Mechanosensitive Ca2 Research Articles

Transient Receptor Potential Channels in Cardiovascular Function and Disease

Sustained elevation in the intracellular Ca2+ concentration via Ca2+ influx, which is activated by a variety of mechanisms, plays a central regulatory role for cardiovascular functions. Recent molecular biological research has disclosed an unexpectedly diverse array of Ca(2+-entry channel molecules involved in this Ca2+ influx. These include more than ten transient receptor potential (TRP) superfamily members such as TRPC1, TRPC3-6, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, and polycystin (TRPP2). Most of them appear to be multimodally activated or modulated and show relevant features to both acute hemodynamic control and long-term remodeling of the cardiovascular system, and many of them have been found to respond not only to receptor stimulation but also to various forms of stimuli. There is good evidence to implicate TRPC1 in neointimal hyperplasia after vascular injury via store-depletion-operated Ca2+ entry. TRPC6 likely contributes to receptor-operated and mechanosensitive Ca2+ mobilizations, being involved in vasoconstrictor and myogenic responses and pulmonary arterial proliferation and its associated disease (idiopathic pulmonary arterial hypertension). Considerable evidence has also been accumulated for unique involvement of TRPV1 in blood flow/pressure regulation via sensory vasoactive neuropeptide release. New lines of evidence suggest that TRPV2 may act as a Ca2+-overloading pathway associated with dystrophic cardiomyopathy, TRPV4 as a mediator of endothelium-dependent hyperpolarization, TRPM7 as a proproliferative vascular Mg2+ entry channel, and TRPP2 as a Ca2+-entry channel requisite for vascular integrity. This review attempts to provide an overview of the current knowledge on TRP proteins and discuss their possible roles in cardiovascular functions and diseases.

Effects of a Hydroxyapatite-based Biomaterial on Gene Expression in Osteoblast-like Cells

Biostite is a hydroxyapatite-derived biomaterial that is used in periodontal and bone reconstructive procedures due to its osteoconductive properties. Since the molecular effects of this biomaterial on osteoblasts are still unknown, we decided to assess whether it may specifically modulate osteoblast functions in vitro. We found that a brief exposure to Biostite significantly reduced the proliferation of MG-63 and SaOS-2 osteoblast-like cells to approximately 50% of the plateau value. Furthermore, gene array analysis of MG-63 cells showed that Biostite caused a differential expression of 37 genes which are involved in cell proliferation and interaction, and related to osteoblast differentiation and tissue regeneration. Results were confirmed by RT-PCR, Western blot, and by an increase in alkaline phosphatase (ALP) specific activity. Biostite also increased levels of polycystin-2, a mechano-sensitive Ca(2+) channel, a promising new marker of bone cell differentiation. Biostite, therefore, may directly affect osteoblasts by enhancing chondro/osteogenic gene expression and cytoskeleton-related signaling pathways, which may contribute to its clinical efficacy.

Physical and chemical interactions between aphids and plants

Aphids feed from sieve tubes deep inside the host plant. Therefore, aphids must be able to recognize their host plant(s) and to direct their stylets which must be long and thin enough to reach and puncture the sieve tubes at a particular site. Sieve tubes in angiosperms are longitudinal arrays of sieve element/companion cell modules which are highly sensitive to disturbance of any kind. The sieve tubes dispose of elaborate sealing mechanisms such as protein plugging and callose sealing which are triggered by a rise in calcium in the sieve tubes. Aphids seem to have developed a range of physical and chemical measures to limit the amount of calcium influx in response to stylet puncturing. Loss of sieve-element turgor pressure induced by stylet insertion is minimized by the minute stylet volume. Turgor-dependent Ca(2+) influx, possibly mediated by mechano sensitive Ca(2+) channels, must therefore be limited. The components of the sheath and watery saliva play a pivotal role in establishing the physical and chemical constraints on the rise of calcium. Most likely, sheath saliva prevents the influx of calcium from the apoplast by sealing the stylet puncture site while watery saliva may prevent plugging and sealing of sieve plates by potential interaction with SE sap ingredients.

Possible Involvement of Mechanosensitive Ca2+ Channels of Plasma Membrane in Mechanoperception in Chara

When an internodal cell of Chara corallina was stimulated with a mechanical pulse of various amplitudes lasting for 0.1 s (mechanical stimulus), the cell generated a receptor potential, which was highly dependent not only on the strength of the stimulus but also on the extracellular Cl- concentration. Extracellular Ca2+ was indispensable for generating receptor potential, since removal of Ca2+ reversibly inhibited generation of the receptor potential. The cytoplasmic Ca2+ level transiently rose upon mechanical stimulation. The stronger the mechanical stimulus, the larger was the increase in the cytoplasmic level of Ca2+. It is proposed that the first step of receptor potential is an activation of mechanosensitive Ca2+ channels at the plasma membrane.

オオシャジクモの“受容器電位”には機械刺激感受性カルシウムチャンネルが関与する

オオシャジクモの“受容器電位”には機械刺激感受性カルシウムチャンネルが関与する

Role of Ca 2+ signaling in initiation of stretch-induced apoptosis in neonatal heart cells

Abnormal mechanical load, as seen in hypertension, is found to induce heart cell apoptosis, yet the signaling link between cell stretch and apoptotic pathways is not known. Using an in vitro stretch model mimicking diastolic pressure stress, here we show that Ca 2+ signaling participates essentially in the early stage of stretch-induced apoptosis. In neonatal rat cardiomyocytes, the moderate 20% stretch resulted in tonic elevation of intracellular free Ca 2+ ([Ca 2+] i). Buffering [Ca 2+] i by EGTA-AM, suppressing ryanodine-sensitive Ca 2+ release, and blocking L-type Ca 2+ channels all prevented the stretch-induced apoptosis as assessed by phosphatidylserine exposure and nuclear fragmentation. Notably, Ca 2+ suppression also prevented known stretch-activated apoptotic events, including caspase-3/-9 activation, mitochondrial membrane potential corruption, and reactive oxygen species production, suggesting that Ca 2+ signaling is the upstream of these events. Since [Ca 2+] i did not change without activating mechanosensitive Ca 2+ entry, we conclude that stretch-induced Ca 2+ entry, via the Ca 2+-induced Ca 2+ release mechanism, plays an important role in initiating apoptotic signaling during mechanical stress.

Up-regulation of endothelial stretch-activated cation channels by fluid shear stress.

Stretch-activated cation channels (SAC) have been suggested to act as endothelial mechanosensors for hemodynamic forces. Ca(2+) influx through SAC could induce an intracellular Ca(2+) signal stimulating Ca(2+)-dependent synthesis of vasodilators like NO, prostacyclin, or EDHF. In the present study we tested whether laminar shear stress (LSS) regulates SAC function. Electrophysiological properties of SAC were investigated in human umbilical vein endothelial cells (HUVEC) subjected to defined levels of LSS in a flow-cone apparatus. In HUVEC, we identified a Ca(2+) permeable SAC that was activated by membrane stretch. Single-channel current densities of SAC in cell-attached patches were significantly increased in HUVEC exposed to an LSS of 5 dyn/cm(2) for 4 h (1.15+/-0.17 SAC/patch) compared to HUVEC kept in stationary culture (0.46+/-0.07 SAC/patch). Exposure of HUVEC to a higher LSS of 15 dyn/cm(2) for 4 h induced similar up-regulation of SAC (1.27+/-0.21 SAC/patch). After 24 h exposure to LSS of 15 dyn/cm(2), single-channel current densities of SAC remained up-regulated (1.07+/-0.18 SAC/patch) compared to controls. In addition, stretch-sensitivity of SAC (channel activity NP(o) at -30 mmHg) significantly increased after 2 h of exposure to LSS of 5 and 15 dyn/cm(2) and remained up-regulated after 24 h. Inhibition of protein kinases and tyrosine kinases by H7 and genistein, respectively, prevented LSS-induced alteration of SAC function. Single-channel current density and mechanosensitivity of SAC in HUVEC is up-regulated by LSS. Up-regulation of SAC function leads to enhanced mechanosensitive Ca(2+) influx, and represents a novel adaptive mechanism of the endothelium in the presence of altered hemodynamic forces.

Nitric oxide synthase inhibitors enhance mechanosensitive Ca 2+ influx in cultured dorsal root ganglion neurons

Nitric oxide (NO) can have opposite effects on peripheral sensory neuron sensitivity depending on the concentration and source of NO, and the experimental setting. The aim of this study was to determine the role of endogenous NO production in the regulation of mechanosensitive Ca 2+ influx of dorsal root ganglion (DRG) neurons. Adult mouse DRG neurons were grown in primary culture for 2–5 days, loaded with Fura-2, and tested for mechanically mediated changes in [Ca 2+] i by fluorescent ratio imaging. In the presence of the NOS inhibitors l-NAME, TRIM, or 7-NI, but not the inactive analogue d-NAME, peak [Ca 2+] i transients to mechanical stimulation were increased more than 2-fold. Neither La 3+ (25 μM), an inhibitor of voltage activated Ca 2+ channels, or tetrodotoxin (TTX, 1 μM), a selective inhibitor of voltage-gated Na + channels, had an effect on mechanically activated [Ca 2+] i transients under control conditions. However, in the presence of l-NAME, both La 3+ and TTX partially blocked the [Ca 2+] i response. Addition of Gd 3+, a blocker of mechanosensitive cation channels and L-type Ca 2+ channels, at a concentration (100 μM) that markedly inhibited the mechanical response under control conditions, only partially inhibited the response in the presence of l-NAME. The combination of either La 3+ or TTX with Gd 3+ caused near complete inhibition of mechanically stimulated [Ca 2+] i transients in the presence of l-NAME. We conclude that focal mechanical stimulation of DRG neurons causes Ca 2+ influx occurs primarily through mechanosensitive cation channels under control conditions. In the presence of NOS inhibitors, additional Ca 2+ influx occurs through voltage-sensitive Ca 2+ channels. These results suggest that endogenously produced NO in cultured DRG neurons decreases mechanosensitivity by inhibiting voltage-gated Na + and Ca 2+ channels.

Mechanosensitive Ca2+ release from intracellular stores in Nitella flexilis.

We found previously that the cytoplasmic drop isolated from internodal cells of Nitella flexilis releases Ca2+ in response to hypotonic treatment and named the phenomenon hydration-induced Ca2+ release (HICR). The HICR is assumed to be a result of activation of Ca2+ permeable channels in the membrane of Ca2+ stores in a stretch-activated manner. To prove this idea, mechanical stimulus was applied to the drop by means of shooting isotonic/hypnotic medium or silicon oil into the drop, or compressing the drop. All these mechanical stimuli induced a rapid increase in the Ca2+ concentration of the drop. The chloroplast fraction isolated from the cytoplasmic drop released Ca2+ on compression, while the chloroplast-free cytoplasm did not. In Chara corallina, the cytoplasmic drop, which shows a very weak HICR, also responded weakly to the mechanical stimulus, but the chloroplast fraction was inert. When chloroplasts from Chara were added to the chloroplast-free cytoplasm of N. flexilis, the cytoplasm recovered the mechanoresponse. Starch grains were as effective as chloroplasts. The data indicate that Ca2+ permeable channels in the membrane of Ca2+ stores in N. flexilis are really mechano-sensitive.

Stretch-sensitive switching among different channel sublevels of an endothelial cation channel

A mechanosensitive Ca 2+-permeable cation channel was recorded by patch clamp in isolated rat aortic endothelial cells. A low level of channel activity could be observed after seal formation. The channel displayed some inward rectification and had a conductance for inward current of approx. 32 pS in Ca 2+-free pipette and bath solutions. Negative suction of −10 to −20 mmHg increased the probability of the channel being open. When the negative pressure in the pipette was raised to −35 to −45 mmHg, the channel underwent an abrupt transition to a large conductance substate that was interrupted occasionally by two other low conductance levels. Under this condition, the overwhelming majority of openings and closings were between a main level of 83 pS and the closed level. Compared to the 32 pS substate, the 83 pS large conductance substate had shorter mean open and closed times. The two channel substates had similar ionic selectivity and both were sensitive to the inhibition of cGMP and protein kinase G. This is the first demonstration showing that mechanostress can change the single channel conductance level of an ion channel in eukaryotic cells.

Extracellular adenosine modulates a volume-sensitive-like chloride conductance in immortalized rabbit DC1 cells.

Cl(-) currents induced by cell swelling were characterized in an immortalized cell line (DC1) derived from rabbit distal bright convoluted tubule by the whole cell patch-clamp techniques and by (125)I(-) efflux experiments. Exposure of cells to a hypotonic shock induced outwardly rectifying Cl(-) currents that could be blocked by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM DIDS, and by 1 mM diphenylamine-2-carboxylate. (125)I(-) efflux experiments showed that exposure of the monolayer to a hypotonic medium increased (125)I(-) loss. Preincubation of cells with LaCl(3) or GdCl(3) prevented the development of the response. The addition of 10 microM adenosine to the bath medium activated outwardly rectifying whole cell currents similar to those recorded after hypotonic shock. This conductance was inhibited by the A(1)-receptor antagonist 8-cyclopentyl-1,3-diproxylxanthine (DPCPX), LaCl(3), or GdCl(3) and was activated by GTPgammaS. The selective A(1)-receptor agonist N(6)-cyclopentyladenosine (CPA) mimicked the effect of hypotonicity on (125)I(-) efflux. The CPA-induced increase of (125)I(-) efflux was inhibited by DPCPX and external application of LaCl(3) or GdCl(3). Adenosine also enhanced Mn(2+) influx across the apical membrane. Overall, the data show that DC1 cells possess swelling- and adenosine-activated Cl(-) conductances that share identical characteristics. The activation of both conductances involved Ca(2+) entry into the cell, probably via mechanosensitive Ca(2+) channels. The effects of adenosine are mediated via A(1) receptors that could mediate the purinergic regulation of the volume-sensitive Cl(-) conductance.

Physiological characterization of gravitaxis in Euglena gracilis and Astasia longa studied on sounding rocket flights.

Euglena gracilis is a photosynthetic, unicellular flagellate found in eutrophic freshwater habitats. The organisms control their vertical position in the water column using gravi- and phototaxis. Recent experiments demonstrated that negative gravitaxis cannot be explained by passive buoyancy but by an active physiological mechanism. During space experiments, the threshold of gravitaxis was determined to be between 0.08 and 0.12 x g. A strong correlation between the applied acceleration and the intracellular cAMP and Ca2+ was observed. The results support the hypothesis, that the cell body of Euglena, which is denser than the surrounding medium exerts a pressure onto the lower membrane and activates mechanosensitive Ca2+ channels. Changes in the membrane potential and the cAMP concentration are most likely subsequent elements in a signal transduction chain, which results in reorientation strokes of the flagellum.

Whole cell current and membrane potential regulation by a human smooth muscle mechanosensitive calcium channel.

Mechanotransduction is required for a wide variety of biological functions. The aim of this study was to determine the effect of activation of a mechanosensitive Ca(2+) channel, present in human jejunal circular smooth muscle cells, on whole cell currents and on membrane potential. Currents were recorded using patch-clamp techniques, and perfusion of the bath (10 ml/min, 30 s) was used to mechanoactivate the L-type Ca(2+) channel. Perfusion resulted in activation of L-type Ca(2+) channels and an increase in outward current from 664 +/- 57 to 773 +/- 72 pA at +60 mV. Membrane potential hyperpolarized from -42 +/- 4 to -50 +/- 5 mV. In the presence of nifedipine (10 microM), there was no increase in outward current or change in membrane potential with perfusion. In the presence of charybdotoxin or iberiotoxin, perfusion of the bath did not increase outward current or change membrane potential. A model is proposed in which mechanoactivation of an L-type Ca(2+) channel current in human jejunal circular smooth muscle cells results in increased Ca(2+) entry and cell contraction. Ca(2+) entry activates large-conductance Ca(2+)-activated K(+) channels, resulting in membrane hyperpolarization and relaxation.

A protein kinase G-sensitive channel mediates flow-induced Ca(2+) entry into vascular endothelial cells.

The hemodynamic force generated by blood flow is considered to be the physiologically most important stimulus for the release of nitric oxide (NO) and prostacyclin (PGI(2)) from vascular endothelial cells (1). NO and PGI(2) then act on the underlying smooth muscle cells, causing vasodilation and thus lowering blood pressure (2, 3). One critical early event occurring in this flow-induced regulation of vascular tone is that blood flow induces Ca(2+) entry into vascular endothelial cells, which in turn leads to the formation of NO (4, 5). Here we report a mechanosensitive Ca(2+)-permeable channel in vascular endothelial cells. The activity of the channel was inhibited by 8-Br-cGMP, a membrane-permeant activator of protein kinase G (PKG), in cell-attached membrane patches. The inhibition could be reversed by PKG inhibitor KT5823 or H-8. A direct application of active PKG in inside-out patches blocked the channel activity. Gd(3+), Ni(2+), or SK&F-96365 also inhibited the channel activity. A study of fluorescent Ca(2+) entry revealed a striking pharmacological similarity between the Ca(2+) entry elicited by flow and the mechanosensitive Ca(2+)-permeable channel we identified, suggesting that this channel is the primary pathway mediating flow-induced Ca(2+) entry into vascular endothelial cells.

Nitric oxide synthase inhibitors enhance mechanosensitive Ca 2+ influx in cultured DRG neurons

Nitric oxide synthase inhibitors enhance mechanosensitive Ca 2+ influx in cultured DRG neurons

PH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions: cellular responses to stress and their implication for plant water relations.

A paradigm for the response of plants to stress is presented which suggests that plants move towards a state of minimal metabolic activity as a stress intensifies and remain in that state until that stress is relieved. The paradigm is based on the proposition that cells that interface with the transpiration stream employ variations on the following theme to move towards that state. Tension on the apoplastic water opens a mechanosensitive Ca2+ channel, a response that is augmented by apoplastic ABA. The resulting elevated cytoplasmic Ca2+ deactivates a plasmalemma H+/ATPase and also activates a K(+)-H+ symport. The inflow of K+ and H+ depolarizes the membrane and renders the apoplast less acidic, the protons being removed to the vacuole and the K+ ions being re-exported via the K+ outward rectifying channel. The onset of darkness in guard and mesophyll cells deactivates the plasmalemma H+/ATPase and then the events outlined above ensue except that these cells do not appear to utilize either Ca2+ or ABA during these changes. In stressed cells it is proposed that elevated cytoplasmic Ca2+ activates the release of an ABA precursor from a stored form. ABA is then released in the apoplast after export of the precursor if the activity of the K(+)-H+ symport has brought the apoplastic pH close to 7.0. It is proposed that aquaporins in the xylem parenchyma and mesophyll cells are opened by elevated cytoplasmic Ca2+ when the water potential of the transpiration stream is high so that water can be stored in the 'xylem parenchyma reservoir'. The water in this reservoir is then used to increase the water potential in the transpiration stream when the water column is under tension and to help repair embolisms by a mechanism that resembles stomatal closure.

Mechanical stimuli induce intracellular calcium response in a subpopulation of cultured rat sensory neurons

Cultured dorsal root ganglion neurons from newborn rats were mechanically deformed with a fine-tipped glass capillary, and the change in the intracellular Ca 2+ concentration ([Ca 2] i) was recorded by Fura-2-based microfluorimetry. The deformation evoked elevation in [Ca 2+] i from 18.7±5.4 nM (mean±S.E.M., n=35) to 137.1±15.2 nM in some subpopulations of cells, especially those larger than 20 μm in diameter. The largest mechanosensitive cell group was that of cells 20–25 μm in diameter; 56% of the mechanosensitive cells were of this cell size. All of the cells larger than 25 μm in diameter displayed the Ca 2+ increase when prodded. The depletion of extracellular Ca 2+ diminished the Ca 2+ elevation. Verapamil and nickel, blockers of voltage-dependent Ca 2+ channels, did not influence the Ca 2+ response, whereas gadolinium, a relatively selective blocker of mechanosensitive channels, diminished the response. Na +-free conditions did not influence the response. We concluded that the mechanical stimulation induced a Ca 2+ influx in large dorsal root ganglion neurons through mechanosensitive Ca 2+-permeable channels.

O-9 - Effects of glucose overload on mechanosensitive Ca2+ transient in bovine aortic endothelial cells

O-9 - Effects of glucose overload on mechanosensitive Ca2+ transient in bovine aortic endothelial cells

Up-regulation of pressure-activated Ca(2+)-permeable cation channel in intact vascular endothelium of hypertensive rats.

In endothelial cells, stretch-activated cation channels have been proposed to act as mechanosensors for changes in hemodynamic forces. We have identified a novel mechanosensitive pressure-activated channel in intact endothelium from rat aorta and mesenteric artery. The 18-pS cation channel responded with a multifold increase in channel activity when positive pressure was applied to the luminal cell surface with the patch pipette and inactivated at negative pipette pressure. Channel permeability ratio for K+, Na+, and Ca2+ ions was 1:0.98:0.23. Ca2+ influx through the channel was sufficient to activate a neighboring Ca2(+)-dependent K+ channel. Hemodynamic forces are chronically disturbed in arterial hypertension. Endothelial cell dysfunction has been implicated in the pathogenesis of arterial hypertension. In two comparative studies, density of the pressure-activated channel was found to be significantly higher in spontaneously hypertensive rats and renovascular hypertensive rats compared with their respective normotensive controls. Channel activity presumably leads to mechanosensitive Ca2+ influx and induces cell hyperpolarization by K+ channel activity. Both Ca2+ influx and hyperpolarization are known to induce a vasodilatory endothelial response by stimulating endothelial nitric oxide (NO) production. Up-regulation of channel density in hypertension could, therefore, represent a counterregulatory mechanism of vascular endothelium.

A novel type of mechanoreception by the flagella of Chlamydomonas

A novel type of mechanosensory mechanism is found in Chlamydomonas reinhardtii. When a cell is captured with a suction pipette and a negative pressure is applied, the cell produces repetitive Ca2+ impulses at a frequency of 0.5-1.0 Hz. The impulse frequency increases with the applied pressure. The impulses are produced when the flagella are sucked into the pipette but not when the cell body is sucked in leaving the flagella outside the pipette. Cells with short flagella produce impulses of small amplitude. Thus, the site where the cell senses mechanical stimuli and generates the impulse current must be localized at the flagella. The amplitude, shape and ion selectivity of the pressure-induced impulses are distinct from the all-or-none flagellar current that is evoked by photostimulation. The impulses are possibly produced by a combination of currents passing through mechanosensitive channels and Ca2+ channels. This response probably functions to modulate flagellar beating and thereby to regulate the behaviour of the cell.