Hypoxia induces an increase in intracellular magnesium via transient receptor potential melastatin 7 (TRPM7) channels in rat hippocampal neurons in vitro

TRPM7, a divalent cation channel, plays an important role in neurons damaged from cerebral ischemia due to permitting intracellular calcium overload. This study aimed to explore whether magnesium was transported via a TRPM7 channel into the intracellular space of rat hippocampal neurons after 1 h of oxygen-glucose deprivation (OGD) and acute chemical ischemia (CI) by using methods of the Mg(2+) fluorescent probe Mag-Fura-2 to detect intracellular magnesium concentration ([Mg(2+)](i)) and flame atomic absorption spectrometry to measure extracellular magnesium concentration ([Mg(2+)](o)). The results showed that the neuronal [Mg(2+)](i) was 1.51-fold higher after 1 h of OGD at a basal level, and the increase of neuronal [Mg(2+)](i) reached a peak after 1 h of OGD and was kept for 60 min with re-oxygenation. Meanwhile, the [Mg(2+)](o) decreased after 1 h of OGD and recovered to the pre-ischemic level within 15 min after re-oxygenation. In the case of CI, the [Mg(2+)](i) peak immediately appeared in hippocampal neurons. This increase of [Mg(2+)](i) declined by removing extracellular magnesium in OGD or CI. Furthermore, by using Gd(3+) or 2-aminoethoxydiphenyl borate to inhibit TRPM7 channels, the [Mg(2+)](i) increase, which was induced by OGD or CI, was attenuated without altering the basal level of [Mg(2+)](i). By silencing TRPM7 with shRNA in hippocampal neurons, it was found that not only was the increase of [Mg(2+)](i) induced by OGD or CI but also the basal levels of [Mg(2+)](i) were attenuated. In contrast, overexpression of TRPM7 in HEK293 cells exaggerated both the basal levels and increased [Mg(2+)](i) after 1 h of OGD/CI. These results suggest that anoxia induced the increase of [Mg(2+)](i) via TRPM7 channels in rat hippocampal neurons.     

Mechanisms of Mg(2+) transport in cultured ruminal epithelial cells

Net Mg(2+) absorption from the rumen is mainly mediated by a transcellular pathway, with the greater part (62%) being electrically silent. To investigate this component of Mg(2+) transport, experiments were performed with isolated ruminal epithelial cells (REC). Using the fluorescent indicators mag-fura 2, sodium-binding benzofuran isophthalate, and 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, we measured the intracellular free Mg(2+) concentration ([Mg(2+)](i)), the intracellular Na(+) concentration ([Na(+)](i)), and the intracellular pH (pH(i)) of REC under basal conditions, after stimulation with butyrate and HCO(-)(3), and after changing the transmembrane chemical gradients for Mg(2+), H(+), and Na(+). REC had a mean resting pH(i) of 6.83 +/- 0.1, [Mg(2+)](i) was 0.56 +/- 0. 14 mM, and [Na(+)](i) was 18.95 +/- 3.9 mM. Exposure to both HCO(-)(3) and HCO(-)(3)/butyrate led to a stimulation of Mg(2+) influx that amounted to 27.7 +/- 5 and 29 +/- 10.6 microM/min, respectively, compared with 15 +/- 1 microM/min in control solution. The increase of [Mg(2+)](i) was dependent on extracellular Mg(2+) concentration ([Mg(2+)](e)). Regulation of pH(i) has been demonstrated to be Na(+) dependent and is performed, for the most part, by a Na(+)/H(+) exchanger. The recovery of pH(i) was fully blocked in nominally Na(+)-free media, even if [Mg(2+)](e) was stepwise increased from 0 to 7.5 mM. However, an increase of [Mg(2+)](i) was observed after reversing the transmembrane Na(+) gradient. This rise in [Mg(2+)](i) was pH independent, K(+) insensitive, dependent on [Mg(2+)](e), imipramine and quinidine sensitive, and accompanied by a decrease of [Na(+)](i). The results are consistent with the existence of a Na(+)/Mg(2+) exchanger in the cell membrane of REC. The coupling between butyrate, CO(2)/HCO(-)(3), and Mg(2+) transport may be mediated by another mechanism, perhaps by cotransport of Mg(2+) and HCO(-)(3).     

Salvia miltiorrhiza attenuates the changes in contraction and intracellular calcium induced by anoxia and reoxygenation in rat cardiomyocytes

The aim of the present study is to investigate the effect of Salvia miltiorrhiza (SM) on contraction and the intracellular calcium of isolated ventricular myocytes during normoxia or anoxia and reoxygenation using a video tracking system and spectrofluorometry. Cardiac ventricular myocytes were isolated enzymatically by collagenase and exposed to 5 min of anoxia followed by 10 min of reoxygenation. SM (1-9 g/L) depressed both contraction and the [Ca(2+)](i) transient in a dose-dependent manner. SM did not affect the diastolic calcium level and the sarcolemmal Ca(2+) channel of myocytes but decreased the caffeine-induced calcium release. During anoxia, the +/-dL/dtmax, amplitudes of contraction (dL) of cell contraction and [Ca(2+)](i) transients were decreased, while the diastolic calcium level was increased. None of the parameters returned to the pre-anoxia level during reoxygenaton. However, SM (3 g/L) did attenuate the changes in cell contraction and intracellular calcium induced by anoxia and reoxygenation. It is concluded that SM has different effects on normoxic and anoxic cardiomyocytes. The SM-induced reduction of changes in contraction and intracellular calcium induced by anoxia/reoxygenation indicates that SM may be beneficial for cardiac tissue in recovery of mechanical function and intracellular calcium homeostasis.     

Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes.

The effects of anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with SBFI, intracellular pH (pHi) with BCECF, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, ATP by 31P NMR spectroscopy in real time, and intracellular free Mg2+ (Mg2+i) from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. After 1 h of anoxia, beta-ATP fell 66%, and 85% after 2 h, while the Pi/ATP ratio increased 10-fold from 2.75 to 28.3. Under control conditions, the resting cytosolic free calcium was 127 +/- 6 nM. Anoxia increased Ca2+i in two distinct phases: a first rise occurred within 15 min and reached a mean value of 389 +/- 35 nM (p less than 0.001). A second peak reached a maximum value of 1.45 +/- 0.12 microM (p less than 0.001) after 1 h. During the first hour of anoxia, Na+i increased from 15.9 +/- 2.4 mM to 32.2 +/- 1.2 mM (p less than 0.001), Mg2+i doubled from 0.51 +/- 0.05 to 1.12 +/- 0.01 mM (p less than 0.001), and pHi decreased from 7.41 +/- 0.03 to 7.06 +/- 0.1 (p less than 0.001). LDH release doubled during the first hour and increased 6-fold during the second hour of anoxia. Upon reoxygenation, ATP, Ca2+i, Mg2+i, Na+i, and LDH returned near the control levels within 45 min. To determine whether the increased LDH release was related to the rise in Ca2+i, and whether the increased Ca2+i was caused by Ca2+ influx, the cells were perfused with Ca(2+)-free KHB (+ 0.1 mM EGTA) during the anoxic period. After 2 h of anoxia in Ca(2+)-free medium, beta-ATP again fell 90%, but Ca2+i, after the first initial peak, fell below control levels, and LDH release increased only 2.7-fold. During reoxygenation, Ca2+i, ATP, Na+i, and LDH returned near the control levels within 45 min. These results suggest that the rise in Ca2+i induced by anoxia is caused by an influx of Ca2+ from the extracellular fluid, and that LDH release and cell injury may be related to the resulting rise in Ca2+i.     

Effects of docosahexaenoic acid on calcium pathway in adult rat cardiomyocytes

In this study we examined the effect of polyunsaturated fatty acids (PUFAs), in particular of docosahexaenoic acid (DHA), on calcium homeostasis in isolated adult rat cardiomyocytes exposed to KCl, ET-1 and anoxia. Free [Ca(2+)](i) in rat cardiomyocytes was 135.7 +/- 0.5 nM. Exposure to 50 mM KCl or 100 nM ET-1 resulted in a rise in free [Ca(2+)](i) in freshly isolated cells (465.4 +/- 15.6 nM and 311.3 +/- 12.6 nM, respectively) and in cultured cells (450.8 +/- 14.8 nM and 323.5 +/- 14.8 nM respectively). An acute treatment (20 minutes) with 10 microM DHA significantly reduced the KCl- and ET-1-induced [Ca(2+)](i) increase (300.9 +/- 18.1 nM and 232.08 +/- 11.8 nM, respectively). This reduction was greater after chronic treatment with DHA (72 h; 257.7 +/- 13.08 nM and 192.18 +/- 9.8 nM, respectively). Rat cardiomyocytes exposed to a 20 minute superfusion with anoxic solution, obtained by replacing O(2) with N(2) in gas mixture, showed a massive increase in cytosolic calcium (1200.2 +/- 50.2 nM). Longer exposure to anoxia induced hypercontraction and later death of rat cardiomyocytes. Preincubation with DHA reduced the anoxic effect on [Ca(2+)](i) (498.4 +/- 7.3 nM in acute and 200.2 +/- 12.2 nM in chronic treatment). In anoxic conditions 50 mM KCl and 100 nM ET-1 produced extreme and unmeasurable increases of [Ca(2+)](i.) Preincubation for 20 minutes with DHA reduced this phenomenon (856.1 +/- 20.3 nM and 782.3 +/- 7.6 nM, respectively). This reduction is more evident after a chronic treatment with DHA (257.7 +/- 10.6 nM and 232.2 +/- 12.5 nM, respectively). We conclude that in rat cardiomyocytes KCl, ET-1 and anoxia interfered with intracellular calcium concentrations by either modifying calcium levels or impairing calcium homeostasis. Acute, and especially chronic, DHA administration markedly reduced the damage induced by calcium overload in those cells.     

CaMKII-dependent reactivation of SR Ca(2+) uptake and contractile recovery during intracellular acidosis

In hearts, intracellular acidosis disturbs contractile performance by decreasing myofibrillar Ca(2+) response, but contraction recovers at prolonged acidosis. We examined the mechanism and physiological implication of the contractile recovery during acidosis in rat ventricular myocytes. During the initial 4 min of acidosis, the twitch cell shortening decreased from 2.3 +/- 0.3% of diastolic length to 0.2 +/- 0.1% (means +/- SE, P < 0.05, n = 14), but in nine of these cells, contractile function spontaneously recovered to 1.5 +/- 0.3% at 10 min (P < 0.05 vs. that at 4 min). During the depression phase, both the diastolic intracellular Ca(2+) concentration ([Ca(2+)](i)) and Ca(2+) transient (CaT) amplitude increased, and the twitch [Ca(2+)](i) decline prolonged significantly (P < 0.05). In the cells that recovered, a further increase in CaT amplitude and a reacceleration of twitch [Ca(2+)](i) decline were observed. The increase in diastolic [Ca(2+)](i) was less extensive than the increase in the cells that did not recover (n = 5). Blockade of sarcoplasmic reticulum (SR) function by ryanodine (10 microM) and thapsigargin (1 microM) or a selective inhibitor of Ca(2+)-calmodulin kinase II, 2-[N- (2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methyl benzylamine (1 microM) completely abolished the reacceleration of twitch [Ca(2+)](i) decline and almost eliminated the contractile recovery. We concluded that during prolonged acidosis, Ca(2+)-calmodulin kinase II-dependent reactivation of SR Ca(2+) uptake could increase SR Ca(2+) content and CaT amplitude. This recovery can compensate for the decreased myofibrillar Ca(2+) response, but may also cause Ca(2+) overload after returning to physiological pH(i).     

Inhibition of NHE protects reoxygenated cardiomyocytes independently of anoxic Ca(2+) overload and acidosis

We investigated the question of whether inhibition of the Na(+)/H(+) exchanger (NHE) during ischemia is protective due to reduction of cytosolic Ca(2+) accumulation or enhanced acidosis in cardiomyocytes. Additionally, the role of the Na(+)-HCO(3)(-) symporter (NBS) was investigated. Adult rat cardiomyocytes were exposed to simulated ischemia and reoxygenation. Cytosolic pH [2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)], Ca(2+) (fura 2), Na(+) [sodium-binding benzolfuran isophthatlate (SBFI)], and cell length were measured. NHE was inhibited with 3 micromol/l HOE 642 or 1 micromol/l 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and NBS was inhibited with HEPES buffer. During anoxia in bicarbonate buffer, cells developed acidosis and intracellular Na and Ca (Na(i) and Ca(i), respectively) overload. During reoxygenation cells underwent hypercontracture (44.0 +/- 4.1% of the preanoxic length). During anoxia in bicarbonate buffer, inhibition of NHE had no effect on changes in intracellular pH (pH(i)), Na(i), and Ca(i), but it significantly reduced the reoxygenation-induced hypercontracture (HOE: 61.0 +/- 1.4%, EIPA: 68.2 +/- 1.8%). The sole inhibition of NBS during anoxia was not protective. We conclude that inhibition of NHE during anoxia protects cardiomyocytes against reoxygenation injury independently of cytosolic acidification and Ca(i) overload.     

Effect of anoxia and pharmacological anoxia on whole-cell NMDA receptor currents in cortical neurons from the western painted turtle

The mammalian brain undergoes rapid cell death during anoxia that is characterized by uncontrolled Ca(2+) entry via N-methyl-D-aspartate receptors (NMDARs). In contrast, the western painted turtle is extremely anoxia tolerant and maintains close-to-normal [Ca(2+)](i) during periods of anoxia lasting from days to months. A plausible mechanism of anoxic survival in turtle neurons is the regulation of NMDARs to prevent excitotoxic Ca(2+) injury. However, studies using metabolic inhibitors such as cyanide (NaCN) as a convenient method to induce anoxia may not represent a true anoxic stress. This study was undertaken to determine whether turtle cortical neuron whole-cell NMDAR currents respond similarly to true anoxia with N(2) and to NaCN-induced anoxia. Whole-cell NMDAR currents were measured during a control N(2)-induced anoxic transition and a control NaCN-induced transition. During anoxia with N(2) normalized, NMDAR currents decreased to 35.3%+/-10.8% of control values. Two different NMDAR current responses were observed during NaCN-induced anoxia: one resulted in a 172%+/-51% increase in NMDAR currents, and the other was a decrease to 48%+/-14% of control. When responses were correlated to the two major neuronal subtypes under study, we found that stellate neurons responded to NaCN treatment with a decrease in NMDAR current, while pyramidal neurons exhibited both increases and decreases. Our results show that whole-cell NMDAR currents respond differently to NaCN-induced anoxia than to the more physiologically relevant anoxia with N(2).     

Fructose protects rat hepatocytes from anoxic injury. Effect on intracellular ATP, Ca2+i, Mg2+i, Na+i, and pHi.

The effects of fructose on the intracellular ionic changes evoked by anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with sodium-binding benzofuran isophthalate, intracellular pH (pHi) with 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, and viability by trypan blue exclusion. ATP, Pi, phosphomonoesters, and the cell phosphorylation potential assessed by the reciprocal of the Pi/ATP ratio were measured by 31P NMR spectroscopy in real time. Intracellular free Mg2+ (Mg2+i) was calculated from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. When the perfusate contained 5 mM glucose as substrate, anoxia caused a fall in ATP, a rise in Pi, and in the Pi/ATP ratio, a biphasic increase in Ca2+i that reached 1.45 +/- 0.42 microM and a 6-fold increase in LDH. When 15 mM fructose was used as substrate during the anoxic period, intracellular ATP decreased much faster than with glucose, Pi did not increase, and the concentration of phosphomonoesters increased 2.5-fold. During the first hour of anoxia, the Pi/ATP ratio was higher in the fructose than in the glucose group indicating that the hepatocyte phosphorylation potential and ATP decreased faster and to lower levels with fructose than with glucose. On the other hand, ATP and the phosphorylation potential of the fructose group increased during the second hour of anoxia, in contrast to their continuous decline in the glucose group. The major surge in Ca2+i was depressed 52% when glucose was replaced by fructose: Ca2+i reached only 0.7 +/- 0.2 microM instead of 1.45 +/- 0.42 microM (p less than 0.01). Anoxia also caused an increase in Na+i and an intracellular acidosis. The rise in Na+i was significantly greater with fructose than with glucose. Na+i rose from a control value of 15.9 +/- 2.4 to 32.2 +/- 0.4 mM with glucose and to 48.7 +/- 0.7 mM with fructose (p less than 0.001). The decrease in pHi from a control value of 7.43 +/- 0.03 was consistently greater and faster with fructose than with glucose: 6.59 +/- 0.03 and 7.04 +/- 0.01, respectively. At the same time, fructose completely suppressed LDH release and reduced the loss of viability produced by anoxia from 27.7 +/- 2.9 to 14 +/- 3.1% (p less than 0.05).     

Reversible decreases in ATP and PCr concentrations in anoxic turtle brain

A hallmark of anoxia tolerance in western painted turtles is relative constancy of tissue adenylate concentrations during periods of oxygen limitation. During anoxia heart and brain intracellular compartments become more acidic and cellular energy demands are met by anaerobic glycolysis. Because changes in adenylates and pH during anoxic stress could represent important signals triggering metabolic and ion channel down-regulation we measured PCr, ATP and intracellular pH in turtle brain sheets throughout a 3-h anoxic-re-oxygenation transition with ³¹P NMR. Within 30 min of anoxia, PCr levels decrease 40% and remain at this level during anoxia. A different profile is observed for ATP, with a statistically significant decrease of 23% occurring gradually during 110 min of anoxic perfusion. Intracellular pH decreases significantly with the onset of anoxia, from 7.2 to 6.6 within 50 min. Upon re-oxygenation PCr, ATP and intracellular pH recover to pre-anoxic levels within 60 min. This is the first demonstration of a sustained reversible decrease in ATP levels with anoxia in turtle brain. The observed changes in pH and adenylates, and a probable concomitant increase in adenosine, may represent important metabolic signals during anoxia.     

Cocaine induces intracellular free Mg deficits, ischemia and stroke as observed by in-vivo 31P-NMR of the brain.

31P-NMR spectroscopic studies were performed in vivo on brains of rats administered cocaine. Cocaine.HCl (1-5 mg/kg) administered systemically to lightly anesthetized rats resulted in significant and progressive deficits in whole brain intracellular free Mg ([Mg2+]i). Intracellular pH (pHi) also fell in a progressive manner but only after a significant fall in brain [Mg2+]i was noted. Both [Mg2+]i and pHi returned to normal in most rats. Brains of rats that exhibited stroke-like events, however, demonstrated continued intracellular acidosis associated with progressive loss of phosphocreatine and elevation of Pi up until death. These observations are consistent with the tenet that injection of cocaine can result in severe cerebral vasospasm, ischemia and rupture of cerebral blood vessels as a consequence of depletion of brain [Mg2+]i.     

The effects of HCl and CaCl(2) injections on intracellular calcium and pH in voltage-clamped snail (Helix aspersa) neurons

To investigate the mechanisms by which low intracellular pH influences calcium signaling, I have injected HCl, and in some experiments CaCl(2), into snail neurons while recording intracellular pH (pH(i)) and calcium concentration ([Ca(2+)](i)) with ion-sensitive microelectrodes. Unlike fluorescent indicators, these do not increase buffering. Slow injections of HCl (changing pH(i) by 0.1-0.2 pH units min(-1)) first decreased [Ca(2+)](i) while pH(i) was still close to normal, but then increased [Ca(2+)](i) when pH(i) fell below 6.8-7. As pH(i) recovered after such an injection, [Ca(2+)](i) started to fall but then increased transiently before returning to its preinjection level. Both the acid-induced decrease and the recovery-induced increase in [Ca(2+)](i) were abolished by cyclopiazonic acid, which empties calcium stores. Caffeine with or without ryanodine lowered [Ca(2+)](i) and converted the acid-induced fall in [Ca(2+)](i) to an increase. Injection of ortho-vanadate increased steady-state [Ca(2+)](i) and its response to acidification, which was again blocked by CPA. The normal initial response to 10 mM caffeine, a transient increase in [Ca(2+)](i), did not occur with pH(i) below 7.1. When HCl was injected during a series of short CaCl(2) injections, the [Ca(2+)](i) transients (recorded as changes in the potential (V(Ca)) of the Ca(2+)-sensitive microelectrode), were reduced by only 20% for a 1 pH unit acidification, as was the rate of recovery after each injection. Calcium transients induced by brief depolarizations, however, were reduced by 60% by a similar acidification. These results suggest that low pH(i) has little effect on the plasma membrane calcium pump (PMCA) but important effects on the calcium stores, including blocking their response to caffeine. Acidosis inhibits spontaneous calcium release via the RYR, and leads to increased store content which is unloaded when pH(i) returns to normal. Spontaneous release is enhanced by the rise in [Ca(2+)](i) caused by inhibiting the PMCA.     

Simultaneous determination of low free Mg2+ and pH in human sickle cells using 31P NMR spectroscopy

The concentrations of free magnesium, [Mg(2+)](free), [H(+)], and [ATP] are important in the dehydration of red blood cells from patients with sickle cell anemia, but they are not easily measured. Consequently, we have developed a rapid, noninvasive NMR spectroscopic method using the phosphorus chemical shifts of ATP and 2,3-diphosphoglycerate (DPG) to determine [Mg(2+)](free) and pH(i) simultaneously in fully oxygenated whole blood. The method employs theoretical equations expressing the observed chemical shift as a function of pH, K(+), and [Mg(2+)](free), over a pH range of 5.75-8.5 and [Mg(2+)](free) range 0-5 mm. The equations were adjusted to allow for the binding of hemoglobin to ATP and DPG, which required knowledge of the intracellular concentrations of ATP, DPG, K(+), and hemoglobin. Normal oxygenated whole blood (n = 33) had a pH(i) of 7.20 +/- 0.02, a [Mg(2+)](free) of 0.41 +/- 0.03 mm, and [DPG] of 7.69 +/- 0.47 mm. Under the same conditions, whole sickle blood (n = 9) had normal [ATP] but significantly lower pH(i) (7.10 +/- 0.03) and [Mg(2+)](free) (0.32 +/- 0.05 mm) than normal red cells, whereas [DPG] (10.8 +/- 1.2 mm) was significantly higher. Because total magnesium was normal in sickle cells, the lower [Mg(2+)](free) could be attributed to increased [DPG] and therefore greater magnesium binding capacity of sickle cells.     

Low glucose sensitivity and polymodal chemosensing in neonatal rat adrenomedullary chromaffin cells

Glucose is the primary metabolic fuel in mammalian fetuses, yet mammals are incapable of endogenous glucose production until several hours after birth. Thus, when the maternal supply of glucose ceases at birth there is a transient hypoglycemia that elicits a counterregulatory surge in circulating catecholamines. Because the innervation of adrenomedullary chromaffin cells (AMCs) is immature at birth, we hypothesized that neonatal AMCs act as direct glucosensors, a property that could complement their previously established roles as hypoxia and acid hypercapnia sensors. During perforated-patch, whole cell recordings, low glucose depolarized and/or excited a subpopulation of neonatal AMCs; in addition, aglycemia (0 mM glucose) caused inhibition of outward K(+) current, blunted by the simultaneous activation of glibenclamide-sensitive K(ATP) channels. Some cells were excited by each of the three metabolic stimuli, i.e., aglycemia, hypoxia (Po(2) ～30 mmHg), and isohydric hypercapnia (10% CO(2); pH = 7.4). Using carbon fiber amperometry, aglycemia and hypoglycemia (3 mM glucose) induced robust catecholamine secretion that was sensitive to nickel (50 μM and 2 mM) and the L-type Ca(2+) channel blocker nifedipine (10 μM), suggesting involvement of both T-type and L-type voltage-gated Ca(2+) channels. Fura-2 measurements of intracellular Ca(2+) ([Ca(2+)] (i)) revealed that ～42% of neonatal AMCs responded to aglycemia with a significant rise in [Ca(2+)] (i). Approximately 40% of these cells responded to hypoxia, whereas ～25% cells responded to both aglycemia and hypoxia. These data suggest that together with hypoxia and acid hypercapnia, low glucose is another important metabolic stimulus that contributes to the vital asphyxia-induced catecholamine surge from AMCs at birth.     

Anoxia-induced dopamine release from rat striatal slices: involvement of reverse transport mechanism

Incubation of rat striatal slices in the absence of oxygen (anoxia), glucose (aglycemia), or oxygen plus glucose (ischemia) caused significant increases in dopamine (DA) release. Whereas anoxia decreased extracellular 3,4-dihydroxyphenylacetic acid levels by 50%, aglycemia doubled it, and ischemia returned this aglycemia-induced enhancement to its control level. Although nomifensine, a DA uptake blocker, completely protected the slices against anoxia-induced DA depletion, aglycemia- and ischemia-induced increases were not altered. Moreover, hypothermia differentially affected DA release stimulated by anoxia, aglycemia, and ischemia. Involvement of glutamate in DA release induced by each experimental condition was tested by using MK-801 and also by comparing the glutamate-induced DA release with that during anoxia, aglycemia, or ischemia. MK-801 decreased the anoxia-induced DA depletion in a dose-dependent manner. This treatment, however, showed a partial protection in aglycemic conditions but failed to improve ischemia-induced DA depletion. Like anoxia, DA release induced by exogenous glutamate was also sensitive to nomifensine and hypothermia. These results indicate that anoxia enhances DA release by a mechanism involving both the reversed DA transporter and endogenous glutamate. Partial or complete lack of effect of nomifensine, hypothermia, or MK-801 in the absence of glucose or oxygen plus glucose also suggests that experimental conditions, such as the degree of anoxia/ischemia, may alter the mechanism(s) involved in DA depletion.     

Lactic acid buffering by bone and shell in anoxic softshell and painted turtles

We tested two hypotheses: first, that the inferior anoxia tolerance of the softshell turtle, Apalone spinifera, compared to the western painted turtle, Chrysemys picta bellii, is related to its less mineralized shell, and second, that turtle bone, like its shell, stores lactate during prolonged anoxia. Lactate concentrations of blood, hindlimb bone, and shell were measured on normoxic Apalone and Chrysemys and after anoxic submergence at 10 degrees C for 2 and 9 d, respectively. Blood and shell concentrations of Ca(2+), Mg(2+), Na(+), K(+), and inorganic phosphate (P(i); for shell only) were also measured. Because a preliminary study indicated lactate distribution in Chrysemys throughout its skeleton during anoxia at 20 degrees C, we used hindlimb bones as representative skeletal samples. Apalone shell, though a similar percentage of body mass as Chrysemys shell, had higher water content (76.9% vs. 27.9%) and only 20%-25% as much Ca(2+), Mg(2+), CO(2), and P(i). When incubated at constant pH of 6.0 or 6.5, Apalone shell powder released only 25% as much buffer per gram wet weight as Chrysemys shell. In addition, plasma [Ca(2+)] and [Mg(2+)] increased less in Apalone during anoxia at an equivalent plasma lactate concentration. Lactate concentrations increased in the shell and skeletal bone in both species. Despite less mineralization, Apalone shell took up lactate comparably to Chrysemys. In conclusion, a weaker compensatory response to lactic acidosis in Apalone correlates with lower shell mineralization and buffer release and may partially account for the poorer anoxia tolerance of this species.     

Amiloride和Ni2+抑制缺血/复灌引起的大鼠心肌细胞内钙的增加

本文旨在研究Na+/H+交换以及Na+/Ca2 +交换对模拟缺血 /复灌引起的大鼠心肌细胞内游离钙水平变化的调节作用。分别利用模拟缺血液和正常台氏液对大鼠心肌细胞进行缺血 /复灌处理 ,在缺血期间分别应用Na+/H+交换抑制剂阿米洛利 (amiloride)、Na+/Ca2 +交换抑制剂NiCl2 以及无钙液 ,观察它们对细胞内游离Ca2 +浓度变化的影响。利用Zeiss LSM 5 10激光共聚焦显微镜检测、采集细胞内游离Ca2 +的指示剂Fluo 3 AM的荧光信号 ,计算出相对于正常(缺血前 )的相对荧光强度 ,以表示胞内游离Ca2 +浓度的变化。结果显示 ,模拟缺血引起大鼠心肌细胞内游离Ca2 +持续上升 ,缺血前的相对荧光强度值为 10 0 % ,模拟缺血 5min后为 140 3± 13 0 % (P <0 0 5 ) ,复灌 15min后为 142 8±15 5 % (P <0 0 5 )。经 10 0 μmol/Lamiloride、5mmol/LNiCl2 和无钙液分别预处理 ,模拟缺血 5min后的相对荧光强度分别为 10 1 4± 16 3 % (P <0 0 5 )、110 4± 11 1% (P <0 0 5 )和 10 7 1± 10 8(P <0 0 5 ) ;复灌 15min后则分别为 97 8±14 3 % (P <0 0 5 )、10 6 2± 14 5 % (P <0 0 5 )和 10 6 6± 15 7(P <0 0 5 )。另外 ,与对照组细胞相比 ,再灌注期间NiCl2和无钙液处理的细胞钙振荡的产生幅度明显减弱 ,amilorid     

Modulation of Mg2+ efflux from rat ventricular myocytes studied with the fluorescent indicator furaptra

The fluorescent Mg(2+) indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with its acetoxy-methyl ester form. The ratio of furaptra's fluorescence intensity at 382 and 350 nm was used to estimate the apparent cytoplasmic [Mg(2+)] ([Mg(2+)](i)). In Ca(2+)-free extracellular conditions (0.1 mM EGTA) at 25 degrees C, [Mg(2+)](i) averaged 0.842 +/- 0.019 mM. After the cells were loaded with Mg(2+) by exposure to high extracellular [Mg(2+)] ([Mg(2+)](o)), reduction of [Mg(2+)](o) to 1 mM (in the presence of extracellular Na(+)) induced a decrease in [Mg(2+)](i). The rate of decrease in [Mg(2+)](i) was higher at higher [Mg(2+)](i), whereas raising [Mg(2+)](o) slowed the decrease in [Mg(2+)](i) with 50% reduction of the rate at approximately 10 mM [Mg(2+)](o). Because a part of the furaptra molecules were likely trapped inside intracellular organelles, we assessed possible contribution of the indicator fluorescence emitted from the organelles. When the cell membranes of furaptra-loaded myocytes were permeabilized with saponin (25 microg/ml for 5 min), furaptra fluorescence intensity at 350-nm excitation decreased to 22%; thus approximately 78% of furaptra fluorescence appeared to represent cytoplasmic [Mg(2+)] ([Mg(2+)](c)), whereas the residual 22% likely represented [Mg(2+)] in organelles (primarily mitochondria as revealed by fluorescence imaging). [Mg(2+)] calibrated from the residual furaptra fluorescence ([Mg(2+)](r)) was 0.6-0.7 mM in bathing solution [Mg(2+)] (i.e., [Mg(2+)](c) of the skinned myocytes) of either 0.8 mM or 4.0 mM, suggesting that [Mg(2+)](r) was lower than and virtually insensitive to [Mg(2+)](c). We therefore corrected furaptra fluorescence signals measured in intact myocytes for this insensitive fraction of fluorescence to estimate [Mg(2+)](c). In addition, by utilizing concentration and dissociation constant values of known cytoplasmic Mg(2+) buffers, we calculated changes in total Mg concentration to obtain quantitative information on Mg(2+) flux across the cell membrane. The calculations indicate that, in the presence of extracellular Na(+), Mg(2+) efflux is markedly activated by [Mg(2+)](c) above the normal basal level (approximately 0.9 mM), with a half-maximal activation of approximately 1.9 mM [Mg(2+)](c). We conclude that [Mg(2+)](c) is tightly regulated by an Mg(2+) efflux that is dependent on extracellular [Na(+)].     

Capsazepine elevates intracellular Ca2+ in human osteosarcoma cells, questioning its selectivity as a vanilloid receptor antagonist

Capsazepine is thought to be a selective antagonist of vanilloid type 1 receptors; however, its other in vitro effect on different cell types is unclear. In human MG63 osteosarcoma cells, the effect of capsazepine on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Capsazepine caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. Capsazepine-induced [Ca(2+)](i) rise was partly reduced by removal of extracellular Ca(2+), suggesting that the capsazepine-induced [Ca(2+)](i) rise was composed of extracellular Ca(2+) influx and intracellular Ca(2+). In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of capsazepine on [Ca(2+)](i) was inhibited by 75%. Conversely, pretreatment with capsazepine to deplete intracellular Ca(2+) stores totally prevented thapsigargin from releasing more Ca(2+). U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not capsazepine-induced, [Ca(2+)](i) rise. Overnight treatment with 1-100 microM capsazepine inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, capsazepine increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing intracellular Ca(2+) release from the endoplasmic reticulum via a phospholiase C-independent manner. Capsazepine may be mildly cytotoxic.