scholarly journals Spectrophotometric measurements of transmembrane potential and pH gradients in chromaffin granules.

1980 ◽  
Vol 75 (2) ◽  
pp. 109-140 ◽  
Author(s):  
G Salama ◽  
R G Johnson ◽  
A Scarpa
Keyword(s):  
Time Course ◽  
Electrical Potential ◽  
Accurate Method ◽  
Rapid Quenching ◽  
Chromaffin Granules ◽  
Experimental Conditions ◽  
Ph Gradients ◽  
Nernst Potential ◽  
Carbonyl Cyanide ◽  
Granule Membrane

The electrical potential (delta psi) and proton gradient (alpha pH) across the membranes of isolated bovine chromaffin granules and ghosts were simultaneously and quantitatively measured by using the membrane-permeable dyes 3,3'dipropyl-2,2'thiadicarbocyanine (diS-C3-(5)) to measure delta psi and 9-aminoacridine or atebrin to measure delta pH. Increases or decreases in the delta psi across the granular membrane could be monitored by fluorescence or transmittance changes of diS-C3-(5). Calibration of the delta psi was achieved by utilization of the endogenous K+ and H+ gradients, and valinomycin or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), respectively, with the optical response of diS-C3-(5) varying linearly with the Nernst potential for H+ and K+ over the range -60 to +90 mV. The addition of chromaffin granules to a medium including 9-aminoacridine or atebrin resulted in a rapid quenching of the dye fluorescence, which could be reversed by agents known to cause collapse of pH gradients. From the magnitude of the quenching and the intragranular water space, it was possible to calculate the magnitude of the alpha pH across the chromaffin granule membrane. The time-course of the potential-dependent transmittance response of diS-C3-(5) and the delta pH-dependent fluorescence of the acridine dyes were studied simultaneously and quantitatively by using intact and ghost granules under a wide variety of experimental conditions. These results suggest that membrane-permeable dyes provide an accurate method for the kinetic measurement of delta pH and delta psi in an amine containing subcellular organelle.

Measurement of the amount and isotopic composition of the CO2 released from the cyanobacterium Synechococcus UTEX 625 after rapid quenching of the active CO2 transport system

1997 ◽  
Vol 75 (6) ◽  
pp. 981-997 ◽  
Author(s):  
Anthony G. Miller ◽  
Christophe Salon ◽  
David T. Canvin ◽  
George S. Espie
Keyword(s):  
Isotopic Composition ◽  
Cell Volume ◽  
Mass Spectrometer ◽  
Transport System ◽  
Time Course ◽  
Rapid Quenching ◽  
Experimental Conditions ◽  
Extracellular Medium ◽  
Fully Depleted ◽  
Entire Cell

Air-grown cells of the cyanobacterium Synechococcus UTEX 625 were suspended in a cuvette connected to a mass spectrometer and supplied with H13C18O3− to investigate the intracellular interconversion between CO2 and HCO3− as determined from the isotopic composition of CO2 appearing in the extracellular medium under a wide variety of experimental conditions. Upon injection of H13C18O3− to the cell suspension in the light, the extracellular [13C16O2] increased. As the CO2 species were 13C labelled, this demonstrated that the 18O-depleted CO2 was originating from the added H13C18O3−. A comparison of the rates of 13C16O16O appearance in the medium with the formation of 13C16O16O from spontaneous dehydration–hydration in the extracellular medium in the presence of cells demonstrated that most of it had to originate from a series of intracellular dehydration–hydration cycles of H13C18O3− that had been recently transported into the cells. During the time course of the experiments both the m/z (mass to charge) = 49 (i.e., 13C18O18O) and 47 (i.e., 13C18O16O) signals decreased constantly, whereas the m/z = 45 signal (i.e.,13C16O2) always increased. Inhibiting CO2 fixation enhanced the amount of CO2 arising in the medium but did not change its isotopic composition, and the CO2 was always fully depleted of 18O. When the CO2 transport system was inhibited by darkening the cells, adding inhibitors such as Na2S or COS, or quenching the uptake of inorganic 13C with an excess of inorganic 12C, the magnitude of the extracellular [13C16O2] was increased but the CO2 species were still always depleted of 18O. Various incubation times of the illuminated cells in the presence of H13C18O3− were used to obtain a variety of internal Ci pool sizes. When the inhibitor (COS) was added, the amount of 13C16O2 arising during the response time of the mass spectrometer was equivalent to the amount of CO2 that would have been present in the whole cell if CO2 and HCO3− were in equilibrium throughout the entire cell volume, but it was at least 40 times higher than the amount of CO2 that would have been present in the cell if the CO2 was confined to the carboxysomes. Experiments were also conducted at pH 9.0 where the spontaneous rate of 13C16O2 production from H13C1803− dehydration–hydration would be negligible, and again the same features were observed. Results show that intracellular HCO3− and CO2 are in rapid equilibrium throughout the entire cell volume. Key words: Synechococcus UTEX 625, cyanobacteria, CO2 leakage, 18O exchange, active CO2 transport, carboxysomes, inorganic C concentrating mechanism.

scholarly journals Ion permeability of isolated chromaffin granules.

1976 ◽  
Vol 68 (6) ◽  
pp. 601-631 ◽  
Author(s):  
R G Johnson ◽  
A Scarpa
Keyword(s):  
Time Course ◽  
Choline Chloride ◽  
Catecholamine Release ◽  
Ionophore A23187 ◽  
Chromaffin Granules ◽  
Ion Permeability ◽  
Chromaffin Granule ◽  
Internal Ph ◽  
Apparent Concentration ◽  
Granule Membrane

The passive ion permeability, regulation of volume, and internal pH of isolated bovine chromaffin granules were studied by radiochemical, potentiometric, gravimetric, and spectrophotometric techniques. Chromaffin granules behave as perfect osmometers between 340 and 1,000 mosM in choline chloride, NaCl, and KCl as measured by changes in absorbance at 430 nm or from intragranular water measurements using 3H2O and [14C]polydextran. By suspending chromaffin granules in iso-osmotic media of various metal ions and selectively increasing the permeability to either the cation or the anion by intrinsically permeable ions or specific ionophores, it was possible to determine by turbidity and potentiometric measurements the permeability to the counterion. These measurements indicate that the chromaffin granule is impermeable to the cations tested (Na+, K+, and H+). Limited H+ permeability across the chromaffin granule membrane was also shown by means of the time course of pH re-equilibration after pulsed pH changes in the surrounding media. The measurement of [14C]methylamine distribution indicates that a significant deltapH exists across the membrane, inside acidic, which at an external value of 6.85 has a value of 1.16. The deltapH is relatively insensitive to changes in the composition of the external media and can be enhanced or collapsed by the addition of ionophores and uncouplers. Measurement at various values of external pH indicates an internal pH of 5.5. Use of the ionophore A23187 indicates that Ca++ and Mg++ can be accumulated against an apparent concentration gradient with calcium uptake exceeding 50 nmol/mg of protein at saturation. These measurements also show that Ca++ and Mg++ are impermeable. Measurement of catecholamine release under conditions where intravesicular calcium accumulation is maximal indicates that catecholamine release does not occur. The physiological significance of the high impermeability to ions and the existence of a large deltapH are discussed in terms of regulation of uptake, storage, and release of catecholamines in chromaffin granules.

A Simple Method for Estimation of Lipoxygenase and Cyclo-Oxygenase Pathways in Human Platelets - the Use of Thiobarbituric Acid Reaction

1980 ◽  
Vol 44 (02) ◽  
pp. 111-114 ◽  
Author(s):  
Hiroshi Takayama ◽  
Minoru Okuma ◽  
Haruto Uchino
Keyword(s):  
Time Course ◽  
Normal Values ◽  
Human Platelet ◽  
Thiobarbituric Acid ◽  
Human Platelets ◽  
Optimal Conditions ◽  
Experimental Conditions ◽  
Simple Method ◽  
Acid Reaction ◽  
Optimal Ph

SummaryTo develop a simple method for estimation of platelet lipoxygenase (PLO) and cyclo-oxygenase (PCO) pathways, the arachidonic acid (AA) metabolism of human platelet was investigated under various experimental conditions by the use of the thiobarbituric acid (TBA) reaction and a radioisotope technique. A TBA-reactive substance different from malondialdehyde (MDA) via PCO pathway was detected and shown to be derived from the PLO pathway. Since the optimal pH and time course of its formation were different from those of MDA formation via PCO pathway, PLO and PCO pathways were estimated by quantitating the TBA-reactive substances produced by the incubation of AA either with aspirin-treated platelets or with untreated ones, respectively, each under optimal conditions. Normal values expressed in terms of nmol MDA/108 platelets were 1.17±0.34 (M±SD, n = 31) and 0.79±0.15 (n = 31) for PLO and PCO pathways, respectively.

The adrenal paraneurone: tubulin organization

1984 ◽  
Vol 62 (5) ◽  
pp. 502-511 ◽  
Author(s):  
M. F. Bader ◽  
F. Bernier-Valentin ◽  
B. Rousset ◽  
D. Aunis
Keyword(s):  
Cell Body ◽  
Intracellular Transport ◽  
Cultured Cells ◽  
Specific Binding ◽  
Secretory Granules ◽  
Immunofluorescent Staining ◽  
Chromaffin Granules ◽  
Two Dimensional Gel Electrophoresis ◽  
Granule Membrane

When chromaffin cells from the bovine adrenal medulla are maintained in culture, they develop neuritelike processes which end with growth-cone-like structures. Chromaffin granules were found to migrate from the cell body to the neurite endings. Thus, the intracellular transport of secretory granules, existing in vivo, seems to occur in an exaggerated way in the cultured cells. These cells offer an excellent model for studying the mechanism of transport, particularly the role of microtubules. By immunofluorescent staining, we observed that tubulin antibodies decorate a complex network visible along the neurites. Colchicine treatment induced the disappearance of this network followed by a return of granules in the cell body and a retraction of neurites. To test the presence of tubulin in the chromaffin granule membrane, we used two-dimensional gel electrophoresis and a radioimmunoassay. Our results indicate that tubulin is not a significant component of chromaffin granules. However, binding experiments show that granule membranes are able to bind tubulin through high affinity binding sites. These results show that microtubules appear involved in neurite formation and probably in granule transport. Tubulin is not an integral constituent of the granule membrane, but is present as a result of a reversible specific binding. This insertion of tubulin into the membrane might represent a step in the association between microtubules and secretory granules.

scholarly journals Calcium metabolism in rat hepatocytes

1978 ◽  
Vol 170 (3) ◽  
pp. 615-625 ◽  
Author(s):  
S Foden ◽  
P J Randle
Keyword(s):  
Cyclic Amp ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Ionophore A23187 ◽  
Total Calcium ◽  
Free Medium ◽  
Adrenergic Agonists ◽  
Calcium Efflux ◽  
Similar Time ◽  
Carbonyl Cyanide

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.

scholarly journals On the mechanism of boundary lubrication. II. Wear prevention by addition agents

1940 ◽  
Vol 177 (968) ◽  
pp. 103-118 ◽  
Keyword(s):  
High Pressures ◽  
Preceding Paper ◽  
Accurate Method ◽  
Polar Compounds ◽  
Experimental Conditions ◽  
Group V ◽  
Highly Sensitive ◽  
Local Pressures ◽  
Steel Balls ◽  
Long Chain

If two metal surfaces slide over each other in the presence of a lubricant and under high load, high pressures and temperatures prevail a t those isolated spots which actually carry the load, leading to wear and possibly to breakdown. The action of wear preventing agents under these conditions has been studied in detail and it has been found that such agents are effective through their chemical polishing action, by which the load becomes distributed over a larger surface and local pressures and temperatures are decreased. Especially effective are compounds containing phosphorus or other elements of group V of the periodic system. These have been found to form a metal phosphide or homolog on the surface which is able to alloy with the metal surface, lowering its melting point markedly, and by this action aiding greatly in maintaining a polish. The wear experiments were carried out with a highly sensitive and accurate method which uses metal-plated steel balls as its sliding elements. Under the experimental conditions additions of 1.5% triphenyl phosphine or triphenyl arsine in white oil gave wear prevention factors of 7.2 and 12.2 respectively (relative to pure white oil). A further addition of 1% of a long chain polar compound is able to double the wear prevention factor obtained with the polishing agents and wear prevention factors as high as 17.6 have been observed. The specifically physical action of the long-chain polar compounds is discussed in the preceding paper.

scholarly journals Role of Mammalian Auditory Cortex in the Perception of Elementary Sound Properties

2001 ◽  
Vol 85 (6) ◽  
pp. 2350-2358 ◽  
Author(s):  
Sanjiv K. Talwar ◽  
Pawel G. Musial ◽  
George L. Gerstein
Keyword(s):  
Auditory Cortex ◽  
Time Course ◽  
Auditory Evoked Potentials ◽  
Mammalian Species ◽  
Sound Intensity ◽  
Primary Auditory Cortex ◽  
Normal Hearing ◽  
Auditory Task ◽  
Experimental Conditions

Studies in several mammalian species have demonstrated that bilateral ablations of the auditory cortex have little effect on simple sound intensity and frequency-based behaviors. In the rat, for example, early experiments have shown that auditory ablations result in virtually no effect on the rat's ability to either detect tones or discriminate frequencies. Such lesion experiments, however, typically examine an animal's performance some time after recovery from ablation surgery. As such, they demonstrate that the cortex is not essential for simple auditory behaviors in the long run. Our study further explores the role of cortex in basic auditory perception by examining whether the cortex is normally involved in these behaviors. In these experiments we reversibly inactivated the rat primary auditory cortex (AI) using the GABA agonist muscimol, while the animals performed a simple auditory task. At the same time we monitored the rat's auditory activity by recording auditory evoked potentials (AEP) from the cortical surface. In contrast to lesion studies, the rapid time course of these experimental conditions preclude reorganization of the auditory system that might otherwise compensate for the loss of cortical processing. Soon after bilateral muscimol application to their AI region, our rats exhibited an acute and profound inability to detect tones. After a few hours this state was followed by a gradual recovery of normal hearing, first of tone detection and, much later, of the ability to discriminate frequencies. Surface muscimol application, at the same time, drastically altered the normal rat AEP. Some of the normal AEP components vanished nearly instantaneously to unveil an underlying waveform, whose size was related to the severity of accompanying behavioral deficits. These results strongly suggest that the cortex is directly involved in basic acoustic processing. Along with observations from accompanying multiunit experiments that related the AEP to AI neuronal activity, our results suggest that a critical amount of activity in the auditory cortex is necessary for normal hearing. It is likely that the involvement of the cortex in simple auditory perceptions has hitherto not been clearly understood because of underlying recovery processes that, in the long-term, safeguard fundamental auditory abilities after cortical injury.

Electrophysiology of plasma membrane vesicles

1984 ◽  
Vol 246 (4) ◽  
pp. F363-F372
Author(s):  
E. M. Wright
Keyword(s):  
Brush Border ◽  
Epithelial Transport ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Tracer Uptake ◽  
Plasma Membrane Vesicles ◽  
Experimental Conditions ◽  
Optical Signals ◽  
Brush Border Membranes ◽  
Vesicle Preparation

In both renal and gastrointestinal physiology, it has become popular to study epithelial transport phenomena using vesicles isolated from the apical and basolateral cell membranes. Transport in vesicle preparations is usually monitored with radioactive tracers, but more recently attention has been directed to electrophysiological methods. As it is impossible to measure the electrical properties of membranes in small vesicles (less than 500 nm diam) with classical electrophysiological techniques, indirect methods have to be employed. In this review I focus on the application of voltage-sensitive optical probes to measure membrane potentials in brush border membrane vesicles. Optical signals are calibrated with diffusion potentials generated with known ion gradients in the presence of ionophores, e.g., EKS with K gradients in the presence of valinomycin. Membrane potential measurements can be used 1) to illustrate the specificity and kinetics of sugar-, amino acid-, and carboxylic acid-Na cotransport systems in brush border membranes, and 2) to determine the ion permeability of brush border membranes. All organic solutes known to be transported by Na cotransport across brush border membranes depolarize the membrane in a Na-dependent, saturable manner. The results agree, both qualitatively and quantitatively, with electrophysiological data obtained in the intact renal tubule and with tracer uptake in vesicles. Bi-ionic potential measurements demonstrate that brush border membranes are permselective to anions and cations, but there are indications that the permeabilities are somewhat dependent on the method of vesicle preparation and the experimental conditions. However, electrical potential measurements provide insight into the mechanisms of ion transport in vesicle preparations, and the application of patch-clamp techniques should provide further gains in the future.

scholarly journals Comparative biofilm assays using Enterococcus faecalis OG1RF identify new determinants of biofilm formation

2021 ◽  
Author(s):  
Julia L. E. Willett ◽  
Jennifer L. Dale ◽  
Lucy M. Kwiatkowski ◽  
Jennifer L. Powers ◽  
Michelle L. Korir ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Growth Medium ◽  
Biofilm Formation ◽  
Time Course ◽  
Opportunistic Pathogen ◽  
Genetic Determinants ◽  
Experimental Conditions ◽  
Biofilm Reactors ◽  
Biofilm Architecture ◽  
Biofilm Development

AbstractEnterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by 6 Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions, and identifies multiple new genetic determinants of biofilm formation.ImportanceE. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.

The Distribution of Ammonia and H+ Between Tissue Compartments in Lemon Sole (Parophrys Vetulus) at Rest, During Hypercapnia and Following Exercise

1988 ◽  
Vol 136 (1) ◽  
pp. 149-175 ◽  
Author(s):  
P. A. WRIGHT ◽  
D. J. RANDALL ◽  
C. M. WOOD
Keyword(s):  
White Muscle ◽  
Red Cells ◽  
Strenuous Exercise ◽  
Red Cell ◽  
Hydrogen Ions ◽  
Experimental Conditions ◽  
Ph Gradients ◽  
Tissue Compartments ◽  
Parophrys Vetulus ◽  
Red Cell Ph

The distribution of ammonia and [14C]DMO was compared in white muscle, heart, brain, red cells and plasma of lemon sole (Parophrys vetulus Girard) at rest, during hypercapnia and following strenuous exercise. In red cells at rest, measured intracellular ammonia levels were equal to those predicted by the plasma to red cell pH gradient. Red cells are unusual in that hydrogen ions are passively distributed according to membrane potential (EM), whereas in other tissues this is not the case. In white muscle, heart and brain under all experimental conditions, intracellular ammonia levels far exceed those predicted by transmembrane pH gradients. Calculated ENHNH4+ values in these tissues are very close to published resting values of EM. We conclude that, in lemon sole, NH4+ permeates cell membranes and that intracellular ammonia stores are not determined by transmembrane pH gradients.

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