y-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in mammalian brain, is known to interact with two classes of GABA receptors denoted GABAA and GABAB. Using Xenopus oocytes, we compared the electrical and pharmacological properties of GABA receptors expressed by poly(A)+ RNA isolated from mammalian brain and retina. RNA from cerebral cortex expressed GABA responses with features characteristic of currents mediated by GABAA receptors. In contrast, RNA from retina expressed responses mediated by GABAA receptors and, in addition, GABA responses that were insensitive to the GABAA antagonist bicuculline and the GABAB agonist baclofen and showed no modulation by barbiturates or benzodiazepines. The bicuculline/baclofen-insensitive GABA response was a Cl current that was blocked by picrotoxin but showed little desensitization or outward rectification. Our results suggest that mmalian retina contains RNAs encoding GABA receptors with distinct pharmacology.In mammals, there are two well-characterized classes of receptors for the inhibitory neurotransmitter y-aminobutyric acid (GABA). GABAA receptors are ligand-gated Cl-channels that are competitively antagonized by bicuculline, noncompetitively blocked by picrotoxin, and allosterically modulated by barbiturates and benzodiazepines (1-3). Molecular cloning of cDNAs encoding GABAA receptor subunits indicates that the receptors are heteromeric and comprised of up to four different subunits, found in a variety of closely related subtypes (e.g., refs. 4-7). In contrast, GABAB receptors regulate K+ and Ca2+ channels through GTP-binding proteins and intracellular messenger pathways (8). These receptors have not been cloned but are presumed to belong to the superfamily of GTP-binding-protein-coupled receptors. GABAB receptors are selectively activated by baclofen, are antagonized by phaclofen and 2-hydroxysaclofen, and are not affected by bicuculline, picrotoxin, or any of the GABAA modulators (9,10).Xenopus oocytes are now widely used to study receptors and ion channels expressed after microinjection of either heterologous poly(A)+ RNA or RNAs transcribed from cloned cDNAs (for reviews, see refs. 11 and 12). GABAA subunits are readily expressed in oocytes and assemble to form receptors that have electrical and pharmacological properties similar to those reported for cells in situ (e.g., refs. 4-7, 13-15).Almost every neurotransmitter/neuromodulator identified in mammalian brain has also been found in retina (e.g., ref. 16). We used Xenopus oocytes to characterize neurotransmitter receptors expressed by retina RNAs, investigating whether there were any clear differences between the properties of brain and retina receptors. Initial studies, using poly(A)+ RNA isolated from bovine retina, showed that retina RNA primarily expressed receptors to excitatory amino acids, glycine, and substance P (ref. 17 and unpublished results). Herein we report on the GABA receptors encoded by retina RNAs. MATERIALS AND METHODSEleven poly(A)+ RNA preparations were made from ...

A number of pyridazinone derivatives bearing an arylpiperazinylalkyl chain were synthesized and tested icv in a model of acute nociception induced by thermal stimuli in mice (tail flick). The most interesting and potent compound in this series was 6a, which showed an ED(50) = 3.5 microg, a value about 3-fold higher with respect to morphine by the same route of administration. When administered per os, 6a was 4-fold more potent than morphine in the same test, suggesting a significant bioavailability. The same compound also showed high potency in the hot plate test. The antinociceptive effect of 6a was completely reversed by pretreatment with yohimbine both in the hot plate test and in the tail flick test. This demonstrated the involvement of the adrenergic system, which was confirmed by in vitro radioligand binding studies.

Accepted ManuscriptTitle: Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Page 1 of 36 A c c e p t e d M a n u s c r i p *Graphical AbstractPage 2 of 36 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 and 100 M (38±14%, P<0.05, and 69±5%, P<0.01, respectively). Up to 100 M, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE 2 generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH 2 metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.

The action of 5-hydroxytryptamine (5HT) on nicotinic acetylcholine receptor (nAChR) channels was investigated in mouse myotubes, human cloned TE671/RD cells, and Xenopus laevis oocytes. The decay of the ACh-activated whole-cell currents was reversibly accelerated in the presence of 5HT (10(-5) to 10(-3) M), in a dose-dependent manner. 5HT also reduced the size and accelerated the decay of currents elicited by ACh in Xenopus oocytes injected with mRNA extracted from C2 myotubes or Torpedo electroplaques, or oocytes injected with cloned mouse muscle AChR subunit mRNAs. The effect of 5HT was promptly reversed after washout, or by depolarizing the oocyte beyond -10 mV. In patch-clamp recordings from myotubes, bath-application of 5HT did not exert an indirect influence on the ACh-activated channels within the patch membrane. In contrast, when the patch membrane was exposed to 5HT (10(-6) M), ACh unit responses appeared as bursts of short pulses. It is concluded that the regulation of ACh responses by 5HT results from a fast noncompetitive blocking action of nAChR-channels. These results show that ligand-gated channels, activated by their specific neurotransmitter, may be regulated by a different neurotransmitter through a direct action on the receptor molecule.

PGs are potent mediators of pain and inflammation. PGE synthases (PGES) catalyze the isomerization of PGH(2) into PGE(2). The microsomal (m)PGES-1 isoform serves as an inducible PGES and is responsible for the production of PGE(2), which mediates acute pain in inflammation and fever. The present study was designed to investigate the regulation of expression of mPGES-1 in polarized phagocytes, which represent central, cellular orchestrators of inflammatory reactions. Here, we report that human peripheral blood monocytes did not express mPGES-1. Exposure to LPS strongly induced mPGES-1 expression. Alternatively activated M2 monocytes-macrophages exposed to IL-4, IL-13, or IL-10 did not express mPGES-1, whereas in these cells, IL-4, IL-13, and to a lesser extent, IL-10 or IFN-gamma inhibited LPS-induced, mPGES-1 expression. It is unexpected that polymorphonuclear leukocytes expressed high basal levels of mPGES-1, which was up-regulated by LPS and down-regulated by IL-4 and IL-13. Induction of mPGES-1 and its modulation by cytokines were confirmed at the protein level and correlated with PGE(2) production. Cyclooxygenase 2 expression tested in the same experimental conditions was modulated in monocytes and granulocytes similarly to mPGES-1. Thus, activated M1, unlike alternatively activated M2, mononuclear phagocytes express mPGES-1, and IL-4, IL-13, and IL-10 tune expression of this key enzyme in prostanoid metabolism. Neutrophils, the first cells to enter sites of inflammation, represent a ready-made, cellular source of mPGES-1.