Role of nitric oxide in the enhancement of pentylenetetrazole-induced seizures caused by Shigella dysenteriae

Abstract

Convulsions and encephalopathy are frequent complications of childhood shigellosis. We studied the role of nitric oxide (NO) in Shigella-related seizures in an animal model. Pretreatment of mice with Shigella dysenteriae 60R sonicate elevated serum NO levels and enhanced the convulsive response to pentylenetetrazole (PTZ), as indicated by a higher mean convulsion score and a higher number of mice responding with seizures. Treatment of the mice with S-methylisothiourea sulfate (SMT), a potent inhibitor of inducible NO synthase (NOS), prevented the elevation of serum NO levels and concomitantly reduced the enhanced response to PTZ. The mean convulsion scores were 0.7, 0.7, 1.3, and 0.8 for mice treated with saline, saline and SMT, S. dysenteriae 60R sonicate, and S. dysenteriae 60R sonicate with SMT, respectively (P = 0.001 for 60R sonicate versus saline and P = 0.013 for 60R sonicate versus 60R sonicate with SMT). The corresponding seizure rates were 40, 44, 75, and 47% for saline, saline with SMT, S. dysenteriae 60R sonicate, and S. dysenteriae 60R sonicate with SMT, respectively (P = 0.0004 for 60R sonicate versus saline and P = 0.005 for 60R sonicate versus 60R sonicate with SMT). In contrast, injection of N-nitro-L-arginine, a selective inhibitor of constitutive NOS, neither abolished the elevation of serum NO nor attenuated the enhancement of seizures. These findings indicate that NO, induced by S. dysenteriae 60R sonicate, is involved in enhancing the susceptibility to seizures caused by S. dysenteriae.

FIG. 1

Serum NO production in response to S. dysenteriae 60R sonicate (4 LD₅₀ i.p.). Measurement of the stable products of NO, NO⁻₂, and NO⁻₃, was performed by using nitrate reductase and the colorimetric assay with Griess reagent, as described in Materials and Methods. Each point represents the average of three animals.

FIG. 2

Effect of treatment with NOS inhibitors on serum NO levels 7 h after S. dysenteriae administration. Mice were injected (i.p.) with saline alone, saline with NOS inhibitor, S. dysenteriae 60R sonicate with saline, or S. dysenteriae 60R sonicate with NOS inhibitor. Each point represents the mean of five determinations. (A) Mice treated with SMT. P = 0.001 for S. dysenteriae sonicate versus saline and P = 0.0001 for S. dysenteriae sonicate versus S. dysenteriae sonicate with SMT. SMT (2 mg/kg) was administered 2 h after S. dysenteriae sonicate. (B) Mice treated with NNA. P = 0.0001 for S. dysenteriae sonicate versus saline and is nonsignificant for S. dysenteriae sonicate versus S. dysenteriae sonicate with NNA-treated mice. NNA (2.5 mg/kg) was administered 3, 5, and 6 1/2 h after S. dysenteriae 60R sonicate.

FIG. 3

Effect of treatment with NOS inhibitors on the enhanced severity of PTZ-induced seizures by S. dysenteriae 60R sonicate. Mice were injected (i.p.) with saline alone, saline with NOS inhibitor, S. dysenteriae 60R sonicate with saline, or S. dysenteriae 60R sonicate with NOS inhibitor; PTZ was injected (i.p.) at 7 h. (A) Mice treated with SMT. P = 0.001 for S. dysenteriae sonicate (n = 53) versus saline (n = 57), and P = 0.013 for S. dysenteriae sonicate versus S. dysenteriae sonicate with SMT (n = 51). SMT (2 mg/kg) was administered 2 h after S. dysenteriae 60R sonicate. (B) Mice treated with NNA. NNA (2.5 mg/kg) was administered 3, 5, and 6 1/2 h after S. dysenteriae 60R sonicate.