Retinal Macroglial Responses in Health and Disease

Due to their permanent and close proximity to neurons, glial cells perform essential tasks for the normal physiology of the retina. Astrocytes and Müller cells (retinal macroglia) provide physical support to neurons and supplement them with several metabolites and growth factors. Macroglia are involved in maintaining the homeostasis of extracellular ions and neurotransmitters, are essential for information processing in neural circuits, participate in retinal glucose metabolism and in removing metabolic waste products, regulate local blood flow, induce the blood-retinal barrier (BRB), play fundamental roles in local immune response, and protect neurons from oxidative damage. In response to polyetiological insults, glia cells react with a process called reactive gliosis, seeking to maintain retinal homeostasis. When malfunctioning, macroglial cells can become primary pathogenic elements. A reactive gliosis has been described in different retinal pathologies, including age-related macular degeneration (AMD), diabetes, glaucoma, retinal detachment, or retinitis pigmentosa. A better understanding of the dual, neuroprotective, or cytotoxic effect of macroglial involvement in retinal pathologies would help in treating the physiopathology of these diseases. The extensive participation of the macroglia in retinal diseases points to these cells as innovative targets for new drug therapies.

Roles of extracellular ions and pH in 5-HT-induced sperm motility in marine bivalve

Factors that inhibit and stimulate the initiation of sperm motility were determined for Manila clam (Ruditapesphilippinarum), Pacific oyster (Crassostrea gigas), and Japanese scallop (Patinopecten yessoensis). Compared with artificial seawater (ASW), serotonin (5-hydroxytryptamine creatinine sulfate, 5-HT) could fully trigger sperm motility and increase sperm velocity and motility duration. Sperm motility was decreased in ASW at pH 6.5–7.0 and suppressed at pH 4.0. In Manila clam and Pacific oyster, 5-HT could overcome the inhibitory effects of acidic pH on sperm motility. In the presence of nigericin (a K+/H+exchanger), sperm motility was only triggered at pH 8.3. Testicular fluid K+concentrations were two- to fourfold higher than that in ASW. Sperm motility and velocity were decreased in ASW or 5-HT containing ≥40 mM K+or ≥2.5 mM 4-aminopyridine, suggesting K+efflux requirement to initiate motility. Sperm motility and velocity were reduced in ASW or 5-HT containing EGTA or W-7, suggesting that extracellular Ca2+is required for Ca2+/calmodulin-dependent flagellar beating. Ca2+influx occurs via Ca2+channels because sperm motility and velocity were decreased in both ASW and 5-HT containing T-type and L-type Ca2+channel blockers. 5-HT-dependent initiation of sperm motility was associated with intracellular Ca2+rise, which was comparable to that seen in ASW but was not observed in the presence of EGTA or a Ca2+channel blocker. Extracellular Na+is also essential for sperm motility initiation via regulation of Na+/Ca2+exchange. Overall, 5-HT-dependent initiation of sperm motility in marine bivalve mollusks is an osmolality-independent mechanism and regulated by extracellular pH, K+, Ca2+, and Na+.

NEURONAL MULTIFUNCTIONAL ATPase

Here, we review the properties of a suggested mechanism for a neural ATPase complex based on our recent experimental findings. The mechanism represents a multifunctional ATPase: an enzyme that is a chloride pump and a GABA receptor. This enables new views on the ways Cl - channel transports anions and its regulation by the intra- and extracellular ions and molecules (in particular by glucose, ATP, [Formula: see text]). The hydrolytic activity of this GABA A-coupled ATPase provides the [Formula: see text] transport process the energy and determines a certain direction of ions flux across neuronal membrane. This can help with the research regarding several diseases such as epilepsy. [Formula: see text]Special Issue Comment: This project is about a multifunctional ATPase complex. Experiments involving measuring & solving individual ATPases are related with the Special Issue about FRET experiments,1 about enzymes,2 and about treatments when solving single molecules.3,4 The model suggested here is simply tested with these experimental and mathematical methods.

Osmotic adjustment and plant adaptation to environmental changes related to drought and salinity

The salinization and water deficit of soil are widespread environmental problems in limiting plant survival, growth, and productivity. However, some plants could adopt some strategies to resist salinity and drought stresses. Among these strategies, the mechanism of osmotic adjustment could help plants and algae to avoid ion toxicity and maintain water uptake in both stresses by accumulating large quantities of osmolytes. Two types of osmolytes, organic solutes and inorganic ions, play a key role in osmotic adjustment. Different osmolytes and their osmotic adjustment actions are different according to their distribution in different plants. Organic solutes, known as compatible solutes, include amino acids, glycerol, sugars, and other low molecular weight metabolites, serve a function in cells to lower or balance the osmotic potential of intracellular and extracellular ions in resistance to osmotic stresses. Inorganic ions for osmotic adjustment are mainly Na+, K+, Ca2+, and Cl–. Inorganic ions make great contribution in osmotic adjustment by ion transport processes with related ion antiporters and ion channels. The aim of this review is to integrate recent data on the mechanisms of osmotic adjustment by osmolytes in plants and algae, and to illustrate the variety of related molecular mechanisms, to introduce new concepts and techniques into this research field. Genetic manipulation including the application of transgenic techniques in plants provides promising strategies to elevate the tolerance capability of plants under osmotic stress conditions.

Altered gating and regulation of a carboxy-terminal ClC channel mutant expressed in the Caenorhabditis elegans oocyte

CLH-3a and CLH-3b are swelling-activated, alternatively spliced Caenorhabditis elegans ClC anion channels that have identical membrane domains but exhibit marked differences in their cytoplasmic NH2 and COOH termini. The major differences include a 71-amino acid CLH-3a NH2-terminal extension and a 270-amino acid extension of the CLH-3b COOH terminus. Splice variation gives rise to channels with striking differences in voltage, pH, and Cl− sensitivity. On the basis of structural and functional insights gained from crystal structures of bacterial ClCs, we suggested previously that these functional differences are due to alternative splicing of the COOH terminus that may change the accessibility and/or function of pore-associated ion-binding sites. We recently identified a mutant worm strain harboring a COOH-terminal deletion mutation in the clh-3 gene. This mutation removes 101 COOH-terminal amino acids unique to CLH-3b and an additional 64 upstream amino acids shared by both channels. CLH-3b is expressed in the worm oocyte, which allowed us to characterize the mutant channel, CLH-3bΔC, in its native cellular environment. CLH-3bΔC exhibits altered voltage-dependent gating as well as pH and Cl− sensitivity that resemble those of CLH-3a. This mutation also alters channel inhibition by Zn2+, prevents ATP depletion-induced activation, and dramatically reduces volume sensitivity. These results suggest that the deleted COOH-terminal region of CLH-3bΔC functions to modulate channel sensitivity to voltage and extracellular ions. This region also likely plays a role in channel regulation and cell volume sensitivity. Our findings contribute to a growing body of evidence indicating that cytoplasmic domains play key roles in the gating and regulation of eukaryotic ClCs.

A virtual instrumentation based protocol for the automated implementation of the inner field compensation method

One influential parameter which mediates interactions between many types of molecules and biological membranes stems from the lumped contributions of the transmembrane potential, dipole potential and the difference in the surface potentials on both sides of a membrane. With relevance to cell physiology, such electrical features of a biomembrane are prone to undergoing changes as a result of interactions with the aqueous surrounding. Among the most useful tools devoted to exploring changes of electrical parameters of a lipid membrane induced by certain extracellular ions, lipid composition, and embedded membrane peptides and proteins, are spectroscopic imaging and the inner field compensation (IFC) method. In this work we layout the principles of a fully computerized version of the IFC method, which makes it more readily available to users. As a direct application, we deployed this improved version of the IFC method to time-resolve changes induced by alamethicin monomers upon membrane dipole potential, following their aggregation within an artificial lipid membrane. Intriguingly, even prior crossing the membrane core, the membrane-bound alamethicin monomers are shown to significantly increase the dipole potential of the monolayer they reside in. Such data further emphasize the yet less-explored interplay between membrane-based protein and peptides, and the membrane dipole potential.

Cation dysfunction associated with cerebral ischemia followed by reperfusion: a comparison of microdialysis and ion-selective electrode methods

Object. Disruption of ionic homeostasis during ischemia is a well-characterized event and is identified by a rise in the concentration of extracellular potassium [K+]e, with a concomitant reduction in the concentration of extracellular sodium [Na+]e. Results of clinical studies in which microdialysis has been used, however, have shown only modest changes in the levels of extracellular ions. The object of this study was to measure [K+]e and [Na+]e by using ion-selective electrodes (ISEs) and to compare these measurements with those obtained using the well-established method of microdialysis. Methods. Fifteen Sprague—Dawley rats were separated into three groups. Five animals were subjected to a 15-minute period of ischemia, and another five animals to a 60-minute period of ischemia; animals in both of these groups received K+-free microdialysis perfusate. The third group of five rats underwent a 60-minute period of ischemia and received a reduced-Na+ microdialysis perfusate. Transient forebrain ischemia was produced by bilateral carotid artery occlusion combined with hypotension. A custom-fabricated glass Na+ electrode and a flexible plastic K+ and reference electrodes were used to monitor extracellular ion transients. Microdialysis samples were obtained with the aid of a 2-mm microdialysis probe that was perfused with K+-free mock cerebrospinal fluid at a rate of 2 µl/minute. Baseline measurements of [K+]e and [Na+]e, obtained using ISEs, were 3.41 ± 0.09 mM and 145 ± 7.75 mM, respectively. Ischemia resulted in a rapid accumulation of [K+]e (in animals subjected to 15 minutes of ischemia, the concentration was 41.9 ± 13.7 mM; and in animals subjected to 60 minutes of ischemia, the concentration was 66.9 ± 11.5 mM), with a concomitant decrease in [Na+]e (in animals subjected to 15 minutes of ischemia, the concentration was 71.7 ± 2.9 mM; and in animals subjected to 60 minutes of ischemia, the concentration was 74.7 ± 1.9 mM). A comparison of microdialysis and ISE methods revealed that microdialysis underestimated the [K+]e changes and was insensitive to concomitant [Na+]e alterations that occur during ischemia. Conclusions. Our results indicate that the flexible ISE is a reliable and accurate tool for monitoring ionic dysfunction that accompanies brain injury.

Store-operated influx of Ca2+ in pancreatic β-cells exhibits graded dependence on the filling of the endoplasmic reticulum

The store-operated pathway for Ca2+ entry was studied in individual mouse pancreatic β-cells by measuring the cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mn2+ ([Mn2+]i) with the fluorescent indicator fura-2. Influx through the store-operated pathway was initially shut off by pre-exposure to 20 mM glucose, which maximally stimulates intracellular Ca2+ sequestration. To avoid interference with voltage-dependent Ca2+ entry the cells were hyperpolarized with diazoxide and the channel blocker methoxyverapamil was present. Activation of the store-operated pathway in response to Ca2+ depletion of the endoplasmic reticulum was estimated from the sustained elevation of [Ca2+]i or from the rate of increase in [Mn2+]i due to influx of these extracellular ions. Increasing concentrations of the inositol 1,4,5-trisphosphate-generating agonist carbachol or the sarco(endo)plasmatic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) cause gradual activation of the store-operated pathway. In addition, the carbachol- and CPA-induced influx of Mn2+ depended on store filling in a graded manner. The store-operated influx of Ca2+/Mn2+ was inhibited by Gd3+ and 2-aminoethoxydiphenyl borate but neither of these agents discriminated between store-operated and voltage-dependent entry. The finely tuned regulation of the store-operated mechanisms in the β-cell has direct implications for the control of membrane potential and insulin secretion.