Sulfate influx across the rabbit ileal brush border membrane: Sodium and proton dependence,and substrate specificities

Summary In intact ileal mucosa, uptake of SO₄ across the brush border membrane requires the presence of Na and is saturable, withK1/2=1.3mm at 140mm Na (P.L. Smith, S.A. Orellana & M. Field, 1981.J. Membrane Biol. 63:199–206). The present study examines the substrate specificities and transport stoichiometry of the Na-dependent SO₄ uptake process. The effects of variations in medium anion and cation composition on lumen-to-epithelium influx of SO₄ (J _me ^SO4 ) were determined under short-circuit conditions.J _me ^SO4 is inhibited by thiosulfate, but not by phosphate, methylsulfate, vanadate or taurocholate. Cl is weakly inhibitory. Uptake of SO₄ is poorly supported by Li, and is unaffected by K, indicating a specific dependence on Na. At low SO₄ concentration (0.22mm),J _me ^SO4 is a hyperbolic function of medium Na concentration; the corresponding Hill plot is linear with a slope of 1.0, suggesting a transport stoichiometry of 1 Na: 1 SO₄. At high SO₄ concentration (6.7mm), the Na-dependent SO₄ velocity curve is sigmoidal and yields a Hill plot which is again linear but has a slope of 1.56, suggesting transport of more than 1 Na per SO₄. SO₄ uptake in presence of Na exhibits a dependence on medium pH. At 0.22mm SO₄ and 140mm Na,J _me ^SO4 was doubled by lowering pH from 7.4 to 6.8. However, at 6.7mm SO₄ and 140mm Na, changing pH had no effect onJ _me ^SO4 over the range 6.8 to 8.5. The pH dependence ofJ _me ^SO4 at 6.7mm SO₄ was restored when medium Na was lowered to 3mm, suggesting that pH sensitivity is a function of the concentration of preformed NaSO ₄ ^– ion pair. The results suggest that SO₄ influx across the ileal brush border occurs by electroneutral Na⁺/NaSO ₄ ^– or Na⁺/H⁺/SO ₄ ^2– cotransport, the former being favored by high concentrations of Na and SO₄.     

The effects of truncating long-range forces on protein dynamics

This paper considers the effects of truncating long-range forces on protein dynamics. Six methods of truncation that we investigate as a function of cutoff criterion of the long-range potentials are (1) a shifted potential; (2) a switching function; (3) simple atom-atom truncation based on distance; (4) simple atom-atom truncation based on a list which is updated periodically (every 25 steps); (5) simple group-group truncation based on distance; and (6) simple group-group truncation based on a list which is updated periodically (every 25 steps). Based on 70 calculations of carboxymyoglobin we show that the method and distance of long range cutoff have a dramatic effect on overall protein behavior. Evaluation of the different methods is based on comparison of a simulation's rms fluctuation about the average coordinates, the rms deviation from the average coordinates of a no cutoff simulation and from the X-ray structure of the protein. The simulations in which long-range forces are truncated by a shifted potential shows large rms deviations for cutoff criteria less than 14 A, and reasonable deviations and fluctuations at this cutoff distance or larger. Simulations using a switching function are investigated by varying the range over which electrostatic interactions are switched off. Results using a short switching function that switches off the potential over a short range of distances are poor for all cutoff distances. A switching function over a 5-9 A range gives reasonable results for a distance-dependent dielectric, but not using a constant dielectric. Both the atom-atom and group-group truncation methods based on distance shows large rms deviation and fluctuation for short cutoff distances, while for cutoff distances of 11 A or greater, reasonable results are achieved. Although comparison of these to distance-based truncation methods show surprisingly larger rms deviations for the group-group truncation, contrary to simulation studies of aqueous ionic solutions. The results of atom-atom or group-group list-based simulations generally appear to be less stable than the distance-based simulations, and require more frequent velocity scaling or stronger coupling to a heat bath.     

Structural and developmental differences between three types of Na channels in dorsal root ganglion cells of newborn rats

Summary The changes in Na current during development were studied in the dorsal root ganglion (DRG) cells using the whole-cell patch-clamp technique. Cells obtained from rats 1–3 and 5–8 days after birth were cultured and their Na currents were compared. On top of the two types of Na currents reported in these cells (fast-FA current and slow-S current) a new fast current was found (FN). The main characteristics of the three currents are: (i) The voltages of activation are –37, –36, and –23 mV for the FN, FA and S currents, respectively. (ii) The activation and inactivation kinetics of FN and FA currents are about five times faster than those of the S current. (iii) The voltages at which inactivation reaches 50% are –139, –75 and –23 mV for the FN, FA and S currents, respectively.The kinetics and voltage-dependent parameters of the three currents and their density do not change during the first eight days after birth. However, their relative frequency in the cells changes. In the 1–3 day-old rats the precent of cells with S, FA, and mixed S+FN currents is 22, 18, and 60% of the cells, respectively. In the 5–8 day-old, the percent of cells with S, FA, and FN+S is 10, 66 and 22%. The relative increase in the frequency of cells with FA current during development can contribute to the ease of action potential generation compared with cells with FN currents, which are almost completely inactivated under physiological conditions. The predominance of FA cells also results in a significant decrease in the relative frequency of cells with the high-threshold, slow current.Antibodies directed against a part of the S₄ region of internal repeat I of the sodium channel (C ₁ ⁺ , amino acids 210–223, eel channel numbering) were found to shift the voltage dependence of FA current inactivation (but not of FN or S currents) to more negative potentials. The effect was found only when the antibodies were applied externally. The results suggest that FN, FA and S types of Na currents are generated by channels, which are different in the topography of the C ₁ ⁺ region in the membrane.     

Use of Microdialysis for Monitoring Tyrosine Hydroxylase Activity in the Brain of Conscious Rats

An on-line microdialysis system was developed which monitored the 3,4-dihydroxyphenylalanine (DOPA) formation in the striatum during infusion of a submicromolar concentration of an L-aromatic amino-acid decarboxylase inhibitor (NSD 1015). The absence of DOPA in dialysates of 6-hydroxydopamine-pretreated rats and the disappearance of DOPA after administration of alpha-methyl-p-tyrosine indicated that the dialyzed DOPA was derived from dopaminergic nerve terminals. Next we investigated whether the steady-state DOPA concentration in striatal dialysates could be considered as an index of tyrosine hydroxylase activity. The increase in DOPA output after intraperitoneal administration of haloperidol or gamma-butyrolactone and the decrease in DOPA output after intraperitoneal administration of apomorphine are in excellent agreement with results of postmortem studies, in which a decarboxylase inhibitor was used to measure the activity of tyrosine hydroxylase. The effect of haloperidol on DOPA formation was not visible when a U-shaped cannula (0.80 mm o.d.) was used. Some methodological problems related to microdialysis of the haloperidol-induced increase in DOPA formation are discussed. We concluded that the proposed model is a powerful and reliable in vivo method to monitor tyrosine hydroxylase activity in the brain. The method is of special interest for investigating the effect of compounds which are not able to pass the blood-brain barrier. As an application of the method in the latter situation, we report the effect of infusion the neurotoxin 1-methyl-4-phenylpyridinium ion (10 mmol/L infused over 20 min) on the activity of striatal tyrosine hydroxylase.     

[3H]1-Methyl-4-Phenyl-2,3-Dihydropyridinium Ion Binding Sites in Mouse Brain: Pharmacological and Biological Characterization

Because 1-methyl-4-phenyl-2,3-dihydropyridinium ion (MPP+) appears to damage the dopaminergic neuron and cause neuronal death, we characterized [3H]MPP+ binding sites in mouse brain membranes. Among several compounds tested, debrisoquin [3,4-dihydro-2(1H)-isoquinolinecarboxamidine] and some analogues were able to antagonize [3H]MPP+ binding. Debrisoquin is able to block adrenergic transmission and inhibit the activity of monoamine oxidase A (MAO-A). We found a certain correlation between the ability of these agents to displace [3H]MPP+ from its binding sites and their capacity to inhibit MAO-A activity. These data and the finding of a higher number of [3H]MPP+ binding sites in human placenta compared to mouse brain suggest that these sites may correspond to MAO-A enzymes. Recently it has been demonstrated in human brain that neurons in regions rich in catecholamines are positive for MAO-A. Accordingly, we suggest MAO-A as a possible accumulation site of MPP+ within the dopaminergic neuron. We also indicate the chemical structural requirement associated with the best binding of debrisoquin analogues with [3H]MPP+ sites. It would be reasonable to test the effects of debrisoquin-like drugs able to pass the blood-brain barrier on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity.     

Potentiation by the Tetraphenylboron Anion of the Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and Its Pyridinium Metabolite

The 1-methyl-4-phenylpyridinium species (MPP+) is the four-electron oxidation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is widely assumed to be the actual neurotoxic species responsible for the MPTP-induced destruction of dopaminergic neurons. MPTP is oxidized by the enzyme monoamine oxidase-B to a dihydropyridinium intermediate which is oxidized further to MPP+, an effective inhibitor of the oxidation of the Complex I substrates glutamate/malate in isolated mitochondrial preparations. In the present study, the tetraphenylboron anion (TPB) greatly potentiated the inhibitory effects of MPP+ and other selected pyridinium species on glutamate/malate respiration in isolated mouse liver mitochondria. At 10 microM TPB, the potentiation ranged from approximately 50-fold to greater than 1,000-fold for the several pyridinium species tested. In other experiments, TPB greatly enhanced the accumulation of [3H]MPP+ by isolated mitochondrial preparations. This facilitation by TPB of MPP+ accumulation into mitochondria explains, at least in part, the potentiation by TPB of the above-mentioned inhibition of mitochondrial respiration. Moreover, TPB addition increased the amount of lactate formed during the incubation of mouse neostriatal tissue slices with MPTP and other tetrahydropyridines. The administration of TPB also potentiated the dopaminergic neurotoxicity of MPTP in male Swiss-Webster mice. All of these observations, taken together, are consistent with the premise that the inhibitory effect of MPP+ on mitochondrial respiration within dopaminergic neurons is the ultimate mechanism to explain MPTP-induced neurotoxicity.     

Reconstituted voltage-sensitive sodium channels from eel electroplax: Activation of permeability by quaternary lidocaine,N-bromoacetamide,and N-bromosuccinimide

Summary We have investigated the ion permeability properties of sodium channels purified from eel electroplax and reconstituted into liposomes. Under the influence of a depolarizing diffusion potential, these channels appear capable of occasional spontaneous openings. Fluxes which result from these openings are sodium selective and blocked (from opposite sides of the membrane) by tetrodotoxin (TTX) and moderate concentrations of the lidocaine analogue QX-314. Low concentrations of QX-314 paradoxically enhance this channel-mediated flux. N-bromoacetamide (NBA) and N-bromosuccinimide (NBS), reagents which remove inactivation gating in physiological preparations, transiently stimulate the sodium permeability of inside-out facing channels to high levels. The rise and subsequent fall of permeability appear to result from consecutive covalent modifications of the protein. Titration of the protein with the more reactive NBS can be used to produce stable, chronically active forms of the protein. Low concentrations of QX-314 produce a net facilitation of channel activation by NBA, while higher concentrations produce block of conductance. This suggests that rates of modifications by NBA which lead to the activation of permeability are influenced by conformational changes induced by QX-314 binding.     

Antiproliferative Action of Benzodiazepines in Cultured Brain Cells Is Not Mediated Through the Peripheral-Type Benzodiazepine Acceptor

The benzodiazepines, Ro 5-4864, diazepam, clonazepam, and also PK-11195, inhibited, at micromolar concentrations, the proliferation of rat C6 glioma and mouse neuro-2A neuroblastoma cells in culture. The cells possessed high levels of "peripheral-type" high-affinity benzodiazepine binding sites as judged by binding assays and displacement potencies. However, the different potencies and specificities of compounds for the antiproliferative actions and binding affinities for the binding site suggest that the antiproliferative actions were not mediated through the peripheral-type binding site. In support of this, these compounds have also been shown to inhibit proliferation of some nonneuronal cultured cell lines, e.g., mouse SP2/O-Ag 14 hybridoma and rat NCTC epithelial cells, which have no detectable high-affinity peripheral-type benzodiazepine binding sites.     

Effects of pollen donors on seed formation inEspeletia schultzii (Compositae) populations at different altitudes

The influence of different pollen donors on seed formation was investigated in three populations ofEspeletia schultzii that differ in environmental conditions and life history characteristics. Self pollen and pollen from different donors (< 15m apart) within each population was used in a diallel design in order to test the genetic base of seed set variation. Three measures of seed formation were used: (1) achene number; (2) proportion of filled achenes (fruits) that distinguishes between achenes with seeds and empty achenes; (3) proportion of aborted seeds that distinguishes between viable and aborted seeds. Self-pollinations resulted in empty achenes. Achene number did not vary between the different pollen donors. A bimodal pattern of filled achenes was found in two populations in two consecutive years. On the other hand, a unimodal pattern was found in crosses between more distant donors (> 30m). These patterns seems to be the results of a sporophytic incompatibility system. Seed abortion was highest at the higher elevations and seems to be correlated with elevation rather than with any genetic effect.