piperidines and 2-2-6-6-tetramethylpiperidin-4-yl-heptanoate

A variety of endothelial agonist-induced responses are mediated by rises in intracellular Ca(2+), suggesting that different Ca(2+) signatures could fine-tune specific inflammatory and thrombotic activities. In search of new intracellular mechanisms modulating endothelial effector functions, we identified nicotinic acid adenine dinucleotide phosphate (NAADP) as a crucial second messenger in histamine-induced Ca(2+) release via H1 receptors (H1R). NAADP is a potent intracellular messenger mobilizing Ca(2+) from lysosome-like acidic compartments, functionally coupled to the endoplasmic reticulum. Using the human EA.hy926 endothelial cell line and primary human umbilical vein endothelial cells, we show that selective H1R activation increases intracellular NAADP levels and that H1R-induced calcium release involves both acidic organelles and the endoplasmic reticulum. To assess that NAADP links H1R to Ca(2+)-signaling we used both microinjection of self-inactivating concentrations of NAADP and the specific NAADP receptor antagonist, Ned-19, both of which completely abolished H1R-induced but not thrombin-induced Ca(2+) mobilization. Interestingly, H1R-mediated von Willebrand factor (VWF) secretion was completely inhibited by treatment with Ned-19 and by siRNA knockdown of 2-pore channel NAADP receptors, whereas thrombin-induced VWF secretion failed to be affected. These findings demonstrate a novel and specific Ca(2+)-signaling mechanism activated through H1R in human endothelial cells, which reveals an obligatory role of NAADP in the control of VWF secretion.

Topics: Base Sequence; Calcium Channels; Calcium Signaling; Carbolines; Cell Line; Endothelial Cells; Gene Knockdown Techniques; Heptanoic Acids; Histamine; Humans; NADP; Nicotinic Antagonists; Piperazines; Piperidines; Receptors, Histamine H1; RNA, Small Interfering; Second Messenger Systems; von Willebrand Factor

In co-cultures of embryonic rat spinal cord slices and skeletal muscle, spinal motoneurons innervate muscle fibres and drive muscle contractions. However, multi-electrode array (MEA) recordings show that muscle contractions often appear in the absence of population activity in the spinal cord networks. Such uncorrelated muscle activity persists when the population bursts in the neuronal networks are prevented by un-coupling the network with the glutamatergic antagonists CNQX and D-APV. By contrast, the uncorrelated muscle activity is fully suppressed by the muscular nicotinic antagonist D-tubocurarine. Together, these findings confirm the previous finding that motoneurons drive muscle fibres in this preparation and suggest that they are intrinsically spiking in the absence of synaptic input. Intracellular recordings from spinal neurons support this suggestion. Analysing the correlated muscle activity, we found that in 15% of the population bursts, muscle activity appears at the beginning or before neuronal activity, suggesting that in these cases motoneurons initiate the population activity. Both the total number of population bursts and the percentage of such bursts that are initiated by muscle activity are reduced by a block of nicotinic receptors. Uncorrelated muscle and neuronal activity is reduced by the gap junction blocker carbenoxolone, suggesting that electrical coupling is involved in the generation of this activity. Together, these findings suggest that intrinsic firing of motoneurons may contribute to the activation of population bursts through cholinergic positive feedback loops in cultured spinal networks.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Atropine; Carbenoxolone; Choline O-Acetyltransferase; Coculture Techniques; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Feedback, Physiological; Heptanoic Acids; Motor Neurons; Muscarinic Antagonists; Muscle, Skeletal; Nerve Net; Neurofilament Proteins; Nicotinic Antagonists; Patch-Clamp Techniques; Piperidines; Rats; Rats, Wistar; Spinal Cord; Statistics, Nonparametric; Synaptic Transmission; Tissue Culture Techniques; Tubocurarine

The therapeutic targeting of nicotinic receptors in the brain will benefit from the identification of drugs that may be selective for their ability to activate or inhibit a limited range of nicotine acetylcholine receptor subtypes. In the present study, we describe the effects of 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), a novel compound that is a potent inhibitor of neuronal nicotinic receptors. Evaluation of nicotinic acetylcholine receptor (nAChR) subunits expressed in Xenopus laevis oocytes indicated that TMPH can produce a potent and long-lasting inhibition of neuronal nAChR formed by the pairwise combination of the most abundant neuronal alpha (i.e., alpha3 and alpha4) and beta subunits (beta2 and beta4), with relatively little effect, because of rapid reversibility of inhibition, on muscle-type (alpha1beta1gammadelta) or alpha7 receptors. However, the inhibition of neuronal beta subunit-containing receptors was also decreased if any of the nonessential subunits alpha5, alpha6, or beta3 were coexpressed. This decrease in inhibition is shown to be associated with a single amino acid present in the second transmembrane domain of these subunits. Our data indicate great potential utility for TMPH to help relate the diverse central nervous system effects to specific nAChR subtypes.

Topics: Amino Acid Sequence; Animals; Dose-Response Relationship, Drug; Female; Heptanoic Acids; Male; Mice; Molecular Sequence Data; Nicotinic Antagonists; Piperidines; Protein Subunits; Rats; Rats, Sprague-Dawley; Receptors, Nicotinic; Xenopus laevis

There are multiple types of nicotine acetylcholine receptors (nAChR) in the brain associated with synaptic function, signal processing, or cell survival. The therapeutic targeting of nicotinic receptors in the brain will benefit from the identification of drugs, which may be selective for their ability to activate or inhibit a limited range of these receptor subtypes. We previously identified a family of bis-tetramethylpiperidine compounds as selective inhibitors of neuronal-type nicotinic receptors. In the present study we describe the in vivo effects and properties of 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), a novel inhibitor of neuronal nicotinic receptors. Delivered systemically, this drug can block central nervous system effects of nicotine, indicating that this drug is able to cross the blood-brain barrier and access sites in the brain. Unlike the prototype CNS-active nicotinic inhibitor, mecamylamine, TMPH blocked some but not all of the CNS effects of nicotine, indicating that it has a unique selectivity for specific receptor subtypes in the brain. The nAChR subtypes that mediate the locomotor effects and hypothermic effects of nicotine appear to be less sensitive to TMPH than those which mediate analgetic effects and discriminative stimuli. These results indicate that TMPH may possess unique selectivity for specific nicotinic receptor subtypes.

Topics: Analgesics; Animals; Body Temperature; Central Nervous System; Discrimination, Psychological; Dose-Response Relationship, Drug; Heptanoic Acids; Male; Mice; Mice, Inbred ICR; Motor Activity; Nicotine; Pain; Pain Measurement; Piperidines; Rats; Rats, Long-Evans; Receptors, Nicotinic; Time Factors