In vitro and in vivo studies of antitumor effects of the recombinant immunotoxin MSH-PE38KDEL on melanoma.

MSH-PE38KDEL is a chimeric molecule composed of MSH, and fused to a truncated mutant form of Pseudomonas exotoxin (PE38KDEL). Our study aims to evaluate the specific cytotoxicity of recombinant immunotoxin MSH-PE38KDEL on melanoma cells A875 and B16 in vitro, as well as its inhibition of metastatic melanoma in vivo. MSH-PE38KDEL was expressed in Escherichia coli, and greater than 90% purity was obtained. The purified MSH-PE38KDEL was found to be selectively cytotoxic to MSH receptor-positive melanoma cells in vitro. The specific cytotoxicity of recombinant MSH-PE38KDEL to A875 and B16 was over 85% by cell viability assay; however, MSH-PE38KDEL had no cytotoxicity to the human 2BS cells. The anti-tumor activity of MSH-PE38KDEL was evaluated in mice with induced melanoma through intra-tumor or intravenous administration. The results showed that 90% melanoma growths were inhibited, and 40% of the tumors were disappeared completely. Histopathology results showed MSH-PE38KDEL can effectively inhibit intrahepatic metastasis. In conclusion, MSH-PE38KDEL had cytotoxic effects on MSH receptor-positive melanoma cells, and causes significant tumor growth inhibition. These results support a possible new approach for the treatment of melanoma.

Current Aspects of the Role of Autoantibodies Directed Against Appetite-Regulating Hormones and the Gut Microbiome in Eating Disorders.

The equilibrium and reciprocal actions among appetite-stimulating (orexigenic) and appetite-suppressing (anorexigenic) signals synthesized in the gut, brain, microbiome and adipose tissue (AT), seems to play a pivotal role in the regulation of food intake and feeding behavior, anxiety, and depression. A dysregulation of mechanisms controlling the energy balance may result in eating disorders such as anorexia nervosa (AN) and bulimia nervosa (BN). AN is a psychiatric disease defined by chronic self-induced extreme dietary restriction leading to an extremely low body weight and adiposity. BN is defined as out-of-control binge eating, which is compensated by self-induced vomiting, fasting, or excessive exercise. Certain gut microbiota-related compounds, like bacterial chaperone protein Escherichia coli caseinolytic protease B (ClpB) and food-derived antigens were recently described to trigger the production of autoantibodies cross-reacting with appetite-regulating hormones and neurotransmitters. Gut microbiome may be a potential manipulator for AT and energy homeostasis. Thus, the regulation of appetite, emotion, mood, and nutritional status is also under the control of neuroimmunoendocrine mechanisms by secretion of autoantibodies directed against neuropeptides, neuroactive metabolites, and peptides. In AN and BN, altered cholinergic, dopaminergic, adrenergic, and serotonergic relays may lead to abnormal AT, gut, and brain hormone secretion. The present review summarizes updated knowledge regarding the gut dysbiosis, gut-barrier permeability, short-chain fatty acids (SCFA), fecal microbial transplantation (FMT), blood-brain barrier permeability, and autoantibodies within the ghrelin and melanocortin systems in eating disorders. We expect that the new knowledge may be used for the development of a novel preventive and therapeutic approach for treatment of AN and BN.

Immunological block to synthetic alpha-melanocyte-stimulating hormone: melanocyte interaction by antibodies isolated from cell-column immunoadsorbents.

Antibodies to formalin-fixed, syngeneic melanoma cells were prepared in mice, purified by immunoaffinity chromatography, and tested for binding activity to viable melanoma cells. The radiolabeled antibodies detected congruent to 9 X 10(6) melanoma antigenic sites/cell. The calculated average association constant (Ka) for the antibody population was 7 to 10 X 10(7) M-1. The antibody was shown to block the binding of melanocyte-stimulating hormone in competitive cell surface binding studies. Results are discussed conceptually in terms of the potentially important role that the humoral immune response may play in the phenomenon of tumor progression.

Evidence that the pars intermedia and pars nervosa of the pituitary do not secrete functionally significant quantities of ACTH.

We have compared the capacity to secrete ACTH in response to stress or adrenalectomy in control rats and in those with total hypophysectomy (H), adenohypophysectomy (AH) with preservation of the intermediate and the neural lobes, neurohypophysectomy (NH) with removal of the pars nervosa and all or part of the pars intermedia with preservation of the adenohypophysis, or incomplete adenohypophysectomy (IAH) in which a portion of the adenohypophysis and all of the pars intermedia and pars nervosa were left intact. Plasma ACTH measured with an N-terminal antibody that reacts on an equimolar basis with ACTH and alpha-MSH but not with other known pituitary hormones was elevated after ether or tourniquet stress in all except the H group. Three weeks after adrenalectomy there was an elevated basal plasma ACTH and an augmented ACTH response to stress in intact and IAH but not in AH rats. When a more specific alpha11-24 ACTH antibody was used there was a high plasma ACTH after ether stress in the IAH, NH, and intact groups but not in the AH or H groups. Adrenal weight and plasma corticosterone after tourniquet or ether stress were indistinguishable in the AH and H groups and were much higher and nearly identical in the intact, NH and IAH groups. We conclude that only the adenohypophysis secretes functionally significant amounts of ACTH. Plasma ACTH detected by the N-terminal antibody in the AH group is probably related to alpha-MSH or similar peptides and is incapable of maintaining adrenal weight or stimulating corticosterone secretion.

Stress-induced secretion of alpha-melanocyte-stimulating hormone and its physiological role in modulating the secretion of prolactin and luteinizing hormone in the female rat.

The pattern of alpha MSH release during immobilization stress in ovariectomized rats was determined and correlated with that of plasma PRL and LH. Stress induced a marked elevation in plasma immunoreactive alpha MSH, with a time course identical to that of plasma PRL. The increment in plasma PRL was greater than that in plasma alpha MSH. Plasma LH was markedly lowered by stress. Analysis of pituitary and hypothalamic alpha MSH indicated a significant (P less than 0.05) increase in the neurointermediate lobe and anterior lobe content of alpha MSH. The alpha MSH content in the hypothalamus was lowered by stress when expressed as tissue content (P less than 0.025), although no significant differences in content in this area were detected when the results were expressed in terms of tissue protein. Stress induced a marked increase (P less than 0.01) in the median eminence levels of alpha MSH. Intraventricular (third ventricle) injection of the gamma-globulin fraction of a specific antiserum raised against alpha MSH increased basal PRL levels (P less than 0.025) and prevented the decline in plasma PRL that occurred 60 min after the onset of stress in the normal rabbit serum-injected rats. The stress-induced suppression of plasma LH was attenuated and delayed by the administration of alpha MSH antibodies. In conclusion, alpha MSH of brain origin is released during stress and is involved in lowering plasma PRL to basal levels and producing a partial suppression of plasma LH.

Immunoreactive alpha-MSH and ACTH levels in rat plasma and pituitary.

A radioimmunoassay is described for the measurement of alpha-melanocyte-stimulating hormone (alpha-MSH). The antibody was produced in rabbits by immunization with alpha-MSH coupled to bovine serum albumin with carbodiimide. The antibody did not react significantly with ACTH, beta-MSH, or 6 fragments of ACTH. The sensitivity and reliability of the assay were improved by employing a simple plasma extraction procedure. When applied to a 2 ml plasma sample, the detection limit of the radioimmunoassay was 6 pg/ml. ACTH was measured with a sensitive and specific radioimmunoassay previously described for humans and adapted for the rat. The anti-ACTH serum cross-reacted with the biologically active portion of alpha-p ACTH and not with alpha-MSH, beta-MSH or the alpha-p 17-39 and alpha-p 25-39 fragments of ACTH. The detection limit was 20 pg/ml. Plasma and pituitary alpha-MSH and ACTH had the same immunoreactivity as synthetic alpha-MSH and ACTH. alpha-MSH and ACTH contents of the rat neurointermediate lobe were 1398 +/- 360 (SE) ng and 28.2 +/- 2.9 ng, respectively, while in the anterior lobe they were 102 +/- 31 ng and 551 +/- 36 ng, respectively. The plasma alpha-MSH concentration at 8 AM in male rats was 64 +/- 8 pg/ml when the plasma ACTH concentration was 92 +/- 15 pg/ml. Over a 24-hour period two peaks of plasma alpha-MSH were observed, one at 4 AM (142 +/- 35 pg/ml) and the other at 4 PM (139 +/- 26 pg/ml). Plasma ACTH was higher at noon (151 +/- 43 pg/ml) and 4 PM (130 +/- 48 pg/ml). Short-term exposure to ether induced a transient increase in alpha-MSH level 5 min later and a rapid return to normal levels. Plasma ACTH increased significantly 2.5 min after the onset of ether stress and remained high for 30 min. Two hours' exposure to ether did not change plasma alpha-MSH, although a 3-fold increase in plasma ACTH was observed. Haloperidol injection was followed by a large increase in plasma alpha-MSH, whereas ACTH levels increased similarly after saline and Haloperidol injection. Corticoid administration reduced ACTH, but not alpha-MSH. Three weeks after adrenalectomy, alpha-MSH levels had not changed but ACTH levels had increased ten-fold. These data indicate that alpha-MSH is secreted in the rat, and that the regulation of its secretion is different from that of ACTH.

A gamma-melanocyte stimulating hormone-like peptide causes reflex natriuresis after acute unilateral nephrectomy.

Previous studies have shown that acute unilateral nephrectomy stimulates sodium (UNaV) and potassium (UKV) excretion by the remaining kidney through reflex pathways requiring an intact pituitary gland, and the natriuresis is accompanied by an increase in the plasma concentration of a peptide or peptides derived from the adrenocorticotropic hormone-beta-endorphin precursor molecule pro-opiomelanocortin. We tested the hypothesis that gamma-melanocyte stimulating hormone (gamma-MSH) was such a peptide involved in the postnephrectomy natriuresis. In six rats undergoing sham nephrectomy, no change in UNaV or UKV occurred and plasma immunoreactive gamma-MSH-like material was 40 +/- 18 (SD) fmol/ml 2 hours after the sham procedure. In 10 rats undergoing acute unilateral nephrectomy, UNaV and UKV from the remaining kidney increased significantly, and immunoreactive gamma-MSH was 81 +/- 36 fmol/ml (p less than 0.02). In individual studies, the increase in UNaV after nephrectomy correlated with the postnephrectomy concentration of immunoreactive gamma-MSH (r = 0.75, p less than 0.001). In 17 rats injected with serum or globulin from control rabbits, unilateral nephrectomy led to the expected increases in UNaV and UKV. In 23 rats injected with serum or globulin from rabbits immunized against gamma-MSH, no postnephrectomy natriuresis occurred and the kaliuresis was blunted. In hydropenic, mineralocorticoid-treated rats, intravenous infusion of synthetic gamma-MSH led to natriuresis and kaliuresis with no change in inulin clearance; pretreatment with rabbit anti-gamma-MSH antiserum blocked this effect of peptide infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Development and validation of a radioimmunoassay for peptides related to beta-melanocyte-stimulating hormone in human plasma: the lipotropins.

A radioimmunoassay is described for the measurement of human "beta-melanocyte-stimulating hormone" ("betah-MSH"). Two antisera have been used, one of which cross-reacts with synthetic betah-MSH as well as with the two larger pituitary peptides betah- and gammah-lipotropin (betah- and gammah-LPH) and the other mainly with betah-MSH and gammah-LPH. The sensitivity and reliability of the assay have been improved by employing a simple plasma extraction procedure, and the shelf-life of the iodinated betah-MSH tracer has been increased more than five-fold by storage in a concentrated human serum albumin solution. Using a 5 ml plasma sample the detection limit is 6 pg/ml. The mean resting "betah-MSH" level in normal subjects is 21 pg/ml (range 13-38 pg/ml) at 9 AM and 12 pg/ml (range 6-20 pg/ml) at 9 PM. Levels are considerably elevated (51-12,000 pg/ml) in patients with Addison's disease. Nelson's syndrome, Cushing's disease and the "ectopic" ACTH syndrome. After administration of insulin or pyrogen, the concentration of plasma "betah-MSH" increases in parallel with that of ACTH and they are approximately equivalent on a molar basis. The stability of purified betah- and gammah-LPH and endogenous "betah-MSH" when incubated in vitro in fresh blood or plasma are similar, in contrast to the less stable peptide synthetic betah-MSH. It is suggested that "betah-MSH" immunoreactivity in human plasma is due to betah- and gammah-LPH rather than betah-MSH.

Size heterogeneity of beta-MSH in ectopic ACTH-producing tumors: presence of beta-LPH-like peptide.

Gel chromatographic, immunologic and biologic properties of beta-melanocyte-stimulating hormone (beta-MSH) in tumor tissues obtained from eight patients with the ectopic ACTH syndrome were studied and compared to those of pituitary beta-MSH. Size heterogeneity of immunoreactive beta-MSH was found in all the tumors studied as well as in normal human pituitaries. Both the tumors and pituitaries contained immunoreactive beta-MSH of a larger molecular size than the well-characterized beta-MSH of small molecular size. The large molecular weight beta-MSH also predominated in the plasma. It was found to be bioactive by an in vitro MSH assay, immunologically indistinguishable from human beta-MSH, and chromatographically very similar to beta-lipotropic hormone (beta-LPH). Tryptic digestion of the large molecular weight beta-MSH under controlled conditions promptly produced bioactive beta-MSH of small molecular size, followed by the appearance of immunologically active but biologically inert fragments. These results suggest that the ectopic ACTH-producing tumor as well as the pituitary elaborate beta-LPH-like peptide which might be the predominant component of immunoreactive beta-MSH in man.

Pituitary and plasma lipotropins: demonstration of the artificial nature of betaMSH.

Evidence has been presented by others that betaMSH immunoreactivity in human plasma is due to beta and gamma lipotropin rather than betaMSH. We have studied this question in normal subjects utilizing a sensitive human betaMSH radioimmunoassay capable of quantifying betaMSH in unextracted plasma with a sensitivity of 7.5 pg/ml. Purified human beta lipotropin cross-reacted 30% on a molar basis with synthetic human betaMSH in this assay. ACTH related peptides showed less than 0.1% cross-reactivity. Normal values at 0800 h were 19.6+/-2.4 pg/ml and suppressed to 9.3+/-1.3 pg/mol following dexamethasone. Metyrapone increased 0800 h values to 379.6+/-89.9 pg/ml. Chromatographic studies on Sephadex G-50 demonstrated no betaMSH per se in human pituitaries, plasma from metyrapone treated normals, patients with Cushing's disease. Nelson's syndrome, or Addison's disease. betaMSH immunoreactivity showed the elution pattern of beta lipotropin.

Direct measurement of the precursors of adrenocorticotropin in human plasma by two-site immunoradiometric assay.

An immunoradiometric assay (IRMA) for the direct measurement of the precursors of ACTH in unextracted human plasma has been developed and evaluated clinically in normal subjects and patients with disorders of the hypothalamic-pituitary-adrenal axis. The IRMA is based on an iodinated monoclonal antibody to ACTH and a monoclonal antibody to gamma MSH coupled to Sephacryl S300. The assay detects only peptides containing both epitopes, i.e. POMC (31K) and pro-ACTH (22K). The reference standard was partially purified POMC from culture medium of human corticotroph adenoma cells. The detection limit (greater than +2.5SD of the 0 standard) was 2.0 pmol/L and the within-assay coefficient of variation was less than 10% between 29 and 2600 pmol/L. Plasma concentrations of ACTH precursor peptides in 11 normal subjects sampled at 0930 h ranged from 5-34 pmol/L. The concentrations in the patient groups studied were: 260-2300 pmol/L in 5 patients with the ectopic ACTH syndrome associated with small cell lung cancer, less than 2.0-104 pmol/L in 10 patients with pituitary-dependent Cushing's disease, 23 pmol/L in a patient with Nelson's syndrome, and 3.0-230 pmol/L in 5 patients with Addison's disease. We conclude that this IRMA offers a simple and reliable method for measuring ACTH precursors in unextracted plasma. The proportionately greater elevation of ACTH precursors compared to ACTH in patients with the ectopic ACTH syndrome associated with small cell lung cancer but not in pituitary-dependent Cushing's syndrome, suggests that this assay may be clinically useful.

Isolated adrenocorticotropin deficiency associated with an autoantibody to a corticotroph antigen that is not adrenocorticotropin or other proopiomelanocortin-derived peptides.

A 44-yr-old man with hypocortisolism was shown to have an undetectable basal plasma ACTH level and absent or subnormal ACTH and beta-lipotropin responses to provocative testing with insulin, vasopressin, and CRH. Endocrine function after glucocorticoid replacement was otherwise normal, thus establishing the diagnosis of isolated ACTH deficiency. This patient's serum was tested immunohistochemically for the presence of an antipituitary antibody by indirect immunofluorescence of rat pituitary tissue. Positive immunostaining was observed in stellate-shaped cells in the anterior and intermediate lobes. Immunopositive cells were shown by immunoelectron microscopy to have ultrastructural characteristics of corticotrophs. Immunoreactivity was concentrated in secretory granules 120-170 nm in diameter. In a double immunolabeling procedure, staining by the patient's serum was shown to colocalize with rabbit antiserum to ACTH, but not with antisera to PRL, GH, beta TSH, or beta LH. Immunoabsorption of the patient's serum with ACTH-(1-24), ACTH-(1-39), gamma MSH, corticotropin-like intermediate lobe peptide, beta-endorphin, or beta-lipotropin failed to diminish immunolabeling in the pituitary. We conclude that the antipituitary antibody in this patient's serum shows immunohistochemical specificity for a rat corticotroph antigen located in secretory granules that is neither ACTH nor any of the proopiomelanocortin (POMC)-derived peptides tested. The autoantigen could be a cell-specific granular factor involved in the posttranslational processing of POMC or secretion of ACTH. We postulate that an autoimmune process may account for this patient's disease, and that his antipituitary antibody could play a pathogenic role by either inhibiting a POMC-processing enzyme or initiating an antibody-dependent cell-mediated cytotoxicity reaction, resulting in the selective destruction of corticotrophs.

The effect of chlorpromazine on the secretion of immunoreactive beta-MSH and prolactin in man.

The effect of chlorpromazine (50 mg. im) on the plasma concentration of immunoreactive beta-melanocyte-stimulating hormone (beta-MSH) and prolactin was studied in 8 hospitalized subjects with non-endocrine skin disorders. Plasma beta-MSH concentrations remained unchanged over a period of 7 h in 6 subjects. In the remaining 2 subjects there was a slight increase. Plasma prolactin concentrations were greatly increased in all subjects 1 1/2-3 h after the injection and had almost returned to pre-injection levels by 7 h. This suggests that the control of beta-MSH secretion in man, unlike that of prolactin in man and MSH peptides in other mammals, is not predominantly inhibitory. The reason for this discrepancy may be that beta-MSH is not a natural MSH in man and occurs as part of the lipotropic hormone (LPH) or as a breakdown product.

gamma 1-Melanotropin-like immunoreactivity in bovine and human adrenocorticotropin-producing tissues.

gamma 1 MSH-like immunoreactivity in bovine pituitary glands, hypothalami, human pituitary glands, ectopic ACTH-producing tumors, and human gastric antral mucosa was investigated using a newly developed RIA for gamma 1 MSH. The minimum detectable quantity of gamma 1 MSH was 3 pg/tube, and the antibody did not cross-react with gamma 2 MSH, gamma 3 MSH, alpha MSH, beta MSH, ACTH, beta-lipotropin, or beta-endorphin. Bovine anterior pituitary, intermediate-posterior pituitary, and hypothalamus contained more than two molecular weight forms of gamma 1 MSH-like immunoreactivities. On the other hand, gamma 1 MSH-like immunoreactivity was not detected in human pituitary glands or human gastric antral mucosa, but was detected in one of seven ectopic ACTH-producing tumors. These results suggest a limited processing of the ACTH-beta-lipotropin precursor to gamma 1 MSH in human tissues, although a rapid degradation of gamma 1 MSH could not be ruled out completely.

alpha-MSH and neurofilament M-protein share a continuous epitope but not extended sequences. An explanation for neurofibrillary staining with alpha-MSH antibodies.

The long recognized neurofibrillary immunoreactivity of the nervous system with alpha-MSH antibodies arises from an epitope on neurofilament M-protein which we have now characterized. It is situated in the amino terminal residues where M-protein and alpha-MSH exhibit similar but not identical sequences. Their divergence past residue 5 precludes a physiological significance of the crossreactivity which seems to have arisen fortuitously. Our results question previous speculations as to the existence of extrapituitary alpha-MSH-like hormones.

A diffuse alpha MSH-immunoreactive projection to the hippocampus and spinal cord from individual neurons in the lateral hypothalamic area and zona incerta.

The course, distribution, and possible neurotransmitter specificity of a projection from the lateral hypothalamic area (LHA) and zona incerta to the hippocampal formation (dentate gyrus, Ammon's horn, subicular region, and entorhinal area) and spinal cord were examined anatomically in the adult rat. First, small injections of the fluorescent tracer fast blue were made into either the septal part of the dentate gyrus and Ammon's horn or the entorhinal area, and the distribution of retrogradely labeled cells was plotted. In each experiment many cells were labeled in the LHA and zona incerta, and little evidence for a topographically organized projection to different parts of the hippocampal formation was found. Second, a combined retrograde transport-immunofluorescence method was used to show that some 95% of the fast blue-labeled neurons in the LHA and zona incerta were also stained with an antiserum to the opiate peptide alpha-melanocyte-stimulating hormone (alpha MSH), but not an antiserum to adrenocorticotropin (ACTH)1-24. It was also found that small numbers of retrogradely labeled neurons were stained with antisera to somatostatin 14 and 28, dynorphin (1-17), and angiotensin II. Third, the distribution of alpha MSH-immunoreactive fibers was plotted, and they were found to form a diffusely organized plexus throughout all of the subfields of the hippocampal formation. These fibers were virtually eliminated after transections of the fimbria and the region between the entorhinal area and the caudal amygdala. Forth, the course of fibers from the LHA and zona incerta was examined with the anterogradely transported lectin Phaseolus Vulgaris Leucoagglutinin (PHAL). Such fibers reach the hippocampal formation by a dorsal route through the septal region and fimbria, and by a ventral route through the amygdala. And fifth, double retrograde transport and immunohistochemical methods were used to show that at least some alpha MSH-stained neurons in the LHA and zona incerta give rise to collaterals that innervate both the hippocampal formation and the spinal cord. Alpha MSH-stained fibers in the spinal cord also form a widely scattered plexus with no obvious circumscribed terminal fields. It is suggested that the diffusely organized projection from the LHA to the cerebral cortex and spinal cord may play a role in the general arousal associated with a variety of motivated behaviors.

Primary olfactory fibres project to the ventral telencephalon and preoptic region in trout (Salmo trutta): a developmental immunocytochemical study.

We studied the development of the primary olfactory system of a teleost, the brown trout, with the aims of clarifying whether the caudal projection pertains to the olfactory or to the terminal nerve system, of identifying the brain regions receiving this projection, and of investigating its possible functional significance. As olfactory markers (OMs) we used two polyclonal antibodies (to substance P and to alpha-melanocyte-stimulating hormone) that were found to label the olfactory projection strongly after preadsortion of the antibody with the corresponding antigen (OMs), and as a terminal nerve marker we used an antiserum to FMRF-amide peptide. OM labelling was observed in both perikarya and axons of olfactory neurons. In adults, olfactory neurons projected not only to olfactory glomeruli in the olfactory bulb but also, as has been reported previously, to more caudal targets in the forebrain through the medial olfactory tract. Our results show that these targets include the ventral and commissural nuclei of the area ventralis telencephali, the periventricular preoptic region, and the organum vasculosum laminae terminalis. Glomeruli were not observed before hatching, and the extrabulbar olfactory projections appear late in development. Extensive periventricular preoptic olfactory plexuses and olfactory innervation of the organum vasculosum laminae terminalis did not appear until adulthood. The cells of the ganglion nervus terminalis, which form ganglionic groups along the olfactory nerves, were not stained with these olfactory markers at any developmental stage studied, nor was the medial olfactory tract FMRP-amide peptide immunoreactive. Our results thus confirm the existence of primary olfactory projections to extrabulbar targets in trout. The target regions identified in this study are implicated in sexual behaviour: We discuss the related possibility that, in teleosts, these extrabulbar olfactory projections (rather than projections of the terminal nerve, as is widely held) are the primary mediators of neuroendocrine response to pheromones.

Presence of an alpha-melanocyte-stimulating hormone-like epitope in the 150,000 dalton neurofilament subunit from diverse regions of the central nervous system: an immunohistochemical and immunoblot study in guinea pig.

Monoclonal antibodies specific for the two higher molecular weight neurofilament (NF) subunits (NF200 and NF150), and antiserum to alpha-melanocyte-stimulating hormone (alpha-MSH) were used to probe the distribution of an alpha-MSH-like epitope in NF proteins of the guinea pig central nervous system using immunoblot and immunohistochemical methods. The anti-alpha-MSH antiserum recognized the same protein band as an anti-NF150 monoclonal antibody in immunoblots of proteins extracted from guinea pig cerebellum, spinal cord, retina, optic nerve, and neurohypophysis; it also stained axons and dendrites in sections of cerebellum, retina, and optic nerve. Although all cells of the pars intermedia and some in the pars distalis exhibited immunoreactivity with this antiserum, it did not stain axons in the neurohypophysis. Our immunoblot data demonstrate an alpha-MSH-like epitope in NF150 extracted from each of the regions studied. The lack of in situ recognition of this alpha-MSH-like epitope in neurophypophyseal axons, using the same immunohistochemical methods that demonstrate this epitope in axons of the cerebellum, retina, and optic nerve, suggests that NF150 is immunochemically heterogeneous in different regions of the guinea pig central nervous system.

Stimulation of intra-uterine growth in rat by alpha-melanocyte-stimulating hormone.

We have studied whether endogenous alpha-MSH has a function in stimulating intra-uterine growth in the rat. The approach used was to determine whether or not this hormone is present during the intra-uterine growth spurt, and if binding of endogenous foetal alpha-MSH by antibodies would inhibit this growth. Antibodies against alpha-MSH or ACTH 1-24, either purified or non-purified, induced immunofluorescence in the intermediate lobe of adult male control rats. Using purified anti-alpha-MSH, fluorescence appeared in the foetal intermediate lobe on day 18 of pregnancy, the day that biologically active MSH was first seen. A negative correlation was observed between the pituitary MSH content and foetal body weight only on day 19 of pregnancy. Injection of purified anti-alpha-MSH induced a drop in foetal body weight, but no effect on placental weight was observed. Purified anti-acth 1-24 had no effect upon body weight but caused an increase in placental weight. These results support our previous findings and indicate that endogenous MSH has a function in the stimulation of foetal growth.

Immunocytohistochemical characterization of pituitary cells of the bluefin tuna, Thunnus thynnus L.

In this paper we report the first complete mapping of the pituitary in a tuna species. The various different adenohypophysis cell types of the bluefin tuna, Thunnus thynnus L. have been identified and located using different antisera against mammalian and piscine hormones and various histochemical techniques: PAS, Alcian Blue pH 2.5 and lectins -ConA and WGA(Neutral and Acidic Glycoproteins); Bromophenol Blue (Proteins) and Tioglycollate-Ferric-Ferricianide-FeIII (-S-S- groups). Prolactin (PRL) and adrenocorticotrophic (ACTH) cells were located in the rostral pars distalis (RPD) of the pituitary, while the proximal pars distalis (PPD) displayed gonadotrophic (GTH), thyrotrophic (TSH), somatotrophic (GH) and also a few PRL cells. Moreover, somatolactin (SL) and melanotrophic (MSH) cells were identified inside the pars intermedia (PI). Interestingly, some SL-immunoreactive fibers were also detected in the neurohypophysis. Some GTH cells were also located on the exterior surface of the PI. Glycoproteins containing mannose (Man) and/or glucose (Glc); N-acetyl-glucosamine (GlcNAc) and/or sialic acid sugar residues, as well as -S-S- groups, were observed in GTH, TSH and SL cells. The Bromophenol Blue technique stained amphiphilic SL, acidophilic GH cells and weakly ACTH cells. GH and ACTH cells were unreactive to PAS, Alcian Blue, Tioglycollate-Ferric-Ferricianide-FeIII and lectin (Con A and WGA) techniques. Finally, PAS reaction was positive in amphiphilic SL cells, which were PbH unreactive, while MSH and ACTH cells were stained with PbH technique.