Tyrode's Medium Research Articles

Is There a Mast Cell-Noradrenergic Nerve Interaction in the Bovine Splenic Vein?

Release of neurotransmitter from noradrenergic nerve terminals can be modulated by a variety of endogenous and exogenous compounds which act as agonists or antagonists at receptors located on the presynaptic membrane of the terminal.For example, noradrenaline acts as an α-adrenoreceptor agonist to decrease further release. This effect can be reversed by drugs such as phentolamine which blocks the α-adrenoreceptor to increase the outflow of transmitter. Actually, the α-receptor mediated overflow after exposure to an antagonist is one of the most studied mechanisms in transmitter regulation. However, we have been confronted with an exception to the rule of noradrenaline overflow after phentolamine (Fig. 1). In this study isolated bovine splenic vein strips were preincubated in tritiated noradrenaline and mounted in a superfusion apparatus. Oxygenated Tyrodes medium (pH 7.4) containing 10-6M cocaine and 10-4M normetanephrine was used during superfusion.

Cation fluxes and volume regulation by human lymphocytes.

The ionic basis of volume regulation by human peripheral blood lymphocytes in hypotonic Tyrode's medium has been studied. The intracellular water space of lymphocytes increased to a maximum after 1 min in 0.68 X isotonic Tyrode's but returned to the isotonic value by 20 min at 37 degrees C. During this phase of volume regulation (1-20 min) both 42K+ efflux and 42K+ influx were stimulated severalfold, but the increase in 42K+ efflux exceeded the influx, resulting in a net loss of 20% of the lymphocyte K+. The increase in 42K+ efflux during the phase of cell shrinkage was unaffected by ouabain or by quinidine. Hypotonicity increased both the ouabain-sensitive (active) and ouabain-insensitive components of 42K+ influx by 76% and 123% respectively. Hypotonic shock stimulated 22Na+ influx by only 25%, but cell Na+ content was unchanged at 1 min and even decreased after 20 min. Thus active K+ influx and Na+ extrusion is increased by hypotonicity, but greater pumping cannot explain the net decrease in cell cations that leads to volume regulation. The 45Ca2+ uptake was not significantly changed by hypotonicity. Although volume regulation was abolished in a hypotonic high K medium, 42K+ efflux was still stimulated 2-fold by the reduction in tonicity. These findings support the hypothesis that volume regulation in hypotonic media occurs largely by a passive loss of cell K+, which results from a selective increase in membrane permeability to this ion. The increase in K+ permeability in hypotonic media is observed even in the absence of volume regulation by the cell.

The effect of harmaline and related β-carbolines on the acetylcholine-stimulated contractions of guinea-pig ileum

Five members of the Harmala family of alkaloids, four commercially available and one synthetic homologue, were tested for their actions on the acetylcholine-elicited contractions of the guinea-pig ileum. Under normal Tyrode conditions over comparable concentration ranges harmalol was without effect, harmaline and harmaline inhibited competitive-noncompetitive inhibition whilst the related 2-methylated derivatives effected purely competitive antagonism. Binding studies using [ 3H]QNB have further confirmed the competitive aspect of this antagonism. The competitive-noncompetitive inhibitory property of harmaline on smooth muscle muscarinic receptors has not been previously reported. Harmaline is known to compete with Na + binding sites and significantly complete substitution of Na + by K + in the Tyrode medium was found to abolish the noncompetitive component of harmaline inhibition. Explanations are offered for both the competitive and noncompetitive components of the inhibition produced by the Harmala alkaloids.

Release of benzoyl-arginine-ethylester esterase from dispersed cells of mouse submandibular gland

Viable dispersed cells were prepared from male mouse submandibular gland by a tissue-dissociation procedure using collagenase and trypsin, and mild shearing force. These cells were incubated in a fortified Tyrode medium under a variety of experimental conditions and the secretion of benzoyl-arginine-ethylester esterase in response to various secretagogues was investigated. The cells released the esterase time-dependently upon stimulation with norepinephrine (1.5 × 10 −6 M) up to 30min at 30 °C and up to 15 min at 37 °C. The extent of esterase release was dependent on the concentration of agonist and was not influenced by varying the number of cells. The release of esterase was caused only by α-adrenergic agonists such as norepinephrine, phenylephrine and methoxamine, and the release by norepinephrine was completely abolished by α-adrenergic blockers such as phenoxybenzamine, ergotamine and dibenamine. Calcium was essential for the esterase release mediated by norepinephrine. The secretory pattern of esterase was similar to that of kallikrein (kininogenase).

The influence of external sodium and potassium on lithium uptake by primary brain cell cultures at "therapeutic" lithium concentration.

The ionic regulating of lithium homeostasis and steady-state intra:extracellular lithium distribution in the brain can be approached by experimental methods using intact nerve cells in vitro. Primary cultures prepared from chick embryonic brain were applied to study the effect of extracellular sodium and potassium on the lithium uptake of nerve cells at 'therapeutic' lithium concentration (1.5 mM). Lithium influx and the level of steady-state intracellular lithium were significantly reduced by increasing the external sodium concentration. At physiological extracellular sodium level, the steady-state content of lithium in the brain cells was about half of that observed in the presence of 10 mM sodium in the incubation media and the value of the intra:extracellular lithium distribution ratio was below 1. External potassium (0.5 - 3mM) strongly inhibited lithium uptake of the nerve cells. Ouabain (10(-4)M) had no effect on this potassium-sensitive lithium uptake in Tyrode media. Sodium influx studied by isotope tracer methodology was higher in cultures preloaded with lithium as compared to that of the controls. It can be concluded that sodium and potassium ions, at physiological concentrations, significantly influence lithium uptake as well as the intra:extracellular lithium distribution in brain cell cultures.

Progress in behavior therapy with delinquents : Jerome S. Stumphauzer. Charles C. Thomas, Publisher (301–327 East Lawrence Avenue, Springfield, Illinois 63703), 1978, 390 pp., hardcover-$29.75; softcover-$22.75

The aim of this study was to assess extenders for cooling equine semen at 5°C and to be used in assisted reproductive technologies (ARTs). Four ejaculates were obtained from each of four stallions. Gel-free semen was diluted in three different extenders: (1) SMK, an opaque skim milk–based extender; (2) SMT, a skim milk (65%) and Tyrode medium (35%); and (3) BSAG, a clear extender containing 1% bovine serum albumin. Samples were packaged (10 mL; 50 × 106 sperm/mL) and stored in a cooling device at 5°C for 12 hours. Analyses were done at 0, 4, 8, and 12 hours after cooling. Semen was analyzed for sperm motility characteristics using a computer-assisted sperm analysis, for plasma membrane and acrosome integrity and mitochondrial membrane potential, using fluorescent probes (propidium iodide, Hoechst 33342, fluorescein isothiocyanate–conjugated Pisum sativum agglutinin, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide [JC-1]). Morphology was evaluated with differential interference contrast microscopy and sperm chromatin integrity by the toluidine blue technique. Data were analyzed by analysis of variance and by Tukey test, with time as a repeated measure (SAS, 1998), when P < .05 was significant. In general, milk-based extenders (SMT and SMK) showed improved maintenance of semen quality compared with BSAG. Finally, the addition of skim milk to equine semen extender for cooling at 5°C for 12 hours seems to play a crucial role in sperm preservation. Although, optically clearer extenders are desired for use in ARTs, such as sperm sexing, the milk-free extender (BSAG) is less efficient for cooled-stored equine semen.

Increase in passive tension of spontaneously beating atria in hyperosmolar media

Spontaneously beating rabbit atria were used for study of increases in passive tension induced by hyperosmolar Tyrode bathing media. The time course and degree of passive tension change was dependent on the level of osmolality and the osmotic agent used. The effectiveness of various agents added to Tyrode solution in increasing resting tension was in the order sucrose greater than mannitol greater than or equal to NaCl greater than glucose greater than = no change. The hyperosmotically induced "contracture" was similar to contracture induced by high Ca2+ medium, but the maximum hyperosmotic effect was greater than that of high Ca2+. In contrast to active atria, inactive atria showed no increase in passive tension in hyperosmotic solutions or in Tyrode solution containing high Ca2+. After depolarization with Tyrode medium containing high K+ or zero K+, increased Ca2+ caused contracture in inactive left atria greater than that caused by hyperosmotic media (+200 mM sucrose) in active right atria or atrial pairs. Results of the study indicate that although other factors may be involved, increased Ca2+ flux inward could largely account for the passive tension response.

Toxic effects of copper on cultured rat lenses

Lenses from 60-day-old rats were cultured for 24 hr at 35°C in Tyrode's medium containing copper sulfate at concentrations of 0, 10, 20, 50 and 100 μ m. Lenses incubated with 0 or 10 μ m copper remained clear. Lenses incubated with 20, 50 or 100 μ m copper became cloudy, the cloudiness increasing with increasing copper concentration and being more intense in the equatorial than in the central region. Glucose utilization was inhibited, lens sodium and water contents were increased, and lens potassium was decreased with increasing copper concentration. At 10 μ m, the copper concentration at which opacification first became observable, glucose utilization was inhibited 20% and lens sodium content was increased threefold compared to control lenses. Copper (100 μ m) inhibited the active transport of 86Rb and increased its passive transport to a substantially greater degree than it inhibited the glucose utilization of cultured lenses. EDTA in the medium afforded substantial protection from copper toxicity; d-penicillamine (used to treat diseases involving copper toxicity) and histidine afforded partial protection. Noteworthy clinical implications of these studies are discussed.

The effect of pH on rabbit atrial response to histamine.

The chorontropic response of isolated rabbit atria in normal Tyrode's medium increases monotonically with increasing doses of histamine (9 X 10-7 -9 X 10-4 M). Plots of the inverse of response against the inverse of concentration were linear; and from these plots were derived values fro the theoretical maximum response at 'infinite' dose and for pH histamine concentration required to evoke a half maximum response. Alteration of pH by changing (HCO3-) at a constant pCO2, (Na) and osolality did not appreciably affect the response to histamine in the range pH 7.0-7.6. However, at pH below 7.0 the magnitude of histamine response was reduced at all concentrations of histamine tested. In the pH range 7.0-7.6, additions of NaHCO3 at constant pCO2 increased the spontaneous rate of rabbit atria (in the absence of histamine); however, there was little effect of changing pH (in this range) by altering (HCO3-) at constant pCO2 when (Na+) and osmolaity were kept constant. Immersion in solutions at pH's less than 7.0 led to decline in spontaneous rate and force contraction. It is probable that depression of adenyl cyclase activity rather than a specific change in ionization of histamine receptor is responsible for a decreased response to histamine at pH 6.9.

Cataracts induced by the polypeptide antibiotic polymyxin B sulfate

The membranes of the lens fibers contain reactive acidic groups as a part of glycoproteins, phospholipids, protein-SH, polyphosphate nucleic acids and acid mucopolysaccharides. Polycations such as polymyxin B sulfate, neomycin sulfate, cetylpyridinium chloride, cetyltrimetylammonium bromide and polylysine affect the membranes of the lens fibers and produce unilateral cataracts after intravitreal injection. Fifteen minutes after intraocular of polymyxin B sulfate administration, breaks in the membranes of lens fibers and vacuoles between the fibers are found by light and electron microscopy. Increased lens hydration results from in vitro lens incubations in Tyrodes medium containing 0·1–0·5 mg/ml polymyxin B sulfate.

Survival Of Adult Clonorchis Sinensis In The Biliary Passages Of Dead Human Bodies.

Adult Clonorchis sinensis were collected from the biliary passage of nine cases of medico-legal autopsies. The worms were washed repeatedly with tap water and rinsed twice with physiological saline. Incubation took place in Tyrode's medium in order to recover live worms. The number of worms surviving in the medium was determined. The rate of surviving worms showed an unexpectedly high percentage (22.8%) in those cases autopsied 2 days after death. The surviving worm burden dropped sharply to 6.3 and 9.2 percent for those samples taken 5 days after death.

Release of cholinesterase by adult Nippostrongylus brasiliensis in vitro.

Adult N. brasiliensis incubated in Tyrode's medium released between 3% and 11% of their initial total cholinesterase into the medium per hour; release of enzyme continued for at least 4 hours. Immune-damaged worms, which have much higher cholinesterase levels than normal adults, released 2 to 3 times more enzyme per unit wet weight than did the normal worms. There was no significant difference between the amounts of cholinesterase released per unit wet weight by male and female worms. Adult Nematospiroides dubius released about 1% of their total cholinesterase per hour; i. e. about 1/20 as much enzyme as that released by normal adult N. brasiliensis. These results are discussed with reference to possible functions of cholinesterase in the host/parasite relationship.

Autoradiographic Studies On The Uptake Of (14)C-Succinic Acid By Clonorchis Sinensis.

The distribution of exogenous C(14)-succinic acid by Clonorchis sinensis incubated in Tyrode medium was studied by using autoradiographic techniques. A comparison was made between macro and microautoradiographs of this worm showed that black grains derived from labeled substance were distinctly observed in parenchymal tissue, oral sucker, ventral sucker, ovary, testes, and uterine tubules with eggs. In pharynx and intestine, a low density was monitored while negligible activity was found in seminal receptacle and vitelline follicles. In studying the further details, microautoradiography was utilized. The most radioactive regions were the reticular tissue and subcuticular musculature of this fluke, closely compatible with the finding of macroautoradiography. Apparent black grains of radioactivity were also found in oral sucker, ventral sucker, pharynx, uterine tubules, eggs in uterus, and intestinal ceca. Structures showing the least radioactivity included ovary, seminal receptacle and vitelline folliciles.

Autoradiographic Studies On The Uptake And Distribution Of (14)C-Glucose By Paragonimus Westermani.

Autoradiographic study was performed in order to know the distrition of exogenous (14)C-glucose by lung fluke, Paragonimus westermani, incubated in Tyrode medium containing 10 micro Ci/ml of labeled substance. After 1 hour incubation at 37 degrees C, microautoradiographs of this fluke showed that black grains derived from radioactive carbon were accumulated mainly in the parenchyme and subcuticular musculature. The muscular tissues such as oral sucker, pharynx and ventral sucker revaled considerable density of fine grains. Slight radioactivity was also observed in the regions of ovary, testes, vitelline follicles, eggs in uteyrus, intestinal ceca, and even in excretory bladder. Structures showing the least activity included the cuticle and uterine tubules of this fluke.

Macrophage membranes viewed through a scanning electron microscope.

When rabbit peritoneal macrophages were cultured in serum and Tyrode medium, numerous membranous structures varying in size and transparency were observed to cover the surface in complex cascading patterns. Modifications in the methods of fixation and drying probably accounted for the new perspective of macrophage surfaces gained in this study.

Fertilization of Chinese hamster eggs in vitro.

Fertilization of eggs in vitro has been described in three mammalian species\p=m- the rabbit (Dauzier, Thibault & Wintenberger, 1954; Chang, 1959; Suzuki & Mastroianni, 1965; Brackett & Williams, 1965), Syrian hamster (Yanagimachi & Chang, 1964; Barros & Austin, 1967) and laboratory mouse (Whittingham, 1968). Penetration by spermatozoa and pronucleus formation have also been observed in rat eggs, following dissolution of the zona pellucida by chymotrypsin (Toyoda & Chang, 1968). In preliminary attempts to fertilize eggs of the Chinese hamster, Cricetulus griseus, in vitro, no success was achieved using fresh spermatozoa, but 10% of eggs (8 of 80) were penetrated by spermatozoa which had previously been incubated with Syrian hamster cumulus clot. This treatment was used in a larger study in August 1968, as described here. Immature and maturing Chinese hamsters, 5 to 8 weeks old, were injected intraperitoneally with 5 i.u. pmsg (Equinex, Ayerst) followed 48 to 52 hr later by 10 i.u. hcg (A.P.L., Ayerst). Tubal eggs were recovered from females killed 15\m=1/2\to 16\m=1/2\hr after hcg and, from earlier studies, these were considered to be not more than 2 hr post-ovulation. Follicular eggs were obtained from females where ovulation had not begun or only a few follicles ruptured at this time, and from one female killed 14\m=1/2\hr after hcg. Syrian hamster cumulus clot was used for all pre-treatments of spermatozoa. It was obtained from superovulated mature females (Greenwald, 1962) killed on Day 4 of the cycle, 14^ to 15-Jhr after injection of 10 i.u. hcg; about half the cumulus from one oviduct was used for each preparation. Chinese hamster spermatozoa were stripped from the vas deferens into mineral oil and added to 1 ml of either Tyrode's solution or TC medium Ham FIO containing 1% bovine serum albumin. A drop of the suspension, 1 to 2 times the volume of the cumulus and containing approximately 4 to 6 million spermatozoa/ml, was then added to the Chinese hamster eggs or to Syrian hamster material for pre-incubation. Eggs for culture with the pre-treated spermatozoa were recovered from Chinese hamster females 2 to 3 hr later and added to the pre-incubation mixture. They were easily distinguished from the Syrian hamster eggs by their smaller size, as well as a different appearance of the

The lens as an osmometer and the effects of medium osmolarity on water transport, 86Rb efflux and 86Rb transport by the lens

The transport of water into or out of the lens was regulated by the osmotic gradients between lens and incubation media. Rabbit lenses incubated in hyposmotic Tyrode's media gained water while water losses were found in lenses in incubated in hyperosmotic Tyrode's media. Concomitant decreases or increases of intralenticular osmolarities to equilibrate the osmolarity of the bathing fluid were found in lenses incubated in the anisotonic media. The water gains or losses from lenses, induced by osmolarity changes, were reversible. Within the 238–368-mosM range of media osmolarities, the lens behaved as a “perfect osmometer”. Minimals lens swelling resulted in hydropic changes in lens epithelium cells and in lens fibers. Additional overhydration led to fluid collection in the interfibrillar space. The rate of 86Rb efflux from the lens increased in swollen lenses proportionally to the degree of hydration. The net transport of 86Rb into the lens was increased (+8, +18%) in minimally swollen lenses and markedly decreased (−50%) in lenses incubated in media −260 mosM hypotonic to the lens. The water permeability coefficient ( k) of lens was 0.4 μ·min −1·atm −1, a value smaller than the k of erythrocytes, of similar magnitude to the k of leukocytes and slightly higher than the k of marine invertebrate eggs.

Metabolism of C(14)-glycine by Clonorchis sinensis.

Radioactive C(14)-glycine was given to Clonorchis sinensis in Tyrode medium in order to trace the metabolic fate of the labeled carbon. The labeled carbon from glycine enters into every major fraction of Clonorchis sinensis and is highest in the fraction of protein and nucleic acid. Significant amount of C(14)-glycine is incorporated into respiratory carbon dioxide. Relatively high percentage of C(14)-glycine in medium is converted to amino acid fraction and lipid fraction of the worm. In general, glycine is continuously being utilized in the synthesis of proteins and for energy production despite the uptake rate of glycine decreased gradually as incubation proceeds.

HISTAMINE RELEASE BY HOMOLOGOUS PLASMA IN THE DOG

Dogs were injected intracutaneously with their own plasma and with plasma from other dogs. The injected skin in each case was excised and incubated in Tyrode's medium. More free histamine was found in the media containing skin injected with homologous plasma than in the media containing skin injected with autologous plasma. When considered with earlier observations, this result justifies the conclusion that, in the dog, homologous plasma releases histamine on initial injection. Similar experiments with the histamine liberator compound 48/80 showed the method to be extremely sensitive in detecting histamine release.

HISTAMINE RELEASE BY HOMOLOGOUS PLASMA IN THE DOG

Dogs were injected intracutaneously with their own plasma and with plasma from other dogs. The injected skin in each case was excised and incubated in Tyrode's medium. More free histamine was found in the media containing skin injected with homologous plasma than in the media containing skin injected with autologous plasma. When considered with earlier observations, this result justifies the conclusion that, in the dog, homologous plasma releases histamine on initial injection. Similar experiments with the histamine liberator compound 48/80 showed the method to be extremely sensitive in detecting histamine release.