Age-related changes in P2Y receptor signalling in mouse cochlear supporting cells.

Our sense of hearing depends on the function of a specialised class of sensory cells, the hair cells, which are found in the organ of Corti of the mammalian cochlea. The unique physiological environment in which these cells operate is maintained by a syncitium of non-sensory supporting cells, which are crucial for regulating cochlear physiology and metabolic homeostasis. Despite their importance for cochlear function, the role of these supporting cells in age-related hearing loss, the most common sensory deficit in the elderly, is poorly understood. Here, we investigated the age-related changes in the expression and function of metabotropic purinergic receptors (P2Y1 , P2Y2 and P2Y4 ) in the supporting cells of the cochlear apical coil. Purinergic signalling in supporting cells is crucial during the development of the organ of Corti and purinergic receptors are known to undergo changes in expression during ageing in several tissues. Immunolabelling and Ca2+ imaging experiments revealed a downregulation of P2Y receptor expression and a decrease of purinergic-mediated calcium responses after early postnatal stages in the supporting cells. An upregulation of P2Y receptor expression was observed in the aged cochlea when compared to 1 month-old adults. The aged mice also had significantly larger calcium responses and displayed calcium oscillations during prolonged agonist applications. We conclude that supporting cells in the aged cochlea upregulate P2Y2 and P2Y4 receptors and display purinergic-induced Ca2+ responses that mimic those observed during pre-hearing stages of development, possibly aimed at limiting or preventing further damage to the sensory epithelium. KEY POINTS: Age-related hearing loss is associated with lower hearing sensitivity and decreased ability to understand speech. We investigated age-related changes in the expression and function of metabotropic purinergic (P2Y) receptors in cochlear non-sensory supporting cells of mice displaying early-onset (C57BL/6N) and late-onset (C3H/HeJ) hearing loss. The expression of P2Y1 , P2Y2 and P2Y4 receptors in the supporting cells decreased during cochlear maturation, but that of P2Y2 and P2Y4 was upregulated in the aged cochlea. P2Y2 and P2Y4 receptors were primarily responsible for the ATP-induced Ca2+ responses in the supporting cells. The degree of purinergic expression upregulation in aged supporting cells mirrored hearing loss progression in the different mouse strains. We propose that the upregulation of purinergic-mediated signalling in the aged cochlea is subsequent to age-related changes in the hair cells and may act as a protective mechanism to limit or to avoid further damage to the sensory epithelium.

Purinergic Signalling in Physiology and Pathophysiology.

Since its inception by the late Geoffrey Burnstock in the early 1970s [...].

Purinoceptor: a novel target for hypertension.

Hypertension is the leading cause of morbidity and mortality globally among all cardiovascular diseases. Purinergic signalling plays a crucial role in hypertension through the sympathetic nerve system, neurons in the brain stem, carotid body, endothelium, immune system, renin-angiotensin system, sodium excretion, epithelial sodium channel activity (ENaC), and renal autoregulation. Under hypertension, adenosine triphosphate (ATP) is released as a cotransmitter from the sympathetic nerve. It mediates vascular tone mainly through P2X1R activation on smooth muscle cells and activation of P2X4R and P2YR on endothelial cells and also via interaction with other purinoceptors, showing dual effects. P2Y1R is linked to neurogenic hypertension. P2X7R and P2Y11R are potential targets for immune-related hypertension. P2X3R located on the carotid body is the most promising novel therapeutic target for hypertension. A1R, A2AR, A2BR, and P2X7R are all related to renal autoregulation, which contribute to both renal damage and hypertension. The main focus is on the evidence addressing the involvement of purinoceptors in hypertension and therapeutic interventions.

Triggering of Major Brain Disorders by Protons and ATP: The Role of ASICs and P2X Receptors.

Adenosine triphosphate (ATP) is well-known as a universal source of energy in living cells. Less known is that this molecule has a variety of important signaling functions: it activates a variety of specific metabotropic (P2Y) and ionotropic (P2X) receptors in neuronal and non-neuronal cell membranes. So, a wide variety of signaling functions well fits the ubiquitous presence of ATP in the tissues. Even more ubiquitous are protons. Apart from the unspecific interaction of protons with any protein, many physiological processes are affected by protons acting on specific ionotropic receptors-acid-sensing ion channels (ASICs). Both protons (acidification) and ATP are locally elevated in various pathological states. Using these fundamentally important molecules as agonists, ASICs and P2X receptors signal a variety of major brain pathologies. Here we briefly outline the physiological roles of ASICs and P2X receptors, focusing on the brain pathologies involving these receptors.

Triggering of Major Brain Disorders by Protons and ATP: The Role of ASICs and P2X Receptors / 神经科学通报·英文版

Adenosine triphosphate (ATP) is well-known as a universal source of energy in living cells. Less known is that this molecule has a variety of important signaling functions: it activates a variety of specific metabotropic (P2Y) and ionotropic (P2X) receptors in neuronal and non-neuronal cell membranes. So, a wide variety of signaling functions well fits the ubiquitous presence of ATP in the tissues. Even more ubiquitous are protons. Apart from the unspecific interaction of protons with any protein, many physiological processes are affected by protons acting on specific ionotropic receptors-acid-sensing ion channels (ASICs). Both protons (acidification) and ATP are locally elevated in various pathological states. Using these fundamentally important molecules as agonists, ASICs and P2X receptors signal a variety of major brain pathologies. Here we briefly outline the physiological roles of ASICs and P2X receptors, focusing on the brain pathologies involving these receptors.

Role of neuron-derived ATP in paclitaxel-induced HMGB1 release from macrophages and peripheral neuropathy.

We examined the role of ATP and high mobility group box 1 (HMGB1) in paclitaxel-induced peripheral neuropathy (PIPN). PIPN in mice was prevented by HMGB1 neutralization, macrophage depletion, and P2X7 or P2X4 blockade. Paclitaxel and ATP synergistically released HMGB1 from macrophage-like RAW264.7 cells, but not neuron-like NG108-15 cells. The paclitaxel-induced HMGB1 release from RAW264.7 cells was accelerated by co-culture with NG108-15 cells in a manner dependent on P2X7 or P2X4. Paclitaxel released ATP from NG108-15 cells, but not RAW264.7 cells. Thus, PIPN is considered to involve acceleration of HMGB1 release from macrophages through P2X7 and P2X4 activation by neuron-derived ATP.

Interplay between Müller cells and microglia aggravates retinal inflammatory response in experimental glaucoma.

BACKGROUND: Glaucoma, the leading cause of irreversible blindness, is a retinal neurodegenerative disease, which results from progressive apoptotic death of retinal ganglion cells (RGCs). Although the mechanisms underlying RGC apoptosis in glaucoma are extremely complicated, an abnormal cross-talk between retinal glial cells and RGCs is generally thought to be involved. However, how interaction of Müller cells and microglia, two types of glial cells, contributes to RGC injury is largely unknown. METHODS: A mouse chronic ocular hypertension (COH) experimental glaucoma model was produced. Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (q-PCR), transwell co-culture of glial cells, flow cytometry assay, ELISA, Ca2+ image, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) techniques were employed to investigate the interaction of Müller cells and microglia, and its underlying mechanisms in COH retina. RESULTS: We first showed that Müller cell activation in mice with COH induced microglia activation through the ATP/P2X7 receptor pathway. The activation of microglia resulted in a significant increase in mRNA and protein levels of pro-inflammatory factors, such as tumor necrosis factor-α and interleukin-6. These inflammatory factors in turn caused the up-regulation of mRNA expression of pro-inflammatory factors in Müller cells through a positive feedback manner. CONCLUSIONS: These findings provide robust evidence, for the first time, that retinal inflammatory response may be aggravated by an interplay between activated two types of glial cells. These results also suggest that to reduce the interplay between Müller cells and microglia could be a potential effective strategy for preventing the loss of RGCs in glaucoma.

Targeting purinergic receptors to suppress the cytokine storm induced by SARS-CoV-2 infection in pulmonary tissue.

The etiological agent of coronavirus disease (COVID-19) is the new member of the Coronaviridae family, a severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2), responsible for the pandemic that is plaguing the world. The single-stranded RNA virus is capable of infecting the respiratory tract, by binding the spike (S) protein on its viral surface to receptors for the angiotensin II-converting enzyme (ACE2), highly expressed in the pulmonary tissue, enabling the interaction of the virus with alveolar epithelial cells promoting endocytosis and replication of viral material. The infection triggers the activation of the immune system, increased purinergic signaling, and the release of cytokines as a defense mechanism, but the response can become exaggerated and prompt the so-called "cytokine storm", developing cases such as severe acute respiratory syndrome (SARS). This is characterized by fever, cough, and difficulty breathing, which can progress to pneumonia, failure of different organs and death. Thus, the present review aims to compile and correlate the mechanisms involved between the immune and purinergic systems with COVID-19, since the modulation of purinergic receptors, such as A2A, A2B, and P2X7 expressed by immune cells, seems to be effective as a promising therapy, to reduce the severity of the disease, as well as aid in the treatment of acute lung diseases and other cases of generalized inflammation.

A conserved strategy for structure change and energy transduction in Hsp104 and other AAA+ protein motors.

The superfamily of massively large AAA+ protein molecular machines functions to convert the chemical energy of cytosolic ATP into physicomechanical form and use it to perform an extraordinary number of physical operations on proteins, nucleic acids, and membrane systems. Cryo-EM studies now reveal some aspects of substrate handling at high resolution, but the broader interpretation of AAA+ functional properties is still opaque. This paper integrates recent hydrogen exchange results for the typical AAA+ protein Hsp104 with prior information on several near and distantly related others. The analysis points to a widely conserved functional strategy. Hsp104 cycles through a long-lived loosely-structured energy-input "open" state that releases spent ADP and rebinds cytosolic ATP. ATP-binding energy is transduced by allosteric structure change to poise the protein at a high energy level in a more tightly structured "closed" state. The briefly occupied energy-output closed state binds substrate strongly and is catalytically active. ATP hydrolysis permits energetically downhill structural relaxation, which is coupled to drive energy-requiring substrate processing. Other AAA+ proteins appear to cycle through states that are analogous functionally if not in structural detail. These results revise the current model for AAA+ function, explain the structural basis of single-molecule optical tweezer kinetic phases, identify the separate energetic roles of ATP binding and hydrolysis, and specify a sequence of structural and energetic events that carry AAA+ proteins unidirectionally around a functional cycle to propel their diverse physical tasks.

The roles of calcium and ATP in the physiology and pathology of the exocrine pancreas.

This review deals with the roles of calcium ions and ATP in the control of the normal functions of the different cell types in the exocrine pancreas as well as the roles of these molecules in the pathophysiology of acute pancreatitis. Repetitive rises in the local cytosolic calcium ion concentration in the apical part of the acinar cells not only activate exocytosis but also, via an increase in the intramitochondrial calcium ion concentration, stimulate the ATP formation that is needed to fuel the energy-requiring secretion process. However, intracellular calcium overload, resulting in a global sustained elevation of the cytosolic calcium ion concentration, has the opposite effect of decreasing mitochondrial ATP production, and this initiates processes that lead to necrosis. In the last few years it has become possible to image calcium signaling events simultaneously in acinar, stellate, and immune cells in intact lobules of the exocrine pancreas. This has disclosed processes by which these cells interact with each other, particularly in relation to the initiation and development of acute pancreatitis. By unraveling the molecular mechanisms underlying this disease, several promising therapeutic intervention sites have been identified. This provides hope that we may soon be able to effectively treat this often fatal disease.

Role of Primary Afferents in Arthritis Induced Spinal Microglial Reactivity.

Increased afferent input resulting from painful injury augments the activity of central nociceptive circuits via both neuron-neuron and neuron-glia interactions. Microglia, resident immune cells of the central nervous system (CNS), play a crucial role in the pathogenesis of chronic pain. This study provides a framework for understanding how peripheral joint injury signals the CNS to engage spinal microglial responses. During the first week of monosodium iodoacetate (MIA)-induced knee joint injury in male rats, inflammatory and neuropathic pain were characterized by increased firing of peripheral joint afferents. This increased peripheral afferent activity was accompanied by increased Iba1 immunoreactivity within the spinal dorsal horn indicating microglial activation. Pharmacological silencing of C and A afferents with co-injections of QX-314 and bupivacaine, capsaicin, or flagellin prevented the development of mechanical allodynia and spinal microglial activity after MIA injection. Elevated levels of ATP in the cerebrospinal fluid (CSF) and increased expression of the ATP transporter vesicular nucleotide transporter (VNUT) in the ipsilateral spinal dorsal horn were also observed after MIA injections. Selective silencing of primary joint afferents subsequently inhibited ATP release into the CSF. Furthermore, increased spinal microglial reactivity, and alleviation of MIA-induced arthralgia with co-administration of QX-314 with bupivacaine were recapitulated in female rats. Our results demonstrate that early peripheral joint injury activates joint nociceptors, which triggers a central spinal microglial response. Elevation of ATP in the CSF, and spinal expression of VNUT suggest ATP signaling may modulate communication between sensory neurons and spinal microglia at 2 weeks of joint degeneration.

Contribution of ATP-Mediated Positive Feedback to Sympathetic Nerve-Induced Positive Inotropy in Guinea Pig Ventricular Myocardium.

The functional role of ATP released from sympathetic nerve terminals was examined in isolated guinea pig ventricular papillary muscles. The contractile force of papillary muscles was increased by field electrical stimulation of sympathetic nerve endings. This increase was attenuated by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or suramin, blockers of the P2X receptor, and was abolished by propranolol and prazosin. PPADS, suramin, and ATP affected neither the basal contractile force nor the positive inotropic effect of noradrenaline. These results provide functional evidence that ATP released from sympathetic nerve terminals enhances noradrenaline release and contributes to sympathetic nerve-induced inotropy.

Purinergic Signaling Mediates PTH and Fluid Flow-Induced Osteoblast Proliferation.

Both parathyroid hormone (PTH) and mechanical signals are able to regulate bone growth and regeneration. They also can work synergistically to regulate osteoblast proliferation, but little is known about the mechanisms how PTH and mechanical signals interact with each other during this process. In this study, we investigated responses of MC3T3-E1 osteoblasts to PTH and oscillatory fluid flow. We found that osteoblasts are more sensitive to mechanical signals in the presence of PTH according to ERK1/2 phosphorylation, ATP release, CREB phosphorylation, and cell proliferation. PTH may also reduce the osteoblast refractory period after desensitization due to mechanical signals. We further found that the synergistic responses of osteoblasts to fluid flow or ATP with PTH had similar patterns, suggesting that synergy between fluid flow and PTH may be through the ATP pathway. After we inhibited ATP effects using apyrase in osteoblasts, their synergistic responses to mechanical stimulation and PTH were also inhibited. Additionally, knocking down P2Y2 purinergic receptors can significantly attenuate osteoblast synergistic responses to mechanical stimulation and PTH in terms of ERK1/2 phosphorylation, CREB phosphorylation, and cell proliferation. Thus, our results suggest that PTH enhances mechanosensitivity of osteoblasts via a mechanism involving ATP and P2Y2 purinergic receptors.

The role of ATP in the differential ability of Sr2+ to trigger Ca2+ oscillations in mouse and human eggs.

At fertilization in mice and humans, the activation of the egg is caused by a series of repetitive Ca2+ oscillations which are initiated by phospholipase-C(zeta)ζ that generates inositol-1,4,5-trisphophate (InsP3). Ca2+ oscillations and egg activation can be triggered in mature mouse eggs by incubation in Sr2+ containing medium, but this does not appear to be effective in human eggs. Here, we have investigated the reason for this apparent difference using mouse eggs, and human eggs that failed to fertilize after IVF or ICSI. Mouse eggs incubated in Ca2+-free, Sr2+-containing medium immediately underwent Ca2+ oscillations but human eggs consistently failed to undergo Ca2+ oscillations in the same Sr2+ medium. We tested the InsP3-receptor (IP3R) sensitivity directly by photo-release of caged InsP3 and found that mouse eggs were about 10 times more sensitive to InsP3 than human eggs. There were no major differences in the Ca2+ store content between mouse and human eggs. However, we found that the ATP concentration was consistently higher in mouse compared to human eggs. When ATP levels were lowered in mouse eggs by incubation in pyruvate-free medium, Sr2+ failed to cause Ca2+ oscillations. When pyruvate was added back to these eggs, the ATP levels increased and Ca2+ oscillations were induced. This suggests that ATP modulates the ability of Sr2+ to stimulate IP3R-induced Ca2+ release in eggs. We suggest that human eggs may be unresponsive to Sr2+ medium because they have a lower level of cytosolic ATP.

The retrotrapezoid nucleus and the neuromodulation of breathing.

Breathing is regulated by a host of arousal and sleep-wake state-dependent neuromodulators to maintain respiratory homeostasis. Modulators such as acetylcholine, norepinephrine, histamine, serotonin (5-HT), adenosine triphosphate (ATP), substance P, somatostatin, bombesin, orexin, and leptin can serve complementary or off-setting functions depending on the target cell type and signaling mechanisms engaged. Abnormalities in any of these modulatory mechanisms can destabilize breathing, suggesting that modulatory mechanisms are not overly redundant but rather work in concert to maintain stable respiratory output. The present review focuses on the modulation of a specific cluster of neurons located in the ventral medullary surface, named retrotrapezoid nucleus, that are activated by changes in tissue CO2/H+ and regulate several aspects of breathing, including inspiration and active expiration.

Reproductive senescence and energetic metabolism of human luteinized granulosa cells: is it all about ATP? A prospective cohort and critical view.

Mitochondria are known to play a key role in the regulation of reproductive capacity. Senescence is known to impair mitochondrial function and, ultimately, cellular energetic metabolism. Therefore, as women age, a deficient energy supply is likely to affect oocyte quality. The analysis of granulosa cells is considered a valuable noninvasive strategy to assess factors implicated in oocyte competence. Thus, we conducted an observational prospective cohort to evaluate the impact of aging on energy production by luteinized granulosa cells (LGCs). The control group comprised 13 young oocyte donors, whereas the comparison group included 13 infertile women over 38 years of age undergoing in vitro fertilization. Women with diseases that could potentially impact mitochondrial function were excluded. No differences were detected in the ATP levels in LGCs from young donors and infertile patients of advanced reproductive age (1.9 ± 0.99 picomoles in the control group vs. 2.1 ± 0.59 picomoles; p-value = .139). Likewise, the ATP levels in our series did not correlate with either oocyte number or maturity. Despite the similar ATP levels in LGCs, an age effect on the bioenergetic status cannot be excluded. Energy metabolism is very complex, and ATP does not seem to be the most important and reliable parameter.

Myelination increases chemical energy support to the axon without modifying the basic physicochemical mechanism of nerve conduction.

The existence of different conductive patterns in unmyelinated and myelinated axons is uncertain. It seems that considering exclusively physical electrical phenomena may be an oversimplification. A novel interpretation of the mechanism of nerve conduction in myelinated nerves is proposed, to explain how the basic mechanism of nerve conduction has been adapted to myelinated conditions. The neurilemma would bear the voltage-gated channels and Na+/K+-ATPase in both unmyelinated and myelinated conditions, the only difference being the sheath wrapping it. The dramatic increase in conduction speed of the myelinated axons would essentially depend on an increment in ATP availability within the internode: myelin would be an aerobic ATP supplier to the axoplasm, through connexons. In fact, neurons rely on aerobic metabolism and on trophic support from oligodendrocytes, that do not normally duplicate after infancy in humans. Such comprehensive framework of nerve impulse propagation in axons may shed new light on the pathophysiology of nervous system disease in humans, seemingly strictly dependent on the viability of the pre-existing oligodendrocyte.

Purinergic Modulation of Activity in the Developing Auditory Pathway.

Purinergic P2 receptors, activated by endogenous ATP, are prominently expressed on neuronal and non-neuronal cells during development of the auditory periphery and central auditory neurons. In the mature cochlea, extracellular ATP contributes to ion homeostasis, and has a protective function against noise exposure. Here, we focus on the modulation of activity by extracellular ATP during early postnatal development of the lower auditory pathway. In mammals, spontaneous patterned activity is conveyed along afferent auditory pathways before the onset of acoustically evoked signal processing. During this critical developmental period, inner hair cells fire bursts of action potentials that are believed to provide a developmental code for synaptic maturation and refinement of auditory circuits, thereby establishing a precise tonotopic organization. Endogenous ATP-release triggers such patterned activity by raising the extracellular K+ concentration and contributes to firing by increasing the excitability of auditory nerve fibers, spiral ganglion neurons, and specific neuron types within the auditory brainstem, through the activation of diverse P2 receptors. We review recent studies that provide new models on the contribution of purinergic signaling to early development of the afferent auditory pathway. Further, we discuss potential future directions of purinergic research in the auditory system.