Alexander T. McKnight

Nociceptin induced inhibition of K+ evoked glutamate release from rat cerebrocortical slices

Nociceptin, an endogenous ligand for the orphan receptor ORL1, has recently been described. In this study we have shown that nociception inhibits 46 mM K+‐stimulated glutamate release from rat perfused cerebrocortical slices with an IC50 of 51 nM. At 100 nM the inhibition amounted to 68 ± 14% and was naloxone (10 μm)‐insensitive excluding an activation of μ, δ and κ opioid receptors. These data demonstrate the functional coupling of ORL1 in glutamatergic neurones and implicates a role for nociceptin in glutamatergic neurotransmission.

Comparison of the effects of [Phe1Ψ(CH2‐NH)Gly2]Nociceptin (1–13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors

Nociceptin(NC) is the endogenous ligand for the opioid receptor like‐1 receptor (NC‐receptor). [Phe1ΨC(CH2‐NH)Gly2]Nociceptin(1–13)NH2 ([F/G]NC(1–13)NH2) has been reported to antagonize NC actions in peripheral guinea‐pig and mouse tissues. In this study, we investigated the effects of a range of NC C‐terminal truncated fragments and [F/G]NC(1–13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHONCR) and electrically evoked contractions of the rat vas deferens (rVD). In radioligand binding assays, a range of ligands inhibited [125I]‐Tyr14‐NC binding in membranes from rCX and CHONCR cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1–13)NH2 was as potent as NC(1–13)NH2. The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHONCR cells was NCNH2NC=NC(1–13)NH2>NC(1–12)NH2>>NC(1–11)NH2. [F/G]NC(1–13)NH2 was a full agonist with a pEC50 value of 8.65. NCNH2 and [F/G]NC(1–13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5±4.9% and 7.39, 58.9±6.8% respectively. In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1–13)NH2, displayed a small (instrinsic activity α=0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD. The differences between [F/G]NC(1–13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1–13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms.

The interaction of the NK1 receptor antagonist CP‐96,345 with L‐type calcium channels and its functional consequences

1 We investigated the effects of the non‐peptide NK1 receptor antagonist, CP‐96,345, its inactive enantiomer CP‐96,344, and the racemic mixture (±)‐CP‐96,345, on the binding of [3H]‐nimodipine and [3H]‐diltiazem to L‐type calcium channels in rat cerebral cortex membranes. In isolated peripheral tissues containing tachykinin receptors, the effects of (±)‐CP‐96,345 have been compared with those of diltiazem. 2 In guinea‐pig trachea, (±)‐CP‐96,345 produced antagonism of responses to the selective NK1 agonists [Sar9, Met(O2)11]SP and substance P‐methyl ester that was apparently competitive in nature (pKB 7.0–7.5), while in guinea‐pig ileum the antagonism was not surmountable. 3 The reduction of maximum responses by (±)‐CP‐96,345 in the guinea‐pig ileum was not selective; it was obtained with muscarinic agonists and other agents, and was also observed in the portal vein of the rat where NK1 receptors are not present. 4 The tissue‐specific reduction of maximum responses by (±)‐CP‐96,345 in ileum was reproduced by diltiazem. 5 (±)‐CP‐96,345 produced a concentration‐dependent enhancement of [3H]‐nimodipine binding to rat cerebral cortex membranes with a maximal stimulation of 186 ± 29% above control (EC50 83.2 nm). Scatchard analysis revealed that (±)‐CP‐96,345 increased the affinity of [3H]‐nimodipine for its binding sites without affecting Bmax (control: KD = 0.32 nm; with 100 nm (±)‐CP‐96,345: KD = 0.074 nm). 6 CP‐96,345, CP‐96,344, and the racemate all inhibited [3H]‐diltiazem binding in rat cerebral cortex membranes with Ki values of 22.5 nm, 34.5 nm and 29.9 nm respectively; a similar value was obtained for diltiazem itself (33.6 nm). In comparison, CP‐96,345 and (±)‐CP‐96,345 inhibited the binding of [125I]‐Bolton‐Hunter‐conjugated substance P in this tissue with Ki values of 59.6 nm and 82.0 nm respectively, while CP‐96,344 had no measurable affinity (IC50 > 10 μm). 7 Substance P and a range of ligands selective for NK1, NK2, or NK3 receptors had no significant effect at 10 μm on either [3H]‐diltiazem or [3H]‐nimodipine binding. 8 The results indicate that in addition to possessing affinity for the NK1 receptor, the non‐peptide antagonist, CP‐96,345, displays high affinity for [3H]‐diltiazem binding sites on L‐type calcium channels. The functional effect that may be observed in integrated models will be a consequence of either property, or be a composite effect of NK1 receptor antagonism and L‐channel blockade.

Bombesin Receptors as a Novel Anti-Anxiety Therapeutic Target: BB1 Receptor Actions on Anxiety through Alterations of Serotonin Activity

The effects of PD 176252 [3-(1H-indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl-2-[3-(nitro-phenyl)ureido]propionamide], a nonpeptide bombesin (BB) BB1/BB2 receptor antagonist, were assessed in rats using several ethologically relevant tests of anxiety. Consistent with a role for the bombesin family of peptides in subserving anxiety behaviors, the antagonist increased social interaction (3.75 and 7.5 mg/kg, i.p.), dose-dependently attenuated the number of vocalizations emitted by guinea pig pups separated from their mother (1–30 mg/kg, i.p.), reduced latency to approach a palatable snack in an anxiogenic (unfamiliar) environment, and reduced the fear-potentiated startle response (5 and 10 mg/kg, i.p., and 100–200 ng per rat, i.c.v.). When administered directly to the dorsal raphé nucleus (DRN), PD 176252 (20–500 ng) increased social interaction under aversive conditions, as did the 5-HT1A receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (50 ng). Furthermore, intra-DRN microinfusion of the peptide antagonist (PD 176252) suppressed, whereas its agonist [neuromedin B (NMB)-30] promoted, the in vivo release of 5-HT in the ventral hippocampus. In parallel, the suppressed social interaction elicited by intra-DRN administration of NMB was attenuated by a systemically administered 5-HT2C (but not 5-HT1A) receptor antagonist. Together, these findings suggest that endogenous BB-like peptides at the DRN evoke the release of 5-HT from the limbic nerve terminals originating from the raphé, specifically at the ventral hippocampus, resulting in anxiogenesis. The finding that this action was attenuated by BB receptor (BB1 and/or BB2) antagonists suggests that these compounds may represent a novel class of anxiolytic agents.

Effect of gastrin receptor blockade on endocrine cells in rats during achlorhydria

Hyperplasia of the oxyntic enterochromaffinlike cells in response to long-lasting blockade of acid secretion is closely related to hypergastrinemia. In the present study, the effect of a specific gastrin receptor antagonist on proton pump inhibitor-induced changes on serum gastrin levels, mucosal height, as well as gastrin- and enterochromaffin-like cells was investigated in rats. The proton pump inhibitor BY 308 or the vehicle methylcellulose [Methocel (controls)] was administered for 2 weeks in the presence and absence of the gastrin receptor antagonist PD 136450 (CAM 1189). BY 308 significantly increased serum gastrin levels, gastrin cell density, and antral gastrin concentration. Concomitant application of PD 136450 did not alter this response. In the oxyntic stomach, mucosal height, enterochromaffinlike cell density, labeling index of enterochromaffinlike cells, and histamine concentration were elevated after treatment with BY 308. These increases were almost completely abolished by PD 136450. Even in normogastrinemic control rats, PD 136450 significantly decreased mucosal height of the oxyntic part of the stomach and the labeling index of enterochromaffinlike cells. The results show that (a) trophic effects of drug-induced achlorhydria are mediated by gastrin; (b) even in control rats (normogastrinemic), gastrin is a trophic factor for the oxyntic mucosa; and (c) antral gastrin cell hyperplasia in states of chronic achlorhydria is not mediated by gastrin itself.

Nocistatin reverses nociceptin inhibition of glutamate release from rat brain slices

We have examined the effects of the recently described heptadecapeptide nocistatin on K+-evoked glutamate release from rat cerebrocortical slices in vitro. In vivo, nocistatin reverses the action of nociceptin. Nocistatin (100 nM, n = 7) did not inhibit K+-evoked glutamate release alone. Nociceptin (100 nM) inhibited glutamate release by 51.7 +/- 8.3% (P < 0.05, n = 6) and this was fully reversed by nocistatin (100 nM). Nocistatin also appears to be an antagonist of nociceptin action in vitro.

The effect of CCKB/gastrin antagonists on stimulated gastric acid secretion in the anaesthetized rat

1 The urethane‐anaesthetized, vagotomised rat preparation was used to investigate the effects of the histamine H2‐antagonist ranitidine, the proton pump inhibitor omeprazole and the CCKB/gastrin antagonists CI‐988, PD 136450 and L‐365,260 on pentagastrin‐, histamine‐ and bethanechol‐induced gastric acid secretion. 2 The novel CCKB/gastrin antagonists CI‐988 and PD 136450, and L‐365,260 dose‐dependently inhibited pentagastrin‐induced secretion. The ED50 value for PD 136450 was 0.05 μmol kg−1, the same following intravenous or subcutaneous administration. 3 CI‐988 and PD 136450 administered subcutaneously at dose levels highly effective for antagonism of pentagastrin responses had no effect on basal acid secretion. 4 Ranitidine inhibited pentagastrin‐, bethanechol‐, and histamine‐induced acid secretion, whereas the CCKB/gastrin antagonists inhibited only the secretory response to pentagastrin. 5 The selective CCKA antagonist, devazepide, was inactive at up to 300 μmol kg−1 i.p. against the three stimulants of acid secretion. 6 CI‐988 and PD 136450 will be useful research tools with which to investigate the role of CCKB/gastrin receptors in gastric acid secretion and the trophic activities of gastrin and cholecystokinin (CCK) on the gastrointestinal tract.

Neuroprotective Effect of the χ-Agonist Enadoline (CI-977) in Rat Models of Focal Cerebral Ischaemia

The neuroprotective efficacy of the χ‐opioid agonist enadoline (CI‐977) was examined in two acute rat models of focal cerebral ischaemia [non‐recovery (4 h) and recovery (24 h)]. In the non‐recovery model, Sprague ‐ Dawley rats were anaesthetized throughout the study period. Focal ischaemia was produced by the permanent occlusion of the left middle cerebral artery (MCA). The amount of early ischaemic damage was assessed in coronal sections at nine pre‐determined stereotaxic planes. Enadoline at doses of 0.1, 0.3 and 1.0 mg/kg (n= 8), administered s.c. 30 min prior to ischaemia, produced dose‐dependent amelioration of cortical damage. Importantly, enadoline had no significant effect on any of the physiological parameters monitored (blood pressure, blood gases, glucose, pH). In the recovery model the left MCA was permanently occluded under isoflurane anaesthesia. Animals were allowed to recover and were killed 24 h later. The amount of ischaemic brain damage and swelling was assessed histologically. In this model pretreatment with enadoline at either 0.1, 0.3, or 1.0 mg/kg s.c. was followed by continuous s.c. infusion at 0.017, 0.05 or 0.17 mg/kg/h respectively (n= 8–17). Enadoline produced dose‐dependent reductions in the volumes of infarction and brain swelling; the greatest reductions were seen at 1.0 mg/kg plus 0.17 mg/kg/h in both infarction (reduced by 37.4% from controls) and swelling (reduced by 47.8%). Therefore the χ‐opioid agonist enadoline affords dose‐dependent neuroprotection in both the non‐recovery and recovery models of focal cerebral ischaemia in the rat. In the recovery model enadoline was effective in reducing infarction and oedema; the reduction in brain swelling occurs in parallel with the reduction in ischaemic damage.

The rational development of small molecule tachykinin NK3 receptor selective antagonists - the utilisation of a dipeptide chemical library in drug design

Abstract Boc(S)Phe(S)PheNH2 (1c) was identified from the biological screening of an in-house dipeptide chemical library as a micromolar NK3 receptor selective ligand (IC50=1150nM). This lead structure has subsequently been developed into a series of potent and selective NK3 receptor antagonists an example of which is the urea derivative Boc(S)Phe(R)αMePheNH(CH2)7NHCOHNH2 (11d, PD157672) (IC50=16nM).

Brain temperature and the neuroprotective action of enadoline and dizocilpine in the gerbil model of global ischaemia

Mongolian gerbils were subjected to transient forebrain ischaemia by occluding both common carotid arteries for 7 min. Subcutaneous administration of either the kappa-opioid receptor agonist enadoline (CI-977; 1 mg kg-1), or the non-competitive NMDA receptor antagonist dizocilpine (MK-801; 3 mg kg-1), at induction of ischaemia prevented neurodegeneration of CA1-CA2 pyramidal neurones in the dorsal hippocampus. It was shown by continuously monitoring intrahippocampal temperature that brain temperature drops by approximately 4 degrees C during ischaemia, when rectal temperature is maintained normothermic. Enadoline at no time point tested affected brain temperature, whereas dizocilpine statistically lowered brain temperature following ischaemia. These findings suggest that enadoline affords neuroprotection in the absence of any hypothermic episode, whilst in the case of dizocilpine, the small transient hypothermia observed following ischaemia may act synergistically or additively with the drug to yield neuroprotection.