Addition Of Metabolic Inhibitors Research Articles

ALTERATIONS IN SPONTANEOUS ACTIVITY OF THE ISOLATED FROG SPINAL CORD IN CALCIUM-FREE ENVIRONMENT

Spontaneous electrical activity of the isolated frog spinal cord was examined in Ca2+-free environment. Spontaneous discharges from the ventral root altered in four distinguishable steps. The first step was an immediate increase in the rate of spontaneous discharges and the second was a gradual decrease. The third was the occurrence of rhythmical bursts, and the last, the appearance of continuous firing. The rhythmical bursts could be depressed by the addition of metabolic inhibitors (ouabain or dinitrophenol in a concentration of 5 x 10(-5) M) as well as of Ca2+-chelating agents (EDTA or EGTA in a concentration of 10(-3) M). Our results suggest that the occurrence of the rhythmical bursts requires a metabolic pumping process to redistribute Na+ and K+ across the membrane and a small amount of Ca2+ for transmitter secretion.

In vitro measurement of rate of fluid secretion in rat isolated seminiferous tubules: effects of metabolic inhibitors and ions.

1. An in vitro technique for measuring secretory rate in rat isolated seminiferous tubules is described. 2. The basal rate of fluid secretion was 0-44+/-0-06 nl. cm-1 min-1 (S.E.) (n=21). The rate was found to be inhibited by cooling, addition of metabolic inhibitor 2,4-dinitrophenol (2-5 x 10(-4) M) and removal of glucose from the incubating solution. This indicates that fluid secretion in isolated rat seminiferous tubules is an energy dependent process. 3. Removal of K+ from the incubating medium inhibited the secretory rate in the isolated seminiferous tubules, whereas a fivefold increase in [K+]0 to 23-5 mM had no effect. The secretory rate was also unaffected by the absence of Cl- in the peritubular fluid. 4. Removal of Ca2+ from the peritubular medium caused a rise in the secretory rate. 5. Ouabain (10(-3) M) and acetazolamide (4 x 10(-5) M) caused a fall in the rate of fluid secretion in isolated seminiferous tubules. 6. These results are discussed in relation to the nature of the ionic secretion produced in the tubules.

Metabolic component in the epithelial intracellular potential of rabbit cornea.

Effects of the change of external ionic composition and the addition of metabolic inhibitors on rabbit cornea were studied by recording the epithelial intracellular potential. High K and Li Ringer's solutions, applied to the corneal endothelial side, caused a marked depolarization of the epithelial cells, but no potential change was seen when applied to the epithelial side. Ouabain, MIAA and NaCN applied to the endothelial side reduced the epithelial potential, while those applied to the epithelial side did not change the potential. DNP and FDNB also had no effect when applied to the epithelial side only. The thermal dependence of the epithelial intracellular potentials of whole eye (0.85 mV/degrees C) and excised cornea (2.01 mV/degrees C) preparations were greater than about 0.2 mV/degrees C predicted by the Nernst equation. It is concluded that the epithelial cell layer of rabbit cornea act as a tight barrier against diffusion of K ion and metabolic inhibitors from the tear side to the epithelial basal cell. A high thermal dependence of the epithelial intracellular potential may depend greatly on the pump ingibition.

Cholesterol exchange between fat cells, chylomicrons and plasma lipoproteins

Isolated rat adipocytes were incubated with serum lipoproteins or lymphchylomicrons which contained 14C-labeled cholesterol. The specific activity of lipoprotein free cholesterol decreased and that of cellular free cholesterol increased linearly up to 7 h. At this time the cell cholesterol specific activity was only 11% of that of medium cholesterol indicating that the rate of exchange was slow. The specific activity of lipoprotein esterified cholesterol remained unchanged while that of cells showed a slight increase suggesting esterification of incorporated free cholesterol. No detectable change of the lipoprotein or cellular cholesterol concentration occurred indicating that the uptake of radioactive free cholesterol was due to exchange without net movement of sterol. The radioactive cholesterol was incorporated into both membrane fraction and the fat droplet of the adipocytes. The rate of cholesterol exchange was temperature-dependent but it was not influenced by the metabolic state of the cells and not by addition of metabolic inhibitors. Trypsin or pronase treatment of the cells were without influence on the rate of the exchange and denaturation of the plasma lipoproteins with formalin increased the rate of exchange. These results indicate that the exchange of cholesterol is a physical chemical process, which is not linked to energy metabolism of the cells, and which is not mediated by either specific lipoprotein receptors on fat cell membranes or pinocytic uptake of lipoproteins. The rate of free cholesterol exchange showed a linear correlation with the concentration of lipoprotein particles in the medium. The relative transfer rate was highest for chylomicrons and decreased in order chylomicron remnants > very low density lipoprotein > low density lipoprotein > high density lipoprotein. A saturation of the system could be obtained only with high density lipoprotein.

In vitro analysis of the renal handling of paraquat

Accumulation of paraquat by mouse renal cortical slices was related to the concentration of paraquat in the medium and the duration of incubation. Paraquat accumulation was depressed by incubation of slices under nitrogen or by addition of metabolic inhibitors. Accumulation of a second organic base, N-methylnicotinamide (NMN), was depressed by a concentration of paraquat which failed to influence accumulation of the organic acid, p-aminohippurate (PAH). The uptake component of NMN accumulation was inhibited by paraquat. The data suggest that paraquat is accumulated by an energy-requiring process and that this accumulation occurs via the organic base transport system. In addition, an apparently toxic effect of paraquat on cortical slice function was observed when the incubation temperature was raised from 25 to 37°C. At this temperature, 10 −3 m paraquat depressed not only NMN accumulation but PAH accumulation and slice oxygen consumption as well. Thus, paraquat can be toxic to the function of kidney slices and this effect appears to be temperature-dependent.

Early cytoplasmic vacuolization of African green monkey kidney cells by SV40

As early as 3--4 hours after infection with SV40 at a high input multiplicity, African green monkey (Cercopithecus aethiops) kidney (AGMK) cells developed cytoplasmic vacuolization. At 10--20 hours after infection, the vacuolization reached its maximal level, then disappeared and SV40 specific cytopathic change followed. This vacuolization developed before the synthesis of the specific T and V antigens. This early cytoplasmic vacuolization (ECV) was prevented by preincubating the virus with specific antiserum, or by heating the virus with MgCl2. The ECV could be induced by UV-irradiated SV40. Addition of metabolic inhibitors had no effect on the induction of the ECV. These results suggest that the capacity to induce the ECV resides in a structural component(s) of SV40 virion and the vacuolization is not associated with the replication of SV40.

Are Cytotoxicity and Interferon Inducing Activity of Poly(I).Poly(C) Invariably Linked in Interferon-treated L cells?

Interferon-treated L cells exhibit a specific enhanced susceptibility to the cytotoxic and interferon inducing activities of double-stranded RNAs such as poly(1). poly(C). These activities remained closely linked through widely varying assay conditions, involving, for example, different time anddosage schedules of poly(1). poly (C),suggesting that there is at least one common step in the mechanisms leading to interferon formation and toxicity in interferon-primed cells exposed to poly(1).poly(C). However, some procedures such as addition of metabolic inhibitors (actinomycin D, cycloheximide) and repeated administration of poly(1).poly(C) suppressed the interferon inducing capacity of poly(1).poly(C) without a concomitant decrease of toxicity. Other procedures such as brief treatment of the cells with interferon or DEAE-dextran permitted full expression of the interferon inducing activity of poly(1).poly(C) without any sign of toxicity. The latter results suggest that the mechanisms underlying interferon production and toxicity of poly(1).poly(C) in interferon-treated L cells diverge from a certain point onward.

Auxin-induced hydrogen ion excretion: correlation with growth, and control by external pH and water stress

1. The acid-growth theory predicts that the rates of auxin-induced cell elongation and H(+)-excretion should be closely correlated as long as the experimental conditions remain fairly constant. To test this, Avena coleoptiles have been induced to elongate at different rates by varying the concentration of auxin, the age of the tissue, or by addition of metabolic inhibitors. As predicted, in each case there was a close correlation between the rates of H(+)-excretion and growth. 2. The rate and direction of movement of H(+) between the coleoptile and the external medium is regulated by the external pH. Coleoptiles take up H(+) passively from acidic solutions and excrete H(+) into basic solutions. In the absence of auxin, uptake and excretion are at equilibrium when the solution pH is near 5.7, a pH too high to allow rapid cell wall loosening. Auxin stimulates the excretion, but as the external pH drops the excretion is inhibited and a new equilibrium is established near 5.0. This allows amlows maximum wall loosening without causing toxic side-effects. 3. H(+)-excretion is also affected by water stress. Increasing water stress induced by mannitol decreases H(+)-excretion in auxin-treated tissues but stimulates H(+)-excretion in the absence of auxin. At incipient plasmolysis H(+)-excretion is insensitive to auxin, suggesting that even if H(+)-excretion is mediated by an auxin-activated ATPase it may be impossible to demonstrate an effect of auxin on this enzyme in vitro. 4. Three types of H(+)-excretion have been recognized in coleoptiles; auxin-induced, stress-induced, and basal level. All appear to require ATP, but only basal level H(+)-excretion does not also require continual protein synthesis.

The comparative transport of K+ and Rb+ in normal and malignant rat tissuesin vivo and in liver slices, diaphragm, and tumor slicesin vitro

The selectivity in the steady state uptakes of Rb(+) and K(+) has been studied in a number of normal and malignant rat tissues. The selectivity is minimal in erythrocytes and the two fastest-growing of four transplantable tumors, in which there is little discrimination between the two ions, and ranges upwards to a maximum Rb(+) uptake in liver. In each tissue, the selectivity is independent of Rb(+) concentration or of K(+) deficiency (except in skeletal muscle). In liver slicesin vitro, reduction of energy metabolism by lowering the temperature or by the addition of metabolic inhibitors reduces the Rb(+)∶K(+) discrimination proportionately much more than K(+) transport. Diaphragm and slices of a transplantable tumor give similar results. With temperature reduction, there is a logarithmic relation between the Rb(+)∶K(+) discrimination ratio and the respiration rate of liver slices. The results are quantitatively accounted for by simultaneous diffusion and metabolically coupled transport across a homogeneous membrane in which Rb(+) transport is more closely coupled than that of K(+) to a metabolic flux across the membrane. There is evidence that the tissue differences in Rb(+)∶K(+) selectivity originate in the different levels of the coupling metabolic flux in different cell types and thus of the energy expenditure on ion transport. In contrast to the differences in steady state selectivity between Rb(+) and K(+), the initial ratio of uptakes of trace(43)K and(86)Rb, in otherwise steady state conditions, is close to unity in both liver and tumor slices, in agreement with theoretical calculations.

The “Point of no Return” Concept in Cell Division. The Effects of Some Metabolic Inhibitors on Synchronized Chlorella pyrenoidosa

AbstractThe coarse of growth and cell division in synchronized cultures of Chlorella pyrenoidosa was studied after the addition of metabolic inhibitors at differing times during the cell cycle (14 h light ‐ 10 h darkness with nitrate as nitrogen source. 12 h light: 12 h darkness with urea as nitrogen source).Dinitrophenol (DNP) added to a final concentration of 0.3 mM at any time in the synchronization cycle, the compound remaining in the suspension from the time of addition to the end of the dark period, inhibited spore formation completely. Growth measured as increase in cell volume was less sensitive to the action of the inhibitor. Chloramphenicol (CAP) added dining the 0–5 h interval to a final concentration of 0.1 mM resulted in 80 per cent inhibition of cell division. Similar treatment started at successive times thereafter resulted in a gradual decrease of the inhibition. Treatment at the 14th hour and during the dark period did not affect the sporulation.Similar experiments with 0.9 mM puromycin added at various times during the illumination period gave almost complete inhibition of cell division, while the growth was reduced by only 25 per cent. para‐Fluorophenylalanine (p‐FPhe) at 3.3 × 10−2 mM stopped cell division nearly completely irrespective of addition time in the light period. Addition during the dark period also prevented an increase in the number of tree cells. In this case about half of the cells produced spores which were not released.It is concluded that DNP inhibits all stages of preparation for cell division, as well as the division process itself. With CAP a genuine transition point of preparation for cell division was observed, although its interpretation as related to protein synthesis is somewhat uncertain. With puromycin and p‐FPhe no transitions were observed.

Properties of a carrier system to transport L-dopa into brain slices.

L-DOPA(DIHYDROXYPHENYLALANINE) is accumulated in brain cortex slices of guinea pigs against a concentration gradient when the slices are incubated in glucose Ringer at 37° C. The accumulation is reduced by anaerobic condition and by addition of metabolic inhibitors.

Exchange diffusion and runout of Diodrast-I-131 from renal tissue in vitro.

Runout of preaccumulated Diodrast-I131 from isolated goldfish kidneys was investigated with particular attention to metabolic inhibition, competition by related organic acids, and low temperature. Addition of metabolic inhibitors, such as cyanide, to medium bathing the tissue increased Diodrast runout, i.e., rate of net movement into medium. In conjunction with these same inhibitors, competitors either decreased or further increased runout, e.g., probenecid exhibited the former and p-aminohippurate (PAH) the latter action. In the absence of metabolic inhibition, increasing medium concentration of a given competitor first increased and then decreased runout. Low temperature decreased control runout as well as actions of competitors and metabolic inhibitors. Differences in lipid solubility correlate with differences in action of particular competitors. These results are interpreted as evidence for a complex membrane process mediating outward Diodrast movement, e.g., a carrier cycle which exhibits characteristics of both exchange and facilitated diffusion. Speculatively, such a process could be the basis for both active secretion and reabsorption, i.e., bidirectional tubular transport of organic acids.