Involvement of Na+–Ca2+ Exchanger in Reperfusion-induced Delayed Cell Death of Cultured Rat Astrocytes

In some cells, Ca²⁺ depletion induces an increase in intracellular Ca²⁺ ([Ca²⁺]_i) after reperfusion with Ca²⁺-containing solution, but the mechanism for the reperfusion injury is not fully elucidated. Using an antisense strategy we studied the role of the Na⁺-Ca²⁺ exchanger in reperfusion injury in cultured rat astrocytes. When astrocytes were perfused in Ca²⁺-free medium for 15–60 min, a persistent increase in [Ca²⁺]_i was observed immediately after reperfusion with Ca²⁺-containing medium, and the number of surviving cells decreased 3–5 days latter. The increase in [Ca²⁺]_i was enhanced by low extracellular Na⁺ ([Na⁺]_o) during reperfusion and blocked by the inhibitors of the Na⁺-Ca²⁺ exchanger amiloride and 3,4-dichlorobenzamil, but not by the Ca²⁺ channel antagonists nifedipine, Cd²⁺ and Ni²⁺. Treatment of astrocytes with antisense, but not sense, oligodeoxynucleotide to the Na⁺-Ca²⁺ exchanger decreased Na⁺–Ca²⁺ exchanger protein level and exchange activity. The antisense oligomer attenuated reperfusion-induced increase in [Ca²⁺]ⁱ and cell toxicity. The Na⁺-Ca²⁺ exchange inhibitors 3,4-dichlorobenzamil and ascorbic acid protected astrocytes from reperfusion injury partially, while the stimulators sodium nitroprusside and 8-bromo-cyclic GMP and low [Na⁺]_o exacerbated the injury. Pretreatment of astrocytes with ouabain and monensin caused similar delayed glial cell death. These findings suggest that Ca²⁺ entry via the Na⁺–Ca²⁺ exchanger plays an important role in reperfusion-induced delayed glial cell death.     

Differential Stimulation of Noradrenaline Release by Reversal of the Na+/Ca2+ Exchanger and Depolarization in Chromaffin Cells

We compared the effectiveness of Ca²⁺ entering by Na⁺/Ca²⁺ exchange with that of Ca²⁺ entering by channels produced by membrane depolarization with K⁺ in inducing catecholamine release from bovine adrenal chromaffin cells. The Ca²⁺ influx through the Na⁺/Ca²⁺ exchanger was promoted by reversing the normal inward gradient of Na⁺ by preincubating the cells with ouabain to increase the intracellular Na⁺ and then removing Na⁺ from the external medium. In this way we were able to increase the cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) by Na⁺/Ca²⁺ exchange to 325 ± 14 nM, which was similar to the rise in [Ca²⁺]_c observed upon depolarization with 35 mM K⁺ of cells not treated with ouabain. After incubating the cells with ouabain, K⁺ depolarization raised the [Ca²⁺]_c to 398 ± 31 nM, and the recovery of [Ca²⁺]_c to resting levels was significantly slower. Reversal of the Na⁺ gradient caused an −6-fold increase in the release of noradrenaline or adrenaline, whereas K⁺ depolarization induced a 12-fold increase in noradrenaline release but only a 9-fold increase in adrenaline release. The ratio of noradrenaline to adrenaline release was 1.24 ± 0.23 upon reversal of the Na⁺/Ca²⁺ exchange, whereas it was 1.83 ± 0.19 for K⁺ depolarization. Reversal of the Na⁺/Ca²⁺ exchange appeared to be as efficient as membrane depolarization in inducing adrenaline release, in that the relation of [Ca²⁺]_c to adrenaline release was the same in both cases. In contrast, we found that for the same average [Ca²⁺]_c, the Ca²⁺ influx through voltage-gated channels was much more efficient than the Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger in inducing noradrenaline release from chromaffin ceils. This greater effectiveness of membrane depolarization in stimulating noradrenaline release suggests that there is a pool of noradrenaline vesicles which is more accessible to Ca²⁺ entering through voltage-gated Ca²⁺ channels than to Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger, whereas the adrenaline vesicles do not distinguish between the source of Ca²⁺.     

The Na+-Ca2+ exchange system in rat glial cells in culture: Activation by external monovalent cations

Cultured rat glial cells display a Na⁺-Ca²⁺ exchange system located at the plasma membrane levels. This was evidenced by the Na⁺ (i)-dependency of a Na⁺ (o)-inhibitable influx of Ca²⁺, or reversal exchange mode. This antiporter has an external site where monovalent cations (K⁺, Li⁺, and Na⁺ were investigated) stimulate the exchange by a chemical action. The monovalent cation is not transported during the exchange cycle. The mechanism of that stimulation agrees with an increase in the apparent affinity of the carrier for Ca²⁺ (o) without effect on the maximal translocation rate. Two models can equally well account for the data: i) the formation of ECa(o) is essential for the binding of the monovalent cation, or ii) the activating cation can bind even when the carrier is free of Ca²⁺(o). The cations K⁺ and Li⁺ produced only stimulation, although that of K⁺ seem to require actions other than the chemical effect. The response to Na⁺ was biphasic; this can be fully explained considering that at low concentrations, Na⁺(o) binds preferentially to the activating monovalent site while at high concentrations it displaces Ca²⁺ from its external transporting site. Pure type I astrocytes displayed the same Na⁺-Ca²⁺ exchange mechanism. © 1995 Wiley-Liss, Inc.     

Potentiometric study of resting potential, contributing K+ channels and the onset of Na+ channel excitability in embryonic rat cortical cells

Resting membrane potential (RMP), K⁺ channel contribution to RMP and the development of excitability were investigated in the entire population of acutely dissociated embryonic (E) rat cortical cells over E11–22 using a voltage-sensitive fluorescent indicator dye and flow cytometry. During the period of intense proliferation (E11–13), two cell subpopulations with distinct estimated RMPs were recorded: one polarized at ∼–70 mV and the other relatively less-polarized at ∼–40 mV. Ca²⁺_o was critical in sustaining the RMP of the majority of less-polarized cells, while the well-polarized cells were characterized by membrane potentials exhibiting a ∼Nernstian relationship between RMP and [K⁺]_o. Analysis of these two subpopulations revealed that > 80% of less-polarized cells were proliferative, while > 90% of well-polarized cells were postmitotic. Throughout embryonic development, the disappearance of Ca²⁺_o-sensitive, less-polarized cells correlated with the disappearance of the proliferating population, while the appearance of the K⁺_o-sensitive, well-polarized population correlated with the appearance of terminally postmitotic neurons, immuno-identified as BrdU^–, tetanus toxin⁺ cells. Differentiating neurons were estimated to contain increased K⁺_i relative to less-polarized cells, coinciding with the developmental expression of Cs⁺/Ba²⁺-sensitive and Ca²⁺-dependent K⁺ channels. Both K⁺ channels contributed to the RMP of well-polarized cells, which became more negative toward the end of neurogenesis. Depolarizing effects of veratridine, first observed at E11, progressively changed from Ca²⁺_o-dependent and tetrodotoxin-insensitive to Na⁺_o-dependent and tetrodotoxin-sensitive response by E18. The results reveal a dynamic development of RMP, contributing K⁺ channels and voltage-dependent Na⁺ channels in the developing cortex as it transforms from proliferative to primarily differentiating tissue.     

Cationic modulation of 5-HT2 and 5-HT3 receptors in rat sensory neurons: the role of K, Ca and Mg

The effects of changes in external K⁺, Ca²⁺, and Mg²⁺ concentrations on 5-HT₂- and 5-HT₃ receptor-mediated depolarizations of the resting membrane potential in rat dorsal root ganglion (DRG) cells was studied. In cells exhibiting a 5-HT₂-mediated response, 5-HT and α-methyl 5-HT depolarized the resting membrane potential (RMP) and increased the slope of the current–voltage (I/V) relationship. The equilibrium potential (E_r) for the depolarization was linearly related to the logarithm of the [K⁺]_o, indicating the depolarization resulted from a decrease in resting K⁺ conductance. In a subpopulation of large-diameter acutely dissociated DRG neurons recorded from using the whole-cell patch-clamp configuration, 5-HT produced an inward shift in the current required to hold cells at −60 mV. This inward shift in holding current was associated with a reduction in membrane conductance and reversed near E_k. This data suggests that the 5-HT₂ receptor-mediated depolarization and increase in R_in seen in intact DRG preparation is produced by blockade of an outward K⁺ leak current. Increases in [K⁺]_o reduced the increase in R_in and depolarization induced by 5-HT with 50% inhibition of the depolarization occurring at 8.3 mM of [K⁺]_o. Half-normal Ca²⁺ (1.2 mM) produced a downward shift of the 5-HT concentration–response curve, reducing the maximal response by 40%, with minimal effect on the half-maximal response. Mg²⁺ ions did not affect this 5-HT response. In cells exhibiting a 5-HT₃ receptor response, 5-HT and 2-methyl-5-HT produced depolarization with decreased R_in. The E_r for this depolarizing response (−30.2±1.8 mV) became less negative (−11.5 mV) in 10 mM [K⁺]_o with minimal effect on the amplitude of the depolarization. In Na⁺-free superfusate, the 5-HT-induced depolarization was converted to hyperpolarization. This indicated the 5-HT₃ response increased a mixed Na⁺/K⁺ conductance. Elevated Ca²⁺ or Mg²⁺ markedly reduced the 5-HT₃ response. Incubation with 3.5 mM Ca²⁺ shifted the 5-HT concentration–response curve downward and to the right, decreasing the maximal response by 49% and increasing the EC₅₀ by 10-fold. Elevated Mg²⁺ produced similar effects. In cells where both 5-HT₂- and 5-HT₃-mediated responses could be demonstrated, the elevation of K⁺ or the reduction of Ca²⁺ converted a 5-HT₂ response to a 5-HT₃ response. The above data suggest that elevation of [K⁺]_o or reduction of [Ca²⁺]_o produced by rapid firing rates of sensory neurons will favor the expression of 5-HT₃ responses over 5-HT₂ responses.     

Effects of Growth Hormone-Releasing Peptide-2 (GHRP-2) on Membrane Ca2+ Permeability in Cultured Ovine Somatotrophs

The newly synthesized GH-releasing peptide, GHRP-2 (D-Ala-D-βNal-Ala-Trp-D-Phe-Lys-NH₂), was studied in somatotroph-enriched populations of ovine pituitary cells in primary culture. Nystatin-perforated whole-cell recordings were made on identified somatotrophs after 4–14 days of culture. Using a standard bath solution (containing Na⁺, Ca²⁺) and an electrode solution containing K⁺ in current-clamp recordings, GHRP-2 (10 nM) depolarized the membrane potential of the cells triggering a burst of action potentials. Voltage-clamp recordings indicated that GHRP-2 produced a slowly inactivated inward current with a slight reduction in outward current. The inward current was blocked by the Ca²⁺ channel blocker, Co²⁺ (0.5 mM). Ca²⁺ currents were then isolated using tetraethylammonium bath solution and an electrode solution containing Cs⁺. Ovine somatotrophs possess transient (T type) and long lasting (L type) Ca²⁺ currents. The L type current was abolished by addition of nifedipine (3 μM) to the bath solution and T type current was isolated on this basis. Current-voltage relationships indicated an increase in both T and L type Ca²⁺ currents in response to GHRP-2. The voltage-dependent inactivation curve for T type Ca²⁺ current was shifted towards a less negative level by the peptide. Intracellular free Ca²⁺ concentration ([Ca²⁺]i) in somatotroph-enriched populations was specifically increased by GHRP-2 but this effect was totally abolished by blockade of membrane Ca²⁺ channels. These data show that GHRP-2 causes an influx of Ca²⁺ leading to an increase in [Ca²⁺]i in ovine somatotrophs. The Ca²⁺ currents were both L type and T type with a shift in the inactivation curve of the latter by the releasing peptide.     

Intracellular Action of Spermine on Neuronal Ca2+ and K+ Currents

Intra-and extracellular effects of the polyamine spermine on electrical activity and membrane currents of identified neurons in the abdominal ganglion of Aplysia californica were studied under current-and voltage-clamp conditions. Lonophoretic injection of spermine reduced the amplitude of action potentials and altered their time course as well as spontaneous discharge activity. Investigation of membrane currents showed that intracellular spermine suppressed the total outward current but increased the inward rectifier current. After separation of ion currents it was found that the voltage-activated, delayed K⁺ outward current and the Ca²⁺ inward current were reduced by intracellular spermine in a dose- and voltage-dependent manner. The block of the K⁺ current can be described by a voltage-dependent reaction, where one spermine molecule binds to one channel. The binding constant K_b, at zero voltage, and the effective valency, zδ, had values of 176/M and 0.41 for cell R-15, 223/M and 0.64 for cell L-11, and 137/M and 0.42 for cell L-3. Apparently, more than one spermine cation is needed to block one Ca²⁺ channel, since the coefficient n, which absorbs the molecularity and cooperativity of the reaction, had non-integral values between 1.34 and 2.22. The binding constant K_b and the effective valency zδ had values of 265/M and 0.64 for cell R-15, 821M and 0.56 for cell L-4, and 263/M and 0.51 for cell L-6. Intracellular spermine also blocked the Ca²⁺-activated K⁺ current induced by ionophoretic Ca²⁺-injections, but increased the current at prolonged times after spermine injection. Extracellular spermine had no effect on electrical activity or on membrane currents. The results indicate that intracellular spermine affects the electrical discharge activity of neurons by acting as a blocker and/or modulator at voltage-dependent membrane conductances.     

Rectification Properties and Ca2+ Permeability of Glutamate Receptor Channels in Hippocampal Cells

Excitatory amino acids exert a depolarizing action on central nervous system cells through an increase in cationic conductances. Non-NMDA receptors have been considered to be selectively permeable to Na⁺ and K⁺, while Ca²⁺ influx has been thought to occur through the NMDA receptor subtype. Recently, however, the expression of cloned non-NMDA receptor subunits has shown that α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are permeable to Ca²⁺ whenever the receptor lacks a particular subunit (edited GluR-B). The behaviour of recombinant glutamate receptor channels predicts that Ca²⁺ would only permeate through receptors that show strong inward rectification and vice versa, i.e. AMPA receptors with linear current-voltage relationships would be impermeable to Ca²⁺ . Using the whole-cell configuration of the patch-clamp technique, we have studied the Ca²⁺ permeability and the rectifying properties of AMPA receptors, when activated by kainate, in hippocampal neurons kept in culture or acutely dissociated from differentiated hippocampus. Cells were classified according to whether they showed outward rectifying (type I), inward rectifying (type II) or almost linear (type III) current-voltage relationships for kainate-activated responses. AMPA receptors of type I cells (52.2%) were mostly Ca²⁺-impermeable (P_caIP_cs= 0.1) while type II cells (6.5%) expressed Ca²⁺-permeable receptors (P_caIP_cs=0.9).Type III cells (41.3%) showed responses with low but not negligible Ca²⁺ permeability (P_caIP_cs= 0.18). The degree of Ca²⁺ permeability and inward rectification were well correlated in cultured cells, i.e. more inward rectification corresponded to higher Ca²⁺ permeability. In acutely dissociated neurons, the restricted activation of the receptors located either in dendritic or somatic membranes revealed that inward rectifying (i.e. Ca²⁺-permeable) AMPA receptors are preferentially located in the dendritic shaft (i.e. synaptic field). Our results indicate that oligomeric AMPA receptors of different subunit composition are coexpressed in dissimilar proportions in different cells, which would explain the incomplete inward rectification and graded Ca²⁺ permeability. In addition, Ca²⁺-permeable AMPA receptors may exhibit non-homogeneous subcellular distribution.     

Stimulation of a sodium influx by cAMP in Helix neurons

Brief pressure injections of aqueous solutions of cAMP in identified neurons of Helix pomatia caused depolarizations which lasted for tens of seconds. In voltage-clamped neurons an inward current of similar duration was induced which saturated at 10 μA/cm² cell surface. In the range of negative membrane potentials with little voltage-dependent activation, this current was not accompanied by a change in membrane conductance. The inward current was not produced by injection of ATP, ADP, adenosine, inosine or cGMP. cAMP derivatives produced longer-lasting effects. Prolongation of the inward current was also observed after inhibition of the phosphodiesterase by IBMX. Drugs which block active transport had no effect on the response to cAMP injection. The inward current depended on extracellular sodium, and was maximal when all other mono- and divalent cations were replaced by Na⁺. The cAMP-induced current was accompanied by a transient increase in [Na⁺]_i, but there was no change in [Cl⁻]_i. Li⁺ could largely substitute for Na⁺; Ca²⁺ was less effective. Addition of Mg²⁺ or Ca²⁺ to solutions containing a high Na⁺-concentration inhibited the response. Internal acidification with HCl reversibly enhanced the inward current. These data indicate that the depolarizing effect of cAMP can be accounted for by an inward movement of Na-ions, and that the effect is augmented by H⁺-ions.     

Role of Voltage-gated Ca2+ Channels and Intracellular Ca2+ in Rat Sympathetic Neuron Survival and Function Promoted by High K+ and Cyclic AMP in the Presence or Absence of NGF

We have examined how NGF-dependent rat sympathetic neurons maintain Ca²⁺ homeostasis when challenged with high K⁺ or 8-(4-chlorophenylthio)cyclic AMP (CPTcAMP), two survival factors. In the presence of NGF, high K⁺ (55 mM) caused a stable, 65% reduction in the density of cell soma voltage-sensitive Ca²⁺ channels within 2 days. Although resting [Ca²⁺]_i was elevated by 1.6-fold, this was 50% less than the rise in [Ca²⁺]_i measured before down-regulation occurred, suggesting that down-regulation may help prevent the toxic effects of persistently elevated [Ca²⁺]_i. Inhibition of protein synthesis by cycloheximide blocked recovery from down-regulation. Moreover, treatment with cycloheximide or actinomycin-D caused a 2-fold rise in the peak Ca²⁺ current, suggesting that voltage-sensitive Ca²⁺ channel activity may be tonically attenuated during normal growth. In the absence of NGF, neurons survived for several days in high K⁺ medium with no significant rise in resting [Ca²⁺]_i, although neurites did not grow. Neither Ca²⁺ channel density nor resting [Ca²⁺]_i were altered in neurons surviving with CPTcAMP. Moreover, CPTcAMP lowered the dependence on extracellular Ca²⁺. However, the dihydropyridine antagonist nitrendipine blocked both high K⁺- and CPTcAMP-dependent survival although it had no effect in the presence of NGF. Thus, in the absence of NGF, sympathetic neurons do not require elevation of [Ca²⁺]_i above resting levels to survive with either high K⁺ or CPTcAMP, but dihydropyridine-sensitive Ca²⁺ channel activity may be essential for their survival promoting actions.     

Inhibition of [Ca2+]i Transients in Rat Adrenal Chromaffin Cells by Neuropeptide Y Role for a cGMP-dependent Protein Kinase-activated K+ Conductance

The effects of neuropeptide Y on the intracellular level of Ca²⁺ ([Ca²⁺]_i) were studied in cultured rat adrenal chromaffin cells loaded with fura-2. A proportion (16%) of cells exhibited spontaneous rhythmic [Ca²⁺]_i oscillations. In silent cells, oscillations could be induced by forskolin and 1,9–dideoxyforskolin. This action of forskolin was not modified by H-89, an inhibitor of protein kinase A. Spontaneous [Ca²⁺_i fluctuations and [Ca²⁺]_i fluctuations induced by forskolin- and 1,9-dideoxyforskolin were inhibited by neuropeptide Y. Increases in [Ca²⁺]_i induced by 10 and 20 mM KCI but not by 50 mM KCI were diminished by neuropeptide Y. However, neuropeptide Y had no effect on [Ca²⁺]_i increases evoked by (-)BAY K8644 and the inhibitory effect of neuropeptide Y on responses induced by 20 mM KCI was not modified by o-conotoxin GVIA, consistent with neither L- nor N-type voltage-sensitive Ca²⁺ channels being affected by neuropeptide Y. Rises in [Ca²⁺]_i provoked by 10 mM tetraethylammonium were not decreased by neuropeptide Y, suggesting that K⁺ channel blockade reduces the effect of neuropeptide Y. However, [Ca²⁺]_i transients induced by 1 mM tetraethylammonium and charybdotoxin were still inhibited by neuropeptide Y, as were those to 20 mM KCI in the presence of apamin. The actions of neuropeptide Y on [Ca²⁺]_i transients provoked by 20 and 50 mM KCI, 1 mM tetraethylammonium, (-)BAY K8644 and charybdotoxin were mimicked by 8–bromo-cGMP. In contrast, 8–bromo-CAMP did not modify responses to 20 mM KCI or 1 mM tetraethylammonium. The inhibitory effects of neuropeptide Y and 8–bromo-cGMP on increases in [Ca²⁺]_i induced by 1 mM tetraethylammonium were abolished by the Rp-8–pCPT-cGMPS, an inhibitor of protein kinase G, but not by H-89. A rapid, transient increase in cGMP level was found in rat adrenal medullary tissues stimulated with 1 μM neuropeptide Y. Rises in [Ca²⁺]_i produced by DMPP, a nicotinic agonist, but not by muscarine, were decreased by neuropeptide Y. Our data suggest that neuropeptide Y activates a K⁺ conductance via a protein kinase G-dependent pathway, thereby opposing the depolarizing action of K⁺ channel blocking agents and the associated rise in [Ca²⁺]_i.     

Involvement of the glutamate transporter and the sodium–calcium exchanger in the hypoxia-induced increase in intracellular Ca in rat hippocampal slices

Hippocampal slices prepared from adult rats were loaded with fura-2 and the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the CA1 pyramidal cell layer was measured. Hypoxia (oxygen–glucose deprivation) elicited a gradual increase in [Ca²⁺]_i in normal Krebs solution. At high extracellular sodium concentrations ([Na⁺]_o), the hypoxia-induced response was attenuated. In contrast, hypoxia in low [Na⁺]_o elicited a significantly enhanced response. This exaggerated response to hypoxia at a low [Na⁺]_o was reversed by pre-incubation of the slice at a low [Na⁺]_o prior to the hypoxic insult. The attenuation of the response to hypoxia by high [Na⁺]_o was no longer observed in the presence of antagonist to glutamate transporter. However, antagonist to Na⁺–Ca²⁺ exchanger only slightly influenced the effects of high [Na⁺]_o. These observations suggest that disturbance of the transmembrane gradient of Na⁺ concentrations is an important factor in hypoxia-induced neuronal damage and corroborates the participation of the glutamate transporter in hypoxia-induced neuronal injury. In addition, the excess release of glutamate during hypoxia is due to a reversal of Na⁺-dependent glutamate transporter rather than an exocytotic process.     

Hypoxia induced by Na2S2O4 increases [Na]i in mouse glomus cells, an effect depressed by cobalt. Experiments with Na-selective microelectrodes and voltage-clamping

The intracellular sodium concentration ([Na⁺]_i) and resting potential (E_m) of cultured mouse glomus cells (clustered and isolated) were simultaneously measured with intracellular Na⁺-sensitive and conventional, KCl-filled, microelectrodes. Results obtained in clustered and isolated cells were similar. During normoxia (PO₂ 122 Torr), [Na⁺]_i was 12–13 mM corresponding to a Na⁺ equilibrium potential (E_Na) of about 58 mV. Em was about −42 mV. Hypoxia, induced by Na₂S₂O₄ 1 mM (PO₂ 10 Torr), depolarized the cells by about 20 mV, [Na⁺]_i increased by 21 mM and E_Na dropped to about 35 mV. One millimolar of CoCl₂ depressed, or blocked, the effects of Na₂S₂O₄ on [Na⁺]_i but did not affect hypoxic depolarization. Voltage-clamping at −70 mV, while delivering pulses of different amplitudes, produced only small (about 10 pA) and slow TTX-insensitive inward currents. Fast and large (TTX-sensitive) inward currents were not detected. The cell conductance (measured with voltage ramps) was less than 1 nS. It was not affected by hypoxia but was depressed by cobalt. Voltage ramps elicited small inward currents in control and hypoxic solutions that were much smaller than those induced by barium (presumably enhancing calcium currents). Also, normoxic and hypoxic currents had lower thresholds and their troughs were at more negative voltages than in the presence of Ba²⁺. All currents were blocked by 1 mM CoCl₂ suggesting that, at this concentration, cobalt exerted a nonspecific effect on glomus membrane channels. Hypoxia induced a large [Na⁺]_i increase (presumably through inflow), but very small voltage-gated inward currents. Thus, Na⁺ increases (inflow) probably occurred by disturbing a Na⁺/K⁺ exchange mechanism and not by activation of voltage-gated channels.     

Regulation of Ca-activated nonselective cationic currents in rat pituitary GH3 cells: involvement in L-type Ca current

Ionic currents were investigated by a patch clamp technique in a clonal strain of pituitary (GH₃) cells, using the whole cell configuration with Cs⁺ internal solution. Depolarizing pulses positive to 0 mV from a holding potential of −50 mV activated the voltage-dependent L-type Ca²⁺ current (I_Ca,L) and late outward current. Upon repolarization to the holding potential, a slowly decaying inward tail current was also observed. This inward tail current upon repolarization following a depolarizing pulse was found to be enhanced by Bay K 8644, but blocked by nifedipine or tetrandrine. This current was eliminated by Ba²⁺ replacement of external Ca²⁺ as the charge carrier through Ca²⁺ channels, removal of Ca²⁺ from the bath solution, or buffering intracellular Ca²⁺ with EGTA (10 mM). The reversal potential of inward tail current was approximately −25 mV. When intracellular Cl⁻ was changed, the reversal potential of the Ca²⁺-activated currents was not shifted. Thus, this current is elicited by depolarizing pulses that activate I_Ca,L and allow Ca²⁺ influx, and is referred to as Ca²⁺-activated nonselective cationic current (I_CAN). Without including EGTA in the patch pipette, the slowly decaying inward current underlying the long-lasting depolarizing potential after Ca²⁺ spike was also observed with a hybrid current–voltage protocol. Thus, the present studies clearly indicate that Ca²⁺-activated nonselective cationic channels are expressed in GH₃ cells, and can be elicited by the depolarizing stimuli that lead to the activation of I_Ca,L.     

Inositol 1,4,5-trisphosphate activates non-selective cation conductance via intracellular Ca2+ increase in isolated frog taste cells

The effect of intracellular Ca²⁺ increase was analysed in isolated frog taste cells under the whole-cell patch clamp. External application of a Ca²⁺-ionophore, ionomycin (3 μm ) induced the sustained inward current of ?200 ± 17 pA (mean ± SE, n = 23) at – 50 mV in taste cells. The ionomycin-induced response was observed in most of the cells exposed in the drug, but not when 10 mm BAPTA (1,2-bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) was included in the pipette (eight cells). Steady-state I–V relationships of ionomycin-induced currents were almost linear and reversed at – 8 ± 1 mV (n = 23). The simultaneous removal of Na⁺ and Ca²⁺ from the external solution eliminated the response completely (three cells). Intracellular dialysis with 1 mm Ca²⁺ or 50 μm inositol 1,4,5-trisphosphate (IP₃) in K⁺-internal solution also induced an inward current in the taste cells. The Ca²⁺-induced and IP₃-induced responses were observed in 82% and 36% of the cells dialysed with the drugs, respectively. The Ca²⁺-induced and IP₃-induced currents were inhibited by external Cd²⁺ (1–2 mm ). The reversal potentials of the inward currents were – 15 ± 3 mV (n = 9) in Ca²⁺ dialysis and – 11 ± 3 mV (n = 13) in IP₃ dialysis. The half-maximal Ca²⁺ concentration in the pipette to induce the inward current was ≈ 170 μm . The results suggest that IP₃ can depolarize the taste cell with mediation by intracellular Ca²⁺.     

Extracellular sodium removal increases release of neuropeptide Y-like immunoreactivity from rat brain hypothalamic synaptosomes: Involvement of intracellular acidification

Rat hypothalamic synaptosomes were exposed via superfusion to various stimuli and the release of neuropeptide Y-like immunoreactivity (NPY-LI) was mea sured by means of radioimmunoassay procedures. High KCl (15–50 mM) concentration dependently evoked NPY-LI release; the evoked overflow reached a plateau at 30 mM KCl and was abolished in the absence of Ca²⁺ ions. Furthermore, a remarkable NPY-LI overflow was obtained when extracellular Na⁺ ions were removed. Low external Na⁺-evoked NPY-LI release was independent of the presence of Ca²⁺ ions from the superfusion medium. It is well known that the reduction of external Na⁺ ions activates the release of several neurotransmitters through an inversion of the uptake-carrier working direction; but such mechanisms, involving Na⁺-dependent uptake, have never been described for neuropeptides. The alteration of the extracellular Na⁺ concentration is able to modify the concentration of the intracellular Ca²⁺ and H⁺ ions. In fact, the concentrations of these two ions are regulated through Na⁺-dependent exchange mechanisms across the membrane. Amiloride, blocking the Na⁺/H⁺ exchanger, was able to maintain low Na⁺-evoked NPY-LI release, underlying that the blockade of the exchanger preserves the H⁺ accumulation induced by the reduction of the external Na⁺ ions. NPY-LI release could also be stimulated by nigericine, a proton ionophore, showing that the intracellular acidification is responsible for NPY-LI release. Intracellular acidification may stimulate Ca²⁺ ion release from intracellular stores, as has been shown by other workers. Large dense-core vesicles containing the peptide appear to be more sensitive to local intracellular Ca²⁺ release compared with extracellular Ca²⁺ ion entry through voltage-dependent channels. Synapse 27:191–198, 1997. © 1997 Wiley-Liss, Inc.     

Na influx through Ca channels can promote striatal GABA efflux in Ca-deficient conditions in response to electrical field depolarization

Electrical field depolarization releases γ-aminobutyric acid (GABA) in rat striatal slices in the absence of external Ca²⁺. ω-Conotoxin GVIA (ω-CgTx; 1–50 nM), a neuronal Ca²⁺ channel blocker, inhibits electrically evoked efflux of newly taken up [³H]GABA in a concentration-dependent manner in either normal or Ca²⁺-free medium. This suggests that ion influx occurs through Ca²⁺ channels in the absence of external Ca²⁺ and contributes to the efflux of GABA. Reducing external Na⁺ concentration to 27.25 mM (low [Na⁺]₀ medium) by equimolarly substituting choline chloride for sodium chloride has differential effects on electrically evoked GABA efflux depending on the external Ca²⁺ concentrations. In normal Ca²⁺ medium, electrically evoked GABA efflux increases whereas, in Ca²⁺-free medium, it is greatly inhibited when [Na⁺]₀ is reduced to 27.25 mM. In low [Na⁺]₀ medium, GABA efflux is largely tetrodotoxin (TTX)-sensitive, however, spike firing evoked by antidromic stimulation of striatal cells is inhibited. In Na⁺-free medium, resting GABA efflux increases 17-fold whereas evoked GABA efflux diminishes. In Ca²⁺-free medium, 70 min of incubation with 1–2-bis-(2-aminophenoxy)ethane-N,N,N′,N′ tetraacetoxy methyl ester (BAPTA-AM, 1 μM), an intracellular calcium chelator, increases both resting GABA efflux and electrically evoked GABA overflow by 100%. These results suggest that: (1) in Ca²⁺-free conditions, Na⁺ permeability of cells increases via Ca²⁺ channels and this profoundly affects GABA efflux. (2) Electrical field depolarization is likely to release GABA by directly depolarizing axon terminals. (3) Ca²⁺-independent GABA efflux is not promoted by an increase in intracellular free Ca²⁺ concentration via Na⁺/Ca²⁺ exchange processes from internal pools.     

Carbachol Potentiates Q Current and Activates a Calcium-dependent Non-specific Conductance in Rat Hippocampus In Vitro

Intracellular recordings were made from CA1 neurons in rat hippocampal slices maintained in vitro. When Na⁺ currents were blocked with tetrodotoxin and K⁺ conductances known to be sensitive to suppression by muscarinic agonists were blocked by 2 mM Ba²⁺, CA1 cells were depolarized by carbachol (3 – 10 μM) with an attendant conductance increase, whereas prior to Ba²⁺ the agonist produced a decrease or no change in conductance. Under voltage clamp at ～–60 mV and in the presence of tetrodotoxin and Ba²⁺, carbachol (3 – 10 μM) induced a variable-latency biphasic inward current of up to 380 pA associated with a conductance increase of ～50%. The first phase was associated with an increase (more than 2-fold) of the Cs⁺-sensitive, hyperpolarization-activated cationic current, I_Q. Carbachol also accelerated the kinetics of I_Q at – 100 mV with an average 24% reduction in its activation time constant. The second phase reflected an additional inward current that was Cs⁺-resistant, displayed little apparent voltage sensitivity and had a mean extrapolated reversal potential, determined in the presence of external Cs⁺ (>5 mM), of ～–20 mV. In a small proportion of cells the second phase of inward current was followed (or overlapped) by an outward current, also associated with a conductance increase, which reversed at ～–70 mV. These carbachol actions were prevented by extracellular 300 μM Cd²⁺ and 2 mM Mn²⁺, by high levels (>5 mM) of extracellular Mg²⁺ or Ca²⁺, and by omission of Ca²⁺ or reduction of extracellular Na⁺ to 25 mM by substitution of NaCl with Tris or N-methyl-d -glucamine. Carbachol action was not mimicked by oxotremorine (≤60 μM), but was irreversibly blocked by this drug. Likewise, atropine (100 nM) irreversibly and gallamine (10 μM) reversibly antagonized carbachol's action. The action of carbachol was blocked shortly after prior exposure of slices to 2 – 5 mM caffeine. Chronic or acute incubation of slices with 2 mM Li⁺ potentiated (between 1- and 2-fold) carbachol responses. The data indicate that muscarinic activation increases cationic flux by a calcium-dependent potentiation of I_Q and activation of a non-selective conductance. The probability that inositol phospholipid metabolism is involved in triggering these events is discussed.     

Characterization of a Calcium-dependent Current Generating a Slow Afterdepolarization of CA3 Pyramidal Cells in Rat Hippocampal Slice Cultures

A depolarization-induced, slowly decaying inward current was examined in slice-cultured CA3 pyramidal cells by voltage-clamp techniques and microfluorometric measurements of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). Action potentials elicited by intracellular injection of short-lasting (50 – 100 ms) depolarizing current pulses were followed by a slowly decaying afterhyperpolarization (AHP). After switching to voltage-clamp mode, short-lasting (50 – 100 ms) depolarizing voltage jumps from –60 mV to between –30 and 0 mV induced a slowly decaying outward aftercurrent (I_AHP) which was depressed by bath application of muscarine (0.5 μM). In the presence of muscarine, the same depolarizations induced a slowly decaying afterdepolarization (ADP) or inward aftercurrent (I_ADP)in voltage-clamp mode. This current was also induced in the presence of trans(±)-1-aminc-1,3-cyclopenta-nedicarboxylic acid (t-ACPD, 5 μM), an agonist of metabotropic glutamate receptors, but not in the presence of noradrenalin (5 μM), while both of these agonists depressed I_AHP. I_ADP was depressed by reducing the external Ca²⁺ concentration from 3.8 to 0.5 mM, by external Co²⁺ (1 mM) and by external Cd²⁺ (10 – 100 μM). Combined voltage-clamp recordings and microfluorometric measurements of [Ca²⁺]_i using the Ca²⁺ indicator fura-2 revealed that the amplitude of I_ADP was correlated with the amplitude of depolarization-induced Ca²⁺ influx, I_ADP was absent at membrane potentials < –90 mV, and reached maximal amplitudes at ～–55 mV. Raising the extracellular K⁺ concentration from 2.7 to 13.5 mM increased the amplitude of I_ADP and resulted in a positively directed shift of the apparent reversal potential of I_ADP. When the external Na⁺ concentration was reduced from 157 to 33 or 18 mM the current reversed at more negative potentials and was reduced to 40 and 21%, respectively, of control amplitude. Lowering the external Cl^- concentration from 159 to 20 mM did not affect I_ADP. We conclude that I_ADP most likely represents a Ca²⁺-activated cation current, rather than a Ca²⁺ tail current, or an electrogenic Ca²⁺ extrusion current.