Jocelyne Cordier

Gap junctions in cultured astrocytes: Single-channel currents and characterization of channel-forming protein

Currents from gap junction channels were recorded from pairs of astrocytes in primary culture using the double whole-cell recording technique. In weakly coupled pairs, single-channel events could be resolved without pharmacological uncoupling treatment. Under these conditions, unitary conductance was 56 +/- 7 pS, and except for multiples of this value, no other level of conductance was observed consistently. To characterize the type of junctional protein constituting astrocyte gap junction channels, immunological and biochemical experiments were carried out on the same material. Specific cDNA probes for three connexins identified in mammals (Cx26, Cx32, and Cx43) showed that only Cx43 mRNA was expressed in cultured astrocytes. The presence of Cx43 protein in cultured astrocytes was demonstrated by immunoblotting, immunofluorescence, and immunogold labeling using anti-peptide antibodies specific to Cx43. These results strongly suggest that gap junctions in astrocytes have a 50-60 pS unitary conductance associated with channels composed of Cx43 protein.

Sphingosine-1-phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades

Sphingosine‐1‐phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg‐1 and Edg‐3. S1P stimulated thymidine incorporation and induced activation of extracellular signal‐regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P‐evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3‐kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin‐sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood–brain barrier.

Effects of General Anesthetics on Intercellular Communications Mediated by Gap Junctions between Astrocytes in Primary Culture

Background:Astrocytes represent a major nonneuronal cell population in the central nervous system (CNS) and are actively involved in several brain functions. These cells are coupled by gap junctions (GJ) into a syncytial-like network resulting in cellular communication through ionic and metabolic exchange between adjacent astrocytes. Whether anesthetics affect astrocyte function is not known. In the present study, the effects of general anesthetics on GJ permeability were investigated in primary cultures of mouse striatal astrocytes. Methods:Junctional permeability was determined by using the fluorescent probe Lucifer yellow and the scrape loading/dye transfer technique. Confluent cells were preincubated 5 min with various concentrations of anesthetic agents and GJ permeability was estimated by measuring the area occupied by the dye from digitalized images taken 8 min after cell loading. Results:Of the intravenous anesthetics tested, only propofol (P: 10-4M, P > 0.01 and 10>-5M, P > 0.05) and etomidate (ET: 10−4M, P > 0.05, but not 10−5) induced a significant reduction of GJ permeability. In contrast, diazepam (10−5M), morphine (10−4M), ketamine (10−4M), thiopental (10−4M), and clonidine (10−7M) did not affect junctional permeability. In addition, the halogenated anesthetics halothane, enflurane, and isoflurane induced a dose-dependent closure of GJ. For halothane, enflurane, and isoflurane, the maximum effect was achieved with a 10−4M, 1.6 X 10−3M, and 10-3M anesthetic concentration, respectively. Removal of volatile anesthetics resulted in the restoration of the control fluorescence area between 15 and 45 min. The time course of recovery of GJ permeability was examined more precisely for shorter periods of halothane administration (5 min, 1 mM). Under these conditions, the rate of dye spread returned to control values following anesthetic washout, while, during the same period of time, complete uncoupling of GJ was still observed in the presence of a 1 mM halothane concentration. Conclusions:These results indicate that general anesthetics differentially affect GJ permeability in cultured astrocytes. This uncoupling effect (closure of gap junctions) may contribute to the mechanisms of action of some anesthetic agents (primarily volatile anesthetics) at the level of the CNS by altering astrocyte communication.

Cellular Expression, Developmental Regulation, and Phylogenic Conservation of PEA-15, the Astrocytic Major Phosphoprotein and Protein Kinase C Substrate

Abstract: PEA‐15 has recently been identified as a major phosphoprotein in astrocytes and an endogenous substrate for protein kinase C. This 15‐kDa protein exists under three molecular forms, an unphosphorylated form, N, and two phosphorylated forms, Pa and Pb. Ȧntisera were raised against synthetic peptides corresponding to the internal sequences of the mouse protein containing the two specific phosphorylation sites and affinity‐purified antibodies were used for immunoblotting. PEA‐15 was found mainly in the cytosol, but its protein kinase C‐phosphorylated form, Pb, was also detectable in association with the membrane and remained with the fraction that contains stabilized microtubules. Abundant in astrocytes, particularly in the hippocampus, PEA‐15 was also detected in all cultured brain cell types examined, indicating a more ubiquitous distribution of the protein, further demonstrated by its detection in the eye and in the lung. Parallel to the increase in expression levels, phosphorylation of PEA‐15 also increased during development. This paralleled results obtained in primary cultures, where PEA‐15 levels increase with cell maturation. Finally, physiological importance of PEA‐15 phosphorylation was illustrated by immunoreactivity observed in brain homogenates of different mammals, birds, amphibians, and fish.

Anandamide and WIN 55212-2 inhibit cyclic AMP formation through G-protein-coupled receptors distinct from CB1 cannabinoid receptors in cultured astrocytes

The effects of anandamide and the cannabinoid receptor agonists WIN 55212‐2 and CP 55940 on the evoked formation of cyclic AMP were compared in cultured neurons and astrocytes from the cerebral cortex and striatum of mouse embryos. The three compounds inhibited the isoproterenol‐induced accumulation of cyclic AMP in neuronal cells, and these responses were blocked by the selective CB1 receptor antagonist SR 141716A. The three agonists were more potent in cortical than striatal neurons. Interestingly, WIN 55212‐2, CP 55940 and anandamide also inhibited the isoproterenol‐evoked accumulation of cyclic AMP in astrocytes but, in contrast to WIN 55212‐2 and CP 55940, anandamide was much more potent in striatal than cortical astrocytes. Inhibition was prevented by pertussis toxin pretreatment, but not blocked by SR 141716A. Therefore, G‐protein‐coupled receptors, distinct from CB1 receptors, are involved in these astrocytic responses. Moreover, specific binding sites for [3H]‐SR 141716A were found in neurons but not astrocytes. Furthermore, using a polyclonal CB1 receptor antibody, staining was observed in striatal and cortical neurons, but not in striatal and cortical astrocytes. Taken together, these results suggest that glial cells possess G‐protein‐coupled receptors activated by cannabinoids distinct from the neuronal CB1 receptor, and that glial cells responses must be taken into account when assessing central effects of cannabinoids.

Homotypic and Heterotypic Coupling Mediated by Gap Junctions During Glial Cell Differentiation In Vitro

Intercellular communication mediated by gap junctions was investigated during oligodendrocyte differentiation in primary and secondary cell cultures from newborn and adult rats. Two types of communication were considered: ionic coupling and dye‐coupling between similar oligodendrocytes selected at the same stage of differentiation (homotypic) and dye‐coupling between oligodendrocytes and astrocytes (heterotypic), Intercellular diffusion of fluorescent probes and double whole‐cell recordings were used to test the incidence of dye and ionic communication respectively. Progenitor cells, identified with A265 antibodies, were characterized by the absence of ionic and dye‐coupling, whereas oligodendrocytes, identified with galactosylceramide antibodies, exhibited both types of communication. This homotypic coupling was inhibited by various uncoupling agents, but unaffected by treatments which increased the intracellular concentration of CAMP. In cocultures of astrocytes and oligodendrocytes, Lucifer yellow and sulphorhodamine B were exchanged in both directions. This heterotypic dye‐coupling, which could be blocked by octanol, first appeared after 3 weeks in culture and increased to an incidence of 25% after 6 weeks, a developmental pattern comparable to homotypic dye‐coupling between oligodendrocytes. In contrast, during the same period, progenitors and microglia were never observed to be dye‐coupled with astrocytes.

Endothelins inhibit junctional permeability in cultured mouse astrocytes

Endothelins, a family of potent vasoconstrictor peptides initially characterized in peripheral tissues, have also been reported to be synthesized in the brain. In this structure several cell types, including astrocytes, endothelial cells and certain neurons, are potential targets for these peptides. In astrocytes, endothelins induce changes in the concentration of several second messengers (calcium, diacylglycerol, arachidonic acid, cAMP) known to be involved in the regulation of gap junction channels. Using the scrape loading/dye transfer technique we have observed that two isoforms of endothelin, endothelin‐1 and endothelin‐3, strongly inhibit the extent of dye‐coupling between confluent astrocytes, suggesting that gap junction permeability was reduced. This inhibitory effect on dye coupling was reproduced by the snake venom sarafotoxin. When used at 10−7 M, these three compounds had inhibitory effects on gap junction channels which were comparable to those induced by the well known uncoupling agents octanol and halothane. In the absence of extracellular calcium, the effects of endothelins were largely prevented, suggesting that second messengers linked to the activation of phospholipases C and/or A2, which both are dependent on external calcium, could be involved in the uncoupling mechanism.

Stathmin Phosphorylation Is Regulated in Striatal Neurons by Vasoactive Intestinal Peptide and Monoamines via Multiple Intracellular Pathways

Abstract: Stathmin is a ubiquitous soluble protein whose phosphorylation is associated with the intracellular mechanisms involved in the regulations of cell proliferation, differentiation, and functions by extracellular effectors. It is present in the various tissues and cell types as at least two distinct isoforms in their unphosphorylated (Mr∼ 19,000; pI ∼ 6.2–6.0) and increasingly phosphorylated forms. Stathmin is particularly abundant in brain, mostly because of its high concentration in neurons, where the protein is a major phosphorylation substrate. In intact striatal neurons grown in primary culture, the cyclic AMP–increasing drug forskolin and the protein kinase. C–activating agent 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) induced a potent phosphorylation of stathmin. Their actions were at least partially additive, appearing actually most likely “sequential” on various phosphorylated states of stathmin. Vasoactive intestinal peptide (VIP) reproduced the forskolin‐like stimulation but stimulated also other, TPA, and/or Ca2+‐like protein phosphorylations. These actions of VIP were already maximal after 5 min and were long lasting, still important after 2 h. In addition, concentrations as low as 1 nM were enough to obtain a significant effect, on both cyclic AMP‐dependent and independent phosphorylations. Dopamine and the β‐adrenergic agonist isoproterenol were also able to stimulate stathmin phosphorylation, but only with a forskolin‐like pattern. Their actions were not additive to those of VIP, confirming previous results on the colocalization of both dopamine D1 and nor‐adrenaline β1 receptors with VIP receptors on striatal neurons. In conclusion, our results show that VIP regulates the functions and differentiation of embryonic striatal neurons through multiple intracellular pathways and further substantiates the role of stathmin as a cytoplasmic relay integrating multiple second messenger signals.

Synergistic Regulation of Cytosolic Ca2+ Concentration in Mouse Astrocytes by NK1 Tachykinin and Adenosine Agonists

The effects on cytosolic Ca2+ concentration of 2‐chloroadenosine and [L‐Pro9]‐substance P, a selective agonist of NK1 receptors, were investigated on astrocytes from embryonic mice in primary culture. Cells responded to [L‐Pro9]‐ substance P with a transitory increase in cytosolic Ca2+ which was of shorter duration when external Ca2+ was removed. A transient response to 2‐chloroadenosine alone occurred. When simultaneously applied, [L‐Pro9]‐substance P and 2‐chloroadenosine evoked a prolonged elevation of cytosolic Ca2+ (up to 30 min). This phenomenon was dependent on the presence of extracellular Ca2+, but insensitive to dihy‐dropyridines, La3+, and Co2+, excluding the implication of voltage‐operated Ca2+ channels. Arachidonic acid also induced a sustained elevation of cytosolic Ca2+, but did not increase further the response evoked by [L‐Pro9]‐substance P and 2‐chloroadenosine. The activation of protein kinase C by a diacylglycerol analogue mimicked the effect of [L‐Pro9]‐substance P in potentiating the 2‐chloroadenosine‐evoked response. Like 2‐chloroadenosine, pinacidil, which hyper‐polarizes the cells by opening K+ channels, prolonged the elevation of cytosolic Ca2+ concentration induced by [L‐Pro9]‐substance P. Conversely, depolarization with 50 mM KC1 canceled the effects of either pinacidil or 2‐chloroadenosine applied with [L‐Pro9]‐substance P. Pertussis toxin pretreatment suppressed all the effects induced by 2‐chloroadenosine.

p38/SAPK2 controls gap junction closure in astrocytes.

Astrocyte gap junction communication (GJC) is thought to contribute to death signal propagation following central nervous system injury, noteworthy in some ischemia/anoxia models. The inhibition of p38/stress‐activated protein kinase 2 (p38/SAPK2) by a pyrimidyl imidazole derivative has been reported to reduce the extent of the lesion area after cerebral ischemia. Therefore, interleukin‐1β (IL‐1β), which contributes to stroke‐induced brain injury and activates p38/SAPK2, and hyperosmolarity induced by sorbitol, a potent stimulus of p38/SAPK2 in non‐neuronal cells, were used to investigate a possible involvement of p38/SAPK2 in GJC modulation in mouse cultured astrocytes. Both stimuli inhibited dye coupling within minutes. The IL‐1β effect was transient, while that of sorbitol lasted up to 90 min. Both stimuli induced a rapid p38/SAPK2 activation, the kinetic of which matched that of induction of dye coupling inhibition. Immunocytochemical studies showed that IL‐1β and sorbitol induced a p38/SAPK2 translocation from the nucleus to the cytoplasm. The pharmacological agent SB203580 specifically blocked p38/SAPK2 activation, cytoplasmic translocation and reversed the IL‐1β and sorbitol‐induced inhibition of GJC. Further characterization of the p38/SAPK2 mode of action on GJC, performed with sorbitol, revealed an increased phosphorylation of protein kinase C (PKC) substrates abolished by both PKC inhibitors and SB203580. Expression and serine phosphorylation of connexin 43, the main component of astrocyte gap junctions, were unchanged, suggesting the existence of additional intracellular signaling mechanisms modulating the channel gating. Altogether, these results demonstrate that p38/SAPK2 is a central mediator of IL‐1β and sorbitol inhibitory actions on GJC and establish PKC among the distal effectors of p38/SAPK2.