Calcium uptake related to K-depolarization and Na/Ca exchange in sheep brain synaptosomes

The uptake of Ca²⁺ by synaptosomes induced by K⁺-depolarization andby Na⁺/Ca²⁺ exchange was studied in synaptosomes in which the internal Na⁺ and K⁺ contents were varied by prolonged incubation at 30 °C or by inhibiting the Na⁺, K⁺-ATPase with 1 mM ouabain. Increased Na⁺ content of the synaptosomes is associated with an increase in Ca²⁺ uptake when the synaptosomes are placed in depolarizing K⁺ media. Furthermore, reduction in the [Na⁺]_o, when the [K⁺]_o is increased, in substitution for [Na⁺]_o, to depolarize the membrane, further increases the Ca²⁺ uptake. Under these conditions, Ca²⁺ entry probably occurs through voltage-sensitive channels and through the Na⁺/Ca²⁺ exchanger. Destruction of the Na⁺ gradient by monensin, or preloading the synaptosomes with K⁺, completely inhibits the Ca²⁺ uptake in a K⁺-depolarizing medium. It is shown that if the Na⁺ gradient is maintained constant during K⁺-depolarization, the Ca²⁺ uptake is very low and that most of the Ca²⁺ uptake is correlated with the Na⁺ gradient. Evidence is presented that K⁺ may stimulate the Na⁺/Ca²⁺ exchange mechanism. Furthermore, divalent cations, Mg²⁺, Mn²⁺ and Zn²⁺, known to block Ca²⁺ channels, also inhibit Na⁺/Ca²⁺ exchange.     

Calcium homeostasis in rat septal neurons in tissue culture

Septal neutons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca²⁺ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca²⁺]_i) in the neurons was low (50–100 nM). Depolarization of the neurons with 50 mM K⁺ resulted in rapid elevation of [Ca²⁺]_i to 500–1,000 nM showing recovery to baseline [Ca²⁺]_i over several minutes. The increases in [Ca²⁺]_i caused by K⁺ depolarization were completely abolished by the removal of extracellular [Ca²⁺], and were reduced by 80% by the ‘L-type’ Ca²⁺ channel blocker, nimodipine (1 μM). [Ca²⁺]_i was also increased by the excitatory amino andl-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNOX and DNOX. In the absence of extracellular Mg²⁺, large fluctuations in [Ca²⁺]_i were observed that were blocked by removal of extracellular Ca²⁺, by tetrodotoxin (TTX), or by antagonists ofN-methyld-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg²⁺ and TTX, NMDA caused dose-dependent increases in [Ca²⁺]_i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca²⁺]_i in the absence of extracellular Ca²⁺, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca²⁺ stores. Thapsigargin (2 μM) had little effect on [Ca²⁺]_i, or on the recovery from K⁺ depolarization. Removal of extracellular Na⁺ had little effect on basal [Ca²⁺]_i or on responses to high K⁺, suggesting that Na⁺/Ca²⁺ exchange mechanisms do not play a significant role in the short-term control of [Ca²⁺]_i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca²⁺]_i; however, [Ca²⁺]_i recovered as normal from high K⁺ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca²⁺]_i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca²⁺]_i associated with action potential activity was measured. The amplitude of the [Ca²⁺]_i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca²⁺]_i from the modest Ca²⁺ loads imposed on the neuron by action potential trains follows a simple exponential decay (τ = 3–5s).     

Restoration of cholinomimetic activity by clonidine in cholinergic plus noradrenergic lesioned rats

Lactate production (J_lac), oxygen consumption rate (Q_O2), plasma membrane potentials (E_m) and cytosolic free calcium levels [Ca²⁺]_i were studied on symaptosomes isolated from rat brains, incubated in presence of high doses of nicardipine (90 μM), diltiazem (0.5 mM) and verapamil (0.25 mM), and submitted to depolarizing stimulation or inhibition of mitochondrial respiration. Nicardipine was able to completely prevent the veratridine-induced stimulation ofJ_lac, Q_O2andE_m depolarization, whereas diltiazem and verapamil were less effective, although the concentrations used were 5 and 3 times higher, respectively, than nicardipine. Diltiazem, verapamil and nicardipine (9 μM) also prevented the veratridine-induced increase in [Ca²⁺]_i, this effect being much less pronounced if the drugs were added after veratridine. Monensin (20 μM) was also able to increase [Ca²⁺]_i but this effect was not affected by verapamil. Synaptosomes were also submitted to an inhibition of respiration of intrasynaptic mitochondria by incubation with rotenone (5 μM); in this condition of mimicked hypoxiaE_m was more positive of about 11 mV; none of the drugs utilized modified this situation. The rotenone-induced 3-fold increase inJ_lac was barely modified by diltiazem and verapamil but it was completely abolished by nicardipine. The possible mechanism of the counteracting action of the drugs towards veratridine stimulation and rotenone inhibition and the involvement of Na⁺/Ca²⁺ exchanger in affecting [Ca²⁺]_i are discussed.     

Enhanced Ca(2+) influx with mossy fiber stimulation in hippocampal CA3 neurons of spontaneously epileptic rats

This study was performed to determine whether the intracellular Ca²⁺ concentration ([Ca²⁺]_i) is increased in hippocampal CA3 neurons of spontaneously epileptic rats (SER) which show both absence-like and convulsive seizures using hippocampal slices loaded with Calcium Green-1 when a weak single stimulation is given to the mossy fiber. [Ca²⁺]_i in the CA3 area was significantly increased after a single stimulus to mossy fibers in SER, while no changes were detected in normal Wistar rats. These findings suggest the existence of an abnormality in the Ca²⁺ channel in the SER CA3 region and that this is probably responsible for epileptic seizures.     

Inability to restore resting intracellular calcium levels as an early indicator of delayed neuronal cell death

The hippocampus is especially vulnerable to excitotoxicity and delayed neuronal cell death. Chronic elevations in free intracellular calcium concentration ([Ca²⁺]_i) following glutamate-induced excitotoxicity have been implicated in contributing to delayed neuronal cell death. However, no direct correlation between delayed cell death and prolonged increases in [Ca²⁺]_i has been determined in mature hippocampal neurons in culture. This investigation was initiated to determine the statistical relationship between delayed neuronal cell death and prolonged increases in [Ca²⁺]_i in mature hippocampal neurons in culture. Using indo-1 confocal fluorescence microscopy, we observed that glutamate induced a rapid increase in [Ca²⁺]_i that persisted after the removal of glutamate. Following excitotoxic glutamate exposure, neurons exhibited prolonged increases in [Ca²⁺]_i, and significant delayed neuronal cell death was observed. The N-methyl-D-aspartate (NMDA) channel antagonist MK-801 blocked the prolonged increases in [Ca²⁺]_i and cell death. Depolarization of neurons with potassium chloride (KCl) resulted in increases in [Ca²⁺]_i, but these increases were buffered immediately upon removal of the KCl, and no cell death occurred. Linear regression analysis revealed a strong correlation (R = 0.973) between glutamate-induced prolonged increases in [Ca²⁺]_i and delayed cell death. These data suggest that excitotoxic glutamate exposure results in an NMDA-induced inability to restore resting [Ca²⁺]_i (IRRC) that is a statistically significant indicator of delayed neuronal cell death.     

Mitogen stimulated rise of intracellular calcium concentration in single t lymphocytes from patients with major depression is reduced

1. 1. The authors investigated the signal transduction in T-lymphocytes as a peripheral model for central neurons.

2. 2. Intracellular free calcium concentration [Ca²⁺]_i was measured using fura 2 in T-lymphocytes from 6 patients with major depression during and after depression and from 6 healthy controls Patients were treated with interpersonal therapy (IPT) but not with psychotropic medication.

3. 3 Phytohemagglutinin (PHA) triggers an oscillatory [Ca²⁺]_i signal in human T-lymphocytes. This implies two mechanisms for [Ca²⁺]_i regulation: inositol phophate (IP) mediated release from intracellular stores and [Ca²⁺]_i influx from the extracellular medium.

4. 4. PHA stimulates 49% of T cells from controls but only 17% of T cells from depressed patients. This finding explains previous results from cells in suspension indicating that [Ca²⁺]_i signals after PHA-stimulation are reduced in cells from depressed patients.

5. 5 Cells from depressed patients show less [Ca²⁺]_i oscillations. Normal oscillation pattems are restored after clinical recovery from depression.

6. 6. Thus altered [Ca²⁺]_i oscillations in T-lymphocytes are a state phenomenon and may give us clues where to search for altered cellular mechanisms during depression.

     

Porcine pancreatic group IB secretory phospholipase A2 potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels

Secretory phospholipase A₂ (sPLA₂) exhibits neurotoxicity in the central nervous system. There are high-affinity binding sites of the porcine pancreatic group IB sPLA₂ (sPLA₂-IB) in the brain. sPLA₂-IB causes neuronal cell death via apoptosis in the rat cerebral cortex. Although apoptosis is triggered by an influx of Ca²⁺ into neurons, it has not yet been ascertained whether the Ca²⁺ influx is associated with the neurotoxicity of sPLA₂-IB. We thus examined the possible involvement of Ca²⁺ in the neurotoxicity of sPLA₂-IB in the primary culture of rat cortical neurons. sPLA₂-IB induced neuronal cell death in a concentration- and time-dependent manner. This death was accompanied by condensed chromatin and fragmented DNA, exhibiting apoptotic features. Before apoptosis, sPLA₂-IB markedly enhanced the influx of Ca²⁺ into neurons. A calcium chelator suppressed neurons from sPLA₂-IB-induced neuronal cell death in a concentration-dependent manner. An L-type voltage-sensitive Ca²⁺ channel (L-VSCC) blocker significantly protected the sPLA₂-IB-potentiated influx of Ca²⁺. On the other hand, blockers of N-VSCC and P/Q-VSCC did not. An L-VSCC blocker protected neurons from sPLA₂-IB-induced neuronal cell death. In addition, the L-VSCC blocker ameliorated the apoptotic features of sPLA₂-IB-treated neurons. Neither an N-VSCC blocker nor P/Q-VSCC blockers affected the neurotoxicity of the enzyme. In conclusion, these findings demonstrate that the influx of Ca²⁺ into neurons play an important role in the neurotoxicity of sPLA₂-IB. Furthermore, the present study suggests that L-VSCC contribute to the sPLA₂-IB-potentiated influx of Ca²⁺ into neurons.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [³H]cyclohexyladenosine (CHA) to the cellular fractions and P₂ subfractions of the goldfish brain was studied. The A₁ receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P₂), 30 mM K⁺ stimulated glutamate, taurine and GABA release in a Ca²⁺-dependent fashion, whereas the aspartate release was Ca²⁺-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 μM) inhibited K⁺-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A₁ receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K⁺ (30 mM) stimulated Ca²⁺ influx (46.8±6.8%) and this increase was completely abolished by pretreatment with 100 nM ω-conotoxin. Pretreatment with 100 μM R-PIA or 100 μM CHA, reduced the evoked increase of intra-synaptosomal Ca²⁺ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A₁ receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca²⁺]_i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca²⁺]_i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca²⁺]_i studies. The kinetic profile for [Ca²⁺]_i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca²⁺]_i in guinea pigs, had no effect in rat, and increased [Ca²⁺]_i in all other species. Staurosporine inhibited SFLLRN-induced [Ca²⁺]_i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca²⁺]_i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.     

Beta-agkistrodotoxin inhibits large-conductance calcium-activated potassium channels in rat hippocampal CA1 pyramidal neurons

Effect of β-agkistrodotoxin (β-AgTx), a presynaptic neurotoxin purified from snake venom, on large-conductance calcium-activated potassium channels (BK_Ca) was studied in rat hippocampal CA1 pyramidal neurons using inside-out configuration of patch-clamp technique. The results showed that in equimolar K⁺ (150 mM) and 1 μM intracellular Ca²⁺ conditions, internal application of β-AgTx inhibited the activity of BK_Ca by reducing open probability (P_o) of the channels in a concentration-dependent manner. High concentration (74 nM) of β-AgTx completely eliminated opening of the channels. However, 37 nM β-AgTx (at −40 mV) decreased P_o from 0.49±0.07 to 0.03±0.03, switched two open time constants (0.51±0.32 and 8.77±1.63 ms) to be a single time constant of 0.46±0.40 ms. The results indicate that inhibition of BK_Ca by β-AgTx may account for the facilitatory phase of the toxin on acetylcholine release from nerve terminals.     

Interrelationship between secretion, protein phosphorylation and intracellular Ca2+ concentration in platelets stimulated by thrombin or thromboxane A2 analogue

The interrelationship between ATP-secretion, protein phosphorylation and intracellular Ca²⁺ concentration ([Ca²⁺]i) was studied in both ³²P and quin 2 loaded human platelets stimulated by thrombin or thromboxane A₂ analogue (STA₂). In platelets stimulated by thrombin, the degree of 47,000 dalton polypeptides (P47) phosphorylation was observed in completely dose-related manner, regardless of the amount of [Ca²⁺]i. In the same condition, the degree of myosin light chain (P20) phosphorylation, however, was well correlated with ATP secretion and [Ca²⁺]i, when platelets were stimulated by lower dose of thrombin. The similar results were obtained in platelets stimulated by STA₂. These findings suggested that P20, but not P47, phosphorylation in activated platelets is mediated by a rise of [Ca²⁺]i and is well correlated with the secretory reaction. It was unlikely that P47 phosphorylation plays any role in promoting platelet activation.     

Effects of Ca antagonists and aminoglycoside antibiotics on Ca current in isolated outer hair cells of guinea pig cochlea

The effects of various Ca²⁺ antagonists and aminoglycoside antibiotics on the Ca²⁺ channel in isolated outer hair cells of the guinea pig were investigated using a whole-cell patch-clamp technique. The inhibitory action was in the order of La³⁺Cd²⁺Ni²⁺Co²⁺ for inorganic Ca²⁺ antagonists, and flunarizine = nicardipine > ω-Conotoxin > methaxyverapamil = diltiazem amiloride for orgaini ones. Aminoglycoside antibiotics also had antagonistic effects on the Ca²⁺ channel.     

NT-3 and BDNF protect CNS neurons against metabolic/excitotoxic insults

Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) were recently shown to have biological activity in central neurons. In the present study, NT-3 and BDNF attenuated glucose deprivation-induced neuronal damage dose-dependently in rat hippocampal, septal and cortical cultures. Direct measurements of intraneuronal free calcium levels ([Ca²⁺]_i) and manipulations of calcium inlux demonstrated that NT-3 and BDNF each prevented the elevation of [Ca²⁺]_i that mediated glucose deprivation-induced injury. Studies in cultures depleted of glia indicateda direct action of NT-3 and BDNF on neurons. Neurons pretreated with NT-3 or BDNF for 24 hr were more resistant to glutamate neurotoxicity, and showed attenuated [Ca²⁺]_i responses to glutamate. TrkB (BDNF receptor) and trkC (NT-3 receptor) proteins were present in hippocampal, cortical and septal cultures where they were localied to neuronal cell bodies and neurites. The data demonstrate that NT-3 and BDNF can protect neurons against metabolic and excitotoxic insults, and suggest that these neurotrophins may serve [Ca²⁺]_i-stabilizing and neuroprotective functions in the brain.     

μ-Opioid receptor-induced Ca mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca-dependent mechanism

Morphine, a preferential μ-opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellular differentiation. Although morphine affects cellular differentiation through a Ca²⁺-dependent mechanism, few studies have examined whether Ca²⁺ mediates the effect of opioids on cell proliferation, or whether a particular Ca²⁺ signal transduction pathway mediates opioid actions. Moreover, it is uncertain whether one or more opioid receptor types mediates the developmental effects of opioids. To address these questions, the present study examined the role of μ-opioid receptors and Ca²⁺ mobilization in morphine-induced astrocyte development. Morphine (1 gmM) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca²⁺-free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca²⁺ ([Ca²⁺]_o), or in unmodified media containing Ca²⁺ ionophore (A23187), nifedipine (1 μM), dantrolene (10 μM), thapsigargin (100 nM), or l-glutamate (100 μM) for 0-72 h. μ-Opioid receptor expression was examined immunocytochemically using specific (MOR1) antibodies. Intracellular Ca²⁺ ([Ca²⁺]ⁱ) was measured by microfluorometric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillary acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating μ-opioid receptors. Astrocytes expressed MOR1 immunoreactivity and morphine's actions were mimicked by the selective μ, agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca²⁺]_i in developing astroglia. At normal [Ca²⁺]_o, morphine attenuated DNA synthesis by increasing [Ca²⁺]_i; low [Ca²⁺]_o (0.3 mM) blocked this effect, while treatment with Ca²⁺ ionophore or glutamate mimicked morphine's actions. At extremely low [Ca²⁺]_o (< 0.005 mM), morphine paradoxically increased BrdU incorporation. Although opioids can increase [Ca²⁺]_i in astrocytes through several pathways, not all affect DNA synthesis or cellular morphology. Nifedipine (which blocks L-type Ca²⁺ channels) did not prevent morphine-induced reductions in BrdU incorporation or cellular differentiation, while thapsigargin (which depletes IP₃-sensitive Ca²⁺ stores) severely affected inhibited DNA synthesis and cellular differentiation-irrespective of morphine treatment. However, dantrolene (an inhibitor of Ca²⁺-dependent Ca²⁺ release) selectively blocked the effects of morphine. Collectively, the findings suggest that opioids suppress astroglial DNA synthesis and promote cellular hypertrophy by inhibiting Ca²⁺-dependent Ca²⁺ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.     

Independent regulation of activation and inactivation phases in non-contractile Ca transients by nicotinic receptor at the mouse neuromuscular junction

Non-contractile Ca²⁺ mobilization (not accompanied by muscle contraction) occurs by the prolonged activation of nicotinic acetylcholine receptor in mouse diaphragm muscles treated with anticholinesterase. To elucidate the regulation properties of non-contractile Ca²⁺ mobilization by nicotinic receptor, the modes of action of competitive and depolarizing neurmuscular blockers were investigated. (+)-Tubocurarine (0.07–0.1 μM), pancuronium (0.05 μM) and -bungarotoxin (0.03–0.06 μM) decreased decay time (T₂, duration of inactivation phase) without changes in rise time (T₁, duration of activation phase) of non-contractile Ca²⁺ transients. These competitive antagonists also suppressed their peak amplitude at higher concentrations than those affectingT₂. Contractile Ca²⁺ transients were not inhibited by these antagonists at the concentrations used. Decamethonium (1 μM), a depolarizing blocker, suppressed the peak amplitude of non-contractile Ca²⁺ transients without affecting their duration. In contrast, succinylcholine (0.3 μM) suppressed both peak amplitude andT₁ without changingT₂, presumably via the receptor desentization. Succinylcholine but not decamthonium inhibited contractile Ca²⁺ transients at the concentrations used. These results demonstrate that the activation and inactivation phase in non-contractile Ca²⁺ transients are independently regulated by nicotinic acetylcholine receptor.     

Blocking of the receptor-stimulated calcium entry into human platelets by verapamil and nicardipine

Verapamil (ED₅₀=3×10⁻⁶ M) and nicardipine (ED₅₀=10⁻⁶ M) inhibited the platelet activating factor (PAF)-induced increase of free cytosolic calcium concentration ([Ca²⁺]_i) in quin2-loaded human platelets. In a Ca-free medium containing 5 mM BaCl₂, PAF stimulated the inflow of Ba²⁺ ions which is completely abolished by verapamil and nicardipine. Simultaneous determination of quin2 fluorescence and ⁴⁵Ca absorption showed that the action of verapamil is accounted for by blocking of the Ca²⁺ entry. Nicardipine suppresses also Ca²⁺ mobilization from intracellular stores. The effects of verapamil and nicardipine are not competitive with respect to PAF.The blockers reduce the [Ca²⁺]_i increase induced by ADP, vasopressin, and PGH₂ analogue U46619.     

Activin exerts a neurotrophic effect on cultured hippocampal neurons

Activin is a member of the transforming growth factor (TGF)-β superfamily, which comprises a growing list of multifunctional proteins that serve as regulators of cell proliferation and differentiation. Recently, activin was shown to regulate the neurotransmitter phenotype in peripheral neurons. It is also a potent survival factor for neurogenic clonal cell lines, retinal neurons and midbrain dopaminergic neurons. We have studied the effect of activin on hippocampal cells which show abundant expression of activin receptors or binding sites. Exposure of primary cultures of rat hippocampal neurons to activin supported neuronal survival. This neurotrophic action of activin was blocked by treatment with the tyrosine kinase inhibitor genistein or the protein kinase C inhibitor calphostin C. However, the Ca²⁺/calmodulin kinase inhibitor KN-62 had no effect. Nicardipine, a blocker of the

Full-size image

-type Ca²⁺ channel, also inhibited the neurotrophic effect of activin. Furthermore, activin potentiated the depolarization-induced elevation in intracellular Ca²⁺ concentration ([Ca²⁺]_i). The neurotrophic effect and the potentiation of depolarization-induced increase of [Ca²⁺]_i caused by activin were completely abolished by the protein synthesis inhibitor cycloheximide. These results suggest that activin supports neuronal survival by increasing the expression of voltage-dependent Ca²⁺ channel through the action of a tyrosine kinase and of protein kinase C, but not of Ca²⁺/calmodulin kinase.     

Novel Ca currents in mammalian CNS neurons

Akaike, Norio, Hisashi Yamanaka and Mitsutoshi Munakata: Novel Ca²⁺ Currents in Mammalian CNS Neurons. Prog. Neuro-Psychopharmacol. & Biol. Psychiatry. 1992, 16(6): 943–957.

1. 1. Voltage-dependent Ca²⁺ currents (I_Ca) in neurons can be classified into T-, N- and L-types. In the CA1 pyramidal neurons freshly dissociated from rat hippocampus we found an additional tetrodotoxin (TTX)-sensitive Ca²⁺ current (termed ‘TTX-I_Ca’). The TTX-I_Ca showed a heterogeneous distribution, preferentially in the dorsal site of CA1 region.

2. 2. Activation and inactivation processes of the TTX-I_Ca were highly potential-dependent, and the latter was fitted by a double exponential function. The TTX-I_Ca was activated at a threshold potential of about −55 mV and reached full activation at −30 mV. The steady-state inactivation of TTX-I_Ca could be fitted by a Boltzmann equation with a slope factor of 6.0 mV and a half-inactivation voltage of −72.5 mV.

3. 3. When the peak amplitudes of TTX-I_Ca were plotted as a function of extracellular Ca²⁺ concentration [Ca²⁺]_o), the current amplitude increased linearly without showing any saturation.

4. 4. The ratio of peak amplitude in the individual I-V relationships of Ca²⁺, Sr²⁺ and Ba²⁺ currents assing through the TTX-sensitive Ca²⁺-conducting channel was 1 : 0.33 : 0.05, although the current kinetics were much the same.

5. 5. TTX inhibited the TTX-I_Ca in time- and concentration-dependent manner without affecting the current kinetics. Lignocaine inhibited the TTX-I_Ca in a second in a concentration-dependent manner, with accelerating the inactivation process. The concentrations of half-inhibition (IC₅₀) were 3.5 × 10⁻⁹ M for TTX and 3.6 × 10⁻⁴ M for lignocaine.

6. 6. Scorpion toxin prolonged the inactivation phase of TTX-I_Ca in a time- and concentration-dependent manner. In the toxin-treated neurons, both the slow time constant of inactivation (τ_is) and its functional contribution to the total current increased with increasing the toxin concentration.

Aging increases calcium influx at motor nerve terminal

To determine whether increased transmitter release from soleus nerve terminals of old C57BL/6J mice is caused by an altered Ca²⁺ regulation, the time course of post-tetanic potentiation of miniature endplate potential (MEPP) frequency was used as an indicator of the kinetics of Ca²⁺ metabolism in young (10 months) and old (24 months) mice. Post-tetanic potentiation properties were studied in either (1) 0.2 mM Ca²⁺, 5.0 mM Mg²⁺ Krebs; or (2) Ca²⁺-free/EGTA Krebs to eliminate Ca²⁺ influx, and thereby isolated Ca²⁺ buffering. In the 0.2 mM Ca²⁺ Krebs, the time constants of decay of augmentation (T_A) and potentiation (T_P) were longer in old (T_A = 10.3 ± 1.0 sec, T_P = 195.3 ± 5.4 sec) than in young (T_A = 7.0 ± 0.7 sec, T_P = 78.8 ± 6.6 sec) nerve terminals. Evoked transmitter release was measured in 0.4 mM Ca²⁺, 2.75 mM Mg²⁺ Krebs. Quantal content of the endplate potential was positively correlated with T_A (r = 0.95) and with T_P (r = 0.98). In the Ca²⁺-free/EGTA Krebs, there was no difference in post-tetanic potentiation properties between young and old terminals. These results suggest that Ca²⁺ influx into the soleus nerve terminal increases with aging. This may explain, at least in part, the increased quantal content observed at old terminals.     

Protein phosphatase inhibitors prolong Ca-transients and divalent cation-dependent action potentials in leech Retzius cells

Elevation of [K⁺]_o for 30 s from 4 to 120 mM produced a fast and reversible depolarization and transient increase in [Ca²⁺]_i in fura-2 loaded Retzius cells of the leech. The protein phosphatase inhibitor, okadaic acid, significantly slowed the return of [Ca²⁺]_i toward baseline without affecting the amplitude of depolarization or rate of repolarization. Furthermore, okadaic acid and another phosphatase inhibitor, calyculin A, prolonged Ba²⁺-dependent action potentials. These results suggest that the kinetics of Ca²⁺ influx may be regulated by the activity of phosphatases PP-1 and/or PP-2A.