Lead modulation of the neuronal nicotinic acetylcholine receptor in PC12 cells

Lead is known to modulate several ligand- and voltage-gated ion channels, including the nicotinic acetylcholine receptor (AChR) channel. We examined the effects of lead on the nicotinic AChR in rat clonal phaeochromocytoma PC12 cells using whole-cell and single-channel patch-clamp techniques to clarify the detailed mechanism of action. Lead suppressed acetylcholine-induced currents in a dose-dependent manner with an EC₅₀ value of 37 μM and a Hill coefficient of 0.82. At the single-channel level, 1–10 μM lead shortened the opening and burst durations, and increased the duration of mean closed time. The open probability was significantly decreased by lead. These changes of single-channel kinetics result in a significant decrease in the total charge carried through the open AChR channels explaining the suppressive effect of lead on acetylcholine-induced whole-cell currents. © 1997 Elsevier Science B.V. All rights reserved.     

Arachidonic acid and prostaglandin D2 cooperatively accelerate desensitization of nicotinic acetylcholine receptor channel in mouse skeletal muscles

To clarify the effects of arachidonic acid (AA) and its metabolites on desensitization of nicotinic acetylcholine (ACh) receptor channel in mouse skeletal muscle cells, we investigated the time-dependent decrease in the channel opening frequency of ACh (1 μM)-activated channel currents by the cell-attached patch clamp technique. AA (30–100 μM) applied to a patched membrane or to non-patched membrane accelerated the decrease in the channel opening frequency. A cyclooxygenase inhibitor, indomethacin (10 μM), prevented the acceleration elicited by 30 μM AA, but not by 100 μM AA. A lipoxygenase inhibitor, nordihydroguaiaretic acid (10 μM), and a cytochrome P-450 inhibitor, ketoconazole (3 μM), did not affect the acceleration by 30 μM AA. Prostaglandin (PG) D₂ at 10 μM alone and at 25 nM in combination with 10 μM AA accelerated the decrease in the channel opening frequency. No acceleration was observed with PGE₂ at 10 μM alone and at 25 nM in combination with 10 μM AA. Pretreatment with a protein kinase (PK) C inhibitor, staurosporine (10 nM), but not with a PKA inhibitor, H-89 (3 μM), prevented the acceleration elicited by AA+PGD₂. These results suggest that AA, and PGD₂ of its metabolites, cooperatively accelerate desensitization of nicotinic ACh receptor channel. The activation of PKC by AA and PGD₂ may be involved in the mechanism of the cooperative acceleration of desensitization.     

Possible modulatory role of voltage-activated Ca currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus

Voltage-activated Ca²⁺ currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6–11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca²⁺ current on depolarizations positive to −90 mV (the maximum occurred at about −40 mV). The magnitude of the T-current varied considerably from cell to cell (−56 to −852 pA) while its steady-state inactivation was consistent (E₅₀=−88.2±1.7 mV, s=−6.0±0.4 mV). The maximum of high-voltage activated (HVA) Ca²⁺ currents was observed at about −15 mV. At a membrane potential of −10 mV the L-type Ca²⁺ channel blocker nifedipine (10 μM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor ω-Conotoxin GVIA (2 μM) reduced the current by 25% while the P/Q-type channel blocker ω-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca²⁺ channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (200 μM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA_B receptor agonist baclofen (100 μM) reversibly inhibited 25% of the HVA current. Simultaneous application of ω-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca²⁺ currents reported in this work are discussed.     

Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca²⁺-activated potassium (BK_Ca) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK_Ca channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K⁺ in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P_o) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P_o was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca²⁺]_i required to half activate BK_Ca channels was only 1 μM in post-ischemic whereas 2 μM in control neurons, indicating an increase in [Ca²⁺]_i sensitivity after ischemia; and (4) BK_Ca channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK_Ca channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.     

Dibutyryl cGMP raises cytosolic concentrations of Ca in cultured nodose ganglion neurons of the rabbit

The effect of dibutyryl cGMP (dbcGMP), a membrane permeant cGMP analogue, on cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) was studied in cultured nodose ganglion neurons of the rabbit using fura-2AM and microfluorometry. Application of dbcGMP (10–1000 μM) increased [Ca²⁺]_i in 42% of neurons (n=67). The effect was observed in a dose-dependent fashion. The threshold dose was 100 μM and the increase at 500 μM averaged 117±8%. Removal of extracellular Ca²⁺ abolished the dbcGMP effect. Application of Ni²⁺ (1 mM) or neomycin (50 μM), a non-L-type voltage-gated Ca²⁺ channel (VGCC) antagonist, eliminated the dbcGMP effect. ω-conotoxin GVIA (2 μM), the N-type Ca²⁺ channel antagonist, or L-type Ca²⁺ channel antagonists (D600, 50 μM, or nifedipine, 10 μM) did not alter the dbcGMP effect. Ryanodine (10 μM) did not alter the effect of dbcGMP. Therefore, cGMP could play a part of role of an intracellular messenger in primary sensory neurons of the autonomic nervous system.     

Arachidonic acid inhibits both K and Ca currents in isolated type I cells of the rat carotid body

Whole-cell patch-clamp recordings were used to investigate the effects of arachidonic acid (AA) on K⁺ and Ca²⁺ channels in isolated rat type I carotid body cells. AA (2–20 μM) produced a concentration-dependent inhibition of both K⁺ currents and Ca²⁺ channel currents. The effects of AA on K⁺ currents were unaffected by indomethacin (5 μM), phenidone (5 μM) or 1-aminobenzotriazole (3 mM), suggesting that AA did not exert its effects via cyclo-oxygenase, lipoxygenase or cytochrome P-450 (cP-450) metabolism. Our results suggest that AA directly and non-selectively inhibits ionic currents in rat type I carotid body cells.     

Differential effects of the pyrethroid tetramethrin on tetrodotoxin-sensitive and tetrodotoxin-resistant single sodium channels

The differential effects of the pyrethroid tetramethrin on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) single sodium channel currents in rat dorsal root ganglion (DRG) neurons were investigated using the outside-out configuration of patch-clamp technique. Channel conductances were 10.7 and 6.3 pS for TTX-S and TTX-R sodium channels, respectively, at a room temperature of 24–26°C. The single-channel current of TTX-S sodium channels at the test potential of −30 mV was −1.27 ± 0.25 pA, and was not changed after exposure to 10 μM tetramethrin (−1.28 ± 0.23 pA). The open time histogram of TTX-S single-channel currents could be fitted by a single exponential function with a time constant of 1.27 ms. After exposure to 10 μM tetramethrin, the open time histogram could be fitted by the sum of two exponential functions with time constants of 1.36 ms (τ_fast) and 5.73 ms (τ_low). The percentage of contribution of each component to the population was 62% for the fast component representing the normal channels and 38% for the slow component representing the tetramethrin modified channels. The amplitudc of TTX-R single-channel currents was slightly changed from −0.72 ± 0.14 to −0.83 ± 0.07 pA by 10 μM tetramethrin. The open time histogram of TTX-R single-channel currents could be fitted by a single exponential function with a time constant of 1.92 ms. In the presence of 10 μM tetramethrin, the open time histogram could be fitted by the sum of two exponential functions with time constants of 2.07 ms (τ_fast) and 9.75 ms (τ_slow). The percentage of contribution of each component was 15% for the fast, unmodified component and 85% for the slow, modified component. Differential effects of tetramethrin on the open time distribution of single sodium channel currents explains the differential sensitivity of TTX-S and TTX-R sodium channels.     

KATP channel blockers selectively interact with A1-adenosine receptor mediated modulation of acetylcholine release in the rat hippocampus

In this study the role of ATP-sensitive K⁺ channels (K_ATP channels) in the A₁ receptor mediated presynaptic inhibitory modulation of acetylcholine release was investigated in the rat hippocampus. N⁶-Cyclohexyladenosine (CHA), the selective A₁-adenosine receptor agonist, reduced concentration-dependently the stimulation-evoked (2 Hz, 1 ms, 240 shocks) [³H]acetylcholine ([³H]ACh) release, from in vitro superfused hippocampal slices preloaded with [³H]choline, an effect prevented by the selective A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). By themselves, neither K_ATP channel openers, i.e. diazoxide, pinacidil and cromakalim, nor glibenclamide and glipizide, the inhibitors of K_ATP channels, exerted a significant effect on the resting and evoked release of [³H]ACh. Glibenclamide and glipizide (10–100 μM) completely prevented the inhibitory effect of 0.1 μM CHA and shifted the concentration response curve of CHA to the right. 4-Aminopyridine (10–100 μM), the non-selective potassium channel blocker, increased the evoked release of [³H]ACh, but in the presence of 4-aminopyridine, the inhibitory effect of CHA (0.1 μM) still persisted. Oxotremorine, the M₂ muscarinic receptor agonist, decreased the stimulation-evoked release of [³H]ACh, but its effect was not reversed by glibenclamide. 1,3-Diethyl-8-phenylxanthine (DPX), the selective A₁-antagonist, effectively displaced [³H]DPCPX in binding experiments, while in the case of glibenclamide and glipizide, only slight displacement was observed. In summary, our results suggest that K_ATP channels are functionally coupled to A₁ receptors present on cholinergic terminals of the hippocampus, and glibenclamide and glipizide, by interacting with K_ATP channels, relieve this inhibitory neuromodulation.     

The bradykinin receptor — a putative receptor-operated channel in PC12 cells: studies of neurotransmitter release and inositol phosphate accumulation

Bradykinin (BK) induced [³H]norepinephrine ([³H]NE) release and phosphatidylinositol turnover were investigated in PC12 cells. Induction of [³H]NE release by BK is mediated by activation of BK-B₂-receptors, as determined using type specific BK receptor antagonists. BK induces [³H]NE release with a half maximal effective concentration of30 ± 0.5nM, and reaches maximal net fractional release of9.0 ± 1% with 200 nM BK. The BK-induced release is Ca²⁺ dependent, reaching maximal release at 1.0 mM Ca²⁺, is pertussis toxin insensitive (1 μg/ml), slightly increased by a dibutyryl cAMP (1 mM) and not affected by inhibitors of the cyclooxygenase or lipoxygenase pathways. Voltage-sensitive Ca²⁺ channel blockers, verapamil (10 μM), nifedipine (10 μM), and ω-conotoxin (CgTx 10 nM), do not block the BK-induced release. However, a considerable inhibitory effect was obtained by divalent cations Co²⁺ (ED₅₀ = 0.2mM) and Ni²⁺ (ED_50²⁺ = 1mM). These results indicate the involvement of a Ca²⁺ channel in the BK-mediated release which is different from the L- or N-type voltage sensitive calcium channels. Whereas [Ca²⁺]_ex is essential for the BK-induction of catecholamine release, the rise in level of InsP's induced by BK in the presence or in the absence of [Ca²⁺]_ex is similar up to concentration of 1 μM. This indicates that the rise in InsP's induced by BK is not sufficient to cause neurotransmitter release. Moreover, subsequent addition of Ca²⁺ to BK-stimulated cells in Ca²⁺-free medium yields no release. Hence, no activity triggered by BK alone could be further stimulated by Ca²⁺ for induction of release. Protein kinase C inhibitors polymyxin B, K_252a, sangivamicin, and Ara-A, do not affect release induced by BK, indicating that also the diacylglycerol pathway activated by phospholipase C is not involved in the BK-mediated release. Since (a) the receptor-mediated release is absolutely calcium-dependent, with no release detected when Ca²⁺ is omitted from the extracellular medium, and (b) the receptor-triggered release of Ca²⁺ from intracellular stores is independent of [Ca²⁺]_ex⁷, it appears that calcium influx, and not Ca²⁺ released from intracellular stores, is the signal for stimulating release. Therefore, it is suggested that the primary signal stimulating release is Ca²⁺ influx via a specific calcium channel, and that the BK receptor may be coupled to this channel, which could be classified as a receptor-operated channel.     

SnCl(2) reduces voltage-activated calcium channel currents of dorsal root ganglion neurons of rats

Stannous dichloride (SnCl₂) occurs in the environment where it has been especially enriched in aquatic ecosystems. Furthermore, it is used in food manufacturing (e.g. for stabilizing soft drinks or as an anti-corrosive substance) and in nuclear medicine where it is employed as a reducing agent for technecium-99m (99mTc) and therefore is applied intravenously to human beings.SnCl₂ is known to have toxic effects on the nervous system which can be related to alterations of intracellular calcium homeostasis ([Ca²⁺]_i). In this study the whole cell patch clamp technique is used on dorsal root ganglion neurons of 3-week-old “Wistar” rats to evaluate the effects of SnCl₂ on voltage-activated calcium channel currents (I_Ca(V)).I_Ca(V) were reduced concentration-dependently by SnCl₂ (1–50 μM). 1 μM SnCl₂ reduced I_Ca(V) by 8.1 ± 4.5% (peak current) and 19.2 ± 8.9% (sustained current), whereas 50 μM inhibited I_Ca(V) by 50.6 ± 4.3% (peak current) and 55.6 ± 11.3% (sustained current). Sustained currents were slightly but not significantly more reduced than peak currents. The effect appeared not to be reversible. The threshold concentration was below 1 μM.The current–voltage relation did not shift which is an indication that different calcium channel subtypes were equally affected. There was a slight but not significant shift of the activation/inactivation curves towards the depolarizing direction.We conclude that voltage-gated calcium channels are affected by Sn²⁺ similarly to other divalent metal cations (e.g. Pb²⁺ or Zn²⁺).The reduction of I_Ca(V) could be related to the neurotoxic effects of SnCl₂.     

Mercury (Hg) decreases voltage-gated calcium channel currents in rat DRG and Aplysia neurons

Inorganic mercury (Hg²⁺) reduced voltage-gated calcium channel currents irreversibility in two different preparations. In cultured rat dorsal root ganglion (DRG) neurons, studied with the whole cell patch clamp technique, a rapid concentration-dependent decrease in the L/N-type currents to a steady state was observed with an IC₅₀ of 1.1 μM and a Hill coefficient of 1.3 T-currents were blocked with Hg²⁺ in the same concentration range (0.5–2 μM). With increasing Hg²⁺ concentrations a slow membrane current was additionally activated most obviously at concentrations over 2 μM Hg²⁺. This current was irreversible and might be due to the opening of other (non-specific) ion channels by Hg²⁺. The current-voltage (I–V) relation of DRG neurons shifted to more positive values, suggesting a binding of Hg²⁺ to the channel protein and/or modifying its gating properties. In neurons of the abdominal ganglion of Aplysia californica, studied with the two electrode voltage clamp technique, a continous decrease of calcium channel currents was seen even with the lowest used concentration of Hg²⁺ (5 μM). A steady state was not reached and the effect was irreversible without any change on resting membrane currents, even with high concentrations (up to 50 μM). No shift of the I–V relation of the calcium channel currents was observed. Effects on voltage-activated calcium channel currents with Hg²⁺ concentrations such low have not been reported before. We conclude that neurotoxic effects of inorganic mercury could be partially due to the irreversible blockade of voltage-activated calcium channels.     

Up-regulation of functional voltage-dependent sodium channels by cyclic AMP-dependent protein kinase in adrenal medulla

Treatment of cultured bovine adrenal chromaffin cells with dbcAMP increased [³H]STX binding with an EC₅₀ of 126 μM and a half-effective time of 12 h; dbcAMP (1 mM × 18 h) raised theB_max approximately 1.5-fold without altering theK_d value. Forskolin (0.1 mM) or IBMX (0.3 mM) also increased [³H]STX binding, while dbcAMP had no effect. Effects of dbcAMP and forskolin were abolished by H-89, an inhibitor of cAMP-dependent protein kinase. Cycloheximide (10 μg/ml) and actinomycin D (10 μg/ml), inhibitors of protein synthesis, nullified the stimulatory effect of dbcAMP, whereas tunicamycin, an inhibitor of protein glycosylation, had no effect. Treatment with dbcAMP augmented veratridine-induced²²Na influx,⁴⁵Ca influx via voltage-dependent Ca channels and catecholamine secretion, while the same treatment did not alter⁴⁵Ca influx and catecholamine secretion caused by high K (a direct activation of voltage-dependent Ca channels) [25]. Na influx via single Na channel calculated from²²Na influx and [³H]STX binding was quantitatively similar between non-treated and dbcAMP-treated cells. Brevetoxin allosterically enhanced veratridine-induced²²Na influx approximately 3-fold in dbcAMP-treated cells as in non-treated cells. These results suggest that cAMP-dependent protein kinase is involved in the modulation of Na channel expression in adrenal medulla.     

Tolbutamide attenuates diazoxide-induced aggravation of hypoxic cell injury

ATP-dependent potassium (K_ATP) channels of neurons are closed in the presence of physiological levels of intracellular ATP and open when ATP is depleted during hypoxia or metabolic damage. The present study investigates hypoxic alterations of purine and pyrimidine nucleotide levels supposed to intracellularly modulate K_ATP channels. In addition, the effects of the K_ATP channel activator diazoxide and its antagonist tolbutamide were investigated on ATP, GTP, CTP and UTP levels in slices of the parietal cortex. Hypoxia was evoked by saturation of the medium with 95% N₂–5% CO₂ instead of 95% O₂–5% CO₂ for 5 min. Nucleotide contents were measured by anion-exchange HPLC in neutralized perchloric acid extracts obtained from slices frozen immediately at the end of incubation. Hypoxia per se decreased purine and pyrimidine nucleoside triphosphate contents. Thus, ATP and GTP contents were reduced to 69.9 and 77.6% of the respective normoxic levels. UTP and CTP contents were even more decreased (to 60.9 and 41.6%), probably because the salvage pathway of these pyrimidine nucleotides is less effective than that of the purine nucleotides ATP and GTP. While tolbutamide (300 μM) had no effect on the hypoxia-induced decrease of nucleotides, diazoxide at 300, but not 30 μM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 μM) antagonized the effects of diazoxide (300 μM). Nucleoside diphosphate (ADP, GDP and UDP) levels were uniformly increased by hypoxia. There was no hypoxia-induced increase of ADP contents in the presence of tolbutamide (300 μM). The ATP/ADP, GTP/GDP and UTP/UDP ratios uniformly declined at a low pO₂. However, only the ATP/ADP ratio was decreased further by diazoxide (300 μM). The observed alterations in nucleotide contents may be of importance for long- and short-term processes related to acute cerebral hypoxia. Thus, hypoxia-induced alterations of purine and pyrimidine nucleotide levels may influence the open state of K_ATP-channels during the period of reversible hypoxic cerebral injury. Furthermore, alterations during the irreversible period of cerebral injury may also arise, as a consequence of decreased pyrimidine nucleotide contents affecting cell survival via protein and DNA synthesis.     

Partial characterization of K-induced increase in [Ca]cyt and GnRH release in GT1-7 neurons

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca²⁺ and GnRH release in response to K⁺-induced depolarization of GT1-7 neurons. Our results showed that K⁺ concentrations from 7.5 to 60 mM increased [Ca²⁺]_cyt in a concentration-dependent manner. Resting [Ca²⁺]_cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; N = 69). K⁺-induced increases in [Ca²⁺]_cyt ranged from 58.2 nM at 7.5 mM [K⁺] to 347 nM at 60 mM [K⁺]. K⁺-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K⁺] to about 60 pg/ml at 45 mM [K⁺]. K⁺-induced increases in [Ca²⁺]_cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca²⁺ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca²⁺ channel antagonist. Nimodipine (1 μM) alone partially inhibited K⁺-induced increases in [Ca²⁺]_cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K⁺-induced GnRH release or [Ca²⁺]_cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K⁺-induced increase in [Ca²⁺]_cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca²⁺ channels exist in GT1-7 neurons and may be part of the response to K⁺. The response of [Ca²⁺]_cyt to K⁺ was linear with increasing [K⁺] whereas the response of GnRH release to increasing [K⁺] appeared to be saturable. K⁺-induced increase in [Ca²⁺]_cyt and GnRH release required extracellular [Ca²⁺]. These experiments suggest that voltage dependent N- and L-type Ca²⁺ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.     

Glutamate selectively increases the high-threshold Ca channel current in sensory and hippocampal neurons

Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca²⁺ channels in hippocampal neurons. We studied whole-cell Ca²⁺ currents (I_Ca) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1–30 μM) increased high-threshold Ca²⁺ current. It was independent of the charge carriers, Ca²⁺ or Ba²⁺. Low-threshold Ca²⁺ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1–2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in _ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits ‘sleepy’ Ca²⁺ channels. Whatever its mechanism, overlasting increase of I_Ca by glutamate may be important in neuronal plasticity.     

Interactions of dextromethorphan with theN-methyl-d-aspartate receptor-channel complex: single channel recordings

The actions of dextromethorphan (DXM) on the 50 pS conductance state of theN-methyl-d-aspartate (NMDA) receptor-operated channel were studied using outside-out patches obtained from cultured rat hippocampal pyramidal neurons. DXM (5–50 μM) had no effect on the amplitudes of unitary currents but caused concentration-dependent reductions in channel mean open times and the frequency of channel openings. Channel open probability was reduced in a concentration-dependent manner by DXM and was one-half of the control value at a DXM concentration of 6 μM, with the patch potential held at −60 mV. An IC₅₀ value of 4 μM was obtained for the reduction by DXM of NMDA-evoked rises in [Ca²⁺]_i in cultured rat hippocampal pyramidal neurons loaded with Fura-2. The results were consistent with drug block of the open NMDA channel with an onward (blocking) rate constant of 7.7 × 10⁶ M⁻¹ · s⁻¹ (at −60 mV). The estimated unblocking rate constant was about 10 s⁻¹, a value considerably higher compared to the off-rate constant found for dizocilpine block of the NMDA channel.     

Coupling of AMPA receptors with the Na/Ca exchanger in cultured rat astrocytes

Astrocytes exhibit three transmembrane Ca²⁺ influx pathways: voltage-gated Ca²⁺ channels (VGCCs), the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na⁺/Ca²⁺ exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca²⁺ concentration ([Ca²⁺]_i); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca²⁺]_i is not known. We demonstrate here that 100 μM AMPA in the presence of 100 μM cyclothiazide (CTZ) causes an increase in [Ca²⁺]_i in cultured cerebral astrocytes that requires transmembrane Ca²⁺ influx. This increase of [Ca²⁺]_i is blocked by 100 μM benzamil or 0.5 μM U-73122, which inhibit reverse-mode operation of the Na⁺/Ca²⁺ exchanger by independent mechanisms. This response does not require Ca²⁺ influx through VGCCs, nor does it depend upon a significant Ca²⁺ influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca²⁺ stores, although depletion of these Ca²⁺ stores does not decrease the peak [Ca²⁺]_i response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca²⁺]_i to increase through the reverse mode operation of the Na⁺/Ca²⁺ exchanger with an associated release of Ca²⁺ from intracellular stores. This proposed mechanism requires neither Ca²⁺-permeant AMPARs nor the activation of VGCCs to be effective.     

Presynaptic effect of 7-OH-DPAT on evoked [H]-acetylcholine release in rat striatal synaptosomes

The objective of the present experiments was to study the presynaptic effect of 7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT, a D₂-like dopamine receptor agonist) on [³H]-acetylcholine ([³H]-ACh) release induced by potassium (15 mM, 25 mM and 60 mM), potassium channel-blockers (4-aminopyridine, 4-AP; tetraethylammonium, TEA and quinine) and veratridine to gain insight into the mechanisms involved in the activation of the D₂ dopamine-receptor subtype located at striatal cholinergic nerve terminals. 7-OH-DPAT (1 μM) inhibited the evoked [³H]-ACh release induced by K⁺ 15 mM in a similar percentage than that obtained during basal conditions (30% and 27%, respectively). Nevertheless, in the presence of 25 mM and 60 mM of K⁺ the inhibitory effect of 7-OH-DPAT was completely abolished. 4-AP (1–100 μM) and TEA (1 and 5 mM) significantly enhanced [³H]-ACh release, showing 69.32%±7.60% (P<0.001) and 52.27%±5.64% (P<0.001), respectively, at the highest concentrations tested. In these conditions, 7-OH-DPAT (1 μM) inhibited the release induced by potassium channel-blockers 25–27%. Quinine (0.1–1 μM) did not alter [³H]-ACh release either in the presence or absence of 7-OH-DPAT. Veratridine 10 μM evoked [³H]-ACh release in the presence of a low-calcium medium, but in such conditions 7-OH-DPAT (1 μM) did not modify the neurotransmitter release in the absence or presence of veratridine. Present data indicate that activation of the presynaptic D₂ dopamine receptor inhibits the [³H]-ACh release by increasing K⁺ conductance, as high K⁺ concentrations abolished the inhibitory control of 7-OH-DPAT on [³H]-ACh release. This effect could be mediated by potassium channels different from those sensitive to 4-AP, TEA and quinine. In addition, the presynaptic D₂ dopamine-receptor activation seems to not involve changes in intracellular Ca²⁺.     

Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons

Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (ec50=40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca²⁺ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca²⁺ media (0–1.0 mM), but was completely abolished in a low Na⁺ medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na⁺, which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μM). The TTX or flunarizine completely blocked veratridine-induced Na⁺ influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca²⁺ was detected under these conditions. Though Na⁺ channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na⁺-dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na⁺ influx via voltage-dependent Na⁺ channels, and suggests the presence of Na⁺-dependent regulatory mechanism(s) in neuronal survival.