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.

75 years of opioid research: the exciting but vain quest for the Holy Grail.

Over the 75-year lifetime of the British Pharmacological Society there has been an enormous expansion in our understanding of how opioid drugs act on the nervous system, with much of this effort aimed at developing powerful analgesic drugs devoid of the side effects associated with morphine--the Holy Grail of opioid research. At the molecular and cellular level multiple opioid receptors have been cloned and characterised, their potential for oligomerisation determined, a large family of endogenous opioid agonists has been discovered, multiple second messengers identified and our understanding of the adaptive changes to prolonged exposure to opioid drugs (tolerance and physical dependence) enhanced. In addition, we now have greater understanding of the processes by which opioids produce the euphoria that gives rise to the intense craving for these drugs in opioid addicts. In this article, we review the historical pathway of opioid research that has led to our current state of knowledge.

Identification and characterisation of functional bombesin receptors in human astrocytes.

Reverse transcription polymerase chain reaction (RT-PCR) demonstrated the presence of bombesin BB2 receptor mRNA but not bombesin BB1 receptor or bombesin BB3 receptor mRNA in cultured human astrocytes. Neuromedin C hyperpolarised human astrocytes in whole-cell current and voltage clamp recordings and increased the intracellular free Ca(2+) ion concentration ([Ca(2+)](i)) in single astrocytes. Treatment with neuromedin C caused larger and more frequent increases in [Ca(2+)](i) than those triggered by neuromedin B, with 96% and 78% of cells responding, respectively. The stimulatory effects of neuromedin C were inhibited significantly by treatment with U73122 or the bombesin BB2 receptor antagonist [D-Phe(6), des-Met(14)]bombesin-(6-14) ethylester. A Fluorometric Imaging Plate Reader (FLIPR) was used to measure [Ca(2+)](i) in cell populations. Neuromedin C was approximately 50-fold more potent than neuromedin B in elevating [Ca(2+)](i) in astrocytes and Chinese hamster ovary (CHO) cells expressing human bombesin BB2 receptors (hBB2-CHO). However, in CHO cells expressing the bombesin BB1 receptor hBB1-CHO, neuromedin B was 32-fold more potent than neuromedin C. [D-Phe(6), des-Met(14)]bombesin-(6-14) ethylester was a partial agonist in hBB1-CHO cells (E(max)=55%) but was a noncompetitive antagonist in both hBB2-CHO cells and astrocytes. These studies report the first identification of functional bombesin receptors on cultured human astrocytes and have demonstrated that the bombesin BB2 receptor contributes significantly to astrocyte physiology.

Comparison of the ORL1 receptor-mediated inhibition of noradrenaline release in human and rat neocortical slices.

The effects of nociceptin/orphanin (N/OFQ) and the selective ORL1 antagonist J-113397 (1-[(3R,4R)-1-cyclo-octylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one) were studied on electrically-evoked release of [(3)H]-noradrenaline ([(3)H]-NA) from human and rat neocortical slices. Specimens of human tissue were obtained during neurosurgery. Slices were preincubated with 0.1 microM [(3)H]-NA, superfused in the presence of desipramine, idazoxan, and naloxone (1 microM each), and stimulated electrically up to three times under conditions (4 pulses, 100 Hz, 2 ms, 60 mA) that prevent inhibition of evoked [(3)H]-NA release by endogenous modulators accumulating during ongoing stimulation. N/OFQ decreased electrically-evoked [(3)H]-NA release in both human and rat neocortical slices in a concentration-dependent manner. The respective pEC(50) values were 7.74 [CI(95): 7.47, 8.04] and 7.64 [CI(95): 7.48, 7.77], and the maximal inhibitions were 36.9% [CI(95): 32.4%, 41.8%] and 66.4% [CI(95): 61.7%, 72.7%]. N/OFQ (1 microM) inhibited K(+) (15 mM)-evoked [(3)H]-NA release from neocortical slices of both species by a similar magnitude, either in the presence or absence of tetrodotoxin. The nonpeptide ORL1 antagonist J-113397 competitively attenuated, with similar potency, the inhibition of electrically-evoked [(3)H]-NA release by N/OFQ in both species (pA(2) values: human, 8.16 [CI(95): 7.64, 8.64]; rat, 8.47 [CI(95): 8.27, 8.67]). J-113397 (0.1 microM) by itself did not alter either the evoked or spontaneous [(3)H]-NA release, suggesting that presynaptic ORL1 receptors are not activated by endogenous N/OFQ under the stimulation conditions employed. This study provides the first evidence that N/OFQ modulates [(3)H]-NA release in human neocortex via specific ORL1 receptors most likely located on noradrenergic axon terminals.

Gabapentin inhibits the substance P-facilitated K(+)-evoked release of [(3)H]glutamate from rat caudial trigeminal nucleus slices.

The effect of gabapentin on the release of the spinal sensory neurotransmitter glutamate has been investigated in an in vitro model using a perfused thin slice preparation from the rat brainstem containing the spinal trigeminal caudal subnucleus (Sp5C) and pre-incubated with [(3)H]glutamate. Addition of excess K(+) to the perfusing solution increased the content of tritium in the perfusate. The prior addition of substance P increased this index of glutamate release in a concentration-dependent manner, with the mean maximum of around 50% increase obtained at 1-3 microM. The action of substance P to increase the evoked release of glutamate was blocked by the antagonist CP-99994, suggesting a specific involvement of the NK(1) receptor in mediating the facilitatory effect. On its own, gabapentin at up to 100 microM did not modify the baseline level of K(+)-evoked release of glutamate; however, gabapentin caused a concentration-dependent decrease of the facilitatory effect of substance P (EC(50)=6.49 microM). The R-(-)- and S-(+)-isomers of 3-isobutylgaba were then tested against the increase in K(+)-evoked release of glutamate by substance P. S-(+)-3-isobutylgaba (pregabalin) at 30 microM acted like gabapentin to reduce the substance P-mediated increase of release almost to the baseline level of K(+)-evoked release, while in contrast the R-(-)-isomer at this concentration produced no reduction, and rather a trend towards a further enhancement of the potentiating effect of substance P. In conclusion, we have found and characterized an effect of gabapentin that is of possible mechanistic relevance to the anti-hyperalgesic/allodynic actions of this compound.