Role of mitochondrial calcium uniporter in regulating mitochondrial fission in the cerebral cortexes of living rats

The mitochondrial calcium uniporter (MCU) transports Ca²⁺ from the cytoplasm to the mitochondrial matrix and thus maintains Ca²⁺ homeostasis. Previous studies have reported that inhibition of MCU by ruthenium red (RR) protects the brain from ischemia/reperfusion (I/R) injury and that mitochondrial fission plays an important role in I/R injury. However, it is still not known whether MCU affects mitochondrial fission. In the present study, treatment with RR was found to decrease the concentration of free calcium in the mitochondria, calcineurin enzyme activity and dynamin-related protein 1 expression, and treatment with spermine was found to have the opposite effect in organisms subjected to occlusion of the middle cerebral artery lasting 2 h followed by 24 h reperfusion. These results indicate that MCU may be related to mitochondrial fission via modulating mitochondrial Ca²⁺ uptake and this relationship between MCU and mitochondrial fission may protect the brain from I/R injury.     

3‐nitropropionic acid‐induced mitochondrial permeability transition: Comparative study of mitochondria from different tissues and brain regions

The adult rat striatum is particularly vulnerable to systemic administration of the succinate dehydrogenase inhibitor 3‐nitropropionic acid (3NP), which is known to induce degeneration of the caudate‐putamen, as occurs in Huntington's disease. The aim of the present study was to compare the susceptibility of isolated mitochondria from different rat brain regions (striatum, cortex, and cerebellum) as well as from the liver, kidney, and heart to mitochondrial permeability transition (MPT) induced by 3NP and Ca²⁺. In the presence of micromolar Ca²⁺ concentrations, 3NP induces MPT in a dose‐dependent manner, as estimated by mitochondrial swelling and a decrease in the transmembrane electrical potential. A 3NP concentration capable of promoting a 10% inhibition of ADP‐stimulated, succinate‐supported respiration was sufficient to stimulate Ca²⁺‐induced MPT. Brain and heart mitochondria were generally more sensitive to 3NP and Ca²⁺‐induced MPT than mitochondria from liver and kidney. In addition, a partial inhibition of mitochondrial respiration by 3NP resulted in more pronounced MPT in striatal mitochondria than in cortical or cerebellar organelles. A similar inhibition of succinate dehydrogenase activity was observed in rat tissue homogenates obtained from various brain regions as well as from liver, kidney, and heart 24 hr after a single i.p. 3NP dose. Mitochondria isolated from forebrains of 3NP‐treated rats were also more susceptible to Ca²⁺‐induced MPT than those of control rats. We propose that the increased susceptibility of the striatum to 3NP‐induced neurodegeneration may be partially explained by its susceptibility to MPT, together with the greater vulnerability of this brain region to glutamate receptor‐mediated Ca²⁺ influx. © 2009 Wiley‐Liss, Inc.     

Spermine increases paired-pulse facilitation in area CA1 of hippocampus in a calcium-dependent manner

The effect of spermine on neurotransmission was studied in area CA1 of the hippocampal slice preparation. Paired-pulse stimulation (20 ms interpulse interval) was delivered to stratum radiatum; the evoked field potential responses were recorded simultaneously from stratum radiatum and from stratum pyramidale. At mM and sub-mM concentrations, spermine decreased the slope of pEPSP in stratum radiatum and the area of the conditioning population spike in stratum pyramidale. Short-latency paired-pulse inhibition of the population spike was converted to facilitation by spermine. These effects of spermine resembled those observed at low calcium concentration. In addition, dose-response and input-output curves determined at various Ca²⁺ concentrations demonstrated that the depressant effects of spermine were larger at low Ca²⁺ levels. The results support the notion that spermine competitively blocks presynaptic voltage-sensitive Ca²⁺ channels, thus causing a decreased release of neurotransmitter. Since spermine is present in brain, it is likely that it is a natural modulator of Ca²⁺ channels.     

Ethylmalonic Acid Induces Permeability Transition in Isolated Brain Mitochondria

Predominant accumulation of ethylmalonic acid (EMA) in tissues and biological fluids is a characteristic of patients affected by short chain acyl-CoA dehydrogenase deficiency and ethylmalonic encephalopathy. Neurological abnormalities are frequently found in these disorders, but the mechanisms underlying the brain injury are still obscure. Since hyperlacticacidemia is also found in many affected patients indicating a mitochondrial dysfunction; in the present work, we evaluated the in vitro and ex vivo effects of EMA plus Ca²⁺ on mitochondrial integrity and redox balance in succinate-supported brain organelles. We verified that the evaluated parameters were disturbed only when EMA was associated with exogenous micromolar Ca²⁺ concentrations. Thus, we found that this short chain organic acid plus Ca²⁺ dissipated the membrane potential and provoked mitochondrial swelling, as well as impaired the mitochondrial Ca²⁺ retention capacity, resulting in a rapid Ca²⁺ release and decreased NAD(P)H matrix content. In contrast, EMA was not able to stimulate mitochondrial hydrogen peroxide generation. We also observed that all these effects were prevented by the mitochondrial Ca²⁺ uptake inhibitor ruthenium red and the mitochondrial permeability transition (MPT) inhibitors cyclosporin A (CsA) and ADP. Furthermore, mitochondria isolated from rat brains after in vivo intrastriatal administration of EMA was more susceptible to Ca²⁺-induced swelling, which was fully prevented by CsA and ADP. Finally, EMA significantly decreased striatal slice viability, which was attenuated by CsA. The data strongly indicate that EMA reduced the mitochondrial threshold for Ca²⁺-induced MPT reinforcing the role of this cation in EMA-induced disruption of mitochondrial bioenergetics. It is, therefore, presumed that EMA acting synergistically with Ca²⁺ compromises mitochondrial energy homeostasis in the central nervous system that may explain at least in part the neurologic alterations presented by patients with abnormal levels of this organic acid.     

Deficient Cation Channel Regulation in Neurons From Mice With Targeted Disruption of the Extracellular Ca-Sensing Receptor Gene

This study presents evidence that a receptor sensitive to the concentration of extracellular Ca²⁺ (Ca²⁺_o) (CaR) is functionally coupled to ion channels involved in modulation of neuronal excitability. This receptor is expressed in hippocampus and other brain regions, suggesting that it could mediate some of the well-recognized but poorly understood direct actions of extracellular Ca²⁺ (Ca²⁺_o) on neuronal function. The effects of polycationic CaR agonists on the activity of a nonselective cation channel (NCC) in cultured hippocampal neurons from wild-type mice and from mice homozygous for targeted disruption of the CaR gene (CaR −/−) were compared in this study. The CaR agonists, neomycin (100 μM), spermine (300 μM), and elevation of Ca²⁺_o from 0.75 to 3 mM, significantly increased the probability of channel opening (Po) in wild-type neurons. None of these agents, however, produced any effect on Po in neurons from mice lacking the CaR. The same NCC, however, could be activated by thapsigargin in neurons from both wild-type mice and CaR-deficient mice, most likely through an associated increase in the cytosolic free calcium concentration (Ca_i). Thus the CaR regulates the activity of Ca²⁺-permeable NCC in hippocampal neurons and could potentially modulate key neuronal functions, including neurotransmission and neuronal excitability, via membrane depolarization.     

NMDA-induced increase in [Ca]i andCa uptake in acutely dissociated brain cells derived from adult rats

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-d-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca²⁺]_i measured with fluo3 and⁴⁵Ca²⁺ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 μM) and N-methyl-dl-aspartate (200 μM) increased [Ca²⁺]_i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated⁴⁵Ca²⁺ uptake about 16–10% in the same regions. The increases in [Ca²⁺]_i and⁴⁵Ca²⁺ uptake were inhibited by 40% in the presence of 1 mM MgCl₂ and by 90–50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca²⁺]_i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca²⁺]_i increase in adult age.     

Apoptotic markers in the mitochondria, cytosol, and nuclei of brain cells during ammonia toxicity

The effects of toxic doses of ammonia on the induction of pro-apoptotic markers in rat brain cells were studied. It was found that neither induction of caspase-9 and caspase-3, nor changes in the membrane potential in nonsynaptic brain mitochondria, release of cytochrome c from mitochondria to cytosol, or apoptosis-inducing signals in the cell nuclei, were responsible for the ammonia toxicity in vivo. Thus, no evidence was found for any functional significance of mitochondria in cell death associated with acute hyperammonemia. The resistance of cell nuclei to ammonia-induced apoptosis could be caused either by a disordered translocation of the p53 protein from the cytosol into the nucleus or by translocation of its inactive form. The nonsynaptic brain mitochondria were also shown to be more resistant to the formation of nonspecific permeability pores and to Ca²⁺-induced swelling than cardiac and hepatic mitochondria and, as well, supported their membrane potential even under conditions of ammonia-stimulated production of active oxygen metabolites, excessive accumulation of Ca²⁺, oxidative stress, and impaired energy metabolism.     

Effects of Pb and Cd on acetylcholine release and Ca movements in synaptosomes and subcellular fractions from rat brain and Torpedo electric organ

In this work we examined the effects of Pb²⁺ and Cd²⁺ on (a) [³H]ACh release and voltage-sensitive Ca²⁺ channels in rat brain synaptosomes, and (b)⁴⁵Ca²⁺ binding to isolated brain mitochondria and microsomes, and synaptic vesicles isolated from Torpedo electric organs. Pb²⁺ (K_i ≈ 1.1 μM) and Cd²⁺ (K_i ≈ 2.2) competitively block the K⁺-evoked influx of⁴⁵Ca²⁺ through the ‘fast’ calcium channels in synaptosomes. The K_is obtained with synaptosomes are in good agreement with the K_i values obtained from electrophysiological experiments at the frog neuromuscular junction (K_Pb:0.99 μM, K_Cd: 1.7 μM)⁷. The K_i for the inhibition of ACh release from synaptosomes by Cd²⁺ is 4.5 μM. Pb²⁺ is a less effective inhibitor of transmitter release (K_i ≈ 16 μM) because it secondarily augments spontaneous transmitter efflux. Cd²⁺ has no effect on spontaneous release at concentrations ≤ 100 μM. The enhancing effect of Pb²⁺ on spontaneous release is (a) not abolished by omission of Ca²⁺ from the bathing medium, (b) is delayed by 1–2 min after the beginning of Pb²⁺ exposure, (c) is reversed upon the removal of Pb²⁺. In the presence of physiological concentrations of ATP (1 mM), Mg²⁺ (1 mM) and P_i (2 mM), 1–10 μM Pb²⁺ inhibits calcium uptake but Pb²⁺ > 10μM causes a several-fold stimulation of passive binding of calcium to the organelles. This effect is associated with Pb²⁺-induced enhancement of P_i uptake. Cd²⁺ inhibits Ca²⁺ binding at all concentrations tested (1–50 μM) and reduces the Pb²⁺-induced Ca²⁺-binding to organelles. Neither Pb²⁺ nor Cd²⁺ have any discernible effects on spontaneous loss of calcium from mitochondria or microsomes preloaded with⁴⁵Ca. In summary, these data are consistent with the notion that Pb²⁺ and Cd²⁺ are potent blockers of presynaptic voltage-sensitive Ca²⁺ channels and the evoked release of transmitter which is contingent on Ca²⁺ influx through these channels. Our results are not consistent with the hypothesis that Pb²⁺ augments spontaneous release by interfering with intraterminal Ca²⁺-buffering by mitochondria, endoplasmic reticulum, or synaptic vesicles.     

Spermine is neuroprotective against anoxia and N-methyl- -aspartate in hippocampal slices

Polyamines were implicated as either neurotoxic or neuroprotective in several models of stroke. Spermine augments the excitotoxicity mediated by the N-methyl- -aspartate (NMDA) receptor because this receptor is activated at micromolar spermine concentrations. However, at higher concentrations, spermine could be neuroprotective because it blocks the NMDA receptor and voltage-activated Ca²⁺ channels. In this work, acute hippocampal slices were exposed to 1 mM spermine and either 10 min of anoxia or 0.5 mM NMDA. The percent recovery of population spikes was the measure of neuroprotection. One millimolar spermine was robustly neuroprotective; however, 0.1 mM spermine and 1 mM putrescine were not. The neuroprotective concentration of spermine was higher than the physiological concentration of free spermine. However, during an excitotoxic episode, extracellular Ca²⁺ is decreased, enabling the inhibitory activity of lower spermine concentration. In addition, several noxious stimuli trigger the release of intracellular spermine and could raise local levels of spermine. Therefore, it is possible that spermine has a neuroprotective role in vivo.     

Reversal of impaired oxidative phosphorylation and calcium overloading in the skeletal muscle mitochondria of CHF-146 dystrophic hamsters

Membrane-mediated excessive intracellular calcium accumulation (EICA) and diminished cellular energy production are the hallmarks of dystrophic pathobiology in Duchenne and Becker muscular dystrophies. We reported reversal of respiratory damage and Ca²⁺-overloading in the in vitro cardiac mitochondria from CHF-146 dystrophic hamsters (DH) with hereditary muscular dystrophy (Bhattacharya et al., 1993). Here we studied respiratory dysfunctions in the skeletal muscle mitochondria from young and old DH, and whether these abnormalities can be reversed by reducing [Ca²⁺] in the isolation medium, thereby lowering intramitochondrial Ca²⁺-overloading. Age- and sex-matched CHF-148 albino normal hamsters (NH) served as controls. As an index of EICA and cellular degeneration, Ca and Mg levels were assayed in the skeletal muscle and mitochondria. Mitochondria from young and old DH, isolated without EDTA (B_o medium), revealed poor coupling of oxidative phosphorylation, diminished stimulated oxygen consumption rate, and lower respiratory control ratio and ADP/O ratios, compared to NH. Incorporation of 10 mM EDTA (B_E medium) in the isolation medium restored mitochondrial functions of the dystrophic organelles to a near-normal level, and reduced Ca²⁺-overloading. The mitochondrial Ca level in DH was significantly higher than in NH, irrespective of the medium. However, compared to B_o medium, the dystrophic organelles isolated in B_E medium had lower Ca levels and markedly improved oxidative phosphorylation as seen in NH. Muscle Ca contents in the young and old DH were elevated relative to NH, showing a positive correlation with the increased mitochondrial Ca²⁺-sequestration. Dystrophic muscle also revealed Ca deposition with an abundance of Ca²⁺-positive and necrotic myofibers by light microscopy, and intramitochondrial Ca²⁺-overloading by electron microscopy, respectively. However, Mg levels in the muscle and mitochondria did not alter with age or dystrophy. These data parallel our observations in the heart, and suggest that functional impairments and Ca²⁺-overloading also occur in the skeletal muscle mitochondria of DH, and are indeed reversible if EICA is regulated by slow Ca²⁺-channel blocker therapy (Johnson and Bhattacharya, 1993).     

Age- and region-dependent patterns of Ca accumulations following status epilepticus

Elevated Ca²⁺ concentrations have been implicated in cell death mechanisms following seizures, however, the age and brain region of intracellular Ca²⁺ accumulations [Ca²⁺]_i, may influence whether or not they are toxic. Therefore, we examined regional accumulations of ⁴⁵Ca²⁺ by autoradiography from rats of several developmental stages (P14, P21, P30 and P60) at 5, 14, and 24 h after status epilepticus. To determine whether the uptake was intracellular, Ca²⁺ was also assessed in hippocampal slices with the dye indicator, Fura 2AM at P14. Control animals accumulated low homogeneous levels of ⁴⁵Ca²⁺; however, highly specific and age-dependent patterns of ⁴⁵Ca²⁺ uptake were observed at 5 h. ⁴⁵Ca²⁺ accumulations were predominant in dorsal hippocampal regions, CA1/CA2/CA3a, in P14 and P21 rats and in CA3a and CA3c neurons of P30 and P60 rats. Selective midline and amygdala nuclei were marked at P14 but not at P21 and limbic accumulations recurred with maturation that were extensive at P30 and even more so at P60. At 14 h, P14 and P21 rats had no persistent accumulations whereas P30 and P60 rats showed persistent uptake patterns within selective amygdala, thalamic and hypothalamic nuclei, and other limbic cortical regions that continued to differ at these ages. For example, piriform cortex accumulation was highest at P60. Fura 2AM imaging at P14 confirmed that Ca²⁺ rises were intracellular and occurred in both vulnerable and invulnerable regions of the hippocampus, such as CA2 pyramidal and dentate granule cells. Silver impregnation showed predominant CA1 injury at P20 and P30 but CA3 injury at P60 whereas little or no injury was found in extrahippocampal structures at P14 and P20 but was modest at P30 and maximal at P60. Thus, at young ages there was an apparent dissociation between high ⁴⁵Ca²⁺ accumulations and neurotoxicity whereas in adults a closer relationship was observed, particularly in the extrahippocampal structures.     

BK channel openers inhibit ROS production of isolated rat brain mitochondria

To delineate the potential mechanism of neuroprotective effects of potassium channel openers we have investigated, how Ca²⁺-activated large conductance potassium channel (BK_Ca channel) openers influence the production of reactive oxygen species (ROS) by rat brain mitochondria, since mitochondrial generation of ROS is known to have a crucial influence on neuronal survival. We studied the effects of BK_Ca channel openers CGS 7184 and NS 1619 on hydrogen peroxide production rate of isolated rat brain mitochondria. In K⁺-containing media 3 μM of both channel openers reduced the hydrogen peroxide production rates by approximately 20%. This effect was not observed in Na⁺-containing media. This potassium-dependent partial inhibition of hydrogen peroxide production was found to be sensitive to the selective blockers of BK_Ca channel iberiotoxin and charybdotoxin applied in nanomolar concentrations. Taken together, our data are compatible with the viewpoint that the opening of a Ca²⁺-activated large conductance potassium channel being localised in the inner membrane of brain mitochondria inhibits ROS production by respiratory chain complex I. This finding is suggested to explain the beneficial effects of BK potassium channel openers on neuronal survival.     

Regulation of isometric contraction in skeletal muscle

The purpose of this study was to determine whether differences in isometric twitch contraction times of skeletal muscles are related more closely to myosin adenosine triphosphatase (ATPase) activity or to the ability of the sarcotubules to accumulate Ca²⁺. The isometric contraction time was observed to be shorter in the crureus muscle than in the soleus muscle of the rabbit. These muscles were found to have similar myosin ATPase activities, but sarcotubules isolated from crureus had a faster rate of Ca²⁺ uptake than soleus sarcotubules. Furthermore, the yield of sarcotubules was 41% higher from crureus. Likewise, the isometric contraction time of rat soleus decreased during the first 3 weeks of life, but no change was observed in myosin ATPase activity. However, sarcotubular uptake of Ca²⁺ increased as did the yield of sarcotubules (44% higher). It is suggested that the differences in isometric contraction time between skeletal muscles are related more closely to the sarcotubular Ca²⁺ uptake than to the activity of myosin ATPase.     

Stimulation of calcium uptake in cultured astrocytes by hypoosmotic stress — effect of cyclic AMP

To investigate the role of Ca²⁺ in astrocyte volume regulation, we determined Ca²⁺ fluxes following hypoosmotic stress and how these fluxes were modified by cyclic AMP. In isoosmotic conditions treatment with dibutyryl cyclic AMP (dBcAMP) caused almost a twofold increase in ⁴⁵Ca²⁺ uptake. Efflux studies of ⁴⁵Ca²⁺ in dBcAMP-treated cells showed three Ca²⁺ compartments while only two Ca²⁺ compartments were identified in non-dBcAMP-treated cells. Following hypoosmotic stress a twofold stimulation of ⁴⁵Ca²⁺ uptake occurred in both non-dBcAMP-treated and dBcAMP-treated astrocytes. Stimulation of Ca²⁺ uptake begins at 270 mOsm and is half-maximally stimulated at 100 mOsm. This uptake is partly mediated through L-type ‘slow’ inactivating Ca²⁺ channels. Hypoosmotic stress also induces the release of Ca²⁺ from intracellular stores. The influx of extracellular Ca²⁺ and efflux of intracellular Ca²⁺ appear to be important factors in volume regulation after hypoosmotic stress. Cyclic AMP plays an important role in modulating hypoosmotically stimulated Ca²⁺ uptake.     

Effect of cadmium on Ca transport in brain microsomes

The effect of Cd²⁺ on Ca²⁺ transport properties (uptake/release) in rat brain microsomes is examined by the tracer method using ⁴⁵Ca²⁺ Cadmium ion (Cd²⁺) shows a dose-dependent inhibition of Ca²⁺-ATPase activity and consequently, exhibits a reduction in ATP-dependent Ca²⁺ uptake. In addition to this, Cd²⁺ also stimulates a rapid release of Ca²⁺ ( ) from the microsomes in a dose-dependent manner. The effect of Cd²⁺ is reversible by 1 mM cysteine or dithiothreitol (DTT). It is suggested that Cd²⁺ plays an important role in regulating the transmembrane flux of the cations in the microsomes. This effect is dramatically modulated by DTT suggesting a role of sulfhydryl groups in Ca²⁺ -transport.     

Dense-cored vesicles,smooth endoplasmic reticulum,and mitochondria are closely associated with non-specialized parts of plasma membrane of nerve terminals: Implications for exocytosis and calcium buffering by intraterminal organelles

To determine whether there are anatomical correlates for intraterminal Ca²⁺ stores to regulate exocytosis of dense-cored vesicles (DCVs) and whether these stores can modulate exocytosis of synaptic vesicles, we studied the spatial distributions of DCVs, smooth endoplasmic reticulum (SER), and mitochondria in 19 serially reconstructed nerve terminals in bullfrog sympathetic ganglia. On average, each bouton had three active zones, 214 DCVs, 26 SER fragments (SERFs), and eight mitochondria. DCVs, SERFs and mitochondria were located, on average, 690, 624, and 526 nm, respectively, away from active zones. Virtually no DCVs were within “docking” (i.e., ≤50 nm) distances of the active zones. Thus, it is unlikely that DCV exocytosis occurs at active zones via mechanisms similar to those for exocytosis of synaptic vesicles. Because there were virtually no SERFs or mitochondria within 50 nm of any active zone, Ca²⁺ modulation by these organelles is unlikely to affect ACh release evoked by a single action potential. In contrast, 30% of DCVs and 40% of SERFs were located within 50 nm of the nonspecialized regions of the plasma membrane. Because each bouton had at least one SERF within 50 nm of the plasma membrane and most of these SERFs had DCVs, but not mitochondria, near them, it is possible for Ca²⁺ release from the SER to provide the Ca²⁺ necessary for DCV exocytosis. The fact that 60% of the mitochondria had some part within 50 nm of the plasma membrane means that it is possible for mitochondrial Ca²⁺ buffering to affect DCV exocytosis. J. Comp. Neurol. 403:378–390, 1999. © 1999 Wiley-Liss, Inc.     

Homeostasis of mitochondrial calcium is disturbed in the cerebellum but not in other brain areas during chronic hyperammonemia

We studied the effects of chronic hyperammonemia, which was induced by consumption of ammonium acetate with food, on homeostasis of mitochondrial calcium in the neocortex, cerebellum, hippocampus, and striatum of rats. We found that in cerebellar mitochondria chronic moderate hyperammonemia results in an increase in the content of endogenous calcium and a decrease in the calcium capacity, rate of Ca²⁺ uptake, and the rates of Na-dependent and hydrogen peroxide-dependent Ca²⁺ release. Thus, only cerebellar mitochondria in vivo are sensitive to food salts of ammonium and hyperammonemia leads to damage of the mitochondrial system of Ca²⁺ transport only in the cerebellum.     

Differential NMDA receptor-dependent calcium loading and mitochondrial dysfunction in CA1 vs. CA3 hippocampal neurons

Hippocampal CA1 pyramidal neurons are selectively vulnerable to ischemia, while adjacent CA3 neurons are relatively resistant. Although glutamate receptor-mediated mitochondrial Ca²⁺ overload and dysfunction is a major component of ischemia-induced neuronal death, no direct relationship between selective neuronal vulnerability and mitochondrial dysfunction has been demonstrated in intact brain preparations. Here, we show that in organotypic slice cultures NMDA induces much larger Ca²⁺ elevations in vulnerable CA1 neurons than in resistant CA3. Consequently, CA1 mitochondria exhibit stronger calcium accumulation, more extensive swelling and damage, stronger depolarization of their membrane potential, and a significant increase in ROS generation. NMDA-induced Ca²⁺ and ROS elevations were abolished in Ca²⁺-free medium or by NMDAR antagonists, but not by zinc chelation. We conclude that Ca²⁺ overload-dependent mitochondrial dysfunction is a determining factor in the selective vulnerability of CA1 neurons.     

Adenylyl Cyclase mRNA Expression does not Reflect the Predominant Ca2+/Calmodulin-Stimulated Activity in the Hypothalamus

Only three (Types I, II, V) of the six currently-described subtypes of adenylyl cyclase are prominently expressed in the rat brain. These species are differently sensitive to Ca²⁺, βγ subunits of G-proteins and protein kinase C. A knowledge of the susceptibility of the cAMP-signalling system in particular brain regions to these diverse modes of regulation can shed light on the mechanism of action of the neurotransmitters that modify neuronal activity in such regions. Cyclic AMP is extensively involved in the physiological functions of the hypothalamus. We have used in situ hybridization histochemistry with synthetic oligonucleotides to examine the expression in the rat hypothalamus of the three major brain subtypes of adenylyl cyclase-Ca²⁺/calmodulin-stimulable (Type I), Ca²⁺-insensitive (Type II) and Ca²⁺ -inhibitable (Type V). The hypothalamus expresses high levels only of Type II mRNA, particularly in the supraoptic and paraventricular nuclei. Curiously, the strong expression of the Ca²⁺-insensitive Type II mRNA and the lack of expression of the major brain specific Type I mRNA does not correlate with the adenylyl cyclase activity, which is largely Ca²⁺/calmodulin stimulable in plasma membranes prepared from the hypothalamus.     

Specialized distributions of mitochondria and endoplasmic reticulum proteins define Ca2+ wave amplification sites in cultured astrocytes

This study was undertaken to examine the expression and role of the endoplasmic reticulum (ER) proteins calreticulin and ryanodine receptors, and mitochondria, in cultured astrocytes. Using several lines of investigation, we have identified a key role for mitochondria in astrocyte Ca²⁺ signalling: (1) a significant correlation was found between sites of regenerative Ca²⁺ wave amplification (possessing high amplitude ER Ca²⁺ release) and the location of mitochondria in the cell; (2) norepinephrine (2 μM) caused a rapid-onset increase in rhod 2 fluorescence in 34% of astrocyte mitochondria, indicating that cytosolic Ca²⁺ responses result in mitochondrial Ca²⁺ elevation; and (3) pretreatment with the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone to inhibit mitochondrial activity markedly reduced the amplitude of subsequent norepinephrine-evoked cytosolic Ca²⁺ responses. We then investigated the roles of several ER proteins in Ca²⁺ signalling by immunocytochemistry. Ryanodine receptors and calreticulin were found to be expressed in heterogeneous patterns in astrocytes. The expression pattern of calreticulin corresponded closely with the distribution of mitochondria, whereas the expression of ryanodine receptors was not similar to that of either of these cellular factors. We measured Ca²⁺ wave kinetics in a single astrocyte, then assessed protein distribution by immunocytochemistry in the same cell. Cross-correlation between norepinephrine-evoked Ca²⁺ wave amplitude and calreticulin distribution indicated a close spatial relationship between this Ca²⁺-binding protein and sites of regenerative wave amplification. These results demonstrate that amplification sites for Ca²⁺ waves in astrocytes are identifiable by accumulations of calreticulin (and type 2 InsP₃Rs), and by the presence of mitochondria, which may regulate the ER Ca²⁺ release process. J. Neurosci. Res. 52:672–683, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.