Kazue Mizumura

TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP

Transient receptor potential V3 (TRPV3) and TRPV4 are heat-activated cation channels expressed in keratinocytes. It has been proposed that heat-activation of TRPV3 and/or TRPV4 in the skin may release diffusible molecules which would then activate termini of neighboring dorsal root ganglion (DRG) neurons. Here we show that adenosine triphosphate (ATP) is such a candidate molecule released from keratinocytes upon heating in the co-culture systems. Using TRPV1-deficient DRG neurons, we found that increase in cytosolic Ca2+-concentration in DRG neurons upon heating was observed only when neurons were co-cultured with keratinocytes, and this increase was blocked by P2 purinoreceptor antagonists, PPADS and suramin. In a co-culture of keratinocytes with HEK293 cells (transfected with P2X2 cDNA to serve as a bio-sensor), we observed that heat-activated keratinocytes secretes ATP, and that ATP release is compromised in keratinocytes from TRPV3-deficient mice. This study provides evidence that ATP is a messenger molecule for mainly TRPV3-mediated thermotransduction in skin.

Effects of prostaglandins and other putative chemical intermediaries on the activity of canine testicular polymodal receptors studied in vitro

Abstract(1) An in vitro testis-superior spermatic nerve preparation was used to evaluate the effects of chemical agents applied in the bathing solution. Both directly evoked discharges and responses to algesic solutions [bradykinin (BK) 9×10−8 M, hypertonic saline 616 mM and high K+ solution 60 mM] of polymodal receptors were studied. (2) Prostaglandin (PG)-E2 (1.4×10−6–1.4×10−5 M) and serotonin (5-HT) (1.1×10−6 to 1.4×10−4 M) had only a weak excitatory effect. However, test responses to algesic substances were regularly greatly increased by PG-E2,-I2 and 5-HT. Concentrations of PG-E2 of 1.4×10−8 M or grealer augmented BK responses; higher concentrations and/or longer applications were needed to enhance responses to algesic salt solutions. Effective concentrations for the PGs and 5-HT were near those reported for inflamed tissues and exudate. (3) Aspirin (ASA) (5.5×10−4 M or greater, for more than 4 min) suppressed the responses to BK but not those evoked by hypertonic saline. The ASA effect on the BK response was largely restored by an addition of PG-E2. (4) Substance P also had a weak excitatory effect on some polymodal receptors, but no significant enhancement of the response to BK was noted. (5) These results further support a role of polymodal receptors in transmitting nociceptive information, of inflammatory origin.

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.

TRP channels and ASICs mediate mechanical hyperalgesia in models of inflammatory muscle pain and delayed onset muscle soreness.

Abstract The roles of ion channels in sensory neurons were examined in experimental models of muscle pain in the rat. Rats were injected with 50 μl of 4% carrageenan or subjected to an eccentric exercise (ECC) of the gastrocnemius muscle (GM). The Randall–Selitto and von Frey tests were performed on the calves to evaluate mechanical hyperalgesia of the muscle. The changes in expression of four genes and proteins of ion channels in dorsal root ganglia were examined using quantitative PCR and immunohistochemistry, respectively. Effects of antagonists to transient receptor potential (TRP) channels and acid sensing ion channels (ASICs) on the mechanical hyperalgesia induced by carrageenan injection or ECC were evaluated. The mechanical hyperalgesia was observed 6–24 h after carrageenan injection and 1–3 days after ECC in the Randall–Selitto test. Infiltrations of the inflammatory cells in the GM were seen in carrageenan‐injected animals but not in those subjected to ECC. Expressions of genes and proteins in sensory neurons showed no changes. Intramuscular injection of antagonists to TRPV1 showed an almost complete suppressive effect on ECC‐induced muscle hyperalgesia but not a carrageenan‐induced one. Antagonists to TRP channels and ASICs showed suppressive effects for both carrageenan‐ and ECC‐induced muscle hyperalgesia. The carrageenan injection and ECC models are useful models of acute inflammatory pain and delayed onset muscle soreness (DOMS), respectively, and the time course and underlying etiology might be different. TRP channels and ASICs are closely related to the development of muscle mechanical hyperalgesia, and TRPV1 is involved in ECC‐induced DOMS.

Muscular mechanical hyperalgesia revealed by behavioural pain test and c‐Fos expression in the spinal dorsal horn after eccentric contraction in rats

Delayed onset muscle soreness (DOMS) is quite common, but the mechanism for this phenomenon is still not understood; even the existence of muscle tenderness (mechanical hyperalgesia) has not been demonstrated in experimental models. We developed an animal model of DOMS by inducing eccentric contraction (lengthening contraction, ECC) to the extensor digitorum longus muscle (EDL), and investigated the existence of mechanical hyperalgesia in the EDL by means of behavioural pain tests (Randall‐Selitto test and von Frey hair test, applied to/through the skin on the EDL muscle) and c‐Fos expression in the spinal dorsal horn. We found that the mechanical withdrawal threshold measured with the Randall‐Selitto apparatus decreased significantly between 1 and 3 days after ECC, while that measured by von Frey hairs did not. The group that underwent stretching of the muscle only (SHAM group) showed no change in mechanical pain threshold in either test. These results demonstrated that the pain threshold of deep tissues (possibly of the muscle) decreased after ECC. c‐Fos immunoreactivity in the dorsal horn (examined 2 days after ECC/SHAM exercise) was not changed by either ECC or compression (1568 mN) to the EDL muscle by itself, but it was significantly increased by applying compression to the EDL muscle 2 days after ECC. This increase was observed in the superficial dorsal horn of the L4 segment of the ipsilateral side, and was clearly suppressed by morphine treatment (10 mg kg−1, i.p.). These results demonstrated the existence of mechanical hyperalgesia in the muscle subjected to ECC. This model could be used for future study of the neural mechanism of muscle soreness.

Influence of surface anesthesia on the pressure pain threshold measured with different-sized probes

Transcutaneous pressure with pressure probes of arbitrary diameters have been commonly used for measuring the threshold and magnitude of muscle pain, yet this procedure lacks scientific validation. To examine the valid probe dimensions, we conducted physiological experiments using 34 human subjects. Pin-prick pain, pressure pain threshold (PPT) to pressure probes of various diameters, heat pain threshold, and electrical pain threshold of deep tissues were measured before and after application of surface lidocaine anesthesia to the skin surface over the brachioradial muscle in a double-blinded manner. The anesthesia neither affected PPT with larger probes (diameters: 1.6 and 15 mm) nor increased electric pain threshold of deep structures, whereas it diminished pain count in pin-prick test and PPT with a 1.0 mm diameter probe, suggesting that mechanical pain thresholds measured with 1.6 and 15 mm probes reflect the pain threshold of deep tissues, possibly muscle. Pain thresholds to heat did not change after application of the anesthesia. These results suggest that larger pressure probes can give a better estimation of muscular pain threshold.

Persistent deep mechanical hyperalgesia induced by repeated cold stress in rats

Chronic muscle pain of the neck, shoulder and low back is quite common and often related to a stressed condition. In this study we tried to make a model of long‐lasting muscle mechanical hyperalgesia based on one type of stress, repeated cold stress (RCS) (Kita T, Hata T, Yoneda R, Okage T. Stress state caused by alternation of rhythm in environmental temperature, and the functional disorders in mice and rats. Folia Pharmacol Jpn 1975;71:195–210). We first validated a method of measuring the muscle mechanical nociceptive threshold through skin, with surface anesthesia of the skin covering the muscle. We found that a pressure test using a Randall–Selitto analgesiometer equipped with a larger probe (φ 2.6 mm) can measure the deep mechanical withdrawal threshold even under the presence of cutaneous punctuate hyperalgesia. RCS was performed by changing the temperature from 22 °C to either 4 °C (RCS at 4 °C) or −3 °C (RCS at −3 °C) every 30 min, and then maintained at 4 °C/−3 °C from 17:30 to 10:00 the next day. RCS at 4 °C for 5 days induced bilateral deep mechanical hyperalgesia lasting 2–3 weeks without cutaneous punctuate hyperalgesia. Deep mechanical hyperalgesia observed after RCS at −3 °C lasted longer (∼6 weeks) and was severer than RCS at 4 °C. Bilateral cutaneous punctuate hyperalgesia was also observed with RCS at −3 °C. Intramuscular injection of lidocaine confirmed that the muscle was hyperalgesic. RCS might serve as a useful model for study of the mechanism of chronic muscle pain and its treatment.

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.

The effects of bradykinin agonists and antagonists on visceral polymodal receptor activities

&NA; The endogenous algesic agent bradykinin (BK) is a consistent stimulant for the polymodal receptor, a type of nociceptor. Two types of BK receptor, B1 and B2, have been proposed in smooth muscles by Regoli. The type of BK receptor mediating the BK response of the polymodal receptor was studied using 3 BK analogs, des‐Arg9‐BK (a Bl agonist), des‐Arg9‐[Leu8]‐BK (a Bl antagonist), and [Thi5,8, d‐Phe7]‐BK (a B2 antagonist). Single‐ and multi‐fiber activities from testicular polymodal receptors were recorded in vitro using testis‐spermatic nerve preparations excised from dogs anesthetized with pentobarbital (30 mg/kg, i.v.). Neither des‐Arg9‐BK, des‐Arg9‐[Leu8]‐BK, nor [Thi5,8,d‐Phe7]‐BK induced discharges in nociceptors at concentrations up to 9.4 × 10−6 M. Des‐Arg9‐[Leu8]‐BK (up to 9.4 × 10−6 M) did not suppress responses to BK (9.4 × 10−8˜−9M), whereas [Thi5,8,d‐Phe7]‐BK (above 2.8 × 10‐7 M) suppressed the BK response in a concentration‐dependent manner and shifted the concentration‐response curve of BK to the right. It was ascertained that [Thi5,8,d‐Phe7]‐BK had no effect on responses to noxious heat and high K+ solution. These results suggest that the BK receptor mediating the nociceptor response to BK is of the B2 type.

Involvement of EP3 subtype of prostaglandin E receptors in PGE2-induced enhancement of the bradykinin response of nociceptors.

Prostaglandin E2 augments bradykinin-induced discharges of polymodal receptors as studied in vitro preparations. The antagonist and agonists for three subtypes of EP receptors were used to determine which subtype is involved in this phenomenon. The agonist for EP3 (M&B28767) simulated the PGE2-induced effect but not for EP2 (butaprost). The antagonist for EP1 (AH6809) did not suppress the effect. These findings indicate the involvement of the EP3 receptor subtype in the effect.