Crucial involvement of actin filaments in celecoxib and morphine analgesia in a model of inflammatory pain

Figures & data

Figure 1 Colchicine and nocodazole potentiate carrageenan-induced hyperalgesia in rat paws.

Notes: Although neither intraplantar colchicine 8 μg administered 60 minutes before intraplantar saline (vehicle) nor intraplantar nocodazole 10 μg administered 60 minutes before intraplantar saline affected the nociceptive thresholds in noninflamed paws, they both increased the duration of hyperalgesia induced by intraplantar carrageenan 250 μg administered at time zero. Nocodazole extended hyperalgesia by just one hour, but colchicine was more effective, with hyperalgesia prolonged to at least 8 hours after carrageenan injection. Data are shown as the mean ± standard error of the mean for five rats in each treatment group. *P < 0.05, significant effect of the cytoskeletal disruptors. The hyperalgesia induced by carrageenan alone was significantly different from basal values for at least 4 hours but has not been marked in the interests of clarity.
Abbreviations: Veh, vehicle; CCC, colchicine; CG, carrageenan; NDZ, nocodazole.

Table 1 Effects of cytoskeletal disruptors, given locally, on carrageenan-induced edema in rat paws

Treatment	Δ paw volume (mL) after carrageenan (CG) injection/time (h)
	0	1	2	3	4	6
CG only	0.02 ± 0.01	0.4 ± 0.1	0.8 ± 0.2	0.7 ± 0.2	0.6 ± 0.2	0.5 ± 0.2
ACD + CG	0.01 ± 0.02	0.4 ± 0.1	0.6 ± 0.2	0.7 ± 0.1	0.5 ± 0.2	0.4 ± 0.2
LAT + CG	0.02 ± 0.02	0.4 ± 0.1	0.8 ± 0.3	0.7 ± 0.2	0.6 ± 0.2	0.3 ± 0.1
CB + CG	0.04 ± 0.01	0.6 ± 0.1	0.8 ± 0.3	0.8 ± 0.3	0.7 ± 0.2	0.4 ± 0.1
NCZ + CG	0.01 ± 0.01	0.4 ± 0.1	0.7 ± 0.2	0.9 ± 0.1*	0.5 ± 0.1	0.4 ± 0.1
CCC + CG	0.04 ± 0.01	0.6 ± 0.2	0.9 ± 0.2*	1.0 ± 0.2*	1.1 ± 0.1*	1.1 ± 0.1*

Notes: Edema was assessed by measuring paw volume (mL) for six hours after carrageenan (CG) with a hydroplethysmometer (Ugo Basile, Italy) and data (means ± SEM) are presented as the difference (Δ) between right and left paw volumes. Carrageenan (250 μg) was injected into the rat paw at time zero and the cytoskeleton disruptors acrylamide (ACD; 10 μg), latrunculin B (LAT; 0.5 μg), cytochalasin B (CB; 1 μg), nocodazole (NCZ; 10 μg) or colchicine (CCC; 2 μg) were given 30 min before the carrageenan (total volume of 50 μL per paw).

* P < 0.05, different from corresponding value with CG only; N = 4–5 animals per group.

Figure 2 Reversal of celecoxib-induced hypoalgesia by nocodazole or colchicine. Celecoxib 12 mg/kg administered systemically 30 minutes before carrageenan prevented development of hyperalgesia after carrageenan and induced hypoalgesia (λ nociceptive threshold values greater than zero). In (A), pretreatment with intraplantar nocodazole 1 μg or 10 μg administered 60 minutes before carrageenan dose-dependently reversed the analgesic effects of celecoxib. Data are shown as the mean ± standard error of the mean for five rats in each treatment group. *P < 0.05, significant effect of nocodazole. Similarly in (B), intraplantar colchicine 0.8 μg or 8 μg administered 60 minutes before carrageenan reversed the effects of celecoxib. Data are shown as the mean ± standard error of the mean for five rats in each treatment group. *P < 0.05, significant effects of colchicine.

Abbreviations: CCC, colchicine; CX, celecoxib; NDZ, nocodazole; Veh, vehicle; CE, carrageenan.

Figure 3 Effects of latrunculin B, cytochalasin B, and acrylamide on celecoxib-induced hypoalgesia. Although intraplantar latrunculin B 0.005–0.5 μg administered 60 minutes before carrageenan did not modify the hyperalgesia induced by carrageenan (data not shown), in (A), it dose-dependently reversed the hypoalgesic effects of celecoxib 12 mg/kg administered systemically 30 minutes before carrageenan. Similarly in (B), intraplantar cytochalasin B 0.001–1 μg administered 60 minutes before carrageenan, which did not affect carrageenan-induced hyperalgesia (data not shown), dose-dependently reversed celecoxib-induced hypoalgesia. However, intraplantar acrylamide 0.1–10 μg administered 60 minutes before carrageenan neither affected carrageenan-induced hyperalgesia (data not shown) nor, as shown in (C), the corresponding celecoxib-induced hypoalgesia.

Notes: Data are shown as the mean ± standard error of the mean for five rats in each treatment group. *P < 0.05, significant effect of cytoskeletal disruptors.
Abbreviations: ACD, acrylamide; CB, cytochalasin B; CCC, colchicine; CG, carrageenan; CX, celecoxib; LB, latrunculin B; Veh, vehicle.

Figure 4 Reversal by cytochalasin B of celecoxib-induced hypoalgesia is dependent on time of treatment.

Notes: In these experiments, intraplantar cytochalasin B 1 μg was administered 30 or 60 minutes after carrageenan, which corresponded to 60 or 90 minutes after celecoxib 12 mg/kg systemically. Given at 30 minutes after carrageenan, cytochalasin B was able to reverse the analgesic effects of celecoxib completely from one hour onwards. Later treatment at 60 minutes was less effective, although the effects of celecoxib were still significantly reduced at 2, 3, and 4 hours after carrageenan. The data are shown as the mean ± standard error of the mean for five rats in each treatment group. *P < 0.05, significant effect of cytochalasin B.
Abbreviations: CG, carrageenan; CX, celecoxib; LB, latrunculin B; Veh, vehicle; CB, cytochalasin B.

Figure 5 Effects of phalloidin on celecoxib-induced hypoalgesia and interactions with cytochalasin B. In (A), a range of intraplantar doses of phalloidin (0.1–10 μg) administered 60 minutes before carrageenan did not modify the hypoalgesia induced by celecoxib 12 mg/kg administered systemically 30 minutes before carrageenan. The data are shown as the mean ± standard error of the mean for five rats in each treatment group. In (B), intraplantar cytochalasin B 1 μg administered 30 minutes after carrageenan (see arrow on time axis) reversed all analgesic effects of celecoxib from 1–4 hours. Pretreatment with intraplantar phalloidin 1 μg administered 60 minutes before carrageenan blocked this reversal by cytochalasin B. The data are shown as the mean ± standard error of the mean for five rats in each treatment group.

Note: *P < 0.05, significant effect of phalloidin.

Abbreviations: CB, cytochalasin B; CG, carrageenan; CX, celecoxib; PD, phalloidin; Veh, vehicle.

Figure 6 Effects of pretreatment with cytochalasin B on the analgesic effects of other COX inhibitors in carrageenan-induced hyperalgesia.

Notes: The results are summarized here as the AUC of the time course over 6 hours with hyperalgesia represented by negative values of AUC and hypoalgesia by positive values. Carrageenan-induced hyperalgesia (first negative bar) was reversed by the nonselective COX inhibitor, indomethacin, administered at 4 mg/kg systemically 30 minutes before carrageenan or by the selective inhibitor of COX-1, SC-560 administered at 5 mg/kg systemically 30 minutes before carrageenan, but only back to basal nociceptive thresholds (AUC about zero). Pretreatment with intraplantar cytochalasin B 1 μg 60 minutes before carrageenan did not affect these analgesic effects. However, the selective COX-2 inhibitor, SC-236 12 mg/kg administered systemically 30 minutes before carrageenan induced clear hypoalgesia (positive AUC) which was completely reversed by the same pretreatment with cytochalasin B. Data are shown as the mean ± standard error of the mean for five rats in each treatment group. *P < 0.05, significant effect of cytochalasin B.
Abbreviations: AUC, area under the concentration-time curve; CB, cytochalasin B; CG, carrageenan; COX, cyclo-oxygenase; Indo, indomethacin; Veh, vehicle.

Figure 7 Modulation of morphine-induced hypoalgesia after carrageenan by colchicine or cytochalasin B. In (A), morphine (M) 2 mg/kg administered systemically 30 minutes before carrageenan (CG) was clearly analgesic, changing the hyperalgesia after carrageenan to hypoalgesia. Pretreatment with two doses of intraplantar colchicine (CCC) 0.8 μg or 8 μg administered at 60 minutes before carrageenan decreased the hypoalgesia but retained the antihyperalgesic effects of morphine. In (B), data from the left paw (without carrageenan) show that hypoalgesia after morphine was not changed by colchicine. Data are shown as the mean ± standard error of the mean for five rats in each treatment group. Similarly, in (C and D), intraplantar cytochalasin B (CB) 1 μg or 10 μg administered 60 minutes before carrageenan partly reversed morphine-induced analgesia to antihyperalgesia in the inflamed right paw, without modifying the effects of morphine in the left noninflamed paw. Data are shown as the mean ± standard error of the mean for five rats in each treatment group.

Notes: ^≤P < 0.05, significant hypoalgesia; *P < 0.05, significant effect of colchicine.
Abbreviations: CB, cytochalasin B; CCC, colchicine; CG, carrageenan; Veh, vehicle.