Adenosine A1 Receptor Knockout Research Articles

Ethanol Differentially Affects Excitatory and Inhibitory Synaptic Transmission in Visual Cortex of Wild-type and Adenosine A1R Knock-out Mice

Ethanol is one of the most commonly used and abused substances in the world. While the behavioral effects of ethanol are well characterized, mechanisms of its action on neurons and synapses remain elusive. Prior research suggested that ethanol could affect neurons by interfering with metabolism of biologically active molecules, such as adenosine. Here, we explored the involvement of adenosine A1 receptors (A1R) in mediating ethanol’s effects on synaptic transmission to layer 2/3 pyramidal neurons of visual cortex using wild type (WT) and A1R knock-out (KO) mice. Ethanol differentially affected excitatory and inhibitory transmission in WT and KO mice. In slices from WT mice ethanol had heterogeneous effects on excitatory transmission (facilitation, suppression or no change), with no net change. Ethanol’s effects remained heterogeneous during acute blockade of A1Rs with a selective antagonist DPCPX. However, in A1RKO mice ethanol consistently suppressed excitatory transmission, with no cases of enhancement observed. Inhibitory transmission was suppressed by ethanol in both WT and A1RKO mice. At both excitatory and inhibitory synapses, changes of response amplitude correlated with changes of paired-pulse ratio, suggesting involvement of presynaptic mechanisms. We conclude that A1Rs are not involved in mediating effects of ethanol on synaptic transmission in mouse visual cortex. However, A1Rs are necessary for development of mechanisms mediating facilitation at some excitatory synapses. Our results add evidence for the diversity of ethanol’s effects and mechanisms of action on synaptic transmission in different brain structures, and even in the same brain area (visual cortex) in different species, rats vs mice.

Caffeine Impaired Acupuncture Analgesia in Inflammatory Pain by Blocking Adenosine A1 Receptor

Caffeine consumption inhibits acupuncture analgesic effects by blocking adenosine signaling. However, existing evidence remains controversial. Hence, this study aimed to examine the adenosine A1 receptor (A1R) role in moderate-dose caffeine-induced abolishing effect on acupuncture analgesia using A1R knockout mice (A1R−/−). We assessed the role of A1R in physiological sensory perception and its interaction with caffeine by measuring mechanical and thermal pain thresholds and administering A1R and adenosine 2A receptor antagonists in wild-type (WT) and A1R−/− mice. Formalin- and complete Freund's adjuvant (CFA)-induced inflammatory pain models were recruited to explore moderate-dose caffeine effect on pain perception and acupuncture analgesia in WT and A1R−/− mice. Moreover, a C-fiber reflex electromyogram in the biceps femoris was conducted to validate the role of A1R in the caffeine-induced blockade of acupuncture analgesia. We found that A1R was dispensable for physiological sensory perception and formalin- and CFA-induced hypersensitivity. However, genetic deletion of A1R impaired the antinociceptive effect of acupuncture in A1R−/− mice under physiological or inflammatory pain conditions. Acute moderate-dose caffeine administration induced mechanical and thermal hyperalgesia under physiological conditions but not in formalin- and CFA-induced inflammatory pain. Moreover, caffeine significantly inhibited electroacupuncture (EA) analgesia in physiological and inflammatory pain in WT mice, comparable to that of A1R antagonists. Conversely, A1R deletion impaired the EA analgesic effect and decreased the caffeine-induced inhibitory effect on EA analgesia in physiological conditions and inflammatory pain. Moderate-dose caffeine administration diminished the EA-induced antinociceptive effect by blocking A1R. Overall, our study suggested that caffeine consumption should be avoided during acupuncture treatment. PerspectiveModerate-dose caffeine injection attenuated EA-induced antinociceptive effect in formalin- and CFA-induced inflammatory pain mice models by blocking A1R. This highlights the importance of monitoring caffeine intake during acupuncture treatment.

Altered Heterosynaptic Plasticity Impairs Visual Discrimination Learning in Adenosine A1 Receptor Knock-Out Mice.

Theoretical and modeling studies demonstrate that heterosynaptic plasticity-changes at synapses inactive during induction-facilitates fine-grained discriminative learning in Hebbian-type systems, and helps to achieve a robust ability for repetitive learning. A dearth of tools for selective manipulation has hindered experimental analysis of the proposed role of heterosynaptic plasticity in behavior. Here we circumvent this obstacle by testing specific predictions about the behavioral consequences of the impairment of heterosynaptic plasticity by experimental manipulations to adenosine A1 receptors (A1Rs). Our prior work demonstrated that the blockade of adenosine A1 receptors impairs heterosynaptic plasticity in brain slices and, when implemented in computer models, selectively impairs repetitive learning on sequential tasks. Based on this work, we predict that A1R knock-out (KO) mice will express (1) impairment of heterosynaptic plasticity and (2) behavioral deficits in learning on sequential tasks. Using electrophysiological experiments in slices and behavioral testing of animals of both sexes, we show that, compared with wild-type controls, A1R KO mice have impaired synaptic plasticity in visual cortex neurons, coupled with significant deficits in visual discrimination learning. Deficits in A1R knockouts were seen specifically during relearning, becoming progressively more apparent with learning on sequential visual discrimination tasks of increasing complexity. These behavioral results confirm our model predictions and provide the first experimental evidence for a proposed role of heterosynaptic plasticity in organism-level learning. Moreover, these results identify heterosynaptic plasticity as a new potential target for interventions that may help to enhance new learning on a background of existing memories.SIGNIFICANCE STATEMENT Understanding how interacting forms of synaptic plasticity mediate learning is fundamental for neuroscience. Theory and modeling revealed that, in addition to Hebbian-type associative plasticity, heterosynaptic changes at synapses that were not active during induction are necessary for stable system operation and fine-grained discrimination learning. However, lacking tools for selective manipulation prevented behavioral analysis of heterosynaptic plasticity. Here we circumvent this barrier: from our prior experimental and computational work we predict differential behavioral consequences of the impairment of Hebbian-type versus heterosynaptic plasticity. We show that, in adenosine A1 receptor knock-out mice, impaired synaptic plasticity in visual cortex neurons is coupled with specific deficits in learning sequential, increasingly complex visual discrimination tasks. This provides the first evidence linking heterosynaptic plasticity to organism-level learning.

Chaihu plus Longgu Muli Decoction Alleviated Brain Injury in Pentylenetetrazole-Kindled Epileptic Mice by Regulating Cyclooxygenase-2/Prostaglandin E2/Multidrug Transporter Pathway

Objective. To evaluate the effect of CLMD administration on epileptic seizures and brain injury in pentylenetetrazole- (PZT-) kindled mice. Methods. The effect of pretreatment with CLMD (5, 10, and 20 ml/kg (mg/kg) by gavage) for seven days on PTZ-induced kindling, duration and grade of kindling-induced seizures, and pathological injury in the cortex and hippocampus was evaluated. Male BALB/c mice with adenosine A1 receptor knockout were subjected to intraperitoneal injection of PTZ (35 mg/kg) once every day until kindling was successfully induced. Quantitative reverse transcription polymerase chain reaction, immunofluorescence, and western blot were performed to assess the mRNA and protein levels of p-glycoprotein (PGP), multidrug resistance-associated protein 1 (MRP1), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and adenylate kinase (ADK) in the cortex and hippocampus. Results. PTZ successfully induced kindling in mice after 21 days, wherein CLMD showed an obvious dose-dependent antiepileptic effect. High-dose CLMD significantly increased the latency of epileptic seizures, decreased the sustained time of epileptic seizures and the seizure grade, and ameliorated the histopathological changes in the cortex and hippocampus. Furthermore, PTZ kindling induced significantly higher levels of PGP, MRP1, COX-2, PGE2, and ADK, but this effect was inhibited by pretreatment with CLMD in a dose-dependent manner. Conclusion. Pretreatment with CLMD attenuates PTZ-kindled convulsions and brain injury in mice. The mechanism may be related to the cyclooxygenase-2/prostaglandin E2/multidrug transporter pathway.

Podocyte Density and Albuminuria in Aging Diabetic Ins2± Mice with or Without Adenosine A1 Receptor Signaling.

Aim of StudyTo investigate podocyte density in aging diabetic Ins2± and Ins2±, A1AR-/- mouse models in C57Bl/6 background.MethodsIns2± mice and especially Ins2±, adenosine A1 receptor knockout mice (Ins2±, A1AR-/-) are mouse models with a phenotype of diabetic nephropathy. Aged mice (at ~40 weeks) were assessed for glomerular filtration barrier function by measuring albuminuria, glomerular filtration, glomerular damage by electron microscopy, and podocyte numbers by Wilms Tumor protein (WT-1) staining.ResultsCompared to healthy wild-type mice, both diabetic mouse models developed diabetic nephropathy, including hyperfiltration (p<0.01) and albuminuria (p<0.05). Typical diabetic structural glomerular and podocyte damage was visualized by electron microscopy. Podocyte count per glomerular area (podocyte density) was significantly decreased in both diabetic mouse models (p<0.01). In contrast, no significant correlation was detected between albuminuria and absolute podocyte count per glomerulus.ConclusionThe amount of albuminuria as marker of diabetic nephropathy does not correlate with the podocytes density; however, a relative podocyte deficiency became evident with an increase in glomerular area in the diabetic animals, suggesting a relative podocytopenia.

The effect of A1 adenosine receptor in diabetic megalin loss with caspase-1/IL18 signaling.

PurposeIn our previous study, exacerbation of albuminuria was observed in A1 adenosine receptor knockout (A1AR−/-) mice with diabetic nephropathy (DN), but the mechanism was unclear. Here, we investigated the relationship of megalin loss and albuminuria, to identify the protective effect of A1AR in megalin loss associated albuminuria by inhibiting pyroptosis-related caspase-1/IL-18 signaling of DN.MethodsWe successfully collected DN patients' samples and built diabetes mice models induced by streptozotocin. Megalin, cubilin, and A1AR expression were detected in kidney tissue samples from DN patients and mice through immunohistochemical and immunofluorescent staining. A1AR, caspase-1, interleukin-18 (IL-18) expression were analyzed using Western blotting in wild-type and A1AR−/- mice. Human renal proximal tubular epithelial cells (PTC) were cultured with high glucose to observe the effect of A1AR agonist and antagonist on caspase-1/IL-18 and megalin injury.ResultsThe loss of megalin, co-localized with A1AR at PTC, was associated with the level of albuminuria in diabetic patients and mice. The injury of megalin-cubilin was accompanied with the A1AR upregulation (1.30±0.1 vs 0.98±0.2, P=0.042), the caspase-1 (1.33±0.1 vs 1.0±0.2, P=0.036), and IL-18 (1.26±0.2 vs 0.96±0.2, P=0.026) signaling activation in mice with DN. More severe pathological injury, 24 hrs urine albumin excretion (170.8±4.1 μg/d vs 132.0±2.9 μg/d vs 17.9±2.8 μg/d, P<0.001) and megalin-cubilin loss were observed in A1AR−/- DN mice with more pronounced caspase-1 (1.52±0.03 vs 1.20±0.01, P=0.017) and IL-18 (1.42±0.02 vs 1.21±0.02, P=0.018) secretion. High glucose could stimulate the secretion of caspase-1 (1.72 times, P≤0.01) and IL-18 (1.64 times, P≤0.01), which was abolished by A1AR agonist and aggravated by A1AR antagonist.ConclusionA1AR played a protective role in proximal tubular megalin loss associated albuminuria by inhibiting the pyroptosis-related caspase-1/IL-18 signaling in DN.

Adenosine A1 receptor activates background potassium channels and modulates information processing in olfactory bulb mitral cells

Adenosine is a widespread neuromodulator in the mammalian brain, but whether it affects information processing in sensory system(s) remains largely unknown. Here we show that adenosine A1 receptors hyperpolarize mitral cells, one class of principal neurons that propagate odour information from the olfactory bulb to higher brain areas, by activation of background K+ channels. The adenosine-modulated background K+ channels belong to the family of two-pore domain K+ channels. Adenosine reduces spontaneous activity of mitral cells, whereas action potential firing evoked by synaptic input upon stimulation of sensory neurons is not affected, resulting in a higher ratio of evoked firing (signal) over spontaneous firing (noise) and hence an improved signal-to-noise ratio. The study shows for the first time that adenosine influences fine-tuning of the input-output relationship in sensory systems. Neuromodulation by adenosine is of critical importance in many brain regions, but the role of adenosine in olfactory information processing has not been studied so far. We investigated the effects of adenosine on mitral cells, which are projection neurons of the olfactory bulb. Significant expression of A1 and A2A receptors was found in mitral cells, as demonstrated by in situ hybridization. Application of adenosine in acute olfactory bulb slices hyperpolarized mitral cells in wild-type but not in adenosine A1 receptor knockout mice. Adenosine-induced hyperpolarization was mediated by background K+ currents that were reduced by halothane and bupivacaine, which are known to inhibit two-pore domain K+ (K2P) channels. In mitral cells, electrical stimulation of axons of olfactory sensory neurons evoked synaptic currents, which can be considered as input signals, while spontaneous firing independent of sensory input can be considered as noise. Synaptic currents were not affected by adenosine, while adenosine reduced spontaneous firing, leading to an increase in the signal-to-noise ratio of mitral cell firing. Our findings demonstrate that A1 adenosine receptors activate two-pore domain K+ channels, which increases the signal-to-noise ratio of the input-output relationship in mitral cells and thereby modulates information processing in the olfactory bulb.

Isoflurane anesthesia in aged mice and effects of A1 adenosine receptors on cognitive impairment.

Isoflurane may not only accelerate the process of Alzheimer's disease (AD), but increase the risk of incidence of postoperative cognitive dysfunction (POCD). However, the underlying mechanisms remain unknown. This study was designed to investigate whether isoflurane contributed to the POCD occurrence through A1 adenosine receptor (A1AR) in aged mice. We assessed cognitive function of mice with Morris water maze (MWM) and then measured expression level of two AD biomarkers (P-tau and Aβ) and a subtype of the NMDA receptor (NR2B) in aged wild-type (WT) and homozygous A1 adenosine receptor (A1AR) knockout (KO) mice at baseline and after they were exposed to isoflurane (1.4% for 2hours). For cognitive test, WT mice with isoflurane exposure performed worse than the WT mice without isoflurane exposure. However, A1AR KO mice with isoflurane exposure performed better than WT mice with isoflurane exposure. WT mice exposed to isoflurane had increased levels of Aβ and phosphorylated tau (P-tau). Levels of Aβ and P-tau were decreased in A1AR KO mice, whereas no differences were noted between KO mice with and without isoflurane exposure. NR2B expression was inversely related to that of P-tau, with no differences found between KO mice with and without isoflurane exposure. We found an association between isoflurane exposure, impairment of spatial memory, decreasing level of NR2B, and increasing levels of A-beta and P-tau, presumably via the activation of the A1A receptor.

Adenosine A1 Receptors Play an Important Protective Role Against Cognitive Impairment and Long-Term Potentiation Inhibition in a Pentylenetetrazol Mouse Model of Epilepsy.

Epilepsy is a complicated neurological disorder that occurs worldwide and features several kinds of comorbidities in addition to recurrent seizures. One of the most common comorbidities is cognitive impairment, which seriously affects patients' quality of life. Through activating pre- and postsynaptic adenosine A1 receptors (A1Rs), adenosine has demonstrated anticonvulsant and neuroprotective effects in many epileptic animal models. However, whether the neuroprotective effect of A1Rs will protect cognition during epileptogenesis remains unknown. Therefore, by using A1R knockout (KO) mice and establishing a pentylenetetrazole (PTZ)-kindled model of epilepsy, the present study investigated A1Rs' influences on memory and synaptic function. Morris water maze test results indicated that A1R knockout exacerbated the memory impairment induced by PTZ kindling compared with the wild-type group. To further study the synaptic function of epileptic A1Rs KO mice, we recorded long-term potentiation (LTP) in the hippocampal CA3-CA1 pathway, and LTP was highly inhibited in kindled A1R KO mice compared with kindled wild-type mice. To reveal the mechanisms underlying these effects, neuronal loss, cell apoptosis, and relevant synaptic protein levels in hippocampus were assessed. Epileptic A1R KO mice exhibited significant reductions in neuronal cell survival in the CA1 region and a marked increase in the activation of caspase-3 in the hippocampus compared with epileptic wild-type mice. In addition, an obvious decrease in the PSD95 and BDNF expression levels of epileptic A1R KO mice was observed 7days after complete kindling. In conclusion, these findings indicated that A1Rs play an important protective role against cognitive impairment by reducing neuron loss and increasing BDNF and PSD95 levels. Activation of A1Rs during epileptogenesis might be beneficial to the preservation of epileptic individuals' cognitive functions.

Adenosine A1 Receptors Selectively Modulate Oxygen-Induced Retinopathy at the Hyperoxic and Hypoxic Phases by Distinct Cellular Mechanisms.

We critically evaluated the role of the adenosine A1 receptor (A1R) in normal development of retinal vasculature and pathogenesis of retinopathy of prematurity (ROP) by using the A1R knockout (KO) mice and oxygen-induced retinopathy (OIR) model. Mice deficient in A1Rs and their wild-type (WT) littermates were examined during normal postnatal development or after being subjected to 75% oxygen from postnatal day (P) 7 to P12 and to room air from P12 to P17 (OIR model of ROP). Retinal vascularization was examined by whole-mount fluorescence and cross-sectional hematoxylin-eosin staining. Cellular proliferation, astrocyte and microglial activation, and tip cell function were determined by isolectin staining and immunohistochemistry. Apoptosis was determined by TUNEL assay. Genetic deletion of the A1R did not affect normal retinal vascularization during postnatal development with indistinguishable three-layer vascularization patterns in retina between WT and A1R KO mice. In the OIR model, genetic deletion of the A1R resulted in stage-specific effects: reduced hyperoxia-induced retinal vaso-obliteration at P12, but reduced avascular area and attenuated hypoxia-induced intraretinal revascularization without affecting intravitreal neovascularization at P17 and reduced avascular areas in retina at P21. These distinct effects of A1Rs on OIR were associated with A1R control of apoptosis mainly in inner and outer nuclear layers at the vaso-obliterative phase (P12) and the growth of endothelium tip cells at the vasoproliferative phase (P17), without modification of cellular proliferation, astrocytic activation, and tissue inflammation. Adenosine A1 receptor activity is not required for normal postnatal development of retinal vasculature but selectively controls hyperoxia-induced vaso-obliteration and hypoxia-driven revascularization by distinct cellular mechanisms.

The in vivo respiratory phenotype of the adenosine A1 receptor knockout mouse

The nucleoside adenosine has been implicated in the regulation of respiration, especially during hypoxia in the newborn. In this study the role of adenosine A1 receptors for the control of respiration was investigated in vivo. To this end, respiration of unrestrained adult and neonatal adenosine A1 receptor knockout mice (A1R−/−) was measured in a plethysmographic device.Under control conditions (21% O2) and mild hypoxia (12–15% O2) no difference of respiratory parameters was observed between adult wildtype (A1R+/+) and A1R−/− mice. Under more severe hypoxia (6–10% O2) A1R+/+ mice showed, after a transient increase of respiration, a decrease of respiration frequency (fR) and tidal volume (VT) leading to a decrease of minute volume (MV). This depression of respiration during severe hypoxia was absent in A1R−/− mice which displayed a stimulated respiration as indicated by the enhancement of MV by some 50–60%. During hypercapnia–hyperoxia (3–10% CO2/97–90 % O2), no obvious differences in respiration of A1R−/− and A1R+/+ was observed. In neonatal mice, the respiratory response to hypoxia was surprisingly similar in both genotypes. However, neonatal A1R−/− mice appeared to have more frequently periods of apnea during hypoxia and in the post-hypoxic control period.In conclusion, these data indicate that the adenosine A1 receptor is an important molecular component mediating hypoxic depression in adult mice and it appears to stabilize respiration of neonatal mice.

Paeoniflorin Promotes Non-rapid Eye Movement Sleep via Adenosine A1 Receptors.

Paeoniflorin (PF, C23H28O11), one of the principal active ingredients of Paeonia Radix, exerts depressant effects on the central nervous system. We determined whether PF could modulate sleep behaviors and the mechanisms involved. Electroencephalogram and electromyogram recordings in mice showed that intraperitoneal PF administered at a dose of 25 or 50 mg/kg significantly shortened the sleep latency and increased the amount of non-rapid eye movement (NREM). Immunohistochemical study revealed that PF decreased c-fos expression in the histaminergic tuberomammillary nucleus (TMN). The sleep-promoting effects and changes in c-fos induced by PF were reversed by 8-cyclopentyl-1,3-dimethylxanthine (CPT), an adenosine A1 receptor antagonist, and PF-induced sleep was not observed in adenosine A1 receptor knockout mice. Whole-cell patch clamping in mouse brain slices showed that PF significantly decreased the firing frequency of histaminergic neurons in TMN, which could be completely blocked by CPT. These results indicate that PF increased NREM sleep by inhibiting the histaminergic system via A1 receptors.

Increased Signaling via Adenosine A1 Receptors, Sleep Deprivation, Imipramine, and Ketamine Inhibit Depressive-like Behavior via Induction of Homer1a

Major depressive disorder is among the most commonly diagnosed disabling mental diseases. Several non-pharmacological treatments of depression upregulate adenosine concentration and/or adenosine A1 receptors (A1R) in the brain. To test whether enhanced A1R signaling mediates antidepressant effects, we generated a transgenic mouse with enhanced doxycycline-regulated A1R expression, specifically in forebrain neurons. Upregulating A1R led to pronounced acute and chronic resiliencetoward depressive-like behavior in various tests. Conversely, A1R knockout mice displayed an increased depressive-like behavior and were resistant to the antidepressant effects of sleep deprivation (SD). Various antidepressant treatments increase homer1a expression in medial prefrontal cortex (mPFC). Specific siRNA knockdown of homer1a in mPFC enhanced depressive-like behavior and prevented the antidepressant effects of A1R upregulation, SD, imipramine, and ketamine treatment. In contrast, viral overexpression of homer1a in the mPFC had antidepressant effects. Thus, increased expression of homer1a is a final common pathway mediating the antidepressant effects of different antidepressant treatments.

Adenosine A1 receptor-dependent antinociception induced by inosine in mice: pharmacological, genetic and biochemical aspects.

Inosine is an endogenous nucleoside that has anti-inflammatory and antinociceptive properties. Inosine is a metabolite of adenosine, and some of its actions suggest the involvement of adenosine A1 receptors (A1Rs). The purpose of this study was to better understand mechanisms of inosine-induced antinociception by investigating the role of A1Rs and purine metabolism inhibitors. Inosine antinociception was evaluated using the formalin test in mice. An A1R-selective antagonist (DPCPX), A1R knockout mice (gene deletion) and mice with A1R reduced expression (antisense oligonucleotides) were used to assess the role of A1Rs in the antinociceptive action of inosine. Binding assays were performed to compare the affinity of inosine and adenosine for A1Rs. Finally, the role of adenosine and inosine breakdown was assessed using deoxycoformycin (DCF) and forodesine (FDS) as enzymatic inhibitors of adenosine deaminase and purine nucleoside phosphorylase, respectively. Inosine induced antinociception in the formalin test when given by systemic, spinal and peripheral routes. Systemically, inosine exhibited a potency similar to adenosine, and its effects were inhibited by DPCPX. Inosine did not induce antinociception in A1R knockout mice or in mice with reduced A1R expression. In binding studies, inosine bound to A1Rs with an affinity similar to adenosine. DCF had no effect on inosine actions. FDS augmented the antinociceptive effect of a low systemic dose of inosine and, at a higher dose, induced antinociception by itself. Collectively, these data indicate that inosine is an agonist for A1Rs with antinociceptive properties and a potency similar to adenosine and can be considered another endogenous ligand for this receptor.

Adenosine inhibits renin release from juxtaglomerular cells via an A1 receptor-TRPC-mediated pathway

Renin is synthesized and released from juxtaglomerular (JG) cells. Adenosine inhibits renin release via an adenosine A1 receptor (A1R) calcium-mediated pathway. How this occurs is unknown. In cardiomyocytes, adenosine increases intracellular calcium via transient receptor potential canonical (TRPC) channels. We hypothesized that adenosine inhibits renin release via A1R activation, opening TRPC channels. However, higher concentrations of adenosine may stimulate renin release through A2R activation. Using primary cultures of isolated mouse JG cells, immunolabeling demonstrated renin and A1R in JG cells, but not A2R subtypes, although RT-PCR indicated the presence of mRNA of both A2AR and A2BR. Incubating JG cells with increasing concentrations of adenosine decreased renin release. Different concentrations of the adenosine receptor agonist N-ethylcarboxamide adenosine (NECA) did not change renin. Activating A1R with 0.5 μM N6-cyclohexyladenosine (CHA) decreased basal renin release from 0.22 ± 0.05 to 0.14 ± 0.03 μg of angiotensin I generated per milliliter of sample per hour of incubation (AngI/ml/mg prot) (P < 0.03), and higher concentrations also inhibited renin. Reducing extracellular calcium with EGTA increased renin release (0.35 ± 0.08 μg AngI/ml/mg prot; P < 0.01), and blocked renin inhibition by CHA (0.28 ± 0.06 μg AngI/ml/mg prot; P < 0. 005 vs. CHA alone). The intracellular calcium chelator BAPTA-AM increased renin release by 55%, and blocked the inhibitory effect of CHA. Repeating these experiments in JG cells from A1R knockout mice using CHA or NECA demonstrated no effect on renin release. However, RT-PCR showed mRNA from TRPC isoforms 3 and 6 in isolated JG cells. Adding the TRPC blocker SKF-96365 reversed CHA-mediated inhibition of renin release. Thus A1R activation results in a calcium-dependent inhibition of renin release via TRPC-mediated calcium entry, but A2 receptors do not regulate renin release.

Sex differences and the effects of ovariectomy on the β-adrenergic contractile response

The presence of sex differences in myocardial β-adrenergic responsiveness is controversial, and limited studies have addressed the mechanism underlying these differences. Studies were performed using isolated perfused hearts from male, intact female and ovariectomized female mice to investigate sex differences and the effects of ovarian hormone withdrawal on β-adrenergic receptor function. Female hearts exhibited blunted contractile responses to the β-adrenergic receptor agonist isoproterenol (ISO) compared with males but not ovariectomized females. There were no sex differences in β(1)-adrenergic receptor gene or protein expression. To investigate the role of adenylyl cyclase, phosphodiesterase, and the cAMP-signaling cascade in generating sex differences in the β-adrenergic contractile response, dose-response studies were performed in isolated perfused male and female hearts using forskolin, 3-isobutyl-1-methylxanthine (IBMX), and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Males showed a modestly enhanced contractile response to forskolin at 300 nM and 5 μM compared with females, but there were no sex differences in the response to IBMX or CPT-cAMP. The role of the A(1) adenosine receptor (A(1)AR) in antagonizing the β-adrenergic contractile response was investigated using both the A(1)AR agonist 2-chloro-N(6)-cyclopentyl-adenosine and A(1)AR knockout (KO) mice. Intact females showed an enhanced A(1)AR anti-adrenergic effect compared with males and ovariectomized females. The β-adrenergic contractile response was potentiated in both male and female A(1)ARKO hearts, with sex differences no longer present above 1 nM ISO. The β-adrenergic contractile response is greater in male hearts than females, and minor differences in the action of adenylyl cyclase or the A(1)AR may contribute to these sex differences.

High-protein-induced glomerular hyperfiltration is independent of the tubuloglomerular feedback mechanism and nitric oxide synthases

A high protein intake is associated with increased glomerular filtration rate (GFR), which has been suggested to be mediated by reduced signaling of the tubuloglomerular feedback (TGF) mechanism. Nitric oxide (NO) has been shown to contribute to high protein-induced glomerular hyperfiltration, but the specific NO synthase (NOS) isoform responsible is not clear. In this study, a model for high-protein-induced hyperfiltration in conscious mice was developed. Using this model, we investigated the role of TGF using adenosine A(1)-receptor knockout mice lacking the TGF mechanism. Furthermore, the role of the different NOS isoforms was studied using neuronal-, inducible-, and endothelial-NOS knockout mice, and furthermore, wild-type mice acutely administered with the unspecific NOS inhibitor N(ω)-nitro-l-arginine methyl ester (100 mg/kg). GFR was measured consecutively in mice given a low-protein diet (8% casein) for 10 days, followed by a high-protein diet (50% casein) for 10 days. All mice developed high protein-induced hyperfiltration to a similar degree. These results demonstrate that high protein-induced glomerular hyperfiltration is independent of the TGF mechanism and NOS isoforms.

The brain injury after pentetrazole kindling in adenosine A1 receptor knock-out mice

Objective To evaluate the neuroprotective function of adenosine A1 receptor ( A1R)by observing the dynamic change of brain injury after pentetrazole kindling in adenosine A1R knock-out (KO) mice. Methods The animals were divided into three groups: wild type group, KO group and control group. For each animal, 4 parts of the brain (cortex, cerebellum, hippocampus and brainstem) were chosen. The latent period of kindling was calculated. The dynamic change of the ultrastructure in neurocytes during pentetrazole kindling was examined by a transmission electron microscope at different time points (2 h, 7 day, 1 month). Results The average latent period in KO group was (2. 38 ± 1.66) days,showing statistically significant difference in comparison with the wild type group (P <0. 01 ). Morphologically, there was widespread brain injury in KO mice at the 2nd h after kindling, especially in the mitochondria. Over time, the injury was gradually aggravated. However, in wild type group, the mitochondrial injury mainly in hippocampus and temporal lobe was found at the 7th day after kindling. At the 1st month, the injury still focused on temporal lobe. Conclusion Adenosine A1R acts strong neuroprotective function.This receptor can reduce the damage of the ultrastructures during pentetrazole kindling. Key words: Adenosine A1 receptor; Ultrastructure; Mitochondrion injury; Neuroprotective function

Myocardial adenosine A1-receptor-mediated adenoprotection involves phospholipase C, PKC-ε, and p38 MAPK, but not HSP27

Adenosine via an adenosine A(1) receptor (A(1)R) is a negative feedback inhibitor of adrenergic stimulation in the heart, protecting it from toxic effects of overstimulation. Stimulation of the A(1)R results in the activation of G(i) protein, release of free Gbetagamma-subunits, and activation/translocation of PKC-epsilon to the receptor for activated C kinase 2 protein at the Z-line of the cardiomyocyte sarcomere. Using an anti-Gbetagamma peptide, we investigated the role of these subunits in the A(1)R stimulation of phospholipase C (PLC), with the premise that the resulting diacylglycerol provides for the activation of PKC-epsilon. Inositol 1,4,5-triphosphate release was an index of PLC activity. Chlorocyclopentyl adenosine (CCPA), an A(1)R agonist, increased inositol 1,4,5-triphosphate production by 273% in mouse heart homogenates, an effect absent in A(1)R knockout hearts and inhibited by anti-Gbetagamma peptide. In a second study, p38 MAPK and heat shock protein 27 (HSP27), found by others to be associated with the loss of myocardial contractile function, were postulated to play a role in the actions of A(1)R. Isoproterenol, a beta-adrenergic receptor agonist, increased the Ca(2+) transient and sarcomere shortening magnitudes by 36 and 49%, respectively. In the rat cardiomyocyte, CCPA significantly reduced these increases, an action blocked by the p38 MAPK inhibitor SB-203580. While CCPA significantly increased the phosphorylation of HSP27, this action was inhibited by isoproterenol. These data indicate that the activation of PKC-epsilon by A(1)R results from the activation of PLC via free Gbetagamma-subunits released upon A(1)R-induced dissociation of G(i)alphabetagamma. Attenuation of beta-adrenergic-induced contractile function by A(1)R may involve the activation of p38 MAPK, but not HSP27.

Intracellular acidification causes adenosine release during states of hyperexcitability in the hippocampus.

Decreased pH increases extracellular adenosine in CNS regions as diverse as hippocampus and ventral medulla. However, thus far there is no clear consensus whether the critical pH change is a decrease in intracellular and/or extracellular pH. Previously we showed that a decrease in extracellular pH is necessary and a decrease in intracellular pH alone is not sufficient, to increase extracellular adenosine in an acute hippocampal slice preparation. Here we explored further the role of intracellular pH under different synaptic conditions in the hippocampal slice. When synaptic excitability was increased, either during gamma-aminobutyric acid type A receptor blockade in CA1 or after the induction of persistent bursting in CA3, a decrease in intracellular pH alone was now sufficient to: 1) elevate extracellular adenosine concentration, 2) activate adenosine A1 receptors, 3) decrease excitatory synaptic transmission (CA1), and 4) attenuate burst frequency in an in vitro seizure model (CA3). Hippocampal slices obtained from adenosine A1 receptor knockout mice did not exhibit these pH-mediated effects on synaptic transmission, further confirming the role of adenosine acting at the adenosine A1 receptor. Taken together, these data strengthen and add significantly to the evidence outlining a change in pH as an important stimulus influencing extracellular adenosine. In addition, we identify conditions under which intracellular pH plays a dominant role in regulating extracellular adenosine concentrations.