Keith Finlayson

VPAC and PAC receptors: From ligands to function

Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptides (PACAPs) share 68% identity at the amino acid level and belong to the secretin peptide family. Following the initial discovery of VIP almost four decades ago a substantial amount of knowledge has been presented describing the mechanisms of action, distribution and pleiotropic functions of these related peptides. It is now known that the physiological actions of these widely distributed peptides are produced through activation of three common G-protein coupled receptors (VPAC(1), VPAC(2) and PAC(1)R) which preferentially stimulate adenylate cyclase and increase intracellular cAMP, although stimulation of other intracellular messengers, including calcium and phospholipase D, has been reported. Using a range of in vitro and in vivo approaches, including cell-based functional assays, transgenic animals and rodent models of disease, VPAC/PAC receptor activation has been associated with numerous physiological processes (e.g. control of circadian rhythms) and clinical conditions (e.g. pulmonary hypertension), which underlies on-going research efforts and makes these peptides and their cognate receptors attractive targets for the pharmaceutical industry. However, despite the considerable interest in VPAC/PAC receptors and the processes which they mediate, there is still a paucity of selective and available, non-peptide ligands, which has hindered further advances in this field both at the basic research and clinical level. This review summarises the current knowledge of VIP/PACAP and the VPAC/PAC receptors with regard to their distribution, pharmacology, signalling pathways, splice variants and finally, the utility of animal models in exploring their physiological roles.

Nicotine improves sustained attention in mice: Evidence for involvement of the α7 nicotinic acetylcholine receptor

In humans, nicotine has been shown to improve attention in both normal and impaired individuals. Observations in rats reflect some, but not all aspects of the nicotine-induced improvements in humans. To date these findings have not been replicated in mice. To examine the effect of nicotine on sustained attention in mice, we have established a version of the 5-choice serial reaction-time (5-CSR) task with graded levels of difficulty, based upon spatial displacement and a variable intertrial interval. Using this paradigm, microgram doses of nicotine produced a consistent reduction in the level of omissions and an improvement in proportion correct in normal mice. This improvement in sustained attention was made irrespectively of whether mice had previously received nicotine. In an attempt to elucidate which nicotinic acetylcholine receptor (nAChR) subtype(s) mediate this effect, we examined the performance of α7 nAChR knockout (KO) mice in the 5-CSR task. α7 nAChR KO mice not only acquired the task more slowly than their wild-type littermates, but on attaining asymptotic performance, they exhibited a higher level of omissions. In conclusion, by increasing the level of task difficulty, the performance of mice was maintained at sufficiently low levels to allow a demonstrable improvement in performance upon nicotine administration. Furthermore, as α7 KO mice are clearly impaired in the acquisition and asymptotic performance of this task, the α7 nAChR may be involved in mediating these effects of nicotine.

Impaired attention is central to the cognitive deficits observed in alpha 7 deficient mice

alpha7-Nicotinic acetylcholine receptors (alpha7-nAChR) have been implicated in a range of cognitive deficits in schizophrenia. Therefore we examined alpha7-nAChR knockout (KO), heterozygote (HT) and wildtype (WT) littermate mice in the 5-CSR (a rodent model of sustained attention) and odour span (a novel mouse working memory paradigm) tasks, and related performance to nAChR density. Whilst there was no difference between groups in baseline 5-CSR task performance, alpha7-nAChR KOs exhibited significantly higher omission levels compared to WT mice on increasing the attentional load, with HT mice performing at an intermediate level. Furthermore, alpha7-nAChR KO mice were significantly impaired in the odour span task when compared to WT mice, in a pattern consistent with impaired attention. These behavioural deficits were associated with the loss of alpha7-nAChRs, as alpha4beta2-nAChR density was unaltered in these mice. Thus these studies intimate that the attentional impairment in alpha7-nAChR transgenic mice maybe core to other deficits in cognition.

The role of the purinergic P2X7 receptor in inflammation

The inflammatory process, orchestrated against a variety of injurious stimuli, is composed of three inter-related phases; initiation, propagation and resolution. Understanding the interplay between these three phases and harnessing the beneficial properties of inflammation whilst preventing its damaging effects, will undoubtedly lead to the advent of much needed therapies, particularly in chronic disease states. The P2X7 receptor (P2X7R) is increasingly recognised as an important cell surface regulator of several key inflammatory molecules including IL-1β, IL-18, TNF-α and IL-6. Moreover, as P2X7R-dependent cytokine production is driven by activating the inflammasome, antagonists of this receptor are likely to have therapeutic potential as novel anti-inflammatory therapies. The function of the P2X7R in inflammation, immunity and its potential role in disease will be reviewed and discussed.

A systematic comparison of intracellular cyclic AMP and calcium signalling highlights complexities in human VPAC/PAC receptor pharmacology

VPAC/PAC receptor activation classically results in cyclic-AMP production, with limited reports evaluating calcium signalling. These studies systematically characterise intracellular cyclic-AMP ([cAMP](i)) and calcium ([Ca(2+)](i)) responses in CHO-cells expressing recombinant human (h) VPAC/PAC receptors (hVPAC(1)R, hVPAC(2)R, hPAC(1)R), using two simple, non-radioactive, HT-amenable assays. The rank order of potency (ROP) of the agonists VIP, PACAP-27 and PACAP-38 was similar in both assays for each individual receptor subtype, although potencies (EC(50)) in the [Ca(2+)](i) assay were approximately 100-fold lower. Importantly, this shift was also evident in SHSY-5Y cells endogenously expressing hPAC(1)R. Furthermore, [Ala(11,22,28)]VIP and maxadilan were selective hVPAC(1)R and hPAC(1)R agonists, respectively, and although R3P65 had no demonstrable hVPAC(2)R selectivity, these compounds exhibited comparable reductions in [Ca(2+)](i) EC(50) values. In contrast, PG97-269 and PG99-465, putatively selective hVPAC(1)R and hVPAC(2)R antagonists, respectively, were marginally less potent in [cAMP](i) studies, whereas M65 was equipotent at hPAC(1)R. Moreover, PG99-465 alone increased [cAMP](i) at all three hVPAC/PAC receptor subtypes, with full hVPAC(1)R and hPAC(1)R agonism. With equivalent agonist ROPs generated in both assays, [Ca(2+)](i) signalling provides an alternative approach to examine hVPAC/PAC receptor pharmacology. However, these studies underscore the paucity of receptor selective compounds, complexities in comparing drug potencies across assays, and the pleiotropic nature of VPAC/PAC-receptor signalling.

Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists.

1 The pharmacological profile of adenosine A1 receptors in human, guinea‐pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]‐8‐cyclopentyl‐1,3‐dipropylxanthine ([3H]‐DPCPX). 2 The affinity of [3H]‐DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea‐pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea‐pig or human cortical membranes. 3 The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5′‐N‐ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5–26.2 fold higher in rat cortical membranes than in human and guinea‐pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 μM) reduced 2‐chloro‐N6‐cyclopentyladenosine (CCPA) affinity 7–13.9 fold, whereas the affinity of DPCPX was unaffected. 4 The affinity of six xanthine‐based adenosine receptor antagonists was 2.2–15.9 fold higher in rat cortical membranes compared with human or guinea‐pig membranes. The rank order of potency was species‐independent. In contrast, three pyrazolopyridine derivatives, (R)‐1‐[(E)‐3‐(2‐phenylpyrazolo[1,5‐a]pyridin‐3‐yl) acryloyl]‐2‐piperidine ethanol (FK453), (R)‐1‐[(E)‐3‐(2‐phenylpyrazolo[1,5‐a]pyridin‐3‐yl) acryloyl]‐piperidin‐2‐yl acetic acid (FK352) and 6‐oxo‐3‐(2‐phenylpyrazolo[1,5‐a]pyridin‐3‐yl)‐1(6H)‐pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea‐pig, rat and mouse brain membranes. pKi values (M) for [3H]‐DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively. 5 Drug affinity for adenosine A2A receptors was determined in a [3H]‐2‐[4‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine ([3H]‐CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]‐DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]‐DPCPX binding sites in human, rat, mouse and guinea‐pig brain membranes.

Progressive impairment in olfactory working memory in a mouse model of Mild Cognitive Impairment

Patients with Mild Cognitive Impairment (MCI), exhibiting both working memory and olfactory deficits are likely to progress to Alzheimers disease (AD). Targeting this pre-clinical AD population with disease modifying agents or cognitive enhancers represents the best strategy for halting or delaying the impact of this pernicious disease. However, there is a paucity of animal models of MCI with which to assess putative therapeutic strategies. We describe an odour span task which assesses the ability of mice to remember lists of odours, and report subtle cognitive deficits in human amyloid over-expressing (Tg2576) mice, at an age prior to plaque deposition. Four-month-old Tg2576 mice exhibited normal acquisition and performance in the standard 12-span task, but were significantly impaired when memory load was increased to 22 odours. By 8-months, a performance deficit was apparent in the 12-span task and by 1-year mice also exhibited significant acquisition deficits. Thus, by assessing olfactory working memory in Tg2576 mice we can model aspects of MCI in rodents and aid development of future therapeutic strategies for AD.

The odour span task: a novel paradigm for assessing working memory in mice.

Impoverished odour recognition and memory are amongst the earliest symptoms observed in mild cognitive impairment, Alzheimers disease and schizophrenia, and have been advocated as early disease bio-markers. Although transgenic animals modelling disease pathologies continually emerge, there remains a paucity of tasks to examine olfactory working memory in mice. The present studies describe a mouse odour span task, which assesses the ability to remember increasing numbers of odours. Since caspase-3 is highly expressed throughout the olfactory system, we postulated that mice over-expressing this apoptogenic protein would exhibit impaired performance in the odour span task. Mice over-expressing human caspase-3 (Tg) exhibited age-independent deficits in olfactory working memory (6-18 months) compared with wild-type littermates, requiring longer for task acquisition and exhibiting impaired asymptotic performance, with reduced span lengths, lower accuracy and increased error rates. These impairments appeared to be selective for working memory, as Tg mice had no deficits in odour discriminatory ability or in locomotor measures. Importantly, nicotine, which improves working memory span in man, reversed the deficits exhibited by Tg mice. In conclusion, the mouse odour span task can detect subtle changes in olfactory working memory induced by genetic manipulation and drug administration and therefore should be applied to animal models of neurological disease.

[3H]Dofetilide binding in SHSY5Y and HEK293 cells expressing a HERG-like K+ channel?

The pharmacological characteristics of [3H]dofetilide binding in SHSY5Y, HEK293 and CHO-K1 cells were examined, and in parallel whole cell recordings used to characterise HERG-like K+ currents. Dofetilide affinity was similar in the human cell lines, SHSY5Y (Kd=99.6 nM) and HEK293 (Kd=102.9 nM), but 10 times lower in CHO-K1 cells (Kd=1200 nM). In contrast, clofilium and E4031 had a similar affinity in all three cell lines, whereas WAY 123,398 had no effect. Electrophysiological studies showed that SHSY5Y cells contained a HERG-like K+ current blocked by application of dofetilide to either side of the membrane. Block was faster when dofetilide was applied intracellularly. In contrast, HEK293 and CHO-K1 cells contained no such current, despite the presence of a partial cDNA for HERG in the former. That [3H]dofetilide is specific for I(Kr)/HERG may be questionable, as HEK293 and CHO-K1 cells contain no such functional K+ current.

VIP and PACAP receptor pharmacology: a comparison of intracellular signaling pathways.

Abstract:  VIP/PACAP receptor activation stimulates the production of [cAMP]i and [Ca2+]i by coupling to independent G‐protein subunits, although agonist potencies for the different transduction pathways appear to differ. Using CHO‐K1 cells stably expressing the human VIP/PACAP receptors (hVPAC1R, hVPAC2R, and hPAC1R), functional assays ([cAMP]i and [Ca2+]i) were established and the receptor pharmacology was characterized with five peptide agonists (VIP, PACAP‐27, PACAP‐38, [Ala11,22,28]VIP, and R3P65). The rank order of potency (ROP) was consistent between assays for the individual receptor subtypes, however, higher agonist concentrations (∼100‐fold) were required for stimulating [Ca2+]i when compared to [cAMP]i.