Incubation In Ringer Solution Research Articles

The impact of erythrocyte age on eryptosis

Mature, circulating erythrocytes undergo senescence, which limits their life span to approximately 120 d. Upon injury, erythrocytes may undergo suicidal erythrocyte death or eryptosis, which may accelerate senescence and shorten their survival. Eryptosis is defined as cell shrinkage and exposure of phosphatidylserine at the cell surface. Triggers of eryptosis include oxidative stress. The present study addresses the impact of erythrocyte age on the relative susceptibility to eryptosis. Erythrocytes were separated into five fractions, based on age-associated differences in density and volume. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, the cell volume from forward scatter, and the Ca(2+) level from Fluo-3-dependent fluorescence. In addition, glutathione (GSH) concentrations were measured by an enzymatic/colourimetric method. After 48 h incubation in Ringer solution, Annexin V binding increased significantly with erythrocyte age. The differences were not accompanied by altered GSH concentrations, but were reversed by addition of the antioxidant N-acetyl-L-cysteine in vitro. Also, N-acetyl-L-cysteine significantly prolonged the half-life of circulating mouse erythrocytes in vivo. Thus, the susceptibility to eryptosis increases with the age of the erythrocytes, and this effect is at least partially due to enhanced sensitivity to oxidative stress.

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.

Changes in isometric force of mouse soleus muscle during the oestrous cycle.

Muscles excised from young female mice at known phases of the oestrous cycle were studied in vitro to determine if there are variations in force analogous to those that occur in vivo during the menstrual cycle in women. Oestrous phase was determined from vaginal smears. The maximum isometric and eccentric forces of pairs of isolated soleus muscles were measured. The first muscle was studied immediately after dissection, the second after incubation in Ringer solution for up to 2 h. Normalised isometric muscle force in the first muscle of each pair depended on the oestrous phase, the force being greatest during dioestrus. There was a negative correlation between normalised force and the eccentric/isometric force ratio. Neither of these phenomena was found with the second muscle of each pair. These results show that in mouse soleus muscle cross-bridge function does vary according to the phase of the oestrous cycle. However, the rise in force does not follow the pattern of the rise in blood oestrogen levels as it does in humans, and in the mouse the effect on cross-bridge function washes out after a few hours in vitro.

Relaxation rate of intact striated muscle fibres after flash photolysis of a caged calcium chelator (diazo-2).

Single twitch fibres from lumbrical muscles of Xenopus have been loaded with the photolysable calcium-chelator diazo-2 by incubation in Ringer solution containing the membrane permeable acetoxymethyl ester (AM) form of diazo-2. Incubation caused a progressive slowing of tetanus rise and relaxation which is ascribed to calcium-buffering by unphotolyzed diazo-2 (Kd = 2.2 microM). After incubation, exposure to a brief UV flash caused a three to four fold increase in the rate of tension fall. A flash given 16-18 ms after the last tetanic stimulus (at 22-24 degrees C) resulted in 10% increase in relaxation rate compared with the control before incubation. A much bigger effect was observed when a flash was given half-way into the slow phase, where an 1.8-1.9-fold increase in relaxation rate, above the preincubation slope, was observed. It is concluded that rapid lowering of [Ca]i, and hence more rapid removal of Ca2+ from troponin, speeds up relaxation, indicating that calcium translocation is the major determinant of the rate of tension fall during the isometric phase of relaxation.

Kainic acid-induced release of serotonin from OFF-bipolar cells in the turtles retina

The immunofluorescence of antibody-labelled serotonin was used as an indicator for the amount of intracellular serotonin. Incubation in Ringer solution containing serotonin and pargyline increased the immunofluorescence which was subsequently decreased in a calcium-dependent manner by incubation in high-potassium Ringer. If preloaded retinas were exposed to 10 μM kainic acid, the immunofluorescence decreased in a time-dependent way. This kainic acid-induced release was blocked by piperidine dicar☐ylic acid. These results support the idea that serotonin is a transmitter in OFF-bipolar cells.

The zinc metabolism of the amphibian lens

The amphibian lens contains a substantial amount of Zn; about 10 −4 mol/kg tissue water. The levels in the “nuclear” and “cortical” zones of the lens appear to be similar. Most of the Zn is bound in an inexchangeable form; only 5–8% can exchange with external 65Zn. Interference with metabolism (with CN and iodoacetate) or ion regulation (with Na-free solutions or ouabain) had no effect on the levels of lenticular Zn. Incubation with EDTA or following freezing and thawing the tissue also had no significant effect on the metal levels in the lens (a small effect however cannot be excluded). Zn was accumulated by the lens when the external concentration of the metal was greater than about 10 −6 m. Uptake of 65Zn occurred slowly and had not reached saturation after 4 hr. At 2·5 × 10 −6 m 65Zn was taken up more rapidly than 109Cd or 203Hg but at 2·5 × 10 −4 m the Hg was taken up faster. Accumulated 65Zn, as well as 109Cd and 203Hg, was lost from the lens during incubation in Ringer solution but even after 3 hr about 50% was still retained in the tissue. Accumulation of 65Zn 2+ was reduced in the presence of Cd 2+ and nearly abolished by La 3+ but was unchanged by an SH blocker (NEM), Ca 2+ or Mg 2+. The initial efflux of 65Zn was increased in the presence of EDTA and La 3+ but was unchanged by NEM, Cd 2+ or Cu 2+. The exchangeable Zn appears to be associated with the peripheral regions of the lens as significant accumulation was not observed in decapsulated lenses. Uptake of Zn 2+ (and Cd 2+) occurred more rapidly from the posterior than the anterior side of the lens.

Intracellular potassium activity of isolated human and rabbit corneal epithelium

Single-barrelled open tip microelectrodes containing a potassium-selective liquid ion exchanger (Corning 477 317) were used to determine intracellular K-activity a K i in squamous and basal cells of isolated rabbit and human corneal epithelium. The intracellular potassium activity a K i during the five hours of incubation in Ringer solution was 114·3±23·6 mmol/l and 107·6±26·2 mmol/l for the basal cell layer of rabbit and human epithelia respectively. The corresponding values for the squamous cells of the superficial cell layer were 41·2±14·2 mmol/l and 50·2±11·1 mmol/l. Based on intracellular chemical concentrations of potassium c K i an apparent activity coefficient could be calculated that is close to that of a dilute solution of the same ionic strength. This indicates that intracellular potassium is essentially free in both epithelia. Ouabain (5 × 10 −5 mol/l) added to the bathing solution of the rabbit cornea lowered the transmembranal voltage Ψ M as well as the intracellular K-activities of squamous and basal cells. Three hours after application of ouabain a K i of the basal cells had decreased to about one third of its initial value. Under all experimental conditions the potassium equilibrium potential E K considerably exceeded Ψ M. Our data indicate: (1) The high absolute values for a K i imply that almost no potassium is bound or sequestered within the epithelial cells. (2) An active potassium uptake is necessary to explain the high K-activity in relation to the transmembranal voltage Ψ M. (3) There is an intraepithelial gradient of a K i within the corneal epithelia.

Cyclic AMP and adrenergic receptors in Gambusia affinis melanophore response in vitro

In vitro responses of Gambusia affinis caudal fin melanophores to norepinephrine, epinephrine, and isoproterenol after incubation in Ringer solution and after alpha- and beta-adrenergic blockade are consistent with the presence of both alpha- and beta-adrenergic receptors on these cells. Mediation of aggregation is regulated through alpha receptors and dispersion through beta-adrenergic stimulation. Dispersion of pigment by cyclic AMP (cAMP), dibutyryl cAMP, and caffeine and acceleration of dispersion by simultaneous administration of either caffeine + cAMP or caffeine + dibutyryl cAMP are interpreted as evidence for mediation of dispersion by the cyclic nucleotide. Dispersion of pigment by ACTH and enhanced dispersion by ACTH + cAMP and by ACTH + caffeine suggest participation of the hormone in increasing cAMP levels. An increase in cAMP levels in response to beta-adrenergic stimulation is indicated by (1) dispersing action of 10 −6 M isoproterenol, which is blocked by simultaneous administration of the beta-adrenergic antagonist dichloroisoproterenol (DCI), (2) significant enhancement of the dispersing action of cyclic nucleotides, caffeine, and ACTH by simultaneous treatment with 10 −6 M isoproterenol or by previous exposure to 10 −4 M isoproterenol, which weakly aggregates pigment, and (3) abolition of enhancement of dispersion when cells are exposed simultaneously to 10 −4 M isoproterenol and DCI prior to treatment with dispersing agents. Prior exposure to norepinephrine prevents dispersion by both ACTH and exogenous cAMP, and inhibitory effects of norepinephrine are abolished when cells are pretreated simultaneously with the catecholamine and the alpha-adrenergic blocking agent dibenamine. These observations are interpreted as evidence supporting the view that alpha-adrenergic stimulation decreases cAMP levels and suggesting that, in these cells, it does so by enhancing phosphodiesterase activity rather than by adenyl cyclase inhibition.

The release of lipoproteins by rat liver slices.

The release of the lipid and protein moieties of lipoproteins by liver slices from rats charged in vivo with palmitate-9,10-3H and leucine-1-14C was followed. The replacement of Ringer solution as the incubation medium by serum resulted in an increase in the release of the labelled lipid moiety but had no effect on the radioactivity of the protein moiety. Analysis of the lipoproteins indicated that serum had no effect on the radioactivity of the neutral lipid in the lipoprotein of d < 1.063 or β-lipoproteins prepared by ultracentrifugation or heparin precipitation, respectively. However, the lipoprotein of d < 1.063 released into serum contained considerable amounts of labelled phospholipids and fatty acids which were not present in the β-lipoprotein precipitated by heparin or in the low density lipoproteins prepared by either procedure following incubation in Ringer solution. Following incubation of the slices in serum, but not in Ringer solution, the lipoprotein having d > 1.063 and the supernatant of the heparin precipitate contained considerable amounts of labelled phospholipid. It is concluded that serum may contain a lipoprotein capable of combining with liver phospholipids and that ultracentrifugation results in the separation of low density lipoproteins containing excess lipid.

The effect of blood-borne factors on adrenal steroid synthesis in the golden hamster (Mesocricetus auratus Nehring).

SUMMARY Incubation of hamster adrenal tissue in Krebs bicarbonate Ringer solution gave rapid conversion of added [4-14C]cortisol to cortisone. Moreover, incubation in Ringer solution with [4-14C]progesterone as precursor invariably yielded cortisone in greater amounts than cortisol. On the other hand it has been confirmed that cortisol is the major free ultraviolet absorbing steroid in the adrenal venous blood of the hamster. Incubation of adrenal tissue with [4-14C]progesterone in hamster whole blood gave relatively greater amounts of cortisol which were more in keeping with the findings in vivo. This suggested that the blood contains factors which affect the cortisone-cortisol equilibrium by modifying adrenal enzyme activity. Some reduction of [3H] cortisone to cortisol was also observed during incubation with blood alone.

A CONTRIBUTION TO THE BIOLOGY OF PERIPHERAL NERVES IN TRANSPLANTATION

1. The Wallerian degeneration occurring in peripheral nerves by incubation in Ringer solution and serum does not occur in plasma. 2. Peripheral nerves incubated in plasma give rise to no growth. The same is true of peripheral nerves in a Wallerian degeneration up to the 4th day. 3. Peripheral nerves in Wallerian degeneration from the 5th day give rise to a growth of the syncytium of Schwann. In cultures from later stages there is a progressive growth of the same structure. It is evident that the proliferation of the cells of Schwann is directly produced by the degeneration of the axis cylinder and its myelin sheath. 4. In no case was growth of axis cylinders observed. 5. The growth of the syncytium of Schwann from degenerating nerves affords a basis for an anatomical conception of the centrifugal orientation of growing axis cylinders in regeneration. 6. Morphologically there is a striking resemblance between the syncytium of Schwann and neuroglia growing in plasma.