Geoff Burnstock Research Articles

Geoff Burnstock, purinergic signalling, and chemosensory control of breathing.

This article is the authors' contribution to the tribute issue in honour of Geoffrey Burnstock, the founder of this journal and the field of purinergic signalling. We give a brief account of the results of experimental studies which at the beginning received valuable input from Geoff, who both directly and indirectly influenced our research undertaken over the last two decades. Research into the mechanisms controlling breathing identified ATP as the common mediator of the central and peripheral chemosensory transduction. Studies of the sources and mechanisms of chemosensory ATP release in the CNS suggested that this signalling pathway is universally engaged in conditions of increased metabolic demand by brain glial cells - astrocytes. Astrocytes appear to function as versatile CNS metabolic sensors that detect changes in brain tissue pH, CO2, oxygen, and cerebral perfusion pressure. Experimental studies on various aspects of astrocyte biology generated data indicating that the function of these omnipresent glial cells and communication between astrocytes and neurons are governed by purinergic signalling, - first discovered by Geoff Burnstock in the 70's and researched through his entire scientific career.

The discovery and development of gefapixant.

Gefapixant is the approved generic name for a compound also known as MK-7264, and prior to that AF-219 and RO-4926219. It is the first-in-class clinically developed antagonist for the P2X3 subtype of trimeric ionotropic purinergic receptors, a family of ATP-gated excitatory ion channels, showing nanomolar potency for the human P2X3 homotrimeric channel and essentially no activity at related channels devoid of P2X3 subunits. As the first P2X3 antagonist to have progressed into clinical studies it has now progressed to the point of successful completion of Phase 3 investigations for the treatment of cough, and the NDA application is under review with US FDA for treatment of refractory chronic cough or unexplained chronic cough. The molecule was discovered in the laboratories of Roche Pharmaceuticals in Palo Alto, California, but clinical development then continued with the formation of Afferent Pharmaceuticals for the purpose of identifying the optimal therapeutic indication for this novel mechanism and establishing a clinical plan for development in the optimal patient populations selected. Geoff Burnstock was a close collaborator and advisor to the P2X3 program for close to two decades of discovery and development. Progression of gefapixant through later stage clinical studies has been conducted by the research laboratories of Merck & Co., Inc., Kenilworth, NJ, USA (MRL; following acquisition of Afferent in 2016), who may commercialize the product once authorization has been granted by regulatory authorities.

The role of enteric inhibitory neurons in intestinal motility.

The enteric nervous system controls much of the mixing and propulsion of nutrients along the digestive tract. Enteric neural circuits involve intrinsic sensory neurons, interneurons and motor neurons. While the role of the excitatory motor neurons is well established, the role of the enteric inhibitory motor neurons (IMNs) is less clear. The discovery of inhibitory transmission in the intestine in the 1960's in the laboratory of Geoff Burnstock triggered the search for the unknown neurotransmitter. It has since emerged that most neurons including the IMNs contain and may utilise more than one transmitter substances; for IMNs these include ATP, the neuropeptide VIP/PACAP and nitric oxide. This review distinguishes the enteric neural pathways underlying the 'standing reflexes' from the pathways operating physiologically during propulsive and non-propulsive movements. Morphological evidence in small laboratory animals indicates that the IMNs are located in the myenteric plexus and project aborally to the circular muscle, where they act by relaxing the muscle. There is ongoing 'tonic' activity of these IMNs to keep the intestinal muscle relaxed. Accommodatory responses to content further activate enteric pathways that involve the IMNs as the final neural element. IMNs are activated by mechanical and chemical stimulation induced by luminal contents, which activate intrinsic sensory enteric neurons and the polarised interneuronal ascending excitatory and descending inhibitory reflex pathways. The latter relaxes the muscle ahead of the advancing bolus, thus facilitating propulsion.

ATP as a cotransmitter in sympathetic and parasympathetic nerves - another Burnstock legacy

Geoff Burnstock created an outstanding scientific legacy that includes identification of adenosine 5′-triphosphate (ATP) as an inhibitory neurotransmitter in the gut, the discovery and characterisation of a large family of purine and uridine nucleotide-sensitive ionotropic P2X and metabotropic P2Y receptors and the demonstration that ATP is as an excitatory cotransmitter in autonomic nerves. The evidence for cotransmission includes that: 1) ATP is costored with noradrenaline in synaptic vesicles in postganglionic sympathetic nerves innervating smooth muscle tissues, including the vas deferens and most arteries. 2) When coreleased with noradrenaline, ATP acts at postjunctional P2X1 receptors to elicit depolarisation, Ca2+ influx, Ca2+ sensitisation and contraction. 3) ATP is also coreleased with acetylcholine from postganglionic parasympathetic nerves innervating the urinary bladder, where it stimulates postjunctional P2X1 receptors, and a second, as yet unidentified site to evoke contraction of detrusor smooth muscle. In both systems membrane-bound ecto-enzymes and soluble nucleotidases released from postganglionic nerves dephosphorylate ATP and so terminate its neurotransmitter actions. Currently, the most promising potential area of therapeutic application relating to cotransmission is treatment of dysfunctional urinary bladder. This family of disorders is associated with the appearance of a purinergic component of neurogenic contractions. This component is an attractive target for drug development and targeting it may be a rewarding area of research.

The extraordinary partnership of Geoff Burnstock and Mollie Holman.

Here, we recognise some of the extraordinary accomplishments of the partnership between Geoff Burnstock and Mollie Holman, and the everlasting impact they both made in autonomic neuroscience in Australia. Much of strength today in autonomic neuroscience can be traced back to a time when Geoff and Mollie commenced their seminal studies on autonomic neuroscience, initially at Oxford, then at The University of Melbourne in the mid 1960's. Mollie and Geoff published their first paper together, at Oxford, with their then mentor, and doyenne of smooth muscle, Professor Edith Bülbring. They did not always agree on the interpretation of their own scientific findings. Geoff was convinced early on that Adenosine triphosphate (ATP), or a related purine, was an excitatory neurotransmitter at peripheral sympathetic neuroeffector junctions. Mollie was reticent for decades. However, she began to take the notion seriously that ATP maybe a neurotransmitter, when receptors for purines were identified in the 1990's. What the partnership between Mollie and Geoff taught us in Australia was to not fear respectful criticism, but rather to be receptive to and embrace objective, collegial and constructive scientific peer-review. One of the many great legacies of Geoff and Mollie was the large number of researchers, who were fortunate disciples of their supervision, and who have now themselves gone on to make significant discoveries in autonomic and visceral neuroscience. This review summarizes some of their major legacies and represents a very personal historical perspective of the two authors, pupils respectively of Mollie and Geoff.

That was then, this is now: the development of our knowledge and understanding of P2 receptor subtypes

P2 receptors are present in virtually all tissues and cell types in the human body, and they mediate the physiological and pharmacological actions of extracellular purine and pyrimidine nucleotides. They were first characterised and named by Geoff Burnstock in 1978, then subdivided into P2X and P2Y purinoceptors in 1985 on the basis of pharmacological criteria in functional studies on native receptors. Molecular cloning of receptors in the 1990s revealed P2X receptors to comprise seven different subunits that interact to produce functional homo- and heterotrimeric ligand-gated cation channels. A family of eight P2Y G protein–coupled receptors were also cloned, which can form homo- and heterodimers. Deep insight into the molecular mechanisms of agonist and antagonist action has been provided by more recent determination of the tertiary and quaternary structures of several P2X and P2Y receptor subtypes. Agonists and antagonists that are highly selective for individual subtypes are now available and some are in clinical use. This has all come about because of the intelligence, insight and drive of the force of nature that was Geoff Burnstock.

Obituary for Geoff Burnstock

Obituary for Geoff Burnstock

Geoffrey Burnstock – An accidental pharmacologist

Geoffrey Burnstock, the founder of the field of purinergic signaling research passed away in Melbourne, Australia on June 3rd, 2020, at the age of 91. With his death, the world of biomedical research lost one of its most passionate, creative and unconventional thought leaders. He was an inspiration to the many researchers he interacted with for more than 50 years and a frequent irritation to those in the administrative establishment. Geoff never considered himself a pharmacologist having being trained as a zoologist and becoming an autonomic neurophysiologist based on his evolving interests in systems and disease-related research. By the end of his life he had: published some 1550 papers; been cited more than 125,000 times; had an h-index of 156 and had supervised over 100 Ph.D. students. His indelible legacy, based on a holistic, data-based, multidisciplinary, unconventional “outside the box” approach to research was reflected in two of the seminal findings in late 20th century biomedical research: the purinergic neurotransmitter hypothesis and the concept of co-neurotransmission, both of which were initially received by his peers with considerable skepticism that at times verged on disdain. Nonetheless, while raising hackles and threatening the status quo, Geoff persevered and prevailed, becoming a mentor for several generations of biomedical researchers. In this review we provide a joint perspective on Geoff Burnstock’s legacy in research.

Real-time measurement of adenosine and ATP release in the central nervous system

This brief review recounts how, stimulated by the work of Geoff Burnstock, I developed biosensors that allowed direct real-time measurement of ATP and adenosine during neural function. The initial impetus to create an adenosine biosensor came from trying to understand how ATP and adenosine-modulated motor pattern generation in the frog embryo spinal cord. Early biosensor measurements demonstrated slow accumulation of adenosine during motor activity. Subsequent application of these biosensors characterized real-time release of adenosine in in vitro models of brain ischaemia, and this line of work has recently led to clinical measurements of whole blood purine levels in patients undergoing carotid artery surgery or stroke. In parallel, the wish to understand the role of ATP signalling in the chemosensory regulation of breathing stimulated the development of ATP biosensors. This revealed that release of ATP from the chemosensory areas of the medulla oblongata preceded adaptive changes in breathing, triggered adaptive changes in breathing via activation of P2 receptors, and ultimately led to the discovery of connexin26 as a channel that mediates CO2-gated release of ATP from cells.

Enteric Inhibitory Neurotransmission, Starting Down Under.

The idea of an inhibitory innervation in the gut came from Geoff Burnstock's group at the University of Melbourne in the 1960s. Being resistant to antagonists of norepinephrine and acetylcholine, enteric inhibitory neurotransmission became known as non-cholinergic, non-adrenergic (NANC) neurotransmission. ATP (or a closely related nucleotide) was proposed as the inhibitory neurotransmitter based on release of purines during nerve stimulation and similarities between responses to ATP and transmural nerve stimulation in several gut preparations. Apamin was found to block purinergic responses, providing evidence that small-conductance Ca(2+)-activated K(+) (SK) channels were responsible for inhibitory junction potentials (IJPs). Actually the IJPs in GI muscles are composed of multiple components, and later studies discovered nitric oxide (NO) to be the other major mediator of NANC inhibitory neurotransmission. The purinergic component of enteric inhibitory neurotransmission is mediated by P2Y1 receptors, and this component is absent in P2Y1(-/-) mice. The criteria for a neurotransmitter are better met by β-nicotinamide adenine dinucleotide (β-NAD) or its immediate metabolite ADP-ribose (ADPR) than by ATP. The cells mediating post-junctional responses have been identified. In addition to smooth muscle cells, two classes of interstitial cells express receptors and effectors for NANC neurotransmitters and are electrically coupled to smooth muscle cells. This integrated structure has been named the SIP syncytium. Interstitial cells of Cajal are involved in transduction of cholinergic and nitrergic inputs to GI muscles, and PDGFRα(+) cells mediate purinergic effects. This short symposium report summarizes major historical points of interest and some of the more recent findings related to enteric inhibitory neurotransmission.

Fourth (2012) UK purine club annual symposium report

The fourth UK Purine Club annual symposium was held in Norwich, Norfolk in November 2012. Delegates from across the UK travelled to convene at this “fine city” and the Assembly House—a Grade I listed building situated in the heart of Norwich. Oral communications took place in the Music Room, a room in which the composer and pianist Franz Listz performed in 1840. Generous financial support from Tocris, Stratech, BMG Labtech, Abcam Biochemicals and New England Biolabs helped us hold the event in such an impressive venue. The meeting comprised three plenary lectures, three oral communication sessions (chaired by Richard Evans, Heather Wilson, Ruth Murrell-Lagnado, Susanna Hourani, Tim Arnett and Keith Brain) and some excellent posters by early stage and established researchers. The meeting opened with a plenary lecture by Professor R. Alan North FRS (Manchester)—an inspiring talk and tour de force of P2X receptor structure-function “20 years of P2X”. This was also an occasion to mark Professor North’s seminal contribution to the field of purinergic signaling and indeed ion channel biology in general. After lunch and posters, we were treated to an insight into pyrimidinergic signaling in a plenary lecture by Professor Antony Galione FMedSci (Oxford). We learnt about the signaling and physiology of NAADP, an intracellular ultrapotent calcium mobilizing second messenger. Professor Nick Dale (Warwick) completed the plenary lectures with an enthusiastic talk on purine biosensors and their medical applications. Professor Geoff Burnstock FRS announced the student winners of the poster competition (Lucy Robinson; Cambridge) and best oral communication (Stuart Hanmer; Cardiff). Professor Burnstock closed by leaving delegates with insight into the expanding world of purinergic signaling. We enjoyed hosting the meeting in Norwich and are happy to see the UK Purine Club continuing with such momentum. We hand over to Ruth Murrell-Lagnado who will host the 5th UK Purine Club meeting in Cambridge 2013—Best wishes to Ruth and her team! Samuel J. Fountain

Third (2011) UK Purine Club annual symposium report

The third UK Purine Club annual symposium was held at the Park Thistle Hotel in Cardiff, South Wales on November 2nd, 2011. Despite having a reputation of being a wet city, the weather was kind and it was a dry day! The Park Thistle Hotel (built in 1883) is a grand Victorian building with a distinctive French-influenced facade. It reopened in 2008 following a multi-million pound refurbishment, so the facilities available were very comfortable and modern. Holding the meeting at such a venue was only possible because of the generous financial support given by Tocris Bioscience, Ascent Scientific, Bioline, New England Biolabs, Promocell, Sarissa Biomedical, and VWR. The meeting was attended by over 70 people from all over the UK and further afield (Denmark, Italy, Netherlands, and Russia). Thirty seven abstracts had been received and these had been scored and ranked by five members of the UK Purine Club (Richard Evans, Alison Gartland, Jack Ham, William Wilkinson, Alex Verkhratsky) before being subdivided into 11 short oral communications and 26 poster presentations. The one-day meeting was opened by Professor Ole Petersen, Director of the School of Biosciences at Cardiff University and the rest of the day was split into three sessions (chaired by Bronwen Evans, Jim Gallagher and Maria Abbracchio). The first invited talk was given by a ‘local boy’, Professor Chris McGuigan. He talked about his groundbreaking work in the field of delivering nucleotides into cells, and two drugs (INX189, which is active against hepatitis C and FV100, an anti-shingles drug) originally developed by his group have recently successfully completed initial phase 2 clinical trials There were then four short communications before a break for lunch and a poster session. After this the second invited speaker, Professor Maria Abbracchio, presented her fascinating work on the P2Y-like GPR17 receptor, followed by 4 more short communications. After a break and more discussions around the posters, the third invited speaker, Dr Stuart Mundell, talked on his exciting work on P2Y12 receptor structure/function. Following the final three short communications, Professor Geoff Burnstock closed the meeting by ‘teasing’ the audience with snippets from work soon to be published in a Special Edition of Purinergic Signalling entitled ‘Cell and Molecular Biology of Purinergic Signalling’. He also presented the prizes for the best oral communication (Ms Swati Gupta, University of Sheffield) and poster presentation (Ms Melina Figueiredo, University of Bristol). A networking session with drinks and nibbles closed the daytime events. In the evening 37 delegates enjoyed the conference dinner at the Park Plaza Hotel in Cardiff, although by this time it was raining! It was a well received evening and one well-known delegate wrote on the feedback form that ‘it was the best conference dinner’ he had ever attended. High praise indeed, since this individual will have attended a huge number of such events during his long and successful career. For some of the more energetic delegates, the day ended dancing away the night at a local nightclub! We really enjoyed planning and hosting the 2011 UK Purine Club annual symposium in Cardiff. We would like now to hand over to Samuel Fountain at the University of East Anglia in Norwich, and look forward to attending the 2012 event.

The history of National Purine Clubs and the International Purine Club

The history of National Purine Clubs and the International Purine Club

Cotransmission in the autonomic nervous system

Cotransmission in the autonomic nervous system

Preface: special issue on medicinal chemistry of purines

The concept of preparing a special issue of Purinergic Signalling to highlight progress in the medicinal chemistry of purines arose during the 8th International Symposium on Adenosine and Adenine Nucleotides, which was held in Ferrara, Italy, from 24 May to 28 May 2006. This meeting emphasized the accelerated pace of introduction of new compounds, including preclinical and clinical candidates, that act through purine-related mechanisms. The suggestion to broaden the scope to include the intracellular actions of purines, notably through protein kinases, was made by Dr. Michael Williams, as an Associate Editor of this journal. Consequently, this volume reveals new ligands for modulation of the function of proteins that are involved in purine action, encompassing both receptors and enzymes. This purview could be described under the umbrella of the “purinome,” which represents numerous critical interactions in cellular function. Given the limited space available, we are unable to effectively showcase all major aspects of the purinome, but rather have provided representative segments. The issue is written from the perspective of the medicinal chemist but in a manner comprehensible to those in the biological sciences. Both review chapters and original chemical research reports are included in this volume. We commend Prof. Geoff Burnstock, Editor in Chief of the journal, for enthusiastically supporting the inclusion of a dedicated issue on medicinal chemistry of purines and for recognizing a central role played by medicinal chemistry in the recent exponential growth of this field. The protein targets of compounds in this volume include twelve G protein-coupled receptors that respond to nucleosides and nucleotides, i.e., both adenosine (four subtypes) and P2Y receptors (eight subtypes), and seven nucleotide-gated ion channels, i.e., P2X receptors. A chapter by Carroll et al. reviews the P2X7 receptor. In addition, agonists (Baraldi et al., Gao et al., Kim et al.), antagonists (Kalla and Zablocki), and allosteric modulators (Goblyos and IJzerman) of the P1 family of adenosine receptors are described including compounds currently under active development. It is to be noted that at least four subtypes of the P2Y receptors are activated by pyrimidine nucleotides (Jacobson et al.). Thus, this issue is not exclusively limited to purines. These purine and pyrimidine receptors are widespread in the body and govern critical physiological processes, notably in the immune, cardiovascular, skeletal-muscular, and nervous systems. The volume also covers inhibition of the superfamily of >500 kinases, which are involved in intracellular signaling and are relevant to cancer (Garcia-Echeverria) and chronic inflammatory diseases (Bhagwat). The search for novel kinase inhibitors, which has become the focus of recent drug discovery efforts, has yielded eight small molecule drugs and a handful of protein therapeutics approved for human use to date. The number of kinase inhibitor drugs for the treatment of human disease is likely to increase in the near future. Also, the volume describes novel inhibitors of nucleotidase enzymes that interconvert extracellular nucleotides (Baki et al.), leading to a spectrum of biological effects. In conclusion, these fields deriving from the purinome have become a major focus in the pharmaceutical industry, and the promise of new drugs emerging from them is enormous. We hope that this special issue will be a good source of information and reference for basic and applied researchers in this field. 14 May 2008

Purines: from premise to promise

Geoff Burnstock’s remarkable insight and tenacity has established the area of purinergic research as a bona fide target for drug discovery. While efforts in P1 receptor-based medicinal chemistry and biology efforts over the past 25 years have not reached the level of success that the pharmaceutical industry investment may have anticipated, the P2 area, with knowledge of the selective localization of members of the P2X and P2Y family members and data from transgenic knockouts, has identified several potential therapeutic areas of major promise including cystic fibrosis, chronic bronchitis, male contraception and neurodegeneration. In addition, interest in the potential of purinergic therapeutics has extended outside the major pharmaceutical companies to the ‘biotech industry’ resulting in an environment where the inherent risks of ‘first in field’ in a therapeutic area may be more appropriately nurtured.

Adventures in the pharmacological analysis of P2 receptors

The pharmacological classification of P2 receptors owes its origin to the pioneering efforts of Geoff Burnstock and those who followed him, research that was conducted primarily in physiological experimental systems. Over recent years, the techniques of molecular biology have been increasingly applied in the study of P2 receptors while, at the same time, advances in their pharmacological analysis have been limited by a lack of potent and selective agonist or antagonist ligands. This has resulted in a classification scheme which is largely structural in nature, with relatively little contribution from pharmacology. Our endeavours in this area have been directed towards the discovery of ligands with which the pharmacological analysis and definition of P2 receptors could be advanced, the ultimate goal being the design of therapeutic agents. This article will describe some of our experiences in this challenging but rewarding area.

Purines and receptors

The fifth Elsevier-sponsored Neuropharmacology conference15th Annual Neuropharmacology conference. Purines and their Receptors. Held in New Orleans, USA; 23–25 October 1997.1 was organized by Alan North (Geneva, Switzerland) and Bertil Fredholm (Stockholm, Sweden) on `Purines and their Receptors'. Geoff Burnstock (London, UK) presented an opening review, conveying with his inspired aplomb the excitement of researchers of the purine field in neurotransmission and neuronal growth and development. The latter were illustrated by dramatic pictures of a dense P2Y receptor presence in the limb buds of mouse embryos and in the neuraxis near the time of closure of the neural tube.

Responses of the isolated sympathetic nerve‐ductus deferens preparation of the guinea‐pig

Responses of the isolated sympathetic nerve‐ductus deferens preparation of the guinea‐pig