Patrick P.A. Humphrey

Classification and nomenclature of somatostatin receptors

There is considerable controversy about the classification and nomenclature of somatostatin receptors. To date, five distinct receptor genes have been cloned and named chronologically according to their respective publication dates, but two were unfortunately given the same appellation (SSTR4). Consensually, a nomenclature for the recombinant receptors has been agreed according to IUPHAR guidelines (sst1, sst2, sst3, sst4, and sst5). However, a more informative classification is to be preferred for the future, employing all classification criteria in an integrated scheme. It is already apparent that the five recombinant receptors fall into two classes or groups, on the basis of not only structure but also pharmacological characteristics. One class (already referred to by some as SRIF1) appears to comprise sst2, sst3 and sst5 receptor subtypes. The other class (SRIF2) appears to comprise the other two recombinant receptor subtypes (sst1 and sst4). This promising approach is discussed but it is acknowledged that much more data from endogenous receptors in whole tissues are needed before further recommendations on somatostatin receptor nomenclature can be made.

A proposed new nomenclature for 5-HT receptors

Abstract The revised 5-HT receptor nomenclature published here was established by the Serotonin Club Receptor Nomenclature Committee and approved by IUPHAR. The members of the Serotonin Club Receptor Nomenclature Committee are: P. B. Bradley, T. A. Branchek, D. E. Clarke, M. L. Cohen, J. R. Fozard, M. Gothert, J. P. Green, P. R. Hartig, D. Hoyer, P. P. A. Humphrey J. E. Leysen, G. R. Martin, D. N. Middlemiss, E. J. Mylecharane, S. J. Peroutka and P. R. Saxena.

Receptors for 5-Hydroxytryptamine: Current perspectives on classification and nomenclature

With the increasing number of 5-HT receptors recently identified, using molecular biology techniques, the classification of 5-HT receptors is under review. An integrated approach is proposed to include operational and transductional as well as structural criteria for definitive receptor characterization. On this basis the existence of as many as seven classes of 5-HT receptor are recognized although only the 5-HT1, 5-HT2 and 5-HT3 receptor classes are well defined.

Alignment of receptor nomenclature with the human genome: classification of 5-HT1B and 5-HT1D receptor subtypes

The continuing rapid progress towards a complete database of structural information on the human genome creates a challenge of ensuring that current schemes for classifying and naming receptors and ion channels effectively integrate this information with functional data to provide unambiguous principles for classification. In this article, Paul Hartig and colleagues review the recent deliberations of the Serotonin Club Nomenclature Committee and outline a number of its recommendations aimed at encouraging consistency in current and future receptor nomenclature. Based on these principles, the present classification of 5-HT1B and 5-HT1D receptors is reconsidered, and a revised nomenclature for 5-HT1B, 5-HT1D alpha and 5-HT1D beta receptor subtypes is suggested.

Acute nociception mediated by hindpaw P2X receptor activation in the rat

1 The functional consequences of P2X receptor activation on peripheral sensory neurones have been investigated in vivo. Behavioural indices of acute nociception were monitored in the conscious rat following subplantar injection of adenosine 5′‐triphosphate (ATP), α,β‐methylene ATP, adenosine 5′‐diphosphate (ADP) and adenosine. 2 Signs of overt nociception, i.e. hindpaw lifting and licking, were apparent in animals injected subplantar with the P2X receptor agonist, α,β‐methylene ATP. Nociceptive behaviours continued for 15 min following administration of α,β‐methylene ATP (200 nmol) and were dose‐related (0–5 min hindpaw lifting times after injection of α,β‐methylene ATP 100 nmol and 1000 nmol were 89±26 s and 232±11 s, respectively). Subplantar ATP evoked a modest response only at the highest dose tested (1000 nmol; 0–5 min hindpaw lifting time 66±19 s) whilst ADP or adenosine (both 600 nmol) elicited negligible spontaneous nociceptive activity. 3 Morphine (3 mg kg−1, i.v.) abolished hindpaw licking behaviour induced by subplantar injection of either α,β‐methylene ATP (600 nmol) or bradykinin (1 nmol) and substantially reduced (88±5%) paw licking in formalin (0.5%, 0.1 ml) injected animals. In contrast, hindpaw lifting was only modestly inhibited (34±11%) in morphine‐pretreated animals that had received subplantar bradykinin and was unaffected in rats in which the noxious stimulus was either subplantar α,β‐methylene ATP or formalin. Pretreatment of hindpaws with subplantar bupivacaine (1% w/v, 0.1 ml) abolished α,β‐methylene ATP‐evoked nociceptive behaviours. 4 Hindpaw lifting and licking mediated by α,β‐methylene ATP (600 nmol, subplantar) were inhibited (72±15% and 95±5%, respectively) by 30 min local pretreatment with 600 nmol α,β‐methylene ATP. Subplantar α,β‐methylene ATP pretreatment did not inhibit behaviour stimulated by subsequent bradykinin (1 nmol) or formalin (0.5%, 0.1 ml) injection into the hindpaw. 5 Desensitization of small diameter sensory neurones with a single subplantar injection of capsaicin (100 μg) abolished all behaviours indicative of spontaneous nociceptive sensation in animals subsequently injected with α,β‐methylene ATP (600 nmol), bradykinin (1 nmol) or formalin (0.5%, 0.1 ml). 6 We conclude that activation of P2X receptors present on small diameter (capsaicin‐sensitive) primary afferent neurones in the rat hindpaw mediates behaviour indicative of acute nociception.

Somatostatin receptors in the central nervous system

Somatostatin was first identified chemically in 1973, since when much has been established about its synthesis, storage and release. It has important physiological actions, including a tonic inhibitory effect on growth hormone release from the pituitary. It has other central actions which are not well understood but recent cloning studies have identified at least five different types of cell membrane receptor for somatostatin. The identification of their genes has allowed studies on the distribution of the receptor transcripts in the central nervous system where they show distinct patterns of distribution, although there is evidence to indicate that more than one receptor type can co-exist in a single neuronal cell. Receptor selective radioligands and antibodies are being developed to further probe the exact location of the receptor proteins. This will lead to a better understanding of the functional role of these receptors in the brain and the prospect of determining the role, if any, of somatostatin in CNS disorders and the identification of potentially useful medicines.

Immunohistochemical localization of the somatostatin sst2(a) receptor in the rat brain and spinal cord

The neuropeptide somatostatin is widely distributed in the CNS and is believed to play a role as a neurotransmitter or a neuromodulator. Somatostatin mediates its actions by the binding of the peptide to high affinity membrane receptors. The genes for five somatostatin receptor types have been cloned recently and Northern blotting and in situ hybridization studies have shown that the transcripts of all five types are expressed in the CNS. Here we report the cellular distribution of somatostatin sst2(a) receptor protein in the adult rat CNS, using a polyclonal anti-peptide antibody directed against a portion of the C-terminal domain of the receptor. The specificity of the affinity-purified antibody was demonstrated by Western blotting and immunolabelling of cells transfected with a hemagglutinin epitope-tagged version of the sst2(a) receptor. Immunohistochemistry showed a distinct distribution of the receptor protein in the rat brain. Cells and processes were labelled in a number of areas, including the basolateral amygdala, the locus coeruleus, the endopiriform nucleus, the deep layers of the cerebral cortex, the subiculum, the claustrum, the habenula, the interpenduncular nucleus, the hippocampus and the central grey. In the spinal cord, the substantia gelatinosa showed strongly-labelled cell bodies and their processes. This study provides an improved understanding of the distribution of the sst2(a) receptor in rat brain.

Pharmacological characterization of ATP- and LPS-induced IL-1β release in human monocytes

We have utilized the human monocytic cell line, THP‐1, and freshly isolated adherent human monocytes with the compounds pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disuphonic acid (PPADS), oxidized ATP, and 1‐(N,O‐bis{5‐isoquinolinesufonyll}‐N‐methyl‐L‐tyrosyl)‐4‐phenylpiperazine (KN‐62) to pharmacologically characterize the P2 receptor involved in ATP‐induced release of interleukin 1β (IL‐1β). We have also investigated the involvement of P2 receptors in lipopolysaccharide (LPS)‐induced IL‐1β release from both cell types. ATP caused release of IL‐1β from LPS primed THP‐1 cells in both a time‐ and concentration‐dependent manner, with a minimal effective ATP concentration of 1 mM. Stimulation of cells with 5 mM ATP resulted in detectable concentrations of IL‐1β in cell supernatants within 30 min. The ATP analogue benzoylbenzoyl ATP (DBATP), a P2X7 receptor agonist, was approximately 10 fold more potent than ATP at eliciting IL‐1β release. KN‐62 (1 μM), PPADS (100 μM) or oxidized ATP (100 uM) significantly inhibited 5 mM ATP‐induced IL‐1β release by 81, 90 and 66% respectively, but failed to significantly inhibit LPS‐induced IL‐1β release in both THP‐1 cells and in freshly isolated human monocytes. In both THP‐1 cells and freshly isolated human monocytes, addition of the ATP degrading enzyme apyrase (0.4 U ml−1) to cell supernatants prior to LPS activation failed to significantly inhibit the LPS‐induced IL‐1β release. In addition there was no correlation between extracellular ATP concentrations and IL‐1β release in THP‐1 cells when studied over a 6 h time period. In conclusion our data confirm the involvement of P2X7 receptors in ATP‐induced IL‐1β release in human monocytes. However no evidence was obtained which would support the involvement of either endogenous ATP release or P2X7 receptor activation as the mechanism by which LPS‐induces IL‐1β release in either the THP‐1 cell line or in freshly isolated human monocytes.

P2X receptor-mediated excitation of nociceptive afferents in the normal and arthritic rat knee joint

1 We tested the hypothesis that functional P2X receptors are present on peripheral terminals of primary afferent articular nociceptors in the rat knee joint. Neural activity was recorded extracellularly from the medial articular nerve innervating the knee joint in rats anaesthetized with pentobarbitone. 2 The selective P2X receptor agonist, αβ methylene ATP (αβmeATP), and the endogenous ligand, ATP, caused a rapid short‐lasting excitation of a sub‐population of C and Aδ nociceptive afferent nerves innervating normal knee joints when injected intra‐arterially or intra‐articularly, and this effect was antagonized by the non‐selective P2 receptor antagonist PPADS. 3 Induction of a chronic (14–21 days) unilateral inflammatory arthritis of the knee joint using locally injected Freunds adjuvant neither increased or decreased responsiveness of joint nociceptors to αβmeATP or ATP. 4 Our results support the hypothesis that αβmeATP‐sensitive P2X receptors are expressed on peripheral nociceptive afferents in the rat knee joint suggesting that they may be involved in the initiation of nociception and pain.

Cloning and functional characterisation of the mouse P2X7 receptor

We have isolated a 1785‐bp complementary DNA (cDNA) encoding the murine P2X7 receptor subunit from NTW8 mouse microglial cells. The encoded protein has 80% and 85% homology to the human and rat P2X7 subunits, respectively. Functional properties of the heterologously expressed murine P2X7 homomeric receptor broadly resembled those of the P2X7 receptor in the native cell line. However, marked phenotypic differences were observed between the mouse receptor, and the other P2X7 receptor orthologues isolated with respect to agonist and antagonist potencies, and the kinetics of formation of the large aqueous pore.