Relative stability of α-melanotropin and related analogues to rat brain homogenates

α-Melanotropin (α-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle⁴, D-Phe⁷]-α-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle⁴, D-Phe⁷]-α-MSH_4–10-NH₂, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle⁴, D-Phe⁷]-α-MSH_4–11-NH₂ is totally resistant to inactivation by rat brain homogenate. Both [Nle⁴, D-Phe⁷]-fragments are resistant to degradation by rat serum, but [Nle⁴]-α-MSH, Ac-[Nle⁴]-α-MSH_4–10-NH₂ and Ac-[Nle⁴]-α-MSH_4–11-NH₂ are rapidly inactivated under both conditions. The cyclic melanotropin, [ ]-α-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[ ]-α-MSH_4–10-NH₂ analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[ ]-α-MSH_4–10-NH₂ is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Effects of a Melanotropic Peptide on Melanoma Cell Growth,Metastasis, and Invasion

Melanocyte stimulating hormone (α-MSH, α-melanotropin),Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Ly-Pro-Val-NH₂, regulates melanogenesis within epidermal melanocytes of many animals. An MSH analogue ([Nle4,D-Phe7]α-MSH) that exhibits superpotency and prolonged biological activity has been synthesized, biologically characterized, and is presently in clinical trials to determine its possible clinical use in tanning of the skin. It also has potential for the diagnosis, localization, and chemotherapy of melanoma. The effects of this analogue on the growth, metastatic behavior, and invasive potential of a melanotic variant of Cloudman S-91 murine melanoma are reported here. In an intracutaneous murine model of melanoma cell tumor growth, the analogue did not increase primary tumor growth (size) after the period of administration of the peptide hormone analogue and did not affect spontaneous lung metastases. Survival times for the control and melanotropin-treated groups were similar, suggesting that overall tumor burden was not affected by treatment with the hormone analogue. Last, melanoma cell invasion through a human amniotic basement membrane in vitro was not enhanced compared to untreated cells.     

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. With the exception of ACTH1-24 there is a close resemblance in structure-activity relationships of the fragments and analogs tested in the two bioassays. [Nle4,-D-Phe7]-alpha-MSH is extremely active in both assays. Weak agonists such as [Leu9]-alpha-MSH did not possess antagonistic properties either in the melanophore assay or in the excessive grooming test. The data suggest that the mechanism of action of alpha-MSH-like peptides in rat brain is receptor-mediated like their action on melanophores.     

Hypothermic and antipyretic effects of centrally administered ACTH (1--24) and alpha-melanotropin

ACTH (1--24) and alpha-melanotropin (alpha-MSH), peptides previously shown to influence body temperature when administered centrally and to occur naturally in brain regions important to temperature control, were injected intracerebroventricularly (ICV) in rabbits. The peptides in doses of 1.25, 2.5 and 5.0 micrograms produced dose-related hypothermias in a 23 degrees C environment, and greater decreases in body temperature when the experiments were repeated in the cold (10 degrees C), but the largest dose had no effect on temperature in the heat (30 degrees C). These results indicate that the peptides do not reduce the central set-point of temperature control. Rather, they appear to selectively inhibit heat conservation and production responses. Five microgram of ACTH reversed vasoconstriction and inhibited rises in temperature caused by leukocytic pyrogen (LP) given IV and ICV. The same dose of alpha-MSH also reduced fever produced by IV and ICV LP, but the reduction was not as great as after ACTH. Both peptides (5 micrograms) also reduced temperature rises and vasoconstriction caused by ICV PGE2. ACTH reduced d-amphetamine-induced hyperthermia without altering vasoconstriction which suggests that this peptide can reduce temperature rises by inhibiting heat production alone. One of the most important findings was that the peptides are antipyretic in that they reduce fever at doses (0.25 microgram, ICV) that do not affect normal temperature. The powerful effects of these peptides on resting body temperature, hyperthermia and fever, together with their presence in brain tissue important to temperature control, suggest that the endogenous central peptides participate in thermoregulation, perhaps by limiting fever and influencing normal temperature.     

Changes in plasma,pituitary and brain α-MSH content in rats from birth to sexual maturity

Immunoreactive α-MSH was measured in plasma, pituitary and brain of male and female rats on the day of birth and at intervals afterwards up to 55–70 days of age. Plasma α-MSH concentrations on the day of birth were 528 ± 111 pg/ml and 406 ± 137 pg/ml in famel and male rats, respectively. Plasma α-MSH concentrations then fell and remained low until the onset of sexual maturity when they again rose reaching 406 ± 38 pg/ml in 70 day old females and 312 ± 46 pg/ml in 55 day old males. Pituitary α-MSH concentrations also changed with age and in male rats generally reflected the changes in plasma α-MSH concentrations. In females, on the other hand, pituitary α-MSH concentrations showed a gradual increase with age. Concentrations of α-MSH in the hypothalamus and brain increased with age and as with plasma and pituitary reached peak values in sexually mature animals. These findings are consistent with the idea that both piutiary and brain α-MSH have a role in sexually mature rats. Although α-MSH may have a role in sexual behaviour there was no evidence of any change in brain α-MSH throughout the estrous cycle.     

Immunohistochemical localization of adrenocorticotropin and melanocyte stimulating hormone in pars intermedia of rat hypophysis

Summary The sites of production of adrenocorticotropin (ACTH) and melanocyte stimulating hormone (MSH) are studied by the immunoglobulin-peroxidase bridge technique, using antisera prepared against synthetic porcine 1–24 and 17–39 ACTH, and bovine MSH on the rat adenohypophysis. Presence of ACTH all over the pars intermedia (PI) is indicated by staining with antisera _p 1–24 and _p 17-3-9 ACTH. There are darkly stained ACTH cells in the PI and pars tuberalis (PT), similar to those in the pars distalis (PD). With higher dilutions of the ACTH antiserum, staining intensity disappears or reduces markedly in majority of the PI cells, whereas, the ACTH cells in the PI, PD and PT do not vary much in their staining intensity. Therefore, it is concluded that majority of the PI glandular cells (light glandular and dark cells) contain less corticotropin than the ACTH cells. From these observations, it seems to me that the major amount of corticotropin is supplied by the ACTH cells of the PD, PI and PT, and less by the light glandular and dark cells of the PI. The antiserum is ineffective after absorption, so the staining reaction appears to be specific for _p 1–24 and _b 17–39 ACTH.Presence of MSH all over the PI is indicated by staining with antisera to bovine MSH. Majority of the PI cells are highly stained even with higher dilution of the antiserum. The unstained cells in the PI seem to be ACTH cells and/or marginal cuboidal cells. The antiserum was ineffective after absorption, so the staining reaction appears to be specific for _b MSH.Control over the PD corticotropin through the median eminence portal circulation and the PI and PT control through nervous system is also discussed.This study was supported by MRC of Canada Grant nos. MA-3759, and MA-5160.The author gratefully wishes to thank Drs. P. Desaulles and W. Rittel (CIBA, Basle, Switzerland) for the synthetic _p 1–24 ACTH and _b MSH, Dr. R. F. Phifer for _p 17–39 ACTH, and Dr. S. S. Spicer for providing samples of rabbit anti-porcine 17–39 ACTH and anti-human ACTH sera, Drs. George Sétáló and Paul Nakane for their valuable advice. He also acknowledges the help of Mr. Shankar Nayak to prepare the antisera and the skilful technical assistance of Miss. Elise Poiré.     

Nitric oxide enhances the sensitivity of alpaca melanocytes to respond to α-melanocyte-stimulating hormone by up-regulating melanocortin-1 receptor

Nitric oxide (NO) and α-melanocyte-stimulating hormone (α-MSH) have been correlated with the synthesis of melanin. The NO-dependent signaling of cellular response to activate the hypothalamopituitary proopiomelanocortin system, thereby enhances the hypophysial secretion of α-MSH to stimulate α-MSH-receptor responsive cells. In this study we investigated whether an NO-induced pathway can enhance the ability of the melanocyte to respond to α-MSH on melanogenesis in alpaca skin melanocytes in vitro. It is important for us to know how to enhance the coat color of alpaca. We set up three groups for experiments using the third passage number of alpaca melanocytes: the control cultures were allowed a total of 5 days growth; the UV group cultures like the control group but the melanocytes were then irradiated everyday (once) with 312 mJ/cm² of UVB; the UV + L-NAME group is the same as group UV but has the addition of 300 μM L-NAME (every 6 h). To determine the inhibited effect of NO produce, NO produces were measured. To determine the effect of the NO to the key protein and gene of α-MSH pathway on melanogenesis, the key gene and protein of the α-MSH pathway were measured by quantitative real-time PCR and Western immunoblotting. The results provide exciting new evidence that NO can enhance α-MSH pathway in alpaca skin melanocytes by elevated MC1R. And we suggest that the NO pathway may more rapidly cause the synthesis of melanin in alpaca skin under UV, which at that time elevates the expression of MC1R and stimulates the keratinocytes to secrete α-MSH to enhance the α-MSH pathway on melanogenesis. This process will be of considerable interest in future studies.     

The effect of the estrous cycle and estrogen on the release of immunoreactive alpha-melanocyte-stimulating hormone

Circulating levels and tissue content of alpha-MSH were measured on the morning of various days of the estrous cycle, and on the afternoon of proestrus in freely moving conscious rats. No surges of alpha-MSH were detected by RIA in the morning of various days of the cycle. The neurointermediate lobe content of alpha-MSH was slightly elevated on diestrus 1 as compared to the levels on diestrus 11 and proestrus but not to estrous levels. No changes in alpha-MSH content were detected in the anterior pituitary, the median eminence, mediobasal hypothalamus and the preoptic area at various stages of the estrous cycle. Plasma alpha-MSH levels were slightly elevated at 1500 hr of proestrus which was followed three hours later by a decline. This profile of plasma alpha-MSH on the afternoon of proestrus was reproduced by the SC administration of estradiol benzoate to long-term ovariectomized rats. These data suggest that, contrary to the results obtained by bioassay of alpha-MSH no surges of alpha-MSH occur on any day of the cycle, although a slight elevation on the afternoon of proestrus was detected. The altered pattern of release of this peptide on the afternoon of proestrus may be induced by estrogen.     

Presence of immunoassayable beta-lipotropin in bovine brain and spinal cord: lack of concordance with ACTH concentrations.

Discrete areas of freshly obtained adult bovine brain were assayed for their content of immunoreactive β-lipotropin (β-LPH), ACTH and β-endorphin. Highest concentrations (pg/100ug protein) of β-LPH were present in hypothalamus (517 ± 81), hippocampus (218 ± 60), central grey rostral mesencephalic level, pons, striatum, and spinal cord (163–258). Lesser concentrations (49–138) were present in other parts of the limbic system, brain stem, cortex and thalamus. Immunoreactive ACTH concentrations were highest in hypothalamus (1702 ± 487) and hippocampus (210 ± 40), with markedly lesser concentrations (5–24) being present in all the other aforementioned areas. Immunoreactive β-endorphin concentrations in hypothalamus were 1990 ± 510, in hippocampus 280 ± 50.     

The distribution of alpha-melanocyte stimulating hormone (alpha-MSH) in the central nervous system of the rat: an immunohistochemical study. II. Lower brain stem

The distribution of immunoreactive alpha-melanocyte stimulating hormone (alpha-MSHI) in the rat lower brain stem was examined by indirect immunofluorescence or peroxidase- anti-peroxidase immunohistochemical method using an antiserum against synthetic alpha-MSH. The results confirmed the presence of alpha-MSHI fibers in the midbrain central gray matter and parabrachial area, and demonstrated a much more extensive distribution of these fibers in various parts of the lower brain stem areas previously thought not contain alpha-MSHI fibers. In addition, the commissural nucleus was identified as a new alpha-MSHI neurons-containing site. No alpha-MSHI neurons were seen in other regions of the rat lower brain stem.     

NMDA excitotoxicity and free radical generation in rat brain homogenates: Application of a chemiluminescence assay

NMDA, the specific agonist of glutamate gated ion channels permeable to calcium, is implicated as a causal factor in the pathogenesis of several neurobiological disorders such as stroke, seizures, ischemia, and chronic neurodegenerative disease. On the other hand, evidence on the roles of oxidative mechanisms involved in NMDA-induced neurotoxicity is accumulating. In this study, we have used chemiluminescence measurements as an easy, rapid and sensitive assay to investigate the effects of NMDA and oxidative stress on brain cell vulnerability. Rat brain homogenates were incubated with increasing concentrations of glutamate and NMDA. Production of reactive oxygen species was followed by single photon emission measurements using the specific enhancers luminol and lucigenin. Increases in emission were observed at excitotoxic concentrations of glutamate and NMDA. Other parameters of oxidative stress such as diene conjugates, TBARS and carbonyl groups were also investigated. Our results indicated that chemiluminescence measurements may be used to study involvement of oxidative stress in neurotoxicity.     

Characterization and subcellular localization of GnRH analog binding in rat brain

The binding of a degradation-resistant analog of gonadotropin-releasing hormone, [D-Phe6]GnRH, to rat brain crude particulate preparation was studied. The binding of this analog at 0 degrees C was saturable and Scatchard analysis revealed the presence of 2 binding sites: one with KD = 1.39 x 10(-7) M and Bmax = 265 pmole/mg protein, and another of lower affinity but higher capacity with KD = 5.58 X 10(-6) M and Bmax = 1734 pmoles/mg protein. The binding at 0 degrees C was substantially higher than that obtained at 37 degrees C, due to binding site-inactivation processes occurring at 37 degrees C. The binding sites exhibited a considerable degree of specificity for GnRH as unrelated peptides (with the exception of ACTH) display a much weaker affinity than GnRH and GnRH analogs. Subcellular fractionation demonstrated that most of the binding was associated with the mitochondrial fraction.     

Corticotropin Peptides and Melanotropins Elevate the Level of Adenosine 3'': 5''-Cyclic Monophosphate in Cultured Murine Brain Cells

Cell cultures derived from mouse and rat brain and consisting mainly of astroblasts are known to respond to several hormones by increasing or decreasing their intracellular concentration of cyclic AMP. In the present study these cultures were analyzed for their susceptibility to various additional hormonal and other neuroactive compounds. Only the peptides of the corticotropin (ACTH)/melanotropin (MSH) family were found active. Their potency for elevating the intracellular level of cyclic AMP decreases in the sequence (values for the half-maximally stimulating concentrations, EC50, in parentheses) ACTH-(1-24) (10 m) greater than alpha-,beta-MSH (30 nm) greater than ACTH (greater than or equal to 100 nm) gamma-MSH, ACTH-(1-10), -(4-10), -(4-11) (greater than or equal to 0.5 microM). The lack of additivity of the maximal effects of the peptides suggests that they all act at the same receptor. The stimulation exerted by these peptides is partially suppressed by hormones known to inhibit cyclic AMP formation in that culture, i.e., noradrenaline (acting via an alpha-adrenergic receptor), adenosine (acting via an A1 receptor), and somatostatin. It is concluded that the receptors for the ACTH/MSH peptides and the inhibitory hormones are located on the same cells, presumably the astroblasts. The maximal response to ACTH and alpha- and beta-MSH depends strongly on the age of culture. The results are discussed in view of the facts that (1) peptides of the ACTH/MSH family affect behavior and learning in animals, and (2) ACTH and alpha-MSH occur in brain.     

Quantification of the permeability of the blood-CSF barrier to alpha-MSH in the rat

The ability of alpha-MSH to cross the blood-CSF barrier of the rat was assessed by measurement of the rate of appearance of immunoreactive alpha-MSH in a cerebrospinal fluid (CSF) perfusate following intravenous injection of peptide. Comparisons were made with the rate of appearance of a simultaneously administered dose of 14C-inulin which is poorly permeable at the blood-CSF barrier. Concentrations of drugs measured in plasma were fitted to two-compartment pharmacokinetic models, and those measured in the CSF perfusate to one-compartment open systems receiving an input from the plasma compartment. The rate constant for entry of alpha-MSH into CSF was 0.00087 min-1, which was not significantly different from that for inulin of 0.00055 min-1. As alpha-MSH penetrated into CSF at a rate comparable to inulin, it was concluded that the limited entry of peptide was by aqueous diffusion along with other water-soluble macromolecules.     

Biosynthesis, processing and release of pro-opiomelanocortin related peptides in the intermediate lobe of the pituitary gland of the frog (Rana ridibunda)

The biosynthesis of pro-opiomelanocortin (POMC) and related peptides by the intermediate lobe of the pituitary gland was studied in the frog Rana ridibunda using the pulse-chase technique. Analysis of radioactive proteins by dodecyl sulfate polyacrylamide gel electrophoresis showed that during pulse incubations a 36,000 dalton (36K) glycosylated prohormone was synthesized. It disappeared slowly during chase incubations, giving rise to another glycosylated protein (Mr 18K), identified as the N-terminal fragment of POMC. This latter protein was secreted to the incubation medium. High performance liquid chromatography analysis of peptides synthesized during chase incubations revealed the biosynthesis of two peptides related to gamma-MSH, three peptides related to alpha-MSH, one endorphin-related and one CLIP-related peptides. These newly synthesized peptides were slowly secreted to the incubation medium. Among the alpha-MSH related peptides, only the des-N alpha-acetyl alpha-MSH form of the peptide was found to be present within the cells, in contrast to the incubation medium where the presence of des-N alpha-acetyl alpha-MSH and a modified alpha-MSH was demonstrated.