The effects of air ions of the living mammalian trachea

Studies on the effects of air ions on the functional efficiency of the extirpated tracheal strip have been extended to the trachea of the living rabbit, rat, and mouse. Animals exposed to high mobility (+) air ions administered via a tracheotomy aperture displayed: (a) Decreased ciliary activity. (b) Decline in mucus flow rate, sometimes reversed by prolonged exposure to (+) ions; a frequent drop in the volume of mucous secretion. (c) Contraction of the membranous posterior tracheal wall. (d) Increased vulnerability to trauma of cilia and mucosal blood vessels. Similar treatment with (-) air ions reversed (+) ion effects on ciliary activity, mucus flow, contraction of the tracheal smooth muscle. Continued (-) ion treatment raised the ciliary rate (invariably) and the mucus flow rate (often) above their initial levels. (+) Air ions administered to unoperated resting mice and rats increased the respiratory rate; (-) ions reversed this effect. Long exposure of unoperated ambulatory mice to (+) air ions produced: (a) Decreased ciliary activity. (b) No clear cut effect on mucus flow. (c) Contraction of the posterior tracheal wall. (d) Increased vulnerability of the mucosa to trauma. (-) Air ions increased ciliary activity but had no clear-cut effect on the mucus flow rate.     

Hydroxyl ion movements across the human erythrocyte membrane. Measurement of rapid pH changes in red cell suspensions

A stopped flow rapid reaction apparatus capable of following changes of ±0.02 pH unit in 0.1 ml of solution in less than 0.005 sec has been developed, utilizing a commercially available pH-sensitive glass electrode. Using this instrument, extracellular pH at 37°C was followed from less than 0.025 sec to 300 sec after mixing equal volumes of the following CO₂-free solutions: (A) normal human red cells, washed three times and resuspended in 150 mM NaCl at pH 7.2 with a hematocrit of 18%; and, (B) 150 mM NaCl adjusted with HCl or NaOH to pH 2.1 to pH 10.3. A minimum of 2 ml of mixture had to flow through the electrode chamber to ensure complete washout. The mixing process produced a step change in the pH of the extracellular fluid, after which exchanges across the red cell membrane and buffering by intracellular hemoglobin caused it to return toward pH 7.2 with an approximately exponential time course. Under the assumption that pH changes after mixing represent exchanges of hydroxyl for chloride ions across the cell membrane, hydroxyl ion permeabilities (P _OH ^- in cm/sec) were calculated and found to vary from 2 x 10^-4 at pH 9 to 4 x 10^-1 at pH 4 according to the empirical relationship P _OH ^- = 170 exp (-1.51 pH). The form of the dependence of P _OH ^- on extracellular pH does not appear compatible with a simple fixed charge theory of membrane permselectivity.     

The Biological Mechanisms of Air Ion Action : I. 5-Hydroxytryptamine as the endogenous mediator of positive air ion effects on the mammalian trachea

Intravenous administration of 5-hydroxytryptamine to rabbits and guinea pigs is shown to bring about changes very similar to those produced by (+) air ions, including (1) decreased ciliary rate, (2) contraction of the posterior tracheal wall, (3) exaggerated response of the tracheal mucosa to trauma, (4) marked vasoconstriction in the tracheal wall, and (5) increased respiratory rate. These effects are reversed by (-) air ions. Iproniazid, which raises 5-hydroxytryptamine levels in the animal by blocking monamine oxidase, produces similar but non-reversible effects. Reserpine, which depletes 5-hydroxytryptamine in the animal, causes changes that resemble those produced by (-) air ions, including (1) increased ciliary rate, (2) relaxed posterior sulcus, (3) hyperemia of the tracheal mucosa, (4) lowered respiratory rate, and (5) increased volume and rate of mucus flow. On the basis of these facts, the hypothesis is advanced that (+) air ion effects are mediated by the release of free 5-hydroxytryptamine, while (-) air ion effects depend on the ability of (-) ions to accelerate the enzymatic oxidation of 5-hydroxytryptamine.     

The Interactions of P-Glycoprotein with Antimalarial Drugs,Including Substrate Affinity,Inhibition and Regulation

The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10⁻⁶ cm/sec, followed by amodiaquine around 20 x 10⁻⁶ cm/sec; both mefloquine and artesunate were around 10 x 10⁻⁶ cm/sec. Methylene blue was between 2 and 6 x 10⁻⁶ cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine.     

Filtration Coefficient of the Axon Membrane As Measured with Hydrostatic and Osmotic Methods

The hydraulic conductivity of the membranes surrounding the giant axon of the squid, Dosidicus gigas, was measured. In some axons the axoplasm was partially removed by suction. Perfusion was then established by insertion of a second pipette. In other axons the axoplasm was left intact and only one pipette was inserted. In both groups hydrostatic pressure was applied by means of a water column in a capillary manometer. Displacement of the meniscus in time gave the rate of fluid flowing across the axon sheath. In both groups osmotic differences across the membrane were established by the addition of a test molecule to the external medium which was seawater. The hydraulic conductivity determined by application of hydrostatic pressure was 10.6 ± 0.8.10^-8 cm/sec cm H₂O in perfused axons and 3.2 ± 0.6.10^-8 cm/sec cm H₂O in intact axons. When the driving force was an osmotic pressure gradient the conductivity was 4.5 ± 0.6 x 10^-10 cm/sec cm H₂O and 4.8 ± 0.9 x 10^-10 cm/sec cm H₂O in perfused and intact axons, respectively. A comparable result was found when the internal solution was made hyperosmotic. The fluid flow was a linear function of the hydrostatic pressure up to 70 cm of water. Glycerol outflux and membrane conductance were increased 1.6 and 1.1 times by the application of hydrostatic pressure. These increments do not give an explanation of the difference between the filtration coefficients. Other possible explanations are suggested and discussed.     

An enzymatic ion exchange model for active sodium transport

An enzymatic ion exchange model for active sodium transport is described. Kinetic equations relating net flux to time, and to concentration difference across the actively transporting membrane are derived. The second of these equations is tested, using the isolated frog skin in the "short-circuit" apparatus of Ussing. Reasonable linearity, as predicted by this equation, is observed. The passive permeability coefficient for Na⁺, is calculated as 5.3 x 10^-4 ± 5.3 x 10^-4 cm./hr. If cholinesterase is assumed to be the enzyme responsible for transport, the activity required to account for the observations reported here is 17.7 x 10^-4 mmoles/cm.²/hr.     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. V—Rabbit (Oryctolagus Cuniculus)

• 1.1. The diffusional water permeability (P_d) of rabbit red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulfonate (PCMBS).
• 2.2. The values of P_d were around 6.3 × 10⁻³ cm/sec at 15°C, 7.0 × 10⁻³cm/sec at 20°C, 8.0 × 10⁻³ cm/sec at 25°C, 9.1 × 10⁻³ cm/sec at 30°C and10.7 × 10⁻³ cm/sec at 37°C.
• 3.3. Systematic studies on the effects of PCMBS on water diffusion indicated that the maximal inhibition was reached in 15 min at 37°C with 0.5 mM PCMBS.
• 4.4. The values of maximal inhibition were around 71–74% at all temperatures.
• 5.5. The basal permeability to water was estimated as 1.6 × 10⁻³cm/sec at 15°C, 2.0 × 10⁻³cm/sec at 20°C, 2.4 × 10⁻³cm/sec at 25°C, 2.6 × 10⁻³cm/sec at 30°C, and 3.1× 10^{−3 cm/secat 37°C}.
• 6.6. The activation energy of water diffusion was around 18 kJ/mol and increased to 27 kcal/mol after incubation with PCMBS in conditions of maximal inhibition of water diffusion.
• 7.7. The membrane polypeptide electrophoretic pattern of rabbit RBCs has been compared with its human counterpart.
• 8.8. The rabbit membrane contained a higher amount of spectrin (bands 1 and 2), while the band 6 (glyceraldehyde-3-phosphate dehydrogenase) was markedly less intense.
• 9.9. Considerable differences in the electrophoretic patterns of the two sources of RBC membranes appeared in the bands migrating in the band 4.5 region and in front of band 7, where some polypeptides were apparent in higher amounts in the rabbit RBC membrane.

     

Kinetics of ion movement in the squid giant axon

The loss of Na²², K⁴², and Cl³⁶ from single giant axons of the squid, Loligo pealii, following exposure to an artificial sea water containing these radioisotopes, occurs in two stages, an initial rapid one followed by an exponential decline. The time constants of the latter stage for the 3 ion species are, respectively, 290, 200, and 175 minutes. The outflux of sodium is depressed while that of potassium is accelerated in the absence of oxygen; the emergence of potassium is slowed by cocaine, while that of sodium is unaffected. One cm. ends of the axons take up about twice as much radiosodium as the central segment; this difference in activity is largely preserved during exposure to inactive solution. Such marked differences are not observed with radiopotassium. From the experimental data estimates are given of the influxes and outfluxes of the individual ions. The kinetics of outflux suggests a cortical layer of measureable thickness which contains the ions in different proportions from those in the medium and which governs the rate of emergence of these ions from the axon as though it contained very few but large (relative to ion dimensions) pores.     

Inhibitory effects of cadmium on carbonic anhydrase activity and ionic regulation of the estuarine crab Chasmagnathus granulata (Decapoda,Grapsidae)

This work was aimed at evaluating the gill carbonic anhydrase (CA) activity of the estuarine crab Chasmagnathus granulata exposed in vivo to cadmium, at different salinities. The in vivo effect of the specific inhibitor acetazolamide (AZ) was also assayed. Besides, the inhibition of CA activity by different heavy metals (cadmium, copper, zinc) and AZ were evaluated under in vitro conditions. For the in vivo assays, adult males were acclimated to salinities of 2.5 or 30‰. The corresponding 96-h LC₅₀ of cadmium was 2.69 mg l⁻¹ at 2.5‰, and >50 mg l⁻¹ at 30‰. Cadmium only caused a significant lower CA activity than control at 2.5‰. EC₅₀ for CA inhibition was estimated to be 1.59 mg l⁻¹ at 2.5‰. Statistical differences in Na⁺ hemolymphatic levels (P<0.05) were only detected at 2.5‰, between 0 and 1.25 mg l⁻¹ of cadmium, but no statistical differences were observed for Cl⁻ levels at any assayed salinity. As CA inhibition registered at 2.5‰ was followed by only changes in Na⁺ concentration, it is likely that cadmium exposure could differentially affect ions permeability, among others factors. The concentrations that inhibited in vitro 50% of enzymatic activity (IC₅₀) were 2.15×10⁻⁵, 1.62×10⁻⁵, 3.75×10⁻⁶ and 4.4×10⁻¹⁰ M for cadmium, copper, zinc and AZ, respectively. The comparison with IC₅₀ values of other aquatic species, indicates a higher CA sensitivity for C. granulata to pollutants.     

THE RATE OF ESCAPE OF HEMOGLOBIN FROM THE HEMOLYZED RED CORPUSCLE

A theoretical treatment is given of the rate of escape of hemoglobin from the hemolyzed red corpuscle. For complete permeability of the surface, as may perhaps be produced by strong lysins, the time taken for the hemoglobin to decrease to 10 per cent of its original concentration is calculated to be 0.16 seconds (for the human cell). For dilute saponin, giving complete lysis of human cells in 3 minutes, Ponder found a time of escape of 4 seconds, from which the permeability of the membrane to the pigment is calculated to be µ_H = 5 x 10^–5 cm./sec.     

Cation-dependent light-induced structural changes in visual receptor membranes

A stearamide spin probe was used to study the light-induced structural changes in Rod Outer Segment Membranes in the presence of sodium and calcium ions. The correlation time (τ_C) for the reorientation of the probe was calculated in the dark and light. In the presence of sodium ions, τ_C = 3.3 × 10^?9 sec in the dark, and 2.7 × 10^?9sec in the light while the opposite was noticed in the presence of calcium ions, τ_C = 2.9 × 10^?9 sec in the dark and 3.6 × 10^?9 sec in the light. The correlation times for reorientation of the probe were also calculated in aqueous glycerol solutions of varying viscosities at 20°C. Comparison of the values of τ_C (dark and light) suggests a change in local mobility in the ROS corresponding to a macroscopic viscosity difference of approximately 150 cp. The significance of calcium ion interaction with negatively charged groups and the formation of a Schiff base is emphasized.     

Effect of leukotriene C4 exposure on ciliated cells of the nasal mucosa

To clarify the effects of leukotriene C4 (LTC4) on human ciliated epithelium, ciliary activity of the ethmoid sinus mucosa was measured photoelectrically in tissue culture. At concentrations ranging from 10⁻⁶M to 10⁻⁹M, LTC4 showed minimal effects on the ciliated epithelium during the initial 30 minutes of exposure; thereafter, ciliary inhibition was observed in a concentration- and time-dependent manner. Irrigation of the mucosa with culture medium 15 minutes after exposure prevented the LTC4-induced ciliary inhibition. However, irrigation 60 minutes after exposure failed to inhibit 10⁻⁸M LTC4-induced ciliary dysfunction and mucosal damage. The LTC4-induced ciliary inhibition was blocked in the presence of FPL-55712 and/or Ly-171883, both leukotriene receptor antagonists. L-serine and sodium tetraborate complex (SBC), a γ-glutamyl transpeptidase (γ-GTP) inhibitor, also inhibited the LTC4-induced ciliary inhibition. These findings indicate that LTC4 is converted to LTD4 by γ-GTP during 60 minutes of exposure, and LTC4 itself has minimal direct effects on the ciliated cells.     

Water Permeability in Resting and Stimulated Crayfish Nerve

The hydraulic conductivity, L_p, was determined in single axons of the crayfish, Procambarus clarkii, by injecting a hypertonic sample between two drops of silicone oil and photographing the volume increase of the sample. The method has the advantage of minimizing errors due to hydrostatic pressure differences across the membrane. In resting axons an L_p of 0.236 x 10^-8 cm/ sec per cm H₂O was found and similar values were obtained with low external calcium concentration and when the nerve was continuously stimulated at 20–30 impulses/sec. Thus the experiments have failed to demonstrate any change of water permeability in cases in which the ionic conductance is known to change. Some possible implications of this are discussed.     

Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed toMycoplasma pneumoniae cells or membranes

Summary The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10⁵ to 10⁷ CFU of virulentMycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 μg per ml of purifiedM. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures. This investigation was supported in part by the National Institutes of Health (PHS Grant AI 12559), by a Biomedical Sciences Support Grant made to the University of Illinois School of Life Sciences, and by the University Research Board.     

Diffusion of Weak Acids across the Toad Bladder : Influence of pH on non-ionic permeability coefficients

Studies have been carried out, using the toad bladder, to determine the influence of pH on the permeability coefficients (K_trans) of the non-ionic species of (a) a series of aliphatic acids ranging from propionic to octanoic and (b) the aromatic acids, benzoic and acetylsalicylic. The data demonstrate that as the acidity of the mucosal bathing solution is increased by changing pH from 6 to 4, the fluxes of propionic, butyric, and acetylsalicylic acids increase in direct proportion to the increase in the calculated non-ionic concentration; the permeability coefficients, therefore, remain constant. However, the fluxes of the six, seven, and eight carbon aliphatic acids and benzoic acid rise only slightly despite an almost tenfold increase in non-ionic concentration, the K_trans falling from approximately 20,000 x 10^-7 cm sec.^-1 at pH 6 to approximately 2500 x 10^-1 cm sec.^-1 at pH 4. It has been tentatively proposed that the common characteristic of the compounds exhibiting this anomalous behavior is their non-polarity and high degree of lipid solubility. Possible explanations for the differences observed between the more lipid-soluble and less lipid-soluble compounds have been considered.     

The cAMP signal from Dictyostelium discoideum amoebae.

Dictyostelium discoideum cells were allowed to differentiate on agar for 600 min at room temperature. All of the cells were then competent to relay or amplify a cAMP signal, but none to produce a cAMP signal autonomously. The cells were stimulated with cAMP concentrations ranging from 10^?9 to 3.5 × 10^?7M. Populations of 10⁶ cells could amplify an initial cAMP concentration of 2.5 × 10^?9M with a low probability, while an initial cAMP concentration of 5 × 10^?8M always induced a response. An initial cAMP concentration of 1.2 × 10^?7M induced the maximum cellular release of cAMP observed; this corresponded to 3 × 10⁷ molecules per cell. No cellular release of cAMP was detected for initial cAMP concentrations of 3 × 10^?7M or more. The amplification of a 10^?7M cAMP stimulus was complete within 8 sec, indicating the pulsatile nature of the cellular release of cAMP. The phosphodiesterase (PDE) activities of D. discoideum cells were measured over a wide range of cell densities. At densities above 7.5 × 10⁴ cells/cm², both cell-bound and extracellular (ePDE) activities declined, per cell, as cell density increased. These results are compared to ePDE activities derived from critical density measurements. We found that PDE activities were in the range of 10^?13–10^?14 moles of cAMP converted/cell/min under culture conditions consistent with normal aggregation.     

Divalent metal ion effects on a mutant histidinol phosphate phosphatase from Salmonella typhimurium

The effect of divalent metal ions on the activity of a mutant histidinol phosphate phosphatase has been studied. The enzyme was isolated from strain TA387, a mutant of Salmonella typhimurium with a nonsense lesion near the midpoint of the bifunctional hisB gene. Mn²⁺, Mg²⁺, Co²⁺, and Zn²⁺ shift the optimal pH of phosphatase activity to 6.5 while Be²⁺ and Ca²⁺ have no effect on the shape of the pH profile. In the absence of divalent metal ions, the pH optimum is 7.5. Four Me²⁺ ions, Mn²⁺, Co²⁺, Zn²⁺, and Fe²⁺ decreased the K_m of histidinol phosphate at pH 6.5 from 5.5 mm (without Me²⁺) to 0.14 mm. Ni²⁺ and Be²⁺ increased the K_m to 22.2 and 25.0 mm, respectively, and Ca²⁺ and Mg²⁺ had an intermediate effect. Changes in maximal velocity were substantially less, only about 2-fold changes being observed. It was shown that the maximal velocity at optimal pH was the same in the absence and presence of Mn²⁺. Kinetic analysis indicated that there was a rapid equilibrium-ordered addition of Mn²⁺ to the enzyme before the addition of the substrate, histidinol phosphate. A k_imn²⁺ of 4.3 μm was calculated for the metal ion activation at both pH 6.5 and 7.5. Addition of ethyl-enediaminetetracetate (EDTA) strongly inhibited the phosphatase; inhibition could be reversed by addition of several Me²⁺ ions, Mg²⁺ being the most efficient followed by Mn²⁺. Prolonged incubation with EDTA led to irreversible inactivation.     

ELECTRON BOMBARDMENT OF BIOLOGICAL MATERIALS : II. THE RATE OF DEATH OF FUNGUS SPORES BOMBARDED IN VACUUM WITH CATHODE RAY BEAMS FROM 4 KV.–15 KV.

A study has been undertaken of the rate of inactivation of spores of the ascomycete fungus Aspergillus niger when bombarded in vacuum, with homogeneous beams of cathode rays of energies from 4 to 15 electron kv. and current densities of 1 x 10^–7 to 3 x 10^–6 amperes per square cm. These velocities and densities are in the range of those of showers of secondary electrons produced in biological materials irradiated with moderately soft x-rays, and so may be made to serve as quantitative indicators of the mechanics of x-ray action. Four qualitative effects are described.     

Lanthanide ions inhibit the activity of dihydrofolate reductase from chicken liver

The influences of mono-, bi- and trivalent metal ions (as chloride salts) on the activity of dihydrofolate reductase (DHFR) from chicken liver have been studied to elucidate the mechanism of ion-activation of this enzyme. The results show that monovalent ions (Na⁺ and K⁺) activate DHFR at low concentration reaching a maximum activation of about 2.5 fold at 0.4–0.5 M and declining at higher concentrations. Ca²⁺ shows similar activation but at lower concentration, reaching a maximum at 0.1 M; activity declines with further increases in concentration. At very high concentration (>0.4 M), Ca²⁺ is inhibitory. The trivalent lanthanide ions, however, show a dramatic inhibition of activity of DHFR even at very low concentration. The activity of DHFR declines to 50% of that of the control at 0.02 mM EuCl₃. Intrinsic fluorescence measurements show that the ion-dependent activation in the presence of mono- and bivalent metal ions is due to the conformational changes in the protein. Energy transfer phenomenon suggests that the specific interaction of Eu³⁺ with Trp24 located in a loop at the active site of DHFR is responsible for the strong inhibition. The possible mechanism for the ion-inhibition is proposed and discussed.     

The effects of varying concentrations of colchicine on the progression of grasshopper neuroblasts into metaphase

The effects of four concentrations of colchicine (2.5 x 10^-7, x 10^-5, x 10^-3, and x 10^-2 M) on the cell cycle of grasshopper neuroblasts have been determined by direct observations on living cells. The lowest concentration, 2.5 x 10^-7 M, does not completely disorganize the spindle but does retard its action. The three higher concentrations disorganize the spindle, so that all cells reaching metaphase are blocked in a c-mitotic condition throughout the period of observations (308 min at 38°C, the minimum duration of the cell cycle in untreated neuroblasts). Continuous treatment with all concentrations reduces the rate at which neuroblasts enter metaphase, the extent of the reduction being a function of increasing concentration and time of exposure. After a short exposure to 2.5 x 10^-5 M colchicine, the neuroblasts recover from the inhibiting effects on progression through the cycle to metaphase, but they show no recovery from the inhibiting effects on spindle formation for more than 3 hr. Apparent stimulation of progression rate occurs early in exposure to all concentrations and during recovery from a short exposure to 2.5 x 10^-5 M. Morphological alterations in the chromatin of telophase, interphase, and prophase cells are induced by the higher concentrations of colchicine. The data indicate that caution should be exercised in the use of colchicine for determining cell cycle duration and/or the effects of physical and chemical agents on the cycle.