Apolipoprotein E polymorphism in various dementias in Taiwan Chinese population.

To verify the association between APOE4 frequency and various dementias in Taiwan Chinese individuals, APOE genotypes were determined in patients with dementia of Alzheimer's type (AD), vascular dementia (VD), dementias due to other general medical conditions (OD), and dementia of Alzheimer's type with cerebral vascular disease (mixed type dementia; MD). Only AD patients exhibited higher APOE4 frequency (OR = 2.95, p = 0.001) than controls after Bonferroni correction to control the overall type I error rate for the multiple testing. No such difference was observed among VD, OD, MD and control groups. The lack of association between VD and APOE4 allele frequency suggests that APOE4 allele does not associate with cerebrovascular pathology related dementia in Taiwan Chinese.

Differential alteration of catecholamine release during chemical hypoxia is correlated with cell toxicity and is blocked by protein kinase C inhibitors in PC12 cells.

Release of neurotransmitters, including dopamine and glutamate, has been implicated in hypoxia/ischemia-induced alterations in neuronal function and in subsequent tissue damage. Although extensive studies have been done on the mechanism underlying the changes in glutamate release, few have examined the mechanism that is responsible for the changes in catecholamines. Rat pheochromocytoma-12 (PC12) cells synthesize, store, and release catecholamines including DA and NE. Therefore, we used HPLC and ED to evaluate extracellular DA and NE concentrations in a medium during chemical hypoxia in PC12 cells. Chemical hypoxia produced by KCN induced differential release of DA and NE. Under normal glucose conditions, KCN induced release of NE, but not DA. Under glucose-free conditions, KCN-induced release of DA was elevated transiently, whereas the release of NE increased progressively. Under parallel conditions, KCN biphasically elevated the level of cytosolic free calcium ([CA(2+)](i)) in glucose-free DMEM, peaking at 95 +/- 18 nM at 1,107 +/- 151 s, followed by a new plateau level at 249 +/- 24 nM sustained from 4,243 +/- 466 to 5,263 +/- 440 s. Cell toxicity, as measured by LDH release, was increased significantly by KCN in glucose-free DMEM but was diminished in the presence of glucose, and was correlated with DA release by chemical hypoxia. The protein kinase C (PKC) inhibitor GO6976 or staurosporine inhibited KCN-induced LDH release as well as the release of NE and DA. Taken together, selective activation of DA but not NE was correlated with the LDH release by chemical hypoxia, and was diminished with GO6976 or staurosporine. These results suggest that selective activation of PKC isoforms is involved in the chemical hypoxia-induced DA release, which may lead to neuronal cell toxicity.

Noise damage in the C57BL/CBA mouse cochlea.

The present study was designed to determine the response to noise of the auditory system of a genetically well-defined laboratory mouse in preparation for examining the effect of noise on mice with specific genetic mutations. The mice were C57BL/CBA F1 hybrids. Eight mice served as non-noise-exposed controls and 39 mice were exposed for 1-24 h to an octave band of noise with a center frequency of 2, 4 or 8 kHz and a sound pressure level of 100-120 dB. Auditory brainstem response thresholds were measured pre-exposure and several times post-exposure (i.e., 0-27 days) to determine the magnitude of the temporary threshold shift (TTS) and permanent threshold shift (PTS). After fixation by cardiac perfusion, the cochleas from each mouse were embedded in plastic, dissected into quarter turns of the cochlear duct and analyzed quantitatively. Immediately post-exposure, all mice had sizable TTSs at the tested frequencies (i.e., 3-50 kHz). At this time, two mice were killed. Thresholds of the other 37 mice recovered somewhat in the first 4 days post-exposure. One mouse fully recovered from its TTS; 10 mice were left with PTSs at all frequencies; 26 mice recovered at some frequencies but not others. Most mice with PTSs for 30-50 kHz had focal losses of inner and outer hair cells in the basal 20% of the organ of Corti, often with degeneration of adjacent myelinated nerve fibers in the osseous spiral lamina. On the other hand, mice with PTSs for the lower frequencies (i.e., 3-20 kHz) had stereocilia disarray without significant hair cell losses in the second and first turns. Considerable variability was found in the magnitude of hair cell losses in those mice that received identical noise exposures, despite their genetic homogeneity.

An anatomically based frequency-place map for the mouse cochlea.

An anatomically based frequency-place map was created for the mouse using C57BL/CBA F1 hybrids by matching noise-induced lesions in the organ of Corti with permanent hearing losses as determined by auditory brainstem response (ABR) thresholds. Twenty-six mice developed 'notched' ABR threshold shifts after exposure to an octave band of noise with a center frequency of 2 kHz at 120 dB SPL for 24 h, 4 kHz at 110 dB SPL for 4 h or 8 kHz at 100 dB SPL for 1 or 2 h. ABR thresholds were determined at several intervals post-exposure until thresholds stabilized (14-27 days). Once thresholds had stabilized, the mice were killed and their cochleas were prepared for phase-contrast microscopic examination as plastic-embedded flat preparations. Hair cell loss, stereocilia damage, and myelinated nerve fiber degeneration as a function of percentage distance from the cochlear apex were determined. Frequency-position matches could be made for 22 of the 26 mice by correlating areas of hair cell loss/stereocilia damage with permanent changes in ABR thresholds. These frequency-position data were fitted with the equation: % Distance from apex=56.6 log (f(Hz))-179.1; r(2)=0.810. This frequency-place function agrees well with Ehret's (1975) theoretical function based on critical bands and masked auditory thresholds.

Blockage of amyloid beta peptide-induced cytosolic free calcium by fullerenol-1, carboxylate C60 in PC12 cells.

Amyloid beta protein (Abeta) alters signal transduction systems, including increases in the cytosolic free calcium ([Ca2+]i) response which have pathophysiological significance in Alzheimer's disease (AD). The purposes of this study were to elucidate the mechanism involved in Abeta's effect on the Ca2+ signal and to evaluate the effect of fullerenol-1, a water-soluble hydroxyl and superoxide radical scavenger, on the Abeta-induced Ca2+ response. Both Abeta and bradykinin (BK) dose-dependently elevated [Ca2+]i in PC12 cells. Fullerenol-1, at a concentration range between 100 nM and 1 microM, dose-dependently reduced the Abeta-induced [Ca2+]i response, but did not alter the subsequent BK-mediated process. Thapsigargin, an inhibitor of Ca2+-ATPase, released Ca2+ from the internal store and diminished the BK-mediated calcium spike but did not affect the Abeta-induced Ca2+ response. In the absence of extracellular calcium, the Abeta-induced, but not BK-induced, calcium spike was completely abolished. The Ca induced by Abeta did not enter through the voltage-dependent calcium channels or ligand gated calcium channels, because the peak of Abeta-evoked Ca2+ was not significantly altered by various Ca2+ channel blockers or a NMDA receptor antagonist MK801. In addition, neither cholera toxin nor pertussis toxin altered the Abeta-induced Ca response. The results demonstrated that Abeta-stimulated [Ca2+]i increase is due to Ca influx from an extracellular source rather than from the intracellular store. Alteration of the membrane lipid structure and permeability by free radicals generated by Abeta may be a major cause of Ca -influx. Furthermore, fullerenol-1, a novel antioxidant, may provide therapeutic benefits in neurodegenerative diseases such as AD.

Antioxidants prevent amyloid peptide-induced apoptosis and alteration of calcium homeostasis in cultured cortical neurons.

Beta-amyloid ((A)beta) is a peptide of 39-42 amino acids that is the primary component of plaques in Alzheimer's disease (AD). The mechanism by which (A)beta expresses its neurotoxic effects may involve induction of reactive oxygen species (ROS) and elevation of intracellular free calcium levels. Cultured cortical cells were utilized to study the alterations in calcium homeostasis underlying the neurotoxic effect of (A)beta. Serum supplement B27 and vitamin E were effective in preventing neuronal death as assessed by lactate dehydrogenase (LDH) release, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and number of apoptotic nuclei. In addition, (A)beta-induced cytosolic free calcium ([Ca2+]i) was blocked by antioxidants vitamin E and U83836E, but not by N-methyl-D-aspartic acid (NMDA) receptor antagonist MK-801, or by voltage-gated calcium channel blocker nimodipine. Taken together, the results suggest that NMDA receptor and voltage-gated calcium channels are not involved in (A)beta-induced [Ca2+]i increase. This increase appeared to be the result of extracellular calcium influx by some unknown mechanisms. In addition, antioxidants such as B27 were effective in protecting cultured cortical neurons against (A)beta, and correlated with (A)beta attenuation of early calcium response.

Amyloid beta peptide impaired carbachol but not glutamate-mediated phosphoinositide pathways in cultured rat cortical neurons.

Signal transduction systems, including cholinergic pathways, which are likely to be of pathophysiological significance are altered in Alzheimer's disease (AD). Muscarinic cholinergic receptors are linked to the hydrolysis of phosphoinositide, involving the production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and the mobilization of cytosolic free calcium concentrations ([Ca2+]i). Effects of amyloid peptide (A(beta)) on these signals prior to neuronal degeneration were examined in cultured rat cortical cells. A(beta) increased the release of lactate dehydrogenase (LDH) in a concentration-dependent manner, however, it was blocked by B27 supplement. Prolonged exposure to a sublethal dose of A(beta) 25-35 or 1-42 disrupted carbachol-mediated release of Ins(1,4,5)P3 and [Ca2+]i, which was inhibited in media supplemented with B27 or the antioxidant vitamin E. In order to determine the specificity of the effect of A(beta), various agonists glutamate or KCl but not bradykinin which utilize the phosphoinositide cascade were investigated. Our results indicated that A(beta) did not affect the stimulation of glutamate or KCl-mediated production of Ins(1,4,5)P3 or cause elevation in [Ca2+]i. Furthermore, metabotropic agonist trans-1-amino-cyclopentane-1,3,-dicarboxylate (ACPD) elevated calcium level was not inhibited by A(beta) pre-treatment. Taken together, the results demonstrate that a sublethal dose of A(beta) selectively impaired cholinergic receptor-mediated signal transduction pathways, and antioxidant or B27 supplement attenuated this effect of A(beta). Alterations of cholinergic signaling by prolonged exposure to A(beta) could be involved in cortical neurodegeneration that occurs in AD. Because functional loss of cholinergic pathways is an important aspect of AD, the differences in susceptibility of these two types of receptors prior to other signs of A(beta) action is important and requires further investigation.

Amyloid beta peptide enhanced bradykinin-mediated inositol (1,4,5)trisphosphate formation and cytosolic free calcium.

Deposition of amyloid beta protein (A beta) and alteration in signal transduction systems may have pathophysiological significance in Alzheimer's disease (AD). This study tested the hypothesis that bradykinin (BK) receptor-mediated signal transduction systems in PC12 cells are altered after treatment with A beta at a concentration not toxic to cells. Exposure to varying doses of A beta 25-35 (1-10 microM) for 18 hrs significantly reduced the number of viable cells, while lower concentrations (0.01-0.1 microM) and control peptide in scramble sequence had no effect. In addition, prolonged exposure of PC12 cells to a sublethal dose of A beta 25-35 (0.1 microM) affected the receptor-mediated signal transduction pathways. BK induced both accumulation of Ins(1,4,5)P3 and elevation in cytosolic free calcium concentration ([Ca2+]i) in the control cells. These responses were further enhanced in the cells treated with A beta. Under similar conditions, A beta-treated cells also demonstrated alterations in the number and affinity of BK receptors. Alternatively, extracellular addition of A beta elevated [Ca2+]i rapidly, without detectable alterations in Ins(1,4,5)P3. This rapid elevation was dependent on extracellular calcium, suggesting that A beta induced calcium influx. Taken together, the results demonstrated that treatment with a sublethal dose of A beta peptide for 18 hrs enhanced BK receptor mediated Ins(1,4,5)P3 formation and mobilization of intracellular calcium, associated with a modification in BK receptors. Changes in the balance of these receptor-mediated signals prior to cell injury could be an important underlying mechanism for A beta peptide-induced degenerative alteration in AD.

Sodium butyrate decreases histamine-stimulated calcium mobilization in C6 glioma cells.

The aim of this study was to investigate the effect of sodium butyrate on calcium mobilization. Histamine was found to stimulate a dose-dependent increase in intracellular calcium concentrations ([Ca2+]i) through H1 receptors, but this effect was attenuated in C6 cells pretreated with 1-5 mM sodium butyrate. Evidence is provided that release of Ca2+ from intracellular stores is decreased in a dose-dependent manner. Experiments with BAPTA that show lower levels of [Ca2+]i in cells pretreated with higher concentrations of sodium butyrate suggest that sodium butyrate also decreases Ca2+ influx. These results suggest that changes in Ca2+ mobilization are at least partially responsible for sodium butylate-induced C6 cell differentiation.

Altered phospholipid metabolism in sodium butyrate-induced differentiation of C6 glioma cells.

We examined the changes in phospholipid metabolisms in sodium butyrate-treated C6 glioma cells. Treatment of 2.5 mM sodium butyrate for 24 h induced an increase in the activity of glutamine synthetase, suggesting that these cells were under differentiation. Similar treatment was associated with (i) increased arachidonic acid incorporation into phosphatidylcholine, and (ii) decreased arachidonic acid incorporation into phosphatidylinositol and (iii) phosphatidylethanolamine. These effects were subsequently investigated by examining the acylation process, de novo biosynthesis, and the agonist-stimulated phosphoinositides hydrolysis in these cells. Our results indicated that sodium butyrate stimulated the acylation of arachidonic acid into lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylinositol. The glycerol incorporation into these lipids was not affected, but the inositol incorporation into total chloroform extracts and Pl and phosphatidylinositol 4-phosphate was decreased in the sodium butyrate-treated cells. Moreover, the accumulation of the rapid histamine-stimulated phosphoinositide metabolites, i.e., inositol monophosphate, inositol diphosphate, and inositol triphosphate (IP3) was decreased in these cells. To elucidate whether the decreased inositol phosphates were due to a decrease in the phosphoinositides hydrolysis, we measured the transient IP3 production directly by a receptor-binding assay. Our results indicated that histamine-stimulated transient IP3 formations were decreased. Taken together, these results indicated that multiple changes by multiple mechanisms of phospholipid metabolisms were found in sodium butyrate-treated C6 glioma cells. The decreased IP3 formation and its subsequent action, i.e., Ca2+ mobilization, may play an early but pivotal role by which sodium butyrate induces C6 glioma cell differentiation.

Allopurinol blocks shock-wave-induced rises in cytosolic calcium levels in MDCK cells.

Allopurinol has been reported to ameliorate the side effects in patients following shock wave lithotripsy (SWL); however, the mechanism has not been studied. We have examined the protective effect of allopurinol on Madin-Darby canine kidney (MDCK) cells after shock wave exposure (SWE) by determining the release of aspartate aminotransferase (ASAT) and lactate dehydrogenase (LD), and the resting cytosolic Ca2+ concentration ([Ca2+]i). In SWE-treated cells, the release of ASAT and LD increased immediately, but largely transiently, by approximately 23% and 5-fold over control, respectively. Within 1-6 h after SWE there was a gradual rise in the resting [Ca2+]i of 16-137% above control. Allopurinol did not affect the transient enzyme release but blocked the long-term rises in the resting [Ca2+]i. The transient changes in [Ca2+]i evoked by two hormones, ATP and bradykinin, and a drug that releases Ca2+ from internal Ca2+ stores, thapsigargin, were only slightly affected in allopurinol-treated cells. We conclude that the protection conferred by allopurinol on patients treated with SWL might involve a direct protection of the kidney cells by maintaining a normal resting [Ca2+]i.

A model for investigating effect of shock waves on intracellular calcium mobilization.

BACKGROUND: Shock wave lithotripsy (SWL) has become a non-invasive standard treatment for urolithiasis; however, it has some unwanted bioeffects. A cell model using calcium (Ca) imaging of cultured Madin-Darby canine kidney (MDCK) cells has been established in this laboratory to study the effects of shock waves on intracellular free Ca mobilization of renal tubular cells. METHODS: Digital video imaging of fura-2 fluorescence was used to measure both resting and stimulated intracellular free Ca concentrations in single cultured MDCK cells. Pharmacological agents including adenosine-5'-trisphosphate (ATP), bradykinin and thapsigargin, were used as Ca mobilizing agents. RESULTS: ATP, bradykinin and thapsigargin all elicited a robust transient increase in intracellular Ca concentration. CONCLUSIONS: A cell model was established to investigate the effect of shock waves on single kidney cells. This provided an opportunity to determine how shock waves affect the regulation of intracellular Ca concentrations in kidney cells; in addition, it allows investigation, for the first time at the single cell level, of whether blocking Ca entry in kidney cells plays any role in the mechanism by which some Ca channel blockers, e.g. nifedipine and verapamil, protect patients of urolithiasis from shock wave-induced renal damage.

Genetic basis of glycogen storage disease type 1a: prevalent mutations at the glucose-6-phosphatase locus.

Diagnosis of glycogen storage disease (GSD) type 1a currently is established by demonstrating the lack of glucose-6-phosphatase (G6Pase) activity in the patient's biopsied liver specimen. Recent cloning of the G6Pase gene and identification of mutations within the gene that causes GSD type 1a allow for the development of a DNA-based diagnostic method. Using SSCP analysis and DNA sequencing, we characterized the G6Pase gene of 70 unrelated patients with enzymatically confirmed diagnosis of GSD type 1a and detected mutations in all except 17 alleles (88%). Sixteen mutations were uncovered that were shown by expression to abolish or greatly reduce G6Pase activity and that therefore are responsible for the GSD type 1a disorder. R83C and Q347X are the most prevalent mutations found in Caucasians, 130X and R83C are most prevalent in Hispanics, and R83H is most prevalent in Chinese. The Q347X mutation has thus far been identified only in Caucasian patients, and the 130X mutation has been identified only in Hispanic patients. Our results demonstrate that the DNA-based analysis can accurately, rapidly, and noninvasively detect the majority of mutations in GSD type 1a. This DNA-based diagnosis now permits prenatal diagnosis among at-risk patients and serves as a database in screening and counseling patients clinically suspected of having this disease.