Changes In Intracellular Ion Concentration Research Articles

Formation mechanism and regulation analysis of trumpet leaf in Ginkgo biloba L.

The research on plant leaf morphology is of great significance for understanding the development and evolution of plant organ morphology. As a relict plant, the G. biloba leaf morphology typically exhibits bifoliate and peltate forms. However, throughout its long evolutionary history, Ginkgo leaves have undergone diverse changes. This study focuses on the distinct "trumpet" leaves and normal fan-shaped leaves of G. biloba for analysis of their phenotypes, photosynthetic activity, anatomical observations, as well as transcriptomic and metabolomic analyses. The results showed that trumpet-shaped G. biloba leaves have fewer cells, significant morphological differences between dorsal and abaxial epidermal cells, leading to a significantly lower net photosynthetic rate. Additionally, this study found that endogenous plant hormones such as GA, auxin, and JA as well as metabolites such as flavonoids and phenolic acids play roles in the formation of trumpet-shaped G. biloba leaves. Moreover, the experiments revealed the regulatory mechanisms of various key biological processes and gene expressions in the trumpet-shaped leaves of G. biloba. Differences in the dorsal and abdominal cells of G. biloba leaves can cause the leaf to curl, thus reducing the overall photosynthetic efficiency of the leaves. However, the morphology of plant leaves is determined during the primordia leaf stage. In the early stages of leaf development, the shoot apical meristem (SAM) determines the developmental morphology of dicotyledonous plant leaves. This process involves the activity of multiple gene families and small RNAs. The establishment of leaf morphology is complexly regulated by various endogenous hormones, including the effect of auxin on cell walls. Additionally, changes in intracellular ion concentrations, such as fluctuations in Ca2+ concentration, also affect cell wall rigidity, thereby influencing leaf growth morphology.

Acclimation of marine invertebrate osmolyte systems to low salinity: A systematic review & meta-analysis

Salinity is a major environmental factor shaping the distribution and abundance of marine organisms. Climate change is predicted to alter salinity in many coastal regions due to sea level rise, evaporation, and changes in freshwater input. This exerts significant physiological stress on coastal invertebrates whose body fluid osmolality follows that of seawater (‘osmoconformers’). In this study, we conducted a systematic review and meta-analysis of osmolytes (both organic and inorganic) utilized by osmoconforming marine invertebrates during a >14-day acclimation to reduced salinity. Of the 2,389 studies screened, a total of 56 fulfilled the search criteria. Thirty-eight studies reported tissue osmolyte. Following acclimation to reduced salinity, tissue concentrations of six organic compounds and sodium were consistently reduced across phyla. This suggests that intracellular inorganic ions are not only utilized as a rapid response system during acute exposure to low salinity stress but also, in concert with reductions in organic osmolyte concentrations, during longer-term acclimation. Our systematic review demonstrates that only a few studies (n = 13) have quantified salinity-induced long-term changes in intracellular ion concentrations. In addition, no study has compiled a complete intracellular osmolyte budget. Alanine, betaine, glycine, and taurine are the major organic osmolytes that are universally employed across five phyla. The characterization of organic osmolytes was heavily weighted towards free amino acids (FAAs) and derivatives—neglecting methylamines and methylsulfonium compounds, which can be as important as FAAs in modulating intracellular osmolality. As a consequence, we suggest best-practice guidelines to streamline experimental designs and protocols in osmoregulation research in order to better understand the conserved mechanisms that define the limits of salinity acclimation in marine invertebrates. To our best knowledge, this is the first systematic review and meta-analysis on osmolyte concentrations in osmoconformers acclimated to low salinity. It creates a valuable baseline for future research and reveals large research gaps. Our meta-analysis suggests that there are common osmolyte actors employed across phyla but no uniform concept since osmolyte pool composition and proportions were taxon-specific. In light of future salinity changes and their potential consequences, it becomes more important to understand salinity tolerance capacities and limits.

Evaluation of pH Measurement Using Electron‐Beam‐Induced Current Detection

The study of biological functions requires measurements of changes in intracellular ion concentrations and their distribution. Ion‐sensitive field‐effect transistors and light‐addressable potentiometric sensors (LAPSs) operate on the basis of ion‐sensitive membranes and semiconductors that can measure the ion concentration around cells. In this study, the authors develop an ion sensors based on an operating principle similar to that of LAPSs. An electron beam rather than light is utilized. When a semiconductor is irradiated with an electron beam, electron–hole pairs are generated, and an induced current is produced with an amplitude that depends on the ion concentration. The bias voltage characteristics of the capacitance and photocurrent of the fabricated sensor substrate are evaluated and it is demonstrated that the proton concentration could be measured with an electron beam based on the same principle as in LAPSs.

A four-electrode method to study dynamics of ion activity and transport in skeletal muscle fibers.

Ion movements across biological membranes, driven by electrochemical gradients or active transport mechanisms, control essential cell functions. Membrane ion movements can manifest as electrogenic currents or electroneutral fluxes, and either process can alter the extracellular and/or intracellular concentration of the transported ions. Classic electrophysiological methods allow accurate measurement of membrane ion movements when the transport mechanism produces a net ionic current; however, they cannot directly measure electroneutral fluxes and do not detect any accompanying change in intracellular ion concentrations. Here, we developed a method for simultaneously measuring ion movements and the accompanying dynamic changes in intracellular ion concentrations in intact skeletal muscle fibers under voltage or current clamp in real time. The method combines a two-microelectrode voltage clamp with ion-selective and reference microelectrodes (four-electrode system). We validate the electrical stability of the system and the viability of the preparation for periods of ∼1 h. We demonstrate the power of this method with measurements of intracellular Cl-, H+, and Na+ to show (a) voltage-dependent redistribution of Cl- ions; (b) intracellular pH changes induced by changes in extracellular pCO2; and (c) electroneutral and electrogenic Na+ movements controlled by the Na,K-ATPase. The method is useful for studying a range of transport mechanisms in many cell types, particularly when the transmembrane ion movements are electrically silent and/or when the transport activity measurably changes the intracellular activity of a transported ion.

Inter-Species Differences in the Response of Sinus Node Cellular Pacemaking to Changes of Extracellular Calcium.

Changes of serum and extracellular ion concentrations occur regularly in patients with chronic kidney disease (CKD). Recently, hypocalcaemia, i.e. a decrease of the extracellular calcium concentration [Ca2+]o, has been suggested as potential pathomechanism contributing to the unexplained high rate of sudden cardiac death (SCD) in CKD patients. In particular, there is a hypothesis that hypocalcaemia could slow down natural pacemaking in the human sinus node to fatal degrees. Here, we address the question whether there are inter-species differences in the response of cellular sinus node pacemaking to changes of [Ca2+]o. Towards this end, we employ computational models of mouse, rabbit and human sinus node cells. The Fabbri et al. human model was updated to consider changes of intracellular ion concentrations. We identified crucial inter-species differences in the response of cellular pacemaking in the sinus node to changes of [Ca2+]o with little changes of cycle length in mouse and rabbit models (<; 83 ms) in contrast to a pronounced bradycardic effect in the human model (up to >1000 ms). Our results suggest that experiments with human sinus node cells are required to investigate the potential mechanism of hypocalcaemia-induced bradycardic SCD in CKD patients and small animal models are not well suited.

Tight Coupling of Astrocyte pH Dynamics to Epileptiform Activity Revealed by Genetically Encoded pH Sensors.

Astrocytes can both sense and shape the evolution of neuronal network activity and are known to possess unique ion regulatory mechanisms. Here we explore the relationship between astrocytic intracellular pH dynamics and the synchronous network activity that occurs during seizure-like activity. By combining confocal and two-photon imaging of genetically encoded pH reporters with simultaneous electrophysiological recordings, we perform pH measurements in defined cell populations and relate these to ongoing network activity. This approach reveals marked differences in the intracellular pH dynamics between hippocampal astrocytes and neighboring pyramidal neurons in rodent in vitro models of epilepsy. With three different genetically encoded pH reporters, astrocytes are observed to alkalinize during epileptiform activity, whereas neurons are observed to acidify. In addition to the direction of pH change, the kinetics of epileptiform-associated intracellular pH transients are found to differ between the two cell types, with astrocytes displaying significantly more rapid changes in pH. The astrocytic alkalinization is shown to be highly correlated with astrocytic membrane potential changes during seizure-like events and mediated by an electrogenic Na(+)/HCO3 (-) cotransporter. Finally, comparisons across different cell-pair combinations reveal that astrocytic pH dynamics are more closely related to network activity than are neuronal pH dynamics. This work demonstrates that astrocytes exhibit distinct pH dynamics during periods of epileptiform activity, which has relevance to multiple processes including neurometabolic coupling and the control of network excitability. Dynamic changes in intracellular ion concentrations are central to the initiation and progression of epileptic seizures. However, it is not known how changes in intracellular H(+) concentration (ie, pH) differ between different cell types during seizures. Using recently developed pH-sensitive proteins, we demonstrate that astrocytes undergo rapid alkalinization during periods of seizure-like activity, which is in stark contrast to the acidification that occurs in neighboring neurons. Rapid astrocytic pH changes are highly temporally correlated with seizure activity, are mediated by an electrogenic Na(+)/HCO3- cotransporter, and are more tightly coupled to network activity than are neuronal pH changes. As pH has profound effects on signaling in the nervous system, this work has implications for our understanding of seizure dynamics.

Expression Level Dependence of the Gating and Permeation Properties of P2X Receptor Channels

P2X receptors are cation channels that are activated by extracellular ATP, and that are widely expressed in virtually every tissue. Seven subtypes of P2X receptors have been identified in mammals, and they play important roles in sensory signaling and inflammation. The expression levels of P2X receptors have been reported to change in response to various stimuli. For instance, the expression level of P2X4 receptors increased strikingly in microglia of spinal cord after nerve injury, and these receptors were suggested to play important roles for tactile allodynia1. Interestingly, it has also been reported that both gating and permeation properties of some P2X receptors change in a channel-density dependent manner2,3. Here we transfected P2X2 receptor channels into HEK293 cells with either high or low expression vectors, and then studied the properties of these channels at varying expression levels. Our results suggest that the selectivity, rectification and ATP sensitivity of P2X receptor channels exhibit expression level dependence, as if distinct open states predominate at different expression levels. These changes do not appear to result from artefactual changes in intracellular ion concentrations or voltage errors, and we are currently studying the underlying mechanism.

In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress

Genetically encoded, fluorescent biosensors have been developed to probe the activities of various signaling molecules inside cells ranging from changes in intracellular ion concentrations to dynamics of lipid second messengers. HyPer is a member of this class of biosensors and is the first to dynamically respond to hydrogen peroxide (H2O2), a reactive oxygen species that functions as a signaling molecule. However, detailed characterization of HyPer's signal is not currently available within the context of bacteria exposed to external oxidative stress, which occurs in the immunological response of higher organisms against invasive pathogenic bacteria. Here, we performed this characterization, specifically in Escherichia coli exposed to external H2O2. We found that the temporal behavior of the signal does not correspond exactly to peroxide concentration in the system as a function of time and expression of the sensor decreases the peroxide scavenging activity of the cell. We also determined the effects of cell number, both before and after normalization of externally added H2O2 to the number of cells. Finally, we report quantitative characteristics of HyPer's signal in this context, including the dynamic range of the signal, the signal-to-noise ratio, and the half saturation constant. These parameters show statistically meaningful differences in signal between two commonly used strains of E. coli, demonstrating how signal can vary with strain. Taken together, our results establish a systematic, quantitative framework for researchers seeking to better understand the role of H2O2 in the immunological response against bacteria, and for understanding potential differences in the details of HyPer's quantitative performance across studies.

Visualizing Cell Signaling: Current Trends and New Technologies

Techniques for the study of intracellular ions are used widely in biology, including for the tracking of calcium waves or ions affecting pH within living cells. The ability to monitor changes in intracellular ion concentrations over time is vital for our understanding of signaling and functional pathways in cellular systems. These pathways are central to many fundamental processes such as muscle contraction and synaptic nerve signal transmission. Ion channels that span the outer cell membrane open or close in response to extracellular and intracellular signals, potentially altering how the cell behaves. These fluctuations can be visualized and quantified using ratiometric microscopy and special fluorescent dyes designed to bind specific ions, such as the FURA-2 indicator dye specific to calcium ions. Changes in the photophysics of the dye as it binds to its target ion allows for quantitation of the bound and unbound ratio, and thus the concentration of the ion under investigation. This webinar will introduce the viewer to the current state of technology in this important field as well as cover the latest advances in microscopy for ion signaling research.

Emerging role of WNK1 in pathologic central nervous system signaling

WNK1 (with no lysine (K)) is a widely expressed serine/threonine protein kinase. The role of this kinase was first described in the kidney where it dynamically controls ion channels that regulate changes in cell volume. WNK1, through intermediates oxidative stress-responsive kinase-1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK), phosphorylates the inwardly directed Na(+)-K+-Cl(-)--cotransporter 1 (NKCC1) and the outwardly directed K(+)-Cl(-)-cotransporter 2 (KCC2), activating and deactivating these channels, respectively. WNK1, NKCC1 and KCC2 are also expressed in the central nervous system (CNS). Growing evidence implicates WNK1 playing a critical role in pathologic nervous system signaling where changes in intracellular ion concentration in response to γ-aminobutyric-acid (GABA) can activate otherwise silent pathways. This review will focus on current research about WNK1, its downstream effectors and role in GABA signaling. Future perspectives include investigating WNK1 expression in the CNS after spinal cord injury (SCI), where altered neuronal signaling could underlie pathological states such as neuropathic pain (NP).

Effects of the anthelmintic drug PF1022A on mammalian tissue and cells

Nematode infections cause human morbidity and enormous economic loss in livestock. Since resistance against currently available anthelmintics is a worldwide problem, there is a continuous need for new compounds. The cyclooctadepsipeptide PF1022A is a novel anthelmintic that binds to the latrophilin-like transmembrane receptor important for pharyngeal pumping in nematodes. Furthermore, PF1022A binds to GABA receptors, which might contribute to the anthelmintic effect. Like other cyclodepsipeptides, PF1022A acts as an ionophore. However, no correlation between ionophoric activity and anthelmintic properties was found. This is the first study describing the effect of PF1022A on mammalian cells and tissues. While channel-forming activity was observed already at very low concentrations, changes in intracellular ion concentrations and reduction of contractility in isolated guinea pig ileum occurred at multiples of anthelmintically active concentrations. PF1022A did not induce necrotic cell death indicated by complete lack of cellular lactate dehydrogenase release. In contrast, apoptosis induction via the mitochondrial pathway was suggested for long-term drug treatment at high concentrations due to numerous apoptotic morphological changes as well as mitochondrial membrane depolarisation. Short time effects were based on cell cycle blockade in G 0/G 1 phase. Additionally, the cell cycle and apoptosis regulating proteins p53, p21 and bax, but not Bcl-2 were shown to impact on PF1022A-induced cytotoxicity. However, since PF1022A-induced cytotoxicity was found at drug concentrations higher than those used in anthelmintic treatment, it can be suggested that PF1022A intake might not impair human or animal health. Thus, PF1022A seems to be a safe alternative to other anthelmintic drugs.

Decreased aquaporin expression leads to increased resistance to apoptosis in hepatocellular carcinoma

Cells undergoing apoptosis are characterized by decreased cell size due to changes in intracellular ion concentration and rapid, aquaporin (AQP)-dependent water movement out of the cell, events required for the activation of pro-apoptotic enzymes. The current study demonstrates AQP 8 and 9 expression is significantly decreased in hepatocellular carcinoma (HCC) versus normal liver. Isolation of hepatic tumor cells (H4IIE) and hepatocytes confirmed a lack of water movement across the H4IIE cell membrane via AQPs and identified an inherent resistance of H4IIE cells to apoptotic stimuli. In contrast, hepatocytes rapidly responded to osmotic challenge through AQP-dependent water movement and underwent cell death following apoptotic stimulation.

Interaction between organophosphate compounds and cholinergic functions during development

Organophosphate (OP) compounds exert inhibition on cholinesterase (ChE) activity by irreversibly binding to the catalytic site of the enzymes. For this reason, they are employed as insecticides for agricultural, gardening and indoor pest control. The biological function of the ChE enzymes is well known and has been studied since the beginning of the XXth century; in particular, acetylcholinesterase (AChE, E.C. 3.1.1.7) is an enzyme playing a key role in the modulation of neuromuscular impulse transmission. However, in the past decades, there has been increasing interest concerning its role in regulating non-neuromuscular cell-to-cell interactions mediated by electrical events, such as intracellular ion concentration changes, as the ones occurring during gamete interaction and embryonic development. An understanding of the mechanisms of the cholinergic regulation of these events can help us foresee the possible impact on environmental and human health, including gamete efficiency and possible teratogenic effects on different models, and help elucidate the extent to which OP exposure may affect human health. The chosen organophosphates were the ones mainly used in Europe: diazinon, chlorpyriphos, malathion, and phentoate, all of them belonging to the thionophosphate chemical class. This research has focused on the comparison between the effects of exposure on the developing embryos at different stages, identifying biomarkers and determining potential risk factors for sensitive subpopulations. The effects of OP oxonisation were not taken into account at this level, because embryonic responses were directly correlated to the changes of AChE activity, as determined by histochemical localisation and biochemical measurements. The identified biomarkers of effect for in vitro experiments were: cell proliferation/apoptosis as well as cell differentiation. For in vivo experiments, the endpoints were: developmental speed, size and shape of pre-gastrula embryos; developmental anomalies on neural tube, head, eye, heart. In all these events, we had evidence that the effects are mediated by ion channel activation, through the activation/inactivation of acetylcholine receptors (AChRs).

TRP channels as new pharmacological targets

Changes in intracellular ion concentrations are universal triggers of cell activation. Ca, for instance, is one of the most versatile intracellular second messengers in eukaryotic cells involved in a wide range of cellular processes including exocytosis, contraction, cell proliferation, development and apoptosis. Stimulation of target cells with neurotransmitters, hormones or growth factors leads to an elevation of the intracellular Ca concentration brought about by release from intracellular stores and influx via Ca-permeable cation channels in the cell membrane (Clapham et al. 2001). While excitable cells like neurons are well equipped with voltage-gated ion channels mediating the bulk of cation influx, the molecular identity of cation entry channels in non-excitable cells has been completely mysterious for quite some time. A major leap forward in our understanding of agonist-triggered cation influx was achieved by the discovery of Drosophila melanogaster visual transduction channels associated with the transient receptor potential (trp) and the trp-like (trpl) mutant phenotypes. Since then a new mammalian gene family of TRP homologs has been identified by homology screening (Montell et al. 2002; Clapham et al. 2003). Based on structural homology and on systematic glycosylation scanning, TRP proteins are allocated to the structural superfamily of six-transmembrane spanning ion channels like voltage-gated K channels, the cyclic nucleotide-gated channel family, and single cassettes of voltage-activated Ca and Na channels (Fig. 1). Both Nand C-termini of TRP proteins are assumed to be localised intracellularly, and a channel pore loop appears to be bordered by transmembrane domains 5 and 6. In specialised cells, some members of the TRP family participate in sensory processes like vision and hearing as well as temperature, pain, gustatory and pheromone perception. In addition, many TRP proteins are involved in ubiquitous, very basic cellular processes like sensing of osmotic or oxidative stress and mediating agonist-induced cation entry (Clapham 2003). Based on primary amino acid homology, the conventional TRP proteins can be classified into three subfamilies: TRPC, TRPV, and TRPM (see Fig. 1). Three additional, more distantly related subfamilies have recently been defined: ANKTM1, a Ca-permeant, non-selective cation channel, is the only mammalian member of the TRPA branch of TRP proteins. It is activated by noxious cold temperature, pungent mustard oil compounds and may additionally serve as a mechanosensitive transduction channel in auditory hair cells as well as an ionotropic cannabinoid receptor. The three mucolipins, TRPML1, 2, and 3, appear to be ion channels in intracellular vesicles. Mutations in TRPML1 cause mucolipidosis type IV, a neurodegenerative lysosomal storage disorder, while genetic defects in TRPP2, a member of the TRPP subfamily, are frequently encountered in patients suffering from autosomal dominant polycystic kidney disease. The current compilation of review articles covers salient features of TRPC, TRPV, TRPM, and TRPP proteins at the cutting edge of science. In particular, the authors continue to explore the pharmacotherapeutic potential of this new class of ion channels, a fruitful endeavor that was started at the 25th Symposium at Rauischholzhausen Castle on “TRP channels as Pharmacological Targets” which took place on June 10–12, 2004. TRPC proteins all share a common gating mechanism that involves the activation of phospholipase C isoforms and thus constitute prime molecular candidates responsible for phospholipase C-dependent cation influx. The molecThis revised version was published online in June 2005 with corrections to the Reference list

Investigation of the electrophysiological properties of enniatins

Enniatins are cyclohexadepsipeptides produced by various species of the genus Fusarium, and are known to have ionophoric, antibiotic, and in vitro hypolipidaemic properties. With the patch clamp technique in the inside-out mode it could be shown that enniatin easily incorporates into the cell membrane in which it forms cation-selective pores. This feature is characterized by unitary transitions to conductance levels typical for channels, ion selectivity, rectification, conductivity in the pS range, and block. A model of vertically stacked enniatin molecules that form sandwich complexes is suggested. Like gramicidin enniatin is a passive channel. Single channel properties for the isomers enniatin A1, B, and B1 differed from each other. This implies an influence of the substituted moieties on the complexation of cations. Electrical activity was followed by changes in intracellular ion concentrations, which are consistent with depolarization of the membrane resting potential, shortening of action potential duration, and reduced contractility.

Amino Acids as Regulators and Components of Nonproteinogenic Pathways

Amino acids are not only important precursors for the synthesis of proteins and other N-containing compounds, but also participate in the regulation of major metabolic pathways. Glutamate and aspartate, for example, are components of the malate/aspartate shuttle and their concentrations control the rate of mitochondrial oxidation of glycolytic NADH. Glutamate also controls the rate of urea synthesis, not only as the precursor of ammonia and aspartate, but as substrate for synthesis of N-acetylglutamate, the essential activator of carbamoyl-phosphate synthase. This mechanism allows large variations in urea synthesis at relatively constant ammonia concentrations. Increases in intracellular amino acid concentration increase cell volume. Cell swelling per se has anabolic effects on protein, carbohydrate and lipid metabolism: enhanced synthesis of macromolecules compensates for increases in intracellular osmolarity. Mechanisms responsible for cell swelling-induced changes in pathway fluxes include changes in intracellular ion concentrations and in signal transduction. Specific amino acids (e.g., leucine) stimulate protein synthesis and inhibit (autophagic) protein degradation independent of changes in cell volume because they stimulate mTOR (mammalian target of rapamycin), a protein kinase, which is one of the components of a signal transduction pathway used by insulin. When the cellular energy state is low, stimulation of mTOR by amino acids is prevented by activation of AMP-dependent protein kinase. Amino acid-dependent signaling also promotes insulin production by beta-cells. This further adds to the anabolic properties of amino acids. It is concluded that amino acids are important regulators of major metabolic pathways.

Ionic Charge Conservation and Long-Term Steady State in the Luo–Rudy Dynamic Cell Model

It has been postulated that cardiac cell models accounting for changes in intracellular ion concentrations violate a conservation principle, and, as a result, computed parameters (e.g., ion concentrations and transmembrane potential, V m) drift in time, never attaining steady state. To address this issue, models have been proposed that invoke the charge conservation principle to calculate V m from ion concentrations (“algebraic” method), rather than from transmembrane current (“differential” method). The aims of this study are to compare model behavior during prolonged periods of pacing using the algebraic and differential methods, and to address the issue of model drift. We pace the Luo–Rudy dynamic model of a cardiac ventricular cell and compare the time-dependent behavior of computed parameters using the algebraic and differential methods. When ions carried by the stimulus current are taken into account, the algebraic and differential methods yield identical results and neither shows drift in computed parameters. The present study establishes the proper pacing protocol for simulation studies of cellular behavior during long periods of rapid pacing. Such studies are essential for mechanistic understanding of arrhythmogenesis, since cells are subjected to rapid periodic stimulation during many arrhythmias.

Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation

Cryopreserved spermatozoa demonstrate reduced conception rates compared with fresh spermatozoa when used for artificial insemination. The preliminary stage of cryopreservation of spermatozoa involves cooling to 5 degrees C, during which spermatozoa experience a capacitation-like change, which may be partially responsible for the reduced conception rate observed. The aim of this study was to determine the nature of these capacitation-like changes and how much this process resembles true capacitation. Boar spermatozoa, cooled to 5 degrees C and re-warmed to physiological temperatures (39 degrees C), were compared with spermatozoa capacitated in Tyrode's complete medium (TALP) for 2 h at 39 degrees C. Fluorescent probes, and SDS-PAGE and western blotting were used to visualize events known to occur during capacitation in vitro. Chlortetracycline staining of membrane domains and Fluo-3 detection of changes in intracellular free calcium by flow cytometry in cooled and re-warmed spermatozoa showed similarities to those of capacitated spermatozoa. Alterations to lipid bilayer fluidity assessed by merocyanine fluorescence staining and intracellular signalling pathways detected by tyrosine phosphorylation of cooled and re-warmed spermatozoa, did not completely reflect the changes detected during capacitation in vitro. Thus, cooling spermatozoa to 5 degrees C results in a similar endpoint to that observed in capacitated cells in terms of reactive membranes and changes in intracellular ion concentrations, which may account for their comparable functionality. However, these modifications are not completely analogous and should not be considered true capacitation, but rather a by-passing of the capacitation process.

Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation

Cryopreserved spermatozoa demonstrate reduced conception rates compared with fresh spermatozoa when used for artificial insemination. The preliminary stage of cryopreservation of spermatozoa involves cooling to 5 degrees C, during which spermatozoa experience a capacitation-like change, which may be partially responsible for the reduced conception rate observed. The aim of this study was to determine the nature of these capacitation-like changes and how much this process resembles true capacitation. Boar spermatozoa, cooled to 5 degrees C and re-warmed to physiological temperatures (39 degrees C), were compared with spermatozoa capacitated in Tyrode's complete medium (TALP) for 2 h at 39 degrees C. Fluorescent probes, and SDS-PAGE and western blotting were used to visualize events known to occur during capacitation in vitro. Chlortetracycline staining of membrane domains and Fluo-3 detection of changes in intracellular free calcium by flow cytometry in cooled and re-warmed spermatozoa showed similarities to those of capacitated spermatozoa. Alterations to lipid bilayer fluidity assessed by merocyanine fluorescence staining and intracellular signalling pathways detected by tyrosine phosphorylation of cooled and re-warmed spermatozoa, did not completely reflect the changes detected during capacitation in vitro. Thus, cooling spermatozoa to 5 degrees C results in a similar endpoint to that observed in capacitated cells in terms of reactive membranes and changes in intracellular ion concentrations, which may account for their comparable functionality. However, these modifications are not completely analogous and should not be considered true capacitation, but rather a by-passing of the capacitation process.

Interactions of bioactive glasses with osteoblasts in vitro: effects of 45S5 Bioglass ®, and 58S and 77S bioactive glasses on metabolism, intracellular ion concentrations and cell viability

In a cell culture model of murine osteoblasts three particulate bioactive glasses were evaluated and compared to glass (either borosilicate or soda-lime-silica) particles with respect to their effect on metabolic activity, cell viability, changes in intracellular ion concentrations, proliferation and differentiation. 45S5 Bioglass ® caused extra- and intracellular alkalinization, a rise in [Ca 2+] i and [K +] i, a small plasma membrane hyperpolarization, and an increase in lactate production. Glycolytic activity was also stimulated when cells were not in direct contact with 45S5 Bioglass ® particles but communicated with them only through the medium. Similarly, raising the pH of culture medium enhanced lactate synthesis. 45S5 Bioglass ® had no effect on osteoblast viability and, under most conditions, did not affect either proliferation or differentiation. Bioactive glasses 58S and 77S altered neither the ion levels nor enhanced metabolic activity. It is concluded that: (1) some bioactive glasses exhibit well-defined effects in osteoblasts in culture which are accessible to experimentation; (2) 45S5 Bioglass ® causes marked external and internal alkalinization which is, most likely, responsible for enhanced glycolysis and, hence, cellular ATP production; (3) changes in [H +] could contribute to alternations in concentrations of other intracellular ions; and (4) the rise in [Ca 2+] i may influence activities of a number of intracellular enzymes and pathways. It is postulated that the beneficial effect of 45S5 ® on in vivo bone growth and repair may be due to some extent to alkalinization, which in turn increases collagen synthesis and crosslinking, and hydroxyapatite formation.