Biomedical subjects

H Pasantes-Morales

Publications and source records attributed to H Pasantes-Morales.

At least 19 recordsLinked to original sources

PubMed2005

Mechanisms of chloride influx during KCl-induced swelling in the chicken retina.

An increase in extracellular KCl ([KCl]o) occurs under various pathological conditions in the retina, leading to retinal swelling and possible neuronal damage. The mechanisms of this KCl o-induced retinal swelling were investigated in the present study, with emphasis on the Cl- transport mechanisms. Increasing [KCl]o (from 5 to 70 mM) led to concentration-dependent swelling in chicken retinas. The curve relating the degree of swelling to [KCl]o was biphasic, with one component from 5 to 35 mM [KCl]o and another from 35 to 100 mM. As Cl- omission prevented swelling in all conditions, the effect of cotransporter or Cl- channel blockers was examined to investigate the mechanisms of Cl- influx. The cotransporter blockers bumetanide and DIOA reduced swelling by 68% and 76%, respectively at [KCl]o 25 mM (K25), and by 14-17% at [KCl]o 54 mM (K54). The Cl- channel blockers NPPB and niflumic acid did not affect swelling at K25 but reduced it by 90-95% at K54 (NPPB IC50 60.7 microM). Furosemide showed an atypical effect, decreasing swelling by 14% at K25 and by 95% at K54 (IC50 173.9 microM). Na+ omission decreased swelling at K25 but not at K54. These results suggest the contribution of cotransporters to Cl- influx at K25 and of Cl- channels at K54. At K25, swelling was found in the ganglion cell layer and in the lower half of the inner nuclear layer. With K54, swelling occurred in all inner retinal layers. The ganglion cell layer swelling was due to both Muller cell end-foot and ganglion cell soma swelling. K54 also induced ganglion cell damage as shown by disorganized, pyknotic and refringent nuclei.

Acetates↗

PubMed2005

Epidermal growth factor receptor is a common element in the signaling pathways activated by cell volume changes in isosmotic, hyposmotic or hyperosmotic conditions.

Changes in external osmolarity, including both hyper- or hyposmotic conditions, elicit the tyrosine phosphorylation of a number of tyrosine kinase receptors (TKR). We show here that the epidermal growth factor receptor (EGFR) is activated by both cell swelling (hyposmolarity, isosmotic urea, hyperosmotic sorbitol) or shrinkage (hyperosmotic NaCl or raffinose) and discuss the mechanisms by which these apparently opposed conditions come to the same effect, i.e., EGFR activation. Evidence suggests that this results from early activation of integrins, p38 and tyrosine kinases of the Src family, which are all activated in the two anisosmotic conditions. TKR transactivation by integrins and p38 is likely occurring via an effect on the metalloproteinases. Information discussed in this review, points to TKR as elements in osmotransduction as a useful mechanism to amplify and diversify the initial response to anisosmolarity and cell volume changes, due to their privileged situation as convergence point for numerous intracellular signaling pathways. The variety of effector pathways connected to TKR is advantageous for the cell to cope with the changes in cell volume including adaptation to stress, cytoskeleton remodeling, adhesion reactions, cell survival and the adaptive mechanisms to ultimately restore the original cell volume.

Animals↗

PubMed2005

Hyposmolarity evokes norepinephrine efflux from synaptosomes by a depolarization- and Ca2+ -dependent exocytotic mechanism.

Osmolarity reduction (20%) elicited 3H-norepinephrine (NE) efflux from rat cortical synaptosomes. The hyposmotic NE release resulted from the following events: (i) a Na+-dependent and La3+-, Gd3+- and ruthenium red-sensitive depolarization; (ii) a cytosolic Ca2+ ([Ca2+]i) rise with contributions from external Ca2+ influx and internal Ca2+ release, probably through the mitochondrial Na+-Ca2+ exchanger; and (iii) activation of a [Ca2+]i-evoked, tetanus toxin (TeTX)-sensitive, PKC-modulated NE efflux mechanism. This sequence was established from results showing a drop in the hyposmotic [Ca2+]i rise by preventing depolarization with La3+, and by the inhibitory effects of Ca2+-free medium (EGTA; 50%), CGP37157 (the mitochondrial Na+-Ca2+ exchanger blocker; 48%), EGTA + CGP37157 or by EGTA-AM (> 95% in both cases). In close correspondence with these effects, NE efflux was 92% decreased by Na+ omission, 75% by La3+, 47% by EGTA, 50% by CGP37157, 90% by EGTA + CGP37157 and 88% by EGTA-AM. PKC influenced the intracellular Ca2+ release and, mainly through this action, modulated NE efflux. TeTX suppressed NE efflux. The K+-stimulated NE release, studied in parallel, was unaffected by Na+ omission, or by La3+, Gd3+ or ruthenium red. It was fully dependent on external Ca2+, insensitive to CGP37157 and abolished by TeTX. These results suggest that the hyposmotic events, although different from the K+-evoked depolarization and [Ca2+]i rise mechanisms, are able to trigger a depolarization-dependent, Ca2+-dependent and TeTX-sensitive mechanism for neurotransmitter release.

Animals↗

PubMed2004

Volume changes and whole cell membrane currents activated during gradual osmolarity decrease in C6 glioma cells: contribution of two types of K+ channels.

Volume changes and whole cell ionic currents activated by gradual osmolarity reductions (GOR) of 1.8 mosM/min were characterized in C6 glioma cells. Cells swell less in GOR than after sudden osmolarity reductions (SOR), the extent of swelling being partly Ca(2+) dependent. In nominally Ca(2+)-free conditions, GOR activated predominantly whole cell outward currents. Cells depolarized from the initial -79 mV to a steady state of -54 mV reached at 18% osmolarity reduction [hyposmolarity of -18% (H-18%)]. Recordings of Cl(-) and K(+) currents showed activation at H-3% of an outwardly rectifying Cl(-) current, with conductance of 1.6 nS, sensitive to niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, followed at H-18% by an outwardly rectifying K(+) current with conductance of 4.1 nS, inhibited by clofilium but insensitive to the typical K(+) channel blockers. With 200 nM Ca(2+) in the patch pipette, whole cell currents activated at H-3% and at H-13% cells depolarized from -77 to -63 mV. A K(+) current activated at H-1%, showing a rapid increase in conductance, suppressed by charybdotoxin and insensitive to clofilium. These results show the operation of two different K(+) channels in response to GOR in the same cell type, activated by Ca(2+) and osmolarity and with different osmolarity activation thresholds. Taurine and glutamate efflux, monitored by labeled tracers, showed delayed osmolarity thresholds of H-39 and H-33%, respectively. This observation clearly separates the Cl(-) and amino acid osmosensitive pathways. The delayed amino acid efflux may contribute to counteract swelling at more stringent osmolarity reductions.

Animals↗

PubMed2001

Reduction of phospholemman expression decreases osmosensitive taurine efflux in astrocytes.

The role of phospholemman (PLM) in taurine and Cl(-) efflux elicited by 30% hyposmotic solution was studied in cultured cerebellar astrocytes with reduced PLM expression by antisense oligonucleotide (AO) treatment. PLM, a substrate for protein kinases (PK) C and A, is a protein that increases an anion current in Xenopus oocytes and forms taurine-selective channels in lipid bilayers. Taurine contributes as an osmolyte to regulatory volume decrease (RVD) and is highly permeable through PLM channels in bilayers. Two antisense oligonucleotides (AO1 and AO2) effectively decreased the expression of the PLM protein by 40% and 30%, respectively, and markedly reduced [(3)H]taurine efflux by 67% and 62%. AO treatment also decreased the osmosensitive release of Cl(-), followed as (125)I. The inhibition of Cl(-) efflux (23% for AO1 and 13% for AO2) was notably lower than for [(3)H]taurine. The contribution of PKC and PKA in the function of PLM was also evaluated in astrocytes. Pharmacological activation or inhibition of PKC and PKA revealed that the osmosensitive taurine efflux is essentially PKC-independent while (125)I efflux is reduced by the PKC blockers H-7 (21%) and Gö6983 (41%). The PKA activator forskolin and dbcAMP increased taurine efflux by 66-70% and (125)I efflux by 21-45%. Norepinephrine increased the osmosensitive taurine efflux at about the same extent as dbcAMP and forskolin, and this was reduced by PKA blockers. These results suggest that PLM plays a role in RVD in astrocytes by predominantly influencing taurine fluxes, which are modulated by PKA but not PKC.

Animals↗

PubMed2001

Evidence for two mechanisms of amino acid osmolyte release from hippocampal slices.

A 30% decrease in osmolarity stimulated 3H-taurine, 3H-GABA and glutamate (followed as 3H-D-aspartate) efflux from rat hippocampal slices. 3H-taurine efflux was activated rapidly but inactivated slowly. It was decreased markedly by 100 microM 5-nitro-(3-phenylpropylamino)benzoic acid (NPPB) and 600 microM niflumic acid and inhibited strongly by tyrphostins AG18, AG879 and AG112 (25-100 microM), suggesting a tyrosine kinase-mediated mechanism. Hyposmolarity activated the mitogen-activated protein kinases (MAPK) extracellular-signal-related kinase-1/2 (ERK1/ERK2) and p38, but blockade of this reaction did not affect 3H-taurine efflux. Hyposmosis also activated phosphatidylinositide 3-kinase (PI3K) and its prevention by wortmannin (100 nM) essentially abolished 3H-taurine efflux. 3H-taurine efflux was insensitive to the protein kinase C (PKC) blocker chelerythrine (2.5 microM) or to cytochalasin E (3 microM). The release of 3H-GABA and 3H-D-aspartate occurred by a different mechanism, characterized by rapid activation and inactivation, insensitivity to NPPB, niflumic acid, tyrphostins or wortmannin. 3H-GABA and 3H-D-aspartate efflux was not due to external [NaCl] decrease, cytosolic Ca2+ increase or depolarization, or to reverse operation of the carrier. This novel mechanism of amino acid release may be mediated by Ca2+-independent exocytosis and modulated by PKC and actin cytoskeleton disruption, as suggested by its inhibition by chelerythrine and potentiation by 100 nM phorbol-12-myristate-13 acetate (PMA) and cytochalasin E. GABA and glutamate osmosensitive efflux may explain the hyposmolarity-elicited increase in amplitude of inhibitory and excitatory postsynaptic potentials in hippocampal slices as well as the hyperexcitability associated with hyponatraemia.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2001

Influence of protein kinases on the osmosensitive release of taurine from cerebellar granule neurons.

The role of phosphorylation events on the activation and modulation of the osmosensitive (3)H-taurine release (OTR) was examined in cultured cerebellar granule neurons (CGN) stimulated with 30% hyposmotic solutions. OTR was not decreased when [Ca(2+)](i) rise evoked by hyposmolarity was prevented by EGTA-AM (50 microM) or depleted by treatment with 1 microM ionomycin in Ca(2+)-free medium. Accordingly, OTR was not inhibited by Ca(2+)-dependent signaling events. The calmodulin (CAM) blocker W-7 (50 microM) potentiated OTR while the Ca(2+)/CAM kinase blocker KN-93 (10 microM) was without effect. Blockade of PKC by H-7, H-8 (50 microM) and Gö6976 (1 microM), as well as activation by phorbol myristate acetate (PMA) (100 nM) did not influence OTR, but chronic treatment to down regulate PKC decreased it by 30%. Forskolin (20 microM) and 8-BrcAMP (10 microM) did not change OTR. Protein tyrosine phosphorylation seems to be of crucial importance in the activation and modulation of OTR, as it was markedly inhibited (90%) by tyrphostine A23 (50 microM) and potentiated by the tyrosine phosphatase inhibitor ortho-vanadate (100 microM). The PI3 kinase blocker wortmannin 100 nM essentially abolished OTR but LY294002 (10-100 microM) was without effect. This difference may be accounted for PI3K isoforms in neurons with different sensitivity to the blockers. Alternatively, the effect of wortmannin may be exerted not in PI3 kinase but instead on phospholipases, which are also sensitive to this blocker. The hyposmotic stimulus induced activation of Erk1/Erk2, but blockade of this effect by PD 98059 (50 microM) only marginally decreased OTR suggesting that the Erk1/Erk2 is an epiphenomenon, not directly involved in OTR activation.

Androstadienes↗

PubMed2001

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 (pi(o)), 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 pi(o) -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 pi(o). Cl--free medium impaired IVR when pi(o) decreased to -24% and Ba2+ blocked it only at a late phase of -30% pi(o). 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 pi(o) 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% pi(o)); and (iv), K+ fluxes do not contribute to IVR until a late phase (< -30% pi(o)).

Amino Acids↗

PubMed2000

Efflux of osmolyte amino acids during isovolumic regulation in hippocampal slices.

The efflux of potassium (K(+)) and amino acids from hippocampal slices was measured after sudden exposure to 10% (270 mOsm), 25% (225 mOsm) or 50% (150 mOsm) hyposmotic solutions or after gradual decrease (-2.5 mOsm/min) in external osmolarity. In slices suddenly exposed to 50% hyposmotic solutions, swelling was followed by partial (74%) cell volume recovery, suggesting regulatory volume decrease (RVD). With gradual hyposmotic changes, no increase in cell water content was observed even when the solution at the end of the experiment was 50% hyposmotic, showing the occurrence of isovolumic regulation (IVR). The gradual decrease in osmolarity elicited the efflux of (3)H-taurine with a threshold at -5 mOsm and D-[(3)H]aspartate (as marker for glutamate) and at -20 mOsm for [(3)H]GABA. The efflux rate of [(3)H]taurine was always notably higher than those of [(3)H]GABA and D-[(3)H]aspartate, with a maximal increase over the isosmotic efflux of about 7-fold for [(3)H]taurine and 3- and 2-fold for [(3)H]GABA and D-[(3)H]aspartate, respectively. The amino acid content in slices exposed to 50% hyposmotic solutions (abrupt change) during 20 min decreased by 50. 6% and 62.6% (gradual change). Taurine and glutamate showed the largest decrease. An enhancement in (86)Rb efflux and a corresponding decrease in K(+) tissue content was seen in association with RVD but not with IVR. These results demonstrate the contribution of amino acids to IVR and indicate their involvement in this mechanism of cell volume control.

Amino Acids↗

PubMed2000

Volume sensitive efflux of taurine in HEK293 cells overexpressing phospholemman.

The role of the phospholemman (PLM) on the efflux of taurine and chloride induced by swelling was studied in HEK293 cells overexpressing stable transfected PLM. PLM, a substrate for protein kinases C and A, is a protein that induces an anion current in Xenopus oocytes and forms taurine-selective channels in lipid bilayers. Taurine contributes as an osmolyte to regulatory volume decrease (RVD) and is highly permeable through PLM channels in bilayers. In PLM-overexpressing cells the process of RVD was more rapid and efficient (75%) than in control cells (44%). Also, [(3)H]taurine and (125)I efflux induced by hyposmolarity were markedly increased (30-100%) in two subclones of cells overexpressing PLM. This increased efflux was sensitive to the Cl channel blockers DDF, NPPB and DIDS. Acute treatment of control cells with isoproterenol and norepinephrine induced a significant potentiation (50-60%) of [(3)H]taurine release induced by hyposmolarity. In PLM-overexpressing cells the potentiation by these drugs was higher (100%). Insulin induced also an increase in [(3)H]taurine release, but only in PLM-overexpressing cells (50%). These results indicate that PLM may play a role in the RVD and that its phosphorylation may have a physiological significance during this process. The mechanisms involved in this process could include the activation of PLM itself as channel or the modulation of other preexisting channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Release of taurine in apoptotic cerebellar granule neurons in culture.

Cell shrinkage is a distinctive feature of apoptotic death, but the mechanisms leading to cell volume loss are unclear at present. Activation of pathways extruding intracellular osmolytes such as K+, Cl- and organic molecules may be part of these mechanisms. This was examined in the present work measuring the release of taurine, gamma-amino-butyric acid (GABA) and glutamate in cerebellar granule neurons cultured in conditions resulting in apoptotic death after 4-7 days in vitro (DIV). The basal release of [3H]taurine from cells started to increase (38%) after 3 DIV and reached a maximal enhancement (250%) at 5 DIV. The increase in taurine efflux closely followed the occurrence of apoptotic death markers such as caspase induction and chromatin condensation. The efflux of glutamate (traced as D-aspartate) and [3H]GABA also increased but notably less than that of taurine (90% and 75%, respectively) at 5 DIV. Taurine release associated with apoptosis was unaffected by 4,4'-diisothiocyanatostilbene 2,2'-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), blockers of the diffusive pathway activated during cell volume regulation in hyposmotic conditions. Taurine efflux was increased in Cl(-)-free (replaced by gluconate) and decreased in Na+-free media. Blockers of the energy-dependent glutamate and taurine carriers, dihydrokainate and guanidinoethane sulfonate, respectively, did not affect the release associated with apoptosis. These results implicate taurine in the mechanism of cell shrinkage during apoptosis.

Animals↗

PubMed2000

Signaling events during swelling and regulatory volume decrease.

Brain cell swelling compromises neuronal function and survival by the risk of generation of ischemia episodes as compression of small vessels occurs due to the limits to expansion imposed by the rigid skull. External osmolarity reductions or intracellular accumulation of osmotically active solutes result in cell swelling which can be counteracted by extrusion of osmolytes through specific efflux pathways. Characterization of these pathways has received considerable attention, and there is now interest in the understanding of the intracellular signaling events involved in their activation and regulation. Calcium and calmodulin, phosphoinositides and cAMP may act as second messengers, carrying the information about a cell volume change into signaling enzymes. Small GTPases, protein tyrosine kinases and phospholipases, also appear to be part of the signaling cascades ultimately modulating the osmolyte efflux pathways. This review focus on i) the influence of hyposmotic and isosmotic swelling on these signaling events and molecules and ii) the effects of manipulating their function on the osmolyte fluxes, particularly K+, CI- and amino acids, and on the consequent efficiency of cell volume adjustment.

Animals↗

PubMed2000

Amino acid osmolytes in regulatory volume decrease and isovolumetric regulation in brain cells: contribution and mechanisms.

Brain adaptation to hyposmolarity is accomplished by loss of both electrolytes and organic osmolytes, including amino acids, polyalcohols and methylamines. In brain in vivo, the organic osmolytes account for about 35% of the total solute loss. This review focus on the role of amino acids in cell volume regulation, in conditions of sudden hyposmosis, when cells respond by active regulatory volume decrease (RVD) or after gradual exposure to hyposmotic solutions, a condition where cell volume remains unchanged, named isovolumetric regulation (IVR). The amino acid efflux pathway during RVD is passive and is similar in many respects to the volume-activated anion pathway. The molecular identity of this pathway is still unknown, but the anion exchanger and the phospholemman are good candidates in certain cells. The activation trigger of the osmosensitive amino acid pathway is unclear, but intracellular ionic strength seems to be critically involved. Tyrosine protein kinases markedly influence amino acid efflux during RVD and may play an important role in the transduction signaling cascades for osmosensitive amino acid fluxes. During IVR, amino acids, particularly taurine are promptly released with an efflux threshold markedly lower than that of K(+), emphasizing their contribution (possibly as well as of other organic osmolytes) vs inorganic ions, in the osmolarity range corresponding to physiopathological conditions. Amino acid efflux also occurs in response to isosmotic swelling as that associated with ischemia or trauma. Characterization of the pathway involved in this type of swelling is hampered by the fact that most osmolyte amino acids are also neuroactive amino acids and may be released in response to stimuli concurrent with swelling, such as depolarization or intracellular Ca(++) elevation.

Amino Acids↗

PubMed2000

Influence of calcium on regulatory volume decrease: role of potassium channels.

In most cell types, hyposmotic swelling consistently elicits an increase in the concentration of cytosolic Ca2+ - [Ca2+]i - with contributions of extracellular and intracellular sources. The mechanisms of Ca2+ entry and release from endogenous sources are not fully clarified and may be cell specific. The ubiquity of the swelling-evoked [Ca2+]i rise makes Ca2+ a likely candidate for a role as osmotransducing signal. However, the regulatory volume decrease (RVD) which follows swelling and the osmolyte fluxes involved in this process are not always Ca2+ dependent. It was found that, with a few exceptions, in most cell types the osmosensitive Cl- efflux pathway and the swelling-activated organic osmolyte fluxes are Ca2+ independent. In contrast, Ca2+-dependent or Ca2+-independent K+ fluxes activated by swelling are detected, depending on the cell type. The close correlation found in this review between the Ca2+ dependence of RVD and that of the K+ channels activated by swelling led to the conclusion that it is the type of osmosensitive K+ pathway which largely confers the Ca2+ dependence to RVD. Interestingly, this coincidence of Ca2+-dependent K+ efflux and RVD is found predominantly in epithelial cells, whereas in nonepithelial cells both processes are largely Ca2+ independent. In these cells, the [Ca2+]i rise elicited by swelling may be an epiphenomenon.

Animals↗

PubMed1999

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 (86)Rb 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 (86)Rb 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 Ba(2+) (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 (86)Rb 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 (86)Rb was 30% reduced. The Ca2+ entry blockers Cd2+, Ni2+, La3+, and Gd3+ did not affect (86)Rb 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 (86)Rb efflux. Blockers of Ca2+/calmodulin W7 and KN-93 decreased (86)Rb efflux to the same extent as EGTA-AM. Ionomycin markedly potentiated (86)Rb release in hyposmotic conditions only when [Ca2+]i was raised to about 1 microM, suggesting the implication of maxi-K+ channels at this [Ca2+]i threshold, which nonetheless, was not attained during hyposmotic swelling. It is concluded that (86)Rb 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.

Animals↗

PubMed1999

Activation and inactivation of taurine efflux in hyposmotic and isosmotic swelling in cortical astrocytes: role of ionic strength and cell volume decrease.

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.

Animals↗

PubMed1999

Amino acids as osmolytes in the retina.

Amino acids play a role as osmolytes during the regulatory volume decrease subsequent to hyposmotic swelling, but less is known about its role when swelling occurs in isosmotic conditions. In this work we examined the efflux of labelled GABA, taurine and glutamate (traced as D-aspartate) from the chick retina, after isosmotic swelling evoked by KCl-containing solutions, and compared its features to those in hyposmotic swelling. In both conditions, GABA and taurine efflux were more sensitive to swelling than glutamate, as assessed by the activation threshold and the amount released. The amino acid efflux in hyposmotic media was decreased by DIDS, tamoxifen and NPPB, agents acting as Cl channels blockers, which also inhibit the osmosensitive Cl efflux. The component associated with swelling in the KCl-stimulated efflux was assessed by the reduction observed when Cl is replaced by an impermeant anion, or by the influence of hyperosmotic media. GABA and taurine efflux exhibited a large swelling-dependent component, which was lower for D-aspartate. This component was markedly decreased by NPPB, but this was due to an effect of the blocker preventing swelling. These results suggest that the influx of Cl, acting as K counterion, which is responsible for cell swelling, occurs through a pathway sensitive to NPPB, similarly to that activated by hyposmolarity. This finding may be of interest in studies aiming at preventing the cell edema which occurs in a number of pathologies.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1998

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+]0, 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.

Animals↗