Alter Ion Fluxes Research Articles

Mechanisms controlling plant proteases and their substrates.

In plants, proteolysis is emerging as an important field of study due to a growing understanding of the critical involvement of proteases in plant cell death, disease and development. Because proteases irreversibly modify the structure and function of their target substrates, proteolytic activities are stringently regulated at multiple levels. Most proteases are produced as dormant isoforms and only activated in specific conditions such as altered ion fluxes or by post-translational modifications. Some of the regulatory mechanisms initiating and modulating proteolytic activities are restricted in time and space, thereby ensuring precision activity, and minimizing unwanted side effects. Currently, the activation mechanisms and the substrates of only a few plant proteases have been studied in detail. Most studies focus on the role of proteases in pathogen perception and subsequent modulation of the plant reactions, including the hypersensitive response (HR). Proteases are also required for the maturation of coexpressed peptide hormones that lead essential processes within the immune response and development. Here, we review the known mechanisms for theactivationof plant proteases, including post-translational modifications, together with the effects of proteinaceous inhibitors.

Fluorescent nano- and microparticles for sensing cellular microenvironment: past, present and future applications.

The tumor microenvironment (TME) demonstrates distinct hallmarks, including acidosis, hypoxia, reactive oxygen species (ROS) generation, and altered ion fluxes, which are crucial targets for early cancer biomarker detection, tumor diagnosis, and therapeutic strategies. Various imaging and sensing techniques have been developed and employed in both research and clinical settings to visualize and monitor cellular and TME dynamics. Among these, ratiometric fluorescence-based sensors have emerged as powerful analytical tools, providing precise and sensitive insights into TME and enabling real-time detection and tracking of dynamic changes. In this comprehensive review, we discuss the latest advancements in ratiometric fluorescent probes designed for the optical mapping of pH, oxygen, ROS, ions, and biomarkers within the TME. We elucidate their structural designs and sensing mechanisms as well as their applications in in vitro and in vivo detection. Furthermore, we explore integrated sensing platforms that reveal the spatiotemporal behavior of complex tumor cultures, highlighting the potential of high-resolution imaging techniques combined with computational methods. This review aims to provide a solid foundation for understanding the current state of the art and the future potential of fluorescent nano- and microparticles in the field of cellular microenvironment sensing.

Voltage-dependent activation of Rac1 by Nav 1.5 channels promotes cell migration.

Ion channels can regulate the plasma membrane potential (Vm) and cell migration as a result of altered ion flux. However, the mechanism by which Vm regulates motility remains unclear. Here, we show that the Nav1.5 sodium channel carries persistent inward Na+ current which depolarizes the resting Vm at the timescale of minutes. This Nav1.5‐dependent Vm depolarization increases Rac1 colocalization with phosphatidylserine, to which it is anchored at the leading edge of migrating cells, promoting Rac1 activation. A genetically encoded FRET biosensor of Rac1 activation shows that depolarization‐induced Rac1 activation results in acquisition of a motile phenotype. By identifying Nav1.5‐mediated Vm depolarization as a regulator of Rac1 activation, we link ionic and electrical signaling at the plasma membrane to small GTPase‐dependent cytoskeletal reorganization and cellular migration. We uncover a novel and unexpected mechanism for Rac1 activation, which fine tunes cell migration in response to ionic and/or electric field changes in the local microenvironment.

TRPV4 Activation ‐ Implications in Hydrocephalic Neurodegenerative Disease

Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and traumatic brain injury, often share similar pathologies, one of which is hydrocephalus. Hydrocephalus is characterized by an imbalance in cerebrospinal fluid (CSF) production and/or reabsorption or by a blockage in the CSF circulation resulting in enlarged ventricles and an increase in brain hydrostatic pressure leading to neuronal destruction. The choroid plexus (CP) is the main producer of CSF and is composed of a high resistance barrier epithelium that surrounds a network of capillaries. The CP epithelium regulates the transport of ions and water between the ventricles and capillaries, thus controlling the production of CSF. Transient Receptor Potential Vanilloid‐4 (TRPV4) is a mechano‐ and osmo‐sensitive, cation channel present in CP epithelia. TRPV4 activation leads to an influx of calcium, which can stimulate calcium activated ion channels leading to the potential for significant transepithelial ion flux. If TRPV4 is overstimulated, it is possible that excess ion movement can cause an increase in CSF volume, resulting in hydrocephalus. Current research in our laboratory has shown that treatment with a TRPV4 antagonist ameliorates hydrocephalus in the Wpk rodent model. We have also shown that TRPV4 expression is increased in the late‐stage hydrocephalic animal compared to normal. TRPV4 can be activated through a number of stimuli, including physical means osmotic changes, increases in temperature and chemical activators, such as cytokines and inflammatory mediators. In particular, the latter activators can be seen in the symptomologies of neurodegenerative diseases exhibiting hydrocephalus. Utilizing a high resistance porcine choroid plexus (PCP‐R) cell line, we investigated the effects of various cytokines, inflammatory mediators, and inflammation pathways on TRPV4 activity using immunohistochemistry and electrophysiology techniques. We found that pro‐inflammatory cytokines, such as Tumor Necrosis Factor (TNF)‐α and interleukin (IL)‐1β, had no effect on TRPV4 stimulated ion flux in vitro; pro‐inflammatory mediators, arachidonic acid and its metabolite 5,6‐epoxyeicosatrienoic acid (EET), had inhibitory effects. Furthermore, the hormone epinephrine, used to combat inflammatory anaphylactic reactions, potentiated TRPV4 activity. Immunofluorescence of the kinase SPAK, which plays a role in Nuclear Factor (NF)‐κB – dependent inflammation and epithelial barrier function, was increased in the CP of hydrocephalic rats compared to the wild type, and inhibitors of NF‐kB altered TRPV4‐mediated ion flux. Together these data imply that selected components of an inflammatory response can play a role in TRPV4 activation and can potentially contribute to the subsequent development of hydrocephalus.Support or Funding InformationDepartment of Defense office of the Congressionally Directed Medical Research Programs (CDMRP) and an Indiana Clinical and Translational Sciences AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Altered swelling and ion fluxes in articular cartilage as a biomarker in osteoarthritis and joint immobilization: a computational analysis.

In healthy cartilage, mechano-electrochemical phenomena act together to maintain tissue homeostasis. Osteoarthritis (OA) and degenerative diseases disrupt this biological equilibrium by causing structural deterioration and subsequent dysfunction of the tissue. Swelling and ion flux alteration as well as abnormal ion distribution are proposed as primary indicators of tissue degradation. In this paper, we present an extension of a previous three-dimensional computational model of the cartilage behaviour developed by the authors to simulate the contribution of the main tissue components in its behaviour. The model considers the mechano-electrochemical events as concurrent phenomena in a three-dimensional environment. This model has been extended here to include the effect of repulsion of negative charges attached to proteoglycans. Moreover, we have studied the fluctuation of these charges owning to proteoglycan variations in healthy and pathological articular cartilage. In this sense, standard patterns of healthy and degraded tissue behaviour can be obtained which could be a helpful diagnostic tool. By introducing measured properties of unhealthy cartilage into the computational model, the severity of tissue degeneration can be predicted avoiding complex tissue extraction and subsequent in vitro analysis. In this work, the model has been applied to monitor and analyse cartilage behaviour at different stages of OA and in both short (four, six and eight weeks) and long-term (11 weeks) fully immobilized joints. Simulation results showed marked differences in the corresponding swelling phenomena, in outgoing cation fluxes and in cation distributions. Furthermore, long-term immobilized patients display similar swelling as well as fluxes and distribution of cations to patients in the early stages of OA, thus, preventive treatments are highly recommended to avoid tissue deterioration.

Hypoxia‐inducible factor signalling mechanisms in the central nervous system

In the CNS, neurones are highly sensitive to the availability of oxygen. In conditions where oxygen availability is decreased, neuronal function can be altered, leading to injury and cell death. Hypoxia has been implicated in a number of central nervous system pathologies including stroke, head trauma and neurodegenerative diseases. Cellular responses to oxygen deprivation are complex and result in activation of short- and long-term mechanisms to conserve energy and protect cells. Failure of synaptic transmission can be observed within minutes following this hypoxia. The acute effects of hypoxia on synaptic transmission are primarily mediated by altering ion fluxes across membranes, pre-synaptic effects of adenosine and other actions at glutamatergic receptors. A more long-term feature of the response of neurones to hypoxia is the activation of transcription factors such as hypoxia-inducible factor. The activation of hypoxia-inducible factor is governed by a family of dioxygenases called hypoxia-inducible factor prolyl 4 hydroxylases (PHDs). Under hypoxic conditions, PHD activity is inhibited, thereby allowing hypoxia-inducible factor to accumulate and translocate to the nucleus, where it binds to the hypoxia-responsive element sequences of target gene promoters. Inhibition of PHD activity stabilizes hypoxia-inducible factor and other proteins thus acting as a neuroprotective agent. This review will focus on the response of neuronal cells to hypoxia-inducible factor and its targets, including the prolyl hydroxylases. We also present evidence for acute effects of PHD inhibition on synaptic transmission and plasticity in the hippocampus.

Flufenamic acid promotes angiogenesis through AMPK activation

Angiogenesis plays critical roles in development, tumor growth and metastasis. Flufenamic acid (FFA) is an anti-inflammatory agent known to alter ion fluxes across the plasma membrane. Its role in angiogenesis has not been fully addressed to date. Here, we report that FFA treatment promotes angiogenesis both in vitro and in vivo. Applying FFA for 12 h promoted tube formation of human umbilical vein endothelial cells (HUVECs) without affecting cell proliferation. Three angiogenesis-related genes, VEGF, e-NOS and AAMP, were analyzed by RT-PCR. A significant difference was found between the FFA group and the control; the FFA group had significantly higher mRNA accumulation levels of all the three genes (p<0.05). Moreover, in the chick embryo chorioallantoic membrane (CAM) assay, FFA promoted the formation of macroscopic blood vessels. Finally, western blotting showed that the FFA-treated group had significantly higher phosphorylated AMPK levels, compared with the control (p<0.05). These results suggest that FFA promotes angiogenesis both in vitro and in vivo likely via promoting tube formation through AMPK activation.

Cross-talk between abscisic acid-dependent and abscisic acid-independent pathways during abiotic stress

Salinity, drought and low temperature are the common forms of abiotic stress encountered by land plants. To cope with these adverse environmental factors, plants execute several physiological and metabolic responses. Both osmotic stress (elicited by water deficit or high salt) and cold stress increase the endogenous level of the phytohormone abscisic acid (ABA). ABA-dependent stomatal closure to reduce water loss is associated with small signaling molecules like nitric oxide, reactive oxygen species and cytosolic free calcium, and mediated by rapidly altering ion fluxes in guard cells. ABA also triggers the expression of osmotic stress-responsive (OR) genes, which usually contain single/multiple copies of cis-acting sequence called abscisic acid-responsive element (ABRE) in their upstream regions, mostly recognized by the basic leucine zipper-transcription factors (TFs), namely, ABA-responsive element-binding protein/ABA-binding factor. Another conserved sequence called the dehydration-responsive element (DRE)/C-repeat, responding to cold or osmotic stress, but not to ABA, occurs in some OR promoters, to which the DRE-binding protein/C-repeat-binding factor binds. In contrast, there are genes or TFs containing both DRE/CRT and ABRE, which can integrate input stimuli from salinity, drought, cold and ABA signaling pathways, thereby enabling cross-tolerance to multiple stresses. A strong candidate that mediates such cross-talk is calcium, which serves as a common second messenger for abiotic stress conditions and ABA. The present review highlights the involvement of both ABA-dependent and ABA-independent signaling components and their interaction or convergence in activating the stress genes. We restrict our discussion to salinity, drought and cold stress.

Exposure of colonic epithelial cells to oxidative and endoplasmic reticulum stress causes rapid potassium efflux and calcium influx

Endoplasmic reticulum (ER) stress and oxidative stress have recently been linked to the pathogenesis of inflammatory bowel diseases. Under physiological conditions, intestinal epithelial cells are exposed to ER and oxidative stress affecting the cellular ionic homeostasis. However, these altered ion flux 'signatures' during these stress conditions are poorly characterized. We investigated the kinetics of K(+) , Ca(2+) and H(+) ion fluxes during ER and oxidative stress in a colonic epithelial cell line LS174T using a non-invasive microelectrode ion flux estimation technique. ER and oxidative stress were induced by cell exposure to tunicamycin (TM) and copper ascorbate (CuAsc), respectively, from 1 to 24 h. Dramatic K(+) efflux was observed following acute ER stress with peak K(+) efflux being -30·6 and -138·7 nmolm(-2) s(-1) for 10 and 50 µg ml(-1) , respectively (p < 0·01). TM-dependent Ca(2+) uptake was more prolonged with peak values of 0·85 and 2·68 nmol m(-2) s(-1) for 10 and 50 µg ml(-1) TM, respectively (p < 0·02). Ion homeostasis was also affected by the duration of ER stress. Increased duration of TM treatment from 0 to 18 h led to increases in both K(+) efflux and Ca(2+) uptake. While K(+) changes were significantly higher at each time point tested, Ca(2+) uptake was significantly higher only after prolonged treatment (18 h). CuAsc also led to an increased K(+) efflux and Ca(2+) uptake. Functional assays to investigate the effect of inhibiting K(+) efflux with tetraethylammonium resulted in increased cell viability. We conclude that ER/oxidative stress in colonic epithelial cells cause dramatic K(+) , Ca(2+) and H(+) ion flux changes, which may predispose this lineage to poor stress recovery reminiscent of that seen in inflammatory bowel diseases.

The anti-inflammatory agent flufenamic acid depresses store-operated channels by altering mitochondrial calcium homeostasis

Fenamates like flufenamic acid (FFA) are anti-inflammatory drugs known to alter ion fluxes through the plasma membrane. They are for instance potent blockers of cation and anion channels, and FFA is now commonly used to block currents through TRP channels and receptor-operated channels. However, FFA exerts complex and multifaceted actions on ion transport systems and, in most instances, a molecular understanding of these FFA-dependent modulations is lacking. In addition, FFA is also to known to perturb the homeostasis of Ca 2+. In the present report, we investigated whether the FFA-induced alterations of the Ca 2+ homeostasis could play a role in the FFA-dependent modulation of transmembrane ion fluxes. Experiments performed with the Ca 2+ indicator Fluo-4 on cultured cortical neurons and HEK-293 cells showed that FFA increased the cytosolic concentration of Ca 2+ even in cells kept in a Ca 2+-free medium or when the endoplasmic reticulum was depleted with thapsigargin. The FFA-dependent Ca 2+ responses were, however, strongly reduced by bongkrekic acid, a specific ligand of the mitochondrial ADP/ATP carrier which, in addition, inhibits the permeability transition pore. Like FCCP, FFA released Ca 2+ from isolated brain mitochondria and indirectly modulates store-operated Ca 2+ channels. We suggest that some of the effects of FFA on plasma membrane ion channels could be explained, at least partially, by its ability to modulate the mitochondrial Ca 2+ homeostasis.

Chloroquine uptake, altered partitioning and the basis of drug resistance: evidence for chloride-dependent ionic regulation.

The biochemical mechanism of chloroquine resistance in Plasmodium falciparum remains unknown. We postulated that chloroquine-resistant strains could alter ion fluxes that then indirectly control drug accumulation within the parasite by affecting pH and/or membrane potential ('altered partitioning mechanism'). Two principal intracellular pH-regulating systems in many cell types are the amiloride-sensitive Na+/H+ exchanger (NHE), and the sodium-independent, stilbene-sensitive Cl-/HCO3- antiporter (AE). We report that under physiological conditions (balanced CO2 and HCO3-) chloroquine uptake and susceptibility are not altered by amiloride analogues. We also do not detect a significant difference in NHE activity between chloroquine-sensitive and chloroquine-resistant strains via single cell photometry methods. AE activity is dependent on the intracellular and extracellular concentrations of Cl- and HCO3- ions. Chloroquine-resistant strains differentially respond to experimental modifications in chloride-dependent homeostasis, including growth, cytoplasmic pH and pH regulation. Chloroquine susceptibility is altered by stilbene DIDS only on chloroquine-resistant strains. Our results suggest that a Cl(-)-dependent system (perhaps AE) has a significant effect on the uptake of chloroquine by the infected erythrocyte, and that alterations of this biophysical parameter may be part of the mechanism of chloroquine resistance in P. falciparum.

ClC‐3: more than just a volume‐sensitive Cl− channel

Pulmonary vascular medial hypertrophy due to enhanced pulmonary artery smooth muscle cell (PASMC) proliferation and/or decreased PASMC apoptosis is a primary cause of increased pulmonary vascular resistance and arterial pressure in patients with pulmonary arterial hypertension. While many factors can contribute to this form of vascular remodeling, it is generally agreed upon that altered transmembrane ion flux via ion channels is involved. While much focus has centered on the role of cations and cation channels in controlling PASMC contraction and proliferation, anion efflux via Cl- channels has recently gained interest for its role in SMC proliferation, differentiation, migration, contraction, and angiogenesis. In this issue, Dai et al. report that the putative volume-sensitive ClC-3 channel is upregulated in PASMC from monocrotaline-induced pulmonary hypertensive rats and in inflammatory cytokine-treated canine PASMC. They also provide evidence that ClC-3 upregulation may protect against oxidative stress-induced PASMC necrosis, thereby improving PASMC survival and promoting medial hypertrophy.

Perioperative Stroke Prevention

The definitions of commonly used terms are given in Table 1 (1–8). Stroke has been simply defined as any form of cerebral infarction (2), which includes those attributable to hemorrhage. The latter group is more likely to occur as a complication of specific surgical procedures (e.g., carotid endarterectomy) or the use of anticoagulants and is otherwise rare in the perioperative period (3). Hence, much of the information in this review pertains to ischemic cerebral events.

Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity.

In guard cells, the plant hormone abscisic acid (ABA) inhibits stomatal opening and induces stomatal closure through the coordinated regulation of ion transport. Despite this central role of ABA in regulating stomatal function, the signal transduction events leading to altered ion fluxes remain incompletely understood. We report that the activity of the enzyme phospholipase D (PLD) transiently increased in guard cell protoplasts at 2.5 and 25 min after ABA application. Treatment of guard cell protoplasts with phosphatidic acid (PtdOH), one of the products of PLD activity, led to an inhibition of the activity of the inward K+ channel. PtdOH also induced stomatal closure and inhibited stomatal opening when added to epidermal peels. Application of 1-butanol (1-buOH), a selective inhibitor of PtdOH production by PLD, inhibited the increase in PtdOH production elicited by ABA. 1-BuOH treatment also partially prevented ABA-induced stomatal closure and ABA-induced inhibition of stomatal opening. This inhibitory effect of buOH was enhanced by simultaneous application of nicotinamide, an inhibitor of cADP ribose action. These results suggest that in the guard cell, ABA activates the enzyme PLD, which leads to the production of PtdOH. This PtdOH is then involved in triggering subsequent ABA responses of the cell via a pathway operating in parallel to cADP ribose-mediated events.

The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins

In incompatible host-pathogen interactions, damage caused by the pathogen remains restricted as a result of the plant's defensive response. Most effective is the hypersensitive reaction, in which the cells around the infection site rapidly necrose. This response is associated with a coordinated and integrated set of metabolic alterations that are instrumental in impeding further pathogen ingress, as well as in enhancing the capacity of the host to limit subsequent infection by different types of pathogens [27, 77]. Altered ion fluxes across the plant cell membrane, generation of active oxygen species, changes in the phosphorylation state of regulatory proteins and transcriptional activation of plant defense systems culminate in cell death at the site of infection, local accumulation of phytoalexins and cell wall rigidification as a result of callose, lignin and suberin deposition [31, 89]. In addition, various novel proteins are induced which are collectively referred to as ``pathogenesis-related proteins '' (PRs). These PRs, defined as proteins coded for by the host plant but induced specifically in pathological or related situations [4, 81], do not only accumulate locally in the infected leaf, but are also induced systemically, associated with the development of systemic acquired resistance (SAR) against further infection by fungi, bacteria and viruses. Induction of PRs has been found in many plant species belonging to various families [78], suggestive of a general role for these proteins in adaptation to biotic stress conditions. SAR, likewise, is a generally occurring phenomenon, that engenders an enhancement of the defensive capacity of plants in response to necrotizing infections [70]. Since some of the tobacco PRs were identified as chitinases [45] and b-1,3-glucanases [38] with potential antifungal activity, it has often been suggested that the collective set of PRs may be effective in inhibiting pathogen growth, multiplication and/or spread, and be responsible for the state of SAR [42, 65]. Originally, five main classes of PRs (PR-1-5) were characterized by both biochemical and molecular-biological techniques in tobacco [9, 80]. Thereupon, in 1994 a unifying nomenclature for PRs was proposed based on their grouping into families sharing amino acid sequences, serological relationship, and/or enzymatic or biological activity. By then 11 families (PR-1--11) were recognized and classified for tobacco and tomato [81] (cf. Table 1). Criteria used for the inclusion of new families of PRs were that (i) protein(s) must be induced by a pathogen in tissues that do not normally express the protein(s), and (ii) induced expression must have been shown to occur in at least two different plant-pathogen combinations, or expression in a single plant-pathogen combination must have been confirmed independently in diaerent laboratories.

ABSCISIC ACID SIGNAL TRANSDUCTION.

The plant hormone abscisic acid (ABA) plays a major role in seed maturation and germination, as well as in adaptation to abiotic environmental stresses. ABA promotes stomatal closure by rapidly altering ion fluxes in guard cells. Other ABA actions involve modifications of gene expression, and the analysis of ABA-responsive promoters has revealed a diversity of potential cis-acting regulatory elements. The nature of the ABA receptor(s) remains unknown. In contrast, combined biophysical, genetic, and molecular approaches have led to considerable progress in the characterization of more downstream signaling elements. In particular, substantial evidence points to the importance of reversible protein phosphorylation and modifications of cytosolic calcium levels and pH as intermediates in ABA signal transduction. Exciting advances are being made in reassembling individual components into minimal ABA signaling cascades at the single-cell level.

Phospholipid subclass specific alterations in the passive ion permeability of membrane bilayers: separation of enthalpic and entropic contributions to transbilayer ion flux.

Alterations in phospholipid class, subclass, and individual molecular species contribute to the diversity of biologic membranes, but their effects on membrane passive ion permeability have not been systematically studied. Herein, we developed a simple and efficient fluorescence technique based upon the loss of valinomycin-inducible membrane potential to characterize the passive flux of ions across phospholipid bilayers. Detailed kinetic characterization of ion flux across membrane bilayers composed of discrete chemical entities demonstrated that the class, subclass, and individual molecular species of each phospholipid have substantive effects on membrane passive ion permeability properties. Increasing the degree of unsaturation in either the sn-1 or sn-2 aliphatic chains in phosphatidylcholine markedly enhanced transmembrane ion flux, with over 10-fold differences in the first-order rate constant manifested in molecular species containing four double bonds in comparison to those possessing three double bonds (e.g., kapp = 0.0014 min-1 for 1-octadec-9'-enoyl-2-octadec-9', 12'-dienoyl-sn-glycero-3-phosphocholine (18:1-18:2 phosphatidylcholine) while kapp = 0.021 min-1 for 1,2-dioctadec-9', 12'-dienoyl-sn-glycero-3-phosphocholine (18:2-18:2 phosphatidylcholine)). Moreover, although the apparent first-order rate constants for transmembrane ion flux in vesicles composed of phosphatidylcholine or plasmanylcholine containing palmitate at the sn-1 position and arachidonate at the sn-2 position were similar (kapp = 0.04 min-1 at 22 degreesC for both), the kapp for corresponding vesicles composed of plasmenylcholine was 20-fold less (kapp = 0.002 min-1 at 22 degreesC). Examination of the temperature dependence of passive membrane ion permeability demonstrated that altered ion flux across membranes composed of choline glycerophospholipids was primarily due to entropic effects without substantial changes in the activation energy for ion translocation. For example, Ea = 19.7 +/- 0.5 and 20.7 +/- 0.6 kcal.mol-1 for 1-hexadecanoyl-2-eicosa-5',8',11', 14'-tetraenoyl-sn-glycero-3-phosphocholine (16:0-20:4 phosphatidylcholine) and 1-O-(Z)-hexadec-1'-enyl-2-eicosa-5',8',11', 14'-tetraenoyl-sn-glycero-3-phosphocholine (16:0-20:4 plasmenylcholine), respectively, while their difference in the entropies of activation (DeltaS) was 4.3 +/- 0.5 cal.mol-1.K-1. Collectively, these results identify substantial differences in the membrane passive ion permeability properties of phospholipid classes, subclasses, and molecular species present in biologic membranes of eukaryotic cells and identify entropic alterations as an important contributor to these differences.

Ca2+-independent insulin exocytosis induced by alpha-latrotoxin requires latrophilin, a G protein-coupled receptor.

alpha-Latrotoxin (alpha-LTX) induces exocytosis of small synaptic vesicles (SSVs) in neuronal cells both by a calcium-independent mechanism and by opening cation-permeable pores. Since the basic molecular events regulating exocytosis in neurons and endocrine cells may be similar, we have used the exocytosis of insulin-containing large dense core vesicles (LDCVs) as a model system. In primary pancreatic beta-cells and in the derived cell lines INS-1 and MIN6, alpha-LTX increased insulin release in the absence of extracellular calcium, but the insulin-secreting cell lines HIT-T15 and RINm5F were unresponsive. alpha-LTX did not alter membrane potential or cytosolic calcium, and its stimulatory effect on exocytosis was still observed in pre-permeabilized INS-1 cells kept at 0.1 microM Ca2+. Consequently, pore formation or ion fluxes induced by alpha-LTX could be excluded. The Ca2+-independent alpha-LTX-binding protein, latrophilin, is a novel member of the secretin family of G protein-coupled receptors (GPCR). Sensitivity to alpha-LTX correlated with expression of latrophilin, but not with synaptotagmin I or neurexin Ialpha expression. Moreover, transient expression of latrophilin in HIT-T15 cells conferred alpha-LTX-induced exocytosis. Our results indicate that direct stimulation of exocytosis by a GPCR mediates the Ca2+-independent effects of alpha-LTX in the absence of altered ion fluxes. Therefore, direct regulation by receptor-activated heterotrimeric G proteins constitutes an important feature of the endocrine exocytosis of insulin-containing LDCVs and may also apply to SSV exocytosis in neurons.

Indapamide accentuates cardiac chronotropic responses to epidermal growth factor in chick cardiomyocytes

We have previously shown that indapamide [chloro-4-N-(methyl-2-indolinyl-1)-sulfamoyal-3-benzamide] has a direct action on the heart to alter ion fluxes. This study sought to examine the potential interaction between indapamide and epidermal growth factor (EGF). Cardiomyocytes were prepared as primary culture from 7-day-old chick embryo hearts as aggregates that have a pattern of consistent spontaneous contraction. Indapamide enhanced the positive chronotropic response to EGF observed in chick embryonic ventricular myocyte aggregates while indapamide itself did not alter cardiac contractile frequency. Taken in conjunction with data that calcium channel blockade, inhibition of sodium entry or Na +-Ca 2+ exchange in the cardiomyocyte opposes the positive chronotropic action of EGF on the cardiomyocyte, this study has identified an agent, indapamide, that accentuates the cardiomyocyte response to EGF.

Mechanical Basis of Cell Morphogenesis and Volume Control

Osmotic differences between the cytoplasm and the surrounding fluid belong to the most important factors in shaping an animal tissue cell. This implies an elastic and contractile fibrillar system attached to the cell membrane which counteracts ionic imbalances and determines cell morphology. Therefore cell volume and cell shape are functionally coupled. Many cell types, epithelial cells in particular, are able to control their volume: ie, swelling (or shrinking) induced by varying the osmolarity of the surrounding fluid is compensated and the previous volume is achieved during a time interval of 10-40 min. This compensation reaction includes the opening of ion channels, activation of the Na/K/2C1 co-transport mechanism, and activities of the cytoskeleton. The changes in ion fluxes have been investigated for various cell types. The main question which is still under debate refers to the signal involved in telling a cell to have reached its appropriate volume. One of the most probable factors is the tension in the cell membrane. By action of tension-sensitive ion channels (K+, Ca++-channels) ion fluxes could be modified. Mechanical forces acting on the cell membrane always affect the cortical cytoplasm - membrane complex: In many cell types a fibrillar meshwork is closely connected to the membrane, thus determining its mechanical properties. Destruction of this complex by drugs like cytochalasins alters the mechanics of the cortex and destroys the ability of the cells for volume regulation. If mechanical forces in the cortex are involved in volume control, the loss of this ability would be a reasonable consequence of cytochalasin treatment. Measurement of the forces at the surface of cells during swelling and during the compensation phase is a direct approach to the problem of volume regulation. Scanning acoustic microscopy (SAM) is an elegant method to determine elastic properties on a subcellular level. Measuring the reflection of sound at the surface of a cell gives a good indication of local elasticity (and thus “tension at the surface”). Using the cell line HaCat which was derived from human epidermis, elasticity changes have been followed by SAM during hypotonicity-induced swelling and the subsequent compensatory volume decrease. On the basis of the present results the regulatory volume decrease (RVD) may be achieved by an increase of forces in the cortex by actomyosin-based contractions in addition to altered ion fluxes. Volume increase first results in a destruction of the actin fibrillar system which is immediately restored by stimulated actin polymerisation which strengthens the complex of the plasma membran with the cortical fibrillar layer. The results support the view that tension in the cortex may participate in RVD and be a signal for volume regulation.