Structural and functional organization of ACTH: synthesis and properties of analogs of ACTH-(11-24)-tetradeca- and ACTH-(1-24)-tetracosapeptides containing hexa-amino acids instead of a natural sequence of ACTH 19-24 amino acids

To assess the role of amino acid sequence ACTH 19-24 in the corticotropin structure and steroidogenic activity, the analogues of ACTH-(11-24)-tetradeca- and ACTH-(1-24)-tetracosapeptides containing hexaglycine, hexaphenylalanine, hexaglutamic acid or hexalysine instead of the natural 19-24 sequence have been synthesized by conventional methods. All these compounds in water have the CD curves characteristic of random coil, CD spectra of analogue ACTH-(1-24)-tetracosapeptide and hexalysine-containing analogue ACTH-(11-24)-tetradecapeptide in trifluoroethanol indicate the presence of alpha-helices. The latter compound manifested higher steroidogenic activity than ACTH-(11-24)-tetradecapeptide. All the other analogues were either less active than ACTH-(1-24)-tetracosapeptide or inactive over the concentration range 10(-5)-10(2) mg/ml, thereby testifying to functional importance of the 19-24 sequence for manifesting full steroidogenic activity.     

Hormone-like activity of a synthetic decapeptide with the adrenocorticotropin-like sequence of human immunoglobulin G1

The antiproliferative and immunosuppressive in vitro effects of immunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11-20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10(-11)-10(-7) M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strain Salmonella typhimurium 415. By using a 125I-labeled "addressing" fragment of ACTH ?[125I]ACTH-(13-24)?, we showed that MT-4 cells express specific receptors for ACTH (Kd 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [125I]ACTH-(13-24) to these receptors with Ki1 of 0.38 and Ki2 of 0.34 nM, respectively. Specific receptors for ACTH (Kd 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to complete with labeled ACTH-(13-24) for binding to these receptors (Ki = 1.8 nM) and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.     

Effects of ACTH-(11-24) on the corticosteroid production of isolated adrenocortical cells

The steroidogenic action of ACTH-(11-24) was studied on isolated zona glomerulosa and zona fasciculata cells dispersed by collagenase. ACTH-(11-24) stimulated the corticosterone production of zona fasciculata cells and the aldosterone production of zona glomerulosa cells; in addition, it potentiated the effects of ACTH-(1-39) on both cell systems. It is suggested that the ACTH molecule contains more active sites for steroidogenesis than usually acknowledged, as has been found for lipolysis and behavior.     

ACTH-(1-24) and alpha-MSH antagonize dopamine receptor-mediated inhibition of striatal dopamine and acetylcholine release

The effects of ACTH-(1-24), alpha-MSH and ACTH-(4-10) were studied on the electrically evoked release of 3H-dopamine and 14C-acetylcholine from striatal slices in the absence and presence of the dopamine receptor agonist TL-99. None of the peptides affected transmitter release when TL-99 was not present. ACTH-(1-24) and alpha-MSH concentration-dependently antagonized the inhibition of striatal transmitter release induced by dopamine receptor stimulation due to the presence of TL-99. ACTH-(1-24), 10(-7)M, reduced the TL-99-induced inhibition of the release of both dopamine and acetylcholine by approximately 50%, and 5 X 10(-6) M ACTH-(1-24) restored the release fully to control values. alpha-MSH was less effective by a factor 20-30 in counteracting the release-inhibiting effect of TL-99. ACTH-(4-10) had no effect at any of the concentrations tested. These results show that ACTH/MSH-like neuropeptides may act by modulating dopamine receptor functions in rat striatum.     

Synthetic peptide KKRR corresponding to the human ACTH fragment 15-18 is an antagonist of the ACTH receptor

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [3H]ACTH (11-24) and [3H]ACTH (15-18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [3H]ACTH (11-24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (Kd 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) were synthesized, and their ability to inhibit the specific binding of [3H]ACTH (11-24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15-18) (KKRR) (Ki 2.3 +/- 0.2 nM), whose [3H] labeled derivative binds to rat adrenocortical membranes (Kd 2.1 +/- 0.1 nM) with a high affinity. The specific binding of [3H]ACTH-(15-18) was inhibited by 100% by unlabeled ACTH (11-24) (Ki 2.0 +/- 0.1 nM). ACTH (15-18) in the concentration range of 1-1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.     

Interaction of adrenocorticotropin-(11-24)-tetradecapeptide with neutral lipid membranes revealed by infrared attenuated total reflection spectroscopy

Infrared attenuated total reflection spectroscopy (IR-ATR) revealed that the hydrophilic adrenocorticotropin-(11-24)-tetradecapeptide ( ACTH11 -24, net charge 6+) assumed an irregular secondary structure when incorporated into the aqueous layers between equilibrated multibilayers of planar membranes prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ( POPC ). This structure was characterized by a perpendicular orientation of the peptide bonds on the bilayer surfaces, as observed earlier for the corresponding segment of adrenocorticotropin-(1-24)-tetracosapeptide (ACTH1-24, 6+). Once incorporated, ACTH11 -24 was not removed by washing, in agreement with its strong positive charge. In contrast to ACTH1-24, ACTH11 -24 was not measurably adsorbed to the neutral membranes from 0.1 mM aqueous solutions. The more hydrophobic adrenocorticotropin-(1-10)-decapeptide is also not adsorbed. We therefore concluded that adsorption of ACTH1-24 to neutral membranes was dependent on its amphiphilic primary (amphipathic primary) structure that resulted from the covalent combination of the hydrophobic ACTH1-10 segment with the hydrophilic ACTH11 -24 segment. This conclusion was consistent with the results obtained by vesicle-mediated hydrophobic photolabeling and equilibrium dialysis.     

Synthesis of [cyclo(Glu gamma-----epsilon Lys(Gly)]ACTH-(5-14) undecapeptide. Biological and physico-chemical properties of analogs of ACTH-(5-10)- and ACTH-(5-14)-peptides

Modified corticotropin fragment - [Lys11 (Gly)]ACTH-(5-14)- and its cyclic analogue - [cyclo (Glu gamma----epsilon Lys (Gly)] ACTH-(5-14)-undecapeptides have been synthesized by classical approach. The cyclic structure has been fixed by amide bond between gamma-COOH group of glutamic acid and alpha-NH2 group of glycine coupled to the epsilon-NH2 group of lysine. Fragment condensation has been achieved by azide or dicyclohexylcarbodiimide methods. Cyclization has been performed using diphenylphosphorylazide. The melanotropic activity of the cyclicanalogue on isolated frog skin exceeds by two orders of magnitude that of the linear undecapeptide, however the steroidogenic activity in isolated cells of rat adrenal cortex is diminished by an order of magnitude as compared with that of the linear precursor. A similarity of the CD spectra for the cyclic ACTH peptides and their linear counterparts in water and trifluoroethanol points to the similarity and relative rigidity of their structures.     

Stress-induced hypokinesia is facilitated by ACTH-(7-10)

Subcutaneous treatment with the neuropeptide ACTH-(4-10) induced hypokinesia in rats subjected to a mild stress induced by placing the animals on a non-functional "hot" plate (21 degrees C) for 30 sec, but not in control animals not exposed to this stress-inducing environment. The lowest effective dose of ACTH-(4-10) was 5 micrograms/kg, administered 50 min before testing. The combination of peptide treatment and the mild stress-inducing procedure mimicked the effect of a short intense stress induced by placing the rats on a hot plate (57 degrees C) for 30 sec, suggesting that this stress-induced hypokinesia is mediated by ACTH neuropeptides. Structure-activity relationship studies revealed that the active core for the ACTH-(4-10)-induced hypokinesia is located in the C-terminal tetrapeptide Phe-Arg-Try-Gly (ACTH-(7-10)). Pretreatment with the opioid antagonist naltrexone did not influence the effect of ACTH-(4-10) indicating that activation of opioid systems is not implicated in this behavioral effect of the peptide.     

Influence of synthetic peptide corresponding to the ACTH-like sequence of human immunoglobulin G1 on proliferation of lymphoblastoid cells

Influence of the ACTH-like peptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH corresponding to the sequence 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain on growth of MT-4 human T-lymphoblastoid cell line was investigated. It was found that the ACTH-like peptide at concentration range 10(-11) -10(-7) M inhibits the proliferation of MT-4 cells. Labeled ACTH 'address segment' [(125)I]ACTH (13-24) was used to establish that MT-4 cells express specific receptors for ACTH (K(d) = 97 pM). The ACTH-like peptide and human ACTH (but not IgG1 heavy chain) were shown to compete with [(125)I]ACTH (13-24) for binding to these receptors (K(i1) = 0.38 nM and K(i2) = 0.34 nM).     

In vitro interaction of ACTH with rat brain muscarinic receptors

ACTH-(1-24) inhibits the in vitro binding of the muscarinic antagonist [3H]QNB to membranes from rat brain. The magnitude of inhibition is dependent on the concentration of ACTH-(1-24). Kinetic analysis indicates a pure competitive inhibition which is suggestive of a reversible interaction of ACTH with muscarinic receptors. A mechanism involving an interaction of ACTH-(1-24) with the phospholipid core of the receptors is suggested. Structure activity studies point to a relation with reported effects of intracerebroventricularly administered ACTH on the turnover rate of acetylcholine and the ACTH-induced stretching and yawning syndrome.     

ACTH-(1-24) and alpha-MSH antagonize feeding behavior stimulated by kappa opiate agonists

ACTH-(1-24) and alpha-MSH, intracerebroventricularly (ICV) injected at the doses of 4 and 10 micrograms/animal, respectively, markedly inhibited spontaneous feeding in adult Sprague-Dawley rats, the effect remaining significant for 6-9 hours. At these same doses, ACTH-(1-24) and alpha-MSH abolished the feeding-stimulatory effect of the kappa opiate receptor agonist pentazocine, intraperitoneally (IP) injected at the dose of 10 mg/kg. The same antagonism was obtained by ICV injection of ACTH-(1-24) into rats IP treated with other kappa opiate agonists, bremazocine and tifluadom, at the doses of 1 and 5 mg/kg, respectively. These data suggest that melanocortin peptides play an inhibitory role in the complex regulation of food intake, and further support and extend the hypothesis of a melanocortin-opioid homeostatic system, its two neuropeptide components usually having opposite, mutually-balancing effects.     

Hormone activity of a synthetic decapeptide with the adrenocorticotropin-like sequence of human immunoglobulin G1

The antiproliferative and immunosuppressivein vitro effects ofimmunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11–20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10⁻¹¹−10⁻⁷ M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strainSalmonella typhimurium 415. By using a¹²⁵I-labeled “addressing” fragment of ACTH {[¹²⁵I]ACTH (13–24)}, we showed that MT-4 cells express specific receptors for ACTH (K _d 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [¹²⁵I]ACTH-(13–24) to these receptors withK _i1 of 0.38 andK _i2 of 0.34 nM, respectively. Specific receptors for ACTH (K _d 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to compete with labeled ACTH-(13–24) for binding to these receptors (K _i=1.8 nM), and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.     

Synthetic peptide KKRR corresponding to the human ACTH fragment 15–18 is an antagonist of the ACTH receptor

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [³H]ACTH (11–24) and [³H]ACTH (15–18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [³H]ACTH-(11–24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (K _d 1.8 ± 0.1 nM). Twenty nine fragments of ACTH (11–24) were synthesized, and their ability to inhibit the specific binding of [³H]ACTH (11–24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15–18) (KKRR) (K _i 2.3 ± 0.2 nM), whose [³H] labeled derivative binds to rat adrenocortical membranes (K _d 2.1 ± 0.1 nM) with a high affinity. The specific binding of [³H]ACTH-(15–18) was inhibited by 100% by unlabeled ACTH (11–24) (K _i 2.0 ± 0.1 nM). ACTH (15–18) in the concentration range of 1–1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.     

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.     

Adrenocorticotropin/α-Melanocyte-Stimulating Hormone (ACTH/MSH)-Like Peptides Modulate Adenylate Cyclase Activity in Rat Brain Slices: Evidence for an ACTH/MSH Receptor-Coupled Mechanism

Abstract: The regulation of adenylate cyclase activity by adrenocorticotropin/α-melanocyte–stimulating hormone (ACTH/MSH)-like peptides was investigated in rat brain slices using a superfusion method. Adenylate cyclase activity was concentration-dependently increased by ACTH-(1–24), α-MSH (EC₅₀ values 16 and 6 nM, respectively), and [Nle⁴,D-Phe⁷]α-MSH (EC₅₀ value 1.6 nM), in the presence of forskolin (1 μM, optimal concentration). 1-9-Dideoxy-forskolin did not augment the response of adenylate cyclase to ACTH-(1–24). Various peptide fragments were tested for their ability to enhance [³H]cyclic AMP production. [Nle⁴,D-Phe⁷]α-MSH increased [³H]cyclic AMP formation with a maximal effect of 30% and was more potent than ACTH-(1–24), ACTH-(1–16)-NH₂, α-MSH, ACTH-(1–13)-NH₂, [MetO⁴]α-MSH, [MetO₂⁴,D-Lys⁸,Phe⁹]ACTH-(4–9), ACTH-(7–16)-NH₂, ACTH-(1–10), and ACTH-(11–24), in order of potency. This structure–activity relationship resembles that found for the previously described peptide-induced display of excessive grooming. ACTH-(1–24) stimulated adenylate cyclase activity in both striatal (maximal effect, ?20%) and septal slices (maximal effect, ?40%), but not in hippocampal or cortical slices. Lesioning of the dopaminergic projections to the striatum did not result in a diminished effect of [Nle⁴,D-Phe⁷]α-MSH on [³H]cyclic AMP accumulation, which indicates that the ACTH/MSH receptor–stimulated adenylate cyclase is not located on striatal dopaminergic terminals. ACTH-(1–24) did not affect the dopamine D₁ or D₂ receptor–mediated modulation of adenylate cyclase activity. Based on the present data, we suggest that the binding of endogenous ACTH or α-MSH to a putative ACTH/MSH receptor in certain brain regions leads to the activation of a signal transduction pathway using cyclic AMP as a second messenger.     

Influence of genetic obesity in mice on the lipolytic response of isolated adipocytes to isoproterenol and ACTH-(1-24).

The lipolytic response of isolated adipocytes from genetic obese (C57/BL/64 ob/ob) and lean (C57BL/6J +/?) mice to ACTH-(1-24), isoproterenol and glucagon has been studied. The mean cell idameter of adipocytes form ob/ob mice was approximately twice that of lean controls. The adipocytes from obese mice contained on the average approximately six times the amount of triacylglycerol present in the smaller lean mouse adipocyte. Lipolysis was calculated both on a per cell basis (10(5) cells) and per mu mole of triacylglycerol and when expressed on a cell number basis, the larger adipocytes from obese mice showed an ACTH-(1-24) stimulated glycerol release which was quantitatively similar to that of smaller adipocytes from lean mice. When expressed per mu mole of triacylglycerol, the smaller cells from lean animals appeared to be dramatically more responsive to either isoproterenol or ACTH-(1-24). On either basis, ACTH-(1-24) stimulated glycerol release from obese mouse cells was greater than the isoproterenol response. The obese mouse of adipocyte showed selective loss of response to isoproterenol compared to its lean control.     

Presence of ACTH-potentiating factors in rat anterior pituitary glands

High molecular weight ACTH fractions, obtained through gel filtration of boiled rat anterior pituitary extract, induced a marked increase in corticosterone production from isolated rat adrenal cells in the presence of low concentrations of ACTH-(1-24). This indicates the presence of heat-stable factors augmenting the steroidogenic action of ACTH in the rat anterior pituitary. We also noted that these factors potentiated the activity not only of ACTH-1(1-24) but also of ACTH-(1-8). The ACTH-potentiating factors in rat anterior pituitary extract are possibly present in heterogeneous forms according to their molecular weights (8,000, 10,000 and 15,000), their mobility in ion-exchange chromatography and their content in RIA-ACTH activity. Of these three forms, the former comigrated with biological ACTH activity. The remaining two forms were free of it. Since the effect of potentiating factors on modified ACTH-(1-9), shown to be less susceptible to proteolytic degradation from ACTH-(1-24), was similar to the effect on ACTH-(1-24), it is suggested that potentiation was not due to an inhibition of ACTH proteolysis.     

Synthesis and biological properties of a cyclic analog of ACTH-(5-14)-decapeptide

A cyclic analogue and the corresponding linear segment of the corticotropin molecule, namely ACTH-(5-14)- and [cyclo (Glu gamma-epsilon Lys)]ACTH-(5-14)-decapeptide, both including the specific and unspecific active centers of the ACTH molecule, have been synthesized and studied. The cyclic structure is fixed by amide bond between the glutamic acid and lysine side chains. Condensation of fragments has been realized by azide or DCC/HOBT methods. Cyclization has been achieved using diphenylphosphorylazide. The cyclic analogue has full steroidogenic activity, while its melanotropic activity is 3 orders of magnitude higher than that of the linear decapeptide.     

In vivo distribution of radioiodinated ACTH1-24 in the rat

After intravenous injection of 125I-ACTH1-24 into rats, the highest concentration of 125I was found in kidneys, adrenal and liver. Addition of 5- and 30-fold excess unlabelled ACTH reduced the uptake of 125I by 50 and 68%, respectively, indicating that the adrenal uptake was specific. Pretreatment with dexamethasone decreased the adrenal uptake of 125I and caused adrenal atrophy. Chronic ACTH treatment increased the size of the adrenals, but did not affect the adrenal uptake of 125I. These experiments demonstrate selective uptake of 125I by the adrenals after administration of 125I-ACTH1-24.     

Structure-function organization of ACTH: fragment ACTH 11-24--a functionally important site of the hormone molecule

The steroidogenic and lipolytic activities of ACTH fragments (ACTH11-24--I, ACTH11-19--II, ACTH11-16--III and ACTH 17-24--IV) were studied. Fragments I--IV exert a steroidogenic effect in isolated fasciculata rat adrenal cells at concentrations of 1--500 micrograms/ml. The inner activity (alpha) and concentration at which a half-maximum effect is achieved (EC50) for fragments I and IV are 0.64+/-0.09 and 0.5--2.0 micrograms/ml, for fragment III--0.49+/-0.07 and 0.7 microgram/ml, respectively. Fragments I--IV have no effect on the lipolysis in isolated rat fat cells. The results obtained are indicative of the functional importance of fragment ACTH11-24 in manifestation of steroidogenic action of ACTH and suggest that the second active site of ACTH is enclosed within this amino acid sequence.