Maria Broccardo

PHARMACOLOGICAL DATA ON DERMORPHINS, A NEW CLASS OF POTENT OPIOID PEPTIDES FROM AMPHIBIAN SKIN

1 Dermorphin and Hyp6‐dermorphin are the first representatives of a new class of potent opioid peptides occurring in amphibian skin. They present the unique feature of having a D‐Ala residue incorporated in the peptide molecule. 2 Dermorphin displayed a potent depressive action on electrically stimulated contractions of the guinea‐pig ileum and mouse vas deferens preparations. Dermorphin was respectively 57,294, 18 and 39 times more potent than Met‐enkephalin, Leu‐enkephalin, β‐endorphin, and morphine on the guinea‐pig ileum opiate receptors. On the vas deferens receptors, dermorphin was about as potent as the enkephalins and 40 times more potent than morphine. Naloxone was a powerful antagonist to dermorphin in both preparations. 3 Dermorphin produced potent and long‐lasting analgesia in mice by intravenous injection, and in rats by intracerebroventricular injection, the ED50 being here of the order of 13–23 pmol/rat. Morphine was 752 and 2170 times less potent, depending on the analgesia test used. At high intracerebroventricular doses analgesia was accompanied by catalepsy. 4 Intracerebroventricular infusion of dermorphin induced development of tolerance and precipitation of withdrawal symptoms upon administration of naloxone. Both tolerance and physical dependence were consistently less marked with dermorphin than with morphine. 5 The minimum sequence requirement for full dermorphin activity was represented by the N‐terminal tetrapeptide. The presence of the D‐Ala2 residue was of crucial importance.

RELATIVE POTENCY OF BOMBESIN‐LIKE PEPTIDES

1 The pharmacological activity of two natural bombesin‐like peptides, alytesin and litorin, and 25 related synthetic peptides has been compared to that of bombesin. 2 The minimum length of the amino acid chain required for the first appearance of bombesin‐like effects was represented by the C‐terminal heptapeptide, and the minimum length for maximal effects by the C‐terminal nonapeptide. The latter possessed approximately the same activity as bombesin and may be considered a good substitute. 3 Both the tryptophan and histidine residues seemed to be essential for bombesin‐like activity. 4 The C‐terminal octapeptide was less active than either bombesin or the C‐terminal nonapeptide and its action was more rapid in onset and less sustained. 5 Litorin apparently has an intermediate position between bombesin octapeptide and bombesin nonapeptide in the speed and duration of its effects. The relationship between structure and activity is discussed.

The brain-gut-skin triangle: New peptides

New data on tachykinins and bombesins are displayed and the present situation of research on the novel amphibian skin peptides sauvagine and dermorphin is illustrated. The potent stimulant effect of sauvagine on ACTH and beta-endorphin release has been confirmed both in vivo and on columns of isolated and dispersed rat pituitary cells, and similarly the potent inhibitory effect on PRL and GH release, both in the rat and man. Particular emphasis is laid on the occurrence of sauvagine-like immunoreactivity in fish urophysis and in amphibian nervous structures, including the retina. It is suggested that the long-searched corticotropin releasing factor and PRL release-inhibiting factor may be a sauvagine-like peptide. Dermorphin, in its turn, has been found to cause, by intracerebroventricular injection, not only analgesia and catalepsy, but also conspicuous EEG and behavioral changes in the rabbit and chick, as well as a sharp reduction in gastric emptying time and gastric acid output in the rat, together with marked stimulation of PRL release.

Central effect of SNC 80, a selective and systemically active δ-opioid receptor agonist, on gastrointestinal propulsion in the mouse

We investigated the effects of SNC 80 ((+)-4-[alphaR)-alpha-((2S,5R)-4-ally1-2,5-dimethyl-1-pipera zinyl)-3-methoxybenzyl]-N,N-diethylbenzamide), a new highly selective, non-peptidic and systemically active delta-opioid receptor agonist, on gastrointestinal and colonic propulsion in mice. Intraperitoneally (i.p.) SNC 80 (1, 10 and 30 mg/kg) significantly decreased gastrointestinal propulsion measured as transit of an orally administered charcoal meal. Pretreatment with the delta-opioid receptor antagonist, naltrindole (1 mg/kg) subcutaneously (s.c.), with the non-selective opioid antagonist, naloxone (5 mg/kg, s.c.) or the mu1-opioid receptor antagonist, naloxonazine (10 mg/kg, i.p.), significantly decreased the antitransit effect of SNC 80 but pretreatment with the non-selective opioid antagonist, naloxone methiodide (5 mg/kg, s.c.), a quaternary salt of naloxone that does not cross the blood-brain barrier, did not. SNC 80 (1, 5 and 10 mg/kg, i.p.), produced dose-related inhibition of colonic propulsion measured as the increase in mean expulsion time of a 3 mm glass bead placed in the distal colon. Naloxone (5 mg/kg, s.c.) and naltrindole (1 mg/kg, s.c.), completely antagonized the colonic antipropulsive effect of SNC 80. In contrast, naloxone methiodide (5 mg/kg, s.c.), left the inhibitory effect of i.p. SNC 80 on colonic function unchanged. These results suggest that peripherally injected SNC 80 inhibits gastrointestinal transit and colonic propulsion. It does so mainly through a central mechanism. Although the gastrointestinal antitransit effect of SNC 80 is naltrindole- and naloxonazine-sensitive, we cannot exclude an opioid-independent mechanism. The colonic antipropulsive effect of SNC 80 confirms the inhibitory role of the central delta-opioid receptor system on colonic motility.

Antidiarrheal and colonic antipropulsive effects of spinal and supraspinal administration of the natural δ opioid receptor agonist, [D-Ala2]deltorphin II, in the rat

The ability of the natural selective delta opioid receptor agonist, [D-Ala2]deltorphin II (DADELT II), to inhibit the diarrhea induced by castor oil and colonic glass bead expulsion, was studied in rats after supraspinal and spinal administration. When injected intracerebroventricularly, DADELT II (0.2, 1, 10 micrograms/rat) inhibited diarrhea and colonic bead expulsion in a dose-related fashion but did not affect the rate of small intestine transit. Similar results were obtained when the same dose of DADELT II was administered spinally. The antidiarrheal and colonic antipropulsive effects of supraspinally and spinally DADELT II were partially or completely antagonized by subcutaneous pretreatment with 1 and 10 mg/kg, respectively, of naltrindole, a selective delta opioid receptor antagonist. These findings indicate that, in the rat, supraspinal and spinal delta opioid receptors play a role in modulating diarrhea and colonic propulsion and that DADELT II is a useful tool for investigating the role of the delta opioid system in gastrointestinal function.

Peripheral anti-nociceptive effect of nociceptin/orphanin FQ in inflammation and stress-induced colonic hyperalgesia in rats

ABSTRACT Nociceptin/orphanin FQ (N/OFQ) and its NOP receptors are present in the central nervous system and in the periphery playing important roles in the modulation of gastrointestinal functions and pain. The aim of this study was to investigate the role of central and peripheral N/OFQ–NOP receptor system in the nociceptive response to colorectal distension (CRD) in basal condition and in two models of gut hypersensitivity triggered by both inflammation and stress. Male Wistar rats were tested in basal and in post‐inflammatory conditions, i.e., 5 days after IC TNBS instillation (80 mg/Kg) and received N/OFQ (2 nmol/Kg IP), UFP‐101 (a selective NOP receptor antagonist, 10 nmol/Kg IP), N/OFQ+UFP‐101, N/OFQ (0.5 nmol/rat ICV) or vehicle. Female rats were tested in basal and after partial restraint stress receiving the same pharmacological treatment. CRD was performed using barostat and abdominal contractions were recorded by electromyography. In basal condition, N/OFQ, ICV and IP injected, did not modify basal visceral sensitivity. Both in TNBS and stress‐induced hyperalgesia, IP but not ICV injection of N/OFQ significantly decreased the number of abdominal contractions. Peripheral injection of UFP‐101 antagonized N/OFQ effect. Moreover, in post‐inflammatory colitis, UFP‐101, injected alone, exacerbated visceral hyperalgesia to CRD compared with vehicle. These findings indicate that in rats, N/OFQ, only peripherally injected, reduces visceral hypersensitivity triggered by inflammation or stress without affecting basal sensitivity. N/OFQ visceral anti‐hyperalgesic effect involves peripheral NOP receptors. In a post‐inflammatory, but not in an acute stress colitis model, N/OFQergic system is endogenously activated.

In vitro and in vivo pharmacological role of TLQP-21, a VGF-derived peptide, in the regulation of rat gastric motor functions.

Background and purpose:  Vgf gene expression has been detected in various endocrine and neuronal cells in the gastrointestinal tract. In this study we investigated the pharmacological activity of different VGF‐derived peptides. Among these, TLQP‐21, corresponding to the 556–576 fragment of the protein was the unique active peptide, and its pharmacological profile was further studied.

Pituitary-adrenal and vagus modulation of sauvagine- and CRF-induced inhibition of gastric emptying in rats

Sauvagine and CRF significantly delayed gastric emptying in rats: sauvagine was 21 times more potent than CRF in inhibiting gastric emptying when injected subcutaneously and 59 times more potent when injected intracerebroventricularly. The inhibitory effect of intracerebroventricular and subcutaneous injections of sauvagine and CRF was always reversed by vagotomy. Hypophysectomy significantly reduced the inhibitory effect of subcutaneously injected sauvagine and CRF, but not that of intracerebroventricularly injected peptides. Adrenalectomy completely prevented the inhibition of gastric emptying only if it was performed 7 days before peptide administration, whereas the central and peripheral inhibitory effect of sauvagine and CRF was retained after acute (1 h) adrenalectomy. In animals adrenalectomized 7 days previously, chronic administration or a single dose of corticosterone at different intervals (1, 24 and 72 h) before the test caused the peptides to recover their inhibitory activity. These results demonstrate that the inhibitory action of sauvagine and CRF clearly depends on the integrity of the parasympathetic nervous system (vagus), and on corticosterone secretion by the adrenal gland.

Evidence of Central and Peripheral Sensitization in a Rat Model of Narcotic Bowel-Like Syndrome

BACKGROUND & AIMS Narcotic bowel syndrome (NBS) is a subset of opioid bowel dysfunctions that results from prolonged treatment with narcotics and is characterized by chronic abdominal pain. NBS is under-recognized and its molecular mechanisms are unknown. We aimed to (1) develop a rat model of NBS and (2) to investigate its peripheral and central neurobiological mechanisms. METHODS Male Wistar rats were given a slow-release emulsion that did or did not contain morphine (10 mg/kg) for 8 days. Visceral sensitivity to colorectal distension (CRD) was evaluated during and after multiple administrations of morphine or vehicle (controls). The effects of minocycline (a microglia inhibitor), nor-binaltorphimine (a kappa-opioid antagonist), and doxantrazole (a mast-cell inhibitor) were observed on morphine-induced visceral hyperalgesia. Levels of OX-42, P-p38 mitogen-activated protein kinase, rat mast cell protease II, and protein gene product 9.5 were assessed at different spinal segments (lumbar 6 to sacral 1) or colonic mucosa by immunohistochemistry. RESULTS On day 8 of morphine administration, rats developed visceral hyperalgesia to CRD (incipient response) that lasted for 8 more days (delayed response). Minocycline reduced the incipient morphine-induced hypersensitivity response to CRD whereas nor-binaltorphimine and doxantrazole antagonized the delayed hyperalgesia. Levels of OX-42 and P-p38 increased in the spinal sections, whereas rat mast cell protease II and protein gene product 9.5 increased in the colonic mucosa of rats that were given morphine compared with controls. CONCLUSIONS We developed a rat model of narcotic bowel-like syndrome and showed that spinal microglia activation mediates the development of morphine-induced visceral hyperalgesia; peripheral neuroimmune activation and spinal dynorphin release represent an important mechanism in the delayed and long-lasting morphine-induced colonic hypersensitivity response to CRD.

Gastrointestinal effects of intracerebroventricularly injected nociceptin/orphaninFQ in rats.

Nociceptin/orphanin FQ/(N/OFQ), a novel heptadecapeptide recently isolated from porcine and rat brain, is the endogenous ligand of the N/OFQ peptide receptor (NOP, previously known as ORL-1). In this study we examined the effects of intracerebroventricularly (icv) injected N/OFQ on gastric emptying, gastrointestinal transit, colonic propulsion and gastric acid secretion in rats. N/OFQ (0.01-10 nmol/rat) significantly delayed gastric emptying of a phenol red meal, inhibited transit of a non-absorbable charcoal marker through the small intestine and increased the mean colonic bead expulsion time. These N/OFQ-motor effects were abolished by the NOP receptor selective antagonist [NPhe(1)]N/OFQ(1-13)-NH(2) (50 nmol/rat), but were unaltered by the classical opioid receptor antagonist, naloxone (9.2 micromol/kg). Icv injected N/OFQ (10 nmol/rat) decreased gastric acid secretion in 2-h pylorus ligated rats in a naloxone sensitive manner. [NPhe(1)]N/OFQ(1-13)-NH(2) (100 nmol/rat) icv administered alone stimulated gastric acid secretion. These results indicate that N/OFQ activates via NOP receptor stimulation a central inhibitory pathway modulating gastrointestinal propulsive activity and gastric acid secretion in rats.