Native ACTH Research Articles

Spontaneous and tetracosactide-induced anti-ACTH antibodies in man.

SummaryContextDuring a clinical trial of regular tetracosactide depot injections, four of 13 patients with autoimmune Addison's disease (AAD) developed adverse reactions immediately following tetracosactide injections. We wished to investigate whether these adverse effects could be due to the production of circulating antitetracosactide (ACTH1–24) antibodies.DesignAnti‐ACTH binding activity was investigated using immunoblotting and ELISA on sera from participants in the trial (n = 13; baseline and after tetracosactide exposure), 131 unrelated patients with AAD, 92 patients with Graves’ disease (GD), 15 patients with isolated ACTH deficiency and 102 controls. Immunohistochemistry of human pituitary tissue sections was also performed using pooled sera.ResultsBands at approximately 4 and 6 kDa, corresponding to ACTH1–24 and full‐length ACTH1–39, respectively, were found in 10 of 13 (77%) of sera from trial patients exposed to tetracosactide, including all those who had an adverse reaction. This is in contrast with healthy control sera, which showed no binding. The same 10 subjects also showed high levels of binding to tetracosactide by ELISA, along with 21% of patients with AAD, 14% of patients with GD (both P < 0·001 compared to controls) and 1 isolated ACTH deficiency patient (7% of 15). These sera also recognized native ACTH in human pituitary sections.ConclusionOur study demonstrates that repeated administration of depot tetracosactide can lead to anti‐ACTH1–24 autoreactivity. In addition, a significant number of patients with AAD and GD also had similar, spontaneous, anti‐ACTH reactivity. The presence of these antibodies could mediate some of the adverse effects or explain the well‐described phenomenon of resistance to chronic ACTH therapy.

Further evidence for ancient role of ACTH peptides at melanocortin (MC) receptors; pharmacology of dogfish and lamprey peptides at dogfish MC receptors

The cloning of melanocortin (MC) receptors in distant species has provided us tools to get insight in how the ligand–receptors interactions in the MC system have evolved. We have however lacked studies on pharmacology of native ancient melanocortin peptides at the ancient MC receptors. In this paper we synthesized melanocortin peptides from both the sea lamprey ( Petromyzon marinus) and spiny dogfish ( Squalus acanthias) and tested them on the MC3 and MC4 receptors from spiny dogfish. The results show that both the dogfish and lamprey ACTH peptides have similar or higher affinity than the dogfish α-, β- and γ-MSH peptides to the dogfish MC3 and MC4 receptors. Moreover, both the dogfish and lamprey ACTH peptides have more than 10-fold higher affinity than α-MSH to the dogfish MC4 receptor. We also show that dogfish δ-MSH is able to bind to MC receptors and its potency is higher than of dogfish β-MSH, which is considered to be its precursor. Our results provide the first evidence that native ACTH ligands from dogfish and lamprey have a preference above native MSH peptides to ancient version of the MC3 and MC4 receptors. This further strengthens the hypotheses that the ligand contributing to the first version of the melanocortin ligand-receptor system resembled ACTH.

Mutational analysis of evolutionarily conserved ACTH residues

α-Melanocyte stimulating hormone (MSH) and adrenocorticotropin (ACTH)1–24, the minimal ACTH sequence required for full activity, differ only by the 10 C-terminal amino acids of ACTH1–24. Interestingly, these ten C-terminal residues have been highly conserved throughout vertebrate evolution. To understand the functional constraints of these 10 amino acids we analyzed the effects of mutating these residues on steroidogenic activity in vivo and in vitro. Alanine substitutions of some of the first four amino acid residues (the basic core residues KKRR, 15–18) greatly reduces ACTH activity in vitro and in vivo; replacement of mutant alanines at residues 15 and 17 with glutamine residues partially restores ACTH activity. Thus, for ACTH receptor binding and activation, the amino acid residues 15–18 are important for their side chains. Surprisingly, conversion of the five C-terminal residues (20–24) to alanines increases ACTH activity in vivo over that of native ACTH. With respect to receptor binding and activity, the last five amino acid residues are important only for the peptide length they contribute; however, with respect to serum stability, their side chains are significant.

Improving Pharmacokinetic Properties of Adrenocorticotropin by Site-Specific Lipid Modification

Although many peptides are potentially good therapeutic agents for treating various diseases, only a few have been developed for limited applications. A major shortcoming is that peptides have generally very short serum half lives. In the present study, we use adrenocorticotropin (ACTH) as a model and explore the potential of combining site-specific amino acid substitution and lipid modification to increase the circulating half-lives of peptides. Phe39 of ACTH was substituted by Cys, which has a free sulfhydryl group that can react specifically with iodoacetamide derivatives of lipophilic groups. The biological activities of lipophilized ACTH(F39C)s were higher than native ACTH. Lipophilized ACTH(F39C)s bound more tightly to human serum albumin and cell membranes in vitro and had longer serum half-lives in vivo than native ACTH. These results indicate that the pharmacokinetic properties of peptides can be improved by site-specific substitution with cysteine residues and subsequent conjugation with lipophilic moieties. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association.

Steroidogenic activity of a peptide specified by the reversed sequence of corticotropin mRNA.

The molecular recognition theory predicts that a reversed (3'----5') reading of an mRNA should yield a peptide that is structurally and functionally similar to that specified in the 5'----3' direction. We tested this idea by synthesizing a corticotropin (ACTH) analogue using a reverse reading of bovine mRNA for ACTH-(1-24). This peptide, designated ACTH-3'----5', had a similar hydropathic profile to native ACTH-5'----3' but had only 30% sequence homology and eight different charge substitutions. ACTH-3'----5' specifically bound to the surface of mouse Y-1 adrenal cells and to polyclonal anti-ACTH antibody. Additionally, ACTH-3'----5' stimulated cAMP synthesis and steroidogenesis in adrenal cells. These findings show that ACTH-3'----5' mimics the corticotropic properties of native ACTH, thereby further validating the molecular recognition theory.

Corticotropin Releasing Hormone and Arginine Vasopressin Stimulation of Acth and Substance P in Human Mononuclear Leukocytes

Bacterial lipopolysaccharide (LPS) and corticotropin releasing hormone (CRH) plus arginine vasopressin (AVP) induce immunoassayable (1-13)ACTH (alpha MSH) from mononuclear leukocytes. We studied the ability of LPS and CRH + AVP to in vitro stimulate native ACTH (not alpha MSH) and substance P (SP) production and thymidine incorporation in human mononuclear leukocytes. Neither CRH + AVP nor LPS stimulated detectable amounts of intracellular or extracellular ACTH (less than 15 pg/8 x 10(6) cells or total medium) or SP (less than 50 pg/8 x 10(6) cells or total medium) at 1, 2, 3 or 4 days of incubation. LPS, but not CRF + AVP, increased the amount of 3H-thymidine incorporation over controls. This data questions the importance of an immunoadrenal axis and the synthesis of SP by mononuclear leukocytes.

Stoichiometric methylation of porcine adrenocorticotropin by protein carboxyl methyltransferase requires deamidation of asparagine 25. Evidence for methylation at the alpha-carboxyl group of atypical L-isoaspartyl residues.

The enzymatic methylation of porcine adrenocorticotropin (ACTH) in both its native form and a form which is deamidated at asparagine 25 has been compared using purified protein carboxyl methyltransferase from bovine brain. Incubation of deamidated ACTH with high concentrations of methyltransferase resulted in near stoichiometric levels of methyl incorporation (78 mol %), while the methylation of native ACTH was highly substoichiometric (3-12 mol %). The Km and Vmax for deamidated ACTH were 1.9 microM and 11,200 pmol/min/mg, respectively, making this peptide the most specific substrate known for the mammalian methyltransferase. Deamidation of asparagine 25 leads to the formation of an atypical isopeptide bond in which the resulting aspartyl residue is linked to the adjacent glycine 26 via its side-chain beta-carboxyl group rather than the usual alpha-carboxyl linkage (Gráf, L., Bajusz, S., Patthy A., Barát, E., and Cseh, G. (1971) Acta Biochim. Biophys. Acad. Sci. Hung. 6, 415-418; Bornstein, P., and Balian, G. (1977) Methods Enzymol. 47, 132-145). A synthetic isopeptide (beta-linked) analog of deamidated ACTH serves as a highly effective substrate for the methyltransferase, but the corresponding normal (alpha-linked) peptide does not, indicating that this enzyme selectively recognizes the alpha-carboxyl group of atypical beta-linked L-aspartyl residues (see also accompanying paper (Murray, E.D., Jr., and Clarke, S. (1984) J. Biol. Chem. 259, 10722-10732]. Methylation of atypical beta-linked L-aspartyl residues resulting from deamidation can account for previous observations that in vitro protein carboxyl methylation in mammalian systems almost always occurs with a low stoichiometry and that these protein methyl esters are considerably less stable than most chemically formed protein methyl esters.

13C‐Nmr studies of ACTH: Assignment of resonances and conformational features

AbstractThe biologically active ACTH(1–32) and ACTH(1–24) and other shorter peptide segments of the native hormone ACTH(1–39) were studied in aqueous solution by 13C‐nmr. In order to identify the 13C resonances—except those of the carbonyls—both high‐field nmr spectroscopy measurements and substitution of residues with amino acids enriched to 85% in 13C were carried out. The main results are (1) the direct characterization of the cis–trans isomerism of proline 24 and its effects on the directly connected and sequentially neighboring residues and (2) findings that the conformational features agree with an α‐helix type organization in the N‐terminal part of the ACTH molecule which is responsible for the biological activity.

Ultrastructural changes in rat adrenal cortical cells after chronic stimulation with ?1?24-corticotropin

Adult rats were given 15 daily subcutaneous injections either of synthetic β1–24-corticotropin or of the corresponding placebo (controls) and were sacrificed 1 h after the final injection. In stimulated animals, the adrenal glands were increased in weight as compared to those of controls. Stereological analysis at light microscopic level of the outer zona fasciculata cells showed moderate volumetric increases of nuclei, cytoplasm and capillaries and a marked volumetric increase of lipid droplets in stimulated animals. Stereologic analysis of electron micrographs confirmed the marked increase in relative volume and surface density of lipid droplets, while volume fractions alone were increased for the Golgi apparatus and decreased for the endoplasmic reticulum and mitochondria. Biochemical analysis of the whole adrenal gland showed that the corticotropin injections produced a moderate increase in protein concentration, a marked increase in triglycerides and no appreciable changes in either phospholipid or cholesterol concentrations. The synthetic polypeptide therefore appears to have stimulating trophic effects on adrenal cortical cells, as shown by the increase in protein and cell size. However, it depresses the activity of the two types of organelle, endoplasmic reticulum and mitochondria, which have a major functional role in steroid synthesis. The increase of lipid droplets was interpreted as being primarily due to neutral fat accumulation, and secondarily to a diminished utilization of cholesterol for steroid synthesis. These findings suggest that, using this regime of administration, synthetic β1–24 corticotropin, unlike native ACTH, inhibits steroid synthesis.

Evidence for separate peptide sequences related to the lipolytic and magnesium-accumulating activities of ACTH. Analogy with adrenergic receptors

Native adrenocorticotropin [ACTH-(1-39)] and ACTH-(1-24) stimulate both lipolysis and magnesium accumulation in rat adipocyte plasma membrane vesicles. ACTH-(1-20) retains full lipolytic activity but has a minimal effect on magnesium accumulation. In contrast ACTH-(11-24) stimulates magnesium accumulation but not lipolysis. These findings indicate that within the ACTH molecule the peptide sequence responsible for stimulation of magnesium accumulation is distinctly separate from the core sequence (residues 4-10) essential for stimulation of adenylyl cyclase activity and cAMP mediated lipolysis. Phentolamine, an alpha-adrenergic antagonist, blocks the bulk of magnesium accumulation stimulated by native ACTH and norepinephrine; propranolol, a beta-adrenergic antagonist, blocks the earliest phase of Mg2+ uptake by these hormones but has little effect on net uptake. Isoproterenol, a beta-adrenergic agonist, stimulates magnesium uptake only minimally. The pattern of uptake stimulated by methoxamine, an alpha-adrenergic agonist, or ACTH-(11-24) is quite similar to that produced by native ACTH in the presence of propranolol. The receptor through which ACTH mediates stimulation of the bulk of magnesium appears to be analogous to the alpha-adrenergic receptor through which norepinephrine stimulates this same process.

Study of adrenocorticotropic hormone conformation by evaluation of intramolecular resonance energy transfer in N -dansyllysine 21 -ACTH-(1-24)-tetrakosipeptide.

The solution conformation of ACTH was studied by intramolecular resonance energy transfer and fluorescence depolarization with the synthetic, biologically active, derivative N(epsilon)-dansyllysine(21)-ACTH-(1-24)-tetrakosipeptide. The Förster parameters involved in energy transfer from the donor Trp(9) to the dansyl acceptor attached to the side chain of Lys(21) were determined from measurements with ACTH fragments containing either the donor or the acceptor alone. From determinations of the fluorescence quantum yields of the donor in the derivative and in the native ACTH-(1-24)-tetrakosipeptide, it was shown that besides energy transfer no additional quenching of the donor fluorescence is introduced by the presence of the acceptor. The short measured rotational relaxation time of the dansyl chromophore reflects the flexibility of the lysine side chain and justifies the use of an average value for the orientation factor in the distance calculations. The calculated intramolecular distance is remarkably independent of solvent, indicating a "random coil" situation with regard to residues 9-21.

Structure-Melanotropic Activity Relationship in Synthetic Polypeptides Related to ACTH

Melanotropic (MSH) activities of synthetic octadeca and tetracosa-peptides were compared with those of α-MSH, β-human-MSH, and porcine ACTH. The MSH activity was determined by in vivo approach using a newly devised reflectance meter to measure the skin colour of Xenopus frog. From the shapes of their time-response curves, α-MSH, Gly1-ACTH (1-18) amide (I), and β-Ala1-ACTH (1-18) amide (II) werefound to be of short action, whereas two long chain peptides, porcine ACTH and ACTH (1-24)-OH were of comparatively long action.β-human-MSH, Orn15-ACTH (1-18) amide (III), and βA-Ala1, D-Phe7, Orn15-ACTH (1-18) amide (IV) possessed extremely prolonged activity. The MSH potencies of these peptides were calculated from the dose required to give a minimal melanotropic response and were ranked as IV>β-human-MSH=II=III>ACTH=ACTH (1-24) OH>I.Compound IV was found to be about 400 times as potent as native ACTH. This ranking differs slightly from that found in the in vitro MSH assay proposed by Inouye et al.(1970 b) and is markedly different from the ranking in steroidogenic and lipolytic activities proposed by Tanaka (1971). The structure-activity relation and possible mechanism for these properties are discussed.

Structure-activity relationships of synthetic adrenocorticotropic octadecapeptides with special reference to biological half-life.

Adrenocorticotropic activities of eight octadecapeptide analogues related in structure to the N-terminal sequence of ACTH were compared. The extravascular: intravasuclar potency ratios of three peptides, Ser1-ACTH (1-18)(I), Ser1-ACTH (1-18) amide (II) and Gly1-ACTH (1-18) amide (III), were 1/5-1/8, whereas those of five peptides, β-Ala1-ACTH (1-18) amide (IV), Ibu1-ACTH (1-18) amide (V), β-Ala1, Orn15-ACTH (1-18) amide (VI), Ibu1, Orn15-ACTH (1-18) amide (VII), and β-Ala1, D-Phe7, Orn15-ACTH (1-18) amide (VIII), were 1-1/2. In vivo half-lives of the steroidogenic and lipolytic activities of the peptides injected intravenously differed slightly, but the ranking of the peptides for both criteria was the same. The in vitro half-lives of the lipolytic activity of peptides, III, IV and V, incubated with fresh plasma were similar and about a half of that of native ACTH, while those of peptides, VI, VII and VIII were much longer than that of native ACTH. When the peptide III was incubated with a medium containing muscle slices, the half-life in lipolytic activity was about 1/6 of that of native ACTH, but those of other peptides were prolonged in the order III<IV<V=VI<VII=native ACTH. These results indicate that (1) an N-terminal β-Ala or Ibu residue resists aminopeptidase activity, which is higher in tissue than in blood, consequently causing a potentiation of the extravascular potency of the peptide.(2) An ornithine residue in the 15th position of the peptide increases the resistance to endopeptidase activity, which is high in circulating blood, consequently prolonging the half-life in plasma in vitro. The relationships between in vitro half-life and two in vitro half-lives in lipolytic activity are considered.

A comparison of the lipolytic effects of β1–24-corticotropin and extracted porcine ACTH

The lipolytic effects of highly purified native porcine ACTH and synthetic β 1–24-corticotropin were compared on basis of their corticotropic potency. Both hormone preparations induced release of FFA from rat adipose tissue when added in vitro. The dose response curves were similar. The minimal effective concentration of the native ACTH was 2.4 × 10 −7 IU/ml.; that of the synthetic preparation was 2.3 × 10 −7 IU/ml. During prolonged incubation of both preparations hormone dependent lipolysis decreased to a similar degree, probably due to a similar instability of both peptides in the in vitro system. Both preparations caused a significant elevation of plasma FFA levels when injected into human subjects. Again, there was no difference in potency between the two preparations.

Stability of ACTH Preparations in Human Plasma Incubatedin Vitro

The stability of the steroidogenic activity of ACTH was studied in human plasma incubated in vitro using a series of native and synthetic ACTH preparations. Native ACTH was stable over a period of 4 hr when incubated in fresh human plasma, although it was significantly inactivated during the same period in plasma stored overnight in a freezer. Synthetic 39 amino acid ACTH (αp1–39-ACTH) was as stable as native ACTH in fresh plasma. However, synthetic preparations with 26, 19 and 18 amino acids (αb1–26-ACTH, α1–19-ACTH and α1–18-ACTH) were progressively less stable in that order, than was the whole molecule. Octadecapeptide amide (α1–18 NH2-ACTH) was significantly more stable than α1–18-ACTH. These results may explain at least partly the differences in steroidogenic potency of these ACTH preparations. It would appear, therefore, that the chain length of the C-terminal portion of the ACTH molecule is an important factor in protecting ACTH from inactivation. It suggests also that the presence of a carboxamide...

Incompatibility of synthetic tetraicosapeptide corticotrophin with the present USP and International Corticotrophin Standard.

COBTICOTROPHIN, unlike insulin or the octapeptides of the posterior lobe of the pituitary gland, is known to exist in different biological forms, due to inconsistencies in bioassays in which various routes of administration are used. The first hormone preparations used were of the same type as the first and second International Standard, that is, crude Corticotrophin—a glacial acetic acid extract of pituitary glands. When in 1951 the oxycellulose adsorption method of Astwood et al.1 yielded a highly purified hormone, the bioassays carried out by subcutaneous injections into hypophysectomized rats showed, on the average, potency estimates three times greater than the classical assay method (Sayers test) which involves intravenous injection. This phenomenon led to a type distinction of oxycel-purified ACTH from crude Corticotrophin, the former being called Corticotrophin A (Wolfson2). Another attempt to purify Corticotrophin involving peptic or strongly acid hydrolysis yielded preparations of lower molecular size (about 28 amino-acid residues), which also differed biologically from crude Corticotrophin3. In this case the ratio of potencies observed after subcutaneous and intravenous comparison with the latter was reversed and of the order of 1 : 4. This third biological ACTH type was designated Corticotrophin B or actide (ref. 2). The foregoing ratios were obtained in rats but appear, in the main, to agree with those in man so far as crude Corticotrophin and Corticotrophin A are concerned. Bearing in mind that ACTH is mainly given subcutaneously (extravascularly), Corticotrophin A may be regarded as the most important type of native ACTH due to its high degree of purity and favourable efficiency when given extravascularly. It is partly for these reasons that the now official International and U.S.P. Corticotrophin Standard preparations are of the corticotrophin A type, a fact which has practically eliminated bioassay discrepancies (crude corticotrophin is now rarely used). The reasons for the foregoing bioassay discrepancies are unknown, but it is most likely that they lie in the different rates of inactivation of the different types of ACTH at the site of extravascular administration.