TRPC Channels Mediate a Muscarinic Receptor-Induced Afterdepolarization in Cerebral Cortex

Hai-Dun Yan, Claudio Villalobos, and Rodrigo Andrade
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48230

Abstract

Activation of muscarinic cholinergic receptors on pyramidal cells of the cerebral cortex induces the appearance of a slow afterdepolarization that can sustain autonomous spiking after a brief excitatory stimulus. Accordingly, this phenomenon has been hypothesized to allow for the transient storage of memory traces in neuronal networks. Here we investigated the molecular basis underlying the muscarinic receptor-induced afterdepolarization using molecular biological and electrophysiological strategies. We find that the ability of muscarinic receptors to induce the inward aftercurrent underlying the slow afterdepolarization is inhibited by expression of a Gq-11 dominant negative and is also markedly reduced in a phospholipase C 1 (PLC1) knock-out mouse. Furthermore, we show, using a genetically encoded biosensor,that activation ofmuscarinic receptorinducesthe breakdown of phosphatidylinositol 4,5-bisphosphatein pyramidal cells. These results indicate that the Gq-11 /PLC1 cascade plays a key role in the ability of muscarinic receptors to signal the inward aftercurrent. We have shown previously that the muscarinic afterdepolarization is mediated by a calcium-activated nonselective cation current, suggestingthe possible involvement of TRPC channels.Wefindthat expression of a TRPC dominant negative inhibits, and overexpression of wild-type TRPC5 or TRPC6 enhances, the amplitude of the muscarinic receptor-induced inward aftercurrent. Furthermore, we find that coexpression of TRPC5 and T-type calcium channels is sufficient to reconstitute a muscarinic receptor-activated inward aftercurrent in human embryonic kidney HEK-293 cells. These results indicate that TRPC channels mediate the muscarinic receptor-induced slow afterdepolarization seen in pyramidal cells of the cerebral cortex and suggest a possible role for TRPC channels in mnemonic processes.

Modified Proteinase-Activated Receptor-1 and -2 Derived Peptides Inhibit Proteinase-Activated Receptor-2 Activation by Trypsin

BAHJAT AL-ANI, MAHMOUD SAIFEDDINE, SURANGA J. WIJESURIYA and MORLEY D. HOLLENBERG

ABSTRACT
Trypsin activates proteinase-activated receptor-2 (PAR2) by a mechanism that involves the release of a tethered receptor activating sequence. We have identified two peptides, FSLLRY-NH2 (FSY-NH2) and LSIGRL-NH2 (LS-NH2) that block the ability of trypsin to activate PAR2 either in PAR2-expressing Kirsten virus-transformed kidney (KNRK) cell lines or in a rat aorta ring preparation. The reverse PAR2 peptide, LRGILS-NH2(LRG-NH2) did not do so and FSY-NH2 failed to block thrombin activation of PAR1 in the aorta ring or in PAR1-expressing human embryonic kidney cells. Half-maximal inhibition (IC50) by FSY-NH2 and LS-NH2 of the activation of PAR2 by trypsin in a PAR2 KNRK calcium-signaling assay was observed at about 50 and 200 M, respectively. In contrast, the activation of PAR2 by the PAR2-activating peptide, SLIGRL-NH2 (SL-NH2) was not inhibited by FSY-NH2, LS-NH2, or LRG-NH2. In a casein proteolysis assay, neither FSY-NH2 nor LS-NH2 inhibited the proteolytic action of trypsin on its substrate. In addition, FSY-NH2
and LS-NH2 were unable to prevent trypsin from hydrolyzing a 20-amino acid peptide, GPNSKGR/SLIGRLDTPYGGC representing the trypsin cleavage/activation site of rat PAR2. Similarly, FSY-NH2 and LS-NH2 failed to block the ability of trypsin to release the PAR2 N-terminal epitope that is cleaved from the receptor upon proteolytic activation of receptor-expressing KNRK cells. We conclude that the peptides FSY-NH2 and LSNH2 block the ability of trypsin to activate PAR2 by a mechanism that does not involve a simple inhibition of trypsin proteolytic activity, but possibly by interacting with a tethered ligand receptor-docking site.

Presenter: Gi Baek Lee

IL-31 levels correlate with pruritus in patients with cholestatic and metabolic liver diseases and is farnesoid X receptor responsive in NASH

Jun Xu , Ya Wang , Mina Khoshdeli , Matt Peach , Jen-Chieh Chuang , Julie Lin , Wen-Wei Tsai , Sangeetha Mahadevan , Wesley Minto , Lauri Diehl , Ruchi Gupta , Michael Trauner , Keyur Patel , Mazen Noureddin , Kris V Kowdley , Aliya Gulamhusein , Christopher L Bowlus , Ryan S Huss , Robert P Myers , Chuhan Chung , Andrew N Billin 

Abstract

Background and aims: Pruritus is associated with multiple liver diseases, particularly those with cholestasis, but the mechanism remains incompletely understood. Our aim was to evaluate serum IL-31 as a putative biomarker of pruritus in clinical trials of an farnesoid X receptor (FXR) agonist, cilofexor, in patients with NASH, primary sclerosing cholangitis (PSC), and primary biliary cholangitis (PBC).

Approach and results: Serum IL-31 was measured in clinical studies of cilofexor in NASH, PSC, and PBC. In patients with PSC or PBC, baseline IL-31 was elevated compared to patients with NASH and healthy volunteers (HVs). IL-31 correlated with serum bile acids among patients with NASH, PBC, and PSC. Baseline IL-31 levels in PSC and PBC were positively correlated with Visual Analog Scale for pruritus and 5-D itch scores. In patients with NASH, cilofexor dose-dependently increased IL-31 from Week (W)1 to W24. In patients with NASH receiving cilofexor 100 mg, IL-31 was higher in those with Grade 2-3 pruritus adverse events (AEs) than those with Grade 0-1 pruritus AEs. IL-31 weakly correlated with C4 at baseline in patients with NASH, and among those receiving cilofexor 100 mg, changes in IL-31 and C4 from baseline to W24 were negatively correlated. IL-31 messenger RNA (mRNA) was elevated in hepatocytes from patients with PSC and NASH compared to HVs. In a humanized liver murine model, obeticholic acid increased IL-31 mRNA expression in human hepatocytes and serum levels of human IL-31.

Conclusions: IL-31 levels correlate with pruritus in patients with cholestatic disease and NASH, with FXR agonist therapy resulting in higher serum levels in the latter group. IL-31 appears to derive in part from increased hepatocyte expression. These findings have therapeutic implications for patients with liver disease and pruritus.