Acupuncture is an invasive procedure commonly used to relieve pain. Acupuncture is practiced worldwide, despite difficulties in reconciling its principles with evidence-based medicine. We found that adenosine, a neuromodulator with anti-nociceptive properties, was released during acupuncture in mice and that its anti-nociceptive actions required adenosine A1 receptor expression. Direct injection of an adenosine A1 receptor agonist replicated the analgesic effect of acupuncture. Inhibition of enzymes involved in adenosine degradation potentiated the acupuncture-elicited increase in adenosine, as well as its anti-nociceptive effect. These observations indicate that adenosine mediates the effects of acupuncture and that interfering with adenosine metabolism may prolong the clinical benefit of acupuncture.

Adenosine is a determinant of metabolic control of organ function increasing oxygen supply through the A2 class of adenosine receptors and reducing oxygen demand through A1 adenosine receptors (A1AR). In the kidney, activation of A1AR in afferent glomerular arterioles has been suggested to contribute to tubuloglomerular feedback (TGF), the vasoconstriction elicited by elevations in [NaCl] in the macula densa region of the nephron. To further elucidate the role of A1AR in TGF, we have generated mice in which the entire A1AR coding sequence was deleted by homologous recombination. Homozygous A1AR mutants that do not express A1AR mRNA transcripts and do not respond to A1AR agonists are viable and without gross anatomical abnormalities. Plasma and urinary electrolytes were not different between genotypes. Likewise, arterial blood pressure, heart rates, and glomerular filtration rates were indistinguishable between A1AR ؉/؉ , A1AR

To investigate the role of aquaporin-1 (AQP1) water channels in proximal tubule function, in vitro proximal tubule microperfusion and in vivo micropuncture measurements were done on AQP1 knockout mice. The knockout mice were generated by targeted gene disruption and found previously to be unable to concentrate their urine in response to water deprivation. Unanesthetized knockout mice consumed 2.8-fold more f luid than wild-type mice and had lower urine osmolality (505 ؎ 40 vs. 1081 ؎ 68 milliosmolar). An important function of the kidney proximal tubule is the near-isosmolar reabsorption of a significant fraction of fluid that is filtered by the glomerulus. The proximal tubule also reabsorbs nearly all of the filtered glucose, amino acids, and bicarbonate. The apical and basolateral plasma membranes of proximal tubule cells contain water channel protein aquaporin-1 (AQP1), which is thought to provide an important water-selective pathway for transcellular fluid transport (1-3). However, there is conflicting evidence that significant paracellular water transport occurs (4), and it has been suggested that other water channels (AQP7, ref. 5) and transporters (glucose transporter GLUT1, refs. 6, 7; sodium-glucose cotransporter SGLT1, ref. 8) might contribute to transcellular water movement. It is generally believed, but without direct evidence, that high proximal tubule water permeability is important to permit the efficient coupling of solute and water transport to accomplish near-isosmolar fluid absorption.The AQP1 water channel is a water-selective transporter (9, 10) that is found in membranes as tetramers (11) in which each functionally independent monomer (12) contains six transmembrane, tilted helical domains surrounding a putative aqueous pore (13-15). In kidney, AQP1 is strongly expressed in apical and basolateral plasma membranes of epithelial cells in proximal tubule and thin descending limb of Henle and in endothelial cells of descending vasa recta (1-3, 16, 17). Recently, a transgenic AQP1 knockout mouse was generated by targeted gene disruption and shown to manifest a severe defect in urinary concentrating ability (18). When given access to water, the mice appeared grossly normal except for mild growth retardation compared with wild-type mice. When deprived of water, the mice were unable to concentrate their urine and conserve fluid, resulting in marked dehydration and serum hyperosmolality in 1-2 days.The purpose of this study was to define the role of AQP1 in proximal tubule water transport and fluid reabsorption. Isolated tubule microperfusion was used to measure transepithelial osmotic water permeability and fluid absorption under defined in vitro conditions. Free-flow micropuncture was used to determine the in vivo consequences of decreased proximal tubule water permeability. A remarkable decrease in proximal tubule water permeability and fluid reabsorption was found in the AQP1 knockout mice. The results have important implications regarding the mechanisms of proximal tubule fluid reabsorpt...

The present studies were undertaken to determine the effect of dietary salt intake on the renal expression of cyclooxygenase-1 (COX-1) and -2 (COX-2). Protein levels were assessed by Western blotting, and mRNA expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR) on cDNA prepared from kidney regions, dissected nephron segments, and cultured renal cells. Both isoforms were expressed at high levels in inner medulla (IM), with low levels detected in outer medulla and cortex. COX-1 mRNA was present in the glomerulus and all along the collecting duct, whereas COX-2 mRNA was restricted to the macula densa-containing segment (MD), cortical thick ascending limb (CTAL), and, at significantly lower levels, in the inner medullary collecting duct. Both isoforms were highly expressed at high levels in cultured medullary interstitial cells and at lower levels in primary mesangial cells and collecting duct cell lines. Maintaining rats on a low- or high-NaCl diet for 1 wk did not affect expression of COX-1. In IM of rats treated with a high-salt diet, COX-2 mRNA increased 4.5-fold, and protein levels increased 9.5-fold. In contrast, cortical COX-2 mRNA levels decreased 2.9-fold in rats on a high-salt diet and increased 3.3-fold in rats on a low-salt diet. A low-salt diet increased COX-2 mRNA 7.7-fold in MD and 3.3-fold in CTAL. Divergent regulation of COX-2 in cortex and medulla by dietary salt suggests that prostaglandins in different kidney regions serve different functions, with medullary production playing a role in promoting the excretion of salt and water in volume overload, whereas cortical prostaglandins may protect glomerular circulation in volume depletion.

The neuromodulator adenosine regulates immune activation and neuronal survival through specific G-protein-coupled receptors expressed on macrophages and neurons, including the A1 adenosine receptor (A1AR). Here we show that A1AR null (A1AR Ϫ/Ϫ ) mice developed a severe progressive-relapsing form of experimental allergic encephalomyelitis (EAE) compared with their wild-type (A1AR ϩ/ϩ ) littermates. Worsened demyelination, axonal injury, and enhanced activation of microglia/macrophages were observed in A1AR Ϫ/Ϫ animals. In addition, spinal cords from A1AR Ϫ/Ϫ mice demonstrated increased proinflammatory gene expression during EAE, whereas anti-inflammatory genes were suppressed compared with A1AR ϩ/ϩ animals. Macrophages from A1AR Ϫ/Ϫ animals exhibited increased expression of the proinflammatory genes, interleukin-1␤, and matrix metalloproteinase-12 on immune activation when matched with A1AR ϩ/ϩ control cells. A1AR Ϫ/Ϫ macrophage-derived soluble factors caused significant oligodendrocyte cytotoxicity compared with wild-type controls. The A1AR was downregulated in microglia in A1AR ϩ/ϩ mice during EAE accompanied by neuroinflammation, which recapitulated findings in multiple sclerosis (MS) patients. Caffeine treatment augmented A1AR expression on microglia, with ensuing reduction of EAE severity, which was further enhanced by concomitant treatment with the A1AR agonist, adenosine amine congener. Thus, modulation of neuroinflammation by the A1AR represents a novel mechanism that provides new therapeutic opportunities for MS and other demyelinating diseases.

Summary Hypertension affects more than 1.5 billion people worldwide but the precise cause of elevated blood pressure (BP) cannot be determined in most affected individuals. Nonetheless, blockade of the renin-angiotensin system (RAS) lowers BP in the majority of patients with hypertension. Despite its apparent role in hypertension pathogenesis, the key cellular targets of the RAS that control BP have not been clearly identified. Here we demonstrate that RAS actions in the epithelium of the proximal tubule have a critical and non-redundant role in determining the level of BP. Abrogation of AT1 angiotensin receptor signaling in the proximal tubule alone is sufficient to lower BP, despite intact vascular responses. Elimination of this pathway reduces proximal fluid reabsorption and alters expression of key sodium transporters, modifying pressure-natriuresis and providing substantial protection against hypertension. Thus, effectively targeting epithelial functions of the proximal tubule of the kidney should be a useful therapeutic strategy in hypertension.

Adenosine is a potent anticonvulsant acting on excitatory synapses through A1 receptors. Cellular release of ATP, and its subsequent extracellular enzymatic degradation to adenosine, could provide a powerful mechanism for astrocytes to control the activity of neural networks during high-intensity activity. Despite adenosine's importance, the cellular source of adenosine remains unclear. We report here that multiple enzymes degrade extracellular ATP in brain tissue, whereas only Nt5e degrades AMP to adenosine. However, endogenous A1 receptor activation during cortical seizures in vivo or heterosynaptic depression in situ is independent of Nt5e activity, and activation of astrocytic ATP release via Ca 2+ photolysis does not trigger synaptic depression. In contrast, selective activation of postsynaptic CA1 neurons leads to release of adenosine and synaptic depression. This study shows that adenosine-mediated synaptic depression is not a consequence of astrocytic ATP release, but is instead an autonomic feedback mechanism that suppresses excitatory transmission during prolonged activity.purinergic signaling | purine | glia | calcium signaling S everal lines of work over the past three decades have documented that adenosine acts as an endogenous anticonvulsant (1-3). The extracellular concentration of adenosine increases during seizures, and it has been proposed that status epilepticus is a result of loss of adenosine signaling (4). Conversely, mice lacking A1 receptors exhibit a decreased threshold for seizure propagation (5-7). Adenosine can be generated in the cytosol of neurons as a consequence of the metabolic exhaustion and released directly, as adenosine, via the equilibrative nucleoside transporters (ENTs)-e.g., the ubiquitously expressed ENT1 and ENT2 (8, 9). Alternatively, adenosine is released indirectly, as ATP followed by extracellular enzymatic catabolism to adenosine. The CNS expresses several ectoenzymes, including nucleoside triphosphate diphosphohydrolases (NTPDases; e.g., CD39, NTPDase-2), ectonucleotide pyrophosphatase/phosphodiesterases (e.g., autotaxin), and ecto-5′-nucleotidases (CD73/Nt5e, prostatic acid phosphatase, and alkaline phosphatase) (10-12). Although the regional and cellular activity patterns for each of these enzymes have not been fully explored, exogenous addition of ATP to ex vivo preparations has shown that all regions of the mammalian brain can dephosphorylate ATP to AMP through an ADP intermediate, whereas dephosphorylation of AMP to adenosine occurs primarily in the striatum and olfactory bulb (11). However, the presence of ectoenzymes does not prove that extracellular conversion of ATP to adenosine plays a physiological role, because extracellular adenosine is rapidly recirculated back into the cytosol by ENT1 and ENT2 (13). Further, ATP is released in small quantities during cell-cell signaling, and the effective diffusion of adenosine is limited because of the potent reuptake mechanisms (14). Thus, it is possible that extracellularly generated adenosine is transported back...

Deep brain stimulation (DBS) is a widely used neurosurgical approach to treating tremor and other movement disorders. In addition, the use of DBS in a number of psychiatric diseases, including obsessive-compulsive disorders and depression, is currently being tested. Despite the rapid increase in the number of individuals with surgically implanted stimulation electrodes, the cellular pathways involved in mediating the effects of DBS remain unknown. Here we show that DBS is associated with a marked increase in the release of ATP, resulting in accumulation of its catabolic product, adenosine. Adenosine A1 receptor activation depresses excitatory transmission in the thalamus and reduces both tremor- and DBS-induced side effects. Intrathalamic infusion of A1 receptor agonists directly reduces tremor, whereas adenosine A1 receptor-null mice show involuntary movements and seizure at stimulation intensities below the therapeutic level. Furthermore, our data indicate that endogenous adenosine mechanisms are active in tremor, thus supporting the clinical notion that caffeine, a nonselective adenosine receptor antagonist, can trigger or exacerbate essential tremor. Our findings suggest that nonsynaptic mechanisms involving the activation of A1 receptors suppress tremor activity and limit stimulation-induced side effects, thereby providing a new pharmacological target to replace or improve the efficacy of DBS.