6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2h-pyran-2-one and Disease-Models--Animal

There is a major clinical need for new therapies for the treatment of chronic itch. Many of the molecular components involved in itch neurotransmission are known, including the neuropeptide NPPB, a transmitter required for normal itch responses to multiple pruritogens in mice. Here, we investigated the potential for a novel strategy for the treatment of itch that involves the inhibition of the NPPB receptor NPR1 (natriuretic peptide receptor 1). Because there are no available effective human NPR1 (hNPR1) antagonists, we performed a high-throughput cell-based screen and identified 15 small-molecule hNPR1 inhibitors. Using in vitro assays, we demonstrated that these compounds specifically inhibit hNPR1 and murine NPR1 (mNPR1). In vivo, NPR1 antagonism attenuated behavioral responses to both acute itch- and chronic itch-challenged mice. Together, our results suggest that inhibiting NPR1 might be an effective strategy for treating acute and chronic itch.

Topics: Animals; Behavior, Animal; Cell-Free System; Dermatitis, Contact; Disease Models, Animal; Ganglia, Spinal; Humans; Mice, Inbred C57BL; Mice, Knockout; Neurons; Pruritus; Receptors, Atrial Natriuretic Factor; Reproducibility of Results; Signal Transduction; Small Molecule Libraries

The mechanism of FFA-induced insulin resistance is not fully understood. We have searched for effector molecules(s) in FFA-induced insulin resistance. Palmitic acid (PA) but not oleic acid (OA) induced insulin resistance in L6 myotubes through C-Jun N-terminal kinase (JNK) and insulin receptor substrate 1 (IRS-1) Ser307 phosphorylation. Inhibitors of ceramide synthesis did not block insulin resistance by PA. However, inhibition of the conversion of PA to lysophosphatidylcholine (LPC) by calcium-independent phospholipase A₂ (iPLA₂) inhibitors, such as bromoenol lactone (BEL) or palmitoyl trifluoromethyl ketone (PACOCF₃), prevented insulin resistance by PA. iPLA₂ inhibitors or iPLA₂ small interfering RNA (siRNA) attenuated JNK or IRS-1 Ser307 phosphorylation by PA. PA treatment increased LPC content, which was reversed by iPLA₂ inhibitors or iPLA₂ siRNA. The intracellular DAG level was increased by iPLA₂ inhibitors, despite ameliorated insulin resistance. Pertussis toxin (PTX), which inhibits LPC action through the G-protein coupled receptor (GPCR)/Gα(i), reversed insulin resistance by PA. BEL administration ameliorated insulin resistance and diabetes in db/db mice. JNK and IRS-1Ser307 phosphorylation in the liver and muscle of db/db mice was attenuated by BEL. LPC content was increased in the liver and muscle of db/db mice, which was suppressed by BEL. These findings implicate LPC as an important lipid intermediate that links saturated fatty acids to insulin resistance.

Topics: Animals; Blood Proteins; Cells, Cultured; Diabetes Mellitus, Type 2; Disease Models, Animal; Gene Silencing; Glucose; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; JNK Mitogen-Activated Protein Kinases; Liver; Lysophosphatidylcholines; Mice; Mice, Knockout; Muscle Fibers, Skeletal; Naphthalenes; Palmitic Acid; Pertussis Toxin; Phospholipases A2, Calcium-Independent; Phosphorylation; Pyrones; Receptors, G-Protein-Coupled; RNA, Small Interfering; Signal Transduction

Phospholipase A(2) (PLA(2)), a regulatory enzyme found in most mammalian cells, catalyzes the breakdown of membrane phospholipids to arachidonic acid. There are two major cytosolic types of the enzyme, calcium-dependent (cPLA(2)) and calcium-independent (iPLA(2)) PLA(2). The present study investigated whether or not iPLA(2) plays a role in the endothelium-dependent contractions of the aorta of the spontaneously hypertensive rat and its normotensive counterpart, the Wistar-Kyoto rat. The presence of iPLA(2) in the endothelial cells was identified by using immunochemistry and immunoblotting. Aortic rings with and without the endothelium were suspended in organ chambers for isometric tension recording. The production of prostanoids was measured by using enzyme immunoassay kits. iPLA(2) was densely distributed in endothelial cells of the aorta of both strains. At 3 x 10(-6) M, the selective iPLA(2) inhibitor, bromoenol lactone (BEL), abrogated endothelium-dependent contractions induced by acetylcholine but not those evoked by the calcium ionophore A-23187. The effects of BEL were similar in the aortae of Wistar-Kyoto and spontaneously hypertensive rats. The nonselective PLA(2) inhibitor quinacrine abolished the contractions triggered by both acetylcholine and A-23187, whereas the store-operated calcium channel inhibitor SKF-96365 prevented only the acetylcholine-induced contraction. The acetylcholine- but not the A-23187-induced release of 6-keto prostaglandin F(1alpha) was inhibited by BEL. The release of thromboxane B(2) by either acetylcholine or A-23187 was not affected by BEL. In conclusion, iPLA(2) plays a substantial role in the generation of endothelium-derived contracting factor evoked by acetylcholine.

Topics: Acetylcholine; Animals; Aorta; Calcimycin; Disease Models, Animal; Endothelium, Vascular; Hypertension; Imidazoles; Ionophores; Male; Muscle Contraction; Muscle, Smooth, Vascular; Naphthalenes; Phosphodiesterase Inhibitors; Phospholipases A2, Calcium-Independent; Pyrones; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Vasodilator Agents