INTRODUCTION

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Hyperalgesia to mechanical and thermal intervention is often observed in the skin, joint, muscle, and many visceral organs after inflammation and ischemia. Various kinds of inflammatory mediators appear in inflamed tissues (see Mizumura 1998 for review), and nociceptor sensitization by these mediators is considered to be one of the mechanisms for hyperalgesia in these conditions (Raja et al. 1999). Previous studies into the sensitization of nociceptors have shown that heat (burn) and various inflammatory mediators [bradykinin, prostaglandin (PG) E2 and I2, 5-hydroxytryptamine, histamine, and others] can sensitize nociceptors to heat. Sensitization by inflammatory mediators was found not only in cutaneous nociceptors (Beck and Handwerker 1974a; Campbell and Meyer 1983; Handwerker 1976; Mizumura 1998 for review) but also in visceral polymodal receptors (Mizumura et al. 1991, 1992; Mizumura 1998 for review). Quantitative study on the sensitizing effects of inflammatory mediators was conducted with visceral polymodal receptors and demonstrated clear differences among various inflammatory mediators in the threshold concentration to induce sensitization (Koda et al. 1996; Kumazawa et al. 1991; Mizumura et al. 1987, 1993b). In addition, the intracellular mechanism of sensitization to heat by bradykinin has been largely demonstrated (Cesare et al. 1999; Mizumura et al. 1997).

In contrast, little evidence is available on nociceptor sensitization to mechanical stimuli. A change in sensitivity to mechanical stimulation of cutaneous nociceptors is not evident after burn (Bessou and Perl 1969; Fitzgerald and Lynn 1977). Reeh et al. (1987) reported that prolonged strong mechanical stimulation induced a decrease in mechanical threshold and an increase in receptive field size of A-delta high-threshold mechanoreceptor fibers (but not C-polymodal receptor fibers) in the rat's tail. Inflammatory mediators such as LTB4 (Martin et al. 1988) and a mixture of mediators (Davis et al. 1993) were reported to sensitize cutaneous nociceptors to mechanical stimulation in vivo. With the exception of these reports, however, studies have failed to find such effects either in vivo or in vitro (Khan et al. 1992; Khasar et al. 1993; Lang et al. 1990; Manning et al. 1991), and it is claimed that only protons can reduce the threshold to von Frey hair stimulation in vitro (Steen et al. 1992).

A possibility worth considering is that the von Frey method often used to detect threshold changes is insufficiently sensitive to detect changes induced by inflammation or other tissue injury. Supporting this is a recent report by Andrew and Greenspan (1999) that showed that cutaneous nociceptors were sensitized to suprathreshold mechanical stimuli after inflammation. Similarly, thin fiber joint afferents were sensitized to mechanical stimulation by inflammatory mediators (bradykinin and prostaglandins) (Neugebauer et al. 1989; Schaible and Schmidt 1988). In visceral organs, sensitization (or recruitment) of silent nociceptors to mechanical stimulation in organs inflamed with mustard oil, turpentine (Habler et al. 1993) or acetic acid (Gebhart 1996) has been reported. Sensitization by inflammatory mediators was reported only when a mixture of inflammatory mediators was used (inflammatory soup) (Su and Gebhart 1998).

To determine which inflammatory mediators sensitize nociceptors to mechanical stimulation and how they modulate the nociceptor response to mechanical stimulation, it would seem to be necessary to have a quantitative understanding of the effect of each individual inflammatory mediator. However, no such data are currently available. In the present study, therefore we attempted to quantitatively clarify the sensitizing effects of four inflammatory mediators (bradykinin, PGE2, histamine, and protons) on visceral nociceptor response to suprathreshold mechanical stimulation and then compare the potencies of these effects. Potencies are also compared with that of thermal injury in inducing sensitization to mechanical stimulation. Finally, the effects of possible intracellular mediators of inflammatory mediators were examined.

Preliminary accounts have appeared elsewhere (Mizumura and Koda 2000).

	METHODS

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Single-fiber recording

Single polymodal receptor activities were recorded as described previously (Kumazawa et al. 1987) in testis-spermatic nerve preparations in vitro. Twenty-five adult male beagle and mongrel dogs (1-3 yr old, 10-14 kg) were used. The bilateral testis and epididymis were excised with the spermatic cord attached under deep anesthesia with pentobarbital sodium (30 mg/kg iv). After removal of the preparation, animals were killed with an overdose of anesthetics.

The testis and epididymis were placed in a bath in which modified Krebs-Henseleit solution [which contained (in mM) 110.9 NaCl, 4.8 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 24.4 NaHCO₃, and 20 glucose] was circulated. The spermatic cord was maintained in an oil bath where single-fiber activities were recorded from the spermatic nerve. The polymodal receptors were identified by their responses to mechanical stimulation and sensitivity to heat. Conduction velocity was measured using a conventional method, measuring the latency of action potential arrival after stimulation of the spermatic nerve. The measured conduction velocity was corrected to the value at 37°C based on the report by Paintal (1965). The temperature of the Krebs solution bathing the receptive field was continuously monitored with a thermocouple and kept at 34 ± 0.5°C.

Action potentials were counted after discrimination with a window-type discriminator. The clock of the spike counter was synchronized with the mechanical stimulator described in the next section.

Mechanical stimulation

Mechanical stimulation was applied by means of a servo-controlled linear motor mechanical stimulator with a long stroke (Dia Medical, Tokyo). To allow perpendicular contact of the mechanical stimulator with the receptive field, polymodal receptors with a receptive field near the center of the testis were chosen. The tip diameter of the probe was 3 mm. The stimulation pattern was a ramp-and-hold type, in which the duration of the ramp was 1 s and the hold time was 9 s. Mechanical threshold was determined as the strength that induced an increase in mean discharge rate during the 10-s stimulation period of 0.2 imp/s (namely, 2 imp/stimulus), by calculating the regression line using the responses to several stimulus strengths. Stimulus strength for testing the sensitizing effects of inflammatory mediators was chosen so that an increase of 0.5-1 imp/s during whole stimulation period was obtained. Before examining the effects of drugs or heat, at least three stimulations were applied, and the mean of the last two responses was used as the control. The mechanical response was calculated as follows: (MDR during the mechanical stimulation period)  (MDR during 30 s before mechanical stimulation), where MDR is the mean discharge rate (per second) during a certain period.

Drug application

Stock solutions of prostaglandin (PG) E2, forskolin, and phorbol 12, 13-dibutyrate (PDBu) were prepared by dissolving these compounds in ethanol (PGE2 and forskolin) or dimethyl sulfoxide (PDBu) at concentrations of 10 mM. They were kept deep frozen and then diluted to the final concentration (PGE2, 0.1-10 µM; forskolin, 10 µM; and PDBu, 0.1 µM) with Krebs solution shortly before usage. Bradykinin (0.001-1 µM) and histamine (histamine hydrochloride, 1-100 µM) were dissolved in Krebs solution on the day of the experiment. Solutions were prewarmed at 34°C. All agents except bradykinin (from Peptide Institute, Minoh-shi, Osaka, Japan) were obtained from Sigma (St. Louis, MO).

Low pH solutions (pH: 4.5 or 4.8, 5.5 or 5.9, 6.5 or 6.6) were made by adding either acetic acid (maximum 0.22%) to Krebs solution or 0.1 M phosphate buffer (pH: 4.5, 5.5, or 6.5) to Krebs solution without NaHCO₃ (mixing ratio, 1 phosphate buffer:9 Krebs solution). pH was finally adjusted by adding a small amount of either 1 N NaOH or HCl solution.

These drugs were applied during the period starting 5 min before mechanical stimulation until it was ended, then washed away. Excitatory effects were represented by mean discharge rate (MDR) during a 5-min period of application. As histamine- and bradykinin-induced discharges usually decrease slowly (Koda et al. 1996; Mizumura et al. 1990), it is assumed that the same level MDR as that during the 30 s before mechanical stimulation continued during the mechanical stimulation period. The mechanical response after drug application, therefore was calculated by subtracting the MDR during the last 30 s of drug application (that is, MDR for 30 s before mechanical stimulation) from the MDR during the mechanical stimulation. The sensitizing effects were represented by changes in the mechanical response after drug application calculated in this way from the control mechanical response. Different concentrations of a certain mediator or different mediators were applied at intervals of 30 min and only when the mechanical response had returned to near the level before the treatment.

55°C heat stimulation

Heat stimulation was applied by continuously superfusing prewarmed (55°C) Krebs solution for 30 s. The sensitizing effect of heat on the following mechanical response was examined 5 and 15 min after heat stimulation. This series of tests was usually carried out at the end of the experiment, and no further drugs were examined after these tests.

Statistics

Data were presented as means ± SE. Statistical analysis for the effects of mediators and others on mechanical response was conducted using one-way ANOVA with repeated measures followed by Bonferroni's multiple comparison test (for testing reproducibility of mechanical response) and Dunnett's test (for drug effects on the mechanical response). The Friedman test followed by Dunn's multiple comparison test was used for the sensitizing effect of 55°C heat stimulation. Drug-induced excitation was analyzed by paired t-test. P < 0.05 was considered to be a significant change.

All the experimental procedures were approved by the Committee for Animal Experiments, Research Institute of Environmental Medicine, Nagoya University.

	RESULTS

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General

A total of 50 units were examined. The conduction velocity ranged 0.6-9.2 m/s with a median of 3.9 m/s (mean, 4.0 ± 0.26 m/s). The mechanical threshold ranged between 49 and 635 mN (mode, 145 mN). Spontaneous activities were observed for 60-120 s before any mechanical stimulations except for search stimuli. In 29 of 50 units studied, low-level spontaneous activities varying between 0.02 and 0.88 imp/s were observed; namely, 15 units had a discharge rate <0.1 imp/s and only 2 units had a discharge rate >0.5 imp/s.

Characteristics of mechanical response

STIMULUS-RESPONSE RELATIONSHIP. The stimulus response relationship (784 mN) was examined in eight units. As seen in the sample recording shown in Fig. 1A, discharges were induced only during the early part of the stimulation period when the stimulus strength was low. As the stimulus strength increased, the period during which the receptor was discharging became longer (see response to 784 mN in Fig. 1A for example) and peak discharge rate increased. The peak discharge rate was usually observed at the beginning of the hold phase of the stimulation. We applied stimulation only 784 mN, and in this range, the stimulus-response relation was roughly linear (Fig. 1B), although the slope of the fitted line (not shown) varied. As will be shown, a stronger stimulation (for example, 588 mN) tended to weaken the subsequent responses, so the stimulus-response relation must have been slightly compromised.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Stimulus-response relationship. A: sample recording. Peristimulus time histograms (1 bin is 1 s) are shown in the order of testing. Mechanical stimulation was applied during the time period marked with a line, and the stimulus strength was presented in multiples of 98 mN. One minute elapsed between applications of stimuli with the 2 different strengths; //, recording was discontinued for a certain time at this point. · · ·, the 0 level of the discharge rate. B: stimulus-response relationship of 8 units studied. Ordinate: net increase in discharge rate during 10 s of mechanical stimulation; abscissa: stimulus strength in multiples of 98 mN. Each represents the response of a different receptor.

REPRODUCIBILITY. To examine whether the response to mechanical stimulation was reproducible, stimulation of the same strength was repetitively applied at 1 min intervals to seven (for 196 mN stimulation) and six (for 588 mN stimulation) polymodal receptors. When 196 mN stimulation was applied, the response tended to decrease slightly on repetition; specifically, the first response was 1.19 ± 0.28 imp/s, while the fifth response was 0.85 ± 0.32 imp/s (Fig. 2), but this change was not statistically significant (P > 0.2 with 1-way ANOVA). In contrast, the 588 mN stimulation induced a larger response (5.71 ± 1.34 imp/s) at the first challenge, but the response clearly decreased on repetitive stimulations (P < 0.001 with 1-way ANOVA). The responses to the fourth and fifth stimulations were significantly different from the first response (Fig. 2, P < 0.01, post hoc analysis with Bonferroni's multiple comparison test). To avoid this change in response, the stimulus strength used in the following experiments never exceeded 392 mN, well below 588 mN.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Change in the mechanical response on repeated stimulation. Ordinate: net increase of mean discharge rate induced by mechanical stimulation. Abscissa: order of testing. , response to 196 mN stimulation; , response to 588 mN stimulation. Data are shown as means ± SE. There was a significant change in the response to 588 mN stimulation (P < 0.001, 1-way ANOVA with repeated measures, n = 7), but no change in the response to 196 mN stimulation (P > 0.2, 1-way ANOVA with repeated measures, n = 7). **P < 0.01 when compared with the 1st response (Bonferroni's multiple comparison test).

Effects of bradykinin

Bradykinin excited polymodal receptors as previously reported (Kumazawa et al. 1991) and facilitated the mechanical response. In the receptor shown in Fig. 3A, bradykinin at 0.001 µM neither excited the receptor nor sensitized it to the mechanical stimulation (data not shown), while bradykinin at 0.01 and 0.1 µM excited the receptor and clearly sensitized it to mechanical stimulation. Discharges tended to continue during the mechanical stimulation period after bradykinin application. With an average of seven units for each concentration, significant sensitization was observed at 0.01 µM. A clear concentration dependency could not be determined. It must be mentioned that both excitation by bradykinin alone and sensitization to mechanical stimulation were observed in the same concentration range (Fig. 3B).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Bradykinin excited the testicular polymodal receptor and facilitated its mechanical response at 0.01 µM. A: a sample recording. Peristimulus time histograms are shown according to the order of testing. Time after bradykinin application is shown at the top left of the histogram. P: mechanical stimulation with 196 mN. *, the recording is continuous at this point. The period during which bradykinin was applied is marked with a shaded bar. The conduction velocity of this unit was 4.4 m/s. Bradykinin both at 0.01 and 0.1 µM clearly facilitated the subsequent mechanical response, but facilitation by bradykinin at 0.1 µM was smaller in this case. B: summary of sensitizing effect () and excitation induced by bradykinin () of different concentrations. *P < 0.05, **P < 0.01 compared with the control mechanical response ( mech. response, Dunnett's test) or discharge rate during the control period (bradykinin-induced discharge, paired t-test). The stimulus strength used was between 147 and 392 mN, n = 7. There was no significant difference among the control responses to mechanical stimulation before bradykinin application (0.78 ± 0.17 imp/s for 0.001 µM, 0.85 ± 0.09 imp/s for 0.01 µM, and 1.05 ± 0.15 imp/s for 0.1 µM, P > 0.37 with 1-way ANOVA).

The sensitizing effect of bradykinin in two receptors lasted 15-35 min after bradykinin was washed away (1 example in Fig. 3A), but in other instances it disappeared quickly, in just 5 min. On average no significant increase in mechanical response was seen 5 min after washing bradykinin away (P > 0.05, Dunnett's test).

Effects of histamine

The previous results showed that polymodal receptor units with conduction velocities slower than 10 m/s were more sensitive to histamine and that the majority of them were excited by histamine 10 µM (Koda et al. 1996). Therefore in the present study we examined the effects of histamine on these more slowly conducting receptors only.

Confirming a previous result (Koda et al. 1996), in this experiment histamine at 1 µM was ineffective in inducing excitation, whereas at 10 µM or higher it induced excitation (sample in Fig. 4A and summary in Fig. 4B, ). The mechanical response was facilitated at the same concentration range as that for excitation. It must be noted that the discharge pattern was different after histamine (100 µM) application; as shown in Fig. 4A, substantial discharges were seen in the later half of the mechanical stimulation period after histamine application. 89 ± 9% of the total discharges induced were seen in the first half of the stimulation period in the control mechanical response, while only 60 ± 9% were seen after histamine application. These values were significantly different (P < 0.03, Wilcoxon signed-rank test). On average, a significant increase in the mechanical response was observed at 10 µM (P < 0.05 and 0.01 for 10 and 100 µM, respectively, compared with the control mechanical response; Dunnett's test). A clear concentration-dependency in sensitizing effects was observed (Fig. 4B, P < 0.05 with linear regression analysis). Five minutes after washing away the histamine, a significant facilitatory effect on the mechanical response was observed only with 100 µM.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Histamine excited the polymodal receptor and facilitated its mechanical response in a concentration-dependent manner. A: a sample recording. The presentation is similar to that in Fig. 3. P: mechanical stimulation with 294 mN. The conduction velocity of this unit was 3.7 m/s. Note that histamine 100 µM induced greater facilitation than 10 µM, and the mechanical response 5' after rinsing histamine away was still facilitated. B: summary of sensitizing effect () and excitation induced by bradykinin () of different concentrations. The stimulus strength used was between 98 and 392 mN. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control mechanical response ( mechanical response, Dunnett's test) or the mean discharge rate during the control period (histamine-induced excitation, paired t-test), n = 6. It was confirmed that there was no difference in the control responses (1.02 ± 0.19 imp/s for 1 µM, 0.62 ± 0.11 imp/s for 10 µM, and 0.90 ± 0.19 imp/s, P > 0.24 with 1-way ANOVA). Clear concentration-dependent sensitization was observed.

Effects of prostaglandin E2

Prostaglandin (PG) E2 rarely induced excitation in the testicular polymodal receptors (change in discharge rate by PGE2 itself was 0.00 ± 0.04, 0.02 ± 0.01, and 0.14 ± 0.06 imp/s for 0.1, 1, and 10 µM PGE2, respectively, not a significant increase), confirming our previous findings (Mizumura et al. 1987). Despite the absence of excitation by PGE2 alone, it sensitized the polymodal receptors to mechanical stimuli. One sample recording is shown in Fig. 5A, and the differences of the responses before and after PG application are illustrated in Fig. 5B. There does not appear to be any difference in discharge pattern (Fig. 5A). PGE2 (0.1 µM) slightly increased the subsequent mechanical response, but it was not statistically significant. Significant facilitation was observed at the concentration of 1 and 10 µM (Fig. 5B). Note that the magnitude of the induced sensitization tended to be smaller than that induced by bradykinin or histamine (see preceding text).

View larger version (14K): [in this window] [in a new window]   Fig. 5. PGE2 facilitated the following mechanical response without exciting the polymodal receptor by itself. A: a sample recording. The presentation is similar to that in Fig. 3. The stimulus strength used was 196 mN, and the conduction velocity of this unit was 5.2 m/s. Note that facilitation of the mechanical response was induced without excitation by PGE2 alone. B: summary of sensitizing effect. The stimulus strength used was between 98 and 392 mN. **P < 0.01 compared with the control mechanical response (Dunnett's test, n = 6 for PGE2 1 µM, n = 7 for 0.1 and 10 µM). It was confirmed that there was no difference in the control response in these cases (0.83 ± 0.05 imp/s for 0.1 µM, 0.85 ± 0.17 imp/s for 1 µM, and 0.72 ± 0.12 imp/s for 10 µM, P > 0.72 with 1-way ANOVA).

Effects of phorbol ester and forskolin

We examined the effects of two possible second messengers, cAMP and protein kinase C (PKC). The former is thought to be involved in the action of PGE2 and the latter in the actions of histamine and bradykinin. The effect of increasing intracellular cAMP was studied using forskolin (10 µM), which activates adenylyl cyclase. Forskolin by itself did not induce a significant change in the discharge rate (0.09 ± 0.04 imp/s, not significantly different from that of the control period, 0.08 ± 0.05 imp/s), whereas it facilitated the mechanical response. One sample recording is shown in Fig. 6A. This facilitatory effect disappeared 5 min later. The response pattern was not different after treatment with forskolin (Fig. 6A), as most of the discharges appeared in the early part of the stimulation period. On average, the mechanical response was facilitated from 1.35 ± 0.16 imp/s (control response) to 2.41 ± 0.20 imp/s (response after forskolin application, P < 0.001 compared with the control mechanical response, Dunnett's test, n = 7, Fig. 6B). The facilitatory effect disappeared 5 min later (Fig. 6, A and B; P > 0.05, Dunnett's test).

View larger version (12K): [in this window] [in a new window]   Fig. 6. Forskolin facilitated the mechanical response reversibly without inducing excitation by itself. A: a sample recording. P, 147 mN. Conduction velocity of this receptor was 4.4 m/s. B: summary. Cont, mechanical response before forskolin application; Fo, response after 5 min forskolin application; aft, response 5 min after washing forskolin away. The stimulus strength used was between 98 and 392 mN. ***P < 0.001 compared with the control response (Dunnett's test, n = 7). The facilitatory effect of forskolin did not last long.

A PKC-activating phorbol ester, PDBu (0.1 µM), facilitated the mechanical response. The response pattern was not modified by PDBu (Fig. 7A). This effect was reversible, disappearing 5 min after washing PDBu away. Again, PDBu at this concentration did not induce any significant excitation by itself (1 sample in Fig. 7A, average change 0.03 ± 0.05 imp/s). On average the mechanical response was increased by PDBu from 1.06 ± 0.19 imp/s (control response) to 2.10 ± 0.33 imp/s (after application; P < 0.001, Dunnett's test, n = 7, Fig. 7B).

View larger version (11K): [in this window] [in a new window]   Fig. 7. Phorbol 12,13-dibutyrate facilitated the mechanical response. A: a sample recording. P, 147 mN. Conduction velocity of this receptor was 3.5 m/s. B: summary. The method of presentation is similar to Fig. 6B. The stimulus strength used was between 98 and 392 mN. ***P < 0.001 compared with the control response (Dunnett's test, n = 7). The facilitatory effect of PDBu was reversible.

Effects of protons

A total of 19 polymodal receptors were examined. Seven units were examined with low-pH Krebs solution acidified with acetic acid 4.8, 2 units 5.5, and one only 6.6. None were excited by low-pH solution. The mechanical response was also not sensitized after 5 min application of low-pH solution (data not shown).

Another nine testicular polymodal receptors were examined with low-pH solutions acidified with low-pH phosphate buffer 4.5. Three of them were excited (0.23-1.11 imp/s, net increase in discharge rate during 5 min of application period): one at pH 6.5 only, one at  pH 6.5, and the remaining one at pH 4.5 only. In these cases, mechanical stimulation was applied twice, 1 and 5 min after starting application of low-pH solution, so that a short-lasting effect could be detected. The response pattern to low-pH solution varied among these receptors: one unit showed a transient type response with rapid onset, one a slowly increasing discharges. In the third unit, which had the greatest response to low-pH solution, mechanical stimulation applied 1 min after commencement of low-pH application apparently triggered continuous excitation, which was observed long after the first mechanical stimulation ended. We failed to detect any sensitizing effects of low-pH solutions up to pH 4.5 applied for 1 or 5 min (data with 1 min application are summarized in Fig. 8), even in the receptor that showed the greatest excitation. The response tended to be rather depressed, although it was not significantly different from the control (Fig. 8, P > 0.3 for all pH, paired t-test).

View larger version (13K): [in this window] [in a new window]   Fig. 8. Protons failed to facilitate the subsequent mechanical response. Each symbol represents a different unit. The difference from the control response (pre) is plotted. The stimulus strength used was between 98 and 392 mN.

Effects of heat (55°C)

In a previous study, we reported that 55°C, 30-s stimulation induced a great increase in discharges by itself and consistently sensitized the subsequent heat responses 3 h in vitro (Mizumura et al. 1992). Therefore we used the same stimulus condition in this experiment. Figure 9A shows one sample of burn-induced sensitization to mechanical stimulation. In this case 55°C, 30-s stimulation induced a vigorous response, and thereafter the mechanical response was clearly, though not vigorously, facilitated. The response pattern was somewhat modified after heat stimulation (Fig. 9A), and fewer discharges were seen in the first half of the mechanical stimulation period (the percentage of discharges observed in the first 5 s of mechanical stimulation was 83 ± 4% before heat vs. 56 ± 10% after heat, for a significant difference, P < 0.04, Wilcoxon signed-rank test). With an average of eight units, this heat stimulation induced an increase of 6.58 ± 1.43 imp/s in the discharge rate and the mechanical response was increased from 0.99 ± 0.11 imp/s (control response) to 1.86 ± 0.23 imp/s (5 min after 55°C stimulation). This increase was statistically significant (P < 0.05, n = 8, Dunn's test, Fig. 9B). The response to mechanical stimulation applied again 10 min later was closer to the level before the 55°C stimulation in four of eight units, but the average response was still significantly greater than the control response (P < 0.05, Dunn's test).

View larger version (12K): [in this window] [in a new window]   Fig. 9. Heat stimulation at 55°C facilitated the subsequent response to mechanical stimulation. A: a sample recording. P, 294 mN. Conduction velocity of this receptor was 3.0 m/s. , period that 55°C stimulation (heat) was applied. B: summary of sensitizing effect. The stimulus strength used was between 98 and 392 mN. The mechanical responses 5 and 15 min after 55°C stimulation (5' aft and 15' aft) were significantly greater than the control response *P < 0.05 compared with the control response (Dunn's test, n = 8).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

One of the major findings of the present study is that bradykinin, histamine, and PGE2 each sensitized testicular polymodal receptors to mechanical stimulation. The presently observed magnitude of sensitization in the mechanical response was not much different from that in previous reports for the heat response in testicular nociceptors (Koda et al. 1996; Kumazawa et al. 1991; Mizumura et al. 1993b). In contrast, this magnitude of sensitization in the mechanical response was much smaller than that observed in pelvic afferents with a mixture of inflammatory mediators (inflammatory soup) (Su and Gebhart 1998). A mixture of inflammatory mediators may not always produce more pronounced sensitization than a single mediator in the testicular nociceptors because inhibitory interaction between mediators also exists (Mizumura et al. 1995). Sensitization to mechanical intervention of nociceptors located in the hollow viscera (e.g., colon) might be exaggerated by additional contraction of smooth muscles directly induced by inflammatory mediators and indirectly induced through reflex by nociceptor activation.

Sensitizing mechanism by inflammatory mediators

The concentration of bradykinin needed for sensitization to mechanical stimulation was the same as that needed to excite testicular polymodal receptors. Histamine also excited nociceptors and sensitized them at the same concentration. This observation might suggest that sensitization to mechanical stimulation is induced indirectly through depolarization of the receptor terminal by bradykinin or histamine. On the other hand, there is accumulating evidence that excitation and sensitization to heat by bradykinin is mediated by activation of PKC (Cesare et al. 1999; Leng et al. 1996; Mizumura et al. 1997); we have also demonstrated the possibility that PKC activation is involved in the histamine-induced excitation and sensitization to heat (Mizumura et al. 2000). Our present observation that PKC-activating phorbol ester sensitized nociceptors to mechanical stimulation may be explained by direct modulation by PKC of mechanotransducer(s) yet to be identified. The concentration of bradykinin needed to sensitize to mechanical stimulation was 100 times higher than that needed to sensitize nociceptors to heat (Kumazawa et al. 1991). This difference in the effective concentration needed for sensitization to mechanical and thermal stimulations might be due to the difference in sensitivity of the mechanotransducer(s) to PKC-induced modulation.

cAMP, which has long been considered the second messenger of PG receptors (Ferreira and Nakamura 1979), facilitated both heat (Mizumura et al. 1993a) and mechanical responses (present experiment). A lowered activation threshold and increased rate of activation, plus inactivation and deactivation of TTX-resistant Na channels through activation of protein kinase A have been suggested as a cAMP-mediated sensitizing mechanism (England et al. 1996; Gold et al. 1996, 1998). Such a mechanism might underlie the sensitization to mechanical stimuli by PGE2 as well; this would also explain sensitization without excitation by PGE2 itself. Alternatively, the sensitizing effect of PGE2 might be mediated by direct phosphorylation of mechanotransducer(s) by protein kinase A.

Protons

We failed to find significant excitation of nociceptors or facilitatory effects on the mechanical response by protons alone. This result contradicts the findings in skin-nerve in vitro preparations by Steen et al. (1992). They reported that nociceptors were excited by protons and the mechanical threshold, as measured by von Frey hairs, decreased. These differences might have resulted from the use of different animal species or tissues or from the fact that we did not examine the threshold change of the mechanical response. However, our present result agrees with the results for rat colon polymodal receptors reported by Su and Gebhart (1998). Colonic polymodal receptors were not excited by infusion of low-pH solution into the luminal cavity nor were their responses to distension of the colon facilitated by low pH. In this case, however, protons may not have been able to pass the mucosal barrier to reach the nociceptors.

Sensitization by burn

We also demonstrated in the present study that exposure to 55°C heat sensitized testicular polymodal receptors to mechanical stimulation. A comparison of this sensitization with sensitization to heat after the same 55°C heat exposure revealed that the magnitude of the mechanical response sensitization was smaller, increasing from 0.99 ± 0.11 to 1.86 ± 0.23 imp/s (present data), against an increase from 1.08 ± 0.16 imp/s (control response) to 4.88 ± 0.62 imp/s (response after 55°C exposure, n = 24) in response to 48°C stimulation (Mizumura et al. 1992). This is surprising, because bradykinin, PGE2, and histamine all sensitized polymodal receptors to mechanical stimulation with magnitudes not much different from those to heat (Koda et al. 1996; Kumazawa et al. 1991; Mizumura et al. 1993b). One possible reason for this is that the number of receptive points was limited in the mechanical stimulation used in this experiment, while all the receptive points were stimulated in our previous studies on the heat response. However, this observation is compatible with previous observations in that there is no evident change in sensitivity to mechanical stimulation of cutaneous nociceptors after burn (Bessou and Perl 1969; Campbell and Meyer 1983; Fitzgerald and Lynn 1977). Possible reasons for this difference between sensitization to heat and mechanical stimulations by burn include the following. 1) Inflammatory mediators sensitized heat as well as mechanical transducers. In addition, heat and/or mediators might have influenced parts of the spike generation mechanism, such as voltage-gated Na channels (Gold et al. 1996, 1998), which are common to heat and mechanical responses. 2) Heat transducing ion channels might have been sensitized by heat, while some mechanotransducing ion-channel that is yet to be identified might have been desensitized. Heat-transducing ion channels VR-1 and VRL-1 were shown to change sensitivity to heat after heat stimulation (Caterina et al. 1999). The slowly adapting, low-threshold mechanoreceptors in the primate reportedly have a reduced response to mechanical stimuli after a burn to their receptive field (Beck and Handwerker 1974b), so the mechanical transducer of nociceptors might also be desensitized after burn. Thus a weaker sensitization to mechanical stimulus would result as a sum of these effects. Alternatively, supposing that bradykinin played a major role in sensitizing nociceptors after burn, a weaker sensitization of the mechanical response would result because a higher concentration of bradykinin would be needed to sensitize to mechanical stimulation. It is interesting that the response pattern to mechanical stimulation changed after both burn and treatment with histamine so that a larger proportion of discharges was seen in the latter half of the stimulation period. This might suggest that histamine plays an important role in sensitization to mechanical stimulation after burn, although it could also be simply coincidence.

Significance of the present observation

This is the first report to demonstrate that visceral nociceptors are sensitized to mechanical stimulation by individual inflammatory mediators as well as by burn, something that has long been suspected from mechanical hyperalgesia in inflamed tissues. Before this, mechanical hyperalgesia in visceral organs was explained by observations of sensitization of nociceptors to heat (Mizumura 1998), by the recruitment of silent nociceptors (Habler et al. 1990), or by increased sensitivity of spinal nociceptive neurons (see Mayer and Gebhart 1994 for review). The present report provides evidence that sensitization of visceral nociceptors that are not silent in normal conditions does occur with treatment of inflammatory mediators or burn. This study may serve as a basis for future exploration of mechanism of sensitization to mechanical stimulation.