Transmembrane Ion Gradients Research Articles

Loss of Na+/K+-ATPase in Drosophila photoreceptors leads to blindness and age-dependent neurodegeneration

The activity of Na+/K+-ATPase establishes transmembrane ion gradients and is essential to cell function and survival. Either dysregulation or deficiency of neuronal Na+/K+-ATPase has been implicated in the pathogenesis of many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and rapid-onset dystonia Parkinsonism. However, genetic evidence that directly links neuronal Na+/K+-ATPase deficiency to in vivo neurodegeneration has been lacking. In this study, we use Drosophila photoreceptors to investigate the cell-autonomous effects of neuronal Na+/K+ ATPase. Loss of ATPα, an α subunit of Na+/K+-ATPase, in photoreceptors through UAS/Gal4-mediated RNAi eliminated the light-triggered depolarization of the photoreceptors, rendering the fly virtually blind in behavioral assays. Intracellular recordings indicated that ATPα knockdown photoreceptors were already depolarized in the dark, which was due to a loss of intracellular K+. Importantly, ATPα knockdown resulted in the degeneration of photoreceptors in older flies. This degeneration was independent of light and showed characteristics of apoptotic/hybrid cell death as observed via electron microscopy analysis. Loss of Nrv3, a Na+/K+-ATPase β subunit, partially reproduced the signaling and degenerative defects observed in ATPα knockdown flies. Thus, the loss of Na+/K+-ATPase not only eradicates visual function but also causes age-dependent degeneration in photoreceptors, confirming the link between neuronal Na+/K+ ATPase deficiency and in vivo neurodegeneration. This work also establishes Drosophila photoreceptors as a genetic model for studying the cell-autonomous mechanisms underlying neuronal Na+/K+ ATPase deficiency-mediated neurodegeneration.

Microfluidic remote loading for rapid single-step liposomal drug preparation.

Microfluidic-directed formation of liposomes is combined with in-line sample purification and remote drug loading for single step, continuous-flow synthesis of nanoscale vesicles containing high concentrations of stably loaded drug compounds. Using an on-chip microdialysis element, the system enables rapid formation of large transmembrane pH and ion gradients, followed by immediate introduction of amphipathic drug for real-time remote loading into the liposomes. The microfluidic process enables in-line formation of drug-laden liposomes with drug : lipid molar ratios of up to 1.3, and a total on-chip residence time of approximately 3 min, representing a significant improvement over conventional bulk-scale methods which require hours to days for combined liposome synthesis and remote drug loading. The microfluidic platform may be further optimized to support real-time generation of purified liposomal drug formulations with high concentrations of drugs and minimal reagent waste for effective liposomal drug preparation at or near the point of care.

Ammonia, like K(+), stimulates the Na(+), K(+), 2 Cl(-) cotransporter NKCC1 and the Na(+),K(+)-ATPase and interacts with endogenous ouabain in astrocytes.

Brain edema during hepatic encephalopathy or acute liver failure as well as following brain ischemia has a multifactorial etiology, but it is a dangerous and occasionally life-threatening complication because the brain is enclosed in the rigid skull. During ischemia the extracellular K(+) concentration increases to very high levels, which when energy becomes available during reperfusion stimulate NKCC1, a cotransporter driven by the transmembrane ion gradients established by the Na(+),K(+)-ATPase and accumulating Na(+), K(+) and 2 Cl(-) together with water. This induces pronounced astrocytic swelling under pathologic conditions, but NKCC1 is probably also activated, although to a lesser extent, during normal brain function. Redistribution of ions and water between extra- and intracellular phases does not create brain edema, which in addition requires uptake across the blood-brain barrier. During hepatic encephalopathy and acute liver failure a crucial factor is the close resemblance between K(+) and NH4(+) in their effects not only on NKCC1 and Na(+),K(+)-ATPase but also on Na(+),K(+)-ATPase-induced signaling by endogenous ouabains. These in turn activate production of ROS and nitrosactive agents which slowly sensitize NKCC1, explaining why cell swelling and brain edema generally are delayed under hyperammonemic conditions, although very high ammonia concentrations can cause immediate NKCC1 activation.

Bistable dynamics underlying excitability of ion homeostasis in neuron models.

When neurons fire action potentials, dissipation of free energy is usually not directly considered, because the change in free energy is often negligible compared to the immense reservoir stored in neural transmembrane ion gradients and the long–term energy requirements are met through chemical energy, i.e., metabolism. However, these gradients can temporarily nearly vanish in neurological diseases, such as migraine and stroke, and in traumatic brain injury from concussions to severe injuries. We study biophysical neuron models based on the Hodgkin–Huxley (HH) formalism extended to include time–dependent ion concentrations inside and outside the cell and metabolic energy–driven pumps. We reveal the basic mechanism of a state of free energy–starvation (FES) with bifurcation analyses showing that ion dynamics is for a large range of pump rates bistable without contact to an ion bath. This is interpreted as a threshold reduction of a new fundamental mechanism of ionic excitability that causes a long–lasting but transient FES as observed in pathological states. We can in particular conclude that a coupling of extracellular ion concentrations to a large glial–vascular bath can take a role as an inhibitory mechanism crucial in ion homeostasis, while the pumps alone are insufficient to recover from FES. Our results provide the missing link between the HH formalism and activator–inhibitor models that have been successfully used for modeling migraine phenotypes, and therefore will allow us to validate the hypothesis that migraine symptoms are explained by disturbed function in ion channel subunits, pumps, and other proteins that regulate ion homeostasis.

Pediatric MRI of the Brain: A Primer

1. Kamakshya P. Patra, MD* 2. Jeffrey D. Lancaster, MD* 3. Jeffery Hogg, MD† 4. Jeffrey S. Carpenter, MD† 1. *Department of Pediatrics, Section of Hospital Pediatrics, West Virginia University Children’s Hospital, Morgantown, WV. 2. †Department of Neuroradiology, West Virginia University Health Sciences Center, Morgantown, WV. * Abbreviations: ADHD: : attention-deficit/hyperactivity disorder CSF: : cerebrospinal fluid CT: : computed tomography DWI: : diffusion-weighted magnetic resonance imaging FLAIR: : fluid-attenuated inversion recovery fMRI: : functional magnetic resonance imaging MRI: : magnetic resonance imaging MRV: : magnetic resonance venography NAA: : N -acetylaspartate SDH: : subdural hematoma Because of recent advances in magnetic resonance imaging (MRI) techniques, pediatricians should be aware of the different modalities and their unique advantages and appropriateness in different clinical situations. After completing this article, readers should be able to: 1. Understand the pros and cons of MRI and computed tomography of the brain. 2. Know the basic principles of MRI and its different image modalities. 3. Be aware of the appropriateness of different modalities in specific clinical situations. Magnetic resonance imaging (MRI) is based on the absorption and emission of radiofrequency energy by hydrogen protons whose spin is influenced by changing magnetic fields (0.3 to 1.5 T). Unlike computed tomography (CT), there is no radiation exposure. T1-weighted images cause fat (eg, myelin in white matter) to appear bright and water (eg, cerebrospinal fluid [CSF] or edema) to appear dark on this sequence. The gray-white interfaces of the brain are well depicted on these sequences, especially if with the images are thinly sliced. T2-weighted images cause water (eg, CSF and edema) to appear bright and fat to appear dark. The MRI-based intravenous contrast agents (eg, gadolinium) are frequently used in T1-weighted images (Fig 1A and B) to make serum appear bright. The blood-brain barrier typically serves to limit the passage of many molecules out of the blood vessels. If disease processes break down this barrier (such as infection, tumors, or inflammation), intravenous contrast agents can cross into the brain, causing areas of contrast entry to appear very bright. Figure 1. T1-weighted image at the level of midbrain. A. The cerebrospinal fluid (CSF) appears dark. B. The CSF appears bright. Note the gray and white matter differentiation …

Transport Reversal during Heteroexchange: A Kinetic Study.

It is known that secondary transporters, which utilize transmembrane ionic gradients to drive their substrates up a concentration gradient, can reverse the uptake and instead release their substrates. Unfortunately, the Michaelis-Menten kinetic scheme, which is popular in transporter studies, does not include transporter reversal, and it completely neglects the possibility of equilibrium between the substrate concentrations on both sides of the membrane. We have developed a complex two-substrate kinetic model that includes transport reversal. This model allows us to construct analytical formulas allowing the calculation of a “heteroexchange” and “transacceleration” using standard Michaelis coefficients for respective substrates. This approach can help to understand how glial and other cells accumulate substrates without synthesis and are able to release such substrates and gliotransmitters.

Decreased Na+/K+ ATPase α1 (ATP1A1) gene expression in major depression patients’ peripheral blood

Abstract Major depression affects the central nervous system and thereafter the autonomic nervous system, immune system, and endocrine system. Na+/K+ ATPase, as a major mediator of cellular transmembrane ionic gradients, plays an important role in nervous signal transduction. Three types of Na+/K+ ATPase α subunit isoforms (ATP1A1, ATP1A2, and ATP1A3) are found in brain but vary in the type of cell and level of expression. It has been confirmed that reduced expression of ATP1A2 and ATP1A3 are related to depressive disorder. However, there is no reported correlation between ATP1A1 and major depression. This study investigated the potential correlation between ATP1A1 gene expression level and major depression. The expression levels of ATP1A1 gene in the peripheral circulation of both depressive patients and healthy human controls were quantified by using reverse transcripted quantitative polymerase chain reaction. Statistical analysis showed a significant decrease of ATP1A1 expression level in major depression patients when compared to that of healthy controls (P<0.01). The differences of gene nucleotide sequences and protein structures among ATP1A1, ATP1A2, and ATP1A3 were also illustrated. This study demonstrates, for the first time, that ATP1A1 gene expression level is significantly associated with major depression and suggests that ATP1A1 could be a significant molecular marker for diagnosis.

Preparation of berberine hydrochloride long-circulating liposomes by ionophore A23187-mediated ZnSO4 gradient method

The aim of the study was to prepare berberine hydrochloride long-circulating liposomes and optimize the formulation and process parameters, and investigate the influence of different factors on the encapsulation efficiency. Berberine hydrochloride liposomes were prepared in response to a transmembrane ion gradient that was established by ionophore A23187. Free and liposomal drug were separated by cation exchange resin, and then the amount of intraliposomal berberine hydrochloride was determined by UV spectrophotometry. The optimized encapsulation efficiency of berberine hydrochloride liposomes was 94.3% ± 2.1% when the drug-to-lipid ratio was 1:20, and the mean diameter was 146.9 nm ± 3.2 nm. As a result, the ionophore A23187-mediated ZnSO4 gradient method was suitable for the preparation of berberine hydrochloride liposomes that we could get the desired encapsulation efficiency and drug loading.

Spreading Depression and Neurovascular Coupling

Spreading depression, discovered by Leao in 1944,1 is an intense depolarization wave that slowly propagates (≈3 mm/min) in gray matter by way of contiguity, regardless of functional divisions or arterial territories. The depolarization is associated with massive transmembrane ionic and water shifts coupled to a surge in extracellular K+ and neurotransmitter levels, including glutamate. Transmembrane ionic gradients are restored within less than a minute in otherwise normal tissue without causing permanent injury. Dating back to Leao’s discovery, spreading depression has been implicated in migraine because of the similarities between electrophysiological characteristics of spreading depression and symptomatology of migraine aura.2–5 Although it has been known that ischemia could also trigger spreading depression,6 it was not until the 1980s that recurrent spreading depression-like depolarization waves were demonstrated in focal ischemic brain.7 Often termed peri-infarct depolarizations, or simply injury depolarizations, these recurrent waves seem to originate at the junction between the severely ischemic core (ie, tissue that has developed anoxic depolarization) and the moderately ischemic penumbra where all synaptic activity has ceased, but membrane potentials are preserved. Once initiated, injury depolarizations propagate along the infarct rim and often into the nonischemic tissue, and exert profound metabolic and hemodynamic effects to acutely exacerbate the supply–demand mismatch and worsen the ischemic tissue outcome. Over the past decade, injury depolarizations have been detected in human brain not only after ischemic stroke but also after subarachnoid hemorrhage, intracerebral …

Electric Field Modulation of the Membrane Potential in Solid-State Ion Channels

Biological ion channels are molecular devices that allow a rapid flow of ions across the cell membrane. Normal physiological functions, such as generating action potentials for cell-to-cell communication, are highly dependent on ion channels that can open and close in response to external stimuli for regulating ion permeation. Mimicking these biological functions using synthetic structures is a rapidly progressing yet challenging area. Here we report the electric field modulation of the membrane potential phenomena in mechanically and chemically robust solid-state ion channels, an abiotic analogue to the voltage-gated ion channels in living systems. To understand the complex physicochemical processes in the electric field regulated membrane potential behavior, both quasi-static and transient characteristics of converting transmembrane ion gradients into electric potential are investigated. It is found that the transmembrane potential can be adequately tuned by an external electrical stimulation, thanks to the unique properties of the voltage-regulated selective ion transport through a nanoscale channel.

Increased glucose availability does not restore prolonged spreading depression durations in hypotensive rats without brain injury

Maintenance of transmembrane ionic gradients and their restoration after cortical spreading depression (CSD) are energy dependent. We recently showed an inverse relationship between blood pressure and CSD duration that is independent of tissue oxygenation. Here, we tested the alternative hypothesis that glucose availability becomes rate-limiting for CSD recovery upon reduced blood pressure in anesthetized rats under full systemic physiological monitoring. Hypotension induced by controlled exsanguination significantly prolonged CSD durations, reduced propagation speeds, and diminished the blood flow response. Hyperglycemia failed to restore the prolonged CSD durations in hypotensive rats and did not significantly alter the propagation speed or the blood flow response. These data suggest that prolonged CSD durations during reduced cerebral perfusion pressure are independent of tissue energy status, and implicate alternative mechanisms of CSD recovery such as vascular clearance of extracellular K+.

Expression of the Sodium/Calcium/Potassium Exchanger, NCKX4, in Ameloblasts

Transcellular calcium transport is an essential activity in mineralized tissue formation, including dental hard tissues. In many organ systems, this activity is regulated by membrane-bound sodium/calcium (Na⁺/Ca²⁺) exchangers, which include the NCX and NCKX [sodium/calcium-potassium (Na⁺/Ca²⁺-K⁺) exchanger] proteins. During enamel maturation, when crystals expand in thickness, Ca²⁺ requirements vastly increase but exactly how Ca²⁺ traffics through ameloblasts remains uncertain. Previous studies have shown that several NCX proteins are expressed in ameloblasts, although no significant shifts in expression were observed during maturation which pointed to the possible identification of other Ca²⁺ membrane transporters. NCKX proteins are encoded by members of the solute carrier gene family, Slc24a, which include 6 different proteins (NCKX1–6). NCKX are bidirectional electrogenic transporters regulating Ca²⁺ transport in and out of cells dependent on the transmembrane ion gradient. In this study we show that all NCKX mRNAs are expressed in dental tissues. Real-time PCR indicates that of all the members of the NCKX group, NCKX4 is the most highly expressed gene transcript during the late stages of amelogenesis. In situ hybridization and immunolocalization analyses clearly establish that in the enamel organ, NCKX4 is expressed primarily by ameloblasts during the maturation stage. Further, during the mid-late maturation stages of amelogenesis, the expression of NCKX4 in ameloblasts is most prominent at the apical poles and at the lateral membranes proximal to the apical ends. These data suggest that NCKX4 might be an important regulator of Ca²⁺ transport during amelogenesis.

Traumatic Alterations in GABA Signaling Disrupt Hippocampal Network Activity in the Developing Brain

Severe head trauma causes widespread neuronal shear injuries and acute seizures. Shearing of neural processes might contribute to seizures by disrupting the transmembrane ion gradients that subserve normal synaptic signaling. To test this possibility, we investigated changes in intracellular chloride concentration ([Cl(-)](i)) associated with the widespread neural shear injury induced during preparation of acute brain slices. In hippocampal slices and intact hippocampal preparations from immature CLM-1 mice, increases in [Cl(-)](i) correlated with disruption of neural processes and biomarkers of cell injury. Traumatized neurons with higher [Cl(-)](i) demonstrated excitatory GABA signaling, remained synaptically active, and facilitated network activity as assayed by the frequency of extracellular action potentials and spontaneous network-driven oscillations. These data support a more inhibitory role for GABA in the unperturbed immature brain, demonstrate the utility of the acute brain slice preparation for the study of the consequences of trauma, and provide potential mechanisms for both GABA-mediated excitatory network events in the slice preparation and early post-traumatic seizures.

The iron transporter ferroportin can also function as a manganese exporter

The present study examined the hypothesis that the iron exporter ferroportin (FPN1/SLC40A1) can also mediate cellular export of the essential trace element manganese, using Xenopus laevis oocytes expressing human FPN1. When compared to oocytes expressing only the divalent metal transporter-1 (DMT1/NRAMP2), 54Mn accumulation was lower in oocytes also expressing FPN1. FPN1-expressing oocytes exported more 54Mn than control oocytes (26.6±0.6% versus 7.1±0.5%, respectively, over 4h at pH 7.4 when preloaded with approximately 16μM 54Mn); however, there was no difference in 54Mn uptake between control and FPN1-expressing oocytes. FPN1-mediated Mn export was concentration dependent and could be partially cis-inhibited by 100μM Fe, Co, and Ni, but not by Rb. In addition, Mn export ability was significantly reduced when the extracellular pH was reduced from 7.4 to 5.5, and when Na+ was substituted with K+ in the incubation media. These results indicate that Mn is a substrate for FPN1, and that this export process is inhibited by a low extracellular pH and by incubation in a high K+ medium, indicating the involvement of transmembrane ion gradients in FPN1-mediated transport.

MG ATP AND ANTIOXIDANTS AUGMENT THE RADIOPROTECTIVE EFFECT OF SURFACTANT COPOLYMERS

Mediated by reactive oxygen species, the damaging effects of high-intensity ionizing irradiation on tissues are dose, frequency, oxygen concentration, and tissue property dependent. Intense ionizing irradiation exposure may cause rapid cellular necrosis by peroxidation of membrane lipids leading to membrane disruption. This leads to a loss of the transmembrane ionic gradients and a subsequent depletion of the cellular ATP store, followed by cellular generation of reactive oxygen species. When membrane disruption is extensive, acute cellular necrosis follows. Triblock copolymer surfactants, such as Poloxamer 188 (P188), are able to seal damaged rhabdomyocyte membranes, increasing post-irradiation viability. Separated rat rhabdomyocytes were exposed to 40 Gy (Co 1.5 Gy min) irradiation and treated at 20 min intervals with combination permutations of P188, N-acetylcysteine (NAC), and Mg-ATP. Cell viability at 18 and 48 h was determined using Calcein-AM and Ethidium Homodimer-1 staining. At 18 h after irradiation, the combined administration of P188, ATP, and NAC restored cell viability rates to near sham-exposed levels of 60%. At 48 h post-irradiation, cell viability dropped substantially to the 7-20% range, regardless of attempted intervention. Nevertheless, the combination of P188, ATP, and NAC more than doubled cell viability at the 48-h time point. Neither 8 kDa polyethylene glycol nor 10 kDa neutral dextran was as effective in enhancing cell viability. These results indicate that antioxidants and cellular energy substrates improve the efficacy of membrane-sealing copolymer surfactants in prolonging cellular viability following massive radiation exposure.

Modeling neuronal dynamics during brain ischemia

Neurons are critically dependent on sufficient supply of oxygen and glucose When this supply is subcritical, as in ischemia, the signaling processes of the neurons are altered due to synaptic transmission failure [1] and decreased rates of the molecular ion pumps needed to maintain transmembrane ion gradients [2]. These changes are partially reflected in the electroencephalogram (EEG), as this is a sensitive measure for global network function, reflecting extracellular currents, mainly from synchronous activity of pyramidal cells [3]. Modeling a neural network suffering from ischemia may further improve our understanding of the effects of ischemia and the relation with the EEG. This may contribute to improved methods for diagnosing pathologies related to ischemia and hypoxia. The electrophysiological behavior of a single neuron, which we calculate with a Hodgkin-Huxley type model, depends heavily on the intra- and extracellular ion concentrations. These are usually assumed to be constant, because the molecular ion pumps (ATPases) of the neurons and glia maintain the concentration differences needed for the generation of action potentials. During ischemia however, the required energy (ATP) for the pumping process is not fully supplied anymore and these concentrations change. This is calculated by integrating the average fluxes of the individual ion species across the cell membranes, following an approach similar to those used by Dronne [4] and Cressman [5]. The pump rates are assumed to be linear with the total energy use of a cell, for which Michaelis-Menten kinetics depending on the oxygen concentration are assumed. These concentrations depend on the position of the cell relative to the blood vessels. With a model for the behavior of a single neuron during hypoxia, the behavior of a network of these neurons, perhaps with different properties, and the resulting EEG signals can be simulated. We will present our first results of the influence on the electrical activity of the oxygen supply to the cells in the network.

The influence of the chloride currents on action potential firing and volume regulation of excitable cells studied by a kinetic model

In excitable cells, the generation of an action potential (AP) is associated with transient changes of the intra- and extracellular concentrations of small ions such as Na +, K + and Cl −. If these changes cannot be fully reversed between successive APs cumulative changes of trans-membrane ion gradients will occur, impinging on the cell volume and the duration, amplitude and frequency of APs. Previous computational studies focused on effects associated with excitation-induced changes of potassium and sodium. Here we present a model based study on the influence of chloride on the fidelity of AP firing and cellular volume regulation during excitation. Our simulations show that depending on the magnitude of the basal chloride permeability two complementary types of responsiveness and volume variability exist: (i) At high chloride permeability (typical for muscle cells), large excitatory stimuli are required to elicit APs; repetitive stimuli of equal strength result in almost identical spike train patterns (Markovian behavior), however, long excitation may lead to after discharges due to an outward directed current of intracellular chloride ions which accumulate during excitation; cell volume changes are large. (ii) At low chloride permeability (e.g., neurons), small excitatory stimuli are sufficient to elicit APs, repetitive stimuli of equal strength produce spike trains with progressively changing amplitude, frequency and duration (short-term memory effects or non-Markovian behavior); cell volume changes are small. We hypothesize that variation of the basal chloride permeability could be an important mechanism of neuronal cells to adapt their responsiveness to external stimuli during learning and memory processes.

Substrate and Inhibitor Binding to a Glutamate Transporter Homologue

Glutamate transporters tightly control extracellular glutamate concentration by pumping the transmitter into neurons and glia. Active transport is achieved by coupling the energy of the transmembrane ionic gradients to the conformational changes of the transporter, which alternates between outward- and inward-facing states with the substrate-binding site accessible from the extra- and intracellular solutions, respectively. In order to concentrate the substrate into the cytoplasm, the transporter's binding site must exhibit a high affinity from the outside and a low affinity from the inside. To better understand this change, we have characterized the binding properties of a prokaryotic homologue of the glutamate transporters, GltPh, by means of isothermal titration calorimetry. We designed double cysteine mutants that, upon cross-linking, lock the transporter in either the outward- or the inward-facing state and measured the thermodynamic binding parameters of the substrate aspartate and a competitive inhibitor TBOA. Surprisingly, the free energies of aspartate binding to the outward- and inward-facing states of the transporter are very similar at 25 °C, although the enthalpic and entropic contributions differ significantly. GltPh affinity for aspartate depends steeply on the concentration of sodium ions (Na), consistent with binding of ∼3 Na being thermodynamically coupled to binding of each substrate molecule. In contrast to the substrate, TBOA shows significantly lower affinity for the inward-facing state compared to the outward-facing state. In both states, TBOA exhibits a weaker sodium-dependence with an apparent number of coupled Na near 1. Our results show that in the absence of a sodium gradient, GltPh binds aspartate and Na with the same affinity in the outward- and inward-facing states. Therefore, the higher substrate affinity on the extracellular side of the membrane is determined by the higher Na concentration in the extracellular space compared to the cytoplasm.

Influence of aging on membrane permeability transition in brain mitochondria

The mitochondrial inner membrane permeability transition (MPT) plays an important role in the pathophysiology of acute disorders of the central nervous systems, including ischemic and traumatic brain injury, and possibly in neurodegenerative diseases. Opening of the permeability transition pore (PTP) by a combination of abnormally elevated intramitochondrial Ca2+ and oxidative stress induces the collapse of transmembrane ion gradients, resulting in membrane depolarization and uncoupling of oxidative phosphorylation. This loss of ATP synthesis eventually results in cellular metabolic failure and necrotic cell death. Drugs, e.g., cyclosporin A, can inhibit the permeability transition through their interaction with the mitochondria-specific protein, cyclophilin D, and demonstrate neuroprotection in several animal models. These characteristics of the MPT were developed almost exclusively from experiments performed with young, mature rodents whereas the neuropathologies associated with the MPT are most prevalent in the elderly population. Some evidence indicates that the sensitivity of mitochondria to Ca2+-induced PTP opening is greater in the aged compared to the young mature brain; however, the basis for this difference is unknown. Based on knowledge of factors that regulate the MPT and on other comparisons between cells and mitochondria from young and old animals, several features may be important. These aging-related features include impaired neuronal Ca2+ homeostasis, increased oxidative stress, increased cyclophilin D protein levels, oxidative modification of the adenine nucleotide translocase and of cardiolipin, and changes in the levels of anti-death mitochondrial proteins, e.g., Bcl-2. The influence of aging on both the contribution of the MPT to neuropathology and the neuroprotective efficacy of MPT inhibitors is a substantial knowledge gap that requires extensive research at the subcellular, cellular, and animal model levels.

Purinergic signalling in epithelial ion transport: regulation of secretion and absorption

Intracellular ATP, the energy source for many reactions, is crucial for the activity of plasma membrane pumps and, thus, for the maintenance of transmembrane ion gradients. Nevertheless, ATP and other nucleotides/nucleosides are also extracellular molecules that regulate diverse cellular functions, including ion transport. In this review, I will first introduce the main components of the extracellular ATP signalling, which have become known as the purinergic signalling system. With more than 50 components or processes, just at cell membranes, it ranks as one of the most versatile signalling systems. This multitude of system components may enable differentiated regulation of diverse epithelial functions. As epithelia probably face the widest variety of potential ATP-releasing stimuli, a special attention will be given to stimuli and mechanisms of ATP release with a focus on exocytosis. Subsequently, I will consider membrane transport of major ions (Cl(-) , HCO(3)(-) , K(+) and Na(+) ) and integrate possible regulatory functions of P2Y2, P2Y4, P2Y6, P2Y11, P2X4, P2X7 and adenosine receptors in some selected epithelia at the cellular level. Some purinergic receptors have noteworthy roles. For example, many studies to date indicate that the P2Y2 receptor is one common denominator in regulating ion channels on both the luminal and basolateral membranes of both secretory and absorptive epithelia. In exocrine glands though, P2X4 and P2X7 receptors act as cation channels and, possibly, as co-regulators of secretion. On an organ level, both receptor types can exert physiological functions and together with other partners in the purinergic signalling, integrated models for epithelial secretion and absorption are emerging.