Dietary Mg2+ regulates the epithelial Mg2+ channel TRPM6 in rat mammary tissue

The epithelial Mg(2+) channel TRPM6 is considered a pivotal component in active Mg(2+)absorption and re-absorption in the intestine and kidney, but its expression and function in other tissues are largely unknown. We have previously demonstrated that extracellular Mg(2+) availability modulates TRPM6, but not the ubiquitous TRPM7, in cultured mammary epithelial cells; in addition, TRPM6 protein expression correlated to Mg(2+) influx capacities. Our results closely remind the modulation of TRPM6 described by others in murine kidney and colon following Mg(2+) dietary restriction. We sought to validate our observations by investigating whether TRPM6 modulation by extracellular Mg(2+)also occurs in vivo. To this aim, we exploited a model consisting of rats fed either with a Mg(2+)-deficient or a Mg(2+)-enriched diet, and studied TRPM6 expression in breast and kidney tissues. Immunohistochemical and western blot analyses confirmed that rat mammary tissues express TRPM6 protein levels similar to those found in the kidney, and that protein expression is modulated by dietary Mg(2+). In particular, Mg(2+) restriction upregulated TRPM6 expression, while Mg(2+) supplementation resulted in a significant decrease in protein levels. This work confirms and extends our previous results on TRPM6 modulation by Mg(2+) availability in mammary tissues. Further studies are required to clarify the functional significance of these findings, and the role of TRPM6 in tissue-specific magnesium homeostasis.     

Hypobaric-hypoxia-induced pulmonary damage in rats ameliorated by antioxidant erdosteine

Free radical-mediated injury to lung and pulmonary vasculature is an important mechanism in hypoxia-induced lung damage. In this study, we aimed to investigate the potential protective effects of erdosteine as an antioxidant agent on hypobaric hypoxia-induced pulmonary hypertension. Adult male rats were assigned randomly to three groups. The first group of rats was exposed to hypobaric-hypoxia and the second group was treated with erdosteine (20mg/kg, daily) for 2 weeks, during which time they were in a hypoxic chamber. These groups were compared with normoxic controls. All rats were sacrificed after 2 weeks. The hypoxia-induced increase in right ventricle to left ventricle plus septum weight ratio (from 0.20+/-0.01 to 0.26+/-0.01) was reduced significantly in the erdosteine-treated group (0.23+/-0.01). Malondialdehyde levels were elevated (from 0.33+/-0.11 to 0.59+/-0.02) and total antioxidant status was not changed significantly (from 1.77+/-0.42 to 2.61+/-0.23) by hypoxia. In contrast to the hypoxia-exposed group, malondialdehyde levels were significantly decreased in the erdosteine-treated group (0.37+/-0.02). Total antioxidant status (4.03+/-0.22) was significantly higher in erdosteine-treated rats when compared to non-treated rats. Histopathological examination demonstrated that erdosteine prevented inflammation and protected lung parenchyma and pulmonary endothelium of hypoxia-exposed rats.     

Type 1 metalloproteinase is selectively expressed in adult rat brain and can be rapidly up-regulated by kainate

The expression of metalloproteinase MMP-1 was traced in frontal sections of the rat brain in normal conditions and 4 h after an intraperitoneal injection of kainate. In the olfactory lobe, immunoreactivity was normally detected in the lateral olfactory tract. Kainate treatment led to the appearance of additional immunoreactivity in the neuropilar tracts. In the hippocampal part of brain, immunoreactive neurons were found exclusively after the kainate treatment in several hypothalamic and amygdalar nuclei, and in the restricted cortex areas (clusters of neurons in layers 3–4 of cortex, and a stripe of cells in layer 6). In the area between the hippocampus and cerebellum, MMP-1-like immunoreactivity was normally present in the entorhinal cortex, in the lateral periaqueductal gray, and in the pontine nucleus. After kainate treatment, the immunoreactive neurons were also found in the medial entorhinal cortex and in the dorsal raphe nucleus. In the brain stem, the immunoreactive cells were normally found in six nuclei. After kainate treatment, additional immunoreactivity appeared in the inferior olive neurons and in tracts supplying the cerebellar cortex. Thus, MMP-1 is present in several brain areas in normal conditions at a detectable level, and its expression increases after kainate-induced seizures.     

Stimulation of choline/Mg2+ antiport in rat erythrocytes by mefloquine.

In non Mg(2+)-loaded and non malaria-infected rat erythrocytes, mefloquine (100 micromol x l (-1)) stimulated choline/Mg2+ antiport without affecting the Na+/Mg2+ antiport. The stimulation of the choline/Mg2+ antiport by mefloquine, found in this study, and by trifluoperazine and fluvoxamine, reported previously [Ebel et al. Biochim Biophys Acta 2004; 1167: 132-40], was associated with CF3 groups attached to the quinoline or benzene ring. The effect of mefloquine on choline/Mg2+ antiport in vitro was not related to the antimalarial action of mefloquine in vivo. In rat erythrocytes, the choline/Mg2+ antiport can be differentiated from the Na+/Mg2+ antiport through the use of cinchonine that inhibited the choline/Mg2+ antiport [Ebel et al. Biochim Biophys Acta 2002; 1559: 135-44], and mefloquine that stimulated the choline/Mg2+ antiport, whereby the Na+/Mg2+ antiport was not affected by either drug at proper concentrations. The Na+/Mg2+ antiport and choline/Mg2+ antiports behave as different molecular entities.     

Age-related decline in cold tolerance can be retarded by brain stimulation

The sharp decline in capacity of old C57BL/6J male mice to maintain body temperature during 3-hr cold exposure can be delayed and even partly restored after 15, 30-min, daily sessions of hypothalamic self-stimulation. In control experiments it was demonstrated that electrical stimulation of “rewarding” areas of hypothalamus itself is sufficient for improvement of the age-related deterioration of thermoregulatory ability.     

Activation of Na+-independent Mg2+ efflux by 20-hydroxyeicosatetraenoic acid in rat renal epithelial cells

Renal epithelial cells may have Mg(2+) transport pathways that regulate intracellular free Mg(2+) concentration ([Mg(2+)](i)) and reabsorption into the body. In mag-fura 2 fluorescent measurement, extracellular Mg(2+) removal induced a Na(+)-independent [Mg(2+)](i) decrease. The [Mg(2+)](i) decrease was suppressed by methyl arachidonyl fluorophosphonate, a cytosolic and Ca(2+)-independent phospholipase A(2) (iPLA(2)) inhibitor, and bromoenol lactone, an iPLA(2) inhibitor, but it was not suppressed by a secretory phospholipase A(2) inhibitor. On the contrary, the [Mg(2+)](i) decrease was enhanced by the addition of exogenous arachidonic acid (AA). Next, we examined the effect of AA metabolite inhibitors on the [Mg(2+)](i) decrease. 17-octadecynoic acid inhibited the [Mg(2+)](i) decrease, but indomethacin and nordihydroguaiaretic acid did not. In the 17-octadecynoic acid-treated cells, 20-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (20-HETE) recovered the [Mg(2+)](i) decrease. Nicardipine inhibited both the basal and the 20-HETE-enhanced [Mg(2+)](i) decrease. These results suggest that 20-HETE is a key mediator in the activation of Na(+)-independent Mg(2+) efflux.     

Ca2+ signals mediated by bradykinin type 2 receptors in normal pancreatic stellate cells can be inhibited by specific Ca2+ channel blockade

Key points

• Bradykinin may play a role in the autodigestive disease acute pancreatitis, but little is known about its pancreatic actions.
• In this study, we have investigated bradykinin‐elicited Ca²⁺ signal generation in normal mouse pancreatic lobules.
• We found complete separation of Ca²⁺ signalling between pancreatic acinar (PACs) and stellate cells (PSCs). Pathophysiologically relevant bradykinin concentrations consistently evoked Ca²⁺ signals, via B2 receptors, in PSCs but never in neighbouring PACs, whereas cholecystokinin, consistently evoking Ca²⁺ signals in PACs, never elicited Ca²⁺ signals in PSCs.
• The bradykinin‐elicited Ca²⁺ signals were due to initial Ca²⁺ release from inositol trisphosphate‐sensitive stores followed by Ca²⁺ entry through Ca²⁺ release‐activated channels (CRACs). The Ca²⁺ entry phase was effectively inhibited by a CRAC blocker.
• B2 receptor blockade reduced the extent of PAC necrosis evoked by pancreatitis‐promoting agents and we therefore conclude that bradykinin plays a role in acute pancreatitis via specific actions on PSCs.

Abstract

Normal pancreatic stellate cells (PSCs) are regarded as quiescent, only to become activated in chronic pancreatitis and pancreatic cancer. However, we now report that these cells in their normal microenvironment are far from quiescent, but are capable of generating substantial Ca²⁺ signals. We have compared Ca²⁺ signalling in PSCs and their better studied neighbouring acinar cells (PACs) and found complete separation of Ca²⁺ signalling in even closely neighbouring PACs and PSCs. Bradykinin (BK), at concentrations corresponding to the slightly elevated plasma BK levels that have been shown to occur in the auto‐digestive disease acute pancreatitis in vivo, consistently elicited substantial Ca²⁺ signals in PSCs, but never in neighbouring PACs, whereas the physiological PAC stimulant cholecystokinin failed to evoke Ca²⁺ signals in PSCs. The BK‐induced Ca²⁺ signals were mediated by B2 receptors and B2 receptor blockade protected against PAC necrosis evoked by agents causing acute pancreatitis. The initial Ca²⁺ rise in PSCs was due to inositol trisphosphate receptor‐mediated release from internal stores, whereas the sustained phase depended on external Ca²⁺ entry through Ca²⁺ release‐activated Ca²⁺ (CRAC) channels. CRAC channel inhibitors, which have been shown to protect PACs against damage caused by agents inducing pancreatitis, therefore also inhibit Ca²⁺ signal generation in PSCs and this may be helpful in treating acute pancreatitis.     

Extracellular magnesium regulates intracellular free Mg2+ in vascular smooth muscle cells

Summary Regulatory effects of extracellular magnesium ions ([Mg²⁺]_o) on intracellular free ionized magnesium ([Mg²⁺]_i) were exmained in cultured vascular smooth muscle cells (VSMCs) fromrat aorta by digital imaging microscopy using the Mg²⁺ fluorescent probe, Mag-fura-2. With normal Mg²⁺(1.2 mM)-containing incubation media, [Mg²⁺]_i in VSMCs was 0.63±0.09 mM. The ratio of [Mg²⁺]_i/[Mg²⁺]_o was 0.52±0.07. Elevation of [Mg²⁺]o up to 4.8 mM induced consistent increments in [Mg²⁺]_i (to a mean values of 1.63±0.08 mM) in 5 min and lowered the ratio of [Mg²⁺]_i/[Mg²⁺]_o to 0.34±0.02. Our data suggest that [Mg²⁺]_o can regulate [Mg²⁺]_i, which may be related to its effects on intracellular Ca²⁺ ([Ca²⁺]_i) and tone of VSMCs.     

Topographic analysis in brain mapping can be compromised by the average reference

Summary The average reference introduces ghost potential fields at the latencies for which the integral of scalp-recorded potentials differs from zero. These spurious effects occur because the average reference is computed from a limited number of (scalp) electrodes which do not survey the bottom half of the head. By arbitrarily re-setting the zero at each latency in the maps to be compared, it can also obliterate or even reverse topographical differences in the case of focal brain potentials enhancements thereby defeating the purpose of brain mapping.Acknowledgement: This work was supported by grants from the Fonds de la Recherche Scientifique Médicale and the Fonds National de la Recherche Scientifique, Belgium.     

Growth inhibitory factor (GIF) can protect from brain damage due to stab wounds in rat brain

We examined the effect of growth inhibitory factor (GIF), also called metallothionein-III (MT-III), in brain damage using a stab wound model. The administration of 3 microM purified rat GIF (prGIF) provided significantly improved brain repair compared with controls, whereas the administration of 15 microM prGIF reduced brain repair compared with controls. To maintain the continuous effect of GIF, we generated an adenoviral vector encoding rat GIF and the myc epitope (AxCArGIFM) and administered an appropriate amount (1 x 10(8) pfu) of AxCArGIFM on the basis of the optimal dosage determined in a previous study on avulsion of the facial nerve. The administration of AxCArGIFM provided significantly improved histological and biochemical parameters of brain repair compared with controls administered AxCALacZ (adenovirus encoding bacterial beta-galactosidase gene as a reporter; 1 x 10(8) pfu). These results show that GIF can protect from brain damage in certain appropriate conditions in vivo and in vitro. The optimal dosage is very important for the treatment in vivo, particularly that for GIF. Our findings show the double-edged effects of GIF. MTs including MT-III are promising as therapeutic agents not only for tissue repair following acute brain injury, but also for some neurodegenerative diseases because they have multifunctional potential including anti-oxidation effects and may have some effect on neurogenesis.     

Extracellular Mg2+ regulates activation of rat eag potassium channel

The rat homologue of Drosophila ether à gogo cDNA (rat eag) encodes voltage-activated potassium (K) channels with distinct activation properties. Using the Xenopus expression system, we examined the importance of extracellular Mg²⁺ on the activation of rat eag. Extracellular Mg²⁺ at physiological concentrations dramatically slowed the activation in a dose- and voltage-dependent manner. Other divalent cations exerted similar effects on the activation kinetics that correlated with their enthalpy of hydration. Lowering the external pH also resulted in a slowing of the activation. Protons competed with Mg²⁺ as the effect of Mg²⁺ was abolished at low pH. A kinetic model for rat eag activation was derived from the data indicating that all four channel subunits undergo a Mg²⁺-dependent conformational transition prior to final channel activation. The strong dependence of rat eag activation on both the resting potential and the extracellular Mg²⁺ concentration constitutes a system for fine-tuning K channel availability in neuronal cells. Received: 21 August 1995/Received after revision: 2 November 1995/Accepted: 29 November 1995     

Pharmacological and electrographic properties of epileptiform activity induced by elevated K2+ and lowered Ca2+ and Mg2+ concentration in rat hippocampal slices

We studied some of the physiological and pharmacological properties of an in vitro model of epileptic seizures induced by elevation of [K⁺]₀ (to 8 mM and 10 mM) in combination with lowering of [Mg²⁺]₀ (to 1.4 mM and 1.6 mM) and [Ca²⁺]₀ (to 0.7 mM and 1 mM) in rat hippocampal slices. These concentrations correspond to the ionic constitution of the extracellular microenvironment during seizures in vivo. The resulting activity was rather variable in appearance. In area CA3 recurrent discharges were observed which resulted in seizure-like events with either clonic-like or tonic-cloniclike ictaform events in area CA1. With ion-sensitive electrodes, we measured the field potential and the changes in extracellular ion concentrations which accompany this activity. The recurrent discharges in area CA3 were accompanied by small fluctuations in [K⁺]₀ and [Ca²⁺]₀. The grouped clonic-like discharges in area CA1 were associated with moderate increases in [K⁺]₀ and small decreases in [Ca²⁺]₀ in the order of 2 mM and 0.2 mM, respectively. Large, negative field-potential shifts and increases in [K⁺]₀ to 13 mM, as well as decreases in [Ca²⁺]₀ by up to 0.4 mM, accompanied the tonic phase of ictaform events. The ictaform events were not blocked by D-2-aminophosphonovalerate (2-APV) but were sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) alone and in combination with 2-APV and ketamine. In order to determine the pharmacological characteristics of the ictaform events we bath-applied most clinically employed anticonvulsants (carbamazepine, phenytoin, valproate, phenobarbital, ethosuximide, trimethadione) and some experimental anticonvulsants (losigamone, vinpocetine, and apovincaminic acid). Carbamazepine, phenytoin, valproate, and phenobarbital were effective at clinically relevant doses. The data suggest that the high-K⁺ model of epileptiform activity is a good model of focal convulsant activity.     

Mg2+ administered up to twenty-four hours following reperfusion prevents ischemic damage of the Ca1 neurons in the rat hippocampus

Inductively coupled plasma emission spectrometry analysis was applied to determine ischemia-induced changes of Mg2+ and Ca2+ in vulnerable regions of rat brain. This method can provide an accurate quantification and lower detection limits, as compared to atomic absorption spectrophotometry or several other methods. In the hippocampus, Mg2+ content significantly increases 24 h following 20 min of ischemia, followed by a gradual decrease between 48 and 72 h. Ca2+ accumulation was found at 48 and 72 h. At the cell membrane, Mg2+ plays a role as an endogenous calcium channel blocker of both the receptor-operated and voltage-dependent gates and, in the mitochondria, Mg2+ inhibits Ca2+ uptake processes. We propose that the mobilization of Mg2+ after 24 h reperfusion may counteract the process of ischemia-induced neuronal damage and that decreases of Mg2+ may be correlated with the degree of brain injury. However, in the natural concentration of Mg2+, the counteraction may not be sufficient for a neuroprotective effect. Therefore, after 24 h reperfusion, an artificial enhancement of Mg2+ is necessary for neuroprotection. In order to test the above hypothesis, MgCl2, (50 mM) was administered directly to the CA1 sector of the rat hippocampus before and at various intervals following 20 min of ischemia. Pyramidal cells were evaluated seven days later and neuronal density was determined. Consistent with the hypothesis, a neuroprotective effect was observed, even when MgCl2 was administered 24 h, but not 48 h, after the ischemic episode.     

Ethacrynic acid inhibitable Ca2+ and Mg2+-activated membrane adenosine triphosphatase in rat mast cells.

A crude plasma membrane fraction from the homogenate of purified rat mast cells demonstrates a high degree of Ca2+-dependent and Mg2+-dependent adenosine triphosphatase (ATPase) activity. The microsomal and mitochondrial fractions show negligible amounts of the Ca2+ and Mg2+-activated ATPases. The broad ATPase inhibitor, ethacrynic acid, effectively blocks the mast cell ATPase activity while ouabain demonstrates little inhibitory effect. Correspondingly, ethacrynic acid inhibits histamine release from antigen-challenged mast cells while ouabain does not. Both ATPase inhibition and histamine release inhibition by ethacrynic acid require the presence of the olefinic bond in the ethacrynic acid molecule.     

Induction of nestin synthesis in rat brain cells by ischemic damage

Nestin, an intermediate filament protein, is known to be expressed in proliferating and provisional cells in the forming mammalian brain, disappearing on differentiation. The aim of the present work was to identify the morphological types and locations of cells regaining the ability to synthesize nestin after transient total brain ischemia in rats. Transient ischemia was found to be followed by the induction of nestin synthesis in astrocytes in the damaged area; these cells acquired structural features not characteristic of the adult brain, and these persisted in the long term. Nestin synthesis was also induced in proliferation-capable undifferentiated cells in the subventricular zone. The acquisition by astrocytes of some of the phenotypic features of immature glial cells, however, does not provide grounds for the notion that they were transformed into neural stem cells. Director: Corresponding Member of the Russian Academy of Medical Sciences Professor V. A. Otellin Director: Professor S. A. Polenov Director: Professor N. N. Petrishchev __________ Translated from Morfologiya, Vol. 131, No. 1, pp. 23–26, January–February, 2007.     

Mg-ATP binding: its modification by spermine, the relevance to cytosolic Mg2+ buffering, changes in the intracellular ionized Mg2+ concentration and the estimation of Mg2+ by 31P-NMR

It is now generally accepted that the intracellular ionized magnesium concentration ([Mg²⁺]_i) in muscle cells is around 1 mmol l⁻¹; in heart muscle this means that from the total some 90-95% is bound (see McGuigan et al. 1991 a ). Although binding will include sequestration by intracellular organelles, a large part of the binding is by ATP in the cytosol and an equilibrium exists in the cytosol between free ATP, ionized magnesium and Mg-ATP. The extend of this equilibrium depends on the equilibrium constant of the reaction, which is a function of pH, temperature and ionic strength. This equilibrium constant is also important in the estimation of [Mg²⁺]_i using ³¹P-NMR. In this method the difference between the α and β peaks of ATP is measured and from this shift and the equilibrium constant between Mg²⁺ and ATP in the cytosol, the [Mg²⁺]_i can be calculated (Vink, 1993).     

Intracellular calibration of the fluorescent Mg2+ indicator furaptra in rat ventricular myocytes

Single ventricular myocytes enzymatically isolated from rat hearts were loaded with the Mg2+ indicator furaptra, and the relationship between the fluorescence ratio signal (R) and the intracellular free concentration of Mg2+ ([Mg2+]i) was studied in situ at 25 degrees C. After the application of ionophores (ionomycin, monensin, nigericin and valinomycin), an immediate change in furaptra R was noted, followed by a slow change in R that reached a steady level in 2-4 h. The direction of the early change in R that accompanied rigor contraction was independent of the extracellular Mg2+ concentration ([Mg2+]o), and was consistent with the breakdown of ATP and release of bound Mg2+. The intracellular calibration curve was constructed from the steady levels of R obtained at various [Mg2+]o between 0 and 47 mM. The dissociation constant of intracellular furaptra was estimated to be 5.3 mM, which was 44% higher than that determined in salt solutions (3.7 mM). The basal [Mg2+]i of rat ventricular myocytes calculated with the intracellular curve averaged 0.91 mM.     

大鼠脑缺血后血管内皮生长因子和血管生成素的表达变化及其作用

目的 探讨大鼠脑缺血后血管内皮生长因子(VEGF)和血管生成素(angiopoietin,Ang)的表达变化及其在血管生成和血管通透性变化中的作用.方法 选择160只雄性SD大鼠,采用随机区组法分为假手术组,缺血2h、6h、12h、1d、3d、7d和14d组,用线栓法制作大脑中动脉阻塞脑缺血损伤模型,分别于脑缺血后不同时间点处死大鼠.通过逆转录聚合酶链反应(RT-PCR)、Western blot及免疫组织化学方法检测大鼠脑缺血后不同时间点VEGF、Ang-1及Ang-2 mRNA和蛋白的表达变化.CD31标记鼠脑缺血后不同时间点的血管数量.Evans blue检测血脑屏障通透性.结果 大鼠脑缺血后VEGF mRNA表达逐渐增高,12 h达第一个高峰(0.7249 ±0.1933,P＜0.01),7d达第二个高峰(0.5264±0.1519,P＜0.01);VEGF蛋白表达也逐渐增高,于12 h达高峰(1.1017±0.1302,P＜0.01).Ang-2 mRNA和蛋白在脑缺血组织也逐渐增高,均12 h达高峰(0.6747±0.2416,P＜0.01;1.1197±0.1780,P＜0.01).与之相反,Ang-1 mRNA和蛋白表达逐渐降低,分别于3 d(0.3220±0.1427,P＜0.01)和1 d(0.1298±0.0293,P＜0.01)达最低水平,这些因子在脑缺血后的表达促进血管生成.脑缺血后血管通透性逐渐增加,EB含量在缺血1d达高峰[(6.219±0.887) μg/g,P＜0.01].结论 大鼠脑缺血后VEGF、Ang-1和Ang-2共同协调作用促进血管生成,在组织损伤修复过程中发挥着重要的作用.     

Chronic dietary Mg2+ deficiency induces cardiac apoptosis in the rat heart.

Severe Mg2+ deficiency provokes pro-oxidative and pro-inflammatory changes, and also has been shown to be pro-apoptotic in thymus and certain cell cultures. In this study we examined the extent that chronic severe dietary Mg2+ deficiency induces apoptosis in the heart. Sprague-Dawley rats were fed during three weeks with normal (25 mM, Mg-control) or magnesium deficient (2.25 mM, Mg-deficient) diets, after which, hearts were harvested and frozen. DNA fragmentation was examined in heart tissue sections, and while < 1% of nuclei were positive for apoptosis in Mg-control rat tissue, over 32% of nuclei gave positive for Klenow fragments in hearts from Mg-deficient rats. Caspase 3 activity measurements in heart homogenates showed a 3.9-fold increase in enzyme activity in Mg-deficient rat hearts compared to Mg-controls (p < 0.002); and furthermore, western blot analysis of cleaved PARP (caspase 3 substrate), showed a 4.6-fold increase of cleaved PARP in Mg-deficient rat hearts (p < 0.002). In summary, our data indicate that chronic Mg2+ deficiency induces apoptosis of myocardium in vivo.