Holly A. Thomas

d-amino acid-substituted analogs of corticotropin-releasing hormone (CRH) and urocortin with selective agonist activity at CRH1 and CRH2β receptors

Abstract The activities of corticotropin-releasing hormone (CRH)-related peptides and several analogs were examined in cells transfected with either CRH1 or CRH2β receptors, in suppression of heat-induced rat paw edema in pentobarbital-anesthetised animals and in stimulation of release of immunoreactive corticotropin (ir-ACTH) from rat anterior pituitary tissue in vitro. The peptides tested were human/rat (h/r)-CRH, r-urocortin, h-urocortin, white sucker fish or maggy sole urotensin I and some analogs of these peptides substituted with d -amino acids at residues 4 (urocortin), 5 (CRH and urotensin I) and 20 (CRH). In cells transfected with CRH1 receptors, these peptides were similar in potency in stimulation of cAMP accumulation. By contrast, at CRH2β receptors peptides of the urocortin and urotensin series were more potent than h/r-CRH while [ d -Glu20]-h/r-CRH was 6.5-fold less active than h/r-CRH. IV administration of h/r-CRH or related peptides 10 min prior to a thermal stimulus produced a significant dose-dependent inhibition of rat paw edema formation. Comparison of the ED50’s showed that urocortins ([ d -Ser4]-h-urocortin, h-urocortin, [ d -Pro4]-r-urocortin, r-urocortin) were approximately 2 to 3 times more active than h/r-CRH, but [ d -Glu20]-h/r-CRH was 18.5-fold less active. In the assay for ir-ACTH release, the activity of h/r-CRH and [ d -Glu20]-h/r-CRH was similar but [ d -Pro5]-h/r-CRH and [ d -Pro4]-r-urocortin was less potent than the native peptide. These results provide further evidence that d -amino acid substition at residue 20 reduces the potency of h/r-CRH at endogenous (anti-edema effect) and transfected (cAMP accumulation) CRH2β receptors whilst activity at the CRH1 receptor is retained (ACTH-release and cAMP accumulation). On the other hand substitutions at residues 4 or 5 in r-urocortin or h/r-CRH respectively appear to decrease activity at CRH1 but not CRH2β receptors The modified CRH and urocortin analogs described here may provide clues for the further design of receptor selective ligands.

Correlation of neuroendocrine and anti-edema activities of alanine-corticotropin-releasing factor analogs.

Thirty-three ovine corticotropin-releasing factor (CRF) analogs, systematically substituted with alanine (Ala) residues, were tested for inhibitory activity on edema induced in the pentobarbital-anesthetized rat paw by heat (immersion in 58 degrees C water for 1 min). The activity of each analog, administered 0.1 mg/kg i.v. 10 min before heat, was compared to the rank order of the analogs potency in stimulating adrenocorticotropin (ACTH) release from cultured rat pituicytes. A strong positive rank correlation was found between the anti-edema and neuroendocrine activities of these analogs.

Dynorphin A(6-12) Analogs Suppress Thermal Edema

Dynorphin A (Dyn A) is a 17-residue opioid peptide derived from prodynorphin precursors found in mammalian tissues. Removal of Tyr1 from Dyn A produces a peptide that is more potent than Dyn A in attenuating the acute phase of the inflammatory response, as measured by inhibition of heat-induced edema in the anesthetized rats paw (exposure to 58 degrees C water for 1 min). Dyn A(2-17), however, no longer interacts with opioid receptors. It was postulated that the non-opioid anti-inflammatory actions of Dyn A(2-17) may reside in Dyn A(6-12); that is, Arg-Arg-Ile-Arg-Pro-Lys-Leu. here we report on the activities of Dyn A(6-12) analogs modified by substitutions on the N terminus, by single N-methyl substitution and by single replacement of residues by alanine. The results indicated that the minimal sequence required for an anti-edema ED50 of <1.0 micromol/kg i.v. was anisoyl-Arg6-Arg7-Xaa8-Arg9-Pro10)-Xaa11-+ ++Xaa12-NH2. A prototype, p-anisoyl-[D-Leu12] Dyn A(6-12)-NH2, with an ED50 of 0.20 micromol/kg i.v. compared to an ED50 of 0.08 micromol/kg i.v. for Dyn A(2-17), was selected for further tests of biological activity. This analog, like Dyn A(2-17), lowered blood pressure in anesthetized rats. In a model of neurogenic inflammation, produced by antidromic stimulation of the vagus in the anesthetized rat, p-anisoyl-[D-Leu12] Dyn A(6-12)-NH2, 0.23 micromol/kg i.v., attenuated the negativity of tracheal tissue interstitial pressure (Pif), which normally develops after nerve stimulation. Modulation of interstitial pressure may be the mechanistic basis for the anti-edema properties of these Dyn A(6-12) analogs.

Bipolar-shape response of human neutrophils to corticotropin-releasing factor

Human neutrophils in whole blood become bipolar in shape after exposure to chemokinetic stimuli. In normal blood, the proportion of non-spherical neutrophils was 1.2 +/- 0.07% (n = 101). After incubation of blood samples with corticotropin-releasing hormone (CRF, 1 to 20 microM) 36 of 101 subjects exhibited a > or = 10% bipolar-shape ellipsoid response. This ellipsoid response was more frequent in female than in male subjects (32/75 vs. 4/26, p < 0.01). Female Caucasian subjects were more sensitive to CRF than female East Asian subjects (25/48 vs. 2/15, p < 0.01). Age was not a factor in sensitivity to CRF. In young female East Asian subjects (23 +/- 0.4 years, n = 8) that did not manifest the ellipsoid response to CRF, formyl-Met-Leu-Phe (fMLP), a chemotactic peptide, 10(-9) M increased non-spherical neutrophils to 31 +/- 0.8%. In these individuals, the fMLP response was inhibited in a dose-dependent manner by CRF. The pharmacological profile of the stimulatory and fMLP-inhibitory actions of CRF on neutrophil shape was consistent with that of a CRF1-receptor mediated response. Expression of mRNA for the CRF1-receptor was detected in hematopoietic cell lines (e.g., HL-60) using a reverse transcriptase polymerase chain-reaction method. The bipolar-shape response of human neutrophils to CRF has the potential to be a useful indicator of the functional state of this hormone-receptor system in inflammation.

[d-Pro5]Corticotropin-releasing factor analogs as selective agonists at corticotropin-releasing factor receptors

Corticotropin-releasing factor (CRF) acts on at least two types of CRF receptors. To search for selective CRF receptor agonists, 37 ovine CRF analogs, systematically substituted with D-amino acids, were tested for inhibitory activity on edema induced in the pentobarbital-anesthetized rat paw by heat (immersion in 58 degrees C water for 1 min). The activity of each analog, administered 21 nmol/kg i.v. 10 min before heat, was compared to published data on the analogs potency in stimulating adrenocorticotropin (ACTH) release from cultured rat pituitary cells. In general, a positive rank correlation was found between the anti-edema and neuroendocrine activities of these analogs, however, one outlier, [D-Pro5]ovine CRF, exhibited greater selectivity for anti-edema activity. The human/rat analog of [D-Pro5]CRF was synthesized and found to be equipotent to human/rat CRF for suppression of heat-edema. In cells transfected with two types of cloned CRF receptors, the intracellular cAMP response to [D-Pro5]human/rat CRF was equipotent to human/rat CRF in the heart-muscle CRF (CRF2 beta) receptor assay but was five times less potent than human/rat CRF in the pituitary-central nervous system CRF (CRF1) receptor assay. We conclude that changing residue Pro5 in the CRF molecule from a L- to a D-configuration confers selectivity by decreasing second messenger activation at the CRF1 receptor whilst retaining full potency at the CRF2 beta receptor.

Potent inhibition of thermal edema in rat by des-Tyr-dynorphin A

In an earlier study, dynorphin A(1-13) [Dyn A(1-13)] was shown to inhibit heat-induced edema in the anesthetized rats paw but the potency of this action was low, with effective doses in the range of 3-4 mg/kg i.v. In this study, Dyn A and related fragments were tested. Thermal edema was elicited in anesthetized male albino rats by immersion of the hindpaw in 58 degrees C water for 1 min. The median effective dose (ED50 and 95% confidence limits) in mg/kg i.v. for inhibition of edema were: Dyn A, Dyn A(2-17), and Dyn A(1-13), 1.7 (1.2-2.4), 0.15 (0.09-0.24), and 3.2 (1.9-5.5), respectively. The ED50 values of [D-Ala2]Dyn A, [D-Ala2]Dyn A(2-17), and [D-Ala2]Dyn A(2-17)-amide were found to be 0.92 (0.40-2.10), 1.25 (0.60-2.63), and 0.65 (0.36-1.16) mg/kg i.v., respectively. Dyn A(2-17), 0.5 mg/kg i.v., also inhibited pulmonary edema produced by i.v. injection of epinephrine. The anti-edema action of Dyn A(2-17) was not blocked by naloxone, an opioid receptor antagonist, or dependent on the hypotensive action of this peptide. It is postulated that the antiedema activity of Dyn A resides in the core fragment Dyn A(6-12). Two peptides, N-acetyl-Dyn A(6-12)-amide and N-acetyl-[D-Leu12]Dyn A(6-12)-amide, were synthesized and, when tested, were effective in reducing thermal edema with ED50 values of 1.4 (0.6-3.7) and 2.2 (1.2-4.1) mg/kg i.v., respectively.