No abstract

External osmolarity reduction (20%) led to labelled glutamate, GABA and taurine release from rat brain cortical synaptosomes. A Cl--independent, Na+-dependent, La3+-sensitive and tetrodotoxin (TTX) reduced depolarization of synaptosomes occurred upon hyposmolarity, suggestive of Na+ entry through nonselective cation channels. This depolarization, together with cytosolic Ca2+ ([Ca2+]I) increase, resulted in exocytosis, monitored by FM1-43. The release fraction resulting from these phenomena was estimated, by its decrease, by La3+, EGTA-AM and tetanus toxin (TeTX), as 34-44% for glutamate, 21-29% for GABA and 18-22% for taurine. Protein kinase C (PKC) activation by phorbol-12-myristate-13-acetate (PMA) increased the hyposmolarity-elicited exocytosis and this activation increased glutamate (80%), GABA (51%) and taurine (42%) hyposmotic efflux. Inhibition by chelerythrine reduced glutamate, GABA and taurine efflux by 64%, 50% and 24%, respectively. The Na+-dependence of amino acid release (glutamate 63%, GABA 46% and taurine 29%) may result from both, prevention of the depolarization-exocytosis efflux, and blockade of the carrier reversal operation. Carrier blockade by dl-threo-beta-benzyloxy aspartate (TBOA) and NO-711 resulted in 37% and 28% reduction of glutamate and GABA release, respectively. Contribution of the osmolyte leak pathway to amino acid release, estimated by the influence of Cl- (NPPB) and tyrosine kinase (AG18) blocker, was up to 55% for taurine, but only 10-18% for GABA, with apparently no contribution for glutamate. The predominant osmolyte-type mechanism of taurine release suggest its function in volume control in nerve endings, while glutamate and GABA respond to events concurrent with hyposmolarity by a neurotransmitter-like release mechanism. The hyposmolarity-induced amino acid efflux from nerve endings may have consequences for neuronal excitability during hyponatremia.

A decrease in external osmolarity results in cell swelling and the immediate activation of a mechanism to restore cell volume, known as regulatory volume decrease (RVD). When exposed to a gradual osmolarity decrease (GODE), some cells do not swell. This reflects the operation of an active regulatory process known as isovolumetric regulation (IVR). The mechanisms underlying IVR appear similar to those activated during RVD, namely the extrusion of K+, Cl-, amino acids, and other organic molecules. A previous study has documented IVR in cerebellar granule neurons, parallel to an early efflux of taurine and Cl-, whereas K+ efflux is delayed. In this work we briefly review the importance of amino acids in the mechanisms of cell volume control in the brain, with emphasis on IVR. We also present experiments showing the response to GODE in cerebellar astrocytes. The currents activated during GODE, recorded in the whole-cell configuration of the patch clamp technique, indicate the early activation of an anion current, followed by a more delayed cation current. A correlation between the time course of amino acid efflux during GODE and the occurrence or not of IVR in various cell types, suggest the importance of these osmolytes in the volume regulatory process in this model.

The biological roles of the three natural FF-ATPase inhibitors, ε, ζ, and IF, on cell physiology remain controversial. The ζ subunit is a useful model for deletion studies since it mimics mitochondrial IF, but in the FF-ATPase of Paracoccus denitrificans (PdFF), it is a monogenic and supernumerary subunit. Here, we constructed a P. denitrificans 1222 derivative (PdΔζ) with a deleted ζ gene to determine its role in cell growth and bioenergetics. The results show that the lack of ζ in vivo strongly restricts respiratory P. denitrificans growth, and this is restored by complementation in trans with an exogenous ζ gene. Removal of ζ increased the coupled PdFF-ATPase activity without affecting the PdFF-ATP synthase turnover, and the latter was not affected at all by ζ reconstitution in vitro. Therefore, ζ works as a unidirectional pawl-ratchet inhibitor of the PdFF-ATPase nanomotor favoring the ATP synthase turnover to improve respiratory cell growth and bioenergetics.

The osmolyte function of amino acids and Cl in native NIH/3T3 cells not expressing the P-glycoprotein was examined by investigating the free amino acid concentration and the swelling-activated efflux of [3H]taurine, as representative of amino acids, and of 125I, as a tracer for Cl. Taurine and 125I efflux was activated by 20 and 30% hyposmotic solutions. At 50% hyposmotic solutions, the osmolyte pool was essentially depleted. The Cl channel blockers 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid inhibited the release of the two osmolytes by 80-95%. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (400 microM) decreased the efflux of taurine 80% without affecting that of 125I. Linolenic and arachidonic acids (5-20 microM) showed a concentration-dependent inhibitory effect on taurine and 125I fluxes. Omission of Ca decreased osmolyte fluxes by 16%. Verapamil inhibited the osmolyte release only at 500 microM. Nimodipine at 25 and 50 microM decreased the release of [3H]taurine and 125I by approximately 60 and 80%, respectively, but this effect was independent of the presence of extracellular Ca. These results indicate that amino acids and Cl function as osmolytes during regulatory volume decrease in native NIH/ 3T3 cells.

A decrease in intracellular ionic strength appears involved in the activation of swelling-elicited 3H-taurine efflux in cortical cultured astrocytes. Hyposmotic (50%) or isosmotic urea-induced swelling leading to a decrease of intracellular ionic strength, activated 3H-taurine efflux from a rate constant of about 0.008 min(-1) to 0.33 min(-1) (hyposmotic) and 0.59 min(-1) (urea). This efflux rate was markedly lower (maximal 0.03 min(-1)) in isosmotic swelling caused by K+ accumulation, where there is no decrease in ionic strength, or in cold (10 degrees C) hyposmotic medium (maximal 0.18 min(-1)), where swelling is reduced and consequently intracellular ionic strength is less affected. Also, astrocytes pretreated with hyperosmotic medium, which recover cell volume by ion accumulation, did not release 3H-taurine when they swelled by switching to isosmotic medium, but when volume was recovered by accumulation of urea, taurine release was restored. These results point to a key role of ionic strength in the activation of osmosensitive 3H-taurine efflux. In contrast, its inactivation was independent of the change in ionic strength but appears related to the reduction in cell volume after swelling, since despite the extent or direction of the change in ionic strength, the 3H-taurine efflux did not inactivate in isosmotic KCl-elicited swelling when cell volume did not recover nor in hyposmotic swelling when RVD was impaired by replacing NaCl in the medium by permeant osmolytes.

The hydrolytic activity from three partially purified family II pyrophosphatases from the photosynthetic bacteria Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodovulum sulfidophilum, as well as the recombinant cytoplasmic pyrophosphatase from Rba. sphaeroides, was tested with Mg 2+-PPi, Mn 2+-PPi, and PPi 4-. Unlike family I pyrophosphatases that hydrolyze only the Mg 2+-PPi complex like those from Rhodospirillum rubrum, all family II enzymes tested showed hydrolytic activity with Mg 2+-PPi, Mn 2+-PPi, and PPi 4without cation. The activity without added cation remained the same, even under exhaustive dialysis or after desalting the enzyme through a Sephadex G-25 column. However, this activity disappeared upon the addition of ethylenediaminetetraacetic acid and could not be restored by adding Mg 2+. Moreover, the enzyme inactivation was not related to dissociation into lower molecular subunits as in other family II enzymes. This is the first report on pyrophosphatases that can hydrolyze pyrophosphate without a divalent cation added and that presumably contain a tightly bound divalent cation in their structure.