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Many important fields of research had a humble origin. In the distant past, A J P Martin’s discovery that amino acids could be separated by paper chromatography and Moore and Stein’s use of columns for quantitative amino acid analysis provided the first steps towards the determination of structure in complex biologically active molecules. They opened the door to reveal the essential relationship that exists between structure and function. In molecular endocrinology, for example, striking advances have been made by chemists with their expertise in the identification of structure working with biologists who contributed valuable knowledge and experience. Advantage was gained from the convergence of different background, and it is notable that the whole is greater than the sum. In the determination of structure, it may be recalled that four of the world’s great pioneers (Archibald Martin, Rodney Porter, Fred Sanger and Vincent du Vigneaud) were acknowledged for their fundamental contributions when individually they were awarded the Nobel Prize. They foresaw that the identification of structure would prove of outstanding importance in the future. Indeed, study of the structures of β-endorphin and enkephalin and the different forms of opiate activity they engender has led to a transformation in our understanding of chemical transmission in the brain.

The synthesis of des-1-tyrosine-α-endorphin and fourteen fragments of this psychostimulant type of neuropeptide is described. In these syntheses use has been made of the classical fragment condensation approach.

The peptide hormones ACTH, MSHs, β-lipotropin (β-LPH), and β-endorphin are all derived from the precursor molecule proopiomelanocortin (POMC). Using confocal laser microscopy and immunoelectron microscopy in human pituitary gland, we demonstrate a peroxisomal localization of β-endorphin and β-LPH in cells expressing the peroxisomal ATP-binding cassette-transporter adrenoleukodystrophy protein (ALDP). The peroxisomal localization of β-LPH and β-endorphin was not restricted to the pituitary gland but was additionally found in other human tissues that express high levels of ALDP, such as dorsal root ganglia, adrenal cortex, distal tubules of kidney, and skin. In contrast to the peptide hormones β-LPH and β-endorphin, which are derived from the C terminus of POMC, the N-terminal peptides ACTH, α-MSH, and γ-MSH were never detected in peroxisomes. This novel peroxisomal localization of β-endorphin and β-LPH in ALDP-positive cells was confirmed by costaining with ALDP and the peroxisomal marker catalase. Moreover, peroxisomal sorting of β-LPH could be modeled in HeLa cells by ectopic expression of a POMC variant, modified to allow cleavage and release of β-LPH within the secretory pathway. Although β-LPH and β-endorphin were only associated with peroxisomes in cells that normally express ALDP, the transporter activity of ALDP is not necessary for the peroxisomal localization, as demonstrated in tissues of X-linked adrenoleukodystrophy patients lacking functional ALDP. It remains to be elucidated whether and how the peroxisomal localization of POMC-derived hormones has a role in the endocrine dysfunction of peroxisomal disease.

The amino acid sequence of beta-lipotropin from the ostrich pituitary has been determined. It consists of 79 amino acids. The amino acid sequence has been determined as follows: H-(1)AlA-Leu-Pro-Pro-Ala-Ala-Met-Leu-Pro-(10)Ala-Ala-Ala-Glu-Glu-Glu-Glu-Gly-Gl u-Glu-(20)Glu-Glu-Glu-Gly-Glu-Ala-Glu-Lys-Glu-Asp-(30)Gly-Gly-Ser-Tyr-Arg-Met-A rg-His-Phe-Arg-(40)Trp-Gln-Ala-Pro-Leu-Lys-Asp-Lys-Arg-Tyr-(50)Gly-Gly-Phe-Met- Ser-Ser-Glu-Arg-Gly-Arg-(60)Ala-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-(70)Ile-Val -Lys-Ser-Ala-Tyr-Lys-Lys-Gly-(79)Gln-OH. When compared with the primary structures of other known beta-lipotropins, the sequence at the NH2-terminal, beta-melanotropin and beta-endorphin portions of the molecule exhibit considerable variability.

The isolation of two peptides similar in amino acid composition to that of human beta-lipotropin is presented. Peptide patterns after enzymatic digestions of these two peptides by Staphylococcus aureus protease and by trypsin were nearly identical. Paper electrophoresis and amino acid analyses of acidic peptides generated from the enzymatic digestions of these two peptides indicate that there is an amide difference between the two peptides. It is proposed that this amide difference is in amino acid residue number nine, and that one is the human beta-lipotropin and the other its [Gln9] analog.

A novel, simple technique using reversed phase thin-layer chromatography has been developed to separate beta-endorphin, beta-lipotropin and Met- and Leu-enkephalins. This method is useful for the rapid determination of the purity of these opioid peptides. The effectiveness of several solvent systems has been assessed. The resolution between beta-endorphin and beta-lipotropin achieved by this method exceeds that reported using high performance liquid chromatography or a variety of column techniques. This system also eliminates the problem of unrecognized loss of peptide due to binding on HPLC or other types of columns since the entire stationary phase is visualized using fluorescamine detection. Efficient, inexpensive and reliable simultaneous separation with visualization of several opioid peptides, their peptide precursors and their degradation products is now possible using the new technique. Such separations are required for many purposes including preparation of samples for radioimmunoassay of opioid peptides.

The 51-residue peptide ovine beta-lipotropin-(41--91) has been synthesized by the solid-phase method in about 5% overall yield. The synthetic product was characterized by partition chromatography on agarose gel, thin-layer chromatography in two solvent systems, paper electrophoresis at two pH values, polyacrylamide gel electrophoresis, amino acid analyses of acid and enzymic hydrolysates, and bioassay for lipolytic and melanotropic activities. The synthetic peptide is about 5.4 times as active on a weight basis as ovine beta-lipotropin in the lipolytic assay. In the melanotropic assay, it was about 2.4 times more active than the beta-lipotropin but only 5% as active as bovine beta-melanotropin. It had negligible opiate activity in the guinea pig ileum assay.

Adrenocorticotropin and beta-lipotropin have been isolated and purified from elephant pituitary glands. The primary structures of these two hormones were determined by amino acid and sequence analyses of enzymatically cleaved peptides from the hormones. Peptide purification involved the use of gel filtration, reverse phase high performance liquid chromatography, and paper electrophoresis.

Submit Manuscript | http://medcraveonline.com Corticosteroids are a versatile group of drugs widely being used in cancer for a range of indications. They are used in conditions with proven evidence of their role like metastatic spinal cord compression [3], superior vena caval syndrome, raised intracranial pressure, bowel obstruction as well in those conditions lacking definitive role of steroids like chronic cancer pain, nausea, fatigue, and anorexia. They are also used to prevent immediate pain flare up following radiotherapy in vertebral metastases [4]. In relation to chronic cancer pain, it is being used in bony pain, visceral pain due to obstruction of hollow viscus, organ capsule distension, neuropathic pain due to infiltration or compression of neural structures and metastatic spinal cord compression. The possible role of steroids in every step of nociception: transduction, transmission, modulation and pain perception has been raised though the exact mechanism of action remains unclear. The mechanisms suggested include-inhibition of collagenase expression (key enzyme involved in tissue destruction during inflammation), reduction of pro-inflammatory cytokines and stimulation of lipotropin synthesis (inhibition of eicosanoid production). Hence steroids are most effective in inflammatory pain. They also inhibit spontaneous discharges from injured nerve and modulate neuroimmune interactions thereby decreasing neuropathic pain. However, corticosteroids are often “double edged swords”; though they provide rapid symptomatic relief they also have side effects that are dose and time dependent. Hence, clear guidelines regarding the “when, what, how, how much and how long” of steroid therapy must be established. Among the steroids, dexamethasone has been used more frequently because of its low mineralocorticoid activity, longer half-life, higher potency, availability of oral and parenteral formulations, higher glucocorticoid activity, 7 times more anti-inflammatory effect than prednisone and affordability. Numerous groups have assessed the utility of dexamethasone in cancer pain using a wide range of doses without arriving at conclusive objective evidence regarding its efficacy (Table 1).

The increases in the level of plasma lipotropin (LPH) and in the LPH/ACTH ratio are considered diagnostic tools in ectopic ACTH syndrome. However, plasma ACTH is also elevated in this syndrome. We report a case of a small carcinoid tumor with an increase in both ACTH and LPH in plasma before surgery. Eight months after the tumoral resection, plasma LPH alone was increased again, whereas plasma ACTH and plasma and urinary cortisol remained normal in this apparently cured patient. This repeated abnormality was the only available feature that allowed successful removal of the occult tumoral residue.

The present study was undertaken to examine the mechanism whereby beta-lipotropin stimulates adrenal steroidogenesis. In guinea pig adrenal cells, beta-lipotropin (10(-8) M) increased basal steroid production 6-, 4-, and 5-fold for cortisol, androstenedione, and dehydroepiandrosterone (DHEA), respectively, whereas the corresponding responses to adrenocorticotropin (ACTH) (10(-9) M) were 12-, 8-, and 7-fold. The conversion of cholesterol to pregnenolone was studied in cells treated with trilostane, an inhibitor of 3 beta-hydroxysteroid delta 4-5 isomerase. beta-Lipotropin (10(-10) and 10(-8) M) and ACTH (10(-9) M) stimulated pregnenolone production in trilostane-treated cells. The production of cortisol and androgens from precursor steroids was also studied in cells treated with aminoglutethimide, an inhibitor of cholesterol side chain cleavage, after addition of exogenous pregnenolone, 17-hydroxypregnenolone, progesterone, or DHEA. Neither ACTH nor beta-lipotropin stimulated cortisol, androstenedione, or DHEA production in the presence of exogenous precursors in aminoglutethimide-treated cells. No inhibition of the beta-lipotropin- or ACTH-stimulated cortisol or androstenedione responses was demonstrated with the opioid receptor antagonist naloxone (10(-11) to 10(-5) M). The results suggest that beta-lipotropin stimulates steroidogenesis by acting on the conversion of cholesterol to pregnenolone and that its effects are not mediated via an opioid receptor but may be mediated via an ACTH receptor.

The use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in identifying proopiomelanocortin (POMC) processing products in melanotrope cells of the pituitary intermediate lobe of Xenopus laevis was explored. Mass spectra were obtained with such a high sensitivity of detection that the peptides could be identified in a single melanotrope cell. In addition to known POMC processing products of the Xenopus melanotrope cell, t he presence of previously unidentified POMC-derived peptides was demonstrated. Together these POMC processing products accounted for the entire length of the POMC precursor. Furthermore, Xenopus possesses two genes for POMC and the sensitivity and accuracy of the MALDI-MS technique allowed identification of processing products of both the POMCA and POMCB gene. In addition, differences were obtained between the mass spectra of melanotrope cells from Xenopus laevis adapted to different conditions of background illumination. These results show that MALDI-MS is a valuable tool in the study of the expression of peptides in single (neuroendocrine) cells. Chemicals/CAS: Amino Acids; Melanocyte-Stimulating Hormones, 9002-79-3; Peptide Fragments; Pro-Opiomelanocortin, 66796-54-1

OBJECTIVE To explore the feedback regulation of glucocorticoids on corticotropin secretion in patients with septic and nonseptic circulatory shock. DESIGN Prospective study. SETTING An intensive care unit of a general hospital. PATIENTS Two groups of patients with septic shock (n = 11) or nonseptic shock (n = 7). A control group (n = 20) was also studied. INTERVENTIONS Intravenous dexamethasone (1 mg/hr for 4 hrs) suppression test. MEASUREMENTS Plasma concentrations of corticotropin-releasing factor, beta-lipotropin, and corticosteroid-binding globulin measured by radioimmunoassays, and plasma cortisol measured by radiocompetition assay; the ratio of cortisol to corticosteroid-binding globulin calculated as the free cortisol index. MAIN RESULTS In both groups of patients, the concentrations of plasma cortisol and beta-lipotropin, and the ratio of cortisol to corticosteroid-binding globulin, were higher than normal subjects (p < .001) and were not different between septic and nonseptic shock patients, whereas the plasma corticosteroid-binding globulin concentration was significantly (p < .001) lower in septic shock patients than in normal subjects (444 +/- 154 vs. 696 +/- 56 nmol/L [22.0 +/- 7.6 vs. 34.5 +/- 2.8 mg/L]), but not significantly lower in nonseptic shock patients (607 +/- 157 nmol/L [30.0 +/- 7.8 mg/L]). In contrast to the complete suppressive effect of dexamethasone infusion on cortisol and beta-lipotropin concentrations in normal subjects, dexamethasone did not suppress cortisol or lipotropin in either septic or nonseptic shock patients. CONCLUSIONS During circulatory shock, hypercortisolism is associated with high concentrations of lipotropin, and is not suppressible by intravenous dexamethasone infusion.

The hypothalamic-pituitary system controls homeostasis during feed energy reduction. In order to examine which pituitary proteins and hormone variants are potentially associated with metabolic adaptation, pituitary glands from ad libitum and energy restrictively fed dairy cows were characterized using RIA and 2-DE followed by MALDI-TOF-MS. We found 64 different spots of regulatory hormones: growth hormone (44), preprolactin (16), luteinizing hormone (LH) (1), thyrotropin (1), proopiomelanocortin (1) and its cleavage product lipotropin (1), but none of these did significantly differ between feeding groups. Quantification of total pituitary LH and prolactin concentrations by RIA confirmed the results obtained by proteome analysis. Also, feed energy restriction provoked increasing non-esterified fatty acid, decreasing prolactin, but unaltered glucose, LH and growth hormone plasma concentrations. Energy restriction decreased the expression of glial fibrillary acidic protein, triosephosphate isomerase, purine-rich element-binding protein A and elongation factor Tu, whereas it increased expression of proline synthetase co-transcribed homolog, peroxiredoxin III, β-tubulin and annexin A5 which is involved in the hormone secretion process. Our results indicate that in response to feed energy restriction the pituitary reservoir of all posttranslationally modified hormone forms remains constant. Changing plasma hormone concentrations are likely attributed to a regulated releasing process from the gland into the blood.

SUMMARY Cushing's Disease is often associated with a depressive syndrome, with mood, vegetative, and cognitive abnormalities of variable severity. In 11 patients with (pituitary ACTH-dependent) Cushing's disease (10 women, 1 man), we studied the relationship between severity of the depressive syndrome and concordance of changes in ACTH and 13-1ipotropin/13-endorphin ([~-LPH/13-E) levels at baseline and in response to metyrapone and dexamethasone. For each condition, blood samples were drawn at 0800h, 1200h, 1600h, and 2200h. Six patients were categorized as mildly depressed (mean [+ SD] depressed mood score = 0.17+ 0.4; modified Hamilton Depression scale score = 7.6 + 4.5) and five as severely depressed (mean depressed mood score = 2.4 + 0.5; modified Hamilton Depression scale score = 15 + 5.6) (p< 0.05). ACTH and ~-LPI-I/[3-E were measured by radioimmunoassay. For each experimental condition, changes in levels were scored as concordant if the two peptides moved in parallel between sampling points. There was a relationship between greater severity of depression and more frequent discordant changes in ACTH and [3-LPI-I/~I-E levels: The six patients with mild depression exhibited 23 concordant and 3 discordant change patterns, while the five patients with severe depression showed 8 concordant and 15 discordant patterns. The mean percentage of concordant patterns per patient differed significantly between the two groups (mildly depressed=90.0+ 16.7; severely depressed=34.6 + 8.7 (p

The relationship between pituitary corticotropin-releasing hormone (CRH) receptor changes and corticotrope responsiveness was studied during chronic cold stress. Exposure of rats to 4°C caused a gradual increase in plasma adrenocorticotropic hormone (ACTH), reaching 3-fold over the basal levels by 6 h (P< 0.005), followed by a reduction to levels only 1.3-fold over basal by 60 h. Plasma corticosterone was significantly increased after 1 h (P

A 42-year-old man and a 51-year-old woman with a positive history of weakness and gastrointestinal complaints were shown to have low basal plasma cortisol and ACTH levels, and low daily urinary excretion of free cortisol. An empty sella was found in patient no. 1, while patient no. 2 was hypothyroid. Both patients showed a normal plasma cortisol response to ACTH and an increment in plasma ACTH and lipotropin levels after ovine CRH (oCRH), lysine vasopressin (LVP) and oCRH-LVP stimulation tests. These studies clearly report an isolated idiopathic ACTH deficiency due to a deficit in CRH in two adult subjects.

Any biological function is at least bimolecular and its evolution therefore is at least dual, with variations in two lines of molecules. The hormone specificity results from a particular fit between the three-dimensional structure of the agent and that of the receptor but, because receptors are not known at the structural level, a discussion on the evolution of the polypeptide hormones is mainly limited to the possible progressive changes of the latter. As for other proteins (enzymes, oxygen carriers etc.) two degrees of complexity can be distinguished according to whether the hormone comprises one or several polypeptide chains. Protein assembly can bring new biological properties, each subunit playing a particular role. In this case, the 'internal' evolution (chain-chain interactions) overlaps the 'external' evolution (hormone-receptor contacts). The 'monomeric' hormones present the following problems: evolution of the prohormone and of the converting enzyme (for insulin), duplication and differentiation of two lines of hormones either by amino acid substitutions (neurohypophysial hormones and neurophysins) or by substitutions and size modifications (corticotropin and lipotropin), duplication and fusion leading to internal homology in the single polypeptide chain (somatotropin, prolactin, placental lactogen). The 'dimeric' hormones lead to several problems: successive duplications giving different subunits, selective associations between subunits, unequal rates of evolution of the subunits, the function of each subunit (lutropin, follitropin, thyrotropin, choriogonadotropin). An attempt is made to integrate the evolution of polypeptide hormones in the frame of the evolution of proteins.

A new procedure is described for isolation of four forms of beta-endorphin from bovine pituitary. The four peptides are: the C-fragment of lipotropin (bovine lipotropin residues 63-93, or beta-endorphin, the alpha, N-acetyl derivative of the C-fragment, the C'-fragment (bovine lipotropin residues 63-89) and the alpha, N-acetyl derivative of the C'-fragment. Of these peptides, beta-endorphin alone possesses potent analgesic activity. The procedure has been applied in studying the distribution of beta-endorphin-related peptides in two regions of the pituitary. The results show that in the anterior pituitary of the pig and the rat, beta-endorphin is produced with a high degree of specificity in its opiate active form. In contrast, in the pars intermedia of both species at least six peptides related to beta-endorphin are elaborated and beta-endorphin represents only a minor component. The principal peptides in the pars intermedia have been identified as acetylated derivatives of lipotropin C'-fragment: in the pig the predominant peptide is alpha,N-acetyl C'-fragment and in the rat the major peptide appears to be an epsilon-acetylated derivative of alpha,N-acetyl C'-fragment. Thus, beta-endorphin is activated in the anterior pituitary and inactivated in the pars intermediate. The results demonstrate selective and specific processing of the 31K ACTH-endorphin prohormone in the different regions of the pituitary. In the anterior pituitary two biologically active peptides, ACTH and beta-endorphin, are generated together; in the pars intermedia alpha-melanotropin (alpha-MSH) is accompanied by forms of beta-endorphin that have been inactivated by acetylation and proteolysis.

Proopiomelanocortin (POMC) is a common precursor of adrenocorticotropic hormone (ACTH), melanophore-stimulating hormone (MSH), and endorphin (END). In pituitary gland, POMC receives posttranslational processing by which different peptides are generated in the pars distalis (PD) and pars intermedia (PI). Recently, we cloned three subtypes of the POMC gene in pituitary gland of barfin flounder. The present study was undertaken to elucidate whether the three POMC genes are expressed in both the PD and PI of barfin flounder pituitary, and to identify peptides derived from POMCs in these lobes. We amplified the transcripts of POMC-A, -B and -C in both the PD and PI by the reverse transcription-polymerase chain reaction. In situ hybridization also detected signals for these three subtypes in the PD and PI. These results demonstrated that all three POMC genes are expressed in both the PD and PI of barfin flounder pituitary. By mass spectrometric analyses, ACTH-A, Des-acetyl (Ac)-alpha-MSH-A/B (amino acid sequence of alpha-MSH-A is identical to that of alpha-MSH-B), beta-MSH-A, corticotropin-like intermediate lobe peptide (CLIP)-A, and N-terminal peptide (N-POMC)-A were identified in the PD. Moreover, Des-Ac-alpha-MSH-A/B, alpha-MSH-A/B, beta-MSH-A and -B, N-beta-lipotropin-A, CLIP-A, N-Ac-beta-END-A(1-41) (C-terminally truncated form of N-Ac-beta-END-A), and N-POMC-A were identified in the PI. Predominant detection of POMC-A-derived peptides indicates the greatest production of POMC-A and no detection of POMC-C-derived peptides indicates the lowest production of POMC-C in both the PD and PI. ACTH-A is specifically produced in the PD, however, the occurrence of Des-Ac-alpha-MSH-A, CLIP-A, and beta-MSH-A shows that the entire POMC-A is further cleaved into small peptides as in the PI. In the PI, some peptides receive modification or truncation as shown by the occurrence of alpha-MSH-A/B and N-Ac-beta-END-A(1-41). These results show differential posttranslational processing of POMC between the PD and PI in barfin flounder pituitary.

The hormones of the anterior pituitary gland play a significant role in the maintenance of normal health and body function. This master gland produces hormones involved in the regulation of somatic growth, metabolic rate, carbohydrate and lipid metabolism, lactation, reproduction, and response to stress. Eleven anterior pituitary hormones have been extensively characterized at the protein and genomic levels. These hormones fall into three classic categories, ie, pro-opiomelanocortin-related (POMC-related) hormones, proteins structurally related to prolactin and growth hormone (PRL/GH-related), and glycoproteins. Structural similarities exist within each biochemical category, and the structural data presented represent listings in the Protein Identification Resource (PIR) database, compiled by the National Biomedical Research Foundation. Summary information on the principal biologic roles of each of these hormones is provided, as are commercial availability and source. Keywords: Adrenocorticotropic hormone; Metabolism; Melanotropins; Beta-endorphins; Lipotropin; Growth hormone; Prolactin; Glycoprotein hormones; Thyroid; Skin pigmentation

This study aimed to characterize the beta-endorphin-immunoreactive material (betaE-IR) detectable in normal human keratinocytes (NHK). The effects of different culturing conditions and UV-irradiation on production of betaE-IR by NHK were assessed by radioimmunoassay and HPLC. All culture systems contained low levels of betaE-IR that was increased in conditioned media after UV-irradiation under certain conditions. NHK grown in nutrient-poor medium contained highest levels of betaE-IR that exhibited beta-lipotropin-like properties after HPLC analysis. The other culturing conditions displayed no authentic betaE-related peptides. Our results indicate that under certain culturing conditions NHK can produce POMC peptides like beta-lipotropin, which can be induced by UV-radiation.

Excess secretion of cortisol in depressed patients has been documented by a number of investigators, which is presumed secondary to increased corticotropin (ACTH) and ACTH-releasing hormone (CRH) secretion. To unmask the proposed increased central (CRH) drive, we administered metyrapone in the AM to 13 depressed and 13 age- and sex-matched normal control subjects. Metyrapone administration resulted in a prompt decrease in plasma cortisol and in an increase in 11-deoxycortisol, the inactive precursor, in all subjects. Both depressed patients and normal control subjects demonstrated clear increases in ACTH and beta-lipotropin/beta-endorphin production. There were no significant differences between patients and controls in any hormonal measures following metyrapone administration. These data suggest that: 1) in the absence of negative feedback (cortisol blockade), mildly to moderately depressed outpatients do not manifest increased central drive in the morning; and 2) the secretory capacity of the corticotropes do not differ between such depressed patients and controls.

The amino terminus of bovine pro-opiomelanocortin (N-POMC1–77) is partially processed in the intermediate lobe of the pituitary to N-POMC1–49 and lys-γ3 -melanotropin. Two pools of N-POMC1–77 were isolated which were differentially glycosylated at threonine45, while N-POMC1–49 isolated from bovine intermediate lobe extracts existed in a non-glycosylated form. This suggested that differential O-linked glycosylation of N-POMC1–77 may regulate cleavage at the Arg49-Lys50 processing site. We tested this hypothesis by incubating N-POMC1–77 glycoforms with purified pro-opiomelanocortin converting enzyme. Only non-O-glycosylated N-POMC1–77 and O-glycosylated N-POMC1–77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys-γ3 -melanotropin, identified by high-performance liquid chromatography. These data provide the first functional evidence to support a role for differential O-linked glycosylation in the regulation of the processing of the N-terminus of bovine POMC.

beta-Lipotropin, a pituitary peptide, is a potent stimulator of lipolysis in rabbit adipose tissue in vitro and in vivo. Insulin inhibited the beta-lipotropin (1-100 nM)-stimulated glycerol release from rabbit adipocytes and fat pads significantly at concentrations of 10 and 100 microM. Both these concentrations of insulin also decreased the degradation of beta-lipotropin in intact adipose tissue to the same extent as the lipolytic activity. Furthermore, insulin reduced the degradation of beta-lipotropin in rabbit adipose tissue homogenate. Like insulin, several lysosomotropic agents also decreased significantly the degradation and the lipolytic activity of beta-lipotropin. On the other hand, insulin-like growth factor I in lower concentrations (1-100 nM) did not effect degradation and lipolytic activity of beta-lipotropin in rabbit adipose tissue. Thus, a direct influence of insulin on lysosomal enzymes degrading beta-lipotropin in rabbit adipose tissue can be suggested.

beta-Lipotropin, a pituitary peptide, is a strong stimulator of lipolysis in rabbit adipose tissue. This polypeptide is shown to be degraded by intact fat pads, homogenized adipose tissue and adipocytes of the rabbit dependent on the amount of adipose tissue, time and the pH of the incubation medium. In subcellular fractions of rabbit adipocytes the proteolytic activity could be localized into the cytosol and the microsomal fraction. To obtain information about the processing of beta-lipotropin in its target cell lipolysis and degradation of this polypeptide were investigated in the presence of inhibitors of distinct cellular mechanisms and in different physiological states such as obesity and starvation. Thus, the stronger lipolytic response in adipocytes from obese rabbits respectively animals fed ad libitum was accompanied by a significantly increased degradation in comparison to lean respectively starved rabbits. The six lysosomotropic agents (chloroquine, NH4Cl, propranolol, quinacrine, acridine orange and tetracaine), the proteinase inhibitors alpha 2-macroglobulin and monodansylcadaverine, cellular ATP depletion by 2-deoxy-D-glucose and 2,4-dinitrophenol and the omission of Ca2+ ions from the incubation medium inhibited dose-dependently the lipolytic activity as well as the degradation of beta-lipotropin in intact and homogenized adipose tissue. Inhibitors of the cytoskeleton such as colchicine, cytochalasin B, vinblastine and concanavalin A also reduced lipolysis but only the degradation in intact adipose tissue. It can be concluded that after receptor-mediated uptake the cytoskeleton and lysosomal proteases are involved in the processing of beta-lipotropin.

In teleost fishes, the melanotropes of the neurointermediate lobe of the pituitary gland release numerous peptides—adrenocorticotropin (ACTH), melanotropin (MSH), lipotropin (LPH), corticotropin-like intermediate lobe peptide (CLIP), and endorphin—which are derived from the precursor molecule proopiomelanocortin. Superfused, isolated, dispersed goldfish neurointermediate lobe cell columns were used to investigate the release of immunoreactive (ir) α-MSH and ir ACTH from goldfish melanotropes. Stimulation of neurointermediate lobe cell columns with pulses of the structurally homologous peptides, Catostomus urotensin I (UI), ovine corticotropin-releasing factor (oCRF), or sauvagine, produced a significant increase in the concomitant release of ir α-MSH and ir ACTH. UI was two to three times as potent as ovine CRF or sauvagine. These studies suggest that CRF- and UI-like peptides stimulate the secretory activity of teleost melanotropes.

Studies in depression using a maximal stimulatory dose of corticotropin releasing factor have concluded that elevated resting cortisol levels in depressed patients exert a negative feedback effect on the corticotroph, resulting in a decreased corticotropin response. In this preliminary report, we examine the effects of a submaximal dose of corticotropin releasing factor on the release of another corticotroph secretory product, beta-lipotropin-beta-endorphin. We observed a decreased beta-lipotropin-beta-endorphin response in depressed subjects, but a normal adrenal cortisol response. Although the total beta-lipotropin-beta-endorphin response was decreased, the initial secretory response did not differ between patients and normal controls. Rather, the patients appeared to turn off secretion faster. This rapid shutoff was seen in all patients regardless of resting cortisol levels, suggesting that resting cortisol levels alone do not explain the decreased response seen in depressed patients.

Using indirect immunofluorescence with fourteen different antisera raised against pituitary hormones and peptides, we characterized immunochemically the cells of the sheep pars tuberalis. The presence of LH- and FSH-containing cells, shown in previous studies, was also observed in the present investigation. In addition, we found TSH-containing cells, never observed in sheep, and beta LPH-containing cells. The latter hormone has never been found in any studied species. It appeared that a small amount of perikarya (less than 20%) were immunolabelled and, that the sheep pars tuberalis contained a majority of immunonegative cells as in the guinea-pig rabbit and rhesus monkey. This study may contribute to a better knowledge of the function of the sheep pars tuberalis.

RU 486 antagonizes both progesterone and glucocorticoids at the receptor level. To study the duration of RU 486 antiglucocorticoid activity on corticotropic function and to establish the means of overcoming it with dexamethasone, plasma corticolipotropic hormones and cortisol were measured in 10 healthy male patients during the 3 days after intake, at 2200 h, of a single 400-mg dose of RU 486, alone or combined with a single dexamethasone dose (1, 2, or 4 mg) given at 2400 h. On the first day after RU 486 alone, ACTH, lipotropin, and cortisol plasma levels were significantly higher than basal values. The 1-mg dose of dexamethasone totally abolished the stimulatory effect of RU 486, and higher doses of dexamethasone (2 and 4 mg) further depressed hormone levels. During the succeeding days, antiglucocorticoid activity of RU 486 alone was still present 34 h after administration, while on the third day all hormone levels returned to normal. After the combined administration, the RU 486 effect reappeared as early as the first day with the 1-mg dose of dexamethasone, while it was delayed until the third day with the higher doses. These results showed that a single 400-mg dose of RU 486 induced a response that lasted at least 34 h. Thus, a dose-dependent competition between RU 486 and dexamethasone was demonstrated. However, the suppressive effect of dexamethasone was only transient, after which the antiglucocorticoid activity of RU 486 reappeared.

Whole neurointermediate lobes were used to study the processing of the common precursor to β-endorphin and α-melanotropin (melanotropin) during pulse and pulse-chase incubations with various radioactive amino acids. After a 30-min pulse with radioactive amino acids, two major radioactive protein bands with apparent molecular weights of 34000 and 36000 were observed on a 10–30% acrylamide/sodium dodecylsulfate slab gel electrophoretogram. After a 2-h chase, most of the radioactivity associated with these two protein bands had disappeared and was recovered in smaller peptides. Analysis of the tryptic fragments from the 34000-Mr and 36000-Mr proteins showed that both contained amino acid sequences characteristic of adrenocorticotropin (corticotropin) and β -lipotropin. Processing of the two precursor forms involves several proteolytic steps. The first occurring between 30 min and 1h after the start of the incubation, releases the β-lipotropin sequence from the rest of the molecule, leaving two polypeptides with apparent molecular weights of 25000 and 27000. These two peptides are thought to have identical or closely related amino acid sequences differing only in the number of carbohydrate side chains. Analysis of the tryptic peptides of these [3H]phenylalanine-labeled peptides showed that they contained fragments characteristic of corticotropin plus at least another fragment from the non-corticotropin, non-β -lipotropin part of the precursor molecule. Further maturation of the β-lipotropin and the two high-molecular-weight forms of corticotropin occurred between 1 h and 2 h after the start of incubation. β-Lipotropin served as a precursor for β-endorphin while α-melanotropin was generated by cleavage of the two high-molecular-weight forms of corticotropin. Peptides with apparent molecular weights of 17000 and 19000 were also recovered as major and stable end products of the maturation process. Tryptic peptide analysis showed that they were related to the portion of the precursor which does not contain either corticotropin or β-lipotropin. When these peptides, labeled with [3H]leucine or [3H]serine, were analyzed by automated Edman degradation, it was shown that their sequence was identical in both cases to the N-terminal sequence of the initial precursor molecule prepared during the 30-min pulse incubation. From these data, it clearly appears that the 19000-Mr and 17000-Mr peptides are related to the N-terminal fragment of the original precursor molecule.

Levels of beta-endorphin-like immunoreactivity (beta END-LI) in uterine secretions were studied in pseudopregnant and ovariectomized gilts treated with ovarian steroids. Pseudopregnancy was induced in 7 gilts by daily injection of estradiol valerate (5 mg/day) between days 11 and 15 of the estrous cycle. Between days 60 and 90 of pseudopregnancy, uterine secretions were collected by flushing the uterus with 0.9% saline. Uterine flushings were extracted with a Sep-Pak C-18 column and assayed for beta END-LI by a specific RIA. The RIA cross-reacted only with beta-lipotropin (15%). Significant amounts of total immunoreactive beta END-LI were detected in the uterine flushings. The inhibition curve for extracts was parallel to the standard END inhibition curve. Extracted uterine flushings were applied to a Sephadex G-50 column. Three peaks of immunoreactive beta END-LI were detected. One peak eluted at the void volume. The second peak eluted at a Kav of 0.21, which was near the Kav of a lipotropin standard (0.23). The highest peak (Kav = 0.53) eluted similar to standard END (Kav = 0.51). In the second experiment, 12 gilts were ovariectomized on day 4 of the estrous cycle and randomly assigned to 4 groups of 3 gilts each. Each group received 1 of the following daily injections: vehicle (corn oil), 0.1 mg 17 beta-estradiol (E2), 200 mg progesterone (Prog), or a combination of E2 and Prog (E2 + Prog). After treatment for 30 days, beginning on the day of ovariectomy, the uterine flushings were assayed for beta END-LI and total protein. Treatment with Prog increased (P less than 0.05) total beta END-LI 532% (12.4 ng) compared to corn oil treatment (2.3 ng), and levels were significantly higher than with E2 (2.9 ng) or E2 + Prog treatment (5.1 ng). Opiate receptor assay showed that extracts of uterine flushing had a displacement curve parallel to that of standard porcine beta END. Results suggest that significant amounts of immunoreactive beta END-LI are present in uterine secretions of gilts and that the accumulation of beta END-LI is influenced by ovarian steroids.

The concentrations of β-endorphin- and γ-lipotropin-immunoreactivities were determined simultaneously in the extracts of thirteen human hypothalami obtained at autopsy. Tissue concentrations (fmol per mg wet weight, mean ± S E M) were 11.7 ± 2.0 and 51.2 ± 7.1 for the β-endorphin- and γ-lipotropin-immunoreactivities. respectively. The two immunoreactivities were significantly correlated (r = 0.85, p

By immunocytochemical techniques, the neuronal cell bodies containing ACTH, β-LPH and α-MSH have only been found in the area of the arcuate nucleus of the hypothalamus, whereas positive nerve fibers have been observed in many hypothalamic and extra-hypothalamic areas. The possible contribution of neurons which could be located in other brain areas has been studied in the rat by experiments involving hypothalamic deafferentation or destruction of the acurate nucleus. Fourteen days after deafferentation, no immunoreactive fibers could be detected in extrahypothalamic areas whereas the concentration of positive cell bodies and fibers remained unchanged within the hypothalamic island. In rats which had been injected in the neonatal period with monosodium glutamate (MSG), which selectively destroys the arcuate nucleus, only a few immunostained cell bodies were observed in hypothalamic region lateral to the arcuate nucleus. As compared to control animals, the concentration of immunostained fibers in both hypothalamic and extrahypothalamic regions was markedly decreased. These results strongly suggest that neuronal cell bodies producing ACTH, β-LPH and α-MSH are located in the region of the arcuate nucleus and send axonal projections into many brain areas.

Lipotropin, beta-endorphin and a series of peptides related to beta-endorphin were extracted from rat antrum and resolved by gel filtration, ion exchange chromatography and high pressure liquid chromatography; the concentrations of the peptides were determined by radioimmunoassay. The major peptide with beta-endorphin immunoreactivity present in the antrum was lipotropin but it was accompanied by substantial quantities of beta-endorphin in its biologically active form; in addition there were minor quantities of a number of inactive beta-endorphin related peptides. The experiments demonstrate that in rat antrum gastrin can be accompanied by both active and inactive forms of beta-endorphin. The implications of post-translational processing mechanisms common to gastrin and beta-endorphin are discussed.

The beta-endorphin (BE) hypothesis of premenstrual symptomatology was assessed in 15 healthy, drug-free normally cycling women. Cycle-phase assignment was aided by measurement of basal body temperature and plasma progesterone. For the entire group, peripheral plasma BE concentrations by radioimmunoassay (with substantial beta-lipotropin (BL) cross-reactivity) were generally unvarying across the menstrual cycle. High and low symptom subgroups were defined by retrospective and prospective daily self-reports of premenstrual changes. For the cycle studied, moderate to extreme symptom severity was defined according to premenstrual increases in impaired concentration, water retention and negative affect. No between-groups difference was observed for any cycle-phase for absolute values of beta-endorphin (BE/BL). When difference scores were examined, there was a positive time-lag correlation between follicular-to-periovulatory changes in BE/BL and the subsequent luteal increase in impaired concentration over the periovulatory baseline (rs = 0.82); considering the number of comparisons, however, the correlation fails to reach significance. The salience of various types of stressors on mood was assessed; there was a trend for "task-demands" to account for a greater percentage of negative attributions premenstrually compared to post-menstrually, for the entire group. The relative inconsistency in plasma BE/BL from cycle to cycle may be related in part, to the lack of stability in certain cycle characteristics from month to month. Future studies of BE/BL across the menstrual cycle may benefit from a demanding task in a provocative stress-paradigm.

The effects of beta-endorphin (lipotropin 61-91) and related naturally-occurring peptides upon acetylcholinesterase activity in rat hind-limb muscles was investigated. beta-endorphin weakly inhibited the activity in a plasma membrane-enriched fraction. The inhibition by beta-endorphin of the membrane-associated acetylcholinesterase was less marked when the fractions were prepared from muscles which had been denervated 4-6 days previously. The membrane-associated acetylcholinesterase was solubilised from normal muscle preparations and separated by sucrose density gradient centrifugation into three major peaks (16S, 10S and 4S). beta-Endorphin inhibited the activity in the 16S peak but not that in the 10S and 4S peaks, whilst tensilon, a competitive inhibitor of acetylcholinesterase, inhibited the activity of all three peaks. beta-Endorphin inhibited the activity in the 16S peak but not that in the 10S and 4S peaks, whilst tensilon, a competitive inhibitor of acetylcholinesterase, inhibited the activity of all three peaks. beta-Endorphin inhibited the 16S activity in a concentration-dependent manner and its action was partly prevented if naloxone was added simultaneously. Purified natural porcine and bovine beta-endorphin were equipotent in terms of effective concentration range but the maximum inhibition was greater with the bovine peptide. beta-Lipotropin was approximately 4 times less potent than beta-endorphin, whilst C-fragment (lipotropin 61-87) was 100 times less potent. Prolonged treatment with collagenase did not reduce the catalytic activity of 16S acetylcholinesterase, but it was no longer susceptible to the inhibitory action of beta-endorphin. Kinetic studies indicated a complex type of inhibition by beta-endorphin (hyperbolic Lineweaver-Burke plot). Methionine enkephalin inhibited acetylcholinesterase in a weakly non-competitive manner and its action was not abolished if the enzyme was predigested with collagenase. beta-Endorphin produces a novel form of inhibition of acetylcholinesterase, acting only on the 16S (A12 or 'motor endplate-specific') form of the enzyme. The findings are discussed in the light of evidence that beta-endorphin-related immunoreactivity is expressed in motor nerve axons in the immature rat.

Secretory granules (SGs) from rat intermediate lobes (IL) were isolated in a highly purified form by differential centrifugation, followed by sucrose density gradient centrifugation. The purified IL-SGs were lysed by freezing and thawing. The granule lysate was then centrifuged to generate membrane and soluble fractions. Proopiocortin -converting enzyme (PCE) activity was assayed by incubation of [3H]arginine- or [3H] phenylalanine-labeled toad proopiocortin with the total granule lysate, the membrane, or the soluble fraction at pH 5.0. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea-gel electrophoresis. The PCE activity in rat IL-SG lysate cleaved proopiocortin to 21,000 mol wt ACTH, 21,000 mol wt ACTH/lipotropin (LPH), 13,000 mol wt ACTH, beta LPH, beta-endorphin-like peptides, and alpha MSH-like peptides, similar to those synthesized by the toad intermediate lobe in situ. Treatment of the PCE cleavage products with carboxypeptidase B resulted in the liberation of free arginine. This observation together with the nature of the products formed suggest that the PCE activity cleaved at pairs of basic residues of proopiocortin , yielding one or more products that terminated with an arginine or an arginine-lysine. PCE activity was found in membrane and soluble granule fractions, and both activities were inhibited by leupeptin, p-chloromercuribenzoate, dithiodipyridine, and pepstatin A, but not by chloroquine or N-alpha-p-tosyl-L-lysine-chloromethylketone HCl. Diisopropyl fluorophosphate and other thiol protease reagents (p-chloromercuriphenyl sulfonic acid, iodoacetic acid, and HgCl2) had a small inhibitory effect. The products formed by PCE activities in the membrane and soluble fractions were similar to those cleaved by the total granule lysate. The membrane fraction primarily cleaved proopiocortin between ACTH and beta LPH to form 21,000 (21 K) mol wt ACTH and beta-LPH, similar to the first processing step in the IL in situ. The soluble fraction, however, showed a greater tendency to cleave proopiocortin between the 16 K N-terminal glycopeptide and ACTH, to yield twice as much 21 K ACTH/LPH product as the membrane fraction. The membrane-associated PCE activity was found to be easily solubilized by extraction with high salt (1 M NaCl), suggesting that it is not an integral granule membrane protein.

Using HeLa whole cell extracts, we have demonstrated that transcription in vitro of the cloned human and bovine corticotropin/beta-lipotropin precursor genes is initiated accurately and efficiently. DNA sequences required for promoter function have been assessed by using a series of 5'-deletion mutants of a fusion gene that contains the 5'-flanking sequence and capping site of the human corticotropin/beta-lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The results obtained have shown that the region between 22 base pairs and 35 base pairs upstream from the capping site is essential for the correct and efficient transcriptional initiation in vitro. Thus, the 'TATA box' present in this region seems to be the main promoter element for transcription of the human corticotropin/beta-lipotropin precursor gene in the HeLa cell-free system. We have also developed a transcription system in vitro from the corticotropin-producing mouse pituitary tumor cell line AtT-20 in culture. Deletion mapping of the fusion gene promoter has indicated that the 'TATA box' region is required for the accurate and efficient transcriptional initiation in this system as well. Characteristic of this system is that the deletion of the sequence lying between 53 base pairs and 59 base pairs upstream from the capping site increases the transcriptional efficiency. Because this effect is observed in the AtT-20 cell-free system, but hardly in the HeLa cell-free system, it seems reasonable to assume that the interaction of this upstream sequence with some factor(s) in the AtT-20 cell extract is responsible for the modulation of transcription of the human corticotropin/beta-lipotropin precursor gene.

The accumulation of immunoreactive ACTH (ACTHi), alpha MSH (alpha MSHi), and gamma-lipotropin (gamma LPHi) as a function of age (10-120 days) was determined in three regions of the brain of male rats: the medial basal hypothalamus (MBH), the preoptic anterior hypothalamus (POA), and the thalamus. In each region of the brain, the concentrations of ACTHi, alpha MSHi, and gamma LPHi increased with age. In the MBH, the increase occurred in such a manner that the molar ratio of alpha MSHi to ACTHi remained constant regardless of the age of the animals. In contrast, in the POA and thalamus, the increase occurred disproportionately in favor of alpha MSHi, and thus the molar ratio of alpha MSHi to ACTHi was 3 times higher in the adult (120 days old) than in the young (10 or 21 days old) animals. Nevertheless, the ratio of (ACTHi plus alpha MSHi) to gamma LPHi was constant at a level of about 2 regardless of the age of the animal or the region of the brain. Extracts of the MBH or POA were fractionated on columns of Sephadex G-75 superfine. The gel filtration profiles of ACTHi were indicative of the presence of five molecular weight forms of ACTH: greater than 40K, 30-40K, 20-30K, 5.7K, and 4.5K. We tentatively identified greater than 40K ACTH as a large form of proopiocortin, 30-40K ACTH as proopiocortin, 20-30K ACTH as ACTH biosynthetic intermediate, 5.7K as glycosylated ACTH(1-39), and 4.5K ACTH as ACTH-(1-39). Regardless of the age of the animals, the fractional amount of 30-40K ACTH the age of the animals, the fractional amount of 30-40K ACTH was high in the MBH compared to that in the POA. Moreover, the small fractional amount of 30-40K ACTH in the POA was associated with a large fractional amount of small molecular weight forms of ACTHi. However, the predominant form of ACTHi in the POA changed with age: 20-30K ACTH was the major form in the young, whereas 4.5K ACTH was the major form in the adult. These results support the proposal that the production of proopiocortin increases with age, and there is enhanced processing of proopiocortin to ACTH-(1-39) and alpha MSH in the brain of the maturing rat.

Chromatography under acid dissociating conditions in conjunction with radioimmunoassay has been employed to investigate the nature of peptides related to opiocortin in human cerebrospinal fluid. Samples of cerebrospinal fluid (CSF) were collected for chromatography from 15 patients prior to air encephalography. 2 patients had pituitary dependent Cushing's disease, 3 non-endocrine neurological disease and 10 non-ACTH related pituitary disease. The column fractions were assayed for N- and C-terminal beta-lipotropin, N-terminal ACTH and gamma-MSH immunoreactivity. Elution profiles obtained from chromatography on Sephadex G-50 demonstrated peaks of immunoreactivity corresponding to the elution positions of synthetic human beta-endorphin, highly purified beta-lipotropin and highly purified gamma-lipotropin in all CSF samples. A peak of a large molecular weight material with N and C terminal beta-lipotropin immunoreactivity was also detected. Chromatography of CSF on Sephadex G-75 showed this large molecular weight peak to be comprised of peptides eluting in the positions of a 31K molecular weight marker with beta-lipotropin and ACTH immunoreactivity and a 16K molecular weight marker with gamma-MSH immunoreactivity. This suggests the presence of the common precursor to ACTH and LPH in the CSF.

Addition of radiolabeled β-endorphin, β-lipotropin and γ-lipotropin to plasma at acid pH results in an apparent reduction in size of these molecules as evidenced by gel permeation chromatography. These acid plasma-treated molecules, however, are indistinguishable from the untreated radiolabeled polypeptides when subjected to high performance liquid chromatographic separations suggesting no differences in molecular composition. As these apparent changes in polypeptide molecular volume are prevented by addition of Trasylol or sodium azide to the plasma, a likely explanation would appear to be an enzyme-dependent production of anionic lipids in plasma which at acid pH bind to the lipotropins and endorphins reducing their molar volume.

Distribution constants of 14 acetylamino acid amides were measured in two solvent systems useful for partition chromatography of peptides. Free energies of transfer of side-chains, expressed by the chromatographic parameter Δ RM, linearly correlated with calculated surface areas for amino acids with hydrocarbon side-chains. Values for a variety of side-chains linearly correlated with Δ RM (side-chain) values previously measured for the 31-residue peptide β-endorphin. It was concluded that accessibility of the amino acid residues in β-endorphin to the solvent environment can explain its separation from some very closely related analogues. High-performance liquid chromatography was inadequate for effecting these separations.

[Met]enkephalin and [Leu]enkephalin are derived from a protein in bovine adrenal medulla that contains multiple copies of [Met]enkephalin [Kilpatrick, D. L., Taniguchi, T., Jones, B. N., Stern, A. S., Shively, J. E., Hullihan, J., Kimura, S., Stein, S. & Udenfriend, S. (1981) Proc. Natl. Acad. Sci. USA 78, 3265--3268.] Here we characterize pro-enkephalin mRNA from bovine and human tissue by use of an oligodeoxynucleotide pentadecamer probe complementary to codons for Tyr-Gly-Gly-Phe-Met ([Met]enkephalin). This probe hybridizes specifically to a species of poly(A)-RNA from adrenal medulla and human pheochromocytoma, (1400--1450 bases), and also to [Met]enkephalin-containing pro-opiomelanocortin mRNAs from bovine pituitary (1200 bases) and from mouse pituitary tumor cell (1100 bases). A cloned cDNA probe (144 bases) complementary to the region of pro-opiomelanocortin mRNA that codes for lipotropin does not hybridize to the RNA from bovine adrenal medulla, demonstrating that the latter RNA is not pro-opiomelanocortin mRNA. The pentadecamer probe was extended to make cDNA with reverse transcriptase after hybridizing it to adrenal poly(A)-RNA. The sequence of an extended cDNA, 62 bases in length, was found to correspond exactly to that expected from the amino acid sequence of peptide E (a bovine adrenal peptide containing [Met]- and [Leu]enkephalin sequences). This cDNA also forms a specific hybrid with the RNA from bovine adrenal and human pheochromocytoma, confirming that these species of RNA are pro-enkephalin mRNA.

Using the peroxidase/antiperoxidase unlabeled antibody enzyme technique pituitaries of several vertebrate species: man, rat, pig, cat, duck, snake, toad, and fish immunostained with an antibody directed against a portion of the cryptic region of the corticotropin/lipotropin common precursor. In all of them, pars distalis corticotropes and pars intermedia melanotropes were positively stained. Specificity studies and controls performed ruled out the possibility of cross-reaction with other known pituitary hormones or peptides. The wide distribution of cross-reaction with the antibody among vertebrate species suggests that a sequence of the cryptic region may be responsible for a yet undiscovered biological effect.