Relative roles of pneumolysin and hydrogen peroxide from Streptococcus pneumoniae in inhibition of ependymal ciliary beat frequency.

Ciliated ependymal cells line the ventricular system of the brain and the cerebral aqueducts. This study characterizes the relative roles of pneumolysin and hydrogen peroxide (H(2)O(2)) in pneumococcal meningitis, using the in vitro ependymal ciliary beat frequency (CBF) as an indicator of toxicity. We have developed an ex vivo model to examine the ependymal surface of the brain slices cut from the fourth ventricle. The ependymal cells had cilia beating at a frequency of between 38 and 44Hz. D39 (wild-type) and PLN-A (pneumolysin-negative) pneumococci at 10(8) CFU/ml both caused ciliary slowing. Catalase protected against PLN-A-induced ciliary slowing but afforded little protection from D39. Lysed PLN-A did not reduce CBF, whereas lysed D39 caused rapid ciliary stasis. There was no effect of catalase, penicillin, or catalase plus penicillin on the CBF. H(2)O(2) at a concentration as low as 100 microM caused ciliary stasis, and this effect was abolished by coincubation with catalase. An additive inhibition of CBF was demonstrated using a combination of both toxins. A significant inhibition of CBF at between 30 and 120 min was demonstrated with both toxins compared with either H(2)O(2) (10 microM) or pneumolysin (1 HU/ml) alone. D39 released equivalent levels of H(2)O(2) to those released by PLN-A, and these concentrations were sufficient to cause ciliary stasis. The brain slices did not produce H(2)O(2), and in the presence of 10(8) CFU of D39 or PLN-A per ml there was no detectable bacterially induced increase of H(2)O(2) release from the brain slice. Coincubation with catalase converted the H(2)O(2) produced by the pneumococci to H(2)O. Penicillin-induced lysis of bacteria dramatically reduced H(2)O(2) production. The hemolytic activity released from D39 was sufficient to cause rapid ciliary stasis, and there was no detectable release of hemolytic activity from the pneumolysin-negative PLN-A. These data demonstrate that D39 bacteria released pneumolysin, which caused rapid ciliary stasis. D39 also released H(2)O(2), which contributed to the toxicity, but this was masked by the more severe effects of pneumolysin. H(2)O(2) released from intact PLN-A was sufficient to cause rapid ciliary stasis, and catalase protected against H(2)O(2)-induced cell toxicity, indicating a role for H(2)O(2) in the response. There is also a slight additive effect of pneumolysin and H(2)O(2) on ependymal toxicity; however, the precise mechanism of action and the role of these toxins in pathogenesis remain unclear.

Interaction of ketamine with mu2 opioid receptors in SH-SY5Y human neuroblastoma cells.

PURPOSE: Ketamine is known to interact with opioid receptors. However, because this agent does not produce opioid-like respiratory depression, it might not interact with mu(2) opioid receptors. Therefore, we have studied the interaction of ketamine with mu(2) opioid receptors expressed in SH-SY5Y cells. METHODS: SH-SY5Y cells (passage 70-80) were used to obtain ketamine dose-response curves for inhibition of 0.4 nM [(3)H][D-Ala(2),MePhe(4),Gly(ol)(5)] enkephalin (DAMGO) binding to mu(2) opioid receptors and of forskolin (1 microM)-stimulated cyclic AMP (cAMP) formation. RESULTS: Ketamine displaced [(3)H]DAMGO binding in SH-SY5Y cells with a K(i) of 12.1 microM. However, this concentrations did not inhibit forskolin-stimulated cAMP formation, although at supraclinical concentrations, significant inhibition was observed with an estimated IC(50) of 700 microM. CONCLUSION: The present study indicates that a clinically relevant concentration of ketamine interacts with mu(2) opioid receptors. However, no agonist activity was observed.

Etomidate inhibits [3H]noradrenaline release from SH-SY5Y human neuroblastoma cells.

We have examined the effects of the intravenous anaesthetic induction agent etomidate on K+ and carbachol evoked [3H]noradrenaline ([3H]NA) release and the associated increase in [Ca2+]i in SH-SY5Y human neuroblastoma cells in a attempt to study potential anaesthetic target site(s). Preincubation with etomidate produced a dose-dependent inhibition of both K+ and carbachol evoked [3H]NA release with estimated IC50 values of 88 and 69 microM, respectively. Only K+ stimulated increase in [Ca2+]i was inhibited by etomidate preincubation with an IC50 of 146 microM. Acute addition of etomidate after K+ challenge also inhibited the increase in [Ca2+]i with an IC50 of 99 microM. In addition etomidate displaced the binding of [3H]PN200-110 to L-type voltage sensitive Ca2+ channels with a Ki of 48 microM. As K+ but not carbachol evoked [3H]NA release is extracellular Ca2+ dependent and was inhibited by etomidate these data coupled with the PN200-110 displacement studies suggest that etomidate may interact with L-type voltage sensitive Ca2+ channels. The inhibition of carbachol evoked release without affecting the associated increase in [Ca2+]i suggests that etomidate may exert additional effects at either the muscarinic receptor or the secretory machinery in these cells.