Glucose and NAADP trigger elementary intracellular ß-cell Ca2+ signals.

Pancreatic ß-cells release insulin upon a rise in blood glucose. The precise mechanisms of stimulus-secretion coupling, and its failure in Diabetes Mellitus Type 2, remain to be elucidated. The consensus model, as well as a class of currently prescribed anti-diabetic drugs, are based around the observation that glucose-evoked ATP production in ß-cells leads to closure of cell membrane ATP-gated potassium (KATP) channels, plasma membrane depolarisation, Ca2+ influx, and finally the exocytosis of insulin granules. However, it has been demonstrated by the inactivation of this pathway using genetic and pharmacological means that closure of the KATP channel alone may not be sufficient to explain all ß-cell responses to glucose elevation. We have previously proposed that NAADP-evoked Ca2+ release is an important step in stimulus-secretion coupling in pancreatic ß-cells. Here we show using total internal reflection fluorescence (TIRF) microscopy that glucose as well as the Ca2+ mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP), known to operate in ß-cells, lead to highly localised elementary intracellular Ca2+ signals. These were found to be obscured by measurements of global Ca2+ signals and the action of powerful SERCA-based sequestration mechanisms at the endoplasmic reticulum (ER). Building on our previous work demonstrating that NAADP-evoked Ca2+ release is an important step in stimulus-secretion coupling in pancreatic ß-cells, we provide here the first demonstration of elementary Ca2+ signals in response to NAADP, whose occurrence was previously suspected. Optical quantal analysis of these events reveals a unitary event amplitude equivalent to that of known elementary Ca2+ signalling events, inositol trisphosphate (IP3) receptor mediated blips, and ryanodine receptor mediated quarks. We propose that a mechanism based on these highly localised intracellular Ca2+ signalling events mediated by NAADP may initially operate in ß-cells when they respond to elevations in blood glucose.

Microplastics as contaminants in the soil environment: A mini-review.

Microplastics (MPs) have become a global environmental concern because of their ubiquitous presence. While extensive microplastic researches have focused on the marine environment, pervasive MPs contamination in soil and their detrimental impacts have been largely overlooked. Excessive concentrations of MPs and additives have been found in soil derived from the use of plastic mulches and the application of sewage sludge to fields. They may pose directly or indirectly as adverse effects on flora and fauna. The objectives of this review are (1) to summarize the abundance, sources, and properties of MPs in soil; (2) to analyze combined effects of MPs and various other environmental pollutants on soil system; and (3) to discuss the possible risks posed by MPs to soil biodiversity, food safety and human health. This review will highlight key future research areas for scientists and policymakers, and increase overall understanding of soil MPs pollution and its potential environmental impacts.

A human ventricular myocyte model with a refined representation of excitation-contraction coupling.

Cardiac Ca(2+)-induced Ca(2+) release (CICR) occurs by a regenerative activation of ryanodine receptors (RyRs) within each Ca(2+)-releasing unit, triggered by the activation of L-type Ca(2+) channels (LCCs). CICR is then terminated, most probably by depletion of Ca(2+) in the junctional sarcoplasmic reticulum (SR). Hinch et al. previously developed a tightly coupled LCC-RyR mathematical model, known as the Hinch model, that enables simulations to deal with a variety of functional states of whole-cell populations of a Ca(2+)-releasing unit using a personal computer. In this study, we developed a membrane excitation-contraction model of the human ventricular myocyte, which we call the human ventricular cell (HuVEC) model. This model is a hybrid of the most recent HuVEC models and the Hinch model. We modified the Hinch model to reproduce the regenerative activation and termination of CICR. In particular, we removed the inactivated RyR state and separated the single step of RyR activation by LCCs into triggering and regenerative steps. More importantly, we included the experimental measurement of a transient rise in Ca(2+) concentrations ([Ca(2+)], 10-15 µM) during CICR in the vicinity of Ca(2+)-releasing sites, and thereby calculated the effects of the local Ca(2+) gradient on CICR as well as membrane excitation. This HuVEC model successfully reconstructed both membrane excitation and key properties of CICR. The time course of CICR evoked by an action potential was accounted for by autonomous changes in an instantaneous equilibrium open probability of couplons. This autonomous time course was driven by a core feedback loop including the pivotal local [Ca(2+)], influenced by a time-dependent decay in the SR Ca(2+) content during CICR.

Expression of Ca²âº-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells.

The second messenger NAADP triggers Ca(2+) release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2(-/-)), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca(2+) responses as assessed by single-cell Ca(2+) imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2(-/-) cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca(2+)-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca(2+) release. High-affinity [(32)P]NAADP binding still occurs in Tpcn1/2(-/-) tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca(2+)-permeable channels indispensable for NAADP signalling.

TBI 13 years on: factors associated with post-traumatic growth.

PURPOSE: To investigate factors associated with post-traumatic growth (PTG) 13 years after severe traumatic brain injury (TBI) and to see if PTG had remained consistent between 11 and 13 years after injury. METHOD: TBI survivors (n=21), were interviewed and completed face-to face administration of questionnaires measuring PTG and factors potentially associated with PTG. The design was a longitudinal follow-up study. RESULTS: Factors significantly associated with PTG included: having a sense of personal meaning (purpose and coherence), high life satisfaction now, social support, high-activity levels, a high number of life events, having paid work, new stable relationships after injury, milder disability, and having religious faith. Having a high level of "purpose" was the best predictor of PTG. There was no change in PTG between 11 and 13 years after injury suggesting PTG is a relatively stable phenomenon once established after the early years. CONCLUSION: Clinicians should be aware of PTG and how it is associated with factors such as "meaning" and "purpose" as well as demographic factors such as, social support, activity such as work, new and stable relationships, milder disability and a shift towards spiritual values. Clinicians can focus advice, resource and effort on supporting these developments. [ IMPLICATIONS FOR REHABILITATION: • People with TBI do perceive benefits or post-traumatic growth (PTG), after time, which once established remains stable.• Factors such as having a sense of "meaning" and "purpose", are predictors of PTG.• Social support, activity such as work, new and stable relationships, a shift toward spiritual values, and milder disability are also associated with PTG.• Clinicians can focus effort into supporting these developments.]

Analyzing electrical activities of pancreatic ß cells using mathematical models.

Bursts of repetitive action potentials are closely related to the regulation of glucose-induced insulin secretion in pancreatic ß cells. Mathematical studies with simple ß-cell models have established the central principle that the burst-interburst events are generated by the interaction between fast membrane excitation and slow cytosolic components. Recently, a number of detailed models have been developed to simulate more realistic ß cell activity based on expanded findings on biophysical characteristics of cellular components. However, their complex structures hinder our intuitive understanding of the underlying mechanisms, and it is becoming more difficult to dissect the role of a specific component out of the complex network. We have recently developed a new detailed model by incorporating most of ion channels and transporters recorded experimentally (the Cha-Noma model), yet the model satisfies the charge conservation law and reversible responses to physiological stimuli. Here, we review the mechanisms underlying bursting activity by applying mathematical analysis tools to representative simple and detailed models. These analyses include time-based simulation, bifurcation analysis and lead potential analysis. In addition, we introduce a new steady-state I-V (ssI-V) curve analysis. We also discuss differences in electrical signals recorded from isolated single cells or from cells maintaining electrical connections within multi-cell preparations. Towards this end, we perform simulations with our detailed pancreatic ß-cell model.

Mitofusin 2 regulates STIM1 migration from the Ca2+ store to the plasma membrane in cells with depolarized mitochondria.

Store-operated Ca2+ channels in the plasma membrane (PM) are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER) and constitute a widespread and highly conserved Ca2+ influx pathway. After store emptying, the ER Ca2+ sensor STIM1 forms multimers, which then migrate to ER-PM junctions where they activate the Ca2+ release-activated Ca2+ channel Orai1. Movement of an intracellular protein to such specialized sites where it gates an ion channel is without precedence, but the fundamental question of how STIM1 migrates remains unresolved. Here, we show that trafficking of STIM1 to ER-PM junctions and subsequent Ca2+ release-activated Ca2+ channel activity is impaired following mitochondrial depolarization. We identify the dynamin-related mitochondrial protein mitofusin 2, mutations of which causes the inherited neurodegenerative disease Charcot-Marie-Tooth IIa in humans, as an important component of this mechanism. Our results reveal a molecular mechanism whereby a mitochondrial fusion protein regulates protein trafficking across the endoplasmic reticulum and reveals a homeostatic mechanism whereby mitochondrial depolarization can inhibit store-operated Ca2+ entry, thereby reducing cellular Ca2+ overload.

Post-traumatic growth after head injury: a long-term follow-up.

PRIMARY OBJECTIVE: To investigate the time course of any positive psychological changes after traumatic brain injury (TBI) by comparing questionnaire responses in two groups of TBI survivors, early, 1-3 years post-injury and late, 10-12 years post-injury. RESEARCH DESIGN: A cross-sectional, between-group design. METHOD AND PROCEDURE: TBI survivors were selected from the Reading Head Injury out-patient records by date of registration, early survivors from 2002-2004 and late survivors from 1993-1996 and sent a battery of postal questionnaires. These included The Post-traumatic Growth Inventory, The Life Satisfaction Checklist and The Hospital Anxiety and Depression Scale. Individuals were also given the opportunity to answer questions about their perception of their condition and to provide free text responses to reflect best and worst advice received, to describe positive and negative changes in themselves and to identify their most useful coping strategies. RESULTS: 61 'early' and 65 'late' people were contacted with study details and 52 consented to the study, receiving questionnaires which provided 23 'early' and 25 'late' responses. There was a significantly greater degree of post-traumatic growth, as measured on 'The Post-traumatic Growth Inventory', reported by the later group (*p = 0.000), but no other significant differences between the groups in terms of all the other variables. Both groups reported greater life satisfaction pre-injury. CONCLUSION: Measures of Post-traumatic Growth (Relating to Others, Personal Strength, New Possibilities, Appreciation of Life and Spirituality) appear to increase over time after head injury. This is a positive message that clinicians should note and reinforce and could help to shape future adjustment.

The effect of deoxygenation on whole-cell conductance of red blood cells from healthy individuals and patients with sickle cell disease.

Red blood cells from patients with sickle cell disease (SCD) exhibit increased electrogenic cation permeability, particularly following deoxygenation and hemoglobin (Hb) polymerisation. This cation permeability, termed P(sickle), contributes to cellular dehydration and sickling, and its inhibition remains a major goal for SCD treatment. Nevertheless, its characteristics remain poorly defined, its molecular identity is unknown, and effective inhibitors have not been established. Here, patch-clamp methodology was used to record whole-cell currents in single red blood cells from healthy individuals and patients with SCD. Oxygenated normal red blood cells had a low membrane conductance, unaffected by deoxygenation. Oxygenated HbS cells had significantly increased conductance and, on deoxygenation, showed a further rise in membrane conductance. The deoxygenation-induced pathway was variable in magnitude. It had equal permeability to Na(+) and K(+), but was less permeable to NMDG(+) and Cl(-). Conductance to Ca(2+) was also of a similar magnitude to that of monovalent cations. It was inhibited by DIDS (100 microM), Zn(2+) (100 microM), and by Gd(3+) (IC(50) of approximately 2 microM). It therefore shares some properties with P(sickle). These findings represent the first electrical recordings of single HbS cells and will facilitate progress in understanding altered red blood cell cation transport characteristics of SCD.

Electrophysiological demonstration of voltage- activated H+ channels in bovine articular chondrocytes.

Matrix synthesis by articular chondrocytes is sensitive to changes in intracellular pH (pH(i)), so characterising the membrane transport pathways that determine pH(i) is important for understanding how chondrocytes regulate the turnover of cartilage matrix. In the present study, the whole-cell patch-clamp technique has been employed to demonstrate the operation of voltage-activated H(+) channels (VAHC) in bovine articular chondrocytes. Using solutions designed to minimise the contribution of ions other than H(+), the application of step voltage-protocols elicited whole-cell currents. These currents were slow activating, observed only in the outward direction, dependent on both extracellular pH (pH(o)) and pH(i), and inhibited by Zn(2+). The reversal potential values, measured by tail current analysis, over a range of different pHo and pHi values, were in good agreement with predicted values for membrane channels having a high selectivity for protons. The results presented here are consistent with the operation of VAHC in articular chondrocytes.

Solute transport via the new permeability pathways in Plasmodium falciparum-infected human red blood cells is not consistent with a simple single-channel model.

After infection of a red blood cell (RBC), the malaria parasite, Plasmodium falciparum, increases the permeability of the host's plasma membrane by inducing new permeability pathways (NPPs). Single-channel patch-clamp experiments have shown the presence in infected RBCs of novel anion-selective channel types with low open-state probabilities at positive membrane potentials. These channels have been postulated to form the NPPs. Here, we have used a range of transport techniques to study whether electroneutral solutes use these channels or altered/separate pathways. Transport of the electroneutral solute sorbitol via the NPPs was found to increase by a small but significant amount after gross membrane depolarization. This is inconsistent with transport via a channel with a reduced open-state probability at positive membrane potentials. As has been demonstrated previously for parasite-induced anion currents, sorbitol transport in infected RBCs was found to be sensitive to the presence of bovine serum albumin (BSA). However, it remains to be shown whether the effect is due to serum/BSA altering a single channel type or activating a new pathway. In addition, the study highlights problems that can occur when using different transport techniques to study the NPPs.

Electrophysiological demonstration of Na+/Ca2+ exchange in bovine articular chondrocytes.

Altered fluxes of Ca2+ across the chondrocyte membrane have been proposed as one pathway by which mechanical load can modulate cartilage turnover. In many cells, Na+/Ca2+ exchange (NCX) plays a key role in Ca2+ homeostasis, and recent studies have suggested it is operative in articular chondrocytes. In this study, an electrophysiological characterisation of NCX in articular bovine chondrocytes has been performed, using the whole-cell patch clamp technique, and the effects of inhibitors and the transmembrane electrochemical gradients of Na+ and Ca2+ on NCX function have been assessed. A Ni2+-sensitive current (I(NCX)) which exhibited outward rectification, was elicited by a voltage ramp protocol. The current was also attenuated by the NCX inhibitors benzamil and KBR7943, without significant differences between the effect of these two compounds upon outward and inward currents. The Ni2+-sensitive current was modulated by changes in extracellular and pipette Na+ and Ca2+ in a manner characteristic of I(NCX). Measured values for the reversal potential differed significantly from those predicted for an exchanger stoichiometry of 3Na+ : 1Ca2+, implying that accumulation of intracellular Ca2+ (from influx or release from stores) or more than one transport mode is occurring. These results demonstrate the operation of NCX in articular chondrocytes and suggest that changes in its turnover rate, as might occur in response to mechanical load, may modify cell composition and thereby dictate cartilage turnover.

Plasmodium falciparum-induced channels.

To survive within a red blood cell, the malaria parasite alters dramatically the permeability of the host's plasma membrane (allowing the uptake of essential nutrients and the removal of potentially hazardous metabolites). The pathway(s) responsible for the increased permeability have been proposed as putative chemotherapeutic targets and/or selective routes for antimalarial agents that target the internal parasite. This review covers our current understanding of this parasite-induced phenomenon in Plasmodium falciparum-infected human red blood cells. In particular, recent electrophysiological studies, using the patch-clamp technique, are reviewed.

Modulation of whole-cell currents in Plasmodium falciparum-infected human red blood cells by holding potential and serum.

Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum-infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole-cell configuration of the patch-clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole-cell recordings in malaria-infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4-fold and inward currents by approximately 2-fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.