Methanol elevates cytosolic calcium ions in cultured canine cerebral vascular smooth muscle cells: possible relation to CNS toxicity.

Acute exposure of cultured canine cerebral vascular smooth muscle cells to methanol (10-400 mM) results in concentration-dependent elevation of the concentration of intracellular free calcium ion ([Ca2+]i) as measured with the fluorescent indicator, fura-2, and digital imaging microscopy. The resting level of [Ca2+]i in the cerebral vascular smooth muscle cells was 89.3+/-5.3 nM. Exposure of these cells to 10 mM methanol for only 5 min resulted in significant elevation in [Ca2+]i (i.e., to 105.7+/-4.6) (p < 0.05). Methanol (10 mM) is a concentration found in the blood of victims demonstrating early CNS toxicity. Other, higher concentrations of methanol rapidly raised [Ca2+]i upwards of 60% over basal resting levels. These result suggest that methanol-induced cerebral vasospasm is a consequence of large rises in intracellular Ca2+. These events could play a crucial role in methanol-induced cerebral edema, brain hemorrhage, and cerebral and retinal infarcts, eventuating in severe deficits in brain blood flow and the known, subsequent CNS disturbances.     

Effects of magnesium on cardiac excitation-contraction coupling

OBJECTIVE: Magnesium regulates a large number of cellular processes. Small changes in intracellular free Mg(2+) ([Mg(2+)](i)) may have important effects on cardiac excitability and contractility. We investigated the effects of [Mg(2+)](i) on cardiac excitation-contraction coupling. METHODS: We used our ionic-metabolic model that incorporates equations for Ca(2+) and Mg(2+) buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na(+)-K(+) pump, sarcolemmal and sarcoplasmic Ca(2+) pumps). RESULTS: Model results indicate that variations in cytosolic Mg(2+) level might sensitively affect diastolic and systolic Ca(2+), sarcoplasmic Ca(2+) content, Ca(2+) influx through L-type channels, efficiency of the Na(+)/Ca(2+) exchanger and action potential shape. The analysis suggests that the most important reason for the observed effects is a modified normal function of sarcoplasmic Ca(2+)-ATPase pump by altered diastolic MgATP levels. CONCLUSION: The model is able to reproduce qualitatively a sequence of events that correspond well with experimental observations during cardiac excitation-contraction coupling in mammalian ventricular myocytes.     

Acute ethanol treatment decreases intracellular calcium-ion transients in mouse single skeletal muscle fibres in vitro

Alcohol misuse frequently leads to muscle weakness, which may also occur in the setting of acute and chronic alcoholic myopathies. At the cellular level, ethanol has been found to interfere with signalling mechanisms in cardiac myocytes, skeletal myotubes, and smooth muscle cells. In this study, we focused on the effects of ethanol on the intracellular calcium ([Ca(2+)](i)) transients responsible for excitation-contraction (EC) coupling in isolated mouse skeletal fibres loaded with the fluorescent Ca(2+) indicator fura-2. Following electrical stimulation, ethanol caused a significant reversible dose-dependent reduction in [Ca(2+)](i) transient amplitude, already significant at 100 mM ethanol (P = 0.03), without modifying resting [Ca(2+)](i). Evaluating the potential loci for the effects of ethanol, we indirectly measured sarcolemmal Ca(2+) entry by monitoring Mn(2+)-quenching of intracellular fura-2 via the nitrendipine-sensitive Ca(2+) channels during electrical pacing. Ethanol at doses of 20 mM and greater caused a dose-dependent reduction in the rate of fura-2 quenching (all at P<0.05). Moreover, the intracellular pool of Ca(2+) releasable by caffeine was found to be reduced at a minimum of 300 mM ethanol (P = 0.05). We conclude that ethanol reduces the [Ca(2+)](i) transients underlying EC coupling in single mouse skeletal muscle fibres. This acute effect of ethanol was primarily due to an inhibitory effect of ethanol on sarcolemmal Ca(2+) influx via voltage-operated Ca(2+)-channels and, to a lesser extent, to a reduction in the Ca(2+) sarcoplasmic reticulum loading state. This inhibitory effect of ethanol may be implicated in the development of muscle weakness with alcohol consumption.     

Ethanol impairs CCK-8-evoked amylase secretion through Ca2+-mediated ROS generation in mouse pancreatic acinar cells.

In the present study, we have investigated the effect of ethanol on amylase release in response to cholecystokinin octapeptide (CCK-8). We have also studied the effect of ethanol on cytosolic free Ca(2+) concentration ([Ca(2+)](c)) and reactive oxygen species (ROS) production by loading of cells with fura-2 and 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H(2)DCFDA), respectively. Our results show that stimulation of pancreatic acinar cells with CCK-8 induced a dose-dependent amylase secretion, resulting in a maximum at 0.3nM of 19.39+/-2.73% of the total content of amylase. Treatment of pancreatic acini with ethanol did not induce any significant effect on amylase release at a wide range of concentrations (1-50mM). In contrast, incubation of cells with 50mM ethanol clearly reduced amylase release stimulated by CCK-8. The inhibitory effect of ethanol on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulfhydryl reducing agent. Ethanol induced an increase in [Ca(2+)](c) resulting in a level higher than the prestimulation level both in the presence and in the absence of extracellular Ca(2+). Additionally, ethanol led to an increase in fluorescence of CM-H(2)DCFDA, reflecting an increase in oxidation. A decrease in oxidation was observed in the absence of extracellular Ca(2+) and in the presence of ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid. Similarly, when the cells were challenged in the presence of the intracellular Ca(2+) chelator 1,2-Bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and in the absence of extracellular Ca(2+), the responses to ethanol were reduced, although not completely inhibited. Taken together, our results suggest that ethanol induces generation of ROS by a Ca(2+)-dependent mechanism and reduces CCK-8-evoked amylase secretion in exocrine pancreatic cells.     

Roles of tyrosine kinase-, 1-phosphatidylinositol 3-kinase-, and mitogen-activated protein kinase-signaling pathways in ethanol-induced contractions of rat aortic smooth muscle: possible relation to alcohol-induced hypertension.

Insights into the relations between and among ethanol-induced contractions in rat aorta, tyrosine kinases (including src family of cytoplasmic tyrosine kinases), 1-phosphatidylinositol 3-kinases (PI-3Ks), mitogen-activated protein kinases (MAPKs), and regulation of intracellular free Ca(2+) ([Ca(2+)](i)) were investigated in the present study. Ethanol-induced concentration-dependent contractions in isolated rat aortic rings were attenuated greatly by pretreatment of the arteries with low concentrations of an antagonist of protein tyrosine kinases (genistein), an src homology domain 2 (SH2) inhibitor peptide, a highly specific antagonist of p38 MAPK (SB-203580), a potent, selective antagonist of two specific MAPK kinases-MEK1/MEK2 (U0126)-and a selective antagonist of mitogen-activated protein kinase kinase (MAPKK) (PD-98059), as well as by treatment with wortmannin or LY-294002 (both are selective antagonists of PI-3Ks). Inhibitory concentration 50 (IC(50)) levels obtained for these seven antagonists were consistent with reported inhibition constant (Ki) values for these tyrosine kinase, MAPK, and MAPKK antagonists. Ethanol-induced transient and sustained increases in [Ca(2+)](i) in primary single smooth muscle cells from rat aorta were markedly attenuated in the presence of genistein, an SH2 domain inhibitor peptide, SB-203580, U0126, PD-98059, wortmannin, and LY-294002. A variety of specific antagonists of known endogenously formed vasoconstrictors did not inhibit or attenuate either the ethanol-induced contractions or the elevations of [Ca(2+)](i). Results of the present study support the suggestion that activation of tyrosine kinases (including the src family of cytoplasmic tyrosine kinases), PI-3Ks, and MAPK seems to play an important role in ethanol-induced contractions and the elevation of [Ca(2+)](i) in smooth muscle cells from rat aorta. These signaling pathways thus may be important in hypertension in human beings associated with chronic alcohol consumption.     

溴氰菊酯对原代培养大鼠星形胶质细胞存活率和胞内游离钙浓度的影响

目的　观察溴氰菊酯 (DM)对原代培养大鼠神经星形胶质细胞存活率及胞内游离钙离子浓度 ([Ca2 + ]i)的影响。方法　以锥虫蓝染料排斥试验检测细胞染DM后存活率的变化 ;以Fura 2 /AM为荧光指示剂 ,RF 5 30 1PC荧光分光光度计测定细胞内 [Ca2 + ]i的变化。结果　 1× 10 -5mol/L组染毒DM 72h后细胞存活率下降为 91.9% ,与对照组的差异有显著性 (P <0 .0 5 ) ,1× 10 -4mol/L组DM染毒4、12、4 8、72h后细胞存活率分别为 89.0 %、84 .8%、81.2 %和 79.2 % ,差异有显著性 (P <0 .0 1) ;1× 10 -7、1× 10 -6、1× 10 -5mol/L组DM染毒 5min后胞内 [Ca2 + ]i分别上升至 (45 1.4± 4 2 .3)、(5 36 .9± 4 7.5 )、(870 .9± 10 0 .5 )nmol/L ,与染毒前及对照组比 ,差异有显著性 (P <0 .0 1)。以后各染毒组胞内 [Ca2 + ]i逐渐下降 ,15min时 1× 10 -8、1× 10 -7、1× 10 -6、1× 10 -5mol/L染毒组胞内 [Ca2 + ]i分别降至 (12 4 .3± 6 .0 )、(131.3± 19.1)、(118.9± 1.4 )、(136 .6± 3.8)nmol/L ,与染毒前及对照组比差异均有显著性 (P <0 .0 5或P <0 .0 1)。结论　DM在体外可降低神经胶质细胞的存活率并短时升高胞内 [Ca2 + ]i。     

50-Hertz magnetic field and calcium transients in Jurkat cells: results of a research and public information dissemination (RAPID) program study

An effect on intracellular calcium continues to be proposed as a biochemical pathway for the mediation of biologic effects of electrical-power-frequency magnetic fields (MF). However, reproducible results among laboratories are difficult to attain and the characteristics of magnetic field effects on intracellular free calcium ([Ca(2+)](i)) are not well understood. We attempted to repeat the studies of Lindström et al. [Intracellular Calcium Oscillations in a T-Cell Line by a Weak 50 Hz Magnetic Field. J Cell Physiol 156:395-398 (1993)] by investigating the effect of a 1.5-G 50-Hz MF on [Ca(2+)](i) in the Jurkat lymphocyte T-cell line. Changes in [Ca(2+)](i) were determined using microscopic imaging of fura-2 loaded Jurkat cells on poly-l-lysine-coated glass coverslips. The MF was generated by a single coil constructed with bifilar wire and located in the same plane as the cells. Cells were randomly exposed for 8 min to MF, sham field (SF), or no field (NF) conditions. The exposure condition remained coded until data analysis was complete. Each exposure period was preceded by an 8-min data collection to establish a baseline for [Ca(2+)](i). After each exposure condition, cells were exposed to anti-CD3 antibody that induced a rapid increase in [Ca(2+)](i) in responsive cells; this provided a positive control. [Ca(2+)](i) was analyzed for individual cells as spatially-averaged background-corrected 340/380 nm ratios, and a [Ca(2+)](i) transient was considered significant for positive deviations from baseline of 3 [multiple] an estimate of noise in the baseline. Typically, 25-50 cells/field were viewed and approximately 50% had no [Ca(2+)](i) transients in the baseline period and also responded to positive control. Only cells responding to positive control and lacking changes in [Ca(2+)](i) during the baseline period were considered qualified for assessment during the exposure period. The incidences of [Ca(2+)](i) transients during the exposure period for two experiments (40 [multiple] objective) were 16.5, 14.6, and 14.2% for MF, SF, and NF, respectively, and were not statistically significantly different. Previous studies by Lindström et al. [Intracellular Calcium Oscillations in a T-Cell Line after Exposure to Extremely-Low-Frequency Magnetic Fields with Variable Frequencies and Flux Densities. Bioelectromagnetics 16:41-47 (1995)] showed a high response rate (92%) for exposure to 1. 5-G 50-Hz MF when individual cells were preselected for investigation. We found no such effect when examining many cells simultaneously in a random and blind fashion. These results do not preclude an effect of MF on [Ca(2+)](i), but suggest that responsive cells, if they exist, were not identified using the approaches that we used in this study.     

Cation modulation of GABA(A) receptors in brain sections of AT and ANT rats.

Changes in magnesium ion (Mg(2+)) concentration may be implicated in alcohol-related behaviors through modulation of neuronal excitability by actions on ligand-gated ion channels. To study whether putative Mg(2+)-binding sites differ between two rat lines, alcohol-insensitive (AT) and alcohol-sensitive (ANT) rats, selectively outbred for differential sensitivity to the motor-impairing effect of ethanol, we compared the effect of Mg(2+) on [35S]tert-butylbicyclophosphorothionate ([35S]TBPS) binding to GABA(A) receptors with the use of ligand autoradiographic analyses of brain sections from these rats. There were some slight differences between the rat lines in modulation of the binding in the forebrain. A low concentration of Mg(2+) (0.1 mM) inhibited basal [35S]TBPS binding more efficiently in the central gray matter and hippocampus in the ANT rats than in the AT rats. In the presence of gamma-aminobutyric acid, the effect of a low concentration of Mg(2+) was higher in the caudate-putamen and inner layer of the cerebral cortex in the AT rats than in the ANT rats. No difference between the rat lines was found at a higher (3 mM) Mg(2+) concentration. Furosemide, a GABA(A) antagonist selective for cerebellar granule cell-specific alpha6beta2/3 subunit-containing receptors, was less efficient in antagonizing the Mg(2+)-induced inhibition of [35S]TBPS binding in the ANT rats than in the AT rats. Another divalent cation, zinc ion, was less efficient in displacing [35S]TBPS binding from the cerebellar granule cell layer in the ANT rats than in the AT rats, whereas a trivalent cation, lanthanum ion, produced identical modulation of the binding in the two rat lines. The results indicate that the alcohol-sensitive ANT rats have altered cerebellar granule cell--specific alpha6 subunit--containing GABA(A) receptors and seem to indicate that these receptors might be implicated in the sensitivity difference of the rat lines to ethanol and sedative drugs.     

Fura-2 measurement of cytosolic free calcium in rat brain cortical synaptosomes and the influence of ethanol

The effects of ethanol on resting and KCl-depolarized cytosolic calcium concentrations ([Ca2+]i) were measured in purified rat cortical synaptosomes using the calcium indicator fura-2. Ethanol (500-700 mM) significantly elevated the resting [Ca2+]i by 13-25% in the presence of 100 microM external calcium. In the absence of external Ca2+, ethanol (50-200 mM) significantly increased [Ca2+]i by 17-23%. Ethanol (200-700 mM) also significantly decreased KCl-induced rise in [Ca2+]i (delta K) by 40-82% in fura-2 loaded preparations incubated in the absence or presence of external calcium. Ethanol did not produce a significant change in delta K at 50 and 100 mM concentrations. These results suggest that ethanol may cause an elevation in [Ca2+]i via mobilization of intracellular calcium stores which may be linked to a calcium-dependent inactivation of voltage-sensitive calcium channels.     

Importance of extracellular Ca2+ and intracellular Ca2+ release in ethanol-induced contraction of cerebral arterial smooth muscle.

The present study was designed to investigate the roles of extracellular Ca2+ ([Ca2+]0) influx and intracellular free Ca2+ ([Ca2+]i) release in ethanol-induced contractions of isolated canine cerebral arteries and primary cultured, cerebral vascular smooth muscle cells. Ethanol (20-200 mM) produced significant contractions in isolated canine basilar arterial rings in a concentration-dependent manner. Removal of [Ca2+]0 and pretreatment of canine basilar arterial rings with verapamil (an antagonist of voltage-gated Ca2+ channels), thapsigargin (a selective antagonist of the sarcoplasmic reticulum Ca2+ pump), caffeine plus ryanodine (a specific antagonist of ryanodine-sensitive Ca2+ release), or heparin (an inositol 1,4,5,-trisphosphate [InsP3]-mediated Ca2+ release antagonist) markedly attenuated (approximately 50%-80%) ethanol-induced contractions. The absence of [Ca2+]0 and preincubation of primary single smooth muscle cells obtained from canine basilar arteries with verapamil, thapsigargin, heparin, or caffeine plus ryanodine markedly attenuated (approximately 50%-80%) the transient and sustained elevations in [Ca2+]i induced by ethanol. Results of the present study suggest to us that both Ca2+ influx through voltage-gated Ca2+ channels and Ca2+ release from intracellular stores (both InsP3 sensitive and ryanodine sensitive) are required for ethanol-induced contractions of isolated canine basilar arteries.     

Effects of quercetin and rutin on vascular preparations

BACKGROUND: Several studies have indicated that quercetin promotes relaxation of vascular smooth muscle both in vivo and in vitro. However, Saponara et al. [(2002) Br J Pharmacol 135: 1819-1827] have demonstrated that quercetin is an activator of vascular L-type Ca(2+) channels. AIM OF THE STUDY: We investigated the mechanical and electrophysiological properties of quercetin and its rutoside, rutin, in an attempt to clarify how Ca(2+) channel activation might be related to the myorelaxing activity. METHODS: Aorta ring preparations and single tail artery myocytes were employed for functional and patch-clamp experiments, respectively. RESULTS: Rutin was found to relax intact rat aorta rings, which had been precontracted with phenylephrine (pIC(50) = 5.65 +/- 0.31) but in contrast had no effect on depolarised (60 mM K(+)) preparations or on those from which the endothelium had been removed. Furthermore, rutin did not affect L-type Ca(2+) current recorded in rat tail artery myocytes. The quercetin-induced relaxation of intact rings precontracted with phenylephrine exhibited two components characterised by 6.23 +/- 0.38 and 4.66 +/- 0.09 pIC(50), respectively. Removal of the endothelium abolished the first component, leaving the second unaltered. Moreover, quercetin was found to relax 60 mM K(+) depolarised rings with a pIC(50) of 4.59 +/- 0.03. The application of quercetin in isolated smooth muscle cells brought about a marked increase of L-type Ca(2+) current (pEC(50) = 5.09 +/- 0.05). Unlike quercetin, Bay K 8644 contracted aorta rings preincubated with 10, 20 or 30 mM K(+). The myotonic effect of Bay K 8644 was observed both in the absence or presence of 30 microM quercetin. The application of Bay K 8644 (10-100 nM) caused a further significant increase in L-type Ca(2+) current in rat tail artery myocytes stimulated with 30 microM quercetin. CONCLUSIONS: Quercetin is a naturally occurring L-type Ca(2+) channel agonist. This effect, however, is overwhelmed by quercetin-induced vasorelaxation taking place via pathways which are more relevant than L-type Ca(2+) influx in the hierarchy of functional competencies.     

溴氰菊酯对大鼠神经细胞胞内游离钙浓度及凋亡的影响

目的　观察溴氰菊酯 (DM )对神经细胞游离钙浓度 ([Ca2 + ]i)及凋亡的影响。方法　Wistar大鼠随机分为对照组与 3个染毒组 ,染毒组腹腔注射 1 2 .5mg/kg的DM ;以Fura 2 /AM为荧光指示剂 ,RF 530 1PC荧光分光光度计测定染毒后 5、2 4、48h后大鼠神经细胞 [Ca2 + ]i浓度的变化 ;FACS42 0型流式细胞仪测定细胞凋亡率。结果　PM染毒 5、2 4、48h组大鼠海马及皮层细胞 [Ca2 + ]i分别为 :5h组海马 :(389.94± 43 .64)nmol/L、皮层 :(449.33± 2 3 .2 3)nmol/L ;2 4h组海马 :(340 .47±32 .36)nmol/L、皮层 :(31 1 .62± 2 5 .48)nmol/L ;48h组海马 (2 87.1 3± 2 4 .2 9)nmol/L、皮层 :(346 .55±36 .87)nmol/L ,均高于对照组 [海马 :(2 0 3 .2 4± 1 8.53)nmol/L、皮层 :(2 2 6 .85± 1 4 .81 )nmol/L] ,差异有显著性 (P <0 .0 1 )。染毒 48h组大鼠此二项指标值较 5h组明显下降 ,差异有显著性 (P <0 .0 5)。染毒 2 4、48h组大鼠神经细胞凋亡率 [海马 :(8.45± 1 .0 2 ) %、(9.44± 1 .1 4 ) % ,皮层 :(7.90± 0 .49) %、(8.0 1± 0 .87) % ]均明显高于对照组 [海马 :(2 .97± 0 .36) %、皮层 :(3 .50± 0 .48) % ] ,差异有显著性 (P<0 .0 1 ) ,且有时间 反应关系 (r=0 .940、0 .893)。结论　DM可影响大鼠神     

Opposing effects of ethanol and nicotine on hippocampal calbindin-D28k expression.

Long-term ethanol exposure produces multiple neuroadaptations that likely contribute to dysregulation of Ca(2+) balance and neurotoxicity during ethanol withdrawal. Conversely, nicotine exposure may reduce the neurotoxic consequences of Ca(2+) dysregulation, putatively through up-regulation of the Ca(2+)-buffering protein calbindin-D(28k). The current studies were designed to examine the extent to which 10-day ethanol exposure and withdrawal altered calbindin-D(28k) expression in rat hippocampus. Further, in these studies, we examined the ability of nicotine, through action at alpha(7)(*)-bearing nicotinic acetylcholine receptors (nAChRs), to antagonize the effects of ethanol exposure on calbindin-D(28k) expression. Organotypic cultures of rat hippocampus were exposed to ethanol (50-100 mM) for 10 days. Additional cultures were exposed to 500 nM (-)-nicotine with or without the addition of 50 mM ethanol, 100 nM methyllycaconitine (an alpha(7)*-bearing nAChR antagonist), or both. Prolonged exposure to ethanol (>/=50 mM) produced significant reductions of calbindin-D(28k) immunolabeling in all regions of the hippocampal formation, even at nontoxic concentrations of ethanol. Calbindin-D(28k) expression levels returned to near-control levels after 72 h of withdrawal from 10-day ethanol exposure. Extended (-)-nicotine exposure produced significant elevations in calbindin-D(28k) expression levels that were prevented by methyllycaconitine co-exposure. Co-exposure of cultures to (-)-nicotine with ethanol resulted in an attenuation of ethanol-induced reductions in calbindin-D(28k) expression levels. These findings support the suggestion that long-term ethanol exposure reduces the neuronal capacity to buffer accumulated Ca(2+) in a reversible manner, an effect that likely contributes to withdrawal-induced neurotoxicity. Further, long-term exposure to (-)-nicotine enhances calbindin-D(28k) expression in an alpha(7)* nAChR-dependent manner and antagonizes the effects of ethanol on calbindin-D(28k) expression.     

Enhanced [3H]-noradrenaline release in synaptosomes from ethanol-tolerant animals: the role of nerve terminal calcium ion concentrations

Cortical slices from brains of ethanol-tolerant rats have previously been shown to release a greater fraction of uptake [3H]-noradrenaline ([3H]-NA) on K+-depolarisation than slices from control animals. In the present experiments, when stimulated with the Ca2+ ionophore A23187 (30 microM), untreated cortical synaptosomes release only a very small fraction of uptaken [3H]-NA. Under these conditions synaptosomal preparations from ethanol-tolerant animals released a greater fraction of uptaken [3H]-NA but the difference was not significant. However, after incubation with ouabain (100 microM, to increase intrasynaptosomal [Ca2+]) A23187 now produced a much greater enhancement of [3H]-NA release in preparations from ethanol-tolerant rats. Further, after superfusion of the synaptosomal preparation with 1mM EGTA and A23187 for 30 min (a procedure which should reduce intrasynaptosomal [Ca2+]) the reintroduction of Ca2+ to the superfusing fluid caused a marked release of [3H]-NA which was also significantly greater in preparations from ethanol-tolerant animals. Ca2+/Mg2+-ATPase activity was also higher in these synaptosomes but no difference could be detected in the release of [3H]-NA from a combined synaptic vesicle: synaptic plasma membrane preparation. The results suggest that the development of ethanol tolerance is associated with a fundamental change in the dynamic control of Ca2+ concentrations within the nerve terminal which potentiates the depolarisation-induced release of neurotransmitters.     

Predicting acute zinc toxicity for Daphnia magna as a function of key water chemistry characteristics: development and validation of a biotic ligand model

The individual effect of different major cations (Ca2+, Mg2+, Na+, K+, and H+) on the acute toxicity of zinc to the waterflea Daphnia magna was investigated. The 48-h median effective concentration (EC50) in the baseline test medium (i.e., a standard medium with very low ion concentrations) was about 6 microM (Zn2+). An increase of Ca2+ (from 0.25 mM to 3 mM), Mg2+ (from 0.25 mM to 2 mM), and Na+ activity (from 0.077 mM to 13 mM) reduced zinc toxicity by a factor of 6.3, 2.1, and 3.1, respectively. No further toxicity reduction was observed when Ca2+ and Mg2+ activities exceeded 3.0 and 2.0 mM, respectively. Both K+ and H+ did not significantly alter zinc toxicity (expressed as Zn2+ activity). From these data, conditional stability constants for Ca2+ (log K = 3.24), Mg2+ (log K = 2.97), Na+ (log K = 2.16), and Zn2+ (log K = 5.31) were derived and incorporated into a biotic ligand model (BLM) predicting acute zinc toxicity to D. magna in surface waters with different water quality characteristics. Validation of the developed BLM using 17 media with different pH, hardness, and dissolved organic carbon (DOC) content resulted in a significant correlation coefficient (R2 = 0.76) between predicted and observed 48-h EC50. Eighty-eight percent of the predictions were within a factor of 1.3 of the observed 48-h EC50.     

Synthesis and biological evaluation of new 4H-pyrano[2,3-b]quinoline derivatives that block acetylcholinesterase and cell calcium signals, and cause neuroprotection against calcium overload and free radicals

The synthesis and biological evaluation of ethyl 5-amino-4-(3-pyridyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinoline-3-carboxylates (9-11) is described. We have found that these compounds inhibit AChE with a mild potency, mitigates the [Ca(2+)](c) triggered by high K(+), and cause neuroprotection against Ca(2+) overloading and free radical-induced neuronal death.     

Mechanism of cell swelling of HeLa cells in an isosmotic Na(+)-free, high-K+ medium: study on the K+ transport pathways.

The K+ content and the free Ca2+ concentration ([Ca2+]c) in HeLa cells began to increase after a latent period following medium change to an isosmotic Na(+)-free, high-K+ medium in the presence of 10-100 microM ouabain. These increases were accompanied by cell swelling, and stimulated by the addition of 1 microM ionomycin, whereas treatment with 1 mM quinine significantly inhibited the net K+ uptake. [Ca2+]c was slightly decreased, but the K+ uptake was unaffected in the Ca(2+)-free, high-K+ medium. However, when [Ca2+]c was extremely reduced by preloading 5 microM BAPTA-AM, the K+ uptake decreased to a level attained in the presence of quinine. Addition of DIDS and substitution with NO3- or SCN- for medium Cl- inhibited the K+ uptake to similar extents, and their effects were less strong than that of quinine. Substitution of medium Cl- with an impermeable anion gluconate completely inhibited the K+ uptake. Therefore, we conclude that cell swelling in the high-K+ medium is associated with K+ uptake mediated in greater part by quinine-sensitive and in smaller part by -insensitive pathway. The former pathway depends on cellular Ca2+ and its major component is Cl(-)-dependent, whereas the latter is independent of the Ca2+ and unselective for anions.     

Association of alcohol in brain injury, headaches, and stroke with brain-tissue and serum levels of ionized magnesium: a review of recent findings and mechanisms of action.

Although there is general agreement that chronic ingestion of alcohol poses great risks for normal cardiovascular functions and peripheral-vascular homeostasis, a direct cause and effect between the real phenomena of alcohol-induced headache and risk of brain injury and stroke is not appreciated. "Binge drinking" of alcohol is associated with an ever-growing number of strokes and sudden death. It is becoming clear that alcohol ingestion can result in profoundly different actions on the cerebral circulation (e.g., vasodilation, vasoconstriction-spasm, vessel rupture), depending upon dose and physiologic state of host. Using rats, it has been demonstrated that acute, high doses of ethanol can result in stroke-like events concomitant with alterations in brain bioenergetics. We review recent in vivo findings obtained with 31P-NMR spectroscopy, optical reflectance spectroscopy, and direct in vivo microcirculatory studies on the intact brain. Alcohol-induced hemorrhagic stroke is preceded by a rapid fall in brain intracellular free magnesium ions ([Mg2+]i) followed by cerebrovasospasm and reductions in phosphocreatine (PCr)/ATP ratio, intracellular pH, and the cytosolic phosphorylation potential (CPP) with concomitant rises in deoxyhemoglobin (DH), mitochondrial reduced cytochrome oxidase aa3 (rCOaa3), blood volume, and intracellular inorganic phosphate (Pi). Using osmotic mini-pumps implanted in the third cerebral ventricle, containing 30% ethanol, it was found that brain [Mg2+]i is reduced 30% after 14 days; brain PCr fell 15%, whereas the CPP fell 40%. Such animals became susceptible to stroke from nonlethal doses of ethanol. Human subjects with mild head injury have been found to exhibit early deficits in serum ionized Mg (IMg2+); the greater the degree of early head injury (30 min-8 h), the greater and more profound the deficit in serum IMg2+ and the greater the ionized Ca (ICa2+) to IMg2+ ratio. Patients with histories of alcohol abuse or ingestion of alcohol prior to head injury exhibited greater deficits in IMg2+ (and higher ICa2+/IMg2+ ratios) and, unlike the subjects without alcohol, did not leave the hospital for at least several days. Women, for some unknown reason, exhibit a much higher incidence of morbidity and mortality from subarachnoid hemorrhage (SAH) than men. Data on 105 men and women with different types of stroke indicate that, on the average, a 20% deficit in serum IMg2+ is seen; total Mg (TMg) or blood pH is usually near normal. Women with SAH, however, exhibit much lower IMg2+ and higher ICa2+/IMg2+ ratios; the presence of ethanol in the blood is associated with even more depression in IMg2+ in SAH in women. It is possible that prior alcohol ingestion is, in large measure, responsible for a great deal of this unexplained higher incidence of SAH in women. It has recently been reported that the cyclical changes in estrogenic hormones appear to control the serum IMg2+ level in young women. A surge in estrogenic levels prior to SAH could thus precipitate, in part, the SAH. In other human studies, it has been shown that migraines and headache, dizziness, and hangover, which accompany ethanol ingestion, are associated with rapid deficits in serum IMg2+ but not in TMg. The former, and the alcohol-associated headache, can be ameliorated with IV administration of MgSO4. Premenstrual tension-headache (PTH) and its exacerbation by alcohol in women is also accompanied by deficits in IMg2+, and elevation in serum ICa2+/IMg2+; IV MgSO4 corrects the PTH and the serum deficit in IMg2+. Animal experiments show that IV Mg2+ can prevent alcohol-induced hemorrhagic stroke and the subsequent fall in brain [Mg2+]i, [PCr], pHi, and CPP. Other recent data indicate that alcohol-induced cellular loss of [Mg2+]i is associated with cellular Ca2+ overload and generation of oxygen-derived free radicals; chronic pretreatment with vitamin E prevents alcohol-induced vascular injury and pathology in the brain. (ABSTRACT TRUNCATED)     

EPC syntheses and structure-activity relationships of hypoglycaemic semicyclic amidines

A series of homochiral sterically hindered mono- and bicyclic amidines was prepared as hypoglycaemic agents by lethargic reaction of O-methylcaprolactim and 3-ethoxy-2-azabicyclo[2.2.2]oct-2-ene, respectively, with homochiral cis-2-substituted cyclopentane amines provided by asymmetrical reductive amination of racemic 2-substituted cyclopentanones. All compounds, except the cyclohexylmethyl-isoquinuclidone derivative which inhibited secretion at 100 microM, significantly stimulated insulin secretion 2-8-fold at 10 microM and 100 microM in INS-1 cells. The most potent activator was the 2-cyclopentyl-substituted caprolactam derivative 5e. The stimulatory effects on secretion increased with rising steric hindrance of both the amidine alpha-carbon and the bicyclic amidine moiety itself. Enantiomeric discrimination was observed for the 2-?(cis-2-bulkysubstituted cyclopentyl)iminohexahydroazepine halides 5e and 5f and for the 3-?(cis-2-substituted cyclopentyl)imino-2-azabicyclo?2.2.2?ctane halides 6a and 6c. The amidines depolarized INS-1 cells and generated action potentials, accompanied by a decrease of membrane conductance. Simultaneously [Ca(2+)](i) increased, probably due to Ca(2+)-entry through voltage-dependent Ca(2+)-channels. At high concentrations, where inhibition of secretion was observed, ?Ca(2+)(i) still rose upon application of the amidines, indicating an additional inhibitory pathway downstream to the elevation of ?Ca(2+)(i). Even at high concentrations (100 microM), the amidines had no toxic effects on insulin secreting INS-1 cells.     

Synergistic effect of 1,25-dihydroxyvitamin D3 and retinoic acid in inducing U937 cell differentiation.

We examined the effects of retinoic acid (RA), 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), and its synthetic analogue, 22-oxa-1,25-(OH)2D3, on differentiation of U937 cells by studying the cellular growth, surface marker expression and cytosolic free Ca2+ concentration ([Ca2+]i). RA inhibited cellular growth but did not induce expression of Mo2 (CD14), a monocyte/macrophage specific surface marker. To the contrary, 1,25-(OH)2D3 did not inhibit cellular growth, but increased CD 14-positive cells. Simultaneous addition of 1,25-(OH)2D3 and RA had no additive effect on cellular growth inhibition or CD14 expression. With regard to [Ca2+]i, however, 5 days' incubation with either of them increased the basal [Ca2+]i level and induced U937 cells to respond to formyl-methionyl-leucyl-phenylalanine (FMLP). When the cells were incubated with both 10(-6) M RA and 10(-8) M 1,25-(OH)2D3, basal [Ca2+]i was higher and FMLP caused a greater increase in [Ca2+]i than when only RA or 1,25-(OH)2D3 was added. These data suggest that RA and 1,25-(OH)2D3 induce monocytoid differentiation in U937 cells through different pathways and act synergistically in the differentiation process. The 22-oxa-1,25-(OH)2D3 induced CD14 expression, basal [Ca2+]i increase and [Ca2+]i response to FMLP, but did not cause cellular growth inhibition in U937 cells, and in these points, 22-oxa-1,25-(OH)2D3 exhibited no significantly different effects from 1,25-(OH)2D3. Thus, 22-oxa-1,25-(OH)2D3 has the same potent activity as 1,25-(OH)2D3 in inducing differentiation of U937 cells.