Electron microscopic and biochemical research on the vacuoles formed in the erythrocytes of frogs as affected by neutral red and novocaine

No lysosomes were found in the frog intact erythrocytes with electron microscope. Under the influence of neutral red (NR-8.7.10(-5) M) and novocaine (N-4.6.10(-3) M) segregation zones (vacuoles) including these substances are formed. Using electron microscopy and morphometry the action of NR and N for 5 minutes up to 48 hours was found to provoke the formation of four types of vacuoles differing in their morphology: with electron-transparent content, with amorphous inclusions and membrane whorls. The dynamics of vacuole formation, of their changes and amount were followed depending on the time of exposition of these substances. Biochemical investigation of both NR and N isolated vacuoles showed in these some activities of lysosomal marker enzymes--acid phosphatase and N-acetyl-beta,D-glucosaminidase. Ultrastructural investigation of acid phosphatase localization in the isolated vacuoles revealed the histochemical reaction product mainly in electron-translucent vacuoles (primary lysosomes) and partly in electron dense ones (secondary lysosomes). On the ground of the above studies a conclusion is made that in frog erythrocytes treated with NR and N lysosome formation is induced to be followed by the induced autophagocytosis and heterophagocytosis. Some possible ways of the vacuolar system formation in frog erythrocytes and the origin of lysosomal hydrolases are discussed.     

ATPase activity of the novocaine segregation zones isolated from the erythrocytes of the common frog

Novocaine segregation zones in frog's erythrocytes, isolated by differential centrifugation, were shown to be ATPase active. The enzyme displays half of its maximum activity at 0.18 Mm ATP concentration to be inhibited by high concentrations of ATP. ATPase is activated by both Mg2+ and Ca2+ (in a lesser degree), with the maximum activity being at pH 7.5. A 5 minutes heating without the substrate results in decreasing the enzyme activity at 30 degrees, and in the total inhibition at 50 degrees C. Along with ATP, the enzyme can hydrolyse GTP and, in a lesser degree, ADP and sodium pyrophosphate. The ATPase activity is not effected with oligomycin (0.5-1.5 mkg/ml) or ouabaine (0.1 mM). Oligomycin in concentration 5 micrograms/ml induced non-specific inhibition of ATPase. Uncouplers, like 2,4-dinitrophenol and carbonyl cyanid p-trifluorometoxyphenylhydrazone, stimulate the enzyme activity. The lack in the ATP-ase sensitivity to oligomycin (specific inhibitor of mitochondrial F1-ATPase) and ouabaine (specific inhibitor of Na+, K+-ATPase) may suggest that the ATPase activity of novocaine segregation zones in frog's erythrocytes is not associated with a random contamination with mitochondria or cytoplasmic membranes. The ATPase under study has much in common with the lysosomal +H-ATPase. The results obtained support a hypothesis that +H-ATPase may function as a course of protones for maintaining acidic medium in segregation zones and promote accumulation of weak bases by means of their protonation.     

Effect of inhibitors of energy metabolism and protein synthesis on the process of neutral red segregation in frog erythrocytes

Effects of inhibitors of energy metabolism and protein synthesis on Neutral red segregation in frog erythrocytes were studied. Inhibitors of both glycolysis and respiration significantly reduced formation of segregation zones. This influence was most striking with antimycin A, rotenone and cyanide. This indicates that intact respiratory pathways may play an important part in the process of Neutral red segregation. Such uncouplers as FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and 2,4-dinitrophenol (DNP) as well as inhibitors of oxidative phosphorylation (arsenate and azide) are also very effective in inhibiting the Neutral red segregation at low concentrations. The effects of these uncouplers and of olygomycin suggest an important role of ATP as an energy source for the segregation process. An inhibitor of protein synthesis, such as cycloheximide, produces some reduction in segregation zones formation. Trapping of Neutral red by protonation could readily explain the high level of this dye accumulation in nucleated erythrocytes. The fact that low concentrations of FCCP and DNP inhibit the process of segregation brings a supporting evidence for the possibility of the ATP-driven proton pump involved in Neutral red segregation.     

Effect of metabolic inhibitors on formed novocaine and neutral red segregation zones in frog erythrocytes

Effects of some metabolic inhibitors, as well as of biologically active compounds (diakarb, ethidium bromide and a phenanthridine alkaloid sanguinarine) on the formed novocaine and neutral red segregation zones were studied. The volume of granules diminished under the influence of a glycolytic inhibitor iodoacetate, uncouplers of oxidative phosphorylation (2,4-dinitrophenol and carbonyl cyanide trifluoromethoxyphenylhydrozone), and respiratory inhibitors (antimycin A and rotenone), as well as under the influence of cycloheximide - an inhibitor of protein synthesis. Diakarb, ethidium bromide or sanguinarine also provoked a regression of the segregation zones. It has been found that all these compounds are inhibitors of ATPase activity of the isolated segregation zones. A possible mechanism of volume decreasing in segregation zones under the influence of both the metabolic inhibitors and diakarb, ethidium bromide and sanguinarine is discussed.     

Autophagocytosis in mouse seminal vesicle cells in vitro. Temperature dependence and effects of vinblastine and inhibitors of protein synthesis

Spontaneous autophagocytosis was observed in mouse seminal vesicle cells during incubation for 2 h in vitro. The number of autophagic vacuoles formed was greatest at 37 degrees C and decreased when the temperature was lowered. At 22 degrees C it reached the near-zero value characteristic of non-incubated control cells. Incubation of the cells at 37 degrees C in the presence of 0.1 mg/ml vinblastine sulfate resulted in a marked increase in the number of autophagic vacuoles, but the drug was ineffective at 22 degrees C. Puromycin (10(-3) M) exerted no influence on spontaneous autophagocytosis, but cycloheximide in concentrations from 10(-7) M to 10(-3) M inhibited both spontaneous and vinblastine-induced autophagocytosis. The formation of tubulin paracrystals in vinblastine treated cells was not prevented either by low (22 degrees C) temperature or in the presence of cycloheximide.     

Effect of hibernation on sodium and chloride ion transport in isolated frog skin

The aim of the study was to evaluate the effect of hibernation on electrophysiological parameters of isolated frog skin under control incubation (Ringer solution) and after inhibition of Na+ and CI- transepithelial transport by application of amiloride and bumetanide. The transepithelial electrical potential difference (PD in mV) was measured before and after mechanical stimulation of isolated frog skin. The tissues were mounted in a modified Ussing chamber. The results revealed a reduced PD of frog skin during hibernation. In February, as compared with November, PD of frog skin incubated in Ringer solution decreased by about 50%. Hibernation also affected hyperpolarization (dPD) of frog skin after mechanical stimulation. In November and December, dPD was about 50% and 30% lower, respectively, compared with the subsequent two months of the experiment. The incubation of frog skin with amiloride, a sodium ion channel blocker, resulted in reduced values of all measured electrophysiological parameters irrespective of the phase of hibernation. After application of chloride ion transport inhibitor (bumetanide), the PD in November and December decreased compared with the control incubation by about 80% and 75%, while in January and February by about 40% and 25%, respectively. In January and February dPD increased by four times and three times as compared with November and December. Hibernation reduces net ion flow in isolated frog skin. During the initial period of hibernation the sensitivity of the skin to mechanical stimulation also decreases. Towards the end of hibernation, on the other hand, excitation of mechanosensitive ion channels takes place.     

Participation of Na, K-ATPase in the mechanism of isotonic fluid absorption by the epithelium of the frog gallbladder]

In experiments on the isolated frog gallbladders it was shown that addition of ouabain (1.10(-4) M) or noradrenaline 3.10(-6) M) into the incubation Ringer solution from the serous surface of the gallbladder and also replacement of K+ in the solution for the equivalent quantity of Na+ ions caused a reduction of the absorption of isotonic fluid by the epithelium and a fall of the Na,K-ATPase activity in its cells. Noradrenaline also cased a reduction of Mg-ATPase activity. A significant positive correlation was found between the transport rate of the isotonic fluid by the epithelium and the Na,K-ATPase activity in its cells.     

Effect of low-molecular nonelectrolytes and hypotonia on the contractile responses of frog sartorius muscles

The potentiation of contractile responses during prolonged incubation of frog sartorius muscles in a conventional Ringer solution and in a calcium-free Ringer solution which contained 400 mM of urea, acetamide or ethylene glycol was observed when stimulation with single electric impulses and with caffeine was carried out. When the muscles were exposed to a hypotonic solution the reduction in the amplitude of caffeine contractures or their complete disappearance were registered, whereas the electrical stimulation of muscles in the same medium exerted an increase in the amplitude of contractile responses. The addition of 400 mM glycerol to the hypotonic solution caused an increase in the amplitude of contractions under both types of stimulation.     

Effect of primycin on some electric properties of the frog skeletal muscle

The effect of primycin, a guanidine-type antibiotic was studied on the electric properties and 42K+ uptake of the frog sartorius and semitendinosus muscle. Both in normal and choline chloride Ringer solution, primycin evoked a concentration and time dependent depolarization of the surface membrane of the muscle. This depolarization was significantly increased by Na ions. Primycin treatment was shown to evoke a dose-dependent decrease of the depolarization induced by 20 mM K+-Ringer. When the muscles were incubated in a Ringer solution containing choline chloride, during an incubation period of 30 min the uptake of 42K+ was decreased to 12% upon the exposure to 5 x 10(-6) mol primycin as compared to the control value. As the primycin-induced depolarization increased, the shape and amplitude of the action potentials elicited by square-wave electric impulses were altered and decreased, respectively. In sodium isaethionate Ringer 1--2 x 10(-6) M primycin induced a slow depolarization resulting in firing potentials. The results suggest that primycin depolarizes the surface membrane exclusively through the blockade of the resting K+ channels, the other phenomena being the results of this depolarizing effect.     

Arterial perfusion of frog tongue for intracellular recording of taste cell receptor potential

The frog tongue was perfused through its artery with a Ringer solution using a peristaltic pump, and a method was developed to record stable intracellular receptor potentials of taste cells. Perfusing at 0.05 ml/min with a Ringer solution containing 5% dextran did not cause tongue edema, but perfusing at the same rate with Ringer without dextran caused edema. After perfusion at 0.05 ml/min with 100 mM K Ringer, the membrane potential of taste cells gradually decreased and reached a constant level in about 30 min, indicating that the intercellular fluid of the tongue could be replaced within this time period. While the artery of the frog tongue was perfused at 0.05 ml/min with Ringer containing 5% dextran, intracellular receptor potentials of taste cells elicited by four basic taste stimuli (1 M NaCl, 10 mM quinine-HCl (Q-HCl), 1 mM acetic acid and 1 M galactose) were similar to those obtained from the control taste cells under normal blood flow.     

Effect of vacuolization on the sodium concentration in an isolated muscle fiber]

Using the Perkin Elmer flame photometer sodium and potassium concentrations have been measured in muscle fibers from the m. ileofibularis of Rana temporaria. After 30 minutes preincubation in the Ringer solution, made hypertonic by the addition of 0.22M glycerol, the muscle fibers were incubated in the normal Ringer solution for 30 min. These fibers showed a vacuolation and an increase in total fiber sodium up to 37.2 mmol/l +/- 5.9 S. E., or 45.8 mmol/kg H2O +/- 7.3 S. E. No significant changes in potassium concentration were observed. Then, the fibers were exposed again to the Ringer solution containing 0.22 M glycerol. This procedure caused the disappearance of vacuoles and decrease in fiber sodium concentration down to 17.7 mmol/l +/- 1.6 S. E., or 21.8 mmol/kg H2O +/- 2.0 S. E. The effect of vacuolation was not blocked by ouabain (1.10(-4) M). It is suggested that the vacuoles have a high NaCl concentration. A model for NaCl and water accumulation in T-tubules is presented.     

Study of Effect of Autacoids on Water Absorption and Ion Transport by Skin of the Frog Rana temporaria

The experiments on the frog Rana temporaria isolated skin showed participation of autacoids in regulation of the epithelium water permeability and of the transepithelial ion transport. The removal of autacoids secreted by the cells into the Ringer solution at its internal surface with the aid of frequent replacements of this solution leads to an increased water permeability and to a decreased transepithelial potential difference. Inhibition of prostaglandin synthesis with 1 × 10^–5 M indomethacin produces the frog skin depolarization. Addition of prostaglandin E₂ to the Ringer solution at the internal surface of the frog skin is accompanied by a decrease of the osmotic permeability, hyperpolarization, and an increase of short-circuit current. The non-contradictory model is described of the role of autacoids in regulation of the frog skin functions connected with participation of the skin in the water–salt homeostasis.     

Structural alterations in nerve fibers produced by hypotonic and hypertonic solutions

In sections of KMnO(4)-fixed, developing mouse sciatic nerves, the central gap of mesaxons in myelinating fibers is normally closed with close apposition of the outside approximately 20 A dense strata of the two approximately 75 A Schwann cell membranes. The two combined outside strata make the intraperiod line bisecting each myelin lamella. The approximately 150 A mesaxon is elaborated spirally around the axon in either a right hand or left hand spiral, and its inside (cytoplasmic) approximately 20 A strata in apposition form the major dense lines of myelin. In hypotonic solutions the lamellae of adult frog sciatic myelinated fibers split apart along the outside membrane strata apposed at the intraperiod line throughout the spiral. Under similar conditions the inside (cytoplasmic) strata of the membranes, in apposition at the major dense lines, do not separate. The approximately 150 A membranous structure resulting from this is called an "internal compound membrane." The double membranes of normal and control frog sciatic unmyelinated fibers have a central gap approximately 100 to 150 A wide. After soaking in 4 to 10 times normal strength Ringer solution or 10 N sucrose-Ringer solution, this gap closes and a membranous structure approximately 150 A wide resembling developing mouse mesaxons results. This is designated by the term "external compound membrane." The latter membranes resemble internal compound membranes, but their central dense zones, each consisting of two apposed outside membrane strata, are less dense.     

The effect of serotonin on epinephrine modulation of transepithelial ion transport in isolated frog skin

The aim of this study was to examine the effect of serotonin and epinephrine on ion transport of isolated frog skin. The addition of serotonin after incubation in Ringer solution (RH), bumetanide (BUME), and after initial incubation in amiloride and subsequently in RH, reduced hyperpolarization and did not effect the mechanosensitivity of frog skin. Following incubation of the frog skin with amiloride (AMI), serotonin did not affect the value of hyperpolarization and increased mechanosensitivity. The addition of epinephrine (EPI) on frog skin incubated in RH and AMI did not affect hyperpolarization, but repeated application of this compound after serotonin increased hyperpolarization. After incubation with bumetanide, addition of EPI before and after application of serotonin did not affect the value of the examined parameters of the frog skin. Initial incubation with AMI and later in RH caused a drop in reaction to EPI and no effect on mechanosensitivity. Repeated addition of epinephrine in this group did not affect the reaction value, while it decreased the reaction value during mechanical stimulation. The experimental data presented in this study indicate that serotonin inhibits the sodium ion current. Epinephrine inhibits the chloride ion current, however, after the application of serotonin, EPI stimulates sodium ion transport.     

Effect of capsaicin and dimethyl sulphoxide (DMSO) on ion transport in isolated skin of frog (Rana esculenta L.)

The effects ofcapsaicin, dimethyl sulphoxide and pH changes on transport of sodium and/or chlorine ions in an isolated frog skin, were studied using electrophysiological methods, adapted to evaluation of ionic currents occurring in the epithelial tissues and organs. The experiment consisted in measuring potential difference (PD in mV) of an isolated skin of the aquatic frog, Rana esculenta L., placed in a Ussing apparatus. The ionic transport processes were modified through incubation of the tissue in Ringer solution and in Ringer solution supplemented with amiloride, bumetanide, and also with dimethyl sulphoxide. The direct effect of capsaicin and dimethyl sulphoxide (DMSO) on frog skin was assessed while these compounds were added to the Ussing chamber with a pipette and a peristaltic pump. Adaptive reactions of the tissue were assessed following at least 60-min exposure to those compounds. It has been demonstrated that amiloride-inhibited sodium ion transport and acidification of the incubation medium (pH 6.4) inhibited mechanically induced epithelium reactions. Both compounds, capsaicin and DMSO modified ionic transport processes depending on the mechanical stimulation.     

A new method for excitation-contraction uncoupling in frog skeletal muscle

The mechanical activity of frog sartorius muscle fibers can be uncoupled from the electrical activity of their surface membranes by immersing the preparation in Ringer solution containing either 1.5 or 2.0 M of formamide for 15--20 min. This uncoupling is not reversed when the muscle is transferred to normal frog Ringer solution. Formamide does not affect the electrical activity of the sciatic nerve branch, and both endplate potentials and miniature endplate potentials may be recorded from the uncoupled muscles. Prolonged exposure to formamide, beyond the time needed to paralyze, causes neuromuscular block.     

Tension and instantaneous stiffness of single muscle fibers immersed in Ringer solution of decreased tonicity.

Isometric tension and instantaneous stiffness were measured in frog semitendinosus single muscle fibers in both isotonic and hypotonic Ringer solution. In 0.7 and 0.5 x normal Ringer tension increased 17 and 20%, respectively. There was no corresponding increase in the measured stiffness. The increase in tension in hypotonic Ringer could be reversed by the addition of an osmotic equivalent of sucrose to the bathing solution. These findings suggest that the potentiated tension observed in hypotonic Ringer is due to an increased tension per cross-bridge and not to an increase in the number of attached cross-bridges.     

Influence of culture medium osmolality on maturation and ovulation of Rana temporaria oocytes stimulated in vitro by the pituitary gland extract or progesterone

The influence of diluted Ringer solution on ovulation and maturation of common frog oocytes stimulated in vitro by homologous pituitary extract (0.005 pit/ml) or progesterone (1 pg/ml) was studied. During wintering, the dilution of Ringer solution led to a decreased percentage of oocytes ovulated and matured under the influence of both inducers. As the season of reproduction approached, the dependence of oocyte maturation and ovulation on the Ringer solution dilution weakened. Possible causes of different dependence of the ovulation of amphibian and sturgeon oocytes stimulated by gonadotropic hormones or progesterone on the culture medium osmolality is discussed.     

Action of high hydrostatic pressure on the fluorescence of respiratory metabolic components and on the staining capacity of the skeletal muscles in the frog

The effect of high hydrostatic pressure (100-1400 at) on frog skeletal muscle was studied. Superthreshold pressures (above 600 at) accompanied by muscle contracture results in the intensification of sorption of both basic (neutral red) and acid (phenol red) dyes, and in the growth of the number of oxidated forms of respiratory metabolic components--flavoproteins and pyridine nucleotides under study. Subthreshold pressures reduce the tinctorial ability of muscles and decrease oxidative processes caused by the maintenance of muscle in small volume of the Ringer solution.     

AUTOPHAGIC VACUOLES PRODUCED IN VITRO : I. Studies on Cultured Macrophages Exposed to Chloroquine

Mouse macrophages exposed to 30 µg/ml of chloroquine in vitro develop autophagic vacuoles containing various cytoplasmic components and acid phosphatase. The early toxic vacuoles appear in the perinuclear region within 15 min; on electron microscopy, they show irregular shape, amorphous moderately dense content, apparent double membranes, and in some instances curved thin tubular extensions with a central, dark linear element. Cytoplasmic structures are probably transported into the vacuoles by invagination of the vacuolar membrane. After exposure to chloroquine for 1–4 hr, macrophages display large vacuoles containing degraded cytoplasmic structures, membranous whorls, and amorphous material. When chloroquine is removed by changing the culture medium after 4 hr, the cells survive and 24 hr later they exhibit no abnormality except for large cytoplasmic dense bodies packed with membrane lamellae. During recovery chloroquine disappears from the cells. 24 hr after exposure to chloroquine the macrophages have accumulated less hydrolases than control cells.