Unaccustomed strenuous exercise that includes lengthening contraction (LC) often causes delayed-onset muscle soreness (DOMS), a kind of muscular mechanical hyperalgesia. The substances that induce this phenomenon are largely unknown. Peculiarly, DOMS is not perceived during and shortly after exercise, but rather is first perceived after ϳ1 d. Using B 2 bradykinin receptor antagonist HOE 140, we show here that bradykinin released during exercise plays a pivotal role in triggering the process that leads to muscular mechanical hyperalgesia. HOE 140 completely suppressed the development of muscular mechanical hyperalgesia when injected before LC, but when injected 2 d after LC failed to reverse mechanical hyperalgesia that had already developed. B 1 antagonist was ineffective, regardless of the timing of its injection. Upregulation of nerve growth factor (NGF) mRNA and protein occurred in exercised muscle over a comparable time course (12 h to 2 d after LC) for muscle mechanical hyperalgesia. Antibodies to NGF injected intramuscularly 2 d after exercise reversed muscle mechanical hyperalgesia. HOE 140 inhibited the upregulation of NGF. In contrast, shortening contraction or stretching induced neither mechanical hyperalgesia nor NGF upregulation. Bradykinin together with shortening contraction, but not bradykinin alone, reproduced lasting mechanical hyperalgesia. We also showed that rat NGF sensitized thin-fiber afferents to mechanical stimulation in the periphery after 10 -20 min. Thus, NGF upregulation through activation of B 2 bradykinin receptors is essential (though not satisfactory) to mechanical hyperalgesia after exercise. The present observations explain why DOMS occurs with a delay, and why lengthening contraction but not shortening contraction induces DOMS.

3beta-Hydroxysteroid-Delta24 reductase (DHCR24) is an endoplasmic reticulum-resident, multifunctional enzyme that possesses antiapoptotic and cholesterol-synthesizing activities. To clarify the molecular basis of the former activity, we investigated the effects of hydrogen peroxide (H(2)O(2)) on embryonic fibroblasts prepared from DHCR24-knockout mice (DHCR24(-/-) mouse embryonic fibroblasts). H(2)O(2) exposure rapidly induced apoptosis, which was associated with sustained activation of apoptosis signal-regulating kinase-1 and stress-activated protein kinases, such as p38 MAPK and c-Jun N-terminal kinase. Complementation of the mouse embryonic fibroblasts by adenovirus expressing DHCR24 attenuated the H(2)O(2)-induced kinase activation and apoptosis. Concomitantly, intracellular generation of reactive oxygen species (ROS) in response to H(2)O(2) was also diminished by the adenovirus, suggesting a ROS-scavenging activity of DHCR24. Such antiapoptotic effects of DHCR24 were duplicated in pheochromocytoma PC12 cells infected with adenovirus. In addition, it was found that DHCR24 exerted cytoprotective effects in the tunicamycin-induced endoplasmic reticulum stress by eliminating ROS. Finally, using in vitro-synthesized and purified proteins, DHCR24 and its C-terminal deletion mutant were found to exhibit high H(2)O(2)-scavenging activity, whereas the N-terminal deletion mutant lost such activity. These results demonstrate that DHCR24 can directly scavenge H(2)O(2), thereby protecting cells from oxidative stress-induced apoptosis.

Bradykinin is an endogenous nonapeptide known to induce pain and hyperalgesia to heat and mechanical stimulation. Correspondingly, it excites nociceptors in various tissues and sensitizes them to heat, whereas sensitizing effect on the mechanical response of nociceptors is not well established. Protein kinase C and TRPV1 contribute to the sensitizing mechanism of bradykinin to heat. In addition, TRPA1 and other ion channels appear to contribute to excitation caused by bradykinin. Finally, prostaglandins sensitize bradykinin-induced excitation in normal tissues by restoring desensitized responses due to the inhibition of protein kinase A.

AMP-activated protein kinase (AMPK) is a key regulator of glucose and fatty acid homeostasis. In muscle cells, AMPK stimulates mitochondrial fatty acid oxidation and ATP production. The thyroid hormone T3 increases cellular oxygen consumption and is considered to be a major regulator of mitochondrial activities. In this study, we examined the possible involvement of AMPK in the stimulatory action of T3 on mitochondria. Treatment of C2C12 myoblasts with T3 rapidly led to phosphorylation of AMPK. Acetyl-coenzyme A carboxylase, a direct target of AMPK, was also phosphorylated after T3 treatment. Similar results were obtained with 3T3-L1, FRTL-5, and HeLa cells. Stable expression of T3 receptor (TR)-alpha or TRbeta in Neuro2a cells enhanced this effect of T3, indicating the involvement of TRs. Because HeLa cells express only Ca2+/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta), one of two known AMPK kinases, it was suggested that the effect of T3 is mediated by CaMKKbeta. Indeed, experiments using a CaMKK inhibitor, STO-609, and an isoform-specific small interfering RNA demonstrated the CaMKKbeta-dependent phosphorylation of AMPK. Furthermore, T3 was found to rapidly induce intracellular Ca2+ mobilization in HeLa cells, and a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suppressed T3- as well as ionomycin-dependent phosphorylation of AMPK. In addition, T3-dependent oxidation of palmitic acids was attenuated by BAPTA, STO-609, and the small interfering RNA for CaMKKbeta, indicating that T3-induced activation of AMPK leads to increased fatty acid oxidation. These results demonstrate that T3 nontranscriptionally activates AMPK via intracellular Ca2+ mobilization and CaMKKbeta activation, thereby stimulating mitochondrial fatty acid oxidation.