Biological Activity of Complexes Involving Nitro-Containing Ligands and Crystallographic-Theoretical Description of 3,5-DNB Complexes.

Drug resistance in infectious diseases developed by bacteria and fungi is an important issue since it is necessary to further develop novel compounds with biological activity that counteract this problem. In addition, new pharmaceutical compounds with lower secondary effects to treat cancer are needed. Coordination compounds appear to be accessible and promising alternatives aiming to overcome these problems. In this review, we summarize the recent literature on coordination compounds based on nitrobenzoic acid (NBA) as a ligand, its derivatives, and other nitro-containing ligands, which are widely employed owing to their versatility. Additionally, an analysis of crystallographic data is presented, unraveling the coordination preferences and the most effective crystallization methods to grow crystals of good quality. This underscores the significance of elucidating crystalline structures and utilizing computational calculations to deepen the comprehension of the electronic properties of coordination complexes.

Insights into the candidacidal mechanism of Ctn[15-34] - a carboxyl-terminal, crotalicidin-derived peptide related to cathelicidins.

PURPOSE: Ctn[15-34], a carboxyl-terminal fragment of crotalicidin (a cathelicidin from the venom gland of a South American rattlesnake), has shown antifungal activity against clinical and standard strains of Candida species. The aim of the present work was to investigate the underlying mechanisms of the candidicidal activity of Ctn[15-34]. METHODOLOGY: The time-kill profile and drug synergism were evaluated by means of a microdilution assay and multi-parametric flow cytometry. The presumptive interaction of Ctn[15-34] with lipid membranes was estimated in vitro with a lipid-mimic compound, the chromogenic substance 4-nitro-3-(octanoyloxy)benzoic acid (4N3OBA).Results/Key findings. The absorbance increment (at 425 nm) indicated a concentration- and time-dependent in-solution association between Ctn[15-34] and 4N3OBA. The interaction of Ctn[15-34] with Candida cells was confirmed by flow cytometric measurements with the 5(6)-carboxyfluorescein-labelled peptide (CF-Ctn[15-34]). Analysis of the killing time of Candida exposed to Ctn[15-34] and amphotericin B (AMB) showed that both the peptide and polyene drug reduce the number of c.f.u. but in mechanistically different ways. The Ctn[15-34] peptide alone caused yeast cell membrane disruption, which was confirmed by lactate dehydrogenase leakage and biomarkers of cell death mediated by necrosis. CONCLUSION: Overall, Ctn[15-34] displays a synergistic antifungal activity with AMB, an effect that can be further developed into a multi-target therapeutic option with other antimycotics currently in use.

HCC prevalence and histopathological findings in liver explants of patients with hereditary tyrosinemia type 1.

BACKGROUND: Untreated tyrosinemia type 1 (HT1) is manifested by liver failure associated with renal tubular dysfunction, growth failure, and rickets. The indication for liver transplantation (LT) is restricted to non-responders to 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC) treatment, patients not treated with NTBC or for patients with HCC. The aim of this study is to report on a series of NTBC naive HT1 patients submitted to LT along with the prevalence of HCC in their liver explants. PROCEDURE: This is a retrospective study of 16 children with HT1 who underwent liver transplantation between January 1993 and December 2012. CLINICAL FEATURES: liver failure in 12 (75%), growth failure in 4 (25%), rickets in 5 (31.2%), hypertrophic cardiomyopathy in three (18.7%), and renal tubulopathy in seven patients (43.7%). Median AFP level was 64,335 ng/ml. Abdominal CT scans showed multiple nodules in most patients. Histopathology of the explants showed cirrhosis in all patients and HCC in 12 (75%), 3 with microvascular invasion. The majority of the tumors were well differentiated. Patient survival rate was 86% at a median follow-up of 6.6 years. All survivors were tumor-free with no adjuvant chemotherapy. CONCLUSION: In countries where neonatal screening programs are not effective and NTBC treatment is not widely available, LT still plays an important role in the treatment of children with HT1. An early indication in patients who present with multinodular livers can also serve to treat an otherwise underdiagnosed HCC condition.

Peripheral antinociception induced by δ-opioid receptors activation, but not µ- or κ-, is mediated by Ca²âº-activated Cl⁻ channels.

Studies have demonstrated that the L-arginine/NO/cGMP pathway and the potassium and calcium channels are involved in the mechanisms underlying opioid receptor activation. As additional pathways may participate in the observed antinociceptive effects following opioid exposure, the aim of our study was to determine whether Ca(2+)-activated Cl(-) channels (CaCCs) are involved in peripheral antinociception induced by µ-, δ- and κ-opioid receptor activation. Hyperalgesia was induced by intraplantar injection of prostaglandin E(2) (PGE(2), 2 µg). Nociceptive thresholds to pressure (grams) were measured using an algesimetric apparatus 3h following injection. The µ-opioid receptor agonist morphine (200 µg), δ-opioid receptor agonist (+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80, 80 µg), κ-opioid receptor agonist bremazocine (50 µg), CaCCs blocker niflumic acid (8-64 µg), CaCCs blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 32-128 µg), nitric oxide donor sodium nitroprusside (SNP, 500 µg) and cGMP exogenous analogs dibutyryl cGMP (db-cGMP, 100 µg) were also administered into the paw. The CaCCs blocker niflumic acid and NPPB partially reversed the peripheral antinociception induced by exposure to the SNC80 in a dose-dependent manner. In contrast, niflumic acid did not modify the antinociceptive effect observed following exposure to morphine or bremazocine. Additionally, the peripheral antinociception induced by the NO donor SNP or by db-cGMP was not inhibited by niflumic acid. These results provide evidence for the involvement of CaCCs in the peripheral antinociception induced by SNC80. CaCCs activation does not appear to be involved when µ- and κ-opioid receptors are activated. In addition, we did not observe a link between CaCCs and the L-arginine/NO/GMPc pathway.

Ion channels in volume regulation of clonal kidney cells.

OBJECTIVES: Clonal kidney cells (Vero cells) are extensively utilized in the manufacture of biological preparations for disease diagnostics and therapeutics and also in preparation of vaccines. In all cells, regulation of volume is an essential function coupled to a variety of physiological processes and is a topic of interest. The objective here was to investigate involvement of ion channels in the process of volume regulation of Vero cells. METHODS: Involvement of ion channels in cell volume regulation was studied using video-microscopy and flow cytometry. Pharmacologically unaltered cells of different sizes, which are presumably at different phases of the cell cycle, were used. RESULTS: Ion transport inhibitors altered all phases of regulatory volume decrease (RVD) of Vero cells, rate of initial cell swelling, V(max) and volume recovery. Effects were dependent on type of inhibitor and on cell size (cell cycle phase). Participation of aquaporins in RVD was suggested. Inhibitors decelerated growth, arresting Vero cells at the G(0) /G(1) phase boundary. Electrophysiological study confirmed presence of volume-activated Cl(-) channels and K(+) channels in plasmatic membranes of the cells. CONCLUSION: Vero cells of all sizes maintained the ability to recover from osmotic swelling. Activity of ion channels was one of the key factors that controlled volume regulation and proliferation of the cells.

Role of CFTR and ClC-5 in modulating vacuolar H+-ATPase activity in kidney proximal tubule.

BACKGROUND/AIMS: It has been widely accepted that chloride ions moving along chloride channels act to dissipate the electrical gradient established by the electrogenic transport of H(+) ions performed by H(+)-ATPase into subcellular vesicles. Largely known in intracellular compartments, this mechanism is also important at the plasma membrane of cells from various tissues, including kidney. The present work was performed to study the modulation of plasma membrane H(+)-ATPase by chloride channels, in particular, CFTR and ClC-5 in kidney proximal tubule. METHODS AND RESULTS: Using in vivo stationary microperfusion, it was observed that ATPase-mediated HCO(3)(-) reabsorption was significantly reduced in the presence of the Cl(-) channels inhibitor NPPB. This effect was confirmed in vitro by measuring the cell pH recovery rates after a NH(4)Cl pulse in immortalized rat renal proximal tubule cells, IRPTC. In these cells, even after abolishing the membrane potential with valinomycin, ATPase activity was seen to be still dependent on Cl(-). siRNA-mediated CFTR channels and ClC-5 chloride-proton exchanger knockdown significantly reduced H(+)-ATPase activity and V-ATPase B2 subunit expression. CONCLUSION: These results indicate a role of chloride in modulating plasma membrane H(+)-ATPase activity in proximal tubule and suggest that both CFTR and ClC-5 modulate ATPase activity.

Volume-activated chloride channels in mice Leydig cells.

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume-activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of approximately 75 mOsm. These currents display the typical biophysical signature of volume-activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(- )> Cl(- )> F(-)). Staurosporine (200 nM) did not block the activation of I(Cl,swell). The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 microM), SITS (200 microM), ATP (500 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 100 miccroM), and Suramin (10 microM) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(do) and fractional distance of the binding site (delta) of 334 microM and 47 %, 880 microM and 35 %, 2,100 microM and 49%, 188 microM and 27%, and 66.5 microM and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that I(Cl,swell) in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

Aortas isolated from sinoaortic-denervated rats exhibit rhythmic contractions that are regulated by pharmacologically distinct calcium sources.

Sinoaortic denervation is characterized by arterial pressure lability, without sustained hypertension. Aortas isolated from rats with sinoaortic denervation present rhythmic contractions. We studied the participation of distinct Ca(2+) sources in the maintenance of the oscillations. Three days after the surgeries, aortic rings were placed in an organ chamber, and the incidence of aortas presenting rhythmic contractions was measured. Specific drugs were employed to analyse the participation of the Ca(2+) released from the sarcoplasmic reticulum [2-APB (diphenylborinic acid 2-aminoethyl ester), thapsigargin and ryanodine] and external Ca(2+) entry [Bay K 8644, verapamil and DMB (dimethylbenzyl amiloride)] on the rhythmic contractions. Additionally, we verified the effects of chloride channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] on the maintenance of the rhythmic contractions. Under phenylephrine stimulus, sinoaortic-denervated rat aortas exhibited rhythmic contractions in the frequency of 4.5 +/- 0.50 cycles/min. and an amplitude of 0.465 +/- 0.05 g. 2-APB, thapsigargin and ryanodine inhibited the rhythmic contractions. Bay K 8644 increased the oscillations, reaching maximum values with a concentration of 50 nM (18.5 +/- 2.5 cycles/min.). The rhythmic contractions were inhibiting by verapamil and Ca(2+)-free solution. DMB and NPPB did not alter the oscillations. In conclusion, we observed that aorta isolated from sinoaortic-denervated rats present rhythmic contractions. Moreover, drugs that impaired intracellular Ca(2+) release from sarcoplasmic reticulum interrupted the oscillations. The oscillations also depend on the extracellular Ca(2+) entry through L-type Ca(2+).

Differentiation of Mycobacterium tuberculosis from other mycobacteria with rho-nitrobenzoic acid using MGIT960.

SETTING: Mycobacteria growth in media with the addition of inhibitory substances has been used in species identification. Growth of the Mycobacterium tuberculosis complex (MTC) is inhibited by rho-nitrobenzoic acid (PNB), whereas non-tuberculous mycobacteria (NTM) are resistant. OBJECTIVE: To develop a rapid PNB test using the automated BACTEC MGIT960 system and to evaluate its usefulness in the screening of mycobacterial isolates. DESIGN: PNB tests were performed in 93 MTC strains and 61 NTM strains from the Instituto Adolfo Lutz Culture Collection. PNB was added to Löwenstein-Jensen (LJ) medium and to BACTEC MGIT960 medium. RESULTS: The MTC strains were all PNB-susceptible, confirming the original identification. Among 10 NTM species, all were found to be resistant to PNB, except for one strain of M. kansasii and another of M. marinum. The median time to obtain presumptive identification of MTC by inhibition test in the BACTEC MGIT960 system was 6.3 days and for NTM it was 2.5 days. The presumptive identification of MTC in LJ was mostly obtained after day 20. CONCLUSION: The key finding of this analysis was the possibility of combining the traditionally accepted method proposed by Tsukamura and Tsukamura in 1964 with the modern, safe and rapid BACTEC MGIT960 methodology.

The anion channel blocker, 4,4'-dinitrostilbene-2,2'-disulfonic acid prevents neuronal death and excitatory amino acid release during glycolysis inhibition in the hippocampus in vivo.

Neuronal death associated with cerebral ischemia and hypoglycemia is related to increased release of excitatory amino acids (EAA) and energy failure. The intrahippocampal administration of the glycolysis inhibitor, iodoacetate (IOA), induces the accumulation of EAA and neuronal death. We have investigated by microdialysis the role of exocytosis, glutamate transporters and volume-sensitive organic anion channel (VSOAC) on IOA-induced EAA release. Results show that the early component of EAA release is inhibited by riluzole, a voltage-dependent sodium channel blocker, and by the VSOAC blocker, tamoxifen, while the early and late components are blocked by the glutamate transport inhibitors, L-trans-pyrrolidine 2,4-dicarboxylate (PDC) and DL-threo-beta-benzyloxyaspartate (DL-TBOA); and by the VSOAC blocker 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Riluzole, DL-TBOA and tamoxifen did not prevent IOA-induced neuronal death, while PDC and DNDS did. The VSOAC blockers 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) and phloretin had no effect either on EAA efflux or neuronal damage. Results suggest that acute inhibition of glycolytic metabolism promotes the accumulation of EAA by exocytosis, impairment or reverse action of glutamate transporters and activation of a DNDS-sensitive mechanism. The latest is substantially involved in the triggering of neuronal death. To our knowledge, this is the first study to show protection of neuronal death by DNDS in an in vivo model of neuronal damage, associated with deficient energy metabolism and EAA release, two conditions involved in some pathological states such as ischemia and hypoglycemia.

Transcriptome analysis of Paracoccidioides brasiliensis cells undergoing mycelium-to-yeast transition.

Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), a systemic mycosis prevalent in South America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and transform into the yeast parasitic form. Thus, the mycelium-to-yeast transition is of particular interest because conversion to yeast is essential for infection. We have used a P. brasiliensis biochip carrying sequences of 4,692 genes from this fungus to monitor gene expression at several time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). The results revealed a total of 2,583 genes that displayed statistically significant modulation in at least one experimental time point. Among the identified gene homologues, some encoded enzymes involved in amino acid catabolism, signal transduction, protein synthesis, cell wall metabolism, genome structure, oxidative stress response, growth control, and development. The expression pattern of 20 genes was independently verified by real-time reverse transcription-PCR, revealing a high degree of correlation between the data obtained with the two methodologies. One gene, encoding 4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD), was highly overexpressed during the mycelium-to-yeast differentiation, and the use of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione], a specific inhibitor of 4-HPPD activity, as well as that of NTBC derivatives, was able to inhibit growth and differentiation of the pathogenic yeast phase of the fungus in vitro. These data set the stage for further studies involving NTBC and its derivatives as new chemotherapeutic agents against PCM and confirm the potential of array-based approaches to identify new targets for the development of alternative treatments against pathogenic microorganisms.

Evaluation of a rapid differentiation test for the Mycobacterium tuberculosis complex by selective inhibition with rho-nitrobenzoic acid and thiophene-2-carboxylic acid hydrazide.

SETTING: Mycobacterial growth in media to which inhibitory substances are added has been used in species identification. Growth of the Mycobacterium tuberculosis complex (MTC) is inhibited by rho-nitrobenzoic acid (PNB), whereas non-tuberculous mycobacteria (NTM) are resistant. Thiophene-2-carboxylic acid hydrazide (TCH) is useful in the differentiation of MTC when performed together with other tests. OBJECTIVE: To develop a test using PNB or TCH added to culture medium, and to evaluate its usefulness in the screening of mycobacteria isolates. DESIGN: In 2001, PNB testing was performed in 109 M. tuberculosis strains identified by Instituto Adolfo Lutz (IAL) and 52 NTM strains from the institute's culture collection. The drugs were added to Löwenstein-Jensen (LJ) medium and to BBL-MGIT. RESULTS: Species differentiation of MTC with the MGIT/TCH method was similar to that observed using the conventional LJ/TCH method. The accuracy of the MGIT/PNB method to differentiate NTM and MTC strains was 99.4%. The BBL-MGIT system allowed presumptive identification in 3-11 days, compared to > or =12 days with LJ medium. CONCLUSION: A simple, low-cost test using growth inhibitors may be incorporated into a modern, safe and quick methodology enabling differentiation of MTC and NTM.

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.

Factors affecting ammonium uptake by C11 clone of MDCK cells.

In several tissues ammonium ions are able to use the transport pathways of other ions, particularly of K+. We investigated this possibility in the C11 clone of MDCK cells, thought to represent intercalated cells, in control and 0 Cl- conditions. Cell pH was measured by ratiometric fluorescence microscopy using the pH indicator BCECF. After preincubating the cells for 10 min in control or 0 Cl- (substituted by gluconate) Ringer, an ammonium pulse was applied to induce cell acidification. The magnitude of the initial alkalinization (DeltapH) was 0.24+/-0.03 ( n=28) pH units in controls, which fell to 0.023+/-0.01 ( n=12) in 0 Cl-, suggesting uptake of NH4+ balancing the alkalinization by NH3. Addition of 10(-3) M bumetanide or furosemide to the 0 Cl- medium, or 10(-4 )M hexamethylene amiloride, did not alter DeltapH. However, with 5 mM Ba+, DeltapH increased to 38% of control. When 2.5x10(-4) M ouabain, an inhibitor of Na+-K+ ATPase, was used, DeltapH increased to 46% of control. Inhibition of H+-K+ ATPase by SCH28080 or by omeprazol caused significant increase in DeltapH. In 0 Cl- solution, these cells underwent a mean volume reduction (-d V) of -10.24+/-1.96% per 10 min as measured by confocal microscopy. To investigate if NH4+ influx was regulated by cell volume or by cell Cl-, volume reduction was avoided by two procedures. When preincubating with NPPB, a Cl- channel blocker, in 0 Cl-, volume reduction was inhibited (d V=-2.12% per 10 min), and DeltapH was 0.24+/-0.04 ( n=5). When the cells were preincubated in hypotonic 0 Cl- (260 mosmol/l), cell volume reduction was abolished (d V=+2.6% per 10 min) and DeltapH was 0.52+/-0.07 ( n=7). Thus, activation of NH4+ influx by several transporters was due to volume reduction rather than to [Cl-] alteration.

Tyrosine kinases and amino acid efflux under hyposmotic and ischaemic conditions in the chicken retina.

The chicken retina was exposed to 20% hyposmotic or ischaemia-like (54 mM KCl and 1 mM ouabain) conditions and changes in cell volume, amino acid release and activation of protein tyrosine kinases measured. To investigate possible connection between these cellular events, the effect of tyrosine kinase blockers on (3)H-taurine, (3)H-GABA and (3)H- D-aspartate (as a tracer for glutamate) efflux was examined. Both hyposmotic and ischaemic conditions increased phosphorylation of the tyrosine kinase p125 focal adhesion kinase (p125(FAK)) and the mitogen-activated protein kinase-p38 (MAPK-p38), but not of the extracellular-signal-related kinases-1/2 (ERK1/ERK2), and markedly activated the tyrosine kinase target enzyme phosphatidylinositide 3-kinase (PI3K). Hyposmolarity and ischaemia both led to rapid retinal swelling followed by active volume recovery of 84% (hyposmolarity) and 40% (ischaemia), together with rapid release of taurine, GABA and D-aspartate. Taurine and GABA efflux under both conditions was reduced markedly by tyrosine kinase and PI3K blockers (50 microM tyrphostin A23, 50 microM genistein, 100 nM wortmannin, 25 microM LY294002) and was decreased by 85% when ischaemia-induced swelling was prevented. About 65% of D-aspartate efflux occurred irrespective of swelling in ischaemia and was either less sensitive (hyposmotic) or largely resistant (ischaemia) to the blockers. These results suggest that in ischaemia, GABA and taurine react primarily to swelling with a typical osmolyte response, while glutamate differs in its release mechanisms under both hyposmotic and ischaemic conditions. These findings suggest new strategies for evaluating the contribution of swelling to excitotoxicity in ischaemia.

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.

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.

Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. I. Regulatory volume decrease.

Exposure of NIH/3T3 fibroblasts not expressing P-glycoprotein to 50, 30, 20, and 10% hyposmotic solutions led to cell volume increases of 70, 32, 21, and 12%, respectively. After swelling, NIH/3T3 cells exhibited regulatory volume decrease (RVD), attaining complete volume recovery after 30 min except in 50% hyposmotic solution, in which volume recovery was 76%. RVD was accelerated by gramicidin and inhibited by the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid and by the K channel, blocker quinidine. RVD was reduced 15% by removal of extracellular Ca. The pathway opened by hypotonicity was highly permeable to K and Rb and only partly permeable to other cations. Most anions were able to permeate, with a permeability ranking of nitrate > benzoate = iodide > thiocyanate > chloride > > gluconate. The pathway was permeable to neutral amino acids, with a permeability ranking of glycine > alanine > glutamate > taurine > gamma-aminobutyric acid > glutamine. The pathway was not permeable to basic amino acids. These results show that, despite the absence of P-glycoprotein, NIH/3T3 cells exhibit RVD with properties similar to those expressed in most cell types.

Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. II. Chloride and amino acid fluxes.

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.