Nanobodies as probes to investigate purinergic signaling.

Nanobodies (VHHs) are the single variable immunoglobulin domains of heavy chain antibodies (hcAbs) that naturally occur in alpacas and other camelids. The two variable domains of conventional antibodies typically interact via a hydrophobic interface. In contrast, the corresponding surface area of nanobodies is hydrophilic, rendering these single immunoglobulin domains highly soluble, robust to harsh environments, and exceptionally easy to format into bispecific reagents. In homage to Geoffrey Burnstock, the pioneer of purinergic signaling, we provide a brief history of nanobody-mediated modulation of purinergic signaling, using our nanobodies targeting P2X7 and the NAD+-metabolizing ecto-enzymes CD38 and ARTC2.2 as examples.

Purinergic signalling in host innate immune defence against intracellular pathogens.

Purinergic signalling is an evolutionarily conserved signalling pathway mediated by extracellular nucleotides and nucleosides. Tri- and diphosphonucleotides released from host cells during intracellular pathogen infections activate plasma membrane purinergic type 2 receptors (P2 receptors) that stimulate microbicidal mechanisms in host innate immune cells. P2X ion channels and P2Y G protein-coupled receptors are involved in activating host innate immune defence mechanisms, phagocytosis, phagolysosomal fusion, production of reactive species, acidification of parasitophorous vacuoles, inflammasome activation, and the release of cytokines, chemokines, and other inflammatory mediators. In this review, as part of a special issue in tribute to Geoffrey Burnstock, we discuss advances in understanding the importance of P2 receptors in the host antimicrobial innate mechanisms against intracellular pathogen infections.

Purinergic smooth muscle contractions in the human prostate: estimation of relevance and characterization of different agonists.

Non-adrenergic prostate smooth muscle contractions may account for the limited effectiveness of α1-adrenoceptor antagonists, which are the first-line option for medical treatment of voiding symptoms suggestive of benign prostatic hyperplasia. In non-human prostates, purinergic agonists induce contractions reaching similar magnitudes as α1-adrenergic contractions. However, evidence for the human prostate is highly limited, and pointed to much weaker purinergic contractions. Here, we examined contractions of different purinergic agonists in human prostate tissues. Tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath, and expression of purinergic receptors was studied by RT-PCR. Electric field stimulation (EFS)-induced contractions amounted to 104% of KCl-induced contractions (95% CI: 84-124%). From all tested agonists, only ATP induced concentration-dependent contractions, reaching an average maximum of 18% (12-24%) of KCl. Maximum tensions following application of other agonists averaged to 7.1% of KCl for α,ß-methylene-ATP (1.8-12.4%), 3.9% for ß,γ-methylene-ATP (2.0-5.4%), 3.1% for 2-methylthio-ATP (- 0.1-6.3%), and 5.1% for ATPγS (1.0-9.2%). Responses were not affected by the P2X antagonist NF023 or the P2Y antagonist PPADS. mRNA expression of P2X1-4 correlated with expression of a marker for catecholaminergic nerves, although neither ATP, NF023, nor PPADS changed EFS-induced contractions. Correlation between expression of receptors and the smooth muscle marker calponin was not observed. Our findings point to a low relevance of purinergic contractions in the human prostate, compared to other contractile stimuli in the human prostate and compared to purinergic contractions in non-human prostates. Purinergic contractions in the human prostate are not sensitive to NF023 or PPADS.

Purinergic signalling in the kidney: In physiology and disease.

Historically, the control of renal vascular and tubular function has, for the most part, concentrated on neural and endocrine regulation. However, in addition to these extrinsic factors, it is now appreciated that several complex humoral control systems exist within the kidney that can act in an autocrine and/or paracrine fashion. These paracrine systems complement neuroendocrine regulation by dynamically fine-tuning renal vascular and tubular function to buffer rapid changes in nephron perfusion and flow rate of tubular fluid. One of the most pervasive is the extracellular nucleotide/P2 receptor system, which is central to many of the intrinsic regulatory feedback loops within the kidney such as renal haemodynamic autoregulation and tubuloglomerular feedback (TGF). Although physiological actions of extracellular adenine nucleotides were reported almost 100 years ago, the conceptual framework for purinergic regulation of renal function owes much to the work of Geoffrey Burnstock. In this review, we reflect on our >20-year collaboration with Professor Burnstock and highlight the research that is still unlocking the potential of the renal purinergic system to understand and treat kidney disease.

From astrocytes to satellite glial cells and back: A 25 year-long journey through the purinergic modulation of glial functions in pain and more.

Fundamental progresses have been made in pain research with a comprehensive understanding of the neuronal pathways which convey painful sensations from the periphery and viscera to the central nervous system and of the descending modulating pathways. Nevertheless, many patients still suffer from various painful conditions, which are often associated to other primary pathologies, and get no or poor relief from available painkillers. Thus, the interest of many researchers has concentrated on new and promising cellular targets and biochemical pathways. This is the case of glia cells, both in the peripheral and in the central nervous system, and of purinergic receptors. Starting from many intuitions and hypotheses raised by Prof. Geoffrey Burnstock, data have accumulated which clearly highlight the fundamental role exerted by several nucleotide and nucleoside receptors in the modulation of glial cell reaction to pain triggers and of their cross-talk with sensory neurons which significantly contributes to the transition from acute to chronic pain. The purinergic system has therefore become an appealing pharmacological target in pain research, also based on the quite unexpected discovery that purines are involved in ancient analgesic techniques such as acupuncture. A more in-depth understanding of the complex and intricated purine-orchestrated scenario in pain conditions will hopefully lead to the identification and clinical development of new and effective analgesics.

Purinergic signaling in diabetes and metabolism.

Purinergic signaling, a concept originally formulated by the late Geoffrey Burnstock (1929-2020), was found to modulate pathways in every physiological system. In metabolic disorders there is a role for both adenosine receptors and P2 (nucleotide) receptors, of which there are two classes, i.e. P2Y metabotropic and P2X ionotropic receptors. The individual roles of the 19 receptors encompassed by this family have been dissected - and in many cases the effects associated with specific cell types, including adipocytes, skeletal muscle, liver cells and immune cells. It is suggested that ligands selective for each of the four adenosine receptors (A1, A2A, A2B and A3), and several of the P2 subtypes (e.g. P2Y6 or P2X7 antagonists) might have therapeutic potential for treating diabetes and obesity. This is a developing story with some conflicting conclusions relevant to drug discovery, which we summarize here.

Purinergic receptors in airway hydration.

Airway epithelial purinergic receptors control key components of the mucociliary clearance (MCC), the dominant component of pulmonary host defense. In healthy airways, the periciliary liquid (PCL) is optimally hydrated, thus acting as an efficient lubricant layer over which the mucus layer moves by ciliary force. When the hydration of the airway surface decreases, the mucus becomes hyperconcentrated, the PCL collapses, and the "thickened" mucus layer adheres to cell surfaces, causing plaque/plug formation. Mucus accumulation is a major contributing factor to the progression of chronic obstructive lung diseases such as cystic fibrosis (CF) and chronic bronchitis (CB). Mucus hydration is regulated by finely tuned mechanisms of luminal Cl- secretion and Na+ absorption with concomitant osmotically driven water flow. These activities are regulated by airway surface liquid (ASL) concentrations of adenosine and ATP, acting on airway epithelial A2B and P2Y2 receptors, respectively. The goal of this article is to provide an overview of our understanding of the role of purinergic receptors in the regulation of airway epithelial ion/fluid transport and the mechanisms of nucleotide release and metabolic activities that contribute to airway surface hydration in healthy and chronically obstructed airways.

History of ectonucleotidases and their role in purinergic signaling.

Ectonucleotidases are key for purinergic signaling. They control the duration of activity of purinergic receptor agonists. At the same time, they produce hydrolysis products as additional ligands of purinergic receptors. Due to the considerable diversity of enzymes, purinergic receptor ligands and purinergic receptors, deciphering the impact of extracellular purinergic receptor control has become a challenge. The first group of enzymes described were the alkaline phosphatases - at the time not as nucleotide-metabolizing but as nonspecific phosphatases. Enzymes now referred to as nucleoside triphosphate diphosphohydrolases and ecto-5'-nucleotidase were the first and only nucleotide-specific ectonucleotidases identified. And they were the first group of enzymes related to purinergic signaling. Additional research brought to light a surprising number of ectoenzymes with broad substrate specificity, which can also hydrolyze nucleotides. This short overview traces the development of the field and briefly highlights important results and benefits for therapies of human diseases achieved within nearly a century of investigations.

Adenosine kinase: A key regulator of purinergic physiology.

Adenosine (ADO) is an essential biomolecule for life that provides critical regulation of energy utilization and homeostasis. Adenosine kinase (ADK) is an evolutionary ancient ribokinase derived from bacterial sugar kinases that is widely expressed in all forms of life, tissues and organ systems that tightly regulates intracellular and extracellular ADO concentrations. The facile ability of ADK to alter ADO availability provides a "site and event" specificity to the endogenous protective effects of ADO in situations of cellular stress. In addition to modulating the ability of ADO to activate its cognate receptors (P1 receptors), nuclear ADK isoform activity has been linked to epigenetic mechanisms based on transmethylation pathways. Previous drug discovery research has targeted ADK inhibition as a therapeutic approach to manage epilepsy, pain, and inflammation. These efforts generated multiple classes of highly potent and selective inhibitors. However, clinical development of early ADK inhibitors was stopped due to apparent mechanistic toxicity and the lack of suitable translational markers. New insights regarding the potential role of the nuclear ADK isoform (ADK-Long) in the epigenetic modulation of maladaptive DNA methylation offers the possibility of identifying novel ADK-isoform selective inhibitors and new interventional strategies that are independent of ADO receptor activation.

[Influence of intraoperative sodium adenosine triphosphate infusion on leukocyte dynamics following colonic resection in cancer patients].

The paper evaluates effects of different methods of systemic intraoperative anesthesia on the dynamics of leukocytes following colonic resection in tumor patients. Lymphocytes levels rose on day 2 after operation in patients receiving non-selective purine receptor agonists (sodium adenosine triphosphate) as an intraoperative analgesic and were significantly higher than in controls treated with conventional drugs (p

[Purine analgesia: experimental results and clinical perspectives].

The high rate of dissatisfaction with analgesia in patients undergoing surgery and by those with chronic pain prompts a search for new pain relief methods. Purine analgesia may considerably ease suffering in these patient groups. This review considers the results of the studies dealing with the analgesic activity of non-selective purine receptor agonists, such as adenosine and adenosine triphosphate, on experimental models and the feasibilities of their clinical application. The paper points to the experimental studies, the results of which are the basis for the clinical use of purines. Much attention is given to the studies using purine analgesia as a component of general anesthesia. It is no less than important that good results of treatment were obtained after administration of purine agonists to patients with neuropathic pain syndromes. Thus, purine analgesia should be regarded as a promising line of the development of anesthesiology.