Samuel J Fountain

• BSc, PhD
• Professor at University of East Anglia

There is growing interest in the P2X4 receptor as a therapeutic target for several cardiovascular, inflammatory and neurological conditions. Key to exploring the physiological and pathophysiological roles of P2X4 is access to selective compounds to probe function in cells, tissues and animal models. There has been a recent growth in selective antag...

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus l...

Neuropeptide Y (NPY) is co-released with norepinephrine and ATP by sympathetic nerves innervating arteries. Circulating NPY is elevated during exercise and cardiovascular disease, though information regarding the vasomotor function of NPY in human blood vessels is limited. Wire myography revealed NPY directly stimulated vasoconstriction (EC50 10.3...

P2X receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on P2X Receptors [49, 146]) have a trimeric topology [118, 128, 144, 197] with two putative TM domains per P2X subunit, gating primarily Na+, K+ and Ca2+, exceptionally Cl-. The Nomenclature Subcommittee has recommended that for P2X receptors, structural criteria should be the init...

Background and Purpose Senescent preadipocytes promote adipose tissue dysfunction by secreting pro‐inflammatory factors, although little is known about the mechanisms regulating their production. We investigated if up‐regulated purinoceptor function sensitizes senescent preadipocytes to cognate agonists and how such sensitization regulates inflamma...

Junctional adhesion molecules (JAMs; comprising JAM-A, -B and -C) act as receptors for viruses, mediate cell permeability, facilitate leukocyte migration during sterile and non-sterile inflammation and are important for the maintenance of epithelial barrier integrity. As such, they are implicated in the development of both communicable and non-comm...

The P2X4 receptor is a ligand-gated ion channel activated by extracellular ATP. P2X4 activity is associated with neuropathic pain, vasodilation and pulmonary secretion and therefore of therapeutic interest. The structure-activity relationship of P2X4 antagonists is poorly understood. Here we elucidate the structure-activity of 5-BDBD at human P2X4...

Background and Purpose P2X4 is a ligand‐gated cation channel activated by extracellular ATP involved in neuropathic pain, inflammation and arterial tone. Experimental Approach Natural products were screened against human or mouse P2X4 activity using fura‐2 loaded 1321N1 cells for measurement of intracellular Ca²⁺ responses. Whole‐cell currents wer...

The known seven mammalian receptor subunits (P2X1–7) form cationic channels gated by ATP. Three subunits compose a receptor channel. Each subunit is a polypeptide consisting of two transmembrane regions (TM1 and TM2), intracellular N‐ and C‐termini, and a bulky extracellular loop. Crystallization allowed the identification of the 3D structure and g...

ATP, norepinephrine and NPY are co-released by sympathetic nerves innervating arteries. ATP elicits vasoconstriction via activation of smooth muscle P2X receptors. The functional interaction between neuropeptide Y (NPY) and P2X receptors in arteries is not known. In this study we investigate the effect of NPY on P2X1-dependent vasoconstriction in m...

P2X receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on P2X Receptors [48, 141]) have a trimeric topology [124, 139, 188] with two putative TM domains, gating primarily Na+, K+ and Ca2+, exceptionally Cl-. The Nomenclature Subcommittee has recommended that for P2X receptors, structural criteria should be the initial criteria for nome...

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the blood vessel wall. Changes in VSMC actomyosin activity and morphology are prevalent in cardiovascular disease. The actin cytoskeleton actively defines cellular shape and the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, comprised of nesprin and the Sad1p, UNC-84 (...

Tissues differentially secrete multiple colony stimulating factors under conditions of homeostasis and inflammation, orientating recruited circulating monocytes to differentiate to macrophage with differing functional phenotypes. Here, we investigated ATP-evoked intracellular Ca2+ responses in human macrophage differentiated with macrophage colony-...

Background and Purpose CCL2 is an inflammatory chemokine that stimulates the recruitment of monocytes into tissue via activation of the GPCR CCR2. Experimental Approach Freshly isolated human monocytes and THP‐1 cells were used. Fura‐2 loaded cells were used to measure intracellular Ca²⁺ responses. Transwell migration to measure chemotaxis. siRNA‐...

Background and purpose CCL2 is an inflammatory chemokine that stimulates the recruitment of monocytes into tissue via activation of the GPCR CCR2. Experimental approach Freshly isolated human monocytes and THP-1 cells are used; Fura-2 loaded cells used to measure intracellular Ca2+ responses; transwell migration; siRNA-mediated gene knockdown....

Background and Purpose The P2X3 receptor is an ATP‐gated ion channel expressed by sensory afferent neurons and is used as a target to treat chronic sensitisation conditions. The first‐in‐class, selective P2X3 and P2X2/3 receptor antagonist, the diaminopyrimidine MK‐7264 (gefapixant), has progressed to Phase III trials for refractory or unexplained...

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSC...

White adipocytes are key regulators of metabolic homeostasis, which release stored energy as free fatty acids via lipolysis. Adipocytes possess both basal and stimulated lipolytic capacity, but limited information exists regarding the molecular mechanisms that regulate basal lipolysis. Here, we describe a mechanism whereby autocrine purinergic sign...

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal cells (AD-MSCs). The p...

Background and purpose: The Gi-coupled, ADP-activated P2Y12 receptor is well characterised as playing a key role in platelet activation via crosstalk with P2Y1 in ADP-evoked intracellular Ca2+ response. There is limited knowledge on the role of P2Y12 in ADP-evoked Ca2+ responses in other blood cells. Here we investigate the role of P2Y12 receptor...

Leukocytes sense extracellular ATP, a danger-associated molecular pattern, released during cellular stress and death, via activation of cell surface P2X and P2Y receptors. Here, we investigate P2 receptor expression in primary human monocyte-derived macrophages and receptors that mediate ATP-evoked intracellular [Ca2+]i signals and cytokine product...

Monocytes and macrophages express a repertoire of cell surface P2 receptors for adenosine 5′-triphosphate (ATP) a damage-associated molecular pattern molecule (DAMP), which are capable of raising cytoplasmic calcium when activated. This is achieved either through direct permeation (ionotropic P2X receptors) or by mobilizing intracellular calcium st...

Adenosine 5′-triphosphate is a well-known extracellular signaling molecule and neurotransmitter known to activate purinergic P2X receptors. Information has been elucidated about the structure and gating of P2X channels following the determination of the crystal structure of P2X4 (zebrafish), however, there is still much to discover regarding the ro...

Mechanisms controlling endoplasmic reticulum (ER) Ca²⁺ homeostasis are important regulators of resting cytoplasmic Ca²⁺ concentration ([Ca²⁺]cyto) and receptor-mediated Ca²⁺ signalling. Here we investigate channels responsible for ER Ca²⁺ leak in THP-1 macrophage and human primary macrophage. In the absence of extracellular Ca²⁺ we employ ionomycin...

ATP is omnipresent in biology and acts as an extracellular signalling molecule in mammals. Information regarding the signalling function of extracellular ATP in single celled eukaryotes is lacking. Here we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP cou...

The chemokine CCL2 serves to target circulating monocytes and other leukocytes to tissue during innate immune responses and the progression of chronic inflammatory disease via activation of CCR2 receptor. Here we show that co-activation of the P2Y6 purinergic receptor occurs when THP-1 cells and human peripheral blood mononuclear cells sense CCL2 t...

Adenosine 5-triphosphate (ATP) is omnipresent in biology. It is therefore no surprise that organisms have evolved multifaceted roles for ATP, exploiting its abundance and restriction of passive diffusion across biological membranes. A striking role is the emergence of ATP as a bona fide transmitter molecule, whereby the movement of ATP across membr...

Release and reception of extracellular ATP by leukocytes plays a critical role in immune responses to infection, injury and cardiovascular disease. Leukocytes of both the innate, adaptive immune and central nervous system express a repertoire of cell surface receptors for ATP (P2X and P2Y receptors) and its metabolites. ATP acts as a damage-associa...

P2X receptors are calcium permeable ligand-gated ion channels activated by ATP. Their role as cell surface receptors for extracellular ATP released physiologically by mammalian cells is well established. However, the cellular function of P2X receptor subtypes that populate the membranes of intracellular compartments is not defined. An initial repor...

Elucidating mechanisms by which Ca(2+) signals are generated by monocytes is important for understanding monocytic function in health and disease. Here we investigate mechanisms underlying Ca(2+) signals generated following disruption of lysosomes by exposure to the cathepsin C substrate glycyl-L-phenylalanine-β-napthylamide (GPN). Exposure to 0.2...

P2X receptors (P2XRs) are ATP-activated calcium-permeable ligand-gated ion channels traditionally viewed as sensors of extracellular ATP during diverse physiological processes including pain, inflammation, and taste. However, in addition to a cell surface residency P2XRs also populate the membranes of intracellular compartments, including mammalian...

Statins have both cholesterol lowering and anti-inflammatory activities, whether mechanisms underlying their activities are independent remains unclear. The ATP-gated P2X(4) receptor is a pro-inflammatory mediator. Here, we investigate the action of fluvastatin and other cholesterol depleting agents on native and recombinant human P2X(4) receptor....

The social amoeba Dictyostelium discoideum is a genetically amenable eukaryotic cell which displays many animal cell traits and has been used to study cellular signalling for over 30 years. Recently studies have highlighted the roles that molecules associated with synaptic transmission in animals, such as glutamate, GABA and ATP play in cellular di...

Voltage-gated K+ channels (K(V) channels) are encoded by the KCNx gene family and have such a wide range of properties that it is necessary to identify the precise expression profile that is instrumental in governing the electrical phenotype of a cell and its response to extrinsic factors. Real-time quantitative RT-PCR methodology has been develope...

Adenosine triphosphate (ATP) is an ancient and fundamentally important biological molecule involved in both intracellular and extracellular activities. P2X ionotropic and P2Y metabotropic receptors have been cloned and characterised in mammals. ATP plays a central physiological role as a transmitter molecule in processes including the sensation of...

P2X receptors are ATP-gated nonselective cation channels with important physiological roles. However, their structures are poorly understood. Here, we analyzed the architecture of P2X receptors using fluorescence resonance energy transfer (FRET) microscopy and direct structure determination using electron microscopy. FRET efficiency measurements in...

We have cloned a P2X receptor (OtP2X) from the green algae Ostreococcus tauri. The 42-kDa receptor shares ∼28% identity with human P2X receptors and 23% with the Dictyostelium P2X receptor. ATP application evoked flickery single channel openings in outside-out membrane patches from human embryonic kidney 293 cells expressing OtP2X. Whole-cell recor...

P2X2 receptors are ATP-gated ion channels widely expressed by neurons. Thr339 lies in the second of the two transmembrane domains of the rat P2X2 receptor protein, and is likely to be close to the narrowest part of the pore. Single-channel and whole-cell recording after expression in human embryonic kidney 293 cells showed that P2X2[T339S] receptor...

K(V)1.5, a voltage-gated potassium channel, has functional importance in regulating blood vessel tone and cardiac action potentials and is a target for numerous therapeutic drug development programs. Despite the importance of K(V)1.5, there is little knowledge of the mechanisms controlling expression of its underlying gene, Kcna5. We identified a 5...

P2X receptors are membrane ion channels gated by extracellular ATP that are found widely in vertebrates, but not previously in microbes. Here we identify a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and show, with the use of heterologous expression in human embryonic kidney cells, that it encodes a membrane ion...

Receptor desensitization can determine the time course of transmitter action and profoundly alter sensitivity to drugs. Among P2X receptors, ion currents through homomeric P2X4 receptors exhibit intermediate desensitization when compared with P2X1 and P2X3 (much faster) and P2X2 and P2X7 (slower). We recorded membrane currents in HEK293 cells trans...

Here we explore the activation mechanisms of human TRPC5, a putative cationic channel that was cloned from a region of the X chromosome associated with mental retardation. No basal activity was evident but activity was induced by carbachol stimulation of muscarinic receptors independently of Ca2+ release. This is 'receptor activation', as described...

This study focused on the hypothesis that KCNA genes (which encode K(V)alpha1 voltage-gated K(+) channels) have enhanced functional expression in smooth muscle cells of a primary determinant of peripheral resistance - the small mesenteric artery. Real-time PCR methodology was developed to measure cell type-specific in situ gene expression. Profiles...