Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts.

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilatation at labor. Incubation of cervical fibroblasts with [3H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [3H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (Kd) of 12 nM, and the binding capacity (Bmax) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [3H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilation at labor. Incubation of cervical fibroblasts with [³H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [³H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (K _d) of 12 nM, and the binding capacity (B _max) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [³H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

On the synthesis of serine and homoserine samples asymmetrically labelled with tritium and deuterium in the hydroxymethylene group.

(1R) [1-3H, 2H1] 3-Phenylpropanol, the key intermediate in the synthesis of (4R) [4-3H, 2H1] D,L-homoserine and of the (4S)-isomer, is obtained from (1S) [1-2H1] 3-phenylpropanol and (1RS) [1-3H] ethanol upon incubation with yeast alcohol dehydrogenase and NAD+; under similar conditions 2-phenylethanol undergoes very small exchange with [1-2H2] ethanol.     

Studies on the serotonin transporter in platelets

[3H]-Imipramine and [3H]-paroxetine label with high affinity a recognition site which is associated with the serotonergic transporter in blood platelets. The pharmacological profile of [3H]-imipramine and [3H]-paroxetine binding is highly correlated with the potency of drugs to inhibit the uptake of serotonin. Dissociation kinetic experiments suggest that the substrate recognition site for serotonin may be different from the modulatory site which is labeled with [3H]-imipramine or [3H]-paroxetine. The existence of an endocoid acting on the imipramine receptor to modulate the serotonin transporter has been proposed by several laboratories. In clinical studies most laboratories have reported a decrease in Bmax of [3H]-imipramine binding in platelets from depressed untreated patients when compared with matched healthy volunteers. The Bmax of [3H]-imipramine binding in platelets appears to be a state-dependent biological marker in depression.     

The effect of the phytoalexins, lubimin, (-)-maackiain, pinosylvin, and the related compounds dehydroloroglossol and hordatine M on human lymphoblastoid cell lines

We have tested the effect of the phytoalexins lubimin, (-)-maackiain and pinosylvin and the related compounds dehydroloroglossol and hordatine M on the growth of the human lymphoblastoid cell lines Molt and Raji. (-)-maackiain, pinosylvin and dehydroloroglossol showed significant growth inhibitory action on the cells. Suppression of [3H] thymidine and [3H] leucine uptake was tested and noted in pinosylvin and dehydroloroglossol. The phytoalexins and related compounds are widespread in plants and provide a potential source of antineoplastic substances.     

Lipoperoxidation rates and drug-oxidizing enzyme activities in the liver and placenta of some mammal species during the perinatal period.

Lipoperoxidation and drug-metabolizing enzymes were measured in livers and placentas of different mammal species during the perinatal perios. In placentas and fetal livers of rat, rabbit and guinea-pig, cofactor-supported lipoperoxidation was negligible, as were the activities of drug-oxidizing enzymes. Human fetal liver contained an intact drug-oxidizing electron transport chain, and lipoperoxidation activity was accordingly abserved. It is suggested that lesions mediated by lipoperoxidation may be possible in human fetus, but they are less probable in animal fetuses.     

The effect of the phytoalexins,lubimin, (−)-maackiain,pinosylvin, and the related compounds dehydroloroglossol and hordatine M on human lymphoblastoid cell lines

Summary We have tested the effect of the phytoalexins lubimin, (–)-maackiain and pinosylvin and the related compounds dehydroloroglossol and hordatine M on the growth of the human lymphoblastoid cell lines Molt and Raji. (–)-maackiain, pinosylvin and dehydroloroglossol showed significant growth inhibitory action on the cells. Suppression of [³H] thymidine and [³H] leucine uptake was tested and noted in pinosylvin and dehydroloroglossol. The phytoalexins and related compounds are widespread in plants and provide a potential source of antineoplastic substances.We would like to acknowledge the assistance of J. Hux in preparing the phytoalexins and related compounds. This work was supported by a grant from National Health and Welfare Canada. Correspondence to Dr. L. Skinnider, Department of Pathology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7M OWO.     

3[H]-Sulpiride labels mesolimbic non-dopaminergic sites that bind antidepressant drugs

3[H]-(-)-Sulpiride and 3[H]-spiperone binding was compared in rat amygdala, nucleus accumbens and striatum, using (+/-)-sulpiride to define specific binding. 3[H]-(-)-Sulpiride bound to twice as many sites in amygdala and nucleus accumbens as 3[H]-spiperone. 3[H]-(-)-Sulpiride binding was directed to these additional sites by using 1 microM spiperone to mask dopaminergic binding. The binding of 3[H]-(-)-sulpiride to these sites was high affinity, reversible, Na+-dependent, but not stereospecific. Metoclopramide, tiapride and antidepressant medications, but not other neuroleptics, ADTN, or serotonin displaced 3[H]-(-)-sulpiride binding to these sites. These data suggest that 3[H]-(-)-sulpiride labels mesolimbic sites other than dopamine receptors which may mediate antidepressant effects.     

Inhibition by tetanus and botulinum A toxin of the release of [3H]noradrenaline and [3H]GABA from rat brain homogenate

Rat brain homogenate was preloaded with [3H]noradrenaline or [3H]GABA and stimulated with high K+. Tetanus toxin and botulinum A neurotoxin partially prevent the evoked [3H]noradrenaline release in the same range of toxin concentrations starting below 10(-10) M. In contrast, release of gamma-amino butyric acid (GABA) is much more sensitive to tetanus than to botulinum A toxin.     

The effect of a bradykinin antagonist on vasodilator responses with particular reference to the submandibular gland of the cat

A bradykinin analogue, D-Arg[Hyp3, Thi5,8, D-Phe7]-Bk, antagonized the vasodilator effect of bradykinin injected close-arterially in the submandibular salivary gland of the cat, without affecting that due to acetylcholine or nerve stimulation. The same analogue also antagonized the hypotensive response to bradykinin injected intravenously in cats and rabbits. We conclude that functional hyperaemia in the submandibular gland of the cat is not due to the release of bradykinin by salivary kallikrein.     

Serum-induced stimulation of snRNA synthesis in mouse 3T3 fibroblasts

Small nuclear RNAs (snRNAs) from quiescent and serum-stimulated 3T3 cultures, labeled with [3H]uridine [( 3H]U), were electrophoresed in polyacrylamide-urea slab gels and revealed by staining with ethidium bromide and by fluorography. Judged by labeling with [3H]U, synthesis of 7S and U1-U6 RNAs was very low or absent in quiescent cultures. The serum-induced transition of 3T3 cells from a resting to a growing state was accompanied by an early, apparently sequential stimulation of snRNA synthesis; stimulated synthesis of 7S, U1, U2, U3, U4 and U6 RNAs coincided in time with serum-induced stimulation of 45S pre-ribosomal RNA (pre-rRNA) and heterogeneous nuclear RNA (hnRNA) synthesis.     

Lipoperoxidation rates and drug-oxidizing enzyme activities in the liver and placenta of some mammal species during the perinatal period

Summary Lipoperoxidation and drug-metabolizing enzymes were measured in livers and placentas of different mammal species during the perinatal period. In placentas and fetal livers of rat, rabbit and guinea-pig, cofactor-supported lipoperoxidation was negligible, as were the activities of drug-oxidizing enzymes. Human fetal liver contained an intact drug-oxidizing electron transport chain, and lipoperoxidation activity was accordingly observed. It is suggested that lesions mediated by lipoperoxidation may be possible in human fetus, but they are less probable in animal fetuses.The skillful technical assistance of Ms.Liisa Tuhkanen and Ms.Vuokko Väisänen is gratefully acknowledged.     

Stimulation of G-6-P independent glycogen synthase activity in the human placenta]

In immature human placentas, the activity of the I (glucose-6-phosphate-independent) form of glycogen synthase is significantly increased by insulin, glucose and by both compounds associated. In full-term placentas, the same kind of results has been found in each organ studied; due to the great variability observed in synthetase I control activities, the stimulation is significant only in the presence of insulin.     

A practicable variant of the ion exchange method for the radiometric estimation of ornithine decarboxylase activity

A known ornithine decarboxylase assay working with ion exchange separation of [3H]ornithine and [3H]putrescine has been revised. The assay can be performed in disposable 1.5 ml vessels with a total of four pipetting steps. The separation of enzyme substrate and product, respectively, requires 3 h per 50 samples. The detection limit is about 50 pmoles [3H]putrescine formed.     

Antihypertensive and cardiac effects of two novel beta-adrenoceptor blocking drugs.

Two new beta-adrenoceptor blocking drugs with acute antihypertensive and positive inotropic effects are described: Compound A (2-[4-(3-tert.butylamino-2-hydroxypropoxy)phenyl]-4-trifluoromethylimidazole) and MK-761 (2-(3-tert.butylamine-2-hydroxypropoxy)-3-cyanopyridine hydrochloride). In SH rats both compounds, given orally, lowered arterial pressure and were more potent than hydralazine. The antihypertensive effect of compound A but not of MK-761 was antagonized by timolol. Both compounds had positive inotropic activity on cat heart papillary muscles; these effects were antagonized by timolol. The pretreatment of animals with reserpine greatly reduced the positive inotropic effect of MK-761 but not of compound A. The acute antihypertensive and positive inotropic effects of compound A are like to be at least partially due to stimulation of beta-adrenoceptors, e.g. intrinsic sympathomimetic activity. The effects of MK-761 on the same parameters appear to be mediated by different mechanisms.     

Relationship between regeneration of cell surface glycoproteins in trypsin-treated chick embryo fibroblasts and cell adhesion to the substratum

The ability of cells to adhere to a substratum was altered by treatment with trypsin but was restored after a 1.5-h culture. A concomitant incorporation of [3H] leucine and [14C] glucosamine in the trypsin-sensitive cell surface glycoproteins was observed and almost reached a plateau within 1.50 h following the treatment with trypsin.     

Serum-induced stimulation of snRNA synthesis in mouse 3T3 fibroblasts

Summary Small nuclear RNAs (snRNAs) from quiescent and serum-stimulated 3T3 cultures, labeled with [³H]uridine ([³H]U), were electrophoresed in polyacrylamide-urea slab gels and revealed by staining with ethidium bromide and by fluorography, Judged by labeling with [³H]U, synthesis of 7S and U1-U6 RNAs was very low or absent in quiescent cultures. The serum-induced transition of 3T3 cells from a resting to a growing state was accompanied by an early, apparently sequential stimulation of snRNA synthesis; stimulated synthesis of 7S, U1, U2, U3, U4 and U6 RNAs coincided in time with serum-induced stimulation of 45S pre-ribosomal RNA (pre-rRNA) and heterogeneous nuclear RNA (hnRNA) synthesis.     

A practicable variant of the ion exchange method for the radiometric estimation of ornithine decarboxylase activity

Summary A known ornithine decarboxylase assay working with ion exchang separation of [³H]ornithine and [³H]putrescine has been revised. The assay can be performed in disposable 1.5 ml vessels with a total of four pipetting steps. The separation of enzyme substrate and product, respectively, requires 3 h per 50 samples. The detection limit is about 50 pmoles [³H]putrescine formed.     

The effect of a bradykinin antagonist on vasodilator responses with particular reference to the submandibular gland of the cat

Summary A bradykinin analogue, D-Arg[Hyp³, Thi^{5, 8}, D-Phe⁷]-Bk, antagonized the vasodilator effect of bradykinin injected close-arterially in the submandibular salivary gland of the cat, without affecting that due to acetylcholine or nerve stimulation. The same analogue also antagonized the hypotensive response to bradykinin injected intravenously in cats and rabbits. We conclude that functional hyperaemia in the submandibular gland of the cat is not due to the release of bradykinin by salivary kallikrein.     

Glucose-evoked recovery of hepatic thyroxine 5'-deiodinase independent of de novo protein synthesis in fasted rat

The glucose-evoked recovery of Type I thyroxine 5'-deiodinase activity in the hepatic microsomes of fasted rat was not inhibited by either cycloheximide, puromycin or actinomycin D during 3 h after glucose feeding; however, [3H]-leucine uptake by the liver or the hepatic microsomal fraction was significantly inhibited by cycloheximide and puromycin but not by actinomycin D. These results indicate that the glucose-evoked recovery of deiodinase activity may be independent of de novo protein synthesis.