P2Y(1) purinoceptor-mediated Ca(2+) signaling and Ca(2+) wave propagation in dorsal spinal cord astrocytes.
ABSTRACT: ATP is known to act as an extracellular messenger mediating the propagation of Ca(2+) waves in astrocyte networks. ATP mediates Ca(2+) waves by activating P2Y purinoceptors, which mobilize intracellular Ca(2+) in astrocytes. A number of P2Y purinoceptor subtypes have been discovered, but it is not known which P2Y subtypes participate in transmitting astrocyte Ca(2+) waves. Here, we show that ATP analogs that are selective agonists for the P2Y(1) subtype of purinoceptor caused release of intracellular Ca(2+) in astrocytes from the dorsal spinal cord. The Ca(2+) responses were blocked by adenosine-3'-phospho-5'-phosphosulfate, an antagonist known to selectively inhibit P2Y(1) but not other P2Y purinoceptor subtypes. Also, we show that P2Y(1) mRNA is expressed in dorsal spinal cord astrocytes. Furthermore, expression of P2Y(1) in an astrocytoma cell line lacking endogenous purinoceptors was sufficient to permit propagation of intercellular Ca(2+) waves. Finally, Ca(2+) wave propagation in dorsal spinal cord astrocytes was suppressed by pharmacologically blocking P2Y(1) purinoceptors. Together, these results indicate that dorsal spinal astrocytes express functional P2Y(1) purinoceptors, which participate in the transmission of Ca(2+) waves. Ca(2+) waves in astrocytes have been implicated as a major signaling pathway coordinating glial and neuronal activity; therefore, P2Y(1) purinoceptors may represent an important link in cell-cell signaling in the CNS.
Project description:In order to elucidate the mechanisms of purinergic transmission of calcium (Ca(2 + )) waves between microglial cells, we have employed micro-photolithographic methods to form discrete patterns of microglia that allow quantitative measurements of Ca(2 + ) wave propagation. Microglia were confined to lanes 20-100 [Formula: see text] wide and Ca(2 + ) waves propagated from a point of mechanical stimulation, with a diminution in amplitude, for about 120 [Formula: see text]. The number of cells participating in propagation also decreased over this distance. Ca(2 + ) waves could propagate across a cell-free lane from one microglia lane to another if this distance of separation was less than about 60 [Formula: see text], indicating that propagation involved diffusion of a chemical transmitter. This transmitter was identified as ATP since all Ca(2 + ) wave propagation was blocked by the purinoceptor antagonist suramin, which blocks P2Y(2) and P2Y(12) at relatively low concentrations. Antibodies to P2Y(12) showed these at very high density compared with P2Y(2), indicating a role for P2Y(12) receptors. These observations were quantitatively accounted for by a model in which the main determinants are the diffusion of ATP released from a stimulated microglial cell and differences in the dissociation constant of the purinoceptors on the microglial cells.
Project description:Intercellular spread of Ca2+ waves is the primary manifestation of cell-to-cell communication among astrocytes. Ca2+ waves propagate via the release of a diffusible extracellular messenger that has been identified as ATP. In dorsal spinal astrocytes, Ca2+ waves are mediated by activation of two functionally distinct subtypes of metabotropic purinoceptor: the P2Y1 receptor and a receptor previously classified as P2U. Here, we show that the P2U receptor is molecularly and pharmacologically identical to the cloned P2Y2 receptor. Both P2Y1 and P2Y2 receptors are necessary for full Ca2+ wave propagation in spinal astrocytes. Conversely, heterologous expression of either P2Y1 or P2Y2 receptors is sufficient for Ca2+ waves, and expressing these receptor subtypes together recapitulates the characteristics of Ca2+ waves in spinal astrocytes. Thus, P2Y1 and P2Y2 receptors are both necessary and sufficient for propagation of Ca2+ waves. Furthermore, we demonstrate that there are dramatic differences in the characteristics of Ca2+ waves propagating through each receptor subtype: Ca2+ waves propagating via P2Y2 receptors travel faster and further than those propagating via P2Y1 receptors. We find that the nucleotidase apyrase selectively blocks Ca2+ wave propagation through P2Y2 receptors but accelerates Ca2+ waves propagating through P2Y1 receptors. Taking our results together with those from the literature, we suggest that mediation of Ca2+ waves by ATP leading to activation of two subtypes of receptor, P2Y1 and P2Y2, may be a general principle for gliotransmission in the CNS. Thus, processes that alter expression or function of these receptors may control the rate and extent of astrocyte Ca2+ waves.
Project description:ATP is a key extracellular messenger that mediates the propagation of Ca 2+ waves in astrocyte networks in various regions of the CNS. ATP-mediated Ca 2+ signals play critical roles in astrocyte proliferation and differentiation and in modulating neuronal activity. The actions of ATP on astrocytes are via two distinct subtypes of P2Y purinoceptors, P2Y1 and P2Y2 receptors (P2Y1Rs and P2Y2Rs), G-protein coupled receptors that stimulate mobilization of intracellular Ca 2+ ([Ca 2+]i) via the phospholipase Cbeta-IP3 pathway. We report here that P2Y1R-mediated and P2Y2R-mediated Ca 2+ responses differentially show two forms of activity-dependent negative feedback. First, Ca 2+ responses mediated by either receptor exhibit slow depression that is independent of stimulation frequency. Second, responses mediated by P2Y1Rs, but not those mediated by P2Y2Rs, show rapid oscillations after high-frequency stimulation. We demonstrate that the oscillations are mediated by recruiting negative feedback by protein kinase C, and we map the site responsible for the effect of protein kinase C to Thr339 in the C terminus of P2Y1R. We propose that frequency-dependent changes in ATP-mediated Ca 2+ signaling pathways may modulate astrocyte function and astrocyte-neuron signaling in the CNS.
Project description:BACKGROUND AND PURPOSE: This study analysed the contribution of the purinergic system to enteric neurotransmission in the longitudinal muscle of mouse distal colon. EXPERIMENTAL APPROACH: Motor responses to exogenous ATP and to nerve stimulation in vitro were assessed as changes in isometric tension. KEY RESULTS: ATP induced a concentration-dependent contraction, reduced by 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzene disulphonic acid (PPADS), suramin, P2Y purinoreceptor desensitisation with adenosine 5'-O-2-thiodiphosphate (ADPbetaS), and atropine, but unaffected by P2X purinoceptor desensitisation with alpha,beta-methylene ATP (alpha,beta-meATP) and by 2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl ester (MRS 2395), a P2Y(12) selective antagonist. The response to ATP was increased by 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS 2179), a P2Y(1) selective antagonist, tetrodotoxin (TTX) or N(omega)-nitro-L-arginine methyl ester (L-NAME). ADPbetaS, a P2Y-purinergic agonist, induced muscular contraction, with the same pharmacological profile as the ATP-induced contraction. ADP, a natural ligand for P2Y(1) receptors, induced muscular relaxation, antagonized by MRS 2179 and by TTX or L-NAME. Nerve stimulation elicited a transient nitrergic relaxation, followed by contraction. Contractile responses was reduced by atropine, PPADS, suramin, P2Y purinoceptor desensitisation, but not by P2X purinoceptor desensitisation, MRS 2179 or MRS 2395. None of the purinergic antagonists modified the nerve-evoked relaxation. CONCLUSIONS AND IMPLICATIONS: In the longitudinal muscle of mouse distal colon, ATP, through ADPbetaS-sensitive P2Y purinoceptors, contributed to the excitatory neurotransmission acting directly on smooth muscle and indirectly via activation of cholinergic neurons. Moreover, P2Y1 purinoceptors appear to be located on nitrergic inhibitory neurons. This study provides new insights into the role of purines in the mechanism inducing intestinal transit in mouse colon.
Project description:Single rat hepatocytes microinjected with aequorin generate oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway. The duration of these transients has been shown to be characteristic of the stimulating agonist, so that transients of very different duration can be induced in the same individual hepatocyte by different agonists. In a previous study we have shown that ADP and ATP, which are believed to act through a single P2y-purinoceptor species, elicit very different [Ca2+]i responses in most of the hepatocytes. We have interpreted this as evidence for two Ca(2+)-mobilizing purinoceptors. The methylated derivative of ATP, adenosine 5'-[alpha beta-methylene]-triphosphate (pp[CH2]pA), is only a weak P2y-purinoceptor agonist. When 100 microM pp[CH2]pA was supplied to aequorin-injected hepatocytes, there was no effect on [Ca2+]i. However, 25 microM pp[CH2]pA co-supplied with ATP causes a potentiation of the [Ca2+]i response in most of the hepatocytes. The effect was specific for ATP-induced transients; [Ca2+]i transients induced by other agonists, and importantly by ADP, were not affected by addition of pp[CH2]pA. This further illustrates differences in the actions of ADP and ATP, strengthening the argument for separate receptors for these nucleotides.
Project description:Extracellular nucleotides have been implicated as signaling molecules used by microglia to sense adverse physiological conditions, such as neuronal damage. They act through purinoceptors, especially the G-protein-coupled P2Y receptor P2Y(12)R. Emerging evidence has indicated that activated spinal microglia responding to nerve injury are key cellular intermediaries in the resulting highly debilitating chronic pain state, namely neuropathic pain. However, the role of microglial P2Y(12)Rs in neuropathic pain remains unknown. Here, we show that the level of P2Y(12)R mRNA expression was markedly increased in the spinal cord ipsilateral to the nerve injury and that this expression was highly restricted to ionized binding calcium adapter molecule 1-positive microglia. An increase in the immunofluorescence of P2Y(12)R protein in the ipsilateral spinal cord was also observed after nerve injury, and P2Y(12)R-positive cells were double labeled with the microglial marker OX-42. Blocking spinal P2Y(12)R by the intrathecal administration of its antagonist AR-C69931MX prevented the development of tactile allodynia (pain hypersensitivity to innocuous stimuli), a hallmark of neuropathic pain syndrome. Furthermore, mice lacking P2ry(12) (P2ry(12)(-/-)) displayed impaired tactile allodynia after nerve injury without any change in basal mechanical sensitivity. Moreover, a single intrathecal administration of AR-C69931MX or oral administration of clopidogrel (a P2Y(12)R blocker clinically in use) to nerve-injured rats produced a striking alleviation of existing tactile allodynia. Together, our findings indicate that activation of P2Y(12)Rs in spinal microglia may be a critical event in the pathogenesis of neuropathic pain and suggest that blocking microglial P2Y(12)R might be a viable therapeutic strategy for treating neuropathic pain.
Project description:Spinal cord injury increases inhibitory factors that may restrict neurite outgrowth after trauma. The expression of repulsive molecules in reactive astrocytes and the formation of the glial scar at the injury site produce the non-permissive environment for axonal regeneration. However, the mechanism that triggers this astrogliotic response is unknown. The release of nucleotides has been linked to this hypertrophic state. Our goal is to investigate the temporal profile of P2Y(2) nucleotide receptor after spinal cord injury in adult female Sprague-Dawley rats. Molecular biology, immunofluorescence studies, and Western Blots were used to evaluate the temporal profile (2, 4, 7, 14, and 28 days post-injury) of this receptor in rats injured at the T-10 level using the NYU impactor device. Real time RT-PCR showed a significant increase of P2Y(2) mRNA after 2 days post-injury that continues throughout 28 days post-injury. Double labeling studies localized P2Y(2) immunoreactivity in neuronal cell bodies, axons, macrophages, oligodendrocytes and reactive astrocytes. Immunofluorescence studies also demonstrated a low level of P2Y(2) receptor in sham samples, which increased after injury in glial fibrillary acidic protein positive cells. Western Blot performed with contused spinal cord protein samples revealed an upregulation in the P2Y(2) 42 kDa protein band expression after 4 days post-injury that continues until 28 days post-injury. However, a downregulation of the 62 kDa receptor protein band after 2 days post-injury that continues up to 28 days post-injury was observed. Therefore, the spatio-temporal pattern of P2Y(2) gene expression after spinal cord injury suggests a role in the pathophysiology response generated after trauma.
Project description:Mouse L-fibroblast cells stably transfected with either type 1 Ins(1, 4,5)P(3) receptor (InsP(3)R) cDNA (L15) or the vector control (Lvec) have been used to investigate the functional consequences of increased InsP(3)R density on receptor-mediated Ca(2+) signalling. L15 cells express approx. 8-fold higher levels of the type 1 InsP(3)R compared with Lvec cells, which endogenously express essentially only the type 1 InsP(3)R protein. Stimulation of Lvec and L15 cells with UTP or ATP increased cytosolic Ca(2+) concentration to a greater extent in L15 cells at all agonist concentrations. UTP and ATP were equipotent, suggestive of the presence of endogenous cell-surface metabotropic P2Y(2)-purinoceptors. In both cell clones the purinoceptors were coupled via pertussis-toxin-insensitive G-protein(s) to phospholipase C activation, resulting in similar concentration-dependent accumulations of InsP(3). Single-cell microfluorimetry revealed that overexpression of InsP(3)Rs reduced the threshold for purinoceptor-mediated Ca(2+) signalling. L-fibroblasts also exhibited temporally complex sinusoidal cytosolic Ca(2+) oscillations in response to submaximal agonist concentrations, with significant increases in oscillatory frequencies exhibited by cells overexpressing InsP(3)Rs. Sustainable oscillatory responses were dependent on Ca(2+) entry and, at higher agonist concentrations, cytosolic Ca(2+) oscillations were superseded by biphasic peak-and-plateau Ca(2+) responses. Overexpression of InsP(3)Rs in L15 cells resulted in a 4-fold reduction in the threshold for this change in the temporal pattern of Ca(2+) mobilization. These data provide the first direct evidence demonstrating that altering the expression of the type 1 InsP(3)R significantly affects receptor-mediated InsP(3)-induced Ca(2+) mobilization.
Project description:Aequorin measurements of cytosolic free Ca2+ in single rat hepatocytes show that ADP and ATP, thought to act through the same P2Y purinoceptor, elicited very different responses in the majority of cells tested. ADP invariably induced transients of short duration (approx. 9 s), whereas ATP induced either similar transients or transients with a much longer duration (approx. 49 s). We explain this variability in terms of two separate purinoceptors on rat hepatocytes, one of which responds to either ATP or ADP to generate free-Ca2+ transients of short duration, and the other responds to ATP only, with transients of longer duration.
Project description:Diadenosine polyphosphates present in the extracellular environment can, through interaction with appropriate purinoceptors, influence a range of cellular activities. Here we have investigated the nature of the ligand:receptor interactions involved in diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A)-mediated stimulation of glycogen breakdown in isolated rat liver cells. [2-3H]Ap4A showed specific binding to both intact isolated liver cells and plasma membrane fractions prepared from isolated liver cells. HPLC analysis confirmed that binding was mediated by intact Ap4A and not by potential breakdown products (e.g. ATP, adenosine, etc.). Binding of [2-3H]Ap4A, to isolated liver cell plasma membrane preparations, was successfully displaced by a range of both naturally occurring and synthetic diadenosine polyphospates with the rank order potency Ap4A > or = Ap5A > Ap6A > Ap3A > Ap2A. [2-3H]Ap4A binding was not displaced by P1 effectors but was successfully displaced by a range of P2 effectors with the rank order potency 2-methylthio-ATP > ATP > ATP > or = adenosine 5'-[alpha beta-methylene]triphosphate > adenosine 5'-[beta gamma-methylene]triphospate. These findings are consistent with the interaction of Ap4A with a P2y-like subclass of purinoceptor and are discussed in relation to (1) the known purinoceptor populations in liver cell plasma membranes and (2) observations concerning the binding of diadenosine polyphosphates to purinoceptors in other tissues.