Jason P. Hannon

Molecular, pharmacological and functional diversity of 5-HT receptors

Serotonin (5-hydroxytryptamine, 5-HT) is probably unique among the monoamines in that its effects are subserved by as many as 13 distinct heptahelical, G-protein-coupled receptors (GPCRs) and one (presumably a family of) ligand-gated ion channel(s). These receptors are divided into seven distinct classes (5-HT(1) to 5-HT(7)) largely on the basis of their structural and operational characteristics. Whilst this degree of physical diversity clearly underscores the physiological importance of serotonin, evidence for an even greater degree of operational diversity continues to emerge. The challenge for modern 5-HT research has therefore been to define more precisely the properties of the systems that make this incredible diversity possible. Much progress in this regard has been made during the last decade with the realisation that serotonin is possibly the least conservative monoamine transmitter and the cloning of its many receptors. Coupled with the actions of an extremely avid and efficient reuptake system, this array of receptor subtypes provides almost limitless signalling capabilities to the extent that one might even question the need for other transmitter systems. However, the complexity of the system appears endless, since posttranslational modifications, such as alternate splicing and RNA editing, increase the number of proteins, oligomerisation and heteromerisation increase the number of complexes, and multiple G-protein suggest receptor trafficking, allowing phenotypic switching and crosstalk within and possibly between receptor families. Whether all these possibilities are used in vivo under physiological or pathological conditions remains to be firmly established, but in essence, such variety will keep the 5-HT community busy for quite some time. Those who may have predicted that molecular biology would largely simplify the life of pharmacologists have missed the point for 5-HT research in particular and, most probably, for many other transmitters. This chapter is an attempt to summarise very briefly 5-HT receptor diversity. The reward for unravelling this complex array of serotonin receptor--effector systems may be substantial, the ultimate prize being the development of important new drugs in a range of disease areas.

Molecular biology of 5-HT receptors

Serotonin (5-hydroxytryptamine; 5-HT) is a monoamine neurotransmitter whose effects are mediated by at least 13 distinct G protein-coupled receptors (GPCRs) of the type A family which includes the monoamine receptors and a combination of ligand-gated ion channels (5-HT3) of the Cys loop family which constitutes heteropentamers. 5-HT receptors are currently divided into seven classes (5-HT1 to 5-HT7), based on structural, transductional and operational features. While this degree of physical diversity clearly underscores the physiological importance of serotonin, evidence for an even greater degree of operational diversity is supported by the existence of a great number of splice and editing variants for several 5-HT receptors, their possible modulation by accessory proteins and chaperones, as well as their potential to form homo or heteromers both at the GPCR and at the ligand-gated channel level.

Drug design at peptide receptors

Somatostatin (SRIF, somatotropin release inhibiting factor), discovered for its inhibitory action on growth hormone (GH) secretion from pituitary, is an abundant neuropeptide. Two forms, SRIF14 and SRIF28 exist. Recently, a second family of peptides with very similar sequences and features was described; the cortistatins (CST), CST17 and CST29 which are brain selective. The five cloned SRIF receptors (sst1–5) belong to the G-protein coupled/heptathelical receptor family. Structural and operational features distinguish two classes of receptors; SRIF1-sst2/sst3/sst5 (high affinity for octreotide or seglitide) and SRIF2=sst1/sst4 (very low affinity for the aforementioned ligands). The affinity of SRIF receptors for somatostatins and cortistatins is equally high, and it is not clear whether selective receptors do exist for one or the other of the peptides. Several radiologlands label all SRIF receptors, e.g., [125I]LTT-SRIF28, [125I]CGP23996, [125I]Tyr10cortistatin or [125I]Tyr11SRIF14. In contrast, [125I]Tyr3octreotide, [125I]BIM23027, [125I]MK678 or [125I]D-Trp8SRIF14 label predominantly SRIF1 sites, especially sst2 and possibly sst5 receptors. In brain, [125I]Tyr3octreotide binding equates with sst2 receptor mRNA distribution. Native SRIF2 receptors can be labeled with [125I]SRIF14 in the presence of high NaCl in brain (sst1) or lung (sst4) tissue. Short cyclic or linear peptide analogs show selectivity for sst2/sst5 (octreotide, lanreotide, BIM 23027), sst1 (CH-275), sst3 (sst3-ODN-8), or sst5 receptors (BIM 23268); although claims for selectivity have not always been confirmed. Beta peptides with affinity for SRIF receptors are also reported. The general lack of SRIF receptor antagonists is unique for peptide receptors, although CYN 154806 is a selective and potent sst2 antagonist. Nonpeptide ligands are still rare, although a number of molecules have been reported with selectivity and potency for sst1 (L 757,519), sst2 (L 779,976), sst3 (L 796,778), sst4 (NNC 26-9100, L 803,087) or sst1/sst5 receptors (L 817,018). Such molecules are essential to establish the role of SRIF receptors, e.g., sst1 in hypothalamic glutamate currents: sst2 in inhibiting release of GH, glucagon, TSH, gastric acid secretion, pain, seizures and tumor growth, and sst5 in vascular remodeling and inhibition of insulin and GH release.

Somatostatin receptor subtypes 2 and 4 affect seizure susceptibility and hippocampal excitatory neurotransmission in mice

We have investigated the role of somatostatin receptor subtypes sst2 and sst4 in limbic seizures and glutamate‐mediated neurotransmission in mouse hippocampus. As compared to wild‐type littermates, homozygous mice lacking sst2 receptors showed a 52% reduction in EEG ictal activity induced by intrahippocampal injection of 30 ng kainic acid (P < 0.05). The number of behavioural tonic–clonic seizures was reduced by 50% (P < 0.01) and the time to onset of seizures was doubled on average (P < 0.05). Seizure‐associated neurodegeneration was found in the injected hippocampus (CA1, CA3 and hilar interneurons) and sporadically in the ipsilateral latero‐dorsal thalamus. This occurred to a similar extent in wild‐type and sst2 knock‐out mice. Intrahippocampal injection of three selective sst2 receptor agonists in wild‐type mice (Octreotide, BIM 23120 and L‐779976, 1.5–6.0 nmol) did not affect kainate seizures while the same compounds significantly reduced seizures in rats. L‐803087 (5 nmol), a selective sst4 receptor agonist, doubled seizure activity in wild‐type mice on average. Interestingly, this effect was blocked by 3 nmol octreotide. It was determined, in both radioligand binding and cAMP accumulation, that octreotide had no direct agonist or antagonist action at mouse sst4 receptors expressed in CCl39 cells, up to micromolar concentrations. In hippocampal slices from wild‐type mice, octreotide (2 µm) did not modify AMPA‐mediated synaptic responses while facilitation occurred with L‐803087 (2 µm). Similarly to what was observed in seizures, the effect of L‐803087 was reduced by octreotide. In hippocampal slices from sst2 knock‐out mice, both octreotide and L‐803087 were ineffective on synaptic responses. Our findings show that, unlike in rats, sst2 receptors in mice do not mediate anticonvulsant effects. Moreover, stimulation of sst4 receptors in the hippocampus of wild‐type mice induced excitatory effects which appeared to depend on the presence of sst2 subtypes, suggesting these receptors are functionally coupled.

Pharmacological profile of somatostatin and cortistatin receptors

Somatostatin (SRIF) and cortistatin (CST) are two endogenous peptides with high sequence similarities that act as hormones/neurotransmitters both in the CNS and the periphery; their genes although distinct result from gene duplication. Their receptors appear to be common, since the five known SRIF receptors (sst1-sst5) have similar subnanomolar affinity for SRIF and CST, whether the short (SRIF-14, CST-14, CST-17) or the long versions (SRIF-28, CST-29) of the peptides. Whether CST targets specific receptors not shared by SRIF, is still debated: MrgX2 has been described as a selective CST receptor, with submicromolar affinity for CST but devoid of affinity for SRIF; however the distribution of CST and MrgX2 is largely different, and there is no MrgX2 in rodents. A similar situation arises with the GHS receptor GHS-R1a, which displays some preferential affinity for CST over SRIF, but for which there is no evidence that it is activated by CST in vivo. In both cases, one may argue that submicromolar affinity is not the norm of a GPCR for its endogenous neuropeptide. On the other hand, all receptors known to bind SRIF have similar high affinity for CST and both peptides act as potent agonists at the sst1-sst5 receptors, whichever transduction pathway is considered. In addition, [(125)I][Tyr(10)]CST(14) labels sst1-sst5 receptors with subnanomolar affinity, and [(125)I][Tyr(10)]CST(14) binding in the brain is overlapping with that of [(125)I][Tyr(0)]SRIF(14). The functional differences reported that distinguish CST from SRIF, have not been explained convincingly and may relate to ligand-driven transductional selectivity, and other complicating factors such as receptor dimerisation, (homo or heterodimerisation), and/or the influence of accessory proteins (GIPs, RAMPS), which remain to be studied in more detail.

Mechanism of airway hyperresponsiveness to adenosine induced by allergen challenge in actively sensitized Brown Norway rats

We have explored the role of allergen sensitization and challenge in defining the response of the airways of the Brown Norway (BN) rat to adenosine. In naïve animals or in rats sensitized to ovalbumin (OA) adenosine induced only weak bronchoconstrictor responses. Challenge of sensitized animals with OA induced a marked airway hyperresponsiveness to adenosine which was not seen with methacholine or bradykinin. The augmented bronchoconstrictor response to adenosine was not affected by acute bivagotomy or atropine nor mimicked by an i.v. injection of capsaicin. It was, however, blocked selectively by disodium cromoglycate methysergide or ketanserin and reduced in animals treated sub‐chronically with compound 48/80. The augmented response to adenosine was associated with increases in the plasma concentrations of both histamine and 5‐hydroxytryptamine (5‐HT), which were attenuated by pretreatment with disodium cromoglycate, and degranulation of mast cells in the lung. Parenchymal strips from lungs removed from sensitized rats challenged with OA gave augmented bronchoconstrictor responses to adenosine relative to strips from sensitized animals challenged with saline. Responses were inhibited by methysergide and disodium cromoglycate. These data demonstrate a marked augmentation of the bronchoconstrictor response to adenosine in actively sensitized BN rats challenged with OA. The augmented response is primarily a consequence of mast cell activation, leading to the release of 5‐HT, which in turn induces bronchoconstriction. Our data further suggest the involvement of a discrete lung‐based population of mast cells containing and releasing mainly 5‐HT and brought into play by prior exposure to allergen.

Somatostatin sst2 receptor knock-out mice: localisation of sst1–5 receptor mRNA and binding in mouse brain by semi-quantitative RT–PCR, in situ hybridisation histochemistry and receptor autoradiography

The peptide hormone/neurotransmitter somatostatin (somatotropin release inhibiting factor; SRIF) and its receptors (sst(1)-sst(5)) appear to regulate many physiological functions in the CNS. Semi-quantitative analysis of the densities of mRNA expression for sst(1-5) receptors and SRIF receptor binding sites were established in sst(2) receptor knock-out (KO) mice. Patterns of sst(1-5) receptor mRNA expression were largely conserved for sst(1,3,4) and sst(5) selective oligonucleotide probes; whereas sst(2) signals were completely absent in KO mouse brain. Autoradiographic analysis demonstrated [(125)I]LTT SRIF(28), [(125)I]CGP 23996 (two radioligands known to label all five recombinant SRIF receptors) and [(125)I]Tyr(3)-octreotide (sst(2) and sst(5) receptor selective) binding in wild type (WT) mouse brain sections; yet no specific binding of [(125)I]Tyr(3)-octreotide in KO mice. In contrast, [(125)I]LTT SRIF(28) and [(125)I]CGP 23996 binding was still present in a number of brain areas in KO mice, although to a lesser degree than in those regions where [(125)I]Tyr(3)-octreotide binding was found, in WT animals. The present data suggest first, that both sst(2) receptor protein and mRNA were completely absent in the brain of these KO animals. Second, there was little evidence of compensatory regulation, at the mRNA level, of the other SRIF receptors as a consequence of the sst(2) KO. Third, the absence of any [(125)I]Tyr(3)-octreotide binding, in KO mice, suggests that this particular ligand is selective for the sst(2) receptor subtype (under the conditions utilised); or that sst(5) receptors are only marginally expressed in brain. Fourth, there were regions where the binding of [(125)I]LTT SRIF(28) and [(125)I]CGP 23996 were moderately affected by the sst(2) KO, suggesting that additional SRIF receptors may well contribute to the binding of the aforementioned radioligands. Finally, since the relative distribution of these two ligands were not entirely superimposable, it suggests that their respective selectivity profiles towards the different SRIF receptor subtypes in situ are not identical.

Further pharmacological characterization of the adenosine receptor subtype mediating inhibition of oxidative burst in human isolated neutrophils

The aims of this study were to characterize the adenosine receptor subtype mediating inhibition of superoxide anion generation induced by N‐formyl‐methionyl‐leucylphenylalanine (fMLP) in human neutrophils and to test the hypothesis that adenosine 3′:5′‐cyclic monophosphate (cAMP) is the second messenger mediating such inhibition. Superoxide anion generation induced by a submaximal concentration of fMLP (1 μM) was inhibited in a concentration‐dependent manner by adenosine receptor agonists with a rank order of potency ofN‐ethylcarboxamidoadenosine (NECA) > 2(4‐[(2‐ carboxyethyl)phenyl]ethylamino)‐5′‐N‐ethylcarboxamido adenosine (CGS 21680) > (R)‐N6‐phenyl‐2‐propyladenosine ((R)‐PIA) > 2‐Chloro‐N6‐(3‐iodobenzyl)9‐[5‐methylcarbamoyl)‐β‐D‐ribofuranosyl] adenine (2‐Cl‐IB‐MECA) > N6‐cyclopentyladenosine (CPA) > (S)‐N6‐phenyl‐2‐propyladenosine ((S)‐PIA) ≥ N6‐(4‐amino‐3‐iodobenzyl) adenosine‐5′‐N‐methyl‐uronamide (AB‐MECA); this order of potency is consistent with the activation of A2A adenosine receptors. The nonselective A1, A2A, and A2B receptor antagonist 8‐p‐(sulphophenyl) theophylline (8‐SPT; 10 μM) produced blockade of each of the agonists (pKB values 4.79–5.68). The selective A1 adenosine receptor antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX; 100 nM) and the selective A3 adenosine receptor antagonist 3‐(3‐iodo‐4‐aminobenzyl)‐8‐(4‐oxyacetate)phenyl‐1‐propylxanthine (I‐ABOPX; 200 nM) also produced blockade of all the agonists (PKB values 7.37–7.61 and 6.75–7.52, respectively); however, the concentrations required were higher than those which are considered selective for the A1 or A3 receptors. The selective A2A receptor antagonist, 4‐(2‐[7‐amino‐2‐(2‐furyl) [1,2,4] triazolo [2,3‐a] [1,3,5] triazin‐5‐yl amino]ethyl) phenol (ZM 241385; 100 nM), powerfully suppressed the inhibition of the oxidative burst induced by each of the agonists. A Schild analysis of the effects of ZM 241385, 1–100 nM, against NECA and CGS 21680 was carried out. ZM 241385 produced concentration‐dependent, parallel shifts of the concentration–effect curves to both NECA and CGS 21680, with pA2 values of 9.62 and 9.59, respectively. Together, these data establish that inhibition of the oxidative burst in human isolated neutrophils, induced by adenosine receptor agonists, is mediated by the A2A receptor. NECA (0.01–10 μM) induced a concentration‐dependent increase in the intracellular cAMP content of neutrophils. This effect was inhibited in a dose‐dependent manner by ZM 241385 (0.001–10 μM), consistent with activation of A2A adenosine receptors. The results clearly demonstrate that in human neutrophils inhibition of the fMLP—induced oxidative burst by adenosine receptor agonists is mediated via activation of A2A adenosine receptors linked positively to cAMP. No evidence of A1, A2B, or A3 adenosine receptor‐mediated modulation of oxidative burst was found. Drug Dev. Res. 43:214–224, 1998.

BW-A522 blocks adenosine A3 receptor-mediated hypotensive responses in the rat.

The effects of 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)-1-propylxanthine (I-ABOPX; BW-A522), which has nanomolar affinity for the recently cloned human and sheep adenosine A3 receptor, on the putative A3 receptor mediated hypotensive response to N6-2-(4-aminophenyl)ethyl adenosine (APNEA) in the rat have been investigated. Following blockade of A1 and A2 receptors with 8-(p-sulphophenyl)theophylline, BW-A522, 10 and 40 mg/kg i.v., blocked dose-dependently and surmountable the hypotensive response to APNEA. The results provide direct evidence of an A3 receptor in the cardiovascular system of the rat which induces hypotension when activated.

SRA880, in vitro characterization of the first non-peptide somatostatin sst1 receptor antagonist

Abstract This report describes the in vitro features of the first somatostatin sst 1 receptor selective non-peptide antagonist, SRA880 ([3R,4aR,10aR]-1,2,3,4,4a,5,10,10a-Octahydro-6-methoxy-1-methyl-benz[g] quinoline-3-carboxylic-acid-4-(4-nitro-phenyl)-piperazine-amide, hydrogen malonate). SRA was evaluated in a number of in vitro systems of various species, both at native and recombinant receptors, using radioligand binding and second messenger/transduction studies. SRA880 has high affinity for native rat, mouse, monkey and human cerebral cortex somatostatin sst 1 receptors (p K d =7.8−8.6) and for human recombinant sst 1 receptors (p K d =8.0−8.1). SRA880 displayed significantly lower affinity for the other human recombinant somatostatin receptors (p K d ≤6.0) or a wide range of neurotransmitter receptors, except for the human dopamine D4 receptors. SRA880 was characterized in various transduction assays: somatotropin release inhibiting factor (SRIF) induced inhibition of forskolin-stimulated cAMP accumulation, SRIF stimulated-GTPγS binding, and SRIF stimulated luciferase gene expression; in all tests, SRA880 was devoid of intrinsic activity and acted as an apparently surmountable antagonist with p K B values of 7.5–7.7. Combined with the data from binding studies, these results suggest that SRA880 acts as a competitive antagonist. Thus, SRA880 is the first non-peptide somatostatin sst 1 receptor antagonist to be reported; SRA880 will be a useful tool for the characterization of somatostatin sst 1 receptor-mediated effects both in vitro and in vivo.