ACTH (1-39), rat

The lipolytic action of natural porcine ACTH1(-39) and of a number of highly purified synthetic ACTH peptide fragments was studied using rat adipocytes. Of the analogues tested, only ACTH1(-24) exhibited full lipolytic activity with respect to intrinsic activity and affinity. Several shorter fragments appeared to be full agonists but had lower affinity. Fragments ACTH5(-10) and ACTH7(-10) were inactive. No antagonistic effects against the lipolytic action of ACTH could be demonstrated with substimulatory doses of ACTH1(-16), ACTH1(-10), ACTH7(-24) and ACTH11(-24). Based on the relative potency derived from dose-response curves, a more refined model with respect to the active centers being encoded in various sequences of the hormone, is proposed.

The antagonist potency of a series of bivalent adrenocorticotropic hormone (ACTH) peptides was examined using suspensions of inner zone rat adrenocortical cells. Bivalent antagonists were prepared by bis(maleimide) covalent cross-linking of carboxyl terminal cysteine sulfhydryl groups of synthetic ACTH peptides, Cys25 ACTH(7-25) and Cys39 ACTH(7-39). Antagonist potency enhancement was defined by shifts in ACTH(1-39) concentration-steroidogenic response curves relative to monovalent antagonist analogs. The EC50 values measured in the presence of 0.5 microM monovalent antagonist were 110 +/- 28 pM for Cys25 ACTH(7-25)-S-N-ethylsuccinimide and 44 +/- 9 pM for Cys39 ACTH(7-39)-S-N-ethylsuccinimide. Some bivalent ACTH antagonists displayed much greater antagonist potency than their monovalent analogs, which supports the findings of Stolz and Fauchere (Helv Chim Acta 71: 1421-1428, 1988). The level of potency enhancement, however, was found to be dependent upon the spacer used to link receptor binding domains and the length of the ACTH peptide used in bivalent antagonist synthesis. The most potent inhibitor, bis(Cys25 ACTH(7-25)-S-succinimidopropionyl)2-hydroxy-1,3-propanediamine, was 28 times more potent than its monovalent analog, Cys25 ACTH(7-25)-S-N-ethylsuccinimide. However, a bivalent Cys25 ACTH(7-25) peptide containing two bis(succinimidopropionyl)2-hydroxy-1,3-propanediamine spacers that had been linked end-to-end via dithioerythritol showed no potency enhancement. Cys25 ACTH(7-25) based peptides containing one receptor binding domain and having the structure peptide-spacer-cysteine displayed no enhancement in antagonist potency. Bivalent Cys39 ACTH(7-39) linked by bis(succinimidopropionyl)2-hydroxy-1,3-propanediamine spacer exhibited only 4-fold enhancement in antagonist potency relative to Cys39 ACTH(7-39)-S-N-ethylsuccinimide. We therefore conclude that the potency enhancement observed with bivalent ACTH peptides: (1) is optimal with spacers less than approximately 40 A in length, (2) is not due to direct interactions between the spacer and cell surface, and (3) is dependent on the length of the ACTH peptide component. In addition, these results indicate that electrostatic interaction between bivalent ACTH peptides and plasma membrane lipids does not adequately account for the potency enhancements observed.

Intracerebroventricular (i.c.v.) injection of adrenocorticotropic hormone (ACTH) elicits grooming in the rat, but the neural organization of this response is still obscure. Electrical stimulation (EHS) in an area around the hypothalamic paraventricular nucleus (PVH) also elicits grooming. This hypothalamic area contains many ACTH-immunoreactive fibres. Injection of ACTH1-24 (0.3 microgram/0.3 microliters) in the same area elicits intense grooming responses in the rat. Latency, intensity and precise patterning of the grooming response are dependent upon the exact site of injection. Comparison of grooming responses elicited by EHS, ACTH injected i.c.v. and ACTH injected in the PVH reveals that these are slightly dissimilar. This may provide clues as to the brain mechanisms involved in the organization of the different components of grooming. EHS does not elicits scratching and even reduces 'spontaneous' scratching. Also, EHS-elicited grooming is characterized by short pauses. The time-course of appearance of yawning differs between ACTH-PVH and ACTH-i.c.v. injections. Excited locomotion elicited only by ACTH-i.c.v. is apparently caused by ACTH-sensitive systems outside the PVH. The results suggest that the ACTH-containing part of the hypothalamus around the PVH is crucially involved in the organization of grooming behaviour. We believe that at this level in the brain, the subroutines of grooming, scratching and yawning are integrated into one skin maintenance behaviour.

Intracerebroventricularly applied pilocarpine (2.4 mg/2 microliters) immediately produced symptoms of epilepsy, ranging from akinesia to motor seizures, in rats. Whereas ACTH-(1-39), ACTH-(1-24), ACTH-(1-18), ACTH-(1-16) and ACTH-(18-39) were not active, subcutaneous pretreatment with smaller ACTH-like fragments, such as ACTH-(4-9), ACTH-(4-10), ACTH-(4-10)(7D-Phe), ACTH-(7-16), and Org2766, reduced the severity of the epilepsy. Moreover, fewer rats developed motor seizures. Thus, ACTH fragments devoid of peripheral endocrine activity reduce pilocarpine-induced epileptiform activity in rats. A narrow bell-shaped dose-response relationship was found. Except for ACTH-(7-16), which was active in a dose of 1 and 10 micrograms/rat s.c., the other fragments were only active at one dose (10 micrograms/rat). The anti-epileptic properties appeared to reside in the sequence 1-16, and more specifically in the sequences 4-7 and 7-16, of the ACTH molecule.