Autoradiographische Untersuchungen über das Wachstum des Reissnerschen Fadens von Cyprinus carpio (L.)

Summary The incorporation of³⁵S-l-cysteine into the hypothalamic-hypophyseal neurosecretory system was studied in normal male albino rats and in similar rats which had received thiouracil or excess thyroxine during 18 days. The study was performed by autoradiography. Labelled cysteine was administered by intraperitoneal injections. The animals were sacrificed 30 min, 3 and 24 hours after injection. From the autoradiographs the grain counts were determined in the supraoptic nucleus, paraventricular nucleus, median eminence and infundibular process. The thyroxine-treated rats in the 30 minute group displayed a statistically almost significantly higher grain count in the supraoptic nucleus than the corresponding controls; the same was observed in the 3 hour group in the median eminence and infundibular process. In the 24 hour group, however, the grain count was clearly lower at all sites examined than in the controls. With the rats that had been under thiouracil treatment statistically significant differences were only established in the 24 hour group. Their grain count was higher at all sites than that of the corresponding controls. From the results the inference is drawn that thyroxine accelerates the protein synthesis in the ganglion cells of the supraoptic nucleus and accelerates the flow of NSM to the infundibular process and its release there into the blood flow. Thiouracil, again, retards the flow and the release.This work was supported by a grant from the Sigrid Jusélius Foundation, Helsinki.     

Effect of hydrocortisone on phenylhydrazine-induced Heinz-body anemia

The effect of hydrocortisone on phenylhydrazine-induced anemia has been examined in adrenalectomized rats. The extent of hemolysis in adrenalectomized rats treated with phenylhydrazine was significantly higher than in normal and adrenalectomized rats supplemented with hydrocortisone.     

Ribonucleic acid synthesis in regenerating liver of irradiated adrenalectomized rats.

The effect of whole-body irradiation on RNA synthesis in regenerating and non-growing livers was studied in intact and adrenalectomized rats. The animals were divided into four sub-groups: (1) control; (2) irradiation only; (3) partially-hepatectomized; and (4) irradiated partially-hepatectomized. Newly-synthesized RNA was determined by 30 or 40 min uptake of (6-14C) orotic acid. Both nuclear and polyribosomal RNA synthesis in regenerating livers of adrenalectomized rats were depressed below their control levels, regardless of whether irradiation was 2 hours before or 2 hours after partial hepatectomy. Specific radioactivity values of regenerating livers of adrenal intact and adrenalectomized rats were elevated above those of the non-growing livers from irradiated and unirradiated rats.     

Effects of acute and subacute cocaine administration on the CNS dopaminergic system in Wistar-Kyoto and spontaneously hypertensive rats: III. Dopamine uptake

The characteristics of dopamine uptake after acute and subacute cocaine administration were determined in striata from WKY and SHR. In acutely-treated (40 mg/kg, s.c.) rats, significant increases in the V_max of dopamine uptake were observed 30 min after the cocaine injection in both strains, without changes in K_m values. The in vitro IC₅₀ for cocaine was significantly decreased at 30 min in WKY and at 2 h in SHR. However, the in vitro IC₅₀ for GBR-12909 was significantly increased at 30 min and at 2 h in both strains following cocaine administration. In both strains, the density (B_max) of the [³H]GBR-12935 binding site was significantly increased at 30 min and at 2 h with no charges in K_d. In subacutely-treated (20 mg/kg, twice daily for 3 or 7 days) rats, a significant increase in the K_m for dopamine uptake was observed in 7 day treated SHR. The in vitro IC₅₀ for GBR-12909 was significantly increased in 3 day treated WKY. The results suggest that cocaine administration alters dopamine uptake and characteristics of dopamine uptake sites in the rat brain.     

Light and dark uptake and loss of 14C: methodological problems with productivity measurements in oceanic waters

Measurements of the uptake and loss of ⁴C in the light and in the dark in the Tasman and Coral Seas have revealed methodological problems with the estimation of productivity in these waters. Rates of productivity estimated without replication, time series incubations and dark controls frequently overestimated the true rates of autotrophic production. The data showed unexpectedly high rates of both uptake and loss in the dark in oligotrophic waters. In oligotrophic oceanic waters, dark incorporation of ¹⁴C sometimes equalled the uptake of ¹⁴C in the light bottle. Rapid uptake of isotope in the dark controls appeared to be the result of rapid bacterial growth and metabolism. This problem was exacerbated by agitation of the sample before or during the incubation. Tropical samples were particularly susceptible to problems arising from the agitation of the samples. Latitudinal gradients of dark uptake and loss were revealed in these incubations. The loss of label during 8–12 hours in the dark (after 12 hr in the light) was as high as 50% in subtropical waters. The loss was frequently unmeasurable (< 10%) in temperate waters. The time course of ¹⁴C uptake indicated active grazing in the bottles and suggested that most of the nighttime losses of label were due to grazing by microheterotrophs. Respiratory losses appeared to be small. Calculated values of the assimilation number (or photosynthetic capacity) which did not correct for dark ¹⁴C uptake were too high to be biochemically realistic. The errors were due to the heterotrophic uptake of label and the lack of dark controls. Rapid release of ¹⁴C in the dark after incubation in the light meant that the estimate of productivity was dependant on the trophic state of the sample and on the period of incubation.     

Comparative efficacy on dogs of a single topical treatment with the pioneer fipronil/(S)-methoprene and an oral treatment with spinosad against Ctenocephalides felis

In the study reported here, the pioneer fipronil/(S)-methoprene topical product (FRONTLINE^® PLUS, Merial Limited, Duluth, GA) was compared to the oral spinosad product (COMFORTIS^® Elanco, Greenfield, IN) for efficacy against adult fleas and preventing egg production. The product presentations, doses and labelling were the one applicable in the USA. Using a standard protocol, 200 cat fleas of mixed sex were applied to dogs on Days 1, 7, 14, 21, 28, 35, and 42. Dogs were combed to remove fleas 24 hours post-infestation, the fleas were counted, collected, and then reapplied to each dog following completion of their respective count. At 48 hours post-infestation, comb counts were performed and fleas were removed. No fleas were collected from any dog in the fipronil/(S)-methoprene group at any 24 or 48 hours post-infestation assessment throughout the six weeks study, yielding a preventive efficacy of 100%. For the spinosad treatment, efficacy was 100% at 24 hours and 48 hours through Day 16, and thereafter declined. The results observed in the spinosad-treated dogs were highly variable between animals. At the 24 and 48 hours counts following the Day 21 infestation, only five of eight spinosad-treated dogs (62.5%) were flea-free. Following the Day 28 infestation, spinosad efficacy fell to 85% and 89%, for the 24 hours and 48 hours counts, and only two dogs (25%) were flea free, compared to 100% flea-free dogs in the fipronil/(S)-methoprene group. No fleas were collected from the fipronil/(S)- methoprene treated dogs throughout the entire study, therefore, no eggs were collected at any time from any dog in the group. However, in the spinosad group adult fleas were found on dogs starting on Day 21 and by Day 30, 42 eggs were collected from one dog that had 107 adult fleas counted at 48 hours. At Day 37 and Day 49, more than 100 eggs were collected from each dog in the spinosad-treated and control groups.     

Studies on the radiosensitivity of the function of the Golgi complex.

The uptake of 3H-glucosamine into primary human-embryo fibroblasts and into the Golgi-rich fraction isolated from liver of mice labelled in vivo was studied, after various doses of X-radiation, by autoradiography and biochemical methods. A dose of 90 rad resulted in an increased precursor uptake in interphase cells at 24 hours and in mitotic cells at 48 hours after irradiation; 226 rad had virtually no effect on the grain counts of interphase cells, but reduced the labelling of mitotic forms. The characteristic intracellular localization of the grains were not influenced by these doses. Although no immediate radiation-induced reaction could be observed in liver cells either, significant stimulation of the 3H-glucosamine incorporation was measured in isolated Golgi-rich fractions 24 hours after whole-body irradiation with 90, 450, or 905 rad. This phenomenon is discussed as a part of the somatic regeneration of membrane structures.     

Early effects of excessive retinol intake on gluconeogenesis. Involvement of adrenals in the increased activities on Gluconeogenic Enzymes of rat.

A stimulation of gluconeogenesis by excessive intake of retinol is suggested on the basis of enhanced incorporation of [2-¹⁴C]glycine into liver glycogen in rats fed excess retinol. Among the key gluconeogenic enzymes studied, activities of hepatic PEP-carboxykinase, fructose-1,6-bisphosphatase, glucose-6-phosphatase, and alanine aminotransferase were markedly increased, whereas that of pyruvate carboxylase remained unaltered. However, feeding of retinol to bilaterally adrenalectomized rats or rats treated with actinomycin D failed to cause significant increase in the activities of these enzymes. It is concluded that (i) gluconeogenesis is stimulated by excess retinol due to, perhaps, increased activities of key gluconeogenic enzymes, and (ii) adrenals are directly or indirectly involved in the retinol-mediated increase in the activities of the gluconeogenic enzymes. Also, data are presented that indicate a requirement for protein synthesis for the expression of retinol-mediated alterations in the activities of gluconeogenic enzymes.     

Autoradiographic assessment of 3H-proline uptake by osteoblasts following guanethidine-induced sympathectomy in the rat

Summary Sympathectomy was carried out in rats by injections of guanethidine-sulfate from birth to 14 days of age. At 45 days of age, the activity of osteoblastic cells was monitored by ³H-proline autoradiography. Effectiveness of sympathectomy was verified by light-microscopic examination of superior cervical and celiac ganglia. Grain counts over periosteal osteoblasts of the femoral diaphysis and osteoblasts mesial to the first molar in the mandible demonstrated a significantly reduced uptake of ³H-proline in the sympathectomized rats. The data provide direct evidence of sympathetic influence on osteoblastic activity and suggest that sympathectomy may result in the loss of a trophic influence which is important in the regulation of osteogenesis.Supported by N.I.D.R. grant DEO 4557 (R.M.K.), N.I.H. grant 5-SO1RR-5373 to K.U.M.C., and N.I.H. grant RR-05332 to N.Y.U. (U.S.). We thank Charles A. Brownley of CIBA-Geigy, Summit, N.J. for the guanethidine sulfate     

Changes in the blood of newts,Notophthalmus viridescens,following the administration of hydrocortisone

Summary Adult newts,Notophthalmus viridescens, were injected with suspensions of hydrocortisone acetate (experimentais) or with distilled water (controls). Forty-eight and 72 hours after treatment, blood smears were prepared, and differential counts of leucocytes were made for the experimental and control animals. At 48 hours, the distributions of neutrophils, eosinophils, basophils, monocytes and lymphocytes were much the same in the two groups of newts (Table 1). However, by 72 hours after injection, increases in neutrophils and decreases in lymphocytes were obvious in the animals which had received hydrocortisone. Such changes were not seen in the controls (Table 2). The changes in the distribution of the white cells seen 72 hours after treatment are very similar to those known to occur in mammals treated with adrenal steroids and to those described earlier in two species of frogs injected with hydrocortisone. Details of some differences in the responses of the amphibians are discussed.Supported by National Science Foundation COSIP grant, GY-7661, to Sweet Briar College.     

Cultured human skin fibroblasts absorb65Zn

The radioactive isotope⁶⁵Zn was used to study the incorporation of zinc by cultured human skin fibroblasts. The development of the method for studying cell uptake of⁶⁵Zn in a minimal synthetic medium is presented. Kinetics carried out on control cultures up to 240 min indicated that zinc uptake occurred in three phases, the first being the most rapid. Temperature and pH affect zinc uptake, in favor of an active transport process. In addition, the rate of incorporation is considerably decreased during the first phases after adding potassium cyanide, during the last phases after adding sodium iodoacetate, and during all the phases if dithioerythritol is used. A hypothesis is therefore proposed according to which several types of mechanisms would be involved in zinc uptake by fibroblasts. At least a part of these mechanisms is energy-dependent.     

Effect of glucocorticoids on the endocrine system of the pancreas of the pigeon and rat after a food load

The level of 35S-methionine incorporation (in 15, 30 min, 1, 2, 3, 6, 12, 24 h) has been investigated in A- and B-cells of the pigeon and rat pancreatic islets against the background of excessive injection of hydrocortisone. The pigeon and rat A- and B-endocrinocytes respond in a similar was to the excess of hydrocortisone. An accelerated elimination of the isotope from the pigeon A- and B-endocrinocytes is noted, while in the rat the effect of the excessive hydrocortisone is opposite.     

Effects of diabetes on glucose oxidation in rat aorta after hypophysectomy and adrenalectomy

The influence of diabetes, hypophysectomy and adrenalectomy on glucose oxidation in rat aorta was studied. Diabetes was induced in normal, adrenalectomized and hypophysectomized-cortisone substituted rats by streptozotocin (65 mg/kg body weight). The oxidation of glucose to CO2 was determined during incubation of rat aorta in vitro for 2-3 hours. The aortic glucose oxidation was reduced after hypophysectomy but was unaffected by adrenalectomy. After streptozotocin treatment the rise in blood glucose concentration was similar in normal, adrenalectomized and hypophysectomized-cortisone substituted rats. In shamoperated diabetic rats the aortic glucose oxidation was reduced after a diabetes duration of 4 days. In adrenalectomized diabetic rats the aortic glucose oxidation was not significantly affected after 4 days but was reduced after a diabetes duration of 14 days. When adrenalectomized diabetic rats were treated with hydrocortisone the aortic glucose oxidation was reduced after diabetes for 4 days. After incubation of normal rat aorta in vitro for 6 hours with cortisol (1 microgram/ml) in the incubation medium a decrease in the aortic glucose oxidation was found. Incubation of aorta with only growth hormone had no effect. These results suggest that cortisol is of importance for the lowered glucose oxidation in diabetic rat aorta.     

Effect of prostaglandin E1 administration on the heart glycogen synthase and phosphorylase systems

The effects of intravenous administration of PGE₁ on the glycogen synthase and phosphorylase system in rat heart were studied.Unlike the consistent effects of PGE₁ on glycogen synthase in liver, the response in heart was variable. A significant decrease in the per cent synthase occurred in fasted intact rats while a significant increase was seen in adrenalectomized hydrocortisone treated fasted rats. No significant effect was seen on the synthase system in either fed intact or fasted adrenalectomized rats.Phosphorylase activity was increased significantly following PGE₁ administration in fed intact rats and slightly increased in adrenalectomized fasted rats. The phosphorylase system was not affected in fasted intact and fasted adrenalectomized rats given glucocorticoid replacement. With our present state of knowledge an adequate explanation for the response of these heart enzymes to PGE₁ under the various conditions of this study does not appear possible.     

Evaluation of the effects of sensory denervation on osteoblasts by 3H-proline autoradiography

Summary The inferior alveolar nerve was unilaterally resected in 30-day-old mice; other animals were unilaterally sham-operated. At 15, 30, 60, 90, or 150 days after surgery, the mice were injected with 2Ci of ³H-proline (sp. act. 1.0 Ci/mM) per g of body weight and killed 15, 30, or 60 min later. Autoradiographs were prepared from 5m decalcified sagittal sections of mandibles and grain counts made over periosteal osteoblasts mesial to the first molar. In denervated mandibles, osteoblasts incorporated less isotope compared to controls with differences being maximal at the early intervals. These differences became attenuated with time, possibly due to an intrinsic compensatory mechanism, secondary to neurotrophic regulation.Supported in part by NSF grant GU-3566     

Effect of hydrocortisone and dexamethasone on xylose uptake by isolated rat soleus muscle

To determine the effects of glucocorticoids on sugar uptake, xylose uptake by isolated rat soleus muscle of bilaterally adrenalectomized animals was studied. The results indicate that in vitro addition of 10⁻⁴ M hydrocortusine, dexamethasone or hydrocortisone sodium succinate had no inhibitory effect on basal xylose uptake. In the presence of both low and high medium insulin, the above steroids failed to inhibit insulin-stimulated uptake. When the concentration of hydrocortisone sodium succinate was increased to 10⁻² M, insulin-stimulated uptake was decreased. The results thus indicate that glucocorticoids at concentrations observed under physiological or pathological conditions do not inihibit basal or insulin-stimulated sugar uptake.     

Effect of angiotensin-II on renal Na/H exchanger-NHE3 and NHE2

The purpose of the present study was to determine the effect of angiotensin II (A-II) on membrane expression of Na⁺/H⁺ exchange isoforms NHE3 and NHE2 in the rat renal cortex. A-II (500 ng/kg per min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. Arterial pressure and circulating plasma A-II level were significantly increased in A-II rats as compared to control rats. pH-dependent uptake of ²²Na⁺ study in the presence of 50 μM HOE-694 revealed that Na⁺ uptake mediated by NHE3 was increased ∼88% in the brush border membrane from renal cortex of A-II-treated rats. Western blotting showed that A-II increased NHE3 immunoreactive protein levels in the brush border membrane of the proximal tubules by 31%. Northern blotting revealed that A-II increased NHE3 mRNA abundance in the renal cortex by 42%. A-II treatment did not alter brush border NHE2 protein abundance in the renal proximal tubules. In conclusion, chronic A-II treatment increases NHE3-mediated Na⁺ uptake by stimulating NHE3 mRNA and protein content.     

Quantitative electron microscopic autoradiography on the mouse thyroid gland after administration of 3H-L-DOPA

Summary The cellular and subcellular distribution of radioactivity in the mouse thyroid gland different times (20 min — 8 hours) after intravenous administration of ³H-L-DOPA was studied by means of quantitative electron microscopic autoradiography.High concentrations of autoradiographic silver grains occur over parafollicular cells and adrenergic nerves while the labelling of follicular cells and lumina is low or absent and similar to the labelling of connective tissue cells at all observation times.Over the parafollicular cells high levels of radioactivity can be recorded already 20 min after administration of the labelled amino acid. The grain counts are highest at 1 hour and decrease then at 2.5 and 8 hours.The intracellular distribution of label is similar at all observation times; thus, the concentration of silver grains over the typical cytoplasmic granules of the parafollicular cells is 4–5 times higher compared to the concentration over the remainder of the cytoplasm and the nucleus.Treatment with a decarboxylase inhibitor prior to the injection of ³H-L-DOPA results in a low and uniform labelling of all thyroid cells. This finding, taken together with the observation that also pretreatment with reserpine abolishes the autoradiographic reaction over the cytoplasmic granules, gives strong support to the idea that the great majority of silver grains observed over parafollicular cells represents dopamine formed by decarboxylation of the labelled precursor.This study was supported by grant K71-12X-3352-01 from the Swedish Medical Research Council. The author wishes to express his gratitude to Mrs. Gunnel Bokhede and Miss Dala Sjögren for expert technical assistance.     

Effects of DMSO on SGOT during hypothermia in adrenalectomized rats

A study of changes in SGOT levels was made in hypothermic Sprague-Dawley rats (rectal temperatures 23°C) which were adrenalectomized and/or treated with DMSO (41% aqueous solution injected IP) prior to hypothermia. Adrenalectomized animals exhibited slight initial increases in SGOT, but returned to control levels within 24 hours after exposure. Animals treated with DMSO alone or adrenalectomized and treated with DMSO showed significant increases in SGOT, which persisted 24 hours after exposure. Data suggests that DMSO exerts its action directly upon the cell membrane, causing permeability changes rather than indirectly by influencing the release of adrenal hormones which in turn affect membrane permeability.     

Absence of hydrocortisone from cytoplasmic hormone-protein complexes formed in vivo after administration of biologically active doses of [3H]hydrocortisone

After administration of [³H]hydrocortisone to adrenalectomized rats, hormone-protein complexes were isolated from liver cytosol by DEAE-cellulose chromatography. After application of biologically active and inactive doses of hydrocortisone five binding components were detected eluting at the same salt concentrations as the hormone-protein complexes observed after incubation of cytosol with [³H]hydrocortisone in vitro. The isolated hormone-protein fractions were acidified and extracted with ethylacetate and the steroids were analyzed by thin-layer chromatography. No significant amount of hydrocortisone could be detected in any of the complexes formed in vivo 5–60 min after administration of biologically active doses of hydrocortisone. 3ξ,11β,17α,20ξ, 21-Pentahydroxypregnane, steroidal carboxy acids, glucuronides and a very polar conjugate of hydrocortisone were found in the different fractions. After an in vivo dose of hydrocortisone of about 1/5000th of the minimal dose required for enzyme induction, hydrocortisone could be found in all cytoplasmic hormone-protein complexes formed. In contrast to the cytoplasmic hormone-protein complexes, hydrocortisone could be readily demonstrated in nuclei isolated after the administration of biologically active doses of hormone, although acid metabolites were found to represent the main part of the radioactive compounds present in the nuclei. These acid metabolites were located in ronide on the basis of its chromatographic behavior. The biological significance of this conjugate of hydrocortisone as well as that of the extremely polar conjugate found in fraction DE-3 cannot be understood on the basis of the published data pertaining to biological functions and metabolism of glucocorticosteroids.Our finding that no ‘classical’ glucocorticosteroid receptor can be detected in rat liver cytosol raises again the question of the way in which hydrocortisone and its active metabolites enter the nucleus. On the basis of the published data, the possibility cannot be ruled out that glucocorticosteroids are transported via the endoplasmic reticulum. A transport by this way has been inferred for the uptake of sodium and inulin by liver nuclei [40–42].