Inhibition of Impulse Activity in a Sensory Neuron by an Electrogenic Pump

The crayfish tonic stretch receptor neuron manifests three phenomena: (a) Impulse frequency in response to a depolarizing current decays exponentially to half the initial rate with a time constant of about 4 sec. (b) One or more extra impulses superimposed on steady activity result in a lengthening of the interspike interval immediately following the last extra impulse which is proportional to the number of extra impulses. However, above a "threshold' number of impulses the proportionality constant becomes abruptly larger. (c) Following trains of impulses, the resting potential of the cell is hyperpolarized by an amount proportional to impulse number. Such posttetanic hyperpolarization (PTH) decays approximately exponentially with a time constant of 11 sec, but this varies with membrane potential. These effects are attributed to the incremental increase of an inhibitory (hyperpolarizing) current with a long (relative to interspike interval) decay constant. We suggest that this inhibitory current is the result of increased electrogenic Na pumping stimulated by Na entering with each impulse. Evidence is presented that the three effects are reversibly inhibited by conditions which depress active Na transport: (a) Li substituted for Na in the bath; (b) application of strophanthidin; (c) K removal; (d) treatment with cyanide; (e) cooling. We conclude that a single process is responsible for the three responses described above and identify that process as electrogenic Na pumping. Our observations also indicate that electrogenic pumping contributes to this neuron's resting potential.     

The rate of uptake of carbon monoxide and of nitric oxide by normal human erythrocytes and experimentally produced spherocytes

The uptake of gases such as oxygen, carbon monoxide, or nitric oxide by the erythrocyte involves: (a) diffusion across the cellular membrane, (b) intraerythrocytic diffusion, and (c) chemical combination with hemoglobin. The aim of this investigation was to obtain data which would permit an analysis of each of these factors in limiting the rate of gas uptake. The initial over-all rate of uptake of gases which combine chemically with hemoglobin to produce a color change can be measured by a modified version of the Hartridge-Roughton-Millikan constant flow, rapid reaction apparatus. If nitric oxide is the reactant gas, only (a) and (b) are measured since the chemical combination of this gas with hemoglobin is extremely rapid. Our studies have shown that human biconcave discoidal erythrocytes at 38 and 48°C., have the same initial rate of carbon monoxide and nitric oxide uptake as the same cells converted into spherocytes of equal volume. Similarly there was no difference between discs and cells sphered with a 30 per cent increase in volume. Shrunken erythrocytes showed a marked decrease in rate of gas uptake. This suggests that surface area and maximum linear distance for intracellular diffusion of this magnitude do not measurably retard gas uptake. In the shrunken cells, a change in the orientation and concentration of intraerythrocytic hemoglobin and/or of the membrane components may have impeded gas diffusion.     

Dipole Theory of Heat Production and Absorption in Nerve Axon

Exact formulas are derived for the energy change of a dipole system with two energy states (or bands) in a changing field in two cases: (a) no dipole flip-flop and (b) dipole flip-flop caused by stimulation. Based on these formulas, the positive and negative heats are calculated. The results are in good agreement with experiment in case b but are 60-180% larger in case a. Furthermore, the theory shows that the negative heat cannot be less than the positive heat in case a but can be either way in case b, the latter result being found prevalent in experiment. It is concluded that nerve excitation is most likely to involve dipole flip-flop at the membrane surface. The theory is consistent in the interpretations and correlations of the electrical, optical, and thermal effects observed in nerve axon.     

ADENINE NUCLEOTIDE-INDUCED CONTRACTION OF THE INNER MITOCHONDRIAL MEMBRANE : II. Effect of Bongkrekic Acid

In bovine heart mitochondria bongkrekic acid at concentrations as low as about 4 nmol/mg protein (a) completely inhibits phosphorylation of exogenous adenosine diphosphate (ADP) and dephosphorylation of exogenous adenosine triphosphate (ATP), (b) completely reverses atractyloside inhibition of inner membrane contraction induced by exogenous adenine nucleotides, and (c) decreases the amount of adenine nucleotide required to elicit maximal exogenous adenine nucleotide-induced inner membrane contraction to a level which appears to correspond closely with the concentration of contractile, exogenous adenine nucleotide binding sites Bongkrekic acid at concentrations greater than 4 nmol/mg protein induces inner membrane contraction which seems to depend on the presence of endogenous ADP and/or ATP. The findings appear to be consistent with the interpretations (a) that the inner mitochondrial membrane contains two types of contractile, adenine nucleotide binding sites, (b) that the two sites differ markedly with regard to adenine nucleotide affinity, (c) that the high affinity site is identical with the adenine nucleotide exchange carrier, (d) that the low affinity site is accessible exclusively to endogenous adenine nucleotides and is largely unoccupied in the absence of bongkrekic acid, and (e) that bongkrekic acid increases the affinity of both sites in proportion to the amount of the antibiotic bound to the inner membrane.     

The structure of verdohemochrome and its implications for the mechanism of heme catabolism

The synthesis, purification as a tetrafluoroborate salt and structural elucidation of the verdohemochrome 2a derived from the coupled oxidation of octaethylhemochrome 1 is described. Based on elemental analyses, spectroscopic studies (visible and infrared absorption, ¹H-NMR) and fast atom bombardment mass spectrometry, the assignment of the iron(II) oxaporphyrin structure for the verdohemochrome 2a and the blue monocarbonyl species 2b, obtained upon treatment of 2a with carbon monoxide, has been accomplished. This assignment raises a number of questions regarding the iron oxidation state of intermediates in the pathway of heme catabolism both in vitro and in vivo. Furthermore, the implications of the occurrence of an iron oxaporphyrin intermediate in the pathway of heme metabolism, which is suggested by the similarity of the visible absorption spectrum of the CO species 2b with that of a new intermediate recently observed in the heme oxygenase-catalyzed degradition of heme and mesoheme, is considered.     

Analysis of the effects of calcium or magnesium on voltage-clamp currents in perfused squid axons bathed in solutions of high potassium

Isolated axons from the squid, Dosidicus gigas, were internally perfused with potassium fluoride solutions. Membrane currents were measured following step changes of membrane potential in a voltage-clamp arrangement with external isosmotic solution changes in the order: potassium-free artificial seawater; potassium chloride; potassium chloride containing 10, 25, 40 or 50, mM calcium or magnesium; and potassium-free artificial seawater. The following results suggest that the currents measured under voltage clamp with potassium outside and inside can be separated into two components and that one of them, the predominant one, is carried through the potassium system. (a) Outward currents in isosmotic potassium were strongly and reversibly reduced by tetraethylammonium chloride. (b) Without calcium or magnesium a progressive increase in the nontime-dependent component of the currents (leakage) occurred. (c) The restoration of calcium or magnesium within 15–30 min decreases this leakage. (d) With 50 mM divalent ions the steady-state current-voltage curve was nonlinear with negative resistance as observed in intact axons in isosmotic potassium. (e) The time-dependent components of the membrane currents were not clearly affected by calcium or magnesium. These results show a strong dependence of the leakage currents on external calcium or magnesium concentration but provide no support for the involvement of calcium or magnesium in the kinetics of the potassium system.     

The deamination of methyl 5-amino-5,6-dideoxy-2,3-O-isopropylidene-α-L-talo- and -β-D-allo-furanoside with nitrous acid

Deamination of methyl 5-amino-5,6-dideoxy-2,3-O-isopropylidene-α-L-talofuranoside (6) with sodium nitrite in 90% acetic acid at ≈0° gave methyl 6-deoxy-2,3-O-isopropylidene-α-L-talofuranoside (8a) and methyl 6-deoxy-2,3-O-isopropylidene-β-D-allofuranoside (9a) (combined yield, 12.3%), the corresponding 5-acetates 8b (2.9%) and 9b (26.4%), and the unsaturated sugar methyl 5,6-dideoxy-2,3-O-isopropylidene-β-D-ribo-hex-5-enofuranoside (10) (43.5%). Similar deamination of methyl 5-amino-5,6-dideoxy-2,3-O-isopropylidene-β-D-allofuranoside (7) gave 8a and 9a (combined yield, 20.4%), 8b (12.5%), 9b (25.8%), 10 (7.7%), and the rearranged products 6-deoxy-2,3-O-isopropylidene-5-O-methyl-L-talofuranose (13a, 17.5%) and the corresponding 1-acetate 13b (14.1%). A synthesis of 13a was accomplished by successive methylation and debenzylation of the conveniently prepared benzyl 6-deoxy-2,3-O-isopropylidene-α-L-talofuranoside (15b). Differences between the two sets of deamination products can be rationalized by assuming that the carbonium-ion intermediate reacts in the initial conformation assumed, before significant interconversion to other conformations occurs.     

Glycosyl esters of amino acids : Part IV. Synthesis of 1-O-(acylaminoacyl)-2,3,4,6-tetra-O-benzyl-D-glucopyranoses by the imidazole-promoted active ester and dicyclohexylcarbodiimide methods

1-O-(Acylaminoacyl)-2,3,4,6-tetra-O-benzyl-D-glucopyranoses were prepared in high yields by two routes involving direct participation of imidazole in the ester linkage formation; namely, (a) the accelerated active-ester method, and (b) the imidazole-promoted dicyclohexylcarbodiimide condensation. The compounds were synthesized from 2,3,4,6-tetra-O-benzyl-α-D-glucopyranose and pentachlorophenyl esters of optically active benzyloxycarbonyl- and tert-butyloxycarbonyl-amino acids in method (a) or benzyloxycarbonyl- and acetyl-amino acids in method (b). By both methods, anomeric mixtures of D-glucosyl esters were obtained; they were resolved by column chromatography and the α and β anomers were fully characterized. The retention of configuration of the amino acid moiety was determined from optical rotations of acylamino acid methyl esters formed from D-glucosyl esters with methanolic sodium methoxide. With benzyloxycarbonyl and tert-butyloxycarbonyl protecting groups, a high degree of retention of optical activity was established in both methods-method (a) being slightly superior.     

Reduction of nitric oxide in bacterial nitric oxide reductase—a theoretical model study

The mechanism of the nitric oxide reduction in a bacterial nitric oxide reductase (NOR) has been investigated in two model systems of the heme-b₃-Fe_B active site using density functional theory (B3LYP). A model with an octahedral coordination of the non-heme Fe_B consisting of three histidines, one glutamate and one water molecule gave an energetically feasible reaction mechanism. A tetrahedral coordination of the non-heme iron, corresponding to the one of Cu_B in cytochrome oxidase, gave several very high barriers which makes this type of coordination unlikely. The first nitric oxide coordinates to heme b₃ and is partly reduced to a more nitroxyl anion character, which activates it toward an attack from the second NO. The product in this reaction step is a hyponitrite dianion coordinating in between the two irons. Cleaving an NO bond in this intermediate forms an Fe_B (IV)O and nitrous oxide, and this is the rate determining step in the reaction mechanism. In the model with an octahedral coordination of Fe_B the intrinsic barrier of this step is 16.3 kcal/mol, which is in good agreement with the experimental value of 15.9 kcal/mol. However, the total barrier is 21.3 kcal/mol, mainly due to the endergonic reduction of heme b₃ taken from experimental reduction potentials. After nitrous oxide has left the active site the ferrylic Fe_B will form a μ-oxo bridge to heme b₃ in a reaction step exergonic by 45.3 kcal/mol. The formation of a quite stable μ-oxo bridge between heme b₃ and Fe_B is in agreement with this intermediate being the experimentally observed resting state in oxidized NOR. The formation of a ferrylic non-heme Fe_B in the proposed reaction mechanism could be one reason for having an iron as the non-heme metal ion in NOR instead of a Cu as in cytochrome oxidase.     

MYONEURAL JUNCTIONS OF TWO ULTRASTRUCTURALLY DISTINCT TYPES IN EARTHWORM BODY WALL MUSCLE

The longitudinal muscle of the earthworm body wall is innervated by nerve bundles containing axons of two types which form two corresponding types of myoneural junction with the muscle fibers Type I junctions resemble cholinergic neuromuscular junctions of vertebrate skeletal muscle and are characterized by three features: (a) The nerve terminals contain large numbers of spherical, clear, ~500 A vesicles plus a small number of larger dense-cored vesicles (b) The junctional gap is relatively wide (~900 A), and it contains a basement membrane-like material, (c) The postjunctional membrane, although not folded, displays prominent specializations on both its external and internal surfaces The cytoplasmic surface is covered by a dense matrix ~200 A thick which appears to be the site of insertion of fine obliquely oriented cytoplasmic filaments The external surface exhibits rows of projections ~200 A long whose bases consist of hexagonally arrayed granules seated in the outer dense layer of the plasma membrane The concentration of these hexagonally disposed elements corresponds to the estimated concentration of both receptor sites and acetylcholinesterase sites at cholinergic junctions elsewhere. Type II junctions resemble the adrenergic junctions in vertebrate smooth muscle and exhibit the following structural characteristics: (a) The nerve fibers contain predominantly dense-cored vesicles ~1000 A in diameter (b) The junctional gap is relatively narrow (~150 A) and contains no basement membrane-like material, (c) Postjunctional membrane specialization is minimal. It is proposed that the structural differences between the two types of myoneural junction reflect differences in the respective transmitters and corresponding differences in the mechanisms of transmitter action and/or inactivation.     

Structure-based design,synthesis, and biological evaluation of lipophilic-tailed monocationic inhibitors of neuronal nitric oxide synthase

Selective inhibitors of neuronal nitric oxide synthase (nNOS) have the potential to develop into new neurodegenerative therapeutics. Recently, we described the discovery of novel nNOS inhibitors (1a and 1b) based on a cis-pyrrolidine pharmacophore. These compounds and related ones were found to have poor blood–brain barrier permeability, presumably because of the basic nitrogens in the molecule. Here, a series of monocationic compounds was designed on the basis of docking experiments using the crystal structures of 1a,b bound to nNOS. These compounds were synthesized and evaluated for their ability to inhibit neuronal nitric oxide synthase. Despite the excellent overlap of these compounds with 1a,b bound to nNOS, they exhibited low potency. This is because they bound in the nNOS active site in the normal orientation rather than the expected flipped orientation used in the computer modeling. The biphenyl or phenoxyphenyl tail is disordered and does not form good protein–ligand interactions. These studies demonstrate the importance of the size and rigidity of the side chain tail and the second basic amino group for nNOS binding efficiency and the importance of the hydrophobic tail for conformational orientation in the active site of nNOS.     

Differential effects of metabolites on the active and inactive forms of hepatic acetyl CoA carboxylase

Partially purified acetyl CoA carboxylase was converted in vitro to its predominately phosphorylated (less active, b) or dephosphorylated (active, a) form. Studies of the properties of the two forms of carboxylase indicated that the a-form had a greater V than the b-form in the presence of different concentrations of citrate, pyruvate, MgATP^2?, MnATP^2?, acetyl CoA, and palmityl CoA. The concentration required for half-maximum stimulation of the a-form was less for citrate and the same as the b-form for MgATP^2?, MnATP^2?, and acetyl CoA. The concentration required for half-maximum inhibition of the a-form was higher for palmityl CoA, avidin, and ATP. The b-form was more strongly inhibited by palmityl CoA and avidin and this inhibition was partially reversed by citrate. These studies indicate that under normal physiological concentrations of metabolites, the b-form is virtually inactive. The physiological significance of the interconversion between the two forms of acetyl CoA carboxylase thus appears to lie in their differential response to the various metabolites which regulate the enzyme activity.     

Changes in membrane characteristics of heart muscle during inhibition

Membrane characteristics were studied in isolated muscle strands from auricles of frogs using the "square pulse" technique. Changes in the time course and spatial spread of subthreshold electrotonic potentials were measured. If acetylcholine is applied in concentrations which cause slowing or stoppage of the heart beat, the following changes are produced: (a) the length constant (λ) of the membrane is reduced, (b) the time constant is shortened. The effects are reversible and increase with acetylcholine concentration. The membrane changes caused by acetylcholine dimmish with time. It is concluded that during acetylcholine inhibition, as well as during vagal inhibition, the conductance of the muscle membrane is increased. Appreciable changes in the resting membrane potential need not accompany inhibition.     

(R)- and (S)-4-Amino-3-(trimethylsilyl)methylbutanoic acids ameliorate neuropathic pain without central nervous system-related side effects

Neuropathic pain is a chronic pain condition resulting from neuronal damage, and is usually treated with pregabalin or gabapentin, which are structurally related to γ-aminobutyric acid (GABA) and are originally developed as anticonvulsant drugs. Here, we report the synthesis and pharmacology of (R)- and (S)-4-amino-3-(trimethylsilyl)methylbutanoic acids (1a and 1b), which showed analgesic activity as potent as that of pregabalin in the Chung spinal nerve ligation model. However, unlike pregabalin, 1a and 1b do not have antiepileptic effects, and they are therefore promising candidates for selective therapeutic agents to treat neuropathic pain without central nervous system-related side effects.     

Depsidones and other constituents from Phomopsis sp. CAFT69 and its host plant Endodesmia calophylloides with potent inhibitory effect on motility of zoospores of grapevine pathogen Plasmopara viticola

In our search for secondary metabolites regulating the motility behavior of zoospores of the grapevine downy mildew pathogen Plasmopara viticola, we found that extracts from an endophytic fungus Phomopsis sp. CAFT69 and its host plant Endodesmia calophylloides remarkably impaired motility of zoospores followed by lysis. The active principles in the extracts were isolated and identified as two new compounds, namely excelsional (1a) and 9-hydroxyphomopsidin (2a), together with excelsione (1b), phomopsidin (2b), alternariol (3a), alternariol-5-O-methyl ether (3b), the hitherto undescribed 5′-hydroxyalternariol (3c), altenusin (4) from the fungus, xanthochymol (5) and 1,5-dihydroxy-3-methoxyxanthone (mesuaxanthone, 6) from the plant. Bioassays revealed that compounds 1a/b, 2a/b, and 3a–6 displayed motility inhibition and lytic activities against zoospores of the grapevine downy mildew pathogen P. viticola in a dose- and time-dependent manner from 1 to 10 μg/mL. Their structures were elucidated by extensive spectroscopic analyses including 2D NMR techniques. This is the first report of an endophyte and its natural products from E. calophylloides and the first isolation of compounds 5 and 6 from this plant.     

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker^1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain^3,4 and ventral nerve cord of embryonic^5,6, larval^7,8,9,10, and adult Drosophila^11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN5¹⁵), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.     

Studies on chlorogenic Acid biosynthesis in sweet potato root tissue in special reference to the isolation of a chlorogenic Acid intermediate

Marked polyphenol production takes place in root tissue of sweet potato, Ipomoea batatas Lam. cv. Norin 1, in response to slicing. A possible intermediate, tentatively termed compound V, of chlorogenic acid biosynthesis was isolated from the root tissue administrated with t-cinnamic acid-2-¹⁴C. Compound V was proved to be an ester whose acid moiety was t-cinnamic acid, and the hydroxyl group-bearing moiety appeared to be a carbohydrate. Compound V was suggested to be the first intermediate after t-cinnamic acid involved in the chlorogenic acid biosynthetic pathway by the following three results. (a) label of t-cinnamic acid-2-¹⁴C was distributed in compound V first, then transferred to chlorogenic acid and isochlorogenic acid, isomers of dicaffeoylquinic acid; (b) specific radioactivity of compound V increased prior to that of the fraction containing chlorogenic acid and isochlorogenic acids and decreased prior to that of the latter; and (c) label of compound V was efficiently incorporated into chlorogenic acid and isochlorogenic acid.     

A novel method for the methylation of heterocyclic amino groups. Conversion of guanosine into its 2-N-methyl- and 2-N,2-N-dimethyl derivatives

The heterocyclic amino-compounds 11a, 13a, 13b, and 17 reacted with formaldehyde and p-thiocresol (14) in alcoholic solution to give the corresponding N-methylphenylthiomethyl derivatives (16, 15a, 15b, and 18a, respectively) in satisfactory to good yields. The reactions were catalyzed by acetic acid. 2-N-Methylguanosine (6a) was obtained in good yield by treatment of 15b with sodium borohydride followed by acidic hydrolysis, or alternatively by Raney nickel desulfurization of 15a followed by ammonolysis of the product. Sodium borohydride reduction of 18a gave 21 in good yield. 2-N,2-N-Dimethylguanosine (6b) was obtained from 19a in three steps.     

Differences between Pacemaker and Nonpacemaker Neurons of Aplysia on Voltage Clamping

The responses of pacemaker and nonpacemaker Aplysia neurons to voltage clamp commands of less than 200 msec duration are essentially identical. For moderate depolarizing commands there is an early inward transient current followed by a late outward current and an outward tail current when the membrane is clamped back to resting potential. On long (1–2 sec) commands in pacemakers there is a marked sag in the late current and an inward tail current. E_tail, the potential of the membrane at which there is no net current flow under the conditions prevailing at the end of the clamp, shifts from about -9.0 mv on short commands to +5.0 mv on long commands. In contrast there is no marked sag of the late current or inward tail current on long commands in nonpacemakers, and E_tail is near -9.0 mv for both short and long commands. The current sag and shift in E_tail can be ascribed to a decreased conductance (presumably to K⁺) at the end of the long as compared to the short command in half of the pacemaker neurons. In the remaining cells the essential difference from nonpacemakers appears to be either a greater restricted extracellular space or a more active potential-dependent electrogenic Na⁺ pump in pacemakers.     

LYSOSOMES IN THE RAT SCIATIC NERVE FOLLOWING CRUSH

Peripheral nerves undergoing degeneration are favorable material for studying the types, origins, and functions of lysosomes. The following lysosomes are described: (a) Autophagic vacuoles in altered Schwann cells. Within these vacuoles the myelin and much of the axoplasm which it encloses in the normal nerve are degraded (Wallerian degeneration). The delimiting membranes of the vacuoles apparently form from myelin lamellae. Considered as possible sources of their acid phosphatase are Golgi vesicles (primary lysosomes), lysosomes of the dense body type, and the endoplasmic reticulum which lies close to the vacuoles. (b) Membranous bodies that accumulate focally in myelinated fibers in a zone extending 2 to 3 mm distal to the crush. These appear to arise from the endoplasmic reticulum in which demonstrable acid phosphatase activity increases markedly within 2 hours after the nerve is crushed. (c) Autophagic vacuoles in the axoplasm of fibers proximal to the crush. The breakdown of organelles within these vacuoles may have significance for the reorganization of the axoplasm preparatory to regeneration. (d) Phagocytic vacuoles of altered Schwann cells. As myelin degeneration begins, some axoplasm is exposed. This is apparently engulfed by the filopodia of the Schwann cells, and degraded within the phagocytic vacuoles thus formed. (e) Multivesicular bodies in the axoplasm of myelinated fibers. These are generally seen near the nodes of Ranvier.