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).     

Function of serotonin in physiologic secretion of growth hormone and prolactin: Action of 5,7-dihydroxytryptamine, fenfluramine and p-chlorophenylalanine

The effect of 4-aminopyridine (4-AP) on the release of labeled transmitters in mouse brain synatosomes was studied in a superfusion system. 4-AP at μM concentrations notably stimulated the spontaneous release of labeled GABA and glutamate, and of acetylcholine (ACh) derived from tritiated choline. No effects on the release of labeled -aminoisobutyric acid were observed. The stimulation of GABA and ACh release was dependent on the presence of Ca²⁺ in the superfusion media, whereas the effect on glutamate release was more variable and no clear Ca²⁺-dependence was observed. In contrast to these results, 4-AP did not have any effect on the release of the above transmitters by K⁺-depolarization in the presence of Ca²⁺. These results are discussed in terms of the possible participation of Ca²⁺ in the action of 4-AP on spontaneous transmitter release in isolated nerve endings.     

Effects of opioids on glutamate induced calcium intracellular increase in individually perfused rat hippocampal neurons in vitro

1. 1. Superfusion of cultured hippocampal cells with glutamate (Glu) 0.5 mM for 5 min induces an increase of [Ca²⁺_i] that is quickly followed by a recovery to control level. Addition of dynorphin or D-pen²-D-pen⁵-enkephalin (DPDPE) induces a persistence of the elevated [Ca²⁺_i], while [D-Ala²,N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO) does not influence it.

2. 2. Superfusion with Glu for 10 min induces a persistent increase of [Ca²⁺_i], that is partially reverted by DAMGO, but not affected by dynorphin or DPDPE.

3. 3. The author suggests a differential influence of selective opioids on the Glu-induced [Ca²⁺_i] increase.

     

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.

     

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.     

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.     

Removal of Ca(2+) following depolarization-evoked cytoplasmic Ca(2+) transients in freshly dissociated pyramidal neurones of the rat dorsal cochlear nucleus

Cytoplasmic [Ca²⁺] ([Ca²⁺]_i) was measured using Fura-2 in pyramidal neurones isolated from the rat dorsal cochlear nucleus (DCN). The kinetic properties of Ca²⁺ removal following K⁺ depolarization-induced Ca²⁺ transients were characterized by fitting exponential functions to the decay phase. The removal after small transients (<82 nM peak [Ca²⁺]_i) had monophasic time course (time constant of 6.43±0.48 s). In the cases of higher Ca²⁺ transients biphasic decay was found. The early time constant decreased (from 3.09±0.26 to 1.46±0.11 s) as the peak intracellular [Ca²⁺] increased. The value of the late time constant was 18.15±1.60 s at the smallest transients, and showed less dependence on [Ca²⁺]_i. Blockers of Ca²⁺ uptake into intracellular stores (thapsigargin and cyclopiazonic acid) decreased the amplitude of the Ca²⁺ transients and slowed their decay. La³⁺ (3 mM) applied extracellularly during the declining phase dramatically changed the time course of the Ca²⁺ transients as a plateau developed and persisted until the La³⁺ was present. When the other Ca²⁺ removal mechanisms were available, reduction of the external [Na⁺] to inhibit the Na⁺/Ca²⁺ exchange resulted in a moderate increase of the time constants. It is concluded that in the isolated pyramidal neurones of the DCN the removal of Ca²⁺ depends mainly on the activity of Ca²⁺ pump mechanisms.     

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.     

Mn and Mg influxes through Ca channels of motor nerve terminals are prevented by verapamil in frogs

At frog neuromuscular junctions immersed in solutions containing 0.5 mM Mn²⁺, verapamil (40 μM) reduced the increase in miniature end-plate potential (MEPP) frequency produced by tetanic stimulation (50 Hz, 2 min) of the motor nerve to 5% of that in the absence of verapamil. In solutions containing 5 mM Mg²⁺, verapamil reduced the tetanic increase in MEPP frequency to 8% of that in the absence of verapamil. Verapamil added to solutions containing 0.15 mM Ca²⁺ decreased the tetanic rise in MEPP frequency to 6% of the control value. In low Ca²⁺ (nominally Ca²⁺-free) solutions, verapamil decreased the tetanic rise to 70% of the control value. The present results suggest that Mn²⁺ and Mg²⁺, as well as Ca²⁺, enter the nerve terminal through Ca²⁺ channels during nerve stimulation and promote transmitter release. In addition to its effect on the Ca²⁺ channel, verapamil at higher concentrations appears to have inhibitory effects on the acetylcholine-gated end-plate channel and on the Na⁺ channel as suggested by its depressive effects on the amplitudes of MEPPs, end-plate potentials and nerve terminal action potentials.     

Human skeletal muscle has a voltage-gated proton current

A voltage-gated proton current, I_H, was studied with the whole-cell patch-clamp technique in human myotubes obtained from biopsies of human muscle. Studies of the reversal potential of I_H during substitution of K⁺, Na⁺, Ca²⁺, Cl⁻, Cs⁺, and H⁺ in the extracellular solution indicated that protons were the major charge carriers of I_H. This current is similar in many respects, but not identical, to the proton currents already described in other cell types. I_H is activated by depolarization and it can be affected by extracellular pH. I_H can be blocked by external divalent cations including Ca²⁺. This block is voltage-dependent, being more efficient at hyperpolarized than at depolarized voltages. The voltage-dependent properties of I_H and its ability to be affected by pH and extracellular Ca²⁺ suggest that I_H might be used by muscle cells to extrude protons during action potentials.     

The action of valproate on spontaneous epileptiform activity in the absence of synaptic transmission and on evoked changes in [Ca2+]o and [K+]o in the hippocampal slice

The effects of valproate (VPA) on neuronal excitability and on changes in extracellular potassium ([K⁺]₀) and calcium ([Ca²⁺]₀) were investigated with ion selective-reference electrode pairs in area CA₁ of rat hippocampal slices. Field potential responses to single ortho- and antidromic stimuli were unaltered by VPA (1–5 mM). The afferent volley evoked in the Schaffer-commissural fibers was also unaffected. In contrast, VPA (1 mM) depressed frequency potentiation and paired pulse facilitation markedly. Decreases in [Ca²⁺]₀ induced either by repetitive stimulation or by application of the excitatory amino acids N-methyl-d-aspartate and quisqualate were reduced, and the latter results suggest that VPA interferes with postsynaptic Ca²⁺ entry. When synaptic transmission was blocked by lowering [Ca²⁺]₀ (0.2 mM) and elevating [Mg²⁺]₀ (7 mM), prolonged afterdischarges elicited by antidromic stimulation were blocked by VPA. VPA also suppressed the spontaneous epileptiform activity seen when [Ca²⁺]₀ was lowered to 0.2 mM, without elevating [Mg²⁺]₀. The amplitudes of the rises in [K⁺]₀ induced by repetitive orthodromic stimulation were only slightly depressed and those elicited by antidromic stimulation were generally unaltered by VPA, as were laminar profiles of stimulus-evoked [K⁺]₀ signals. These results indicate that VPA has membrane actions in addition to known effects on excitatory and inhibitory transmitter pools.     

-Receptor and cholinergic receptor-linked changes in cytosolic Ca and membrane potential in primary rat astrocytes

Both phenylephrine and carbachol caused a sustained increase in Ca²⁺ influx and intracellular free Ca²⁺ of primary astrocytes as measured with ⁴⁵Ca²⁺ and fura-2. The responses to phenylephrine and carbachol were additive, suggesting that they use different releasable pools of Ca²⁺. If extracellular Ca²⁺ was removed by EGTA only a transient rise in cytosolic Ca²⁺ was seen upon application of the agonists. Both compounds caused depolarization of the astrocyte membrane as determined with the optical probe 3,3-diethylthiadicarboxyamineiodide. Activation of protein kinase C with 12-tetradecanoylphorbol myristate acetate (TPA) or the diacylglycerol analogue dioctanoylglycerol (DiC₈) also depolarized the cells. A prior activation of protein kinase C with TPA or DiC₈ abolished the depolarizing effect of phenylephrine suggesting that they act through the same mediators. If the cells were made ideally permeable to K⁺ with the ionophre valinomycin, or the K⁺ channels had been blocked with Ba²⁺, neither TPA nor phenylephrine had any significant effect on the membrane potential. Neither TPA nor phenylephrine had any effect on the ⁸⁶Rb⁺ equilibrium potential across the cell membrane. The results suggest that the depolarizing effect of these substances could be through a blocking of K⁺ channels.     

Analysis of potential shifts associated with recurrent spreading depression and prolonged unstable spreading depression induced by microdialysis of elevated K in hippocampus of anesthetized rats

The potential shifts (ΔV_o) associated with spreading depression (SD) were analysed with the help of multiple extracellular recording and ion-selective microelectrodes in the CA1 region of the dorsal hippocampus of anesthetized rats. Recurrent waves of SD were induced by perfusing high K⁺ solution through microdialysis probes. SD-related ΔV_o had a composite wave shape, consisting of an early, rapidly shifting part (phase I) followed by a slower shift to a second negative maximum (phase II). ΔV_o shifts in stratum radiatum usually started earlier, always lasted longer and had lartger amplitude than those recorded in stratum pyramidale. The ΔV_o responses in stratum radiatum had an inverted saddle shape created by a transient relatively positive “hump” interposed between phases I and II. During this “hump”, the potentials in the two layers transiently approached one another. During continuous high K⁺ dialysis, successive ΔV_o waves episodes evolved according to a consistent pattern: while phase I remained unchanged, phase II increased in amplitude and duration with each episode. Eventually, a depressed state developed which lasted for many minutes, termed here prolonged unstable spreading depression. During phase I, ΔV_o and extracellular K ([K⁺]_o) changes were correlated. During phase II, [K⁺]_o decreased even as ΔV_o continued to increase. During SD, [Ca²⁺]_o decreased to <0.01 mM. During phases I and II, both [Ca²⁺]_o and [Na⁺]_o remained low. the recoverries of [Ca²⁺]_o and [Na⁺]_o had an initial fast and a later much slower phase and took several minutes longer than the recoveries of [K⁺]_o and ΔV_o. Depth profiles of ΔV_o and Δ[K⁺]_o revealed strikingly steep gradients early and late during a wave; but voltage and ion gradients were not precisely correlated either in time or in space. We conclude that ΔV_o of phases I and II are generated by different processes. Membrane ion currents cannot fully explain the ΔV_o responses. The possible contributions by ion diffusion and by active ion transport are discussed. The extremely low level to which [Ca²⁺]_o sinks during SD, and its two-phase recovery, indicate intracellular sequestration or binding of substantial amounts of Ca²⁺ ions. The residual deficit of [Ca²⁺_o following recovery of SP shifts may account for the persistent depression of synaptic transmission after repolarization of neurons.     

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.     

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.     

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.     

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.     

The influx of Ca and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels

We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca²⁺ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca²⁺]_i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker ω-conotoxin (0.1 μmol/1) diminished the release of noradrenaline and the increase of intraterminal Ca²⁺ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 μmol/1) diminished the release of noradrenaline by 25% and the increase of [Ca²⁺]_i by 39%. The P-type channel blocker ω-agatoxin (0.3 μmol/1) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca²⁺ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca²⁺]_i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca²⁺ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.     

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.     

Modification of the Na,K-pump of glial cells within cobalt-induced epileptogenic cortex of rat

The characteristics of a glial Na⁺,K⁺-pump dependent on extracellular K⁺ within epileptogenic cortex were studied electrophysiologically, biochemically and histochemically in vitro using slices from cobalt-induced epileptogenic cortex of rat. When the extracellular K⁺ concentration ([K⁺]_o) was varied between 4 and 40 mM, the mean slope of membrane potential plotted against [K⁺]_o was about 57 mV in glia from the normal cortex (tissue A) and about 44 mV in glia from the epileptogenic cortex (tissue B); whereas no significant difference in the resting membrane potential of these tissues was observed. In glia from tissue B, a marked transient hyperpolarization above control level was caused by replacement of elevated [K⁺]_o with the normal medium. Ouabain abolished these phenomena observed in glia from tissue B, but had no effect on the membrane potential during normal [K⁺]_o. Reduction of extracellular Na⁺, Ca²⁺ and Cl⁻ did not significantly affect the membrane potential of glia from either tissue. In tissue A, the cells marked by intracellular injection of horseradish peroxidase after intracellular recording were protoplasmic astrocytes; in tissue B, fibrous astrocytes with abnormal processes predominated. K⁺-dependent stimulation of Na⁺,K⁺-ATPase activity of the astrocyte-enriched fraction and its membrane preparation from tissue B was much larger than that from tissue A. A certain amount of the reaction product of K⁺-pNPPase activity was seen on glial plasma membrane within tissue B but not on that from tissue A. The above findings suggest that a glial Na⁺,K⁺-pump within actively firing epileptogenic cortex may be modified to increase in its activity.