Yasuko Kozaki

Bradykinin and Nerve Growth Factor Play Pivotal Roles in Muscular Mechanical Hyperalgesia after Exercise (Delayed-Onset Muscle Soreness)

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 ∼1 d. Using B2 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. B1 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 B2 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.

3β-Hydroxysteroid-Δ24 Reductase Is a Hydrogen Peroxide Scavenger, Protecting Cells from Oxidative Stress-Induced Apoptosis

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.

Excitation and sensitization of nociceptors by bradykinin: What do we know?

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.

Compression-induced ATP release from rat skeletal muscle with and without lengthening contraction.

Adenosine triphosphate (ATP) is well known to be released from injured or inflamed tissues, and to excite/sensitize nociceptors in response to heat and mechanical stimulation. To determine whether muscle releases ATP when it is compressed, we measured ATP release from the extensor digitorum longus muscle (EDL). In addition, we investigated whether there is any difference in ATP release from the EDL of rats 2 days after lengthening contraction (LC), since the condition of the muscle is different, i.e., mechanically hyperalgesic and swollen. The EDL was put in a small chamber and superfused with Krebs-Henseleit solution equilibrated with a gas mixture of 95% oxygen and 5% carbon dioxide. The muscle was quantitatively stimulated with a servo-controlled mechanical stimulator. Reproducibility of ATP release was examined with stimulation using a 20 g force. Stimulus intensity-dependency of ATP release was also examined with 5 time compression with intensities of 5, 10, 20 and 40 g force. Bioluminescent determination by the luciferin-luciferase method was used to quantify ATP in the sample. The ATP release was decreased by repetitive mechanical stimulation of the EDL with 30 min intervals, and it was stimulus intensity (5-40 g force)-dependent. The amount of ATP released from the muscle preparations was not different between the non-treated control and the LC group. These results provide clear evidence that ATP is released from rat skeletal muscle by compression.

Molecular cloning of prostaglandin EP3 receptors from canine sensory ganglia and their facilitatory action on bradykinin-induced mobilization of intracellular calcium

We previously demonstrated that the activation of prostaglandin E‐prostanoid‐3 (EP3) receptor sensitized the canine nociceptor response to bradykinin (BK). To elucidate the molecular mechanism for this sensitization, we cloned two cDNAs encoding EP3s with different C‐terminals, from canine dorsal root ganglia, and established the transformed cell lines stably expressing them. In both transformants, EP3 agonist did not increase intracellular cAMP levels, but it attenuated forskolin‐dependent cAMP accumulation in a pertussis toxin (PTX)‐sensitive manner and increased intracellular calcium levels in a PTX‐resistant manner, indicating that both EP3s can couple with Gi and Gq, but not with Gs proteins. As the nociceptor response to BK is mediated by BK B2 receptor, it was transfected into the transformants and the effects of EP3 agonist on BK‐dependent calcium mobilization were investigated. When BK was applied twice with a 6‐min interval, the second response was markedly attenuated. Pre‐treatment with EP3 agonist had no effect on the initial response, but restored the second response in a PTX‐sensitive manner. A protein kinase A inhibitor mimicked the effect of EP3 agonist. These results demonstrate that the activation of EP3 restores the response to BK by attenuating the desensitization of BK B2 receptor activity via Gi protein.

Prostaglandin E2 negatively regulates AMP-activated protein kinase via protein kinase A signaling pathway.

We investigated possible involvement of prostaglandin (PG) E2 in regulation of AMP-activated protein kinase (AMPK). When osteoblastic MG63 cells were cultured in serum-deprived media, Thr-172 phosphorylation of AMPK alpha-subunit was markedly increased. Treatment of the cells with PGE2 significantly reduced the phosphorylation. Ser-79 phosphorylation of acetyl-CoA carboxylase, a direct target for AMPK, was also reduced by PGE2. On the other hand, PGE2 reciprocally increased Ser-485 phosphorylation of the alpha-subunit that could be associated with inhibition of AMPK activity. These effects of PGE2 were mimicked by PGE2 receptor EP2 and EP4 agonists and forskolin, but not by EP1 and EP3 agonists, and the effects were suppressed by an adenylate cyclase inhibitor SQ22536 and a protein kinase A inhibitor H89. Additionally, the PGE2 effects were duplicated in primary calvarial osteoblasts. Together, the present study demonstrates that PGE2 negatively regulates AMPK activity via activation of protein kinase A signaling pathway.

A possible role of prostaglandin in the recycling of internalized bradykinin B2 receptor through EP3 receptor

Varicella-zoster virus (VZV) expresses immediate-early protein 62 (IE62), and zoster is associated with neuropathic pain. Brain-derived neurotrophic factor (BDNF) is involved in the neuronal mechanism underlying pain hypersensitivity. Zoster is associated with prodrome and the robust production of booster antibody to VZV. We hypothesized that the production of antibody to IE62 cross-reacting with BDNF may augment allodynia in zoster and postherpetic neuralgia (PHN) by enhancing BDNF activity. One of three monoclonal antibodies against the 268-556 peptide of IE62 recognized BDNF. Immunological cross-reactivity between IE62 and BDNF and the effects of anti-IE62 monoclonal antibody (anti-IE62 MAb) cross-reactivity with BDNF on BDNF activity in cultured neurons were examined. Anti-IE62 MAb and anti-BDNF MAbs recognized the 414-429 peptide of IE62 and the BDNF dimer. AntiIE62 MAb significantly augmented BDNF-related transcription in neurons and the morphological development of spinal dorsal horn neurons. Sera from patients with zoster and PHN recognized IE62 and BDNF and enhanced BDNF activity in neurons. The effect of anti-IE62 antibody on mechanical allodynia induced by spinal nerve injury in mice was examined. The intrathecal administration of anti-IE62 MAb to or immunization with cross-reacting IE62 protein to mice significantly enhanced mechanical allodynia. The results suggest that the intrathecal production of anti-IE62 antibody augmenting BDNF activity contributes to the pathogenesis of allodynia in zoster and PHN.

Increased expression of mRNA for B1 and B2 Bradykinin receptors in the skin of adjuvant inoculated rats

Bradykinin is the most established endogenous algesic substance. It has two receptors subtypes, B 1 and B2, which are classified based on their pharmacological properties. It is generally accepted that the B2 receptor (B2R) is normally expressed in many tissues, whereas the B1 receptor (B1R) exists only in limited tissues and is synthesized de noun following tissue injury. We recently demonstrated that B2R-mediated bradykinin responses of peripheral nociceptors were increased in skin-saphenous nerve preparations in vitro in adjuvant inflamed rats, but involvement of B1R could not be confirmed. To examine the possibility that B1R is expressed in cells other than neurons and that we missed B1R-mediated responses while using in vitro preparations, we evaluated the mRNA expression of B1R in rat hind-paw skin and compared it with that of B2R after adjuvant inoculation. B2R mRNA was expressed in control and inflamed animals, and the level of its expression was increased 3 weeks after adjuvant inoculation. B1R mRNA, in contrast to the general belief, was expressed in control animals and increased 3 weeks after adjuvant inoculation, similar to B2R mRNA. Possible meanings of this finding are discussed.

Possible Implication of N. Parabrachialis in Opioid-Mediated Respiratory Suppression Induced by Thin-Fiber Muscular Afferents

The great majority of thin-fiber muscular afferents are of the polymodal receptor type, signaling nociceptive information. Arterial injection of various algesic substances into the gastrocnemius muscle of anesthetized, spontaneously ventilated dogs, causes an increase in minute respiratory volume similar to increases observed in discharge rates of muscular polymodal receptors in response to the same stimulus.2 Such findings suggest an involvement of the muscular polymodal receptors in the respiratory response.