The effect of MSH/ACTH 4-10 on delayed response performance and post-test locomotor activity in rats

Delayed response performance was measured in male, Long-Evans rats 1 hr after IP administration of various doses of MSH/ACTH 4-10 or control in a Hunter delayed reaction apparatus. Additional treatments consisting of naloxone 500 micrograms/kg (IP) and naloxone 500 micrograms/kg in conjunction with MSH/ACTH 4-10 95 micrograms/kg were also administered. Directly after delayed response performance was assessed, gross locomotor activity was determined. MSH/ACTH 4-10, at a dose of 95 micrograms/kg, significantly enhanced retention of a visual stimulus, while MSH/ACTH 4-10, at doses of 195 and 285 micrograms/kg, significantly impaired delayed response performance. Naloxone treatment resulted in significantly impaired delayed response performance when compared to control. However, naloxone plus MSH/ACTH 4-10 treatment failed to produce a significant difference from control in the delayed response performance paradigm. In post-test locomotor activity determination, an apparent dose-response existed for MSH/ACTH 4-10 with the two highest doses (190 and 285 micrograms/kg) resulting in significantly increased locomotor activity. The observed delayed response performance data support theories implicating MSH/ACTH peptides in attentional processes involving visual stimuli. The fact that large doses of MSH/ACTH 4-10 disrupt delayed response performance while increasing post-test activity suggest that an optimum level of effect caused by the MSH/ACTH peptide exists in this paradigm.     

MSH/ACTH4-10: a tool to differentiate between the role of vasopressin in memory consolidation or retrieval processes

MSH/ACTH4-10 induces a dose dependent increase of latency scores during retention of a passive avoidance response, when injected SC prior to retention but not when administered immediately after the learning trial. Intracerebroventricular administration of anti-vasopressin serum immediately after the learning trial or 1 hr prior to retention induces marked deficits in passive avoidance behavior as indicated by low latencies during retention. SC injection of MSH/ACTH4-10 increased latency scores in animals which received anti-vasopressin serum prior to retention, but did not alter latencies in animals, which received anti-vasopressin serum after the learning trial. These results suggest that MSH/ACTH4-10 is involved in retrieval processes and is able to differentiate between the effects of vasopressin on memory consolidation and on retrieval.     

Pressor and cardioaccelerator effects of gamma MSH and related peptides

We have recently demonstrated that the hypertensinogenic and natriuretic actions of ACTHI-39 can be found in a non-steroidogenic fragment of ACTH, ACTH4-10. These effects of ACTH or ACTH4-10 may be due to their ability to act as weak agonists of gamma MSH. gamma MSH is found in the 16K N-terminus of pro-opiocortin, and contains a sequence analogous to ACTH4-10, gamma MSH3-9. We investigated the cardiovascular effects of gamma 2MSH, gamma MSH3-9, and sterically restricted analogs of ACTH4-10. The results indicate that gamma MSH3-9, had essentially the same activities as ACTH4-10. The addition of five other amino acid residues to gamma MSH3-9 (gamma 2MSH) resulted in significant enhancement of pressor and cardioaccelerator activity. Steric restriction of the ACTH4-10 sequence by the substitution of a D-Phe in place of an L-Phe residue in position #7, or cyclization of the peptide by a half-Cys4, half Cys10 intramolecular disulfide-bridge derivatization, resulted in increased cardiovascular activities. Based on these data, the cardiovascular actions of ACTH4-10, gamma MSH3-9, and gamma 2MSH are predicted to be due to the assumption of a reverse-turn three-dimensional structure. The additional residues in gamma 2MSH appear to specifically enhance the cardiovascular activities of gamma MSH3-9. The results suggest the existence of a new class of hypophyseal peptides with cardiovascular activities, which require the assumption of a defined three-dimensional structure.     

Evidence for increased alpha MSH receptor binding in the F1 variant of B16 melanoma cells grown in dialyzed fetal calf serum

The specific binding of an alpha MSH analogue (Ac-[Nle4, D-Phe7] alpha MSH4-11 NH2) was enhanced in the presence of 10% dialyzed fetal calf serum (FCS) as compared with 10% FCS (nondialyzed) in the F1 variant of B16 melanoma cells. The replenishment of dialyzed serum with adrenocorticotrophic hormone (ACTH) or insulin had no effect on the increased level of alpha MSH receptor binding in these cells. However, 10 nM alpha MSH or 1 microM ACTH under identical conditions significantly decreased the level of alpha MSH binding. Competitive binding studies involving the alpha MSH analogue revealed that the specificity of the receptor was restricted to the complete molecule of alpha MSH, our analogue, beta MSH and ACTH1-24, ACTH4-10, which contains the amino acid sequence responsible for biological activity, showed a very low affinity for the receptor. Furthermore, we observed an interesting phenomenon unique to dialyzed FCS in that once the cells were grown to confluence and melanin was produced, the cells were no longer viable. However, in McCoy's medium, which is deficient in tyrosine, the cells did not produce melanin and remained viable.     

ACTH/MSH like peptides in the treatment of cisplatin neuropathy.

The neurological toxicity seen in patients treated with cisplatin in most cases concerns ototoxicity and peripheral neuropathy. Thus far, the pathogenesis of cisplatin neuropathy remains obscure. Yet the fact that cisplatin affects mainly the sensory peripheral nerve fibers points towards an involvement of the dorsal root ganglia. In a rat model of cisplatin neuropathy, following a cumulative dose of approx. 12 mg/kg cisplatin the sensory nerve conduction velocity began to slow as compared to age-matched controls. Peptides derived from ACTH and MSH are known to exert neurotrophic effects. In vivo they facilitate postlesion repair mechanisms in the peripheral nervous system by enhancing the early sprouting response of the damaged nerve. Surprisingly, chronic treatment with a synthetic ACTH4-9 analog not only prevented cisplatin neurotoxicity following a low or high dose regimen, but also counteracted already existing cisplatin-induced neurotoxicity. Stimulated by these findings a randomized, double blind, placebo-controlled study was performed to assess the efficacy of the peptide in the prevention of cisplatin neuropathy in women suffering from ovarian cancer. The threshold of vibration perception (VPT) was used as the principal measure of neurotoxicity. Following 6 cycles of chemotherapy the VPT had increased more than 8-fold in women receiving placebo as co-medication. Whereas the VPT in women receiving 1 mg/m2 body surface ACTH4-9 analog before and after each cisplatin cycle only increased less than 2-fold. No side effects of the peptide treatment were observed and the clinical response to the chemotherapy was similar in all treatment groups. Collectively these preclinical and clinical data suggest that treatment based on non-endocrine fragments of ACTH/MSH may be a therapeutic option in the treatment of cisplatin neuropathy.     

Can ACTH analogs support discriminative learning in rats?

Ten rats were trained to discriminate between the stimulus properties of subcutaneously (SC) administered MSH/ACTH4-10 and saline in a two-lever, food-motivated operant task. After 12 weeks of discriminative training with 100 micrograms/kg MSH/ACTH4-10, half the rats received 200 micrograms/kg MSH/ATCH4-10, whereas the other half were administered 400 micrograms/kg, for 6 additional weeks. Subsequently, all rats continued training on 50 micrograms/kg ORG 2766 (SC) and, after 12 weeks of training, were randomly assigned to receive either 100 or 200 micrograms/kg ORG 2766. The results of this extensive 36 week training schedule indicate that only 1 of the 10 rats learned to discriminate the interoceptive cues produced by the ACTH analogs. However, this rat's performance was so sustained and errorless that the possibility exists that it was relatively more sensitive to the effects of MSH/ACTH4-10 and its analogs and that these substances may support discriminative learning in the rat.     

ACTH/MSH(4-10) peptide increases postnatal metabolic activity of EDL muscle following early prenatal administration

ACTH/MSH(4-10) (10 micrograms/kg/b.i.d.; IP), administered to pregnant Sprague-Dawley rats during gestational days (GD) 3 to 12, significantly increased the metabolic activity of extensor digitorum longus (EDL) muscle at postnatal day 14. ACTH/MSH peptide, administered from day of birth to postnatal day 13, had no effect on EDL muscle metabolic activity using the 2,3,5-triphenyltetrazolium chloride indicator. By postnatal day 30, no differences were seen between the early prenatally treated group and saline controls. These results confirm our previous electrophysiological studies that showed that early prenatal ACTH/MSH(4-10) administration accelerates EDL muscle maturation.     

Are learning and attention related to the sequence of amino acids in ACTH/MSH peptides?

Learning and attention were examined in rats after injections of one of several molecules related to adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH). The initial phase of the learning process was linearly related to the length of the peptide with the smallest fragment (MSH/ACTH 4–10) improving learning the most and the largest molecule (ACTH 1–24) exerting no effect. Later stages of the learning problem, which were sensitive to the attentional state of the organism, resulted in U-shaped relations with the length of the same peptides. Enhancement of attention was significant only for the MSH compounds. These data indicate that some behaviors may be influenced as a function of the redundant information contained in the molecule while other behaviors may be discretely related to the specific conformation of the molecule.     

Differences in cortisol, aldosterone and testosterone responses to ACTH 1-17 administered at two different times of day

The effects of 100 micrograms, i.m. of the analog ACTH 1-17 administered at 0800 and 1800 on the secretion of cortisol, aldosterone and testosterone have been studied in normal subjects: 8 male and 8 female. The group as a whole and the males had significantly greater absolute and percent increments in plasma cortisol after administration at 1800. In the females, there was only a greater percent increment in cortisol after the evening administration. The heptadecapeptide always significantly stimulated serum aldosterone, with no difference between the two times of administration. In the females, ACTH 1-17 significantly stimulated testosterone, with a more protracted secretion after the evening administration. In the males, there was always a significant testosterone decrease after the administration of the drug, with no difference between morning and evening. In conclusion, 100 micrograms i.m. of the analog ACTH 1-17 stimulates cortisol secretion more when given during the circadian nadir of plasma cortisol, but only in men. ACTH 1-17 increases testosterone in women and decreases it in men, whereas it seems to increase aldosterone secretion in both sexes.     

ACTH 4-9 effects on the human visual event-related potential

Three oral doses (5, 10 and 20 mg) of an analog of ACTH 4-9 were compared with a vehicle control and d-amphetamine (10 mg). In a double-blind procedure, five men and five women were tested at weekly intervals with each treatment. In each session, four visual event-related potentials (ERPs) were recorded at hourly intervals. Visual ERPs were averaged from the electroencephalogram recorded from the left and right hemisphere. Dosage, time after administration, hemisphere of the brain and sex of the subject influenced the ERP. The ACTH 4-9 analog decreased amplitude of P100 but increased integrated activity of the ERP. This effect peaked at 60 minutes then "recovered." The effects of the peptide were more pronounced with doses of 5 and 10 mg, in the right hemisphere of men and in the left hemisphere of women. The findings indicated that the ACTH 4-9 analog influenced components of ERP commonly related to the perceptual/attentional state of the organism in a sexually dimorphic manner.     

Sequence of pituitary-adrenal cortical hormone responses to low-dose physostigmine administration in young adult women and men

We previously demonstrated greater HPA axis activation in adult men compared to adult women following low-dose administration of the anticholinesterase inhibitor, physostigmine (PHYSO). Because blood sampling was done infrequently following PHYSO, the rise times of AVP, ACTH1-39, and cortisol could not be determined. In the present study, we determined the sequence of hormone increases by frequent blood sampling following PHYSO. Twelve adult women and 12 adult men underwent three test sessions 5-7 days apart: PHYSO, saline control, and repeat PHYSO. As in the earlier study, PHYSO produced no side effects in half the subjects and mild side effects in the other half, with no significant female-male differences. None of the hormone responses was significantly correlated with the presence or absence of side effects. In both women and men, the AVP increase preceded the ACTH1-39 increase, which in turn preceded the cortisol increase. The AVP and ACTH AUCs were significantly positively correlated in both women and men, supporting AVP as an acute stimulus to ACTH secretion. Also as in the earlier study, the AVP response to PHYSO was more than twice as great in men as in women, but the difference was not statistically significant. We therefore analyzed the results of both studies combined (N=26 women and 26 men). The men had a significantly greater AVP response and a trend toward a greater ACTH1-39 response compared to the women. These findings further support the concept of sexual diergism (functional sex difference) in the influence of CNS cholinergic systems on HPA hormone secretion.     

The MSH/ACTH(4-9) analog Org2766 counteracts isolation-induced enhanced social behavior via the amygdala.

MSH/ACTH-like peptides influence social behavior induced by isolation It has been previously demonstrated that changes in locomotor activity as a result of isolation can be counteracted by Org2766 via the amygdala. The present study investigates whether isolation-induced changes in social behavior can also be affected by this peptide via the amygdala. A fully automated observation system was applied for detailed registration and analysis of movements of group-housed and 7-day isolated rats in a social interaction test. Administration of the MSH/ACTH(4-9) analog into the central nucleus of the amygdala elicited decreased locomotion, approach, and avoidance behaviors after isolation as compared to placebo-treated controls. However, general activity and social interest of group-housed rats were not affected by the MSH/ACTH(4-9) fragment. It is hypothesized that the amygdala is a site of action for neuropeptides in modulating social behavior.     

Role of chain termini in selective steroidogenic effect of ACTH/MSH(4-10) on isolated adrenocortical cells

To investigate the role of charged chain ends in the corticosteroidogenic effect of ACTH/MSH(4-10), acetyl and amide derivatives of ACTH/MSH(4-10) were synthesized and tested in isolated zona glomerulosa and zona fasciculata cells. ACTH/MSH(4-10)-NH2, Ac-ACTH/MSH(4-10) and Ac-ACTH/MSH(4-10)-NH2 (10 microM to 1 mM) stimulated the aldosterone production of zona glomerulosa cells, whereas these peptides did not stimulate the corticosterone production of zona fasciculata cells, even at 1 mM concentration. As ACTH/MSH(4-10) has been shown to have a steroidogenic effect on both types of adrenocortical cells, both charged chain termini seem to be essential for triggering of the corticosterone production of zona fasciculata cells, but for aldosterone production their presence appears not to be important.     

Intracranial administration of corticotropin-like peptides increases incidence of amphibian reproductive behavior

Male rough-skinned newts (Taricha granulosa) exhibit an increase in sexual behavior (amplectic clasping) following intracerebroventricular (ICV) injection of adrenocorticotropin (ACTH 1–39), ACTH 4–10, or melanocyte-stimulating hormone (αMSH). In contrast, intraperitoneal (IP) administration of ACTH 1–39 or corticosterone significantly decreases the incidence of sexual behavior in male newts. These results suggest that a corticotropin-like peptide acts centrally to enhance sexual behavior and that systemic ACTH acts on the interrenal tissue to inhibit sexual behavior by stimulating the release of corticosterone.     

Neonatal neuromuscular parameters vary in susceptibility to postnatal ACTH/MSH 4-10 administration

Neonatal Sprague-Dawley rats administered the fragment of adrenocorticotropic hormone ACTH/MSH 4-10 (10 micrograms/kg/daily, SC) postnatally, show marked differences in the plasticity of the functional and morphological parameters of their neuromuscular system. Initial contraction durations of the immature fast muscle, extensor digitorum longus (EDL), are shorter than saline-treated controls indicating accelerated development. Qualitative studies of the developing EDL neuromuscular junctions as viewed by the scanning electron microscope and quantitative analysis permitted by light microscopy confirms that ACTH/MSH 4-10 affects the maturation of the endplate region. Motor behavior of rat pups demonstrates an age-related difference in the susceptibility to this peptide fragment; one week old neonates showing no response to ACTH/MSH 4-10, two week old pups showing an increase in motor activity. The results indicate that while the developing neuromuscular system is sensitive to the input of ACTH/MSH peptide treatment, this susceptibility is age-related.     

ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in serum-free primary culture

It is well known that alpha-melanocyte stimulating hormone (MSH) induces the differentiation of mouse epidermal melanocytes in vivo and in vitro. Although adrenocorticotropic hormone (ACTH) possesses the same amino acid sequence as MSH does, it is not clear whether the peptide and its fragments induce the differentiation of mouse epidermal melanocytes. In this study, the differentiation-inducing potencies of human ACTH and its fragments were investigated by adding them into a culture medium (0.001-1,000 nM) from the initiation of primary culture of epidermal cell suspensions. Their potencies were compared with the potency of alpha-MSH. After 2-4 days of primary cultures with ACTH(1-13), ACTH(1-17), ACTH(1-24), ACTH(1-39), ACTH(4-12), ACTH(4-13), and alpha-MSH, pigment granules appeared in the cytoplasms and dendrites of melanoblasts that were in contact with the adjacent keratinocyte colonies. By 14 days, cultures contained mostly pigmented melanocytes. The order of potencies of ACTH fragments and alpha-MSH shown by the ED(50) value was as follows: alpha-MSH = ACTH(1-13) = ACTH(1-17) = ACTH(4-12) = ACTH(4-13) > ACTH(1-24) > ACTH(1-39). The length of their peptide chains was inversely proportional to the potency. On the contrary, ACTH(1-4), ACTH(11-24), and ACTH(18-39) failed to induce the differentiation of melanocytes. In contrast, ACTH(1-10), ACTH(4-10), ACTH(4-11), and ACTH(5-12) possessed a weak potency at high doses only (100 and 1,000 nM). These results suggest that ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in culture completely. The amino acids of Met(4) and Pro(12) are suggested to be important for its potency.     

Cultured Human Melanocytes Respond to MSH Peptides and ACTH

Although the administration of melanocyte-stimulating hormone (MSH) peptides results in skin darkening in man, cultured human melanocytes have been reported to be unresponsive to these peptides. This may be a consequence of the conditions under which the cells were maintained in vitro, particularly the use of phorbol esters and cholera toxin as melanocyte mitogens. By culturing the cells in the absence of these additives, we demonstrate that α-MSH and its synthetic analogue Nle⁴DPhe⁷α-MSH (NDP-MSH) induce dose-related increases in melanin content and tyrosinase activity and affect cell morphology in the majority of human melanocyte cultures. In addition, NDP-MSH induces increases in tyrosinase mRNA and tyrosinase-related protein-1 (TRP-1) mRNA. The dose-response curves for the MSH peptides are sigmoidal and the two peptides are equipotent in their effects on human melanocytes. Adrenocorticotropic hormone (ACTH) also affects morphology and stimulates melanogenesis and tyrosinase activity in human melanocytes. However, the dose-response curves for ACTH are biphasic, and the melanocytes respond to lower concentrations of ACTH than MSH peptides, similar to those normally present in human plasma. These findings may be important in understanding the role of these pro-opiomelanocortin peptides in human skin pigmentation.     

The utility of antisera to different synthetic adrenocorticotrophins (ACTH) and melanotrophins (MSH) for immunocytochemical staining of the dog pituitary gland

Using the immunoperoxidase technique and specific antisera to synthetic ACTH beta (1-24), ACTH beta (17-39) and bMSHbeta1, selective immunocytochemical staining was localized in a distinctive cell type in the pars distalis and pars tuberalis of the dog pituitary gland. Except for a rare cell, the pars distalis and pars tuberalis did not stain with an anti-bMSH alpha serum. In the pars intermedia immunoreactive cells containing ACTH beta(1-24), ACTHbetap(17-39), bMSHbeta and/or bMSH alpha were observed. The specificity and validity of the antisera were demonstrated by elimination of their immunostaining capacity after prior absorption with their respective antigens, while absorption with other antigens failed to decrease staining intensity. The cytoplasm of the ACTH/MSH cells showed a positive reaction to periodic-acid-Schiff and assumed a pale aniline blue colour, whilst the granules were stained with carmoisine L and acid alizarine blue. These ACTH/MSH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunochemical double staining. It is concluded that ACTH and MSH beta were present and most probably produced by the corticomelanotrophs of the pars distalis and pars tuberalis. In addition to corticomelanotrophs analogous to those of the pars distalis and pars tuberalis, the pars intermedia showed many cells which contain MSH alpha alone or together with MSH beta and/or ACTH.     

Melanocortins and fast axonal transport in intact and regenerating sciatic nerve

The effect of ACTH/MSH peptides on fast axonal transport along intact or regenerating sciatic nerve was examined following injection of tritiated leucine into the rat lumbar spinal cord. The rate of fast axonal transport was not significantly changed by treatment with ACTH/MSH(4-10), the ACTH(4-9) analog ORG 2766, hypophysectomy, or adrenalectomy. Fast axonal transport was unchanged in regenerating nerves and in regenerating, ACTH(4-10)-treated nerves. However, treatment with ORG 2766 in dosages of either 1 or 10 micrograms/kg/day IP for seven days significantly reduced (62% and 64%, respectively) the crest height of the fast axonal transport curve of intact sciatic nerve. The results suggest that the reported peptide-induced enhancement of nerve regeneration is not due to changes in the rate of fast axonal transport.     

Towards the therapeutic use of intranasal neuropeptide administration in metabolic and cognitive disorders

The nose provides an effective way for delivering neuropeptides to the central nervous system, bypassing the blood-brain barrier and avoiding systemic side effects. Thereby intranasal neuropeptide administration enables the modulation of central nervous signaling pathways of body weight regulation and cognitive functions. Central nervous control of energy homeostasis is assumed to rely on hypothalamic neuropeptidergic pathways that are triggered by the peripheral adiposity signals insulin and leptin conveying the amount of body fat to the brain. Melanocortins, including alpha-melanocyte stimulating hormone (alpha-MSH), are essential for inducing anorexigenic/catabolic effects, i.e. for inhibiting caloric intake and increasing energy expenditure. Insulin, in addition to its function as an adiposity signal, also influences memory formation. Here we present a series of studies on the intranasal administration of MSH/ACTH(4-10), a melanocortin receptor agonist, and of insulin. Prolonged administration of MSH/ACTH(4-10) induced weight loss in normal-weight, but not in overweight humans. Intranasal insulin reduced body fat and improved memory functions in the absence of adverse peripheral side effects. Our results may contribute to the future development of therapeutic strategies in disorders like obesity and cognitive impairments that derive from dysfunctions of central nervous neuropeptidergic pathways.