Mechanisms of protease-activated receptor 2-evoked hyperexcitability of nociceptive neurons innervating the mouse colon

Agonists of protease-activated receptor 2 (PAR(2)) evoke hyperexcitability of dorsal root ganglia (DRG) neurons by unknown mechanisms. We examined the cellular mechanisms underlying PAR(2)-evoked hyperexcitability of mouse colonic DRG neurons to determine their potential role in pain syndromes such as visceral hyperalgesia. Colonic DRG neurons were identified by injecting Fast Blue and DiI retrograde tracers into the mouse colon. Using immunofluorescence, we found that DiI-labelled neurons contained PAR(2) immunoreactivity, confirming the presence of receptors on colonic neurons. Whole-cell current-clamp recordings of acutely dissociated neurons demonstrated that PAR(2) activation with a brief application (3 min) of PAR(2) agonists, SLIGRL-NH(2) and trypsin, evoked sustained depolarizations (up to 60 min) which were associated with increased input resistance and a marked reduction in rheobase (50% at 30 min). In voltage clamp, SLIGRL-NH(2) markedly suppressed delayed rectifier I(K) currents (55% at 10 min), but had no effect on the transient I(A) current or TTX-resistant Na(+) currents. In whole-cell current-clamp recordings, the sustained excitability evoked by PAR(2) activation was blocked by the PKC inhibitor, calphostin, and the ERK(1/2) inhibitor PD98059. Studies of ERK(1/2) phosphorylation using confocal microscopy demonstrated that SLIGRL-NH(2) increased levels of immunoreactive pERK(1/2) in DRG neurons, particularly in proximity to the plasma membrane. Thus, activation of PAR(2) receptors on colonic nociceptive neurons causes sustained hyperexcitability that is related, at least in part, to suppression of delayed rectifier I(K) currents. Both PKC and ERK(1/2) mediate the PAR(2)-induced hyperexcitability. These studies describe a novel mechanism of sensitization of colonic nociceptive neurons that may be implicated in conditions of visceral hyperalgesia such as irritable bowel syndrome.

The novel Syk inhibitor R406 reveals mechanistic differences in the initiation of GPVI and CLEC-2 signaling in platelets.

The inhibitory effect of R406 provides direct evidence of a role for Syk in GPVI, CLEC-2 and integrin alphaIIbbeta3 signaling in human platelets. Further, the results demonstrate a critical role for Syk in mediating tyrosine phosphorylation of CLEC-2, suggesting a novel model in which both Src and Syk kinases regulate tyrosine phosphorylation of the C-type lectin receptor leading to platelet activation.

The p38-MAPK inhibitor, SB203580, inhibits cardiac stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs).

SB203580 is a recognised inhibitor of p38-MAPKs. Here, we investigated the effects of SB203580 on cardiac SAPKs/JNKs. The IC50 for inhibition of p38-MAPK stimulation of MAPKAPK2 was approximately 0.07 microM, whereas that for total SAPK/JNK activity was 3-10 microM. SB203580 did not inhibit immunoprecipitated JNK1 isoforms. Three peaks of SAPK/JNK activity were separated by anion exchange chromatography, eluting in the isocratic wash (44 kDa), and at 0.08 M (46 and 52 kDa) and 0.15 M NaCl (54 kDa). SB203580 (10 microM) completely inhibited the 0.15 M NaCl activity and partially inhibited the 0.08 M NaCl activity. Since JNK1 antibodies immunoprecipitate the 46 kDa activity, this indicates that SB203580 selectively inhibits 52 and 54 kDa SAPKs/JNKs.