Evaluation of a nonlinear Hertzian-based model reveals prostate cancer cells respond differently to force than normal prostate cells.

Understanding how the mechanical properties of cells alter with disease may help with the development of novel diagnostics and treatment regimes. The emergence of tools such as the atomic force microscope (AFM) has enabled us to physically measure the mechanical properties of cells. However, suitable models for the analysis of real experimental data are either absent, or fail to provide a simple analysis tool in which experimental data can be analyzed quickly and reliably. The Hertz model has been widely used to study AFM data on living cells, however it makes assumptions that are untrue for cells, namely that cells behave as linear elastic bodies. This article presents and evaluates an alternative nonlinear Hertz model, which allows the Young's modulus to vary according to a second order polynomial function of indentation depth. Evaluation of the model revealed that prostate cancer cells (PC3) responded more uniformly to force compared to the normal PNT2 cells. Also, more energy (J) was needed to deform the normal prostate cells compared to the prostate cancer cells. Finally, the model described here suggests that overall the normal prostate cells behave in a more linear fashion to applied force compared to the prostate cancer cells.

Differential regulation of calcitonin secretion in normal and neoplastic pulmonary neuroendocrine cells in vitro.

Within the mammalian lung, cells with a neuroendocrine phenotype are few in number and are sparsely distributed. In contrast, neuroendocrine neoplasms represent a major group of lung cancers. The aim of this study was to develop a model of mammalian PNECs and to compare glucocorticoid regulation of calcitonin secretion in normal and neoplastic cells with neuroendocrine differentiation. Cell cultures of PNECs were initiated after the disaggregation of neonatal hamster lungs with 0.1% collagenase and fractionation of the resultant cell suspension on a gradient of iodixanol (1.320 g/mL). Cell fractions enriched in PNECs were identified by positive staining for 5-hydroxytryptamine and the presence of calcitonin. Calcitonin secretion was investigated after exposure to hydrocortisone (0 to 1,000 nM). A dose-dependant inhibition of calcitonin secretion was seen after 7 days between 10 nM (55% of control), and 1,000 nM (29%) hydrocortisone. Cell cultures grown in the presence of hydrocortisone also contained significantly fewer PNECs between 10 nM (90% of control), and 1,000 nM (45%). Human bronchial carcinoid cells (NCIH727) cultured under identical conditions showed a similar inhibition of calcitonin secretion between 10 nM (53%) and 1,000 nM (52%), although at these concentrations, no reduction in cell number was seen. In contrast, 2 human small cell lung cancer cell lines (DMS-79 and COR-L24 cells) showed no dose-dependent inhibition of calcitonin secretion and no effect on cell proliferation in response to hydrocortisone. These results show that enriched cultures of mammalian PNECs can be used to investigate functional aspects of their biology, including peptide secretion in response to potential regulators. Furthermore, calcitonin secretion is inhibited in normal PNECs and bronchial carcinoid cells at physiological concentrations of glucocorticoids, but this feature appears not to be present in the 2 more invasive neuroendocrine neoplasms (small cell lung cancer cells) investigated in this study.

Impaired processing of proopiomelanocortin in corticotroph macroadenomas.

The regulation and secretion of the ACTH precursors POMC and pro-ACTH were assessed directly using a 2-site immunoradiometric assay in six patients with pituitary macroadenomas (> or = 1.2 cm in diameter) and 27 patients with Cushing's disease due to a microadenoma. ACTH precursor levels were elevated in patients with macroadenomas (150-3690 pmol/L; normal range, < 5-40 pmol/L) and significantly higher than those in microadenoma patients (median, 29 pmol/L; range, 9-104 pmol/L; P < 0.001). Patients with macroadenomas also had increased ACTH precursor/ACTH ratios (15-181:1) compared with microadenoma patients (median, 5:1, range, 0.7-18.5:1; P < 0.001). ACTH precursors were unresponsive to high dose dexamethasone in patients with macroadenomas, whereas ACTH and cortisol responses varied. After CRH administration, ACTH precursors were unchanged, whereas cortisol increased significantly, suggesting the release of biologically active ACTH. This study clearly demonstrates reduced processing of POMC to ACTH in large pituitary tumors, a characteristic usually associated with tumors causing the ectopic ACTH syndrome, and provides evidence for differential regulation of ACTH precursors and ACTH by glucocorticoid and CRH. Variation in the clinical symptoms of patients with corticotroph macroadenomas may be attributable to differences in biological potency between the ACTH precursors and ACTH.

Elevated levels of adrenocorticotropin (ACTH) precursors in post-adrenalectomy Cushing's disease and their regulation by glucocorticoids.

The ACTH precursor pro-opiomelanocortin (POMC) undergoes specific endoproteolytic cleavages in the anterior pituitary gland to give ACTH-(1-39). ACTH precursors circulate in normal subjects and are high both in the ectopic ACTH syndrome and in patients with aggressive pituitary adenomas. This study examines plasma levels of ACTH precursors and ACTH in 24 patients with post-adrenalectomy Cushing's disease, in 10 of whom computed tomography showed evidence of tumor progression. ACTH precursors were higher in post-adrenalectomy Cushing's disease (median 97.5 pmol/L, range 26-647 pmol/L) than in untreated Cushing's disease (median 29 pmol/L, range 9-104 pmol/L) and normal controls (5-40 pmol/L) (P < 0.001) and were significantly higher in patients with larger tumors (median 175 pmol/L, range 52-647 pmol/L) than in the remainder (median 41 pmol/L, range 26-510 pmol/L) (P = 0.02). Surprisingly, pro-opiomelanocortin (POMC) processing to ACTH was enhanced in post-adrenalectomy patients (ratio 1 +/- 0.5) compared with Cushing's disease (5.6 +/- 0.8) and normal subjects (5.3 +/- 0.9). After hydrocortisone ACTH precursors rose in 36% of post-adrenalectomy patients (increase 15-78%) and remained within 10% of basal levels in 46%. Six patients had a rise in precursors and a fall in ACTH suggesting differential regulation of these peptides. In conclusion, whereas ACTH precursors are high in post-adrenalectomy Cushing's disease and higher levels correlate with tumor progression, processing of precursors to ACTH is enhanced.

Biological activity of adrenocorticotropic hormone precursors on ovine adrenal cells.

Adrenocorticotropic hormone (ACTH) is synthesized in the corticotrophs as a precursor, pro-opiomelanocortin (POMC), which is processed via proACTH to ACTH. Both precursors and ACTH are secreted. Although the steroidogenic activity of ACTH is well characterized, that of the precursors is not. This study assessed the capacity of POMC and proACTH to alter cortisol synthesis. POMC and proACTH were prepared by subjecting medium, conditioned by exposure to DMS-79 cells, to Sephadex chromatography, and the bioactivity was assessed in cultured-dissociated ovine adrenal cells. Alone neither POMC (< or = 2.6 nM) nor proACTH (< or = 0.7 nM) showed any consistent acute (6 h) stimulatory or inhibitory action on cortisol in either fetal or adult cells. In contrast, in fetal cells the precursors inhibited steroidogenic response to ACTH-(1-24). POMC at 2.6 nM, but not lower concentrations, decreased the cortisol responses to 0.01, 0.1, and 1 nM ACTH by at least 50%. ProACTH (0.70 and 0.23 nM) decreased the responses to ACTH at 0.01 nM by 89 and 67%, respectively, and at 0.1 nM by 49 and 34%, respectively. At 1 nM ACTH only 0.7 nM proACTH decreased the response to ACTH (by 69%). In contrast, in adult adrenal cells, the precursors did not significantly reduce the response to ACTH (range 0.01-1 nM). Therefore, these data indicate that POMC and proACTH can inhibit the cortisol response to ACTH in fetal adrenal cells, an effect that is concentration dependent. The data suggest that precursors may play a physiological role, possibly regulating fetal plasma cortisol concentrations.

Cerebrospinal fluid levels of beta endorphin in painful and painless diabetic polyneuropathy.

beta endorphin (beta-EP) is an important modulator of central pain pathways. To examine whether changes in central production of beta-EP contribute to the pathogenesis of diabetic neuropathic pain, we compared the cerebrospinal fluid (CSF) levels of beta-EP and its precursor proopiomelanocortin (POMC) between 15 diabetic patients with chronic painful diabetic polyneuropathy, eight patients with severe painless diabetic neuropathy, and ten nondiabetic controls. Both peptides were measured by specific monoclonal antibody-based two-site immunoradiometric assays (IRMAs). In the diabetic patients with painful neuropathy, mean +/- SD CSF beta-EP concentrations (5.7 +/- 2.2 pmol/L) were comparable to those of the diabetic patients with painless neuropathy (6.0 +/- 2.3 pmol/L) and did not correlate with the severity of neuropathic pain. CSF beta-EP, but not POMC, concentrations were lower in the diabetic neuropathic patients overall (5.8 +/- 1.9 pmol/L) compared to the control subjects (7.6 +/- 2.2 pmol/L) (p < 0.05). CSF POMC showed no intergroup differences. However, POMC levels were 80-fold higher than those of beta-EP and should always be considered when interpreting immunoreactive beta-EP or other derivative peptide levels in CSF. We conclude that CSF beta-EP levels appear to be reduced in diabetic polyneuropathy but they do not relate to the presence of neuropathic pain. This might explain why opioid analgesics are of little, if any, help in alleviating diabetic neuropathic pain.

Choice of treatment affects plasma levels of insulin-like growth factor-binding protein-1 in noninsulin-dependent diabetes mellitus.

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) modulates the metabolic and mitogenic effects of IGFs. Although IGFBP-1 levels are abnormally high in insulin-dependent diabetes (IDDM), relatively little is known in NIDDM; conflicting data have suggested both high and low levels. We investigated whether treatment modifies IGFBP-1 levels in two groups of NIDDM patients. Study 1 examined fasting concentrations in groups of patients with NIDDM, comparable except for treatment type (sulfonylurea, n = 23; once daily insulin, n = 15; sulfonylurea plus once daily insulin, n = 14; multiple insulin injections, n = 9) and 25 nondiabetic subjects. In sulfonylurea-treated patients there were markedly reduced plasma IGFBP-1 concentrations (median, interquartile range in parentheses): control, 61.0 (36-96) micrograms/L; sulfonylureas alone, 31.5 (21-61) micrograms/L (P < 0.01); and sulfonylureas plus insulin, 31.5 (9-53) micrograms/L (P < 0.01). Once daily insulin was associated with values similar to those in the control group [62.0 (27-103) micrograms/L; P = NS], whereas IGFBP-1 levels were higher with multiple insulin injection therapy [156.0 (71-184) micrograms/L; P < 0.05]. Proinsulin levels were higher in sulfonylurea-treated patients, but there was no significant correlation between IGFBP-1 and proinsulin within any individual group. Study 2 examined the effects of treatment on the dynamics of IGFBP-1 levels between 0800-1900 h. In control subjects (n = 8), levels fell from 0800 h (mean +/- SEM, 22.4 +/- 5.2 micrograms/L) to 1000 h (14 +/- 5.2 micrograms/L), followed by a rise, more rapid after food, to a peak at 1240 h (20.6 +/- 3.7 micrograms/L). Levels then declined until 1500 h (10.7 +/- 2.9 micrograms/L), with a further postprandial peak at 1840 h (23.1 +/- 3.2 micrograms/L). Sulfonylurea therapy (n = 6) resulted in a complete loss of this pattern, with a marked fall in IGFBP-1 from 0800 h (22 +/- 2.7 micrograms/L) to less than 7 micrograms/L for the remainder of the study (area under the curve, 1150-1400 h, P < 0.001 vs. control). By contrast, in metformin-treated patients (n = 7), neither IGFBP-1 levels nor postprandial peaks were significantly different from those in the control group. Our findings suggest that in patients with NIDDM, the regulation of IGFBP-1 is markedly influenced by the choice of treatment.

Differential release of proopiomelanocortin-derived peptides from the human pituitary: evidence from a panel of two-site immunoradiometric assays.

In humans, proopiomelanocortin (POMC) and the peptides derived from it have been individually identified in plasma under differing conditions. However, direct quantitative comparison has proved difficult because of the limitations of RIAs. Using a panel of monoclonal antibodies recognizing different regions of POMC, we have developed specific two-site immunoradiometric assays (IRMAs) for the ACTH precursors (POMC and pro-ACTH), ACTH, beta-lipotropin (beta LPH), beta-endorphin (beta EP), and the N-terminal POMC fragment (N-POC). We have quantified these peptides directly in plasma from normal subjects under basal conditions and in response to different regulatory factors. Basal levels of ACTH precursors, 5-40 pmol/L, were greater than or equal to ACTH, less than 0.9-11.3 pmol/L; N-POC, 5.6-16.8 pmol/L; beta LPH, 2.5-6.7 pmol/L; and beta EP less than or equal to 1.7 pmol/L. ACTH, N-POC, beta LPH, and beta EP levels increased in parallel in response to metyrapone (n = 8) and decreased in response to dexamethasone (n = 8), whereas ACTH precursor concentrations did not respond. After human CRH administration, peripheral concentrations of ACTH, N-POC, and beta LPH showed similar increments (median increment, 163%, 145%, and 172%, respectively; n = 6). POMC peptide responses to human CRH were also assessed in inferior petrosal sinuses draining the pituitary in 20 patients with pituitary-dependent Cushing's disease. In these patients, the increment in ACTH after CRH exceeded that in ACTH precursors by 4-fold (median, 459% and 96%). An increase in the ratios of ACTH/N-POC and ACTH/beta LPH was also apparent after CRH stimulation. The increment in beta EP after CRH always exceeded the increments in POMC and beta LPH. In summary, these data suggest that significant concentrations of ACTH precursors are present in the circulation of normal subjects, that ACTH precursors are not regulated in the same way as the processed POMC peptides, and that ACTH and beta EP are preferentially released from the pituitary in response to CRH.

Effect of late hypothalamo-pituitary disconnection on the development of the HPA axis in the ovine fetus and the initiation of parturition.

The studies of Liggins et al. (1) in which fetuses stalk-sectioned from day 116 onwards delivered at or near term, suggested that a connection between the fetal hypothalamus and pituitary is not essential for parturition to occur. The objective of this study was to repeat these experiments on the effects of pituitary stalk sections at different gestational ages and include information on the plasma concentrations of key fetal hormones. We have used the more sophisticated technique of hypothalamo-pituitary disconnection (HPD) at either of two gestational age ranges (123-127 days or 133-135 days). Completeness of the procedure was assessed by demonstrating an attenuated prolactin response to chlorpromazine challenge. Following HPD, gestation was prolonged for at least eight days beyond term (146.2 +/- 1.5 days) in 9 of the 10 fetuses operated. Fetal plasma ACTH1-39 concentrations were not different between the HPD and control fetuses, increasing in all groups with increasing gestation. Fetal plasma cortisol concentrations increased (P < 0.01) in control fetuses over gestation. Cortisol concentrations did not change significantly in the day 125 HPD group following HPD but increased in the day 135 HPD group (P < 0.05) with advancing gestation. These latter concentrations, however were markedly less (P < 0.001) than those for control fetuses prior to parturition. Fetal and maternal plasma PGE2 concentrations increased (P < 0.01) in the control group over gestation but did not change following HPD. Maternal plasma progesterone concentrations decreased (P < 0.05) after day 143 in the control group but did not change in the HPD group.(ABSTRACT TRUNCATED AT 250 WORDS)

ACTH precursors characterize the ectopic ACTH syndrome.

OBJECTIVE: ACTH is secreted by the pituitary following processing of larger molecular weight precursors, proopiomelanocortin and pro-ACTH. Ectopic ACTH syndrome refers to the secretion of ACTH by non-pituitary tumours, but the predominant circulating form of proopiomelanocortin-related peptides remains unclear. PATIENTS: Fifteen patients with ectopic ACTH syndrome were compared to 20 patients with pituitary-dependent Cushing's syndrome, 22 patients with small cell lung carcinoma but no evidence of Cushing's syndrome, and 25 controls. DESIGN AND MEASUREMENTS: Measurement of plasma ACTH and ACTH precursors using specific monoclonal-based immunoradiometric assays at 0900 h and, in five patients with ectopic ACTH syndrome, at 15-minute intervals for 6-24 hours. RESULTS: ACTH precursors were grossly elevated in patients with ectopic ACTH syndrome (median 2194, range 139-18000 pmol/l) compared to patients with Cushing's disease (median 33, 8-73 pmol/l, P < 0.001), patients with small cell lung carcinomas (38, 8-117 pmol/l, P < 0.001) and controls (26, 10-39 pmol/l, P < 0.001). ACTH levels were also elevated in ectopic ACTH syndrome (0900 h median 34, 11-152 pmol/l) compared to patients with Cushing's disease (0900 h median 8, 3-19 pmol/l), but not to the same degree as ACTH precursors. In contrast with Cushing's disease, ACTH was secreted in a non-pulsatile fashion. ACTH precursors but not ACTH itself correlated with plasma cortisol in patients with ectopic ACTH syndrome (r = 0.65, P < 0.05). Chromatographic analysis of plasma from a patient with ectopic ACTH syndrome confirmed ACTH precursors and not ACTH to be the predominant circulating form. With the cross-reactivity of proopiomelanocortin and pro-ACTH in the ACTH IRMA of < 1 and < 10% respectively, ACTH precursors could represent all the ACTH immunoreactivity in patients with ectopic ACTH syndrome. CONCLUSIONS: Ectopic 'ACTH' is characterized by aberrant processing of proopiomelanocortin and should be more accurately referred to as 'ectopic ACTH precursor syndrome'.

Discrimination between beta-endorphin and beta-lipotrophin in human plasma using two-site immunoradiometric assays.

OBJECTIVE: We wished to discriminate between the opioid peptide beta-endorphin (beta-EP) and its non-opioid precursor beta-lipotrophin (beta-LPH) in normal subjects and patients with ACTH-related disorders. DESIGN: We produced monoclonal antibodies to beta-EP and beta-LPH for the development of two-site immunoradiometric assays (IRMAs) which specifically quantitate beta-EP and beta-LPH. PATIENTS: Samples were obtained from patients with a range of ACTH-related disorders and compared with 18 normal subjects. Peptide levels were also compared in six patients with Cushing's syndrome undergoing bilateral inferior petrosal sinus sampling with corticotrophin releasing hormone administration. MEASUREMENTS: In the beta-EP IRMA, antibody 6B2, specific for beta-EP 18-27, is radiolabelled and antibody 2E10, recognizing beta-EP 1-5, is coupled to Sephacryl S-300 as solid phase. The IRMA is specific for beta-EP (beta-LPH cross-reacts < 0.02%), has a detection limit of 1.4 +/- 0.7 pmol/l (n = 7) and has a within-assay coefficient of variation of < 10% between 4.9 and 1200 pmol/l. In the beta-LPH IRMA, antibody 6B2, which recognizes an epitope common to beta-LPH and beta-EP, is radiolabelled and paired with solid-phase antibody 5C11 which recognizes beta-LPH 39-56. The binding site of this antibody ensures that beta-EP cannot be measured in the beta-LPH assay. The detection limit is 0.8 +/- 0.1 pmol/l (n = 9) and the within-assay coefficient of variation is < 10% at concentrations 1.7-870 pmol/l. RESULTS: In normal subjects, beta-EP and beta-LPH levels were < 1.4-1.7 pmol/l (< 5-6 ng/l) and 2.5-6.7 pmol/l (29-77 ng/l) at 0930 h and < 1.4-1.7 pmol/l (< 5-6 ng/l) and 1.9-4.5 pmol/l (22-49 ng/l) at 1600 h, respectively. In patients with ACTH-related pathologies concentrations of beta-EP and beta-LPH paralleled those of ACTH. The ratio of beta-LPH:beta-EP in plasma varied between 3.2:1 and 38:1 in these patients demonstrating that beta-LPH is the major circulating peptide derived from the C-terminal of pro-opiomelanocortin in man. However, in two patients undergoing bilateral inferior petrosal sampling with corticotrophin releasing hormone for diagnosis of Cushing's disease beta-EP concentrations increased rapidly during the first 5 minutes of the test, resulting in a sharp decrease in the beta-LPH: beta-EP ratio. These results suggest that beta-EP is preferentially released in response to acute corticotrophin releasing hormone stimulation. CONCLUSIONS: It is concluded that two-site IRMAs for beta-EP and beta-LPH provide an easy approach to study the dynamic changes in processing of beta-LPH to beta-EP.

Measurement of insulin-like growth factor-II in human plasma using a specific monoclonal antibody-based two-site immunoradiometric assay.

An immunoradiometric assay (IRMA) for the measurement of insulin-like growth factor-II (IGF-II) in human plasma has been developed, optimized and evaluated clinically in normal subjects and patients with disorders of the GH/IGF-I axis. Six monoclonal antibodies (MAbs) to recombinant human IGF-II (rhIGF-II) were produced, all of which had low cross-reactivity with rhIGF-I (< 0.01%) and insulin (< 0.01%). Compatibility of pairs of MAbs was tested in two-site IRMAs using three radioiodinated MAbs and three MAbs linked to Sephacryl S-300 (with separation of bound and free radiolabelled MAb by sucrose layering). Seven pairs of MAbs bound rhIGF-II and the combination of 125I-labelled W3D9 and W2H1 linked to solid phase was selected. The optimized assay had a completion time of 4 h, a minimum detection limit of 30 ng/ml (2.5 standard deviations from the zero standard) and detected a single peak of endogenous IGF-II in normal plasma which co-eluted with rhIGF-II after acid gel chromatography. IGF-II was measured in formic acid/acetone extracts of plasma from 16 normal subjects (mean 685, range 516-1008 micrograms/l), four acromegalic patients (mean 637, range 553-700 micrograms/l), fourteen patients with type-1 diabetes (mean 635, range 247-753 micrograms/l), nine patients with uraemia (mean 423, range 78-850 micrograms/l), and three patients with Laron-type GH insensitivity (75, 35 and 36 micrograms/l). No significant fluctuations were detected between samples obtained hourly from 08.00 to 19.00 h in normal subjects. Low levels of IGF-binding proteins (IGFBPs) remaining in plasma extracts may interfere with the measurement of IGF-II and give rise to falsely elevated IGF-II levels in radioimmunoassays or falsely suppressed levels in IRMAs. Such interference did not occur with the IRMA when used to measure IGF-II in extracts from normal subjects, acromegalic patients and patients with type-1 diabetes, and the addition of excess rhIGF-I in order to displace IGF-II from residual IGFBPs had no effect on IGF-II measurements in these samples. However, levels of IGF-II measured in extracts from patients with Laron-type GH insensitivity and patients with uraemia increased markedly after preincubation with excess rhIGF-I. The accurate measurement of IGF-II by IRMA in extracts from these subjects therefore requires the displacement of IGF-II from IGFBPs prior to assay. We conclude that, in contrast to radioimmunoassays, the two-site IRMA developed here provides a practical, rapid and specific method for the measurement of IGF-II in human plasma.

Proopiomelanocortin is the predominant adrenocorticotropin-related peptide in human cerebrospinal fluid.

High mol wt forms of immunoreactive ACTH and beta-endorphin (beta EP) are present in cerebrospinal fluid (CSF). We have quantified these peptides directly in the CSF of 26 patients undergoing routine myelography, using a panel of monoclonal antibody-based two-site immunoradiometric assays, specific for ACTH precursors (both POMC and pro-ACTH cross-react 100%), POMC, ACTH, and beta EP. The mean +/- SD levels of POMC in CSF were 530 +/- 150 pmol/L similar to total ACTH precursor immunoreactivity (414 +/- 83 pmol/L). By comparison, the CSF levels of ACTH (3.2 +/- 0.6 pmol/L) and beta EP (6.7 +/- 2.9 pmol/L) were 100-fold lower. POMC, by virtue of its 1% cross-reactivity in the ACTH immunoradiometric assay, could have also accounted for the ACTH immunoreactivity in CSF. Sephadex G-75 chromatography of CSF confirmed the presence of a single major peak of ACTH precursors eluting at the position of POMC (31K), while ACTH immunoreactivity was not detected at the position of ACTH-(1-39) (4.5K). We also studied the effect of exogenous glucocorticoids on CSF POMC peptides by giving 2.5 mg dexamethasone (0.5 mg, orally, every 6 h for 24 h) to a similar group of age-matched patients before lumbar puncture. No significant differences in CSF peptide content were observed between the two groups. These data suggest that the unprocessed precursor molecule POMC is the predominant peptide of the POMC family in human CSF and should always be considered when interpreting data involving ACTH or other component peptide immunoreactivity in this biological fluid.

Glucocorticoid inhibition of ACTH peptides: small cell lung cancer cell lines are more resistant than pituitary corticotroph adenoma cells.

In the normal pituitary, glucocorticoids are the principal negative regulatory of the pro-opiomelanocortin (POMC) gene which gives rise to the biologically active peptides ACTH and beta-endorphin. In Cushing's syndrome, ACTH-secreting pituitary tumours show a degree of glucocorticoid resistance, whilst ACTH-secreting extra-pituitary tumours have an even greater resistance to glucocorticoid excess. In an attempt to understand the mechanism of this phenomenon, we have compared the effects of glucocorticoids on POMC mRNA and peptide secretion in human and mouse corticotroph adenoma cells and in small cell lung carcinoma (SCLC) cells. ACTH precursor peptides were inhibited within 24 h by 25-50 nM hydrocortisone in primary cultures from a human corticotroph adenoma. In the mouse corticotroph adenoma cell line (AtT20), inhibition of both ACTH precursors and ACTH was not observed after 24 h but, by 10 days, glucocorticoids suppressed peptide levels with a concentration causing 50% inhibition of 50 nM hydrocortisone and maximal inhibition at 500 nM hydrocortisone. In marked contrast, there was no response to 500 nM hydrocortisone in the five SCLC cell lines (COR L103, COR L42, COR L24, COR L31, DMS 79) all of which secrete ACTH precursors. However, two of the five SCLC cell lines (COR L31 and DMS 79) were responsive to 1000 nM hydrocortisone. POMC mRNA, quantitated by slot-blot analysis, gave similar results for the five SCLC cell lines, implying that the abnormality may occur at the level of gene expression. When one of the three resistant cell lines (COR L103) was incubated with 2000 nM hydrocortisone or 2000 nM dexamethasone a clear suppression of precursor peptides and POMC mRNA was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple bioassay for epidermal growth factor using pig thyrocytes.

A bioassay for epidermal growth factor (EGF) is described using an eluted stain assay (ESTA) of dehydrogenase activity in pig thyrocytes. Optimal responsiveness to EGF was obtained in confluent cultures of primary pig thyrocytes cultured with EGF or biological samples containing EGF for either 24 or 48 h. Dehydrogenase activity was determined by measuring the production and then the release of a coloured formazan product produced by reduction of a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide substrate added to the cells. The assay responded equally to mouse, human and recombinant EGF and was suitable for measuring EGF activity in some but not all biological fluids. Specificity of detection of EGF activity was confirmed using antibody to EGF. The ESTA assay compared favourably with the radioreceptor assay for EGF in terms of sensitivity to EGF with half-maximal activation at 0.24 +/- 0.06 nmol/l (mean +/- S.E.M., n = 22 experiments) for the ESTA assay and 0.60 +/- 0.13 nmol/l (n = 7 experiments) for the radioreceptor assay.

Bromocriptine inhibits pro-opiomelanocortin mRNA and ACTH precursor secretion in small cell lung cancer cell lines.

We have previously reported that a human small cell lung cancer (SCLC) cell line (COR L103) that expresses the proopiomelanocortin (POMC) gene and secretes ACTH precursor peptides is relatively resistant to glucocorticoid regulation. Using this model, we have now examined alternative regulatory mechanisms of the POMC gene and found that both the mRNA and ACTH precursor peptides were stimulated four- and two-fold, respectively, after 48 h incubation with db-cAMP. Next, we examined the dopamine agonist, bromocriptine, which acts predominantly through D2 receptors linked to adenyl cyclase to cause a reduction in intracellular cAMP. Bromocriptine suppressed cAMP levels and inhibited precursor peptide secretion within 24 h in a dose-dependent manner (0.15-15 microM). At the highest dose, peptide secretion was inhibited from 95 to 53 pmol/mg protein, and POMC mRNA was reduced by 50%, while beta-actin mRNA remained unchanged. This effect could not be mimicked by incubation of cells with the alpha-adrenergic antagonist, phenoxybenzamine, suggesting that the alpha-adrenergic effects of bromocriptine were not responsible for this observation. These cells also secrete estradiol, but the secretory rate was unaffected by bromocriptine, suggesting, with the beta-actin data, that the POMC inhibition was not a cytotoxic effect. No recovery in precursor peptide secretion was seen in a 48-h period after the removal of bromocriptine. However, when the postchallenge incubation was extended to 8 d, there was a recovery in secretory potential between day 3 and day 8 and normal growth kinetics in the 4 d after removal of the drug. In contrast to these findings, the mouse corticotroph cell line, AtT20, showed no response to bromocriptine, in keeping with reports that this agonist has no effect on anterior lobe corticotrophs. We conclude that bromocriptine effectively inhibits POMC expression in SCLC cells, and that this phenomenon might be of useful clinical application.

Elevated plasma insulin-like growth factor binding protein-1 levels in type 1 (insulin-dependent) diabetic patients with peripheral neuropathy.

Previous studies have suggested that nerve regeneration may be defective in patients with diabetic polyneuropathy. Since insulin-like growth factor I (IGF-I) has been shown to stimulate nerve regeneration, and IGF binding protein-1 is acutely regulated by plasma insulin we have investigated the relationships between plasma IGF-I, IGFBP-1, glucose and insulin in Type 1 (insulin-dependent) diabetic patients with peripheral polyneuropathy. Plasma samples were taken at hourly intervals over an 11-h period (08.00-19.00 hours) in order to characterise secretory profiles for 15 Type 1 diabetic patients (eight neuropathic and seven non-neuropathic) and eight non-diabetic control subjects. In the non-diabetic subjects, mean plasma IGF-I levels were stable throughout the 11-h period with a range of 97 micrograms/l-169 micrograms/l. In contrast, mean plasma IGFBP-1 levels declined steadily from a high level of 1.99 micrograms/l at 08.00 hours to approximately one half (0.86 microgram/l) at 15.00 hours. Comparison of areas under the curves revealed significant negative correlations between IGFBP-1 and glucose (-0.88, p = 0.01), IGFBP-1 and insulin (-0.75, p = 0.016), and IGFBP-1 and IGF-I (-0.68, p = 0.03). A significant positive correlation was found between insulin and IGF-I (+0.89, p = 0.001). The diabetic patients had markedly elevated plasma IGFBP-1 levels (area under curve, p = 0.01) and lower plasma IGF-I levels (p = 0.033) even though these patients were hyperinsulinaemic throughout the study period.(ABSTRACT TRUNCATED AT 250 WORDS)

Central actions of CRF on thermogenesis are mediated by pro-opiomelanocortin products.

Corticotrophin-releasing factor (CRF) causes central activation of thermogenesis. The aim of this study was to investigate whether this action is mediated by ACTH or other peptides derived from the ACTH precursor pro-opiomelanocortin (POMC) within the CNS. Central (intracerebroventricular) injection of rat CRF caused dose-dependent increases in resting oxygen consumption (VO2) in conscious rats (maximal 26 +/- 5% at 2 nmol CRF). These responses were significantly attenuated by pretreatment (i.c.v.) with either a monoclonal antibody raised to gamma 1MSH or with naloxone which antagonises beta-endorphin (beta-EP) actions. The increases were not affected by pretreatment with monoclonal antibodies to ACTH or the N-terminal of POMC. Central injections of gamma 1-melanocyte-stimulating hormone (MSH) or beta-EP caused dose-dependent increases in VO2 (maximal at 0.5-1.5 pmol) and these were markedly inhibited by pretreatment with the anti-gamma 1-MSH antibody or naloxone respectively. Injection of ACTH or alpha MSH did not significantly affect VO2 at doses up to 2 nmol. These data indicate that the central actions of CRF on thermogenesis may be mediated, at least in part, by release of gamma MSH and/or beta-EP.

Corticotrophin releasing hormone (CRH1-41) stimulates the secretion of adrenocorticotrophin, vasopressin and oxytocin but not adrenocorticotrophin precursors: evidence from petrosal sinus sampling in man.

To examine the relationship between corticotrophin releasing hormone (CRH), arginine vasopressin (AVP) and oxytocin (OXT) we have studied the responses of adenohypophyseal and neurohypophyseal hormones to CRH in eight patients (age 26-64 years, six female) with suspected pituitary-dependent Cushing's syndrome during bilateral, simultaneous inferior petrosal sinus catheterization. Blood samples were taken from both petrosal sinuses and a peripheral vein before, and at 5-min intervals for 15 min after, an intravenous injection of 100 micrograms human CRH1-41. CRH increased sinus AVP concentrations in all eight patients and OXT concentrations in four of five patients studied. Although AVP concentrations often increased in both sinuses, the side of maximal AVP rise was termed side(max-AVP). CRH did not affect peripheral or petrosal sinus mean concentrations of LH, FSH, GH or TSH. While there was no change in mean peripheral concentrations of AVP, OXT, ACTH, ACTH precursors or prolactin after CRH, sinus concentrations of OXT, ACTH and prolactin on side(max-AVP) were markedly elevated over contralateral values. CRH did not increase mean sinus concentrations of ACTH precursors. In seven patients with either no radiological abnormality or the pituitary fossa or a small adenoma the mean ACTH precursor/ACTH ratio in blood sampled from all sites was 2.1 +/- 0.16 (mean +/- SEM, n = 50). In a patient with a large, locally invasive tumour the mean ACTH precursor/ACTH molar ratio was 32.1 +/- 1.3 (n = 12; P less than 0.001), suggesting that alterations in this molar ratio may reflect the biological properties of the tumour. The source of CRH-stimulatable AVP and OXT remains uncertain.(ABSTRACT TRUNCATED AT 250 WORDS)

The responses of arginine vasopressin and adrenocorticotrophin to nausea induced by ipecacuanha.

Ipecacuanha syrup induces emesis by an early peripheral (gastric irritant) action and a later central effect at the chemoreceptor trigger zone (CTZ). We have studied the responses of plasma AVP, ACTH and ACTH-precursors to early and late ipecacuanha-induced nausea in nine healthy male subjects. Symptom severity was assessed using a linear analogue scale. All subjects reported 'early' nausea (N1) with a latency of 16 +/- 2 min (mean +/- SEM) and eight subjects vomited. Six subjects experienced recurrent nausea (N2) (latency 106 +/- 10.4 min) of whom five also vomited. The interval between the cessation of N1 and the onset of N2 was 55 +/- 10.8 min (range 25-80 min). The severity of nausea at the onset of N1 or N2 was similar but the AVP and ACTH responses were highly variable. Thus, while mean plasma AVP concentrations increased during both symptom periods, in three subjects during N1 and in three subjects during N2 plasma AVP concentrations did not rise above the normal range, despite marked symptoms. No clear pattern of AVP response to distinguish early peripheral from late central ipecacuanha-induced emesis was demonstrated. Whilst mean plasma ACTH concentrations increased during both N1 and N2 there were no changes in mean plasma ACTH-precursor concentrations. Analysis of pooled data for N1 and N2 demonstrated direct correlations between the nausea score and the peak incremental plasma responses of either AVP or ACTH and, despite the variability, peak incremental concentrations of AVP and of ACTH were also correlated. The data indicate that there is no difference in the AVP responses to peripherally or centrally stimulated ipecacuanha-induced nausea.(ABSTRACT TRUNCATED AT 250 WORDS)