Evidence for opening of hair-cell transducer channels after tip-link loss

The mechanosensitive transducer channels of hair cells have long been proposed to be gated directly by tension in the tip links. These are thin, elastic extracellular elements connecting the tips of adjacent stereocilia located on the apical surface of the cell. If this hypothesis is true, the channels should close after destruction of tip links. The hypothesis was tested pharmacologically using receptor currents obtained in response to mechanical stimulation of the stereociliary bundle of outer hair cells isolated from the adult guinea pig cochlea. Application of elastase (20 U/ml) or 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid (BAPTA; 5 mM), both of which are known to disrupt tip links in other hair-cell preparations, led to the expected irreversible loss of receptor currents. However, the cells then displayed a maintained inward current, implying that channels were left permanently open. This current was similar in magnitude to the receptor current before treatment and was reduced reversibly by known blockers of mechanosensitive channels, namely, dihydrostreptomycin (100 microM), amiloride (300 microM), and gadolinium ions (1 mM). These observations suggest that the maintained current flows through the mechanosensitive channels. Electron microscopical analysis of isolated hair cells, exposed to the same concentrations of elastase or BAPTA as in the electrophysiological experiments, demonstrated an almost total loss of tip links in hair bundles that showed no evidence of other mechanical damage. It is concluded that although the tip links are required for mechanoelectrical transduction, the channels are not gated directly by the tip links.     

Pathophysiological mechanisms of hearing loss

An understanding of auditory transduction in the ear can contribute to a better comprehension of the pathophysiological mechanisms which give rise to hearing loss. The incoming sound sets up a mechanical traveling wave which begins at the base and progresses along the basilar membrane, reaching a point of maximal displacement. The region of maximal displacement is a function of stimulus frequency. The mechanical displacement, by directly opening ion channels in the stereocilia of the hair cells, induces changes in the electrical potential of the hair cells. This initial stage is called mechano-electrical transduction, and in the normal ear, is followed by a stage of electro-mechanical transduction based on the ability of the outer hair cells to respond to the electrical changes induced in them with a change in their length. This "electromotility" presumably provides mechanical feedback to the basilar membrane, augmenting its mechanical displacement. This is called the cochlear amplifier, providing the ear with improved sensitivity and frequency discrimination. Most forms of sensori-neural hearing losses (affecting the inner ear) are due to a lesion to some part of this cochlear amplifier (e.g. noise induced hearing loss, ototoxic drugs) and are therefore characterized by auditory threshold elevations and poorer frequency discrimination.     

The eastern Asian and eastern and western North American floristic disjunction: congruent phylogenetic patterns in seven diverse genera

Calcium (Ca2+) channels are present in non-excitable as well as in excitable cells. In bone cells of the osteoblast lineage, Ca2+ channels play fundamental roles in cellular responses to external stimuli including both mechanical forces and hormonal signals. They are also proposed to modulate paracrine signaling between bone-forming osteoblasts and bone-resorbing osteoclasts at local sites of bone remodeling. Calcium signals are characterized by transient increases in intracellular Ca2+ levels that are associated with activation of intracellular signaling pathways that control cell behavior and phenotype, including patterns of gene expression. Development of Ca2+ signals is a tightly regulated cellular process that involves the concerted actions of plasma membrane and intracellular Ca2+ channels, along with Ca2+ pumps and exchangers. This review summarizes the current state of knowledge concerning the structure, function, and role of Ca2+ channels and Ca2+ signals in bone cells, focusing on the osteoblast.     

Mechanical strength of acrylic bone cements impregnated with antibiotics

Admixing of several antibiotic powders which were insoluble in methyl methacrylate did not decrease the compressive and diametral tensile strengths of two acrylic bone cements when tested after setting for 1 day and after leaching 40 days in water at 37 degrees C. When antibiotics were added as water solutions, the included water resulted in a significant decrease in these bulk mechanical properties. Storage in water for 40 days caused surface irregularities only on specimens of the set antibiotic admixtures. Approximately 0.5% of the admixed dosage of these water-soluble antibiotics could be leached from the set cements. The observed surface channels, presumably left by the loss of antibiotic, suggest further study of surface-sensitive mechanical properties may be needed. The bulk mechanical strengths presented here are conclusive only for the particular combinations of antibiotics and cements investigated, and should not be generalized at this time to any or all antibiotic admixtures or other mechanical properties.     

Modulation of voltage-activated calcium currents by mechanical stimulation in rat sensory neurons

We examined the effects of mechanical stress, induced by a stream of bath solution, on evoked action potentials, electrical excitability, and Ca2+ currents in rat dorsal root ganglion neurons in culture with the use of the whole cell patch-clamp technique. Action-potential duration was altered reversibly by flow in 39% of the 51 neurons tested, but membrane potential and excitability were unaffected. The flow-induced increases and decreases in action-potential duration were consistent with the different effects of flow on two types of Ca2+ channel, determined by voltage-clamp recordings of Ba2+ currents. Current through omega-conotoxin-sensitive (N-type) Ca2+ channels increased by an estimated 74% with flow, corresponding to 23% increase in the total high voltage-activated current, whereas current through low-threshold voltage-activated (T-type) channels decreased by 14%. We conclude that modulation of voltage-activated Ca2+ currents constitutes a route by which mechanical events can regulate Ca2+ influx in sensory neurons.     

Calcium signalling in glial cells

Glial cells respond to a variety of external stimuli such as neurotransmitters, hormones or even mechanical stress by generating complex changes in the cytoplasmic Ca2+ concentration. This Ca2+ signal is controlled by an interplay of different mechanisms including plasmalemmal and intracellular Ca2+ channels, Ca2+ transporters and cytoplasmic Ca2+ buffers. In astrocytes, the Ca2+ signal can travel as waves within the syncytium spreading via gap junctions which might be regarded as a possible means for interglial communication. Ca2+ signalling is also an important medium for neurone-glia interaction: neuronal activity can trigger Ca2+ signals in glial cells and, in turn, there is evidence that glial Ca2+ signals can elicit responses in neurones. While glial cells are not equipped with the proper channels to generate action potentials, Ca2+ signalling could be the instrument by which these cells integrate and propagate signals in the CNS.     

Mechanosensitive ion channels of the archaeon Haloferax volcanii

Mechanosensitive (MS) ion channels have been documented in a variety of cells belonging to Eukarya and Eubacteria. We report the novel finding of two types of MS ion channels in the cell membrane of the halophilic archaeon Haloferax volcanii, a member of the Archaea that comprise the third phylogenetic domain. The two channels, MscA1 and MscA2, differed in their kinetic properties with MscA1 exhibiting more frequent open-closed transitions than MscA2. Both channels have large conductances that rectify between -40 mV and +40 mV where the conductance of MscA1 ranged from 380 to 680 picosiemens, whereas MscA2 ranged from 850 to 490 picosiemens. Both channels were blocked by submillimolar gadolinium. In addition, the channels of either membrane vesicles or detergent-solubilized membrane proteins remained functional upon reconstitution into artificial liposomes, a result that indicates that these channels are activated by mechanical force transmitted via the lipid bilayer alone. Subsequently a 37-kDa protein corresponding to the MscA1 channel activity was purified. With the possible functional similarity to bacterial MS channels, our finding of MS channels in Archaea emphasizes the ubiquity and importance of these channels in all domains of the evolutionary tree.     

Role of natriuretic peptides in ion transport mechanisms

Natriuretic peptides (NP) act as ligands on the guanylyl cyclase family of receptors. The NP binding site on these receptors is extracellular and the guanylyl cyclase and protein kinase domains are intracellular. The guanylyl cyclase receptor catalyzes the synthesis of the second messenger molecule, cGMP, which activates protein kinase. This in turn is involved in the phosphorylation of various ion transport proteins. Ion transport proteins, which are modulated by NP and are thought to underlie the natriuretic and diuretic actions of NP, include: (a) calcium-activated K+ channels; (b) ATP-sensitive K+ channels; (c) inwardly-rectifying K+ channels; (d) outwardly-rectifying K+ channels; (e) L-type Ca2+ channels; (f) Cl- channels including cystic fibrosis transmembrane conductance regulator Cl- channels; (g) Na+- K+ 2Cl- co-transporter; (h) Na+- K+ ATPase; (i) Na+ channels; (j) stretch-activated channels; and (k) water channels. It appears that NP modulate the kinetics, rather than the conductance, of ion channels. Some of these channels, like the Ca2+, ATP-sensitive K+ and stretch-activated channels, are also involved in NP secretion. In addition, the structural properties of the NP, e.g., ovCNP-22 and ovCNP-39, appear to confer on them the ability to form ion channels. These CNP-formed ion channels can modify the trans-membrane signal transduction and second messenger systems underlying NP-induced pathological effects.     

Duality of the voltage-dependent calcium influx in adrenal glomerulosa cells

Both T- and L-type calcium channels are expressed in bovine adrenal glomerulosa cells and both channels are sensitive to moderate depolarizations of the cell membrane induced by angiotensin II (AngII) or physiological concentrations of extracellular K+. These channels present distinct pharmacology, L-type channels being more sensitive to dihydropyridines, whereas T channels are inhibited by lower concentrations of mibefradil, a new type of calcium antagonist currently used for treating hypertension. The activity of these channels is also differently modulated by AngII, which inhibits T channels through activation of protein kinase C and L channels through a Pertussis toxin-sensitive G protein. Finally, whereas the activity of L-type channels is directly reflected on the levels of the cytosolic calcium concentration ([Ca2+]c), T-type channels are more closely related to the control of steroidogenesis, possibly through a kind of "calcium pipeline" linking the plasma membrane to the mitochondria. In conclusion, two types of calcium channels, with distinct functions and differential modulation by AngII, are activated by agonists of aldosterone biosynthesis in adrenal glomerulosa cells. Most importantly, these channels have distinct sensitivities to currently used antihypertensive therapeutic drugs.     

Structure and function of the Mec-ENaC family of ion channels

The epithelial sodium channel (ENaC) is the prototype of a new family of ion channels known as the Mec-ENaC superfamily. This new family of proteins are involved in a wide variety of functions that range from maintenance of sodium homeostasis to transduction of mechanical stimuli and nociceptive pain by specialized neurons. They show distinct tissue- and cell type-dependent expression and differential sensitivity to inhibition by the diuretic amiloride and its analogs. Despite the very little amino acid identity shared by these proteins, they all have the same common structure that has become a hallmark of the Mec-ENaC superfamily. The efforts to understand the structure and regulation of these ion channels have been stimulated by the recent discovery of severe disturbances in the maintenance of blood pressure caused by gain- or loss-of-function mutations in the genes that encode the subunits of ENaC in humans. Moreover, cloning of the ion channels that mediate pain elicited by tissue injury and inflammation will facilitate the development of new drugs to treat these common ailments.     

Modeling study of the effects of overlapping Ca2+ microdomains on neurotransmitter release

Although single-channel Ca2+ microdomains are capable of gating neurotransmitter release in some instances, it is likely that in many cases the microdomains from several open channels overlap to activate vesicle fusion. We describe a mathematical model in which transmitter release is gated by single or overlapping Ca2+ microdomains produced by the opening of nearby Ca2+ channels. This model accounts for the presence of a mobile Ca2+ buffer, provided either that the buffer is unsaturable or that it is saturated near an open channel with Ca2+ binding kinetics that are rapid relative to Ca2+ diffusion. We show that the release time course is unaffected by the location of the channels (at least for distances up to 50 nm), but paired-pulse facilitation is greater when the channels are farther from the release sites. We then develop formulas relating the fractional release following selective or random channel blockage to the cooperative relationship between release and the presynaptic Ca2+ current. These formulas are used with the transmitter release model to study the dependence of this form of cooperativity, which we call Ca2+ current cooperativity, on mobile buffers and on the local geometry of Ca2+ channels. We find that Ca2+ current cooperativity increases with the number of channels per release site, but is considerably less than the number of channels, the theoretical upper bound. In the presence of a saturating mobile buffer the Ca2+ current cooperativity is greater, and it increases more rapidly with the number of channels. Finally, Ca2+ current cooperativity is an increasing function of channel distance, particularly in the presence of saturating mobile buffer.     

Osmotic dilution stimulates axonal outgrowth by making axons more sensitive to tension

Mechanical tension is a potent stimulator of axonal growth rate, which is also stimulated by osmotic dilution. We wished to determine the relationship, if any, between osmotic stimulation and tensile regulation of axonal growth. We used calibrated glass needles to apply constant force to elongate axons of cultured chick sensory neurons. We find that a neurite being pulled at a constant force will grow 50-300% faster following a 50% dilution of inorganic ions in the culture medium. That is, osmotic dilution appears to cause axons to increase their sensitivity to applied tensions. Experimental interventions suggest that this effect is not mediated by dilution of extracellular calcium, or to osmotic stimulation of adenylate cyclase, or to osmotic stimulation of mechanosensitive ion channels. Rather, experiments measuring the static tension normally borne by neurites suggest a direct mechanical effect on the cytoskeletal proteins of the neurite shaft. Our results are consistent with a formal thermodynamic model for axonal growth in which removing a compressive load on axonal microtubules promotes their assembly, thus promoting axonal elongation.     

Regulation of gamma-aminobutyric acid (GABA) transporters by extracellular GABA

Hydrophobic interactions between a bilayer and its embedded membrane proteins couple protein conformational changes to changes in the packing of the surrounding lipids. The energetic cost of a protein conformational change therefore includes a contribution from the associated bilayer deformation energy (DeltaGdef0), which provides a mechanism for how membrane protein function depends on the bilayer material properties. Theoretical studies based on an elastic liquid-crystal model of the bilayer deformation show that DeltaGdef0 should be quantifiable by a phenomenological linear spring model, in which the bilayer mechanical characteristics are lumped into a single spring constant. The spring constant scales with the protein radius, meaning that one can use suitable reporter proteins for in situ measurements of the spring constant and thereby evaluate quantitatively the DeltaGdef0 associated with protein conformational changes. Gramicidin channels can be used as such reporter proteins because the channels form by the transmembrane assembly of two nonconducting monomers. The monomerleft arrow over right arrow dimer reaction thus constitutes a well characterized conformational transition, and it should be possible to determine the phenomenological spring constant describing the channel-induced bilayer deformation by examining how DeltaGdef0 varies as a function of a mismatch between the hydrophobic channel length and the unperturbed bilayer thickness. We show this is possible by analyzing experimental studies on the relation between bilayer thickness and gramicidin channel duration. The spring constant in nominally hydrocarbon-free bilayers agrees well with estimates based on a continuum analysis of inclusion-induced bilayer deformations using independently measured material constants.     

Localized deformation and hardening in irradiated metals: Three-dimensional discrete dislocation dynamics simulations

When irradiated, metals undergo significant internal damage accumulation and degradation of mechanical properties. Damage takes the form of a high number density of nanosize defect clusters (stacking-fault tetrahedrons (SFTs) or interstitial loops). The alteration of mechanical properties is manifested in a hardening behavior and localized plastic deformation in defect-free channels. This work uses discrete dislocation dynamics (DD) to capture these effects. It sets the framework for the elastic interaction between gliding dislocations and defect clusters and details a scheme for loop unfaulting and absorption into dislocations. Here, it is shown that SFTs represents weaker pinning points for dislocation motion than parent dislocation loops. It is also shown that appreciable yield drop can be attributed to high density of defects decorating the dislocations. Strong obstacles cause dislocations in Cu to continually double cross slip causing the formation of defect-free channels. Finally, the correlation between yield stress increase and defect number density is in excellent agreement with the experiment.     

Phase-field simulation of micropores constrained by a solid network

A 2D phase-field model has been developed in order to describe the morphology of a pore forming within interdendritic liquid channels and the geometrical effect of mechanical contacts with neighboring solid. The distribution of the solid, liquid and gas phases is calculated with a multiphase-field approach which accounts for the pressure difference between the liquid and gas phases, as well as diffusion of dissolved gases in the liquid. The model incorporates the perfect gas and Sievert’s laws to describe the concentration and partitioning of gas molecules or atoms at the pore/liquid interface. The results show that the presence of solid can substantially influence the volume and pressure of the pore. A pore constrained to grow in narrow liquid channels exhibits a substantially higher mean curvature, a larger pressure and a smaller volume as compared with a pore grown under unconstrained conditions.     

Airway responses in healthy and asthmatic subjects following exposure to low ambient air temperature

Agonist-activated Ca2+ entry is important in many biological responses such as secretion and cell growth(1,2). In nonexcitable cells which have no voltage-operated Ca2+ channels (VOCC), agonist-receptor interaction can trigger Ca2+ entry across the plasmalemma via several entry pathways(1-3) (Fig 1): (A) channels which are intrinsic structures of the receptor (receptor-operated channels), (B) channels which are coupled to receptors via a G-protein (G-protein-operated channels), (C) channels which are activated by some second messengers (second-messenger-operated channels), and (D) channels which open upon intracellular nonmitochondrial Ca2+ store depletion (Ca2+ release-activated channels) resulting from inositol 1, 4, 5-trisphosphate-induced Ca2+ release or inhibition of Ca2+ re-uptake (see next section). Ca2+ entry via the 4th type of channel, also known as "capacitative Ca2+ entry" (CCE)[4], has aroused much interest in the past decade because of its intriguing nature as retrograde signalling. In this brief review, we present the evidence for and the possible biochemical processes involved in CCE. We also discuss the use of 2 novel Ca2+ entry blockers: tetrandrine and SK&F 96365. Emphasis will be put on the human leukemic HL-60 cell line, a popular cell system for intracellular Ca2+ homeostasis studies and also a model the signal transduction of which we have been investigating during the past few years.     

Involvement of different ion channels in osteoblasts' and osteocytes' early responses to mechanical strain

The involvement of functional ion channels in previously documented early responses of osteocytes and osteoblasts to mechanical strain in bone tissue was investigated in explants of rat ulnae by the use of ion channel blockers. Gadolinium chloride (a blocker of stretch/shear-sensitive cation channels) elevated basal prostaglandin (PG) E2 and prostacyclin (PGI2) release and osteocyte glucose-6-phosphate dehydrogenase (G6PD) activity, but was associated with a reduction in basal nitric oxide (NO) production. Gadolinium abolished loading-related increases in the release of PGI2 and NO and osteocyte G6PD activity. Gadolinium also reduced the loading-related release of PGE2 assumed to originate from osteoblasts and the magnitude of loading-related increases in G6PD activity in these cells. Nifedipine (a blocker of L-type voltage-dependent calcium channels) had no effect on basal levels of prostanoid or NO release, or G6PD activity in osteocytes or osteoblasts, and did not affect loading-related release of PGI2 or increase in osteocyte G6PD. However, nifedipine prevented loading-related increases in PGE2 and NO release and osteoblast G6PD activity. These results are consistent with osteocytes' response to bone loading requiring activatable ion channels sensitive to gadolinium, but not those sensitive to nifedipine. In osteoblasts, the early responses to bone loading appear to be associated with ion channels sensitive to gadolinium and nifedipine; however, the nifedipine-sensitive channels seem to have the dominant effect.     

Neonatal treatment with tamoxifen causes immediate alterations of the sexually dimorphic nucleus of the preoptic area and medial preoptic area in male rats

We have characterized two different types of Cl- currents in calf pulmonary artery endothelial (CPAE) cells by using a combined patch-clamp and Fura-2 microfluorescence technique to measure simultaneously ionic currents and the intracellular Ca2+ concentration, [Ca2+]i. Exposure of CPAE cells to 28% hypotonic solution induces cell swelling without a change in membrane capacitance and [Ca2+]i, and concomitantly activates a current. This current, I(Cl, vol), is closely correlated with the changes in cell volume and shows a modest outward rectification. It slowly inactivates at potentials more positive than +60 mV but is time- and voltage-independent at other potentials. Increase in [Ca2+]i by different maneuvers, such as application of vasoactive agonists (ATP), ionomycin, or loading of the cells directly with Ca2+ also activates a Cl- current, I(Cl, Ca). This current slowly activates at positive potentials, inactivates quickly at negative potentials and shows strong outward rectification. A time-independent component of the current activated by elevation of [Ca2+]i alone can be inhibited by cell shrinking by exposing the cells to hypertonic solution, indicating that an increase in [Ca2+]i also co-activates I(Cl, vol). Forskolin or cAMP never activated a current in CPAE cells, which indicates the lack of cAMP-activated channels in these cells. There is also no evidence for the existence of voltage-gated Cl- channels in resting, nonstimulated cells. Challenging a cell with elevated [Ca2+]i and hypotonic solutions activated I(Cl, vol) on top of I(Cl, Ca), suggesting that I(Cl, Ca) and I(Cl, vol) are different channels. We conclude that CPAE cells do not express voltage-gated (ClC-type) or cAMP-gated (CFTR-type) Cl- channels, but activate large Cl- currents after volume (mechanical?) or chemical (Ca2+) stimulation.     

3D screen printing for the fabrication of small intricate Ti-6Al-4V parts

For the production of complex shaped titanium parts huge advances have been made by powder metallurgy (P/M) during the last years. However, when it comes to miniaturized parts with intricate inner structures like closed channels without entrapped powder and high aspect ratios conventional P/M methods are stretched to their limits. To remedy this shortcoming, Fraunhofer IFAM Dresden uses 3D screen printing, a modified screen printing process based on a powder-binder mixture which is printed layer-by-layer to the desired shape followed by debinding and a conventional sintering step. Especially for small parts a productivity of several million parts per year is achievable. The objective of this work is to demonstrate the possibility to manufacture complex designs based on titanium including thin walls down to 120 μm. Therefore, pre-alloyed gas atomized Ti-6Al-4V powder was used. Analysis of part accuracy, resulting porosity, impurity level and mechanical properties were performed.