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ABSTRACT

The sympathetic nervous system is believed to play a part in the control of insulin release from the pancreatic islets of Langerhans. Stimulation of α-adrenoceptors is thought to inhibit the release of insulin whereas stimulation of β-adrenoceptors enhances insulin release. The present experiments were conducted to establish the existence of β-adrenergic receptors on guinea-pig and rat islet cells and to quantify them using the selective β-adrenergic ligands [³H]dihydroalprenolol (DHA) and [¹²⁵I]cyanoiodopindolol (CYP).

Guinea-pig islets had 62 fmol β-adrenoceptors/mg protein using [³H]DHA, corresponding to 43 700 binding sites/cell and 25 fmol β-adrenoceptors/mg protein using [¹²⁵I]CYP, corresponding to 17 400 sites/cell. Rat islet cells were found to have 4·6 fmol β-adrenoceptors/mg protein using [¹²⁵I]CYP, corresponding to 7200 sites/cell. Adenylate cyclase activation exhibited a positive dose–response relationship when exposed to the β-adrenoceptor agonist isoprenaline, with a maximum response (190 ± 21% above basal) at 10 μmol isoprenaline/l. This response was abolished with 1 μmol/l of the β-adrenergic antagonist 1-alprenolol. Insulin secretion in the presence of 10 mmol glucose/l, but in the absence of the α-adrenoceptor blocker phentolamine, was not affected by 10 μmol isoprenaline/l. However, perifusion experiments showed that secretion of insulin from isolated rat islets in the presence of 10 mmol glucose/l was significantly increased (332%) by 10 μmol isoprenaline/l in the presence of 10 μmol phentolamine/l.

These results suggest that binding of selective radio-labelled ligands occurs to β-adrenergic receptors on the B cell surface of the islets of Langerhans, and that these receptors are functionally coupled to insulin secretion through modulation of adenylate cyclase activity.

J. Endocr. (1986) 111, 263–270

SUMMARY

Human granulosa cells from Graafian follicles aspirated 3–4 h before the expected time of ovulation were incubated with various steroid substrates, including pregnenolone, androstenedione, testosterone and dehydroepiandrosterone (DHA). Steroid production after 3 and 10 h of incubation was determined by radioimmunoassay.

Progesterone and 17α-hydroxyprogesterone were the major products of granulosa cells in control short-term cultures with endogenous substrates. The addition of pregnenolone increased the synthesis of progesterone and 17α-hydroxyprogesterone compared with the controls, although the response varied considerably between paired short-term cultures. Little or no oestradiol-17β was produced from endogenous precursors or short-term cultures to which pregnenolone had been added; one follicle, however, produced similar amounts of oestradiol-17β in the control cultures and after incubation with pregnenolone.

When granulosa cells were cultured with various amounts of androstenedione, DHA or testosterone, large amounts of oestradiol-17β were produced, especially in short-term cultures in which larger amounts of substrate were added. Progesterone production continued and progesterone was synthesized more rapidly or in greater amounts in some short-term test cultures than in the controls.

The results indicate that human granulosa cells are one source of oestradiol-17β during the preovulatory phase. The data support the two-cell theory for oestradiol synthesis, for granulosa cells do not appear to undertake steroid conversion via the 5-unsaturated pathway, but aromatize androgens known to be produced by thecal cells. It is also suggested that either androgens or oestradiol-17β stimulate progesterone production by granulosa cells, at least in vitro.

SUMMARY

The placenta, foetal membranes and uterine mucosa of mice (pregnant for 8–17 days) have been investigated by histochemical methods for NAD-dependent 3β-hydroxysteroid dehydrogenase (3β-HSDH), and for NAD-and NADP-dependent 17α- and 17β-hydroxysteroid dehydrogenases (17α- and 17β-HSDH), 11α- and 11β-hydroxysteroid dehydrogenases (11α- and 11β-HSDH), and 20β-hydroxysteroid dehydrogenase (20β-HSDH).

3β-HSDH was found to be distributed in the trophoblastic giant cells of the first generation with both pregnenolone and DHA as substrates, and in the giant cells of the second generation and of the labyrinth and the endodermal cells of the inverted yolk sac placenta, but only with DHA as substrate.

17α-HSDH and 17β-HSDH, NAD-dependent, were present in both the first and second generation giant cells and in the giant cells of the labyrinth as well as in the endodermal cells of the inverted yolk sac placenta. With NADP as cofactor, 17α-HSDH and 17β-HSDH were weakly positive with all the substrates used in the giant cells of the second generation and of the labyrinth, while NADP-dependent 17β-HSDH was present in the first generation giant cells and in the endodermal cells of the inverted yolk sac placenta but only with oestradiol-17β as substrate.

The histochemical reaction for 11α-HSDH, both NAD- and NADP-dependent, was limited to trophoblastic giant cells of the second generation and of the labyrinth; 11β-HSDH, both NAD- and NADP- dependent, was distributed in the giant cells of the second generation and of the labyrinth, the epithelial cells of the uterine mucosa and the decidua basalis.

The histochemical reaction for 20β-HSDH, NAD- and NADP-dependent, was weakly positive in the giant cells of the first generation only.

SUMMARY

A longitudinal study was made of the urinary excretion of a number of C₁₉ and C₂₁ steroids in 11 healthy boys and 9 healthy girls aged 8–12 yr. Urine collections were made every 6 months, over periods ranging from 1 to 2½ yr. in different children. The excretions of most substances showed surprisingly regular increments in individual children. Differences between individual children were marked and often consistent; some children excreted high amounts of one substance and low amounts of another, and other childen the reverse. The variation between individuals in the excretion of substances such as androsterone and aetiocholanolone was reduced when the results were plotted against skeletal rather than chronological age. DHA was excreted by all subjects, but at a very low level before the skeletal age of 10 yr. The glucuronide: sulphate ratio of the 11-deoxy-17-oxosteroids fell consistently from 8 to 12 yr. whereas the ratio of 5α-: 5β-11-deoxy-17-oxosteroids increased. Boys excreted more C₁₉ steroids than girls with the same skeletal maturity score. This comparison is physiologically more meaningful than comparing boys and girls of the same chronological age.

SUMMARY

The presence of hydroxysteroid dehydrogenases in the pig placenta and associated uterine structures is described between 26 days of gestation and term.

Activity with androsterone, aetiocholanolone, DHA, pregnenolone, 17α-hydroxypregnenolone, 3β-hydroxy-5β-androstan-17-one, cortisol, 3β,16β-dihydroxyandrost-5-ene 3-methyl-ether, 3,16β-dihydroxyoestra-1,3,5(10)-triene 3-methyl-ether, oestradiol-17β, and 20β-hydroxyprogesterone in the trophoblast increased in mid-pregnancy, and thereafter decreased towards term, except with androsterone, cortisol, 3β,16β-dihydroxyandrost-5-ene 3-methyl-ether, 3,16β-dihydroxyoestra-1,3,5(10)-triene 3-methyl-ether and oestradiol whose increased level of activity persisted. No activity was found in the trophoblast with 1 1β-hydroxyandrostenedione, 3β-hydroxyandrost-5-en-16-one 3-methyl-ether, 17α-oestradiol, testosterone, or 20α-hydroxyprogesterone. Activity in the trophoblast of the areolae was consistently absent or greatly decreased.

In the maternal epithelium, intense activity with oestradiol-17β, and testosterone as substrates (but no others) was observed early in pregnancy, but decreased markedly later. Activity was consistently higher in the epithelium related to the opening of the areolae. No other uterine or foetal structures gave a positive reaction.

Some attempt is made to correlate the presence of the enzymes with their function, with biochemical findings regarding isolation of steroids from the placenta, and with the effects of ovariectomy on pregnancy in the pig.

SUMMARY

Intact pregnant rats in which gonadotrophin secretion was suppressed by administration of the tranquillizer trifluoperazine (Stelazine) were very sensitive to testosterone propionate (TP), dehydroepiandrosterone (DHA) and androstenedione as inducers of nidation but insensitive to 5α-androstan-3α-ol-17-one. The minimum effective dose of the former steroids was 0·025 mg. intramuscularly or 0·005 mg. injected under the ovarian capsule. This effect of androgens seems to be indirect, inasmuch as 0·005 mg. TP was ineffective when applied locally to the uterine mesometrium. Moreover, 2 mg. TP failed to induce nidation after ovariectomy (followed by daily administration of progesterone), although adrenalectomy did not prevent the action of this dose of TP. MER-25, 20 mg., given simultaneously with 2 mg. TP invariably prevented nidation.

Nidation was also induced in lactating pregnant rats by intramuscular injection of 1–2 mg. TP. In the absence of the ovaries such animals failed to respond to 1·0 mg. TP. Lactating pregnant rats also failed to respond to 0·05 mg. TP applied to the ovaries but nidation was induced when this dose was injected into the spleen.

It is concluded that the ovaries of pregnant rats under the influence of Stelazine more readily convert androgens into oestrogens than the ovaries of lactating pregnant animals. It is suggested that the suppression of gonadotrophin secretion caused by trifluoperazine or lactation induces a deficiency in the secretion of androgenic steroids by both the ovaries and adrenals. However, the ovaries do not lose the enzyme system capable of converting androgens into the oestrogens required for nidation. Finally, it is suggested that the oestrogen surge from the ovaries during the critical period before nidation is not necessarily due to an acute release of pituitary luteinizing hormone, as is ovulation, since androgens from the adrenals can be converted by ovarian tissue(s) to the oestrogen required for nidation.

SUMMARY

1. With rat diaphragm in vitro, addition of insulin to the medium so as to give a concentration as low as 0·05 mu./ml. of the hormone, stimulated the incorporation of [¹⁴C]glycine into protein of tissue. Simultaneous addition of glucose to the medium did not affect either the minimal amount of insulin required to produce a significant stimulation of incorporation of glycine, or the magnitude of the effect of the small concentration of insulin used.

2. Addition of a mixture of oxidized A and B chains of the insulin molecule did not affect incorporation of a mixture of labelled amino acids into the protein of isolated diaphragm, but a degraded insulin (DHA-insulin), which has about 15% of the activity of insulin in stimulating glucose uptake by diaphragm, was found to stimulate incorporation of [¹⁴C]glycine to an extent comparable with its effect in stimulating glucose-uptake.

3. Addition of rat serum, or the dipping of diaphragm in a medium containing insulin, stimulated incorporation of [¹⁴C]glycine into protein of diaphragm. Both these effects and the stimulation produced by insulin in vitro were abolished when the medium contained an antiserum to insulin.

4. Addition in vitro of growth hormone (GH) stimulated incorporation of [¹⁴C]glycine into protein of diaphragm from the hypophysectomized rat but had no effect on diaphragm from the normal rat, whether or not a small dose of insulin was also added in vitro. The action of GH in promoting incorporation of [¹⁴C]glycine into protein of diaphragm from the hypophysectomized rat was not neutralized by insulin antiserum.

5. Corticotrophin, cortisol, thyroxine, vitamin B₁₂, vitamin D₂ and linoleic acid all had no observable effect on incorporation of labelled amino acids into diaphragm. Glucagon stimulated incorporation, but the stimulation was abolished by the in vitro addition of antiserum to insulin and was probably attributable to the presence of a trace of insulin in the glucagon.

6. Anaerobiosis, and the addition of various metabolic inhibitors, were found to suppress incorporation of [¹⁴C]glycine into diaphragm protein almost entirely.