10 Amino Acids and Brain Volume Regulation Contribution and Mechanisms

8/13/2019 10 Amino Acids and Brain Volume Regulation Contribution and Mechanisms

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10 Amino Acids and Brain Volume

Regulation: Contribution andMechanismsH. Pasantes Morales

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

2 Hyposmotic Swelling and Regulatory Volume Decrease: Amino Acid Contribution . . . . . . . . . 227 2.1 Amino Acid Levels in Brain During Volume Adjustment to Hyposmolarity . . . . . . . . . . . . . . . . . . 2272.2 The Mechanism of Regulatory Volume Decrease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2282.2.1 The Amino Acid Efflux Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2292.2.2 The Osmotransduction Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2302.3 Hyposmolarity Induced Release of Amino Acids from Nerve Endings . . . . . . . . . . . . . . . . . . . . . . . . . 2312.4 Volume Regulation After Gradual Decreases in Osmolarity: The Relevance of Amino Acids . 2332.5 Taurine as an Osmotransmitter of Neurohormone Output . .. . .. . . .. . .. . .. .. .. . .. .. .. . .. . . .. .. . 235

3 Isosmotic Swelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 3.1 Amino Acids and Isosmotic Swelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

4 Brain Cell Volume Decrease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 4.1 Mechanism of the Compensatory Increase in Amino Acid Levels During Hypernatremia . . . . 2394.1.1 Hypertonicity and Osmolyte Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2394.1.2 Hypertonicity and Osmolyte Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

5 Proliferation, Apoptosis and Cell Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 5.1 Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2425.2 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

# Springer-Verlag Berlin Heidelberg 2007


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Abstract: Cell volume is continuously compromised by the generation of local and transient osmoticmicrogradients associated with uptake of nutrients, secretion, cytoskeletal remodeling and transynapticionic gradients. It is also disturbed in pathological conditions including those leading to hyponatremia orthose associated with ion redistribution such as ischemia, trauma and epilepsy. Changes in cell volume,swelling or shrinkage, appear as key signals in directing the cell death type to necrosis or apoptosis and assignals for proliferation. Cell swelling in brain is critical since the limited expansion imposed by the rigidcranium results in vascular rupture and the consequent ischemic episodes and neuronal death. Besides,disturbing the extracellular/intracellular ionic equilibrium in the brain, as occurs in isosmotic swelling orduring volume recovery after hyposmotic swelling results in hyperexcitability and hypersynchrony of neuronal activity. Therefore, the role for amino acids as osmolytes in volume regulation, particularly those being synaptically inhibitory or inert is of particular importance. However, others such as glutamateexacerbate neuronal excitability and lead ultimately to excitotoxicity and neuronal death. Understandingthe implication of amino acids in cell volume control, and elucidating the signals and mechanismsunderlying their participation is crucial to the design of strategies to prevent swelling and to protect

brain cells, neurons particularly, from the deleterious effects of ionic disequilibrium and excitotoxicity. Thisis important also for avoiding the dangers of a rapid correction of the osmolarity of external uids inhyponatremic conditions. This review presents an overview of the available information about the aminoacid contribution to volume regulation after swelling in hyposmotic and isosmotic conditions, their role involume recovery after cell shrinkage and their implication in cell volume changes during apoptosis andproliferation.

List of Abbreviations: CaMKII, calcium-calmodulin kinase II; CGN, cerebellar granule neurons; EAAC1,excitatory amino acid transporter-1; EAAT, excitatory amino acid transporter; EGTA-AM, EGTA Acetox- ymethyl ester; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; JNK, Jun N-terminalkinase; MAP, mitogen-activated protein; PI3K, phosphatidyl inositol 3-kinase; PKA, protein kinase A;

PKC, protein kinase C; RVD, regulatory volume decrease; TAUT, taurine transporter; TBOA, DL-threo- b-benzyloxyaspartate; TonEBP, tonicity-responsive enhancer binding protein; TonE, tonicity-responsiveenhancer element

1 Introduction

The ability to regulate cell volume is a trait preserved in essentially all species throughout evolution. Themaintenance of a constant cell volume is a homeostatic imperative in animal cells, since even small changesin cell water content may modify the concentration of messenger molecules and disturb the complex signaling network, crucial for cell functioning and intercellular communication. Although in physiologicalconditions the extracellular uids have a highly controlled osmolarity, numerous diseases lead to alterationsof systemic osmolarity. The intracellular volume constancy is also continuously compromised by thegeneration of local and transient osmotic microgradients associated with uptake of nutrients, secretion,cytoskeletal remodeling, and transynaptic ionic gradients (Lang et al., 1998a).

Cell volume perturbation is a challenge for cells in all animal organs but has particularly dramaticconsequences in brain, since the rigid skull gives narrow margins for buffering intracranial volume changes.As expansion occurs, the constraining and eventual rupture of small vessels, generate episodes of ischemia,infarct, excitotoxicity, and neuronal death. In extreme conditions, caudal herniation of the brain parenchy-ma affects brain stem nuclei, resulting in death by respiratory and cardiac arrest. Cellular edema in brainoccurs by a decrease in external osmolarity (anisosmotic swelling) or in isosmotic conditions, by changes in

ion redistribution or by accumulation of ammonia or lactate. This is named cytotoxic or cellular edema.These two types of swelling are characteristically different from the vasogenic edema in which the hallmark is the brainblood barrier disruption. However, in most pathologies, one type of edema gradually results inthe development of the other type.

226 10 Amino acids and brain volume regulation: Contribution and mechanisms


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2 Hyposmotic Swelling and Regulatory Volume Decrease:Amino Acid Contribution

The role of amino acids in cell volume control in brain was evident since the early studies in chronichyponatremia, the most common cause of hyposmotic swelling in brain cells. Hyponatremia occurs inpathologies such as renal or hepatic failure, inappropriate secretion of antidiuretic hormone, glucocorticoiddeciency, hypothyroidism, excessive use of thiazide diuretics, and psychotic polydipsia. Conditions such ashead trauma, brain tumor, and cerebrovascular accidents also result in hyponatremia associated withinappropriate secretion of antidiuretic hormone or the cerebral salt wasting syndromes. Hyponatremiamay also be caused by rapid correction of uremia by excessive hemodialysis or by infusion of hypotonicsolutions in the perioperative period. It is a common condition in the elderly and during pregnancy. Fatalhyponatremia induced cerebral edema has been recently associated with ecstasy use.

2.1 Amino Acid Levels in Brain During Volume Adjustmentto Hyposmolarity

Early studies in experimental hyponatremia showed that brain is not behaving as a perfect osmometerbut that the initial swelling is followed by progressive water loss until almost complete normalization,despite the persistence of hyponatremia. A compensatory displacement of uids from the interstitialto the ventricular spaces rst occurs, followed by the decrease of intracellular electrolytes. This how-ever, was found not sufcient to compensate for the loss of water observed, particularly in the longterm, and the contribution of other osmotically active solutes had to be considered. These moleculesinitially referred as idiogenic osmolytes were further identied as organic molecules, including myoi-nositol, phosphocreatine/creatine, glycerophosphorylcholine, betaine, N acetylaspartate, and the amino

acids, glutamate, glutamine, taurine, GABA, and glycine. Creatine, myoinositol, glutamate, and taurineare those more importantly contributing to volume control ( > Figure 10-1) (Verbalis and Gullans,

. Figure 10-1Decrease of osmolyte levels in brain of hyponatremic rats. (a) Change in the concentration of K , Cl , and of themain organic osmolytes in the brain of rats during moderate chronic hyponatremia. (b) Brain content of osmolytes in the normotremic condition. Abbreviations: Glu, glutamate; Cr, creatine; Gln, glutamine; myo I,myoinositol. Other organic molecules with marginal contribution to the hyposmolarity corrections in brain areN acetylaspartate, aspartate, GABA, glycerophosphorylcholine, and betaine. Data from Verbalis and Gullans(1991)

Amino acids and brain volume regulation: Contribution and mechanisms 10 227


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1991). Interestingly, while decreases of electrolyte reverse with time, those of organic osmolytes, particu-larly that of taurine, are sustained as long as the hyponatremic conditions persists, suggesting thatthe electrolyte loss is an emergency mechanism to rapidly counteract brain swelling, but may bepotentially harmful on long term basis, in contrast to the relatively innocuous organic osmolytes. Taurinein particular, may be a perfect osmolyte because it is metabolically inert and exhibits only weak synapticinteractions.

In experimental chronic hyponatremia in vivo, the contribution of electrolytes and organic osmolytesto the total brain osmolarity change has been estimated as 6270% and 2329%, respectively. Amongorganic osmolytes, creatine and myoinositol are the major contributors to volume adjustment, togetherwith the amino acids glutamate, glutamine, and taurine, which show a level reduction of 39%, 54%, and89%, respectively, after 7 days in chronic moderate hyponatremia ( > Figure 10-1) (Verbalis and Gullans,1991). This pattern of amino acid change is found, in general, in all brain regions (Massieu et al., 2004) aswell as in a variety of brain preparations, including slices, cultured cells, and in vivo superfusion. Taurine isnot only the amino acid showing the largest reduction in response to the osmolarity decrease, but it also

exhibits the highest sensitivity to the stimulus (Solis et al., 1998; Estevez et al., 1999; Olson, 1999). This may be due to a higher efciency of the taurine efux pathway or to the fact that taurine pools are more availablefor release than those of amino acids involved in other functions such as neurotransmitters or metabolicelements.

This marked change in the brain content of amino acids and other organic osmolytes resulting from theadaptation to external osmolarity should be considered during the correction of hyponatremia to avoidbrain injury (Berl, 1990). In a chronic hyponatremic condition, the intracellular osmolarity is in equilibri-um with the external hyposmotic environment and therefore, when the correction in plasma tonicity restores the normal, isosmotic condition, this condition is now sensed as hyperosmotic by brain cells, whichdehydrates until activation of new adaptive mechanisms. The main risk of this situation is a neurologicalsequel of demyelinating lesions, a pathology known as osmotic demyelination syndrome, or pontine

demyelination (because of its preferential location at the basis pontis), whose salient clinical features aremotor abnormalities, progressing to accid quadriplegia, occasional respiratory paralysis, mental statedisturbances, lethargy, and coma. Although not yet fully understood, it seems that dehydration is affectingthe tight junctions of the bloodbrain barrier exposing oligodendrocytes to substances normally excludedfrom the brain, such as complement, that could be the precipitous factor of demyelination (Baker et al.,2000).

2.2 The Mechanism of Regulatory Volume Decrease

Insights into the role of amino acids in cell volume regulation and the mechanisms involved, have derivedfrom in vitro studies showing the activation of amino acid efux during the process known as regulatory volume decrease (RVD). Following the pattern in vivo, neurons and astrocytes swell when exposed tohyposmotic external solutions, and then recover their initial volume by extrusion of intracellular osmolytesfollowed by water. This is illustrated in > Figure 10-2a for cultured astrocytes (Pasantes Morales, 1996). Theosmolytes involved in RVD are the same as in the brain in vivo, i.e., K and Cl and organic osmolytes,including amino acids (Pasantes Morales et al., 1996; Lang et al., 1998a). RVD has been studied in detailin cultured astrocytes and neurons, in neuroblastoma and glioma cell lines, and in the snail neurons(Pasantes Morales, 1996). RVD is a complex chain of events formed by a volume sensor(s), a signalingcascade for transducing the information about volume changes into the activation of pathways for osmolyteextrusion, leading to volume correction. The sensor machinery has memory of the original cell volume and

sets the timing for inactivation of the regulatory process. In the last years, most efforts have been directed toidentify and characterize the osmolyte efux pathways, and it is only recently that interest has emerged forunderstanding the osmotransduction mechanisms. There is so far only scarce information about the natureof the volume sensing mechanisms.



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2.2.1 The Amino Acid Efux Pathway

In most cells so far examined, the corrective osmolyte uxes occur via diffusive pathways. K and Clpermeate through separate channels, with marginal participation of electroneutral cotransporters( > Figure 10-2c and > d ) (Nilius et al., 1997; Niemeyer et al., 2001), and organic osmolytes through leak

pathways, with essentially no contribution of energy dependent carriers (Kirk, 1997). Amino acid efux during RVD has been reported in a large number of brain cells and preparations, including glial cells and

neurons in culture ( > Figure 10-2b ) (Pasantes Morales and Schousboe, 1988; Kimelberg et al., 1990;Pasantes Morales et al., 1993; PasantesMorales and Schousboe, 1997), slices from brain regions (Lehmann,

. Figure 10-2Regulatory volume decrease (RVD) and osmolyte efux pathways in cultured glial cells. (a) When exposed tohyposmotic solutions, cells rst swell and then activate a process of volume recovery, which occurs despite thepersistence of the hyposmotic condition. (b) RVD occurs by the extrusion via leak pathways, of intracellularosmolytes including amino acids, here illustrated by the release of labeled taurine, glutamate, alanine, andglycine from cultured astrocytes. Cells loaded with the labeled amino acids are superfused with isosmoticmedium and then with a medium of reduced osmolarity. Samples are collected every minute and the pointsrepresent the radioactivity released per minute as percent of total incorporated radioactivity. (c, d) Thehyposmolarity evoked efux of K and Cl in glial cells occurs through volume sensitive channels. Cl andK currents activated by hyposmolarity reduction were measured in the whole cell recording mode of patch clamp technique with a holding potential of 70 mV. For Cl currents (c) the pipette was lled with140 mM CsCl and for K currents (d) with K aspartate. Data from Pasantes Morales et al. (1993) andOrdaz et al. (2004a, b)



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1989; Law, 1994; Deleuze et al., 2000) and in the brain in vivo using paradigms of microdialysis orsuperfusion (Solis et al., 1998; Estevez et al., 1999; Massieu et al., 2004). In accordance with the decreasein brain content in vivo, the amino acids preferentially released by hyposmolarity are taurine and glutamate(Pasantes Morales et al., 1993; Massieu et al., 2004). N acetylaspartate seems to be also released, particularly from neurons (Davies et al., 1998). In most preparations, taurine is the most sensitive to the osmoticperturbation, with the lowest release threshold and the largest amount released. For these reasons, togetherwith some particular features such as its metabolic inertness and high intracellular concentrations, taurineseems a suitable molecule for the study of RVD mechanisms and has been often considered as representativeof organic osmolytes. However, this has to be handled with caution since particularly in brain, the featuresof hyposmotic taurine efux do not always match those of other organic osmolytes, including amino acids(Mongin et al., 1999; Franco et al., 2001).

The osmosensitive taurine release occurs via a leak pathway, and taurine translocation is driven by theconcentration gradient (Pasantes Morales and Schousboe, 1997; Hoffmann et al., 1988). In most cell types,including cultured astrocytes and neurons, the swelling activated taurine efux is sensitive to Cl channel

blockers, a nding which raised the proposal of an anion channellike molecule as a common pathway forthe corrective uxes of Cl and amino acids during RVD (Strange and Jackson, 1995). However, recent

evidence favors the notion of two separate pathways for taurine and Cl (Stutzin et al., 1999). In this case,the effect of Cl channel blockers on taurine efux may reect a close interconnection between the twopathways or the need of a specic change in intracellular Cl for taurine efux activation. Other candidatesto act as taurine translocating molecules are the anion exchanger (band 3) and the phospholemman. Theanion exchanger seems involved mainly in sh erythrocytes (Perlman and Goldstein, 2004), and its possiblerole in brain cells has not been examined. The phospholemman is a member of a superfamily of proteinswith single transmembrane domains, forming homomeric channels. Phospholemman, rst found in heart,is present in a variety of cells and tissues, including neurons and astrocytes (Moorman and Jones, 1998;Moran et al., 2001). A characteristic of phospholemman is its markedly high permeability to taurine, a

feature due possibly to the presence within the pore of binding sites for cations and anions, facilitating thetransport of zwitterionic molecules. Besides the high taurine permeability, other ndings are in support of phospholemman as an osmosensitive taurine efux pathway such as taurine efux inhibition by antisenseoligonucleotide blockade of phospholemman function in cultured astrocytes and increased RVD efciency and taurine release by phospholemman overexpression (Moran et al., 2001).

Besides taurine, hyposmolarity activates uxes of GABA, glutamate, and glycine in cultured astrocytesand neurons (Pasantes Morales et al., 1993). It has been often assumed that a pathway similar to that of taurine serves for other amino acid efux, but recent results challenge this hypothesis by showingremarkable differences between taurine and glutamate. These differences include the time course, showingslower activation and inactivation for taurine efux as compared to glutamate and the sensitivity toCl channel blockers and tyrosine kinase blockers (Mongin et al., 1999; Franco et al., 2001). Thesedifferences appear to be restricted to brain cells or tissue since they are not found in other cell types(unpublished results).

2.2.2 The Osmotransduction Signaling

Identication of the signaling chains connecting cell swelling and the activation of osmolyte efux pathwaysfor volume adjustment has been complicated by the fact that during the volume increase and recovery, anumber of reactions occur which are unrelated to osmotransduction itself. The change in cell volume is acomplex process, with numerous concurrent phenomena such as stress, reorganization of the cytoskeleton,

and adhesion or retraction mechanisms, among others. All of them activate their own signals, which may ormay not be implicated in the activation of corrective osmolyte uxes. In brain cells, in addition, swellingleads to depolarization and [Ca 2 ] i rise, two conditions involved in neurotransmitter release. Since themain neurotransmitter amino acids, i.e., GABA and glutamate, are playing also a role as osmolytes, thesemay be the signals for their release. Besides the [Ca 2 ] increase, hyposmotic swelling leads to changes in the



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concentration of second messengers, such as cAMP, IP 3 , and arachidonic acid, as well as activation of enzymes, mainly protein tyrosine kinases and phospholipases (Pasantes Morales and Morales Mulia, 2000;Wehner et al., 2003; Lambert, 2004). The role of each one of them in osmotransduction and their possibleinterplay are now actively investigated (Lambert, 2004). Despite the swelling evoked [Ca 2 ] i rise, theosmosensitive amino acid uxes are often Ca 2 -independent (Pasantes Morales and Morales Mulia,2000), although a modulatory role for Ca 2 on the taurine and glutamate efux pathway has now beenfound. An increase of [Ca 2 ] i over that elicited by hyposmolarity as that elicited by ATP or ionomycinpotentiates taurine and glutamate efux once the pathway has been activated (Cardin et al., 2003; Francoet al., 2004a; Mongin and Kimelberg, 2005).

Protein tyrosine kinases play a role in the hyposmotic taurine efux, as documented by the markedinhibitory effect of tyrosine kinase blockade on taurine release, as well as by the corresponding potentiationby tyrosine phosphatase inhibition (Pasantes Morales and Franco, 2002). A number of protein tyrosinekinases activate by hyposmolarity including p125 FAK , p38, JNK, p56lck , p72syk, and ERK1/ERK2 (Hubertet al., 2000; van der Wijk et al., 2000). This, however, does not necessarily imply a link with osmolyte uxes.

In fact, hyposmotic activation of ERK1/ERK2, p38, and JNK appears unrelated to amino acid uxes incultured astrocytes and neurons (Pasantes Morales and Franco, 2002). In contrast, phosphorylation of thetyrosine kinase membrane receptors appears closely involved in the taurine efux pathway. Hyposmolarity activates the epidermal growth factor receptor in broblasts and the ErbB4 receptor in cerebellar gra-nule neurons ( > Figure 10-3a ), and blockade of this reaction reduces the osmosensitive taurine efux ( > Figure 10-3b ) (Franco et al., 2004b; Lezama et al., 2005). In erythrocytes, the swelling induced tyrosinephosphorylation of band 3 (anion exchanger) is linked to p72syk and p56lyn (Hubert et al., 2000), thisbeing the rst report showing direct tyrosine phosphorylation of the osmolyte translocation molecule.The activity of the phosphoinositide 3kinase (PI3K), a target of protein tyrosine kinases, has also aninuence on the taurine efux pathway. In cultured neurons and astrocytes, in hippocampal brain slices,and in the retina, PI3K activates by hyposmolarity, and its blockade with wortmannin impairs volume

regulation and inhibits the hyposmotic taurine efux ( > Figure 10-3a and > b ) (Franco et al., 2004b). Incerebellar granule neurons, phosphorylation by hyposmolarity of ErbB4 and PI3K are interconnectedreactions (Lezama et al., 2005).

Other possible elements of the osmotransduction signaling are the small GTPase p21Rho, and itsdownstream kinase Rho kinase connecting with the light myosin chain. The role of phospholipases onamino acid uxes related to hyposmolarity is still unclear, with conicting results about the effectof blockers of several phospholipases (Lambert, 2004). The connection between all these enzymes andtheir hierarchy remain to be established. An association between PI3K, Rho GTPases, and phospholipaseshas been shown in a variety of pathways, some of them regulating the dynamics of the cortical andcytoplasmic actin cytoskeleton, which may be modulatory of the amino acid efux pathway (van derWijk et al., 2000).

2.3 Hyposmolarity Induced Release of Amino Acids from Nerve Endings

The effect of volume changes on subcellular compartments, such as dendrites or nerve endings, has notbeen studied in detail. This is of interest, since dendrites are particularly sensitive to swelling and nerveendings are continuously exposed to osmotic gradients due to constant ion redistribution and neuro-transmitter uptake and release. Nerve ending swelling occurs in conditions of damage and hyperexcita-bility such as in head trauma and seizures. The question is raised on whether nerve ending swelling may result in the release of amino acids, this being a most important subject since amino acids acting as

osmolytes and neurotransmitters, such as glutamate and GABA, have a prominent role in the control of brain excitability.A study in isolated nerve endings (synaptosomes) showed a hyposmolarity evoked release of taurine,

glutamate, and GABA (Tuz et al., 2004) ( > Figure 10-4a ). The release mechanism differs for the threeamino acids. Most of the hyposmotic taurine efux shows the typical features of the osmolyte translocation,



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such as inhibition by Cl channel blockers, tyrosine kinase blockers, and PI3K blockers, while glutamateefux is essentially insensitive to these blockers but is markedly Na dependent, blocked by toxinsinterfering with the exocytotic release and modulated by protein kinase C (PKC). GABA efux shows thetwo types of release ( > Figure 10-4b ). In nerve endings, hyposmolarity evokes depolarization and [Ca 2 ]increase and thus, glutamate, but not taurine, efux may be a response to these events and mediated by exocytosis or carrier reversal operation (Tuz et al., 2004).

The nerve ending release of amino acids may explain the increased susceptibility to seizures associatedwith hyponatremia. Also the hyposmotic release of GABA and glutamate from nerve endings may beresponsible for the increase in duration and amplitude of excitatory and inhibitory postsynaptic potentialsin hyposmotic conditions (Chebabo et al., 1995; Baraban and Schwartzkroin, 1998). Thus, swelling may beaffecting brain excitability and synaptic transmission. The hyposmotic taurine release from nerve endings,and likely also from synaptic vesicles, is an interesting nding since taurine even when it is highly concentrated in nerve endings (Kontro et al., 1980) has a marginal function as neurotransmitter. The

presence of this swellingresponsive synaptic taurine pool may reect a need for mechanisms to correctvolume in the nerve terminal and/or in synaptic vesicles, disturbed by ion and neurotransmitter redistri-

bution during synaptic activity. In pathological conditions, the release of taurine may have a dual benet,relieving swelling rst and once translocated into the extracellular space, acting as a neuroprotectant, awidely documented action of this amino acid.

. Figure 10-3The volume sensitive taurine efux pathway is inuenced by the activity of tyrosine kinase membrane recep-tors and the associated tyrosine kinase target phosphoinositide 3 kinase (PI3K). (a) Illustrates the effect of hyposmolarity in cerebellar granule neurons activating phosphorylation of the tyrosine kinase membrane recep-tor ErbB4 and of PI3K. The upper panel shows ErbB4 phosphorylation in the following conditions: Isos, isosmoticmedium; H30%, hyposmotic medium 30%; Her b , effect of the receptor ligand heregulin b (200 ng/ml).(b) Blockade of these reactions as shown in (a), with 50 mM AG 213 (ErbB4) or 100 nM wortmannin (PI3K),markedly reduces the volume sensitive taurineefux. Taurine release was measured as in > Figure 10.2b . ErbB4phosphorylation was assayed by immunoblotting with the appropriate antibodies, and PI3K activity wasassayed by measuring phosphorylation of its main target AKT. Data are from Lezama et al. (2005)



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2.4 Volume Regulation After Gradual Decreases in Osmolarity: The Relevanceof Amino Acids

In most studies about volume regulation, cells are exposed to sudden and large osmolarity reductions, of amagnitude probably never occurring in brain under physiological conditions or even in pathologicalsituations. An approach closer to normal was devised by Lohr and Grantham (1986) in renal cells, whichconsisted in exposing the cells to small and gradual changes in external osmolarity. Cells were then able to

. Figure 10-4Hyposmolarity induced release of amino acids from rat brain synaptosomes. (a) Reducing osmolarity by 20%elicits a rapid efux of glutamate, taurine, and GABA (followed by 3 Htracers) from isolated nerve endings.Synaptosomes loaded with the labeled amino acids were superfused with isosmotic medium, and at the arrow the superfusion medium was changed to a 20% hyposmotic medium. Results are expressed as radioactivityreleased per min as percentage of the total radioactivity incorporated. (b) Contribution of depolarization

exocytosis [estimated by the inhibitory effect of La 3 , ethyleneglycol bis (b aminoethylether) N ,N 0tetraaceticacid AM (EGTAAM) and tetanus toxin], carrier reversal {efux sensitive to the carrier blockers for GABA and DL

threo b benzyloxyaspartate (TBOA) for glutamate} or leak pathway (efux reduced by Cl channel andtyrosine kinase blockers). Data in (b) correspond to the amino acid efux during the time of stimulation withthe hyposmotic medium (10 min). For details see Tuz et al. (2004)



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maintain a constant volume over a wide range of tonicities if the rate of change in osmolarity was lowerthan 2.2 mOsm/min. This response was named isovolumetric regulation, a term reecting the activenature of this process, as the constant volume was not due to the absence of swelling but to a continuousvolume adjustment accomplished by the extrusion of intracellular osmolytes. This paradigm has beenapplied to other cell types and marked differences have been found with respect to the efciency of theprocess. Interestingly, these differences appear to be related to the contribution of amino acids.

According to their behavior facing gradual osmolarity decreases, three types of cell response have beenso far observed. Renal cells and cerebellar granule neurons show a volume constancy, even when the externalosmolarity has dropped by 50% (Lohr and Grantham, 1986; Van Driessche et al., 1997; Tuz et al., 2001)( > Figure 10-5a ). Glioma C6 cells, cultured myocytes, and astrocytes (Souza et al., 2000; Ordaz et al., 2004a, b)respond to gradual osmolarity changes by a volume increase, which nevertheless is lower than when an

. Figure 10-5Changes in cell volume and taurine efux in cerebellar granule neurons or astrocytes, exposed to conditionsof gradual osmolarity reduction. Cells were exposed to media in which the osmolarity decrease rate was1.8 mOsm/min. At the end of the experiment, the osmolarity of the medium has decreased by 50% (150mOsm). Cell volume change was measured by large angle light scattering as described in Ordaz et al. (2005)(astrocytes) or by dilution of calcein AM followed by spectrouorometry (granule neurons) as in Tuz et al.(2001). Volume is expressed as V t / V 0 . (a) Cerebellar granule neurons ( ) maintained a constant volume facingthe osmolarity reduction while in astrocytes ( ) volume increased but still it is lower than in cells exposed to asudden decrease in osmolarity of the same magnitude, as shown in the upper curve ( ), suggesting a moreefcient volume control. (b, c) Taurine efux in response to gradual reductions in osmolarity. Taurine efux wasmeasured as described in > Figure 10-2 , following the release of [ 3 H]taurine. The taurine efux threshold is

notably lower in neurons (b) than in astrocytes (c)



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osmotic stimulus of the same magnitude is suddenly imposed ( > Figure 10-5a ). Finally, trout erythrocytes(Godart et al., 1999) respond with similar swelling to gradual or sudden exposure to hyposmotic solutions.

The osmolytes involved in volume corrective mechanisms during gradual osmolarity reductions are K ,Cl , and organic molecules, but the activation threshold and the efcacy of the osmolyte translocationpathways show differences in the various cell types, which appears to correspond with the efciency tocounteract changes in cell volume. Thus, a correlation is observed between the taurine and glutamate efux threshold and the extent of swelling in conditions of gradual osmolarity changes. In cerebellar granuleneurons, which show the typical isovolumetric regulation, the efux of taurine and glutamate activates very early after the osmotic stimulus, as early as a 2% osmolarity reduction for taurine (Tuz et al., 2001)( > Figure 10-5b ), whereas in astrocytes, taurine efux occurs later ( > Figure 10-5c ) (Ordaz et al., 2004a),and in trout erythrocytes (Godart et al., 1999), the efux of taurine is markedly delayed and seemsinsufcient to contribute to volume regulation. The higher ability of neurons as compared to astrocytesto resist changes in external osmolarity, which seems based primarily on the contribution of organicosmolytes, may represent a protective mechanism to spare neurons from the deleterious consequences of

swelling.

2.5 Taurine as an Osmotransmitter of Neurohormone Output

Besides the role of taurine as a ubiquitous osmolyte, in some specic cells the osmosensitive taurineefux may have additional functions. In the hypothalamic neurohypophysial system, a role for taurinehas been proposed in the regulation of the whole body uid balance through the control of plasmaticlevels of vasopressin and oxytocin, two hormones critically involved in the body water balance. This effectof taurine is suggested by experiments showing a regulatory role of the glial hyposmotic release of taurineon the neuroendocrine cells at the supraoptic nucleus, resulting in vasopressin release by a mechanismlikely involving activation of glycine receptors by the high extracellular taurine levels, reached after itshyposmolarity induced release from glial cells (Hussy et al., 2000). Moreover, recent work has shown anosmotic regulation of the depolarization evoked vasopressin secretion in the neurohypophysis, similarly mediated by the osmosensitive release of taurine from pituicytes, an effect involving also glycine receptors(Hussy et al., 2001).

3 Isosmotic Swelling

Isosomotic (cytotoxic) swelling occurs in association with various brain pathologies including epilepsy,ischemia, and head trauma. A number of causal factors of brain cell swelling are common to these three condi-tions, such as energy failure, elevated extracellular K levels, depolarization, extracellular glutamate increase,lactacidosis, and in the late phases, reactive oxygen species generation, membrane lipid peroxidation, andexcitotoxicity (Arundine and Tymianski, 2004). In hepatic encephalopathy, another pathology in whichbrain edema is a fatal outcome, other mechanisms are involved initially, but the energy failure andoxidative stress also concur in the late phases of edema (Felipo and Butterworth, 2002; Butterworth,2003).

In epilepsy, the abnormally high ring rate of neurons elevates K levels, and the resulting depolariza-tion activates the release of glutamate from the widespread glutamatergic synapses. The astrocyte capacity of K clearance by spatial buffering is exceeded and K accumulates intracellularly, followed by Cl and

water (Walz, 2000). During the early phases of ischemia, the interruption of glucose and oxygen supply leads to energy failure, ATP depletion, and the progressive collapse of transmembrane ionic gradients,resulting in glial and neuronal depolarization, the increase in extracellular K , and glutamate rise (Hanssonet al., 2000; Walz, 2000; Nishizawa, 2001). Additional elements for cell swelling in brain, particularly inastrocytes, result from the activation of anaerobic glycolysis, a pathway which remains operating inastrocytes under hypoxic conditions as long as there is a remnant delivery of glucose. This alternate route



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contributes to swelling by generation of protons and lactate. In the late ischemic phases, the mechanism of glutamate removal is inefciently operating, as the disturbed ionic gradients make the carriers to work inreverse, further increasing the concentration of extracellular glutamate (Rossi et al., 2000; Nelson et al.,2003; Phillis and ORegan, 2003). At this moment, the cascade of excitotoxicity is triggered, with its sequelof intracellular [Ca 2 ] rise, phospholipid degradation, free fatty acid release, and formation of reactiveoxygen species (Arundine and Tymiansky, 2004). All these factors substantially contribute, by differentmechanisms, to further aggravate cell swelling. The situation may be different at the ischemic penumbra,where energy depletion and ionic disruption are less severely affected. The normal rather than reversedoperation of the glutamate transporter, particularly the astrocyte type, may then lead to swelling (Feustelet al., 2004).

Traumatic brain injury is a most common situation leading to cytotoxic swelling, resulting from anumber of mechanical and biochemical events, with different time course and strong interplaying. Themost directly related to swelling is the posttraumatic widespread depolarization, with its associated increasein intracellular Na , Cl , and water inux. Additional swelling factors derive from the transient sheer forces

that mechanically deform membranes, resulting in ion permeability changes. When, as often happens,restriction of cerebral blood ow concurs with trauma, the cascade of reactions characteristic of ischemia isalso triggered. An indirect mechanism of swelling after brain trauma is the hyponatremia, evolving as aconsequence of a disturbed secretion of the antidiuretic hormone.

In hepatic encephalopathy, brain edema is a most characteristic neuropathological feature resultingfrom the acute liver failure, and it is the major cause of death. Brain cellular edema, in hepatic encephalop-athy is essentially restricted to astrocytes (Haussinger et al., 2000). The initial and causal factor is an increasein brain ammonia levels subsequent to its blood rise (Felipo and Butterworth, 2002; Butterworth, 2003).Ammonia detoxication in brain, in both normal or hyperammonemic conditions is essentially carried outby astrocytes. Since the brain lacks the key enzymes to remove ammonia in the form of urea, ammoniametabolism occurs basically via the synthesis of glutamine, through the amidation of glutamate by the

glutamine synthetase, an enzyme localized almost exclusively in astrocytes (Martinez Hernandez et al.,1997). Consequently, brain glutamine production in astrocytes is dramatically increased following hyper-ammonemia. Besides the accumulation of glutamine, which may be per se a swelling inductor, severalmechanisms are put in motion in association with the ammonium/glutamine rise in astrocytes, includingblockade of key enzymes in the oxidative metabolism and lactate production, free radical generation, andmitochondrial dysfunction by induction of the permeability transition pore, all of them leading to cellswelling (Norenberg et al., 2004).

3.1 Amino Acids and Isosmotic Swelling

In contrast to chronic hyponatremia, cytotoxic swelling in brain seems not being followed by efcientmechanisms of volume adjustment. This is not unexpected, since as discussed in the preceding section,volume regulation relies on water extrusion carried by the efux of K , Cl , and organic molecules. Thisoccurs via diffusion pathways, with the osmolytes moving in their concentration gradient direction.In ischemic conditions, the imbalance of K , Na , and Cl gradients due to the energy failure excludesion efux as an effective mechanism for volume correction, since gradients may favor inux instead of efux. Besides, the swellingsensitive Cl channel, which is crucial for an adequate process of volumeregulation, requires ATP for activation and will thus be inoperative in conditions of ATP depletion.Furthermore, a recent study (Mori et al., 2002) has shown impaired activity of the volume sensitiveCl channel during lactacidosis, a condition associated with various situations generating cellular astrocyte

swelling.The release of amino acids and other organic osmolytes may occur in isosmotic swelling if the extrusionpathways are not impaired. This may reduce the extent of cell swelling but seems insufcient to permit cellvolume recovery. A marked increase of amino acid efux, of manyfold over basal release, has been observedin essentially all models of ischemia, either in vivo, in models of total or focal ischemia, or in vitro, in a



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variety of brain preparations, in experimental models of chemical ischemia (Benveniste et al., 1984;Saransaari and Oja, 1999; Phillis and OReagan, 2003). Aspartate, glutamate, GABA and taurine are theamino acids preferentially released, with manyfold increases in the ischemic condition. Efux of glycine,alanine, serine, and phosphoethanolamine occurs mainly upon reperfusion (Phillis and ORegan, 2003).Some differences are observed between brain regions but the general pattern is essentially similar.

For amino acids, such as GABA, aspartate, glutamate, and glycine, which besides serving as osmolytes,have a widespread function as neurotransmitters, the key point is to identify whether the efux responds toswelling or to other events concurrent with the ischemic situation such as depolarization, Ca 2 inux, andion redistribution. Glutamate release is of special interest, since the rise in glutamate is largely responsiblefor neuronal death by excitotoxicity during ischemia. Moreover, the survival chances of cells around theischemic focus, at the penumbra area, appear to depend also on the control of extracellular glutamate levels(Feustel et al., 2004). As mentioned before, the ischemic condition is a multifactorial situation, in which asequential series of responses are evoked as the ischemic episode proceeds, with events characteristic foreach step, which may put in motion different mechanisms for amino acid release and/or their persistence at

the extracellular space ( >

Figure 10-6 ). Thus, amino acid efux may occur by one or several events, one of which may be swelling. Most studies on the mechanisms leading to amino acid overow in ischemia havefocused on glutamate. Available evidence supports the involvement of at least three mechanisms: (1) Ca 2 dependent exocytotic release, (2) the reverse operation of the energy dependent glutamate transporters,due to intracellular Na accumulation as a consequence of the energy failure, and (3) swelling activatedefux (Benveniste et al., 1984; Seki et al., 1999; Rossi et al., 2000; Nelson et al., 2003). The second option isconsidered at present, as the main contributor to the elevated extracellular glutamate, with less participa-tion of the swelling related mechanisms. The same considerations are valid for other amino acids with thedual role of osmolyte and neurotransmitters (Allen et al., 2004).

The contribution of swelling and the reverse carriers to the extracellular glutamate increase has beenexamined in brain regions less severely affected by ischemia. In a model of middle cerebral artery occlusion

in rats, the efux of glutamate collected by microdialysis from regions of incomplete ischemia was found tobe reduced by tamoxifen, a blocker of the volume sensitive Cl channel, but not by dihydrokainate, aglutamate carrier blocker, leading to the conclusion that during incomplete ischemia, glutamate overow occurs predominantly by swelling and not by the reversal carrier operation (Feustel et al., 2004). This is animportant notion for a rational design of strategies to improve survival of brain cells in the ischemicpenumbra. In the same line, a reduction of ischemia evoked amino acid uxes by anion channel inhibitorshas been reported in cortical superfusates of a four vessel cerebral ischemia. The blockers markedly reducedthe efux of aspartate, glutamate, taurine, and phosphoethanolamine, with less effect on GABA, and noeffect on serine, alanine, or glutamine (Phillis and ORegan, 2003). It is worthy to notice that the glutamateefux decrease by Cl channel blockers may reect the inhibition of the glutamate efux pathway or aneffect of the blockers preventing Cl inux and swelling. The efux of inhibitory amino acids, such astaurine, GABA, or glycine, which in contrast to glutamate, do not generate per se a secondary volumeincrease, nor excitotoxicity, may contribute not only to attenuate swelling but also to counteract thehyperexcitability generated during ischemia. However, recent results suggest an effect of GABA releasedduring ischemia as a factor of neuronal swelling (Allen et al., 2004).

Amino acid efux including excitotoxic amino acids is also observed in brain during cytotoxic swellingby hyperamonemia or head trauma (Stover and Unterberg, 2000). The mechanism of this release is stillunclear, but at least for taurine in hepatic encephalopathy models, it seems not directly caused by swelling(Zielin ska et al., 1999), and in vivo, no clear correlation between taurine efux has been established neitherin experimental models of hepatic encephalopathy nor in patients (Butterworth, 2003). Extracellular brainglutamate levels are enhanced possibly due to the ammonia effect decreasing the expression of EAAT1, the

glial glutamate transporter (Filipo and Butterworth, 2002). Then, hyperactivation of glutamate receptors by both glutamate and depolarization triggers the cascade of reactive oxygen species generation and mito-chondrial dysfunction, all this contributing to brain edema. A microdialysis study on NH 4 inducedacidosis, reports an increase in extracellular N acetylaspartate, an amino acid present in large amountsin neurons, which may have an important role as an osmolyte. (Davies et al., 1998).



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It has been consistently observed that cytotoxic edema in vivo is more prominent in astrocytes than inneurons, being so far unclear whether this difference is due to a selective localization of the swelling

generating mechanisms in astrocytes or to the presence of more efcient mechanisms of cell volumecontrol in neurons (Pasantes Morales and Franco, 2005). In this respect, a most interesting observation isthe transfer of taurine and glutamate from neurons to astrocytes during experimental ischemia (Torpet al., 1991). By this mechanism, neurons are spared and protected from the deleterious effects of swelling.

. Figure 10-6Isosmotic swelling and glutamate efux from astrocytes and neurons during hypoxia and ischemia. At earlyischemic phases or during hypoxia, the energy failure, ATP depletion and progressive collapse of ionicgradients, leads to glutamate efux by the reversal operation of the neuronal carrier. The glial transportermay be still working for glutamate inux, due to ATP generation by glycolysis, which operates if a remnantglucose supply persists. This glutamate inux may cause astrocyte swelling by the Na inux and watercotransport intrinsic to the carrier. At late ischemic phases, also the astrocyte transporter operates forglutamate efux. The increased extracellular glutamate activates ionotropic glutamate receptors, primarily inneurons, precipitating a chain of uncontrolled swelling by generation of reactive oxygen species, membranelipid peroxidation, ion overload, and further swelling. At this point, the volume sensitive glutamate pathway inboth neurons and astrocytes contributes to glutamate efux. EAAT, excitatory amino acid transporter; ROS,reactive oxygen species



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4 Brain Cell Volume Decrease

Hypernatremia is the main clinical condition resulting in brain cell shrinkage. It is most prevalent in thegeriatric population. Hypernatremia is caused by a loss of water or a gain in Na , the two conditions beingrelated in most cases. The mechanisms for defective water intake involve mainly impaired thirst mechan-isms, due to diseases or defects in the hypothalamic thirst center or in the frontal lobe thirst perception.Decient water intake may occur also during impaired mental function by coma or confusion, duringanesthesia, or by unavailability of water. Other situations associated with or causing hypernatremia includenephrogenic diabetes insipidus when the kidney fails to respond to vasopressin, in diuretic therapy, or by salt poisoning. Central diabetes insipidus due to failure of the hypothalamus to make vasopressin may occur as a consequence of cerebrovascular diseases, ischemia, and head trauma, resulting in hypernatremia.In all cases, the brain cell water content decreases as a consequence of the osmotic gradient between bloodand brain. This is then followed by an adaptive response of brain, consisting of the accumulation of osmotically active intracellular solutes, decreasing the osmotic gradient and tending to restore brain water.

The brain osmolytes involved in the volume regulatory increase are K

, Cl

, and Na

, as well as a number of organic molecules (Heilig et al., 1989).The contribution of organic osmolytes is particularly important in chronic hypernatremia. The same

three major groups of organic molecules involved in the adjustment of brain water content in hypona-tremia, i.e., polyols, trimethylamines, amino acids and their derivatives, serve this function of osmolytes inhypernatremia. Myoinositol, betaine, and glycerophosphoryl choline rise in brain in moderate or severehypernatremia, while betaine only accumulates in severe hypernatremia (Lien et al., 1990). The change inthe brain amino acid content in hypernatremia has been examined in chronic or acute hypernatremiccondition. A study in rats showed that amino acid levels are not substantially modied in acute hyperna-tremia, but in the chronic moderate condition, there is a substantial increase in glutamate, glutamine, andtaurine of 45%, 70%, and 39%, respectively ( > Figure 10-7a ). In severe chronic hypernatremia, the aminoacid increase is of 84%, 143%, and 78%, respectively (Lien et al., 1990). In some animal models, the braintaurine increase accounts for as much as 50% of the osmolytes required for brain volume adjustment.

4.1 Mechanism of the Compensatory Increase in Amino Acid LevelsDuring Hypernatremia

4.1.1 Hypertonicity and Osmolyte Transporters

The increase in brain content of organic osmolytes, including amino acids, may occur by an osmoregulationof the transport or/and by increasing biosynthesis. The rst studies on the mechanisms of hyperosmoticregulation of organic osmolyte transporters were carried out in renal cells (Garc aPerez and Bourg, 1991),but other studies soon followed, showing similar mechanisms in brain cells, particularly in astrocytes.Hyperosmolarity induced increased levels and transporter activity in astrocytes have been described mainly for myoinositol (Strange et al., 1991; Paredes et al., 1992; Isaacks et al., 1994), betaine (Garc aPerez andBourg, 1991), and taurine (Olson and Goldnger, 1990; Beetsch and Olson, 1996) ( > Figure 10-7b ). Ingeneral, hyperosmolarity increases V max without signicant changes in K m , suggesting an increase in thenumber of transporters. The effect of hyperosmolarity on the specic Na dependent transporter fortaurine, TAUT, has been studied in detail. TAUT is specic for the bamino acids taurine, hypotaurine,and balanine. It is a 620 amino acid protein of a molecular weight of 70 kDa, 12 transmembrane domains,and more than 90% homology in mammalian cells. The molecule requires two Na ions and one Cl to

transport one taurine molecule (Tappaz, 2004). The transporter has several putative consensus sites forphosphorylation by PKC and protein kinase A (PKA). Increased expression of TAUT by hyperosmoticconditions, and a resultant increase in taurine levels by a more efcient accumulation, has been found witha rather similar pattern in numerous mammalian cell types, including hepatoma cells, endothelial cells fromliver, aorta, and brain capillaries, corneal and lens epithelium, cultured astrocytes as well as in retinal cells



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(Tappaz, 2004). The hypertonicity induced expression of TAUT seems to result from the transcriptionactivation of osmosensitive genes. Genes responsive to hyperosmolarity have been identied for themyoinositol and the betaine/GABA transporters (Uchida et al., 1992; Burg et al., 1997). The 5 0ankingregion of these genes contains a short 11 base pair consensus sequence considered as a tonicity responsiveenhancer element (TonE) whose mutation or deletion prevents the hypertonicity induced transcription

. Figure 10-7Increase of osmolyte levels in brain of hypernatremic rats. (a) Change in the concentration of the main organicosmolytes in the brain of rats during moderate chronic hypernatremia. Abbreviations: Glu, glutamate;Cr, creatine; Gln, glutamine; myo I, myoinositol. (b) Increased taurine content, taurine transport, and TAUTgene expression induced by hyperosmolarity in rat brain cultured astrocytes. (a) Results are expressed aspercentage increase in each case of the hyperosmotic over the normosmotic condition. Data are from Lien et al.(1990), Olson et al. (1996), and Bitoun and Tappaz (2000)



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of the osmosensitive genes (Takenaka et al., 1994; Ferraris et al., 1999). Furthermore, a protein namedTonEBP, which is induced by hypertonicity, is acting as the main transcriptional activator of theosmosensitive genes by binding to TonE (Ferraris et al., 1999; Miyakawa et al., 1999). The osmosensitivity of the TAUT gene has not yet been conclusively demonstrated, but a sequence in the 5 0anking regionshowing a sequence that ts the functional consensus sequence for TonE (Han et al., 2000) suggest amechanism of gene osmoregulation similar to that found for other organic osmolyte transporters.TonEBP is rapidly and strongly overexpressed in the nucleus of neurons (Loyher et al., 2004) inhippocampus and cerebral cortex following acute systemic hypertonicity. Interestingly, the tonicity expression of TonEBP in neurons is not bidirectionaly regulated since hyposmolarity does not result ina reduction in its expression. This study showed that TonEBP is very weakly expressed in nonneuronalcells, including astrocytes. Since a robust hypertonicity induced expression of organic osmolyte trans-porters, including TAUT, has been found in astrocytes, this observation is puzzling and requires furtherstudies on the time course and specic features of the osmosensitivity of the transporter genes inastrocytes. Osmoregulation of the EAAC1 glutamate transporter has been found in a line of renal cells

NBL1, with features similar to those described for TAUT and the other organic osmolyte transporters, i.e., a large increase in EAAC1 mRNA levels and in the immunoreactive EAAC1 protein (McGivan and

Nicholson, 1999). However, these adaptative changes may be restricted to renal cells, not being observedin several other cell types, including possibly the brain cells.

The hyperosmotic regulation of TAUT seems to be modulated by Ca 2 and the Ca 2 /calmodulin chain.A study in the human intestinal cell line Caco 2 shows a signicant inhibition of TAUT induction by Ca 2

chelators or by blockers of Ca 2 /calmodulin, and its total suppression by Ca 2 blockers (Tappaz, 2004).Protein tyrosine kinases may also participate. Hypertonicity has been shown to induce at least three MAPkinase pathways: (1) the ERK pathway which is rapidly activated by hypertonicity and could extend thesignal cascade to the nucleus, (2) the JNK pathway which has c jun and AP 1 transcription factors aspathway elements, and (3) the p38 pathway. A specic role for these signaling pathways in the regulation of

gene expression for organic osmolytes/TAUT transporters has not yet been ascribed. Heat shock proteinsmay also be involved since gene expression for those proteins is activated by hypertonicity but link withTAUT has not yet been established (Tappaz, 2004). All these enzymes may be involved in the activation of events associated with the cell volume change, which are numerous and complex, involving cell responsessuch as cytoskeleton organization, adhesion reactions, and stress.

4.1.2 Hypertonicity and Osmolyte Biosynthesis

The intracellular increase of organic osmolytes as a mechanism to counteract cell shrinkage may be achieved

also by adaptive changes in the rate of synthesis. This is known to occur in renal cells, where the increase insorbitol during hypernatremia results essentially from the enhanced activity of aldose reductase, the enzymeforming sorbitol from glucose (Garc aPerez and Bourg, 1991), following an effect of hypertonicity on thealdose reductase gene transcription (Ferraris et al., 1994). The question is whether a change in biosynthesisalso contributes to the increase of other organic osmolytes, in particular that of taurine. Taurine biosyn-thesis in the nervous tissue occurs from the precursor cysteine to cysteinesulnate and hypotaurine. Thekey enzyme in this biosynthetic route is the cysteinesulnate decarboxylase. A study in cultured astrocytesfrom rat brain has shown that taurine synthesis is enhanced by hypertonicity (Beetsch and Olson, 1998).However, hypertonicity did not increase the mRNA levels of either cysteine dehydrogenase or cysteinesul-nate decarboxylase, suggesting that the enzyme genes may not be tonicity sensitive (Tappaz, 2004).Moreover, the hypertonicity evoked increase in cell taurine content is essentially dependent on the presenceof the amino acid in the extracellular milieu. Furthermore, the activity of cysteinesulnate decarboxylase,the rate limiting enzyme for taurine biosynthesis is very small in the human brain, thus making marginal if any, the contribution of this mechanism for taurine adaptation to hypertonicity in humans.



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5 Proliferation, Apoptosis and Cell Volume

5.1 Proliferation

Cells have to reach a certain size before initiating division. Cell size is attained after an interplay of eventsduring the various phases of the cell cycle. Mitogens and growth factors stimulate the activity or expressionof carriers and channels involved in nutrient transport and in numerous other aspects of the dynamics of the growing cell. This nally results in accumulation of a variety of molecules, some of them acting asosmolytes and driving water inux, changing the concentration of intracellular elements involved in the cellcycle progression, and affecting the rate of cell proliferation. The increase in cell volume may, in turn, act astrigger for growth factor receptor activation (Franco et al., 2004b; Lezama et al., 2005), thus recycling thechain of volume related proliferative events The cellular ion content resulting from these events seems toplay a key role in proliferation as pointed out by the potent effect of Cl channel blockers modulatingproliferation in different cell types (Voets et al., 1995; Lang et al., 2000; Wondergem et al., 2001). This effect

may be related either to a decrease in Cl inux reducing swelling or to a specic requirement of Clchannel functional expression at some point of the proliferation cycle. As mentioned in the above sections,Cl channel blockers are potent inhibitors of the volume sensitive efux of organic osmolytes, notably amino acids, thus raising the question of whether these osmolytes are participating in the proliferativeprocess. Studies on the effect of taurine addition show either inhibition of cell proliferation as in hepaticstellate cells and aortic vascular smooth muscle cells (Imada et al., 2003; Chen et al., 2004) or increasing orrestoring proliferation as in human fetal neurons (Chen et al., 1998) or fetal pancreatic cells (Boujendaret al., 2002). The effect of decreasing intracellular taurine levels on cell volume and proliferation rate has notbeen examined.

In physiological conditions, taurine may play a role in the homeostasis of the growing and proliferatingcells. This is suggested by the markedly higher taurine concentration found in the developing brain ascompared to the adult brain, the difference being up to vefold higher taurine levels in the immature brain(Sturman and Gaull, 1975). The reason for this difference has not been fully explained. Acting as anosmolyte, taurine may contribute to maintain the cell size required for the progress of proliferation. Asdevelopment progresses, there is an increase in protein expression and a parallel decrease in taurine levels,giving some support to this possible role of taurine in regulating the cell size in face of the continuouschanges in metabolite cell content occurring during development. Taurine deciency is known to impairthe maturation and subsequent migration of neurons in cerebellum and visual cortex by a still unknownmechanism (Sturman et al., 1985; Neuringer et al., 1990). The sequencial steps of brain ontogeny includeneuronal proliferation, maturation, migration, differentiation, and synaptogenesis. In the taurine decientcats, the presence of numerous mitotic gures at a time in which they are not found anymore in the normalcats suggests a delay in the process of proliferation. This delayed proliferation occurring when taurine levelsare lower than normal, may t the hypothesis discussed above of its requirement to attain the cell volumenecessary for a normal cell proliferation rate.

5.2 Apoptosis

A decrease in cell volume has been always recognized as a hallmark of apoptosis, but it is only recently thatthis volume reduction as been considered as an element in the signaling mechanisms of the program for celldeath. Cell shrinkage normally precedes apoptotic events such as cytochrome c release, caspase3 activation,nuclear condensation, and DNA fragmentation (Maeno et al., 2000). The mechanisms of cell shrinkage

during apoptosis include K

and Cl loss through activated K

and Cl channels. It is then likely thatexperimental manipulation of the intracellular concentration of these ions, including channel blockade,exert a profound inuence on apoptosis (Maeno et al., 2000; Bortner and Cidlowski, 2004). The question israised of whether organic osmolytes including amino acids may have a similar role in modulatingapoptosis. The release of taurine in connection with apoptosis has been reported in cerebellar granuleneurons grown in the absence of depolarizing K and in Jurkat lymphocytes after stimulation of the



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CD95receptors (Lang et al., 1998b; Moran et al., 2000) ( > Figure 10-8 ). Taurine efux in these cells is notreduced but rather notably increased by Cl channel blockers ( > Figure 10-8 ) and is insensitive to tyrosinekinase blockers, in contrast to the diffusive pathway activated by swelling. The Na and temperature

dependence of apoptotic taurine release suggests the involvement of a carrier mediated transport. Incerebellar granule neurons, apoptosis increases also the efux of glutamate and GABA, which may thenalso contribute to cell shrinkage.

Effects of manipulating K and Cl cell content on apoptosis appear mediated by two main events of the process, i.e., the caspase pathway and the mitochondrial cytochrome c release (Maeno et al., 2000;

. Figure 10-8Taurine efux from apoptotic cerebellar granule neurons (CGN) and Jurkat lymphocytes. Apoptotic death wasinduced in CGN by growing cultures in K 5 mM instead of the depolarizing K concentrations (K 25 mM),required for cell survival, and in Jurkat lymphocytes by activation of the Fas (CD95) receptor. (a) Evolution of apoptosis followed by activation of caspase 3 in CGN. (b) Taurine efux from CGN at different days in culture inapoptosis inducing conditions. Taurine efux was followed by the labeled tracer [ 3 H]taurine. The efux wasmeasured at the indicated time in culture, and the efux rate was obtained from samples collected every30 min during 5 h. (c) Effect of Na or Cl omission and of Cl channel blockers on the efux of taurine fromCGN and Jurkat cells. In Na free or Cl free media, NaCl was replaced by choline chloride or by Na gluconate,

respectively. Bars represent the percentage change in taurine efux with respect to efux control cells notexposed to the apoptotic conditions. Data are from Lang et al. (1998a, b) and Moran et al. (2000)



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Bortner and Cidlowski, 2004). Taurine has also some effects attenuating apoptosis induced by differentfactors in several cell types (Verzola et al., 2002). In the ischemia induced apoptosis in cardiomyocytes,taurine affects the caspase pathway, and inhibits the assembly of the Apaf 1/caspase9 apoptosome(Takatani et al., 2004), but has no inuence on the mitochondrial potential and cytochrome c release. As yet, there is not sufcient information to clarify whether the effect of taurine attenuating apoptosis is linkedto a reduction of the apoptotic activated taurine efux or to an effect preventing cell shrinkage. Theconcentration of external taurine used in most studies, of about 2040 mM, would presumably block theefux of intracellular taurine and possibly also the apoptotic Cl efux and might thus prevent or reducethe cell shrinkage.

Taurine efux, cell shrinkage, and apoptosis may be associated with the photoreceptor death knownto occur during taurine deciency. This link is suggested by the photoreceptor appearance observed inearly studies of taurine deciencyinduced retinal degenerations, showing the typical shrinkage precedingapoptotic cell death (Lake and Malik, 1987). A recent report, which seems to conrm this possibility, showsa severe and progressive photoreceptor apoptotic death in TAUT knockout mice (Heller Stilb et al., 2002).

Acknowledgments

Work in the authors laboratory has been supported by grants No. 46465 from CONACYT and IN209507from DGAPA, UNAM.

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10 Amino Acids and Brain Volume Regulation Contribution and Mechanisms

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