Sodium-independent binding of [H]cocaine in mouse striatum is serotonin related

There was a highly significant correlation between IC₅₀ values of various drugs in inhibiting the Na⁺-independent [³H]cocaine binding in the mouse striatum and their values in inhibiting the synaptosomal uptake of [³H]serotonin. In contrast, there was no correlation between the inhibition of binding in the absence of Na⁺ and the inhibition of [³H]dopamine uptake. Lesioning of serotonergic nerve terminals with 5, 7-dihydroxytryptamine reduced the Na⁺-independent [³H]cocaine binding, without affecting the Na⁺-dependent binding. These results indicate that the bulk of the Na⁺-independent [³H]cocaine binding in the mouse striatum is associated with serotonergic nerve terminals.     

Effects of lithium therapy on Na+-K+-ATPase activity and lipid peroxidation in bipolar disorder

Objectives

Oxidative stress induced lipid peroxidation along with a reduced Na⁺–K⁺-ATPase activity has been implicated in the pathophysiology of bipolar disorders (BPD). Although, lithium therapy results in significant improvement in the symptoms of the disease, studies regarding its effect on the altered sodium pump activity and lipid peroxidation status have come out with conflicting results. The present study was undertaken to evaluate the status of lipid peroxidation and analyze the role of lithium and Na⁺–K⁺-ATPase activity in its regulation in BPD patients in our region.

Method

We measured RBC membrane Na⁺–K⁺-ATPase activity and serum thiobarbituric acid reacting substances (TBARS) level in 73 BPD patients and serum lithium, in addition, in 48 patients receiving lithium therapy among them.

Results

Na⁺–K⁺-ATPase activity and serum TBARS level were significantly decreased and increased respectively in all BPD patients compared to age and sex matched healthy controls. Same trend was observed in the BPD patients stabilized on lithium therapy compared to the lithium naive ones. Although, the enzyme activity showed a reciprocal relationship with TBARS in all patients of BPD, a significant positive correlation and dependence of the enzyme activity was evident with serum lithium level only in the lithium stabilized BPD group.

Conclusions

BPD patients showed significantly compromised Na⁺–K⁺-ATPase activity and increased lipid peroxidation. Lithium induced improvement in the enzyme activity was associated with significant reduction in lipid peroxidation. Enhancement of the Na⁺–K⁺-ATPase activity by optimum dosage of lithium may be a potential contributing factor for reducing oxidative stress in BPD patients.     

Insulin affects synaptosomal GABA and glutamate transport under oxidative stress conditions

In this study, we investigated the in vitro effect of exogenously administered insulin on the susceptibility to oxidative stress and on the accumulation of the amino acid neurotransmitters gamma-aminobutyric acid (GABA) and glutamate in a synaptosomal fraction isolated from male Wistar rat brain cortex. Insulin (1 microM) did not affect synaptosomal lipid peroxidation induced by the oxidant pair ascorbate/Fe(2+), although under these conditions an increase in thiobarbituric acid reactive substances (TBARS) levels was observed. Under control conditions, the presence of insulin did not change the uptake of [3H]GABA or [3H]glutamate. In contrast, under oxidizing conditions, we observed a 1.8- and a 2.2-fold decrease in [3H]GABA and [3H]glutamate accumulation, respectively, and insulin reverted the lower levels of both [3H]GABA and [3H]glutamate accumulation (to 86.74+/-6.26 and 67.01+/-6.65% of control, respectively). Insulin also increased the extrasynaptosomal levels of GABA and glutamate, determined both in control and oxidizing conditions. From this study, we can conclude that insulin is a modulator of amino acid neurotransmitter transport, either directly, as seems to occur under normal conditions, or via the decrease in ATP levels and the subsequent reversion of the amino acid transporters, as seems to occur under oxidative stress conditions. The modulation of both GABA and glutamate transport might be implicated in the neuroprotective role of insulin.     

Effect of oxidative stress on the release of [H]GABA in cultured chick retina cells

The effect of ascorbate (1.5 mM)/Fe²⁺ (7.5 μM)-induced oxidative stress on the release of pre-accumulated [³H]γ-aminobutyric acid ([³H]GABA) from cultured chick retina cells was studied. Depolarization of control cells with 50 mM K⁺ increased the release of [³H]GABA by 1.01 ± 0.16% and 2.5 ± 0.3% of the total, in the absence and in the presence of Ca²⁺, respectively. Lipid peroxidation increased the release of [³H]GABA to 2.07 ± 0.31% and 3.6 ± 0.39% of the total, in Ca²⁺-free or in Ca²⁺-containing media, respectively. The inhibitor of the GABA carrier, 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711) blocked almost completely the release of [³H]GABA due to K⁺-depolarization in the absence of Ca²⁺, but only 65% of the release occurring in the presence of Ca²⁺ in control and peroxidized cells. Under oxidative stress retina cells release more [³H]GABA than control cells, being the Ca²⁺-independent mechanism, mediated by the reversal of the Na⁺/GABA carrier, the most affected. MK-801 (1 μM), a non-competitive antagonist of the NMDA receptor-channel complex, blocked by 80% the release of [³H]GABA in peroxidized cells, whereas in control cells the inhibitory effect was of 40%. The non-selective blocker of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), inhibited the release of [³H]GABA by 30% and 70% in control and peroxidized cells, respectively. Glycine (5 μM) stimulated [³H]GABA release evoked by 50 mM K⁺-depolarization in control but not in peroxidized cells. The release of -[³H]aspartate (a non-metabolized analog of -glutamate) evoked by 50 mM K⁺, in the absence of Ca²⁺, was significantly higher in peroxidized cells (6.76 ± 0.64% of the total) than in control cells (3.79 ± 0.27% of the total). The results suggest that oxidative stress induced by ascorbate/Fe²⁺ causes an excessive release of endogenous excitatory amino acids upon K⁺-depolarization. The glutamate released may activate NMDA and non-NMDA receptors, raising the intracellular Na⁺ concentration and consequently stimulating the release of [³H]GABA by reversal of the Na⁺/GABA carrier.     

Control of the Na+, K+-ATPase under normal and pathological conditions

The Na⁺, K⁺-ATPase is an important enzyme in determining the ionic milieu of the cerebromicrovasculature and neurons. The effect of hypertension or aging on this enzyme, as well as its susceptibility to regulation by fatty acids or aluminum, is the focus of this study. A significant increase (34%) in the apparent affinity constant (K _D) but no change in the maximum binding capacity (B _max) for [³H]ouabain binding to the cerebromicrovascular Na⁺, K⁺-ATPase occurs after induction of acute hypertension. In addition, long chain unsaturated fatty acids stimulate the binding of [³H]ouabain to the enzyme in microvessels from normotensive and hypertensive rats. The synaptosomal Na⁺, K⁺-ATPase is sensitive to aluminum. AlCl₃ (1–100 μM) inhibits the K⁺-dependent-p-nitrophenylphosphatase (K⁺-NPPase) activity of the Na⁺, K⁺-ATPase in a dose-dependent manner. AlCl₃ (100 μM) decreases theV _max by 14% but does not alter theK _M, suggestive of noncompetitive inhibition. The enzyme from aged brain displays a greaterV _max, but shows the same susceptibility to AlCl₃ as the enzyme from younger brain. In summary, disruption of the Na⁺, K⁺-ATPase may underlie, at least in part, abnormalities of nerve and vascular cell function in disorders where elevated concentrations of fatty acids or metal ions are involved.     

Aluminum-induced alterations in [3H]Ouabain binding and ATP hydrolysis catalyzed by the rat brain synaptosomal (Na+ + K+)-ATPase°

The (Na⁺ + K⁺)-ATPase is responsible for maintenance of the ionic milieu of cells. The objective of this study is to investigate the effect of aluminum, an ion implicated in several neurological disorders, on ATP hydrolysis catalyzed by the rat brain synaptosomal (Na⁺ + K⁺)-ATPase and on the binding of [³H]ouabain to this enzyme. AlCl₃ (25–100 μM) inhibits the phosphatase activity of the (Na⁺ + K⁺)-ATPase in a dose-dependent manner. AlCl₃ appears to act as a reversible, noncompetitive inhibitor of (Na⁺ + K⁺)-ATPase activity by decreasing the maximum velocity of the enzyme without significantly affecting the apparent dissociation constant with respect to ATP. AlCl₃ may affect Mg²⁺ sites on the (Na⁺ + K⁺)-ATPase but does not appear to interact with Na⁺ or K⁺ sites on the enzyme. In contrast to this inhibitory effect on the phosphatase function of the enzyme, AlCl₃ (1–100 μM) stimulates the binding of [³H]ouabain to the (Na⁺ + K⁺)-ATPase. This effect is due to an increase in the maximum [³H]ouabain binding capacity of the enzyme with no change in the [³H]ouabain binding affinity. These data support the hypothesis that AlCl₃ may stabilize the phosphorylated form of the synaptosomal (Na⁺ + K⁺)-ATPase which increases [³H]ouabain binding while inhibiting the phosphatase activity of the enzyme.     

Differential long-term effect of AF64A on [H]ACh synthesis and release in rat hippocampal synaptosomes

The activities of various presynaptic cholinergic parameters were determined in hippocampal synaptosomes of rats 29 weeks after intracerebroventricular injection of ethylcholine aziridinium (AF64A) (3 nmol/2 μl/side) or vehicle (saline). Synaptosomes were preloaded with [³H]choline ([³H]Ch), treated with diisopropyl fluorophosphate to inhibit cholinesterase activity and then were assayed for their content of [³H]Ch and [³H]acetylcholine ([³H]ACh) and for their ability to synthesize and release [³H]ACh. In synaptosomes from AF64A-treated rats compared with synaptosomes from vehicle-treated rats we observed that: (i) specific uptake of [³H]ACh was reduced to 60% of control; (ii) residing [³H]ACh levels were 43% of control while residing [³H]Ch levels were 72% of control; (iii) basal and K⁺-induced [³H]ACh release were 77% and 73% of control, respectively; (iv) high K⁺-induced synthesis of [³H]ACh was only 9% of control; (v) but, choline acetyltransferase activity remained relatively high, being 80% of control. These results suggest that AF64A-induced cholinergic hypofunction is expressed by both loss of some cholinergic neurons and impairment in the functioning of the spared neurons.     

Release of γ-[H]aminobutyric acid from synaptosomes: effect of external cations and of ouabain

In the present study we have investigated the effect of cations and ouabain on Ca²⁺-independent and Ca²⁺-dependent release of γ-[³H]aminobutyric acid ([³H]GABA) from sheep brain synaptosomes. The presence of Na⁺ in the external medium is essential for the Ca²⁺-independent release induced by K⁺ or ouabain. Thus, in the absence of Ca²⁺, ouabain of K⁺ causes the release of [³H]GABA provided that Na⁺ is present in the external medium. Under K⁺-depolarizing conditions, in a Na⁺ medium, either ouabain or Ca²⁺ further increases the [³H]GABA release induced by depolarization, but their effects are not additive. The presence of external Na⁺ is not required for the Ca²⁺-dependent release of [³H]GABA due to K⁺ depolarization, and this release, which occurs in a choline medium, is not modified by ouabain. Under these conditions (choline medium) K⁺-depolarization dependent release is absolutely dependent on external Ca²⁺, which suggests that this release of [³H]GABA occurs only by exocytosis, without the carrier-mediated efflux which normally co-exists with exocytosis due to K⁺-depolarization in a Na⁺ medium. It is likely that the release induced by ouabain or K⁺ involves the membrane carrier which responds to changes in membrane potential.     

High-affinity uptake system for cysteine in crude synaptosomal fractions of rat cerebral cortex

The in vitro uptake of [³⁵S] cysteine was studied in crude synaptosomal preparation of the cerebral cortex of rat. The accumulation of cysteine was found to be temperature- and time-dependent. It was linear at least for four minutes at 37 C with characteristics of saturable kinetics. Uptake of cysteine was Na⁺- and K⁺-dependent. Increasing the Na⁺ ion concentration increased the accumulation of cysteine in synaptosomal preparations; unlike the Na⁺ ion, an increase in the K⁺ ion, an increase in the K⁺ ion inhibited cysteine uptake. Cysteine was accumulated against concentration gradients by a saturable mechanism. Double reciprocal plot of the cysteine uptake suggests two types of affinity systems, with K_m values for the high-affinity uptake of about 12.2 μM and for the low-affinity uptake of about 4 mM. The high-affinity uptake was also significantly inhibited by ouabain, a potent inhibitor of the Na⁺-K⁺-dependent ATPase, and other metabolic inhibitors. The results of the effects of cysteine analogues on uptake also suggested that it is a substrate-specific high-affinity uptake system for cysteine.     

A comparison of sodium-dependent glutamate binding with high-affinity glutamate uptake in rat striatum

The specific sodium-dependent binding of [³H]glutamate to membranes of the rat striatum was examined and a comparison made with high affinity glutamate uptake. In the presence of sodium, [³H]glutamate binding was saturable and of high affinity. No binding could be detected in the absence of sodium. Removal of the cortical afferents to the striatum resulted in a parallel decrease in Na⁺-dependent glutamate binding and in high-affinity glutamate uptake. After the injection of the neurotoxin kainic acid into the striatum, the density of Na⁺-dependent glutamate binding sites was reduced by 40%, while high-affinity uptake showed no significant decrease. Drugs which inhibit high-affinity uptake were also effective at inhibiting Na⁺-dependent binding. The results suggest that about half the Na⁺-dependent glutamate binding sites in the striatum represent high-affinity uptake sites on the corticostriatal terminals. The remainder of the binding sites are located on striatal neurons and may also be uptake sites.     

In Vivo and In Vitro Effects of Phenytoin (PHT) on ATPases and [14C]-PHT Binding in Synaptosomes and Mitochondria from Rat Cerebral Cortex

Summary: The effect of phenytoin (PHT) on Na⁺-K⁺-ATPase and Mg²⁺-ATPase activities and on [¹⁴C]-PHT binding in vitro to synaptosomal and mitochondrial sub cellular fractions from rat cerebral cortex was studied after chronic PHT treatment. Synaptosomal and mitochondrial fractions were characterized with plasma membrane and mitochondrial enzymatic markers. Synaptoso-mal Na⁺-K⁺-ATPase was not affected in vitro by PHT 1–200 μM or by chronic treatment with 2–50 mg/kg/day of the unlabeled drug for 8 days. Mitochondria1 Mg²⁺-ATPase was significantly stimulated by PHT after chronic treatment with 5 mg/kg/day for 8 days; reaching maximal effect (76%), at 10–25 mg/kg. PHT had no effect on mitochondrial Mg²⁺-ATPase when added in vitro. [¹⁴C]-PHT binding in vitro to the subcellular fractions was determined by dialysis to assess in vivo binding of the unlabeled PHT during chronic treatment. Indeed, [¹⁴C]-PHT bound to synaptosomes was significantly reduced by chronic PHT treatment from 218 ±10 to 119 & 11 pmol/mg protein after 1 week of treatment; a similar effect was obtained after 2–3 weeks with 10 mg/kg/day. Mitochondrial fraction bound 117 ±10 pmol/mg protein labeled PHT. Chronic treatment with unlabeled PHT also reduced the amount of [¹⁴C]-PHT bound to 19.9 ± 2.2 pmol/mg protein. These results show slow reversible PHT in vivo binding to synaptosomes and mitochondrias from rat cerebral cortex, supporting the idea that the modulatory action of PHT on Na+ and Ca2+ permeabilities are mediated through these slow reversible binding proteins. The data also suggest a possible role of intra synaptosomal mitochondria in [Ca²⁺]_i buffering.     

Evidence for a hippocampal-septal glutamatergic pathway in the rat.

Kainic acid lesions of the hippocampal formation resulted in degeneration of axons and terminals in the lateral septal nuclei and in the nucleus accumbens. The degeneration of the hippocampal-septal projection was associated with a selective reduction in the synaptosomal sodium-dependent uptake of l-[³H]glutamate (?30 to 40%) and in the concentration of l-glutamate in the P₂ fraction (?20%). Presynaptic neurochemical markers for cholinergic, GABAergic, and noradrenergic neurons in the septal nuclei were not reduced by the hippocampal lesion. Transection of the hippocampal-septal projection at the fornix reduced the synaptosomal uptake of l-[³H]glutamate 40% but did not significantly reduce [³H]GABA uptake in the septal nuclei. The hippocampal-septal pathway, an excitatory projection, may thus be glutamatergic.     

Quantitative autoradiography of [H]ouabain binding sites in rat brain

In vitro quantitative autoradiography was used to localize in rat brain binding sites for [³H]ouabain, an inhibitor of the Na⁺,K⁺-ATPase. High levels of [³H]ouabain binding sites were found in the superior and inferior colliculi, the mammillary nucleus, the interpeduncular nucleus, and in various divisions of the olfactory, auditory and somatomotor systems. The heterogeneous distribution of [³H]ouabain binding closely parallels the regional brain glucose consumption as determined by the [¹⁴C]deoxyglucose method. Lesion studies of the rat hippocampus using the excitotoxin, ibotenic acid, showed both a marked decrease of neuronal cell types on the injected side and a corresponding decrease in [³H]ouabain binding, indicating that some of the [³H]ouabain binding sites are localized to neurons. The close correlation between [³H]-ouabain binding and regional glucose utilization provides further evidence for a linkage between glucose utilization and the neuronal Na⁺,K⁺-ATPase.     

Biochemical evidence thatl-glutamate is a neurotransmitter of primary vagal afferent nerve fibers

To determine in rat if vagal afferent fibers projecting into the intermediate one third of the nucleus tractus solitarius (NTS), the site of termination of baroafferents, utilize glutamate as a neurotransmitter, the high-affinity uptake of [³H]l-glutamate and content of glutamate were analyzed in micropunches of rat brain stem. The intermediate NTS contains a high-affinity synaptosomal uptake system for [³H]l-glutamate that is greater in capacity than that in areas adjacent to the NTS; it is almost two-fold higher than uptake in medial septum and nucleus accumbens and equal to that of hippocampal regions purportedly containing a rich glutamatergic innervation. Unilateral ablation of the nodose ganglion (i.e. cells of origin of vagal afferents) resulted, within 24 h in a prolonged significant reduction, to 56% of control, of [³H]l-glutamate uptake, bilaterally in the NTS. The reduction of Na⁺-dependent synaptosomal uptake of [³H]l-glutamate, resulted from a decrease in V_max without change in theK_m of the process, was anatomically restricted to the intermediate NTS, and was not associated with changes in [³H]GABA uptake. The content of glutamate in the NTS was significantly (P < 0.01) decreased by 30% 7 days following unilateral extirpation of the nodose ganglion without changes in the concentrations of aspartate, glycine, glutamine, or GABA. A population of vagal afferent fibers projecting to NTS are glutamatergic. The results are consistent with the hypothesis obtained by physiological and pharmacological techniques that glutamate is a neurotransmitter of baroafferents.     

Synaptosomal neurotransmitter uptake systems in the retina and brain nuclei of light- and dark-adapted rabbits

High affinity uptake rate for [14C]aspartate and [3H]dopamine by retinal homogenate (H), Pl (outer plexiform layer, OPL), and P2 (inner plexiform layer, IPL) retinal synaptosomal fractions were not significantly different between light- and dark-adapted rabbits. However, there were significant increases in the dark in [3H]gamma-aminobutyric acid high affinity uptake rate by retinal H and P2 but not that of Pl. There was a significantly higher [3H]choline uptake rate by retinal H, Pl and P2 in the dark-adapted compared to light-adapted rabbits, but there was no significant change in this rate for synaptosomal fractions from the lateral geniculate body, superior colliculus, visual cortex (VA I + II), caudate nucleus (CN) and hippocampus (HP). Data obtained in this study, along with reports of others, indicate that the change in retinal neurotransmission functions may not always be parallel with the change in high affinity uptake rates of neurotransmitters by retinal synaptosomal fractions. Data obtained indicate an increase in retinal cholinergic neuronal activities in the dark and indicate that optic nerves are not cholinergic and cholinergic neurons in brain nuclei, such as VA, CN and HP, are not significantly influenced by optic nerve inputs in light and dark conditions.     

Erythrocyte membrane abnormalities in human myotonic dystrophy

Membrane-bound enzyme activities and cardiac glycoside binding were determined in red blood cell membrane preparations from patients with myotonic dystrophy and in age matched controls. Na⁺-K⁺-activated ATPase activity was signficantly increased in myotonic patients. [³H]Ouabain binding to erythrocyte membranes was also significantly increased in myotonic dystrophy patients. The Mg²⁺-ATPase (ouabain-insensitive) was, however, unchanged. The K⁺-stimulated paranitrophenyl phosphatase (KPNPPase) activity was markedly enhanced in myotonic patients as compared to controls. The kinetic analysis showed a marked change in V_max of Na⁺-K⁺ ATPase with respect to the activation by Na⁺, K⁺ and ATP. However, the K_m values were the same in control as well as in myotonic groups. The increased erythrocyte membrane Na⁺-K⁺-ATPase activity, KPNPPase and [³H]ouabain binding in myotonic patients supports the hypothesis that generalized membrane abnormality may be involved in pathogenesis of the human myotonic dystrophy.     

Thallium-Induced Toxicity in Rat Brain Crude Synaptosomal/Mitochondrial Fractions is Sensitive to Anti-excitatory and Antioxidant Agents

The mechanisms by which the heavy metal thallium (Tl⁺) produces toxicity in the brain remain unclear. Herein, isolated synaptosomal/mitochondrial P2 crude fractions from adult rat brains were exposed to Tl⁺ (5–250 μM) for 30 min. Three toxic endpoints were evaluated: mitochondrial dysfunction, lipid peroxidation, and Na⁺/K⁺-ATPase activity inhibition. Concentration-response curves for two of these endpoints revealed the optimum concentration of Tl⁺ to induce damage in this preparation, 5 μM. Toxic markers were also estimated in preconditioned synaptosomes incubated in the presence of the N-methyl-d-aspartate receptor antagonist kynurenic acid (KYNA, 50 μM), the cannabinoid receptor agonist WIN 55,212-2 (1 μM), or the antioxidant S-allyl-L-cysteine (SAC, 100 μM). All these agents prevented Tl⁺ toxicity, though SAC did it with lower efficacy. Our results suggest that energy depletion, oxidative damage, and Na⁺/K⁺-ATPase activity inhibition account for the toxic pattern elicited by Tl⁺ in nerve terminals. In addition, the efficacy of the drugs employed against Tl⁺ toxicity supports an active role of excitatory/cannabinoid and oxidative components in the toxic pattern elicited by the metal.     

Calcium-independent release of [H]spermine from chick retina

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [³H]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [³H]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca²⁺-independent manner. Inhibition of Na⁺/K⁺-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na⁺ electrochemical gradients, since nigericin and veratrine did not induce release in Na⁺ containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.     

Autoradiography of high affinity uptake of catecholamines by primary astrocyte cultures

Uptake ofd,l-[³H]norpinephrine (³H]NE and [³H]dopamine (³H]DA) by primary astrocyte cultures prepared from neonatal rat brains, which are 95% glial fibrillary acidic protein (GFAP(+)), was studied by measuring accumulation of tritium label, and localizing such uptake at the cellular level by autoradiography. Uptake of [³H]NE was 95% Na⁺ dependent at 10⁻⁷ M and 80% Na⁺ dependent at 7.5 × 10⁻⁷M [³H]NE. Uptake of [³H]DA at 7.5 × 10⁻⁷M was 58% Na⁺ dependent, but total uptake of [³H]NE or [³H]DA showed that a high proportion of all the cells in these cultures had a grain density which was clearly above background. When Na⁺ was omitted from the medium, the temperature was lowered to 4 °C, or 10⁻⁷ M desmethyllimipramine or 10⁻⁷ M amitryptyline were present, cellular grain density after exposure to both [³H]NE and [³H]DA was greatly reduced, to close to background levels. It also appeared necessary to have inhibitors of both monoamine oxidase (pargyline) and catecholamine-O-methyltransferase (tropolone) present to see clear cellular localization for [³H]DA. In the case of [³H]NE the presence of tropolone alone was adequate to observe cellular localization. These results confirm our previous findings of the existence of a high affinity uptake process for catecholamines in primary astrocyte cultures based on uptake properties, and in the present study also localizes such uptake to the major, astrocytic cell type.     

Membrane desmosterol and the kinetics of the sarcolemmal Na,K-ATPase in myotonia induced by 20,25-diazacholesterol

The kinetics of the sarcolemmal Na⁺,K⁺-ATPase were studied in rats made myotonic by treatment with 20,25-diazacholesterol (20,25-D; 200 mg/kg every 14 days). Overall Na⁺,K⁺-ATPase activity in purified sarcolemma increased from 44.6 ± 8.7 nmol P_i/mg protein⁻¹ min⁻¹ in controls to 77.7 ± 7.9 nmol mg⁻¹ min⁻¹ in treated animals whose membrane desmosterol concentrations exceeded 70% of total membrane sterol. In general the activity of this enzyme paralleled the desmosterol content of the sarcolemma. The total number of enzyme units, however, was not changed as evidenced by [³H]ouabain binding and by measurements of steady-state phosphorylated intermediate in the absence of K⁺. Independent measurements of the Na⁺,K⁺-ATPase dephosphorylation partial reaction in 20,25-D sarcolemma revealed no change from control rats at any temperature between 5 and 40°C; the K⁺ dependence of this reaction was also unchanged. Rates of phosphoprotein formation inferred from measurements of steady-state phosphorylated intermediate under various ionic conditions were also not altered by increasing desmosterol. Kinetic analysis suggests that the increase in overall Na⁺,K⁺-ATPase activity in membranes with high concentrations of desmosterol may reflect an increase in the rate of conformational interchange between two states of the enzyme during its activity cycle.