A comparison of structure-activity relationships within alpha-MSH on melanophores of Anolis carolinensis and Rana pipiens.

We have compared the structure-function relationship of the tridecapeptide alpha-melanocyte stimulating hormone (alpha-MSH) on the melanophores of the lizard Anolis carolinensis and the frog Rana pipiens by determining the melanosome-dispersing potency of 15 shorter peptide sequences and 8 substituted alpha-MSH analogues. Major differences were found between the lizard and the frog in their response to alpha-MSH peptide fragments and analogues. In Anolis, the sequence Ser-Tyr-Ser- is not as important for the pigmentary response as in Rana since alpha-MSH-(4-13) was nearly as potent (89%) as alpha-MSH-(1-13) (100%), whereas in Rana alpha-MSH-(4-13) potency was reduced to 7.

alpha-MSH-induced changes in protein phosphorylation of Cloudman S91 mouse melanoma cells.

The role of protein phosphorylation in MSH-induced melanogenesis was investigated with an in vivo phosphorylation assay using intact cultured Cloudman S91 mouse melanoma cells preincubated with [32P]orthophosphate. Exposure of the cells to alpha-MSH increased the extent of labelling of two protein bands on SDS gel electrophoresis with estimated molecular weights of 43 and 34 kDa, respectively. The 32P incorporation was concentration-dependent and reached a maximal value at 10(-8) M alpha-MSH for the 43 kDa band (156% of controls) and at 10(-5) M alpha-MSH for the 34 kDa band (250% of controls).

Dihydrotestosterone heptanoate: synthesis, pharmacokinetics, and effects on hypothalamic-pituitary-testicular function.

Dihydrotestosterone heptanoate (DHT-hp), a seven-carbon fatty acid ester of DHT, was synthesized, and its pharmacokinetics and effects on hypothalamic-pituitary-testicular function were determined in men and pubertal boys. Plasma DHT levels markedly increased 24 h after im injection of DHT-hp, reached their peak during the first week, and fell to baseline levels after 4-6 weeks. An estimated 43-55% of DHT-hp was converted to DHT 4-6 weeks after injection.

Coexistence of melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) in the preoptic nucleus of the frog brain.

Coexistence of MCH- and alpha-MSH-like peptides in specific neurons of the frog hypothalamus has been investigated on serial frozen sections using the indirect immunofluorescence method. In the anterior region of the preoptic nucleus, perikarya containing MCH- and alpha-MSH-immunoreactive materials were co-distributed and the two peptides were generally co-sequestered within the same neurons. In contrast, alpha-MSH immunoreactive neurons of the ventral infundibular nucleus did not contain any MCH-like peptide.

Melanin concentrating hormone inhibits the release of alpha MSH from teleost pituitary glands.

A radioimmunoassay was developed for salmonid melanin concentrating hormone (MCH) and used to measure immunoreactive (ir)MCH in the hypothalamus and pituitary of trout (Salmo gairdneri) and eels, (Anguilla anguilla) maintained under different regimes of background color. In trout, 95% of the total irMCH was located in the pituitary gland. The amount of MCH in both pituitary and hypothalamus was increased when white-adapted trout were transferred to a black background.

Growth hormone in urine: development of an ultrasensitive assay applicable to plasma and urine.

A radiometric assay for human growth hormone (HGH) was developed based on a polyclonal goat anti-HGH antiserum covalently coupled to nonsedimenting polyacrylamide particles. HGH can be specifically immunoextracted from sample volumes of up to 10 ml. Subsequently, bound HGH is identified and quantitatively measured by a 125I-labelled monoclonal anti-HGH antibody.

Changes in alpha-melanocyte-stimulating hormone content in discrete hypothalamic areas of the male rat during a twenty-four-hour period.

Circadian variations in alpha-melanocyte-stimulating hormone (alpha-MSH) content of discrete hypothalamic areas of the male rat were observed either with radioimmunoassay or bioassay. In the medial basal hypothalamus and preoptic area the alpha-MSH content increased sharply between 02.00 and 06.

Corticotrophin-releasing factor-test used with bilateral, simultaneous inferior petrosal sinus blood-sampling for the diagnosis of pituitary-dependent Cushing's disease.

Bilateral, simultaneous inferior petrosal sinus blood-sampling for determinations of ACTH levels has improved the ability to establish a differential diagnosis of Cushing's disease, particularly in patients whose endocrinological studies show equivocal results and whose computed tomography scans yield negative or inconclusive findings. Individual anatomical variations in the configuration of the sinus and insignificant differences between the ACTH levels obtained from its two sides may be a problem. Seven patients with clinically and biochemically typical Cushing's disease and one with atypical Cushing's disease were examined.

In situ melanin assay for MSH using mouse B16 melanoma cells in culture.

A sensitive in situ melanin assay using cultured mouse B16 melanoma cells is described for structure-activity studies with melanocyte-stimulating hormone (MSH) peptides. B16 Cells were seeded at a density of 2500 cells per well in 96-well microtest tissue culture plates; after 24 h the cells were incubated in the presence of serial dilutions of MSH peptides for 3 to 5 days. The melanin released into the medium of each well was then determined spectrophotometrically at a wavelength of 405 nm using an automatic microplate reader calibrated against synthetic melanin.

Localization of melanin-concentrating hormone-like immunoreactivity in the brain and pituitary of the frog Rana ridibunda.

The distribution of melanin-concentrating hormone (MCH) in the central nervous system of the frog Rana ridibunda was determined by the indirect immunofluorescence technique using antibodies against synthetic salmon MCH, generated in rabbits. The most prominent group of MCH-like containing perikarya was detected in the preoptic nucleus. Comparatively, a moderate number of cell bodies was observed in the dorsal infundibular nucleus and in the ventral thalamic area.

Steroidogenic activity of highly potent melanotropic peptides in the adrenal cortex of the rat.

Highly purified ACTH and MSH peptides were studied in isolated rat glomerulosa and inner zone cells and their activity compared with that in an Anolis melanophore assay. While both ACTH1-39 and ACTH1-24 were almost equally potent steroidogenic peptides in the two cell types (ED50 between 1 and 4 X 10(-12) M), alpha-MSH displayed only weak steroidogenic activity. Although it was a full agonist, it was about 10(4)-fold less potent in both capsular and inner zone cells.

Treatment of persistent pubertal gynecomastia with dihydrotestosterone heptanoate.

Four boys with persistent pubertal gynecomastia were given intramuscular dihydrotestosterone heptanoate (DHT-hp) at 2 to 4-week intervals for 16 weeks. By the end of treatment, breast size in all four boys had decreased 67% to 78%. Initial plasma levels of gonadotropins, estradiol, testosterone, and dihydrotestosterone (DHT) were normal.

In vivo and in vitro development of alpha-MSH and ACTH in the embryonic and postnatal rat brain.

The appearance of immunoreactive alpha-melanotropin (alpha-MSH) and adrenocorticotropin (ACTH) during development was studied in 3 areas of the rat brain--cerebral hemispheres, midbrain and hindbrain--from embryonic day (ED) 13-14 until day 21 postnatally. The alpha-MSH content in vivo was always highest in the midbrain; a peak content at birth was followed by a transient decline and a later, higher plateau from postnatal day 7 onwards. The alpha-MSH content in the cerebral hemispheres rose progressively after birth reaching a peak at day 21.

Plasma ACTH in diagnosis and control of adrenal disorders.

Unstimulated plasma ACTH concentrations remain at or below the detection limit of conventional immunoassays. Grossly elevated ACTH concentrations are diagnostic in suspected adrenal insufficiency, remain elevated well above 200 ng/l during substitution therapy and obviate the need of further tests. For the diagnosis of secondary adrenal failure, plasma ACTH, cortisol and 11-desoxycortisol response to a single midnight dose of metyrapone (1.

ACTH1-4 potentiates alpha-MSH-induced melanophore dispersion and excessive grooming.

The biological activity and a possible modulatory role of the N-terminal tetrapeptide Ser-Tyr-Ser-Met from alpha-MSH/ACTH was tested in the Anolis melanophore assay, the Xenopus melanophore assay, tyrosinase stimulation in mouse melanoma cells and in excessive grooming in the rat. ACTH1-4 did not exhibit biological activity in any of these four assays nor did it have modulatory properties in the Xenopus and the melanoma cell assay. However, in the Anolis assay ACTH1-4 potentiated pigment dispersion induced by alpha-MSH, alpha-MSH5-13 and ACTH1-24 by a factor of about 2.

Effect of melanin concentrating hormone on pigment and adrenal cells in vitro.

Highly purified synthetic salmonid melanin concentrating hormone (MCH) and some analogs were investigated for their ability to concentrate the pigment in scale melanophores of the Chinese grass carp, Ctenopharyngodon idellus, to produce melanin dispersion in frog or lizard melanophores and to inhibit alpha-MSH in its action on mouse melanoma and rat adrenal glomerulosa cells in vitro. In the grass carp, MCH produced half-maximal pigment aggregation at 6 X 10(-11) M and its oxidized form at 7 X 10(-11) M. Replacement of the two methionines at position 3 and 6 with norvaline lowered the potency by a factor of 2.

Comparison of structural requirements of alpha-MSH and ACTH for inducing excessive grooming and pigment dispersion.

alpha-MSH and ACTH-like peptides are known to play an important role in the adaptation of many vertebrates to a new environment. These peptides induce pigment dispersion in amphibian melanophores through a receptor-mediated mechanism. In this study we compared the structural requirements of these peptides for melanotropic activity on Xenopus laevis melanophores with those for inducing excessive grooming in the rat.

An Anolis skin melanophore assay suitable for photoaffinity labeling studies with alpha-MSH.

Visual determination of MSH-induced pigment migration in melanophores of small pieces of Anolis carolinensis skin is standardized by first measuring photoelectrometrically the change in reflection/transmission of the whole dorsal skin in response to different hormone concentrations. This method allows the rapid and precise recording of time-response curves after photo-affinity labeling of MSH receptors or of dose-response curves of large series of synthetic compounds.

Inhibition of the ACTH adrenal response to stress by treatment with hydrocortisone, prednisolone and dexamethasone in the rat.

16- and 4-week-old intact and adrenalectomized rats have been treated with different doses of the three glucocorticoids hydrocortisone, prednisolone and dexamethasone by gavage. The delayed feedback effect on plasma ACTH and corticosterone response to an ether stress have been assessed. Almost complete suppression of corticosterone response 20 min after an ether stress and an ACTH suppression to 20% of control values 5 min after an ether stress were observed with 25 micrograms of dexamethasone, 10 mg of prednisolone and 20 mg of hydrocortisone.

Photoaffinity labelling of MSH receptors on Anolis melanophores: effects of catecholamines, calcium and forskolin.

Photoaffinity labelling of MSH receptors on Anolis melanophores was used as a tool for studying the effects of catecholamines, calcium and forskolin on hormone-receptor interaction and receptor-adenylate cyclase coupling. Covalent attachment of photoreactive alpha-MSH to its receptor was suppressed in calcium-free buffer but was hardly influenced by catecholamines or forskolin. The longlasting signal generated by the covalent MSH-receptor complex was readily and reversibly abolished by adrenaline, noradrenaline, dopamine or clonidine or by the absence of calcium.

DNA sequence around the Escherichia coli unc operon. Completion of the sequence of a 17 kilobase segment containing asnA, oriC, unc, glmS and phoS.

The nucleotide sequence is described of a region of the Escherichia coli chromosome extending from oriC to phoS that also includes the loci gid, unc and glmS. Taken with known sequences for asnA and phoS this completes the sequence of a segment of about 17 kilobases or 0.4 min of the E.

Photoaffinity labelling of MSH receptors on Anolis melanophores: irradiation technique and MSH photolabels for irreversible stimulation.

Excised dorsal skin of Anolis carolinensis was exposed to high intensity UV-irradiation in the presence of different photoreactive alpha-MSH derivatives. The resulting covalent binding of the hormone to its receptor induced irreversible pigment dispersion. The duration of the longlasting response depended on the type and length of irradiation; it was maximal after two 5 min irradiation phases with a light intensity of approximately 180 mW/cm2 and a spectrum from 310 to 550 nm, fresh hormone being added after the first phase.

Peptides of the neurointermediary lobe of rat pituitary stimulate GH secretion in vitro.

alpha-MSH and other fragments of ACTH are potent stimulators of GH release in vivo. The action of such peptides and of extracts of the neurointermediary lobe (NIL) of rat pituitary, a source of endogenous MSH-related peptides, on GH release was investigated in vitro. Peptides with the core sequence of alpha-MSH stimulate GH secretion by primary cultures of rat anterior pituitary cells; however, both the absolute and the relative potencies of these peptides exclude the involvement of melanotropic receptors comparable in specificity to the extrapituitary receptors for these hormones.

Photoaffinity labelling of peptide hormone receptors.

Photoreactive peptide derivatives for the labelling of hormone receptors are usually prepared by inserting a chemically stable aryl azide or nitroaryl azide into a specific site of the molecule, such as an alpha or omega amino or carboxyl group, or into the side-chain of an Arg, Cys, His, Trp or Tyr. With p-azidophenylalanine (Pap), a more or less isosteric replacement of Tyr or Phe can be achieved when other alterations would impair the biological activity of the hormone. Reversible attachment of photoreactive groups via S-S linkage to, e.