Benito Ordaz

Mechanisms Counteracting Swelling in Brain Cells During Hyponatremia

Water gain in the brain consequent to hyponatremia is counteracted by mechanisms that initially include a compensatory displacement of liquid from the interstitial space to cerebrospinal fluid and systemic circulation and subsequently an active reduction in cell water accomplished by extrusion of intracellular osmolytes to reach osmotic equilibrium. Potassium (K+), chloride (Cl-), amino acids, polyalcohols, and methylamines all contribute to volume regulation, with a major contribution of ions at the early phase and of organic osmolytes at the late phase of the regulatory process. Experimental models in vitro show that osmolyte fluxes occur via leak pathways for organic osmolytes and separate channels for Cl- and K+. Osmotransduction signaling cascades for Cl- and taurine efflux pathways involve tyrosine kinases and phosphoinositide kinases, while Ca2+ and serine-threonine kinases modulate K+ pathways. In-depth knowledge of the cellular and molecular adaptive mechanisms of brain cells during hyponatremia contributes to a better understanding of the associated complications, including the risks of inappropriate correction of the hyponatremic condition.

Osmosensitive Release of Neurotransmitter Amino Acids: Relevance and Mechanisms

Hyposmolarity activates amino acid efflux as part of the corrective volume process in a variety of cells. This review discusses the mechanism of amino acid release in brain cells preparations. Results present evidence of substantial differences between the efflux of taurine and that of GABA and glutamate, which besides a possible role as osmolytes, have a main function as synaptic transmitters. The differences found concern the efflux time course, the sensitivity to Cl− channel blockers, the modulation by tyrosine kinases, the influence of PKC and the effect of cytoskeleton disruptive agents. While taurine efflux features fit well with the mechanisms so far described in most cell types, the efflux of GABA and glutamate does not. Alternate mechanisms for the release of these two amino acids are discussed, including a PKC-modulated, actin-dependent exocytosis.

Isovolumetric regulation mechanisms in cultured cerebellar granule neurons

Cultured cerebellar granule neurons exposed to gradual reductions in osmolarity (− 1.8 mOsm/min) maintained constant volume up to − 50% external osmolarity (πo), showing the occurrence of isovolumetric regulation (IVR). Amino acids, Cl−, and K+ contributed at different phases of IVR, with early efflux threshold for [3H]taurine, d‐[3H]aspartate (as marker for glutamate) of πo− 2% and − 19%, respectively, and more delayed thresholds of − 30% for [3H]glycine and − 25% and − 29%, respectively, for Cl− (125I) and K+ (86Rb). Taurine seems preferentially involved in IVR, showing the lowest threshold, the highest efflux rate (five‐fold over other amino acids) and the largest cell content decrease. Taurine and Cl− efflux were abolished by niflumic acid and 86Rb by 15 mm Ba2+. Niflumic acid essentially prevented IVR in all ranges of πo. Cl−‐free medium impaired IVR when πo decreased to − 24% and Ba2+ blocked it only at a late phase of − 30% πo. These results indicate that in cerebellar granule neurons: (i) IVR is an active process of volume regulation accomplished by efflux of intracellular osmolytes; (ii) the volume regulation operating at small changes of πo is fully accounted for by mechanisms sensitive to niflumic acid, with contributions of both Cl− and amino acids, particularly taurine; (iii) Cl− contribution to IVR is delayed with respect to other niflumic acid‐sensitive osmolyte fluxes (osmolarity threshold of − 25% πo); and (iv), K+ fluxes do not contribute to IVR until a late phase (< − 30% πo).

Osmolytes and Mechanisms Involved in Regulatory Volume Decrease Under Conditions of Sudden or Gradual Osmolarity Decrease

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.

Hyposmolarity-induced ErbB4 phosphorylation and its influence on the non-receptor tyrosine kinase network response in cultured cerebellar granule neurons.

Exposure of cultured cerebellar granule neurons (24 h serum‐starved) during 3 min to 30% hyposmotic medium activated the tyrosine kinase receptor ErbB4 in the absence of its ligand. Hyposmolarity also activated the non‐receptor tyrosine kinases, Src, focal adhesion kinase (FAK), extracellular signal‐regulated protein kinase (ERK)1/2, and the tyrosine kinase target phosphatidyl‐inositol‐3‐kinase (PI3K). The hyposmotic‐induced activation of these kinases required the prior phosphorylation of ErbB4 as shown by the effect of ErbB4 blockade with AG213 reducing by 85–95% the phosphorylation of FAK and ERK1/2, by 74% and 36% that of PI3K and Src, respectively. These results suggest a key role of ErbB4 as a signal integrator of events associated with hyposmolarity. PI3K seems to be an important connecting element in the signaling network evoked by the hyposmolarity/ErbB4 activation as: (i) the p85 regulatory subunit of PI3K co‐immunoprecipitates with ErbB4 and with FAK; (ii) PI3K blockade with wortmannin reduced the hyposmotic activation of FAK (90%) and ERK1/2 (84–91%). Inhibition of Src with PP2 reduced ErbB4 phosphorylation and inhibited the subsequent cytosolic kinase activation with the same potency as ErbB4 blockade. These results point to Src and ErbB4 and as early targets of the hyposmotic stimulus and osmosignaling. The functional significance for cell volume regulation of the ErbB4‐Src‐PI3K signaling cascade is indicated by the 48–66% decrease of the hyposmotic taurine efflux observed by inhibition of these kinases.

Somatostatin modulates generation of inspiratory rhythms and determines asphyxia survival

Breathing and the activity of its generator (the pre-Bötzinger complex; pre-BötC) are highly regulated functions. Among neuromodulators of breathing, somatostatin (SST) is unique: it is synthesized by a subset of glutamatergic pre-BötC neurons, but acts as an inhibitory neuromodulator. Moreover, SST regulates breathing both in normoxic and in hypoxic conditions. Although it has been implicated in the neuromodulation of breathing, neither the locus of SST modulation, nor the receptor subtypes involved have been identified. In this study, we aimed to fill in these blanks by characterizing the SST-induced regulation of inspiratory rhythm generation in vitro and in vivo. We found that both endogenous and exogenous SST depress all preBötC-generated rhythms. While SST abolishes sighs, it also decreases the frequency and increases the regularity of eupnea and gasping. Pharmacological experiments showed that SST modulates inspiratory rhythm generation by activating SST receptor type-2, whose mRNA is abundantly expressed in the pre-Bötzinger complex. In vivo, blockade of SST receptor type-2 reduces gasping amplitude and consequently, it precludes auto-resuscitation after asphyxia. Based on our findings, we suggest that SST functions as an inhibitory neuromodulator released by excitatory respiratory neurons when they become overactivated in order to stabilize breathing rhythmicity in normoxic and hypoxic conditions.

Influence of CA2+ on K+ efflux during regulatory volume decrease in cultured astrocytes

The calcium (Ca2+) dependence of potassium (K+) efflux activated by hyposmolarity in cultured cerebellar astrocytes was investigated, measuring in parallel experiments 86Rb release and changes in cytosolic Ca2+ ([Ca2+]i). Hyposmotic (50%) medium increased [Ca2+]i from 117 to 386 nM, with contributions of extracellular Ca2+ and Ca2+ from the endoplasmic reticulum. Hyposmotic medium increased 86Rb efflux rate from 0.015 min−1 to a maximal of 0.049 min−1 and a net release of 30%. This osmosensitive efflux was inhibited by Ba2+ (0.028 min−1), quinidine (0.024 min−1), and charybdotoxin (0.040 min−1), but was unaffected by TEA, 4‐AP, or apamin. Removal of external Ca2+ from the hyposmotic medium increased 86Rb efflux to a maximal rate constant of 0.056 min−1 and a net release of 38% and caused a delay of inactivation. These changes were due to the overlaping of an efflux activated by Ca2+ removal in isosmotic medium. This isosmotic 86Rb efflux was unaffected by TEA or 4‐AP, reduced by verapamil, and abolished by Ba2+, nitrendipine, and Mg2+. With the swelling‐induced [Ca2+]i rise suppressed by ethyleneglycoltetraacetic acid‐acetoxy‐methyl ester (EGTA‐AM), hyposmotic 86Rb was 30% reduced. The Ca2+ entry blockers Cd2+, Ni2+, La3+, and Gd3+ did not affect 86Rb efflux. A 40% decrease observed with verapamil and nitrendipine was found unrelated to Ca2+, because these agents did not affect the [Ca2+]i rise and the inhibition persisted in the absence of external Ca2+. The phospholipase C blocker U‐73122 did not affect [Ca2+]i nor 86Rb efflux. Blockers of Ca2+/calmodulin W7 and KN‐93 decreased 86Rb efflux to the same extent as EGTA‐AM. Ionomycin markedly potentiated 86Rb release in hyposmotic conditions only when [Ca2+]i was raised to about 1 μM, suggesting the implication of maxi‐K+ channels at this [Ca2+]i threshold, which nonetheless, was not attained during hyposmotic swelling. It is concluded that 86Rb efflux in cerebellar astrocytes is largely (70%) Ca2+‐independent and the Ca2+‐dependent fraction is sustained essentially by Ca2+ released from the endoplasmic reticulum and mediated by a mechanism involving Ca2+/calmodulin. J. Neurosci. Res. 57:350–358, 1999.

Amyloid beta 1-42 inhibits entorhinal cortex activity in the beta-gamma range: role of GSK-3.

Oscillatory activity in the entorhinal cortex has been associated with several cognitive functions. Accordingly, Alzheimer Disease-associated cognitive decline has been related to amyloid beta-induced disturbances in several of these oscillatory patterns. We have previously shown that acute application of amyloid beta inhibits the generation of slow frequency oscillations (7-20 Hz). In contrast, alterations in faster oscillations recorded in Alzheimer Disease-transgenic mice that over-express amyloid beta have been controversial. Since transgenic mice may produce complex responses due to compensatory mechanisms, we tested the effect of acute application of amyloid beta on fast oscillations (beta-gamma bursts) generated by entorhinal cortex slices in vitro in a Mg2+ -ree solution. We also explored the participation of the enzyme glycogen synthase kinase 3 (GSK-3) in this effect. Our results show that bath application of a clinically relevant concentration of amyloid beta (10 nM) activates GSK-3 and reduces the power of beta-gamma bursts in the entorhinal cortex. The reduction of beta-gamma bursts by amyloid beta is blocked by inhibiting GSK-3 either with lithium or with SB 216763. Our results suggest that amyloid beta-induced inhibition of entorhinal cortex beta-gamma activity involves GSK-3 activation, which may provide a molecular mechanism for amyloid beta-induced neural network disruption and support the use of GSK-3 inhibitors to treat Alzheimer Disease.

Amyloid Beta Peptides Differentially Affect Hippocampal Theta Rhythms In Vitro

Soluble amyloid beta peptide (Aβ) is responsible for the early cognitive dysfunction observed in Alzheimers disease. Both cholinergically and glutamatergically induced hippocampal theta rhythms are related to learning and memory, spatial navigation, and spatial memory. However, these two types of theta rhythms are not identical; they are associated with different behaviors and can be differentially modulated by diverse experimental conditions. Therefore, in this study, we aimed to investigate whether or not application of soluble Aβ alters the two types of theta frequency oscillatory network activity generated in rat hippocampal slices by application of the cholinergic and glutamatergic agonists carbachol or DHPG, respectively. Due to previous evidence that oscillatory activity can be differentially affected by different Aβ peptides, we also compared Aβ 25−35 and Aβ 1−42 for their effects on theta rhythms in vitro at similar concentrations (0.5 to 1.0 μM). We found that Aβ 25−35 reduces, with less potency than Aβ 1−42, carbachol-induced population theta oscillatory activity. In contrast, DHPG-induced oscillatory activity was not affected by a high concentration of Aβ 25−35 but was reduced by Aβ 1−42. Our results support the idea that different amyloid peptides might alter specific cellular mechanisms related to the generation of specific neuronal network activities, instead of exerting a generalized inhibitory effect on neuronal network function.

CA2+ CHANGES AND 86RB EFFLUX ACTIVATED BY HYPOSMOLARITY IN CEREBELLAR GRANULE NEURONS

Hyposmotic swelling increased 86Rb release in cultured cerebellar granule neurons (1 day in vitro [DIV]) with a magnitude related to the change in osmolarity. 86Rb release was partially blocked by quinidine, Ba2+, and Cs+ but not by TEA, 4‐AP, or Gd3+. 86Rb efflux decreased in Cl−‐depleted cells or cells treated with DDF or DIDS, suggesting an interconnection between Cl− and K+ fluxes. Swelling induced a substantial increase in [Ca2+]i to which both external and internal sources contribute. However, 86Rb efflux was independent of [Ca2+]o, unaffected by depleting the endoplasmic reticulum (ER) by ionomycin or thapsigargin and insensitive to charybdotoxin, iberiotoxin, and apamin. Swelling‐activated 86Rb efflux in differentiated granule neurons after 8 DIV, which express Ca2+‐sensitive K+ channels, was not different from that in 1 DIV neurons, nor in time course, net release, Ca2+‐dependence, or pharmacological sensitivity. We conclude that the swelling‐activated K+ efflux in cerebellar granule neurons is not mediated by Ca2+‐sensitive large conductance K+ channels (BK) as in many cell types but resembles that in lymphocytes where it is possibly carried by voltage‐gated K+ channels. J. Neurosci. Res. 53:626–635, 1998.