Ion-sensitive Microelectrodes Research Articles

Resting membrane potential and intracellular [Na+] at rest, during fatigue and during recovery in rat soleus muscle fibres in situ.

Large trans-sarcolemmal ionic shifts occur with fatiguing exercise or stimulation of isolated muscles. However, it is unknown how resting membrane potential (EM) and intracellular sodium concentration ([Na+]i) change with repeated contractions in living mammals. We investigated (i) whether [Na+]i (peak, kinetics) can reveal changes of Na+-K+ pump activity during brief or fatiguing stimulation and (ii) how resting EM and [Na+]i change during fatigue and recovery of rat soleus muscle in situ. Muscles of anaesthetised rats were stimulated with brief (10s) or repeated tetani (60Hz for 200ms, every 2s, for 30s or 300s) with isometric force measured. Double-barrelled ion-sensitive microelectrodes were used to quantify resting EM and [Na+]i. Post-stimulation data were fitted using polynomials and back-extrapolated to time zero recovery. Mean pre-stimulation resting EM (layer 2-7 fibres) was -71mV (surface fibres were more depolarised), and [Na+]i was 14mM. With deeper fibres, 10s stimulation (2-150Hz) increased [Na+]i to 38-46mM whilst simultaneously causing hyperpolarisations (7.3mV for 2-90Hz). Fatiguing stimulation for 30s or 300s led to end-stimulation resting EM of -61 to -53mV, which recovered rapidly (T1/2, 8-22s). Mean end-stimulation [Na+]i increased to 86-101mM with both fatigue protocols and the [Na+]i recovery time-course (T1/2, 21-35s) showed no difference between protocols. These combined findings suggest that brief stimulation hyperpolarises the resting EM, likely via maximum Na+-induced stimulation of the Na+-K+ pump. Repeated tetani caused massive depolarisation and elevations of [Na+]i that together lower force, although they likely interact with other factors to cause fatigue. [Na+]i recovery kinetics provided no evidence of impaired Na+-K+ pump activity with fatigue. KEY POINTS: It is uncertain how resting membrane potential, intracellular sodium concentration ([Na+]i), and sodium-potassium (Na+-K+) pump activity change during repeated muscle contractions in living mammals. For rat soleus muscle fibres in situ, brief tetanic stimulation for 10s led to raised [Na+]i, anticipated to evoke maximal Na+-induced stimulation of the Na+-K+ pump causing an immediate hyperpolarisation of the sarcolemma. More prolonged stimulation with repeated tetanic contractions causes massive elevations of [Na+]i, which together with large depolarisations (via K+ disturbances) likely reduce force production. These effects occurred without impairment of Na+-K+ pump function. Together these findings suggest that rapid activation of the Na+-K+ pump occurs with brief stimulation to maintain excitability, whereas more prolonged stimulation causes rundown of the trans-sarcolemmal K+ gradient (hence depolarisation) and Na+ gradient, which in combination can impair contraction to contribute to fatigue in living mammals.

CrossTalk proposal: Apical NKCC1 of choroid plexus epithelial cells works in the net inward flux mode under basal conditions, maintaining intracellular Cl- and cell volume.

CrossTalk proposal: Apical NKCC1 of choroid plexus epithelial cells works in the net inward flux mode under basal conditions, maintaining intracellular Cl- and cell volume.

Clearance of activity-evoked K+ transients and associated glia cell swelling occur independently of AQP4: A study with an isoform-selective AQP4 inhibitor.

The mammalian brain consists of 80% water, which is continuously shifted between different compartments and cellular structures by mechanisms that are, to a large extent, unresolved. Aquaporin 4 (AQP4) is abundantly expressed in glia and ependymal cells of the mammalian brain and has been proposed to act as a gatekeeper for brain water dynamics, predominantly based on studies utilizing AQP4-deficient mice. However, these mice have a range of secondary effects due to the gene deletion. An efficient and selective AQP4 inhibitor has thus been sorely needed to validate the results obtained in the AQP4-/- mice to quantify the contribution of AQP4 to brain fluid dynamics. In AQP4-expressing Xenopus laevis oocytes monitored by a high-resolution volume recording system, we here demonstrate that the compound TGN-020 is such a selective AQP4 inhibitor. TGN-020 targets the tested species of AQP4 with an IC50 of ~3.5 μM, but displays no inhibitory effect on the other AQPs (AQP1-AQP9). With this tool, we employed rat hippocampal slices and ion-sensitive microelectrodes to determine the role of AQP4 in glia cell swelling following neuronal activity. TGN-020-mediated inhibition of AQP4 did not prevent stimulus-induced extracellular space shrinkage, nor did it slow clearance of the activity-evoked K+ transient. These data, obtained with a verified isoform-selective AQP4 inhibitor, indicate that AQP4 is not required for the astrocytic contribution to the K+ clearance or the associated extracellular space shrinkage.

Catalytically inactive carbonic anhydrase-related proteins enhance transport of lactate by MCT1.

Carbonic anhydrases (CA) catalyze the reversible hydration of CO2 to protons and bicarbonate and thereby play a fundamental role in the epithelial acid/base transport mechanisms serving fluid secretion and absorption for whole‐body acid/base regulation. The three carbonic anhydrase‐related proteins (CARPs) VIII, X, and XI, however, are catalytically inactive. Previous work has shown that some CA isoforms noncatalytically enhance lactate transport through various monocarboxylate transporters (MCT). Therefore, we examined whether the catalytically inactive CARPs play a role in lactate transport. Here, we report that CARP VIII, X, and XI enhance transport activity of the MCT MCT1 when coexpressed in Xenopus oocytes, as evidenced by the rate of rise in intracellular H+ concentration detected using ion‐sensitive microelectrodes. Based on previous studies, we suggest that CARPs may function as a ‘proton antenna’ for MCT1, to drive proton‐coupled lactate transport across the cell membrane.

Developmental maturation of activity-induced K+ and pH transients and the associated extracellular space dynamics in the rat hippocampus.

Neuronal activity induces fluctuation in extracellular space volume, [K+ ]o and pHo , the management of which influences neuronal function The neighbour astrocytes buffer the K+ and pH and swell during the process, causing shrinkage of the extracellular space In the present study, we report the developmental rise of the homeostatic control of the extracellular space dynamics, for which regulation becomes tighter with maturation and thus is proposed to ensure efficient synaptic transmission in the mature animals The extracellular space dynamics of volume, [K+ ]o and pHo evolve independently with developmental maturation and, although all of them are inextricably tied to neuronal activity, they do not couple directly. Neuronal activity in the mammalian central nervous system associates with transient extracellular space (ECS) dynamics involving elevated K+ and pH and shrinkage of the ECS. These ECS properties affect membrane potentials, neurotransmitter concentrations and protein function and are thus anticipated to be under tight regulatory control. It remains unresolved to what extent these ECS dynamics are developmentally regulated as synaptic precision arises and whether they are directly or indirectly coupled. To resolve the development of homeostatic control of [K+ ]o , pH, and ECS and their interaction, we utilized ion-sensitive microelectrodes in electrically stimulated rat hippocampal slices from rats of different developmental stages (postnatal days 3-28). With the employed stimulation paradigm, the stimulus-evoked peak [K+ ]o and pHo transients were stable across age groups, until normalized to neuronal activity (field potential amplitude), in which case the K+ and pH shifted significantly more in the younger animals. By contrast, ECS dynamics increased with age until normalized to the field potential, and thus correlated with neuronal activity. With age, the animals not only managed the peak [K+ ]o better, but also displayed swifter post-stimulus removal of [K+ ]o , in correlation with the increased expression of the α1-3 isoforms of the Na+ /K+ -ATPase, and a swifter return of ECS volume. The different ECS dynamics approached a near-identical temporal pattern in the more mature animals. In conclusion, although these phenomena are inextricably tied to neuronal activity, our data suggest that they do not couple directly.

Interstitial ion homeostasis and acid-base balance are maintained in oedematous brain of mice with acute toxic liver failure

Acute toxic liver failure (ATLF) rapidly leads to brain oedema and neurological decline. We evaluated the ability of ATLF-affected brain to control the ionic composition and acid-base balance of the interstitial fluid. ATLF was induced in 10–12 weeks old male C57Bl mice by single intraperitoneal (i.p.) injection of 100 μg/g azoxymethane (AOM). Analyses were carried out in cerebral cortex of precomatous mice 20–24 h after AOM administration. Brain fluid status was evaluated by measuring apparent diffusion coefficient [ADC] using NMR spectroscopy, Evans Blue extravasation, and accumulation of an intracisternally-injected fluorescent tracer. Extracellular pH ([pH]e) and ([K+]e) were measured in situ with ion-sensitive microelectrodes. Cerebral cortical microdialysates were subjected to photometric analysis of extracellular potassium ([K+]e), sodium ([Na+]e) and luminometric assay of extracellular lactate ([Lac]e). Potassium transport in cerebral cortical slices was measured ex vivo as 86Rb uptake. Cerebral cortex of AOM-treated mice presented decreased ADC supporting the view that ATLF-induced brain oedema is primarily cytotoxic in nature. In addition, increased Evans blue extravasation indicated blood brain barrier leakage, and increased fluorescent tracer accumulation suggested impaired interstitial fluid passage. However, [K+]e, [Na+]e, [Lac]e, [pH]e and potassium transport in brain of AOM-treated mice was not different from control mice. We conclude that in spite of cytotoxic oedema and deregulated interstitial fluid passage, brain of mice with ATLF retains the ability to maintain interstitial ion homeostasis and acid-base balance. Tentatively, uncompromised brain ion homeostasis and acid-base balance may contribute to the relatively frequent brain function recovery and spontaneous survival rate in human patients with ATLF.

The Effects of Alamandine on the NHE3 Exchanger in in vivo Proximal Tubule of Spontaneously Hypertensive Rats.

The action of Alamandine on the NHE3 exchanger contributes to understanding the etiology of hypertension in spontaneously hypertensive rats (SHR) and their controls, Wistar‐Kyoto rats (WKY). Angiotensin‐(1–7) [Ang‐(1–7)], a heptapeptide member of the renin‐angiotensin system, acts in opposition to the vasoconstrictor, proliferative, and profibrotic actions of Angiotensin II (Ang II) in the cardiovascular system and proximal renal tubule. In normotensive Wistar rats, the dose‐dependent biphasic action of Ang‐(1–7) on NHE3 of renal proximal tubular epithelium is also in opposition of the dose‐dependent biphasic action of Ang II. The presently reported effects of Alamandine were similar to those obtained with Ang‐(1–7), including vasodilation, antifibrosis, reduction of blood pressure in spontaneously hypertensive rats and control of cardiovascular functions. This study was designed to determine the effects of Alamandine on reabsorptive bicarbonate flow (JHCO3−) in in vivo middle proximal tubules. This action was evaluated by bicarbonate reabsorption (JHCO3−) of in vivo proximal renal tubules, using microperfusion with H+ ion‐sensitive microelectrodes. By means tail‐cuff plethysmography the mean values of systolic pressure were obtained in WKY [136 ± 1 mmHg (N=15)] and SHR [181± 3 mmHg (N=9)]. In WKY rats, the mean control value of JHCO3− was 2.34 ± 0.13 nmol.cm−2.s−1 (N= 14). Alamandine [10−12 M] in luminally perfused tubules caused a significant decrease of the JHCO3− to 1,10 ± 0,07 nmol.cm−2.s−1 (28); the following perfusions also significantly decreased JHCO3−: Alamandine [10−9 M] to 1.20 ± 0.13 nmol.cm−2.s−1 (22); Alamandine [10−6 M] to 1.58 ± 0,10 nmol.cm−2.s−1 (17). In SHR rats, the mean control value of JHCO3− was 2.31 ± 0.15 nmol.cm−2.s−1 (12). Alamandine [10−12 M] in luminally perfused tubules causes a significant decrease of the JHCO3− to 1.38 ± 0.07 nmol.cm−2.s−1 (18); the following perfusions also significantly decreased JHCO3−: Alamandine [10−9 M] to 1.42 ± 0.16 nmol.cm−2.s−1 (15); Alamandine [10−6 M] to 1.58 ± 0.13 nmol.cm−2.s−1 (20). So, our data are showing that in WKY and SHR rats, the inhibitory dose‐dependent effect of Alamandine on JHCO3−, demonstrates a possible inhibitory beneficial effect of Alamandine in hypertensive rats opposite to the deleterious stimulatory effects of Ang II. It is therefore reasonable to assume that, in the intact animal, the interaction of the opposite dose‐dependent effects of Alamandine and Ang II on the Na+/H+ exchanger may represent an important physiological regulation in terms of intra‐ and extracellular volume and/or pH changes.Support or Funding InformationFAPESP; CNPq and CAPESThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activity-dependent astrocyte swelling is mediated by pH-regulating mechanisms.

During neuronal activity in the mammalian brain, the K+ released into the synaptic space is initially buffered by the astrocytic compartment. In parallel, the extracellular space (ECS) shrinks, presumably due to astrocytic cell swelling. With the Na+ /K+ /2Cl- cotransporter and the Kir4.1/AQP4 complex not required for the astrocytic cell swelling in the hippocampus, the molecular mechanisms underlying the activity-dependent ECS shrinkage have remained unresolved. To identify these molecular mechanisms, we employed ion-sensitive microelectrodes to measure changes in ECS, [K+ ]o and [H+ ]o /pHo during electrical stimulation of rat hippocampal slices. Transporters and receptors responding directly to the K+ and glutamate released into the extracellular space (the K+ /Cl- cotransporter, KCC, glutamate transporters and G protein-coupled receptors) did not modulate the extracellular space dynamics. The HCO3--transporting mechanism, which in astrocytes mainly constitutes the electrogenic Na+ / HCO3- cotransporter 1 (NBCe1), is activated by the K+ -mediated depolarization of the astrocytic membrane. Inhibition of this transporter reduced the ECS shrinkage by ∼25% without affecting the K+ transients, pointing to NBCe1 as a key contributor to the stimulus-induced astrocytic cell swelling. Inhibition of the monocarboxylate cotransporters (MCT), like-wise, reduced the ECS shrinkage by ∼25% without compromising the K+ transients. Isosmotic reduction of extracellular Cl- revealed a requirement for this ion in parts of the ECS shrinkage. Taken together, the stimulus-evoked astrocytic cell swelling does not appear to occur as a direct effect of the K+ clearance, as earlier proposed, but partly via the pH-regulating transport mechanisms activated by the K+ -induced astrocytic depolarization and the activity-dependent metabolism.

Glutamate transporter activity promotes enhanced Na+ /K+ -ATPase-mediated extracellular K+ management during neuronal activity.

Management of glutamate and K+ in brain extracellular space is of critical importance to neuronal function. The astrocytic α2β2 Na+ /K+ -ATPase isoform combination is activated by the K+ transients occurring during neuronal activity. In the present study, we report that glutamate transporter-mediated astrocytic Na+ transients stimulate the Na+ /K+ -ATPase and thus the clearance of extracellular K+ . Specifically, the astrocytic α2β1 Na+ /K+ -ATPase subunit combination displays an apparent Na+ affinity primed to react to physiological changes in intracellular Na+ . Accordingly, we demonstrate a distinct physiological role in K+ management for each of the two astrocytic Na+ /K+ -ATPase β-subunits. Neuronal activity is associated with transient [K+ ]o increases. The excess K+ is cleared by surrounding astrocytes, partly by the Na+ /K+ -ATPase of which several subunit isoform combinations exist. The astrocytic Na+ /K+ -ATPase α2β2 isoform constellation responds directly to increased [K+ ]o but, in addition, Na+ /K+ -ATPase-mediated K+ clearance could be governed by astrocytic [Na+ ]i . During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na+ -coupled glutamate transporters, thereby elevating [Na+ ]i . It thus remains unresolved whether the different Na+ /K+ -ATPase isoforms are controlled by [K+ ]o or [Na+ ]i during neuronal activity. Hippocampal slice recordings of stimulus-induced [K+ ]o transients with ion-sensitive microelectrodes revealed reduced Na+ /K+ -ATPase-mediated K+ management upon parallel inhibition of the glutamate transporter. The apparent intracellular Na+ affinity of isoform constellations involving the astrocytic β2 has remained elusive as a result of inherent expression of β1 in most cell systems, as well as technical challenges involved in measuring intracellular affinity in intact cells. We therefore expressed the different astrocytic isoform constellations in Xenopus oocytes and determined their apparent Na+ affinity in intact oocytes and isolated membranes. The Na+ /K+ -ATPase was not fully saturated at basal astrocytic [Na+ ]i , irrespective of isoform constellation, although the β1 subunit conferred lower apparent Na+ affinity to the α1 and α2 isoforms than the β2 isoform. In summary, enhanced astrocytic Na+ /K+ -ATPase-dependent K+ clearance was obtained with parallel glutamate transport activity. The astrocytic Na+ /K+ -ATPase isoform constellation α2β1 appeared to be specifically geared to respond to the [Na+ ]i transients associated with activity-induced glutamate transporter activity.

Modulation of the glial response via the platelet-derived growth factor receptors (PDGFR) on the diencephalic dopaminergic neurons in zebrafish larvae

s / Parkinsonism and Related Disorders 22 (2016) e149ee192 e180 P 6.017. MODULATION OF THE GLIAL RESPONSE VIA THE PLATELET-DERIVED GROWTH FACTOR RECEPTORS (PDGFR) ON THE DIENCEPHALIC DOPAMINERGIC NEURONS IN ZEBRAFISH LARVAE Anwar Norazit, David Wong, Nadia Abdul Halim, Suzita Mohd Noor. Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Objectives: Activated astrocytes express PDGF receptors on their surface. PDGF may have a role in modulating the expression of astrocytes to act as neuroprotectors on the dopaminergic neurons. Methods: Zebrafish larvae were treated with either 0.1% DMSO (vehicle control), Imatinib (PDGFR inhibitor), 0.1 mg/ml MPTP (dopaminergic neurotoxin), or a combination of Imatinib and MPTP at 4 dpf. Solutions were refreshed daily and larvae were maintained till 7 dpf. Behavioural analyses was conducted to measure response to tail touch and posture before and after tail touch. The presence of spinal curvature was also observed. RT-PCR was done to determine the expression levels of th (dopaminergic neurons) and gfap (astrocytes). Results: Behaviour analyses revealed marked slow responses in larvae treated with MPTP, Imatinib and Imatinib in combination with MPTP. Furthermore, a significant (p<0.05) number of Imatinib-treated larvae and larvae treated in combination with Imatinib and MPTP exhibited spinal curvatures compared to other treatment groups. Despite the behavioural changes, gene expression analysis did not show any qualitative change for all treated groups. Conclusion: These findings suggest PDGF may have a role in modulating astrocytes' neuroprotective response via the PDGF receptors. The changes in behavioural indices needs to be confirmed by qRT-PCR and wholemount in situ immunofluorescence. P 6.018. INTEGRATION OF CORTICAL AND BASAL GANGLIA INPUTS IN MOTOR THALAMUS OF PARKINSONIAN ANIMAL MODELS Gerg Orban, Wei Song, Alain Kaelin-Lang, Salvatore Galati. Neurocenter of Southern Switzerland, Lugano, Switzerland Objectives: Pathological erratic motor thalamus (MTh) activity has been considered to contribute to the generation of Parkinson's disease (PD) symptoms. Basal ganglia (BG) structures afferent to MTh shown synchronized oscillatory activity in dopamine (DA) -depleted BG reflecting underlying pathophysiological mechanisms of Parkinsonism. Acute or chronic DA depletion, drives pathological cortical-substantia nigra pars reticulata (SNr) coherences in urethane-anesthetized rats. Since SNr exerts direct control on the BG-receiving MTh relaying information to the cortex, its disorganized patterns of activity may have a strong influence on the activity of the downstream thalamocortical relay. We aim to characterized the impact of these changes on MTh. Methods: We investigate the firing properties of MTh cells following i) chronic DA depletion in 6-hydroxydopamine (6-OHDA) animal models of PD and ii) in acute DA depleted animals after tetrodotoxin (TTX)-mediated blockade of the nigro-striatal pathway. Results: MTh neurons showed a firing reduction in 6-OHDA animals. In TTX group we observed a time-looked inhibition of MTh cells (Figure1). Conclusions: The MTh is a structure strategically situated between BG and cortex whose activity was conjectured to play a crucial role in PD. Our preliminary data are in agreement with the classical basal ganglia scheme. P 6.019. SPREADING DEPRESSION SUSCEPTIBILITY IS REDUCED IN PARKINSON RAT MODEL Mahmoud Lotfinia. Shefa Neuroscience Research Center, Tehran, Islamic Republic of Iran Objectives: To evaluate whether administration of 6-hydroxydopamine in the medial forebrain bundle and creating a Parkinson rat model, can have an effect on Spreading depression Susceptibility or not? Parkinson disease (PD) is known by amajor loss of dopaminergic nigrostriatal neurons and by an increased turnover of neurotransmitter by surviving neurons of the nigrostriatal tract. The clinical diagnosis of PD is based on the identification of some combination of the cardinal motor signs of bradykinesia, rigidity, tremor, and postural instability. Spreading depression (SD) known as an evoked neuronal activity and changes in ionic, metabolic and hemodynamic characteristics of the brain. Pronounced release of dopamine during SD and the probable role of dopamine in SD process suggests that disruption of dopaminergic pathway in PD may cause SD to behave differently. Methods: To test this possibility, we induced dopaminergic lesion by bilateral intracerebral stereotactic injection of 6 mL of 6-hydroxydopamine in the medial forebrain bundle (MFB). After 4 days, SD was induced by the injection of 3M KCl and SD propagation was followed using two ion-sensitive microelectrodes placed in the parietal and occipital cortex. Results: Eliciting SD in ratmodel was associatedwith a significant increase in the threshold of SD and a decrease in the propagation velocity and duration of accompanying extracellular DC changes. Conclusions: The present data show that rat model of Parkinson's disease are less prone to SD.

Biological system development for GraviSat: A new platform for studying photosynthesis and microalgae in space

Microalgae have great potential to be used as part of a regenerative life support system and to facilitate in-situ resource utilization (ISRU) on long-duration human space missions. Little is currently known, however, about microalgal responses to the space environment over long (months) or even short (hours to days) time scales. We describe here the development of biological support subsystems for a prototype “3U” (i.e., three conjoined 10-cm cubes) nanosatellite, called GraviSat, designed to experimentally elucidate the effects of space microgravity and the radiation environment on microalgae and other microorganisms. The GraviSat project comprises the co-development of biological handling-and-support technologies with implementation of integrated measurement hardware for photosynthetic efficiency and physiological activity in support of long-duration (3–12 months) space missions. It supports sample replication in a fully autonomous system that will grow and analyze microalgal cultures in 120 μL wells around the circumference of a microfluidic polymer disc; the cultures will be launched while in stasis, then grown in orbit. The disc spins at different rotational velocities to generate a range of artificial gravity levels in space, from microgravity to multiples of Earth gravity. Development of the biological support technologies for GraviSat comprised the screening of more than twenty microalgal strains for various physical, metabolic and biochemical attributes that support prolonged growth in a microfluidic disc, as well as the capacity for reversible metabolic stasis. Hardware development included that necessary to facilitate accurate and precise measurements of physical parameters by optical methods (pulse amplitude modulated fluorometry) and electrochemical sensors (ion-sensitive microelectrodes). Nearly all microalgal strains were biocompatible with nanosatellite materials; however, microalgal growth was rapidly inhibited (∼1 week) within sealed microwells that did not include dissolved bicarbonate due to CO2 starvation. Additionally, oxygen production by some microalgae resulted in bubble formation within the wells, which interfered with sensor measurements. Our research achieved prolonged growth periods (>10 months) without excess oxygen production using two microalgal strains, Chlorella vulgaris UTEX 29 and Dunaliella bardawil 30 861, by lowering light intensities (2–10 μmol photons m−2 s−1) and temperature (4–12 °C). Although the experiments described here were performed to develop the GraviSat platform, the results of this study should be useful for the incorporation of microalgae in other satellite payloads with low-volume microfluidic systems.

Flexible Polyimide Platform based on the Integration of Potentiometric Multi-sensor for Biomedical Applications

The selective detection of cations is commonly required in a wide range of biomedical applications. We have fabricated a low-cost flexible polyimide multi-sensor platform that integrates K+ and Na+ ion sensitive microelectrodes (μISE) for biomedical applications in organic media. The platform is fabricated using rapid prototyping techniques based on KAPTON polymeric substrate. The novel multi-sensor exhibits a good behavior regarding the detection of different cations with a high selectivity, high sensitivity, low detection limit and fast response time. An electropolymerized polypyrrole film is used in order to achieve a good adhesion between the gold electrodes and the membrane.

Dose-dependent effects of angiotensin-(1–7) on the NHE3 exchanger and [Ca2+]iin in vivo proximal tubules

The acute direct action of angiotensin-(1–7) [ANG-(1–7)] on bicarbonate reabsorption ( JHCO3−) was evaluated by stationary microperfusions on in vivo middle proximal tubules in rats using H ion-sensitive microelectrodes. The control JHCO3−is 2.82 ± 0.078 nmol·cm−2·s−1(50). ANG-(1–7) (10−12or 10−9M) in luminally perfused tubules decreases JHCO3−(36 or 60%, respectively), but ANG-(1–7) (10−6M) increases it (80%). A779 increases JHCO3−(30%) and prevents both the inhibitory and the stimulatory effects of ANG-(1–7) on it. S3226 decreases JHCO3−(45%) and changes the stimulatory effect of ANG-(1–7) to an inhibitory effect (30%) but does not affect the inhibitory effect of ANG-(1–7). Our results indicate that in the basal condition endogenous ANG-(1–7) inhibits JHCO3−and that the biphasic dose-dependent effect of ANG-(1–7) on JHCO3−is mediated by the Mas receptors via the Na+/H+exchanger 3 (NHE3). The control value of intracellular Ca2+concentration ([Ca2+]i), as monitored using fura-2 AM, is 101 ± 2 nM ( 6 ), and ANG-(1–7) (10−12, 10−9, or 10−6M) transiently (3 min) increases it (by 151, 102, or 52%, respectively). A779 increases the [Ca2+]i(25%) but impairs the stimulatory effect of all doses of ANG-(1–7) on it. The use of BAPTA or thapsigargin suggests a correlation between the ANG-(1–7) dose-dependent effects on [Ca2+]iand JHCO3−. Therefore, the interaction of the opposing dose-dependent effects of ANG II and ANG-(1–7) on [Ca2+]iand JHCO3−may represent an physiological regulatory mechanism of extracellular volume and/or pH changes. However, whether [Ca2+]imodification is an important direct mechanism for NHE3 activation by these peptides or is a side effect of other signaling pathways will require additional studies.

Low-pH and Aluminum Resistance in Arabidopsis Correlates with High Cytosolic Magnesium Content and Increased Magnesium Uptake by Plant Roots

Low-pH stress and Al(3+) toxicity affect root growth in acid soils. It was hypothesized that the capacity of genotypes to maintain Mg(2+) uptake in acidic environments may contribute to low-pH and Al resistance, but explicit evidence is lacking. In this work, an Al-resistant alr104 mutant and two Al-sensitive mutants (als5 and als3) of Arabidopsis thaliana were compared with the wild type (Col-0) for Mg(2+) uptake and intracellular Mg(2+) concentration under low-pH and combined low-pH/Al stresses. Magnesium accumulation in roots was measured in long-term (7 d) experiments. The Mg(2+) fluxes were measured using ion-sensitive microelectrodes at the distal elongation and the mature root zones in short-term (0-60 min) experiments. Intracellular Mg(2+) concentrations were measured in intact root cells at the distal elongation zone using magnesium-specific fluorescent dye and fluorescent lifetime imaging (FLIM) analysis. Under low-pH stress, Arabidopsis mutants als5 and alr104 maintained a higher Mg concentration in roots, and had greater Mg(2+) influx than the wild type and the als3 mutant. Under combined low-pH/Al treatment, Al-resistant genotypes (wild type and alr104) maintained a higher Mg(2+) accumulation, and had a higher Mg(2+) influx and higher intracellular Mg(2+) concentration than Al-sensitive genotypes (als3 and als5). Overall, these results show that increased Mg(2+) uptake correlates with an enhanced capacity of Arabidopsis genotypes to cope with low-pH and combined low-pH/Al stresses.

The K+–Cl−cotransporter KCC2 promotes GABAergic excitation in the mature rat hippocampus

GABAergic excitatory [K(+)](o) transients can be readily evoked in the mature rat hippocampus by intense activation of GABA(A) receptors (GABA(A)Rs). Here we show that these [K(+)](o) responses induced by high-frequency stimulation or GABA(A) agonist application are generated by the neuronal K(+)-Cl() cotransporter KCC2 and that the transporter-mediated KCl extrusion is critically dependent on the bicarbonate-driven accumulation of Cl() in pyramidal neurons. The mechanism underlying GABAergic [K(+)](o) transients was studied in CA1 stratum pyramidale using intracellular sharp microelectrodes and extracellular ion-sensitive microelectrodes. The evoked [K(+)](o) transients, as well as the associated afterdischarges, were strongly suppressed by 0.5-1 mm furosemide, a KCl cotransport inhibitor. Importantly, the GABA(A)R-mediated intrapyramidal accumulation of Cl(), as measured by monitoring the reversal potential of fused IPSPs, was unaffected by the drug. It was further confirmed that the reduction in the [K(+)](o) transients was not due to effects of furosemide on the Na(+)-dependent K(+)-Cl() cotransporter NKCC1 or on intraneuronal carbonic anhydrase activity. Blocking potassium channels by Ba(2+) enhanced [K(+)](o) transients whereas pyramidal cell depolarizations were attenuated in further agreement with a lack of contribution by channel-mediated K(+) efflux. The key role of the GABA(A)R channel-mediated anion fluxes in the generation of the [K(+)](o) transients was examined in experiments where bicarbonate was replaced with formate. This anion substitution had no significant effect on the rate of Cl() accumulation, [K(+)](o) response or afterdischarges. Our findings reveal a novel excitatory mode of action of KCC2 that can have substantial implications for the role of GABAergic transmission during ictal epileptiform activity.

Slc26a9—Anion Exchanger, Channel and Na+ Transporter

The SLC26 gene family encodes anion transporters with diverse functional attributes: (a) anion exchanger, (b) anion sensor, and (c) anion conductance (likely channel). We have cloned and studied Slc26a9, a paralogue expressed mostly in lung and stomach. Immunohistochemistry shows that Slc26a9 is present at apical and intracellular membranes of lung and stomach epithelia. Using expression in Xenopus laevis oocytes and ion-sensitive microelectrodes, we discovered that Slc26a9 has a novel function not found in any other Slc26 proteins: cation coupling. Intracellular pH and voltage measurements show that Slc26a9 is a nCl(-)-HCO(3)(-) exchanger, suggesting roles in gastric HCl secretion or pulmonary HCO(3)(-) secretion; Na(+) electrodes and uptakes reveal that Slc26a9 has a cation dependence. Single-channel measurements indicate that Slc26a9 displays discrete open and closed states. These experiments show that Slc26a9 has three discrete physiological modes: nCl(-)-HCO(3)(-) exchanger, Cl(-) channel, and Na(+)-anion cotransporter. Thus, the Slc26a9 transporter channel is uniquely suited for dynamic and tissue-specific physiology or regulation in epithelial tissues.

Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule

The direct action of aldosterone (10(-12) M) on net bicarbonate reabsorption (J(HCO(3)(-))) was evaluated by stationary microperfusion of an in vivo middle proximal tubule (S2) of rat kidney, using H ion-sensitive microelectrodes. Aldosterone in luminally perfused tubules caused a significant increase in J(HCO(3)(-)) from a mean control value of 2.84 +/- 0.08 [49/19 (n degrees of measurements/n degrees of tubules)] to 4.20 +/- 0.15 nmol.cm(-2).s(-1) (58/10). Aldosterone perfused into peritubular capillaries also increased J(HCO(3)(-)), compared with basal levels during intact capillary perfusion with blood. In addition, in isolated perfused tubules aldosterone causes a transient increase of cytosolic free calcium ([Ca(2+)](i)), monitored fluorometrically. In the presence of ethanol (in similar concentration used to prepare the hormonal solution), spironolactone (10(-6) M, a mineralocorticoid receptor antagonist), actinomycin D (10(-6) M, an inhibitor of gene transcription), or cycloheximide (40 mM, an inhibitor of protein synthesis), the J(HCO(3)(-)) and the [Ca(2+)](i) were not different from the control value; these drugs also did not prevent the stimulatory effect of aldosterone on J(HCO(3)(-)) and on [Ca(2+)](i). However, in the presence of RU 486 alone [10(-6) M, a classic glucocorticoid receptor (GR) antagonist], a significant decrease on J(HCO(3)(-)) and on [Ca(2+)](i) was observed; this antagonist also inhibited the stimulatory effect of aldosterone on J(HCO(3)(-)) and on [Ca(2+)](i). These studies indicate that luminal or peritubular aldosterone (10(-12) M) has a direct nongenomic stimulatory effect on J(HCO(3)(-)) and on [Ca(2+)](i) in proximal tubule and that probably GR participates in this process. The data also indicate that endogenous aldosterone stimulates J(HCO(3)(-)) in middle proximal tubule.

Carbon Dioxide Transport through Membranes

Several membrane channels, like aquaporin-1 (AQP1) and the RhAG protein of the rhesus complex, were hypothesized to be of physiological relevance for CO(2) transport. However, the underlying assumption that the lipid matrix imposes a significant barrier to CO(2) diffusion was never confirmed experimentally. Here we have monitored transmembrane CO(2) flux (J(CO2)) by imposing a CO(2) concentration gradient across planar lipid bilayers and detecting the resulting small pH shift in the immediate membrane vicinity. An analytical model, which accounts for the presence of both carbonic anhydrase and buffer molecules, was fitted to the experimental pH profiles using inverse problems techniques. At pH 7.4, the model revealed that J(CO2) was entirely rate-limited by near-membrane unstirred layers (USL), which act as diffusional barriers in series with the membrane. Membrane tightening by sphingomyelin and cholesterol did not alter J(CO2) confirming that membrane resistance was comparatively small. In contrast, a pH-induced shift of the CO(2) hydration-dehydration equilibrium resulted in a relative membrane contribution of about 15% to the total resistance (pH 9.6). Under these conditions, a membrane CO(2) permeability (3.2 +/- 1.6 cm/s) was estimated. It indicates that cellular CO(2) uptake (pH 7.4) is always USL-limited, because the USL size always exceeds 1 mum. Consequently, facilitation of CO(2) transport by AQP1, RhAG, or any other protein is highly unlikely. The conclusion was confirmed by the observation that CO(2) permeability of epithelial cell monolayers was always the same whether AQP1 was overexpressed in both the apical and basolateral membranes or not.

Development of a miniaturised electrochemical cell integrated on epoxy-glass laminate

The development of the miniaturised electrochemical cell integrated on epoxy-glass laminate support is reported. Potassium-selective microelectrodes based on valinomycin and two polymeric matrices [plasticized poly(vinyl chloride) and polyurethane] have been fabricated on planar Au or Ag/AgCl transducers. Polymeric layers containing ionic liquid were cast on the surface of Ag/AgCl microelectrodes to form the reference half-cells. Performances of the ion-sensitive and reference microelectrodes (i.e., ion-selectivity, slopes of calibration curves, signal stability and repeatability, long-term stability) were studied. The influence of the method of membrane deposition on sensor characteristics was not significant, however the microelectrodes with polyurethane membranes exhibited better durability. Theoretical calibration curves were obtained when K+-selective electrodes and reference electrodes were integrated on a single substrate. The designed electrochemical cell may be helpful in further studies devoted to the development of miniaturised electrode arrays for multi-component analysis or electronic tongues purposes.

A miniaturized and integrated galvanic cell for the potentiometric measurement of ions in biological liquids

A procedure for an all-plastic electrochemical cell comprising miniaturized planar indicator and reference electrodes is described. All electrodes are based on conducting polymers, are fully integrated, and contain no internal electrolyte. The reference microsensors were deposited via electrochemical polymerization from a water solution containing the monomer 3,4-ethylenedioxythiophene (EDOT) or 1-methylpyrrole (MPy) and a biochemical buffer 3-(N-morpholino) propanesulfonic acid [MOPS], 2-(N-morpholino) ethanesulfonic acid [MES], or 2-hydroxy-5-sulfobenzoic acid [SSA]). Ion-sensitive microelectrodes were prepared by the deposition of the ion-sensitive membrane solution (Ca2+, K+, and Cl−) directly onto the mediating poly-EDOT (PEDOT), PEDOT–SSA, PEDOT–MES, PEDOT–MOPS, or poly-MPy–MOPS layers.