Structural and conformational modifications of α-MSH/ACTH4–10 provide melanotropin analogues with highly potent behavioral activities

Previous studies have identified the (4–10) heptapeptide sequence as the central core of α-MSH/ACTH peptides required for mediation of important biological activities. In the present study, the structure-activity relationships of Nle⁴-substituted and -bridged cyclic α-MSH analogues, which were previously shown to exhibit a wide range of melanotropic potencies from weak agonism to super potency, were examined for grooming behavioral activity in the rat following intracerebroventricular injections. The results showed that stepwise C-terminal elongation of the linear Nle⁴-substituted Ac-α-MSH_4–10-NH₂ increased grooming potencies of the peptides in a manner similar to their actions on melanocytes. The most interesting finding was the observation that cyclization of the inactive linear “central (4–10) core” of α-MSH (Ac-α-MSH_4–10) to form Ac-[ ]-α-MSH_4–10-NH₂ resulted in a super potent agonist in the grooming assay. However, while cyclization of the (4–10) heptapeptide produced potent agonists on grooming behavior, the structure-activity relationships were different than the frog skin bioassay. These findings support the hypothesis that appropriate structural and confirmational modifications of α-MSH-related peptides can produce profound effects on the bioactivities of the peptides, and suggest that different structural-conformational requirements exist for α-MSH interactions with its various receptors.     

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.     

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

α-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 ACTH_1–24 there is a close resemblance in structure-activity relationships of the fragments and analogs tested in the two bioassays. [Nle⁴,-D-Phe⁷]-α-MSH is extremely active in both assays. Weak agonists such as [Leu⁹]-α-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 α-MSH-like peptides in rat brain is receptor-mediated like their action on melanophores.     

Enzymatic synthesis of α- -fucosyl-N-acetyllactosamines and 3′-O-α- -fucosyllactose utilizing α- -fucosidases

An α- -fucosidase from porcine liver produced α- -Fuc-(1→2)-β- -Gal-(1→4)- -GlcNAc (2′-O-α- -fucosyl-N-acetyllactosamine, 1) together with its isomers α- -Fuc-(1→3)-β- -Gal-(1→4)- -GlcNAc (2) and α- -Fuc-(1→6)-β- -Gal-(1→4)- -GlcNAc (3) through a transglycosylation reaction from p-nitrophenyl α- -fucopyranoside and β- -Gal-(1→4)- -GlcNAc. The enzyme formed the trisaccharides 1–3 in 13% overall yield based on the donor, and in the ratio of 40:37:23. In contrast, transglycosylation by Alcaligenes sp. α- -fucosidase led to the regioselective synthesis of trisaccharides containing a (1→3)-linked α- -fucosyl residue. When β- -Gal-(1→4)- -GlcNAc and lactose were acceptors, the enzyme formed regioselectively compound 2 and α- -Fuc-(1→3)-β- -Gal-(1→4)- -Glc (3′-O-α- -fucosyllactose, 4), respectively, in 54 and 34% yields, based on the donor.     

Evidence for a common precursor for αMSH and β-endorphin in the intermediate lobe of the pituitary of the reptile Anolis carolinensis

Immunohistochemical studies on the pituitary of Anolis carolinensis detected ACTH-like, β-endorphin-like, and 16K fragment-like immunoreactivity in distinct clusters of cells in the anterior lobe; ACTH-like, αMSH-like, β-endorphin-like, and 16K fragment-like immunoreactivity was detected in all the cells of the intermediate lobe. Crude acid extracts of both lobes, when alayzed by radioimmunoassay, gave displacement curves in ACTH and β-endorphin assays which were parallel to the appropriate synthetic standard. Only extracts of the intermediate lobe gave parallel displacement curves in an αMSH radioimmunoassay. Extracts of both lobes crossreacted with antiserum to 16K fragment, but the displacement curves were not parallel to that of mouse 16K fragment standard. The levels of immunoreactive ACTH and β-endorphin in the intermediate lobe were approximately 8-fold higher than in the anterior lobe. Fractionation of anterior lobe and intermediate lobe extracts by either gel filtration on Sephadex G-75 in 10% formic acid or sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed multiple forms of ACTH-related and β-endorphin-related substances in both lobes. In the anterior lobe the major forms of immunoreactivity were, respectively, ACTH-sized and β-endorphin-sized. In the intermediate lobe the major forms of immunoreactivity were αMSH-sized, CLIP-sized, and β-endorphin-sized. In both lobes, antisera directed against ACTH and β-endorphin detected high molecular weight material with an apparent molecular weight slightly less than that of mouse pro-ACTH/endorphin; this material probably represents the putative common precursor for ACTH and β-endorphin in this species.     

Intracellular acetylation of desacetyl αMSH in the xenopus laevis neurointermediate lobe

High performance liquid chromatography (HPLC) followed by radioimmunoassay (RIA) of the chromatographic fractions were used to separate and quantify, respectively, the αMSH-like peptides stored in the neurointermediate lobe (NIL) of the Xenopus laevis (X. laevis) pituitary gland and released from the X. laevis NIL, in vitro. Immunoreactive (IR) material eluting with a similar HPLC retention time as desacetyl αMSH was the major IR peptide in the NIL. Material with a retention time similar to αMSH and immunological properties equivalent to αMSH was also present in the NIL. However, the retention times of the X. laevis and mammalian αMSH-like peptides were not identical, suggesting species difference in these peptides. Following incubation of NILs in the presence of [³H]-acetyl CoA, the X. laevis variant of αMSH was the major [³H]-labeled, immunoprecipitable material present. Following an incubation of NILs in the presence of [³H]-amino acids for 21 hours, immunoprecipitable [³H]-αMSH was detected in the NILs and the ratio of [³H]-desacetyl αMSH to [³H]-αMSH was similar to the ratio of IR-desacetyl αMSH to IR-αMSH. The X. laevis variant of αMSH was the major αMSH-like peptide released from the NILs into the incubation medium. Dopamine (50 μM) significantly inhibited the release of IR-αMSH but not IR-desacetyl αMSH. No net increase in total αMSH (sum of release and NIL content) was observed in the actively secreting (control) NIL group versus the dopaminetreated group. These results indicate that acetylation of desacetyl αMSH occurs intracellularly.     

Bovine intermediate pituitary α-amidation enzyme: Preliminary characterization

A secretory granule-associated enzymatic activity that converts mono-[¹²⁵I]-D-Tyr-Val-Gly into mono-[¹²⁵I]-D-Tyr-Val-NH₂ has been studied. The activity is primarily soluble and shows optimal activity at pH 7 to pH 8. Amidation activity was stimulated 9-fold by addition of optimal amounts of copper (3 μM). In the presence of optimal copper, ascorbate stimulated the reaction 7-fold; none of the other reduced or oxidized cofactors tested was as effective. Taking into account the dependence of the reaction on ascorbate and molecular oxygen and the production of glyoxylate [2], it is suggested that the α-amidation enzyme is a monooxygenase. Lineweaver Burk plots with D-Tyr-Val-Gly as the varied substrate demonstrated Michelis-Menten type kinetics with the values of K_m and V_max increasing with the addition of ascorbate to the assay. A variety of peptides ending with a COOH-terminal Gly residue act as inhibitors of the reaction. Two synthetic peptides, γ₂MSH and ACTH(1–14), with carboxyl termini similar to the presumed physiological substrates for the enzyme, act as competitive inhibitors with similar K₁ values. It is likely that this secretory granule α-amidation activity is involved in the physiological biosynthetic α-amidation of a wide range of bioactive peptides.     

Enzymatic syntheses of T antigen-containing glycolipid mimicry using the transglycosylation activity of endo-α-N-acetylgalactosaminidase

Thomsen–Friedenreich antigen (T antigen) disaccharide, β- -galactose-(1→3)-α-N-acetyl- -galactosamine (β- -Gal-(1→3)-α- -GalNAc), containing glycolipid mimicry was synthesized using the transglycosylation activity of endo-α-N-acetylgalactosaminidase from Bacillus sp. This enzyme could transfer the disaccharide from a p-nitrophenyl substrate to water-soluble 1-alkanols and other alcohols at a transfer ratio of 70% or more. Although the transfer ratios were lower for water-insoluble than water-soluble alcohols, they were shown to increase by adding sodium cholate to the reaction mixtures. The enzyme also transferred the disaccharide directly from asialofetuin to 1-alkanols. The anomeric bond between the disaccharide and 1-alkanols of the transglycosylation product is in the α configuration as determined by sequential digestion of jack bean β-galactosidase and Acremonium α-N-acetylgalactosaminidase. Since the transglycosylation product, β- -Gal-(1→3)-α- -GalNAc-(1→O)-hexyl, efficiently inhibits the binding of anti-T antigen monoclonal antibody to asialofetuin, it has potential as an agent for blocking T antigen-mediated cancer metastasis.     

Synthesis of monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside for epitope mapping of anti-Candida albicans antibodies

A panel of six complementary monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (1) were synthesized by stereoselective glycosylation of monodeoxy and mono-O-methyl monosaccharide acceptors with a 2-O-acetyl-glucosyl trichloroacetimidate donor, followed by a two-step oxidation–reduction sequence at C-2′. The β-manno configurations of the final deprotected congeners 2–7 were confirmed by measurement of ¹J_C1,H1 heteronuclear and ³J_1′,2′ homonuclear coupling constants. These disaccharide derivatives will be used to map the protective epitope recognized by a protective anti-Candida albicans monoclonal antibody C3.1 (IgG3) and to determine its key polar contacts with the binding site.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Differential in vitro metabolism of β-endorphin in schizophrenia

Biologically active peptide fragments derived from the proteolytic cleavage of β-endorphin (βE) have been shown to be present in the brain. Based on clinical results using some of these fragments in neuropsychiatric disease studies we investigated the in vitro metabolism of βE by twice-washed membrane homogenates of postmortem putamen from sex and age matched controls versus subjects with a diagnosis of schizophrenia. The present study demonstrates that frozen (−80°C) postmortem human tissues are viable for these studies and that metabolism in control tissue proceeds similarly to fresh tissues. Furthermore, a significant increase in the formation of the putative neuroleptic-like peptide fragment desenkephalin-γ-endorphin in postmortem schizophrenic putamen versus controls was shown. A significant decrease in the formation of βE 6–21 was also reported. These data suggest that an approach using postmortem human brain is possible in studying β-endorphin catabolism and is therefore applicable to other neuropeptide systems.     

β-Endorphin and ACTH related peptides in primary cultures of rat anterior pituitary cells: evidence for different intracellular pools

Acid extracts of rat anterior pituitary cells and cell-derived culture media were shown to contain three forms of β-endorphin immunoreactive peptides, corresponding in molecular size to the prohormone pro-opiomelanocortin (POMC), β-lipotropin and 3.5 kDa β-endorphin, and essentially two forms of adrenocorticotropin (ACTH) immunoreactivity, representing a 20 kDa intermediate fragment and 4.5 kDa ACTH. Under basal conditions the intracellular peptides contained a high proportion of the bioactive forms of β-endorphin and ACTH whereas the extracellular peptides contained a higher proportion of the inactive precursors. When the cells were incubated for 3 h in the presence of 10⁻⁸ M CRF, the levels of intracellular β-endorphin and ACTH immunoreactivity were reduced by 15–30% and there was a 4–5-fold increase in the level of the secreted peptides; furthermore, unlike the peptides released under basal conditions, the peptides secreted under the influence of CRF contained much higher proportions of 4.5 kDa ACTH and 3.5 kDa β-endorphin, reflecting the intracellular patterns of these peptides. Similar results were obtained when secretion was stimulated by 10⁻⁷ M epinephrine, which produced a 2-fold increase in peptide release. In the presence of 10⁻⁶ M dexamethasone the basal secretion of ACTH and β-endorphin related peptides, and the intracellular levels of these peptides, remained unaltered. The results point to the existence of different intracellular compartments from which peptides at different states of maturation can be released selectively.β-EndorphinACTHPituitary cell cultureProcessingCRFEpinephrine     

α-MSH and coat color changes in the mouse

The effect of α-MSH on coat color was examined in viable yellow mice (C3H/He-A*^vy). These mice normally grow a coat of darkly pigmented hair at puberty. This darkening effect was also evident in hair that grew in a region that had been plucked at 13 days of age. Administration of α-MSH increased the darkness of this hair and the hair which grew naturally in an unplucked area. However, the natural coat darkening that occurred at puberty was not associated with an increase in plasma immunoreactive α-MSH levels. Moreover, although bromocryptine, a dopamine agonist that inhibits α-MSH release from the pituitary reduced the darkness of the coat that grew after plucking the reduction in coat darkening was unrelated to changes in plasma α-MSH. Nevertheless, this effect of bromocryptine was reversed when α-MSH was administered together with the drug. Apomorphine had no effect on coat darkening and produced only a slight decrease in plasma α-MSH. Melatonin reduced coat darkening slightly but, like apomorphine, had little effect on plasma α-MSH concentrations. Although α-MSH may have a physiological role in coat darkening in the C3H/He-A*^vy mouse at puberty the response seems to be unrelated to an increase in circulating α-MSH. Thus, other factors, such as changes in melanocyte sensitivity to α-MSH or inhibitory mechanisms that prevent coat darkening during prepubertal and adult life may be involved in regulation of coat color in the viable yellow mouse.     

Molecular forms of α-melanocyte-stimulating hormone in the canine pituitary anterior and intermediate lobe

Reverse-phase high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) were used to determine the distribution of naturally occurring forms of α-melanocyte-stimulating hormone (α-MSH) in acid extracts of pars intermedia (PI) and anterior lobe (AL) tissue from canine and rat pituitary. Similarly, intracellular and secreted forms of α-MSH were determined using cultured canine PI and AL cells. Rat PI tissue contained predominantly diacetyl-α-MSH, while monoacetyl-α-MSH was the most abundant form in canine PI. In both canine and rat AL tissue extracts desacetyl-α-MSH was the major form of α-MSH. The profile of α-MSH contained in and secreted into culture medium by canine PI cells was found to be very similar to that in PI tissue extracts. The proportion of monoacetyl-α-MSH and diacetyl-α-MSH secreted by cultured canine AL cells and contained in extracts of AL cells in culture, however, was much higher than that in tissue extracts. These results indicate that in the dog, as in all other mammalian species studied, acetylated forms of α-MSH predominate in PI tissue, while nonacetylated α-MSH is the major form in AL tissue. It appears, however, that acetylation of α-MSH may occur in cultured canine AL cells, possibly as a result of the absence of factors that normally inhibit acetyltransferase in vivo or as a consequence of culture conditions.     

Conversion of α-methyltropate to optically active α-phenylpropionate by tropate-degrading Rhodococcus sp. KU1314

Microorganisms which can assimilate tropate were screened from soil. Among them, we found a microorganism which has an ability to convert α-methyltropate to optically active α-phenylpropionate, and it was identified as Rhodococcus sp. KU1314. Substrate specificity of the microorganism has been studied. When the aryl group was phenyl, 4-methoxyphenyl and 2-naphthyl, the substrate gave optically active α-propionate in good yields. To estimate the reaction mechanism, some compounds considered to be the intermediates were subjected to the reaction. Both enantiomers of α-methyltropate were converted to (R)-α-phenylpropionate with almost the same enantiomeric excess (68 and 72% from R-and S-enantiomers, respectively) and yield (605 and 48% from R-and S-enantiomers, respectively).     

Biotransformation of (±)-α-ionone and β-ionone by cultured cells of Caragana chamlagu

Suspension cultures of Caragana chamlagu (Leguminosae) convert (±)-α-ionone (1) into (±)-3-oxo-α-ionone (3) as the major product and β-ionone (2) into 5,6-epoxy-β-ionone (6) as the sole product. It is interesting to note that the cultured cells of C. chamlagu convert regioselectively the cycloolefinic part of 1 into the corresponding unsaturated carbonyl compound, allylic alcohol and epoxide as the oxidation products, whereas the suspension cultures of Nicotiana tabacum (Solanaceae) convert the unsaturated carbonyl of 1 into the corresponding saturated ketones and alcohols as reduction products.     

Characterization of in vitro proteolytic processing of β-endorphin by reversed-phase HPLC

In vitro, central and peripheral proteolytic processing of β-endorphin by membrane-bound enzymes results in the formation of specific active fragments that have been recently shown to function in behavior, intestinal motility and in the central control of urinary bladder activity. A high resolution, reversed phase high performance liquid chromatography system capable of separating 28 β-endorphin related fragments simultaneously was used to study the time-course processing of β-endorphin by membrane associated peptidases in the brain and regions of the small intestine. The hypothesis we tested was that a homeostatic balance between α- and γ-type endorphins exists in these tissues. The results of the study show that the rate and quantity of fragments produced between the mucosa and nerve-muscle regions of the small intestine are significantly different. Metabolic rates, pattern, and the ratio of α/γ-type endorphins in the brain were very similar to the nerve-muscle region of the small intestine. This suggests that β-endorphin processing to active fragments is occurring at the nerves of the small intestine and that a specific and similar balance of α/γ-type endorphin exists in the brain and gastrointestinal system at neutral pH.     

Antinoceptive effect of triterpenoid α,β-amyrin in rats on orofacial pain induced by formalin and capsaicin

The effects of α,β-amyrin, a pentacyclic triterpene isolated from Protium heptaphylum was investigated on rat model of orofacial pain induced by formalin or capsaicin. Rats were pretreated with α,β-amyrin (10, 30, and 100 mg/kg, i.p.), morphine (5 mg/kg, s.c.) or vehicle (3% Tween 80), before formalin (20 μl, 1.5%) or capsaicin (20 μl, 1.5 μg) injection into the right vibrissa. In vehicle-treated controls, formalin induced a biphasic nociceptive face-rubbing behavioral response with an early first phase (0–5 min) and a late second phase (10–20 min) appearance, whereas capsaicin produced an immediate face-rubbing (grooming) behavior that was maximal at 10–20 min. Treatment with α,β-amyrin or morphine significantly inhibited the face-rubbing response in both test models. While morphine produced significant antinociception in both phases of formalin test, α,β-amyrin inhibited only the second phase response, more prominently at 30 mg/kg, in a naloxone-sensitive manner. In contrast, α,β-amyrin produced much greater antinociceptive effect at 100 mg/kg in the capsaicin test, which was also naloxone-sensitive. These results provide first time evidence to show that α,β-amyrin attenuates orofacial pain atleast, in part, through a peripheral opioid mechanism but warrants further detailed study for its utility in painful orofacial pathologies.     

Simultaneous determination of retinol, β-carotene and α-tocopherol in adipose tissue by high-performance liquid chromatography

A method is described for evaluation of fat-soluble vitamin in human adipose tissue with the aim to obtain, accurately and within the shortest analysis time, a time-integrated measure of exposure to vitamins from the diet. Fat tissue was deproteinized with ethanol and extracted with n-hexane. Normal-phase HPLC was performed in a Lichrosorb Si60 column with a gradient of n-hexane–2-propanol at 1 ml/min. Detection was accomplished using a diode-array system (for retinol and β-carotene) in series with a fluorescence detector (α-tocopherol). The method was validated and applied to human adipose tissue in a total of 140 subjects. The mean contents found were 0.43, 0.84, 240.3 μg/g for retinol, β-carotene and α-tocopherol, respectively. The method is sensitive enough for detecting the compounds in 1.6 mg of adipose tissue considering the lowest concentration found.     

Chronic exposure to antibodies directed against anti-opiate peptides alter δ-opioid receptor levels

The development of addictive states in response to chronic opioid use may be regulated partially by the release of endogenous peptides. These anti-opiate peptides (AOP) are secreted or released into the CNS and produce diverse actions that counterbalance the effects of prolonged opiate exposure. Though the mechanism(s) by which these peptides exert their physiological properties remain largely unknown, there is some indication that AOP’s modulate opioid receptor levels. In this study, we investigated the effects of chronically infused α-melanocyte stimulating hormone (α-MSH), dynorphin_1-8 (DYN_1-8), dynorphin A (DYNA), and NPFF antibodies on δ-opioid receptor expression in rat brains. Quantitative autoradiographic experiments revealed that antibodies directed against α-MSH and DYNA produced significant increases in delta receptor levels in the caudate, claustrum, and cingulate cortex of the rat brain. Conversely, NPFF monoclonal antibodies caused significant decreases in the caudate, nucleus accumbens, olfactory tubercle, and cingulate cortex. These results suggest that the density of δ-opioid receptors is affected by changes in the levels of the anti-opioid peptides in the extracelluar fluid in the rat brain.