KT5720

The suprachiasmatic nuclei (SCN) of the hypothalamus house the main mammalian circadian pacemaker. Cell bodies in the rat SCN contain the neuropeptide neurotensin (NT), and two NT receptor types, NTS1 and nts2. Because the role of NT in the circadian rhythm processes is unknown, we studied the phase-shifting effects of NT on the firing rate rhythm of rat SCN neurons in vitro. Additionally, the NT receptor antagonists SR142948a and SR48692 were used to try and block any NT-induced phase shifts. To elucidate the second messenger pathway responsible for mediating the phase-resetting actions of NT, we utilized the phospholipase C (PLC) and protein kinase A (PKA) inhibitors U-73122 and KT5720, respectively. Application of NT during the projected day resulted in a large advance in the time of peak in FRR, whereas treatments during the projected night had no effect. Both NT receptor antagonists blocked the NT-induced phase shifts, as did the PLC inhibitor U-73122. The PKA inhibitor KT5720 had no influence on the magnitude of the phase shift caused by NT during the middle of the projected day. These results provide the first evidence that NT may play a role in regulating the rat circadian pacemaker, using NTS1 and nts2 receptors presumably coupled to PLC.

Cyclic adenosine monophosphate (cAMP) modulates various agent-induced apoptosis. In this study, we observed that cAMP had a significantly protective effect on nitric oxide (NO)-induced cytotoxicity in H9c2 cardiac muscle cells. Pretreatment with DBcAMP (cAMP analogue) or forskolin (adenylyl cyclase activator) also significantly prevented the SNP-induced apoptosis in H9c2 cells. In contrast, H-89 or KT5720 (PKA inhibitor) reversed the protective effects of DBcAMP. In this study, DBcAMP or forskolin reduced SNP-induced JNK/SAPK activation to the basal level, but KT5720 reversed the inhibitory effects of these two agents. In contrast to JNK/SAPK activation, DBcAMP and forskolin significantly enhanced SNP-activated p38 MAPK phosphorylation and did not affect SNP-mediated ERK activation. KT5720 reversed the effects of DBcAMP and forskolin on p38 MAPK phosphorylation. The inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK/SAPK markedly reduced the extent of SNP-induced cell death. Taken together, we suggest that JNK/SAPK is related to cAMP-protective effect in SNP-induced apoptosis. In addition, c-AMP relating agents protected SNP-induced cell death in neonatal rat ventricular cardiomyocytes.

The present study was designed to characterize the urinary bladder-derived relaxant factor that was demonstrated by acetylcholine-induced relaxation response in a coaxial bioassay system consisting of rat bladder as the donor organ and rat anococcygeus muscle as the assay tissue. The concentration-dependent relaxation to acetylcholine (10 nM-1 mM) was inhibited by atropine but was not altered by the antagonists of calcitonin gene-related peptide (CGRP 8-37), vasoactive intestinal peptide (VIP 6-28), tachykinin NK1 (L-732138), tachykinin NK2 (MEN-10376), tachykinin NK3 (SB-218795), purinergic P2 (PPADS) and adenosine (CGS 15943) receptors as well as alpha-chymotrypsin. Adenylate cyclase inhibitor SQ-22536 and protein kinase A inhibitor KT-5720 significantly inhibited the acetylcholine response while guanylate cyclase inhibitor ODQ, and protein kinase C inhibitor H-7 did not have any effect. The P2X agonist alpha,beta-methylene ATP (10 nM-0.1 mM) also produced concentration-dependent relaxation response that was inhibited by PPADS, SQ-22536 and KT-5720 in the coaxial bioassay system. In bladder strips, acetylcholine and alpha,beta-methylene ATP elicited concentration-dependent contractions that were not altered in the presence of SQ-22536 and KT-5720.