IGF-I bioactivity better reflects growth hormone deficiency than total IGF-I.

CONTEXT: GH is considered the main regulator of circulating IGF-I. Total (extractable) IGF-I is therefore routinely used for diagnosis of GH deficiency (GHD) and for monitoring treatment. Methods currently used for measurement of circulating total IGF-I may be hampered by interferences of IGF-binding proteins. Recently a kinase receptor activation assay was developed to determine IGF-I bioactivity in human serum. The principle of this assay is based on quantification of IGF-I receptor activation after stimulation with serum in vitro. OBJECTIVE: The objective of the study was to investigate the diagnostic potential of IGF-I bioactivity in adults with GHD. DESIGN: This was a single-center observational study. STUDY PARTICIPANTS: Ninety-four GH-untreated patients diagnosed with GHD by GH-provocative tests were included. MAIN OUTCOME MEASURES: IGF-I bioactivity (determined by the IGF-I kinase receptor activation assay) and total IGF-I (determined by immunoassay) were measured in fasting blood samples. RESULTS: IGF-I bioactivity was more frequently below the normal range (<-2 sd) in untreated GH-deficient patients than total IGF-I levels (81.9 vs. 61.7%, respectively), especially in patients older than 40 years of age. IGF-I bioactivity decreased with the duration of GHD, whereas total IGF-I did not. With a decreasing number of additional pituitary deficits, total IGF-I levels more frequently remained within the normal range, whereas the percentage below the normal range was high for IGF-I bioactivity, independent of additional deficits. CONCLUSION: Determination of IGF-I bioactivity may offer advantages in the evaluation of adult GHD compared with total IGF-I as bioactivity better reflects GHD as defined by GH stimulation tests, especially in subjects older than 40 years of age.

Expression and functional analysis of dopamine receptor subtype 2 and somatostatin receptor subtypes in canine cushing's disease.

Cushing's disease (CD) is a severe disorder characterized by chronic hypercortisolism due to an ACTH-secreting pituitary adenoma. Transsphenoidal adenomectomy is the treatment of choice in humans with CD, but recurrences occur frequently. Finding an effective and safe medical treatment for CD may improve long-term clinical outcome. The recent demonstration of expression of somatostatin receptor subtypes (mainly sst5) and dopamine receptor subtype 2 (D2) in human corticotroph adenomas offers the possibility for medical treatment of CD with novel somatostatin analogs and dopamine agonists. Investigation of the effects of these drugs is hampered by the low incidence of CD in humans. Interestingly, CD is a frequent disorder in dogs with striking clinical similarities with CD in humans. Therefore, we investigated the expression and functional role of D2 and somatostatin receptors in corticotroph adenoma cells from 13 dogs with active CD that underwent therapeutic hypophysectomy and normal anterior pituitary cells from five dogs. Quantitative RT-PCR and immunohistochemistry revealed that both in CD and normal anterior pituitary, sst2 was the predominant receptor subtype expressed, whereas D2 was modestly expressed and sst5 was expressed only at very low levels. In primary cultures of canine adenomas (n = 7), the sst2-preferring agonist octreotide also showed the strongest ACTH-suppressive effects. In conclusion, canine corticotroph adenomas provide an interesting model to study CD, but differences in somatostatin and dopamine receptor expression between humans and dogs should be taken into account when using dogs with CD as a model to evaluate efficacy of novel somatostatin analogs and dopamine agonists for human CD.

Postoperative evaluation of patients with acromegaly: clinical significance and timing of oral glucose tolerance testing and measurement of (free) insulin-like growth factor I, acid-labile subunit, and growth hormone-binding protein levels.

CONTEXT: It is not exactly known when patients with acromegaly should be evaluated for cure after transsphenoidal adenomectomy (TA). OBJECTIVE: The objective of this study was to define the optimal time point of postoperative evaluation by serial measurements of glucose-suppressed GH levels [oral glucose tolerance test (OGTT)] and the GH-dependent parameters IGF-I, free IGF-I, acid labile subunit (ALS), and GH-binding protein (GHBP). DESIGN: We describe a prospective study with 1-yr follow-up. SETTING: The study was conducted at a university hospital. PATIENTS: Seventeen patients with acromegaly were included in the study. MAIN OUTCOME MEASURES: The main outcome measures were OGTT results at 1, 2, 3, 8, and 12 wk after TA; weekly measured GH, (free) IGF-I, ALS, and GHBP levels up to 12 wk; and total IGF-I levels measured at 52 wk. RESULTS: Postoperatively, nine patients were in remission with an OGTT GH nadir of less than 0.5 microg/liter and normalized IGF-I levels, whereas eight patients had persistent acromegaly. In both cured and noncured patients, OGTT results at 1 wk after TA were highly reproducible over time. In contrast, early postoperative IGF-I levels fluctuated and only stabilized at 12 wk. In all cured patients, free IGF-I levels rapidly normalized within 2 wk after TA (specificity, 100%). Preoperative ALS levels were elevated in all patients and normalized only in the cured patients after TA (specificity, 89%). Preoperative GHBP levels were low and increased from 2 wk after surgery. CONCLUSIONS: We show that in the postoperative evaluation of patients with acromegaly, already 1 wk after surgery, an OGTT using 0.5 microg as the GH nadir cutoff value has a high predictive value for cure, whereas early IGF-I levels show varying patterns toward stabilization. Therefore, IGF-I should be measured as a predictive parameter not within 3 months after surgery. Free IGF-I and ALS levels may have an additional value in the postoperative assessment of disease activity.

Acute stress response in children with meningococcal sepsis: important differences in the growth hormone/insulin-like growth factor I axis between nonsurvivors and survivors.

Septic shock is the most severe clinical manifestation of meningococcal disease and is predominantly seen in children under 5 yr of age. Very limited research has been performed to elucidate the alterations of the GH/IGF-I axis in critically ill children. We evaluated the GH/IGF-I axis and the levels of IGF-binding proteins (IGFBPs), IGFBP-3 protease, glucose, insulin, and cytokines in 27 children with severe septic shock due to meningococcal sepsis during the first 3 d after admission. The median age was 22 months (range, 4-185 months). Eight patients died. Nonsurvivors had extremely high GH levels that were significant different compared with mean GH levels in survivors during a 6-h GH profile (131 vs. 7 mU/liter; P < 0.01). Significant differences were found between nonsurvivors and survivors for the levels of total IGF-I (2.6 vs. 5.6 nmol/liter), free IGF-I (0.003 vs. 0.012 nmol/liter), IGFBP-1 (44.3 vs. 8.9 nmol/liter), IGFBP-3 protease activity (61 vs. 32%), IL-6 (1200 vs. 50 ng/ml), and TNFalpha (34 vs. 5.3 pg/ml; P < 0.01). The pediatric risk of mortality score correlated significantly with levels of IGFBP-1, IGFBP-3 protease activity, IL-6, and TNFalpha (r = +0.45 to +0.69) and with levels of total IGF-I and free IGF-I (r = -0.44 and -0.55, respectively). Follow-up after 48 h in survivors showed an increased number of GH peaks, increased free IGF-I and IGFBP-3 levels, and lower IGFBP-1 levels compared with admission values. GH levels and IGFBP-1 levels were extremely elevated in nonsurvivors, whereas total and free IGF-I levels were markedly decreased and were accompanied by high levels of the cytokines IL-6 and TNFalpha. These values were different from those for the survivors. Based on these findings and literature data a hypothetical model was constructed summarizing our current knowledge and understanding of the various mechanisms.

Interferon-alpha-2a is a potent inhibitor of hormone secretion by cultured human pituitary adenomas.

Interferon-alpha (IFN alpha) may exert direct inhibitory effects on cell proliferation and on the production of different peptide hormones. We investigated the effect of IFN alpha on hormone production by 15 GH-secreting pituitary adenomas, 4 clinically nonfunctioning or gonadotroph pituitary adenomas, and 4 prolactinomas in vitro. In the GH-secreting pituitary adenoma cultures, a short term (72-h) incubation with IFN alpha (50-100 U/mL) significantly inhibited GH secretion in 3 of 7 cases and PRL secretion in 6 of 7 cultures. During prolonged incubation (14 days) with IFN alpha, GH and/or PRL secretion was significantly inhibited in 7 of 8 cultures (GH, 17-78% inhibition; PRL, 39-88% inhibition). In the clinically nonfunctioning or gonadotroph cultures, incubation with IFN alpha resulted in inhibition of the secretion of gonadotropins and/or alpha-subunit in all cases (27-62%), whereas in the prolactinoma cultures PRL secretion was inhibited by IFN alpha in all cases (37-76%). The effect of IFN alpha was additive to the inhibitory effects of the dopamine agonist bromocriptine (10 nmol/L) or the somatostatin analog octreotide (10 nmol/L). The inhibition of hormone secretion by IFN alpha was accompanied by inhibition of the intracellular hormone concentrations. The effect of IFN alpha was dose dependent, with an IC50 for inhibition of hormone secretion of 2.3 +/- 0.3 U/mL (n = 5), which is relatively low compared with the concentrations that are reached in patients treated with IFN alpha for various malignancies. In conclusion, the potent antihormonal effect of IFN alpha on cultured pituitary adenomas suggests that this drug might be of benefit in the treatment of selected patients with secreting pituitary adenomas. As treatment with IFN alpha is associated with considerable adverse reactions, studies with this drug should only be considered in inoperable, invasive aggressive, and dopamine agonist- and/or somatostatin analog-resistant functioning pituitary macroadenomas.

The IGF-I/IGFBP system in congenital partial lipodystrophy.

BACKGROUND AND OBJECTIVES: Insulin and IGF-I interact at many levels. Little is known about the insulin-like growth factor-I/insulin-like growth factor binding proteins (IGF-I/IGFBP) system in congenital partial lipodystrophy, a syndrome characterized by insulin resistance, hyperinsulinaemia and absence of truncal and limb fat. Some cases have acromegaloid features with thick skin and large hands and feet in association with normal levels of circulating growth hormone. METHODS: In four females known with congenital partial lipodystrophy, hyperinsulinaemia with acromegaloid features, the number and affinity of the IGF-I receptors on peripheral blood mononuclear cells (PBMCs), and the concentration of circulating insulin, total and free IGF-I, IGFBP-1 and IGFBP-3 levels were measured in the fasting and the fed state. Cultures of PBMCs of the patients with lipodystrophy were also used to study the effect of IGF-I stimulation on thymidine uptake in vitro. MEASUREMENTS: In the subjects with lipodystrophy the affinity and the number of the IGF-I receptors on peripheral mononuclear cells (PBMCs) and erythrocytes did not differ significantly from controls in the fasting state. Insulin levels were significantly higher in subjects with lipodystrophy both in the fasting as well in the fed state. Total IGF-I, free IGF-I and IGFBP-3 levels did not differ but serum IGFBP-1 levels were lower in lipodystrophy subjects than in healthy controls. The free IGF-I/IGFBP-1 ratio was increased in lipodystrophy subjects both in the fasting and the fed states. The effects of IGF-I stimulation on thymidine uptake by PBMCs of lipodystrophy subjects in the absence of IGFBP-1 were not different from healthy controls cultures in vitro. When a combination of IGFBP-1 (in a concentration comparable to the fasting serum IGFBP-1 levels in lipodystrophy patients found in our study) and IGF-I was added to PBMC cultures from lipodystrophy patients no decrease in thymidine uptake by PBMCs was found. CONCLUSIONS: In the four subjects with lipodystrophy hyperinsulinaemia, lowered free IGF-I and IGFBP-1 levels, but increased free IGF-I/IGBP-1 ratios were observed. Low IGFBP-1 concentrations in culture media did not reduce the stimulating IGF-I effect on thymidine uptake by PBMCs from lipodystrophy patients. Our data suggest that the observed increased IGF-I/IGFBP-1 ratio in lipodystrophy patients contributes to an unopposed biological effect of IGF-I on IGF-I receptors, thereby inducing the development of acromegaloid features, acanthosis nigricans and polycystic ovaries in some patients with congenital partial lipodystrophy.

Insulin-like growth factor I receptors on blood cells: their relationship to circulating total and "free" IGF-I, IGFBP-1, IGFBP-3 and insulin levels in healthy subjects.

The relationships between the insulin-like growth factor I/insulin-like growth factor binding protein (IGF-I/IGFBP) system and the IGF-I receptor characteristics on erythrocytes and PBMCs in healthy subjects in the fasting state were studied to establish whether this would be a valid way of examining IGF-I receptors in vivo. The K(d) of the IGF-I receptor on erythrocytes was positively related to circulating "free" IGF-I levels. For the IGF-I receptor on PBMCs no relationship was observed with "free" IGF-I levels. IGFBP-3 levels were inversely related to the number of IGF-I binding sites on erythrocytes and to the K(d) of the IGF-I receptor on PBMCs. Total IGF-I, insulin and IGFBP-1 levels showed no relation to the IGF-I receptor on erythrocytes and PBMCs in the fasting state. This report suggests that studies of IGF-I receptor characteristics on erythrocytes and PBMCs in the fasting state are cell-specific and cannot be extrapolated to other cell types, which may be more relevant target tissues for IGF-I action in vivo.

Dissociation between the effects of somatostatin (SS) and octapeptide SS-analogs on hormone release in a small subgroup of pituitary- and islet cell tumors.

The effects of somatostatin (SS-14 and/or SS-28) and of the three octapeptide SS-analogs that are available for clinical use (octreotide, BIM-23014 and RC-160) on hormone release by primary cultures of 15 clinically nonfunctioning pituitary adenomas (NFA), 7 prolactinomas, and 2 insulinomas were investigated. In the pituitary adenoma cultures, a comparison was made with the effects of the dopamine (DA) agonists bromocriptine and/or quinagolide. In 5 NFAs, 2 prolactinomas and 1 insulinoma somatostatin receptor (subtype) expression was determined by ligand binding studies and by in situ hybridization to detect sst1, sst2, and sst3 messenger RNAs (mRNAs). Four NFA cultures did not secrete detectable amounts of alpha-subunit, FSH, and/or LH. In the other cultures, hormone and/or subunit release was inhibited by DA-agonists (10 nM) in 9 of 11, by SS (10 nM) in 7 of 11, and by octapeptide SS-analogs (10 nM) in 3 of 10 cultures. In three NFA cultures, hormone release was sensitive to SS but not to SS-analogs. In all cultures, except for one, DA-agonists were the most effective in inhibiting hormone release. In the prolactinoma cultures, PRL release was inhibited by DA-agonists (10 nM) in 7 of 7, by SS in 4 of 4, and by octapeptide SS-analogs in 3 of 7 cultures. A dissociation between the effects of SS and SS-analogs was found in 3 cases. In the cultures sensitive to both bromocriptine and SS-28, bromocriptine was the most potent compound in 2 out of 4 cultures. In the 2 other cultures, both compounds were equally effective. In 2 insulinoma cultures, insulin release was inhibited by SS, and by octapeptide SS-analogs in only one. The presence or absence of an inhibitory effect by octreotide was in all cases in parallel with the presence or absence of the inhibitory effect by BIM-23014 and RC-160. Autoradiographic studies using [125I-Tyr0]SS28 showed specific binding in 4 of 5 NFAs, 1 of 2 prolactinomas, and 1 of 1 insulinoma. Specific [125I-Tyr3]octreotide binding was found in 2 of 5 NFAs, in 1 of 2 prolactinomas, and in the insulinoma. Two NFAs showed binding of SS28, but not of the sst2.5 specific ligand octreotide. The tumors showed variable sst1 and/or sst3 mRNA expression, whereas no sst2 expression was found. In conclusion, a dissociation between the inhibitory effects of SS on the one hand and of the octapeptide SS-analogs octreotide, BIM-23014 and RC-160 on the other hand, is observed in a small subgroup of NFAs, prolactinomas, and insulinomas, suggesting that novel sst subtype specific SS-analogs might be of benefit in the treatment of selected patients with somatostatin receptor positive secreting tumors not responding to octapeptide SS-analogs. However, in the majority of NFAs and prolactinomas, DA-agonists were equally or more effective than SS in the suppression of tumoral secretion products.

17-beta-estradiol-dependent regulation of somatostatin receptor subtype expression in the 7315b prolactin secreting rat pituitary tumor in vitro and in vivo.

UNLABELLED: In the present study, we have investigated the role of estrogens in the regulation of somatostatin receptor subtype (sst) expression in 7315b PRL-secreting rat pituitary tumor cells in vitro and in vivo. sst were undetectable in freshly dispersed cells of the transplantable 7315b tumor. When 7315b cells were cultured in medium containing 10% FCS, the number of high affinity sst increased with prolonged culture time. However, when the medium was supplemented with 10% horse serum (HS) instead of FCS, no sst were detectable on 7315b cells even after three weeks of culturing. In contrast to HS, FCS contains high E2-levels (HS, 8 pM; FCS, 134 pM). The antiestrogen tamoxifen (0.5 microM) significantly inhibited the sst number to 50.5% of the value of untreated FCS-grown cells, suggesting that E2 stimulates sst expression in 7315b rat pituitary tumor cells. E2 (10 nM) induced a rapid increase in sst number in HS-grown 7315b cells. Octreotide (1 microM) significantly inhibited PRL release and the intracellular PRL concentration of 7315b cells that were cultured in medium supplemented with FCS or with HS + 10 nM E2 but not in HS alone. This indicates that the sst present on these cells are biologically active. RT-PCR analysis revealed that none of the five currently known sst subtypes were present in freshly dispersed 7315b pituitary tumor cells. The expression of sst2- and sst3-messenger RNA (mRNA) was unequivocally correlated to the presence of E2 because these sst subtypes were detected only in cells that were cultured for 7 and 14 days in medium supplemented with FCS or with HS + 10 nM E2. sst1, sst4 and sst5 messenger RNA could not be detected. The 7315b tumor itself synthesizes and secretes huge amounts of PRL. The high PRL levels in tumor-bearing rats inhibit the ovarian E2-production. No detectable E2 levels could be measured in the serum of 7315b tumor-bearing rats. The sc administration of 20 micrograms/day E2-benzoate normalized the circulating E2 levels in 7315b tumor-bearing rats. Moreover, E2-treatment indeed induced sst expression in vivo as shown by ligand binding studies using membrane homogenates and [125I-Tyr3]-octreotide as radioligand and by autoradiography on tissue sections. In agreement with the in vitro studies, the expression of the sst2 subtype was established by RT-PCR analysis in 7315b tumors of E2-treated rats. However, in contrast to the in vitro studies, E2-treatment did not effectuate the expression of the sst3 subtype, suggesting that the in vitro stimulus of E2 is stronger. IN CONCLUSION: 1) sst2 and sst3 expression in the 7315b rat prolactinoma model is primarily dependent upon the presence of estrogens; 2) the antihormonal action of octreotide in 7315b tumor cells in vitro is mediated via the sst2 and/or sst3 subtypes; 3) the absence of sst expression in vivo can be explained by the hormonal environment of the 7315b tumor cells. The 7315b tumor cells in vivo may down regulate their own receptor status via their host, because of the ensuring hyperprolactinemia results in a hypo-estrogenic state.

Food-dependent Cushing's syndrome resulting from abundant expression of gastric inhibitory polypeptide receptors in adrenal adenoma cells.

We studied a 45-yr-old woman with food-dependent Cushing's syndrome. Plasma cortisol levels were subnormal (4-47 nmol/L) after an overnight fast and increased after a mixed meal to values between 500-1000 nmol/L. There was a close correlation between circulating gastric inhibitory polypeptide (GIP) and cortisol levels during normal food intake (r = 0.92; P < 0.0002). Plasma corticotropin (ACTH) levels were undetectable. Nonfasting plasma cortisol levels were not suppressed by low or high doses of dexamethasone. Plasma ACTH and cortisol levels did not increase after human CRH administration, but fasting plasma cortisol levels increased after ACTH treatment. The infusion of GIP increased plasma cortisol levels to 7.8 times above baseline. Radiological and cholesterol uptake studies pointed to a unilateral adrenal adenoma. Treatment with octreotide initially prevented the meal-induced increases in cortisol and GIP levels and decreased urinary cortisol excretion. Unilateral adrenalectomy was performed. Cortisol production by cultured adrenal adenoma cells from the patient was stimulated by GIP and ACTH. In situ hybridization studies using a GIP receptor probe showed an abundant expression of GIP receptor messenger ribonucleic acid in the adrenocortical adenoma. We conclude that food-dependent Cushing's syndrome results from the expression of GIP receptors on adrenocortical adenoma cells.

IGFBP-3 is a poor parameter for assessment of clinical activity in acromegaly.

OBJECTIVE: Elevated serum IGF-I and IGF binding protein-3 (IGFBP-3) levels have been found in patients with active acromegaly. We have studied the relative diagnostic merits of measurements of IGFBP-3 compared with IGF-I as a parameter of disease activity in these patients. DESIGN/PATIENTS: Thirty untreated patients with acromegaly were compared with 30 healthy adults. MEASUREMENTS: Twenty-four-hour sampling for serum GH in patients with acromegaly, serum IGF-I and IGFBP-3. RESULTS: Mean IGF-I levels were 22.0 nmol/l (range 6.5-38.4) in the healthy adults and 118.7 nmol/l (range 67.7-206.0) in patients with acromegaly. Mean IGFBP-3 levels were 3.5 mg/l (range 2.1-4.8) in controls and 5.4 mg/l (range 4.2-6.6) in patients with acromegaly. Mean IGF-I/IGFBP-3 ratios were 6.5 nmol/mg (range 1.9-14.5) in the healthy adults and 22.0 nmol/mg (range 14.3-32.7) in patients with acromegaly. There was a considerable overlap for IGFBP-3 levels but not for IGF-I levels, between normals and acromegalics. The IGF-I/IGFBP-3 ratio also showed overlap between normals and acromegalics. There was a significant correlation between the mean 24-hour GH and IGFBP-3 levels (P = 0.036) and between the IGF-I and IGFBP-3 levels (P < 0.002) in acromegaly. In patients with acromegaly, the IGFBP-3 levels showed a decrement, but the IGF-I/IGFBP-3 ratio did not change significantly with age. CONCLUSIONS: IGFBP-2 has no additional discriminatory value over IGF-1 measurements for the assessment of clinical activity in acromegaly. In acromegaly, IGFBP-3 decreases with increasing age. In acromegaly, IGFBP-3 levels significantly correlate with mean 24-hour GH levels and IGF-I levels.

Octreotide, but not bromocriptine, increases circulating insulin-like growth factor binding protein 1 levels in acromegaly.

Twenty-three patients with active acromegaly underwent serum sampling for growth hormone (GH), insulin and insulin-like growth factor binding protein 1 (IGFBP-1) after placebo or single doses of octreotide or bromocriptine. Integrated 24-h serum GH levels decreased by 90% after octreotide and 49% after bromocriptine. A statistically significant correlation between the course of GH levels after octreotide and bromocriptine was observed (p < 0.001). Octreotide, but not bromocriptine, induced a significant increase in integrated 24-h serum IGFBP-1 levels to 37.4 times the baseline values. Bromocriptine caused a non-significant increase in integrated 24-h serum IGFBP-1 levels, which argues against a direct regulatory effect of GH on IGFBP-1 production in acromegaly. In conclusion, octreotide induces in acromegaly the production of IGFBP-1, which occurs independently of the number of somatostatin receptors on the GH-secreting pituitary adenoma. The supposed inhibitory effect of IGFBP-1 on the biological effect of IGF-1 might result in an additional clinical benefit in acromegalic patients as compared to treatment directed at the pituitary level.

Pituitary tumour localization in patients with Cushing's disease by magnetic resonance imaging. Is there a place for petrosal sinus sampling?

OBJECTIVE: We wished to analyse the relative value and diagnostic accuracy of bilateral simultaneous inferior petrosal sinus blood sampling for plasma ACTH measurements when compared with pituitary magnetic resonance imaging (MRI) for the preoperative localization of microadenoma (tumour diameter < 10 mm) within the pituitary fossa in patients with Cushing's disease. DESIGN: Pituitary MRIs were assessed blind and independently. The sinus blood sampling was performed before and after administration of corticotrophin releasing hormone (CRH). The ratios of the ACTH concentrations in plasma samples from the inferior petrosal sinuses to the concentrations in peripheral blood plasma samples (the IPS:P ratio) and the ratios of the ACTH concentrations in samples from both inferior petrosal sinuses (the intersinus gradient) were calculated. PATIENTS: Twenty consecutive patients with Cushing's disease were prospectively studied. All but two patients subsequently underwent transsphenoidal exploration of the pituitary fossa. RESULTS: In three of 20 patients (15%), positioning of catheter tips in both inferior petrosal sinuses was unsuccessful. The diagnosis of Cushing's disease was confirmed by the greater basal IPS:P ratio amounting to > or = 2.0 in 13 of 17 patients (76%), and amounting to > or = 3.0 in CRH-stimulated peak samples in 15 of 17 patients (88%). Anatomical variations of the inferior petrosal sinus, precluding reliable conclusions about lateralization of pituitary venous ACTH drainage, were observed in five of 20 patients (25%). Adding the three patients with technical failure and one patient who presented with a macroadenoma (tumour diameter 11 mm), this left interpretable data with regard to lateralization of the microadenomas in only 11 of 20 patients (55%). In 15 of 20 patients (75%) a pituitary microadenoma was found at MRI. In 14 of these 15 patients (93%) a tumour was indeed found at that position at subsequent transsphenoidal operation. Concordance between the lateralization by the intersinus gradient and microadenoma localization by MRI was observed in six of 11 cases (55%) when using basal samples and in seven of 11 cases (64%) when using peak samples obtained after stimulation with CRH. Concordance between the lateralization by the intersinus gradient and subsequent microadenoma localization at surgery was observed in seven of 11 patients (64%) before and in eight of 11 cases (73%) after CRH stimulation. Reversal of the intersinus gradient after CRH stimulation, suggesting a shift in the lateralization to the contralateral side of the gland, was found in three of 12 cases (25%). CONCLUSIONS: Bilateral simultaneous inferior petrosal sinus blood sampling for plasma ACTH measurements before and after CRH stimulation successfully confirmed the diagnosis of pituitary dependent Cushing's disease in 15 of 17 patients (88%) in whom this diagnosis was suspected on the basis of conventional biochemical testing. Magnetic resonance imaging, however, is superior to bilateral simultaneous inferior petrosal sinus blood sampling for the localization/lateralization of pituitary microadenomas in patients with Cushing's disease. Therefore, bilateral simultaneous inferior petrosal sinus blood sampling should be reserved for the assessment of those patients with Cushing's syndrome in whom either the results of biochemical tests are equivocal and/or subsequent pituitary magnetic resonance imaging gives unconvincing results.

Caffeine enhances the speed of the recovery of the hypothalamo-pituitary-adrenocortical axis after chronic prednisolone administration in the rat.

Chronic administration of corticosteroids results in a suppression of the hypothalamo-pituitary-adrenocortical (HPA) axis. The time course of the recovery of the HPA axis depends on the dose and duration of corticosteroid administration. We investigated the recovery of the HPA axis after 14 days of prednisolone administration to rats at a dose of 2.0 mg/rat/day via the drinking water (188 mumol/l). The in vitro corticosterone production by dispersed adrenal cells in response to increasing concentrations of ACTH had recovered 3 days after stopping prednisolone administration. In parallel the initially suppressed plasma corticosterone concentrations had recovered after 3 days, while the pituitary ACTH content had recovered after 5 days. We investigated the possibility to enhance the speed of the recovery of the HPA axis by the simultaneous administration of two drugs with known CRF-stimulating activity via the drinking water. Caffeine in a dose of 100 mg/kg body weight enhanced the recovery of the prednisolone-suppressed HPA axis significantly. One day after the end of prednisolone administration a significant increase in the adrenal weight, in the corticosterone production by dispersed adrenal cells, as well as in the plasma corticosterone concentrations, and in the pituitary ACTH content was observed in the caffeine-treated rats. Chlorimipramine (20 mg/kg body weight), on the other hand, did not influence the prednisolone-mediated suppression of the HPA axis.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro and in vivo effects of human growth hormone administration on tumor growth of rats bearing a transplantable rat pituitary tumor (7315b).

The direct effects of human GH and IGF-I on PRL secretion and cell proliferation were studied on PRL secreting rat pituitary tumor 7315b cells in vitro, as well as the effects in vivo of human GH administration on body weight, IGF-I levels and tumor size in rats bearing this transplantable tumor. In the in vitro studies IGF-I levels above 5 nM stimulated PRL release in a dose-dependent manner while GH, in concentrations of 0.23-45 nM, did not affect PRL release. Cell proliferation was stimulated by IGF-I in a dose-dependent manner from 0.5 nM onwards, while GH did not have an effect. The in vivo studies showed that 1 mg GH/rat/day prevented tumor-induced cachexia and normalized the suppressed IGF-I levels without stimulating tumor growth. It is concluded that tumor-induced cachexia can be prevented by exogenous GH administration without an increase in tumor mass, even if a tumor model is used whose cultured tumor cells respond to exposure to IGF-I with a mitotic response.

Suramin prevents ACTH-stimulated corticosterone release by dispersed adrenocortical cells.

Suramin, a polyanionic compound which has been used in the treatment of trypanosomiasis and oncocerciasis, has recently been used in treatment of AIDS, while preliminary success has been reported in the treatment of cancer. However, suramin also causes adrenal insufficiency. The present study was undertaken in order to investigate the effect of suramin on ACTH-stimulated steroid production by dispersed rat adrenal cells. It was shown that suramin at concentrations of 10(-4)-10(-3) M inhibits ACTH-stimulated corticosterone release in a dose-dependent manner IC50 (2.10(-4) M). In addition, suramin caused a parallel decrease in ACTH-stimulated pregnenolone, progesterone and corticosterone release, suggesting that suramin does not affect corticosteroidogenesis via an inhibition of its regulatory enzymes. Suramin at 10(-4) M did not inhibit cholera toxin (10 mg/l)-, forskolin (5 microM)- and dbcAMP (5 mM)-stimulated corticosterone release, while cholera toxin completely overcame the inhibitory effects of very high concentrations of suramin (up till 10(-3) M), on ACTH-stimulated corticosterone release. Finally, chromatographic studies with a matrex gel showed that suramin directly interacted with the ACTH molecule. In conclusion, suramin at "therapeutic" concentrations (10(-4) M and higher) prevents ACTH-stimulated corticosterone release probably via a direct interaction with the ACTH molecule.

Characterization of adrenal autonomy in Cushing's syndrome: a comparison between in vivo and in vitro responsiveness of the adrenal gland.

We measured cortisol and precursor steroid production in response to ACTH, cholera toxin, and forskolin by the dispersed adrenocortical cells prepared from the adrenal glands of 10 patients with different forms of Cushing's syndrome. The cells prepared from the hyperplastic adrenal glands from 4 patients with Cushing's disease responded in a dose-dependent manner to ACTH, cholera toxin, and forskolin. The adrenal cells prepared from 4 encapsulated adrenal adenomas showed no (n = 2), a lowered (n = 1), or a clear (n = 1) response of cortisol release to ACTH. The cells prepared from the adrenal glands of 1 patient with dysplastic micronodular adrenal glands showed a limited response to ACTH, while the cells from an adrenocortical carcinoma, which secreted very little cortisol per cell, were unresponsive to ACTH, cholera toxin, and forskolin. The reaction of the dispersed adrenal cells from these 10 patients to ACTH, cholera toxin, and forskolin showed a close correlation (P less than 0.001 in all instances). This suggests that the defect in autonomous glands is not located at the level of the ACTH receptor, but, rather, involves the adenylate cyclase complex as a whole or its coupling to cAMP-dependent protein kinase. The release into the medium of the cortisol precursors deoxycortisol, 17-hydroxyprogesterone, and progesterone showed that the four autonomous nodules were characterized by a significantly higher deoxycortisol/cortisol ratio in the medium (P less than 0.01), suggesting a relative blockade of 11 beta-hydroxylase in these adrenal adenomas. This was further substantiated in cells from several adrenals by a significant increase in the release of these precursors in response to ACTH in the absence of a cortisol response. We conclude the following. 1) Adrenal adenoma formation in patients with Cushing's syndrome is accompanied by a parallel decrease in the stimulation of the release of steroid hormones in response to ACTH, cholera toxin, and forskolin. This points to a defect in the adenoma cells beyond the ACTH receptor. 2) Adrenal adenoma formation in patients with Cushing's syndrome is accompanied by a relative blockade of 11 beta-hydroxylase activity. 3) By comparing the preoperative dynamic tests of the pituitary-adrenal axis, the plasma ACTH concentration, the morphology of the adrenal glands, and their in vitro responsiveness, a gradual transition from pituitary to (partial) adrenal autonomy could be recognized in several patients.

The new aromatase inhibitor CGS-16949A suppresses aldosterone and cortisol production by human adrenal cells in vitro.

CGS-16949A is a new orally active nonsteroidal aromatase inhibitor which is more than 100-fold more potent than aminoglutethimide. This compound is an imidazole derivative, and therefore, its possible effect on cytochrome P-450-dependent enzyme activities in the adrenal gland was evaluated. In vitro investigations with dispersed normal and hyperplastic human adrenocortical cells showed that CGS-16949A at 10(-7)-10(-6) M is a potent 11 beta-hydroxylase inhibitor, which inhibits ACTH-stimulated cortisol release to a similar extent as an equimolar concentration of metyrapone (IC50 for both compounds, 10(-7)-5 X 10(-7) M). Etomidate was a more potent 11 beta-hydroxylase inhibitor (IC50, approximately 10(-8) M), while 10(-7)-10(-6) M ketoconazole caused (via 17 alpha-hydroxylase inhibition) a similar inhibition of cortisol release as 10(-7) M CGS-16949A (IC50, 10(-7)-5 X 10(-7) M). The 11 beta-hydroxylase inhibition by CGS-16949A was accompanied by a dose-dependent increase in the release of precursor steroids by the adrenocortical cells in vitro, including deoxycortisol, 17-hydroxyprogesterone, and androstenedione. Aldosterone release was suppressed 50% by 10(-9) M CGS-16949A, while the IC50 for cortisol in the same cells was 10(-7) M. Aldosterone release by the dispersed adenoma cells obtained from a patient with primary aldosteronism was also significantly suppressed by CGS-16949A. We concluded that 1) the new nonsteroidal aromatase inhibitor CGS 16949A is an inhibitor of 11 beta-hydroxylase which is equipotent to metyrapone. At present it is unclear whether the compound at the dose that causes complete aromatase inhibition in vivo also affects stress-induced cortisol release in man. 2) CGS-16949A exerts a very potent inhibitory effect on normal aldosterone release (IC50, 10(-9) M) and on tumorous aldosterone secretion. CGS-16949A might, therefore, be a drug that can be used in the treatment of primary hyperaldosteronism.

The effect of the long-acting somatostatin analogue SMS 201-995 on ACTH secretion in Nelson's syndrome and Cushing's disease.

Chronic therapy of a patient with Nelson's syndrome for 2 years with 300 micrograms SMS 201-995 per day resulted in a significant decrease in circulating ACTH levels, normalization of the visual field defect and of loss of visual acuity of one eye, and stabilization of tumour growth, without radiological evidence of shrinkage of the pituitary tumour. In two other patients with Nelson's syndrome, SMS 201-995 acutely inhibited circulating ACTH levels. This effect could be shown best if cortisol replacement was temporarily withheld. SMS 201-995 did not affect plasma ACTH and cortisol levels in three patients with untreated Cushing's disease.

Effects of gonadotropin-releasing hormone and its agonists on prolactin secretion from normal and tumorous pituitary cells.

Previous studies on the effect of gonadotropin-releasing hormone (LHRH) agonist on prolactin (PRL) secretion from normal and tumorous pituitary cells have not been conclusive as to the mechanism of action of these agonists. In this study the short-term administration of a LHRH agonist did not affect circulating PRL levels, but depleted the PRL content of the pituitary gland by 24, 49 and 73% after 2, 3 and 4 days, respectively, in normal female rats and by 75% after 4 days in normal male rats. This effect of the agonist could not be attributed to changes in the sex steroid environment: although plasma 17 beta-estradiol concentrations were significantly suppressed in female rats, circulating testosterone levels had not changed yet in the male rats. Interestingly, the pituitary luteinizing hormone (LH) content was depleted already from day 2 of LHRH agonist administration onwards, while the follicle-stimulating hormone (FSH) content of the pituitary glands had not changed even after 4 days. Culture studies with pituitary cells from normal adult male and female rats for 4-7 days did not reveal a direct effect of synthetic LHRH or an agonist on PRL release. Chronic systemic administration of a LHRH agonist greatly inhibited the growth of the transplantable PRL-secreting rat pituitary tumor 7315a in female rats, while circulating PRL levels were also suppressed. However, no direct effect of the LHRH agonist was observed on PRL release from a tumor cell clone, derived from the 7315a tumor, and no LHRH-binding sites were detectable on the tumor.(ABSTRACT TRUNCATED AT 250 WORDS)