Preincubation Of Tissue Research Articles

Interaction of EPTC and 2,4-D on Excised Tissue Growth

Soybean(Glycine max(L.) Merr., ‘Hawkeye 63′) seedling segments from the zone of elongation and corn(Zea maysL., ‘Pioneer 3306) seedling segments were utilized to evaluate the antagonistic interaction ofS-ethyl dipropylthiocarbamate (EPTC) and (2,4-dichlorophenoxy)-acetic acid (2,4-D). EPTC inhibited growth of the soybean and corn tissue, and 2,4-D in combination with EPTC caused an increase in growth compared to EPTC alone. The 2,4-D-enhanced growth in the presence of EPTC was due to an antagonism of the EPTC inhibition. Inhibition of soybean tissue growth byS-ethyl diisobutylthiocarbamate (butylate) also was antagonized by 2,4-D. Preincubation of soybean tissue in EPTC for 4 hr prior to the addition of 2,4-D did not prevent 2,4-D enhancement of growth. Conversely, the rate of growth was inhibited when EPTC was added after 4 hr of 2,4-D-enhanced growth. The addition of uridine to the incubation media with EPTC did not decrease the EPTC-inhibition of soybean tissue growth. This indicated that EPTC did not inhibit the synthesis of pyrimidine precursors. EPTC vapor losses from the incubation media reduced the inhibitory effect of EPTC to soybean tissue. Also, a growth reduction occurred when the amount of soybean tissue per unit of EPTC was increased.

Permissive effects of hormones on lypolysis.

Hypophysectomy severely reduced glycerol and free fatty acid (FFA) production by segments of epididymal fat. Preincubation of adipose tissue from either hypophysectomized or normal rats with dexamethasone (.016 μg/ml) increased glycerol and FFA production in response to low concentrations of epinephrine (.01–0.05 μg/ml). This effect of dexamethasone had a lag period in excess of 2 hr, and was blocked by actinomycin d. Glucose was not required. Even greater potentiation was seen when growth hormone was also present. It is suggested that acute effects of dexamethasone on lipolysis may result from some modification in the metabolism of cyclic adenosine monophosphate, secondary to the induction of synthesis of some new species of protein. Pretreatment of hypophysectomized rats with triiodothyronine (T3, 5 μg/rat) also markedly potentiated the lipolytic response of segments of adipose tissue to epinephrine and theophylline when tested in vitro 16 hr later. This effect of T3 was not inhibited by actinomycin d. ...

Effect of prostaglandin and dietary fats on lipolysis and esterification in rat adipose tissue in vitro

1. 1. A possible role of prostaglandin (PGE 1) in lipid metabolism was investigated. It was found to inhibit the release of free fatty acids and glycerol from adipose tissue of fat-fed rats, the amount depending upon the nature of the fat ingested. The release of less glycerol and free fatty acids from tissue of rats fed a polyunsaturated fat (corn oil), as compared to those fed lard, was in effect similar to that in the presence of prostanlandin. 2. 2. Preincubation of tissue with prostaglandin produced a similar response suggesting that prostaglandin may have entered the adipose tissue cells during preincubation causing an inhibition of lipolysis. There was also a greater incorporation of [1- 14C]palmitic acid into the lipids and triglycerides of this tissue in the corn oil-fed rat and in prostaglandin-treated tissues. 3. 3. An inhibition of lipolysis and increased rate of removal of fatty acids from the medium by adipose tissue in the presence of prostaglandin or by this tissue of corn oil-fed rats, both associated with an increased esterification of fatty acids in the tissue, suggests the possibility that the effect of the latter may be the promotion of the formation by these rats of greater amounts of prostaglandin from the unsaturated fatty acids ingested.

Effects of growth hormone on the lipolytic response of adipose tissue to theophylline.

Adipose tissue from hypophysectomized rats has a reduced capacity to carry on lipolysis in response to theophylline and other lipolytic agents. Three hr after a single injection of growth hormone (100 μg), FFA and glycerol production in response to theophylline are increased, but basal lipolysis is unchanged. Preincubation of adipose tissue for 3 hr in the presence of 0.01–1.0 μg/ml of growth hormone significantly increased the lipolytic response to theophylline, again without affecting the basal rate of lipolysis. This action of growth hormone has a lag period of more than 1 hr, is blocked by insulin and augmented by dexamethasone, which has a similar, but less consistent effect. Actinomycin failed to block this response to growth hormone and cycloheximide significantly enhanced it. These data are thought to illustrate an effect of growth hormone on the level of 3′,5′-cyclic AMP in adipose tissue, and suggest a biochemical explanation for the “permissive” effect of growth hormone on lipolysis. (Endocrinology81: 1027, 1968)

Dilution effects on the measurement of blood cholinesterase inhibition by carbamates.

UNDER “steady state” conditions in the absence of its normal substrate (acetylcholine), or when the substrate concentration can be neglected, the inhibition of acetyl-cholinesterase (ChE) by insecticidal or related carbamates, such as 1-naphthyl N-methylcarbamate, o-isopropoxy-phenyl N-methylcarbamate (IPMC) and eserine, can be defined by the equation The symbols v′ and v are the rates of substrate hydrolysis by the inhibited and uninhibited enzyme respectively, i is the concentration of carbamate, K1 the effective bimolecular carbamylation rate constant, and K2 the effective unimolecular rate constant for the hydrolytic regeneration of free enzyme1,2. It followed from (1) that if v′0/v0 represents the steady state inhibition obtaining in vivo or as a result of the pre-incubation of tissue and inhibitor in vitro, and v′z/vz that estimated after diluting the tissue z times and allowing a new steady state to be achieved When estimating human blood ChE inhibition as an index of accidental or occupational exposure to insecticidal carbamates it is therefore important to keep z minimal or to carry out the assay so rapidly that the change towards the new steady state can be neglected. Fortunately, the values of K2 for typical insecticidal carbamates2,3 are such that, if assays can be completed within a few minutes of sample dilution, the changes before and during the measurement are relatively small. A simple radiometric method for estimating blood ChE under field conditions has been described4 which enables an assay to be made within 2 min of dilution and involves diluting a 20 µl. blood sample a nominal eleven times. When this method was applied to samples of undiluted human blood pre-incubated with IPMC or eserine to simulate conditions in vivo, it was found that the new steady state effects of subsequent dilution were invariably less than those indicated by equation (2). The steady state inhibition obtaining at the instant of dilution was estimated by extrapolating the time-inhibition curves obtained after dilution. The results obtained in experiments with IPMC and eserine at 25° C are summarized in Table 1. In these experiments the actual dilution was ten times, because of a small volume of aqueous inhibitor that was present in the whole blood. The observed steady state inhibition by eserine has been corrected for the slow enzymatic decomposition of the inhibitor itself which was expected at the relatively high enzyme concentrations used2. This decomposition tends to exaggerate and extend the dilution effect so that reactivation of the diluted enzyme appeared to continue almost to completion after 18 h. Because of its lower carbamylation rate, no correction was required for the steady state inhibition by IPMC, and this did not change significantly over a period of 15 h. The discrepancy between the observed and calculated effects of dilution illustrated in Table 1 may be explained as a partition phenomenon resulting in a lowering of the concentration of free inhibitor in the aqueous phase as follows.

Stimulation of metabolic activity of adipose tissue from fasted rats by prolonged incubation in vitro: I. requirement for glucose and insulin

1. 1. Rat epididymal adipose tissue may be incubated in Eagle's tissue-culture medium for periods as long as 36 h without evidence of serious metabolic deterioration. Tissue transfer to new medium was carried out every 4.5 or 6 h. Sterile media were used, although without strictly sterile transfer technique; penicillin and streptomycin were present throughout. 2. 2. Continued viability of the tissue was evidenced by persistent glucose uptake, oxidation of glucose to CO 2, and fatty acid as well as protein synthesis from glucose, together with intact sensitivity to insulin throughout. However, from 24 h on there was a tendency to increased production of lactate by the tissue, together with a slight decrease in glucose oxidation, suggesting increasing occurrence within the tissue of anaerobic glycolysis. 3. 3. The grossly depressed metabolic activity of adipose tissue obtained from animals fasted 96 h was restored toward normal by preincubation of the tissue for periods ranging from 4.5 to 24 h, provided that the preincubation medium contained glucose and insulin or human serum. Increased lipogenesis, together with increased ability to oxidize glucose to CO 2 and to incorporate glucose carbon into protein, reached a plateau after approx. 9 h of preincubation. 4. 4. Although tissue-culture medium was used in most of these studies, the presence of essential amino acids and co-factors required for tissue cell growth in vitro was not required for the occurrence of the preincubation effect upon lipogenesis and glucose oxidation in adipose tissue. 5. 5. Lipogenesis from pyruvate, and pyruvate oxidation to CO 2 a were not affected by preincubation of epididymal adipose tissue from severely fasted rats. The presence of insulin was without effect upon pyruvate metabolism either before or after pre-incubation with glucose. However, preincubation without glucose in the presence of pyruvate with or without insulin, restored the lipogenetic activity of adipose tissue from glucose in a manner similar to that observed when preincuuation was carried out in the presence of glucose and insulin. 6. 6. Several enzymic activities concerned with glucose and fatty acid synthesis were measured in the homogenate of adipose tissue from severely fasted rats, both immediately after killing the animal and following preincubation with glucose and insulin for 9 h. In no instance was an increase in any of the enzymic activities measured noted; in most instances a very slight decrease, of the order of magnitude of 5%, was seen. 7. 7. The most likely but still hypothetical explanation for the increased lipo-genetic activity of adipose tissue of fasted rats after preincubation is the accumulation (or re-accumulation) within the tissue of metabolic products or metabolites which in themselves stimulate lipogenesis. These metabolites would require the presence of insulin to arise in sufficient quantity from the metabolism of glucose, but would arise from the metabolism of pyruvate in adequate amounts even in the absence of insulin.

Esterification of intra- and extra-cellular free fatty acids by rat adipose tissue.

The esterification of intracellular and extracellular FFA by rat adipose tissue in vitro was investigated. The rate of incorporation of FFA into neutral lipids was proportional to the FFA concentration in the incubation medium. Both in the presence and absence of a lipolytic agent (epinephrine), heptadecanoate-1-C14, which is not specifically diluted by tissue fatty acids, was esterified in the same manner as palmitate-9,10-H3. Stearate, palmitate, and oleate were esterified at similar rates by adipose tissue taken from fed animals and incubated with glucose. The rate of esterification of one fatty acid was not significantly affected by the presence of another. Similar results were obtained when tissues were taken from fasted animals and incubated in the absence of glucose, except that the overall rate of esterification was diminished and FFA accumulated in the tissue. It is concluded that long-chain fatty acids do not compete for esterification or entry into the adipose tissue cell.In some experiments tissue FFA esterification was studied by measuring the incorporation of glucose-U-C14carbons into glyceride–glycerol. Esterification, assayed in this manner, increased when albumin was present in the incubation medium and allowed FFA to diffuse from the tissue. However, pre-incubation of adipose tissue in medium containing labelled FFA indicates that much of the intracellular FFA may be esterified without its mixing with the extracellular FFA pool.

Effect of thyrotrophic hormone on thyroxine uptake by abdominal muscle of mouse.

By in vitro study with surviving abdominal muscle of mouse, an enhancing effect of TSH on uptake of I131-labeled Lthyroxine (T4) was demonstrated. Muscle pieces were incubated in Krebs-Ringer phosphate solution containing I131-T4. The T4- binding capacity proved to be smaller in tissue from TSH-injected mice, and larger in tissue from T4-injected mice, than in muscle taken from control animals. However, the rate at which T4 uptake approached maximal was the same in all treatment groups. These results suggest that T4 uptake by peripheral tissue in vivo is not always increased by a rise in circulating T4 level. The decrease in the binding capacity of tissue from TSH-treated mice is ascribed to a decrease in the number of intracellular T4-binding sites in vivo, due to increased uptake of T4. Results obtained in experiments involving preincubation of muscle tissue with stable T4 or TSH confirmed that the decrease in binding capacity was due to the partial presaturation of the intracellular T4-binding sites wi...