Brain cell membrane Na,K-ATPase activity following severe hypoxic injury in the newborn piglet

This study tests the hypothesis that severe brain hypoxia causes decreased Na⁺,K⁺-ATPase activity, resulting in permanent alterations in the neuronal cell membranes. Seventeen anesthetized piglets (normoxic control (NC), no recovery after hypoxia (Group 1), 6 h normoxic recovery (Group 2), and 48 h normoxic recovery (Group 3) were studied. Hypoxia was induced by lowering the FiO₂ to maintain PCr/P_i ratio at 25% of baseline for 1 h as monitored by ³¹P-NMR spectroscopy. PCr/P_i returned to 57% of baseline by 6 h and was normal by 48 h. At termination, cortical tissue Na⁺,K⁺-ATPase activity was determined. Na⁺,K⁺-ATPase activity was measured in cortical membrane preparations by determining the rate of ATP hydrolysis. NC membranes had Na⁺,K⁺-ATPase activity of 58.3 ± 1.3 μM P_i/mg protein/h (mean ± S.E.M.). Na⁺,K⁺-ATPase activity was reduced in Groups 1, 2, and 3 (45.8 ± 1.3, 47.4 ± 3.6, 48.7 ± 2.9 μM P_i/mg protein/h) (P < 0.05 compared to NC). There was no differene in enzyme activity among Groups 1, 2, or 3. The data show that in spite of recovery of neuronal oxiditive phosphorylation (PCr/P_i) by 48 h, there is a permanent decrease in Na⁺,K⁺-ATPase activity in cells that have undergone severe hypoxic injury. The persistent decrease in Na⁺,K⁺-ATPase activity indicates ongoing cell injury following severe cerebral hypoxia, and that recovery of oxidative phosphorylation as indicated by PCr/P_i values cannot be used as an index of recovery of cell function.     

The effect of acute hypoglycemia on the cerebral NMDA receptor in newborn piglets

The effects of acute insulin-induced hypoglycemia on the cerebral NMDA receptor in the newborn were examined by determining [³H]MK-801 binding as an index of NMDA receptor function in 6 control and 7 hypoglycemic piglets. In hypoglycemic animals, the glucose clamp technique with constant insulin infusion was used to maintain a blood glucose concentration of 1.2 mmol/l for 120 min before obtaining cerebral cortex for further analysis; controls received a saline infusion. Concentrations of glucose, lactate, ATP, and PCr were measured in cortex, and Na⁺,K⁺-ATPase activity was determined in a brain cell membrane preparation. [³H]MK-801 binding was evaluated by: (1) saturation binding assays over the range of 0.5–50 nM [³H]MK-801 in the presence of 100 μM glutamate and glycine; and (2) binding assays at 10 nM [³H]MK-801 in the presence of glutamate and/or glycine at 0, 10, or 100 μM. Blood and brain glucose concentrations were significantly lower in hypoglycemic animals than controls. There was no change in brain ATP with hypoglycemia, but PCr was decreased 80% compared to control (P < 0.05). Na⁺,K⁺-ATPase activity was 13% lower in hypoglycemic animals (P < 0.05). Based on saturation binding data, hypoglycemia had no effect on the number of functional receptors (B_max), but the apparent affinity was significantly increased, as indicated by a decrease in the K_d (dissociation constant) from the control value of 8.1 ± 1.6 nM to 5.5 ± 2.1 nM (P < 0.05). Augmentation of [³H]MK-801 binding by glutamate and glycine alone or in combination was also significantly greater in the hypoglycemic animals. These data suggest that acute hypoglycemia may enhance the excitotoxic effects of glutamate in the newborn.     

5-HT inhibits lateral entorhinal cortical neurons of the rat in vitro by activation of potassium channel-coupled 5-HT1A receptors

Serotonin (1–40 μM) reduced input resistance by 20.6±6% and hyperpolarized stellate and pyramidal neurons of layers two and three of the lateral entorhinal cortex. 5-Carboxamidotryptamine, a 5-HT₁ agonist, and the selective 5-HT_1A agonist 8-hydroxy-dipropylaminotetralin mimicked the action of serotonin. The reversal potential of 5-HT-mediated hyperpolarizations was sensitive to the extracellular K⁺ concentration, indicating a potassium conductance change. Serotonin treatment suppressed excitatory amino acid-mediated synaptic potentials (by 48%, K_d=6.9 μM) and responses to exogenously applied glutamate (70.1±17% of control, n=7), but did not alter paired-pulse facilitation, indicating a postsynaptic site of action. Intracellular application of QX-314, a blocker of potassium conductance, significantly reduced depression of synaptic potentials by 5-HT agonists. In cells filled with QX-314, responses to exogenously applied glutamate were not reduced by serotonin or 5-carboxamidotryptamine application. These results indicate that the observed conductance increase associated with 5-HT application accounts for most if not all of the observed depressant effects of 5-HT_1A agonists on excitatory amino acid-mediated neurotransmission.     

Blood-brain barrier transport of choline is reduced in the aged rat

An age-related impairment in choline transport across the blood-brain barrier (BBB) may contribute to the cholinergic mechanisms of geriatric memory dysfunction. To test this hypothesis, the brain choline uptake in male Fisher 344 rats at 2, 18 and 24 months of age was studied using the Oldendorf technique. The V_max of cholie transport in the 24-month-old rats (0.05 ± 0.04 nmol/min/g) was significantly lower than that in the 2-month-old rat (2.5 ± 1.0 nmol/min/g) (P < 0.05). The K_m of transport in old rats (13 ± 35 μM) was also significantly smaller than the value in 24-month-old rats (450 ± 195 μM), while the constant of the non-saturable component of the transport, K_d, was not significantly different in older rats (1.2 ± 0.1 μl/min/g). These results indicate that the carrier in old rats has reduced capacity and increased affinity to choline. The reduced choline carrier capacity explains the significant decrease in BBB choline transport in aged rats.     

Sumatriptan modifies cortical free radical release during cortical spreading depression. A novel antimigraine action for sumatriptan?

Increases in concentration of brain NO are proposed to initiate and mediate migraine headache. Triggered by focal depolarisation, spreading depression (SD) represents a suitable mechanism for eliciting widespread release of nitric oxide. The current study examines the effect of sumatriptan, a 5-HT_1B/1D agonist and effective antimigraine therapy, on free radical release (nitric oxide and superoxide) in SD in the simple and complex cortices of the rat and cat. Following initiation of SD, sumatriptan pretreatment (300 μg kg⁻¹ i.v., 15 min prior to SD) modulated all phases of nitric oxide release associated with each SD in both cats and rats. As a result, superoxide levels were observed to significantly (ANOVA, post hoc LSD) increase versus vehicle treated animals (saline 1 ml kg⁻¹ i.v. 15 min prior to SD) during specific phases of each SD depolarisation. Averaged over all SD depolarisations, mean peak SD nitric oxide levels per depolarisation were 0.73±0.23 μM (n=29) in cats, and 0.42±0.09 μM (n=34) in rats. Sumatriptan significantly (Students t-test, P<0.05, two tailed hypothesis, P<0.05) modulated this increase in cortical nitric oxide concentrations to 0.32±0.06 μM (n=25) and 0.22±0.07 μM (n=37) in cats and rats. Sumatriptan appears to decrease the amplitude of nitric oxide release but enhances extracellular superoxide concentrations in both lissencephalic and gyrencephalic cortices during SD.     

Electrophysiological characterization of sodium channel types in the HCN-1A human cortical cell line

Electrically evoked sodium currents were recorded under whole-cell patch clamp from undifferentiated HCN-1A cells. Peak sodium currents had a half-maximal activation, V_m0.5, of −22.6 ± 1.0 mV with a voltage dependence, K_m, of 7.28 ± 0.39 mV⁻¹. Steady-state inactivation indicated the presence of two types of sodium channel. One type inactivated with V_h0.5 = −93.8 ± 1.2 mV and k_h = −6.8 ± 0.4 mV⁻¹. The second type of sodium channel inactivated w V_h0.5 = −44.6 ± 1.5 mV and k_h = −7.3 ± 0.4 mV⁻¹. The occurrence of each channel type varied from cell to cell and ranged from 0 to 100% of the total sodium current. No variation in the rate of inactivation was seen when the holding potential was adjusted to eliminate the more negative of the two inactivation components. Application of tetrodotoxin (TTX) or saxitoxin (STX) revealed channel types with two different affinities for each toxin. TTX blocked peak sodium conductance with apparent IC50s of 22 nM and 5.3 μM. STX was more potent, with apparent IC₅₀s of 1.6 nM and 1.2 μM. There was no statistical correlation between toxin sensitivity and steady-state inactivation voltage, suggesting that these properties varied independently among sodium channel types.     

Differences in uptake kinetics of cis-3-aminocyclohexane carboxylic acid into neurons and astrocytes in primary cultures

The uptake of [³H]ACHC and [³H]GABA into cultured neurons and astrocytes was studied. [³H]ACHC uptake was less efficient than that of GABA in both cell types and K_m values for ACHC uptake into neurons and astrocytes were 40.3 μM and 210.8 μM, respectively. The corresponding V_max values were 0.321 and 0.405 nmol·min⁻¹·mg⁻¹ cell protein, respectively. Kinetic studies of the effects of GABA on ACHC uptake and vice versa showed that GABA is a linear competitive inhibitor of ACHC uptake in both cell types with a K_i value of 15 μM. On the other hand, ACHC turned out to be a complex inhibitor of astrocytic GABA uptake being competitive at lower concentrations and non-competitive at higher concentrations. ACHC inhibited GABA uptake into neurons competitively with a K_i of 69 μM. It is concluded that ACHC acts primarily on neuronal GABA uptake sites but its uptake is much more complicated than hitherto anticipated.     

The kinetic and structural characterization of the reaction of nafamostat with bovine pancreatic trypsin

Nafamostat mesilate (FUT-175), a synthetic serine protease inhibitor, is active against a number of the serine proteases involved in coagulation. This has been proposed as the basis of its anticoagulant activity. We investigated the reaction of Nafamostat with bovine pancreatic trypsin as a model system. It was shown to act as a time-dependent competitive inhibitor, and the inhibition constants for the binding of Nafamostat to trypsin (i.e., K_i) and the overall inhibition constants (i.e., K_i^*) were calculated to be 11.5 μM and 0.4±0.14 nM, respectively. The second-order rate constant for the reaction was 4.5±0.19×10⁵ M⁻¹s⁻¹, and the product released following the acylation step, 6-amidino-2-naphthol, showed mixed-type inhibition. The competitive (K_ic) and uncompetitive (K_iu) inhibition constants were 14.7 μM and 19.5 μM, respectively. Formation of the acyl-enzyme intermediate was dissected into at least two steps, with rates of 0.9 s⁻¹ and 195 s⁻¹. The deacylation step was relatively much slower (3.2±0.19×10⁻⁵ s⁻¹), enabling the mass spectroscopic analysis of the acyl-enzyme intermediate, which confirmed the covalent attachment of 4-guanidinobenzoic acid to trypsin. The product of the deacylation step, 4-guanidinobenzoic acid, showed no inhibition up to a concentration of 200 μM. These data strongly suggest that while Nafamostat is a potent inhibitor of trypsin, it is actually an extremely poor substrate, and that apparent inhibition is due to the competitive formation of a very stable acyl-enzyme intermediate, analogous to some other active site titrants.     

Response to cyanide of two types of glomoid cells in mature rat carotid body

Cells belonging to glomoids of mature rat carotid bodies were studied using the whole-cell patch clamp technique following acute dissociation. The recorded population encompassed two subtypes: one type (n=202), termed G(out), was characterized by a small voltage-dependent inward current (43±9pA, mean ±S.E.M.), large outward current (671±31 pA@⁺40 mV), high membrane resistance (1910 ± 110M Ω) and low capacitance (5.1 ± 0.1pF). A second subtype (n=56), termed G(in), had significantly lower membrane resistance (177 ± 35 MΩ), membrane capacitance (15.0 ± 1.0 pF) and little voltage-dependent current. Neither subtype supported generation of multiple action potentials during depolarization in the current clamp mode. Intracellular staining of the recorded cells by Lucifer yellow showed co-localization of both subtypes to clusters of cells which stained positively for catecholamines. Somal diameter was slightly, but significantly, larger for G(in) cells 8.7 ± 0.4 μM, n=7) compared to G(out) cells (7.8±0.2 μM, n=31) and all cells had fine cytoplasmic process s extending around neighboring cells. During recordings using the perforated patch technique, histotoxic hypoxia significantly decreased a voltage-dependent outward current in G(out) cells by 113±60pA (n=13), and decreased the holding current by 10±4pA (n=13) from a control value of −32±6pA. In G(in) cells, cyanide significant decreased membrane resistance and decreased holding current by 55±28pA from a control value of +120±42pA (n=7), but caused no significant change in outward current. These results show that glomoids of mature rat carotid bodies contain at least two types of cells which differ in their morphologic and electrophysiologic characteristics. The subtypes rapidly respond to histotoxic hypoxia and thus may mediate separate roles in the organ response to chemostimuli.     

Halothane's effects on GABA-gated chloride flux in mice selectively bred for sensitivity or resistance to diazepam

The DS (diazepam-sensitive) and DR (diazepam-resistant) lines of mice, selected on the basis of their ataxic response to diazepam, also diverge in the physiologic response of their brain γ-aminobutyric acid_A (GABA_A) receptors to benzodiazepines, as indicated by augmentation of GABA-mediated chloride flux. Cross-sensitivity and -resistance to other sedatives known to interact with the GABA_A-receptor have also been demonstrated in DS and DR mice. Based on the finding that these mice also show cross-sensitivity and -resistance to obtundation by halothane, we predicted that their GABA_A-receptors would also exhibit a differential response to halothane as assayed by an in vitro³⁶Cl⁻ influx assay using purified brain microvesicles. Consistent with this prediction, therapeutic concentrations of halothane enhanced 1 μmol/1 GABA-gated flux with significantly greater potency in DS than in DR mice (halothane EC₅₀ 336±64 μmol/1 (S.E.M.) vs. 605±110 μmol/1, respectively, P = 0.03), but there was no difference in maximal flux enhancement between the two lines (DS 4.7±0.4 nmol·mg⁻¹·3⁻¹, vs. DR 4.7±0.5nmol·mg⁻¹·3s⁻). Halothane (500 μmol/1) also shifted the entire GABA concentration-flux relationship significantly to the left, decreasing the EC₅₀ for GABA in both the DS and DR lines. Importantly, the shift in the GABA concentration-flux response in the presence of halothane was more pronounced in the DS mice (GABA EC₅₀ 1.8±0.4 μmol/1vs.14.7±0.9 μmol/1 without halothane) than in the DR mice (GABA EC₅₀ 4.7±0.6 μmol/1vs.14.7±0.9 μmol/1 without halothane). This effect of halothane was highly significant, both when compared to control, and between the selected lines (P < 0.001). The findings that halothane enhances GABA-gated flux and enhances GABA's channel gating potency support the hypothesis that differential enhancement of agonist-stimulated chloride permeability at GABA_A receptors could be a mechanism underlying the differential obtunding potency of halothane in DS and DR mice. However, at high GABA concentrations halothane decreased maximal chloride flux, more in DS than in DR mice (P < 0.001), which is not consistent with such a mechanism.     

Selective sensitivity of synaptosomal membrane function to cerebral cortical hypoxia in newborn piglets

The effect of hypoxia on the structure and function of the synaptosomal membranes and myelin fraction (glial cells, neuronal cell bodies and axonal membranes) was investigated by measuring Na⁺,K⁺-ATPase activity and levels of lipid peroxidation products in cerebral cortical synaptosomal membranes and myelin fractions obtained from newborn piglets. Hypoxic hypoxia was induced and cerebral hypoxia was documented as a decrease in the ratio of phosphocreatine to inorganic phosphate (PCr/Oi) using³¹P-NMR spectroscopy. PCr/Pi decreased from baseline of2.93 ± 0.76to0.61 ± 0.36 during hypoxia. The synaptosomal membrane Na⁺,K⁺-ATPase activity decreased from a control value of56.6 ± 3.7to40.4 ± 6.0 μgmol Pi/mg protein/h during hypoxia. The level of conjugated dienes increased from zero (reference value) to4.5 ± 2.7 nmol/mg lipid and the level of fluorescent compounds increased from23.5 ± 2.2to92.6 ± 46.4 ng quinine sulfate/mg lipid in the synaptosomal membranes during hypoxia. No change in myelin fraction Na⁺,K⁺-ATPase activity or levels of lipid peroxidation products were noted. These data indicate that sunaptosomal membranes, rich in polyunsaturated fatty acids, are more susceptible to oxygen free radical mediated lipid peroxidative damage during hypoxia.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [³H]cyclohexyladenosine (CHA) to the cellular fractions and P₂ subfractions of the goldfish brain was studied. The A₁ receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P₂), 30 mM K⁺ stimulated glutamate, taurine and GABA release in a Ca²⁺-dependent fashion, whereas the aspartate release was Ca²⁺-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 μM) inhibited K⁺-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A₁ receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K⁺ (30 mM) stimulated Ca²⁺ influx (46.8±6.8%) and this increase was completely abolished by pretreatment with 100 nM ω-conotoxin. Pretreatment with 100 μM R-PIA or 100 μM CHA, reduced the evoked increase of intra-synaptosomal Ca²⁺ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A₁ receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

Characterization of electrogenic Na/K pump in rat neostriatal neurons

The electrogenic Na/K pump current (Ip) was studied in the dissociated neostriatal neurons of the rat by using the nystatin-perforated patch recording mode. The Ip was activated by external K⁺ in a concentration-dependent manner with an EC₅₀ of 0.7 mM at a holding potential (V_H) of −40 mV. Other monovalent cations also caused Ip and the order of potency was Tl⁺>K⁺, Rb⁺>NH₄⁺, Cs⁺>>>Li⁺. The Ip decreased with membrane hyperpolarization in an external solution containing 150 mM Na⁺, while the Ip did not show such voltage dependency without external Na⁺. Ouabain showed a steady-state inhibition of Ip in a concentration- and temperature-dependent manner at a V_H of −40 mV. The IC₅₀ values at 20 and 30°C were 7.1×10⁻⁶ and 1.3×10⁻⁶ M, respectively. The decay of Ip after adding ouabain well fitted with a single exponential function. At a V_H of −40 Mv, the association (k₊₁) and dissociation (k₋₁) rate constants estimated from the time constant of the current decay at 20°C were 4.0×10² s⁻¹ M⁻¹ and 6.3×10⁻³ s⁻¹, respectively. At 30°C, k₊₁ increased to 2.8×10³ s⁻¹ M⁻¹ while k₋₁ showed no such change with a value of 1.8×10⁻³ s⁻¹. A continuous Na⁺ influx was demonstrated by both the Na⁺-dependent leakage current and tetrodotoxin-sensitive Na⁺ current, which resulted in the continuous activation of the Na/K pump. It was thus concluded that the Na/K pump activity was well-maintained in the dissociated rat neostriatal neurons with distinct functional properties and that the activity of the pump was tightly connected with Na⁺ influxes.     

Quantitative microdialysis of neuropeptide Y

The feasibility of using the difference method of quantitative microdialysis to measure neuropeptide Y (NPY) was evaluated in vitro and in vivo. The accuracy of this method was tested in vitro under steady-state conditions for 3 test solutions containing known concentrations of NPY. The estimated concentrations of NPY were 1.2 ± 0.6, 3.7 ± 0.9, and 15.1 ± 0.7 pg/μl (mean ± SEM) in agreement with the actual concentrations of NPY in the test solutions which were 1.1 ± 0.8, 4.6 ± 0.6, and 14.6 ± 0.5 pg/μl (mean ± SEM of solution samples), respectively. The responsiveness of the estimated NPY_ext measure to changes in the external concentration of NPY was also evaluated in vitro. An accurate estimate of NPY_ext was obtained within the first sampling period (within 15 min) after a 2–3-fold increase in the test solution concentration of NPY and within 2–3 sampling periods (15–45 min) in response to a 2–3-fold decrease in the test solution concentration of NPY. In vivo, the estimated basal concentration of NPY in dialysis samples from probes in the medial basal hypothalamus of anesthetized female rats (n = 4) was 4.0 ± 1.6 pg/μl and increased to 9.5 ± 0.3 pg/μl during K⁺ stimulation. Relative recovery was 22% in vivo under steady-state conditions and ranged from 14% to 30% during dynamic conditions. These results demonstrate that the difference method of quantitative microdialysis accurately estimates picomolar concentrations of NPY in vitro, and is sufficiently sensitive to detect basal and increasing concentrations of NPY in vivo.     

Impairment of endothelium-dependent dilatation of the basilar artery during diabetes mellitus

The goal of this study was to determine whether responses of the basilar artery are altered during diabetes mellitus. We measured the diameter of the basilar artery in vivo in non-diabetic and diabetic rats (streptozotocin; 50–60 mg/kg i.p.). Responses of the basilar artery to agonists, which presumably produce dilatation by releasing endothelium-derived relaxing factor (EDRF), were impaired in diabetic rats compared to non-diabetic rats. Acetylcholine (1.0 and 10 μM) dilated the basilar artery by 13 ± 2 and 26 ± 4% (means±S.E.M.), respectively, in non-diabetic rats, but by only 13 ± 1 and 9 ± 2%, respectively, in diabetic rats (P < 0.05). Bradykinin (1.0 and 10 μM) dilated the basilar artery by14 ± 2 and 35 ± 6 % (means±S.E.M.), respectively, in non-diabetic rats, but by only5 ± 1 and 6± 2%, respectively, in diabetic rats (P < 0.05). The response to nitroglycerin was similar in non-diabetic and diabetic rats. Thus, impairment of vasodilatation in diabetic rats in response to acetylcholine and bradykinin is not related to non-specific impaired of vasodilation than impaired dilator responses of the basilar artery in response to acetylcholine and bradykinin in diabetic rats may be related to the activation of the throm☐ane A₂-prostaglandin H₂ receptor. SQ 29548 (a specific throm☐ane A₂-prostaglandin H₂ receptor antagonist) did not alter responses of the basilar artery to acetylcholine and bradykinin. These findings suggest that diabetes mellitus impairs endothelium-dependent dilation of the basilar artery. In addition, the mechanism of impaired responses of the basilar artery during diabetes mellitus does not appear to be related to the activation of the throm☐ane A₂-prostaglandin H₂ receptor.     

Restoration of cholinomimetic activity by clonidine in cholinergic plus noradrenergic lesioned rats

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.     

Removal of Ca(2+) following depolarization-evoked cytoplasmic Ca(2+) transients in freshly dissociated pyramidal neurones of the rat dorsal cochlear nucleus

Cytoplasmic [Ca²⁺] ([Ca²⁺]_i) was measured using Fura-2 in pyramidal neurones isolated from the rat dorsal cochlear nucleus (DCN). The kinetic properties of Ca²⁺ removal following K⁺ depolarization-induced Ca²⁺ transients were characterized by fitting exponential functions to the decay phase. The removal after small transients (<82 nM peak [Ca²⁺]_i) had monophasic time course (time constant of 6.43±0.48 s). In the cases of higher Ca²⁺ transients biphasic decay was found. The early time constant decreased (from 3.09±0.26 to 1.46±0.11 s) as the peak intracellular [Ca²⁺] increased. The value of the late time constant was 18.15±1.60 s at the smallest transients, and showed less dependence on [Ca²⁺]_i. Blockers of Ca²⁺ uptake into intracellular stores (thapsigargin and cyclopiazonic acid) decreased the amplitude of the Ca²⁺ transients and slowed their decay. La³⁺ (3 mM) applied extracellularly during the declining phase dramatically changed the time course of the Ca²⁺ transients as a plateau developed and persisted until the La³⁺ was present. When the other Ca²⁺ removal mechanisms were available, reduction of the external [Na⁺] to inhibit the Na⁺/Ca²⁺ exchange resulted in a moderate increase of the time constants. It is concluded that in the isolated pyramidal neurones of the DCN the removal of Ca²⁺ depends mainly on the activity of Ca²⁺ pump mechanisms.     

Chronic depolarization induced by veratridine increases the survival of rat retinal ganglion cells ‘in vitro’

During the last decades it has been shown that trophic molecules released by target, afferent and glial cells play a pivotal role controlling neuronal cell death. Trophic molecules are able to inhibit this regressive event during development as well as during degenerative diseases. One of the mechanisms involved in the control of neuronal survival by afferent cells requires the release of trophic molecules stimulated by electrical activity. It has been demonstrated that veratridine (a depolarizing agent that keeps the Na⁺ channels opened) induces an increase in neuronal survival. In the present work we show that 3 μM veratridine induced a two-fold increase on the survival of retinal ganglion cells after 48 h in culture. The veratridine effect was inhibited by 50 μM amiloride (an inhibitor of Ca²⁺ channels), 25 μM benzamil (an inhibitor of Na⁺ channels), 30 μM dantrolene and 7.5 μM caffeine (both inhibitors of Ca²⁺ release from the endoplasmatic reticulum) and 10 μM BAPTA-AM (an intracellular Ca²⁺ chelator). However, 5 μM nifedipine (a selective inhibitor of voltage-dependent -type Ca²⁺ channels) and 100 μM MK 801 (an inhibitor of NMDA receptors) did not block the veratridine effect. On the other hand, treatment with 10 μM genistein (an inhibitor of tyrosine kinase enzymes), 20 μM fluorodeoxyuridine (an inhibitor of cell proliferation) or 10 μM atropine (an antagonist of muscarinic receptors) completely abolished the effect of veratridine. Taken together, our results indicate that veratridine increases the survival of rat retinal ganglion cells through mechanisms involving Na⁺ influx, intracellular Ca²⁺ release, activation of tyrosine kinase enzymes and cellular proliferation. They also indicate that cholinergic activity plays an important role in the veratridine effect.     

Nitric oxide induces an increased Na conductance in identified neurons of Aplysia

The ionic mechanism of the effects of micropressure ejections of hydroxylamine (HOA) and sodium nitroprusside (SNP), nitric oxide (NO) generators, on the membrane of identified neurons (R9–R12) of Aplysia kurodai was investigated with conventional voltage-clamp, micropressure ejection, and ion-substitution techniques. Micropressure ejection of HOA and SNP onto the neurons caused a marked depolarization in the unclamped neurons. Clamping the same neurons at their resting potential level (−60 mV) and reejecting HOA and SNP with the same dose produced a slow inward current (I_i(HOA) and I_i(SNP), 3–7 nA in amplitude, 15–60 s in duration) associated with an increase in input membrane conductance. Bath-applied hemoglobin (50 μM), a nitric oxide scavenger, almost completely blocked I_i(HOA) and I_i(SNP), and 3-isobutyl-1-methylxanthine (IBMX, 50 μM) prolonged and enhanced both I_i(HOA) and I_i(SNP). An intracellular injection of cyclic guanosine 3′,5′-monophosphate (cGMP) into the same neurons produced a slow inward current (I_i(cGMP)) which resembled the responses to HOA and SNP, and this current was enhanced in IBMX. Bath-applied methylene blue (10 μM), an inhibitor of guanylate cyclase, significantly reduced I_i(HOA) and I_i(SNP). The inward currents induced by HOA, SNP and cGMP were sensitive to changes in the external Na⁺ concentration. These results suggest that extracellular NO can induce a slow inward current associated with an increase in Na⁺ conductance, mediated by an increase in intracellular cGMP.