Cytosolic Ca2+ under high glucose with suppressed Na+/K+ pump activity in rat sensory neurons

Cytosolic Ca2+ concentration ([Ca2+]i) was measured in isolated rat dorsal root ganglion (DRG) neurons using the fluorescent Ca2+ indicator fura-2. Exposure to high (50 mM) extracellular K+ evoked a robust increase in [Ca2+]i, which was almost totally abolished by concomitant presence of nisoldipine (10 microM) and omega-conotoxin GVIA (10 microM). Whereas either high (30 mM) D-glucose alone or ouabain (100 microM) alone did not appreciably affect the high K+-induced [Ca2+]i elevation, neurons pretreated with high D-glucose together with ouabain exhibited a significantly larger [Ca2+]i response to high K+ stimulation, which was almost completely inhibited by nisoldipine and omega-conotoxin GVIA. These results suggest that a combination of high glucose and suppressed Na+/K+ pump activity potentiates the [Ca2+]i elevation stimulated by activation of the voltage-gated Ca2+ channels in rat DRG neurons.     

Dendritic electrogenesis in rat hippocampal CA1 pyramidal neurons: functional aspects of Na+ and Ca2+ currents in apical dendrites

The regenerative properties of CA1 pyramidal neurons were studied through differential polarization with external electrical fields. Recordings were obtained from somata and apical dendrites in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-amino-5-phosphonovaleric acid (APV), and bicuculline. S+ fields hyperpolarized the distal apical dendrites and depolarized the rest of the cell, whereas S÷ fields reversed the polarization. During intradendritic recordings, S+ fields evoked either fast spikes or compound spiking. The threshold response consisted of a low-amplitude fast spike and a slow depolarizing potential. At higher field intensities the slow depolarizing potential increased in amplitude, and additional spikes of high amplitude appeared. During intrasomatic recordings, S+ field evoked repetitive firing of fast spikes, whereas S÷ fields evoked a slow depolarizing, potential on top of which high- and low-amplitude spikes were evoked. Tetrodotoxin (TTX) blocked all types of responses in both dendrites and somata. Perfusion with Ca²⁺-free, Co²⁺-containing medium increased the frequency and amplitude of fast spikes evoked by S+ field and substantially reduced the slow depolarizing potential evoked by S÷ fields. Antidromic stimulation revealed that an all-or-none dendritic component was activated in the distal apical dendrites by back-propagating somatic spikes. The dendritic component had an absolute refractory period of about 4 ms and a relative refractory period of 10–12 ms. Ca²⁺-dependent spikes in the dendrites were followed by a long-lasting afterhyperpolarization (AHP) and a decrease in membrane input resistance, during which dendritic excitability was selectively reduced. The data suggest that generation of fast Na⁺ currents and slow Ca²⁺ currents in the distal part of apical dendrites is highly sensitive to the dynamic state of the dendritic membrane. Depending on the mode and frequency of activation these currents can exert a substantial influence on the input-output behavior of the pyramidal neurons. © 1996 Wiley-Liss, Inc.     

Na+/Ca2+ exchanger 1 alters in pyramidal cells and expresses in astrocytes of the gerbil hippocampal CA1 region after ischemia

Alterations of immunoreactivity and protein contents of Na(+)/Ca(2+) exchanger 1 (NCX1) were observed in the gerbil hippocampus proper after 5 min of transient forebrain ischemia. NCX1 immunoreactivity was significantly changed in the hippocampal CA1 region, but not in the CA2/3 region after ischemia/reperfusion. In the sham-operated group, NCX1 immunoreactivity was mainly detected in CA1 pyramidal cells. However, 30 min after ischemia/reperfusion, NCX1 immunoreactivity was significantly decreased and then increased at 1 day after ischemia. At this time, NCX1 immunoreactivity in CA1 pyramidal cells was similar to that of the sham-operated group. At 3 days after ischemia, NCX1 immunoreactivity was significantly reduced in the CA1 region compared to that of the sham-operated group and NCX1 immunoreactivity was significantly increased again 4 days after ischemia. Thereafter, NCX1 immunoreactivity was decreased time-dependently in ischemia groups. Between 15 min and 6 h post-ischemia, NCX1 immunoreactivity was expressed in astrocytes in the strata oriens and radiatum of the CA1 region. From 3 days post-ischemia, NCX1 immunoreactivity was expressed in astrocytes in the strata oriens and radiatum. Ischemia-induced changes in NCX1 protein contents in the hippocampus proper concurred with immunohistochemical data post-ischemia. Our results suggest that changes in NCX1 in CA1 pyramidal cells and astrocytes after ischemia are associated with intracellular Na(+) concentrations and that NCX1 may induce an intracellular calcium overload, which may be related to neuronal death.     

Effects of adrenalectomy on Ca2+ currents and Ca2+ channel subunit mRNA expression in hippocampal CA1 neurons of young rats

Previously it was found that the amplitude of Ca currents in CA1 hippocampal neurons increases after adrenalectomy (ADX) of young adult rats. Preliminary data suggested that this effect of ADX is age-dependent. In the present study we therefore investigated the effect of ADX on Ca currents in three age groups: rats that were 1, 3, or 6 months old. It appeared that ADX of the youngest age group resulted in a selective enhancement of sustained, high threshold Ca currents. By contrast, ADX of the oldest rats enhanced only the low threshold, transient Ca current amplitude. The age dependency of the ADX-induced effects can be explained by developmental changes in Ca current properties in the adrenally intact rats with which ADX animals were compared. Data from acutely dissociated cells, which lack most of their dendrites, suggest that the ADX-induced changes of Ca current properties are at least partly targeted at currents generated in the distal dendrites. No significant changes were observed with age or after ADX for the mRNA expression of the α1D Ca channel subunit, which forms the ionpore of the sustained high threshold L-type Ca channel. We can therefore presently not exclude that the altered amplitude of Ca current with age and ADX is due to changes in ion channel function rather than in the total number of these channels. Synapse 26:155–164, 1997. © 1997 Wiley-Liss, Inc.     

T-type Ca2+ channel activity increases in rat hippocampal CA1 region during kindling epileptogenesis

Epileptogenesis is a dynamical process that involves synaptic plasticity changes such as synaptic reorganization of excitatory and inhibitory systems and axonal sprouting in the hippocampus, which is one of the most studied epileptogenic regions in the brain. However, the early events that trigger these changes are not understood well. We investigated short-term and long-term synaptic plasticity parameters and T-type Ca²⁺ channel activity changes in the early phase of a rat kindling model. Chronic pentylenetetrazole (PTZ) application was used in order to induce the kindling process in rats. The recordings were obtained from hippocampal slices in the CA1 region at 25th day of PTZ application. Tetraethylammonium was used in order to induce long-term potentiation and T-type Ca²⁺ channel activity was assessed in the presence of mibefradil. We found that tetraethylammonium-induced long-term potentiation was not prevented by mibefradil in the kindling group in contrast to control group. We also found an increase in paired-pulse ratios in the PTZ-applied group. Our findings indicate an increase in the “T-type Ca²⁺ channel component of LTP” in the kindling group, which may be an early mechanism in epileptogenesis.     

Calpain activation and Na+/Ca2+ exchanger degradation occur downstream of calcium deregulation in hippocampal neurons exposed to excitotoxic glutamate

Delayed calcium deregulation (DCD) plays an essential role in glutamate excitotoxicity, a major detrimental factor in stroke, traumatic brain injury, and various neurodegenerations. In the present study, we examined the role of calpain activation and Na⁺/Ca²⁺ exchanger (NCX) degradation in DCD and excitotoxic cell death in cultured hippocampal neurons. Exposure of neurons to glutamate caused DCD accompanied by secondary mitochondrial depolarization. Activation of calpain was evidenced by detecting NCX isoform 3 (NCX3) degradation products. Degradation of NCX isoform 1 (NCX1) was below the detection limit of Western blotting. Degradation of NCX3 was detected only after 1 hr of incubation with glutamate, whereas DCD occurred on average within 15 min after glutamate application. Calpeptin, an inhibitor of calpain, significantly attenuated NCX3 degradation but failed to inhibit DCD and excitotoxic neuronal death. Calpain inhibitors I, III, and VI also failed to influence DCD and glutamate‐induced neuronal death. On the other hand, MK801, an inhibitor of the NMDA subtype of glutamate receptors, added shortly after the initial glutamate‐induced jump in cytosolic Ca²⁺, completely prevented DCD and activation of calpain and strongly protected neurons against excitotoxicity. Taken together, our results suggest that, in glutamate‐treated hippocampal neurons, the initial increase in cytosolic Ca²⁺ that precedes DCD is insufficient for sustained calpain activation, which most likely occurs downstream of DCD. © 2009 Wiley‐Liss, Inc.     

Increases in K+ conductance and Ca2+ influx under high glucose with suppressed Na+/K+-pump activity in rat myelinated nerve fibers

To test the combined effect of high glucose and decreased Na+/K+-pump activity, a condition which closely mimics the diabetic state, on nerve ionic currents, changes in action potential and membrane current induced by high glucose in the presence of ouabain were investigated using voltage clamp analysis in rat single myelinated nerve fibers. In the presence of 0.1 mM ouabain, 30 mM glucose caused a progressive increase in the delayed K+ current as well as persistent decreases in action potential and Na+ current, suggesting that Na+/K+ pump plays an important role in preventing the increase in the K+ current. The latter increase was suppressed by a blocker of Ca2+-activated K+ channels. Two types of voltage-dependent Ca2+ channel blockers (L and N-type) as well as a Na+/Ca2+-exchange blocker diminished the ouabain-induced increase in K+ conductance. These results suggest that high glucose with suppressed Na+/K+ pump activity might induce an increase of Ca2+ influx through either Ca2+ channels or reverse Na+/Ca2+-exchange, possibly leading to the elevation of Ca2+-activated voltage-dependent K+ channels. Both a decrease in inward Na+ current and an increase in K+ conductance may result in decreased nerve conduction. In addition, a possible increase of axoplasmic Ca2+ concentration may lead to axonal degeneration. These results provide a clue for understanding the pathophysiologic mechanism of diabetic neuropathy.     

Amyloid beta peptide 1-40 stimulates the Na+/Ca2+ exchange activity of SNCX

The Na+/Ca2+ exchangers, RNCX and SNCX, were cloned from mesangial cells of salt sensitive and salt resistant Dahl/Rapp rats, respectively, and differ at amino acid 218 (RNCXi/SNCXf) and in the exons expressed at the alternative splice site (RNCXB, D/SNCXB, D, F). These isoforms are also expressed in myocytes, neurons, and astrocytes where they maintain cytosolic calcium homeostasis. We demonstrated that cells expressing SNCX were more susceptible to oxidative stress than cells expressing RNCX. Others demonstrated that amyloid beta peptide (Abeta) augments the adverse effects of oxidative stress on calcium homeostasis. Therefore, we sought to assess the effect of Abeta 1-40 on the abilities of OK-PTH cells stably expressing RNCX and SNCX and human glioma cells, SKMG1, to regulate cytosolic calcium homeostasis. Our studies showed that Abeta 1-40 (1 microM) did not affect RNCX activity, as assessed by changes in [Ca2+]i (Delta[Ca2+]i, 260+/-10 nM to 267+/-8 nM), while stimulating exchange activity 2.4 and 3 fold in cells expressing SNCX (100+/-8 to 244+/-12 nM) and in SKMG1 cells (90+/-11 nM to 270+/-18 nM), respectively. Our results also showed that Abeta 1-40, while not affecting the rate of Mn2+ influx in cells expressing RNCX, stimulated the rate of Mn2+ influx 2.8 and 2.9 fold in cells expressing SNCX and in SKMG1 cells. Thus, our studies demonstrate that Abeta-induced cytosolic calcium increase is mediated through certain isoforms of the Na+/Ca2+ exchanger and reveals a possible mechanism by which Abeta 1-40 can alter cytosolic calcium homeostasis.     

Differential contributions of Ca2+‐activated K+ channels and Na+/K+‐ATPases to the generation of the slow afterhyperpolarization in CA1 pyramidal cells

In many types of CNS neurons, repetitive spiking produces a slow afterhyperpolarization (sAHP), providing sustained, intrinsically generated negative feedback to neuronal excitation. Changes in the sAHP have been implicated in learning behaviors, in cognitive decline in aging, and in epileptogenesis. Despite its importance in brain function, the mechanisms generating the sAHP are still controversial. Here we have addressed the roles of M‐type K⁺ current (I_M), Ca²⁺‐gated K⁺ currents (I_Ca(K)'s) and Na⁺/K⁺‐ATPases (NKAs) current to sAHP generation in adult rat CA1 pyramidal cells maintained at near‐physiological temperature (35 °C). No evidence for I_M contribution to the sAHP was found in these neurons. Both I_Ca(K)'s and NKA current contributed to sAHP generation, the latter being the predominant generator of the sAHP, particularly when evoked with short trains of spikes. Of the different NKA isoenzymes, α₁‐NKA played the key role, endowing the sAHP a steep voltage‐dependence. Thus normal and pathological changes in α₁‐NKA expression or function may affect cognitive processes by modulating the inhibitory efficacy of the sAHP.     

Characterization of Na+/H+ exchange activity in cultured rat hippocampal astrocytes

Astrocytes actively maintain their intracellular pH (pH_i) more alkaline than expected by passive distribution of H⁺. Acid extruding transporters such as the amiloride-sensitive Na⁺/H⁺ exchanger (NHE) are necessary for pH regulation. Currently, four mammalian NHEs (NHE1-NHE4) have been cloned, with a fifth (NHE5) partially cloned. We attempted to determine which isoform(s) of NHE was present in cultured hippocampal astrocytes using amiloride sensitivity and immunospecificity as criteria. In the absence of HCO₃⁻, amiloride blocked pH_i recovery after an acid load with an IC₅₀ of ∼3.18 μM, similar to values reported for the amiloride-sensitive isoforms NHE1 and NHE2. Immunoblotting with a highly specific antibody for NHE1 identified a 100 kDa protein, indicating the presence of NHE1 in whole brain, hippocampus, and cultured hippocampal astrocytes. Further probing for an additional amiloride-sensitive NHE failed to detect evidence of the presence of NHE4. Surprisingly, application of the potent analog of amiloride, ethylisopropylamiloride (EIPA), caused a reversible alkalinization of pH_i, suggesting the presence of an additional acid/base transport mechanism that is EIPA-sensitive. © 1996 Wiley-Liss, Inc.     

Na+/Ca2+ exchange activity is increased in Alzheimer's disease brain tissues.

These studies were performed to determine the changes that occur in Na+/Ca2+ exchange activity in Alzheimer's disease (AD) brain tissues. Cerebral plasma membrane vesicles were purified by sucrose density gradient centrifugation from frozen postmortem hippocampal/temporal cortex tissue slices derived from age matched brains of normal, AD and non-Alzheimer dementia (NAD) origin (autopsy confirmed). Membrane marker assays (Na/K ATPase, muscarinic receptor, cytochrome c oxidase) revealed no change in membrane purity across different preparations. Thin-section electron microscopy revealed predominantly intact unilamellar vesicles. Vesicles were preincubated for 15 min (37 degrees C) in buffer containing 132 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 10 mM glucose and 10 mM HEPES (pH 7.4). Ca2+ uptake was initiated by diluting vesicles 20-fold with buffer containing either 132 mM NaCl or 132 mM choline chloride and 45CaCl2 then terminated by addition of 200 microM LaCl3 and rapid filtration. Ca2+ content increased rapidly at first and then maintained a steady plateau for up to 5 min. When the Ca2+ ionophore A23187 (10 microM) with 100 microM EGTA was added after 4 min, Ca2+ content was reduced to 10% of its original value. Ruthenium red (10 microM) had no effect on Ca2+ content. Na(+)-dependent Ca2+ uptake (Ca2+ content measured in choline chloride minus that measured in NaCl) was increased in AD brains as evidenced by both an increase in the initial rise in Ca2+ content and in elevated values of peak plateau Ca2+ content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical study of Ca2+-ATPase activity in ischemic CA1 pyramidal neurons in the gerbil hippocampus

Although cytosolic Ca²⁺ accumulation plays a pivotal role in delayed neuronal death, there have been no investigations on the role of the cellular Ca²⁺ export system in this novel phenomenon. To clarify the function of the Ca²⁺-pump in delayed neuronal death, the plasma membrane Ca²⁺-ATPase activity of CA1 pyramidal neurons was investigated ultracytochemically in normal and ischemic gerbil hippocampus. To correlate enzyme activity with delayed neuronal death, histochemical detection was performed at various recirculation times after 5 min of ischemia produced by occlusion of the bilateral carotid arteries. At 10 min after ischemia, CA1 pyramidal neurons showed weak Ca²⁺-ATPase activity. Although enzyme activity had almost fully recovered 2 h after ischemia, it was reduced again 6 h after ischemia. Thereafter, Ca²⁺-ATPase activity on the plasma membrance of CA1 pyramidal neurons decreased progressively, losing its localization on day 3. On day 4 following ischemia, reaction products were diffusely scattered throughout the whole cell body. Our results indicate that, after once having recovered from ischemic damage, severe disturbance of the membrane Ca²⁺ export system proceeds from the early stage of delayed neuronal death and disturbs the re-export of accumulated cytosolic Ca²⁺, which might contribute to delayed neuronal death. Occult disruption of Ca²⁺ homeostasis seems to occur from an extremely early stage of delayed neuronal death in CA1 pyramidal cells.     

Deletion of calcineurin from GFAP-expressing astrocytes impairs excitability of cerebellar and hippocampal neurons through astroglial Na+/K+ ATPase

Astrocytes perform important housekeeping functions in the nervous system including maintenance of adequate neuronal excitability, although the regulatory mechanisms are currently poorly understood. The astrocytic Ca²⁺/calmodulin-activated phosphatase calcineurin (CaN) is implicated in the development of reactive gliosis and neuroinflammation, but its roles, including the control of neuronal excitability, in healthy brain is unknown. We have generated a mouse line with conditional knockout (KO) of CaN B1 (CaNB1) in glial fibrillary acidic protein-expressing astrocytes (a stroglial c alcin eurin KO [ACN-KO]). Here, we report that postnatal and astrocyte-specific ablation of CaNB1 did not alter normal growth and development as well as adult neurogenesis. Yet, we found that specific deletion of astrocytic CaN selectively impairs intrinsic neuronal excitability in hippocampal CA1 pyramidal neurons and cerebellar granule cells (CGCs). This impairment was associated with a decrease in after hyperpolarization in CGC, while passive properties were unchanged, suggesting impairment of K⁺ homeostasis. Indeed, blockade of Na⁺/K⁺-ATPase (NKA) with ouabain phenocopied the electrophysiological alterations observed in ACN-KO CGCs. In addition, NKA activity was significantly lower in cerebellar and hippocampal lysates and in pure astrocytic cultures from ACN-KO mice. While no changes were found in protein levels, NKA activity was inhibited by the specific CaN inhibitor FK506 in both cerebellar lysates and primary astroglia from control mice, suggesting that CaN directly modulates NKA activity and in this manner controls neuronal excitability. In summary, our data provide formal evidence for the notion that astroglia is fundamental for controlling basic neuronal functions and place CaN center-stage as an astrocytic Ca²⁺-sensitive switch.     

Voltage-clamp analysis of the potentiation of the slow Ca2+-activated K+ current in hippocampal pyramidal neurons

Exploring the principles that govern activity-dependent changes in excitability is an essential step to understand the function of the nervous system, because they act as a general postsynaptic control mechanism that modulates the flow of synaptic signals. We show an activity-dependent potentiation of the slow Ca2+-activated K+ current (sl(AHP)) which induces sustained decreases in the excitability in CA1 pyramidal neurons. We analyzed the sl(AHP) using the slice technique and voltage-clamp recordings with sharp or patch-electrodes. Using sharp electrodes-repeated activation with depolarizing pulses evoked a prolonged (8-min) potentiation of the amplitude (171%) and duration (208%) of the sl(AHP). Using patch electrodes, early after entering the whole-cell configuration (<20 min), responses were as those reported above. However, although the sl(AHP) remained unchanged, its potentiation was markedly reduced in later recordings, suggesting that the underlying mechanisms were rapidly eliminated by intracellular dialysis. Inhibition of L-type Ca2+ current by nifedipine (20 microM) markedly reduced the sl(AHP) (79%) and its potentiation (55%). Ryanodine (20 microM) that blocks the release of intracellular Ca2+ also reduced sl(AHP) (29%) and its potentiation (25%). The potentiation of the sl(AHP) induced a marked and prolonged (>50%; approximately equals 8 min) decrease in excitability. The results suggest that sl(AHP) is potentiated as a result of an increased intracellular Ca2+ concentration ([Ca2+]i) following activation of voltage-gated L-type Ca2+ channels, aided by the subsequent release of Ca2+ from intracellular stores. Another possibility is that repeated activation increases the Ca2+-binding capacity of the channels mediating the sl(AHP). This potentiation of the sl(AHP) could be relevant in hippocampal physiology, because the changes in excitability it causes may regulate the induction threshold of the long-term potentiation of synaptic efficacy. Moreover, the potentiation would act as a protective mechanism by reducing excitability and preventing the accumulation of intracellular Ca2+ to toxic levels when intense synaptic activation occurs.     

Cytosolic Ca2+ changes during in vitro ischemia in rat hippocampal slices: major roles for glutamate and Na+-dependent Ca2+ release from mitochondria.

This work determined Ca2+ transport processes that contribute to the rise in cytosolic Ca2+ during in vitro ischemia (deprivation of oxygen and glucose) in the hippocampus. The CA1 striatum radiatum of rat hippocampal slices was monitored by confocal microscopy of calcium green-1. There was a 50-60% increase in fluorescence during 10 min of ischemia after a 3 min lag period. During the first 5 min of ischemia the major contribution was from Ca2+ entering via NMDA receptors; most of the fluorescence increase was blocked by MK-801. Approximately one-half of the sustained increase in fluorescence during 10 min of ischemia was caused by activation of Ca2+ release from mitochondria via the mitochondrial 2Na+-Ca2+ exchanger. Inhibition of Na+ influx across the plasmalemma using lidocaine, low extracellular Na+, or the AMPA/kainate receptor blocker CNQX reduced the fluorescence increase by 50%. The 2Na+-Ca2+ exchange blocker CGP37157 also blocked the increase, and this effect was not additive with the effects of blocking Na+ influx. When added together, CNQX and lidocaine inhibited the fluorescence increase more than CGP37157 did. Thus, during ischemia, Ca2+ entry via NMDA receptors accounts for the earliest rise in cytosolic Ca2+. Approximately 50% of the sustained rise is attributable to Na+ entry and subsequent Ca2+ release from the mitochondria via the 2Na+-Ca2+ exchanger. Sodium entry is also hypothesized to compromise clearance of cytosolic Ca2+ by routes other than mitochondrial uptake, probably by enhancing ATP depletion, accounting for the large inhibition of the Ca2+ increase by the combination of CNQX and lidocaine.     

Modulation of two functionally distinct Ca2+ stores in astrocytes: Role of the plasmalemmal Na/Ca exchanger

Mechanisms that regulate the amount of releasable Ca²⁺ in intracellular stores of cultured mouse astrocytes were investigated using digital imaging of fura-2 loaded cells. At rest, the cytoplasmic Ca²⁺ concentration, [Ca²⁺]_cyt, was about 110 nM. In the absence of extracellular Ca²⁺, cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum (ER) Ca²⁺-ATPase, induced a transient, four-fold increase in [Ca²⁺]_cyt due to the release of Ca²⁺ from inositol triphosphate (IP₃) sensitive stores. Caffeine (CAF), which releases Ca²⁺ from Ca²⁺-sensitive stores, induced a two-fold increase in [Ca²⁺]_cyt. The CPA- and CAF-sensitive stores could be released independently. Changes in the amplitudes of the Ca²⁺ transients were taken as a measure of changes in store content. Removal of extracellular Na⁺ or addition of ouabain, which inhibit Ca²⁺ extrusion and promote Ca²⁺ entry across the plasmalemma via the Na/Ca exchanger, caused minimal increases in resting [Ca²⁺]_cyt but greatly potentiated both CPA- and CAF-induced Ca²⁺ transients. The amount of Ca²⁺ releasable from the IP₃ (CPA) sensitive store was directly proportional to cytosolic Na⁺ concentration (i.e., inversely proportional to the transmembrane Na⁺ electrochemical gradient). Under these reduced Na⁺ gradient conditions, little, if any, Ca²⁺ destined for the ER stores enters the cells through voltage-dependent Ca²⁺ channels. These results demonstrate that mouse astrocytes contain two distinct ER Ca²⁺ stores, the larger, IP₃- (CPA-) sensitive, and the smaller, Ca²⁺- (CAF-) sensitive. The Ca²⁺ content of both ER stores can be regulated by the Na/Ca exchanger. Thus, the magnitude of cellular responses to signals that are mediated by Ca²⁺ release induced by the two second messengers, IP₃ and Ca²⁺, can be modulated by factors that affect the net transport of Ca²⁺ across the plasmalemma. © 1996 Wiley-Liss, Inc.     

Effects of Na+ and Ca2+ gradients on intracellular free Ca2+ in voltage-clamped Aplysia neurons

Selected neurons of the abdominal ganglion of Aplysia californica were voltage-clamped and intracellular free Ca [( Ca2+]i) and Na [( Na+]i) concentrations were monitored with ion selective microelectrodes. Reducing [Na+]o from 500 mM (normal seawater, NSW) to 5 mM resulted in a decrease of the potential measured by the Ca electrode (VCa). Increasing [Ca2+]o from 10 to 50 mM increased [Ca2+]i two-fold, keeping [Ca2+]o at 50 mM and decreasing [Na+]o to 5 mM still led to a decrease in VCa. With 100 mM [Ca2+]o, which also increased [Ca2+]i, decreasing [Na+]o increased VCa in two of the eight cells tested. This indicates that in normal or moderately high resting [Ca2+]i, Ca2+ extrusion by Na/Ca exchange (forward mode) is not essential for [Ca2+]i buffering. [Na+]i was 12.9 +/- 3.6 mM (S.E.M., n = 7) in NSW; reducing [Na+]o to 5 mM decreased [Na+]i to 2.0 +/- 1.1 mM (S.E.M.). Keeping [Na+]o at 5 mM and increasing [Ca2+]o from 10 to 20 mM further decreased [Na+]i to about 1.0 mM, evidence of Na/Ca exchange operating in the reverse mode. Attempts to increase [Ca2+]i by bath application of the Ca ionophores A23187, X537A, ionomycin or ETH 1001 resulted in no measurable change of the resting [Ca2+]i. Application of Ouabain caused an apparent increase in [Ca2+]i in two of the six cells tested. In cells injected with the metallochromic indicator arsenazo III (AIII), the rate of the falling phase of the AIII absorbance increase, following a voltage-clamp pulse, was significantly slower in 5 mM [Na+]o. This indicates that in its forward mode Na-Ca exchange is active in clearing large submembrane increases in [Ca2+]i.     

Dendritic Ca2+ accumulations and metabotropic glutamate receptor activation associated with an n-methyl-d-aspartate receptor-independent long-term potentiation in hippocampal CA1 neurons

Bathing hippocampal slices in the potassium channel blocker tetraethylammonium (TEA), while stimulating the Schafer collaterals at a low frequency, induces Ca²⁺ -dependent, N-methyl-D-aspartate (NMDA) receptor-independent long-term potentiation of synaptic transmission (LTP_k) in CA1 neurons. We have combined ratio imaging of fura-2 and mag-fura-5 in hippocampal CA1 neurons with intracellular and field recordings to evaluate postsynaptic Ca²⁺ changes that occur in the induction of LTP_k. Test stimuli were applied at 0.05 Hz to stratum radiatum in the presence of the NMDA receptor antagonists D , L -2-amino-5-phosphonovaleric acid (100μM) or MK-801 (10μM). During TEA exposure (15–25 mM; 10 min), cells fired prolonged action potentials both spontaneously and in response to test stimuli resulting in transient, micromolar Ca²⁺ accumulations in both somata and dendrites. The initial EPSP slope, measured 60 min after TEA wash-out, was potentiated to approximately 200% of control. The Ca²⁺ channel blocker nimodipine (10 μM) greatly reduced Ca²⁺ transients in both magnitude and duration and prevented LTP_k induction. Pretreatment of slices with compounds that block metabotropic glutamate receptor (mGluR)-stimulated phosphoinositide hydrolysis, L -2-amino-3-phosphonopropionic acid (L -AP3, 50-200 μM) or L -aspartate-β-hydroxamate (50–100 μM), as well as protein kinase C (PKC) inhibitors (sphingosine, 20 μM; RO-31-8220, 0.2 μM; or calphostin C, 2 μM) also blocked LTP_k. Ca²⁺ transients were unaffected by L -AP3 or RO-31-8220. These findings suggest that Ca²⁺ influx through voltage-gated channels and co-activation of PKC by mGluRs are both necessary for induction of LTP_k. Activation of mGluRs must also occur in NMDA receptor-dependent induction paradigms, but is possibly of lesser importance owing to the much greater gating of Ca²⁺ directly into the dendritic spines. © 1994 Wiley-Liss, Inc.     

Delayed calcium dysregulation in neurons requires both the NMDA receptor and the reverse Na+/Ca2+ exchanger

Glutamate-induced delayed calcium dysregulation (DCD) is a causal factor leading to neuronal death. The mechanism of DCD is not clear but Ca2+ influx via N-methyl-d-aspartate receptors (NMDAR) and/or the reverse plasmalemmal Na+/Ca2+ exchanger (NCXrev) could be involved in DCD. However, the extent to which NMDAR and NCX(rev) contribute to glutamate-induced DCD is uncertain. Here, we show that both NMDAR and NCX(rev) are critical for DCD in neurons exposed to excitotoxic glutamate. In rat cultured hippocampal neurons, 25 μM glutamate produced DCD accompanied by sustained increase in cytosolic Na+ ([Na+]c) and plasma membrane depolarization. MK801 and memantine, noncompetitive NMDAR inhibitors, added shortly after glutamate, completely prevented DCD whereas AP-5, a competitive NMDAR inhibitor, failed to protect against DCD. None of the tested inhibitors lowered elevated [Na+]c or restored plasma membrane potential. In the experiments with NCX reversal by gramicidin, MK801 and memantine robustly inhibited NCXrev while AP-5 was much less efficacious. In electrophysiological patch-clamp experiments MK801 and memantine inhibited NCXrev-mediated ion currents whereas AP-5 failed. Thus, MK801 and memantine, in addition to NMDAR, inhibited NCXrev. Inhibition of NCXrev either with KB-R7943, or by collapsing Na+ gradient across the plasma membrane, or by inhibiting Na+/H+ exchanger with 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and thus preventing the increase in [Na+]c failed to preclude DCD. However, NCXrev inhibition combined with NMDAR blockade by AP-5 completely prevented DCD. Overall, our data suggest that both NMDAR and NCXrev are essential for DCD in glutamate-exposed neurons and inhibition of individual mechanism is not sufficient to prevent calcium dysregulation.