EP2 receptor plays pivotal roles in generating mechanical hyperalgesia after lengthening contractions.

We previously demonstrated that nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) were upregulated after lengthening contractions (LC) in exercised muscle through B2 bradykinin receptor activation and cyclooxygenase (COX)-2 upregulation, respectively, and that these trophic factors sensitized nociceptors resulting in mechanical hyperalgesia (delayed-onset muscle soreness, DOMS). Here, we examined the prostaglandin receptor subtype involved in DOMS. The mechanical withdrawal threshold of the exercised muscle was measured before and after LC in rats administered prostaglandin E2 receptor (EP) antagonists before LC, or in wild-type (WT), EP2 knockout (EP2-/- ), and IP knockout (IP-/- ) mice. The change in expression of NGF, GDNF, or COX-2 mRNA was examined using real-time RT-PCR in the muscle in EP2-/- and WT mice. None of the antagonists to EP1, EP3, and EP4 receptors (ONO-8713, ONO-AE5-599, and ONO-AE3-208, respectively) induced a significant difference in DOMS compared with controls in rats. WT and IP-/- mice developed mechanical hyperalgesia after LC, but EP2-/- mice did not. Upregulation of NGF, GDNF, and COX-2 mRNA was observed after LC in WT mice but not in EP2-/- mice. Injecting an EP2 agonist (ONO-AE1-259-01) into the mouse muscle increased expression of COX-2 mRNA. These results suggest that EP2 contributes to generating mechanical hyperalgesia through positive feedback upregulation of COX-2 expression in muscle after LC.

Decreased nerve growth factor upregulation is a mechanism for reduced mechanical hyperalgesia after the second bout of exercise in rats.

Delayed onset muscle soreness (DOMS) is reduced when the same exercise is repeated after a certain interval. However, the mechanism for this adaptation, called a repeated bout effect, is still not well understood. Recently, we showed that upregulated nerve growth factor (NGF) triggered by B2 bradykinin receptor (B2R) activation in exercised muscle was responsible for DOMS. In this study, we investigated whether NGF upregulation was reduced after repeated bouts of exercise in rats, and if so, whether this change occurred upstream of B2R. A bout of 500 lengthening contractions (LC) was applied on day 0 and again 5 days later. DOMS was evaluated by the mechanical withdrawal threshold of the exercised extensor digitorum longus (EDL) muscle. Mechanical hyperalgesia and NGF mRNA upregulation in EDL were observed after the first LC, but not after the second LC. We then injected HOE140, a B2R antagonist with effects lasting only several hours, once before the first LC. This blocked the development of mechanical hyperalgesia and NGF mRNA upregulation not only after the first LC but also after the second LC. This suggests that adaptation occurred upstream of B2R, as the influence of the first LC was limited to that area by HOE140.

Muscular mechanical hyperalgesia after lengthening contractions in rats depends on stretch velocity and range of motion.

BACKGROUND: The current study investigated stretch variables and mechanical factors of lengthening contractions (LC) in the processes leading to muscular mechanical hyperalgesia in rats to understand mechanisms underpinning delayed onset muscle soreness (DOMS). METHODS: Under isoflurane anaesthesia, ankle extensor muscles were loaded with repetitive LC with angular stretch velocities (50°, 100°, 200° and 400°/s) at a fixed range of motion (ROM) of 90°, and with ROMs (30°, 60°, 90° and 120°) at a fixed velocity of 200°/s. RESULTS: Mechanical hyperalgesia was observed in a velocity- and ROM-dependent manner. Under the fixed ROM, integrated torque generated during LC (iTq[max] ) was inversely correlated with the velocity, but the rate of torque increase during LC (rTq[max] ) was positively and significantly correlated with the velocity, and the magnitude of hyperalgesia was correlated with rTq[max] (p < 0.001). When the velocity was fixed, iTq[max] was significantly correlated with ROM, and the magnitude of hyperalgesia was correlated with iTq[max] (p < 0.01). Necrotic myofibres were observed only sparsely (<0.8%) after any of the LC protocols tested. Up-regulation of nerve growth factor and glial cell line-derived neurotrophic factor mRNA in the muscle was positively correlated with the increases in the LC velocity and ROM (p < 0.05~0.001). CONCLUSIONS: Both velocity and ROM are pivotal variables determining the initiation of mechanical hyperalgesia. Neurotrophic factor-mediated peripheral mechanisms, but apparently not inflammatory changes caused by myofibre damage, are responsible for the mechanical hyperalgesia. SIGNIFICANCE: Mechanical hyperalgesia appears after LC in a stretch velocity- and range of motion-dependent manner. The rate of torque increase and integrated torque are the crucial factors. Neurotrophic factor-mediated peripheral pain mechanisms without robust inflammatory changes caused by myofibre damage were required for this mechanical hyperalgesia.

Spinal termination patterns of canine identified A-delta and C spermatic polymodal receptors traced by intracellular labeling with Phaseolus vulgaris-leucoagglutinin.

The spinal projection patterns of spermatic polymodal receptors were studied by intracellular labeling of functionally identified canine dorsal root ganglion (DRG) neurons with Phaseolus vulgaris-leucoagglutinin (PHA-L). The processes of 2 C-fiber and 1 A-delta-fiber spermatic polymodal receptor neurons were labeled well enough to trace their central terminations. The labeled C-fiber DRG neurons were of medium size (mean diameter 36.8 and 40.7 microns). On entering the spinal cord, axons of the C-polyclonal receptors divided into rostral and caudal main branches that extended over 3 spinal segments (20 and 25 mm, respectively), and issued a total of 16 and 15 collaterals, respectively. The majority of collaterals ran in or along the lateral surface, but both neurons had 1 or 2 collaterals or terminal branches running through the middle, or along the medial surface of the dorsal horn. Terminal swellings and en passant enlargements were observed mainly in laminae I, V, and VII. Some C-fiber terminations appeared in lamina II and the adjacent lateral column. The A-delta polymodal receptor had a termination pattern similar to that of the C-fiber units with the exception of a shorter distance over which its 13 identified collaterals were issued (10 mm), and continuation of the rostral main branch into Lissauer's tract (traced for 3.6 mm) after all branches appeared. Two terminal branches were found running just above the central canal in another A-delta neuron in which termination could be only partially traced.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermally potentiated responses to algesic substances of visceral nociceptors.

Using spermatic nerve preparations from testis superior, the effects of temperature rise on chemical responses were studied in vitro, within both subthreshold and suprathreshold ranges for testicular polymodal receptors, one type of visceral nociceptor. In the range of temperature subthreshold for polymodal receptors, the responses to algesic substances tested were greater at higher temperatures. The mean discharge rates induced by various concentrations of bradykinin (BK, 9 X 10(-9)-9 X 10(-6) M), were significantly greater at 36 degrees C than at 30 degrees C. Responses to hypertonic saline, tested in the temperature range, 34-43 degrees C, showed similar temperature-dependent increases, and Q10 values between 2.3 and 4.2. In the units exhibiting 'heat sensitization' with repeated testing of suprathreshold temperature rises, the response to hypertonic saline (616 mM) at 34 degrees C also increased. Although calor in the inflamed tissues is in itself not high enough to cause excitation of 'pain receptors,' temperature-dependent augmentation of chemical responses of the polymodal receptor might partly explain peripheral hyperalgesic behaviour observable in inflamed tissue on the basis of sensory receptor activity.

Amantadine inhibits RANTES production by influenzavirus-infected human bronchial epithelial cells.

1. Amantadine can prevent and decrease airway inflammation by inhibiting influenza virus (IV) replication; however, the effect of amantadine on RANTES production by human bronchial epithelial cells (BEC) has not been determined. In the present study, we examined the effect of amantadine on RANTES production and also analysed p38 mitogen-activated protein (MAP) kinase and c-Jun-NH2-terminal kinase (JNK) activation to clarify the mechanism in the effect of amantadine on RANTES production, since we have previously shown that p38 MAP kinase and JNK regulate RANTES production by IV-infected BEC. 2. BEC that had been preincubated with amantadine were infected with IV and then p38 MAP kinase and JNK activation in the cells and RANTES concentrations in the culture supernatants were determined. 3. Amantadine-induced inhibition of virus replication resulted in a decrease in p38 MAP kinase and JNK activity and decreased expression of RANTES in IV-infected cells. 4. Amantadine did not inhibit p38 MAP kinase and JNK activation induced by tumour necrosis factor-alpha (TNF-alpha) as a non-viral stimulus. 5. These results indicate that amantadine inhibits IV infection-induced RANTES production by human BEC and that the inhibition by amantadine of RANTES production might result from an indirect inhibitory effect of amantadine on p38 MAP kinase and JNK activation via the inhibition of virus replication, and we emphasize that amantadine may produce a beneficial effect on controlling bronchial asthma exacerbation caused by IV infection.

Effects of PGE(2) on neurons from rat dorsal root ganglia in intact and adjuvant-inflamed rats: role of NGF on PGE(2)-induced depolarization.

The effects of prostaglandin E(2) (PGE(2)) on primary afferent neurons were studied by intracellular recording from small (<30 microm) dorsal root ganglion (DRG) neurons cultured for up to 3 days. PGE(2) (10(-9)-10(-5) M) depolarized 4-10% of neurons cultured with nerve growth factor (NGF) in intact rats. The percentage of neurons depolarized increased in a concentration dependent manner, while the average amplitude of the depolarization did not change with concentration. The threshold to evoke an action potential was decreased by PGE(2) (10(-9)-10(-5) M) with the maximum percentage at 10(-9) M, and this effect was also observed in neurons not depolarized by PGE(2). Whether a neuron was depolarized by PGE(2) was not related with its capsaicin (CAP) sensitivity. In addition, we examined whether NGF influences the PGE(2) response of neurons in adjuvant-inflamed young adult animals. Removal of NGF from culture medium did not change the percentage of neurons depolarized by PGE(2) in intact rats (20 and 18% for neurons cultured without or with NGF for 2-3 days, respectively). Adjuvant induced inflammation increased the percentage of neurons depolarized by PGE(2) to 38%, but this was not reversed by an addition of anti-NGF antibody to the culture medium, suggesting that NGF does not play a substantial role in the increase in sensitivity to be depolarized by PGE(2).

Increase in spontaneous action potentials and sensitivity in response to norepinephrine in dorsal root ganglion neurons of adjuvant inflamed rats.

To gain an understanding of the cellular mechanisms of hyperalgesia and spontaneous pain in adjuvant-induced chronic inflammation, we investigated the effects of nerve growth factor (NGF), which is known to increase in inflamed tissues and to cause hyperalgesia, on the spontaneous activities and norepinephrine-induced excitation of dorsal root ganglion (DRG) neurons. Intracellular recordings were obtained from freshly dissociated and cultured DRG neurons (<30 microm) from intact and adjuvant inflamed (AI) rats. Of more than 100 freshly dissociated DRG neurons from the intact rats, none produced spontaneous action potentials, whereas 23% of the neurons from the AI rats did. Spontaneous activities were induced in 34% neurons from intact rats when cultivated for one day with NGF. No neurons from the intact rats responded to norepinephrine (NE), irrespective of whether they were freshly dissociated or cultured with NGF. In contrast, 11% of neurons from the AI rats, both freshly dissociated and cultured without NGF, had a small depolarization in response to NE. The present results suggest that, in AI rats NGF plays an important role in inducing spontaneous activities in DRG neurons, but not in inducing sensitivity to NE.

Nerve growth factor increases sensitivity to bradykinin, mediated through B2 receptors, in capsaicin-sensitive small neurons cultured from rat dorsal root ganglia.

To examine the effects of nerve growth factor (NGF) on the response to bradykinin (BK) of primary afferent neurons, intracellular recordings were obtained from small (< 30 microm) and large (> or = 35 microm) neurons in rat dorsal root ganglia (DRG). The response to BK in the small neurons was tested in 23 freshly dissociated neurons (dissociated group), 37 neurons cultured in the absence of NGF (no-NGF group) and 117 neurons in the presence of NGF (NGF group). Application of BK (10(-7) or 10(-5) M) induced a depolarization in a small number of neurons in the freshly dissociated (13%) and the no-NGF (11%) groups. After cultivation with NGF, the percentage of neurons that were depolarized by BK significantly increased to 46% after 2 days of cultivation. In the NGF group, the percentage of neurons sensitive to BK was significantly greater among capsaicin (CAP)-sensitive than among CAP-insensitive neurons (48 vs 20%). This BK-induced depolarization was completely blocked by a B2 receptor antagonist, but not a B1 receptor antagonist. With large neurons, in contrast, NGF did not increase the percentage that were BK-sensitive (9% in the dissociated group vs 0% after being cultured 2 days with NGF). These results demonstrate that NGF increases sensitivity to BK, mediated through B2 receptors only, in capsaicin-sensitive small neurons cultured from rat DRGs.

Opposite effects of increased intracellular cyclic AMP on the heat and bradykinin responses of canine visceral polymodal receptors in vitro.

To clarify the validity of the long standing hypothesis that effects of E series prostaglandin (PG)S are mediated by cyclic AMP (cAMP), we studied the effects of increases in intracellular cAMP on the heat and bradykinin responses of testicular polymodal receptors. Polymodal receptor activities were recorded in vitro from testis-spermatic nerve preparations excised from dogs anesthetized with pentobarbital (30 mg/kg, i.v.). Increases in intracellular cAMP induced by either forskolin (5 or 10 microM), an adenylyl cyclase activator, or a mixture of dibutyryl cAMP (20-100 microM), a membrane permeable cAMP analog, and 3-isobutyl-1-methyl xanthine (20-100 microM), an inhibitor of the cAMP degrading enzyme, significantly augmented the response to heat (42-48 degrees C). In contrast, these substances failed to facilitate the response to bradykinin (0.1 or 1 microM) and instead suppressed it. Dideoxyforskolin (10 microM), an inactive analog of forskolin, had no effects on both the heat and bradykinin responses. These results demonstrate that an increase in intracellular cAMP induces opposite effects on the heat and bradykinin responses. Possible involvement of intracellular cAMP in the facilitatory effects of PGE2 on both responses was discussed in connection with the PGE receptor subtypes involved in the sensitization of the bradykinin and heat responses.

Coexistence of calcitonin gene-related peptide- and substance P-like immunoreactivity in retrogradely labeled superior spermatic neurons in the dog.

The coexistence of calcitonin gene-related peptide (CGRP) and substance P (SP) was determined in primary afferent neurons of the superior spermatic nerve of the dog. Testicular afferent neurons were visualized by retrograde labeling with the fluorescent dye fast blue. CGRP-like immunoreactivity (LI) was found in about 80% of testicular L1 and L2 dorsal root ganglion cells, and 81% of CGRP-positive neurons also contained SP. Conversely, SP-LI was found in 66% of testicular afferents, and 96% of SP-positive neurons simultaneously contained CGRP. Both CGRP- and SP-LI were observed in the whole size range of the testicular afferent neurons. No significant difference in the diameter was detected between CGRP- and SP-positive testicular afferent neurons. In contrast, the diameter of SP-positive cells was significantly smaller than that of CGRP-positive cells in the whole population of dorsal root ganglion (DRG) neurons. Compared to skin and muscle afferents, a larger population of the testicular afferents contain these peptides. This is considered to be one of the characteristic features of visceral afferents. Coexistence of CGRP and SP in testicular afferent neurons suggest a close functional relationship between these two neuropeptides in the sensory nervous system.

Possible contribution of protein kinase C in the effects of histamine on the visceral nociceptor activities in vitro.

To clarify the possible contribution of protein kinase C activation in histamine-induced excitation and sensitization of the heat response of testicular polymodal receptors, the effects of staurosporine, a protein kinase C inhibitor, and phorbol 12,13-dibutyrate, a protein kinase C activating phorbol ester, were studied in visceral polymodal receptors. Single polymodal receptor activities were recorded in vitro from testis-spermatic nerve preparations obtained from deeply anesthetized dogs (pentobarbital sodium, 30 mg/kg, i.v.). Histamine (10 microM)-induced excitation and facilitation of the heat response of polymodal receptors were both suppressed by staurosporine (1 microM), suggesting that activation of protein kinase C is involved in both these effects of histamine. Application of phorbol 12,13-dibutyrate (0.1 microM) mixed with histamine increased the histamine-induced excitation, whereas a 5 min application of phorbol 12,13-dibutyrate before histamine suppressed it. These results suggest that phorbol 12,13-dibutyrate-activated protein kinase C has inactivation as well as activation effects on the intracellular cascade connected to histamine receptors, and that the former has a slower time course.

Temperature dependency of the chemical responses of the polymodal receptor units in vitro.

Thermal effects on chemical responses of polymodal receptors were studied using in vitro testis-superior spermatic nerve preparations excised from anesthetized dogs. Unitary discharge rates of the polymodal receptors responding to bradykinin and hypertonic NaCl solutions were found to be dependent on the temperature: the higher the temperature, the greater the discharge rate induced, although the temperature itself was mostly subthreshold to activate the receptor.

Abnormal activity of polymodal receptors induced by clioquinol (5-chloro-7-iodo-8-hydroxyquinoline).

To know possible involvement of primary afferents in paresthesia of subacute myelo-optico-neuropathy (SMON), influence on polymodal receptor, a type of nociceptor, of its causal agent clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) was tested using in vitro testis-superior spermatic nerve preparations of dogs. Exposure to clioquinol (greater than 1 microM) induced an abnormal bursting activity in polymodal receptors, and thereafter the receptors were sensitized to an algesic stimulus (hypertonic saline) and gained a new sensitivity to cold, while pure mechanoreceptors were not influenced. On the other hand, clioquinol glucuronide (100 microM), a detoxicated form, had no such effects. These results are consistent with several clinical observations of sensory aberrations, suggesting possible involvement of the abnormal activities of polymodal receptors in SMON paresthesia.

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.

Facilitatory effects of opioids on the discharges of visceral nociceptors.

Effects of opioids on the activity of visceral nociceptors were tested using the in vitro testis-spermatic nerve preparations excised from deeply anesthetized dogs. Morphine, DADLE and dynorphin (10 microM) elicited discharges of polymodal receptors in approximately 1/3 of the tested cases. The incidence of the excitatory response of morphine increased at higher concentrations. The excitatory responses were quite variable among preparations and showed a strong tendency for tachyphylaxis. Similar increases in discharges were elicited when morphine was applied during the steady state of the response evoked by bradykinin (BK) or BK mixed with prostaglandin E2. Pretreatment of morphine for 5 min significantly augmented the subsequent BK responses for 30 min or more. Naloxone per se induced neither excitation nor augmentation of the subsequent BK response, however it reversed the augmenting effect of morphine on BK response. In contrast with previous reports proposing peripheral analgesic effects of opioids, suppressive effects on nociceptors were never observed in the present experiment. Peripheral effects of opioids were discussed.

Endogenous nerve growth factor increases the sensitivity to bradykinin in small dorsal root ganglion neurons of adjuvant inflamed rats.

To examine the cellular mechanisms of hyperalgesia observed in an adjuvant-induced chronic inflammation, the role of nerve growth factor (NGF) in the response to bradykinin (BK) in small neurons from dorsal root ganglia (DRG) was studied via intracellular recordings. After 2 days of cultivation in the absence of NGF, the percentage of neurons from adjuvant-inflamed (AI) rats which were depolarized by BK (53%) was significantly higher than that in neurons from intact rats (13%). This higher percentage in AI rat neurons was significantly reduced after culturing with anti-NGF (17%), but was not influenced by the addition of NGF (57%). The present result demonstrated that sensitivity to BK of DRG neurons from AI rats is increased due to the action of endogenous NGF, suggesting that plastic change in primary afferent neurons caused by NGF may be one of the mechanisms involved in hyperalgesia.

Potentiation and suppression of the histamine response by raising and lowering the temperature in canine visceral polymodal receptors in vitro.

It is well known that itch and inflammatory pain are enhanced when tissue is warmed, while they are suppressed when tissue is cooled. To see whether these changed sensations are based on the changed response of sensory receptors, the temperature dependency of the excitation of polymodal receptors induced by histamine, which plays an important role both in itch and inflammatory pain, was studied. Single nerve activities of polymodal receptors were recorded from canine testis-spermatic nerve preparations in vitro. Raising the temperature from 34 to 40 degrees C, a temperature below the threshold for the heat response of polymodal receptors, facilitated the histamine-induced nerve discharge to 268% of that at 34 degrees C, while lowering the temperature to 28 degrees C decreased it to 25%. Facilitation of the histamine response was also observed in the noxious temperature range (48 and 51 degrees C). These results suggest that the potentiation of the histamine-induced sensation by increasing the tissue temperature, as well as its suppression by lowering tissue temperature, can be explained by a temperature-dependent response of peripheral sensory receptors to histamine. However, the suppression of itch by noxious heat reported by Bickford (Bickford, R.G., Experiments relating to the itch sensation, its peripheral mechanism, and central pathways, Clin. Sci. Incorp. Heart, 3 (1937) 377-386) cannot be explained by the noxious heat-induced facilitation of the peripheral receptor response reported in this paper.

Possible involvement of the EP2 receptor subtype in PGE2-induced enhancement of the heat response of nociceptors.

Prostaglandin E2 augments bradykinin- and heat-induced discharges of polymodal receptors as studied in vitro preparations. Our previous study revealed the involvement of the EP3 receptor subtype in the PGE2 induced enhancement of the BK response [Brain Res. 632 (1993) 321-324]. The agonist for EP2 (butaprost; 10(-8) M) significantly augmented heat responses, but did not augment the BK responses at concentrations from 10(-8) to 10(-5) M; however, the agonist for EP3 (M&B28767) or EP1 (17-phenyl-trinor-PGE2) at 10(-7) M did not affect the heat responses. These findings indicate the involvement of the EP2 receptor subtype in the augmenting effect of PGE2 on heat responses.

Evidence that protein kinase C activation is involved in the excitatory and facilitatory effects of bradykinin on canine visceral nociceptors in vitro.

The involvement of protein kinase (PK) C activation in the effects of bradykinin (BK) on peripheral nociceptors, polymodal receptors, was examined using canine testis-spermatic nerve preparations in vitro. Phorbol 12,13-dibutyrate 0.1 microM, which activates PKC, suppressed the BK-induced excitation when applied for 3-5 min prior to BK application, but facilitated it when applied simultaneously with BK. Neither effect was induced by an inactive phorbol ester, 4alpha-phorbol 12, 13-didecanoate, demonstrating that both effects were mediated through the activation of PKC. In addition, staurosporine 1 microM, a PK inhibitor, suppressed both BK-induced excitation and facilitation of the heat response of testicular polymodal receptors without influencing on-going activities and the heat response itself. These results suggest that PKC activation is involved in the excitatory and facilitatory effects of BK on peripheral nociceptors.