Characterization of vinpocetine effects on DA and DOPAC release in striatal isolated nerve endings

The effect of vinpocetine, a nootropic drug with anti-ischemic potential, on the release of DA and its main metabolite, DOPAC, was investigated in striatum isolated nerve endings under resting and depolarized conditions. Vinpocetine does not modify the baseline release of DA or the exocytotic release of DA evoked by high K(+), but inhibits the release of DA evoked by veratridine reversal of the DA transporter. In addition to these results, which confirm the vinpocetine selective blockade of voltage-sensitive presynaptic Na(+) channels (VSSC) previously reported [Neurochem. Res. 24 (1999) 1585], vinpocetine increases DOPAC release either under resting, veratridine or high K(+) depolarized conditions. This latter effect, which does not involve VSSC, was characterized. The parallel determination of the released and retained catecholamine concentrations revealed that vinpocetine increases DOPAC release at the expense of internal DA in a dose-dependent manner (low microM range). In contrast to vinpocetine, the selective MAO-A inhibitor, clorgyline, increases DA and decreases DOPAC formation. The combined action of vinpocetine and clorgyline does not indicate, however, that the activation of MAO is the mechanism responsible for the increase in DOPAC caused by vinpocetine. Reserpine, although more potent and efficient than vinpocetine, qualitatively exerts the same pattern of changes on DA and DOPAC concentrations. It is concluded that, in addition to the inhibition of presynaptic VSSC permeability, which selectively inhibits the transporter-mediated release of all neurotransmitters, vinpocetine increases DOPAC by impairing the vesicular storage of DA. Our results indicate that the cytoplasm extravesicular DA is metabolized by MAO to DOPAC. Most of the DOPAC formed is exported to the extracellular medium.     

Induction of astrocyte metallothioneins (MTs) by zinc confers resistance against the acute cytotoxic effects of methylmercury on cell swelling, Na uptake, and K release

Metallothionein (MT) proteins play an important role in the detoxification of heavy metals. Since methylmercury (MeHg) preferentially accumulates in astrocytes, we investigated the ability of the astrocyte-specific MT isoform, MT-I, to attenuate MeHg-induced cytotoxicity. Increased astrocytic MT expression was achieved by 24-h pretreatment of neonatal rat primary astrocyte cultures with 100 μM zinc (ZnSO₄). Subsequently, the astrocytes were treated with MeHg (10 μM), and its toxic effects on cell volume, Na⁺ uptake, and K⁺ release were investigated and compared to cells treated with or without MeHg, but in the absence of Zn pretreatment. Pretreatment of astrocytes with Zn was associated with a 2.9-fold increase in MT protein levels (P<0.02), and a 5.6-fold increase in MT mRNA levels (p<0.002) compared to control astrocytes. Astrocytes expressing increased MT protein levels were resistant to MeHg-induced swelling. In isotonic buffer the effect of MeHg on swelling was abolished (p<0.01) by 24-h Zn pretreatment, in such a way that volume profiles in these cells did not differ from controls. Zn-induced increased expression of MTs was also associated with significant attenuation of astrocytic Na⁺ uptake (p<0.01) and Rb⁺ (a marker for K⁺) release (p<0.001) in response to treatment with MeHg. These results demonstrate (1) that astrocytes can be induced to express high levels of MT proteins by pretreatment with Zn, and (2) that Zn confers resistance against the acute effect of MeHg on astrocytic swelling and the associated changes in ion (Na⁺ and K⁺) transport. Taken together, the data suggest that astrocytic MT induction offers effective cellular adaptation to MeHg cytotoxicity.     

Modulation of glial glutamate transport through cell interactions with the extracellular matrix

Glial glutamate transport plays a pivotal role in maintaining glutamate homeostasis in the central nervous system. Expression of glutamate transporters is highly regulated during brain development, and a number of pathological conditions are associated with deficits in expression and/or function of glutamate transports. While several soluble factors have been shown to regulate the expression of glutamate transporter, the contribution of cell-cell interaction and cell-environmental interaction in the regulation of glutamate transport is unknown. Extracellular matrix (ECM) molecules are essential components in cell-cell and cell-environmental interactions, and the ECM has been shown to play critical role in normal development and during brain pathogenesis. We, therefore, investigated the possibility that ECM molecules may regulate astrocytic glutamate transport. Therefore, we cultured rat cortical astrocytes with different ECMs and determined expression levels of the two astrocytic glutamate transporters GLT-1 and GLAST by Western Blot and determined transporter activity through measurements of 3H-D-aspartate uptake. Astrocytes grown on poly-ornithine or poly-D/L-lysine showed approximately two-fold higher GLT-1 expression than sister cells grown on plastic dishes without ECM. Naturally occurring ECM's, including laminin and collagen, showed a dose-dependent regulation of GLT-1 protein expression. These effects were specific for GLT-1 as GLAST expression was unaffected by different ECMs. Surprisingly, however, none of the examined ECMs altered the apparent glutamate uptake activity. In probing blots side-by-side for expression of Na(+)/K(+)-ATPase, we found that ECMs affected expression of Na(+)/K(+)-ATPase and GLT-1 in a reciprocal fashion. Poly-ornithine, for example, enhanced GLT-1 expression, but reduced expression of Na(+)/K(+)-ATPase. Na(+) transport may, thus, be a limiting factor for glutamate uptake.     

K efflux pathways and neurotransmitter release associated to hippocampal ischemia: effects of glucose and of K channel blockers

Ischemia of hippocampal slices leads to⁸⁶Rb⁺ efflux and to amino acid neurotransmitter release. This⁸⁶Rb⁺ efflux which corresponds to the massive K⁺ efflux from neuronal cells observed in ischemic animals is inhibited by glucose (IC₅₀ = 1.7mM). Glucose also inhibi the ischemia induced liberation of GABA and aspartate.⁸⁶Rb⁺ efflux is insensitive to any type of known blockers for ATP-sensitive, Ca²⁺-sensitive and voltage-sensitive K⁺ channels.     

Glial contribution to seizure: K activation of (Na, K)-ATPase in bulk isolated glial cells and synaptosomes of epileptogenic cortex

Glial and neuronal (Na⁺, K⁺)-ATPase have dissimilar apparent affinities for K⁺ ions. Glial (Na⁺, K⁺)-ATPase is maximally activated by 20 mM K⁺ while neuronal (Na⁺, K⁺)-ATPase is maximally stimulated by 3–5 mM K⁺. Because this glial property may play an important role in the clearance of [K⁺]₀ during seizures, we investigated the K⁺ activation of (Na⁺, K⁺)-ATPase within bulk isolated glial cells and synaptosomes isolated from epileptogenic brains. The primary focus (F), the homolateral brain area around the focus (PF) and the mirror (M) or secondary focus induced by freezing lesions were studied.Results show that both glial and synaptosomal enzyme activities in the epileptogenic state change in comparison with controls, i.e. sham-operated cats. In F and M., glial enzyme decreased reaction velocities between 3 and 18 mM K⁺. In PF, maximum velocities increased in glial (Na⁺, K⁺)-ATPase. These results indicate that in actively firing epileptogenic tissue (F, M), glial (Na⁺, K⁺)-ATPase decreased rate reactions while the catalytic activity was increased in cortical tissues surrounding the focus. These phenomena appeared early, i.e. 1–3 days after production of the freezing lesion, and was associated with a sharp decrease in absolute levels of enzyme activity.Synaptosomal (Na⁺, K⁺)-ATPase from controls always exhibited a saturation curve at 3–6 mM K⁺. Synaptosomal enzyme activities in the primary (F) lesion increased slightly 24 h after lesion production, then progressively decreased 3 days after lesion production. No significant changes were seen in M and PF.     

Role of opioidergic and monoaminergic neurotransmission in the GnRH release mechanism of EBP-primed OVX rats

We examined the effect of intracerebroventricular (i.c.v.) administration of μ-opioid agonist, morphine, and its antagonist naloxone followed by morphine on the activities of monoamine-metabolizing enzymes, namely tyrosine hydroxylase (TH) and monoamine oxidase (MAO) along with adenosinetriphosphatase (Na⁺, K⁺ -ATPase), the enzyme responsible for the maintenance of ionic gradients across the membrane, in seven discrete regions of brain from estrogen- and progesterone-primed ovariectomized rats. TH activity decreased after morphine treatment in some areas such as the median eminence-arcuate region (ME-ARC), the amygdala, and the thalamus, showing statistically significant change. MAO activity increased in all the areas studied, but more appreciable change was observed in medial preoptic area (mPOA), the ME-ARC region, and the cortex. Pronounced increase in Na⁺, K⁺ -ATPase enzyme activity was observed after the drug treatment. Naloxone given prior to morphine injection resulted in recovery of the enzyme activities in most of the areas studied. Our study may provide insights into the precise opioidergic modulation of gonadotropin releasing hormone (GnRH) release mechanisms through the involvement of monoaminergic system, elucidating the basis of various neuronal dysfunctions and their management in opioid addicts.     

Comparison of the distribution of Na, K-ATPase and myelin-associated glycoprotein (MAG) in the optic nerve, spinal cord and trigeminal ganglion of shiverer (shi/shi) and control (+/+) mice

Na+,K+ ATPase and myelin-associated glycoprotein (MAG) were studied by immunocytochemistry on paraffin sections of the spinal cord, optic nerve and trigeminal ganglion of adult control (+/+) and CNS myelin-deficient shiverer (shi/shi) mice. Immunostaining for Na+, K+-ATPase outlined the periphery of nerve fibers in the spinal cord white matter, optic nerve and trigeminal ganglion of +/+ and shi/shi mice. Immunostaining for Na+,K+-ATPase appeared somewhat denser in the optic nerve and spinal cord lateral funiculi of shi/shi than in +/+ mice. In addition, immunostaining for Na+,K+-ATPase was demonstrated at the plasmalemma of presumed satellite cells situated at the periphery of ganglion cell bodies in the trigeminal ganglion of both species of mice. Immunostaining for MAG was localized along the periphery of nerve fibers in the spinal cord funiculi (with little immunostaining within gray horns), optic nerve and trigeminal ganglion of both +/+ and shi/shi mice. The major differences between shi/shi and +/+ mice were that the number of MAG-immunostained nerve fibers was greatly reduced in the spinal cord funiculi and the density of immunostaining was slightly increased in the optic nerve of shi/shi mice. The numbers of MAG-immunostained nerve fibers in trigeminal ganglion were similar in both species. Also, the cytoplasm of some oligodendrocyte-like cells was found densely immunostained for MAG in the spinal cord and optic nerve of shi/shi mice, but not of +/+ mice. This light microscopic study provides evidence that the defective shiverer gene leads to a decrease in MAG deposition and to aggregations of MAG-like material within perikarya of oligodendrocyte-like cells in regions of the CNS.     

Effects of mammalian brain extracts and chlormadinone acetate on neuronal Na, K-ATPase and electrogenic Na, K-pump activity in vitro

Acid-acetone extracts of brain (from beef and guinea pig) and chlormadinone acetate (CMA) were compared with ouabain for their ability to inhibit the electrogenic Na⁺,K⁺-pump and the Na⁺,K⁺-ATPase of neuronal tissues. The membrane potential of neurones in the paravertebral sympathetic ganglion of the bullfrog was recorded in K⁺-free Ringer's solution by means of the sucrose gap technique. The potassium activated hyperpolarization (K_H⁺), induced by the re-introduction of potassium, was used as an index of electrogenic Na⁺,K⁺-pumping. The K_H⁺ was blocked by 1 μM ouabain. Na⁺,K⁺-ATPase activity was measured in microsomal membrane preparations of frog and beef brain using a continuous spectrophotometric assay. Although ouabain consistently inhibited beef brain Na⁺,K⁺-ATPase (IC₅₀ = 2.2 μM), acid-acetone extracts prepared from guinea pig and beef brain produced only partial inhibition. Neither of the extracts significantly reduced the K_H⁺ of the frog ganglion. CMA inhibited Na⁺,K⁺-ATPase prepared from bullfrog brain and spinal cord with slightly greater potency (IC₅₀ = 4.5 μM) than did ouabain (IC₅₀ = 10 μM). In contrast, electrogenic Na⁺,K⁺-pumping (i.e. the K_H⁺) in the frog ganglion was not affected by this steroid. It is concluded that although both the extracts and CMA inhibited Na⁺,K⁺-ATPase, neither can be considered ouabain-like due to their failure to affect the electrogenic Na⁺,K⁺-pump in situ.     

Effects of inhibitors of Na,K-ATPase on the membrane potentials and neurotransmitter efflux in rat brain slices

The potassium potential EK, of rat brain slices was estimated by determining the uptake of 86Rb+. The ERb was the same for slices prepared from five rostral brain regions, the average value being 66.4 mV. The ERb values in the presence of 20 microM ouabain were only slightly lower than the resting values; increasing concentrations of ouabain above 20 microM resulted in a graded depolarization in all five brain regions. High concentrations (1 mM) of two other inhibitors of Na+,K+-ATPase, dihydro-ouabain and strophanthidin, produced no more depolarization than did 20 microM ouabain. Competitive binding studies indicated that the differential effects were due to the relative binding to brain slices. Erythrosin B, an inhibitor of Na+,K+-ATPase, had no measurable effect on ERb. Intermediate concentrations of the Na+/H+ ionophore monensin slightly hyperpolarized striatal slices, whereas the same monensin concentrations plus 20 microM ouabain, 1 mM strophanthidin or 70 microM erythrosin B resulted in marked depolarization. Measurement of the membrane potential via uptake of methyltriphenylphosphonium cation indicated that ERb was indeed a valid estimation of the membrane potential. EK was measured directly by monitoring 42K+ uptake in striatal slices and was found to be essentially identical to ERb. Uptake of 22Na+ was consistent with the values for ERb or EK. Several conditions that resulted in little or no measurable depolarization of striatal slices did induce efflux of exogenously loaded GABA and dopamine; these conditions included 20 microM ouabain, 1 mM dihydro-ouabain or strophanthidin, and 70 microM erythrosin B. Neurotransmitter efflux in the absence of general cell depolarization was not accompanied by altered rates of respiration or decreased ATP levels.     

Inhibition of purified (Na,K)-ATPase activity from porcine cerebral cortex by NO generating drugs

We tested the effects of several nitric oxide (NO) generating compounds on the activity of sodium-potassium adenosine 5′-triphosphatase [(Na⁺,K⁺)-ATPase] purified from porcine cerebral cortex. Sodium nitroprusside (SNP),S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1) and (dl)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR 3) inhibited the (Na⁺,K⁺)-ATPase activity dose dependently. Superoxide dismutase, a NO scavenger, and sulfhydryl (SH) compounds, reduced-form glutathione (rGSH) and dithiothreitol (DTT), prevented the inhibitory action of SNAP, SIN-1 and NOR 3 but not of SNP, when applied simultaneously with NO generating compounds, and this enzyme inhibition could be reactivated by the incubation with these SH compounds but not with SOD. The inhibitory action by SNP was magnified by simultaneous application of DTT. These results suggest that NO generating compounds, SNAP, SIN-1 and NOR 3 but not SNP, may release NO or NO-derived products and may inhibit (Na⁺,K⁺)-ATPase activity by interacting with a SH group at the active site of the enzyme.     

Intrastriatal hypoxanthine administration affects Na,K-ATPase, acetylcholinesterase and catalase activities in striatum, hippocampus and cerebral cortex of rats

The aim of this study was to investigate the effects of a single intrastriatal injection of hypoxanthine, the major metabolite accumulating in Lesch-Nyhan disease, on Na(+),K(+)-ATPase, acetylcholinesterase and catalase activities in striatum, cerebral cortex and hippocampus of rats at different post-infusion periods. Adult Wistar rats were divided in two groups: (1) vehicle-injected group (control) and (2) hypoxanthine-injected group. For Na(+),K(+)-ATPase activity determination, the animals were sacrificed 3h, 24h and 7 days after drug infusion. For the evaluation of acetylcholinesterase and catalase activities, the animals were sacrificed 30min, 3h, 24h and 7 days after hypoxanthine infusion. Results show regional and time dependent effects of hypoxanthine on Na(+),K(+)-ATPase, acetylcholinesterase and catalase activities. The in vitro effect of hypoxanthine on the same enzymes in striatum was also investigated. Results showed that hypoxanthine inhibited Na(+),K(+)-ATPase, but not the activities of acetylcholinesterase and catalase in rat striatum. We suggest that these modification on cerebral biochemical parameters (Na(+),K(+)-ATPase, acetylcholinesterase and catalase activities) induced by intrastriatal administration of hypoxanthine in all cerebral structures studied, striatum, hippocampus and cerebral cortex, could be involved in the pathophysiology of Lesch-Nyhan disease.     

Influence of lactate accumulation on Na,K-ATPase activity of ischemic and post-ischemic brain

In this study the cerebral Na+, K+-ATPase activity as well as selected parameters of oxidative metabolism and electrophysiological function were assessed in normoglycemic and hyperglycemic rats which were exposed to ischemia produced by electrocautery of the vertebral arteries and reversible occlusion of the carotid arteries. In hyperglycemic animals 0.5 h of ischemia was associated with massive accumulation of lactate (34 mumol X g-1) and enhanced Na+, K+-ATPase activity (116% control), whereas normoglycemic animals showed more moderate lactate accumulation (17 mumol X g-1) and normal Na+, K+-ATPase activity (102% control). In normoglycemic animals release of the carotid clamps and recirculation for 0.5-1.5 h was associated with a normalization of the lactate levels and a decrease in Na+, K+-ATPase activity (68-72% control). Restituted hyperglycemic animals showed metabolic changes which seemed related to the blood pressure, with hypotensive hyperglycemic animals showing continuing massive lactacidosis (30-35 mumol X g-1) and enhanced Na+, K+-ATPase activity (108-110% control), whereas normotensive hyperglycemic animals showed progressive decreases in lactate level (14-20 mumol X g-1) and normal or mildly suppressed Na+, K+-ATPase activity (88-97% control). These patterns of change suggest that the reperfusion of the post-ischemic hyperglycemic-hyperlactacidotic brain was inadequate or non-homogeneous.     

Lead increases tetrodotoxin-insensitive spontaneous release of glutamate and GABA from hippocampal neurons

This study was aimed at investigating the effects of the environmental pollutant lead (Pb2+) on the tetrodotoxin (TTX)-insensitive release of neurotransmitters from hippocampal neurons. Evidence is provided that Pb2+ (>/=100 nM) increases the frequency of gamma-aminobutyric acid (GABA)- and glutamate-mediated miniature postsynaptic currents (MPSCs) recorded by means of the patch-clamp technique from cultured hippocampal neurons. Because Pb2+ changed neither the amplitude nor the decay-time constant of the MPSCs, Pb2+-induced changes in MPSC frequency are exclusively due to a presynaptic action of this heavy metal. Increase by Pb2+ of the action potential-independent release of GABA and glutamate was concentration dependent and was only partially reversible upon washing of the neurons with nominally Pb2+-free external solution. This effect was also Ca2+ independent and began approximately after 1-2-min exposure of the neurons to Pb2+. The latency for the onset of the Pb2+'s effect on the MPSC frequency and the inability of the chelator ethylenediaminetetraacetic acid (100 microM) to reverse the effect that remained after washing of the neurons with external solution suggested that Pb2+ acted via an intracellular mechanism. Of interest also was the finding that Pb2+ simultaneously increased the release of GABA and glutamate, overriding the ability of these neurotransmitters to decrease the release of one another. Given that synaptic activity is a key mechanism for the establishment of stable synaptic connections early in the development, it is possible that, by interfering with spontaneous transmitter release, Pb2+ has lasting effects on neuronal maturation and plasticity.     

The mechanism of the actions of oxaliplatin on ion currents and action potentials in differentiated NG108-15 neuronal cells

Oxaliplatin (OXAL) is a platinum-based chemotherapeutic agent which is effective against advanced or metastatic gastrointestinal cancer. However, the mechanisms responsible for the development of the neuropathy induced by this agent remain unclear. In this study, we attempted to evaluate the possible effects of OXAL on ion currents and action potentials (APs) in NG108-15 cells differentiated with dibutyryl cyclic-AMP. Application of OXAL decreased the peak amplitude of voltage-gated Na⁺ current (I_Na) with no change in the overall current–voltage relations of the currents. This agent also produced a concentration-dependent slowing of I_Na inactivation. A further application of ranolazine reversed OXAL-induced slowing of I_Na inactivation. Unlike ranolazine or riluzole, OXAL had no effect on persistent I_Na elicited by long ramp pulses. OXAL (100 μM) also had little or no effect on the peak amplitude of L-type Ca²⁺ currents in NG108-15 cells, while it suppressed delayed-rectifier K⁺ current. In current-clamp recordings, OXAL alone reduced the amplitude of APs; however, it did not alter the duration of APs. However, after application of tefluthrin, OXAL did increase the duration of APs. Moreover, OXAL decreased the peak amplitude of I_Na with a concomitant reduction of current inactivation in HEK293T cells expressing SCN5A. The effects of OXAL on ion currents presented here may contribute to its neurotoxic actions in vivo.     

Cyclic AMP enhances acetylcholine (ACh)- induced ion fluxes and catecholamine release by inhibiting Na+, K+-ATPase and participates in the responses to ACh in cultured bovine adrenal medullary chromaffin cells

Summary The effects of cyclic AMP (cAMP) on intracellular Na⁺ concentration ([Na⁺]i), membrane depolarization and intracellular Ca²⁺ concentration ([Ca²⁺]i) and the involvement of cAMP in acetylcholine (ACh)-induced such cellular events and catecholamine (CA) release were studied in cultured bovine adrenal medullary chromaffin cells. 8-Bromo-cyclic AMP (8Br-cAMP) and forskolin caused a rise in [Na⁺]i, membrane depolarization and a rise in [Ca²⁺]i and potentiated these responses and CA release to ACh. The effects of 8Br-cAMP or forskolin on ACh-induced changes of but not on basal level of [Na⁺]i, membrane potential and [Ca²⁺]i were blocked by tetrodotoxin (TTX, 1 M). In Na⁺ deprivated medium, forskolin failed to produce an increase in basal [Ca²⁺]i level and to potentiate ACh-induced rise. The similar results as in 8Br-cAMP and forskolin were obtained using ouabain, and 8Br-cAMP or foskolin produced no further effects in the presence of ouabain. Inhibitors of cAMP-dependent protein kinase not only blocked the effects of 8Br-cAMP and forskolin on membrane depolarization, [Ca²⁺]i rise and CA release, but also reduced these responses to ACh. From the similarity between the effects of cAMP and those of ouabain on the cellular events and the counteraction of the effects of cAMP by ouabain, it may be suggested that cAMP produces its effects on ion fluxes and CA release probably via an inhibition of Na⁺, K⁺-ATPase in intact chromaffin and cAMP may participate in the responses to ACh.     

Block of (Na,K)ATPase with ouabain induces spreading depression-like depolarization in hippocampal slices

We used ouabain (100 microM) to block Na+,K(+)ATPase of in vitro rat hippocampal slices. This treatment was sufficient to cause the sudden depolarization that is the hallmark of both spreading depression (SD) and of the SD-like anoxic depolarization (AD). This depolarization was accompanied by a large and sudden increase in [K](o), also reminiscent of that observed during both SD and AD. Ouabain-induced SD did not require a complete inactivation of Na+,K(+)ATPase, as it occurred when the enzyme was still capable of providing recovery of both V(o) and [K](o). The data indicate that functional inactivation of Na+,K(+)ATPase per se initiates events that lead to an SD-like AD. This ouabain-induced depolarization was not affected by block of synaptic transmission, instead it was abolished by hyperosmolarity of the extracellular space. The possible relevance of these findings to the pathophysiology of AD is discussed.     

Dependence of Na,K-ATPase and electrogenic component of Em in cultured myotubes on cell fusion

This study was undertaken in order to determine the relation among cell fusion, [3H]ouabain binding and the membrane potential (Em) of cultured rat skeletal muscle. The amount of ouabain bound and the Em both increased with age, the increases being most dramatic following fusion. Inhibition of fusion prevented the developmental increases in both properties of cultured muscle. After fusion, the size of the electrogenic component of Em, determined by the decrease in Em produced by ouabain within 5-10 min, increased independent of the age at which fusion occurred. It is concluded that the increase in Em with age depends on postfusion appearance and activity of Na,K-ATPase.     

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.     

Veratridine-induced release of acetylcholine from mouse forebrain minces: Dependence on the hydrolysis of cytoplasmic acetylcholine for a source of choline

The importance of depolarization induced hydrolysis of cytoplasmic acetylcholine (ACh) in providing choline for the veratridine- and high K⁺-induced release of acetylcholine was studied in mouse forebrain minces. Results indicated that a loss of hydrolyzable cytoplasmic ACh prior to depolarization reduced the amount of ACh released by veratridine but not the amount released by high K⁺. The reduction in the veratridine-induced release of ACh did not occur during the first 5 min of incubation. Loss of vesicular ACh prior to depolarization reduced both the veratridine- and K⁺-induced release of ACh during the first 5 min of incubation. Blockade of extra-cellular choline transport by hemicholinium (HC-3) did not affect the veratridine-induced release of ACh during a 10 min incubation period unless the cytoplasmic pool of ACh had first been depleted and was unavailable as a source of choline. In contrast, HC-3 reduced the K⁺-induced release of ACh from brain tissue with normal stores of cytoplasmic ACh. These results indicate that both depolarizing agents primarily stimulate the release of preformed ACh from a vesicular fraction during the first 5 min of mince incubation. Thereafter, they both stimulate the release of newly synthesized ACh, however, they differ in one important respect. The principal source of choline for the veratridine-induced release of newly synthesized ACh appears to be the cytoplasmic pool of ACh, whereas the major source of choline for the K⁺-induced release of newly synthesized ACh appears to be extracellular choline.