The effects of ACTH 1-17 on GH secretion in vitro

Recently we demonstrated that ACTH 1-17 infusion in normal subjects is able to stimulate growth hormone (GH) secretion. In order to study the mechanism by which ACTH 1-17 induces this hormonal secretory pattern, we examined the effects of ACTH 1-17 addition to primary cultures of rat anterior pituitary cells and of two human pituitary adenomas (a mixed GH- and PRL-secreting adenoma and a prolactinoma) on GH and PRL secretion. Normal rat pituitary cells responded to rGRF with a dose-dependent increase of rGH: ACTH 1-17 induced a slight not significant increase of rGH secretion even at micromolar concentrations. Furthermore no additive effect of ACTH 1-17 on rGRF-stimulated GH release was observed. No significant stimulatory effect was also documented in the human tumors studied. These results suggest that the GH releasing activity of ACTH 1-17 observed in vivo is mediated via a direct action on CNS.     

Stimulatory effects of a short chain phosphatidate on superoxide anion production in guinea pig polymorphonuclear leukocytes

Treatment of guinea pig polymorphonuclear leukocytes (PMNL) with a phosphatidate containing short-chain fatty acids, 1,2-didecanoyl-3-sn-phosphatidate (PA10), induced substantial superoxide anion (O2-) production in a dose-dependent manner, whereas phosphatidates prepared from egg lecithin and 1,2-dioleoyl-3-sn-phosphatidate (PA18:1) had no such effect. Calcium was not involved in PA10-induced O2- production, since the production was also observed in the case of addition of EGTA prior to PA10 or pretreatment of PMNL with quin-2 and EGTA to eliminate contributions of both extracellular and intracellular calcium. We have reported in previous papers that the phosphorylation of 46K protein(s), which was commonly observed in parallel with an activation of NADPH oxidase in PMNL, was increased by treatment with 10 microM 1-oleoyl-2-acetylglycerol (OAG) with little change in the O2- production (Okamura et al. (1984) Arch. Biochem. Biophys. 228, 270-277; Ohtsuka et al. (1988) Arch. Biochem. Biophys. 260, 226-231). Treatment of PMNL with a combination of PA10, which slightly increased 46K protein phosphorylation, and such a low concentration of OAG induced a marked increase in the O2- production with the increase in 46K protein phosphorylation, which was probably due to OAG action. Thus, it is likely that this protein phosphorylation plays a significant role in the stimulation of the O2- production by phosphatidate in PMNL.     

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.     

Investigating the effects of physiological bile acids on GLP-1 secretion and glucose tolerance in normal and GLP-1R(-/-) mice

Physiological secretion of bile acids has previously been linked to the regulation of blood glucose. GLP-1 is an intestinal peptide hormone with important glucose-lowering actions, such as stimulation of insulin secretion and inhibition of glucagon secretion. In this investigation, we assessed the ability of several bile acid compounds to secrete GLP-1 in vitro in STC-1 cells. Bile acids stimulated GLP-1 secretion from 3.3- to 6.2-fold but some were associated with cytolytic effects. Glycocholic and taurocholic acids were selected for in vivo studies in normal and GLP-1R(-/-) mice. Oral glucose tolerance tests revealed that glycocholic acid did not affect glucose excursions. However, taurocholic acid reduced glucose excursions by 40% in normal mice and by 27% in GLP-1R(-/-) mice, and plasma GLP-1 concentrations were significantly elevated 30 min post-gavage. Additional studies used incretin receptor antagonists to probe involvement of GLP-1 and GIP in taurocholic acid-induced glucose lowering. The findings suggest that bile acids partially aid glucose regulation by physiologically enhancing nutrient-induced GLP-1 secretion. However, GLP-1 secretion appears to be only part of the glucose-lowering mechanism and our studies indicate that the other major incretin GIP is not involved.     

Naloxone-like influence of TRH and ACTH-(4-7) on hypothalamic blood flow autoregulation in the rat

The influence of intracerebroventricularly (ICV) administered thyrotropin-releasing hormone pGlu-His-Pro-NH2 (TRH), pGlu-His-Phe-NH2 (TRH analog, (TRHa)), Met-Glu-His-Phe(ACTH-(4-7)) and His-Phe-Arg-Trp-Gly (ACTH-(6-10)) on autoregulation of cerebral blood flow was studied in anesthetized, ventilated rats. Autoregulatory capacity of the cerebrovascular bed was tested by hypothalamic blood flow (HBF) and total cerebral blood volume (CBV) determinations during consecutive stepwise lowering of the systemic mean arterial pressure to 80, 60 and 40 mmHg, by hemorrhage. None of the peptides caused a change in resting HBF or CBV upon ICV administration (5 micrograms/kg). However, the same dose of TRH, TRHa and ACTH-(4-7) resulted in impairment of autoregulation. ACTH-(6-10) was not effective. Thus, the disturbed autoregulation may be due to the presence of the dipeptide Glu-His which is common to TRH, TRHa and ACTH-(4-7).     

Effect of soman (pinacolyl methylphosphonofluoridate) on the blood levels of corticosterone and adrenocorticotropin in mice

Mice were poisoned by an extremely toxic organophosphate anticholinesterase soman (pinacolyl methylphosphonofluoridate), 50 or 100 micrograms/kg at 1000, and the serum concentrations of corticosterone were determined fluorometrically at 3-h intervals for at least 24 h. The lower soman dose (50 micrograms/kg) produced a modest increase in serum corticosterone concentrations but by 24 h the levels were not significantly different from control. Following the higher soman dose (100 micrograms/kg) the serum corticosterone levels were elevated significantly (p less than 0.05), for at least 27 h. However, ACTH concentrations were not elevated. It is possible that the elevated levels of corticosterone were due to a reduced metabolism and excretion of corticosterone resulting from the intense hypothermia, following soman poisoning which may change cardiac output and organ (liver and kidney) perfusion and not due to an enhanced release from the adrenal gland.     

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.     

The differential effects of galanin-(1-30) and -(3-30) on anterior pituitary hormone secretion in vivo in humans

Intravenous injection of galanin increases plasma growth hormone (GH) and prolactin (PRL) concentrations. In the rat, the effects of galanin on GH appear to be mediated via the hypothalamic galanin receptor GAL-R(1), at which galanin-(3-29) is inactive. In contrast, the effect of galanin on PRL is mediated via the pituitary-specific galanin receptor GAL-R(W), at which galanin-(3-29) is fully active. We investigated the effects of an intravenous infusion of human galanin (hGAL)-(1-30) and -(3-30) on anterior pituitary hormone levels in healthy females. Subjects were infused with saline, hGAL-(1-30) (80 pmol. kg(-1). min(-1)), and hGAL-(3-30) (600 pmol. kg(-1). min(-1)) and with boluses of gonadotropin-releasing hormone, thyrotropin-releasing hormone, and growth hormone-releasing hormone (GHRH). Both hGAL-(1-30) and -(3-30) potentiated the rise in GHRH-stimulated GH levels [area under the curve (AUC), saline, 2,810 +/- 500 vs. hGAL-(1-30), 4,660 +/- 737, P < 0.01; vs. hGAL-(3-30), 6, 870 +/- 1,550 ng. min. ml(-1), P < 0.01]. In contrast to hGAL-(1-30), hGAL-(3-30) had no effect on basal GH levels (AUC, saline, -110 +/- 88 vs. hGAL 1-30, 960 +/- 280, P < 0.002; vs. hGAL-(3-30), 110 +/- 54 ng. min. ml(-1), P = not significant). These data suggest that the effects of galanin on basal and stimulated GH release are mediated via different receptor subtypes and that the human equivalent of GAL-R(W) may exist.     

Comparison between adrenal steroid secretion after stimulation with two synthetic analogues of corticotropin : ACTH 1-24 and ACTH 1-17

Chemical manipulations of the native ACTH (1-39) molecule led to the synthesis of a newly characterized compound which shows several interesting features, such as an enhanced half-life, an increased resistance against enzymic degradation and lower immunogenic activity. This study aimed at comparing the adrenal steroidogenetic response of the 1-17 analogue and of the other commercially available synthetic ACTH (1-24). Results suggest that ACTH 1-17 has a more pronounced and prolonged activity, which is confined to glico- and mineralocorticoid secretion.     

Nociceptin and dynorphin A(1-17) produce antianalgesia through independent systems in mice

The administration of dynorphin A(1-17), Dyn, intrathecally (i.t.) or of nociceptin, intracerebroventricularly (i.c.v.) produces antianalgesic actions against i.t. morphine in the tail flick test in mice. The antianalgesic action of nociceptin is mediated by spinal PGE2 and attenuated by i.t. PGD2 or indomethacin. The Dyn response is mediated by release of IL1beta in the spinal cord to activate an ascending pathway to the brain and in turn releases IL1beta in the brain which activates a descending pathway to the spinal cord. The present work investigated the possibility that the action of IL1beta in the Dyn system might release prostaglandins so that the Dyn and nociceptin antianalgesic systems would overlap at these points. The results indicated that in the Dyn system neither the IL1beta in the spinal cord or brain implicated prostaglandin release because i.t. and i.c.v. PGD2 and indomethacin did not affect Dyn-induced antianalgesia. In addition, nociceptin-induced antianalgesia did not involve components in the Dyn system. Thus, the Dyn and nociceptin antianalgesic systems did not overlap and each were independent systems.     

Glucagon-like peptide-1(1-37) can enhance blood glucose homeostasis in mice

Tyrosyl O-sulfation is a common posttranslational derivatization of proteins that may also modify regulatory peptides. Among these are members of the cholecystokinin (CCK)/gastrin family. While sulfation of gastrin peptides is without effect on the bioactivity, O-sulfation is crucial for the cholecystokinetic activity (i.e. gallbladder emptying) of CCK peptides. Accordingly, the purification of CCK as a sulfated peptide was originally monitored by its gallbladder emptying effect. Since then, the dogma has prevailed that CCK peptides are always sulfated. The dogma is correct in a semantic context since the gallbladder expresses only the CCK-A receptor that requires sulfation of the ligand. CCK peptides, however, are also ligands for the CCK-B receptors that do not require ligand sulfation. Consequently, unsulfated CCK peptides may act via CCK-B receptors. Since in vivo occurrence of unsulfated products of proCCK with an intact α-amidated C-terminal tetrapeptide sequence (-Trp-Met-Asp-PheNH(2)) has been reported, it is likely that unsulfated CCK peptides constitute a separate hormone system that acts via CCK-B receptors. This review discusses the occurrence, molecular forms, and possible physiological as well as pathophysiological significance of unsulfated CCK peptides.     

Effects of an ACTH/MSH(4-9) analog (HOE 427) on the sleep EEG and nocturnal hormonal secretion in humans.

The synthetic ACTH/MSH(4-9) analog HOE 427 ("ebiratide"), which is behaviorally the most potent ACTH-derived peptide but which is devoid of endocrine activity, was administered intravenously in a pulsatile mode 4 times (120 micrograms each) at 2200, 2300, 2400 and 0100 to study its effect on the sleep EEG and on concomitant hormonal secretion of cortisol and growth hormone. In comparison to placebo, the peptide produced signs of general activation associated with specific deteriorating effects on the quality of sleep. Sleep onset latency and intermittent wakefulness were increased, slow wave sleep was reduced, but only during the first 3 hours of the sleep period. The nocturnal secretory patterns of cortisol and growth hormone were unaffected by HOE 427. These effects are different from those reported in similar studies in which corticotropin-releasing hormone (CRH) was applied in humans, and they suggest that peripherally administered neuropeptides have specific nonendocrine behavioral effects.     

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.     

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.     

Anti-inflammatory and anti-arthritic effects of new synthetic 3-(4-hydroxyphenyl)-4-(4-thiomethoxyphenyl)-1H-pyrrole-2,5-dione

We previously reported that 3-(4-hydroxyphenyl)-4-(4-thiomethoxyphenyl)-1H-pyrrole-2,5-dione (1, HMP) has a strong inhibitory effect on prostaglandin E(2) (PGE(2)) production. In this study, the anti-inflammatory and anti-arthritic effects of HMP were evaluated on LPS-induced RAW 264.7 macrophages and rats with carrageenan-induced paw edema and adjuvant-induced arthritis (AIA). The attenuation of PGE(2) production by HMP was found to be caused by the inhibition of cyclooxygenase-2 (COX-2) activity, but not COX-1 activity. However, HMP did not affect COX-2 at the protein or mRNA levels, whereas it suppressed the releases and expressions of inflammatory cytokines, such as, interleukin-1β (IL-1β) and IL-6 in LPS-induced macrophages. Furthermore, HMP suppressed LPS-induced nitric oxide (NO) production by down regulating the protein and mRNA expressions of inducible nitric oxide synthase (iNOS). In rats with carrageenan-injected acute inflammation, oral administration of HMP (25 or 50mg/kg, po) reduced paw swelling, and PGE(2) release and myeloperoxidase (MPO) activity in tissue. Furthermore, HMP (25 or 50mg/kg, po) significantly reduced paw swelling, arthritic indices and plasma PGE(2) concentrations in rat with AIA. These results show that HMP reduces swelling in a model acute inflammation and inhibits arthritic responses in a model of chronic inflammation via the inhibition of PGE(2) production. These results suggest that HMP is a potential therapeutic agent for the treatment of arthritis and associated disorders.     

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.     

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.     

Behavioral and neurotrophic activity of ACTH-(7-16)NH2

The behavioral and neurotrophic effects of ACTH-(7-16)NH2 were assessed in a number of tests in which other ACTH fragments are active. Subcutaneous injection of ACTH-(7-16)NH2 increased motor activity of group-housed rats tested under low light intensity and induced hypokinesia in rats subjected to the mild stress of a nonfunctional "hot" plate. In rats with 6-OHDA lesions in the nucleus accumbens daily subcutaneous treatment with ACTH-(7-16)NH2 during the first week following the lesions reversed the lesion-induced motor hypoactivity. The ED50's for the effects of ACTH-(7-16)NH2 on the environmentally induced changes in motor activity, the stress-induced hypokinesia and the impaired motor activity of 6-OHDA lesioned rats were approximately 8 micrograms/kg. 6.3 micrograms/kg and 0.45 micrograms/kg respectively. It is concluded that ACTH-(7-16)NH2 may mimic the effect of an ACTH-like peptide in the brain involved in brain processes triggered by changes in the environment and by brain damage.