Inhibition of α2/α3 sodium pump isoforms potentiates glutamate neurotoxicity

Excessive stimulation of neurons by glutamic acid initiates a destructive cascade of ion fluxes, cellular swelling, and death. Homeostatic mechanisms which rectify these disturbances depend largely upon transmembrane ion gradients maintained by Na⁺, K⁺-ATPase (NaP). We proposed that the neurotoxicity of glutamate is enhanced when the NaP capacity is exceeded, and therefore, that the degree of neuronal death varies inversely with endogenous NaP activity. To test this concept, we directly reduced NaP activity in cultured rat telencephalic cells using either the specific inhibitor ouabain, or dcAMP, and assessed whether these treatments increased glutamate-induced neuronal death. Since rodent NaP catalytic subunits possess both low (α1) and high (α2/α3) affinity for ouabain, we were able to inhibit selectively the α2 (principally glial) and α3 (neuronal) catalytic subunits without affecting the α1 isoform. Brief exposures (5–60 min) to high ouabain concentrations (1–10 mM), which blocks the activity of all three catalytic subunits, killed differentiated neurons but spared glia. In contrast, differential inhibition of the α2/α3 isoforms (by 1 μM ouabain) was not of itself toxic, but produced a supersensitivity to glutamate. [³H]Ouabain binding studies confirmed that the glutamate neurotoxicity observed varied inversely with the degree of NaP inhibition. Further, this relationship was not absolutely dependent upon ouabain, since reduction in α2/α3 pump activity induced by dcAMP also amplified glutamate toxicity. We conclude glutamate excitotoxicity. Since the distribution of NaP is highly heterogenous in the nervous system, with similar cell types varying greatly in isoform expression, constitutive levels of this isoenzyme could constitute a major factor in the survival of stimulated neurons. Further, factors which directly affect pump activity, such as activation of protein kinase A, may modulate excitotoxicity.     

Non-uniform expression of alpha subunit isoforms of the Na+/K+ pump in rat dorsal root ganglia neurons

Tissue sections and antibodies selectively recognizing isoforms of the alpha subunit of the Na+/K+ pump were used to determine the expression of alpha1, alpha2 and alpha3 pump isoforms in the plasma membrane of adult rat dorsal root ganglia (DRG) neurons. There was no detectable membrane signal from DRG neurons that were probed with antibodies to the alpha2 isoform of the Na+/K+ pump. The alpha1 isoform of the Na+/K+ pump was found in most (77+/-4%) studied DRG neurons, regardless of cell size. Only 16+/-7% of the neurons expressed a detectable level of the alpha3 Na+/K+ pump and all were apparently from a subpopulation of large DRG neurons. Comparison of cell size distributions and a study of neurons identified in serial sections suggested that of the alpha3 positive DRG neurons about 75% coexpressed the alpha1 isoform of the Na+/K+ pump. These data show that the expression of the protein of the alpha subunit isoforms of the Na+/K+ pump is not uniform throughout the population of DRG neurons and that alpha1 is the predominant isoform in the plasma membrane of these neurons.     

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.     

Effects of ralfinamide, a Na+ channel blocker, on firing properties of nociceptive dorsal root ganglion neurons of adult rats

Recent studies revealed that ralfinamide, a Na(+) channel blocker, suppressed tetrodotoxin-resistant Na(+) currents in dorsal root ganglion (DRG) neurons and reduced pain reactions in animal models of inflammatory and neuropathic pain. Here, we investigated the effects of ralfinamide on Na(+) currents; firing properties and action potential (AP) parameters in capsaicin-responsive and -unresponsive DRG neurons from adult rats in the presence of TTX (0.5 microM). Ralfinamide inhibited TTX-resistant Na(+) currents in a frequency- and voltage-dependent manner. Small to medium sized neurons exhibited different firing properties during prolonged depolarizing current pulses (600 ms). One group of neurons fired multiple spikes (tonic), while another group fired four or less APs (phasic). In capsaicin-responsive tonic firing neurons, ralfinamide (25 microM) reduced the number of APs from 10.6+/-1.8 to 2.6+/-0.7 APs/600 ms, whereas in capsaicin-unresponsive tonic neurons, the drug did not significantly change firing (8.4+/-0.9 in control to 6.6+/-2.0 APs/600 ms). In capsaicin-responsive phasic neurons, substance P and 4-aminopyridine induced multiple spikes, an effect that was reversed by ralfinamide (25 microM). In addition to effects on firing, ralfinamide increased the threshold, decreased the overshoot, and increased the rate of rise of the AP. To conclude, ralfinamide suppressed afferent hyperexcitability selectively in capsaicin-responsive, presumably nociceptive neurons, but had no measurable effects on firing in CAPS-unresponsive neurons. The action of ralfinamide to selectively inhibit tonic firing in nociceptive neurons very likely contributes to the effectiveness of the drug in reducing inflammatory and neuropathic pain as well as bladder overactivity.     

Taurine-induced modulation of voltage-sensitive Na+ channels in rat dorsal root ganglion neurons

The physiological role of taurine, an abundant free amino acid in the neural system, is still poorly understood. The aim of this study was to investigate its effect on TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na+ currents in enzymatically dissociated neurons from rat dorsal root ganglion (DRG) with conventional whole-cell recording manner under voltage-clamp conditions. A TTX-S Na+ current was recorded preferentially from large DRG neurons and a TTX-R Na+ current preferentially from small ones. For TTX-S Na+ channel, taurine of the concentration > or = 10 mM shifted the activation curve in the depolarizing direction and the inactivation curve in the hyperpolarizing direction. There was no change in the activation curve for TTX-R Na+ channel and the inactivation curve was shifted in the hyperpolarizing direction slightly in the presence of taurine > or = 20 mM. When the recovery kinetics was examined, the presence of taurine resulted in a slower recovery from inactivation of TTX-S currents and no change of TTX-R ones. All the effects of taurine were weakly concentration-dependent and partly recovered quite slowly after washout. Our data indicate that taurine alters the properties of Na+ currents in intact DRG neurons. These may contribute to the understanding of taurine as a natural neuroprotectant and the potential of taurine as a useful medicine for the treatment of sensory neuropathies.     

The effects of galanin on dorsal root ganglion neurons with high glucose treatment in vitro

The exposure of neurons to high glucose concentrations is considered a determinant of diabetic neuropathy. The extracellular high concentration of glucose can cause neuronal cellular damage. Galanin (Gal) not only plays a role in processing of sensory information but also participates in energy homeostasis and glucoregulation. However, the effects of Gal on dorsal root ganglion (DRG) neurons with high glucose are not clear. Using an in vitro model of high glucose-treated DRG neurons in culture, the effects of Gal on intracellular reactive oxygen species (ROS) expression, cell viability, apoptosis, expression of Gal and its receptors (GalR1 and GalR2) of DRG neurons were investigated. Neurons were dissociated from embryonic day 15 (E15) rat DRG and cultured for 48 h and then maintained in serum-free neurobasal medium containing high glucose (45 mmol/L) or normal glucose (25 mmol/L) for 24 h. Mannitol (20 mmol/L) was also used to create a high osmotic pressure mimicking the high glucose condition. The results showed that high glucose caused a rapid increasing of intracellular ROS, decreases of cell viability, and upregulation of Gal and its mRNA. Exogenous Gal (1 μmol/L) inhibited the above effects caused by high glucose. Interestingly, high glucose caused downregulation of GalR1 and its mRNA and administration of exogenous Gal could further decrease their expression, whereas expression of GalR2 and its mRNA was not affected at different experimental conditions. The results of the present study indicate for the first time that Gal and its receptor system are involved in high glucose-induced DRG neuronal injury. The contribution of exogenous Gal on neuroprotection appears to be quite significant. These results provide rationale and experimental evidence for development and further studies of Gal on therapeutic strategy for improving diabetic neuropathy.     

trkB-like immunoreactivity in rat dorsal root ganglia following sciatic nerve injury

The expression of full-length trkB protein, the functional high affinity receptor for BDNF and NT-4, was examined by immunohistochemistry in adult rat L4–L5 dorsal root ganglia after different types of sciatic nerve lesions. In normal ganglia, 52.5% of the neurons showed trkB-like immunoreactivity. Size measurements demonstrated that trkB-like immunoreactivity was seen predominantly in small- and medium-sized cells. This was confirmed by the finding that 28% of all trkB-positive neurons showed affinity to RT97, an antibody which lanels a neurofilament epitope specific for medium-sized and large primary afferent neurons. After crush, section or neuroma formation of the sciatic nerve, the proportion of trkB-positive cells was 64.5%, 58% and 61.9%, respectively. Since trkB-receptors are present in regenerating primary afferent neurons, these data could indicate that BDNF and/or NT-4 are involved in sensory nerve fiber regeneration after adult injury.     

S100 protein-immunoreactive primary sensory neurons in the trigeminal and dorsal root ganglia of the rat

The cell body size (cross-sectional area) of S100-immunoreactive (-ir) primary neurons was measured in the trigeminal (TG) and lumbar dorsal root ganglia (DRG). About a half of neurons exhibited S100-immunoreactivity (-ir) in the DRG (44.0%) and TG (59.0%). DRG neurons with cell bodies >1200 μm² mostly exhibited S100-ir (96.5%), whereas S100-ir DRG neurons <600 μm² were rare (8.0%). 36.6% of DRG neurons in the cell size range 600–1200 μm² showed the ir. TG neurons >800 μm² mostly exhibited S100-ir (93.1%), whereas those <400 μm² were devoid of it (positive cells 10.5%). 58.3% of TG cells in the range 400–800 μm² contained S100-ir. Double-immunofluorescence method revealed the co-expression of S100 and other calcium-binding proteins. Parvalbumin-ir neurons mostly exhibited S100-ir in the DRG (97.4%) and TG (97.0%). The co-expression of S100 and calbindin D-28k was very rare in the DRG, because the DRG contained few calbindin D-28k-ir neurons. Unlike in the DRG, numerous neurons co-expressed S100- and calbindin D-28k-ir in the TG. Most calbindin D-28k-ir TG neurons were also immunoreactive for S100 (90.7%). Sub-populations of calretinin (CR)-ir neurons co-expressed S100-ir in both the DRG (68%) and TG (50.0%). Virtually all CR-ir neurons >1400 μm² co-expressed S100-ir in the DRG (100%) and TG (95.9%). CR-ir neurons <800 μm² were rarely exhibited S100-ir (DRG 18.0%, TG 21.9%). 71.3 and 60.5% of CR-ir neurons in the range 800–1400 μm² co-expressed S100-ir in the DRG and TG, respectively. The present study indicates that S100 is closely correlated to the primary neuronal cell size in the DRG and TG.     

Early transient expression of somatostatin (SRIF) immunoreactivity in dorsal root ganglia during ontogenesis in the rat

Immunofluorescence was used in the rat to study the early ontogenetic expression of somatostatin (SRIF) in the dorsal root ganglia (DRGs) from gestational day 10.5 to day 15.5. SRIF-immunoreactivity (IR) was not detectable in day-10.5 embryos, was first observed in DRGs at day 11.5, reached a peak in intensity and distribution at around day 13.5 and thereafter decreased to become undetectable by day 15.5 in the DRGs of the trunk region. The dynamic expression of SRIF-IR in DRG perikarya could be correlated with its expression in nerve fibers located in the limbs and the abdominal mesenchyme. Thus, SRIF-IR is expressed at a time when sensory fibers could have established connections with their embryonic targets and when DRG neurons could have undergone their final mitotic phase. These data showing the earliest and transient expression of a neuropeptide in developing DRGs confirm and extend the notion that SRIF plays an important role in developmental processes.     

Neurofibrillary tangles in the neurons of spinal dorsal root ganglia of patients with progressive supranuclear palsy

Summary Neurofibrillary tangles (NFTs) occur in neurons of human central nervous system (CNS) both in aged subjects and patients with several degenerative diseases, with a certain topographical predilection. In surveying the NFT distribution in nervous tissue of patients with progressive supranuclear palsy (PSP), we found silver-positive fibrillary tangles in the neurons of dorsal root ganglia (DRG) in two of five patients. By immunohistochemistry, these tangles were stained with antibodies to human tau protein, paired helical filaments (PHFs) and ubiquitin. Electron microscopy revealed that they were mainly composed of PHFs that were morphologically indistinguishable from PHFs in the NFTs of CNS typically seen in Alzheimer's disease brains. Our data demonstrate for the first time that the neurons of DRG produce NFTs in PSP and suggest that the pathological process(es) leading to tangle formation can occur in the neurons of the peripheral nervous system in this disease condition.Supported in part by Grant-in-Aid for Scientific Research of the Ministry of Education, Science and Culture of Japan     

Effect of noradrenaline and vanadium on Na+K+-activated ATPase in rat cerebral cortex synaptosomal preparation

Summary The effect of l-noradrenaline and vanadate on the activity of Na⁺K⁺-activated ATPase was studied on synaptosomal brain cortex preparation. Using neutron activation analysis it was found that the rat cerebral cortex synaptosomal preparation contains 0.16M. vanadium. The concentration of vanadium needed to reduce enzyme activity by 50% proved to be 2×10^–6 M. Evidence has been provided that the increase by noradrenaline of enzyme activity in synaptosomal preparation depends on the presence of an inhibitory contaminant in commercial ATP preparations. In homogenate, however, noradrenaline was able to enhance enzyme activity even when vanadium-free ATP was used. This fact indicates that noradrenaline removes the inhibitory effect of cytoplasmic factor thereby stimulating enzyme activity.     

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.     

Changes of the Na/K ATPase activity in the cerebral cortical microvessels of rat after single intraperitoneal administration of mercuric chloride: histochemical demonstration with light and electron microscopy

Summary Since inorganic mercury salts only poorly penetrate the cerebral microvascular endothelial cells comprising the blood-brain barrier (BBB), their neurotoxicity may be predicted to result from interference with BBB transport enzymes. In the present study, we tested the effect of mercuric chloride (HgCl₂) on Na⁺/K⁺ ATPase activity, a key enzyme involved in the ion transport in and out of the brain. Routine histochemical staining in conjunction with light and electron microscopy was used to evaluate the changes in the Na⁺/K⁺ ATPase activity in cerebral cortical microvesels of rats who received a single intraperitoneal injection of 6 mg/kg HgCl₂. At 1 h after HgCl₂ administration, light microscopy revealed uniform reduction of the Na⁺/K⁺ ATPase reaction in all cortical layers. Electron microscopy confirmed the enzyme reaction to be very weak to completely absent in both the luminal and abluminal endothelial cell membranes, and the luminal plasmalemma showed invaginations and pinocytic vesicles indicative of changes in its transport functions. The enzyme inhibition coincided with, and was likely to contribute to, profound perivascular swelling, involving mainly the astrocytic endfeet. The enzyme activity showed a partial recovery 18 h after HgCl₂ treatment, mainly in cortical layers II and III. After 5 days, the recovery of the enzyme activity appeared complete as observed by light and electron microscopy. The recovery of the microvascular Na⁺/K⁺ ATPase coincided with the appearance of a strongly positive Na⁺/K⁺ ATPase reaction in the adjacent astrocytic processes and with the diminution of perivascular swelling.Supported by the Medical Research Centre Project no. 7     

Neonatal hypothyroidism alters the kinetic properties of Na+, K+-ATPase in synaptic plasma membranes from rat brain

Neonatal hypothyroidism was induced in rat pups by injecting 131I within two days of birth and the effects on kinetic properties of Na+, K+-ATPase from synaptic plasma membranes were examined. Neonatal hypothyroidism resulted in a generalized decrease in V(max) with ATP, Na+, K+ and Mg2+ together with an increase in the K(m) for ATP, appearance of a low affinity component for Na+ and allosteric characteristic for the Mg2+-dependent activity at high Mg2+ concentrations. Binding pattern for Na+ and Mg2+ changed. Our results suggest that impairment of Na+, K+-ATPase activity together with altered kinetic properties could be one of the underlying biochemical mechanism leading to central nervous system (CNS) dysfunctions as a consequence of thyroid hormone deprivation during critical stages of brain development.     

Glutamate receptors communicate with Na+/K+-ATPase in rat cerebellum granule cells

Two glutamate receptor agonists, NMDA (N-methyl-d-aspartic acid) and ACPD (cis-(1S/3R)-1-aminocyclopentane-1,3-dicarboxylic acid), induce the reactive oxygen species (ROS) production in rat cerebellum granule cells, whereas the third one, 3-HPG (3-hydroxyphenylglycine), decreases this parameter. The simultaneous presence of 3-HPG, together with NMDA or ACPD, prevents the generation of ROS by neuronal cells. A similar effect of these ligands on Na⁺/K⁺-ATPase can be demonstrated: NMDA and ACPD inhibited the enzyme activity, but 3-HPG activated Na⁺/K⁺-ATPase and prevented its inhibition by NMDA or ACPD. In terms of current classification, NMDA is an agonist of ionotropic glutamate receptors of the so-called NMDA class, whereas ACPD and 3-HPG belong to metabotropic agonists, the former primarily being an activator of metabotropic glutamate receptors (mGluRs) of groups 2 and 3, and the latter, that of mGluRs of groups 1 and 5. Thus, the data presented illustrate the existence of diverse mechanisms of the cross talk between Na⁺/K⁺-ATPase and different glutamate receptors, as well as that between glutamate receptors of different classes.     

ATP modulation of sodium currents in rat dorsal root ganglion neurons

The modulation of tetrodotoxin-sensitive (TTX-S) and slow tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons by ATP was studied using the whole-cell patch-clamp method. The effects of ATP on two types of sodium currents were either stimulatory or inhibitory depending on the kinetic parameters tested. At a holding potential of -80 mV ATP suppressed TTX-S sodium currents when the depolarizing potential was positive to -30 mV but it increased them when the depolarizing potential was negative to -30 mV. At the same holding potential slow TTX-R sodium currents were always increased by ATP regardless of the depolarizing potential. In both types of sodium currents ATP shifted both the conductance-voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction, and accelerated the time-dependent inactivation. ATP decreased the maximum conductance of TTX-S sodium currents but increased that of slow TTX-R sodium currents. The results suggest that ATP would decrease the excitability of neurons with TTX-S sodium channels but would increase that of neurons with slow TTX-R sodium channels. The effects of ATP on sodium currents were preserved in the presence of a G-protein inhibitor, GDP-beta-S, or purinergic antagonists, suramin and Reactive Blue-2, suggesting that purinergic receptors might not be involved in ATP modulation of sodium currents.     

Three types of sodium channels in adult rat dorsal root ganglion neurons

Several types of Na+ currents have previously been demonstrated in dorsal root ganglion (DRG) neurons isolated from neonatal rats, but their expression in adult neurons has not been studied. Na+ current properties in adult dorsal root ganglion (DRG) neurons of defined size class were investigated in isolated neurons maintained in primary culture using a combination of microelectrode current clamp, patch voltage clamp and immunocytochemical techniques. Intracellular current clamp recordings identified differing relative contributions of TTX-sensitive and -resistant inward currents to action potential waveforms in DRG neuronal populations of defined size. Patch voltage clamp recordings identified three distinct kinetic types of Na+ current differentially distributed among these size classes of DRG neurons. 'Small' DRG neurons co-express two types of Na+ current: (i) a rapidly-inactivating, TTX-sensitive 'fast' current and (ii) a slowly-activating and -inactivating, TTX-resistant 'slow' current. The TTX-sensitive Na+ current in these cells was almost completely inactivated at typical resting potentials. 'Large' cells expressed a single TTX-sensitive Na+ current identified as 'intermediate' by its inactivation rate constants. 'Medium'-sized neurons either co-expressed 'fast' and 'slow' current or expressed only 'intermediate' current. Na+ channel expression in these size classes was also measured by immunocytochemical techniques. An antibody against brain-type Na+ channels (Ab7493)10 labeled small and large neurons with similar intensity. These results demonstrate that three types of Na+ currents can be detected which correlate with electrogenic properties of physiologically and anatomically distinct populations of adult rat DRG neurons.     

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.     

Ultracytochemical localisation of Na+, K+-ATPase in cerebral endothelium in acute hypertension

Summary This ultracytochemical study was undertaken to determine whether increased arteriolar permeability in acute hypertension is accompanied by altered localisation of the ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase (K⁺-NPPase), a component of the Na⁺, K⁺-ATPase system. Rats were injected with horseradish peroxidase (HRP) intravenously and acute hypertension was induced by a 2-min infusion of angiotensin amide. Rats were killed at 3 and 15 min, following which brains were sliced and reacted for demonstration of K⁺-NPPase and HRP reaction product. Vessels of normotensive and hypertensive rats that were nonpermeable to HRP showed discontinuous distribution of K⁺-NPPase on the outer plasma membranes of endothelial and adventitial cells of arterioles and endothelial cells and pericytes of capillaries. Arterioles of the hypertensive rats which were permcable to HRP showed marked reduction of K⁺-NPPase localisation in their walls at 3 min while at 15 min when the blood pressure had returned to resting levels the enzyme localisation was similar to controls. This study demonstrates transient alteration of the NA⁺, K⁺-ATPase system during increased endothelial permeability in acute hypertension. The implication of this finding and our previous observation of reduced Ca²⁺-ATPase localisation in endothelial plasma membranes in acute hypertension has been discussed.Supported by the Heart and Stroke Foundation of Ontario     

Dexamethasone enhances NT-3 expression in rat hippocampus after traumatic brain injury

The cellular events in traumatic brain injury (TBI) are complicated, and the factors mediating neurotrophins to protect and repair the injured brain cells are only beginning to be identified. This study examined the effect of dexamethasone (DEX) on neurotrophin-3 (NT-3) expression following TBI. Levels of NT-3 mRNA and protein in rat hippocampus were measured using in situ hybridization and immunohistochemistry, respectively. After TBI, the NT-3 mRNA expression was down-regulated during the first 24 h. DEX reversed the post-traumatic reduction of NT-3 mRNA expression at 2, 4, 6, and 12 h in the hippocampus, and also decreased the cell death in hippocampal hilum and supraventricular cerebral cortex after 7 days. The NT-3 protein levels generally corresponded to the mRNA levels in the hippocampal region. DEX enhanced the NT-3 expression after TBI, indicating that post-traumatic neuroprotection in the hippocampus is at least partially mediated by NT-3 and thus can be modulated by DEX treatment.