Synthesis and pharmacological evaluation of 2, 3-dihydro-3-oxo-4H-thieno[3,4-e][1,2,4]thiadiazine 1,1-dioxides as voltage-dependent calcium channel blockers

The synthesis of a novel series of 2,3-dihydro-3-oxo-4H-thieno[3, 4-e][1,2,4]thiadiazine 1,1-dioxides and their pharmacological evaluation as drugs with effects on the rat cardiovascular system are described. The compounds under study were synthesized via Curtius rearrangement of appropriate sulfamoylacylazides which, in turn, were prepared from known starting materials. In isolated rat portal vein, these thienothiadiazines, like verapamil and diazoxide, inhibited the spontaneous motility produced by KCl (20 mM). In addition, the new compounds, like verapamil and unlike diazoxide, also exhibited inhibitory effects in the same preparation when the cell membrane was depolarized by an increased extracellular KCl concentration (80 mM) and, consequently, the membrane potential approached a level close to the K(+) equilibrium potential. Further characterization of this inhibitory activity led to the identification of a selective inhibitory effect of the new compounds on KCl (80 mM)-induced 45Ca(2+) uptake in the same vascular tissue. When tested in vivo (anaesthetized normotensive rats), acute administration of verapamil, diazoxide and some of the most in vitro potent compounds in 45Ca(2+) uptake experiments produced a gradual, dose-dependent and sustained decrease in diastolic arterial blood pressure, devoid of cardiac effects. These results suggest that, like verapamil, the cardiovascular effects produced by the new thienothiadiazines seem to be due, at least in part, to a blockade of transmembrane voltage-dependent calcium channels present in vascular smooth muscle cells and not to an activation of ATP-sensitive K(+) channels. Compounds 5b, 5e and 5i have been selected for further studies as antihypertensive agents.     

Calcium signaling and cytotoxicity

The divalent calcium cation Ca(2+) is used as a major signaling molecule during cell signal transduction to regulate energy output, cellular metabolism, and phenotype. The basis to the signaling role of Ca(2+) is an intricate network of cellular channels and transporters that allow a low resting concentration of Ca(2+) in the cytosol of the cell ([Ca(2+)]i) but that are also coupled to major dynamic and rapidly exchanging stores. This enables extracellular signals from hormones and growth factors to be transduced as [Ca(2+)]i spikes that are amplitude and frequency encoded. There is considerable evidence that a number of toxic environmental chemicals target these Ca(2+) signaling processes, alter them, and induce cell death by apoptosis. Two major pathways for apoptosis will be considered. The first one involves Ca(2+)-mediated expression of ligands that bind to and activate death receptors such as CD95 (Fas, APO-1). In the second pathway, Ca(2+) has a direct toxic effect and its primary targets include the mitochondria and the endoplasmic reticulum (ER). Mitochondria may respond to an apoptotic Ca(2+) signal by the selective release of cytochrome c or through enhanced production of reactive oxygen species and opening of an inner mitochondrial membrane pore. Toxic agents such as the environmental pollutant tributyltin or the natural plant product thapsigargin, which deplete the ER Ca(2+) stores, will induce as a direct result of this effect the opening of plasma membrane Ca(2+) channels and an ER stress response. In contrast, under some conditions, Ca(2+) signals may be cytoprotective and antagonize the apoptotic machinery.     

Effects of multiglycosides of Tripterygium wilfordii (GTW) on rat fertility and Leydig and Sertoli cells.

Primary cultures of rat Leydig and Sertoli cells were used to evaluate the direct effects of GTW on testicular cells and to compare these to the effects of gossypol acetate. Both GTW and gossypol acetate can affect the survival of Leydig and Sertoli cells. But Sertoli cells are much more sensitive than Leydig cells, either to gossypol acetate or GTW. Leydig and Sertoli cells all died when they were exposed to gossypol acetate or GTW at a dose of 3.0 micrograms/ml or 30 micrograms/ml, respectively, for 24 hours. The cell survival-time course demonstrated that the cell numbers were decreased after 2 hours, and especially so after 8 hours. No significant changes were observed in testosterone production in Leydig cells after 24 hours of exposure to 1.0-20 micrograms/ml GTW. The forward motility of epididymal spermatozoa was completely lost and fertility of rat was significantly inhibited after the treatment of GTW in vivo. It is concluded that GTW does affect the fertility of rat and viability of cultured rat Leydig and Sertoli cells.     

甲基汞对大脑皮层神经细胞内游离钙的影响

目的　研究甲基汞对急性分离的大脑皮层神经细胞内游离Ca2 水平的影响。方法　采用钙离子指示剂Fura 2双波长荧光检测技术。结果　 (0 10～ 5 0 0 )× 10 -6mol/L甲基汞可使大脑皮层神经细胞内游离Ca2 水平显著升高 ,且有剂量 效应关系。甲基汞升高神经细胞内游离Ca2 的作用与胞外Ca2 大量内流和胞内储Ca2 释放有关 ,且以胞外Ca2 内流为主。胞外Ca2 内流与N 甲基 D 天冬氨酸受体门控Ca2 通道以及电压门控Ca2 通道有关 ,而与电压依赖性Na 通道无关。结论　甲基汞可使神经细胞内游离Ca2 浓度异常升高 ,且与Ca2 通道有密切关系。     

A relationship between estrogenicity and antifertility activity of 1-diphenylmethylenyl-2-methyl-3-ethyl-4-acetoxycyclohexane (ORF 8511) and similar nonsteroidal anti-implantive agents

Several nonsteroidal estrogens, such as ORF 3858 and F6103, which inhibit pregnancy in experimental animals when given postcoitally, have been previously described. ORF 8511 (1-diphenylmethylenyl-2-methyl-3-ethyl-4-acetoxycyclohexane) which is structurally similar to these compounds was studied for its postcoital antifertility activity and estrogenicity in rats, hamsters, mice and rabbits. Results of these studies suggest a relationship between these two biological endpoints. ORF 8511 produced uterotropic stimulation in the rat at microgram doses and totally inhibited implantation at 250 μg/kg/day administered on days 1–6 of pregnancy. However, the other species were considerably less sensitive to the compound with respect to both parameters. The compound stimulated tubal transport in rats at its minimum effective dose for antifertility activity as did diethylstilbestrol, a known estrogen. In the hamster, a species relatively insensitive to the antifertility effect of ORF 8511, endogenous estrogen titres during early pregnancy were higher than those in the rat. These data suggest that ORF 8511 and similar nonsteroidal compounds may owe their postcoital antifertility activity to their estrogenicity and that estrogens may act as pharmacological agents only in species with low normal endogenous estrogen titres.     

Effect of cadmium on transmembrane Na+ and K+ transport systems in human erythrocytes.

The effects of cadmium (Cd2+) on Na+,K(+)-ATPase in disrupted human erythrocyte membranes and on various transmembrane Na+ and K+ transport systems in intact erythrocyte suspensions were studied. Cadmium2+ inhibited the erythrocyte Na+,K(+)-ATPase enzyme with a 50% inhibition at a Cd2+ concentration of 6.25 microM. The Cd2+ inhibition in the human erythrocyte was non-competitive with respect to Na+,K+, and ATP. Cadmium2+ exerted no acute effect, however, on the Na+,K(+)-ATPase pump activity as measured by the ouabain sensitive 86Rb uptake or Na+ efflux in intact red blood cells. Cadmium2+ also inhibited the Ca2+ dependent K+ channels in human red blood cells, whereas it had no effect on Na+,K+ cotransport, Na+,Li+ countertransport, anion carrier, and the number of active Na+ pump units. The data indicate that in human erythrocytes under acute conditions Cd2+ exerts an inhibitory effect on Na+,K(+)-ATPase enzyme in disrupted erythrocytes and the Ca2+ stimulated K+ efflux in intact red blood cells without affecting the Na+ pump, Na+,K+ cotransport, and Na+,Li+ countertransport activity.     

Spermicidal efficacy of H2-receptor antagonists and potentiation with 2', 4'-dichlorobenzamil hydrochloride: role of intrasperm Ca2+

The present investigation was designed to study the possible role of intrasperm Ca(2+) in spermicidal action of H(2)-receptor antagonists. Influence of commonly used H(2)-receptor antagonists cimetidine, ranitidine and famotidine on sperm viability and intrasperm Ca(2+) was evaluated in ejaculated human semen samples. All these drugs were found to reduce sperm viability in a dose- and time-dependent manner. This action was accompanied with elevation of intrasperm Ca(2+). 2', 4'-dichlorobenzamil hydrochloride (DBZ), a Na(+)-Ca(2+) exchange inhibitor, that is known to elevate intrasperm Ca(2+), potentiated the spermicidal action of H(2)-receptor antagonists. Intrasperm Ca(2+) was found to rise at much faster rate when DBZ was combined with any of the three H(2)-receptor antagonists. Due to this, the maximum Ca(2+) level required to produce death of sperm cells was attained much earlier as compared to the per se effect of any one of these drugs. These results suggest that elevation of intrasperm Ca(2+) by H(2)-receptor antagonists plays a key role in influencing sperm viability.     

Ethanol and voltage- or receptor-mediated increases in cytosolic Ca2+ in brain cells.

Dissociated brain cells were isolated from newborn rat pups and loaded with fura-2. Different mechanisms for stimulating increased free intracellular Ca2+ concentrations [( Ca2+]i) were examined in the absence and presence of ethanol. KCl, carbachol, and kainate concentration-dependently increased [Ca2+]i. Quisqualate also elevated [Ca2+]i but did not produce clear concentration-dependent increases. KCl, carbachol, and quisqualate responses reached peak levels within 10-30 s and then desensitized within 90 s. However, kainate-stimulated increases in [Ca2+]i plateaued and did not decline after 90 s. Of these different [Ca2+]i-mediated processes, only 60 mM KCl stimulation was significantly inhibited by 100 mM ethanol, while lower KCl concentrations were not affected. Carbachol-induced release of intracellular Ca2+ and activation of non-NMDA (i.e., kainate, quisqualate) excitatory amino acid receptor-operated cation channels were also not significantly inhibited by 100 mM ethanol. Thus, in acutely dissociated brain cells from newborn rats, only Ca2+ influx via voltage- and, as reported previously, NMDA-operated Ca2+ channels were sensitive to ethanol inhibition.     

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.     

动脉粥样硬化兔主动脉平滑肌细胞钙激活钾通道变化及其对钙敏感性的研究

[目的]探讨动脉粥样硬化(Atherosclerosis,AS)主动脉平滑肌细胞钙激活钾通道(Calcium-activatedPotas-siumChannel,KCa)变化及其对钙的敏感性。[方法]用酶消化法获取单个主动脉平滑肌细胞,通过膜片钳记录技术检测AS兔主动脉平滑肌细胞KCa通道的活性,并在浴液中分别加入不同浓度的Ca2+,以Pclamp7.0应用软件实时采样记录通道的开放概率(Po)、平均开放时间(To)、平均关闭时间(Tc)和电流幅值(Am),并以加钙前作对照观察上述参数的变化。[结果]AS组KCa通道的活性明显高于对照组;与加Ca2+前比较,对照组Po增加了(22.35±9.43)倍,而AS组仅增加了(5.31±0.42)倍(P<0.01)。[结论]AS兔血管平滑肌细胞KCa通道的活性明显增加,可能是AS血管发生代偿性扩张的机制之一;该通道对Ca2+的敏感性降低,可能是促进AS发展的因素之一。     

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.     

Enhanced antifertility activity of non-steroidal molecules with 3-n-butylamino-2-hydroxypropyloxy side chain

A comparative study of relative binding affinity (RBA) for estradiol-17 beta-receptors, estrogenicity and antifertility activity of compounds 2,2-dimethyl-3-phenyl-4-p-(3-n-butylamino-2-hydroxypropyloxy-pheny l)- 7-methoxycoumarin 4, 2,2-dimethyl-3-phenyl-4-p-(3-n-butylamino-2-hydroxy-propyloxyphenyl++ +)-7-methoxy chromene 5 and trans-2,2-dimethyl-3-phenyl-4-p-(3-n-butylamino-2-hydroxypropyloxyphe nyl)- 7-methoxychroman 6 with the corresponding 4-p-(beta-pyrrolidinoethoxyphenyl) compounds 1-3, is reported. It has been found that the introduction of the novel 3-n-butylamino-2-hydroxypropyloxy moiety in place of the classical tert-beta-aminoethoxy group leads to enhancement of antifertility activity.     

Effect of cadmium on transmembrane Na+ and K+ transport systems in human erythrocytes.

The effects of cadmium (Cd2+) on Na+,K(+)-ATPase in disrupted human erythrocyte membranes and on various transmembrane Na+ and K+ transport systems in intact erythrocyte suspensions were studied. Cadmium2+ inhibited the erythrocyte Na+,K(+)-ATPase enzyme with a 50% inhibition at a Cd2+ concentration of 6.25 microM. The Cd2+ inhibition in the human erythrocyte was non-competitive with respect to Na+,K+, and ATP. Cadmium2+ exerted no acute effect, however, on the Na+,K(+)-ATPase pump activity as measured by the ouabain sensitive 86Rb uptake or Na+ efflux in intact red blood cells. Cadmium2+ also inhibited the Ca2+ dependent K+ channels in human red blood cells, whereas it had no effect on Na+,K+ cotransport, Na+,Li+ countertransport, anion carrier, and the number of active Na+ pump units. The data indicate that in human erythrocytes under acute conditions Cd2+ exerts an inhibitory effect on Na+,K(+)-ATPase enzyme in disrupted erythrocytes and the Ca2+ stimulated K+ efflux in intact red blood cells without affecting the Na+ pump, Na+,K+ cotransport, and Na+,Li+ countertransport activity.     

Dietary (n-3) polyunsaturated fatty acids exert antihypertensive effects by modulating calcium signaling in T cells of rats

After 10 wk of feeding an experimental diet enriched with (n-3) polyunsaturated fatty acids (PUFA), i.e., eicosapentaenoic acid [EPA, 20:5(n-3)] and [DHA, 22:6(n-3)] (EPAX), blood pressure in spontaneously hypertensive rats (SHR), but not in normotensive Wistar-Kyoto (WKY) rats was reduced relative to rats fed an unsupplemented control diet. Concanavalin A-stimulated T-cell proliferation was diminished in both strains of rats fed the PUFA/EPAX diet. The experimental diet lowered secretion of interleukin-2 in SHR, but not in WKY rats compared with rats fed the control diet. To determine whether there was a defect in calcium homeostasis in T cells during hypertension, we employed the following agents: caffeine, which recruits calcium from the cytosolic Ca(2+)-induced Ca(2+)-release pool; ionomycin, which at low concentrations opens calcium channels; and thapsigargin (TG), which mobilizes [Ca(2+)]i from the endoplasmic reticulum (ER) pool. Caffeine-induced increases in [Ca(2+)]i were not modified by the PUFA/EPAX diet. The ionomycin-induced increases in [Ca(2+)]i in T cells from SHR were greater than in those from WKY rats; consumption of the PUFA/EPAX diet did not modify Ca(2+) influx in cells of either strain. The TG-induced increases in [Ca(2+)]i in T cells from SHR were greater than those in cells from WKY rats. Interestingly, consumption of the experimental diet reduced TG-evoked increases in [Ca(2+)]i in T cells from SHR and increased those in T cells from WKY rats, indicating that the PUFA/EPAX diet could reverse the calcium mobilization from the ER pool in T cells. These results suggest that (n-3) PUFA exert antihypertensive effects and modulate T-cell calcium signaling during hypertension in rats.     

Differential effects of Ca2+ on proliferation of stomach, colonic, and pancreatic cancer cell lines in vitro

Calcium intake inhibits growth of colon cancer in vivo, the mechanisms of which are not fully elucidated. The objective of this study was to determine whether Ca2+ directly affects the growth of colon cancer cells in vitro and to compare the effects of Ca2+ on the growth of several gastroenteropancreatic cancer cells, including mouse colon cancer (MC-26), human colon cancer (LoVo and WIDR), human gastric cancer (AGS and SII), and human pancreatic cancer (PANC-1 and MIA) cells. All tumor cell lines tested grew in medium containing low concentration (approx 0.16 mM) of Ca2+. Higher concentrations of Ca2+ significantly inhibited the growth of all three colon cancer cell lines tested but had no significant effect on proliferation of the stomach and pancreatic cancer cell lines. Growth of AGS cells, in the presence of 0.1 or 0.5 mM EGTA (resulting in the loss of the extracellular Ca2+) was similar to that observed in the absence of EGTA, indicating that AGS cells were relatively insensitive to loss of extracellular Ca2+. In the presence of TMB-8, an inhibitor of intracellular Ca2+ release, the growth of colonic cancer cell lines was inhibited in a dose-dependent manner, indicating that a minimum basal level of intracellular Ca2+ was required for continued proliferation of colon cancer cells. The stomach cancer cell lines (AGS) was once again less sensitive to the effects of TMB-8 than were the colon cancer cells, indicating an inherent difference in Ca2+ requirements and sensitivity to Ca2+ for growth of different gastroenteropancreatic cancer cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

三氯化铝对小鼠海马神经元细胞内游离钙浓度及其学习记忆的影响

目的探讨接触不同浓度三氯化铝对小鼠海马神经元细胞内游离钙浓度的影响及其与学习记忆能力之间的关系。方法健康雄性ICR小鼠随机平均分为4组(1个对照组和3个染毒组),每组15只。对照组饮双蒸水,染毒组经饮水中加入三氯化铝染毒,剂量分别为10、50、300mg·kg-1·d-1,饲养100d,以钙敏感的荧光指示剂(Fura2)作为探针,观察三氯化铝对小鼠海马神经元细胞内游离钙的影响,利用Morris水迷宫实验测定小鼠学习记忆能力的变化。结果染铝50、300kg·kg-1·d-1剂量组海马神经元游离钙浓度([Ca2+]i)分别为[(412.25±53.20)、(467.37±32.85)次],与对照组[(293.91±32.21)次]相比明显下降,差异有统计学意义(P<0.01),且染毒剂量与[Ca2+]i呈正相关(rS=0.861,P<0.01);Morris水迷宫50、300mg·kg-1·d-1剂量组与对照组相比潜伏期延长,差异有统计学意义(分别为P<0.05和P<0.01)。结论铝对小鼠海马神经元细胞内游离钙浓度有影响,而其浓度的增加可能是导致小鼠学习记忆能力下降的原因之一。     

Extracellular magnesium regulates nuclear and perinuclear free ionized calcium in cerebral vascular smooth muscle cells: possible relation to alcohol and central nervous system injury.

Quantitative digital imaging microscopy, confocal laser scanning microscopy (CLSM), and multiple molecular fluorescent probes were utilized to test the hypothesis that cerebral vascular muscle cell nuclear ([Ca(2+)](n)), perinuclear ([Ca(2+)](pn)), and cytoplasmic free calcium ([Ca(2+)](i)) levels are regulated by the concentration of extracellular free magnesium ions ([Mg(2+)](o)). Primary cultured canine cerebral vascular smooth muscle cells were loaded with either fura-2/AM, indo-1/AM, or fluo-3/AM, and the subcellular Ca(2+) responses to stepwise reduction in [Mg(2+)](o) (i.e., from 1.36 to 0.17 mM) were analyzed over time. With normal 1.36 mM [Mg(2+)](o)-containing incubation media, basal mean [Ca(2+)](i) was 89.6+/-15 nM. Lowering [Mg(2+)](o) to 1.07, 0.88, 0.48, and 0.17 mM resulted in rapid (<4 min) increments in [Ca(2+)](i) going to 213+/-43, 368+/-67, 471+/-77, and 642+/-98 nM, respectively; the longer the exposure time (up to 30 min) to lowered [Mg(2+)](o), the higher the [Ca(2+)](i). Restoration of [Mg(2+)](o) to normal caused decreases in [Ca(2+)](i) to 215.9+/-42.3 nM, but only complete removal of [Ca(2+)](o) returned [Ca(2+)](i) to basal levels. Results show that basal [Ca(2+)](pn) (282+/-92 nM) exceeds basal cytoplasmic Ca(2+) (61+/-27.8 nM) and [Ca(2+)](n) (20+/-7.6 nM). However, reduction of normal [Mg(2+)](o) to 0.48 mM resulted in dramatic, rapid rises in all subcellular compartments, where [Ca(2+)](pn) (1503+/-102 nM)>cytoplasmic Ca(2+) (688+/-49 nM) approximately equal to [Ca(2+)](n) (674+/-12 nM). Nuclear Ca(2+) rose dramatically (e.g., 35-40 times basal levels). Both verapamil (1 microM) and Ni(2+) (5 mM) prevented, completely, the rises in Ca(2+) in all compartments, suggesting that Mg(2+)-dependent Ca(2+) accumulation may be dependent on nuclear, endoplasmic reticulum-Golgi, and cytoplasmic L-type voltage membrane-regulated Ca(2+) channels. The normally low [Ca(2+)](n) suggests that Ca(2+) does not transport passively across the nuclear membrane in cerebral vascular smooth muscle cells. These results may help to explain much of the impact of hypomagnesemic states on cerebral-central nervous system pathobiology, and, particularly, alcohol-induced strokes.     

Lack of dose-responsive effect of dietary phyto-oestrogens on transepithelial calcium transport in human intestinal-like Caco-2 cells

Ca absorption has been shown to be unaffected by high luminal concentrations of two commonly consumed soyabean phyto-oestrogens (PO) (genistein and daidzein) in Caco-2 cells grown under oestrogen-depleted conditions. However, these compounds exhibit dose-dependent biphasic effects in some tissues, such as reproductive tissue and bone. Thus, in light of this biphasic activity, the effect of lower concentrations of genistein and daidzein on Ca absorption requires further investigation. Therefore, the aim of the present study was to investigate the effect of a range of concentrations of genistein and daidzein on Ca absorption in the human Caco-2 intestinal-like cell model. Caco-2 cells were seeded onto permeable filter supports and allowed to differentiate into monolayers. On day 21, the Caco-2 monolayers (n 12 per treatment), grown in oestrogen-deplete media, were then exposed to 10 nm-1,25-dihydroxycholecalciferol (1,25 (OH)2D3), or 1, 10 and 50 microm-genistein or -daidzein for 24 h. After exposure, transepithelial and transcellular transport of (45)Ca and fluorescein transport were measured. As expected, 1,25 (OH)2D3 stimulated Ca absorption in Caco-2 cells, by up regulating transcellular transport. Ca absorption was unaffected by either PO at luminal concentrations of 1, 10 or 50 microm, typical of intakes by Western and Asian populations as well as supplemental levels, respectively. The results of this model suggest that the proposed beneficial effects of supplemental levels of these PO compounds on bone mass in postmenopausal women more probably arise from direct effects on bone cells, and not by an indirect effect of these compounds on Ca absorption.     

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.