Effect of adrenaline on basal and ovine corticotrophin-releasing factor-stimulated ACTH secretion in man

Six normal male subjects were given, in single blind random order on six separate occasions, i.v. bolus doses of synthetic ovine corticotrophin-releasing factor-41 (oCRF-41; 25 and 50 micrograms) with and without adrenaline (3 micrograms/min) i.v. for 150 min, the adrenaline infusions alone and saline placebo. The adrenaline infusions resulted in plasma adrenaline concentrations of 4.33 +/- 0.82 (S.E.M.) nmol/l and were associated with an increase in blood glucose, heart rate and systolic blood pressure and a reduction of diastolic blood pressure. Despite these evident biological effects at several sites, there was no stimulation of plasma ACTH or cortisol by adrenaline in comparison with the effect of saline, and no enhancement of the stimulatory effect of either dose of oCRF-41 on ACTH or cortisol secretion. The ACTH response to 50 micrograms oCRF-41 was greater than that to 25 micrograms, indicating that the 25 micrograms dose of oCRF-41 was submaximal and capable of further enhancement. As the plasma adrenaline concentrations during the adrenaline infusions reached the upper limit of the physiological range of plasma adrenaline in man, yet failed to enhance the ACTH or cortisol responses to a submaximal dose of oCRF-41, we conclude that circulating adrenaline neither exerts a direct stimulatory effect on pituitary corticotrophs nor enhances the effect of CRF under physiological circumstances. The adrenaline infusions attenuated the ACTH and cortisol responses to oCRF-41 and were associated with a transient reduction of basal concentrations of both hormones.     

Relationship between maternal and fetal corticotrophin-releasing hormone-41 and ACTH levels in human mid-trimester pregnancy

Samples of maternal blood, amniotic fluid and umbilical arterial and venous blood were collected from 11 women at 16-24 weeks of pregnancy. Corticotrophin-releasing hormone-41 (CRH-41) and ACTH were measured by immunoradiometric assay. The mean levels of ACTH were 11 pmol/l in maternal plasma, 12 pmol/l in fetal plasma and 9.7 pmol/l in amniotic fluid. The mean levels of CRH-41 were 1.6 pmol/l in maternal plasma and 0.7 pmol/l in fetal plasma. There was a positive correlation between maternal and fetal plasma CRH-41 and between maternal CRH-41 and ACTH. In fetal plasma there was a weak inverse correlation between CRH-41 and ACTH. This is the first demonstration of CRH-41 in the circulation of the mid-trimester human fetus, but on the basis of the present findings it is not possible to specify the exact source (fetal, placental or maternal).     

Steroid sulfatase activity in amnion tissue and amnion cells maintained in monolayer culture

Phenolic steroid sulfatase activity in amnion tissue, amnion homogenate, subcellular fractions, and amnion epithelial cells in culture was demonstrated with radiolabeled estrone sulfate as the substrate. Sulfatase activity could not be detected in either amnion tissue or cells when evaluated with dehydroisoandrosterone sulfate as the substrate. Phenolic steroid sulfatase activity in amnion tissue was linear with incubation time up to 3 h and with amnion tissue weight up to 800 mg/ml. The rate of estrone sulfate hydrolysis in amnion tissue increased in a linear manner with temperature from 3 to 60 C. The apparent Km of amnion tissue sulfatase for estrone sulfate was 9 microM. The highest specific activity of the enzyme was found in both the mitochondrial-lysosomal and microsomal fractions. In studies with amnion epithelial cells in monolayer culture, phenolic steroid sulfatase activity was linear with incubation time up to 4 h and with cell number up to 2 X 10(5)/ml. The apparent Km of amnion cell sulfatase for estrone sulfate was 5.5 microM. The product of hydrolysis, i.e. estrone, was metabolized in situ to 17 beta-estradiol in both amnion tissue and cells. The hydrolysis of estrone sulfate (and possibly other phenolic steroid sulfates present in amniotic fluid) by amnion cells may be important in providing biologically potent estrogens for in situ action.     

Measurement of circulating corticotrophin-releasing factor in man

A radioimmunoassay was developed to measure corticotrophin-releasing factor (CRF-41) extracted from human plasma using Vycor glass. Assay sensitivity was 20 ng/l and intra- and interassay coefficients of variation were 10.2 and 11.4% respectively. The normal range of plasma CRF-41 was less than 20-110 ng/l (n = 46). Plasma concentrations of CRF-41 in patients with Cushing's disease. Nelson's syndrome and Addison's disease were within the normal range. No correlation was found between CRF-41 and ACTH in these syndromes. Two patients with the ectopic ACTH syndrome had increased plasma concentrations of CRF-41. In normal subjects no changes in plasma CRF-41 occurred after insulin-induced hypoglycaemia, treatment with dexamethasone or feeding, and changes in the concentrations of CRF-41 did not reflect circadian changes in plasma concentrations of cortisol. Concentrations of immunoreactive CRF in plasma of women in the third trimester of pregnancy were increased (550-9300 ng/l) and gel filtration chromatography showed that this comprised CRF-41 and a higher molecular weight form. Reversed-phase high-performance liquid chromatography also revealed multiple peaks of immunoreactive CRF in extracts of plasma and placenta.     

Effects of angiotensin II infusion on the early morning surge of ACTH and o-CRH-provoked ACTH secretion in normal man

The question of whether elevated plasma angiotensin II (AII) levels modulate ACTH secretion in man still awaits a definite answer. We performed two sets of experiments pertinent to that problem: Seven healthy young males each received AII (5 ng/kg/min) and sham infusions on different days in a randomized sequence from 03.00 h to 06.00 h in the morning, while plasma ACTH and cortisol were measured every 20 min. Mean blood pressure rose by about 10 mmHg during AII infusion. Mean plasma ACTH levels were slightly higher with AII than with sham infusion in every single individual (P less than 0.05). Differences in a pre- and post-infusion period were significant. Plasma cortisol levels were almost identical with or without AII infusion. Nine healthy young males received AII (5 ng/kg/min) or sham infusions on different days from 16.30 h to 20.00 h in a randomized sequence and a 100 micrograms o-CRH injection at 17.00 h. Plasma ACTH and cortisol were measured every 15 or 30 min between 16.30 h and 20.00 h. Mean blood pressure rose by about 14 mmHg during AII infusion. The rapid increment and further change in plasma ACTH and cortisol was not significantly different between the AII and sham infusion studies. Conclusions: The dose of AII infused was probably just above the threshold of ACTH stimulation, although AII plasma levels obtained were probably far above the physiological range. On the adrenal level, a vasoconstrictor effect of AII may have prevented stimulation of cortisol. This may be different in states of sodium depletion with reduced vascular effects of AII.(ABSTRACT TRUNCATED AT 250 WORDS)     

The corticotrophin-releasing hormone test is the most reliable noninvasive method to differentiate pituitary from ectopic ACTH secretion in Cushing's syndrome

OBJECTIVE: It has been reported previously that the paired interpretation of the corticotrophin-releasing hormone (CRH) test and the 8-mg dexamethasone suppression test (HDDST) could have higher diagnostic power than any single test in the differential diagnosis of ACTH-dependent Cushing's syndrome. This finding has not been confirmed thereafter in large series. The aim of the present study has been to assess the operating characteristics of either the CRH test or the overnight HDDST and also to evaluate the potential utility of combining the interpretation of both tests in the differential diagnosis of ACTH-dependent Cushing's syndrome. DESIGN AND PATIENTS: We have reviewed the medical records of 59 consecutive cases with ACTH-dependent Cushing's syndrome: 49 patients with proven Cushing's disease (CD) and 10 patients with proven ectopic ACTH syndrome (EAS). Univariate curves of the receiver operating characteristics (ROC) have been performed to define the best cut-off values, the sensitivity and the specificity for CRH and overnight HDDST. A comparison between the areas under the ROC curves has also been performed. RESULTS: For the CRH test, the point on the ROC curve closest to 1 corresponded to a value of ACTH percentage increment of 50%[sensitivity 86% (72.6-94.8) and specificity 90% (55.5-98.3)]. The best threshold for cortisol percentage (30%) increment gave inferior results [sensitivity 61% (45.5-75.6) and specificity 70% (34.8-93.0)]. For the HDDST, the point on the ROC curve closest to 1 corresponded to a value of cortisol decrease from the baseline of 50%[sensitivity 77% (62.7-88.5), specificity 60% (26.4-87.6)]. The area under the ROC curve of the ACTH percentage increment after CRH was significantly greater than the area under the diagonal [0.9 (0.7-1.0), P= 0.0001]. Conversely, the area under the cortisol percentage decrement after dexamethasone was not different from that obtained by chance [0.7 (0.5-0.9), P= ns]. The area under the ROC curve of CRH is significantly greater than that of overnight HDDST (P = 0.03). A correct diagnosis has been achieved by the CRH test in 86.5% of cases and by the HDDST in 73% (P = 0.06). The combination of both tests has given a correct diagnosis in a significantly lower percentage of cases than the CRH test alone (69%, P= 0.04). The bilateral inferior petrosal sinus sampling (BIPSS) has been performed in 29 patients (24 CD, five EAS) who had negative imaging and/or discordant results of the noninvasive tests. Considering the criterion of a central to peripheral ACTH ratio > 3 after CRH stimulation, a correct diagnosis was achieved in all cases. CONCLUSIONS: The present data suggest that the CRH is likely to be the most reliable noninvasive diagnostic procedure for the differential diagnosis of the ACTH-dependent Cushing's syndrome. The criterion for a diagnosis of EAS is an ACTH percentage increment lower than 50%. The use of a combination of tests is not recommended because it does not add valuable information and may even impair the outcome of the CRH test. Cases with discordant results in pituitary imaging and CRH test should undergo BIPSS. The validity of this approach, which is straightforward and easily applicable in clinical practice, should be verified in larger series.     

Effect of two prostaglandin synthesis inhibitors, indomethacin and acetylsalicylic acid, on plasma ACTH and cortisol levels in man.

The aim of this study was to investigate the possible role of prostaglandins (PG) in the control of the hypohtalamic-pituitary-adrenocortical axis in normal volunteers. Acute oral administration of 100 mg indomethacin (ID) or 1.5 g acetylsalicylic acid (ASA) did not alter ACTH and cortisol plasma levels. Administration of 300 mg daily ID for 4 days delayed the onset, but increased the magnitude, of the response of ACTH to insulin hypoglycaemia, while it blunted the cortisol response. Administration of 3.2 g ASA daily depressed ACTH response to hypoglycaemia leaving the cortisol response unchanged, except for a 15 min delay in onset. These results are interpreted assuming that ID and ASA chiefly acted at the pituitary and hypothalamic level, respectively, and that ID, but not ASA, interfered with adrenocortical cortisol production. Our findings support the concept, based on animal studies, that PG enhance hypothalamic CRF release and adrenocortical steroidogenesis and may restrain ACTH secretion in the pituitary.     

Regulation of 15-hydroxy prostaglandin dehydrogenase by corticotrophin-releasing hormone through a calcium-dependent pathway in human chorion trophoblast cells

Prostaglandins (PGs) play a crucial role in mediating parturition events, and their synthesis and metabolism are regulated by PG H synthase and 15-hydroxy-PG dehydrogenase (PGDH), respectively. Within the chorion tissue, it is the actions of PGDH that predominate. Throughout gestation, the fetal membranes secrete increasing amounts of CRH. We hypothesized that CRH, produced locally in the chorion, could act to modulate PGDH activity throughout gestation. To investigate this, we obtained Percoll-purified human chorion and placental trophoblast cells from uncomplicated term pregnancies and cultured them for 72 h. Activity of PGDH was assessed by incubation (4 h) with PGF(2alpha) (282 nM) and measurement of conversion to 13,14-dihydro-15-keto PG F(2alpha). Dose-response curves were constructed for the chorion cell cultures with CRH or 8-bromo-cAMP. To investigate the role of CRH and calcium, cells were treated with either astressin, a CRH antibody, BAPTA, or EGTA. CRH (0-1 micro M) had no effect on PGDH activity; however, cells treated with astressin (10 micro M), with or without exogenous CRH (1 micro M), and cells treated with a CRH antibody showed a significant decrease in PGDH activity. 8-Bromo-cAMP (0-1 mM) had no effect on 13,14-dihydro-15-keto PG F(2alpha) output in chorion trophoblast cells but significantly decreased output from placental trophoblast cells. Cells treated with either BAPTA-AM or EGTA had significantly reduced PGDH activity; and, at intermediate concentrations of chelator, exogenous CRH restored PGDH activity. We suggest that, in chorion trophoblast cells, endogenously produced CRH exerts a tonic stimulatory effect on PGDH activity and may help maintain a metabolic barrier, preventing the transfer of bioactive PGs from the chorioamnion to the myometrium.     

Determination of renin, angiotensin converting enzyme and angiotensin II levels in human placenta, chorion and amnion from women with pregnancy induced hypertension

OBJECTIVE  Pregnancy induced hypertension has been shown to be associated with a normal or low activity of the maternal circulating renin–angiotensin system (RAS) but little is known of the local RAS in placenta and fetal membranes. The present study attempts to determine, at full term of human preeclamptic pregnancies, the activity of the chorioplacental renin–angiotensin system.
PATIENTS AND MEASUREMENTS  We analysed the concentrations of active renin, prorenin, angiotensin converting enzyme (ACE) and angiotensin II in homogenates of human placenta and fetal membranes from preeclamptic patients at full term pregnancy. The values of renin, ACE and angiotensin II found in the patients with moderate preeclampsia (gestosis index 0–1) ( n  = 10) were compared with those of normal pregnant women ( n  = 8).
RESULTS  Our experiments showed that in preeclamptic pregnancies, the chorion membrane contained the highest concentrations of active renin (2905 ± 152 pg/g, mean ± SD), prorenin (21 315 ± 2849 pg/g) and ACE (1258 ± 302 U/g) whereas the placenta had more angiotensin II than the chorion and amnion (741 ± 45 vs 456 ± 40 and 428 ± 64 pg/g, respectively). In the placenta, as in the fetal membranes, no significant difference was found in the levels of active renin, ACE or angiotensin II between hypertensive patients and normal subjects but a slightly lower level of chorionic prorenin ( P  < 0.05) was observed in pregnancy induced hypertension.
CONCLUSION  These findings indicate that in moderate preeclampsia (gestosis index 0–1), the activity of the renin–angiotensin system in term human placenta and fetal membranes remains essentially normal.     

Immunohistochemical localization of relaxin, prolactin and prostaglandin synthase in human amnion, chorion and decidua

Relaxin, prolactin and prostaglandin synthase were localized by the avidin-biotin immunoglucose oxidase method in human amnion, chorion and decidua. Specimens from ten normal spontaneous deliveries and four elective Caesarean section deliveries with no labour were compared. Relaxin was found more consistently in the cells of the chorionic cytotrophoblast than in the cells of the parietal decidua adherent to the fetal membranes. Only half the tissues after spontaneous delivery contained positive relaxin-stained cells, whereas all the tissues from elective Caesarean sections contained cells positively stained with antiserum to relaxin. In both series of tissues prolactin was localized predominantly in the parietal decidual cells and was very infrequently found in the chorionic cytotrophoblast. Polyclonal antiserum to prostaglandin synthase was used to identify those cells producing prostaglandin in amnion, chorion and decidua. The cells of the amnion and chorion showed positive immunolocalization with no differences between tissues collected before or after labour. Double immunostaining using avidin-biotin immunoperoxidase for prolactin, followed by avidin-biotin immunoglucose oxidase for prostaglandin synthase, produced identical results in the same series of tissues examined with the single-staining method.     

Circulating immunoreactive somatostatin in man. Effect of age, pregnancy, growth hormone deficiency and achlorhydria

Plasma immunoreactive somatostatin (IRS) levels were measured fasting at 09.00 h in groups of adult individuals and children of different ages, as well as in pregnant women, in patients with pernicious anaemia documented to be achlorhydric, and in children with growth hormone deficiency. There was a gradual rise in the mean level of IRS from the third decade (mean 35.8 +/- 3.8 pg/ml), which reached significance at the seventh (61.1 +/- 8.4 pg/ml), eighth (66.7 +/- 5 pg/ml) and ninth decade (82.6 +/- 13.8 pg/ml). No change was observed in the second 28.3 +/- 3.8 pg/ml) and third (31.1 +/- 3.2 pg/ml) trimester of pregnancy when compared with matched, non-pregnant controls (29.7 +/- 2.2 pg/ml); however, the children aged under 2 years (69.6 +/- 11.2 pg/ml) had significantly higher values than the eldest group (12 to 16 years old) (46.3 +/- 7.2 pg/ml) (P less than 0.05). In achlorhydric patients, basal (27.2 +/- 3.7 pg/ml; P less than 0.01) and postprandial IRS (42.8 +/- 7.7 pg/ml; P less than 0.001) was significantly lower than in a matched, normal control group (basal 59.4 +/- 7.2; postprandial 132.1 +/- 26.3 pg/ml). Growth hormone deficiency was not associated with any differences in circulating IRS, basally or after insulin hypoglycaemia, when compared with values in normal children.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor binding to human amnion, chorion, decidua, and placenta from mid- and term pregnancy: quantitative light microscopic autoradiographic studies

All of the cell types, but not noncellular elements, found in amnion, chorion, decidua, and placenta of mid- and term pregnancy contained numerous silver grains after incubation with 1 nM [125I]epidermal growth factor ([125I]EGF). These grains completely disappeared when excess unlabeled EGF, but not unlabeled insulin, was added with [125I]EGF, suggesting the presence of specific EGF receptors. Light cells of amnion contained more EGF receptors than dark cells of amnion, and the number of light cells decreased from mid- to term pregnancy. Numerous syncytiotrophoblasts containing a greater number of receptors than darkly stained cells in connective tissue were found in chorion from midpregnancy. The chorionic syncytiotrophoblasts were considerably reduced at term. Dark cells of decidua contained more receptors than light cells of decidua, and these cell numbers did not change during pregnancy. Placental syncytiotrophoblasts contained the EGF receptors. At mid- and term pregnancy, placenta contained the highest number of EGF receptors, followed by chorion greater than decidua greater than amnion. The receptor number in all tissues was higher at mid-compared to term pregnancy. A decrease in number of cells as well as a decrease or fewer receptors per cell during pregnancy may explain the tissue and mid- vs. term pregnancy differences. The higher number of EGF receptors in amnion, chorion, decidua, and placenta at midpregnancy, at a time when the maternal and fetal blood EGF levels are at their peak, suggests that EGF may exert maximal biological effects on the feto-placental unit at midpregnancy.     

Characteristics of the ACTH response to repeated pulses of corticotrophin-releasing factor and arginine vasopressin in vitro

A multi-column perifusion system was used to investigate the dynamics of the dose-response relationships of ACTH release by ovine pituitary cells when stimulated by both corticotrophin-releasing hormone (CRF) and arginine vasopressin (AVP) given alone and in combination. A dose-response relationship was obtained when 10-min pulses were given at 60-min intervals over the range of 0.002-2000 nmol CRF/1 and 1-2000 nmol AVP/1, with a minimum effective concentration of 0.02 nmol CRF/1 or 1 nmol AVP/1. When AVP was given together with CRF, the expected potentiation of the ACTH response occurred when compared with the summed response of these secretagogues given separately. At the higher concentrations of CRF and AVP used, the ACTH responses to repeated pulses decreased with time during the experiment. The rate of this loss of responsiveness was significantly correlated to the size of the response to the first pulse (for CRF: r = 0.89, P less than 0.01; for AVP: r = 0.95, P less than 0.01), being greatest when the response was potentiated by adding the secretagogues together (for CRF plus AVP: r = 0.95, P less than 0.01). Reduced availability of receptors or changes in intracellular transduction processes may contribute to this desensitization. Reduced levels of secretable ACTH do not appear to be implicated because desensitization to pulses of one secretagogue did not cause equivalent desensitization to the other. In addition, cells stimulated continuously with submaximal levels of either secretagogue showed desensitization while more ACTH was still available for release to higher levels of stimulant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the corticotrophin-releasing activity of vasopressin, using ACTH secretion by cultured rat adenohypophyseal cells.

CRF activity of synthetic vasopressins and pitressin was studied in an in vitro system of cultured rat adenohypophyseal cells using direct measurement of ACTH by radioimmunoassay. Pitressin (posterior pituitary extract) induced a dose-related secretion of ACTH whereas synthetic arginine or lysine vasopressin were devoid of CRF activity, even with the largest tested dose (4 mug/ml). No potentiation of the CRF activity of hypothalamic extract was observed with any vasopressin preparation studied. We concluded that: 1) the CRF activity of posterior pituitary extract is not due to vasopressin, and 2) the ACTH secretion induced by vasopressin administration in vivo is unlikely to be due to a direct effect of vasopressin on adenohypophyseal cells.