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Biomedical subjects

A Amsterdam

Publications and source records attributed to A Amsterdam.

At least 19 recordsLinked to original sources
PubMed1979

Effect of modulators of cytoskeletal function on desensitization and recovery of PGE2-responsive ovarian adenylate cyclase.

Exposure of cultured Graafian follicles to PGE2 for 20 h resulted in a loss of the cyclic AMP response to fresh hormone. This desensitization was prevented by addition to the medium of D2O (25--50%) or Li+ (0.6--6 mM), agents believed to stabilize microtubules, as well as by phalloidin (1.0--10 microM), believed to stabilize the polymerized state of actin, in a dose-dependent manner. The spontaneous recovery of responsiveness to PGE2 upon incubation of refractory follicles for 6 h in hormone-free medium was prevented by addition to the medium of cytochalasin B (CB; 3 microgram/ml) or of the actin-binding myosin subfragment HMM S-1 (80 microgram/ml) or of anti-actin serum; viz. by agents likely to interfere with microfilament function. D2O (50%) caused morphological damage to the inner layer of the membrana granulosa and severe depression of protein synthesis. The other drugs used (phalloidin, LiCl and cytochalasin B) had no such effects. Resensitization of refractory follicles was also prevented by cycloheximide (10 micrograms/ml) and by actinomycin D (10 micrograms/ml). It is speculated that the recovery process may involve the insertion of a newly synthesized protein, such as PG-receptor, into the membrane by a mechanism dependent on microfilament action. These findings provide suggestive evidence for the hypothesis that cytoskeletal elements associated with the cell membrane take part in the modulation of the adenylate cyclase response to hormones.

Actins↗
PubMed1978

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. I. Inhibition by heparin of gonadotrophin-stimulated ovarian adenylate cyclase.

Heparin inhibits (I50 = 2 microgram/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5'-(beta,gamma-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by heparin (I50 = 6 microgram/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was not eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Heparin (3 microgram/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did not change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged heparin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.

Adenylyl Cyclases↗
PubMed1978

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues.

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I50 = 2 microgram/ml) when compared to other naturally occurring glycosamin oglycans. This inhibition was also apparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E2. Heparin was also found to inhibit glucagon-sensitive rat hepatic adenylate cyclase, and the prostaglandin E1-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfated polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary and was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.

Adenylyl Cyclases↗
PubMed1978

Distribution of an alpha-bungarotoxin-binding cholinergic nicotinic receptor in rat brain.

Cholinergic nicotinic receptors in rat brain were demonstrated by the use of the potent nicotinic antagonist [125I]alpha-bungarotoxin [125I]alpha-Btx). Biochemical studies on binding of [125I]alpha-Btx to rat hippocampal homogenates revealed saturable binding sites which are protected by nicotine, D-tuborcurarine and acetylcholine but not by atropine or oxotremorine. The hippocampus and hypothalamus displayed relatively high [125I]alpha-Btx specific binding whereas the cerebellum was devoid of specific binding. Other regions displayed intermediate binding levels. Analysis of the regional distribution of [125I]alpha-Btx binding by autoradiography of frontal brain sections revealed high labeling in the hippocampus, hypothalamic supraoptic, suprachiasmatic and periventricular nuclei, ventral lateral geniculate and the mesencephalic dorsal tegmental nucleus. It is suggested that the limbic forebrain and midbrain structures as well as sensory nuclei are the main nicotinic cholinoceptive structures in the brain.

Animals↗
PubMed1978

Localization of acetylcholine receptor in excitable membrane from the electric organ of Torpedo: Evidence for exposure of receptor antigenic sites on both sides of the membrane.

Nicotinic acetylcholine receptor was localized in a receptor-rich membrane preparation from the electric organ of Torpedo californica by applying an immunoferritin technique. The membrane preparation was incubated with (Fab')2 fragments derived from specific rabbit antibodies against the purified acetylcholine receptor and subsequently with ferritin-conjugated goat antiserum to rabbit immunoglobulin. More than 50% of the vesicles were found to be labeled with ferritin while the rest remained unlabeled. Ferritin labeling on both sides of the membrane was evident in open membrane vesicles, whereas in closed vescles the labeling was confined to the outer surface due to the inability of the tracer to penetrate the membrane. These data suggest that antigenic sites of the receptor molecule are exposed on both sides of the excitable membrane, and that acetylcholine receptor may be a transmembrane protein.

Animals↗
PubMed1977

Localization of actin and myosin in the rat oocyte and follicular wall by immunofluorescence.

The distribution of actin and myosin in the rat ovary at different stages of postnatal development was studied by examination of cryostat sections treated with antibodies to chicken smooth muscle (gizzard) myosin or to chicken (pectoral muscle) actin and subsequently with fluoresceinated goat anti-rabbit gamma-globulins. Staining with either antiserum revealed several layers of intensely fluorescent elongated cells within the theca externa, forming a coherent band around the larger Graafian follicles. In smaller follicles, this band of fluorescent cells was incomplete, and ovaries of immature (6-day-old) rats were devoid of strongly fluorescent cells. Corpora lutea contained only scattered fluorescent cells at their circumference. Sections of mature ovaries incubated with antibodies raised against striated (pigeon pectoral) muscle myosin generated no significant fluorescence. Large oocytes stained with either anti-actin or anti-smooth muscle myosin showed a thin fluorescent band just beneath the zona pellucida, suggesting that actin and myosin are associated with the oolemma. The distribution of the two antigens in serial sections of follicles coincided, suggesting that the same cells contained both actin and myosin. It is suggested that follicular growth and maturation is attended by the development of a smooth muscle layer in the theca and that contraction of this layer in response to catecholamines and/or prostaglandins may play a role in the extrusion of the oocyte. The role of contractile elements in the oocyte remains to be elucidated.

Actins↗
PubMed1977

Suppression of 20 alpha-hydroxysteroid dehydrogenase activity in cultured rat luteal cells by prolactin.

20alpha-Hydroxysteroid dehydrogenase (20 alpha-SDH) activity increases in the cycling corpus luteum of the rat, beginning at 14.00 h on the day of diestrus, but remains low in corpora lutea of pregnancy throughout the first 19 days of gestation. When cells derived from 7-day-old corpora lutea of pregnant rats were cultured for 7 or 12 days, there was a spontaneous rise in 20 alpha-SDH activity from an initial value of 0.44 +/- 0.27 to 4.1 +/- 0.7 units/mg supernatant protein. Addition of LH (NIH-S-18; 2.0 mug/ml) or prostaglandin F2alpha (2.8 X 10(-5) M) to the medium from day 4 to the end of incubation period caused a slight but significant reduction in 20 alpha-SDH activity (20%, P less than 0.05). Supplementation of the medium with ovine prolactin (HIH-P-S11; 10.0 mug/ml) from the time of seeding or from the 2nd to 4th day of culture reduced the activity of 20 alpha-SDH measured on day 12 by 61% (P less than 0.001). This finding suggests that the suppression of 20 alpha-SDH by prolactin, hitherto demonstrated only in vivo results from a direct action of the hormone on the luteal cell.

Animals↗
PubMed1977

Differentiation of decidual cells in cultures of rat endometrium.

Endometrial scrapings were collected from rat uteri at various times (0-4 days) after induction of a decidual reaction by i.p. injection of pyrathiazine hydrochloride (20 mg/animal) on the 5th day after a sterile mating. The tissue was dissociated by treatment with trypsin and the cells were cultured as monolayers. The differentiation of decidual cells was followed in these cultures. Two morphologically distinct cell populations were recognized: (i) dispersed spindle-shaped or stellate cells, and (ii) colonies of closely packed polygonal cells, first apparent after 48 h in culture. During culture, [3H] thymidine was incorporated into the nuclei of both cell types, as indicated by autoradiography. There was an increase in the number of cells in the colonies as culture progressed; most mitotic figures and the highest % of labelled nuclei were located within the colonies. Bi- and multinucleated cells, which are a characteristic feature of decidual tissue in vivo, appeared in the colonies on the 3rd day of culture and constituted about 60% of the colony population after 4-5 days. The dispersed cells showed a doubling in nuclear area during 4 dyas in culture, suggesting the formation of polyploid cells; such cells are prominent in fully differentiated decidual cells in vivo. The content of prostaglandin E in the cultured cell, determined by radioimmunoassay, was about 15-fold higher than that in rat embryo fibroblast cultures grown under similar conditions, and was comparable to the level of prostaglandins found in decidual cells in vivo.

Animals↗