Aberrant production and regulation of proopiomelanocortin-derived peptides in ectopic Cushing's syndrome

A 64 year old woman with a pancreatic islet cell tumor developed Cushing's syndrome. Glucocorticoid secretion did not decrease after low or high dose dexamethasone administration, and the Cushing's syndrome was cured by removal of tumor tissue. Immunohistochemistry and radioimmunoassays revealed the presence of immunoreactive ACTH, beta-endorphin and alpha-MSH in the tumor cells. Gel-permeation chromatography confirmed that beta-endorphin was the predominant opioid peptide produced by the tumor. The tumor was shown to contain a single 1.2 kilobase RNA species which hybridized to a 32P human POMC-cDNA; this POMC RNA was identical in size to that isolated from a normal human pituitary. In dispersed monolayer culture, CRF failed to elicit ACTH release from the tumor cells, but dexamethasone caused a paradoxical increase in ACTH secretion in vitro. This study demonstrates that aberrant regulation of POMC synthesis and peptide processing can be seen in tumors which synthesize a POMC RNA identical in size to that made in the pituitary gland.     

The use of the corticotropin-releasing hormone test to monitor the recovery of patients with Cushing's disease or Cushing's syndrome due to an adrenal adenoma after adenomectomy

Six patients with Cushing's disease and three with Cushing's syndrome due to an adrenal adenoma were monitored after their adenomectomy with the corticotropin-releasing hormone test to evaluate the progress of recovery of their pituitary adrenal function. Before surgery the patients with Cushing's disease showed either high, normal or low responses of plasma ACTH and cortisol to 100 micrograms synthetic ovine corticotropin-releasing hormone (CRH) administered intravenously, whereas all three patients with Cushing's syndrome due to an adrenal adenoma showed no response of plasma ACTH or cortisol to CRH. One or two months after surgery, the patients who had Cushing's disease had low levels of basal plasma ACTH and cortisol and their responses to CRH were extremely low. However, the same patients were tested later, it was found that their responses to CRH gradually increased and reached normal ranges approximately within one year after tumor removal, which coincided with the overall improvement in their clinical signs and symptoms due to adrenal insufficiency. In contrast, the recovery of the pituitary adrenal function in patients who had Cushing's syndrome due to an adrenal adenoma was not complete even one year after surgery. Thus the corticotropin-releasing factor test is a useful criteria to evaluate the recovery of the pituitary adrenal function in these patients after surgery, since the responses of plasma ACTH and cortisol to the administered CRH are parallel with the improvements in clinical signs and symptoms due to adrenal insufficiency in patients with Cushing's disease.     

Effect of tunicamycin on the synthesis, processing, and secretion of pro-opiomelanocortin peptides in mouse pituitary cells

Pro-opiomelanocortin (POMC) is glycosylated and proteolytically cleaved to produce a number of smaller peptide hormones including adrenocorticotropic hormone (ACTH) and endorphin in mammalian pituitary and the mouse pituitary cell line AtT-20/D16v. When glycosylation of POMC is inhibited in AtT-20 cells with the drug tunicamycin, a 26,000-dalton protein appears in place of the glycosylated 29,000- and 32,000-dalton forms of POMC. The 26,000-dalton form found in tunicamycin-treated cells has the same [35S]methionine tryptic peptides as 29,000- and 32,000-dalton POMC, indicating that the decrease in apparent mass is most likely due to loss of carbohydrate and not to changes in the peptide backbone. The 4,500-dalton form of alpha(1-39)ACTH and the 3,000- and 11,000-dalton forms of endorphin are all present in tunicamycin-treated cells. The glycosylated form of alpha(1-39)ACTH, however, is missing and the glycosylated ACTH intermediates are replaced by unglycosylated ACTH intermediates. Pulse-chase studies demonstrate that the 26,000-dalton unglycosylated POMC is the precursor of the smaller ACTH and endorphin molecules in tunicamycin-treated cells. Furthermore, all of the forms of ACTH and endorphin found in tunicamycin-treated cells are secreted. Thus, it appears that glycosylation is not an essential step for correct cleavage or secretion of POMC or its products.     

Human corticotropin-releasing hormone test in normal subjects and patients with hypothalamic, pituitary or adrenocortical disorders

Human corticotropin-releasing hormone (hCRH) test was performed in 57 normal volunteers and 102 patients with hypothalamic, pituitary and adrenocortical diseases. Intravenous bolus injection of synthetic hCRH, 100 micrograms for adults or 1.5 micrograms/kg for children, increased plasma ACTH and cortisol levels in about 90% of normal subjects. In 47 patients with Cushing's disease, plasma ACTH tended to show an exaggerated response to hCRH and peak ACTH was the most frequent abnormal component among the several reaction parameters. Poor responders among normal subjects and patients with Cushing's disease had significantly higher plasma cortisol levels before CRH administration. Patients with hypothalamic hypopituitarism showed exaggerated response, whereas patients with primary pituitary lesion, isolated ACTH deficiency or adrenal Cushing's syndrome showed no ACTH response. These differences in the response of patients suggest the value of the hCRH test in their differential diagnosis.     

Diurnal rhythms of proopiomelanocortin-derived N-terminal peptide, beta-lipotropin, beta-endorphin and adrenocorticotropin in normal subjects and in patients with Addison's disease and Cushing's disease

In order to clarify the diurnal pattern of secretion of plasma immunoreactive (IR) proopiomelanocortin (POMC)-derived peptides, IR-N-terminal peptide (Nt), IR-beta-endorphin (Ep), IR-beta-lipotropin (LPH), and IR-ACTH (ACTH) in normal subjects and in patients with Addison's disease and Cushing's disease, we measured these 4 peptides in the same plasma obtained at 0900 h and then every three hours until 0600 h at the next day. All four peptides showed diurnal rhythms with the peaks at 0600 h, and the nadirs of ACTH, LPH, Ep and Nt were at 0000 h, 0000 h, 1800 h and 0300, respectively in normal subjects. In patients with Addison's disease, these four peptides also showed diurnal rhythms with the peaks at 0600 h for ACTH and Ep and at 0900 h for LPH and Nt, and the nadirs at 2100 h for ACTH and Ep and at 0000 h for LPH and Nt. The molar ratios of Ep/ACTH, LPH/ACTH and Nt/ACTH in plasma also presented diurnal variations in normal subjects and in patients with Addison's disease. On the other hand, in patients with Cushing's disease, ACTH, LPH and Nt showed no rhythmicity or change in molar ratios of Ep/ACTH, LPH/ACTH or Nt/ACTH. Only Ep showed diurnal variation. The molar ratios of Ep/ACTH, LPH/ACTH and Nt/ACTH in patients with Cushing's disease were significantly higher than those in normal subjects and in patients with Addison's disease at 0000 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Major role of cathepsin L for producing the peptide hormones ACTH, beta-endorphin, and alpha-MSH, illustrated by protease gene knockout and expression

The pituitary hormones adrenocorticotropic hormone (ACTH), beta-endorphin, and alpha-melanocyte stimulating hormone (alpha-MSH) are synthesized by proteolytic processing of their common proopiomelanocortin (POMC) precursor. Key findings from this study show that cathepsin L functions as a major proteolytic enzyme for the production of POMC-derived peptide hormones in secretory vesicles. Specifically, cathepsin L knock-out mice showed major decreases in ACTH, beta-endorphin, and alpha-MSH that were reduced to 23, 18, and 7% of wild-type controls (100%) in pituitary. These decreased peptide levels were accompanied by increased levels of POMC consistent with proteolysis of POMC by cathepsin L. Immunofluorescence microscopy showed colocalization of cathepsin L with beta-endorphin and alpha-MSH in the intermediate pituitary and with ACTH in the anterior pituitary. In contrast, cathepsin L was only partially colocalized with the lysosomal marker Lamp-1 in pituitary, consistent with its extralysosomal function in secretory vesicles. Expression of cathepsin L in pituitary AtT-20 cells resulted in increased ACTH and beta-endorphin in the regulated secretory pathway. Furthermore, treatment of AtT-20 cells with CLIK-148, a specific inhibitor of cathepsin L, resulted in reduced production of ACTH and accumulation of POMC. These findings demonstrate a prominent role for cathepsin L in the production of ACTH, beta-endorphin, and alpha-MSH peptide hormones in the regulated secretory pathway.     

Steroid-dependent ACTH-produced thymic carcinoid: regulation of POMC gene expression by cortisol via methylation of its promoter region

AIMS: Metyrapone causes a decrease in the serum cortisol level without affecting ACTH production in ectopic tumors. We report a case who presented with Cushing's syndrome due to an ectopic ACTH-producing thymic carcinoid. In the present case, it was demonstrated that metyrapone administration resulted in a significant decrease in the plasma ACTH and serum cortisol levels. We hypothesized that the steroid hormone may promote proopiomelanocortin (POMC) gene expression in the carcinoid cells. METHODS: An 11-year-old boy presented with Cushing's syndrome. Prior to the detection of a thymic tumor, metyrapone was administered to ameliorate the symptoms of Cushing's syndrome. Interestingly, plasma ACTH as well as serum cortisol levels immediately decreased after metyrapone administration. The levels of cortisol and ACTH were observed to be normal after complete surgical resection of the tumor. Biological characterization of the tumor cells was by in vitro analysis. RESULTS: Thein vitro culture of the tumor cells showed an increased expression of POMC in the presence of cortisol. A CpG methylation assay showed that the demethylation of the POMC promoter was induced by a steroid hormone. CONCLUSION: These findings suggest that the ectopic ACTH-producing tumor may partly be regulated by the elevated levels of cortisol.     

Significant gene expression of insulin-like growth factor II and proliferating cell nuclear antigen in a rapidly growing recurrent pituitary acth-secreting adenoma.

BACKGROUND: We quantified the expression of various growth-related factors in an adrenocorticotropic hormone (ACTH)-secreting adenoma that had recurred very rapidly as invasive macroadenoma. METHODS/RESULTS: A 43-year-old woman underwent successful transsphenoidal surgery for Cushing's disease. Seven years later, she was admitted to our ward for further endocrine examinations. In spite of a very high plasma ACTH level, the serum cortisol level was normal. Discrepancies between ACTH and cortisol levels were detected on the basis of diurnal rhythms, dexamethasone suppression tests, and corticotropin-releasing hormone test. The patient showed no clinical features of Cushing's disease. Magnetic resonance imaging of the pituitary showed an almost empty sella, and no microadenoma was found. These results, along with those of Sephadex column gel filtration and high-performance liquid chromatography of plasma-immunoreactive ACTH, suggested that the patient's residual corticotrophs secreted biologically inactive ACTH. Two years later, the patient suddenly developed diplopia and right abducens nerve palsy. She was slightly moonfaced and centrally obese. Her plasma ACTH and serum and urinary free cortisol levels were elevated, although discrepancies between ACTH and cortisol still existed. Magnetic resonance imaging revealed a large pituitary mass with suprasellar and cavernous sinus extensions. The tumor was excised, and the proopiomelanocortin gene and the expression of growth-related factors were analyzed. No mutations were found in the ACTH-coding region of the proopiomelanocortin gene. A significant expression of insulin-like growth factor II and proliferating cell nuclear antigen mRNAs was demonstrated. A high MIB-1 antibody labeling index was also detected in the adenoma tissue, suggesting high Ki-67 expression. CONCLUSION: These growth- and proliferation-related factors might be involved in the rapid growth and aggressiveness of this patient's pituitary adenoma.     

Proopiomelanocortin gene is expressed in many normal human tissues and in tumors not associated with ectopic adrenocorticotropin syndrome

Immunoreactive (IR) POMC peptides have been detected in several human nonpituitary tissues and most pheochromocytomas and lung cancers, including those not associated with ectopic ACTH syndrome. We found IR-ACTH, IR-gamma MSH, IR-beta-endorphin (beta END), and IR-lipotropin in extracts from the following 10 normal human tissues, listed in order of decreasing POMC peptide concentrations: adrenal, testis, spleen, kidney, ovary, lung, thyroid, liver, colon, and duodenum. IR-ACTH, IR-gamma MSH, and IR-beta END were detected in all six pheochromocytomas and all 12 lung tumors (six squamous cell carcinomas, five adenocarcinomas, and one small cell carcinoma) we examined, as well as in a squamous cell carcinoma of the larynx. None of the patients had clinical evidence of ectopic ACTH syndrome. To determine whether these nonpituitary tissues and tumors actually synthesize POMC, rather than simply absorb POMC peptides from plasma, we examined poly(A) RNA prepared from these tissues and total RNA from pituitary by Northern blot hybridization for the presence of POMC-like mRNA with an exon 3 riboprobe. Pituitary contained a single POMC mRNA species of about 1150 bases. A short POMC-like mRNA of about 900 bases was found in all normal nonpituitary tissues, three of five pheochromocytomas, eight of nine lung cancers, and the laryngeal squamous cell tumor. In addition, larger POMC-like mRNA species between 1200 to 1500 bases were detected in adrenal, testis, ovary, placenta, two pheochromocytomas, and three squamous cell lung tumors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticotropin-releasing factor in humans. II. CRF stimulation in patients with diseases of the hypothalamo-pituitary-adrenal axis

A stimulation test with 100 micrograms ovine or human corticotropin-releasing factor (CRF) is a useful diagnostic tool in diseases of the hypothalamo-pituitary-adrenal axis. No serious side effects were observed during the test procedure. The results showed that the CRF test is useful in making the differential diagnosis of established Cushing's syndrome (n = 42). The CRF test was also repeated after transsphenoidal surgery in 25 patients with Cushing's disease. Successfully operated patients exhibit no, blunted or normal adrenocorticotropic hormone (ACTH) responses to CRF (n = 15), whereas patients who did not show remission remained hyperresponsive (n = 10). In patients with autonomous adrenal cortisol secretion, the ACTH response to CRF was suppressed (n = 10). After surgery the ACTH response to CRF can already be demonstrated when cortisol levels are still undetectable. Pulsatile administration of CRF in one patient after unilateral adrenalectomy and another patient under corticoid therapy revealed that ACTH responses to CRF normalize rapidly but cannot be sustained if CRF administration is withdrawn, suggesting that the cause of adrenal failure after unilateral adrenalectomy for Cushing's syndrome or long-term corticoid therapy is due to hypothalamic CRF deficiency. The decrease of the ACTH responses to CRF in glucocorticoid-treated patients correlated directly to the daily corticoid dosage. Since the ACTH hyperresponse to CRF in 6 patients with Cushing's disease was also suppressed by short-term dexamethasone treatment, the pituitary level as target site for the acute feedback inhibition is also demonstrated. The evaluation of the CRF-induced ACTH response in patients with secondary adrenal failure without detectable pathology in the sella and suprasellar region (n = 6) enables the differentiation between hypothalamic and pituitary adrenal insufficiency. In patients with hypothalamic lesions the ACTH response to CRF was normal whereas insulin hypoglycemia failed to induce an ACTH rise.     

Blockade of endothelin-1 release contributes to the anti-angiogenic effect by pro-opiomelanocortin overexpression in endothelial cells

Pro-opiomelanocortin (POMC) is the precursor of several neuropeptides, such as corticotropin (ACTH), alpha-melanocyte-stimulating hormone (MSH), and the endogenous opioid, beta-endorphin (EP). ACTH-dependent Cushing's syndrome is characterized by ACTH overproduction and is associated with an increased risk of cardiovascular disease. Endothelial dysfunction has been recognized as an early marker of cardiovascular disease. However, the mechanism underlying endothelial dysfunction by ACTH overexpression in Cushing's patients remains elusive. Endothelial cells, the primary cells producing endothelin (ET)-1, are both the source and target of POMC-derived peptides. In the present study, we generated adenovirus vectors (Ad) encoding POMC (Ad-POMC) and green fluorescent protein (GFP; Ad-GFP) to investigate whether POMC gene transfer altered the ET-1 homeostasis and angiogenic functions in human EA.hy926 endothelial cells. Via adenovirus gene delivery, the POMC-transduced EA.hy926 cells released significantly elevated ACTH and beta-EP levels (P < 0.001). In addition, POMC gene delivery significantly decreased the ET-1 release (P < 0.001) without affecting the ET-1 messenger RNA (mRNA) level. Despite no effect on the secretion of matrix metalloproteinases (MMPs) and cell proliferation, POMC gene delivery significantly inhibited the migration (P < 0.01) and tube-forming capability (P < 0.01) of endothelial cells. Moreover, the POMC-induced inhibition of tube formation could be partially reversed by adding exogenous ET-1 (P < 0.05). In summary, the attenuated ET-1 release and angiogenic processes by POMC overexpression may contribute to endothelial dysfunction, thereby providing a link between Cushing's syndrome and cardiovascular diseases.     

Androgen secretion in ectopic ACTH syndrome and in Cushing's disease: modifications before and after surgery.

The role of ACTH in the control of adrenal androgen secretion is known, although the possible existence of other regulatory factors has been also suggested. While some data concerning Cushing's disease have been reported, only few studies concerned androgen levels in ectopic ACTH secretion. The aim of this study was to evaluate serum DHEA-S, androstenedione (A) and testosterone (T) levels in 36 women with ACTH-dependent Cushing's syndrome (30 with Cushing's disease and 6 with ectopic ACTH secretion) before and after surgery. Two men with ectopic ACTH production were also studied. In 30 women with Cushing's disease serum DHEA-S (9.6 +/- 0.9 micromol/l), A (15.2 +/- 1.2 nmol/l) and T (4.1 +/- 0.5 nmol/l) were higher than in controls (p < 0.01): elevated DHEA-S, A and T values were found in 8, 18 and 17 cases, respectively. After adenomectomy in 15 apparently cured patients DHEA-S, A and T levels were low at 1 - 3 months and at 6 - 12 months after surgery. At 18 - 24 months, DHEA-S remained low in spite of cortisol normalisation. In ectopic Cushing's syndrome, A levels were significantly higher (23.1 +/- 4.9 nmol/l) than in Cushing's disease (p < 0.05), while no differences were found in DHEA-S and T levels. Two patients had elevated DHEA-S values, 3 women had high T levels and 7 of the 8 patients had very high A concentration that was lowered in 3 operated cases. In conclusion, the pattern of adrenal androgen secretion is rather different in patients with pituitary or with ectopic Cushing's syndrome. While the frequency of DHEA-S and T alterations is similar, androstenedione secretion is greatly increased in the latter condition. It is suggested that in ACTH-secreting non-pituitary tumours, the production of a POMC-derived peptide, although unidentified, may lead to preferentially stimulated androstenedione secretion, without affecting other enzymatic pathways.     

Regulation of lymphokine (gamma-interferon) production by corticotropin

We have shown that corticotropin (ACTH), alpha-endorphin, and enkephalins can regulate antibody responses, which suggested a role for neuropeptides in a regulatory circuit between the immune and neuroendocrine systems. ACTH and structurally related peptides were examined here for regulation of mitogen induction of the lymphokine gamma-interferon (IFN gamma) in C57BL/6 mouse spleen cell cultures. Synthetic ACTH1-39 and a porcine pituitary extract containing ACTH activity were potent suppressors of the IFN gamma response. Synthetic ACTH1-39 suppressed the response by approximately 62% at 1 to 3 microM, whereas the porcine extract suppressed by greater than 90% at 1 to 3 microM ACTH. The greater potency of the pituitary extract was shown to be due to the presence of an additional peptide of Mr 2100 that was reactive with antibodies to the N-terminal region of ACTH (ACTH1-13), possessed potent anti-cellular activity against L cells and various transformed cells, but lacked ACTH biologic activity. The anti-cellular peptide suppressed the IFN gamma response by greater than 99% at 0.05 microM. The ACTH1-39 cleavage products, alpha-melanocyte stimulating hormone (alpha MSH; acetylated and amidated ACTH1-13), and corticotropin-like intermediate lobe peptide (CLIP; ACTH18-39) had no effect on IFN gamma production. ACTH1-24, like ACTH1-39, has full steroidogenesis activity but also had no effect on IFN gamma production, which suggests a dissociation of the immunoregulatory and steroidogenic properties of ACTH1-39. ACTH1-39, and possibly also the anti-cellular 2100 Mr peptide, is initially synthesized as the precursor polyprotein pro-opiomelanocortin (POMC). Enzymatic processing of POMC, first to the active ACTH1-39 or the anti-cellular peptide and then to the inactive smaller peptides, probably plays an important role in regulation of lymphokine and antibody production by ACTH and ACTH-related neuropeptides. This is consistent with the recent demonstration of the production of ACTH-like peptides by lymphocytes.     

Serum total prostate-specific antigen assay in women with Cushing's disease or alcohol-dependent pseudo-Cushing's State

BACKGROUND: The distinction between Cushing's disease (Cushing's syndrome dependent on adrenocorticotropic hormone (ACTH)-secreting tumors of pituitary origin) and pseudo-Cushing's states (Cushingoid features and hypercortisolism sometimes present in alcoholic, depressed or obese subjects) can present a diagnostic challenge in clinical endocrinology. Recently, the availability of a highly sensitive immunofluorometric assay for the measurement of total prostate-specific antigen (PSA) provided the possibility to measure serum PSA levels in women. Interestingly, PSA gene expression and protein production has been found to be upregulated by steroid hormones, such as androgens, glucocorticoids, mineral corticoids and progestins. In fact, serum total PSA concentrations appear to be higher in female patients with Cushing's disease than in normal women. We wondered whether a similar phenomenon also occurs in pseudo-Cushing's state. METHODS: In order to answer this question, we compared the serum total PSA levels measured in 10 female subjects with alcohol-dependent pseudo-Cushing's state with those observed in 8 female patients with Cushing's disease and in 15 age-matched healthy women. Serum testosterone, ACTH and cortisol, and 24-hour urinary cortisol levels were measured; cortisol suppression after dexamethasone was also tested in all subjects. RESULTS: The basal serum levels of ACTH and cortisol were significantly lower in normal subjects than in patients with Cushing's disease or pseudo-Cushing's state; these latter groups showed similar basal hormonal values. Dexamethasone administration was unable to suppress serum cortisol levels in 5 subjects with Cushing's disease and 6 subjects with pseudo-Cushing's state. Serum testosterone values in the group with Cushing's disease were higher than in the other groups. No differences were observed between pseudo-Cushing's and normal subjects. Serum total PSA levels were significantly higher in women with Cushing's disease than in subjects with pseudo-Cushing's state and normal controls; these latter groups showed similar PSA values. When serum total PSA and testosterone levels were considered together, a significant positive correlation was observed in the group with Cushing's disease, but not in the other groups. CONCLUSIONS: These data indicate that the steroid milieu responsible for the elevation in serum PSA in women with Cushing's disease is not present in subjects with alcohol-dependent pseudo-Cushing's state, suggesting the possible use of PSA as a marker of differentiation between these pathological conditions in women.     

Ectopic ACTH syndrome

Ectopic ACTH syndrome represents a cancer-induced amplification of a property [proopiomelanocortin (POMC) peptides production] normally present in the cells from which the cancer originated but with aberrant posttranslational processing of POMC resulting in a greatly elevated secretion of ACTH precursors. The classic ectopic ACTH-producing tumors described in the 1960s were highly malignant but more recently slowly growing tumors such as carcinoids are reported with increasing frequency. Clinical features of patients with ectopic ACTH were analyzed, including biochemical abnormalities, plasma ACTH, cortisol and urinary steroids. Dynamic tests such as high-dose dexamethasone suppression, metyrapone and ovine-CRH (oCRH) stimulation were explored, as well as inferior petrosal sinus ACTH sampling before and after oCRH. Among the tumor markers examined, elevation of ACTH precursors was uniformly present followed by increased output of calcitonin, gut hormones, oncofetal and placental hormones in decreasing order. Since more than 90% of ectopic ACTH tumors are neuroendocrine in nature exhibiting APUD characteristics, their 2 markers, neuron-specific enolase and chromogranins are very useful. The imaging procedures for localization of the tumor ranged from chest X-rays to computed tomography and magnetic resonance of the chest and abdomen. Abdominal ultrasonography was also useful. Finally somatostatin receptor scintigraphy permitted demonstration of unrecognized tumors and/or metastases, even when the tumors were occult. The ACTH content, immunostaining for APUD markers and altered POMC processing were evaluated in ectopic tumors and/or metastases. Occult ectopic ACTH syndrome of more than 4–6 months of symptoms without the emergence of an obvious source was reviewed. Since the tumors are often clinically and biochemically undistinguishable from pituitary-dependent Cushing's disease, inferior petrosal sinus sampling for ACTH after oCRH stimulation established the diagnosis in over 90% of the cases. 60% of the occult tumors were thoracic carcinoids (3/4 bronchial carcinoids), followed by small cell lung cancer and pancreatic neuroendocrine tumors. In 12% the primary etiology was not detected. The rare syndrome of ectopic CRH syndrome (6 published cases) leading to excessive stimulation of the pituitary which became hyperplastic and secreted excessive amounts of ACTH is discussed. Finally, the 12 published cases and 1 unreported patient with ectopic CRH-ACTH tumors were reviewed, the majority being metastatic small cell lung carcinomas, bronchial and thymic carcinoids.     

Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.     

Ectopic Cushing's syndrome in cloacogenic carcinoma of the anal canal: a case demonstrating APUD characteristics

A patient found to have ectopic Cushing's syndrome three months after surgical resection of cloacogenic carcinoma of the anal canal was studied with serial plasma cortisol and ACTH measurements. The effects of therapy on plasma ACTH and cortisol levels were noted. An autopsy was performed immediately after death and liver metastatic tumor tissue was assayed for "small" ACTH, "big" ACTH, PTH, and alpha sub-unit of hCG, Clinical Cushing's syndrome was observed along with nonsuppressible plasma cortisol level. Plasma ACTH only reached the highest normal level but tumor ACTH ("small"), "big" ACTH, alpha sub-unit and PTH were markedly elevated. It was concluded that a case of classic cloacogenic carcinoma of the anal canal produced ectopic Cushing's syndrome. Elevated tumor alpha sub-unit and PTH were not associated with appreciable biologic activity. Ectopic Cushing's syndrome in this disease may imply poorer prognosis.     

Characterisation of ACTH Peptides in Human Skin and Their Activation of the Melanocortin-1 Receptor

α-Melanocyte-stimulating hormone (α-MSH) is a proopiomelanocortin (POMC)-derived peptide, which is produced in the pituitary and at other sites including the skin. It has numerous effects and in the skin has a pigmentary action through the activation of the melanocortin-1 (MC-1) receptor, which is expressed by melanocytes. Recent evidence suggests that the related POMC peptides such as adrenocorticotrophin (ACTH), which is the precursor of α-MSH, is also an agonist at the MC-1 receptor. By using immunocytochemistry, we confirmed the presence of α-MSH in human skin where staining was evident in keratinocytes and especially strong in melanocytes and possibly Langerhans cells. ACTH was also present and tended to show the strongest reaction in differentiated keratinocytes. Immunostaining was also observed for the prohormone convertases, PC1 and PC2, which are involved in the formation of ACTH and its cleavage to α-MSH, respectively. The amounts of immunoreactive ACTH exceeded those of α-MSH. Using HPLC we identified for the first time the presence of ACTH1-39, ACTH1-17, ACTH1-10, acetylated ACTH1-10, α-MSH, and desacetyl α-MSH in epidermis and in cultured keratinocytes. The ability of these peptides to activate the human MC-1 receptor was examined in HEK 293 cells that had been transfected with the receptor. All peptides increased adenylate cyclase in these cells with the following order of potency: ACTH1-17 > α-MSH > ACTH1-39 > desacetyl α-MSH > acetylated ACTH1-10 > ACTH1-10. ACTH1-17 also increased the dendricity and melanin content of cultured human melanocytes indicating that the peptide was able to activate MC-1 receptors when present in their normal location. However, as found with α-MSH, not all cultures were responsive and, as we have previously suggested, we suspect that this was the result of changes at the MC-1 receptor. Nevertheless, it would appear that ACTH peptides can serve as natural ligands of the MC-1 receptor on human melanocytes and their presence in the skin suggests that, together with α-MSH, they may have a role in the regulation of human melanocytes.