Calcium signaling in live cells on elastic gels under mechanical vibration at subcellular levels

A new device was designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured to mechanically stimulate these cells at subcellular locations. A Fluorescence Resonance Energy Transfer (FRET)-based calcium biosensor (an improved Cameleon) was used to monitor the spatiotemporal distribution of intracellular calcium concentrations in the cells upon this mechanical stimulation. A clear increase in intracellular calcium concentrations over the whole cell body (global) can be observed in the majority of cells under mechanical stimulation. The chelation of extracellular calcium with EGTA or the blockage of stretch-activated calcium channels on the plasma membrane with streptomycin or gadolinium chloride significantly inhibited the calcium responses upon mechanical stimulation. Thapsigargin, an endoplasmic reticulum (ER) calcium pump inhibitor, or U73122, a phospholipase C (PLC) inhibitor, resulted in mainly local calcium responses occurring at regions close to the stimulation site. The disruption of actin filaments with cytochalasin D or inhibition of actomyosin contractility with ML-7 also inhibited the global calcium responses. Therefore, the global calcium response in HUVEC depends on the influx of calcium through membrane stretch-activated channels, followed by the release of inositol trisphosphate (IP3) via PLC activation to trigger the ER calcium release. Our newly developed mechanical stimulation device can also provide a powerful tool for the study of molecular mechanism by which cells perceive the mechanical cues at subcellular levels.     

Mechanisms of integrin-mediated calcium signaling in MDCK cells: regulation of adhesion by IP3- and store-independent calcium influx.

Peptides containing Arg-Gly-Asp (RGD) immobilized on beads bind to integrins and trigger biphasic, transient increases in intracellular free Ca2+ ([Ca2+]i) in Madin-Darby canine kidney epithelial cells. The [Ca2+]i increase participates in feedback regulation of integrin-mediated adhesion in these cells. We examined influx pathways and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ store release as possible sources of the [Ca2+]i rise. The RGD-induced [Ca2+]i response requires external Ca2+ (threshold approximately 150 microM), and its magnitude is proportional to extracellular calcium. RGD-induced transients were attenuated by Ca2+ channel inhibitors (Ni2+ and carboxy-amidotriazole) or by plasma membrane depolarization, indicating that Ca2+ influx contributes to the response. Loading cells with heparin reduced the size of RGD-induced [Ca2+]i transients, indicating that IP3-mediated release of Ca2+ from stores may also contribute to the RGD response. Depletion of Ca2+ stores with thapsigargin activated Ni(2+)-sensitive Ca2+ influx that might also be expected to occur after IP3-mediated depletion of stored Ca2-. However, RGD elicited a Ni(2+)-sensitive Ca2+ influx even after pretreatment with thapsigargin, indicating that Ca2+ influx is controlled by a mechanism independent of IP3-mediated store depletion. We conclude that RGD-induced [Ca2+]i transients in Madin-Darby canine kidney cells result primarily from the combination of two distinct mechanisms: 1) IP3-mediated release of intracellular stores, and 2) activation of a Ca2+ influx pathway regulated independently of IP3 and Ca2+ store release. Because Ni2+ and carboxy-amidotriazole inhibited adhesion, whereas store depletion with thapsigargin had little effect, we suggest that the Ca2+ influx mechanism is most important for feedback regulation of integrin-mediated adhesion by increased [Ca2+]i.     

Mechanisms involved in the increase in intracellular calcium following hypotonic shock in bovine articular chondrocytes

The extracellular osmotic environment of chondrocytes fluctuates during joint loading as fluid is expressed from and reimbibed by the extracellular matrix. Matrix synthesis by chondrocytes is modulated by joint loading, possibly mediated by variations in intracellular composition. The present study has employed the Ca2+-sensitive fluoroprobe Fura-2 to determine the effects of hypotonic shock (HTS) on intracellular Ca2+ concentration ([Ca2+]i) and to characterise the mechanisms involved in the response for isolated bovine articular chondrocytes. In cells subjected to a 50% dilution, [Ca2+]i rapidly increased by approximately 250%, a sustained plateau being achieved within 300 s. The effect was inhibited by thapsigargin or by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i reflects both influx from the extracellular medium and release from intracellular stores. Inhibition of the response by neomycin implicates activation of PLC and IP3 synthesis in the mobilisation of Ca2+ from intracellular stores. The rise was insensitive to inhibitors of L-type voltage-activated Ca2+ channels (LVACC) or reverse mode Na+/Ca2+ exchange (NCE) but could be significantly attenuated by ruthenium red, an inhibitor of transient receptor potential vanilloid (TRPV) channels and by Gd3+, a blocker of stretch-activated cation (SAC) channels. The HTS-induced rise in [Ca2+]i was almost completely absent in cells treated with Ni2+, a non-specific inhibitor of Ca2+ entry pathways. We conclude that in response to HTS the opening of SACC and a member of TRPV channel family leads to Ca2+ influx, simultaneously with the release from intracellular stores.     

Aggregating platelets increase intracellular calcium in endothelial cells through release of adenine nucleotides

Aggregating platelets relax isolated coronary arteries through the release of endothelium-derived relaxing factor (EDRF). Since release of EDRF may be calcium dependent, we tested if and how aggregating platelets stimulated a calcium response in cultured endothelial cells. Aggregating platelets caused a transient increase in intracellular calcium in endothelial cells loaded with the fluorescent calcium indicator fura-2. The adenine nucleotides ADP and ATP, but not other platelet-derived mediators, mimicked the platelet-induced calcium response, and inhibition of adenine nucleotides impaired the response to aggregating platelets. Thus, aggregating platelets release adenine nucleotides and stimulate a rise in intracellular calcium in cultured endothelial cells. This calcium response may represent the intracellular transduction mechanism by which aggregating platelets induce endothelial release of EDRF and subsequent relaxation of coronary arteries.     

The involvement of transient receptor potential channels in mast cell activation by microbubbles

This study was to explore the activation of mast cells by microbubbles, with the focus on transient receptor potential (TRP) channels mediated degranulation and calcium influx. Bone marrow-derived mast cells (BMMCs) were primarily obtained from femurs in mice and induced differentiation for 4 weeks. After the purity identification, BMMCs were contacted by homogeneous microbubbles with the diameter of 1 mm for 1 h. β-hexosaminidase and histamine levels in supernatants were assessed by enzyme-linked immunosorbent assay (ELISA) and the CD63 expression was tested by flow cytometry. The intracellular calcium binding with Fluo-4 AM dyes in BMMCs was observed under the fluorescence microscope and the mean fluorescence intensity was quantitatively measured by flow cytometry. β-hexosaminidase release, histamine concentration, CD63 expression and calcium influx were significantly increased in BMMCs group upon microbubble stimulation compared to the control groups. After preconditioning with the available inhibitors and microbubble contact, only transient receptor potential vanilloid 1 (TRPV1) and TRPV4 inhibitors robustly suppressed the microbubble-induced degranulation. Likewise, the elevated fluorescence intensity of cytosolic calcium level was also significantly weaken. The results demonstrated microbubble stimulus effectively promoted BMMCs degranulation, which could be substantially restrained by inhibitors targeted for blocking TRPV1 or TRPV4 channel. The alternation of intracellular calcium level in BMMCs was consistent with the changes of degranulation capacity. It's suggested that the activation of BMMCs by microbubbles may involve specific TRP calcium dependent channels.     

L-type calcium channel modulates mechanosensitivity of the cardiomyocyte cell line H9c2

The application of mechanical stimuli to cells often induce increases in intracellular calcium, affecting the regulation of a variety of cell functions. Although the mechanism of mechanotransduction-induced calcium increases has not been fully resolved, the involvement of mechanosensitive ion channels in the plasma membrane and the endoplasmic reticulum has been reported. Here, we demonstrate that voltage-gated L-type calcium channels play a critical role in the mechanosensitive calcium response in H9c2 rat cardiomyocytes. The intracellular calcium level in H9c2 cells increased in a reproducible dose-dependent manner in response to uniaxial stretching. The stretch-activated calcium response (SICR) completely disappeared in calcium-free medium, whereas thapsigargin and cyclopiazonic acid, inhibitors of sarcoendoplasmic reticulum calcium ATPase, partially reduced the SICR. These findings suggest that both calcium influx across the cell membrane and calcium release from the sarcoendoplasmic reticulum are involved in the SICR. Nifedipine, diltiazem, and verapamil, inhibitors of L-type calcium channels, reduced the SICR in a dose-dependent manner. Furthermore, small interfering RNA against the L-type calcium channel α1c subunit diminished the SICR dramatically. Nifedipine also diminished the mechanosensitivity of Langendorff-perfused rat heart. These results suggest that the SICR in H9c2 cardiomyocytes involves the activation of L-type calcium channels and subsequent calcium release from the sarcoendoplasmic reticulum.     

Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells

The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch clamp and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.     

Endothelin-induced prostacyclin production in rat aortic endothelial cells: role of calcium.

Endothelin (ET) is a potent vasoconstrictor peptide, released from endothelial cells, which is associated with prostaglandin (PG) release. The mechanism by which ET causes the release of PG is not clearly understood. We used rat aortic endothelial cells to investigate the role of calcium (Ca2+) in ET-1-induced prostacyclin (PGI2) release. ET-1 (10(-9) M) produced a significant increase in PGI2 release. Pretreatment of rat aortic endothelial cells with different doses (10(-9) M and 10(-6) M) of diltiazem (voltage-sensitive L-type calcium channel blocker) produced significant inhibition of ET-1- and PDBu-induced PGI2 release. Inhibition was first noted at 10(-9) M and was complete at 10(-6) M. Conversely, pretreatment of rat aortic endothelial cells with different doses (10(-9) M and 10(-6) M) of calcium channel blockers (thapsigargin, an intracellular calcium channel blocker or conotoxin, a voltage-sensitive N-type calcium channel blocker) produced no changes on ET-1- or PDBu-induced PGI2 release. These results provide further support for the concept that PKC mediates ET-induced PGI2 release in rat aortic endothelial cells via an increase in intracellular calcium and this increase is due to the influx of extracellular calcium and not to the release of calcium from the sarcoplasmic reticulum.     

Air Bubble Contact with Endothelial Cells Causes a Calcium-Independent Loss in Mitochondrial Membrane Potential

Objective

Gas microembolism remains a serious risk associated with surgical procedures and decompression. Despite this, the signaling consequences of air bubbles in the vasculature are poorly understood and there is a lack of pharmacological therapies available. Here, we investigate the mitochondrial consequences of air bubble contact with endothelial cells.

Methods and Results

Human umbilical vein endothelial cells were loaded with an intracellular calcium indicator (Fluo-4) and either a mitochondrial calcium indicator (X-Rhod-1) or mitochondrial membrane potential indicator (TMRM). Contact with 50–150 µm air bubbles induced concurrent rises in intracellular and mitochondrial calcium, followed by a loss of mitochondrial membrane potential. Pre-treating cells with 1 µmol/L ruthenium red, a TRPV family calcium channel blocker, did not protect cells from the mitochondrial depolarization, despite blocking the intracellular calcium response. Mitigating the interactions between the air-liquid interface and the endothelial surface layer with 5% BSA or 0.1% Pluronic F-127 prevented the loss of mitochondrial membrane potential. Finally, inhibiting protein kinase C-α (PKCα), with 5 µmol/L Gö6976, protected cells from mitochondrial depolarization, but did not affect the intracellular calcium response.

Conclusions

Our results indicate that air bubble contact with endothelial cells activates a novel, calcium-independent, PKCα-dependent signaling pathway, which results in mitochondrial depolarization. As a result, mitochondrial dysfunction is likely to be a key contributor to the pathophysiology of gas embolism injury. Further, this connection between the endothelial surface layer and endothelial mitochondria may also play an important role in vascular homeostasis and disease.     

Imaging calcium entering the cytosol through a single opening of plasma membrane ion channels: SCCaFTs--fundamental calcium events

Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca(2+) permeable ion channel using Ca(2+) indicators like fluo-3. These Single Channel Ca(2+) Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca(2+) sparks and Ca(2+) puffs caused by Ca(2+) release from intracellular stores (due to the opening of ryanodine receptors and IP(3) receptors, respectively). In contrast to intracellular Ca(2+) release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca(2+) handling in the vicinity of a channel with a known Ca(2+) influx, to obtain the Ca(2+) current passing through plasma membrane cation channels in near physiological solutions, to localize Ca(2+) permeable ion channels on the plasma membrane, and to estimate the Ca(2+) currents underlying those elementary events where the Ca(2+) currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca(2+) channels, and stretch-activated channels. For the L-type Ca(2+) channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca(2+) currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.     

Calcium channels in PDGF-stimulated A172 cells open after intracellular calcium release and are not voltage-dependent.

Using laser image cytometry and Indo-1 fluorescence, we investigated the intracellular free Ca2+ concentration ([Ca2+]i) of confluent A172 human glioblastoma cells stimulated by the BB homodimer of platelet-derived growth factor (PDGF-BB). The shape of the calcium transients and the delay time between stimulation and the beginning of the transient varied considerably. The percentage of responsive cells, the peak [Ca2+]i and the duration of the response were directly related to PDGF-BB dose, while the delay time was inversely related; the maximal response occurred at a PDGF-BB concentration of 20 ng/ml. Studies with EGTA and inorganic calcium-channel blockers (Ni2+, La3+) showed that the increase of [Ca2+]i resulted from initial release of intracellular stores and subsequent calcium influx across the plasma membrane. Opening of calcium channels in the plasma membrane, monitored directly by studying Mn2+ quenching of Indo-1 fluorescence, was stimulated by PDGF-BB and blocked by La3+; the opening occurred 55 +/- 10 s after the initial increase in [Ca2+]i. Therefore, in these tumor cells, intracellular release always occurs before channel opening in the plasma membrane. Depolarization of cells with high extracellular [K+] did not generally induce calcium transients but did decrease calcium influx. L-type calcium-channel blockers (verapamil, nifedipine, and diltiazem) had little or no effect on the calcium influx induced by PDGF-BB. These results indicate that PDGF-BB induces calcium influx by a mechanism independent of voltage-sensitive calcium channels in A172 human glioblastoma cells.     

Pseudomonas aeruginosa cytotoxin stimulates prostacyclin production in cultured pulmonary artery endothelial cells: membrane attack and calcium influx

The effects of highly purified Pseudomonas aeruginosa cytotoxin were investigated on cultured pulmonary artery endothelial cells. This toxin dose-dependently (7.5-60 micrograms/ml) and time-dependently (20-75 minutes) stimulated the release of radiolabeled arachidonic acid and metabolites and the synthesis of prostacyclin in the absence of overt cell damage (no enhanced lactate dehydrogenase [LDH] release). Preincubation of the toxin with neutralizing antibodies abolished the effect. The toxin response on endothelial cells required extracellular calcium but not magnesium and was accompanied by a calcium influx. Interference with intracellular calcium function by TMB 8 or with (calcium)-calmodulin function by trifluoperazine and W7 dose-dependently reduced the cytotoxin mediated synthesis of prostacyclin. Calcium channel blockers (nimodipine, diltiazem, verapamil, D 888), however, were ineffective in this system. Following addition of cytotoxin to endothelial cells, an increased passive permeability for small marker molecules (potassium, 45calcium, 3H-sucrose), but for large ones (3H-inulin, 3H-dextran, LDH) was noted, suggesting that cytotoxin creates discrete hydrophilic transmembrane lesions of about 0.5-1.5 nm in diameter. These data are compatible with the notion that Pseudomonas aeruginosa cytotoxin triggers the arachidonic acid pathway in cultured pulmonary artery endothelial cells by calcium influx and suggest that this calcium influx may proceed through toxin created transmembrane lesions.     

Thrombin-induced activation of calcium transport pathways and their role in platelet functions

In human platelets thrombin-induced calcium release from intracellular stores, the consequent influx of extracellular calcium, as well as their role in the aggregation and ATP-secretion reactions were examined. In indo-1-loaded platelets intracellular calcium release was studied in the presence of excess EGTA in the incubation medium, while calcium influx was followed after a rapid repletion of external calcium. After thrombin-stimulation both calcium release and calcium influx produced about the same peak levels of cytoplasmic free calcium but in the first case it was only a transient response, while in the latter one a sustained calcium signal was observed. Increased calcium influx could be evoked for several minutes after the addition of thrombin, it was selectively inhibited by Mg2+ (20 mM) and Ni2+ (1 mM) ions, by neomycin and by PCMB, a non-penetrating SH-group reagent. This calcium influx was practically insensitive to organic calcium channel blockers. Thrombin-induced platelet aggregation was only partial in the absence of external calcium, even if excess magnesium was present in the media, while the aggregation response became complete if external calcium was repleted. A significantly reduced aggregation could be seen in calcium-containing media if calcium influx was selectively inhibited. Platelet ATP-secretion under the same conditions did not depend on external calcium or on calcium influx. These data indicate that in thrombin-stimulated platelets the opening of specific plasma membrane calcium channels can be selectively modulated and these channels play a major role in the development of a full-scale aggregation.     

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.     

Voltage-independent calcium influx in smooth muscle

In smooth muscle cells, agonists such as neurotransmitters or hormones can induce an increase in [Ca(2+)](i) via a release of intracellular stored calcium or/and an influx of extracellular calcium. The calcium entry pathway operates through a variety of plasmalemmal calcium channels which involve voltage-dependent and voltage-independent calcium channels. Voltage-independent calcium channels include (1) receptor-operated channels (ROCs) activated by agonist-receptor interaction and, in the majority of cases, the downstream signal transduction proteins, (2) store-operated channels (SOCs) activated by the emptying of intracellular Ca(2+) store (mainly the sarcoplasmic reticulum), (3) mechanosensitive or stretch-activated channels (SACs) activated by membrane stretch. Generally, voltage-independent calcium channels are calcium permeable non-selective cation channels with electrophysiological differences, complex regulatory mechanisms and pharmacology. Although the molecular identity of voltage-independent calcium channels is not yet fully elucidated, there are growing evidences that these channels correspond to a new family of membrane proteins encoded by mammalian homologues of specific transient receptor potential (TRP) genes. Several types of TRP proteins are ubiquitously expressed in smooth muscle cells and variations in the expression depend on tissue and species. More recently, other proteins such as Orai1 and STIM1 proteins have been also proposed as participating in the molecular identity of voltage-independent calcium channels. These channels control phenomena such as smooth muscle cells proliferation and/or contraction.     

Cyclosporine A-induced prostacyclin release is maintained by extracellular calcium in rat aortic endothelial cells: role of protein kinase C.

Chronic treatment with the immunosuppressive drug Cyclosporine A (CsA) is associated with increased intracellular calcium in vascular smooth muscle cells, which may activate phospholipase A2. We used rat aortic endothelial cells to investigate the role of protein kinase C (PKC) in CsA-induced prostacyclin (PGI2) release. CsA (10(-9) M) produced a significant increase in PGI2 release. CsA-induced PGI2 release were inhibited 80-85% by 10(-9) M, and 99-100% by 10(-6) M pretreatment doses of any of three different PKC inhibitors, i.e. 1-(5-isoquinolinesulfonylmethyl)piperazine(H7), staurosporine or 1-(5-isoquinolinesulfonyl)piperazine. Pretreatment with (10(-9) M) of diltiazem (a voltage-sensitive L-type calcium channel blocker) completely inhibited both CsA-induced PGI2 release. Conversely, pretreatment with (10(-9) M) of thapsigargin (an intracellular calcium channel blocker) did not alter the action of CsA. These results strongly suggest that PKC, in association with an influx of extracellular calcium, mediates CsA-induced PGI2 release in rat aortic endothelial cells.     

nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells

In Drosophila, nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory synaptic transmission in mushroom body Kenyon cells, a neuronal population involved in generation of complex behaviors, including responses to drugs of abuse. To determine whether activation of nAChRs can induce cellular changes that contribute to functional plasticity in these neurons, we examined nicotine-evoked responses in cells cultured from brains of late stage OK107-GAL4 pupae. Kenyon cells can be identified by expression of green fluorescent protein (GFP+). Nicotine activates alpha-bungarotoxin-sensitive nAChRs, causing a rapid increase in intracellular calcium levels in over 95% of the Kenyon cells. The nicotine-evoked calcium increase has a voltage-gated calcium channel (VGCC) dependent component and a VGCC-independent component that involves calcium influx directly through nAChRs. Thapsigargin treatment reduces the nicotine response consistent with amplification by calcium release from intracellular stores. The response to nicotine is experience-dependent: a short conditioning pulse of nicotine causes a transient 50% reduction in the magnitude of the response to a test pulse of nicotine when the interpulse interval is 4 h. This cellular plasticity is dependent on activation of the VGCC-component of the nicotine response and on cAMP-signaling, but not on protein synthesis. These data demonstrate that activation of nAChRs induces a calcium-dependent plasticity in Kenyon cells that could contribute to adult behaviors involving information processing in the mushroom bodies including responses to nicotine.     

Stretch-elicited calcium responses in the intact mouse thoracic aorta

Stretch-elicited intracellular calcium ([Ca(2+)](i)) changes in individual smooth muscle cells in a ring of aorta were measured simultaneously with the force developed by the ring. A phasic increase in [Ca(2+)](i) was observed in 30% of the cells and a sustained one in 10%. Depletion of intracellular calcium store by thapsigargin and caffeine decreased phasic and increased sustained calcium responses. The inhibition of calcium entry either by stretching the aorta in a calcium-free medium or by the inhibition of stretch-activated, non-selective cationic channels by 5 microM GsMtx-4 toxin, decreased the proportion of sustained [Ca(2+)](i) responses but increased transient responses. In this condition, a third of the cells responded to stretch by a bursts of [Ca(2+)](i) spikes. The decrease of calcium influx triggered the generation of burst of calcium spikes after the application of stretch steps to the vascular wall. We conclude that progressive recruitment of smooth muscle cells is the mechanism underlying the force-generating part of the myogenic response. Two types of stretch-elicited calcium responses were observed during the recruitment of the smooth muscle cells. One was a phasic calcium discharge generated by the sarcoplasmic reticulum. The second was a tonic response produced by the activation of the stretch-sensitive cationic channels allowing extracellular Ca(2+) entry.     

Arachidonate mobilization is coupled to depletion of intracellular calcium stores and influx of extracellular calcium in differentiated U937 cells

We have previously reported that dimethylsulfoxide-differentiation of U937 cells induced significant A23187-stimulatable arachidonate mobilization, consistent with characteristics of cytosolic phospholipase A₂ (Rzigalinski, B.A. and Rosenthal, M.D. (1994) Biochim. Biophys. Acta 1223, 219–225). The present report demonstrates that differentiated cells attained higher elevations of intracellular free calcium in response to A23187 and thapsigargin, consistent with enhancement of the capacitative calcium influx pathway. Differentiation induced a significant increase in the size of the intracellular calcium stores, as well as in the capacity for store-activated calcium influx. Alterations in the capacitative calcium influx pathway were coupled to differentiation-induced activation of cPLA₂ and mobilization of arachidonate in response to thapsigargin and fMLP stimulation. Although cPLA₂ activity is often associated with influx of extracellular calcium, arachidonate mobilization in response to thapsigargin or fMLP was not simply a consequence of calcium influx. Assessment of intracellular free calcium elevations during thapsigargin or fMLP-induced stimulation suggest that a low level of arachidonic acid release was initiated upon release of intracellular store calcium. This initial release of arachidonate was unaffected by inhibition of calcium influx with nickel, EGTA, or SKF96365. Arachidonate release was observed when extracellular calcium was replaced with extracellular strontium, suggesting activation of the cytosolic PLA₂ rather than secretory PLA₂. Inhibition of PLA₂ with prostaglandin B oligomer prevented both thapsigargin and fMLP-stimulated influx of extracellular calcium. Furthermore, exogenous free arachidonate stimulated influx of extracellular calcium in differentiated U937 cells. These results suggest that cPLA₂-mediated release of free arachidonate may participate in the formation of a calcium influx factor which controls influx of extracellular calcium through store-controlled channels in the plasma membrane.     

Modulation of venular microvessel permeability by calcium influx into endothelial cells.

It has been proposed that calcium ion influx into endothelial cells modulates the permeability of venular microvessels via a calcium-dependent contractile process. The results of recent investigations using permeabilized endothelial cell monolayers conform to this hypothesis by demonstrating a calcium-dependent interaction of endothelial actin and myosin during the retraction of adjacent endothelial cells exposed to inflammatory agents. Little is known about the pathway for calcium influx into endothelial cells after exposure to mediators of inflammation, but evidence suggests that the properties of the calcium entry pathways are similar to the calcium entry pathways that regulate the release of endothelium-derived relaxing factor (EDRF). Substances that stimulate EDRF release from arterial endothelium also increase venular microvessel permeability. Recently developed methods to measure cytoplasmic calcium concentration in the endothelial cells forming the walls of individually perfused microvessels enable a direct investigation of the modulation of the permeability of venular microvessels by calcium influx. These experiments demonstrate that the magnitude of the initial increase in the permeability of microvessels after exposure to an agent that increases permeability, such as a calcium ionophore, is determined by the magnitude of calcium ion influx into the endothelial cells. Furthermore, the magnitude of the calcium influx into endothelial cells is modulated by the membrane potential of the endothelial cells. Depolarization of the endothelial cell membrane reduces calcium influx and attenuates increases in permeability whereas hyperpolarization of the endothelial membrane increases calcium influx and potentiates increases in permeability. These data conform to the hypothesis that a passive conductance channel for calcium is a major pathway for calcium ion flux responsible to eliciting an increase in the permeability of the endothelial barrier in microvessels.