Incorporation of [3H]inositol into phosphoinositides and inositol phosphates by rabbit blastocysts

Preimplantation rabbit embryos collected at the early morula stage were cultured to blastocysts in the presence of [³H]inositol. The blastocysts were lysed, and both the aqueous and lipid portions were analysed for incorporated radioactivity. Thin-layer chromatographic separation of the lipid portion indicated that [³H]inositol was incorporated into phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. HPLC anion-exchange chromatography indicated that [³H]inositol was incorporated into inositol phosphates, including the two second messengers, inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, and also inositol monophosphate and inositol 1,4-bisphosphate. These results provide evidence that rabbit blastocysts may have an active phosphatidylinositol second messenger system, which may be responsive to intrauterine factors or intraembryonic paracrine factors. © 1993 Wiley-Liss, Inc.     

Factors in mouse uterine fluid that inhibit the incorporation of [3H]uridine by blastocysts in vitro

Fluid flushed from the uteri of "delayed implanting" and "implanting" mice was fractionated by gel-filtration. The fractions were freeze-dried, individually resuspended in culture medium containing [3H]uridine, and used for incubating "implanting" blastocysts. Several factors were found that reduced incorporation of [3H]uridine by the blastocysts. The amount of inhibitory activity in corresponding factors was generally similar in flushings from both types of uteri, but there was significantly more inhibitory activity in the void volume fractions of flushings from "delayed implanting" animals and it is suggested that this factor may be responsible for the metabolic dormancy of embryos during the diapause associated with delayed implantation.     

Effect of indomethacin in vivo on prostaglandin content of several rabbit tissues

The prostaglandin (PG) content of several tissues and fluids from 6 day pregnant rabbits was evaluated following treatment with indomethacin or vehicle in vivo. PGE and PGF were measured by radioimmunoassay. More complete depletion of PGE and PGF was accomplished by 3 injections of indomethacin (s.c.) given during the 18 h before sacrifice at a dose of 10 mg indomethacin per kg body weight than was accomplished by 1 injection of the same amount of indomethacin (i.v.) 1.5 h before sacrifice. Levels of PGF were more easily depressed by indomethacin than were those of PGE. PG levels in the kidney and blastocysts were depressed to a greater extent by indomethacin than were those in the uterus, uterine fluid or peritoneal fluid. Evaluation of the effect of indomethacin on a particular physiological function should be interpreted with caution unless the extent of PG depletion in that tissue is also measured.     

In vivo conversion of [3H]myoinositol to [3H]chiroinositol in rat tissues.

We report here the in vivo conversion of [3H]myoinositol to [3H]chiroinositol. After labeling intraperitoneally with [3H]myoinositol for 3 days to reach radioisotope equilibrium in urine, [3H]chiroinositol was isolated from tissues and purified after 6 N HCl hydrolysis by two sequential paper chromatographies and high performance liquid chromatography (HPLC). Percent conversion of [3H]myoinositol to [3H]chiroinositol was highest in urine (36%), liver (8.8%), muscle (8.8%), and blood (7.6%) with intestine, brain, kidney, spleen, and heart decreasing in percentage from 2.8 to 0.7%. Labeling of other inositol isomers including scyllo-, neo-, and epi-, and mucoinositol was minimal, approximately 0.06% of [3H]myoinositol. Glucose was unlabeled, but glucuronate, the product of myoinositol oxidation, was labeled up to 1.5% of the [3H] myoinositol. Acid hydrolysates of combined inositol-containing phospholipids contain significant labeled chiroinositol. [3H]Phosphatidylinositols and [3H]glycosylphosphatidylinositols were extracted from liver, muscle, and blood, isolated by thin layer chromatography, and inositols purified by HPLC after acid hydrolysis. Percent conversion of [3H]myoinositol to [3H] chiroinositol was highest in blood (60.4%) followed by muscle (7.7%) and liver (2.2%).     

Rabbit blastocysts accumulate [3H]quinuclidinyl benzilate in vitro

Day-6 rabbit blastocysts were able to accumulate [3H]quinuclidinyl benzilate (QNB) from their environment. This accumulation was reduced approximately 50% in the presence of 1.5 x 10(-4) M atropine (an accepted antagonist for ligands which bind to muscarinic cholinergic receptors). The accumulation of QNB was sensitive to temperature and was apparently saturable. In the presence of 2 nM QNB, Day-6 blastocysts accumulated 30.3 +/- 2.0 fmoles per blastocyst. When the cellular elements alone were examined, lesser amounts of specific binding were detected. Owing to the complexity of this multicompartmental system, Scatchard analysis did not provide meaningful results. This accumulation appears higher than that reported for other tissues such as rabbit heart homogenates or rabbit uterine endometrial cells. This muscarinic cholinergic accumulation may have some roll in blastocyst-maternal recognition.     

3H]dopamine uptake by platelet storage granules in schizophrenia.

[3H]Dopamine (DA) uptake by platelet storage granules was determined in 26 schizophrenic male patients, paranoid type (14 acute stage; 12 in remission) and 20 age-matched, normal controls. Maximum velocity (Vmax) of DA uptake was significantly higher in acute patients, than patients in remission or controls (p less than 0.05). The apparent Michaelis constant (Km) of DA uptake in acute patients was also significantly different from chronic patients (p less than 0.05). Preincubation with reserpine (10(-4), 10(-5) M) produced a substantial diminution of DA uptake, while haloperidol (10(-4), 10(-5) M) did not affect the assay. Considering that a DA dysequilibrium in schizophrenia may be expressed not only in the brain, but also in the periphery and that an increased amount of DA accumulated in the vesicles, implies that an increased quantity of catecholamine is available for release, our findings suggest additional evidence for the role of DA overactivity in the pathophysiology of this disorder.     

Measurement of the protein-synthetic activity in vivo of various tissues in rats by using [3H]Puromycin.

The validity of a new technique was examined for estimating the protein-synthetic activity of various tissues in vivo. The basic assumption underlying the method is that the number of peptide chains growing on each active ribosome would increase as the protein-synthetic activity of each tissue increases. The principle of the procedure, which was devised originally by Wool & Kurihara [(1967) Proc. Natl. Acad. Sci. U.S.A. 58, 2401-2407] to determine in vitro the number of functional ribosomes in skeletal muscle, is as follows. Puromycin is known to bind easily to the C-terminal end of the growing peptide on ribosomes and thus stop further chain elongation. Hence, if the number of puromycin molecules attached to the nascent peptide is determined by using radioactive puromycin as a tracer, one can estimate the number of growing peptides, i.e. the activity of tissue protein synthesis. By using this technique, it is shown that both starvation and the feeding of a protein-free diet caused marked decreases in the relative rate of formation of peptidyl-puromycin, i.e. activity of protein synthesis in liver, skeletal muscle, heart, spleen, testis, lung, kidney and intestine.     

Assessment of in vivo protein synthesis in lamb tissues with [3H]valine flooding doses

Week-old lambs received an intravenous injection of 4.3, 8.5, 12.8 or 17.1 mmol [3H]valine/5 kg body weight, i.e., 3.6-14.4-times the whole-body free valine content. To ensure that protein synthesis measurements in lambs are reliable within a 30-min period, these large amounts of valine must account for at least around 11-times the total free pool of valine. This amounted to 12.8 mmol valine/5 kg body weight. There were no significant variations in plasma insulin and plasma glucagon levels 5, 13 and 30 min after the injection of so much valine. The fractional rates of protein synthesis were determined in tissues of animals receiving either 12.8 or 17.1 mmol valine/5 kg body weight. The rates of protein synthesis in the jejunum (87.5%/day), liver (106.6%/day) and tensor fasciae latae muscle (18.8%/day) of lambs injected with the 12.8 mmol [3H]valine flooding dose, were in the range of data obtained in immature rats. Increasing the flooding amount of valine up to 17.1 mmol/5 kg body weight did not significantly alter protein synthesis rates in the jejunum, liver or skeletal muscle. This suggested that both the flooding-dose method in itself and valine had no effect on in vivo protein synthesis.     

Steroidogenesis and prostaglandin synthesis by cultured bovine blastocysts.

Bovine blastocysts were collected at Days 13, 15 and 16 and placed in TCM-199 supplemented with 5% fetal calf serum; some blastocysts were immediately frozen while the others were cultured for 48 h and then frozen. Samples (tissue + medium, 5--12/group) were thawed, homogenized and analysed by radioimmunoassays. Measurable amounts of progesterone were found in all blastocysts but values were higher (P less than 0.01) after culture. Testosterone was not found in the cultured or uncultured blastocysts at Day 13, but was detectable on Days 15 and 16 and in greater amounts (P less than 0.05) in the cultured blastocysts. PGF and PGE-2 were increased (P less than 0.05) in the cultured blastocysts in all 3 days. Oestradiol was measurable in some but not all blastocysts. It is suggested that PG synthetase and enzymes capable of synthesizing progesterone, testosterone and, possibly, oestradiol are present in these early bovine blastocysts.     

Whole protein uptake and metabolism by mouse blastocysts

Preimplantation mouse embryos take up whole 125I-labelled BSA from their environment. In blastocysts this uptake was temperature-sensitive and reversibly inhibited by trypan blue: properties consistent with an endocytotic mechanism. The uptake kinetics indicate that a saturable component predominates at low protein concentrations, but a non-saturable component is the major uptake route at higher concentrations. This suggests that BSA is pinocytosed probably bound to the membrane and dissolved in the bulk solvent phase. The rate of uptake, equivalent to about 5 pl/min/blastocyst was similar to that reported for non-saturable glycine uptake. In blastocysts the protein is degraded to acid-soluble products. At reported genital tract fluid protein concentrations this would represent a significant contribution to the embryonic pool of fixed nitrogen.     

3H]prostaglandin F2 alpha membrane binding reexamined

Tritiated prostaglandin F2 alpha ([3H]PGF2 alpha) binding to bovine corpora luteal membranes has been reexamined from the viewpoint of eventual PGF2 alpha receptor purification. Several modifications of the literature on PGF2 alpha binding allow for a more stabilized [3H]PGF2 alpha PGF2 alpha receptor complex which should then facilitate the PGF2 alpha receptor purification. Of particular importance were: identification of protease inhibitors which protect [3H]PGF2 alpha binding and protease inhibitors which are detrimental to subsequent [3H]PGF2 alpha binding; the finding that EGTA treatment of tissue homogenates greatly protects subsequent [3H]PGF2 alpha binding; the observation that Mn(+)+ substitutes for Ca(+)+ and, in fact, among the divalent cations Mn(+)+ greater than Mg(+)+ greater than Ca(+)+ in facilitating [3H]PGF2 alpha binding where as Cd(+)+, Cu(+)+ and Zn(+)+ either have no effect or are detrimental to this binding; the lack of effect of ATP, GTP, GDP and cAMP or of kinase and phosphatase inhibitors and activators to alter binding of [3H]PGF2 alpha to isolated membranes; and the ease with which the [3H]PGF2 alpha-PGF2 alpha receptor complex can be removed from the membrane in spite of the receptor being an integral membrane protein. A new simple technique for separating protein bound [3H]PGF2 alpha (PGF2 alpha receptor-[3H]PGF2 alpha complexes) from free [3H]PGF2 alpha by use of hydroxyapatite (HAP) is introduced. This HAP method is of particular use in solubilized membrane preparations (but can also be used during PG radioimmunoassays to separate free PG from antibody bound PG). These changes were required to facilitate subsequent chromatographic steps leading to identification and purification of the PGF2 alpha receptor. (ABSTRACT TRUNCATED AT 250 WORDS)     

[3H]Tyramine binding: A comparison with neuronal [3H]-dopamine uptake and [3H]mazindol binding processes

[³H]Spiperone ([³H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [³H]tyramine ([³H]PTA), thus indicating that [³H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [³H]PTA binding and [³H]DA uptake processes were markedly decreased, with no effect on [³H]mazindol ([³H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo andin vitro inhibitor of [³H]PTA binding, did not affect the [³H]MAZ binding process. This further supported the suggestion that while [³H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [³H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.Special issue dedicated to Dr. Paola S. Timiras     

[3H]GABA binding in developing rabbit retina

We have studied the developmental sequence of the GABA system in the rabbit retina using an in vitro binding assay to monitor developmental changes in the post-synaptic receptor. A variety of tissue treatments including perchlorate and Triton X-100 were employed to optimize binding and remove endogenous factors which inhibit binding. Pre-treatment of the tissue with 0.05% Triton X-100 revealed high affinity binding for [³H]GABA which increased in a sigmoidal fashion with the post-natal age of the animal. A constant level of binding, at about 16% of adult levels, was noted until day 8, at which time a rapid increase occurred. At 16 days post-natal, the amount of specific binding reached a plateau near adult levels. Kinetic analysis of the GABA receptor showed an increase in the number of receptors (B_max) with little or no change in the apparent affinity (K_D). Our results suggest that the onset of post-synaptic receptor activity is delayed approximately 1 to 2 days, relative to the pre-synaptic components, and the period of rapid increase in GABA receptor binding coincides with the period of maximum increase in retinal synaptic density.     

Binding and uptake of [3H]adrenaline by perfused rat liver.

The binding and uptake of [3H]adrenaline by the intact perfused rat liver was investigated. We showed that the administration of [3H]adrenaline to liver resulted in the rapid uptake of the radioligand, and that such uptake was independent of any Ca2+ redistributions induced by the hormone. At low adrenaline concentrations (less than 50 nM) uptake was inhibited by prazosin, whereas at higher hormone concentrations a significant proportion of total [3H]adrenaline uptake could not be inhibited by this antagonist. [3H]Adrenaline uptake could be directly correlated with adrenaline-induced responses such as an increased rate of respiration and glycogenolysis. The partial inhibition (approx. 25%) of [3H]adrenaline uptake by antagonists was sufficient for the total inhibition of hormone-induced responses. The effect of various pharmacological agents on [3H]adrenaline uptake was investigated, and the contribution of tissue-related factors to alpha-adrenergic agonist-antagonist interactions in vivo is discussed.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.