteins was used to show the endocytosis and degradation of heterologous proteins. These results show that isolated EGCs can endocytose and degrade heterologous proteins.

Absorption of intact proteins by the intestinal epithelium of trout, Salmo gairdneri. A luminescence enzyme immunoassay and cytochemical study.

In the rainbow trout, a carnivorous species with a stomach, orally administered horseradish peroxidase (HRP) crossed the intestinal epithelium into the circulation. The protein first appeared in the blood 7 to 8 h after intubation, and could be assayed up to 24 h after administration. The method used, which combines ELISA (enzyme-linked immunoabsorbant assay) and chemiluminescence, enabled the transfer to be measured quantitatively. There was a direct correlation between the quantity ingested and the quantity transferred to the plasma within the experimental limits chosen. The clearance was monophasic and exponential (clearance rate: 3% per minute). Up to 6% of the ingested HRP was transferred to the blood. By cytochemistry it was possible to demonstrate that the protein crossed the intestinal cells at the level of the posterior segment, escaping the particularly intense intracellular lysosomal digestion. After entering the intercellular space, HRP was transferred to the interstitial space of the subepithelial lamina propria. During this transfer the HRP was in close contact with infiltrated macrophages and leukocytes resembling lymphoid cells. Thus, the passage of these protein particles could be the first indispensable step in the possible triggering of a local and/or a systemic immune response.

Is the Golgi apparatus the obligatory final step for lipoprotein secretion by intestinal cells?

It is currently admitted that the synthesis and excretion of triglyceride-rich lipoproteins (chylomicrons and 'small chylomicrons') by intestinal epithelial cells involves the Golgi apparatus as an obligatory final step before exocytosis. The cells of the proximal intestine of the trout are an excellent model for investigating functional compartmentalization in the course of lipid absorption. Using this model, our data invalidate morphological data which were the basis for considering the Golgi apparatus as the mandatory final stage for their secretion. In particular, we show that triglyceride-rich particles can be transported directly from the endoplasmic reticulum to the intercellular space. Two pathways of intestinal lipoprotein excretion appear to coexist. One follows the classical export route, the second functions in a manner that bypasses the Golgi apparatus. The arguments used to affirm the requirement for the Golgi apparatus as a final step (glycosylation of apoprotein B, membrane vehicle for exocytosis) are discussed.

Immunological demonstration of intestinal absorption and digestion of protein macromolecules in the trout (Salmo gairdneri).

An immunofluorescence technique using antibodies against the Fc and Fab fragments of human IgG (IgGH) was used to study the absorption of proteins by the intestinal epithelial cells of rainbow trout after oral or anal administration. Cellular absorption of a high molecular weight protein, hepatitis-B surface antigen (HBsAg), was also studied by using two monoclonal antibodies, one specific for the confirmation of the antigen (implying disulfide bridges), and the other that reacts with the constituent polypeptides. Both absorbed IgGH and HBsAg were seen to be segregated in the apical vacuolar system, a characteristic feature of intestinal epithelial cells. The same antibodies were used with an everted sac technique in conjunction with immunofluorescence, to show the intravacuolar degradation of IgGH and HBsAg following absorption. By using an antibody against cathepsin D, it was possible to demonstrate, by immunofluorescence, the localization of this enzyme in the same vacuolar system. After coupling the antibody to peroxidase or to the protein A/colloidalgold complex, the ultrastructural antigenic sites of cathepsin D could be seen to be localized in the interior of the vacuoles. The vacuolar localization of a cathepsin B activity was determined by incubating sections of intestinal mucosa, or isolated epithelial cells, with a specific synthetic substrate (Z-Ala-Arg-Arg-methoxynaphthylamide). The supranuclear hyaloplasmic vacuoles of intestinal epithelial cells may be considered to be phagolysosomes that assure the degradation of absorbed proteins. This function may be of fundamental importance in the in the nutritional processes of this species.

Local immunological response in the posterior intestinal segment of the rainbow trout after oral administration of macromolecules.

This paper deals with the effect of an orally administered antigen (human immunoglobulin G, IgGH) on the local intestinal immune system of trout kept at 12 degrees C. Significantly increased numbers of antigen-binding (ABC) and plaque forming cells (PFC) were induced in the epithelium by a single oral administration of antigen. In the primary immune response, ABC and PFC first occurred on days 4 and 6 respectively and peak counts were obtained at day 6 and 10 respectively. PFC counts returned to background levels by day 18. Lymphoid cells recovered from the intestinal epithelium 3 days after immunization were able to proliferate and to secrete Ig in vitro. 24 h after administration, the antigen could be visualized by immunofluorescence on the outer surface of the intraepithelial macrophages. An intestinal secretory immune system stimulated by oral administration of antigens is operative in trout, a carnivorous species with a well differentiated stomach.

New views on intestinal absorption of lipids in teleostean fishes: an ultrastructural and biochemical study in the rainbow trout.

Lipid absorption in rainbow trout was studied after gastric administration of [1-14C]linoleic and [1-14C]-palmitic acids. The intestinal epithelial cells were isolated at various times of absorption and the major lipid classes were isolated. Radioactivity was found primarily in the triglycerides. Blood radioactivity was measured at different times after administration of the labeled acids. It was very low until after 6 hr. After 4 hr when it was detectable, it was located essentially in the triglyceride fraction. At various times after feeding (a meal with 60% of unsaturated long chain fatty acids) the absorptive epithelium of the anterior intestine and pyloric caeca were examined by electron microscopy. Surprisingly, the esterification of fatty acids corresponded to the formation of VLDL-like particles, seen in SER, RER, Golgi apparatus, lamellar structures, intercellular space, interstitial space of lamina propria and lumen of lymphatic vessels. The respective roles of endoplasmic reticulum and Golgi apparatus in the lipoprotein synthesis are discussed. The particles are delivered in the intercellular spaces by way of the lamellar structures, whose role until now was unknown. Though the absorption of dietary triglycerides is much slower than in mammals, the mechanism does not differ fundamentally. The long chain fatty acids are esterified by the intestinal cells and transferred as VLDL-like particles to lymph. X

[The fine structure of trout liver glycogen]. / La structure fine du glycogène hépatique de la truite arc-en-ciel.

1. The fine structure of trout liver glycogen has been investigated using an enzymatic method. 2. The total conversion of glycogen into glucose under the action of amyloglucosidase and the percentage of beta-amylolysis before (37.4%) and after (97.8%) isoamylase debranching are similar to the mammalian glycogen. 3. However, the resistance to beta-amylase of certain debranched material leads to an hypothesis during glycogenolysis.

Evolution of the glycogen content and of glucose-6-phosphatase activity in the liver of Salmo gairdneri during development.

The evolution of the liver's glycogenic content, cytochemical characterization of the glycogen and glucose-6-phosphatase activity enable us to define three successive phases up to stage 36, just before the first feed. The grade which was low up to stage 24 is then due to beta-particles of ovule origin. Then, up to stage 27, there is a storage phase: alpha-particles appear and accumulate while the enzymatic activity remains non-existent. From the stage 28 to 36 the grade is progressively increasing, the enzymatic activity appears and increases. When the phase ends the liver is able to ensure glycemic regulation and to deal with exogenous nutritional contributions.