Treponema denticola Outer Membrane Inhibits Calcium Flux in Gingival Fibroblasts

Treponema denticola is a cultivable oral spirochete which perturbs the cytoskeleton in cultured cells of oral origin, but intracellular signalling pathways by which it affects actin assembly are largely unknown. As the outer membrane (OM) of Treponema denticola disrupts actin-dependent processes that normally require precise control of intracellular calcium, we studied the effects of an OM extract on internal calcium release, ligand-gated and calcium release-activated calcium channels, and related mechanosensitive cation fluxes in human gingival fibroblasts (HGF). Single-cell ratio fluorimetry demonstrated that in resting cells loaded with Fura-2, baseline intracellular Ca²⁺ concentration ([Ca²⁺]_i) was not affected by treatment with OM extract, but normal spontaneous [Ca²⁺]_i oscillations were dramatically increased in frequency for 20 to 30 min followed by complete blockade. OM extract inhibited ATP-induced and thapsigargin-induced release of calcium from intracellular stores by 40 and 30%, respectively. Addition of Ca²⁺ to the extracellular pool following depletion of intracellular Ca²⁺ by thapsigargin and extracellular Ca²⁺ by EGTA yielded 59% less replenishment of [Ca²⁺]_i in OM extract-treated than in control HGF. In cells loaded with collagen-coated ferric oxide beads to stimulate integrin-dependent calcium release, baseline [Ca²⁺]_i was nearly doubled but was not significantly different in control and OM extract-treated cells. Magnetically generated tensile forces on the beads induced >300% increases of [Ca²⁺]_i above baseline. Cells preincubated with OM extract exhibited dose-dependent and time-dependent reductions in stretch-induced [Ca²⁺]_i transients, which were due to neither loss of beads from the cells nor cell death. The T. denticola OM inhibitory activity was eliminated by heating the OM extract to 60°C and by boiling but not by phenylmethylsulfonyl fluoride treatment. Thus nonlipopolysaccharide, nonchymotrypsin, heat-sensitive protein(s) in T. denticola OM can evidently inhibit both release of calcium from internal stores and uptake of calcium through the plasma membrane, possibly by interference with calcium release-activated channels.     

Enhanced Na+/H+ exchange activity contributes to the pathogenesis of muscular dystrophy via involvement of P2 receptors

A subset of muscular dystrophy is caused by genetic defects in dystrophin-associated glycoprotein complex. Using two animal models (BIO14.6 hamsters and mdx mice), we found that Na⁺/H⁺ exchanger (NHE) inhibitors prevented muscle degeneration. NHE activity was constitutively enhanced in BIO myotubes, as evidenced by the elevated intracellular pH and enhanced ²²Na⁺ influx, with activation of putative upstream kinases ERK42/44. NHE inhibitor significantly reduced the increases in baseline intracellular Ca²⁺ as well as Na⁺ concentration and stretch-induced damage, suggesting that Na⁺_i-dependent Ca²⁺overload via the Na⁺/Ca²⁺ exchanger may cause muscle damage. Furthermore, ATP was found to be released continuously from BIO myotubes in a manner further stimulated by stretching and that the P2 receptor antagonists reduce the enhanced NHE activity and dystrophic muscle damage. These observations suggest that autocrine ATP release may be primarily involved in genesis of abnormal ionic homeostasis in dystrophic muscles and that Na⁺-dependent ion exchangers play a critical pathological role in muscular dystrophy.     

Different Reactions of Aortic and Venular Endothelial Cell Monolayers to Histamine on Macromolecular Permeability: Role of cAMP, Cytosolic Ca2+ and F-actin

Endothelial cells assume a central role in the one process that the permeation of microvessels is accelerated in case of inflammation. We studied the effect of histamine on endothelial permeability, [Ca²⁺]_i, cAMP and F-actin, using same origin aortic and venular cultured endothelial monolayers. When HUVEC were treated with histamine (10^–7–10^–5 M), permeability of FITC-dextran (molecular weight 70,000) and [Ca²⁺]_i were increased, while cAMP content was unchanged, and F-actin content was reduced. When bovine vein-derived endothelial cells were treated with histamine, [Ca²⁺]_i was increased via H1 receptors, but permeability and F-actin content were not altered. When human aorta-derived endothelial cells were, [Ca²⁺]_i was increased via H1 receptors and cAMP content was increased via H2 receptors, while permeability and F-actin content were not changed. When bovine aorta-derived endothelial cells were, cAMP and F-actin content were increased, while permeability was reduced. These findings suggest that endothelial cells derived from different tissues clearly showed the different reactions to histamine, the increase in [Ca²⁺]_i led to the increase in endothelial permeability, while the increase in cAMP levels led to the reduction in permeability, and finally, F-actin regulated endothelial macromolecular permeability.     

Pasteurella haemolytica A1-Derived Leukotoxin and Endotoxin Induce Intracellular Calcium Elevation in Bovine Alveolar Macrophages by Different Signaling Pathways

Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381–388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237–252, 1995). Furthermore, extracellular Ca²⁺ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca²⁺ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca²⁺ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca²⁺ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca²⁺ in a concentration-dependent fashion by influx of extracellular Ca²⁺ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca²⁺ even in the absence of extracellular Ca²⁺. Leukotoxin-induced intracellular Ca²⁺ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A₂ and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca²⁺ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca²⁺ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A₂ and C and release of arachidonic acid in bovine alveolar macrophages. Ca²⁺ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca²⁺ signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.     

Activation of the volume-sensitive chloride current in vascular endothelial cells requires a permissive intracellular Ca2+ concentration

Combined patch clamp and Ca²⁺-measurements (Fura-2) were used to study the dependence of volume-activated Cl^–-currents (I_Cl,vol) of endothelial cells from bovine pulmonary artery on the intracellular Ca²⁺-concentration [Ca²⁺]_i. Loading the cells with high concentrations of EGTA or BAPTA via ruptured membrane patches or by preincubating them with 50 M BAPTA-AM caused a substantial decrease of I_Cl,vol. This reduction was independent of the activation state of the current: the current amplitude was not only diminished if [Ca²⁺]_i was lowered at the peak of the volume-activated current, but this low Ca²⁺-concentration also prevented activation of the current by a second hypotonic challenge.I_Cl,vol is already maximally activated at intracellular Ca²⁺-concentrations between 50 and 100 nmol/l, a further increase of [Ca²⁺]_i does not affect the size of I_Cl,vol.These results indicate that a sustained full activation of I_Cl,vol in endothelial cells requires submicromolar concentrations of Ca²⁺, and that changes in [Ca²⁺]_i do not modulate the current.     

Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential

We studied the role of the membrane potential in the control of the intracellular free calcium concentration ([Ca²⁺]_i) and release of the two autacoids endothelium-derived relaxing factor (EDRF = nitric oxide) and prostaglandin I₂ in endothelial cells. ATP (3 mol/l) and bradykinin (1 nmol/l) evoked rapid increases (sixfold) in [Ca²⁺]_i in cultured endothelial cells. [Ca²⁺]_i remained elevated over several minutes. When the cells were depolarized, either by K⁺ (70–90 mmol/l) or by preincubation with the blocker of K⁺ channels tetraethylammonium (3 mmol/l), the initial peak of [Ca²⁺]_i remained unaffected but [Ca²⁺]_i returned significantly faster to resting levels, indicating a reduction in Ca²⁺ influx. In native, freshly isolated endothelial cells, K⁺ abolished increases in [Ca²⁺]_i induced by acetylcholine (3 mol/l). Release of EDRF in response to bradykinin (cultured cells) and acetylcholine (native cells) was inhibited by K⁺ (by 70%), whereas release of prostaglandin I₂ was not significantly reduced. Preincubation of cultured endothelial cells with the receptor-independent stimulus thimerosal (5 mol/l, 40 min) evoked a long-lasting release of EDRF and small elevations of [Ca²⁺]_i (twofold) after washout of the drug. Depolarization with K⁺ decreased thimerosal-induced EDRF release and [Ca²⁺]_i in a reversible manner. In patch-clamped endothelial cells, bradykinin (1 nmol/l) induced transient hyperpolarizations that were significantly prolonged by BRL 34915 (1 mol/l), an activator of K⁺ channels. BRL 34915 also elicited increases in [Ca²⁺]_i, particularly in thimerosal-stimulated endothelial cells. These effects were abolished by K⁺. We conclude that the initial rise in [Ca²⁺]_i in response to receptor-binding agonists, caused by mobilization of Ca²⁺ from intracellular stores, activates K⁺ channels, thereby inducing hyperpolarization. This hyperpolarization provides the driving force for transmembrane Ca²⁺ influx into endothelial cells and is thus an important signal for synthesis and release of EDRF.     

Histamine-activated,non-selective cation currents and Ca2+ transients in endothelial cells from human umbilical vein

Permeation properties and modulation of an ionic current gated by histamine were measured in single endothelial cells from human umbilical cord veins by use of the patch-clamp technique in the ruptured-whole-cell mode or using perforated patches. We combined these current measurements with a microfluorimetric method to measure concomitantly free intracellular calcium concentration ([Ca²⁺]_i). Application of histamine induced an intracellular calcium transient and an ionic current that reversed near 0 mV. The amplitude of the current ranged from –0.2 to –2nA at –100mV. The tonic rise in [Ca²⁺]_i and the ionic current are partly due to Ca²⁺ influx. This Ca²⁺ entry pathway is also permeable for Ba²⁺ and Mn²⁺. The amplitude of the histamine-activated current was also closely correlated with the amplitude of the concomitant Ca²⁺ transient, suggesting that the latter is at least partially due to Ca²⁺ influx through histamine-activated channels. The reversal potential of the histamine-induced current was 7.6±4.1 mV (n=14) when the calcium concentration in the bath solution ([Ca²⁺]_o) was 1.5mmol/l. With 10 mmol/l [Ca²⁺]_o it was –13.7±4.7 mV and shifted to +13.0±1.5 mV in nominally Ca²⁺-free solution (n=3 cells). The amplitude of the current in Ca²⁺-free solution was enhanced compared to that in 10 mmol/l [Ca²⁺]_o. The shift of the reversal potential and the concomitant change of the current amplitude suggest that the channel is permeable for calcium but has a smaller permeability for calcium than for monovalent cations. The latency between the application of histamine and the appearance of the current was voltage dependent and was much smaller at more negative potentials. This effect is unlikely to be due to desensitization, but may suggest a voltage-dependent step in the signal transduction chain. Similar histamine-induced Ca²⁺ signals were observed if the currents were measured in patches perforated with nystatin. The onset of the agonist-activated current was, however, much more delayed and its amplitude significantly lower than in ruptured patches. The histamine-induced currents and intracellular Ca²⁺-transients were largely reduced after incubation of endothelial cells with the phorbol ester TPA. H7, a blocker of protein kinase C, induced membrane currents and Ca²⁺ signals in the absence of an agonist. It is concluded that the agonist-activated Ca²⁺-entry in endothelial cells occurs through non-selective cation channels which can be down-regulated by protein kinase C activation.     

炎性环境下内皮微粒介导单核细胞促内皮细胞增殖的作用

目的 探讨炎性环境中内皮微粒(EMPs)在单核细胞参与的促内皮细胞增殖中的作用,并探讨EMPs对单核细胞表达内皮细胞标志物的影响.方法 体外培养人脐静脉内皮细胞(HUVECs),炎性因子肿瘤坏死因子α(TNF-α)刺激内皮细胞,超速离心法获取EMPs;用透射电镜观察其形态和结构;运用酶联免疫吸附法(ELISA)检测经EMPs激活的单核细胞株THP-1对血管内皮生长因子(VEGF)中VEGF-A、VEGF-C的表达情况;将HUVECs和THP-1细胞或者经EMPs诱导的THP-1细胞共培养,检测HUVECs的增殖情况;运用RT-PCR和免疫荧光细胞化学技术,观察经EMPs激活的THP-1细胞对内皮细胞标志物vWF和血管内皮生长因子受体2(VEGFR-2)的表达情况.结果 内皮细胞经炎性因子刺激后释放EMPs,EMPs刺激的THP-1细胞上清中VEGF-A和VEGF-C蛋白水平明显升高;HUVECs细胞增殖实验中,与THP-1细胞或者经EMPs诱导的THP-1共培养的两种情况下,HUVECs均显著增多;与EMPs共培养3d后,THP-1中vWF和VEGFR-2蛋白表达为阳性,基因表达为阴性,共培养8d后,vWF和VEGFR-2的蛋白和基因表达均为阴性.结论 炎性环境中内皮细胞释放EMPs可以促使单核细胞分泌VEGF-A和VEGF-C,后两者可使HUVECs增殖,EMPs仅能使单核细胞一过性呈现内皮表型,而不能将其转分化为内皮细胞.     

Thapsigargin discharges intracellular calcium stores and induces transmembrane currents in human endothelial cells

We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase), on membrane currents and on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single endothelial cells from the human umbilical cord vein. Currents were recorded by means of the patchclamp technique in the whole-cell mode and [Ca²⁺]_i was measured using Fura II. Application of thapsigargin at concentrations between 0.2 and 2 mol/l induced a slow increase in [Ca²⁺]_i to a peak value of 400±110 nmol/l above a resting level of 120±35 nmol/l, and then slowly declined to a new steady-state level of 315±90 nmol/l (n=33). The thapsigargin-induced increase in [Ca²⁺]_i depended on the extracellular Ca²⁺ concentration ([Ca²⁺]_o: it declined after removal of extracellular Ca²⁺, but increased again when [Ca²⁺]_o was augmented, indicating that the response depends on a transmembrane influx of Ca²⁺ ions. The peak amplitude of the histamine-induced Ca²⁺ transient was reduced in the presence of thapsigargin. This reduction was more pronounced when histamine was applied at the peak of the increase in [Ca²⁺]_i induced by thapsigargin than during the rising phase of the changes in [Ca²⁺]_i. The decline of the Ca²⁺ transient induced by histamine after washing out the agonist was also affected by thapsigargin. Before application of thapsigargin, this decline could be described by a single exponential with a time constant equal to 24.5±5 s (n=7). In the presence of thapsigargin, the decline was much slower (n =8 cells), although in four cells a fraction of about 23% still exchanged with a similar fast value of 29.4±4 s. Thapsigargin also induced a slowly developing inward current in endothelial cells at a holding potential of –40 mV. Voltage ramps applied before and during the development of this current indicated that a non-selective cation channel with a reversal potential near 0 mV was activated. In contrast with the Ca²⁺ transients, these currents did not show a declining phase. These results indicate that inhibition of the endoplasmic Ca²⁺ pump in endothelial cells increases [Ca²⁺]_i. The tonic component of this increase might be partly due to opening of non-selective Ca²⁺-permeable cation channels activated by depletion of intracellular stores.     

Epidermal vascular endothelial growth factor production is required for permeability barrier homeostasis, dermal angiogenesis, and the development of epidermal hyperplasia: implications for the pathogenesis of psoriasis

Primary abnormalities in permeability barrier function appear to underlie atopic dermatitis and epidermal trauma; a concomitant barrier dysfunction could also drive other inflammatory dermatoses, including psoriasis. Central to this outside-inside view of disease pathogenesis is the epidermal generation of cytokines/growth factors, which in turn signal downstream epidermal repair mechanisms. Yet, this cascade, if sustained, signals downstream epidermal hyperplasia and inflammation. We found here that acute barrier disruption rapidly stimulates mRNA and protein expression of epidermal vascular endothelial growth factor-A (VEGF-A) in normal hairless mice, a specific response to permeability barrier requirements because up-regulation is blocked by application of a vapor-impermeable membrane. Moreover, epidermal vegf^−/− mice display abnormal permeability barrier homeostasis, attributable to decreased VEGF signaling of epidermal lamellar body production; a paucity of dermal capillaries with reduced vascular permeability; and neither angiogenesis nor epidermal hyperplasia in response to repeated tape stripping (a model of psoriasiform hyperplasia). These results support a central role for epidermal VEGF in the maintenance of epidermal permeability barrier homeostasis and a link between epidermal VEGF production and both dermal angiogenesis and the development of epidermal hyperplasia. Because psoriasis is commonly induced by external trauma [isomorphic (Koebner) phenomenon] and is associated with a prominent permeability barrier abnormality, excess VEGF production, prominent angiogenesis, and epidermal hyperplasia, these results could provide a potential outside-inside mechanistic basis for the development of psoriasis.     

Contribution of Macrophages to Angiogenesis Induced by Vascular Endothelial Growth Factor Receptor-3-Specific Ligands

Vascular endothelial growth factor receptor (VEGFR)-2 is a major stimulator of hemangiogenesis (HA), whereas VEGFR-3 stimulates lymphangiogenesis (LA). Contrary to this understanding, we demonstrate that implantation of pellets containing VEGFR-3-specific ligands (VEGF-C156S and recombinant murine VEGF-D) into the corneal stroma induce not only LA but also robust HA characterized by blood vessels that are positive for VEGFR-3 expression. The implantation of pellets containing VEGFR-3-specific ligands also leads to the recruitment of VEGF-A-secreting macrophages. Depletion of these infiltrating macrophages using clodronate-liposome administration shows a significant reduction in HA as well as LA. Blockade of either VEGFR-2 or VEGFR-3 signaling reduces both HA and LA; however, the percent reduction of HA is greater in the VEGFR-2 blockade group. In addition, in the VEGFR-3 blockade group, the percent reduction of HA is significantly greater with VEGFR-3-specific ligands than that by VEGF-A or VEGF-C. Collectively, our data suggest that VEGFR-3-specific signaling can induce new blood vessels, to which macrophages contribute a major role, and signify its potential as an additional therapeutic target to the existing VEGF-A/VEGFR-2 signaling-based antiangiogenesis strategies.Vascular endothelial growth factors (VEGFs), the key regulators of vasculogenesis, exert their effect via specific transmembrane tyrosine kinases, which include VEGF receptor (VEGFR)-1 (Flt-1), VEGFR-2 (KDR or Flk-1), and VEGFR-3 (Flt-4).^1,2,3 VEGF-A binds to both VEGFR-1 and VEGFR-2 and regulates hemangiogenesis (HA), whereas VEGF-C and VEGF-D bind to VEGFR-3 and regulate lymphangiogenesis (LA).^2,4 VEGF-C can also directly bind to VEGFR-2 and induce HA.⁵ Furthermore, VEGF-D and VEGF-C can be proteolytically processed and bind VEGFR-2 in addition to VEGFR-3.⁴The major function of VEGFR-2 is the stimulation of blood vascular endothelial cell survival/growth and promotion of HA.^2,4 VEGFR-2 is highly expressed in vascular endothelial progenitors in early embryogenesis.⁶ During later stages of vascular development, VEGFR-2 expression declines but can be up-regulated under conditions of pathological angiogenesis such as in tumors and in inflammation. During early embryogenesis, VEGFR-3 mRNA is expressed by most of the endothelial cells, and VEGFR-3 gene inactivation results in embryonic death because of abnormal remodeling of the primary vascular plexus.⁷ In the later stages of development, VEGFR-3 expression becomes gradually restricted to lymphatic vessels (LVs),⁸ although fenestrated blood capillaries of some adult organs continue to express low levels of VEGFR-3.⁹ VEGFR-3 is also expressed by some subsets of bone marrow-derived cells, including monocytes, macrophages, and dendritic cells.^10,11,12 The major functional role of VEGFR-3 during the postnatal period is thought to be limited to the induction of LVs.^3,4,13 It is reported that signaling via VEGFR-3 alone is sufficient for lymphangiogenic signals, because mutant VEGF-C156S, which only activates VEGFR-3^14,15 but not VEGFR-2, induces a similar phenotype to nonmutant VEGF-C-transgenic mice.^16,17On the basis of our current understanding, VEGFR-3-specific ligands, VEGF-C156S, and recombinant murine VEGF-D (rmVEGF-D)^16,17,18 should principally induce new LVs without significant concurrent blood vessel (BV) formation. Herein, we present data demonstrating that contrary to our expectations, VEGF-C156S and rmVEGF-D induce not only LVs but also significant BVs. Moreover, these newly formed blood vascular endothelial cells express copious VEGFR-3. Previously, VEGFR-3 expression has been shown only in neovascularization related to vascular tumors and some nonvascular tumors.^19,20,21 Induction of new BVs by VEGFR-3-mediated signaling alone has not yet been reported, even though one study previously reported the de novo expression of VEGFR-3 in pre-existing iris BVs injected with VEGF-A.²² This is important, because the contribution of VEGFR-3 to HA sheds light on potential limitations to most current antiangiogenic strategies that rely solely on blockade of VEGF-A or VEGFR-2. Our study also demonstrates that innate immune cells, particularly macrophages, are recruited in response to VEGFR-3 stimulation and contribute significantly to angiogenesis. Herein, we delineate the mechanisms of VEGFR-3⁺ newly formed BVs through VEGFR-3-specific signaling and demonstrate the effects of macrophage depletion as well as selective blockade of VEGFR-2 and VEGFR-3 on angiogenesis induced by VEGFR-3-specific ligands VEGF-C156S and rmVEGF-D.     

Chemically, mechanically, and hyperosmolarity-induced calcium responses of rat cortical capillary endothelial cells in culture

 The purpose of the present work was to characterize calcium responses of brain-capillary endothelial cells (BCEC), the cells forming the blood-brain barrier, to chemical, hyperosmolar and mechanical stimulation. Confluent BCEC cultures were grown from capillary fragments isolated from rat cerebral cortex. Intracellular free calcium ([Ca²⁺]_i) was measured using fura-2 and digital imaging. Our experiments show large endothelial calcium responses to substance P and ATP, up to a peak value of approximately 1000 and 600 nM, respectively, and these responses were observed in 2/3 of the cells. Calcium responses to bradykinin, histamine, and hyperosmolar sucrose or mannitol were smaller, attaining a peak in the range 180–340 nM, and were observed in a smaller fraction of the cells. No calcium responses were observed to high-potassium, l-glutamate, serotonin, carbachol, noradrenalin, and nitric-oxide donors. Consecutive superfusion of the cultures with ATP, bradykinin, and histamin showed that cells with a certain response pattern were spatially grouped; the response pattern itself varied widely between experiments. Mechanical stimulation of a single cell caused a calcium response in the stimulated cell in primary cultures and triggered an intercellularly propagating calcium wave in passaged cultures. Given the important effect of endothelial [Ca²⁺]_i on blood-brain barrier permeability and transport, we conclude that substance P and ATP are potential modulators of blood-brain barrier function. Hyperosmolarity-induced blood-brain barrier opening is probably not mediated through endothelial [Ca²⁺]_i. Received: 21 September 1998 / Accepted: 8 February 1999     

Membrane potential governs calcium influx into microvascular endothelium: integral role for muscarinic receptor activation

Key points

• Endothelial function in resistance vessels entails Ca²⁺ and electrical signalling to promote vasodilatation and increase tissue blood flow. Whether membrane potential (V _m) governs intracellular calcium concentration ([Ca²⁺]_i) of the endothelium remains controversial.
• [Ca²⁺]_i and V _m were evaluated simultaneously during intracellular current injection using intact endothelial tubes freshly isolated from mouse skeletal muscle resistance arteries.
• [Ca²⁺]_i did not change during hyperpolarization or depolarization under resting conditions. However in the presence of 100 nM ACh (∼EC₅₀), [Ca²⁺]_i increased during hyperpolarization and decreased during depolarization. These responses required extracellular Ca²⁺ and were attenuated by half with genetic ablation of TRPV4 channels.
• In native microvascular endothelium, half‐maximal stimulation of muscarinic receptors enables V _m to govern [Ca²⁺]_i by activating Ca²⁺‐permeable channels in the plasma membrane. This effect of V _m is absent at rest and can be masked during maximal receptor stimulation.

Abstract

In resistance arteries, coupling a rise of intracellular calcium concentration ([Ca²⁺]_i) to endothelial cell hyperpolarization underlies smooth muscle cell relaxation and vasodilatation, thereby increasing tissue blood flow and oxygen delivery. A controversy persists as to whether changes in membrane potential (V _m) alter endothelial cell [Ca²⁺]_i. We tested the hypothesis that V _m governs [Ca²⁺]_i in endothelium of resistance arteries by performing Fura‐2 photometry while recording and controlling V _m of intact endothelial tubes freshly isolated from superior epigastric arteries of C57BL/6 mice. Under resting conditions, [Ca²⁺]_i did not change when V _m shifted from baseline (∼−40 mV) via exposure to 10 μM NS309 (hyperpolarization to ∼−80 mV), via equilibration with 145 mm [K⁺]_o (depolarization to ∼−5 mV), or during intracellular current injection (±0.5 to 5 nA, 20 s pulses) while V _m changed linearly between ∼−80 mV and +10 mV. In contrast, during the plateau (i.e. Ca²⁺ influx) phase of the [Ca²⁺]_i response to approximately half‐maximal stimulation with 100 nm ACh (∼EC₅₀), [Ca²⁺]_i increased as V _m hyperpolarized below −40 mV and decreased as V _m depolarized above −40 mV. The magnitude of [Ca²⁺]_i reduction during depolarizing current injections correlated with the amplitude of the plateau [Ca²⁺]_i response to ACh. The effect of hyperpolarization on [Ca²⁺]_i was abolished following removal of extracellular Ca²⁺, was enhanced subtly by raising extracellular [Ca²⁺] from 2 mm to 10 mm and was reduced by half in endothelium of TRPV4^−/− mice. Thus, during submaximal activation of muscarinic receptors, V _m can modulate Ca²⁺ entry through the plasma membrane in accord with the electrochemical driving force.

Abbreviations

ACh

acetylcholine

BK_Ca

large‐conductance Ca²⁺‐activated K⁺ channel

[Ca²⁺]_i

intracellular Ca²⁺ concentration

[Ca²⁺]_o

extracellular Ca²⁺ concentration

EC

endothelial cell

EC₅₀

drug concentration giving half‐maximal response

E_K

Nernst equilibrium potential for K⁺

ER

endoplasmic reticulum

FCCP

carbonyl cyanide p‐trifluoromethoxyphenylhydrazone

GSK101

GSK1016790A

GSK219

GSK2193874

ID

internal diameter

[K⁺]_o

extracellular K⁺ concentration

NO

nitric oxide

OD

outer diameter

PSS

physiological salt solution

SEA

superior epigastric artery

SK_Ca/IK_Ca

small‐ and intermediate‐conductance Ca²⁺‐activated K⁺ channels

SMC

smooth muscle cell

TRP

transient receptor potential

TRPV4

transient receptor potential vanilloid type 4 channel

TRPV4^−/−

TRPV4 knockout

V_m

membrane potential

     

Calcium-phosphate metabolism parameters and erythrocyte Ca2+ concentration in autosomal dominant polycystic kidney disease patients with normal renal function

Introduction

The aim of this study was to assess calcium-phosphate metabolism of autosomal dominant polycystic kidney disease (ADPKD) patients with a special consideration to the following serum parameters: calcium (Ca²⁺), inorganic phosphate (Pi), parathyroid hormone (PTH) and intracellular erythrocyte calcium ([Ca²⁺]_i) concentrations.

Material and methods

The study included 49 adult ADPKD patients (19 males, 30 females) aged 36 ±11 years with normal renal function and no diagnosis of diabetes as well as 50 healthy controls (22 males, 28 females) matched for age and gender. Serum concentrations of sodium (Na⁺), potassium (K⁺) and magnesium (Mg²⁺) ions and Pi were determined with an indirect ion-selective method, while Ca2+ concentration was measured with a direct ion-selective method. The PTH was detected using a radioimmunometric method. [Ca²⁺]_i concentration was determined with the Ca²⁺ sensitive fluorescent dye Fura-2 method.

Results

In the ADPKD group, when compared to controls, the following concentrations were significantly higher: serum Ca²⁺ (1.18 ±0.06 mmol/l vs. 1.15 ±0.06 mmol/l, p = 0.0085), [Ca²⁺]_i (146.9 ±110.0 nmol/l vs. 96.5 ±52.7 nmol/l, p = 0.0075), serum Na+ (139.4 ±2.7 mmol/l vs. 138.5 ±2.1 mmol/l, p = 0.060, borderline significance), and PTH (15.5 ±6.8 pg/ml vs. 13.6 ±5.3 pg/ml, p = 0.066, borderline significance), while serum Mg²⁺ was significantly lower (0.81 ±0.09 mmol/l vs. 0.85 ±0.05 mmol/l, p = 0.021). In the ADPKD group we observed significant negative correlations of PTH with Ca²⁺ serum concentrations (Rs = –0.32, p = 0.025) and with estimated glomerular filtration rate (Rs = –0.31, p = 0.033).

Conclusions

The erythrocyte Ca²⁺ concentration is elevated in ADPKD patients with normal renal function. It may result from a dysfunction of mutated polycystins which can affect various aspects of electrolyte metabolism.     

Effects of leukotriene C4 and D4, histamine and bradykinin on cytosolic calcium concentrations and adhesiveness of endothelial cells and neutrophils

We compared the effects of leukotrienes B₄, C₄ and D₄ (LTB₄, C₄ and D₄) in vitro, as well as of histamine and bradykinin, on adhesive interactions between cultured umbilical vein endothelial cells (HUVEC) and polymorphonuclear neutrophils (PMN) and on cytosolic calcium transients, [Ca²⁺]_i. in vitro. LTB₄, but not LTC₄ or LTD₄ (at 1-100 nM), increased HUVEC adhesiveness for PMN, maximally 2·8-fold; in addition, PMN adhesion was augmented by LTB₄ (but not by LTC₄ and LTD₄) to a plastic surface. Rapid, but smaller increments of HUVEC (but not of PMN) adhesiveness were induced by histamine and bradykinin (at 10 μM). Nonetheless, LTC₄ and LTD₄ (at 100 nM) induced rapid rises of [Ca²⁺]_i in HUVEC, whereas ⁺100-fold higher concentrations were needed of histamine and bradykinin for similar rises. In PMN LTD₄ (and LTB₄) induced rapid increases of [Ca²⁺]_i, whereas no significant effect was seen with LTC₄, histamine or bradykinin. The [Ca²⁺]_i responses to LTC₄ and LTD₄ were inhibited by the peptidoleukotriene receptor blocker SKF 104,353. Thus, LTB₄ and the peptidoleukotrienes display disparate profiles as inducers of adhesion and calcium transients in PMN and HUVEC, indicating discrete differences in the stimulus response coupling for these closely related leukotrienes.     

Shear stress induced membrane currents and calcium transients in human vascular endothelial cells

We have measured membrane currents induced by shear stress together with intracellular calcium signals in endothelial cells from human umbilical cord veins. In the presence of extracellular calcium (Ca²⁺]_o), shear stress induced an inward current at a holding potential of 0 mV which is accompanied by a rise in intracellular Ca²⁺ ([Ca²⁺]_i). In the absence of extracellular calcium shear stress was unable to evoke a calcium signal but still induced a membrane current. The voltage dependence of the shear stress induced current was obtained from difference currents evoked by linear voltage ramps before and during application of shear stress. Its reversal potential E_rev shifted from –2.3±0.8 mV (n=4) in a nominally Ca²⁺ free solution to +1.5±1.6 mV at 1.5 mM [Ca²⁺]_o (n=4) and to +21.9±4.4 mV (n=7) at 10 mM [Ca²⁺]_o. From our data we conclude that shear stress opens an ion channel that is 12.5±2.9 (n=7) times more permeable for calcium than for sodium or cesium.     

Evidence for the existence of inositol (1,4,5)-trisphosphate- and ryanodine-sensitive pools in bovine endothelial cells. Ca2+ releases in cells with different basal level of intracellular Ca2+

In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca²⁺ transients in a Ca²⁺-free medium have been revealed, which evidently reflects Ca²⁺ release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca²⁺ ([Ca²⁺]_i) from about 50 to 110 nM, a biphasic dependence of the Ca²⁺ transients on resting [Ca²⁺]_i was shown and spontaneous Ca²⁺ oscillations were observed. At a [Ca²⁺]_i level over 110 nM, a pronounced rise in Ca²⁺ transients occurred and only single transients were observed. Ryanodine (10 μM) produced a transient [Ca²⁺]_i elevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release (IICR) and Ca²⁺-sensitive Ca²⁺ release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca²⁺ oscillations in BAE cells, whereas the ATP-induced (5–100 μM) Ca²⁺ response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous and ATP-induced Ca²⁺ transients was associated with a [Ca²⁺]_i decrease to a level below the initial resting [Ca²⁺]_i (undershoot). Its depth biphasically depended on the resting [Ca²⁺]_i from 50 to 110 nM, suggesting that the lack of a Ca²⁺ leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca²⁺ leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations and in ATP-induced transients. Received: 13 November 1995/Received after revision and accepted 27 March 1996     

噻庚啶和山莨菪碱拮抗TNFα诱导的内皮细胞[Ca2+]i增高

目的：研究噻庚啶（Cyp）和山莨菪碱（Ani）对肿瘤坏死因子（TNFα）诱导单个内皮细胞内Ca²⁺浓度（[Ca²⁺]_i）变化的影响，以探TNFα介导休克和Cyp、Ani的抗休克的机制。方法：人脐静脉内皮细胞株（ECV304）接种于35 mm含2 mL DMEM培养基的组织培养盘中培养。Fluo-3/AM负载细胞，激光扫描共聚焦显微技术（LSCM）测定单个内皮细胞[Ca²⁺]_i。结果：TNFα使单个内皮细胞[Ca²⁺]_i呈剂量依赖性升高，在60 s内达到峰值，然后下降并保持在基础水平之上。共聚焦扫描图像显示细胞核区[Ca²⁺]_i升高比胞浆区明显，下降比胞浆区慢。Cyp（3×10^-5 mol/L或6×10^-5 mol/L）、Ani（2×10^-5 mol/L或4×10^-5 mol/L）均能显著抑制由TNFα（1.2×10^-9 mol/L）诱导的单个内皮细胞[Ca²⁺]_i升高。结论：TNFα诱导内皮细胞[Ca²⁺]_i升高可能是TNFα介导休克的重要机制；Cyp和Ani抑制TNFα诱导的[Ca²⁺]_i升高可能是其抗休克作用的机制之一。     

Expression and localization of the multidrug resistance protein 5 (MRP5/ABCC5), a cellular export pump for cyclic nucleotides, in human heart

The multidrug resistance protein 5 (MRP5/ABCC5) has been recently identified as cellular export pump for cyclic nucleotides with 3′,5′-cyclic GMP (cGMP) as a high-affinity substrate. In view of the important role of cGMP for cardiovascular function, expression of this transport protein in human heart is of relevance. We analyzed the expression and localization of MRP5 in human heart [21 auricular (AS) and 15 left ventricular samples (LV) including 5 samples of dilated and ischemic cardiomyopathy]. Quantitative real-time polymerase chain reaction normalized to β-actin revealed expression of the MRP5 gene in all samples (LV, 38.5 ± 12.9; AS, 12.7 ± 5.6; P < 0.001). An MRP5-specific polyclonal antibody detected a glycoprotein of ~190 kd in crude cell membrane fractions from these samples. Immunohistochemistry with the affinity-purified antibody revealed localization of MRP5 in cardiomyocytes as well as in cardiovascular endothelial and smooth muscle cells. Furthermore, we could detect MRP5 and ATP-dependent transport of [³H]cGMP in sarcolemma vesicles of human heart. Quantitative analysis of the immunoblots indicated an interindividual variability with a higher expression of MRP5 in the ischemic (104 ± 38% of recombinant MRP5 standard) compared to normal ventricular samples (53 ± 36%, P < 0.05). In addition, we screened genomic DNA from our samples for 20 single-nucleotide polymorphisms in the MRP5 gene. These results indicate that MRP5 is localized in cardiac and cardiovascular myocytes as well as endothelial cells with increased expression in ischemic cardiomyopathy. Therefore, MRP5-mediated cellular export may represent a novel, disease-dependent pathway for cGMP removal from cardiac cells.