Purinergic P2 Receptors: Structure and Function 2.0.

This Special Issue of the International Journal of Molecular Sciences (IJMS) is a direct continuation of the previous Special Issue of this journal, entitled "Purinergic P2 Receptors: Structure and Function" https://www [...].

Erythrocytes from patients with ST-elevation myocardial infarction induce cardioprotection through the purinergic P2Y13 receptor and nitric oxide signaling.

Red blood cells (RBCs) are suggested to play a role in cardiovascular regulation by exporting nitric oxide (NO) bioactivity and ATP under hypoxia. It remains unknown whether such beneficial effects of RBCs are protective in patients with acute myocardial infarction. We investigated whether RBCs from patients with ST-elevation myocardial infarction (STEMI) protect against myocardial ischemia-reperfusion injury and whether such effect involves NO and purinergic signaling in the RBCs. RBCs from patients with STEMI undergoing primary coronary intervention and healthy controls were administered to isolated rat hearts subjected to global ischemia and reperfusion. Compared to RBCs from healthy controls, RBCs from STEMI patients reduced myocardial infarct size (30 ± 12% RBC healthy vs. 11 ± 5% RBC STEMI patients, P < 0.001), improved recovery of left-ventricular developed pressure and dP/dt and reduced left-ventricular end-diastolic pressure in hearts subjected to ischemia-reperfusion. Inhibition of RBC NO synthase with L-NAME or soluble guanylyl cyclase (sGC) with ODQ, and inhibition of cardiac protein kinase G (PKG) abolished the cardioprotective effect. Furthermore, the non-selective purinergic P2 receptor antagonist PPADS but not the P1 receptor antagonist 8PT attenuated the cardioprotection induced by RBCs from STEMI patients. The P2Y13 receptor was expressed in RBCs and the cardioprotection was abolished by the P2Y13 receptor antagonist MRS2211. By contrast, perfusion with PPADS, L-NAME, or ODQ prior to RBCs administration failed to block the cardioprotection induced by RBCs from STEMI patients. Administration of RBCs from healthy subjects following pre-incubation with an ATP analog reduced infarct size from 20 ± 6 to 7 ± 2% (P < 0.001), and this effect was abolished by ODQ and MRS2211. This study demonstrates a novel function of RBCs in STEMI patients providing protection against myocardial ischemia-reperfusion injury through the P2Y13 receptor and the NO-sGC-PKG pathway.

ATP induced calcium signaling activity in perivascular cells differ at different vascular branch levels in the porcine retina.

BACKGROUND: The purine adenosine triphosphate (ATP) plays a significant role in retinal blood flow regulation and recent evidence suggests that the vasoactive effect of the compound differs in vessels at different branching level. However, the cellular basis for the regulation of retinal blood flow mediated by ATP has only been scarcely studied. METHODS: Perfused porcine hemiretinas (n = 60) were loaded with the calcium-sensitive fluorophore Oregon Green ex vivo. Spontaneous oscillations in fluorescence were studied in perivascular cells at five different vascular branching levels ranging from the main arteriole to the capillaries, before and after the addition of intra- and extravascular ATP alone or in the presence of a P2-purinergic receptor antagonist. RESULTS: Intravascular ATP induced an overall significant (p < 0.01) constriction of (mean ± SD) -9.79 ± 13.40% and extravascular ATP an overall significant (p < 0.01) dilatation of (mean ± SD) 19.62 ± 13.47%. Spontaneous oscillations of fluorescence in perivascular cells were significantly more intense around third order arterioles than around vessels at both lower and higher branching levels (p < 0.05 for all comparisons). ATP increased intracellular fluorescence in perivascular cells of first and second order arterioles after extravascular application, and the increase correlated with the accompanying vasodilatation (p < 0.03). Blocking of P2-receptors reduced oscillating fluorescence in pre-capillary arterioles secondary to intravascular ATP (p = 0.03). CONCLUSIONS: Spontaneous oscillations of calcium-sensitive fluorescence in perivascular retinal cells differ at different vascular branching levels. Extravascular ATP increases fluorescence in cells around the larger retinal arterioles exposed to the retinal surface. Future studies should investigate calcium signaling activity in perivascular retinal cells during interventions that simulate retinal pathology such as hypoxia.

A Systematic Review and Meta-Analysis on the Impact of High On-Treatment Platelet Reactivity on Clinical Outcomes for Patients Taking ADP Receptor Inhibitors Following Lower Limb Arterial Endovascular Intervention.

OBJECTIVE: Adenosine diphosphate (ADP) receptor inhibitors such as clopidogrel are known to be less effective at reducing platelet function for some patients because of a phenomenon called high on-treatment platelet reactivity (HTPR). However, the clinical effect of this for patients undergoing endovascular intervention for peripheral arterial disease is unclear. The aim of this study was to assess the impact of ADP receptor inhibitor HTPR on clinical outcomes following lower limb arterial endovascular intervention for peripheral arterial disease. METHODS: A systematic review and meta-analysis was performed. Primary outcomes included all cause mortality and major bleeding. Secondary outcomes were major adverse cardiovascular events, major adverse limb events, restenosis, and target lesion revascularisation. Outcome quality was assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) tool. RESULTS: There were 10 eligible studies including 1 444 patients included in the meta-analysis. The most commonly tested ADP receptor inhibitor was clopidogrel (seven studies). The pooled rate of ADP receptor inhibitor HTPR was 29% (95% CI 27 - 32). The meta-analysis showed that ADP receptor inhibitor HTPR was associated with a greater risk of major adverse limb events (OR 6.25, 95% CI 2.09 - 18.68, p = .001) and a trend towards a higher all cause mortality (OR 1.71, 95% CI 0.99 - 2.94, p = .050) and more major adverse cardiovascular events (OR 4.23, 95% CI 0.46 - 38.92, p = .20) after endovascular intervention. Overall strength of evidence was very low for all outcomes. CONCLUSION: ADP receptor inhibitor HTPR was associated with worse clinical outcomes after lower limb endovascular intervention for peripheral arterial disease. Prospective studies are required to determine the impact of modifying the antithrombotic regimen on clinical outcomes.

P2Y2 purinergic receptor modulates virus yield, calcium homeostasis, and cell motility in human cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV) manipulates many aspects of host cell biology to create an intracellular milieu optimally supportive of its replication and spread. Our study reveals that levels of several components of the purinergic signaling system, including the P2Y2 and P2X5 receptors, are elevated in HCMV-infected fibroblasts. Knockdown and drug treatment experiments demonstrated that P2Y2 enhances the yield of virus, whereas P2X5 reduces HCMV production. The HCMV IE1 protein induces P2Y2 expression; and P2Y2-mediated signaling is important for efficient HCMV gene expression, DNA synthesis, and the production of infectious HCMV progeny. P2Y2 cooperates with the viral UL37x1 protein to regulate cystolic Ca2+ levels. P2Y2 also regulates PI3K/Akt signaling and infected cell motility. Thus, P2Y2 functions at multiple points within the viral replication cycle to support the efficient production of HCMV progeny, and it may facilitate in vivo viral spread through its role in cell migration.

Enhanced sympathetic neurotransduction in the superior mesenteric artery in a rat model of heart failure: role of noradrenaline and ATP.

Heart failure (HF) is associated with neurohumoral activation, which in turn leads to an increased peripheral resistance. In mesenteric vasculature, perivascular innervation plays relevant role maintaining vascular tonus and resistance. Therefore, we aimed to determine the possible alterations in superior mesenteric artery (SMA) perivascular innervation function in HF rats. HF was induced by coronary artery occlusion in male Wistar rats, and sham-operated (SO) rats were used as controls. After 12 wk, a greater vasoconstrictor response to electrical field stimulation (EFS) was observed in endothelium-intact and endothelium-denuded SMA of HF rats. Alpha-adrenoceptor antagonist phentolamine diminished this response in a higher magnitude in HF than in SO animals. However, the noradrenaline (NA) reuptake inhibitor desipramine increased EFS-induced vasoconstriction more in segments from HF rats. Besides, EFS-induced NA release was greater in HF animals, due to a higher tyrosine hydroxylase expression and activity. P2 purinoceptor antagonist suramin reduced EFS-induced vasoconstriction only in segments from SO rats, and adenosine 5'-triphosphate (ATP) release was lower in HF than in SO. Moreover, nitric oxide (NO) synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) enhanced EFS-induced vasoconstriction in a similar extent in both groups. HF was not associated with changes in EFS-induced NO release or the vasodilator response to NO donor sodium nitroprusside. In conclusion, HF postmyocardial infarction enhanced noradrenergic function and diminished purinergic cotransmission in SMA and did not change nitrergic innervation. The net effect was an increased sympathetic participation on the EFS-induced vasoconstriction that could help to understand the neurotransduction involved on the control of vascular tonus in HF.NEW & NOTEWORTHY This study reinforces the pivotal role of noradrenergic innervation in the regulation of mesenteric vascular tone in a rat model of heart failure. Moreover, our results highlight the counteracting role of ATP and NA reuptake, and help to understand the signaling pathways involved on the control of vascular tonus and resistance in heart failure postmyocardial infarction.

Targeting P2 receptors in purinergic signaling: a new strategy of active ingredients in traditional Chinese herbals for diseases treatment.

Adenosine triphosphate (ATP) and its metabolites adenosine diphosphate, adenosine monophosphate, and adenosine in purinergic signaling pathway play important roles in many diseases. Activation of P2 receptors (P2R) channels and subsequent membrane depolarization can induce accumulation of extracellular ATP, and furtherly cause kinds of diseases, such as pain- and immune-related diseases, cardiac dysfunction, and tumorigenesis. Active ingredients of traditional Chinese herbals which exhibit superior pharmacological activities on diversified P2R channels have been considered as an alternative strategy of disease treatment. Experimental evidence of potential ingredients in Chinese herbs targeting P2R and their pharmacological activities were outlined in the study.

Lipopolysaccharide suppresses T cells by generating extracellular ATP that impairs their mitochondrial function via P2Y11 receptors.

T cell suppression contributes to immune dysfunction in sepsis. However, the underlying mechanisms are not well-defined. Here, we show that exposure of human peripheral blood mononuclear cells to bacterial lipopolysaccharide (LPS) can rapidly and dose-dependently suppress interleukin-2 (IL-2) production and T cell proliferation. We also report that these effects depend on monocytes. LPS did not prevent the interaction of monocytes with T cells, nor did it induce programmed cell death protein 1 (PD-1) signaling that causes T cell suppression. Instead, we found that LPS stimulation of monocytes led to the accumulation of extracellular ATP that impaired mitochondrial function, cell migration, IL-2 production, and T cell proliferation. Mechanistically, LPS-induced ATP accumulation exerted these suppressive effects on T cells by activating the purinergic receptor P2Y11 on the cell surface of T cells. T cell functions could be partially restored by enzymatic removal of extracellular ATP or pharmacological blocking of P2Y11 receptors. Plasma samples obtained from sepsis patients had similar suppressive effects on T cells from healthy subjects. Our findings suggest that LPS and ATP accumulation in the circulation of sepsis patients suppresses T cells by promoting inappropriate P2Y11 receptor stimulation that impairs T cell metabolism and functions. We conclude that inhibition of LPS-induced ATP release, removal of excessive extracellular ATP, or P2Y11 receptor antagonists may be potential therapeutic strategies to prevent T cell suppression and restore host immune function in sepsis.

Nucleotide signalling during inflammation.

Inflammatory conditions are associated with the extracellular release of nucleotides, particularly ATP. In the extracellular compartment, ATP predominantly functions as a signalling molecule through the activation of purinergic P2 receptors. Metabotropic P2Y receptors are G-protein-coupled, whereas ionotropic P2X receptors are ATP-gated ion channels. Here we discuss how signalling events through P2 receptors alter the outcomes of inflammatory or infectious diseases. Recent studies implicate a role for P2X/P2Y signalling in mounting appropriate inflammatory responses critical for host defence against invading pathogens or tumours. Conversely, P2X/P2Y signalling can promote chronic inflammation during ischaemia and reperfusion injury, inflammatory bowel disease or acute and chronic diseases of the lungs. Although nucleotide signalling has been used clinically in patients before, research indicates an expanding field of opportunities for specifically targeting individual P2 receptors for the treatment of inflammatory or infectious diseases.

Transcranial amelioration of inflammation and cell death after brain injury.

Traumatic brain injury (TBI) is increasingly appreciated to be highly prevalent and deleterious to neurological function. At present, no effective treatment options are available, and little is known about the complex cellular response to TBI during its acute phase. To gain insights into TBI pathogenesis, we developed a novel murine closed-skull brain injury model that mirrors some pathological features associated with mild TBI in humans and used long-term intravital microscopy to study the dynamics of the injury response from its inception. Here we demonstrate that acute brain injury induces vascular damage, meningeal cell death, and the generation of reactive oxygen species (ROS) that ultimately breach the glial limitans and promote spread of the injury into the parenchyma. In response, the brain elicits a neuroprotective, purinergic-receptor-dependent inflammatory response characterized by meningeal neutrophil swarming and microglial reconstitution of the damaged glial limitans. We also show that the skull bone is permeable to small-molecular-weight compounds, and use this delivery route to modulate inflammation and therapeutically ameliorate brain injury through transcranial administration of the ROS scavenger, glutathione. Our results shed light on the acute cellular response to TBI and provide a means to locally deliver therapeutic compounds to the site of injury.

P2 Receptors Influence hMSCs Differentiation towards Endothelial Cell and Smooth Muscle Cell Lineages.

BACKGROUND: Human mesenchymal stem cells (hMSCs) have shown their multipotential including differentiating towards endothelial and smooth muscle cell lineages, which triggers a new interest for using hMSCs as a putative source for cardiovascular regenerative medicine. Our recent publication has shown for the first time that purinergic 2 receptors are key players during hMSC differentiation towards adipocytes and osteoblasts. Purinergic 2 receptors play an important role in cardiovascular function when they bind to extracellular nucleotides. In this study, the possible functional role of purinergic 2 receptors during MSC endothelial and smooth muscle differentiation was investigated. METHODS AND RESULTS: Human MSCs were isolated from liposuction materials. Then, endothelial and smooth muscle-like cells were differentiated and characterized by specific markers via Reverse Transcriptase-PCR (RT-PCR), Western blot and immunochemical stainings. Interestingly, some purinergic 2 receptor subtypes were found to be differently regulated during these specific lineage commitments: P2Y4 and P2Y14 were involved in the early stage commitment while P2Y1 was the key player in controlling MSC differentiation towards either endothelial or smooth muscle cells. The administration of natural and artificial purinergic 2 receptor agonists and antagonists had a direct influence on these differentiations. Moreover, a feedback loop via exogenous extracellular nucleotides on these particular differentiations was shown by apyrase digest. CONCLUSIONS: Purinergic 2 receptors play a crucial role during the differentiation towards endothelial and smooth muscle cell lineages. Some highly selective and potent artificial purinergic 2 ligands can control hMSC differentiation, which might improve the use of adult stem cells in cardiovascular tissue engineering in the future.

P2 Receptors in Cardiac Myocyte Pathophysiology and Mechanotransduction.

ATP is a major energy source in the mammalian cells, but it is an extracellular chemical messenger acting on P2 purinergic receptors. A line of evidence has shown that ATP is released from many different types of cells including neurons, endothelial cells, and muscle cells. In this review, we described the distribution of P2 receptor subtypes in the cardiac cells and their physiological and pathological roles in the heart. So far, the effects of external application of ATP or its analogues, and those of UTP on cardiac contractility and rhythm have been reported. In addition, specific genetic alterations and pharmacological agonists and antagonists have been adopted to discover specific roles of P2 receptor subtypes including P2X4-, P2X7-, P2Y2- and P2Y6-receptors in cardiac cells under physiological and pathological conditions. Accumulated data suggest that P2X4 receptors may play a beneficial role in cardiac muscle function, and that P2Y2- and P2Y6-receptors can induce cardiac fibrosis. Recent evidence further demonstrates P2Y1 receptor and P2X4 receptor as important mechanical signaling molecules to alter membrane potential and Ca2+ signaling in atrial myocytes and their uneven expression profile between right and left atrium.

Neonatal Seizures and Purinergic Signalling.

Neonatal seizures are one of the most common comorbidities of neonatal encephalopathy, with seizures aggravating acute injury and clinical outcomes. Current treatment can control early life seizures; however, a high level of pharmacoresistance remains among infants, with increasing evidence suggesting current anti-seizure medication potentiating brain damage. This emphasises the need to develop safer therapeutic strategies with a different mechanism of action. The purinergic system, characterised by the use of adenosine triphosphate and its metabolites as signalling molecules, consists of the membrane-bound P1 and P2 purinoreceptors and proteins to modulate extracellular purine nucleotides and nucleoside levels. Targeting this system is proving successful at treating many disorders and diseases of the central nervous system, including epilepsy. Mounting evidence demonstrates that drugs targeting the purinergic system provide both convulsive and anticonvulsive effects. With components of the purinergic signalling system being widely expressed during brain development, emerging evidence suggests that purinergic signalling contributes to neonatal seizures. In this review, we first provide an overview on neonatal seizure pathology and purinergic signalling during brain development. We then describe in detail recent evidence demonstrating a role for purinergic signalling during neonatal seizures and discuss possible purine-based avenues for seizure suppression in neonates.

Exploration of purinergic receptors as potential anti-migraine targets using established pre-clinical migraine models.

BACKGROUND: The current understanding of mechanisms behind migraine pain has been greatly enhanced with the recent therapies targeting calcitonin gene-related peptide and its receptor. The clinical efficacy of calcitonin gene-related peptide-blocking drugs indicates that, at least in a considerable proportion of patients, calcitonin gene-related peptide is a key molecule in migraine pain. There are several receptors and molecular pathways that can affect the release of and response to calcitonin gene-related peptide. One of these could be purinergic receptors that are involved in nociception, but these are greatly understudied with respect to migraine. OBJECTIVE: We aimed to explore purinergic receptors as potential anti-migraine targets. METHODS: We used the human middle meningeal artery as a proxy for the trigeminal system to screen for possible anti-migraine candidates. The human findings were followed by intravital microscopy and calcitonin gene-related peptide release measurements in rodents. RESULTS: We show that the purinergic P2Y13 receptor fulfills all the features of a potential anti-migraine target. The P2Y13 receptor is expressed in both the human trigeminal ganglion and middle meningeal artery and activation of this receptor causes: a) middle meningeal artery contraction in vitro; b) reduced dural artery dilation following periarterial electrical stimulation in vivo and c) a reduction of CGRP release from both the dura and the trigeminal ganglion in situ. Furthermore, we show that P2X3 receptor activation of the trigeminal ganglion causes calcitonin gene-related peptide release and middle meningeal artery dilation. CONCLUSION: Both an agonist directed at the P2Y13 receptor and an antagonist of the P2X3 receptor seem to be viable potential anti-migraine therapies.

Purinergic receptor P2Y6 contributes to 1-methyl-4-phenylpyridinium-induced oxidative stress and cell death in neuronal SH-SY5Y cells.

Oxidative stress and neural degeneration have been shown to be involved in the pathogenesis of Parkinson's disease (PD). The P2Y6 purinergic receptor (P2Y6R) has been shown to participate in the activation of microglia and the production of pro-inflammatory factors induced by lipopolysaccharide to cause neuronal loss. However, the function of P2Y6R during oxidative stress in neurons is unclear. In the present study, 1-methyl-4-phenylpyridinium (MPP+ ) treatment increased the level of UDP/P2Y6R on neuronal SH-SY5Y cells. Importantly, pharmacological inhibition of P2Y6R or knockdown of P2Y6R using a siRNA exerted an increased protective effect by preventing MPP+ -induced increases in the levels of reactive oxygen species (ROS), superoxide anion, inducible nitric oxide synthase (iNOS), and malondialdehyde (MDA) and down-regulation of superoxide dismutase 1 (SOD1) expression. UDP, an agonist of P2Y6R, enhanced the effects of MPP+ , which was also inhibited by apyrase or MRS2578. Additionally, P2Y6R knockdown also significantly reversed both the loss of cell viability and the increase in the levels of phosphorylated extracellular signal-regulated protein kinase (p-ERK1/2) and p38 (p-p38) caused by MPP+ stimulation. However, the inhibition of the ERK1/2 and p38 kinase signaling pathways had no effect on P2Y6R expression. Taken together, these results support the hypothesis that P2Y6R expressed on neuronal SH-SY5Y cell is associated with the progression of oxidative stress and cell death induced by MPP+ , suggesting that P2Y6R may play an important role in the pathogenesis of PD.

Caspase-1 inhibition by VX-765 administered at reperfusion in P2Y12 receptor antagonist-treated rats provides long-term reduction in myocardial infarct size and preservation of ventricular function.

Patients with acute myocardial infarction receive a P2Y12 receptor antagonist prior to reperfusion, a treatment that has reduced, but not eliminated, mortality, or heart failure. We tested whether the caspase-1 inhibitor VX-765 given at reperfusion (a requirement for clinical use) can provide sustained reduction of infarction and long-term preservation of ventricular function in a pre-clinical model of ischemia/reperfusion that had been treated with a P2Y12 receptor antagonist. To address, the hypothesis open-chest rats were subjected to 60-min left coronary artery branch occlusion/120-min reperfusion. Vehicle or inhibitors were administered intravenously immediately before reperfusion. With vehicle only, 60.3 ± 3.8% of the risk zone suffered infarction. Ticagrelor, a P2Y12 antagonist, and VX-765 decreased infarct size to 42.8 ± 3.3 and 29.2 ± 4.9%, respectively. Combining ticagrelor with VX-765 further decreased infarction to 17.5 ± 2.3%. Similar to recent clinical trials, combining ticagrelor and ischemic postconditioning did not result in additional cardioprotection. VX-765 plus another P2Y12 antagonist, cangrelor, also decreased infarction and preserved ventricular function when reperfusion was increased to 3 days. In addition, VX-765 reduced infarction in blood-free, isolated rat hearts indicating at least a portion of injurious caspase-1 activation originates in cardiac tissue. While the pro-drug VX-765 only protected isolated hearts when started prior to ischemia, its active derivative VRT-043198 provided the same amount of protection when started at reperfusion, indicating that even in blood-free hearts, caspase-1 appears to exert its injury only at reperfusion. Moreover, VX-765 decreased circulating IL-1ß, prevented loss of cardiac glycolytic enzymes, preserved mitochondrial complex I activity, and decreased release of lactate dehydrogenase, a marker of pyroptosis. Our results are the first demonstration of a clinical-grade drug given at reperfusion providing additional, sustained infarct size reduction when added to a P2Y12 receptor antagonist.

A cyclic pathway of P2 × 7, bradykinin, and dopamine receptor activation induces a sustained articular hyperalgesia in the knee joint of rats.

OBJECTIVE: We investigated whether: (1) P2 × 7 receptor activation by its agonist (BzATP) induces articular hyperalgesia in the rat's knee joint via inflammatory mechanisms and (2) activation of P2 × 7 receptors by endogenous ATP contributes to the articular hyperalgesia induced by bradykinin, TNF-α, IL-1ß, CINC-1, PGE2, and dopamine. METHODS: The articular hyperalgesia was quantified using the rat knee joint incapacitation test. The knee joint inflammation, characterized by the concentration of pro-inflammatory cytokines and by neutrophil migration, was quantified in the synovial lavage fluid by ELISA and myeloperoxidase enzyme activity assay, respectively. RESULTS: BzATP induced a dose-dependent articular hyperalgesia in the rat's knee joint that was significantly reduced by the selective antagonists for P2 × 7, bradykinin B1 or B2 receptors, ß1 or ß2 adrenoceptors, and by pre-treatment with Indomethacin. BzATP induced a local increase of TNF-α, IL-1ß, IL-6, and CINC-1 concentration and neutrophil migration into the knee joint. The co-administration of the selective P2 × 7 receptor antagonist A-740003 significantly reduced the articular hyperalgesia induced by bradykinin and dopamine, but not by TNF-α, IL-1ß, CINC-1, and PGE2. CONCLUSIONS: P2 × 7 receptor activation induces articular hyperalgesia mediated by the previous inflammatory mediator release. P2 × 7 receptor-induced articular hyperalgesia is sustained by the involvement of this purinergic receptor in bradykinin and dopamine-induced hyperalgesia in the knee joint.

Ovariectomy uncovers purinergic receptor activation of endothelin-dependent natriuresis.

We recently reported that natriuresis produced by renal medullary salt loading is dependent on endothelin (ET)-1 and purinergic (P2) receptors in male rats. Because sex differences in ET-1 and P2 signaling have been reported, we decided to test whether ovarian sex hormones regulate renal medullary ET-1 and P2-dependent natriuresis. The effect of medullary NaCl loading on Na+ excretion was determined in intact and ovariectomized (OVX) female Sprague-Dawley rats with and without ET-1 or P2 receptor antagonism. Isosmotic saline (284 mosmol/kgH2O) was infused in the renal medullary interstitium of anesthetized rats during a baseline urine collection period, followed by isosmotic or hyperosmotic saline (1,800 mosmol/kgH2O) infusion. Medullary NaCl loading significantly enhanced Na+ excretion in intact and OVX female rats. ETA+B or P2 receptor blockade did not attenuate the natriuretic effect of medullary NaCl loading in intact females, whereas ETA+B or P2 receptor blockade attenuated the natriuretic response to NaCl loading in OVX rats. Activation of medullary P2Y2 and P2Y4 receptors by UTP infusion had no significant effect in intact females but enhanced Na+ excretion in OVX rats. Combined ETA+B receptor blockade significantly inhibited the natriuretic response to UTP observed in OVX rats. These data demonstrate that medullary NaCl loading induces ET-1 and P2-independent natriuresis in intact females. In OVX, activation of medullary P2 receptors promotes ET-dependent natriuresis, suggesting that ovarian hormones may regulate the interplay between the renal ET-1 and P2 signaling systems to facilitate Na+ excretion.

Fluorocitrate-mediated depolarization of astrocytes in the retrotrapezoid nucleus stimulates breathing.

Evidence indicates that CO2/H+-evoked ATP released from retrotrapezoid nucleus (RTN) astrocytes modulates the activity of CO2-sensitive neurons. RTN astrocytes also sense H+ by inhibition of Kir4.1 channels; however, the relevance of this pH-sensitive current remains unclear since ATP release appears to involve CO2-dependent gating of connexin 26 hemichannels. Considering that depolarization mediated by H+ inhibition of Kir4.1 channels is expected to increase sodium bicarbonate cotransporter (NBC) conductance and favor Ca2+ influx via the sodium calcium exchanger (NCX), we hypothesize that depolarization in the presence of CO2 is sufficient to facilitate ATP release and enhance respiratory output. Here, we confirmed that acute exposure to fluorocitrate (FCt) reversibly depolarizes RTN astrocytes and increased activity of RTN neurons by a purinergic-dependent mechanism. We then made unilateral injections of FCt into the RTN or two other putative chemoreceptor regions (NTS and medullary raphe) to depolarize astrocytes under control conditions and during P2-recepetor blockade while measuring cardiorespiratory activities in urethane-anesthetized, vagotomized, artificially ventilated male Wistar rats. Unilateral injection of FCt into the RTN increased phrenic (PNA) amplitude and frequency without changes in arterial pressure. Unilateral injection of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS, a P2-receptor antagonist) into the RTN dampened both PNA amplitude and frequency responses to FCt. Injection of MRS2179 (P2Y1-receptor antagonist) into the RTN did not affect the FCt-induced respiratory responses. Fluorocitrate had no effect on breathing when injected into the NTS or raphe. These results suggest that depolarization can facilitate purinergic enhancement of respiratory drive from the RTN.NEW & NOTEWORTHY Astrocytes in the retrotrapezoid nucleus (RTN) are known to function as respiratory chemoreceptors; however, it is not clear whether changes in voltage contribute to astrocyte chemoreception. We showed that depolarization of RTN astrocytes at constant CO2 levels is sufficient to modulate RTN chemoreception by a purinergic-dependent mechanism. These results support the possibility that astrocyte depolarization can facilitate purinergic enhancement of respiratory drive from the RTN.

Effects of ATP and adenosine on contraction amplitude of rat soleus muscle at different temperatures.

INTRODUCTION: The aim of this study was to evaluate the effects of adenosine 5'-triphosphate (ATP) and adenosine on the contractility of mammalian skeletal muscle under hypothermic conditions. METHODS: Contractions of isolated rat soleus muscle were induced by either electrical stimulation (ES) or carbachol at physiological temperatures (37°C) and hypothermic conditions (30-14°C) and recorded in the presence of ATP, adenosine, suramin, and 8-(p-sulfophenyl)-theophylline (8-SPT). RESULTS: At 37°C, incubation of the muscles with ATP inhibited ES-induced contractions; the inhibitory effect of ATP disappeared at 14°C. Adenosine inhibited ES-induced contractions at all temperature levels; 8-SPT fully prevented the action of adenosine. ATP and adenosine did not significantly affect carbachol-induced contractions at 37°C, while at lower temperatures ATP potentiated them. Suramin fully prevented effects of ATP. CONCLUSIONS: ATP is involved in both pre- and postsynaptic regulation of rat soleus muscle contractility, and these processes are significantly more pronounced at low temperatures. Muscle Nerve 55: 417-423, 2017.