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

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

Identification in mammalian brain of an endogenous substance with Na channel blocking activities similar to those of tetrodotoxin

A substance with Na+ channel blocking activities has been isolated from pig brain after extraction and purification on sulfopropyl-Sephadex C-25, reversed-phase and carboxymethyl Synchropak high pressure liquid chromatography columns. The peptidic material i) displaces [3H]ethylenediamine tetrodotoxin ([3H]en-TTX) from its binding sites on rat brain membranes, (ii) it blocks 22Na+ influx induced by veratridine and sea anemone toxin on neuroblastoma and embryonic chick heart cells in culture, (iii) it specifically decreases the height of the action potential generated in frog sciatic nerve, and (iv) it blocks the fast Na+ current in voltage-clamped neuroblastoma cells. These properties are similar to those of tetrodotoxin while the endogenous factor is a peptide that is destroyed by proteases. These results suggest the presence in pig brain of a potent Na+ channel modulation activity.     

Down-regulation of norepinephrine transporter function induced by chronic administration of desipramine linking to the alteration of sensitivity of local-anesthetics-induced convulsions and the counteraction by co-administration with local anesthetics

Alterations of norepinephrine transporter (NET) function by chronic inhibition of NET in relation to sensitization to seizures induce by cocaine and local anesthetics were studied in mice. Daily administration of desipramine, an inhibitor of the NET, for 5 days decreased [(3)H]norepinephrine uptake in the P2 fractions of hippocampus but not cortex, striatum or amygdalae. Co-administration of lidocaine, bupivacaine or tricaine with desipramine reversed this effect. Daily treatment of cocaine increased [(3)H]norepinephrine uptake into the hippocampus. Daily administration of desipramine increased the incidence of appearance of lidocaine-induced convulsions and decreased that of cocaine-induced convulsions. Co-administration of lidocaine with desipramine reversed the changes of convulsive activity of lidocaine and cocaine induced by repeated administration of desipramine. These results suggest that down-regulation of hippocampal NET induced by chronic administration of desipramine may be relevant to desipramine-induced sensitization of lidocaine convulsions. Inhibition of Na(+) channels by local anesthetics may regulate desipramine-induced down-regulation of NET function. Repeated administration of cocaine induces up-regulation of hippocampal NET function. Desipramine-induced sensitization of lidocaine seizures may have a mechanism distinct from kindling resulting from repeated administration of cocaine.     

Voltage-dependent sodium channels in synaptoneurosomes: studies with 22Na+ influx and [3H]saxitoxin and [3H]batrachotoxinin-A 20-alpha-benzoate binding. Effects of proparacaine isothiocyanate.

22Na+ influx and binding of [3H]saxitoxin ([3H]STX) and [3H]batrachotoxin-A 20-alpha-benzoate ([3H]BTX-B) were studied in guinea pig cerebral synaptoneurosomes. STX and tetrodotoxin (TTX) completely blocked the stimulation of sodium influx induced by 1 microM BTX. The IC50 values for STX and TTX closely matched the Ki values for inhibition of [3H]STX binding, suggesting that the sites labelled by [3H]STX are associated with a population of BTX-sensitive channels. BTX induced a dose-dependent stimulation of sodium influx in synaptoneurosomes (EC50 280 nM). The potency of BTX for stimulation of sodium influx was increased (EC50 24 nM) in the presence of 0.6 microgram/ml scorpion venom without any change in maximal influx. In contrast, specific binding of [3H]BTX-B to synaptoneurosomes was minimal in the absence of scorpion venom, but it was increased several fold in the presence of 60 micrograms/ml scorpion venom. With proparacaine isothiocyanate (PROPRIT), an irreversible local anesthetic, the inhibition of [3H]BTX-B binding by PROPRIT did not occur in parallel with an inhibition of sodium influx induced by BTX. Preincubation of synaptoneurosomes with 10 microM PROPRIT for 10 min resulted in approximately 70% inhibition of [3H]BTX-B binding in the presence of scorpion venom. Such preincubation did not alter BTX-induced sodium uptake in synaptoneurosomes. Preincubations of synaptoneurosomes with 100 microM PROPRIT for 10 min completely inhibited [3H]BTX-B binding, and under these conditions BTX-induced sodium influx was reduced only by 50%. The results indicate that virtual elimination of binding sites labeled by [3H]BTX-B in the presence of scorpion venom by PROPRIT has little effect on sodium influx induced by BTX.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Slight impairment of Na+,K+-ATPase synergistically aggravates ceramide- and beta-amyloid-induced apoptosis in cortical neurons

Dysfunction of the Na(+),K(+)-ATPase (Na(+),K(+)-pump), due to reduced energy supply or increased endogenous ouabain-like inhibitors, likely occurs under pathological conditions in the central nervous system. In cultured mouse cortical neurons, we examined the hypothesis that a mild non-toxic inhibition of the Na(+),K(+)-ATPase could synergistically sensitize the vulnerability of neurons to normally non-lethal apoptotic signals. Ouabain at a low concentration of 0.1 microM slightly lessened the Na(+),K(+)-pump activity measured as an ouabain-sensitive current, yet did not affect K(+) homeostasis and viability of cortical neurons. Co-exposure to 0.1 microM ouabain plus non-lethal C(2)-ceramide (5 microM) or beta-amyloid 1-42 (5 microM), however, induced marked intracellular K(+) loss, caspase-3 cleavage, DNA laddering, and synergistically triggered neuronal death. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) predominantly blocked the caspase activation and neuronal death. These results suggest that slight impairment of Na(+),K(+)-pump activity may amplify the disruption of K(+) homeostasis in the presence of a non-lethal apoptotic insult, leading to activation of apoptotic cascade and substantial neuronal injury.     

Action of diazepam on the voltage-dependent Na current. Comparison with the effects of phenytoin, carbamazepine, lidocaine and flumazenil

The influence of diazepam, an agonist, and flumazenil (Ro 15-1788), an antagonist of the benzodiazepine receptor, on repetitive firing of action potentials in cultured spinal neurons and on voltage-dependent Na+ currents in cultured N2A neuroblastoma cells was examined. The effects were compared to those of the antiepileptics phenytoin and carbamazepine and the local anesthetic lidocaine. The whole-cell configuration of the patch-clamp technique was used for potential and current recording. Diazepam (10 microM) or phenytoin (10 microM) reduced the duration of repetitive action potential discharges in 50 or 67% of the spinal neurons, respectively. At a concentration of 100 microM repetitive firing was completely blocked. Flumazenil (100 microM) had no effect. In N2A neuroblastoma cells diazepam, phenytoin, carbamazepine and lidocaine, but not flumazenil, at a concentration of 100 microM reduced the Na+ current to 60-67% of control. At 10 microM no or only a weak depression was seen with any drug. In the presence of diazepam (100 microM) the Na+ channel inactivation curve was shifted in the hyperpolarizing direction by -4.8 +/- 0.5 mV. Phenytoin, carbamazepine and lidocaine (all 100 microM) caused stronger shifts of -17.4 +/- 2.1, -10.6 +/- 0.9 and -17.0 +/- 2.1 mV, respectively. Inhibition of the Na+ current by diazepam increased use-dependently over 9 depolarizing pulses repeated at high frequency (200 Hz), whereas use-dependent effects of the other compounds developed less rapidly. At a low stimulation rate (7 Hz) use-dependent block was pronounced with lidocaine, but weak or absent with diazepam and carbamazepine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Na, K-ATPase activity in rat striatum by the metabolites accumulated in Lesch–Nyhan disease

In the present study, we investigated the in vitro effect of hypoxanthine, xanthine and uric acid, metabolites accumulating in tissue of patients with Lesch-Nyhan disease, on Na(+), K(+)-ATPase activity in striatum of neonate rats. Results showed that all compounds significantly inhibited Na(+), K(+)-ATPase activity. We also studied the kinetics of the inhibition of Na(+), K(+)-ATPase activity caused by hypoxanthine. The apparent K(m) and V(max) of Na(+), K(+)-ATPase activity for ATP as the substrate and hypoxanthine as the inhibitor were 0.97 mM and 0.69 nmol inorganic phosphate (Pi) released per min per mg of protein, respectively. K(i)-value was 1.9 microM, and the inhibition was of the non-competitive type. We also observed that the inhibitory effects of hypoxanthine, xanthine and uric acid probably occur through the same mechanism, suggesting a common binding site for these oxypurines on Na(+), K(+)-ATPase. Therefore, it is conceivable that inhibition of brain Na(+), K(+)-ATPase activity may be involved at least in part in the neuronal dysfunction characteristic of patients with Lesch-Nyhan disease.     

Expression and kinetic properties of Na(+) currents in rat cardiac dorsal root ganglion neurons

The expression and properties of voltage-gated Na(+) currents in cardiac dorsal root ganglion (DRG) neurons were assessed in this study. Cardiac DRG neurons were labelled by injecting the Fast Blue fluorescent tracer into the pericardium. Recordings were performed from 138 cells. Voltage-dependent Na(+) currents were found in 115 neurons. There were 109 neurons in which both tetrodotoxin-sensitive (TTX-S, blocked by 1 microM of TTX) and tetrodotoxin-resistant (TTX-R, insensitive to 1 microM of TTX) Na(+) currents were present. Five cells expressed TTX-R current only and one cell only the TTX-S current. The kinetic properties of Na(+) currents and action potential waveform parameters were measured in neurons with cell membrane capacitance ranging from 15 to 75 pF. The densities of TTX-R (110.0 pA/pF) and TTX-S (126.1 pA/pF) currents were not significantly different. Current threshold was significantly higher for TTX-R (-34 mV) than for TTX-S (-40.4 mV) currents. V(1/2) of activation for TTX-S current (-19.6 mV) was significantly more negative than for TTX-R current (-9.2 mV), but k factors did not differ significantly. V(1/2) and the k constant for inactivation for TTX-S currents were -35.6 and -5.7 mV, respectively. These values were significantly lower than those recorded for TTX-R current for which V(1/2) and k were -62.3 and -7.7 mV, respectively. The action potential threshold was lower, the 10-90% rise time and potential width were shorter before than after the application of TTX. Based on this we drew the conclusion that action potential recorded before adding tetrodotoxin was mainly TTX-S current dependent, while the action potential recorded after the application of toxin was TTX-R current dependent. We also found 23 cells with mean membrane capacitance ranging from 12 to 35 pF (the smallest labelled DRG cells found in this study) that did not express the Na(+) current. The function of these cells is unclear. We conclude that the overwhelming majority of cardiac dorsal root ganglion neurons in which voltage-dependent Na(+) currents were present, exhibited both TTX-S and TTX-R Na(+) currents with remarkably similar expression and kinetic properties.     

Intermittent hypoxia modulates Na channel expression in developing mouse brain

Because our previous work showed that intermittent hypoxia alters neuronal excitability and Na+ current density, we examined in this work the effect of intermittent hypoxia on Na+ channel subtypes using 3H-saxitoxin (3H-STX) autoradiography and immunoblotting. Mice were exposed to intermittent hypoxia for 2 or 4 weeks from postnatal day 2 or 3. A 2-week intermittent hypoxia reduced cerebral STX binding density with significant decrease in Na(v)1.2 in the rostral and Na(v)1.1 in the caudal regions. In contrast, a 4-week intermittent hypoxia tended to increase STX binding density in most brain regions. Our data suggest that intermittent hypoxia differentially regulates plasma membrane Na+ channels in the developing brain, depending on duration of intermittent hypoxia.     

4-aminopyridine, a specific blocker of K(+) channels, inhibited inward Na(+) current in rat cerebellar granule cells

The effects of 4-aminopyridine (4-AP), a specific blocker of outward K(+) current, on voltage-activated transient outward K(+) current (I(K(A))) and inward Na(+) current (I(Na)) were investigated on cultured rat cerebellar granule cells using the whole cell voltage-clamp technique. At the concentration of 1-5 mM, 4-AP inhibited both I(K(A)) and I(Na). It reduced the amplitude of peak Na(+) current without significant alteration of the steady-state activation and inactivation properties. The inhibitory effect was not enhanced by repeated depolarizing pulses (0.5 or 0.1 Hz), suggesting that the binding affinity of 4-AP on Na(+) channels is state-independent. In contrast, the effect of 4-AP on Na(+) channels appeared to be voltage-dependent, the weaker inhibition occurred at more depolarization. Moreover, 4-AP slowed both the activation and inactivation kinetics of Na(+) current. These effects were similar to those induced by alpha-scorpion toxin and sea anemone toxins on Na(+) channels in other cell model. Our data demonstrate for the first time that 4-AP is able to block not only A-type K(+) channels, but also Na(+) channels in rat cerebellar granule cells. It is concluded that the inhibition exerted by 4-AP on Na(+) current likely differs from that provoked by local anesthetics. The possibility that the binding site of neurotoxin receptor 3 may be involved is discussed.     

Neurochemical correlates of selective neuronal loss following cerebral ischemia: role of decreased Na, K-ATPase activity

In order to investigate the role of Na+,K(+)-ATPase in the development of neuronal necrosis following cerebral ischemia, ischemia was induced in gerbils by occluding the common carotid artery unilaterally for 10 min. A time-course analysis revealed that significant reductions of the Na+,K(+)-ATPase activity in the cerebral cortex and hippocampus were manifested at 15 min, 30 min, and 1 h, and returned to the control level one day following recirculation. No apparent alterations of the Mg(2+)-ATPase activity, on the other hand, were obtained throughout the experimental period. Furthermore, Scatchard analyses of [3H]ouabain binding to the cerebral cortex membranes disclosed that the Bmax values invariably decreased without any change of Kd values following ischemia. It has also been shown that treatment of the animals with an agent known to mitigate ischemic neuronal necrosis, i.e. BY-1949, significantly reversed such derangements. These results suggest that the recovery of decreased Na+,K(+)-ATPase activity shortly after ischemia exerts a protective effect against ischemic brain damage.     

Evidence for the presence of ‘Ouabain like’ compound in human cerebrospinal fluid

Material extracted and partially purified from human cerebrospinal fluid (CSF) is capable of: a, inhibiting [3H]ouabain binding to rat brain synaptosomes; b, inhibiting the activity of purified pig kidney Na+,K+-ATPase; and c, inhibiting ouabain sensitive induced 86Rb influx to tissue cultured fibroblasts. These results demonstrate the existence of an 'ouabain like' compound (OLC) in human CSF, and are consistent with the hypothesis of the function of this compound as a neuromodulator.     

SM-20220, a Na(+)/H(+) exchanger inhibitor: effects on ischemic brain damage through edema and neutrophil accumulation in a rat middle cerebral artery occlusion model

The Na(+)/H(+) exchanger (NHE) is activated during ischemia-reperfusion in an effort to restore intracellular pH to normal levels. The NHE is recognized to exist as a distinct protein in the plasma membranes of a variety of cells. We investigated the pharmacological effects of a Na(+)/H(+) exchanger inhibitor, SM-20220 (N-(aminoiminomethyl)-1-methyl-1-H-indole-2-carboxamide methanesulfonate), on ischemic brain damage, edema and neutrophil accumulation at 72 h after middle cerebral artery (MCA) occlusion in a rat MCA occlusion model. SM-20220 was intravenously administered as a bolus injection immediately after occlusion, followed by a continuous infusion over 2.5 h. At 72 h after occlusion, the infract area was measured using hematoxylin-eosin staining and, using the same slices, neutrophils in the brain were immuno-stained with anti-myeloperoxidase (n=11). In a separate study, rat behavior was scored and scaled, and brains removed for the determination of water content by the dry-weight method. SM-20220 significantly (P<0.05) attenuated cerebral infarct volume, water content, and the neutrophil accumulation at 72 h after the MCA occlusion, and ameliorated neurological deficits. SM-20220, an NHE inhibitor prevented the progress of cerebral ischemic damage and edema following MCA occlusion in rats though a possible mechanism that may be due to the inhibition of neutrophil accumulation. The NHE in neutrophils may enhance the progress of cerebral damage following cerebral ischemia-reperfusion.     

Sodium overload through voltage-dependent Na(+) channels induces necrosis and apoptosis of rat superior cervical ganglion cells in vitro

Using the failure to exclude trypan blue as a criterion for cell death, we found that veratridine, the voltage-dependent Na(+) channel activator, exerted its toxicity to cultured sympathetic neurons in a dose-dependent manner (half-maximal toxicity occurred at 2 microM). The co-presence of tetrodotoxin completely reversed the toxicity only at concentrations of veratridine < 20 microM. Veratridine neurotoxicity was due to the influx of Na(+); a medium low in Na(+) (36 mM) completely abolished its neurotoxicity, whereas a Ca(2+)-free medium did not attenuate its neurotoxicity. Furthermore, the buffering action of 1, 2-Bis-(2-aminophenoxy)ethane-N,N,N',N',-tetraacetate (BAPTA) on veratridine-induced increase in intracellular Ca(2+) levels neither blocked veratridine neurotoxicity in normal medium, nor attenuated the low Na(+) effect. Elevated K(+) effectively blocked veratridine neurotoxicity in a Ca(2+)-dependent manner. Cytoplasmic pH measurements using a fluorescent pH indicator demonstrated that cellular acidification (from pH 7.0 to pH 6.5) occurred upon treatment with veratridine. Both veratridine-induced acidification and cell death were ameliorated by 5-(N-ethyl-N-isopropyl)amiloride, the specific inhibitor of the Na(+)/H(+) exchanger (IC(50) = 0.5 microM). Finally, necrosis occurred predominantly in veratridine neurotoxicity, but both staining with bis-benzimide and TUNEL analysis showed nuclear features of apoptosis in sympathetic neurons undergoing cell death.     

Effect of cocaine and cocaine congeners on veratridine-induced depolarization in mouse cerebrocortical synaptoneurosomes

Structure-activity relationships were determined for cocaine congeners in counteracting the depolarization induced by the action of veratridine on voltage-dependent sodium channels of synaptoneurosomes from mouse brain cortex, and their potency was compared to those determined previously on Na+ uptake and batrachotoxinin binding. Cocaine, norcocaine, (+)-pseudococaine, (-)-pseudococaine, (+)-neopseudococaine, benzoyltropine, benzoylpseudotropine, ecgonine methylester, atropine, WIN-35,428, WIN-35,140, WIN-35,065-3, WIN-35,004, and procaine were tested for their potency in inhibiting depolarization as measured by the distribution of the lypophilic cation [3H]triphenylmethylphosphonium across the membrane. All of the tested compounds inhibited the veratridine-induced depolarization in a competitive manner. The structure-activity relationships were similar to those for inhibition of 22Na+ uptake in mouse brain homogenates, and the potency of these local anesthetics in inhibiting veratridine-induced uptake of [3H]triphenylmethylphosphonium correlated well with their potency in inhibiting [3H]batrachotoxinin A 20-alpha-benzoate binding in mouse brain synaptosomes.     

Alteration of intracellular pH and activity of CA3-pyramidal cells in guinea pig hippocampal slices by inhibition of transmembrane acid extrusion

Transmembrane acid extruders, such as electroneutral operating Na(+)/H(+)-exchangers (NHE) and Na(+)-dependent Cl(-)/HCO(3)(-)-exchangers (NCHE) are essential for the maintenance and regulation of cell volume and intracellular pH (pH(i)). Both of them are hypothesised to be closely linked to the control of excitability. To get further information about the relation of neuronal pH(i) and activity of cortical neurones we investigated the effect of NHE- and/or NCHE-inhibition on (i) spontaneous action potentials and epileptiform burst-activity (induced by bicuculline-methiodide, caffeine or 4-aminopyridine) and (ii) on pH(i) of CA3-neurones. NHE-inhibition by amiloride (0.25-0.5 mM) or its more potent derivative dimethylamiloride (50 microM) and NCHE-inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 0.25-0.5 mM) induced a biphasic alteration of neuronal activity: an initial, up to 30 min lasting, increase in frequency of action potentials and bursts preceded a growing and partially reversible suppression of neuronal activity. In BCECF-loaded neurones the pH(i), however, continuously decreased during either amiloride- or DIDS-treatment and reached its steady-state (DeltapH(i) up to 0.3 pH-units) when the neuronal activity was markedly suppressed. Combined treatment with amiloride (0.5 mM) and DIDS (0.5 mM) or treatment with harmaline alone (0.25-0.5 mM), which also continuously acidified neurones via inhibition of an amiloride-insensitive NHE-subtype, induced a monophasic and partially reversible suppression of neuronal activity. As an initial excitatory period failed to occur during combined NHE/NCHE-inhibition we speculate that its occurrence during amiloride- or DIDS-treatment resulted rather from disturbances in volume- than in pH(i)-regulation. The powerful inhibitory and anticonvulsive properties of NHE- and NCHE-inhibitors, however, very likely based upon intracellular acidification - as derived from our previous findings that a moderate increase in intracellular free protons is sufficient to reduce membrane excitability of CA3-neurones.     

Plasma from myasthenia gravis patients reduces acetylcholine receptor agonist-induced Na flux into TE671 cell line

Plasma from myasthenia gravis patients was tested for its ability to inhibit agonist-induced 22Na+ influx into the TE671 cell line that expresses human acetylcholine receptors. Reduced 22Na+ influx correlated weakly with the total anti-acetylcholine receptor antibody level in the plasma, and was also related to the presence of antibody directed against the agonist binding site, as detected by inhibition of 125I-alpha-bungarotoxin binding. However, in some cases there was inhibition of 22Na+ flux without evident anti-alpha-bungarotoxin binding site antibody. We conclude that in most patients antibodies that interfere with 22Na+ influx do so by blocking the agonist binding site. However, in some cases antibodies may be directed at the Na+ ion channel or some important functional determinant.     

Development of saxitoxin-sensitive and insensitive sodium channels in cultured neonatal rat astrocytes

Voltage-sensitive Na channels were studied in cultures of neonatal rat cortical astrocytes. These channels were present at all times in culture as determined by tracer 22Na+ influx in the presence of batrachotoxin (BTX) and sea anemone polypeptide toxin (AxTx). The affinity of saxitoxin (STX) binding and sensitivity to STX inhibition of sodium influx were utilized to characterize these channels. Up to 7 d in culture, high-affinity 3H-STX binding (Kd of 0.2 nM at 4 degrees C) was very low, and 22Na+ influx was inhibited only by high concentrations (Ki = 170 nM) of STX. From 7 to 14 d, total specific binding of STX increased to a maximum of over 2 pmol/mg protein and remained constant for 28 d. By 14 d, inhibition of 22Na+ influx by STX was clearly biphasic, indicating the presence of 2 populations of channels with Ki's of 0.2 nM and 150 nM. At 14 d in culture, binding of 3H-STX to astrocyte membranes revealed the presence of 2 specific sites. During this second week, increasing numbers of high-affinity STX binding sites and increasing sensitivity to the inhibition of BTX + AxTx-stimulated 22Na+ influx by STX coincided with the change in morphology of primitive flat polygonal cells to highly branched stellate forms characteristic of mature astrocytes in vivo. Changes in culture conditions modified the time course of the onset of high STX affinity binding. Twenty-four hours after changing to serum-free G5 medium, there was both an 8-fold increase in STX binding sites and a change to a stellate shape in all cells. The results suggest that although low-affinity STX Na channels are always present in astrocytes, after 7 d in culture a different population of channels appears with the high affinity for STX characteristic of adult neuronal sodium channels. This spontaneous process is greatly accelerated by changing to a chemically defined medium.     

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.