Background: Nitric oxide is key to endothelial regeneration, but it is still unknown whether endothelial cell (EC) loss results in an increase in NO levels at the wound edge. We have already shown that endothelial damage induces a long-lasting Ca2+ entry into surviving cells though connexin hemichannels (CxHcs) uncoupled from their counterparts on ruptured cells. The physiological outcome of injury-induced Ca2+ inflow is, however, unknown.Methods: In this study, we sought to determine whether and how endothelial scraping induces NO production (NOP) in the endothelium of excised rat aorta by exploiting the NO-sensitive fluorochrome, DAF-FM diacetate and the Ca2+-sensitive fluorescent dye, Fura-2/AM.Results: We demonstrated that injury-induced NOP at the lesion site is prevented in presence of the endothelial NO synthase inhibitor, L-NAME, and in absence of extracellular Ca2+. Unlike ATP-dependent NO liberation, the NO response to injury is insensitive to BTP-2, which selectively blocks store-operated Ca2+ inflow. However, injury-induced NOP is significantly reduced by classic gap junction blockers, and by connexin mimetic peptides specifically targeting Cx37Hcs, Cx40HCs, and Cx43Hcs. Moreover, disruption of caveolar integrity prevents injury-elicited NO signaling, but not the accompanying Ca2+ response.Conclusions: The data presented provide the first evidence that endothelial scraping stimulates NO synthesis at the wound edge, which might both exert an immediate anti-thrombotic and anti-inflammatory action and promote the subsequent re-endothelialization.

Endothelial injury is the primary event that leads to a variety of severe vascular disorders. Mechanical injury elicits a Ca(2+) response in the endothelium of excised rat aorta, which comprises an initial Ca(2+) release from inositol-1,4,5-trisphosphate (InsP(3))-sensitive stores followed by a long-lasting decay phase due to Ca(2+) entry through uncoupled connexons. The Ca(2+) signal may also adopt an oscillatory pattern, the molecular underpinnings of which are unclear. In the light of the role played by Ca(2+) spiking in tissue regeneration, this study aimed to unveil the mechanisms underlying injury-induced Ca(2+) oscillations. The latter reversibly ceased upon removal of extracellular Ca(2+) or addition of the gap junction blockers heptanol, 18 α,β-glycyrrhetinic acid, La(3+) and Ni(2+), but were insensitive to BTP-2 and SKF 96365. The spiking response was abolished by inhibiting the Ca(2+) entry mode of the Na(+)/Ca(2+) exchanger (NCX). The InsP(3)-producing agonist ATP resumed Ca(2+) oscillations in silent cells, while the phospholipase C inhibitor U73122 suppressed them. Injury-induced Ca(2+) transients were prevented by the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) blockers thapsigargin and cyclopiazonic acid, while they were unaffected by suramin and genistein. These data show for the first time that the coordinated interplay between NCX-mediated Ca(2+) entry and InsP(3)-dependent Ca(2+) release contributes to injury-induced intracellular Ca(2+) concentration oscillations.

The airway smooth muscle (ASM) layer within the airway wall modulates airway diameter and distensibility. Even in the relaxed state, the ASM layer possesses finite stiffness and limits the extent of airway distension by the radial force generated by parenchymal tethers and transmural pressure. Airway stiffness has often been attributed to passive elements, such as the extracellular matrix in the lamina reticularis, adventitia, and the smooth muscle layer that cannot be rapidly modulated by drug intervention such as ASM relaxation by β-agonists. In this study, we describe a calcium-sensitive component of ASM stiffness mediated through the Rho-kinase signaling pathway. The stiffness of ovine tracheal smooth muscle was assessed in the relaxed state under the following conditions: 1) in physiological saline solution (Krebs solution) with normal calcium concentration; 2) in calcium-free Krebs with 2 mM EGTA; 3) in Krebs with calcium entry blocker (SKF-96365); 4) in Krebs with myosin light chain kinase inhibitor (ML-7); and 5) in Krebs with Rho-kinase inhibitor (Y-27632). It was found that a substantial portion of the passive stiffness could be abolished when intracellular calcium was removed; this calcium-sensitive stiffness appeared to stem from intracellular source and was not sensitive to ML-7 inhibition of myosin light chain phosphorylation, but was sensitive to Y-27632 inhibition of Rho kinase. The results suggest that airway stiffness can be readily modulated by targeting the calcium-sensitive component of the passive stiffness within the muscle layer.

Endothelial injury is the primary event that leads to a variety of severe vascular disorders. The signal transduction pathway which drives the subsequent healing process is far from being fully elucidated. Mechanical injury elicits a Ca2+ response in the endothelium of intact rat aorta, which comprises an initial Ca2+ release from inositol-1,4,5-trisphosphate-sensitive (IP3Rs) stores followed by a long-lasting decay phase due to Ca2+ entry through uncoupled connexons. In a minor fraction of cells, the Ca2+ signal adopts an oscillatory pattern whose molecular underpinnings are yet to be elucidated. In the light of the role played by repetitive Ca2+ spikes in regulating tissue regeneration, the present study aims at elucidating the mechanisms underlying injury-induced Ca2+ oscillations. The repetitive Ca2+ signal reversibly ceased upon removal of extracellular Ca2+ or addition of the inorganic cations, La3+ and Ni2+. Moreover, the spiking response was abolished by the gap-junction blockers, heptanol and 18 beta-glycyrrhetinic acid and by interfering with the Ca2+ entry-mode mode of the Na+/Ca2+ exchanger (NCX). The InsP3-producing agonist, ATP, resumed Ca2+ oscillations in silent cells, while the phospholipase C inhibitor, U73122, inhibited the oscillatory signal. The latter was also prevented by the SERCA inhibitors, thapsigargin and cyclopiazonic acid acid. These data show that injury-induced [Ca2+]i oscillations require the coordinated interplay between NCX-mediated Ca2+ entry and InsP3-dependent Ca2+ release. Besides directly gating Ca2+ inflow, uncoupled connexons might let Na+ into the cells and stimulate Ca2+ entry through NCX by increasing submembranal Na+ levels.

In blood vessels, stimulation of the vascular endothelium by the Ca2+-mobilizing agonist ATP initiates a number of cellular events that cause relaxation of the adjacent smooth muscle layer. Although vascular endothelial cells are reported to express several subtypes of purinergic P2Y and P2X receptors, the major isoform(s) responsible for the ATP-induced generation of vasorelaxant signals in human endothelium has not been well characterized. To address this issue, ATP-evoked changes in cytosolic Ca2+, membrane potential and acute nitric oxide production were measured in isolated human umbilical vein endothelial cells (HUVECs) and profiled using established P2X and P2Y receptor probes. Whereas selective P2X agonist (i.e. α,β-methyl ATP) and antagonists (i.e. TNP-ATP and PPADS) could neither mimic nor block the observed ATP-evoked cellular responses, the specific P2Y receptor agonist UTP functionally reproduced all the ATP-stimulated effects. Furthermore, both ATP and UTP induced intracellular Ca2+ mobilization with comparable EC50 values (i.e. 1–3μM). Collectively, these functional and pharmacological profiles strongly suggest that ATP acts primarily via a P2Y2 receptor sub-type in human endothelial cells. In support, P2Y2 receptor mRNA and protein were readily detected in isolated HUVECs, and siRNA-mediated knockdown of endogenous P2Y2 receptor protein significantly blunted the cytosolic Ca2+ elevations in response to ATP and UTP, but did not affect the histamine-evoked response. In summary, these results identify the P2Y2 isoform as the major purinergic receptor in human vascular endothelial cells that mediates the cellular actions of ATP linked to vasorelaxation.

The stimulation of vascular integrins by ECM‐associated molecules can lead to changes in arterial tone through the generation of local vasoactive signals. In this study, we have examined how endothelial cell integrins may contribute to the regulation of vascular tone via nitric oxide (NO) signaling. Using cultured HUVECs (EA.hy926 cell line) loaded with either 2 μM Fluo‐4 or 1 μM DAF‐FM, we observed that acute exposure of HUVECs to purified human vitronectin (~0.5 mg/ml) produced modest elevations in cytosolic free Ca2+ (40–50% above baseline) in 10/12 experiments. Conversely, exposure of HUVECs to human fibronectin (~0.5 mg/ml) evoked Ca2+ increases in only 4/10 experiments. The purinergic agonist UTP (0.5 μM) stimulated robust Ca2+ elevations in all cells examined. Pre‐treatment of cells with the αVβ3 integrin antibody LM609 blocked vitronectin‐induced Ca2+ mobilization, whereas similar pre‐treatment with an antibody recognizing the α5β1 integrin (i.e. JBS5) did not interfere with the vitronectin‐stimulated Ca2+ elevation. Importantly, both vitronectin and UTP were observed to stimulate acute NO production in HUVECs, and the vitronectin‐evoked response was inhibited by pretreatment with the LM609 antibody. Collectively, these data suggest that αVβ3 integrins may be primarily responsible for mediating the observed vitronectin‐evoked increases in cytosolic Ca2+ and acute NO synthesis.

Although the structure of the contractile unit in smooth muscle is poorly understood, some of the mechanical properties of the muscle suggest that a sliding-filament mechanism, similar to that in striated muscle, is also operative in smooth muscle. To test the applicability of this mechanism to smooth muscle function, we have constructed a mathematical model based on a hypothetical structure of the smooth muscle contractile unit: a side-polar myosin filament sandwiched by actin filaments, each attached to the equivalent of a Z disk. Model prediction of isotonic shortening as a function of time was compared with data from experiments using ovine tracheal smooth muscle. After equilibration and establishment of in situ length, the muscle was stimulated with ACh (100 μM) until force reached a plateau. The muscle was then allowed to shorten isotonically against various loads. From the experimental records, length-force and force-velocity relationships were obtained. Integration of the hyperbolic force-velocity relationship and the linear length-force relationship yielded an exponential function that approximated the time course of isotonic shortening generated by the modeled sliding-filament mechanism. However, to obtain an accurate fit, it was necessary to incorporate a viscoelastic element in series with the sliding-filament mechanism. The results suggest that a large portion of the shortening is due to filament sliding associated with muscle activation and that a small portion is due to continued deformation associated with an element that shows viscoelastic or power-law creep after a step change in force.

Purinergic agonists, such as ATP, can produce robust vasodilation, yet the specific receptor(s) (i.e. P2X or P2Y) responsible for these effects remain poorly characterized. In this study, we have examined the purinergic receptors mediating acute Ca2+ mobilization, membrane hyperpolarization and nitric oxide (NO) synthesis in cultured HUVECs (EA.hy926 cell line). Brief application of either ATP or UTP (2 μM) to single HUVECs produced comparable elevations in cytosolic Ca2+, membrane hyperpolarizations and acute NO synthesis. These responses were largely unaffected in the presence of the non‐selective P2X receptor antagonists PPADS (10 μM) and TNP‐ATP (1 μM). In contrast to ATP and UTP, the selective P2X receptor agonist α,β‐methyl ATP (2 μM) had little effect on cytosolic Ca2+, membrane potential or NO production. Collectively, these data suggest that the observed stimulatory actions of ATP and UTP in this cell system occur primarily via P2Y, and not P2X, receptors. A plot of the concentration‐dependent rise in cytosolic Ca2+ evoked by ATP and UTP revealed similar EC50 values for the two agonists (1–3 μM); this equipotent action of ATP and UTP further suggests that P2Y2 receptors are the predominant purinergic sub‐type. Finally, RT‐PCR analysis confirmed the presence of P2Y2 receptor mRNA in this human vascular endothelial cell model.Research support to AP Braun was provided by the CIHR and HSF Alberta

Airway smooth muscle (ASM) is able to generate maximal force under static conditions, and this isometric force can be maintained over a large length range due to length adaptation. The increased force at short muscle length could lead to excessive narrowing of the airways. Prolonged exposure of ASM to submaximal stimuli also increases the muscle's ability to generate force in a process called force adaptation. To date, the effects of length and force adaptation have only been demonstrated under static conditions. In the mechanically dynamic environment of the lung, ASM is constantly subjected to periodic stretches by the parenchyma due to tidal breathing and deep inspiration. It is not known whether force recovery due to muscle adaptation to a static environment could occur in a dynamic environment. In this study the effect of length oscillation mimicking tidal breathing and deep inspiration was examined. Force recovery after a length change was attenuated in the presence of length oscillation, except at very short lengths. Force adaptation was abolished by length oscillation. We conclude that in a healthy lung (with intact airway-parenchymal tethering) where airways are not allowed to narrow excessively, large stretches (associated with deep inspiration) may prevent the ability of the muscle to generate maximal force that would occur under static conditions irrespective of changes in mean length; mechanical perturbation on ASM due to tidal breathing and deep inspiration, therefore, is the first line of defense against excessive bronchoconstriction that may result from static length and force adaptation.

The role of Na(+)-Ca(2+) exchanger (NCX) in vascular endothelium is still matter of debate. Depending on both the endothelial cell (EC) type and the extracellular ligand, NCX has been shown to operate in either the forward (Ca(2+) out)- or the reverse (Ca(2+) in)-mode. In particular, acetylcholine (Ach) has been shown to promote Ca(2+) inflow in the intact endothelium of excised rat aorta. Herein, we assessed the involvement of NCX into the Ca(2+) signals elicited by ATP in such preparation. Removal of extracellular Na(+) (0Na(+)) causes the NCX to switch into the reverse-mode and induced an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which disappeared in the absence of extracellular Ca(2+), and in the presence of benzamil, which blocks both modes of NCX, and KB-R 7943, a selective inhibitor of the reverse-mode. ATP induced a transient Ca(2+) signal, whose decay was significantly prolonged by 0Na(+), benzamil, DCB, and monensin while it was unaffected by KB-R 7943. Notably, lowering extracellular Na(+) concentration increased the sensibility to lower doses of ATP. These date suggest that, unlike Ach-stimulated ECs, NCX promotes Ca(2+) extrusion when the stimulus is provided by ATP in intact endothelium of rat aorta. These data show that, within the same preparation, NCX operates in both modes, depending on the chemical nature of the extracellular stimulus.

The mechanism whereby extracellular Ca2+ exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca2+-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca2+-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd3+, La3+ and neomycin, elicited a heterogeneous intracellular Ca2+ signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP3) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na+/Ca2+ exchanger upon substitution of extracellular Na+ unmasked the Ca2+ signal triggered by an increase in extracellular Ca2+ levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca2+ response to the CaSR agonist La3+. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca2+ from intracellular InsP3-sensitive stores.

Papaya (Carica Papaya L.) fruits index 2 were treated with 1.5, 2.5 and 3.5% solutions of calcium chloride by dipping and vacuum infiltration (-33 Kpa) or untreated (0%) as control. Effects of these treatments were evaluated on storage life and postharvest quality characteristics of papaya. After 21 days of storage at 13±1°C, the fruits were removed from storage for physicochemical analysis. Following additional five days holding in the storage condition for fruits used for evaluation of the rate of disease incidence and storage life. Postharvest dip treatments at different concentrations of calcium prolonged storage life, slowed down the ripening processes and maintained the quality of papaya. Whereas, it was effectively greater with calcium infiltration treatments than that for dip treatments. Calcium infiltration extended the storage life and retained the quality as calcium concentrations increased up to 2.5% and then declined. The desired effect was obtained at 2.5% infiltration compared with other treatments. The least disease incidence was found in those fruits infiltrated with 2.5% calcium. Hence, it can be concluded that postharvest infiltration of calcium at 2.5% has the potential to control disease incidence, prolong the storage life and preserve valuable attributes of postharvest papaya, presumably because of its effects on inhibition of ripening and senescence process and loss of the fruit firmness of papaya.

The inner watt of excised rat aorta was scraped by a microelectrode and Ca2+ signals were investigated by fluorescence microscopy in endothetial cells (ECs) directly coupled with injured cells. The injury caused an immediate increase in the intracellular Ca2+ concentration ([Ca2+](i)), followed by a long-lasting decay phase due to Ca2+ influx from extracellular space. The immediate response was mainly due to activation of purinergic receptors, as shown by the effect of P-2X and P-2Y receptors agonists and antagonists, such as suramin, alpha,beta-MeATP, MRS-2179 and 2-MeSAMP. Inhibition of store-operated Ca2+ influx did not affect either the peak response or the decay phase. Furthermore, the latter was: (i) insensitive to phospholipase C inhibition, (ii) sensitive to the gap junction blockers, palmitoleic acid, heptanol, octanol and oleamide, and (iii) sensitive to La3+ and Ni2+, but not to Gd3+. Finally, ethidium bromide or Lucifer Yellow did not enter ECs facing the scraped area.These results suggest that endothelium scraping: (i) causes a short-tasting stimulation of healthy ECs by extracellular nucleotides released from damaged cells and (ii) uncouples the hemichannels of the ECs facing the injury site; these hemichannels do not fully close and allow a long-lasting Ca2+ entry. (C) 2007 Elsevier Ltd. All rights reserved.