Apnoeic response to stimulation of peripheral GABA receptors in rats

Respiratory effects of intracarotid injection of gamma-amino-butyric acid (GABA) were investigated in two groups of rats. In the first group of 12 rats the effects of GABA were checked in the intact state, following bilateral vagotomy and GABA receptor blockade. The second group consisted of five initially vagotomized rats, challenged with GABA prior to and after bilateral carotid chemodenervation (CSN-cut). All rats were urethane and chloralose anaesthetized and spontaneously breathing. Injection of 39 micromol/kg GABA prior to and after vagotomy induced an expiratory apnoea of, respectively 5.5+/-0.84 sec and 3.9+/-0.6 sec duration (mean+/-S.E.M.), P>0.05 in all 12 rats. In breaths that followed the apnoea tidal volume increased above the control level by 23.3% (P<0.01) and 25.6% (P<0.01) pre- and post-vagotomy, respectively. Blockade of GABA receptors with bicuculline and picrotoxin abolished the inhibition of breathing. In five vagotomized rats with intact carotid sinus nerves (CSNs) intracarotid GABA challenge increased tidal volume by 39% compared with baseline breathing (P<0.05). Section of the CSNs precluded the occurrence of apnoea and undergoing respiratory changes evoked by GABA. Intracarotid GABA caused significant decrease in the mean blood pressure independent of the neural state, but the fall was delayed by CSNs neurotomy. Results of this study indicate that GABA given systemically induces apnoea followed by post-apnoeic hyperventilation. Carotid bodies are required for the ventilatory response to GABA; vagal afferents are not involved in this response.     

P2 receptors modulate respiratory rhythm but do not contribute to central CO2 sensitivity in vitro

Multiple brainstem sites are proposed to contribute to central respiratory chemosensitivity, however, the underlying molecular mechanisms remain unknown. P2X2 subunit-containing ATP receptors, which mediate pH-sensitive currents, appear to contribute to central chemosensitivity in vivo [J. Physiol. 523 (2000) 441]. However, recent data from P2X2 knockout mice [J. Neurosci. 23 (2003) 11315] indicate that they are not essential. To further explore the role of P2 receptors in central chemosensitivity, we examined the effects of P2 receptor agonists/antagonists on respiratory-related activity and CO2-sensitivity of rhythmically-active in vitro preparations from neonatal rat. Our main findings: (i) that putative chemosensitive regions of the ventrolateral medulla are immunoreactive for the P2X2 subunit; (ii) that ATP potentiates respiratory frequency in a dose-dependent, and PPADS-sensitive (P2 receptor antagonist), manner; and (iii) that the increase in burst frequency produced by increasing CO2 is unaffected by PPADS, indicate that ATP is a potent modulator of respiratory activity, but that P2 receptors do not contribute to central chemosensitivity in vitro.     

Adenosine 2A receptors in acute kidney injury

Acute kidney injury (AKI) is an important clinical problem that may lead to death and for those who survive, the sequelae of AKI include loss of quality of life, chronic kidney disease and end‐stage renal disease. The incidence of AKI continues to rise without clear successes in humans for the pharmacological prevention of AKI or treatment of established AKI. Dendritic cells and macrophages are critical early initiators of innate immunity in the kidney and orchestrate inflammation subsequent to ischaemia–reperfusion injury. These innate cells are the most abundant leucocytes present in the kidney, and they represent a heterogeneous population of cells that are capable of responding to cues from the microenvironment derived from pathogens or endogenous inflammatory mediators such as cytokines or anti‐inflammatory mediators such as adenosine. Lymphocyte subsets such as natural killer T cells and Tregs also play roles in regulating ischaemic injury by promoting and suppressing inflammation respectively. Adenosine, produced in response to IR, is generally considered as a protective signalling molecule and elicits its physiological responses through four distinct adenosine receptors. However, its short half‐life, lack of specificity and rapid metabolism limit the use of adenosine as a therapeutic agent. These adenosine receptors play various roles in regulating the activity of the aforementioned hematopoietic cells in elevated levels of adenosine such as during hypoxia. This review focuses on the importance of one receptor, the adenosine 2A subtype, in blocking inflammation associated with AKI.     

Respiratory responses to intermittent hypoxia in unsedated piglets: relation to substance P binding in brainstem

Respiratory responses to single intermittent hypoxia (5 min 21% O(2), 5 min 8% O(2) X6) in 5-6, 10-11, 21-22 and 26-27 day-old piglets, and to recurrent six daily intermittent hypoxia in 10-11 and 26-27 day-old piglets were assessed. Substance P binding in the piglets' brainstem immediately after the last hypoxic episode was measured. All piglets hyperventilated during hypoxia. Weight adjusted inspired ventilation, tidal volume and instantaneous flow decreased with age. The oldest piglets uniquely displayed attenuated ventilation and tidal volume during the sixth versus first hypoxic episode with single intermittent hypoxia, and reduced inspired ventilation and tidal volume during the first hypoxic episode on the sixth daily hypoxia compared to single hypoxia. By contrast, substance P binding was greatly reduced in the solitary, hypoglossal, paraambigual and lateral reticular brainstem nuclei of both younger and older piglets following either single or recurrent intermittent hypoxia. Thus, the reduction in membrane-bound neurokinin receptors by intermittent hypoxia, presumably consequent to endogenously released substance P, does not exclusively determine whether the ventilatory response to that hypoxia will be attenuated or not.     

Muscarinic receptors and alpha2-adrenoceptors interact to modulate the respiratory rhythm in mouse neonates

The respiratory rhythm generator (RRG) is modulated by several endogenous substances, including acetylcholine (ACh) and noradrenaline (NA) that interact in several modulatory processes. To know whether ACh and NA interacted to modulate the RRG activity, we used medullary "en bloc" and slice preparations from neonatal mice where the RRG has been shown to receive a facilitatory modulation from A1/C1 neurons, via a continuous release of endogenous NA and activation of alpha2 adrenoceptors. Applying ACh at 25 microM activated the RRG but ACh had no effects at 50 microM. Applying the ACh receptor agonists nicotine and muscarine facilitated and depressed the RRG, respectively. After yohimbine pre-treatment that blocked the alpha2 facilitation, the nicotinic facilitation was not altered, the muscarinic depression was reversed and ACh 50 microM significantly facilitated the RRG. After L-tyrosine pre-treatment that potentiated the alpha2 facilitation, the muscarinic depression was enhanced. Thus, ACh regulates the RRG activity via nicotinic and muscarinic receptors, the muscarinic receptors interacting with alpha2 adrenoceptors.     

Endogenous 5-HT(1/2) systems and the newborn rat respiratory control. A comparative in vivo and in vitro study

Consequences of 5-HT(1/2) systems blockade by methysergide on newborn rats respiratory drive were evaluated in vivo with unrestrained animals and in vitro using brainstem-spinal cord preparations. A decrease in respiratory frequency until a plateau level was observed under both in vivo (82.8 +/- 0.6% of control values) and in vitro (76.8 +/- 0.8% of control values) conditions whereas an increase in inspiratory amplitude (135.1 +/- 2.1% of control values) was only retrieved in vivo. By the use of the c-fos expression analysis, we correlated these effects with neuronal activity changes, particularly, in vivo in two key structures between the respiratory ponto-medullary network and the peripheral or suprapontine afferences, namely the commissural subnucleus of the nucleus of the solitary tract and the lateral parabrachial nucleus. Thus, peripheral and suprapontine inputs seem to be of a primeval importance in the respiratory influence of endogenous 5-HT. Besides, as 5-HT is involved in the respiratory perturbations that occur in sudden infant death syndrome (SIDS), our results suggest a participation of peripheral and suprapontine inputs in these disorders.     

Adenosine A2A receptors in ventral striatum, hypothalamus and nociceptive circuitry implications for drug addiction, sleep and pain

Adenosine A2A receptors localized in the dorsal striatum are considered as a new target for the development of antiparkinsonian drugs. Co-administration of A2A receptor antagonists has shown a significant improvement of the effects of l-DOPA. The present review emphasizes the possible application of A2A receptor antagonists in pathological conditions other than parkinsonism, including drug addiction, sleep disorders and pain. In addition to the dorsal striatum, the ventral striatum (nucleus accumbens) contains a high density of A2A receptors, which presynaptically and postsynaptically regulate glutamatergic transmission in the cortical glutamatergic projections to the nucleus accumbens. It is currently believed that molecular adaptations of the cortico-accumbens glutamatergic synapses are involved in compulsive drug seeking and relapse. Here we review recent experimental evidence suggesting that A2A antagonists could become new therapeutic agents for drug addiction. Morphological and functional studies have identified lower levels of A2A receptors in brain areas other than the striatum, such as the ventrolateral preoptic area of the hypothalamus, where adenosine plays an important role in sleep regulation. Although initially believed to be mostly dependent on A1 receptors, here we review recent studies that demonstrate that the somnogenic effects of adenosine are largely mediated by hypothalamic A2A receptors. A2A)receptor antagonists could therefore be considered as a possible treatment for narcolepsy and other sleep-related disorders. Finally, nociception is another adenosine-regulated neural function previously thought to mostly involve A1 receptors. Although there is some conflicting literature on the effects of agonists and antagonists, which may partly be due to the lack of selectivity of available drugs, the studies in A2A receptor knockout mice suggest that A2A receptor antagonists might have some therapeutic potential in pain states, in particular where high intensity stimuli are prevalent.     

Adenosine A2A receptors and brain injury: broad spectrum of neuroprotection, multifaceted actions and "fine tuning" modulation

This review summarizes recent developments that have contributed to understand how adenosine receptors, particularly A2A receptors, modulate brain injury in various animal models of neurological disorders, including Parkinson's disease (PD), stroke, Huntington's disease (HD), multiple sclerosis, Alzheimer's disease (AD) and HIV-associated dementia. It is clear that extracellular adenosine acting at adenosine receptors influences the functional outcome in a broad spectrum of brain injuries, indicating that A2A Rs may modulate some general cellular processes to affect neuronal cells death. Pharmacological, neurochemical and molecular/genetic approaches to the complex actions of A2A receptors in different cellular elements suggest that A2A receptor activation can be detrimental or protective after brain insults, depending on the nature of brain injury and associated pathological conditions. An interesting concept that emerges from these studies is A2A R's ability to fine tune neuronal and glial functions to produce neuroprotective effects. While the data presented here clearly highlight the complexity of using adenosinergic agents therapeutically in PD and other neurodegenerative disorders and point out many areas for further inquiry, they also confirm that adenosine receptor ligands, particularly A2A receptor ligands, have many promising characteristics that encourage the pursuit of their therapeutic potential.     

Role of adenosine A2A receptors in parkinsonian motor impairment and l-DOPA-induced motor complications

Adenosine A2A receptors have a unique cellular and regional distribution in the basal ganglia, being particularly concentrated in areas richly innervated by dopamine such as the caudate-putamen and the globus pallidus. Adenosine A2A receptors are selectively located on striatopallidal neurons and are capable of forming functional heteromeric complexes with dopamine D2 and metabotropic glutamate mGlu5 receptors. Based on the unique cellular and regional distribution of this receptor and in line with data showing that A2A receptor antagonists improve motor symptoms in animal models of Parkinson's disease (PD) and in initial clinical trials, A2A receptor antagonists have emerged as an attractive non-dopaminergic target to improve the motor deficits that characterize PD. Experimental data have also shown that A2A receptor antagonists do not induce neuroplasticity phenomena that complicate long-term dopaminergic treatments. The present review provides an updated summary of results reported in the literature concerning the biochemical characteristics and basal ganglia distribution of A2A receptors. We subsequently aim to examine the effects of adenosine A2A antagonists in rodent and primate models of PD and of l-DOPA-induced dyskinesia. Finally, concluding remarks are made on post-mortem human brains and on the translation of adenosine A2A receptor antagonists in the treatment of PD.     

Stoichiometric analysis of the TM2 6' phenylalanine mutation on desensitization in alpha1beta2 and alpha1beta2gamma2 GABA A receptors

The presence of phenylalanine (F) at the 6' position of transmembrane domain 2 (TM2) in the alpha4 subunit of alpha4beta2 nicotinic receptors enhances desensitization. As the GABA A receptor affords the ability to study the influence of as few as one and as many as five Fs at this position, we have used it to investigate potential subunit- and stoichiometry-dependent effects of the TM2 6'F mutation on desensitization. Whereas the presence of one F at this position decreased extent of desensitization, desensitization was increased in all configurations that included two or more Fs at the TM2 6' position; desensitization was particularly rapid with 3 or 4 F residues present. Our results demonstrate the ability of F residues at the TM2 6' position to modulate desensitization is likely conserved in the cys-loop family of ligand-gated ion channels. Moreover, our findings demonstrate both stoichiometric- and subunit-dependent effects of the ability of this mutation to regulate desensitization in GABA A receptors.     

早期应用泡式鼻持续气道正压通气8cmH2O和5cmH2O联合肺表面活性物质防治新生儿呼吸窘迫综合征的临床研究

目的探讨早期应用泡式鼻持续气道正压通气（B—NCPAP）8cmH2O（1cmH2O=0．098kPa）与肺表面活性物质（PS）＋B—NCPAP 5cmH2O防治NRDS的疗效差异。方法B—NCPAP8组选取2007年4月30日至2008年8月30日在中山大学附属第三医院分娩且生后30min内收入新生儿病房的早产儿,胎龄27～37周、羊水泡沫实验（-）～（＋）,生后30min内应用B—NCPAP 8cmH2O,分为B—NCPAP8预防（治疗前未出现呼吸窘迫）和B—NCPAP8治疗亚组（治疗前出现呼吸窘迫,胸部X线检查提示存在不同程度NRDS改变）。Ps＋B—NCPAP5组选取为胎龄27—37周、羊水泡沫实验（一）～（＋）的早产儿,生后6h内气管内注入Ps并给予B—NCPAP5cmH20,分为PS＋B—NCPAP5预防及PS＋B-NCPAP5治疗亚组。结果研究期间,B—NCPAP8预防和治疗亚组分别纳入48和25例;PS＋B—NCPAP5预防和治疗亚组分别纳入36和22例。B—NCPAP8预防亚组41／48例（85．4％）未出现呼吸窘迫;7例在应用B—NCPAP4～6h后出现呼吸窘迫,其中5例为轻度NRDS,继续治疗8h后症状逐渐改善,2例为重度NRDS,在生后8～10h行气管插管注入PS并改用机械通气后存活;应用成功率为95．8％（46／48例）。PS＋B—NCPAP5预防亚组32／36例（88．9％）未出现呼吸窘迫;4例在应用BNCPAP4～6h后出现呼吸窘迫,其中3例为轻度NRDS,继续治疗后症状逐渐改善,应用成功率为97．2％（35／36例）;1／36例（2．8％）为中度NRDS,改用机械通气后死亡。B—NCPAP8治疗亚组24／25例（96．0％）治疗2h后血气分析指标改善,1例改用机械通气。PS＋B—NCPAP5治疗亚组18／22例（81．8％）治疗8h后临床症状及血气指标均改善,1／22例（4．5％）死亡。B—NCPAP8与PS＋B—NCPAP5预防及治疗亚组在防治NRDS的疗效上差异无统计学意义,但B—NCPAP8预防及治疗亚组的肺部感染发生率、住院时间及住院费用较低（P均〈0．05）。结论早期应用B．NCPAP 8cmH2O与PS＋B—NCPAP 5cmH2O预防及治疗NRDS的疗效相近,但前者能减少肺部并发症发生,缩短住院时间,减少住院费用。     

A homozygous variant in RRM2B is associated with severe metabolic acidosis and early neonatal death

RRM2B encodes the crucial p53-inducible ribonucleotide reductase small subunit 2 homolog (p53R2), which is required for DNA synthesis throughout the cell cycle. Mutations in this gene have been associated with a lethal mitochondrial depletion syndrome. Here we present the case of an infant with a novel homozygous p.Asn221Ser mutation in RRM2B who developed hypotonia, failure to thrive, sensorineural hearing loss, and severe metabolic lactic acidosis, ultimately progressing to death at 3 months of age. Through molecular modeling using the X-ray crystal structure of p53R2, we demonstrate that this mutation likely causes disruption of a highly conserved helix region of the protein by altering intramolecular interactions. This report expands our knowledge of potential pathogenic RRM2B mutations as well as our understanding of the molecular function of p53R2 and its role in the pathogenesis of mitochondrial DNA depletion.     

A subpopulation of serotonin1B receptors colocalize with the AMPA receptor subunit GluR2 in the hippocampal dentate gyrus

The serotonin_1B receptor (5-HT_1BR) plays a role in cognitive processes that also involve glutamatergic neurotransmission via amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors. Accumulating experimental evidence also highlights the involvement of 5-HT_1BRs in several neurological disorders. Consequently, the 5-HT_1BR is increasingly implicated as a potential therapeutic target for intervention in cognitive dysfunction. Within the hippocampus, a brain region critical to cognitive processing, populations of pre- and post-synaptic 5-HT_1BRs have been identified. Thus, 5-HT_1BRs could have a role in the modulation of hippocampal pre- and post-synaptic conductance. Previously, we demonstrated colocalization of 5-HT_1BRs with the N-methyl-d-aspartate (NMDA) receptor subunit NR1 in a subpopulation of granule cell dendrites (Peddie et al. [53]). In this study, we have examined the cellular and subcellular distribution of 5-HT_1BRs with the AMPA receptor subunit GluR2. Of 5-HT_1BR positive profiles, 28% displayed colocalization with GluR2. Of these, 87% were dendrites, corresponding to 41% and 10% of all 5-HT_1BR labeled or GluR2 labeled dendrites, respectively. Dendritic labeling was both cytoplasmic and membranous but was not usually associated with synaptic sites. Colocalization within dendritic spines and axons was comparatively rare. These findings indicate that within the dentate gyrus molecular layer, dendritic 5-HT_1BRs are expressed predominantly on GluR2 negative granule cell processes. However, a subpopulation of 5-HT_1BRs is expressed on GluR2 positive dendrites. Here, it is suggested that activation of the 5-HT_1BR may play a role in the modulation of AMPA receptor mediated conductance, further supporting the notion that the 5-HT_1BR represents an interesting therapeutic target for modulation of cognitive function.     

A monoclonal antibody with ability to distinguish between NH2-terminal fragments derived from fibrinogen and fibrin

A stable hybridoma secreting homogeneous antibody (immunoglobulin class IgG2a) has been prepared by fusion using cells of immunoglobulin non-secreter myeloma (P3X63Ag8.653) and spleen cells of mice which had previously been immunized with the NH2-terminal CNBr fragment of human fibrinogen, the so-called N-DSK [(A alpha 1-51, B beta 1-118, gamma 1-78)2]. In competitive ELISA or radioimmunoassay this antibody (MAb/1-8C6) cross-reacted with intact fibrinogen, N-DSK, a des fibrinopeptide A (des FPA) variant of N-DSK, the so-called (B)N-DSK, as well as the intact B beta chain (B beta 1-118) obtained from N-DSK. Also, and mot importantly, cross-reactivity was observed with fibrinogen-free ethanol extracts of plasma obtained from patients known to contain high levels of fibrinogen or fibrin degradation products. In vitro thrombin digestion of any of these competitors resulted in complete loss of cross-reactivity. MAb/1-8C6 did not react with the A alpha or gamma-chains of N-DSK, free fibrinopeptide B(FPB), free B beta 15-42, as well as equimolar mixtures of the latter two peptides. These results suggest that MAb/1-8C6 may be to an epitope in or around the thrombin-susceptible B beta 14 Arg-25 Gly bond. Furthermore, due to its reactivity with patient plasma extracts, this antibody may be useful in clinical investigations dealing with fibrino(geno)lysis.     

Development of CO(2)-response in the early newborn period in rat

We examined the respiratory response to moderate hypercapnia in rat pups during the first 10 days after birth and also studied immediate early gene expression to investigate whether areas described as chemosensitive in the adult rat are activated also in the early postnatal period. Breathing frequency increased in 1- and 3-day-old pups, but decreased in older animals in response to hypercapnia. Tidal volume and ventilation increased significantly in all age groups but relatively more in the 10-day-old pups as compared to younger animals. In situ hybridisation for c-fos mRNA revealed increased expression in several of the areas assigned as chemosensitive in the adult, including the caudal nucleus tractus solitarii and the ventral lateral medulla. In contrast, locus coeruleus and the majority of midline raphe neurons did not exhibit increased expression of c-fos mRNA. We conclude that the hypercapnic respiratory response tends to decrease during the first postnatal week, but thereafter increases on day 10 due to increased tidal volumes rather than changes in respiratory timing. We also speculate that differences in activation of chemosensitive brainstem neurons may be part of the maturation of the hypercapnic ventilatory response.