Dual effects of Rho-kinase inhibitors on a rat model of inflammatory pain

Abstract

Background: Rho-kinases (ROCKs), a family of small GTP-dependent enzymes, are involved in a range of pain models, and their inhibition typically leads to antinociceptive effects.

Objectives: To study the effects of inhibiting ROCKs using two known inhibitors, Y27632 and HA1077 (fasudil), administered locally, on nociception and paw edema in rats.

Methods: A range of doses of Y27632 or HA1077 (2.5 μg to 1000 μg) were injected locally into rat paws alone or in combination with carrageenan, a known proinflammatory stimulus. Nociceptive responses to mechanical stimuli and increased paw volume, reflecting edema formation, were measured at 2 h and 3 h, using a Randall-Selitto apparatus and a hydroplethysmometer, respectively.

Results: Animals treated with either ROCK inhibitor showed biphasic nociceptive effects, with lower doses being associated with pronociceptive, and higher doses with antinociceptive responses. In contrast, a monophasic dose-dependent increase in edema was observed in the same animals. Local injection of 8-bromo-cyclic (c)GMP, an activator of the nitric oxide⁄cGMP⁄protein kinase G pathway, also produced biphasic effects on nociceptive responses in rat paws; however, low doses were antinociceptive and high doses were pronociceptive. Local administration of cytochalasin B, an inhibitor of actin polymerization and a downstream mediator of ROCK activity, reversed the antinociceptive effect of Y27632.

Conclusions: The results of the present study suggest that ROCKs participate in the local mechanisms associated with nociception⁄antinociception and inflammation, with a possible involvement of the nitric oxide⁄cGMP⁄protein kinase G pathway. Also, drug effects following local administration may differ markedly from the effects following systemic administration. Finally, separate treatment of pain and edema may be needed to maximize clinical benefit in inflammatory pain.

Figure 1)

Dual effects of two Rho-kinase (ROCK) inhibitors, Y27632 (A) and HA1077 (B), on nociceptive threshold in rats. Nociceptive thresholds were measured using a pressure algesimeter at 15 min, 30 min, 60 min and 120 min following single injections in rat hind paws. Data were transformed into area under the curve (AUC) over 120 min. Inhibitors were prepared in a solution of dimethyl sulfoxide diluted in iso-tonic saline: vehicle (Veh); Y27632 (Y), 2.5 μg to 1000 μg; HA1077 (HA), 25 μg to 500 μg. ^*Significantly different from vehicle (P<0.01). n=4 to n=5 animals per group

Figure 2)

Dose-response curve of Y27632 (Y) (A) and HA1077 (HA) (B) on paw edema formation. The paw volumes (in mL) were measured using a plethysmometer (Ugo Basile, Italy) at 15 min, 30 min, 60 min and 120 min following single injections in rat hindpaws. Inhibitors were prepared in a solution of dimethyl sulfoxide diluted in isotonic saline: vehicle (Veh); Y27632 (Y), 2.5 μg to 1000 μg; HA1077 (HA), 25 μg to 500 μg. ^*Significantly different from vehicle (P<0.01). n=4 to n=5 animals per group

Figure 3)

Dual effect of a cyclic (c)GMP analogue, 8-bromo-cGMP (8-B-cGMP) (25 μg to 1000 μg), on nociceptive thresholds in rat paws. Low doses of the cGMP analogue (25 μg) induced analgesia and higher doses induced hyperalgesia. ^*Significantly different from vehicle (P<0.01); n=4 animals per group. AUC Area under the curve; Veh Vehicle

Figure 4)

Effects of the combination of Y27632 (Y) (25 μg or 500 μg) with carrageenan (CG) (250 μg) on rat hyperalgesia (A) and paw edema (B). The effects were assessed using a pressure algesimeter and a plethysmometer, respectively. Data were transformed to area under the curve (AUC) over 180 min. ^#Significantly different from CG (vehicle [Veh]-CG); P<0.05. n=5 animals per group

Figure 5)

Reversal of the analgesic effect induced by Y27632 (Y) (25 μg or 500 μg) by cytochalasin B (CB) (1 μg) in rat paws. CB was injected 5 min before administration of a low (25 μg) or a high (500 μg) dose of Y locally. CB and Y were diluted in a solution of dimethyl sulfoxide and isotonic saline (vehicle [Veh]). ^#Significant difference between Veh-Y500 and CB-Y500 (P<0.05). n=3 to n=5 animals per group