In Memoriam Geoffrey Burnstock: Creator of Purinergic Signaling

Geoff Burnstock (1929–2020) discovered purinergic signaling in a fastidious research that started in early 1960 and culminated in a concept of purinergic nerves in 1972. Subsequently, Geoff developed the concept of purinergic transmission and demonstrated ATP storage, release, and degradation in the context of cotransmission, which was another fundamental concept developed by him. Purinergic transmission contributes to the most fundamental physiological functions such as sensory transduction, regulation of heart rate, smooth muscle contraction, bile secretion, endocrine regulation, immune responses, as well as to various pathophysiological conditions, including inflammation, cancer, neuropathic pain, diabetes, and kidney failure.

Purinergic nerves

It was in 1972 that Burnstock laid the foundation of a new nerve type that he called ‘purinergic nerves’. In this article, he presented experimental data using five criteria to establish that adenosine triphosphate can be considered to be a neurotransmitter, including (1) the release of a purinergic molecule from terminal axons, (2) the structures of purinergic nerves, (3) the electrophysiological properties of purinergic transmission, (4) the pharmacology of adenyl compounds and purinergic transmission, and (5) the distribution and evolution of the purinergic nerves. However, in spite of convincing data, it took more than 20 years for the scientific community to accept this hypothesis. Since then, it has been recognized that the purinergic system is involved in multiple short-term actions such as cell proliferation and pain.

Activation of apical P2U purine receptors permits inhibition of adrenaline-evoked cyclic AMP accumulation in cultured equine sweat gland epithelial cells.

Experiments were undertaken using cultured equine sweat gland epithelial cells that express purine receptors belonging to the P2U subclass which allow the selective agonist uridine triphosphate (UTP) to increase the concentration of intracellular free Ca2+ ([Ca2+]i). Experiments using pertussis toxin (Ptx), which inactivates certain guanine-nucleotide-binding proteins (G-proteins), showed that this response consisted of Ptx-sensitive and Ptx-resistant components, and immunochemical analyses of the G-protein alpha subunits present in the cells showed that both Ptx-sensitive (alpha i1-3) and Ptx-resistant (alpha q/11) G-proteins were expressed. P2U receptors may, therefore, normally activate both of these G-protein families. Ptx-sensitive, alpha i2/3 subunits permit inhibitory control of adenylate cyclase, and UTP was shown to cause Ptx-sensitive inhibition of adrenaline-evoked cyclic AMP accumulation, suggesting that the receptors activate Gi2/3. Experiments using cells grown on permeable supports suggested that P2U receptors became essentially confined to the apical membrane in post-confluent cultures. Polarised epithelia may, therefore, express apical P2U receptors which influence two centrally important signal transduction pathways. It is highly improbable that these receptors could be activated by nucleotides released from purinergic nerves, but they may be involved in the autocrine regulation of epithelial function.

Extracellular ATP mediates Ca2+ signaling in cultured myenteric neurons via a PLC-dependent mechanism

In the myenteric plexus, ATP is released as a neurotransmitter by "purinergic" nerves, relaxing visceral smooth muscle. We report a signal transduction mechanism for ATP in cultured myenteric neurons involving receptor-mediated release of intracellular Ca2+ stores. Primary cultures of myenteric neurons from guinea pigs taenia coli were loaded with the Ca2+ indicator fura 2-acetoxymethyl ester (AM) and examined using digital imaging microscopy. Superfusion of single neurons with ATP (0.01-1,000 microM) resulted in concentration-dependent increases in intracellular Ca2+ concentration ([Ca2+]i) that were independent of extracellular Ca2+. Decrements in peak [Ca2+]i were seen with repetitive ATP exposure. Responsiveness of myenteric neurons to purinergic agonists (100 microM) was consistent with action at a neuronal P 2y purinoceptor: 2-chloro-ATP = ATP = 2-methyl-thio-ATP (MeSATP) > ADP > alpha, beta-MeATP = beta,gamma-MeATP > AMP > adenosine. ATP-evoked Ca2+ transients were inhibited dose dependently by suramin, a nonspecific P2 antagonist, and reactive blue 2, a specific P 2y antagonist. ATP and cyclopiazonic acid (30 microM) appear to release an identical intracellular Ca2+ store. Preincubation with the aminosteroid U-73122 (10 microM) inhibited ATP-evoked Ca2+ transients by 71 +/- 7%, whereas phorbol ester pretreatment (phorbol 12-myristate 13-acetate, 100 nM, 5 min) caused a 76 +/- 4% inhibition. Peak [Ca2+]i evoked by ATP was not affected by preincubation with pertussis toxin (100 ng/ml, 24 h) or nifedipine (10 microM). These data suggest a signal transduction mechanism for ATP in cultured myenteric neurons involving purinoceptor-mediated activation of phospholipase C (PLC), with release of D-myo-inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores.

Vasomotion in chicken foot: dual innervation of arteriovenous anastomoses

Blood exits the foot of the domestic chicken via two major venous routes: a counter-current network surrounding the major incoming artery and a large collateral vein. Between these two routes are numerous large collateral vein. Between these two routes are numerous anastomotic veins. Both venous routes drain capillaries and arteriovenous anastomoses (AVAs). Blood flow through the foot was measured on unanesthetized hens. Flow varies with ambient temperature: 0.2 ml/min at 5 degrees C, 2.2 ml/min at thermoneutrality, and 5.4 ml/min at 36 degrees C; the AVAs contribute 8, 26, and 63% to this flow, respectively. Flow through capillaries is reduced by alpha-adrenergic agonists and is increased by beta-adrenergic agonists. Blocking nerve conduction to the foot at thermoneutrality releases alpha-adrenergic tone and increase AVA flow. Faradic stimulation of foot nerves after adrenergic blockage increases AVA flow, but not capillary flow, suggesting active vasodilation of the AVAs. Such AVA vasodilation normally occurs during body heating, since AVA flow decreases after denervation. Dopaminergic or beta-adrenergic nerves are not involved in active vasodilatation, however, purinergic nerves may play a role. Thus AVAs have a functional dual innervation.