Acute downregulation of Cx43 alters P2Y receptor expression levels in mouse spinal cord astrocytes

Propagation of intercellular calcium waves (ICW) between astrocytes depends on the diffusion of signaling molecules through gap junction channels and diffusion through the extracellular space of neuroactive substances acting on plasmalemmal receptors. The relative contributions of these two pathways vary in different brain regions and under certain pathological conditions. We have previously shown that in wild-type spinal cord astrocytes, ICW are primarily gap junction-dependent, but that deletion of the main gap junction protein (Cx43) by homologous recombination results in a switch in mode of ICW propagation to a purinoceptor-dependent mechanism. Such a compensatory mechanism for ICW propagation was related to changes in the pharmacological profile of P2Y receptors, from an adenine-sensitive P2Y(1), in wild-type, to a uridine-sensitive P2U receptor subtype, in Cx43 knockout (KO) astrocytes. Using oligonucleotide antisense to Cx43 mRNA for acute downregulation of connexin43 expression levels, we provide evidence for the molecular nature of such compensatory mechanism. Pharmacological studies and Western blot analysis indicate that there is a reciprocal regulation of P2Y(1) and P2Y(4) expression levels, such that downregulation of Cx43 leads to decreased expression of the adenine-sensitive P2Y(1) receptor and increased expression of the uridine-sensitive P2Y(4) receptor. This change in functional expression of the P2Y receptor subtype population in acutely downregulated Cx43 was paralleled by changes in the mode of ICW propagation, similar to that previously observed for Cx43 KO spinal cord astrocytes. On the basis of these results, we propose that Cx43 regulates both modes of ICW by altering P2Y receptor subtype expression in addition to providing intercellular coupling.     

Long-term potentiation and long-term depression induced by local application of ATP to hippocampal CA1 neurons of the guinea pig

The present study has investigated the role of ATP in the induction of synaptic plasticity, using local application of ATP by picopump administration into the stratum radiatum of guinea pig hippocampal region CA1. Excitatory postsynaptic currents (EPSCs) evoked by stimulation of Schaffer collateral/commissural afferents synapsing on CA1 pyramidal cells of hippocampal slices were monitored in voltage-clamp mode, using whole-cell recording. Brief local application of ATP (1 mM) induced an inward current, usually consisting of early- and late-phase components. Because the late-phase component of an ATP-induced current was largely inhibited by Ca2+-free solution, this component is supposed to depend on extracellular Ca2+. After local application of ATP, long-term synaptic modification of EPSCs was induced: LTP was detected in neurons exhibiting a small late Ca2+ current, while LTD was obtained from recordings showing a large late Ca2+ current in response to ATP application. There was a statistically significant correlation between the magnitude of long-term plastic changes and the size of Ca2+ currents in response to ATP application. Furthermore, there was significant difference between the average size of the Ca2+ current in the LTP group and the size in the LTD group. These results suggest that a small Ca2+ influx in response to ATP application induces LTP, whereas a large one induces LTD in guinea pig hippocampal CA1 neurons.     

Involvement of a purinergic pathway in the sympathetic regulation of motility in rat ileum

We investigated extrinsic neuronal regulation of intestinal motility. The mesenteric nerve stimulation (MNS; duration 0.5 ms, 10 Hz for 30 s) evoked relaxation in the longitudinal muscle direction of the isolated rat ileum. The MNS-induced relaxation was abolished by guanethidine (2 microM) or propranolol (10 microM), but was not affected by prazosin (10 microM), rauwolscine (10 microM), hexamethonium (100 microM) or capsaicin (1 microM). Exposure to a high concentration (100 microM) of ATP (ATP-desensitization) or ADP (ADP-desensitization) reduced the MNS-induced relaxation to 44.7% or 32.5% of the control (P<0.01), respectively. P2 purinoceptor antagonists [suramin (100 microM) and reactive blue-2 (RB-2, 50 microM)] or small conductance Ca(2+)-activated K(+) channel blocker, apamin (0.5 microM), significantly decreased the relaxation to 54.4% and 25.6% or 19.4% of the control (P<0.01), respectively, whereas selective P2Y(1) purinoceptor antagonist MRS2179 (10 microM) failed to affect the relaxation. Furthermore, exogenous ATP (1 microM) or ADP (1 microM) elicited relaxation in the rat ileum, which was almost abolished by reactive blue-2 (50 microM, 9.1% of control remained, P<0.05). In contrast, relaxation induced by noradrenalin (10 microM) was not antagonized by ATP-desensitization, apamin (0.5 microM) or reactive blue-2 (50 microM). From the present results, we conclude that noradrenergic sympathetic nerves might regulate intestinal motility mediated through a purinergic inhibitory neuronal pathway in the rat small intestine.     

Extracellular ATP-induced currents in astrocytes: Involvement of a cation channel

Whole-cell currents were measured with the perforated patch clamp technique in cultured rat astrocytes to analyze the underlying ionic mechanism for a P₂-purinoceptor-mediated depolarization. ATP (100 μM) induced an inward current with a mean amplitude of 130 pA and an EC₅₀ of 17 μM. The response desensitized during a 1 min application. Replacement of extracellular Na⁺ with NMDG or K⁺ abolished the ATP-evoked inward current. Replacement of Na⁺ with choline, however, resulted in an ATP-evoked response of one-third the amplitude in normal solution. This is indicative of a cation rather than Na⁺ channel. However, due to difficulties in voltage-clamping these gap junction-coupled cells at voltages different from the membrane resting potential, the current reversal potential could not be determined. Measurements with K⁺-sensitive microelectrodes showed that 100 μM ATP lowered the intracellular K⁺ concentration. Replacement of extracellular Ca²⁺ or Cl^? did not alter the ATP-induced inward currents. Fura-2 imaging experiments revealed a transient rise of the intracellular Ca²⁺ concentration during ATP application. Removal of extracellular Ca²⁺ did not influence the peak response; it did, however, shorten the time course. These results and previous observations that the permeability changes are caused by a P_2× receptor are indicative of an ATP-sensitive cation conductance. In addition, cytoplasmic Ca²⁺ is increased by mobilization from intracellular stores, and by additional influx across the cell membrane. Extracellular ATP released by neurons could evoke K⁺ release from astrocytes as well as be a mediator for cation changes that signal cell activation processes when released by damaged cells. © 1994 Wiley-Liss, Inc.     

Electroacupuncture improves neuropathic pain Adenosine,adenosine 5'-triphosphate disodium and their receptors perhaps change simultaneously

Applying a stimulating current to acupoints through acupuncture needles-known as electroacupuncture-has the potential to produce analgesic effects in human subjects and experimental animals. When acupuncture was applied in a rat model, adenosine 5-triphosphate disodium in the extracellular space was broken down into adenosine, which in turn inhibited pain transmission by means of an adenosine A1 receptor-dependent process. Direct injection of an adenosine A1 receptor agonist enhanced the analgesic effect of acupuncture. The analgesic effect of acupuncture appears to be mediated by activation of A1 receptors located on ascending nerves. In neuropathic pain, there is upregulation of P2X purinoceptor 3 (P2X3) receptor expression in dorsal root ganglion neurons. Conversely, the onset of mechanical hyperalgesia was diminished and established hyperalgesia was significantly reversed when P2X3 receptor expression was downregulated. The pathways upon which electroacupuncture appear to act are interwoven with pain pathways, and electroacupuncture stimuli converge with impulses originating from painful areas. Electroacupuncture may act via purinergic A1 and P2X3 receptors simultaneously to induce an analgesic effect on neuropathic pain.     

P2X7受体对缺氧诱发小鼠视网膜神经节细胞凋亡的影响

目的：：探讨嘌呤受体P2X7对缺氧诱发小鼠视网膜神经节细胞凋亡的影响。方法：以小鼠视网膜神经节细胞株RGC-5为研究对象,按照不同处理因素将细胞随机分为4组：正常对照组( G1)、缺氧组( G2)、缺氧+激动剂( BzATP )组( G3)、缺氧+拮抗剂(BBG)组(G4);采用四甲基偶氮唑蓝(MTT)法检测细胞的存活率;用Annxin V/PI染色流式细胞术检测细胞凋亡率;Western Blot检测细胞内cleave-caspase-3和cleave-PARP蛋白的表达。结果：与正常对照组相比, RGC-5细胞经缺氧处理后,细胞存活率明显降低;凋亡率显著升高;细胞内 cleave-caspase-3和cleave-PARP蛋白表达增加;P2X7受体激动剂BzATP能明显加重缺氧诱发的细胞凋亡,而BBG预处理可以显著拮抗缺氧所致的细胞凋亡。结论：缺氧能激活视网膜神经节细胞嘌呤受体P2X7,并参与视网膜神经节细胞的凋亡。