Systemic delivery of selective EP1 and EP3 receptor antagonists attenuates pentylenetetrazole-induced seizures in mice

Abstract

Neuroinflammation plays a major role in brain excitability and may contribute to the development of epilepsy. Prostaglandin E₂ (PGE₂) is a direct mediator of inflammatory responses and, through EP receptors, plays an important role in neuronal excitability. Pharmacological evidence supports that centrally-administered EP1 and EP3 receptor antagonists reduced acutely evoked seizures in rats. Translation of these findings would benefit from evidence of efficacy with a more clinically relevant route of delivery and validation in another species. In the current study we investigated whether the systemic administration of EP1 and EP3 agonists and antagonists modulate pentylenetetrazole (PTZ)-induced seizures in mice. In addition, it was examined whether these compounds alter Na⁺, K⁺-ATPase activity, an enzyme responsible for the homeostatic ionic equilibrium and, consequently, for the resting membrane potential in neurons. While the systemic administration of EP1 and EP3 antagonists (ONO-8713 and ONO-AE3-240, respectively) attenuated, the respective agonists (ONO-DI-004 and ONO-AE-248) potentiated PTZ-induced seizures (all compounds injected at the dose of 10 µg/kg, s.c., 30 min before PTZ challenge). Co-administration of either EP1 or EP3 agonist with the respective antagonists nullified the anticonvulsant effects of EP1/3 receptor blockade. In addition, EP1 and EP3 agonists exacerbated PTZ-induced decrease of Na⁺, K⁺-ATPase activity in both cerebral cortex and hippocampus, whereas, EP1 and EP3 antagonists prevented PTZ-induced decrease of Na⁺, K⁺-ATPase activity in both structures. Our findings support and extend evidence that EP1 and EP3 receptors may be novel targets for the development of anticonvulsant drugs.

Keywords: EP1 receptor; EP3 receptor; Epilepsy; pentylenetetrazole; prostaglandin E2.

Figure 1

(A-C) Effect of EP1 receptor agonist and antagonist (ONO-DI-004 and ONO-8713, respectively; 10 µg/kg, s.c.) on PTZ-induced seizures (60 mg/kg, i.p.). (A) Latency to myoclonic jerks. (B) Latency to tonic-clonic generalized seizure. (C) Total time spent in generalized seizures. Data expressed as median and interquartile range (A and B), and mean + SEM (C), for n = 6-12 in each experimental group. (D, E) Effect of EP1 receptor agonist (D) and antagonist (E) (ONO-DI-004 and ONO-8713, respectively; 10 µg/kg, s.c.) on the mean amplitude of EEG recordings in the cortex of animal injected with PTZ (60 mg/kg, i.p.). Mean amplitude of EEG recordings was analyzed by two-way ANOVA followed by the Bonferroni’s test and expressed as mean + S.E.M, for n = 6-12 in each experimental group. (F-H) Representative electrocorticographic recordings of animals after PTZ injection are represented as follows: (F) vehicle, (G) ONO-DI-004, and (H) ONO-8713. Black and white arrowheads indicate PTZ injection and seizures latency, respectively, and the y-axis (amplitude) and x-axis (time) calibration bar is the same for all traces. (I) Effect of ONO-DI-004 (3 µg/kg, s.c.) administration, followed or not by ONO-8713 (10 µg/kg, s.c.), on PTZ-induced seizures, measured as latency to tonic-clonic seizure. ONO-DI-004 (3 µg/kg, s.c.) prevented the protective effect of ONO-8713 (10 µg/kg, s.c.) against PTZ-induced seizures. Data expressed as median and interquartile range for n = 3-7 in each group. A probability of P < 0.05 was considered significant. *P < 0.05, and **P < 0.01, when compared with vehicle or vehicle + vehicle group.

Figure 2

(A-D) EP1 receptors ligand effects on Na⁺, K⁺-ATPase activity in the brain. ONO-DI-004 (10 µg/kg, s.c.) increases (A) and ONO-8713 (10 µg/kg, s.c.) prevents (B) PTZ-induced decrease of Na⁺, K⁺-ATPase activity in cerebral cortex and hippocampus homogenates in mice. Data are presented in nmol Pi/mg protein/min, as mean + S.E.M., for n = 5 in each group. Data were analyzed by a two-way ANOVA followed by the Bonferroni’s test. (C) ONO-DI-004 (3 µg/kg, s.c.) does not present per se effect on Na⁺, K⁺-ATPase (P > 0.05 by Student’s t-test, for n = 5). (D) Two-way ANOVA followed by the Bonferroni’s test revealed that ONO-DI-004 (3 µg/kg, s.c.) accentuated PTZ-induced decrease of Na⁺, K⁺-ATPase activity and prevented ONO-8713 (10 µg/kg, s.c.) effect on this enzyme activity in cerebral cortex and hippocampus homogenates in mice. Data are presented in nmol Pi/mg protein/min, as mean + S.E.M., for n = 5 in each experimental group. *P < 0.05, when compared with the respective control group; #P < 0.05, when compared with vehicle group.

Figure 3

(A-C) Effect of EP3 receptor agonist and antagonist (ONO-AE-248 and ONO-AE3-240, respectively; 10 µg/kg, s.c.) on PTZ-induced seizures (60 mg/kg, i.p.). (A) Latency to myoclonic jerk. (B) Latency to tonic-clonic generalized seizure. (C) Total time spent in generalized seizures. Data expressed as median and interquartile range (A and B), and mean + SEM (C), for n = 8 in each experimental group. (D, E) Effect of EP3 receptor agonist (D) and antagonist (E) (ONO-AE-248 and ONO-AE3-240, respectively; 10 µg/kg, s.c.) on the mean amplitude of EEG recordings in the parietal cortex of animal injected with PTZ (60 mg/kg, i.p.). Mean amplitude of EEG recordings was analyzed by two-way ANOVA followed by the Bonferroni’s test and expressed as mean + S.E.M., for n = 8 in each group. (F-H) Representative electrocorticographic recordings of animals after PTZ injection are represented as follows: (F) vehicle, (G) ONO-AE-248, and (H) ONO-AE3-240. Black and white arrowheads indicate PTZ injection and seizures latency, respectively, and the y-axis (amplitude) and x-axis (time) calibration bar is the same for all traces. (I) Effect of ONO-AE-248 (3 µg/kg, s.c.) administration, followed or not by ONO-AE3-240 (10 µg/kg, s.c.), on PTZ-induced seizures, measured as latency to the first tonic-clonic seizure (I). ONO-AE-248 (3 µg/kg, s.c.) prevented the protective effect of ONO-AE3-240 (10 µg/kg, s.c.) against PTZ-induced seizures. Data expressed as median and interquartile range for n = 3-6 in each group. A probability of P < 0.05 was considered significant. *P < 0.05, and **P < 0.01, when compared with vehicle or vehicle + vehicle group.

Figure 4

(A-D) Effects of EP3 receptor ligands on Na⁺, K⁺-ATPase activity. ONO-AE-248 (10 µg/kg, s.c.) augments (A) and ONO-AE3-240 (10 µg/kg, s.c.) prevents (B) PTZ-induced decrease of Na⁺, K⁺-ATPase activity in cerebral cortex and hippocampus homogenates in mice. Data are presented in nmol Pi/mg protein/min, as mean + S.E.M., for n = 5 in each group. The Na⁺, K⁺-ATPase activity was analyzed by two-way ANOVA followed by the Bonferroni’s test. (C) ONO-AE-248 (3 µg/kg, s.c.) does not present per SE effect on Na⁺, K⁺-ATPase (P > 0.05 by Student’s t-test, for n = 5). (D) Two-way ANOVA followed by the Bonferroni’s test revealed that ONO-AE-248 (3 µg/kg, s.c.) accentuated PTZ-induced decrease of Na⁺, K⁺-ATPase activity and prevented ONO-8713 (10 µg/kg, s.c.) effect on this enzyme activity in cerebral cortex and hippocampus homogenates in mice. Data are presented in nmol Pi/mg protein/min, as mean + S.E.M., for n = 5 in each experimental group. *P < 0.05, when compared with the respective control group; #P < 0.05, when compared with vehicle group.