Metabolic Trafficking ThroughAstrocytic Gap Junctions

CHRISTIAN GIAUME, * ARANTXA TABERNERO,2 AND JOSE M. MEDINA2

1INSERM U114, College de France, Paris, France2Departamento de Bioquimicay Biologia Molecular, Universidad de Salamanca, Salamanca, Spain

KEY WORDS intercellular communication; connexins; glucose; lactate; glutamate

ABSTRACT Astrocytes are interposed between the pericapillary space and neuronalmembranes. Consequently, they may represent an important intermediary elementbetween the source of energetic substrates and the main site of energy-consumingelements, respectively, the blood circulation and the neurons. A typical feature ofastrocytes is the connections they establish between each other by specialized membranestructures, defined as gap junctions. These intercellular junctions allow direct cell-to-cellexchanges of ions and small molecules, including several compounds involved in majormetabolic pathways occuring in astrocytes. The permeability of astrocytes gap junctionchannels is controled by several endogeneous compounds released by astrocytes them-selves or by other brain cell types, including neurons and endothelial cells. In primarycultures of astrocytes, the intercellular diffusion, the utilization and the uptake ofglucose and derivates are modified when gap junctional permeability is inhibited byuncoupling agents. Altogether these observations indicate that intercellular pathwaysconstituted by groups of coupled astrocytes could participate to the metabolism and thedistribution of energetic substrates throughout the brain. GLIA 21:114–123, 1997.r 1997 Wiley-Liss, Inc.

INTRODUCTION

One of the central questions related to energy metabo-lism within the brain concerns the identification ofintermediary steps involved in the traffic of nutrients.Typically, they are delivered by blood circulation, thesource of energetic substrates, to the neurons thatrepresent the main site of energy-consuming elements.The study of brain metabolism includes the identifica-tion of the anatomical elements that actively partici-pate in this traffic and of the different biochemicalreactions leading to the transformation of glucose. Thiscompound, which represents the obligatory energy sub-strate for brain provided by blood circulation, is trans-formed into carbon fuel and then used by neurons.Although a diffusion of glucose through the extracellu-lar cleft from capillaries to neurons cannot be excluded(see Coles, 1995), it seems more likely that astrocytesplay a key role in the supply of energetic compounds toneurons. This hypothesis is strengthened by the lack ofclear evidence for direct relation between neurons andblood vessels. Accordingly, astrocytes could act as inter-mediary elements and thus form the first cellularbarrier that glucose entering the brain encounters after

cerebral endothelial cells that participate in the brain-blood barrier (Magistretti et al., 1993; Tsacopoulos andMagistretti, 1996). In fact, as these cells are physicallyinterposed between the capillaries and neuronal ele-ments, they have been traditionally assigned a role inmetabolic support of neurons (Hamprecht and Dringen,1995). Moreover, astrocytes are ideally suited for such anutrient role. Indeed, glucose is processed glycoliticallyin astrocytes that store glycogen at the point wherethey can be identified at the ultrastructural level by thepresence of glycogen granules, astrocytic end-feet sur-round intraparenchymal blood vessels, and processes ofastrocytes are also in close contact with neurons (Peterset al., 1991). In addition, a different glucose transporterfor neurons and astrocytes may also contribute tocarbohydrate targeting between both cell types, theglucose transporter GLUT-1 being mainly located in

Abbreviations: Cx, connexin; 2-DG, 2-deoxyglucose; TAC, tricarboxylic acidcycle; Cx43, connexin 43.

*Correspondence to: Dr. C. Giaume, INSERM U114, College de France, 11,Place Marcelin Berthelot, 75231 Paris Cedex 05 France. E-mail:giaume@infobiogen.fr

Accepted 11 November 1996.

GLIA 21:114–123 (1997)

r 1997 Wiley-Liss, Inc.

astrocytes while neurons use GLUT-3 (Maher et al.,1994). Thus due to their typical morphology, theirintimate ensheating of blood capillaries, and synapticcontacts, their specific glucose uptake, and enzymaticactivity, astrocytes may participate to several brainfunctions. For example, they can sense synaptic activ-ity, evaluate the neuronal need for energetic metabo-lites, and dispatch metabolites for the maintenance ofenergy balance in neurons.

In situ this metabolic role for astrocytes could beplayed by single and independent astrocytes (Fig. 1).However, since a characteristic feature of in vivo and invitro astrocytes is an abundant expression of intercellu-lar gap junctions (Massa and Mugnaini, 1982, 1985;Mugnaini, 1986), it seems more likely that functionallycoupled astrocytes may be involved in metabolic traffick-ing (Fig. 1). Effectively, gap junction channels arepermeable to molecules of a molecular weight up to1000–1200 Da (Loewenstein, 1981), which provide inter-cellular pathways, allowing groups of cells to communi-cate and thus exchange small molecules through activeor passive processes. Since glucose and most of itsderivates are under this size limit, the participation ofgap junctions to metabolic trafficking could be consid-ered at least in two different ways. Junctional meta-bolic coupling could take place linearly by the connec-tion of two or more astrocytes forming a ‘‘shuttlesystem’’ between the blood vessels and the neurons (Fig.1). Alternatively, the presence of gap junctions betweenastrocytic endfeet and between astrocytic processescould favor lateral intercellular communication be-

tween groups of astrocytes contacting distinct neurons.This later possibility is more relevant in brain regionswhere capillaries are in high density (WagmanBorowsky and Collins, 1989) and are likely presentwithin a small distance from neurons.

The purpose of this chapter is to determine whetheror not intercellular communication, mediated betweenastrocytes by gap junctions, participates in the distribu-tion of glucose and the regulation of its metabolismwithin the brain. First, this has been addressed byfollowing the intercellular diffusion of radiolabeledcompounds in the presence of various pharmacologicaltreatments known to affect the permeability of astro-cytic gap junctions. Second, the consequences of alteredjunctional communication in cultured astrocytes andglioma cells has also been investigated by comparingthe fate and the uptake of glucose in communicatingand non-communicating astrocytes. As a whole theseresults should contribute to define the role played byastrocytes in brain metabolism in a way that takes intoaccount the rather unique network organization theyform within the brain.

METABOLIC FUEL UTILIZATIONBY ASTROCYTES

Glucose is the main metabolic substrate for the brainalthough ketone bodies (Lopes-Cardozo and Klein, 1985;Lopes-Cardozo et al., 1986; Williamson, 1982), lactate(for a review, see Medina et al., 1992), and glutamine(Vicario et al., 1993) may also play a role as a source ofenergy and lipids for brain development. Transport ofglucose into astrocytes is carried out by the glucosetransporter GLUT-1, which is an ubiquitous, non-insulin-sensitive transporter with a high affinity forglucose (Maher et al., 1994). Once glucose is inside theastrocyte, it may undergo transformation through gly-colysis, through the pentose phosphate shunt (PPS) orbe stored as glycogen (Fig. 2). Glucose utilization iscontrolled by the hexokinase-catalyzed reaction, a keystep in glucose utilization in the brain (Turek et al.,1986). When stored, glucose is accumulated in astro-cytes in the form of glycogen, which is the maincarbohydrate reserve throughout the brain (Cataldoand Broadwell, 1986). Although glucose-6-phosphataseactivity has been detected in some astrocytes (Bell etal., 1993), it is commonly accepted that most astrocyteslack glucose-6-phosphatase. This enzyme catalyzes thelast step in the pathway of glycogen breakdown andhence glucose-6-phosphate, but not glucose, is the endproduct of glycogenolysis in astrocytes. Accordingly, theproducts of glycogen breakdown cannot leave the astro-cyte because the plasma membrane is not permeable tophosphorylated compounds. However, glucose-6-phos-phate may be transformed into lactate by glycolysis(Fig. 2), which can freely cross the plasma membraneand thus become available to neurons (Dringen et al.,1993). In fact, it has been shown that lactate transportinto neurons is mediated by a specific carrier (Dringen

Fig. 1. Schematic representation of possible astrocytic pathways forenergetic metabolites from a capillary to a neuron. Two hypotheticalroutes are considered and labeled by arrows. One takes into accountonly a single astrocyte intermediary between the circulation andneurons (upper arrow), while the other supposes that metabolitestransit through a more complex pathway involving interastrocytic gapjunctions (lower arrow). The location of gap junctions is represented bybridges between astrocytes.

115GAP JUNCTIONS AND METABOLISM IN ASTROCYTES

et al., 1993) and that these cells exhibit a high capacityfor lactate utilization, not only as a source of energy(Vicario et al., 1993) but also as a precursor for lipidsynthesis (Tabernero et al., 1993). In agreement withthis, astrocytes also exhibit a high capacity for lactateutilization, suggesting that lactate may be an impor-tant substrate for brain cells, particularly during devel-opment (Tabernero et al., 1993; Vicario et al., 1993). Infact, lactate is used not only for the requirement ofastrocytes themselves but is also exported to the extra-cellular medium (Fig. 3), probably in the form of citrateand/or other tricarboxylic acid cycle intermediates(Sonnewald et al., 1991; Westergaard et al., 1994).Bovine serum albumin (BSA), which has previouslybeen reported to enhance lactate utilization (Vicario etal., 1993), increases the export of carbons from lactateto the extracellular medium (Fig. 3). This phenomenonis not observed in neurons (Fig. 3) because these cells

lack pyruvate carboxylase activity (Cesar and Ham-precht, 1995; Shank et al., 1985), a compulsory step foroxalacetate synthesis (Fig. 2). Indeed, the transfer ofcitrate and other tricarboxylic acid cycle intermediatesfrom astrocytes to neurons is probably designed toreplenish tricarboxylic acid cycle intermediates, whichare depleted in neurons since these compounds areused as neurotransmitter precursors (Kaufman andDriscoll, 1992).

GAP JUNCTIONAL COMMUNICATIONIN ASTROCYTES

Gap junctions are classically described as assembly ofintercellular channels aggreged at close apposition

Fig. 2. Major metabolic pathways in astrocytes. HK, hexokinase;PPS, pentose phosphate shunt; PRPP, phosphoribosyl-pyrophosphate;PDH, pyruvate dehydrogenase complex; PC, pyruvate carboxylase;OAA, oxalacetic acid; a-KG, alpha-ketoglutarate.

Fig. 3. Export of carbons by astrocytes. Rat neurons and astrocytesin primary culture were incubated with 5 mM sodium bicarbonate and500–100 dpm/nmol of 14CO3HNa in the absence or presence of 10 mML-lactate or 10 mM L-lactate plus 2% bovine serum albumin (BSA) andthe radioactivity incorporated into the cells (upper panel) or releasedinto the incubation medium (lower panel) was measured by liquidscintillation counting. ***P , 0.001 as compared to the valuesobtained in the absence of lactate by Student’s t test.

116 GIAUME ET AL.

between cellular membranes of adjacent cells (seeBennett, 1977). Owing to their particular location,these junctional channels allow direct intercellularcommunication between the cytoplasm of neighboringcells. Gap junction channels are poorly selective for ions(Neyton and Trautman, 1985) and permeable for mol-ecules of low molecular weight (see Loewenstein, 1981),which account for functional properties of permeabilitydefined as electrical (or ionic) and chemical (or meta-bolic) coupling. The ultrastructure and framework of ajunctional channel is now well established (see Bennettet al., 1991), each channel being composed by the facingand docking of two hemichannels (or connexons) formedby a ring of six protein subunits, called connexins. Sincethe mid-1980, some 16 members of the connexin familyhave been sequenced and a general model of theirthree-dimensional structure has been proposed (seeBruzzone et al., 1996; Peracchia et al., 1994).

In the central nervous system the distribution of gapjunctions is now becoming largely documented (seeDermietzel and Spray, 1993; Giaume and Venance,1995; Ransom, 1995; Wolburg and Rohlmann, 1995).Besides neurons where they were studied initially (seeBennett, 1977), gap junctions have been found in mosttypes of brain cells including glial (and ependymalcells), pineal, endothelial, and leptomeningial cells.Among these, the expression of gap junctions is particu-larly abundant and widespread in glial cells and moreprecisely in astrocytes (Mugnaini, 1986). Furthermore,astrocytes have the equipment to receive, integrate,and transmit signals to surrounding neurons since theyexpress a wide variety of membrane ionic channels,transporters, and receptors linked to most known signal-transduction pathways (see Kettenmann and Ransom,1995). This is of particular interest since there is nowincreasing evidence that astrocytes may have a moreactive role in brain functions that initially thought(Smith, 1994; Travis, 1994). Such an active participa-tion supposes that astrocytes have developed an effi-cient and reliable way to communicate in order tointeract with each other and to coordinate their behav-ior. Gap junction mediated intercellular communica-tion could play this role by providing the morphologicalbasis for functional astrocytic networks.

In mammals, evidence for astrocytic gap junctionshas been shown by electron-microscopical and freeze-fractures studies performed in both white and graymatter (Dermietzel, 1974; Massa and Mugnaini, 1982).Their functionality has been demonstrated in brainslices mainly by dye-coupling experiments, performedby injecting low molecular weight dye in individualastrocytes and showing its diffusion into neighboringcells (Binmoller and Muller, 1992; Connors et al., 1984;Gutnick et al., 1981). In vitro astrocytes also expressfunctional gap junctions (Kettenmann et al., 1983) andprimary cultures have provided an easier preparationto address the question of their molecular compositionand pharmacology (see Giaume and McCarthy, 1996).Thus, the combination of immunological, biochemical,and electrophysiological techniques performed with

pure (.95%) primary cultures of astrocytes have dem-onstrated that connexin 43 (Cx43) was the majormolecular constituent of gap junctions in this type ofglia (Dermietzel et al., 1991; Giaume et al., 1991a). Thiswas also observed by in situ hybridization and immuno-cytochemistry, since Cx43 mRNAs and immunoreactivesites for Cx43 antibodies were found to be localized inastrocytes (Micevitch and Abelson, 1991; Yamamoto etal., 1990b). Taken together these in situ and in vitrodata indicate than astrocytes are widely coupled to eachother and that the major Cx they express is Cx43.However, this does not exclude that other Cx could bepresent in specific brain regions, in subtypes of astro-cytes, or in certain conditions during development orpathologies. Indeed Cx40 mRNAs have been detected inhippocampal and cerebelar astrocytes (Zheng et al.,1995) and evidence for the expression of another Cx hasbeen reported in astrocytes cultured from Cx43 knock-out mice (Spray et al., 1995), although intercellularcommunication are drastically altered in these cells(Bechberger et al., 1995).

In vivo, localization of astrocytic gap junctions, iden-tified either by morphological criteria or with anti-Cx43antibodies, have been achieved at both cellular andultrastructural levels. They indicated that couplingsites between astrocytes occur near neuronal somata aswell as the neuropil, and that they are located betweenprocesses or cell bodies and between cell bodies andprocesses (Massa and Mugnaini, 1982; Yamamoto et al.,1990a,b). Although mRNA and expression of Cx43 iswidespread within the CNS, their distribution is heter-ologous and exhibits clear difference between nuclei,suggesting that functional delimitation and comparti-mentalization may occur (Micevytch and Abelson, 1991;Yamamoto et al., 1990b). Another remarkable featurefor Cx43 expression is the observation of very intenselystained puncta of 1 to 2 µm diameter that are seenarranged in meandering interconnected linear se-quences forming a ‘‘honeycomb’’ meshwork (Fig. 4).Such an important expression of gap junctions betweenastrocytic end-feet known to ensheath most of braincapillaries, indicates that there is likely a high degreeof intercellular communication at this level, whichcorresponds to the entry in the brain of energeticmetabolites carried by the blood circulation and up-taken by astrocytes.

PERMEABILITY OF ASTROCYTIC GAPJUNCTIONS FOR METABOLIC COMPOUNDS

Classically, most of the studies performed in order todemonstrate the occurrence of gap junction mediatedintercellular communication have been carried outusing either electrophysiological techniques or intracel-lular injection (or loading) of low molecular weighttracers or fluorescent dyes (Loewenstein, 1981; Spray,1994). On the whole, these studies have led to theconclusion that the junctional channel is an aqueouspore of large diameter and low selectivity. While, based

117GAP JUNCTIONS AND METABOLISM IN ASTROCYTES

upon permeability studies, the predicted critical chan-nel diameter was initially estimated to be 16A8 (Flagg-Newton and Loewenstein, 1979), atomic force micros-copy has now led to propose a larger diameter for thepore of the channel, which is about 1.5 to 2 nm (Hoh etal., 1991).

From observations, which were mainly based uponthe use of intercellular probes that are not physiologi-cally relevant (Procion yellow, 6-carboxyfluocein, Luci-fer yellow, biocytin, calcein, etc.), it was assumed thatexchange of signalling molecules and metabolic coopera-tion may also occur. However, in most cases no directdemonstration was provided to confirm this statement(see Sheridan and Atkinson, 1985). For instance, con-cerning the permeability of gap junctions for energeticmetabolites, only a little information is available. Junc-tional permeability for glucose has been mainly testedusing [3H]-2-deoxyglucose (2DG), since this tracer en-ters the cells by substituting for glucose; once inside itis phosphorylated and in this form is neither metabo-lized further nor may it recross the cell membrane. Sofar, junctional permeability for this compound has beendemonstrated in cell lines (Pitts and Finbow, 1977), inlenses (Goodenough et al., 1980), and between uterinesmooth muscle cells (Cole and Gardfield, 1986; Cole etal., 1985). However, these observations were collectedat a time when the diversity in the connexin family wasnot known. As there is now increasing evidence that

indicates that selectivity of junctional communicationdepends on the type of connexin expressed (Elfgang etal., 1995; Steinberg et al., 1994) and their state ofphosphorylation (Kwak et al., 1995), these initial datacannot simply be extended to all coupled cell systems.

Recently, the diffusion through gap junctions of vari-ous energetic metabolites was evaluated by adaptingthe scrape-loading dye transfer technique (El-Fouly etal., 1987), by using several radioactive glucose com-pounds and following their intercellular spread byautoradiography (Tabernero et al., 1996). This method,which has to be performed with a confluent cell popula-tion, results in the loading of a large number of cellsafter they have been transiently open to the extracellu-lar medium devoided of calcium ions and containing ahigh concentration of the probe used to test gap junc-tion permeability. After a wash, if junctional channelsare functional, an intercellular diffusion of the dye isobserved, provided the probe used does not leak outsidethrough the plasma membrane. Such a technique iswell suited for a membrane impermeant dye such asLucifer yellow but has to be modified when glucosecompounds are used. Thus, to prevent diffusion oflabeled metabolites through the plasma membrane, theexperiments have to be carried out at low temperature(4°C) in the presence of a saturating concentration ofthe same non-radioactive metabolite. Moreover, after adefine time (1–2 min) is allowed for intercellular diffu-

Fig. 4. Typical ‘‘honeycomb’’ pattern of connexin43 immunoreactiv-ity along brain blood vessels. A: Low magnification montage of Cx43immunolabeling in cerebral cortex; superficial is to left. B: Highermagnification of vessel in layers III–IV of the cortex showing thehoneycomb meshwork of labeling formed by punctuate Cx43-immuno-

reactivity distributed along the vessel. Areas circumscribed by immu-nostaining puncta represent astrocytic end-feet. C: Interference con-trast of similar labeling along a blood vessel at the cortical surface.Magnifications: A, 390; B, 3200; C, 3150 (illustration provided by Dr.J.I. Nagy, The University of Manitoba, Canada).

118 GIAUME ET AL.

sion, preparations are ‘‘fixed’’ by drying the cells on ahotplate (100°C). Prints of the distribution of the la-beled compounds are finally obtained by exposing thedried cover slips to a X-ray sensitive film, which is thenphotographed and scanned for quantification (Taber-nero et al., 1996).

Figure 5 shows the passage of glutamine and gluta-mate through astrocyte gap junctions with this tech-nique. Diffusion of these metabolites is inhibited byoctanol, a well-known inhibitor of gap junction perme-ability, suggesting that glutamine and glutamate canbe distributed throughout the brain by passage throughastrocytic gap junctions. In fact, glutamine is an excel-lent energetic substrate for both neurons and astro-cytes in primary culture (Vicario et al., 1993). Gluta-mine crosses the blood-brain barrier (Sanchez-del-Pinoet al., 1995) and its carbons are easily decarboxylated inthe tricarboxylic acid cycle (Fig. 2); this is consistentwith the idea that glutamine is a very importantenergetic substrate. Although glutamine is a poor sub-strate for lipid synthesis in both neurons and astrocytes

(Vicario et al., 1993), its carbons can also be used toreplenish tricarboxylic acid cycle intermediates in neu-rons (Schousboe et al., 1993). The passage of glutamineand glutamate through astrocytic gap junctions mayalso play an important role in the operation of theglutamine and glutamate cycle between neurons andastrocytes (Schousboe et al., 1993). The above-described technique has also been used to demonstratethat astrocytic gap junctions are freely permeable toglucose (Tabernero et al., 1996). In addition, the phos-phorylated form of glucose, i.e., glucose-6-phosphate,also crosses astrocytic gap junctions (Tabernero et al.,1996), suggesting that gap junctions are permeable tolow molecular weight phosphorylated compounds. Sincethe plasma membrane is not permeable to phosphory-lated compounds, gap junctions may be responsible forthe traffic of phosphorylated metabolites between astro-cytes. However, glucose phosphorylation is not compul-sory for the passage of glucose through gap junctionssince the non-phosporylisable glucose derivative 3-ortho-methyl-glucose diffuses through gap junctions (Taber-nero et al., 1996).

The main product from glucose and probably glyco-gen metabolism in astrocytes is lactate (Dringen et al.,1993), which is strongly synthesized in both normal andpathological situations. Astrocytic gap junctions arepermeable to lactate, which may contribute to itsdiffusion through brain structures (Tabernero et al.,1996). In fact, both astrocytes and neurons show a highcapacity for lactate utilization as a source of bothenergy and carbon skeletons for lipid synthesis. Thismay explain why lactate is able to support neuronalactivity (Bock et al., 1993; Izumi et al., 1994; Maran etal., 1994).

The passage of glucose-6-phosphate and lactatethrough gap junctions may play a role in the traffickingof glucose carbons between astrocytes. Although brainglycogen is mainly confined in astrocytes (Cataldo andBroadwell, 1986), these lack glucose-6-phosphatase ac-tivity, even though this enzyme may be found in certainastrocytes (Bell et al., 1993). Since the plasma mem-brane is not permeable to phosphorylated compounds,glucose-6-phosphate is confined within the astrocyteand is likely channelled to adjacent astrocytes throughgap junctions. Glucose-6-phosphate can be transformedinto lactate through the glycolytic pathway for releasetowards neurons. Indeed, it has been reported that themain product of glycogen breakdown in cultured astro-cytes is lactate (Dringen et al., 1993), which is releasedinto the medium presumably for use by neurons. Itshould be noted that glycogen has been proposed as asource of energy for memory processes (O’Dowd et al.,1994). If so, the above-described mechanism for traffick-ing glycogen products through brain structures shouldbe stressed.

One consequence of the activation of several astro-cytic membrane receptor subtypes is the up- or down-regulation of gap junction permeability, which indicatesthat junctional communication in astrocytes may ex-hibit some degree of plasticity and remodeling (see

Fig. 5. Passage of glutamine and glutamate through astrocyte gapjunctions. Astrocytes in primary culture on coverslips were scrape-loaded in the presence of 50 mM glutamine or glutamate and 20µCi/mL of [14C]-U-glutamine or [14C]-glutamate. Dried coverslips wereautoradiographed and the digitalized images scanned perpendicularto the scrape. Note that this technique does not allow cellularresolution. The difference in background between control conditionsand uncoupling by octanol indicates a difference in diffusion of theradiolabeled compounds. In profiles of the scanned images, the closevalues measured at the edge of the cut indicate that in all conditionsthe initial loading of the cells were comparable.

119GAP JUNCTIONS AND METABOLISM IN ASTROCYTES

Giaume and McCarthy, 1996). For instance, noradrena-line, ATP, and glutamate have been reported to controlthe gap junctional communication in astrocytes. More-over, endothelins, a family of peptides produced byseveral brain cell types including endothelial cells(Yoshimoto et al., 1990), astrocytes (Mac Cumber et al.,1990) and certain neurons (Fuxe et al., 1991), arepotent inhibitors of astrocytic gap junctions (Giaume etal., 1992). Although the final molecular mechanismsinvolved in these regulations remain to be determined,there are several intracellular signalling pathways thatare suspected to participate in these processes. All thereceptors agonists that lead to an inhibition of junc-tional communication in astrocytes have in common toactivate phospholipases C and/or A2, suggesting thatproducts of the activity of these enzymes could beinvolved in the regulation of astrocytic gap junctionalcommunication (Giaume and McCarthy, 1996). It shouldbe noted that arachidonic acid, an important product ofthese phospholipases, is a potent inhibitor of astrocyticgap junctional communication (Giaume et al., 1991b).These mechanisms may be operative in the regulationof the passage of metabolites through gap junctionsbecause endothelin and its second messenger arachi-donic acid inhibit gap junction permeability for glucoseand its derivatives (Tabernero et al., 1996). This sug-gests that the passage of metabolites through astrocyticgap junctions can be regulated paracrinely.

REGULATION OF GLUCOSE UTILIZATIONAND GAP JUNCTION PERMEABILITY

IN ASTROCYTES

Gap junction permeability could play a role in theregulation of glucose utilization since inhibition of gapjunction communication, by endothelin-1 or uncouplingagents, sharply increases glucose uptake in culturedastrocytes (Tabernero et al., 1996). In addition, theinhibition of gap junction permeability results in asignificant increase in the activity of the pentose phos-phate shunt, whereas the rates of the tricarboxylic acidcycle, pyruvate dehydrogenase, and lipogenesis arescarcely changed (Tabernero et al., 1996). Although themechanism by which the inhibition of gap junctionpermeability increases glucose uptake is not known, itcould be suggested that the enhancement of glucoseuptake caused by the inhibition of gap junction perme-ability would be associated with an increase in Na1/K1

ATPase activity. Thus, glucose uptake may be linked toan inward Na1 gradient (Brookes and Yarowssy, 1985),which would depend on Na1/K1 ATPase activity (Taka-hashi et al., 1995). Consistent with this, it has beenreported that endothelin-1 stimulates Na1/K1 ATPasein brain capillary endothelium (Kawai et al., 1994) andthat ouabain, a specific inhibitor of Na1/K1 ATPase,inhibits glucose uptake in cultured astrocytes (Brookesand Yarowssy, 1985).

The mechanism by which the inhibition of gap junc-tion permeability stimulates pentose phosphate shunt

activity is intriguing. However, mechanisms that trig-ger proliferation are accompanied by an inhibition ofgap junction permeability (for a review, see Klaunigand Ruch, 1990). Indeed, an increase in proliferationdepends on a supply of ribose-5-phosphate for nucleicacid synthesis (Fig. 2). It is important to note that onemolecule of glucose is consumed by the pentose phos-phate shunt to synthesize one molecule of ribose re-quired for each nucleotide and therefore genome replica-tion requires a huge amount of glucose. The ultimatemechanism by which genome replication stimulates thepentose phosphate shunt remains to be elucidated.However, it may be speculated that the plunge innucleotide concentrations due to an enhancement ofnucleic acid synthesis would deinhibit 5-phosphoribosyl-1-pyrophosphate synthetase activity, resulting in ahigher demand for ribose-5-phosphate (Fig. 2).

In conclusion, the inhibition of gap junction permeabil-ity enhances glucose utilization through the pentosephosphate shunt, which is accompanied by an increasein glucose uptake by the astrocyte (Tabernero et al.,1996). This may be designed to fuel ribose-5-phosphatesynthesis. In short, extinction of the syncytial operationof astrocyte metabolism brings about a drastic changein glucose utilization, probably towards fated cellularproliferation.

THE ROLE OF ASTROCYTIC GAPJUNCTIONS IN NORMAL ANDPATHOLOGICAL SITUATIONS

The end-feet of astrocytes completely surround bloodcapillaries and their processes mingle with those ofneurons, suggesting that astrocytes constitute a physi-cal bridge in the supply of metabolic substrates fromblood to neurons. Since these cells are extensivelycoupled by gap junctions, groups of astrocytes maycooperate in the trafficking of metabolites betweenblood and brain cells. Indeed, gap junctions betweenadjacent astrocytes are permeable to metabolites suchas glucose, lactate (Tabernero et al., 1996), glutamine,and glutamate (Fig. 5). It may therefore be suggested,that astrocytic gap junctions would be used as ‘‘cellularlocks’’ that power the channelling of metabolic sub-strates throughout the brain (Fig. 1). This system maybe essential for the transport of glucose-6-phosphate,which coming from glycogen breakdown cannot leaveastrocytes because the plasma membrane is imperme-able to phosphorylated compounds. On the other hand,energy metabolism can control gap junction permeabil-ity because intracellular concentrations of ATP regu-late junctional communication in astrocytes. Indeed,the decrease of ATP concentration promotes the inhibi-tion of gap junction permeability by a calcium-depen-dent mechanism (Vera et al., 1996).

Since quiescent astrocytes are profusely coupled bygap junctions, their metabolism operates syncytially.However, when signals for cell proliferation are trig-gered cellular communication through gap junctions is

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inhibited while metabolism changes to adapt to thisunique situation. In fact, the inhibition of permeabilitythrough gap junctions increases the activity of thepentose phosphate shunt (Tabernero et al., 1996), whichsupplies ribose-5-phosphate for nucleic acid synthesis.In order to account for the enhancement of glucoserequirements, the uptake of glucose sharply increasesas a consequence of the inhibition of gap junctionpermeability (Tabernero et al., 1996). Moreover, duringthe first week of development after birth astrocyte gapjunctions are not permeable to Lucifer yellow until theybegin to differentiate (Binmoller and Muller, 1992), afact consistent with the idea that intercellular commu-nication must be interrupted during proliferation. Inregard to this, it is noteworthy that endothelins, whichhave a mitogenic effect on astrocytes (Mac Cumber etal., 1990), are also potent inhibitors of astrocytic gapjunctions (Giaume et al., 1992) and block the intercellu-lar diffusion of metabolites in association with anincrease in the uptake of 2DG (Tabernero et al., 1996).

Astrocytes respond to an ischemic insult by reorganiz-ing their gap junctions in such a way that Cx43decreases in the damaged focus but increases in thesurrounding astrocytes (Hossain et al., 1994). Thismechanism may be designed to confine reactive gliosiswithin ischemic damaged areas. Likewise, gap junc-tions may also play an important role in the progressionof neurodegenerative disorders. Thus, it has recentlybeen shown that the induction of nitric oxide synthaseby lipopolysaccharide treatment inhibits gap junctionpermeability, a process mediated by free radical com-pounds derived from peroxynitrite (Bolanos and Me-dina, 1996). Under conditions of inflammation causedby sepsis, encephalitis, multiple sclerosis, or Parkin-son’s disease, high brain levels of endotoxins and/orcytokines have been described (Banati et al., 1993;Boka et al., 1994; Merrill, 1987). Some of these condi-tions are known to be associated with the induction ofnitric oxide synthase in astrocytes (Bernatowicz et al.,1995; Bo et al., 1994; Endoh et al., 1994), which mayresult in the inhibition of gap junction permeability.Consequently, this mechanism may contribute to con-fine toxic products and thus prevent the damage fromspreading.

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