The 27-kDa heat shock protein (HSP27) is a reliable hippocampal marker of full development of pilocarpine-induced status epilepticus.

PURPOSE: The pilocarpine-induced status epilepticus (SE) in rodents provides a valuable animal model of temporal lobe epilepsy. Since not all animals enter SE following pilocarpine injection, we aimed to find a biomarker for full development of pilocarpine-induced SE using a proteomic approach. METHODS: Two-dimensional gel electrophoresis and Western blot analysis were performed with protein extracts from hippocampal tissue taken from four different groups of animals: pilocarpine-treated rats with full development of SE, pilocarpine-treated rats without seizures, pilocarpine-treated rats with seizures but without SE, and saline-injected rats. RESULTS: 2D gel electrophoresis revealed two protein spots which were only present after full development of pilocarpine-induced SE, but neither in gels from pilocarpine-treated rats without seizures nor in those of saline-injected animals. The protein in both spots was identified as the small 27-kDa heat shock protein (HSP27) by MALDI-TOF mass spectrometry. Subsequent Western blot analyses confirmed that HSP27 immunoreactivity was only observed with the hippocampal protein extracts in the status epilepticus group, but in none of the other groups. Furthermore, immunocytochemistry showed that HSP27 expression following pilocarpine-induced SE was localized in astrocytes. CONCLUSION: We propose that HSP27 is a highly sensitive and specific hippocampal marker for full development of pilocarpine-induced status epilepticus.

Network excitability in a model of chronic temporal lobe epilepsy critically depends on SK channel-mediated AHP currents.

Hippocampal CA1 pyramidal neurons generate an after-hyperpolarization (AHP) whose medium component is thought to be generated by small-conductance Ca(2+)-activated K(+) channels (SK channels). Neuronal excitability is increased in epilepsy, and the AHP in turn is fundamentally involved in regulation of cellular excitability. We therefore investigated the involvement of the SK channel-mediated AHP in controlling cell and network excitability in the pilocarpine model epilepsy. Both acutely isolated CA1 pyramidal cells and isolated hippocampal slices were investigated in terms of the impact of SK channel-mediated AHP on hyperexcitability. Our findings show that pilocarpine-treated chronically epileptic rats exhibit significantly reduced SK channel-mediated hyperpolarizing outward current which was accompanied by a significant decrease in the somatic AHP. Paradoxically, inhibiting SK channels strongly exacerbated 0-Mg(2+)-induced epileptiform activity in slices from pilocarpine-treated animals, while having a significantly smaller effect in control tissue. This suggests that in chronically epileptic tissue, network excitability very critically depends on the remaining SK-channel mediated AHP. Additional real-time RT-PCR and semiquantitative Western blot experiments revealed that both the SK2 channel transcript and protein were significantly downregulated in the epileptic CA1 region. We conclude that SK2 channels are down-regulated in chronic epilepsy underlying the impaired SK channel function in CA1 pyramidal cells, and a further reduction of the remaining critical mass of SK channels results in an acute network decompensation.

Inhibitory effect of the lectin wheat germ agglutinin on the binding of 125I-CCK-8s to the CCK-A and -B receptors of AR42J cells.

INTRODUCTION: Cholecystokinin (CCK) is a peptide hormone and plays a major role both in the regulation of pancreatic enzyme secretion and growth of the gastrointestinal tract. The pancreatic CCK receptors are highly glycosylated membrane proteins that are able to bind plant lectins such as wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I). AIM AND METHODOLOGY: In preceding papers, we demonstrated an inhibition of CCK-8s induced Ca2+ signaling and secretion of rat pancreatic acini and AR42J cells by the lectins WGA and UEA-I (Pancreas 2001;23:368-374). Here we studied the influence of WGA, UEA-I, and 22 other lectins on 125I-CCK-8s binding on AR42J cells. A binding assay was used with 125I-CCK-8s and dexamethasone-stimulated AR42J cells, bearing CCK-A as well as CCK-B receptors. RESULTS: WGA inhibits 125I-CCK-8s binding in a dose-dependent manner. The binding is affected at concentrations of WGA >1 microg/mL. The EC50 for inhibition is 8 microg/mL. At a concentration of 25 microg/mL, WGA inhibits the hormone binding 70%. This inhibition can be abolished by the specific sugars for WGA N,N',N"-triacetylchitotriose and N-acetylglucosamine, but not by N-acetylneuraminic acid. UEA-I diminished hormone binding but without significance, although UEA-I binds to the fucose residues of receptor glycosylations. All other 22 lectins tested here were ineffective. CONCLUSION: The blockage of CCK receptors by WGA explains the inhibition of CCK-8s induced Ca2+ signaling and the secretion of pancreatic acinar cells and AR42J cells. Although the inhibitory effect of WGA is in agreement with the findings of Santer et al, the results with UEA-I are in contrast to those of Santer et al (1990), who described a strong increase in 125I-CCK-8s binding to isolated crude rat pancreatic cell membranes in the presence of UEA-I.

Inhibitory effects of human and porcine alpha-amylase on CCK-8-stimulated lipase secretion of isolated rat pancreatic acini.

Previously we have demonstrated inhibitory effects of the plant lectin wheat germ agglutinin (WGA) on (125)I-CCK-8 binding to pancreatic AR42J cells as well as on CCK-8-stimulated Ca(2+) release and alpha-amylase secretion of rat pancreatic acini or acinar cells. Therefore, it is entirely conceivable that alpha-amylase having several lectin-like carbohydrate recognition domains can modulate the CCK-8 stimulated lipase secretion. Human alpha-amylase, purified from pancreatic juice by affinity chromatography to homogeneity, and commercial porcine pancreatic alpha-amylase inhibit CCK-8-stimulated lipase secretion of rat pancreatic acini in a concentration-dependent manner. Acarbose, a specific inhibitor of alpha-amylase, was without effect on CCK-8-induced cellular lipase secretion. The data presented here provide evidence for a regulatory function of alpha-amylase in CCK-8-stimulated pancreatic secretion.