Satoko Kubo

Novel antagonists for proteinase-activated receptor 2: inhibition of cellular and vascular responses in vitro and in vivo

Background and purpose:  Proteinase‐activated receptor 2 (PAR2) is a G‐protein coupled receptor associated with many pathophysiological functions. To date, the development of PAR2 antagonists has been limited. Here, we identify a number of novel peptide‐mimetic PAR2 antagonists and demonstrate inhibitory effects on PAR2‐mediated intracellular signalling pathways and vascular responses.

Dual modulation of the tension of isolated gastric artery and gastric mucosal circulation by hydrogen sulfide in rats.

Abstract.To clarify roles of H2S in regulation of gastric circulation, we investigated effects of NaHS, a H2S donor, on tension of isolated rat gastric artery and gastric mucosal blood flow in rats. In the precontracted ring preparations, NaHS caused contraction and relaxation at low and high concentrations, respectively. The NaHS-induced vasorelaxation was only partially blocked by glibenclamide, a K+ATP channel inhibitor. The contractile activity of NaHS disappeared by removal of the endothelium or by inhibition of nitric oxide synthase and the endothelium-derived hyperpolarizing factor (EDHF) pathways. Intravenous injection of NaHS caused transient increase followed by decrease in gastric mucosal blood flow in rats. Collectively, in the gastric artery, NaHS appears to cause relaxation through both K+ATP channel-dependent and -independent pathways and contraction through inhibition of NO and EDHF pathways. Together with the in vivo results, our study implies that H2S plays multiple complex roles in regulation of gastric circulation.

Distinct Activity of Peptide Mimetic Intracellular Ligands (Pepducins) for Proteinase‐Activated Receptor‐1 in Multiple Cells/Tissues

Abstract:  Proteinase‐activated receptor‐1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, can be activated not only by PAR1‐activating peptides (PAR1APs) based on the N‐terminal cryptic tethered ligand sequence but also by an N‐palmitoylated (Pal) peptide, Pal‐RCLSSSAVANRSKKSRALF‐amide (P1pal‐19), based on the intracellular loop 3 of PAR1, designated pepducin, in human platelets or PAR1‐transfected cells. The present article evaluated the actions of P1pal‐19 and also the shorter peptide, Pal‐RCLSSSAVANRS‐amide (P1pal‐12), known as a possible PAR1 antagonist, in multiple cells/tissues that naturally express PAR1. P1pal‐19 as well as a PAR1AP, TFLLR‐amide, evoked cytosolic Ca2+ mobilization in cultured human lung epithelial cells (A549) and rat gastric mucosal epithelial cells (RGM1). P1pal‐19 and TFLLR‐amide, but not a PAR2‐activating peptide, SLIGRL‐amide, caused delayed prostaglandin E2 formation in RGM1 cells. P1pal‐19, like TFLLR‐amide, produced endothelial NO‐dependent relaxation in rat aorta and epithelial prostanoid‐dependent relaxation in mouse bronchus. The P1pal‐19‐induced relaxation remained constant even after desensitization of PAR1 with TFLLR‐amide in either tissue. P1pal‐19 failed to mimic the contractile effects of TFLLR‐amide in the endothelium‐denuded preparations of rat aorta or superior mesenteric artery and the rat gastric longitudinal smooth muscle strips. P1pal‐12 partially inhibited the vasorelaxation caused by TFLLR‐amide and P1pal‐19, but not SLIGRL‐amide, in the rat aorta. Our data thus indicate that P1pal‐19 is capable of mimicking the effects of PAR1APs in the endothelial and epithelial, but not smooth muscle, cells/tissues, and suggest that P1pal‐12 may act as a PAR1 antagonist in the vascular endothelium.

Involvement of ERK in NMDA receptor-independent cortical neurotoxicity of hydrogen sulfide.

Hydrogen sulfide (H(2)S), a gasotransmitter, exerts both neurotoxicity and neuroprotection, and targets multiple molecules including NMDA receptors, T-type calcium channels and NO synthase (NOS) that might affect neuronal viability. Here, we determined and characterized effects of NaHS, an H(2)S donor, on cell viability in the primary cultures of mouse fetal cortical neurons. NaHS caused neuronal death, as assessed by LDH release and trypan blue staining, but did not significantly reduce the glutamate toxicity. The neurotoxicity of NaHS was resistant to inhibitors of NMDA receptors, T-type calcium channels and NOS, and was blocked by inhibitors of MEK, but not JNK, p38 MAP kinase, PKC and Src. NaHS caused prompt phosphorylation of ERK and upregulation of Bad, followed by translocation of Bax to mitochondria and release of mitochondrial cytochrome c, leading to the nuclear condensation/fragmentation. These effects of NaHS were suppressed by the MEK inhibitor. Our data suggest that the NMDA receptor-independent neurotoxicity of H(2)S involves activation of the MEK/ERK pathway and some apoptotic mechanisms.