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1.  Depression of Ventilation by Dopamine in Man 
Journal of Clinical Investigation  1978;61(3):708-713.
Dopamine is present in the carotid body and has been postulated to be an inhibitory neurotransmitter. The purpose of this study was to determine the effects of dopamine on ventilation in man and to examine its mechanism of action. Dopamine (0.5-10 μg/kg per min) was infused in eight normal men at different levels of arterial chemoreceptor activity, produced by varying the inspired Po2. During normoxia dopamine produced a small decrease in minute ventilation (V̇e) and an increase in arterial Pco2. When arterial chemoreceptors were stimulated by hypoxia, infusion of dopamine produced a marked initial depression of V̇e followed by a sustained although less pronounced decrease in V̇e. An increase in Paco2 and a decrease in Pao2 were also observed. When arterial chemoreceptor activity was suppressed by hyperoxia, infusion of dopamine did not affect ventilation. Subjects also breathed a hypercarbic, hyperoxic gas mixture. The hypercarbia produces hyperventilation by stimulating central chemoreceptors, whereas the hyperoxia suppresses peripheral chemoreceptors. Dopamine did not alter ventilation while the subjects were breathing this gas mixture.
These studies suggest that dopamine suppresses ventilation in man through an action on the arterial chemoreceptor reflex. These findings support the hypothesis that dopamine is an inhibitory neurotransmitter in the carotid body, and that release of dopamine may modulate the sensitivity of peripheral arterial chemoreceptors.
PMCID: PMC372584  PMID: 641149
2.  Inhibition of Hippocampal Synaptic Activity by ATP, Hypoxia or Oxygen-Glucose Deprivation Does Not Require CD73 
PLoS ONE  2012;7(6):e39772.
Adenosine, through activation of its A1 receptors, has neuroprotective effects during hypoxia and ischemia. Recently, using transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1), we reported that nucleoside transporter-mediated release of adenosine from neurons was not a key mechanism facilitating the actions of adenosine at A1 receptors during hypoxia/ischemia. The present study was performed to test the importance of CD73 (ecto-5′-nucleotidase) for basal and hypoxic/ischemic adenosine production. Hippocampal slice electrophysiology was performed with CD73+/+ and CD73−/− mice. Adenosine and ATP had similar inhibitory effects in both genotypes, with IC50 values of approximately 25 µM. In contrast, ATP was a less potent inhibitor (IC50 = 100 µM) in slices from mice expressing hENT1 in neurons. The inhibitory effects of ATP in CD73+/+ and CD73−/− slices were blocked by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and were enhanced by the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI), consistent with effects that are mediated by adenosine after metabolism of ATP. AMP showed a similar inhibitory effect to ATP and adenosine, indicating that the response to ATP was not mediated by P2 receptors. In comparing CD73−/− and CD73+/+ slices, hypoxia and oxygen-glucose deprivation produced similar depression of synaptic transmission in both genotypes. An inhibitor of tissue non-specific alkaline phosphatase (TNAP) was found to attenuate the inhibitory effects of AMP and ATP, increase basal synaptic activity and reduce responses to oxygen-glucose deprivation selectively in slices from CD73−/− mice. These results do not support an important role for CD73 in the formation of adenosine in the CA1 area of the hippocampus during basal, hypoxic or ischemic conditions, but instead point to TNAP as a potential source of extracellular adenosine when CD73 is absent.
PMCID: PMC3382561  PMID: 22761898
3.  The effect of combined glutamate receptor blockade in the NTS on the hypoxic ventilatory response in awake rats differs from the effect of individual glutamate receptor blockade 
Physiological Reports  2014;2(8):e12092.
Ventilatory acclimatization to hypoxia (VAH) increases the hypoxic ventilatory response (HVR) and causes persistent hyperventilation when normoxia is restored, which is consistent with the occurrence of synaptic plasticity in acclimatized animals. Recently, we demonstrated that antagonism of individual glutamate receptor types (GluRs) within the nucleus tractus solitarii (NTS) modifies this plasticity and VAH (J. Physiol. 592(8):1839–1856); however, the effects of combined GluR antagonism remain unknown in awake rats. To evaluate this, we exposed rats to room air or chronic sustained hypobaric hypoxia (CSH, PiO2 = 70 Torr) for 7–9 days. On the experimental day, we microinjected artificial cerebrospinal fluid (ACSF: sham) and then a “cocktail” of the GluR antagonists MK‐801 and DNQX into the NTS. The location of injection sites in the NTS was confirmed by glutamate injections on a day before the experiment and with histology following the experiment. Ventilation was measured in awake, unrestrained rats breathing normoxia or acute hypoxia (10% O2) in 15‐min intervals using barometric pressure plethysmography. In control (CON) rats, acute hypoxia increased ventilation; NTS microinjections of GluR antagonists, but not ACSF, significantly decreased ventilation and breathing frequency in acute hypoxia but not normoxia (P <0.05). CSH increased ventilation in hypoxia and acute normoxia. In CSH‐conditioned rats, GluR antagonists in the NTS significantly decreased ventilation in normoxia and breathing frequency in hypoxia. A persistent HVR after combined GluR blockade in the NTS contrasts with the effect of individual GluR blockade and also with results in anesthetized rats. Our findings support the hypotheses that GluRs in the NTS contribute to, but cannot completely explain, VAH in awake rats.
Ventilatory acclimatization to hypoxia involves plasticity in the central nervous system, as well as in arterial chemoreceptors. NMDA and AMPA glutamate receptors in the NTS contribute to different aspects of ventilatory acclimatization to hypoxia. However, total ionotropic glutamate receptor blockade in the NTS does not block acclimatization and the effects are not predictable from those of individual antagonists.
PMCID: PMC4246593  PMID: 25107985
AMPA receptors; hypercapnic ventilatory response; kainate receptors; NMDA receptors
4.  Hypothyroidism Attenuates SCH 23390-mediated Depression of Breathing and Decreases D1 Receptor Expression in Carotid Bodies, PVN and Striatum of Hamsters 
Brain research  2011;1401:40-51.
Hypothyroidism can lead to depressed breathing. We determined if propylthiouracil (PTU)–induced hypothyroidism in hamsters (HH) altered dopamine D1 receptor expression, D1 receptor-modulated ventilation, and ventilatory chemoreflex activation by hypoxia or hypercapnia. Hypothyroidism was induced by administering 0.04% PTU in drinking water for three months. Ventilation was evaluated following saline or 0.25 mg/kg SCH 23390, a D1 receptor antagonist, while awake hamsters breathed normoxic (21% O2 in N2), hypoxic (10% O2 in N2) and hypercapnic (5% CO2 in O2) air. Relative to euthyroid hamsters (EH), HH exhibited decreased D1 receptor protein levels in carotid bodies, striatum, and hypothalamic paraventricular nucleus, but not in the nucleus tractus solitarius. Relative to EH, HH exhibited lower ventilation during exposure to normoxia, hypoxia, or hypercapnia, but comparable ventilatory responsiveness to chemoreflex activation. SCH 23390 decreased ventilation of EH hamsters exposed to normoxia, hypoxia, and hypercapnia. In HH SCH 23390 increased ventilation during baseline normoxia and did not affect ventilation during exposure to hypoxia and hypercapnia, resulting in reduced ventilatory responsivess to chemoreflex activation by hypoxia and hypercapnia. Furthermore, in HH D1 receptor protein levels are decreased in several brain regions and within the carotid bodies. Moreover, D1 receptor-modulation of breathing at rest and during gas exposures were depressed in EH but not HH.
PMCID: PMC3124595  PMID: 21669406
hypothyroidism; carotid body PVN; NTS; hypoxia; hypercapnia; SCH 23390; propylthiouracil
5.  Hemodynamic and ventilatory response to different levels of hypoxia and hypercapnia in carotid body-denervated rats 
Clinics  2013;68(3):395-399.
Chemoreceptors play an important role in the autonomic modulation of circulatory and ventilatory responses to changes in arterial O2 and/or CO2. However, studies evaluating hemodynamic responses to hypoxia and hypercapnia in rats have shown inconsistent results. Our aim was to evaluate hemodynamic and respiratory responses to different levels of hypoxia and hypercapnia in conscious intact or carotid body-denervated rats.
Male Wistar rats were submitted to bilateral ligature of carotid body arteries (or sham-operation) and received catheters into the left femoral artery and vein. After two days, each animal was placed into a plethysmographic chamber and, after baseline measurements of respiratory parameters and arterial pressure, each animal was subjected to three levels of hypoxia (15, 10 and 6% O2) and hypercapnia (10% CO2).
The results indicated that 15% O2 decreased the mean arterial pressure and increased the heart rate (HR) in both intact (n = 8) and carotid body-denervated (n = 7) rats. In contrast, 10% O2 did not change the mean arterial pressure but still increased the HR in intact rats, and it decreased the mean arterial pressure and increased the heart rate in carotid body-denervated rats. Furthermore, 6% O2 increased the mean arterial pressure and decreased the HR in intact rats, but it decreased the mean arterial pressure and did not change the HR in carotid body-denervated rats. The 3 levels of hypoxia increased pulmonary ventilation in both groups, with attenuated responses in carotid body-denervated rats. Hypercapnia with 10% CO2 increased the mean arterial pressure and decreased HR similarly in both groups. Hypercapnia also increased pulmonary ventilation in both groups to the same extent.
This study demonstrates that the hemodynamic and ventilatory responses varied according to the level of hypoxia. Nevertheless, the hemodynamic and ventilatory responses to hypercapnia did not depend on the activation of the peripheral carotid chemoreceptors.
PMCID: PMC3611878  PMID: 23644862
Arterial Pressure; Heart Rate; Pulmonary Ventilation; Chemoreceptor Cells
6.  Respiratory plasticity after perinatal hyperoxia is not prevented by antioxidant supplementation 
Perinatal hyperoxia attenuates the hypoxic ventilatory response in rats by altering development of the carotid body and its chemoafferent neurons. In this study, we tested the hypothesis that hyperoxia elicits this maladaptive plasticity through the increased production of reactive oxygen species (ROS). Rats were born and raised in 60% O2 for the first two postnatal weeks while treated with one of two antioxidants: vitamin E (via milk from mothers whose diet was enriched with 1000 IU vitamin E kg−1) or a superoxide dismutase mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP; via daily intraperitoneal injection of 5–10 mg kg−1); rats were subsequently raised in room air until studied as adults. Peripheral chemoreflexes, assessed by carotid sinus nerve responses to cyanide, asphyxia, anoxia and isocapnic hypoxia (vitamin E experiments) or by hypoxic ventilatory responses (MnTMPyP experiments), were reduced after perinatal hyperoxia compared to those of normoxia-reared controls (all P<0.01); antioxidant treatment had no effect on these responses. Similarly, the carotid bodies of hyperoxia-reared rats were only one-third the volume of carotid bodies from normoxia-reared controls (P<0.001), regardless of antioxidant treatment. Protein carbonyl concentrations in the blood plasma, measured as an indicator of oxidative stress, were not increased in neonatal rats (2 and 8 days of age) exposed to 60% O2 from birth. Collectively, these data do not support the hypothesis that perinatal hyperoxia impairs peripheral chemoreceptor development through ROS-mediated oxygen toxicity.
PMCID: PMC2431464  PMID: 18069076
Hypertension  2010;56(1):151-158.
Recently we discovered that intact kidneys release into the extracellular compartment 2',3'-cAMP (a positional isomer of 3',5'-cAMP with unknown pharmacology) and metabolize 2',3'-cAMP to 2'-AMP, 3'-AMP and adenosine. Because adenosine inhibits growth of vascular smooth muscle cells and mesangial cells, we tested the hypothesis that extracellular 2',3'-cAMP attenuates growth of preglomerular vascular smooth muscle and mesangial cells via production of adenosine. For comparison, all experiments were performed with both 2',3'-cAMP and 3',5'-cAMP.
Study 1
2',3'-cAMP, 3',5'-cAMP, 5'-AMP, 3'-AMP or 2'-AMP were incubated with cells and purines measured in the medium by mass spectrometry. Both preglomerular vascular smooth muscle and mesangial cells metabolized 3',5'-cAMP to 5'-AMP and adenosine, 5'-AMP to adenosine, 2',3'-cAMP to 2'-AMP, 3'-AMP and adenosine and 2'-AMP and 3'-AMP to adenosine. 3-Isobutyl-1-methylxanthine (phosphodiesterase inhibitor) and 1,3-dipropyl-8-psulfophenylxanthine (ecto-phosphodiesterase inhibitor) blocked conversion of 3',5'-cAMP to 5'-AMP and adenosine and α,β-methylene-adenosine-5'-diphosphate (CD73 inhibitor) blocked conversion of 5'-AMP to adenosine. These enzyme inhibitors had little effect on metabolism of 2',3'-cAMP, 2'-AMP or 3'-AMP.
Study 2
2',3'-cAMP and 3',5'-cAMP profoundly inhibited proliferation (thymidine incorporation and cell number) of both cell types with 2',3'-cAMP more potent than 3',5'-cAMP. Antagonism of A2B receptors (MRS-1724), but not A1 (1,3-dipropyl-8-cyclopentylxanthine), A2A (SCH-58261) or A3 (VUF-5574) receptors, attenuated the growth inhibitory effects of 2',3'-cAMP and 3',5'-cAMP.
Extracellular 2',3'-cAMP inhibits growth of preglomerular vascular smooth muscle and mesangial cells more profoundly than does 3',5'-cAMP. Although both cAMPs inhibit growth in part via conversion to adenosine followed by A2B-receptor activation, their metabolism is mediated by different enzymes.
PMCID: PMC2892387  PMID: 20516392
2',3'-cAMP; 3',5'-cAMP; Adenosine; Adenosine Receptors; A2B Receptor; Vascular smooth muscle cells; glomerular mesangial cells
8.  Cocaine exposure modulates dopamine and adenosine signaling in the fetal brain 
Neuropharmacology  2009;58(2):436.
Exposure to cocaine during the fetal period can produce significant lasting changes in the structure and function of the brain. Cocaine exerts its effects on the developing brain by blocking monoamine transporters and impairing monoamine receptor signaling. Dopamine is a major central target of cocaine. In a mouse model, we show that cocaine exposure from embryonic day 8 (E8) to E14 produces significant reduction in dopamine transporter activity, attenuation of dopamine D1-receptor function and upregulation of dopamine D2-receptor function. Cocaine’s effects on the D1-receptor are at the level of protein expression as well as activity. The cocaine exposure also produces significant increases in basal cAMP levels in the striatum and cerebral cortex. The increase in the basal cAMP levels was independent of dopamine receptor activity. In contrast, blocking the adenosine A2a receptor downregulated of the basal cAMP levels in the cocaine-exposed brain to physiological levels, suggesting the involvement of adenosine receptors in mediating cocaine’s effects on the embryonic brain. In support of this suggestion, we found that the cocaine exposure downregulated adenosine transporter function. We also found that dopamine D2- and adenosine A2a-receptors antagonize each other’s function in the embryonic brain in a manner consistent with their interactions in the mature brain. Thus, our data show that prenatal cocaine exposure produces direct effects on both the dopamine and adenosine systems. Furthermore, the dopamine D2 and adenosine A2a receptor interactions in the embryonic brain discovered in this study unveil a novel substrate for cocaine’s effects on the developing brain.
PMCID: PMC2813374  PMID: 19765599
9.  Perinatal hyperoxia exposure impairs hypoxia-induced depolarization in rat carotid body glomus cells 
Chronic post-natal hyperoxia reduces the hypoxic ventilatory response by reducing the carotid body sensitivity to acute hypoxia as demonstrated by a reduced afferent nerve response, reduced calcium response of carotid body glomus cells and reduced catecholamine secretion in response to acute hypoxia. The present study examined whether hyperoxia alters the electrophysiological characteristics of glomus cells. Rats were treated with hyperoxia for 1 week starting at P1 or P7 and for 2 weeks starting at P1 followed by harvesting and dissociation of their carotid bodies for whole cell, perforated-patch recording. As compared to glomus cells from normoxia animals, hyperoxia treated cells showed a significant reduction in the magnitude of depolarization in response to hypoxia and anoxia, despite little change in the depolarizing response to 20 mM K+. Resting cell membrane potential in glomus cells from rats exposed to hyperoxia from P1 to P15 and studied at P15 was slightly depolarized compared to other treatment groups and normoxia-treated cells, but conductance normalized to cell size was not different among groups. We conclude that postnatal hyperoxia impairs carotid chemoreceptor hypoxia transduction at a step between hypoxia sensing and membrane depolarization. This occurs without a major change in baseline electrophysiological characteristics, suggesting altered signaling or alterations in the relative abundance of different leak channel isoforms.
PMCID: PMC4068117  PMID: 23669494
carotid body; chemoreceptor; potassium channel; oxygen sensing; development
10.  Sleep fragmentation attenuates the hypercapnic (but not hypoxic) ventilatory responses via adenosine A1 receptors in awake rats 
Sleep fragmentation (SF) and intermittent hypoxia and hypercapnia are the primary events associated with obstructive sleep apnea (OSA). We previously found that SF eliminates ventilatory long-term facilitation and attenuates poikilocapnic hypoxic ventilatory responses (HVR). This study examined the effect of SF on isocapnic HVR and hypercapnic ventilatory responses (HCVR), and investigated the time course of and the role of adenosine A1 receptors in these SF effects in conscious adult male Sprague-Dawley rats. SF was achieved by periodic, forced locomotion in a rotating drum (30 s rotation/90 s stop for 24 h). Ventilation during baseline, isocapnic hypoxia (11% O2 plus 4% CO2) and hypercapnia (6% CO2) was measured using plethysmography. About 1 h after 24 h SF, resting ventilation, arterial blood gases and isocapnic HVR (control: 169.3 ± 11.5% vs. SF: 170.0 ± 10.3% above baseline) were not significantly changed, but HCVR was attenuated (control: 172.8 ± 17.5% vs. SF: 129.5 ± 9.6%; P=0.003). This attenuated HCVR then returned spontaneously to the control level ~4 h after SF (168.9 ± 12.1%). This HCVR attenuation was also reversed (184.0 ± 17.5%) by systemic injection of the adenosine A1 receptor antagonist 8-CPT (2.5 mg/kg) shortly after SF, while 8-CPT at this dose had little effect on HCVR in control rats (169.9 ± 11.8%). Collectively, these results suggest that: (1) 24 h SF does not change isocapnic HVR but causes an attenuation of HCVR; and (2) this attenuation lasts for only a few hours and requires activation of adenosine A1 receptors.
PMCID: PMC3005083  PMID: 20833276
Respiratory control; Sleep fragmentation; Chemoresponsiveness; Hypercapnia; Adenosine receptors; Plasticity
11.  Adenosine A2B receptors induce proliferation, invasion and activation of cAMP response element binding protein (CREB) in trophoblast cells 
Placental hypoxia is a result of abnormal and shallow trophoblast invasion and involved in the pathophysiology of preeclampsia. Hypoxia increases extracellular adenosine levels and plays an important role in the regulation of angiogenesis, proliferation, vascular tone, endothelial permeability and inflammation. It was shown that adenosine concentrations are higher in preeclamptic patients. We tested the hypothesis that hypoxia and A2B adenosine receptor activation influence cyclic adenosine monophosphate (cAMP) production, proliferation, invasion and cAMP-PKA-CREB signaling in trophoblast cells (HTR-8/SVneo).
HTR-8/SVneo and human uterine microvascular endothelial cells (HUtMVEC) were used as model for experiments. We employed a cAMP assay, invasion assay, proliferation, RT-PCR and Western Blot. Statistical analyses were performed with ANOVA, Kruskal-Wallis-, Wilcoxon signed rank- or Mann–Whitney Test, as appropriate.
Hypoxia (2% O2) in comparison to normoxia (21% O2) led to increased A2B mRNA levels (1.21 ± 0.06 fold, 1 h 2% O2; 1.66 ± 0.2 fold, 4 h 2% O2 and 1.2 ± 0.04 fold, 24 h 2% O2). A2B adenosine receptor activation (NECA) stimulated trophoblast proliferation at 2% O2 (1.27 ± 0.06 fold) and 8% O2 (1.17 ± 0.07 fold) after 24 h and at 2% O2 (1.22 ± 0.05 fold), 8% O2 (1.23 ± 0.09 fold) and 21% O2 (1.15 ± 0.04 fold) after 48 h of incubation. Trophoblast invasion into an endothelial monolayer was significantly expanded by activation of the receptor (NECA) at 8% O2 (1.20 ± 0.07 fold) and 21% O2 (1.22 ± 0.006 fold). A2B adenosine receptor stimulation (NECA) additionally led to increased CREB phosphorylation in trophoblast cells at 2% O2 (2.13 ± 0.45 fold), 8% O2 (1.55 ± 0.13 fold) and 21% O2 (1.71 ± 0.34 fold). Blocking of CREB signaling resulted in reduced proliferation and CREB phosphorylation.
These data expand the recent knowledge regarding the role of adenosine receptor A2B in human placental development, and may provide insight in mechanisms associated with pregnancy complications linked to impaired trophoblast invasion such as preeclampsia.
PMCID: PMC3909477  PMID: 24383849
Adenosine receptors; Trophoblast cells (HTR-8/SVneo cells); Hypoxia; Proliferation; CREB
12.  Restoration of the cAMP second messenger pathway enhances cardiac preservation for transplantation in a heterotopic rat model. 
Journal of Clinical Investigation  1993;92(6):2994-3002.
Current organ preservation strategies subject graft vasculature to severe hypoxia (PO2 approximately 20 Torr), potentially compromising vascular function and limiting successful transplantation. Previous work has shown that cAMP modulates endothelial cell (EC) antithrombogenicity, barrier function, and leukocyte/EC interactions, and that hypoxia suppresses EC cAMP levels. To explore the possible benefits of cAMP analogs/agonists in organ preservation, we used a rat heterotopic cardiac transplant model; dibutyryl cAMP added to preservation solutions was associated with a time- and dose-dependent increase in the duration of cold storage associated with successful graft function. Preservation was also enhanced by 8-bromo-cAMP, the Sp isomer of adenosine 3',5'monophosphorothioate, and types III (indolidan) and IV (rolipram) phosphodiesterase inhibitors. Neither butyrate alone nor 8-bromoadenosine were effective, and the cAMP-dependent protein kinase antagonist Rp isomer of adenosine 3',5'monophosphorothioate prevented preservation enhancement induced by 8-bromo-cAMP. Grafts stored with dibutyryl cAMP demonstrated a 5.5-fold increase in blood flow and a 3.2-fold decreased neutrophil infiltration after transplantation. To explore the role of cAMP in another cell type critical for vascular homeostasis, vascular smooth muscle cells were subjected to hypoxia, causing a time-dependent decline in cAMP levels. Although adenylate cyclase activity was unchanged, diminished oxygen tensions were associated with enhanced phosphodiesterase activity (59 and 30% increase in soluble types III and IV activity, respectively). These data suggest that hypoxia or graft ischemia disrupt vascular homeostasis, at least in part, by perturbing the cAMP second messenger pathway. Supplementation of this pathway provides a new approach for enhancing cardiac preservation, promoting myocardial function, and maintaining vascular homeostatic properties.
PMCID: PMC288503  PMID: 8254053
13.  Dopamine and Full-Field Illumination Activate D1 and D2–D5-Type Receptors in Adult Rat Retinal Ganglion Cells 
The Journal of comparative neurology  2012;520(17):4032-4049.
Dopamine can regulate signal generation and transmission by activating multiple receptors and signaling cascades, especially in striatum, hippocampus, and cerebral cortex. Dopamine modulates an even larger variety of cellular properties in retina, yet has been reported to do so by only D1 receptor-driven cyclic adenosine monophosphate (cAMP) increases or D2 receptor-driven cAMP decreases. Here, we test the possibility that dopamine operates differently on retinal ganglion cells, because the ganglion cell layer binds D1 and D2 receptor ligands, and displays changes in signaling components other than cAMP under illumination that should release dopamine. In adult rat retinal ganglion cells, based on patch-clamp recordings, Ca2+ imaging, and immunohistochemistry, we find that 1) spike firing is inhibited by dopamine and SKF 83959 (an agonist that does not activate homomeric D1 receptors or alter cAMP levels in other systems); 2) D1 and D2 receptor antagonists (SCH 23390, eticlopride, raclopride) counteract these effects; 3) these antagonists also block light-induced rises in cAMP, light-induced activation of Ca2+/calmodulin-dependent protein kinase II, and dopamine-induced Ca2+ influx; and 4) the Ca2+ rise is markedly reduced by removing extracellular Ca2+ and by an IP3 receptor antagonist (2-APB). These results provide the first evidence that dopamine activates a receptor in adult mammalian retinal neurons that is distinct from classical D1 and D2 receptors, and that dopamine can activate mechanisms in addition to cAMP and cAMP-dependent protein kinase to modulate retinal ganglion cell excitability.
PMCID: PMC3538137  PMID: 22678972
retina; immunohistochemistry; dopamine; cAMP; CaMKII; Ca2+; excitability
14.  Recovery of carotid body O2 sensitivity following chronic postnatal hyperoxia in rats 
Chronic postnatal hyperoxia blunts the hypoxic ventilatory response (HVR) in rats, an effect that persists for months after return to normoxia. To determine whether decreased carotid body O2 sensitivity contributes to this lasting impairment, single-unit chemoafferent nerve and glomus cell calcium responses to hypoxia were recorded from rats reared in 60% O2 through 7 d of age (P7) and then returned to normoxia. Single-unit nerve responses were attenuated by P4 and remained low through P7. After return to normoxia, hypoxic responses were partially recovered within 3 d and fully recovered within 7–8 d (i.e., at P14-15). Glomus cell calcium responses recovered with a similar time course. Hyperoxia altered carotid body mRNA for O2-sensitive K+ channels TASK-1, TASK-3, and BKCa, but only TASK-1 mRNA paralleled changes in chemosensitivity (i.e., downregulation by P7, partial recovery by P14). Collectively, these data do not support a role for reduced O2 sensitivity of individual chemoreceptor cells in long-lasting reduction of the HVR after developmental hyperoxia.
PMCID: PMC3081780  PMID: 21420511
control of breathing; developmental plasticity; chemoreceptor; glomus cell; mRNA expression; K+ channel
15.  Cellular signalling pathways mediating dilation of porcine pial arterioles to adenosine A2A receptor activation 
Cardiovascular Research  2013;99(1):156-163.
Adenosine is a potent vasodilator contributing to cerebral blood flow regulation during metabolic stress. However, the distribution of adenosine receptor subtypes and underlying signalling mechanisms for dilation of pial arterioles remain unclear. The present study aimed at addressing these issues.
Methods and results
Isolated porcine pial arterioles were subjected to study of vasomotor function, localization of adenosine receptors, and production of nitric oxide (NO). Concentration-dependent vasodilation to adenosine was inhibited by A2A receptor antagonist ZM241385 but not by A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. A2A receptors were detected in endothelium and smooth muscle of pial arterioles via immunohistochemistry. Adenosine significantly increased arteriolar production of NO, and the induced dilation was insensitive to KATP channel blocker glibenclamide but was attenuated by endothelial denudation, NO synthase inhibitor l-NAME, or guanylyl cyclase inhibitor ODQ in a similar manner. Both inward rectifier potassium (Kir) channel inhibitor barium and cAMP signalling inhibitor Rp-8-Br-cAMPS attenuated adenosine-induced dilation. In the presence of l-NAME or the absence of endothelium, addition of Rp-8-Br-cAMPS but not barium further reduced adenosine-induced responses. Barium diminished endothelium-independent vasodilation to NO donor sodium nitroprusside. Comparable to the adenosine-induced response, vasodilation to A2A receptor agonist CGS21680 was attenuated by endothelial removal, ZM241385, l-NAME, barium, or Rp-8-Br-cAMPS, but not by glibenclamide.
Adenosine evokes dilation of porcine pial arterioles via parallel activation of endothelial and smooth muscle A2A receptors. Stimulation of endothelial NO production activates smooth muscle guanylyl cyclase for vasodilation by opening Kir channels. Adenosine also activates smooth muscle cAMP signalling leading to vasodilation.
PMCID: PMC3687749  PMID: 23539502
Adenosine; Microcirculation; Nitric oxide; Potassium channels; Vasodilation
16.  Receptor crosstalk: haloperidol treatment enhances A2A adenosine receptor functioning in a transfected cell model 
Purinergic Signalling  2010;6(4):373-381.
A2A adenosine receptors are considered an excellent target for drug development in several neurological and psychiatric disorders. It is noteworthy that the responses evoked by A2A adenosine receptors are regulated by D2 dopamine receptor ligands. These two receptors are co-expressed at the level of the basal ganglia and interact to form functional heterodimers. In this context, possible changes in A2A adenosine receptor functional responses caused by the chronic blockade/activation of D2 dopamine receptors should be considered to optimise the therapeutic effectiveness of dopaminergic agents and to reduce any possible side effects. In the present paper, we investigated the regulation of A2A adenosine receptors induced by antipsychotic drugs, commonly acting as D2 dopamine receptor antagonists, in a cellular model co-expressing both A2A and D2 receptors. Our data suggest that the treatment of cells with the classical antipsychotic haloperidol increased both the affinity and responsiveness of the A2A receptor and also affected the degree of A2A–D2 receptor heterodimerisation. In contrast, an atypical antipsychotic, clozapine, had no effect on A2A adenosine receptor parameters, suggesting that the two classes of drugs have different effects on adenosine–dopamine receptor interaction. Modifications to A2A adenosine receptors may play a significant role in determining cerebral adenosine effects during the chronic administration of antipsychotics in psychiatric diseases and may account for the efficacy of A2A adenosine receptor ligands in pathologies associated with dopaminergic system dysfunction.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-010-9201-z) contains supplementary material, which is available to authorized users.
PMCID: PMC3033501  PMID: 21437008
A2A adenosine receptors; D2 dopamine receptor antagonists; A2A binding parameters; Receptor functioning; Receptor crosstalk
Neuroscience  2012;207:103-109.
We tested the hypothesis that antagonism of progesterone receptor (PR) in newborn rats alters carotid body and respiratory responses to hypoxia and nicotinic receptor agonists. Rats were treated with the PR antagonist mifepristone (daily oral gavage 40 μg/g/d) or vehicle between post-natal days 3 and 15. In 11–14-day-old rats, we used in vitro carotid body/carotid sinus nerve preparation and whole body plethysmography to assess the carotid body and ventilatory responses to hypoxia (65 mmHg in vitro, 10% O2 in vivo) and to nicotinic receptor agonists (as an excitatory modulator of carotid body activity—nicotine 100 μM for in vitro studies, and epibatidine 5 μg/kg, i.p., which mainly acts on peripheral nicotinic receptors, for in vivo studies). The carotid body responses to hypoxia and nicotine were drastically reduced by mifepristone. Compared with vehicle, mifepristone-treated rats had a reduced body weight. The ventilatory response to epibatidine was attenuated; however, the hypoxic ventilatory response was similar between vehicle and mifepristone-treated pups. Immunohistochemical staining revealed that mifepristone treatment did not change carotid body morphology. We conclude that PR activity is a critical factor ensuring proper carotid body function in newborn rats.
PMCID: PMC3782486  PMID: 22326965 CAMSID: cams3224
carotid sinus nerve; whole-body plethysmography; hypoxia; nicotine receptor agonist; progesterone receptor antagonist; newborn
18.  Regulation of photoreceptor gap junction phosphorylation by adenosine in zebrafish retina 
Visual neuroscience  2014;31(3):237-243.
Electrical coupling of photoreceptors through gap junctions suppresses voltage noise, routes rod signals into cone pathways, expands the dynamic range of rod photoreceptors in high scotopic and mesopic illumination, and improves detection of contrast and small stimuli. In essentially all vertebrates, connexin 35/36 (gene homologues Cx36 in mammals, Cx35 in other vertebrates) is the major gap junction protein observed in photoreceptors, mediating rod-cone, cone-cone, and possibly rod-rod communication. Photoreceptor coupling is dynamically controlled by the day/night cycle and light/dark adaptation, and is directly correlated with phosphorylation of Cx35/36 at two sites, serine110 and serine 276/293 (homologous sites in teleost fish and mammals respectively). Activity of protein kinase A (PKA) plays a key role during this process. Previous studies have shown that activation of dopamine D4 receptors on photoreceptors inhibits adenylyl cyclase, down-regulates cAMP and PKA activity, and leads to photoreceptor uncoupling, imposing the daytime/light condition. In this study we explored the role of adenosine, a nighttime signal with a high extracellular concentration at night and a low concentration in the day, in regulating photoreceptor coupling by examining photoreceptor Cx35 phosphorylation in zebrafish retina. Adenosine enhanced photoreceptor Cx35 phosphorylation in daytime, but with a complex dose-response curve. Selective pharmacological manipulations revealed that adenosine A2a receptors provide a potent positive drive to phosphorylate photoreceptor Cx35 under the influence of endogenous adenosine at night. A2a receptors can be activated in the daytime as well by micromolar exogenous adenosine. However, the higher affinity adenosine A1 receptors are also present and have an antagonistic though less potent effect. Thus the nighttime/darkness signal adenosine provides a net positive drive on Cx35 phosphorylation at night, working in opposition to dopamine to regulate photoreceptor coupling via a push-pull mechanism. However, the lower concentration of adenosine present in the daytime actually reinforces the dopamine signal through action on the A1 receptor.
PMCID: PMC4109651  PMID: 24844306
adenosine; A2a receptor; A1 receptor; Cx36; photoreceptor
Hypertension  2010;55(5):1123-1128.
In the kidney, defects in the regulation of urine salt excretion can result in extracellular fluid volume expansion, leading to salt-sensitive hypertension. Previous studies have demonstrated that when rats are maintained on a high sodium chloride (NaCl) diet, adenosine production increases in the renal medulla with parallel changes in adenosine receptor expression. These studies suggest that adenosine signaling in the kidney can respond to high NaCl loading; however, the functional consequences of these changes in adenosine signaling are not clear.
We used the immortalized cell line mIMCD-K2, a murine model system for the renal inner medullary collecting duct (IMCD), to study the direct effects of adenosine on NaCl transport across IMCD epithelium with an Ussing chamber system. When epithelial Na+ channels were inhibited, addition of adenosine to the apical side of mIMCD-K2 cell sheets stimulated short-circuit current (Isc) in a dose-dependent manner. This increase in Isc was inhibited by a CFTR Cl− channel inhibitor. Pharmacological studies with a panel of adenosine receptor agonists and antagonists demonstrated that adenosine activates apical A2b adenosine receptors to enhance Isc. Furthermore, adenosine application to mIMCD-K2 cell sheets increased intracellular cAMP, while inhibition of protein kinase A (PKA) completely blocked the adenosine response. Together, our findings indicate that adenosine stimulates Cl− secretion through CFTR in mIMCD-K2 cells by activating apical A2b receptors and signaling through cAMP/PKA. We propose that this adenosine receptor pathway may provide one mechanism for enhancing urine NaCl excretion in the setting of high dietary NaCl intake.
PMCID: PMC2885947  PMID: 20308611
adenosine; CFTR; A2b receptor; renal medulla; collecting duct
20.  The Role of Muscarinic Receptors in the Beneficial Effects of Adenosine against Myocardial Reperfusion Injury in Rats 
PLoS ONE  2011;6(11):e25618.
Adenosine, a catabolite of ATP, displays a wide variety of effects in the heart including regulation of cardiac response to myocardial ischemia and reperfusion injury. Nonetheless, the precise mechanism of adenosine-induced cardioprotection is still elusive. Isolated Sprague-Dawley rat hearts underwent 30 min global ischemia and 120 min reperfusion using a Langendorff apparatus. Both adenosine and acetylcholine treatment recovered the post-reperfusion cardiac function associated with adenosine and muscarinic receptors activation. Simultaneous administration of adenosine and acetylcholine failed to exert any additive protective effect, suggesting a shared mechanism between the two. Our data further revealed a cross-talk between the adenosine and acetylcholine receptor signaling in reperfused rat hearts. Interestingly, the selective M2 muscarinic acetylcholine receptor antagonist methoctramine significantly attenuated the cardioprotective effect of adenosine. In addition, treatment with adenosine upregulated the expression and the maximal binding capacity of muscarinic acetylcholine receptor, which were inhibited by the selective A1 adenosine receptor antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) and the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME). These data suggested a possible functional coupling between the adenosine and muscarinic receptors behind the observed cardioprotection. Furthermore, nitric oxide was found involved in triggering the response to each of the two receptor agonist. In summary, there may be a cross-talk between the adenosine and muscarinic receptors in ischemic/reperfused myocardium with nitric oxide synthase might serve as the distal converging point. In addition, adenosine contributes to the invigorating effect of adenosine on muscarinic receptor thereby prompting to regulation of cardiac function. These findings argue for a potentially novel mechanism behind the adenosine-mediated cardioprotection.
PMCID: PMC3208538  PMID: 22073139
21.  Activation of Adenosine Receptor on Gingival Fibroblasts 
Journal of dental research  2006;85(8):739-744.
CD73 (ecto-5′-nucleotidase) on human gingival fibroblasts plays a role in the regulation of intracellular cAMP levels through the generation of adenosine, which subsequently activates adenosine receptors. In this study, we examined the involvement of ecto-adenosine deaminase, which can be anchored to CD26 on human gingival fibroblasts, in metabolizing adenosine generated by CD73, and thus attenuating adenosine receptor activation. Ecto-adenosine deaminase expression on fibroblasts could be increased by pre-treatment with a lysate of Jurkat cells, a cell line rich in cytoplasmic adenosine deaminase. Interestingly, the cAMP response to adenosine generated from 5′-AMP via CD73 and the ability of 5′-AMP to induce hyaluronan synthase 1 mRNA were significantly decreased by the pre-treatment of fibroblasts with Jurkat cell lysate. This inhibitory effect was reversed by the specific adenosine deaminase inhibitor. These results suggest that ecto-adenosine deaminase metabolizes CD73-generated adenosine and regulates adenosine receptor activation.
PMCID: PMC2225592  PMID: 16861292
adenosine receptor; CD73; ecto-adenosine deaminase
22.  Evaluation of neuronal phosphoproteins as effectors of caffeine and mediators of striatal adenosine A2A receptor signaling 
Brain research  2006;1129(1):1-14.
Adenosine A2A receptors are predominantly expressed in the dendrites of enkephalin-positive γ-aminobutyric acidergic medium spiny neurons in the striatum. Evidence indicates that these receptors modulate striatal dopaminergic neurotransmission and regulate motor control, vigilance, alertness, and arousal. Although the physiological and behavioral correlates of adenosine A2A receptor signaling have been extensively studied using a combination of pharmacological and genetic tools, relatively little is known about the signal transduction pathways that mediate the diverse biological functions attributed to this adenosine receptor subtype. Using a candidate approach based on the coupling of these receptors to adenylate cyclase-activating G proteins, a number of membranal, cytosolic, and nuclear phosphoproteins regulated by PKA were evaluated as potential mediators of adenosine A2A receptor signaling in the striatum. Specifically, the adenosine A2A receptor agonist, CGS 21680, was used to determine whether the phosphorylation state of each of the following PKA targets is responsive to adenosine A2A receptor stimulation in this tissue: Ser40 of tyrosine hydroxylase, Ser9 of synapsin, Ser897 of the NR1 subunit of the N-methyl-D-aspartate-type glutamate receptor, Ser845 of the GluR1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor, Ser94 of spinophilin, Thr34 of the dopamine- and cAMP-regulated phosphoprotein, Mr32,000, Ser133 of the cAMP-response element-binding protein, Thr286 of Ca2+/calmodulin-dependent protein kinase II, and Thr202/Tyr204 and Thr183/Tyr185 of the p44 and p42 isoforms, respectively, of mitogen-activated protein kinase. Although the substrates studied differed considerably in their responsiveness to selective adenosine A2A receptor activation, the phosphorylation state of all postsynaptic PKA targets was up-regulated in a time- and dose-dependent manner by treatment with CGS 21680, whereas presynaptic PKA substrates were unresponsive to this agent, consistent with the postsynaptic localization of adenosine A2A receptors. Finally, the phosphorylation state of these proteins was further assessed in vivo by systemic administration of caffeine.
Section: Cellular and Molecular Biology of Nervous Systems
PMCID: PMC1847645  PMID: 17157277
Caffeine; Striatum; Adenosine A2A receptor PKA; MAPK; Signal transduction
23.  Ethanol Blocks Adenosine Uptake via Inhibiting the Nucleoside Transport System in Bronchial Epithelial Cells 
Adenosine uptake into cells by nucleoside transporters plays a significant role in governing extracellular adenosine concentration. Extracellular adenosine is an important signaling molecule that modulates many cellular functions via four G-protein-coupled receptor subtypes (A1, A2A, A2B, and A3). Previously, we demonstrated that adenosine is critical in maintaining airway homeostasis and airway repair and that airway host defenses are impaired by alcohol. Taken together, we hypothesized that ethanol impairs adenosine uptake via the nucleoside transport system. To examine ethanol-induced alteration on adenosine transport, we used a human bronchial epithelial cell line (BEAS-2B). Cells were preincubated for 10 min in the presence and absence of varying concentrations of ethanol (EtOH). In addition, some cells were pretreated with S - (4-Nitrobenzyl)-6-thioinosine (100 μM: NBT), a potent adenosine uptake inhibitor. Uptake was then determined by addition of [3H]-adenosine at various time intervals. Increasing EtOH concentrations resulted in increasing inhibition of adenosine uptake when measured at 1 min. Cells pretreated with NBT effectively blocked adenosine uptake. In addition, short-term EtOH revealed increased extracellular adenosine concentration. Conversely, adenosine transport became desensitized in cells exposed to EtOH (100 mM) for 24 hr. To determine the mechanism of EtOH-induced desensitization of adenosine transport, cAMP activity was assessed in response to EtOH. Short-term EtOH exposure (10 min) had little or no effect on adenosine-mediated cAMP activation, whereas long-term EtOH exposure (24 hr) blocked adenosine-mediated cAMP activation. Western blot analysis of lysates from unstimulated BEAS-2B cells detected a single 55 kDa band indicating the presence of hENT1 and hENT2, respectively. Real-time RT-PCR of RNA from BEAS-2B revealed transcriptional expression of ENT1 and ENT2. Collectively, these data reveal that acute exposure of cells to EtOH inhibits adenosine uptake via a nucleoside transporter, and chronic exposure of cells to EtOH desensitizes the adenosine transporter to these inhibitory effects of ethanol. Furthermore, our data suggest that inhibition of adenosine uptake by EtOH leads to an increased extracellular adenosine accumulation, influencing the effect of adenosine at the epithelial cell surface, which may alter airway homeostasis.
PMCID: PMC2940831  PMID: 19298329
ethanol; adenosine; airway diseases; nucleoside transporter
24.  Hypoxia and glucose independently regulate the beta-adrenergic receptor-adenylate cyclase system in cardiac myocytes. 
Journal of Clinical Investigation  1991;88(1):204-213.
We explored the effects of two components of ischemia, hypoxia and glucose deprivation, on the beta-adrenergic receptor (beta AR)-adenylate cyclase system in a model of hypoxic injury in cultured neonatal rat ventricular myocytes. After 2 h of hypoxia in the presence of 5 mM glucose, cell surface beta AR density (3H-CGP-12177) decreased from 54.8 +/- 8.4 to 39 +/- 6.3 (SE) fmol/mg protein (n = 10, P less than 0.025), while cytosolic beta AR density (125I-iodocyanopindolol [ICYP]) increased by 74% (n = 5, P less than 0.05). Upon reexposure to oxygen cell surface beta AR density returned toward control levels. Cells exposed to hypoxia and reoxygenation without glucose exhibited similar alterations in beta AR density. In hypoxic cells incubated with 5 mM glucose, the addition of 1 microM (-)-norepinephrine (NE) increased cAMP generation from 29.3 +/- 10.6 to 54.2 +/- 16.1 pmol/35 mm plate (n = 5, P less than 0.025); upon reoxygenation cAMP levels remained elevated above control (n = 5, P less than 0.05). In contrast, NE-stimulated cAMP content in glucose-deprived hypoxic myocytes fell by 31% (n = 5, P less than 0.05) and did not return to control levels with reoxygenation. beta AR-agonist affinity assessed by (-)-isoproterenol displacement curves was unaltered after 2 h of hypoxia irrespective of glucose content. Addition of forskolin (100 microM) to glucose-supplemented hypoxic cells increased cAMP generation by 60% (n = 5; P less than 0.05), but in the absence of glucose this effect was not seen. In cells incubated in glucose-containing medium, the decline in intracellular ATP levels was attenuated after 2 h of hypoxia (21 vs. 40%, P less than 0.05). Similarly, glucose supplementation prevented LDH release in hypoxic myocytes. We conclude that (a) oxygen and glucose independently regulate beta AR density and agonist-stimulated cAMP accumulation; (b) hypoxia has no effect on beta AR-agonist or antagonist affinity; (c) 5 mM glucose attenuates the rate of decline in cellular ATP levels during both hypoxia and reoxygenation; and (d) glucose prevents hypoxia-induced LDH release, a marker of cell injury.
PMCID: PMC296021  PMID: 1647415
25.  Plasticity of Central Chemoreceptors: Effect of Bilateral Carotid Body Resection on Central CO2 Sensitivity 
PLoS Medicine  2007;4(7):e239.
Human breathing is regulated by feedback and feed-forward control mechanisms, allowing a strict matching between metabolic needs and the uptake of oxygen in the lungs. The most important control mechanism, the metabolic ventilatory control system, is fine-tuned by two sets of chemoreceptors, the peripheral chemoreceptors in the carotid bodies (located in the bifurcation of the common carotid arteries) and the central CO2 chemoreceptors in the ventral medulla. Animal data indicate that resection of the carotid bodies results, apart from the loss of the peripheral chemoreceptors, in reduced activity of the central CO2 sensors. We assessed the acute and chronic effect of carotid body resection in three humans who underwent bilateral carotid body resection (bCBR) after developing carotid body tumors.
Methods and Findings
The three patients (two men, one woman) were suffering from a hereditary form of carotid body tumors. They were studied prior to surgery and at regular intervals for 2–4 y following bCBR. We obtained inspired minute ventilation (Vi) responses to hypoxia and CO2. The Vi-CO2 responses were separated into a peripheral (fast) response and a central (slow) response with a two-compartment model of the ventilatory control system. Following surgery the ventilatory CO2 sensitivity of the peripheral chemoreceptors and the hypoxic responses were not different from zero or below 10% of preoperative values. The ventilatory CO2 sensitivity of the central chemoreceptors decreased by about 75% after surgery, with peak reduction occurring between 3 and 6 mo postoperatively. This was followed by a slow return to values close to preoperative values within 2 y. During this slow return, the Vi-CO2 response shifted slowly to the right by about 8 mm Hg.
The reduction in central Vi-CO2 sensitivity after the loss of the carotid bodies suggests that the carotid bodies exert a tonic drive or tonic facilitation on the output of the central chemoreceptors that is lost upon their resection. The observed return of the central CO2 sensitivity is clear evidence for central plasticity within the ventilatory control system. Our data, although of limited sample size, indicate that the response mechanisms of the ventilatory control system are not static but depend on afferent input and exhibit a large degree of restoration or plasticity. In addition, the permanent absence of the breathing response to hypoxia after bCBR may aggravate the pathological consequences of sleep-disordered breathing.
Bilateral carotid body resection in three individuals led to reduced sensitivity of central chemoreceptors to CO2, followed by a gradual return, providing evidence of central plasticity within the ventilatory control system.
PMCID: PMC1925127  PMID: 17676946

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