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PubMed1994

Light-dependent hydration of the space surrounding photoreceptors in chick retina.

PURPOSE: To characterize the light-evoked increase in the volume (or hydration) of the subretinal space (SRS) of chick retina and to investigate its retinal pigment epithelial (RPE) origin. METHODS: The authors perfused chick retina-RPE-choroid preparations with the extracellular space marker, tetramethylammonium (TMA+), and estimated its concentration ([TMA+]o) using double-barreled, ion-selective electrodes. They recorded dark-light changes in subretinal [TMA+]o and studied the effects of drugs that suppress RPE ion transport and conductances. RESULTS: The authors observed a slow, light-evoked decrease in subretinal [TMA+]o, about 7% in amplitude, that slowly returned to baseline and then increased at light OFF. It was 40% to 65% inhibited by bumetanide and furosemide, known to inhibit RPE apical membrane Na-K-Cl cotransport; 70% to 80% inhibited by DIDS, an inhibitor of RPE basal membrane chloride conductance, and 50% inhibited by 0.5 mM BaCl2, a blocker of the K+ conductance of the RPE apical membrane. Decreases in subretinal [TMA+]o also were produced when the authors lowered [Cl-]o or [K+]o in the SRS by reducing concentrations in the retinal perfusate. The effect on [TMA]o was larger for [Cl-]o than for [K+]o and was inhibited by bumetanide and DIDS. CONCLUSIONS: Changes in subretinal ion concentrations in light and darkness, most likely [K+]o, modify SRS hydration by modulating RPE apical membrane Na-K-Cl cotransport, as well as the conductive effluxes of K+ across the apical membrane and Cl- across the basal membrane. These changes depend principally on the movement of water into or out of the RPE. In turn, subretinal hydration modifies local ion concentrations and the composition of the interphotoreceptor matrix.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1973

A critical review of currently used single-dose rodenticides.

The introduction of the anticoagulants in the early 1950s, with their much greater safety to nontarget animals, resulted in a general decline in the use of single-dose rodenticides. However, the appearance of rodent resistance to the anticoagulants, first in the United Kingdom, later elsewhere in Europe, and still more recently in the USA, has revived interest in the use of single-dose rodenticides. Unfortunately, owing to their danger to nontarget mammals, the use of several of these compounds must be restricted; others, despite their long use, are now recognized to be unsatisfactory because of their poor acceptance or reacceptance by rats and mice. Thus, only very few compounds of this type are available for unrestricted use and there is an urgent need for the development of effective alternatives.

Amides↗
PubMed1970

Bacteriolytic enzymes from Staphylococcus aureus. Properties of the endo-beta-N-acetylglucosaminidase.

An extracellular bacteriolytic endo-beta-N-acetylglucosaminidase has been purified and its specificity of action has been investigated (Wadström & Hisatsune, 1970a,b). Some enzymic properties, such as optimum pH for enzyme activity on whole cells and cell walls of Micrococcus lysodeikticus and Staphylococcus aureus and optimum pH for stability, have been studied. The activity was maximum in 0.05m-tris-hydrochloric acid buffer, pH7.0. A higher ionic strength inhibited cell-wall hydrolysis. Since the crude and purified enzymes were found to be unstable on storage, the stabilizing and inhibiting effects of several compounds were investigated. Several heavy metal ions inactivated the enzyme at very low concentrations. Thiol compounds stabilized and thiol-reacting compounds partly inhibited the activity. Crude and purified glucosaminidase was found to be heat-stable at acidic pH and unstable at alkaline pH as previously found for several lysozymes (endo-beta-N-acetylmuramidases). Other properties of the staphylococcal enzyme and hen's-egg-white lysozyme have been compared, since the modes of action of the two are quite similar (Wadström & Hisatsune, 1970b).

Bacteriolysis↗
PubMed2001

Barium oxoaluminate hydride.

The crystal structure of Ba(3)(AlO(4))H is isotypic with Ba(3)SiS(5) and contains AlO(4)(5-) and H(-) anions. The hydride and oxide anions are coordinated by six Ba and five Ba/one Al atoms in an octahedral geometry. The hydrogen content was examined by MAS-NMR experiments of the deuterated compound.

Journal Article↗
PubMed1977

Effect of starvation on excretion, distribution and metabolism of DDT in mice.

Effect of starvation on tissue radioactivity of 14C-DDT was examined in mice 8 days after its single injection. Animals were completely fasted and given barium sulfate by gastric intubation for the last 3 days. The findings obtained from starved mice were as follows: 1) loss of body weight, 2) decrease in organ weight of epedidymal fat and liver, 3) reduction in lipid content of whole body as well as of epididymal fat, 4) a marked elevation of DDT levels in tissues except muscle, and 5) occasional neurotoxidc signs characterized by tremors and convulsions. Excretion of DDT-related metabolites was not changed by starvation. Analysis of metabolic producets of 14C-DDT using thin-layer radiochromatography revealed that there was little or no significant difference between control and starved mice in the metabolic pattern of DDT-related compounds in the tissues and excreta. It was assumed the DDT-ingested animals with dietary energy restriction had a subsequent risk of toxicity resulted from redistribution of DDT, but not from alteration in excretion or metabolism.

Adipose Tissue↗
PubMed1993

Redistribution of hepatocyte chloride during L-alanine uptake.

We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (Vm), intracellular K+, Cl-, and Na+ activities (aiK, aiCl and aiNa), and water volume during L-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 mM L-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 mM tetramethylammonium ion (TMA+) for 10 min. Changes in cell water volume during L-alanine uptake were determined by changes in intracellular, steady-state TMA+ activity measured with the K+ electrode. Hepatocyte control Vm was -33 +/- 1 mV. L-alanine uptake first depolarized Vm by 2 +/- 0.2 mV and then hyperpolarized Vm by 5 mV to -38 +/- 1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, aiNa increased by 30% from 19 +/- 2 to 25 +/- 3 mM (P < 0.01), and aiK did not change significantly from 83 +/- 3 mM. However, with added ouabain (1 mM) L-alanine caused only a 2-mV increase in Vm, but now aiK decreased from 61 +/- 3 to 54 +/- 5 mM (P < 0.05). Hyperpolarization of Vm by L-alanine uptake also resulted in a 38% decrease of aiCl from 20 +/- 2 to 12 +/- 3 mM (P < 0.001). Changes in Vm and VCl-Vm voltage traces were parallel during the time of L-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl- with the Vm in hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1993

Hepatocyte water volume and potassium activity during hypotonic stress.

Hepatocytes exhibit a regulatory volume decrease (RVD) during hypotonic shock, which comprises loss of intracellular K+ and Cl- accompanied by hyperpolarization of transmembrane potential (Vm) due to an increase in membrane K+ conductance, (GK). To examine hepatocyte K+ homeostasis during RVD, double-barrel, K+-selective microelectrodes were used to measure changes in steady-state intracellular K+ activity (aKi) and Vm during hyposmotic stress. Cell water volume change was evaluated by measuring changes in intracellular tetramethylammonium (TMA+). Liver slices were superfused with modified Krebs physiological salt solution. Hyposmolality (0.8 x 300 mosm) was created by a 50 mM step-decrease of external sucrose concentration. Hepatocyte Vm hyperpolarized by 19 mV from -27 +/- 1 to -46 +/- 1 mV and aiK decreased by 14% from 91 +/- 4 to 78 +/- 4 mM when slices were exposed to hyposmotic stress for 4-5 min. Both Vm and aKi returned to control level after restoring isosmotic solution. In paired measurements, hypotonic stress induced similar changes in Vm and aKi in both control and added ouabain (1 mM) conditions, and these values returned to their control level after the osmotic stress. In another paired measurement, hypotonic shock first induced an 18-mV increase in Vm and a 15% decrease in aKi in control condition. After loading hepatocytes with TMA+, the same hypotonic shock induced a 14-mV increase in Vm and a 14% decrease in aTMAi. This accounted for a 17% increase of intracellular water volume, which was identical to the cell water volume change obtained when aKi was used as the marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Effects of K+ channel blockers on inwardly and outwardly rectifying whole-cell K+ currents in sheep parotid secretory cells.

We have used whole-cell patch-clamp techniques to examine the sensitivities of the inwardly and the outwardly rectifying K+ currents in sheep parotid cells to K+ channel blockers. Extracellular tetraethylammonium (ID50 approximately 200 mu mol/liter), quinine (ID50 approximately 100 mu mol/liter), verapamil (ID50 approximately 30 mumol/liter) and charybdotoxin (ID50 < 0.1 mu mol/liter) reduced the outwardly rectifying current but had no effect on the inwardly rectifying current. Quinidine inhibited the outwardly rectifying current (ID50 approximately 200 mu mol/liter) and, at a concentration of 1 mmol/liter, reduced the inwardly rectifying current by 35%. Extracellular Ba2+ inhibited both the inwardly and outwardly rectifying K+ currents but the inwardly rectifying K+ current was more sensitive to it (ID50 approximately 1 mu mol/liter) than was the outwardly rectifying K+ current (ID50 approximately 2 mmol/liter). Extracellular Cs+ reduced the inwardly rectifying K+ current (ID50 approximately 100 mu mol/liter) without affecting the outwardly rectifying current; 4-aminopyridine (1 or 10 mmol/liter), lidocaine (0.1 or 1 mmol/liter) and flecainide (0.01 or 0.1 mmol/liter) affected neither current. In excised outside-out patches, the addition to the bath of quinine (100 mu mol/liter), quinidine (100 mu mol/liter), verapamil (100 mu mol/liter) or charybdotoxin (100 nmol/liter) inhibited Ca(2+)- and voltage-sensitive 250 pS K+ channels (BK channels), but 4-aminopyridine (1 mmol/liter) and lidocaine (0.1 mmol/liter) did not. The pattern of blocker sensitivities is thus consistent with the hypothesis that BK channels are responsible for the outwardly rectifying whole-cell current seen in resting sheep parotid cells.

Animals↗
PubMed1986

Two different presynaptic calcium currents in mouse motor nerve terminals.

Extracellular recordings of potential changes under the perineural sheath of nerve bundles close to some of the nerve terminals were performed using the M. triangularis sterni of the mouse. The nerve signals consisted of a predominant double-peaked negativity which was often preceded by a small positive deflection. While the first negative peak is related to the propagating nerve action potential, the second negative deflection can be attributed to a potassium conductance since it was selectively blocked by tetraethylammonium (TEA) or 3,4-diaminopyridine (3,4-DAP). Combined application of TEA and 3,4-DAP gave rise to a prolonged positive-going wave which was blocked by Cd2+, thus, indicating its underlying cause to be a Ca current. Ionophoretic application of TEA and Cd2+ to the endplates affected potassium and calcium components of the subendothelial signals, respectively, thus indicating their presynaptic origin. This finding is supported by the decrease of the amplitude of these components with increasing distance from the endplate region. Maximal effects on K conductance attainable with 3,4-DAP could still be potentiated by TEA, indicating the presence of at least two distinct sets of K channels. The prolonged positive potential induced by TEA and 3,4-DAP consisted of a fast and slow component, both of which can be attributed to Ca conductances with different characteristics. The fast positive signal component is attributed to the voltage-dependent Ca channel, responsible for the initiation of transmitter release. Its amplitude and duration depend on extracellular Ca2+ -concentration. The fast component is still present when Ca2+ is substituted by Sr2+ or Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Aminopyridine↗
PubMed1988

Ca2+-activated K+ channels in the apical membrane of Necturus choroid plexus.

The properties of Ca2+-activated K+ channels in the apical membrane of the Necturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K+ over Na+ and K+ over Cs+. They were blocked by high cytoplasmic Na+ concentrations (110 mM). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca2+ concentrations. Increasing Ca2+ from 5 to 500 nM, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca2+ was 500 nM. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba2+ reduced both channel-open probability and conductance. The sites for the action of Ba2+ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117 mM were calculated as the apparent Ba2+ dissociation constants (KD(0 mV] for the effects on probability and conductance, respectively. TEA+ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with a KD(0 mV) = 5.6 mM. A second site, with a higher affinity, KD(0 mV) = 0.23 mM, may account for the near total block of channel conductance by 2 mM TEA+ applied to the outside of the membrane. It is concluded that the channels in Necturus choroid plexus exhibit many of the properties of "maxi" Ca2+-activated K+ channels found in other tissues.

Animals↗
PubMed1996

An inactivating inward-rectifying K current present in prolactin cells from the pituitary of lactating rats.

Primary cultures containing a high percentage of lactotrophs were obtained by dissociating the pituitary of rats following 14-18 days of lactation. Lactotrophs with a distinctive appearance were recorded after 1-35 days in vitro and identified by immunocytochemical staining for prolactin. Whole-cell voltage clamp measurements in isotonic KCl solution from a holding potential of -40 mV revealed the presence of inward-rectifying K currents with a time-dependent, Na(+)-independent inactivation at potentials negative to -60 mV. The time for complete inactivation was strikingly different between lactotrophs, varying between 1 sec and more than 5 sec at -120 mV, and was not related to time in culture. The reversal potential shifted 59 mV (25 degree C) for a tenfold change in external K+ concentration, demonstrating the selectivity of the channel for K+ over Na+. The inward-rectifying K current was blocked by 5 mM Ba2+ and partially blocked by 10 mM TEA. Chloramine-T (1 and 2 mM) produced a total block of the inward-rectifying K current in lactotrophs. Thyrotropin-releasing hormone (500 nM) significantly reduced the inward-rectifying K current in about half of the lactotrophs. This current is similar to the inward-rectifying K current previously characterized in clonal somatomammotrophic pituitary cells (GH3B6). The variability of the rate of inactivation of this current in lactotrophs and its responsiveness to TRH is discussed.

Animals↗
PubMed1997

Characterization of an inward-rectifying potassium current in NG108-15 neuroblastoma x glioma cells.

By using the whole-cell patch-clamp technique, an inwardly rectifying potassium current, which resembled the "classic" inward-rectifying potassium current (IKIR) of other cells in terms of electrophysiological and pharmacological properties, was identified in db-cAMP-differentiated NG108-15 cells. First, the current was dependent on voltage and time. It could be elicited by applying an initial depolarizing prepulse and a subsequent hyperpolarizing command pulse to the cell. The amplitude of the current depended on both the prepulse and the command pulse and increased with the hyperpolarization of the command pulse as well as the depolarization and the prolongation of the prepulse. The activation and inactivation of the current could be fitted well by single-exponential functions and increased with the hyperpolarization of the membrane. Second, the current was dependent on the extracellular potassium concentration ([K+]o). Elevation of [K+]o resulted in a marked increase in the current amplitude and a positive shift of the peak-current/voltage curve as well as the reversal potential. A tenfold increase of [K+]o introduced an approximately 43-mV shift of the reversal potential, indicating that the current was carried mainly by K+. The conductance (g/gMax) of the current was also dependent on the [K+]o and increased with increases in [K+]o in a manner approximately proportional to the square-root of [K+]o. Finally, the current was sensitive to Cs+ (1 mmol/l), Ba2+ (1 mmol/l) and quinidine (0.2 mmol/l); whereas, two typical potassium channel inhibitors, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), were weak blockers and reduced the current at high concentration (>10 mmol/l). It was also observed that the current was depressed by Cd2+ (1 mmol/l) and Co2+ (1 mmol/l) and increased by perfusing the cell with Ca2+-free solution. Thus, except for the sensitivity to Cd2+, Co2+ and Ca2+, the current displayed most of the hallmarks described for the "classic" IKIR. In conclusion, there appears to be a voltage-dependent IKIR-type inward rectifier in db-cAMP-differentiated NG108-15 cells.

4-Aminopyridine↗
PubMed1984

Electrophysiological properties of dentate granule cells in mouse hippocampal slices maintained in vitro.

Intracellular recordings were obtained from granule cells of the dentate gyrus in mouse hippocampal slices maintained in vitro. All the spikes observed in standard Krebs solution had a short duration and were tetrodotoxin (TTX)-sensitive. When elicited by synaptic activation or by direct electrical stimulation of the cells, these fast sodium spikes were followed by a brief spike afterhyperpolarization. In contrast, antidromic spikes elicited by electrical stimulation of the hilum as well as spikes arising at the end of hyperpolarizing current pulses passed through the recording microelectrode were followed by depolarizing afterpotentials (DAPs). These DAPs were reversed into brief spike afterhyperpolarizations by depolarization of the cells. After substitution of calcium (Ca) by barium (Ba) or after introduction of tetraethylammonium (TEA) in the bath, the fast spike repolarization became slower and the brief spike afterhyperpolarizations were abolished, suggesting that they involved fast K conductances. Slow spikes and long-lasting depolarizations were also elicited in granule cells in the presence of Ba or TEA. Since these slow events were left unaffected by TTX and were selectively abolished by the Ca channel blockers cobalt or cadmium, they are likely to be mediated by voltage-dependent Ca conductances, unmasked by the reduction of the fast K conductances.

Animals↗
PubMed1995

Persistent activation of min K channels by chemical cross-linking.

Expression of the structurally and functionally distinct min K channel in Xenopus oocytes results in voltage-dependent potassium currents that activate with a characteristic slow time course. Application of a membrane-impermeable chemical cross-linking agent to oocytes expressing min K decreased the time-dependent current, increased its rate of activation, and induced persistently activated inward and outward potassium currents. These effects required membrane depolarization, demonstrating use dependence. Persistently activated channels retained potassium selectivity and sensitivity to block by clofilium and barium. These results suggest that a major conformational change occurs during min K channel gating, which can be stabilized by chemical cross-linking, and are consistent with a model in which min K channels activate by voltage-dependent subunit aggregation.

Animals↗
PubMed1998

Ba2+ does not support synaptic vesicle retrieval in rat cerebrocortical synaptosomes.

To investigate whether any specific requirement for extracellular Ca2+ exists in the control synaptic vesicle retrieval, we examined the ability of the divalent cation Ba2+ to substitute for Ca2+ in both vesicle exocytosis and endocytosis. Ba2+ stimulated glutamate release from rat cerebrocortical synaptosomes. Ba2+-evoked release was inhibited by bafilomycin A1, indicating release was via exocytosis of synaptic vesicles. However, Ba2+ did not stimulate vesicle retrieval, monitored by a FM2-10-based retrieval assay. Therefore synaptic vesicle retrieval in central nerve terminals has a specific requirement for extracellular Ca2+ and the Ca2+ receptor for retrieval has a different cation specificity to the Ca2+ receptor for exocytosis.

4-Aminopyridine↗
PubMed2000

Determination of sugar compounds in olive plant extracts by anion-exchange chromatography with pulsed amperometric detection.

We describe a chromatographic method that uses isocratic elution and pulsed amperometric detection to determine soluble carbohydrates in plant tissues. Such a method provides a rapid and convenient means to obtain a complete profile of the sugar components of leaves and roots from olive (Olea europaea L. cv. Coratina) plants. A simple purification of plant extracts using pure water was developed, which is far less time-consuming and retains a high level of accuracy. Excellent separation of myo-inositol, galactinol, mannitol, galactose, glucose, fructose, sucrose, raffinose, and stachyose was achieved with an anion-exchange column and 12 mM NaOH spiked with 1 mM barium acetate as an eluent. At a flow rate of 1.0 mL/min, the time of analysis was less than 25 min, and repeatability of the method on the order of 2.2% as RSD or better for retention times and lower than 5.2% for peak areas. Recoveries approximated 100% (range 97.2-104.5%), and the method provided good precision with a coefficient of variation which ranged between 0.9 and 3.3%. Among identified carbohydrates extracted from leaves and roots of olive plants, glucose and mannitol were major compounds. Their molar ratio was estimated to be 1.2+/-0.1 and 2.2+/-0.3 for olive leaves and roots, respectively. The occurrence of soluble galactinol in plant tissues was also validated.

Anion Exchange Resins↗
PubMed2000

Nitric oxide modulates a late step of exocytosis.

The effects of nitric oxide (NO) on the late phase of exocytosis have been studied, by amperometry, on Ba(2+)-stimulated chromaffin cells. Acute incubation with NO or NO donors (sodium nitroprusside, spermine-NO, S-nitrosoglutathione) produced a drastic slowdown of the granule emptying. Conversely, cell treatment with N(omega)-nitro-l-arginine methyl ester (a NO synthase inhibitor) or with NO scavengers (methylene blue, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium) accelerated the extrusion of catecholamines from chromaffin granules, suggesting the presence of a NO modulatory tone. The incubation with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine or zaprinast) or with the cell-permeant cGMP analog 8-bromo-cGMP, mimicked the effects of NO, suggesting the involvement of the guanylate cyclase cascade. NO effects were not related to changes in intracellular Ba(2+). NO did not modify the duration of feet. Effects were evident even on pre-fusioned granules, observed under hypertonic conditions, suggesting that the fusion pore is not the target for NO, which probably acts by modifying the affinity of catecholamines for the intragranular matrix. NO could modify the synaptic transmitter efficacy through a novel mechanism, which involves the regulation of the emptying of secretory vesicles.

1-Methyl-3-isobutylxanthine↗