Effects of hypoxia induced by Na2S2O4 on intracellular calcium and resting potential of mouse glomus cells

Isolated and cultured glomus cells, obtained from mouse carotid bodies, were superfused with Ham's F-12 equilibrated with air (mean PO₂, 119 Torr; altitude 1350 m). [Ca²⁺]_o was 3.0 mM. In one experimental series, dual cell penetrations with microelectrodes measured intracellular calcium ([Ca²⁺]_i) and the resting potential (E_m). In another series, [Ca²⁺]_i was measured with Indo-1/AM, dissolved in DMSO. Normoxic cells had a mean E_m of −42.4 mV and [Ca²⁺]_i was about 80 nM (measured with both methods). The calculated calcium equilibrium potential (E_Ca) was 137±0.74 mV. Hypoxia, induced by Na₂S₂O₄ 1 mM, reduced pO₂ to 10–14 Torr. This effect was accompanied by cell depolarization to −19.1 mV. Hypoxia increased [Ca²⁺]_i to 231 nM when detected with Ca-sensitive microelectrodes, but only to 130.2 nM when measured with Indo-1/AM. Calcium increases were preceded by decreases in [Ca²⁺]_i, which also were more pronounced with microelectrode measurements. CoCl₂ 1 mM blocked the hypoxic [Ca²⁺]_i increase and exaggerated the decreases in [Ca²⁺]_i. Correlations between ΔE_m and Δ[Ca²⁺]_i during hypoxia were significant (p<0.05) in 19% of the cells. But, in 29% of them significance was at the p<0.1 level. In the rest (52%), there was no correlation between these parameters. Thus, voltage-gated calcium channels are rare in mouse glomus cells. Their activation by depolarization cannot explain the two to threefold increase in [Ca²⁺]_i seen during hypoxia. More likely, [Ca²⁺]_i increase may be due to hypoxic inactivation of a Ca–Mg ATPase transport system across the cell membrane. The blunting of hypoxic [Ca²⁺]_i increase, seen in Indo-1/AM experiments, is probably due to its solvent (DMSO), which also depresses hypoxic cell depolarization.     

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.     

Suppression of glomus cell K conductance by 4-aminopyridine is not related to [Ca]i, dopamine release and chemosensory discharge from carotid body

The hypothesis that suppression of O₂-sensitive K⁺ current is the initial event in hypoxic chemotransduction in the carotid body glomus cells was tested by using 4-aminopyridine (4-AP), a known suppressant of K⁺ current, on intracellular [Ca²⁺]_i, dopamine secretion and chemosensory discharge in cat carotid body (CB). In vitro experiments were performed with superfused–perfused cat CBs, measuring chemosensory discharge, monitoring dopamine release by microsensors without and with 4-AP (0.2, 1.0 and 2.0 mM in CO₂-HCO₃^- buffer) and recording [Ca²⁺]_i by ratio fluorometry in isolated cat and rat glomus cells. 4-AP decreased the chemosensory activities in normoxia but remained the same in hypoxia and in flow interruption. It decreased the tissue dopamine release in normoxia, and showed an additional inhibition with hypoxia. Also, 4-AP did not evoke any rise in [Ca²⁺]_i in glomus cells either during normoxia and hypoxia, although hypoxia stimulated it. Thus, the lack of stimulatory effect on chemosensory discharge, inhibition of dopamine release and unaltered [Ca²⁺]_i by 4-AP are not consistent with the implied meaning of the suppressant effect on K⁺ current of glomus cells.     

Na+—Ca2+ Exchange Currents in Cortical Neurons: Concomitant Forward and Reverse Operation and Effect of Glutamate

Na⁺-Ca²⁺ exchanger-associated membrane currents were studied in cultured murine neocortical neurons, using whole-cell recording combined with intracellular perfusion. A net inward current specifically associated with forward (Na⁺_o-Ca²⁺_i) exchange was evoked at -40 mV by switching external 140 mM Li⁺ to 140 mM Na⁺. The voltage dependence of this current was consistent with that predicted for 3Na⁺:1Ca²⁺ exchange. As expected, the current depended on internal Ca²⁺, and could be blocked by intracellular application of the exchanger inhibitory peptide, XIP. Raising internal Na⁺ from 3 to 20 mM or switching the external solution from 140 mM Li⁺ to 30 mM Na⁺ activated outward currents, consistent with reverse (Na⁺,-Ca²⁺_o) exchange. An external Ca²⁺-sensitive current was also identified as associated with reverse Na⁺-Ca²⁺ exchange based on its internal Na⁺ dependence and sensitivity to XIP. Combined application of external Na⁺ and Ca²⁺ in the absence of internal Na⁺ triggered a 3.3–fold larger inward current than the current activated in the presence of 3 mM internal Na⁺, raising the intriguing possibility that Na⁺-Ca²⁺ exchangers might concurrently operate in both the forward and the reverse direction, perhaps in different subcellular locations. With this idea in mind, we examined the effect of excitotoxic glutamate receptor activation on exchanger operation. After 3–5 min of exposure to 100–200 μM glutamate, the forward exchanger current was significantly increased even when external Na⁺ was reduced to 100 mM, and the external Ca²⁺-activated reverse exchanger current was eliminated.     

K-current modulated by PO2 in type I cells in rat carotid body is not a chemosensor

According to the membrane channel hypothesis of carotid body O₂ chemoreception, hypoxia suppresses K⁺ currents leading to cell depolarization, [Ca²⁺]_i rise, neurosecretion, increased neural discharge from the carotid body. We show here that tetraethylammonium (TEA) plus 4-aminopyridine (4-AP) which suppressed the Ca²⁺ sensitive and other K⁺ currents in rat carotid body type I cells, with and without low [Ca²⁺]_o plus high [Mg²⁺]_o, did not essentially influence low

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effects on [Ca²⁺]_i and chemosensory discharge. Thus, hypoxia may suppress the K⁺ currents in glomus cells but K⁺ current suppression of itself does not lead to chemosensory excitation. Therefore, the hypothesis that K⁺–O₂ current is linked to events in chemoreception is not substantiated. K⁺–O₂ current is an epiphemenon which is not directly linked with O₂ chemoreception.     

Hyperpolarization induces a rise in intracellular sodium concentration in dopamine cells of the substantia nigra pars compacta

We investigated the effect of changes in membrane-voltage on intracellular sodium concentration ([Na⁺]_i) of dopamine-sensitive neurons of the substantia nigra pars compacta in a slice preparation of rat mesencephalon. Whole-cell patch-clamp techniques were combined with microfluorometric measurements of [Na⁺]_i using the Na⁺-sensitive probe, sodium-binding benzofuran isophthalate (SBFI). Hyperpolarization of spontaneously active dopamine neurons (recorded in current-clamp mode) caused the cessation of action potential firing accompanied by an elevation in [Na⁺]_i. In dopamine neurons voltage-clamped at a holding potential of ?60 mV elevations of [Na⁺]_i were induced by long-lasting (45–60 s) voltage jumps to more negative membrane potentials (–90 to ?120 mV) but not by corresponding voltage jumps to ?30 mV. These hyperpolarization-induced elevations of [Na⁺]_i were depressed during inhibition of I_h, a hyperpolarization-activated inward current, by Cs⁺. Hyperpolarization-induced elevations in [Na⁺]_i might occur also in other cell types which express a powerful I_h and might signal lack of postsynaptic activity.     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.     

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.     

Prostaglandin E2 excites neurons of the nucleus tractus solitarius by activating cation channels

Nucleus tractus solitarius (NTS) has a high density of prostaglandin E₂ (PGE₂)-binding sites. Action of PGE₂ (10⁻⁹–10⁻⁶ M) was tested on neurons in a NTS slice with patch-clamp recording under synaptic blockade. PGE₂ raised the firing rate in approximately half of the neurons in cell-attached patch mode. In whole-cell current clamp, PGE₂ depolarized membrane potential accompanied by an increase in firing rate. In whole-cell voltage clamp (−58 mV), PGE₂ induced the inward current with an increase in conductance. The current was linearly related to voltage from −100 mV to −10 mV and suppressed between −10 mV and 20 mV. The current-voltage curve remained similar under low external Cl⁻ or high internal Cl⁻ conditions and after external Na⁺ was replaced by Cs⁺. It is concluded that PGE₂ excites NTS neurons by activating cation conductance.     

AMPA/kainate receptor activation blocks K+ currents via internal Na+ increase in mouse cultured stellate astrocytes

Cultured mouse cortical astrocytes of the stellate type were studied by using the patch-clamp technique in whole-cell configuration. The astrocytes express at least two types of outwardly rectifying K⁺ channels which mediate a transient and a sustained current. Activation of AMPA receptors by kainate leads to a substantial blockade of both types of K⁺ currents. The blockade is absent when Na⁺ is withdrawn from the external medium, suggesting that it is caused by constant Na⁺ influx through AMPA receptors. The presence of high Na⁺ solutions in the pipette induces a blockade of both K⁺ currents which is very similar to the blockade induced by kainate, supporting thus the view that the mechanism of the blockade of K⁺ channels by kainate involves Na⁺ increases in the submembrane area. The blockade occurs between 20 and 40 mM [Na⁺]_i, which is within the physiological range of [Na⁺]_i in astrocytes. The data may suggest that the blockade of K⁺ channels by high [Na⁺]_i conditions could provide a mechanism to prevent K⁺ leakage from the astrocytes into the extracellular space during periods of intense neuronal activity. GLIA 20:38-50, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Voltage-dependent Ca influx into identified leech neurones

We determined the relationships between the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and the membrane potential (E_m) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca²⁺]_i was monitored by using iontophoretically injected fura-2. The membrane depolarization evoked by raising the extracellular K⁺ concentration ([K⁺]_o) up to 89 mM caused a persistent increase in [Ca²⁺]_i, which was abolished in Ca²⁺-free solution indicating that it was due to Ca²⁺ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca²⁺]_i increase ranged between −40 and −25 mV. The different threshold potentials as well as differences in the relationships between [Ca²⁺]_i and E_m were partly due to the cell-specific generation of action potentials. In Na⁺-free solution, the action potentials were suppressed and the [Ca²⁺]_i/E_m relationships were similar. The K⁺-induced [Ca²⁺]_i increase was inhibited by the polyvalent cations Co²⁺, Ni²⁺, Mn²⁺, Cd²⁺, and La³⁺, as well as by the cyclic alcohol menthol. Neither the polyvalent cations nor menthol had a significant effect on the K⁺-induced membrane depolarization. Our results suggest that different leech neurones possess voltage-dependent Ca²⁺ channels with similar properties.     

Intracellular potassium activity, potassium equilibrium potential and membrane potential of carotid body glomus cells

The intracellular potassium activity (a_i(K)) of glomus cells in isolated rabbit carotid bodies was measured with potassium ion selective microelectrodes (K⁺ electrodes). The measurements yielded a mean a_i(K) value of 33.0 mM in Hepes-buffered Tyrode's solution equilibrated with 100% O₂. This value is significantly higher than that predicted by assuming that K⁺ ions are passively distributed across the membrane of glomus cells. The relationship between the membrane potential (E_M) and the equilibrium potential of K⁺ ions (E_K) at various extracellular potassium activities (a_o(K)) suggests that K⁺ ions are not involved in the maintenance of the carotid body glomus cell E_M.     

Gap junctions equalize intracellular Na+ concentration in astrocytes

Gap junctions between glial cells allow intercellular exchange of ions and small molecules. We have investigated the influence of gap junction coupling on regulation of intracellular Na⁺ concentration ([Na⁺]_i) in cultured rat hippocampal astrocytes, using fluorescence ratio imaging with the Na⁺ indicator dye SBFI (sodium-binding benzofuran isophthalate). The [Na⁺]_i in neighboring astrocytes was very similar (12.0 ± 3.3 mM) and did not fluctuate under resting conditions. During uncoupling of gap junctions with octanol (0.5 mM), baseline [Na⁺]_i was unaltered in 24%, increased in 54%, and decreased in 22% of cells. Qualitatively similar results were obtained with two other uncoupling agents, heptanol and α-glycyrrhetinic acid (AGA). Octanol did not alter the recovery from intracellular Na⁺ load induced by removal of extracellular K⁺, indicating that octanol's effects on baseline [Na⁺]_i were not due to inhibition of Na⁺, K⁺-ATPase activity. Under control conditions, increasing [K⁺]_o from 3 to 8 mM caused similar decreases in [Na⁺]_i in groups of astrocytes, presumably by stimulating Na⁺, K⁺-ATPase. During octanol application, [K⁺]_o-induced [Na⁺]_i decreases were amplified in cells with increased baseline [Na⁺]_i, and reduced in cells with decreased baseline [Na⁺]_i. This suggests that baseline [Na⁺]_i in astrocytes “sets” the responsiveness of Na⁺, K⁺-ATPase to increases in [K⁺]_o. Our results indicate that individual hippocampal astrocytes in culture rapidly develop different levels of baseline [Na⁺]_i when they are isolated from one another by uncoupling agents. In astrocytes, therefore, an apparent function of coupling is the intercellular exchange of Na⁺ ions to equalize baseline [Na⁺]_i, which serves to coordinate physiological responses that depend on the intracellular concentration of this ion. GLIA 20:299–307, 1997. © 1997 Wiley-Liss, Inc.     

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.     

A pharmacological model for studying the role of Na gradients in the modulation of synaptosomal free [Ca]i levels and energy metabolism

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 ouabain-induced [Ca]i increase in leech Retzius neurones is mediated by voltage-dependent Ca channels

In leech Retzius neurones the inhibition of the Na⁺–K⁺ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca²⁺]_i). To elucidate the mechanism of this increase we investigated the changes in [Ca²⁺]_i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na⁺–K⁺ pump in bathing solutions of different ionic composition. The results show that Na⁺–K⁺ pump inhibition induced a [Ca²⁺]_i increase only if the cells depolarized sufficiently in the presence of extracellular Ca²⁺. Specifically, the relationship between [Ca²⁺]_i and the membrane potential upon Na⁺–K⁺ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca²⁺ channels by raising the extracellular K⁺ concentration. It is concluded that the [Ca²⁺]_i increase caused by inhibiting the Na⁺–K⁺ pump in leech Retzius neurones is exclusively due to Ca²⁺ influx through voltage-dependent Ca²⁺ channels.     

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⁺.     

Effect of the removal of extracellular Ca on the response of cytosolic concentrations of Ca to ouabain in carotid body glomus cells of adult rabbits

Effect of the removal of extracellular Ca²⁺ on the response of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to ouabain, an Na⁺/K⁺ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca²⁺]_i (mean±S.E.M.; 38±5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca²⁺]_i increase was abolished by the removal of extracellular Na⁺. D600 (50 μM), an L-type voltage-gated Ca²⁺ channel antagonist, inhibited the [Ca²⁺]_i increase by 57±7% (n=4). Removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i increase, but subsequent washing out of ouabain in Ca²⁺-free solution produced a rise in [Ca²⁺]_i (62±8% increase, n=6, P<0.05), referred to as a [Ca²⁺]_i rise after Ca²⁺-free/ouabain. The magnitude of the [Ca²⁺]_i rise was larger than that of ouabain-induced [Ca²⁺]_i increase. D600 (5 μM) inhibited the [Ca²⁺]_i rise after Ca²⁺-free/ouabain by 83±10% (n=4). These results suggest that ouabain-induced [Ca²⁺]_i increase was due to Ca²⁺ entry involving L-type Ca²⁺ channels which could be activated by cytosolic Na⁺ accumulation. Ca²⁺ removal might modify the [Ca²⁺]_i response, resulting in the occurrence of a rise in [Ca²⁺]_i after Ca²⁺-free/ouabain which mostly involved L-type Ca²⁺ 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.