Sylvia O. Suadicani

P2X7 Receptors Mediate ATP Release and Amplification of Astrocytic Intercellular Ca2+ Signaling

Modulation of synaptic transmission and brain microcirculation are new roles ascribed to astrocytes in CNS function. A mechanism by which astrocytes modify neuronal activity in the healthy brain depends on fluctuations of cytosolic Ca2+ levels, which regulate the release of “gliotransmitters” via an exocytic pathway. Under pathological conditions, however, the participation of other pathways, including connexin hemichannels and the pore-forming P2X7R, have been proposed but remain controversial. Through the use of genetically modified 1321N1 human astrocytoma cells and of spinal cord astrocytes derived from neonatal Cx43- and P2X7R-null mice, we provide strong evidence that P2X7Rs, but not Cx43 hemichannels, are sites of ATP release that promote the amplification of Ca2+ signal transmission within the astrocytic network after exposure to low divalent cation solution. Moreover, our results showing that gap junction channel blockers (heptanol, octanol, carbenoxolone, flufenamic acid, and mefloquine) are antagonists of the P2X7R indicate the inadequacy of using these compounds as evidence for the participation of connexin hemichannels as sites of gliotransmitter release.

Connexin and pannexin mediated cell—cell communication

In this review, we briefly summarize what is known about the properties of the three families of gap junction proteins, connexins, innexins and pannexins, emphasizing their importance as intercellular channels that provide ionic and metabolic coupling and as non-junctional channels that can function as a paracrine signaling pathway. We discuss that two distinct groups of proteins form gap junctions in deuterostomes (connexins) and protostomes (innexins), and that channels formed of the deuterostome homologues of innexins (pannexins) differ from connexin channels in terms of important structural features and activation properties. These differences indicate that the two families of gap junction proteins serve distinct, complementary functions in deuterostomes. In several tissues, including the CNS, both connexins and pannexins are involved in intercellular communication, but have different roles. Connexins mainly contribute by forming the intercellular gap junction channels, which provide for junctional coupling and define the communication compartments in the CNS. We also provide new data supporting the concept that pannexins form the non-junctional channels that play paracrine roles by releasing ATP and, thus, modulating the range of the intercellular Ca(2+)-wave transmission between astrocytes in culture.

Inhibition of Endothelial Cell Migration, Intercellular Communication, and Vascular Tube Formation by Thromboxane A2

The eicosanoid thromboxane A2 (TXA2) is released by activated platelets, monocytes, and the vessel wall and interacts with high affinity receptors expressed in several tissues including endothelium. Whether TXA2 might alter endothelial migration and tube formation, two determinants of angiogenesis, is unknown. Thus, we investigated the effect of the TXA2 mimetic [1S-(1α,2β(5Z),3α(1E,3R),4α]-7-[3-(3-hydroxy-4-(4′-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5′-heptenoic acid (IBOP) on human endothelial cell (HEC) migration and angiogenesis in vitro. IBOP stimulation inhibited HEC migration by 50% and in vitro capillary formation by 75%. These effects of IBOP were time- and concentration-dependent with an IC50 of 25 nm. IBOP did not affect integrin expression or cytoskeletal morphology of HEC. Since gap junction-mediated intercellular communication increases in migrating HEC, we determined whether IBOP might inhibit coupling or connexin expression in HEC. IBOP reduced the passage of microinjected dyes between HEC by 50%, and the effects of IBOP on migration and tube formation were mimicked by the gap junction inhibitor 18β-glycyrrhetinic acid (1 μm) with a similar time course and efficacy. IBOP (24 h) did not affect the expression or phosphorylation of connexin 43 in whole HEC lysates. Immunohistologic examination of HEC suggested that IBOP may impair functional coupling by altering the cellular distribution of gap junctions, leading to increased connexin 43 internalization. Thus, this finding that TXA2 mimetics can prevent HEC migration and tube formation, possibly by impairing intercellular communication, suggests that antagonizing TXA2 signaling might enhance vascularization of ischemic tissue.

Gap junction remodeling and cardiac arrhythmogenesis in a murine model of oculodentodigital dysplasia

Gap junction channels are required for normal cardiac impulse propagation, and gap junction remodeling is associated with enhanced arrhythmic risk. Oculodentodigital dysplasia (ODDD) is a multisystem syndrome due to mutations in the connexin43 (Cx43) gap junction channel gene. To determine the effects of a human connexin channelopathy on cardiac electrophysiology and arrhythmogenesis, we generated a murine model of ODDD by introducing the disease-causing I130T mutant allele into the mouse genome. Cx43 abundance was markedly reduced in mutant hearts with preferential loss of phosphorylated forms that interfered with trafficking and assembly of gap junctions in the junctional membrane. Dual whole-cell patch–clamp studies showed significantly lower junctional conductance between neonatal cell pairs from mutant hearts, and optical mapping of isolated-perfused hearts with voltage-sensitive dyes demonstrated significant slowing of conduction velocity. Programmed electrical stimulation revealed a markedly increased susceptibility to spontaneous and inducible ventricular tachyarrhythmias. In summary, our data demonstrate that the I130T mutation interferes with Cx43 posttranslational processing, resulting in diminished cell–cell coupling, slowing of impulse propagation, and a proarrhythmic substrate.

ATP signaling is deficient in cultured pannexin1-null mouse astrocytes

Pannexins (Panx1, 2, and 3) comprise a group of proteins expressed in vertebrates that share weak yet significant sequence homology with the invertebrate gap junction proteins, the innexins. In contrast to the other vertebrate gap junction protein family (connexin), pannexins do not form intercellular channels, but at least Panx1 forms nonjunctional plasma membrane channels. Panx1 is ubiquitously expressed and has been shown to form large conductance (500 pS) channels that are voltage dependent, mechanosensitive, and permeable to relatively large molecules such as ATP. Pharmacological and knockdown approaches have indicated that Panx1 is the molecular substrate for the so‐called “hemichannel” originally attributed to connexin43 and that Panx1 is the pore‐forming unit of the P2X7 receptor. Here, we describe, for the first time, conductance and permeability properties of Panx1‐null astrocytes. The electrophysiological and fluorescence imaging analyses performed on these cells fully support our previous pharmacological and Panx1 knockdown studies that showed profoundly lower dye uptake and ATP release than wild‐type untreated astrocytes. As a consequence of decreased ATP paracrine signaling, intercellular calcium wave spread is altered in Panx1‐null astrocytes. Moreover, we found that in astrocytes as in Panx1‐expressing oocytes, elevated extracellular K+ activates Panx1 channels independently ofmembrane potential. Thus, on the basis of our present findings and our previous report, we propose that Panx1 channels serve as K+ sensors for changes in the extracellular milieu such as those occurring under pathological conditions.

Bidirectional calcium signaling between satellite glial cells and neurons in cultured mouse trigeminal ganglia

Astrocytes communicate with neurons, endothelial and other glial cells through transmission of intercellular calcium signals. Satellite glial cells (SGCs) in sensory ganglia share several properties with astrocytes, but whether this type of communication occurs between SGCs and sensory neurons has not been explored. In the present work we used cultured neurons and SGCs from mouse trigeminal ganglia to address this question. Focal electrical or mechanical stimulation of single neurons in trigeminal ganglion cultures increased intracellular calcium concentration in these cells and triggered calcium elevations in adjacent glial cells. Similar to neurons, SGCs responded to mechanical stimulation with increase in cytosolic calcium that spread to the adjacent neuron and neighboring glial cells. Calcium signaling from SGCs to neurons and among SGCs was diminished in the presence of the broad-spectrum P2 receptor antagonist suramin (50 muM) or in the presence of the gap junction blocker carbenoxolone (100 muM), whereas signaling from neurons to SGCs was reduced by suramin, but not by carbenoxolone. Following induction of submandibular inflammation by Complete Freunds Adjuvant injection, the amplitude of signaling among SGCs and from SGCs to neuron was increased, whereas the amplitude from neuron to SGCs was reduced. These results indicate for the first time the presence of bidirectional calcium signaling between neurons and SGCs in sensory ganglia cultures, which is mediated by the activation of purinergic P2 receptors, and to some extent by gap junctions. Furthermore, the results indicate that not only sensory neurons, but also SGCs release ATP. This form of intercellular calcium signaling likely plays key roles in the modulation of neuronal activity within sensory ganglia in normal and pathological states.

Gap junction channels coordinate the propagation of intercellular Ca2+ signals generated by P2Y receptor activation

Astrocytes express gap junction proteins and multiple types of P2Y receptors (P2YRs) that contribute to the propagation of intercellular Ca2+ waves (ICW). To gain access to the role played by gap junctional communication in ICW propagation generated by P2YR activation, we selectively expressed P2Y1,2,4R subtypes and Cx43 in the human 1321N1 astrocytoma cell line, which lacks endogenous P2 receptors. Fluorescence recovery after photobleaching revealed that 1321N1 cells are poorly dye‐coupled and do not propagate ICW. Forced expression of Cx43 in 1321N1 cells (which did not show functional hemichannels) increased dye coupling and allowed short‐range ICW transmission that was mainly mediated by intercellular diffusion of Ca2+ generated in the stimulated cells. Astrocytoma clones expressing each of the P2YR subtypes were also able to propagate ICWs that were likely dependent on IP3 generation. These waves exhibited properties particular to each P2YR subtype. Co‐expression of eGFP‐hCx43 and P2Y1R modified the properties of P2Y1R‐generated ICW to those characteristics of P2Y2R. Increased coupling in P2Y4R clones induced by expression of eGFP‐hCx43 abolished the ICWs observed in uncoupled P2Y4R clones. No changes in the behavior of ICWs generated in P2Y2R clones were observed after forced expression of Cx43. These data indicate that in 1321N1 cells gap junctional communication provides intercellular integration of Ca2+ signals generated by P2YR activation, thus coordinating the propagation of intercellular calcium waves.

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 P2Y1, 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 P2Y1 and P2Y4 expression levels, such that downregulation of Cx43 leads to decreased expression of the adenine‐sensitive P2Y1 receptor and increased expression of the uridine‐sensitive P2Y4 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. GLIA 42:160–171, 2003.

Involvement of urinary bladder Connexin43 and the circadian clock in coordination of diurnal micturition rhythm

Summary Nocturnal enuresis in children and nocturia in the elderly are two highly prevalent clinical conditions characterized by a mismatch between urine production rate in the kidneys and storage in the urinary bladder during the sleep phase. Here we demonstrate, using a novel method for automated recording of mouse micturition, that connexin43 (Cx43), a bladder gap junction protein, is a negative regulator of functional bladder capacity. Bladder Cx43 levels and functional capacity show circadian oscillations in wild-type mice, but such rhythms are completely lost in Cry-null mice having a dysfunctional biological clock. Bladder muscle cells have an internal clock, and show oscillations of Cx43 and gap junction function. A clock regulator, Rev-erbα, upregulates Cx43 transcription as a co-factor of Sp1 using Sp1 cis-elements of the promoter. Therefore, circadianoscillation of Cx43 is associated with the biological clock and contributes to diurnal changes in bladder capacity, which avoids disturbance of sleep by micturition.

Promises and pitfalls of a Pannexin1 transgenic mouse line

Gene targeting strategies have become a powerful technology for elucidating mammalian gene function. The recently generated knockout (KO)-first strategy produces a KO at the RNA processing level and also allows for the generation of conditional KO alleles by combining FLP/FRT and Cre/loxP systems, thereby providing high flexibility in gene manipulation. However, this multipurpose KO-first cassette might produce hypomorphic rather than complete KOs if the RNA processing module is bypassed. Moreover, the generation of a conditional phenotype is also dependent on specific activity of Cre recombinase. Here, we report the use of an efficient molecular biological approach to test pannexin1 (Panx1) mRNA expression in global and conditional Panx1 KO mice derived from the KO-first mouse line, Panx1tm1a(KOMP)Wtsi. Using qRT-PCR, we demonstrate that tissues from wild-type (WT) mice show a range of Panx1 mRNA expression levels, with highest expression in trigeminal ganglia, bladder and spleen. Unexpectedly, we found that in mice homozygous for the KO-first allele, Panx1 mRNA expression is not abolished but reduced by 70% compared to that of WT tissues. Thus, Panx1 KO-first mice present a hypomorphic phenotype. Crosses of Panx1 KO-first with FLP deleter mice generated Panx1f/f mice. Further crosses of the latter mice with mGFAP-Cre or NFH-Cre mice were used to generate astrocyte- and neuron-specific Panx1 deletions, respectively. A high incidence of ectopic Cre expression was found in offspring of both types of conditional Panx1 KO mice. Our study demonstrates that Panx1 expression levels in the global and conditional Panx1 KO mice derived from KO-first mouse lines must be carefully characterized to ensure modulation of Panx1 gene expression. The precise quantitation of Panx1 expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of Panx1 under physiological and pathological conditions.