Thrombolysis effect with FIIa from Agkistrodon acutus venom in different thrombosis model.

AIM: A fibrinolytic enzyme from Agkistrodon acutus venom, called FIIa, was tested for thrombolytic activity in animals. METHODS: Carotid thrombosis model in rats and rabbits and middle cerebral artery (MCA) thrombosis model in rats were used. RESULTS: Intravenous administration of FIIa, at a dosage of 0.625 mg/kg, resulted in thrombolysis of carotid thrombi. However, in middle cerebral artery thrombosis, the effective thrombolysis dose was 1.25 mg/kg. When the dosage of FIIa increased, the thrombolytic effect was stronger. Histological examination of kidney, liver, heart, and lung tissue showed no hemorrhage. CONCLUSION: It shows that FIIa from Agkistrodon acutus venom is able to solubilize thrombus in vivo without hemorrhage at an effective dose for thrombolysis.

[Relationship between heat stress suppression of neuroapoptosis and activation of nuclear kappa B in primary cultured rat cerebellar granule cells].

It has been well demonstrated that heat stress response (HSR) plays a crucial role in protecting cells from injury induced by various pathological stimuli. However, the protective mechanism of HSR is only poorly understood. The object of this article was to further investigate the relationship between the protective role of heat stress response and activation of NF-kappa B in primary cultured rat cerebellar granule cells. Heat stress was induced by hyperthermia (43+/-0.5 degrees centigrade), and DNA binding activity of NF-kappa B was determined with electrophoretic mobility shift assay (EMSA). Neuroapoptosis was measured by Hoechst 33258, agarose gel electrophoresis and flow cytometry (FCM) analysis. The results showed that the neurons treated with low potassium for l6 h could induce neuroapoptosis and promote the activity of nuclear kappa B. Heat stress treatment for 30, 60 and 90 min could suppress neuroapoptosis and the activity of nuclear kappa B induced by low potassium in a time-dependent manner. Activation of NF-kappa B using 100 nmol/L phorbol 12-myristate l3-acetate (PMA) could promote antiapoptotic action of heat stress response. In contrast, when NF-kappa B activation was inhibited by 10 micromol/L pyrrolidine dithiocarbamate derivatives (PDTC), heat stress did not provide protection against cell apoptosis induced by low potassium. The results suggest that the neuroprotection of heat stress has no relation to the suppression of NF-kappa B activity, and activation of NF-kappa B may promote antiapoptotic action of heat stress.

Antagonistic action of caffeine against LY294002-induced apoptosis in cerebellar granule neurons.

AIM: To study the effect of caffeine on apoptosis induced by inhibition of 1-phosphatidylinositol 3-kinase in cerebellar granule neurons. METHODS: Cerebellar granule neurons culture, agar gel electrophoresis, and stress-activated protein kinase (SAPK)/c-Jun N-terminal protein kinase (JNK) assay kit to measure SAPK/JNK activity. RESULTS: LY294002 evoked apoptosis concentration-dependently in cerebellar granule neurons. But death resulting from LY294002 was prevented by caffeine in a concentration-dependent manner. The survival effect of caffeine was not affected by inhibitors of ryanodine-sensitive Ca2+ release, nor was it inhibited by L-type channel blockers and N-methyl-D-aspartate (NMDA) receptor blocker. In addition, RP-cAMP, H89, and KN62 were not able to inhibit the protective effect of caffeine. Phosphorylation of c-Jun was necessary for the induction of apoptosis induced by LY294002 in cerebellar granule neurons. But caffeine directly inhibited the activation of JNK and decreased phospho-c-Jun in granule neurons. CONCLUSION: Caffeine inhibited the activation of JNK and decreased the phosphorylation of c-Jun to protect granule neurons from LY294002-induced apoptosis.

[Neuroprotective effect of alpha-dihydroergocryptine depends on activation of nuclear factor kappa B].

AIM: To investigate the relationship between the neuroprotective effect of alpha-dihydroergocryptine (alpha-DHEC) and the activation of nuclear factor Kappa B (NF-Kappa B). METHODS: Adult male rats were subjected to cerebral ischemia induced by middle cerebral artery occlusion (MCAO). DNA binding activity of NF-Kappa B was determined with electrophoretic mobility shift assay (EMSA) in animals subjected to varying durations of cerebral ischemia. Neuroapoptosis induced by ischemic damage was measured by deoxynucleotidy transferase-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry (FCM) analysis. RESULTS: No change was observed in nuclear NF-Kappa B DNA binding in normal animal. A low level of constitutive NF-Kappa B DNA binding was detected in animals subjected to cerebral ischemia of 1 h, and significant increase in the amount of active NF-Kappa B in nuclear extracts was observed after cerebral ischemia of 3 h, 6 h, and 12 h. Peak of NF-Kappa B DNA binding was observed at the time point of 3 h. Animals subjected to cerebral ischemia of 3 h potentially initiates neuroapoptosis and activates NF-Kappa B in nuclear extract. Alpha-DHEC (100 micrograms.kg-1 and 150 micrograms.kg-1) showed significant protective effect on neuroapoptosis-induced by cerebral ischemia of 3 h, and inhibiting NF-Kappa B activation using 100 mg.kg-1 pyrrolidinedithiocarbamate (PDTC) in the continuous presence of alpha-DHEC, the neuroprotective effect of alpha-DHEC was blocked. CONCLUSION: The findings suggest that the neuroprotetive effect of alpha-DHEC may depend on the activation of NF-Kappa B.

The NF-kappaB/Rel family of proteins mediates Abeta-induced neurotoxicity and glial activation.

The beta-amyloid peptide (Abeta) is deposited in neuritic plaques which are characteristic features of Alzheimer's disease (AD). Prominent neurodegeneration and glial activation occurs around these plaques leading to the hypothesis that Abeta may play a causative role in the neuronal loss and the inflammatory response associated with AD. Here we show that Abeta-induced toxicity of cultured fetal rat cortical neurons is associated with internucleosomal DNA fragmentation beginning just 6 h after neurons are exposed to Abeta. Additionally, constitutive NF-kappaB activity readily measured in fetal rat cortical neurons decreases in a concentration- and time-dependent fashion following exposure to Abeta, but there is no corresponding decrease in NF-kappaB mRNA or protein (p65). An upregulation of both IkappaB alpha protein and mRNA which occurs in cortical neurons exposed to Abeta may be responsible for retaining NF-kappaB in the cytoplasm accounting for the observed decrease in activated NF-kappaB. The latter is supported by the observation that pretreatment of cortical cultures with an antisense oligonucleotide to IkappaBalpha mRNA is neuroprotective. In contrast to cortical neurons, exposure of rat primary astroglial cultures to Abeta results in a concentration- and time-dependent activation of NF-kappaB with subsequent upregulation of IL-1beta and IL-6. Our data suggest that Abeta-induced neurotoxicity as well as astrocyte activation may be medicated by the NF-kappaB/Rel family of proteins, and thus alterations in NF-kappaB-directed gene expression may contribute to both the neurodegeneration and inflammatory response which occur in AD.

Transient increase in cerebellar transcriptional activity precedes the expression of GABA(A) receptor alpha6 subunit mRNA during postnatal maturation.

The purpose of this study was to investigate the postnatal expression of GABA(A) receptor alpha6 subunit genes in the context of cerebellar differentiation. We examined steady-state levels of GABA(A) receptor alpha1 and alpha6 subunit mRNAs, polyadenylated (polyA+) mRNA and beta-actin mRNA in7-, 14-, 21-, 28-, 35-, 49- and 120-day-old rats. Messenger RNA expression and splicing were evaluated in parallel using Northern blot analysis and in situ hybridization histochemistry. The expression of mature GABA(A) receptor alpha6 subunit mRNA species (2.7 kb) was found 1 week after birth in cerebellar granule cells. Prior to stable expression of the mature alpha6 subunit gene, we detected large alpha6 subunit premessengers (3.8 and 3.5 kb) by Northern blot analysis. These premessenger species were detected in prenatal day (PND) 15 and neonatal rat cerebellum, when the mature alpha6 subunit mRNAs (2.7 kb) were not yet expressed. The maximal expression of mature alpha6 subunit mRNA species was observed at PND 21 when the peak level of cerebellar transcriptional activity was measured by polyA+ RNA levels. In contrast, beta-actin mRNA expression was decreased at PND 21 compared to birth levels. These major transcriptional events take place during a period of about 1 week (between PND 14 and 21), immediately following the most active phase of cell division in the external granule layer and migration of granule cells to the internal granule cell layer. Comparison between the relative abundance of these genes shows that differential regulation of each gene occurs during postnatal development. The induction of GABA(A) receptor alpha6 subunit gene expression is preceded by a reduction in beta-actin mRNA levels and a transient increase in total transcriptional activity. The expression of alpha6 subunit mRNA is maintained at the PND 21 level through adulthood, but the alpha1 subunit mRNA levels decrease drastically within the following week (from PND 21 to 28). These results suggest that tissue-specific expression of the GABA(A) receptor alpha6 subunit gene is correlated with a series of developmentally regulated morphologic and transcriptional events.

Mastoparan-induced apoptosis of cultured cerebellar granule neurons is initiated by calcium release from intracellular stores.

We have recently reported that mastoparan, a peptide toxin isolated from wasp venom, induces apoptosis in cultured cerebellar granule neurons that can be blocked by cholera toxin, an activator of Gs. Measurements of intracellular free calcium concentration ([Ca2+]i) reveal that mastoparan induces a dramatic elevation of [Ca2+]i that is frequently followed by enhanced leakage of fura-2 out of the neurons, suggesting that this rise in [Ca2+]i may be due to a more generalized change in membrane permeability. However, the mastoparan-induced initial elevation of [Ca2+]i is maintained in the absence of extracellular Ca2+, suggesting that the rise of [Ca2+]i is from intracellular stores. This conclusion is supported by the observation that depletion of [Ca2+]i stores by pretreatment with either caffeine or thapsigargin attenuates both the rise in [Ca2+]i and cell death induced by mastoparan. Phospholipase C (PLC) inhibitors, neomycin and U73122 block mastoparan-induced increases of [Ca2+]i and protect against neuronal death. Pretreatment with cholera toxin, but not pertussis toxin, reduced the mastoparan-induced rise in [Ca2+]i. Taken together, our data suggest that mastoparan initiates cell death in cerebellar granule neurons by inducing Ca2+ release from intracellular stores, probably via activation of PLC and IP3. A secondary or parallel process results in disruption of plasma membrane integrity and may be ultimately responsible for the death of these neurons by mastoparan.

Activation of G proteins bidirectionally affects apoptosis of cultured cerebellar granule neurons.

Cultured cerebellar granule neurons maintained in depolarizing concentrations of K+ (25 mM) and then switched to physiological concentrations of K+ (5 mM) undergo apoptosis. We now report that activation of specific G proteins robustly and bidirectionally affects apotosis of cultured rat cerebellar granule neurons. Stimulation of Gs with cholera toxin completely blocks apoptosis induced by nondepolarizing concentrations of K+, whereas stimulation of Go/Gi with the wasp venom peptide mastoparan induces apoptosis of cerebellar granule neurons even in high (depolarizing) concentrations of K+. Moreover, pretreatment of cerebellar granule neurons with cholera toxin attenuates neuronal death induced by mastoparan. By contrast, pertussis toxin, cell-permeable analogues of cyclic AMP, and activators of protein kinase A do not affect apoptosis of cultured cerebellar granule neurons. These data suggest that G proteins may function as key switches for controlling the programmed death of mammalian neurons, especially in the developing CNS.

Regional expression and cellular localization of the Na(+)-dependent inorganic phosphate cotransporter of rat brain.

We have recently isolated and identified a brain specific Na(+)-dependent inorganic phosphate (Pi) cotransporter cDNA (rBNPI) from a rat brain cDNA library (Ni, et al., 1994). We now report the regional and developmental expression, as well as the cellular localization, of rBNPI mRNA in the rat brain. In situ hybridization histochemistry reveals that rBNPI mRNA is selectively expressed in neuron-enriched regions of the adult rat brain, such as the cerebral cortex, hippocampus, and cerebellum. Cellular localization of rBNPI transcripts reveals expression in both pyramidal and granule neurons in these regions. By contrast, little to no hybridization signal was observed in white matter-enriched areas such as the corpus callosum. The expression of rBNPI mRNA was determined during pre- and postnatal development of the rat CNS. From embryonic day 17 to early postnatal day 10 (PND 10), there is a rather widespread but diffuse pattern of rBNPI expression in brain. During late postnatal development, however, the expression of rBNPI mRNA becomes confined to discrete populations of neurons in the cerebral cortex, hippocampus, and cerebellum. Thus, rBNPI expression is developmentally regulated and abundant levels of mRNA are found in rather discrete populations of neurons in the adult rat brain. The latter suggests that rBNPI may serve to selectively regulate intracellular Pi transport in certain neurons for either metabolic and (or) signaling events.

Diphenylhydantoin induces apoptotic cell death of cultured rat cerebellar granule neurons.

Apoptosis is one form of physiological or programmed cell death responsible for the selective elimination of various cell types during development. We have observed and characterized a delayed-type of neurotoxicity induced in cultured cerebellar granule neurons by diphenylhydantoin. Diphenylhydantoin toxicity of cerebellar granule neurons is time and concentration dependent. Morphological studies using Nomarski optics and staining with the fluorescent dye Hoechst 33258 demonstrate that diphenylhydantoin-induced neurotoxicity of cerebellar granule neurons is associated with cytoplasmic blebbing, heterochromatic clumping and condensation of chromatin that precede cell death. Unlike glutamate toxicity (excitotoxicity) diphenylhydantoin-induced neurotoxicity of cerebellar granule neurons is attenuated by actinomycin D and cycloheximide, and is associated with nucleosomal size DNA fragmentation. Since we have previously reported that depolarization of cultured cerebellar granule neurons with high concentrations of K+ promotes the survival of these neurons by blocking apoptosis, we examined the effects of diphenylhydantoin on the K(+)-evoked increase in intracellular calcium. Using microfluorimetry and fura-2 to measure intracellular calcium we found that neurotoxic concentrations of diphenylhydantoin markedly reduce the increase in intracellular calcium associated with elevated extracellular potassium. Taken together, these data demonstrate that exposure of cultured cerebellar granule neurons to pharmacologically relevant concentrations of diphenylhydantoin results in a delayed type of neurotoxicity characterized by the biochemical and morphological features of apoptosis.

Activation of muscarinic cholinergic receptors blocks apoptosis of cultured cerebellar granule neurons.

We have recently reported that the majority of cultured rat cerebellar granule neurons undergo apoptosis when maintained in the presence of physiological concentrations of K+ (nondepolarizing conditions). We now report that exposure of cultured cerebellar granule neurons, maintained under nondepolarizing conditions, to the muscarinic cholinergic receptor (mAchR) agonists carbachol and muscarine results in a concentration- and time-dependent inhibition of apoptosis. The nicotinic cholinergic receptor agonist (-)-nicotine fails to mimic, and the nicotinic cholinergic receptor antagonist dihydro-beta-erythroidine fails to antagonize, the survival-promoting effects of carbachol. In contrast, relatively low concentrations of atropine completely prevent the effects of carbachol in blocking apoptotic death of cultured granule neurons. Although the m1- and m2-preferring mAchR antagonists pirenzepine and gallamine, respectively, fail to reverse the effects of carbachol, the m3-preferring antagonist 4-diphenylacetoxyl-N- methylpiperidine methiodide completely blocks the survival-promoting effects of carbachol. These data demonstrate that activation of the mAchR (possibly of the m3 subtype) blocks apoptosis of cultured cerebellar granule neurons. The antiapoptotic effects of mAchR agonists are not indirectly mediated via glutamate release from granule neurons, because antagonists of either N-methyl-D-aspartate or non-N-methyl-D-aspartate glutamate receptors fail to affect the antiapoptotic effects of carbachol or muscarine. Moreover, exposure of cultured cerebellar granule neurons to antiapoptotic concentrations of carbachol, in contrast to high concentrations of K+ or glutamate receptor agonists, results in only a small and transient elevation of the intracellular Ca2+ concentration, as measured by fura-2 microfluorimetry. Slow neurotransmitters such as acetylcholine, acting via their cognate G protein-coupled receptors, may prevent neuronal apoptosis in the developing (and perhaps adult) central nervous system.

Depolarization or glutamate receptor activation blocks apoptotic cell death of cultured cerebellar granule neurons.

Cerebellar granule neurons can be readily maintained in culture if depolarized with high concentrations of K+ or subtoxic concentrations of various excitatory amino acids. We now report that these depolarizing stimuli promote cerebellar granule neuron survival by blocking their programmed death via apoptosis. Cerebellar granule neurons maintained in depolarizing conditions and then changed to non-depolarizing conditions, exhibit the morphological and biochemical features of apoptosis, including cytoplasmic blebbing, condensation and aggregation of nuclear chromatin and internucleosomal DNA fragmentation. Inhibitors of RNA or protein synthesis greatly attenuate cell death induced by non-depolarizing culture conditions. In contrast, cerebellar granule neurons, when exposed to fresh serum-containing medium or to high concentrations of glutamate, exhibit a delayed-type of neurotoxicity which is non-apoptotic in nature. Given the actions of excitatory amino acid receptor agonists in preventing apoptosis of cultured cerebellar granule neurons, we hypothesize that the functional innervation of postmigratory granule neurons during cerebellar development may prevent further elimination of these neurons by blocking their programmed death.