Oxidative burst and NO generation as initial response to ischemia in flow-adapted endothelial cells

Shear stress modulates endothelial physiology, yet the effect(s) of flow cessation is poorly understood. The initial metabolic responses of flow-adapted bovine pulmonary artery endothelial cells to the abrupt cessation of flow (simulated ischemia) was evaluated using a perfusion chamber designed for continuous spectroscopy. Plasma membrane potential, production of reactive O2 species (ROS), and intracellular Ca(2+) and nitric oxide (NO) levels were measured with fluorescent probes. Within 15 s after flow cessation, flow-adapted cells, but not cells cultured under static conditions, showed plasma membrane depolarization and an oxidative burst with generation of ROS that was inhibited by diphenyleneiodonium. EGTA-inhibitable elevation of intracellular Ca(2+) and NO were observed at approximately 30 and 60 s after flow cessation, respectively. NO generation was decreased in the presence of inhibitors of NO synthase and calmodulin. Thus flow-adapted endothelial cells sense the altered hemodynamics associated with flow cessation and respond by plasma membrane depolarization, activation of NADPH oxidase, Ca(2+) influx, and activation of Ca(2+)/calmodulin-dependent NO synthase. This signaling response is unrelated to cellular anoxia.     

Ca2+- and phosphatidylinositol 3-kinase-dependent nitric oxide generation in lung endothelial cells in situ with ischemia

Endothelial cells generate nitric oxide (NO) in response to agonist stimulation or increased shear stress. In this study, we evaluated the effects of abrupt cessation of shear stress on pulmonary endothelial NO generation and its relationship to changes in intracellular Ca(2+). In situ endothelial generation of NO and changes in intracellular Ca(2+) in isolated, intact rat lungs were evaluated using fluorescence microscopy with diaminofluorescein diacetate, an NO probe, and Fluo-3, a Ca(2+) probe. The onset of increased NO generation in endothelial cells of subpleural microvessels in situ occurred between 30 and 90 s after onset of ischemia and was preceded by an increase in intracellular Ca(2+) due to both influx of extracellular Ca(2+) and release from intracellular stores. Flow cessation-induced NO generation in endothelial cells in situ was Ca(2+)-, calmodulin-, and PI3-kinase-dependent. The similarity of endothelial cell response (increased NO generation) to either increased flow or cessation of flow suggests that cells respond to an imposed alteration from a state of adaptation. This response to flow cessation may constitute a compensatory vasodilatatory mechanism and may play a role in signaling for cell proliferation and vascular remodeling.     

Activation of endothelial NADPH oxidase during normoxic lung ischemia is KATP channel dependent

Previous studies have shown endothelial cell membrane depolarization and generation of reactive oxygen species (ROS) in endothelial cells with abrupt reduction in shear stress (ischemia). This study evaluated the role of ATP-sensitive potassium (K(ATP)) channels and NADPH oxidase in the ischemic response by using Kir6.2-/- and gp91(phox)-/- mice. To evaluate ROS generation, we subjected isolated perfused mouse lungs labeled with 2',7'-dichlorodihydrofluorescein (DCF), hydroethidine (HE), or diphenyl-1-pyrenylphosphine (DPPP) to control perfusion followed by global ischemia. In wild-type C57BL/6J mice, imaging of subpleural endothelial cells showed a time-dependent increase in intensity for all three fluorescence probes with ischemia, which was blocked by preperfusion with cromakalim (a K(ATP) channel agonist) or diphenyleneiodonium (DPI, a flavoprotein inhibitor). Endothelial cell fluorescence with bis-oxonol, a membrane potential probe, increased during lung ischemia indicating cell membrane depolarization. The change in membrane potential with ischemia in lungs of gp91(phox)-/- mice was similar to wild type, but ROS generation did not occur. Lungs from Kir6.2-/- showed marked attenuation of the change in both membrane potential and ROS production. Thus membrane depolarization during lung ischemia requires the presence of a K(ATP) channel and is required for activation of NADPH oxidase and endothelial ROS generation.     

Measurement of membrane potential and [Ca2+]i in cell ensembles: application to the study of glutamate taste in mice.

We have studied the spectral properties of the voltage-sensitive dye, 1-(3-sulfonatopropyl)-4-[beta [2-(di-n-octylamino)-6-naphtyl]vinyl] pyridinium betaine (di-8-ANEPPS), and the Ca(2+)-sensitive dye, fura-2, in azolectin liposomes and in isolated taste buds from mouse. We find that the fluorescence excitation spectra of di-8-ANEPPS and fura-2 are largely nonoverlapping, allowing alternate ratio measurements of membrane potential and intracellular calcium ([Ca2+]i). There is a small spillover of di-8-ANEPPS fluorescence at the excitation wavelengths used for fura-2 (340 and 360 nm). However, voltage-induced changes in the fluorescence of di-8-ANEPPS, excited at the fura-2 wavelengths, are small. In addition, di-8-ANEPPS fluorescence is localized to the membrane, whereas fura-2 fluorescence is distributed throughout the cytoplasm. Because of this, the effect of spillover of di-8-ANEPPS fluorescence in the [Ca2+]i estimate is < 1%, under the appropriate conditions. We have applied this method to study of the responses of multiple taste cells within isolated taste buds. We show that membrane potential and [Ca2+]i can be measured alternately in isolated taste buds from mouse. Stimulation with glutamate and glutamate analogs indicates that taste cells express both metabotropic and ionotropic receptors. The data suggest that the receptors responding to 2-amino-4-phosphonobutyrate (L-AP4), presumably metabotropic L-glutamate receptors, do not mediate excitatory glutamate taste responses.     

Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling

Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O(2)(.-)) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O(2)(.-), pulmonary microvascular endothelial cells were preloaded with the O(2)(.-)-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O(2)(.-) resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O(2)(.-) triggered Ca(2+) release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O(2)(.-) production and cellular apoptosis. These "signaling" effects of O(2)(.-) were prevented by DIDS treatment, by depletion of intracellular Ca(2+) stores with thapsigargin and by chelation of intracellular Ca(2+). This study demonstrates that O(2)(.-) flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca(2+) release, which activates mitochondrial O(2)(.-) generation.     

Possible use of quercetin, an antioxidant, for protection of cells suffering from overload of intracellular Ca2+: a model experiment

Quercetin is known to protect the cells suffering from oxidative stress. The oxidative stress elevates intracellular Ca(2+) concentration, one of the phenomena responsible for cell death. Therefore, we hypothesized that quercetin would protect the cells suffering from overload of intracellular Ca(2+). To test the hypothesis, the effects of quercetin on the cells suffering from oxidative stress and intracellular Ca(2+) overload were examined by using a flow cytometer with appropriate fluorescence probes (propidium iodide, fluo-3-AM, and annexin V-FITC) and rat thymocytes. The concentrations (1-30 microM) of quercetin to protect the cells suffering from intracellular Ca(2+) overload by A23187, a calcium ionophore, were similar to those for the cells suffering from oxidative stress by H(2)O(2). The cell death respectively induced by H(2)O(2) and A23187 was significantly suppressed by removal of external Ca(2+). Furthermore, quercetin greatly delayed the process of Ca(2+)-dependent cell death although it did not significantly affect the elevation of intracellular Ca(2+) concentration by H(2)O(2) and A23187, respectively. It is concluded that quercetin can protect the cells from oxidative injury in spite of increased concentration of intracellular Ca(2+). Results suggest that quercetin is also used for protection of cells suffering from overload of intracellular Ca(2+).     

Actions of reactive oxygen species on AH/type 2 myenteric neurons in guinea pig distal colon

With conventional intracellular recording methods, we investigated the mechanism of actions of reactive oxygen species (ROS) derived from hypoxanthine and xanthine oxidase (HX/XO) reactions on AH/type 2 myenteric neurons in the guinea pig distal colon. Of the 54 neurons to which HX/XO was applied, 32 neurons showed a transient membrane hyperpolarization(s) followed by a long-lasting membrane depolarization. Two additional groups of 10 myenteric neurons exhibited only a membrane hyperpolarization(s) or a late-onset membrane depolarization, respectively, and the remaining two neurons did not show any response to HX/XO. Analysis of changes of the input resistance induced by HX/XO indicated that suppression and augmentation of the conductance of Ca(2+)-dependent K(+) channels are the ionic mechanisms underlying the membrane hyperpolarization and depolarization, respectively. The effects of HX/XO on myenteric neurons were mimicked by application of caffeine or H(2)O(2). The results suggest that OH(.), but neither H(2)O(2) nor O(2)(.-), is responsible for HX/XO-induced responses. The intracellular Ca(2+) store may be the acting site of ROS in colonic AH/type 2 neurons.     

Hydrogen peroxide alters mitochondrial activation and insulin secretion in pancreatic beta cells.

The effects of a transient exposure to hydrogen peroxide (10 min at 200 microM H(2)O(2)) on pancreatic beta cell signal transduction and insulin secretion have been evaluated. In rat islets, insulin secretion evoked by glucose (16.7 mM) or by the mitochondrial substrate methyl succinate (5 mM) was markedly blunted following exposure to H(2)O(2). In contrast, the secretory response induced by plasma membrane depolarization (20 mM KCl) was not significantly affected. Similar results were obtained in insulinoma INS-1 cells using glucose (12.8 mM) as secretagogue. After H(2)O(2) treatment, glucose no longer depolarized the membrane potential (DeltaPsi) of INS-1 cells or increased cytosolic Ca(2+). Both DeltaPsi and Ca(2+) responses were still observed with 30 mM KCl despite an elevated baseline of cytosolic Ca(2+) appearing approximately 10 min after exposure to H(2)O(2). The mitochondrial DeltaPsi of INS-1 cells was depolarized by H(2)O(2) abolishing the hyperpolarizing action of glucose. These DeltaPsi changes correlated with altered mitochondrial morphology; the latter was not preserved by the overexpression of the antiapoptotic protein Bcl-2. Mitochondrial Ca(2+) was increased following exposure to H(2)O(2) up to the micromolar range. No further augmentation occurred after glucose addition, which normally raises this parameter. Nevertheless, KCl was still efficient in enhancing mitochondrial Ca(2+). Cytosolic ATP was markedly reduced by H(2)O(2) treatment, probably explaining the decreased endoplasmic reticulum Ca(2+). Taken together, these data point to the mitochondria as primary targets for H(2)O(2) damage, which will eventually interrupt the transduction of signals normally coupling glucose metabolism to insulin secretion.     

Endothelial cell signaling during conducted vasomotor responses

ACh and KCl stimulate vasomotor responses that spread rapidly and bidirectionally along arteriole walls, most likely via spread of electric current or Ca2+ through gap junctions. We examined these possibilities with isolated, cannulated, and perfused hamster cheek pouch arterioles (50- to 80-microm resting diameter). After intraluminal loading of 2 microM fluo 3 to measure Ca2+ or 1 microM di-8-ANEPPS to measure membrane potential, photometric techniques were used to selectively measure changes in intracellular Ca2+ concentration ([Ca2+]i) or membrane potential in endothelial cells. Activation of the endothelium by micropipette application of ACh (10-4 M, 1.0-s pulse) to a short segment of arteriole (100-200 microm) increased endothelial cell [Ca2+]i and caused hyperpolarization at the site of stimulation. This response was followed rapidly by vasodilation of the entire arteriole ( approximately 2-mm length). Change in membrane potential always preceded dilation, both at the site of stimulation and at distant sites along the arteriole. In contrast, an increase in endothelial cell [Ca2+]i was observed only at the application site. Micropipette application of KCl, which can depolarize both smooth muscle and endothelial cells (250 mM, 2.5-s pulse), also caused a rapid, spreading response consisting of depolarization followed by vasoconstriction. With KCl stimulation, in addition to changes in membrane potential, increases in endothelial cell [Ca2+]i were observed at distant sites not directly exposed to KCl. The rapid longitudinal spread of both hyperpolarizing and depolarizing responses support electrical coupling as the mode of signal transmission along the arteriolar length. In addition, the relatively short distance between heterologous cell types enables the superimposed radial Ca2+ signaling between smooth muscle and endothelial cells to modulate vasomotor responses.     

Excitation-contraction coupling in airway smooth muscle

Excitation-contraction (EC) coupling in striated muscles is mediated by the cardiac or skeletal muscle isoform of voltage-dependent L-type Ca(2+) channel (Ca(v)1.2 and Ca(v)1.1, respectively) that senses a depolarization of the cell membrane, and in response, activates its corresponding isoform of intracellular Ca(2+) release channel/ryanodine receptor (RyR) to release stored Ca(2+), thereby initiating muscle contraction. Specifically, in cardiac muscle following cell membrane depolarization, Ca(v)1.2 activates cardiac RyR (RyR2) through an influx of extracellular Ca(2+). In contrast, in skeletal muscle, Ca(v)1.1 activates skeletal muscle RyR (RyR1) through a direct physical coupling that negates the need for extracellular Ca(2+). Since airway smooth muscle (ASM) expresses Ca(v)1.2 and all three RyR isoforms, we examined whether a cardiac muscle type of EC coupling also mediates contraction in this tissue. We found that the sustained contractions of rat ASM preparations induced by depolarization with KCl were indeed partially reversed ( approximately 40%) by 200 mum ryanodine, thus indicating a functional coupling of L-type channels and RyRs in ASM. However, KCl still caused transient ASM contractions and stored Ca(2+) release in cultured ASM cells without extracellular Ca(2+). Further analyses of rat ASM indicated that this tissue expresses as many as four L-type channel isoforms, including Ca(v)1.1. Moreover, Ca(v)1.1 and RyR1 in rat ASM cells have a similar distribution near the cell membrane in rat ASM cells and thus may be directly coupled as in skeletal muscle. Collectively, our data implicate that EC-coupling mechanisms in striated muscles may also broadly transduce diverse smooth muscle functions.     

Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria

Impairment of the normal spatiotemporal pattern of intracellular Ca(2+) ([Ca(2+)](i)) signaling, and in particular, the transition to an irreversible "Ca(2+) overload" response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca(2+) overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H(2)O(2)) evokes a Ca(2+) overload response and inhibition of plasma membrane Ca(2+)-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938-C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca(2+)](i) clearance assay in which mitochondrial Ca(2+) uptake was blocked with Ru-360, H(2)O(2) (50 microM-1 mM) markedly inhibited the PMCA activity. This H(2)O(2)-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H(2)O(2)-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca(2+) handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca(2+) overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.     

The essential role of H2O2 in the regulation of intracellular Ca2+ by epidermal growth factor in rat-2 fibroblasts

We have investigated a new mechanism by which epidermal growth factor (EGF) increases intracellular Ca(2+) ([Ca(2+)](i)) in Rat-2 fibroblasts. EGF induced a transient increase of [Ca(2+)](i), and sustained Ca(2+) increase disappeared in the absence of extracellular Ca(2+). However, EGF had no effect on the formation of inositol phosphates. Expression of N17Rac or scrape-loading of C3 transferase blocked the elevation of [Ca(2+)](i) by EGF, but not by lysophosphatidic acid (LPA). EGF increased intracellular H(2)O(2), with a maximal increase at 5 min, which was blocked by catalase, scrape-loading of C3 transferase, or expression of N17Rac. H(2)O(2) scavengers, catalase and N-acetyl-L-cysteine, also blocked the Ca(2+) response to EGF, but not to LPA. In the presence of EGTA, preincubation with EGF completely inhibited subsequent Ca(2+) response to extracellular H(2)O(2) and vice versa. Incubation with EGF or phosphatidic acid abolished subsequent elevation of [Ca(2+)](i) by phosphatidic acid or EGF, respectively. Furthermore, preincubation with LPA inhibited the subsequent Ca(2+) response to EGF, but not vice versa. These results suggested that intracellular H(2)O(2) regulated by Rac and RhoA, but not inositol phosphates, was responsible for the EGF-stimulated elevation of [Ca(2+)](i). It was also suggested that EGF cross talked with LPA in the regulation of [Ca(2+)](i) by producing intracellular H(2)O(2).     

Na(+)/Ca(2+) exchange facilitates Ca(2+)-dependent activation of endothelial nitric-oxide synthase.

Recent evidence suggests the expression of a Na(+)/Ca(2+) exchanger (NCX) in vascular endothelial cells. To elucidate the functional role of endothelial NCX, we studied Ca(2+) signaling and Ca(2+)-dependent activation of endothelial nitric-oxide synthase (eNOS) at normal, physiological Na(+) gradients and after loading of endothelial cells with Na(+) ions using the ionophore monensin. Monensin-induced Na(+) loading markedly reduced Ca(2+) entry and, thus, steady-state levels of intracellular free Ca(2+) ([Ca(2+)](i)) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca(2+)](i), Ca(2+)-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca(2+) concentration response curve in monensin-treated cells. This facilitation of Ca(2+)-dependent activation of eNOS was strictly dependent on the presence of Na(+) ions during treatment of the cells with monensin. Na(+)-induced facilitation of eNOS activation was not due to a direct effect of Na(+) ions on the Ca(2+) sensitivity of the enzyme. Moreover, the effect of Na(+) was not related to Na(+) entry-induced membrane depolarization or suppression of Ca(2+) entry, since neither elevation of extracellular K(+) nor the Ca(2+) entry blocker 1-(beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazol e hydrochloride (SK&F 96365) mimicked the effects of Na(+) loading. The effects of monensin were completely blocked by 3', 4'-dichlorobenzamil, a potent and selective inhibitor of NCX, whereas the structural analog amiloride, which barely affects Na(+)/Ca(2+) exchange, was ineffective. Consistent with a pivotal role of Na(+)/Ca(2+) exchange in Ca(2+)-dependent activation of eNOS, an NCX protein was detected in caveolin-rich membrane fractions containing both eNOS and caveolin-1. These results demonstrate for the first time a crucial role of cellular Na(+) gradients in regulation of eNOS activity and suggest that a tight functional interaction between endothelial NCX and eNOS may take place in caveolae.     

Hydrogen peroxide-induced Ca2+ mobilization in pulmonary arterial smooth muscle cells

Reactive oxygen species (ROS) generated from NADPH oxidases and mitochondria have been implicated as key messengers for pulmonary vasoconstriction and vascular remodeling induced by agonists and hypoxia. Since Ca(2+) mobilization is essential for vasoconstriction and cell proliferation, we sought to characterize the Ca(2+) response and to delineate the Ca(2+) pathways activated by hydrogen peroxide (H(2)O(2)) in rat intralobar pulmonary arterial smooth muscle cells (PASMCs). Exogenous application of 10 microM to 1 mM H(2)O(2) elicited concentration-dependent increase in intracellular Ca(2+) concentration in PASMCs, with an initial rise followed by a plateau or slow secondary increase. The initial phase was related to intracellular release. It was attenuated by the inositol trisphosphate (IP(3)) receptor antagonist 2-aminoethyl diphenylborate, ryanodine, or thapsigargin, but was unaffected by the removal of Ca(2+) in external solution. The secondary phase was dependent on extracellular Ca(2+) influx. It was unaffected by the voltage-gated Ca(2+) channel blocker nifedipine or the nonselective cation channel blockers SKF-96365 and La(3+), but inhibited concentration dependently by millimolar Ni(2+), and potentiated by the Na(+)/Ca(2+) exchange inhibitor KB-R 7943. H(2)O(2) did not alter the rate of Mn(2+) quenching of fura 2, suggesting store- and receptor-operated Ca(2+) channels were not involved. By contrast, H(2)O(2) elicited a sustained inward current carried by Na(+) at -70 mV, and the current was inhibited by Ni(2+). These results suggest that H(2)O(2) mobilizes intracellular Ca(2+) through multiple pathways, including the IP(3)- and ryanodine receptor-gated Ca(2+) stores, and Ni(2+)-sensitive cation channels. Activation of these Ca(2+) pathways may play important roles in ROS signaling in PASMCs.     

Effect of hydrogen peroxide and superoxide anions on cytosolic Ca2+: comparison of endothelial cells from large-sized and small-sized arteries

We compared the Ca(2+) responses to reactive oxygen species (ROS) between mouse endothelial cells derived from large-sized arteries, aortas (aortic ECs), and small-sized arteries, mesenteric arteries (MAECs). Application of hydrogen peroxide (H(2)O(2)) caused an increase in cytosolic Ca(2+) levels ([Ca(2+)](i)) in both cell types. The [Ca(2+)](i) rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP(3)) receptors. Removal of Ca(2+) from the bath also decreased the [Ca(2+)](i) rises in response to H(2)O(2). In addition, treatment of endothelial cells with H(2)O(2) reduced the [Ca(2+)](i) responses to subsequent challenge of ATP. The decreased [Ca(2+)](i) responses to ATP were resulted from a pre-depletion of intracellular Ca(2+) stores by H(2)O(2). Interestingly, we also found that Ca(2+) store depletion was more sensitive to H(2)O(2) treatment in endothelial cells of mesenteric arteries than those of aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca(2+)](i) rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H(2)O(2) but not by hydroxyl radical. H(2)O(2) contribution in HX-XO-induced [Ca(2+)](i) rises were more significant in endothelial cells from mesenteric arteries than those from aortas. In summary, H(2)O(2) could induce store Ca(2+) release via phospholipase C-IP(3) pathway in endothelial cells. Resultant emptying of intracellular Ca(2+) stores contributed to the reduced [Ca(2+)](i) responses to subsequent ATP challenge. The [Ca(2+)](i) responses were more sensitive to H(2)O(2) in endothelial cells of small-sized arteries than those of large-sized arteries.     

NADPH oxidase activation increases the sensitivity of intracellular Ca2+ stores to inositol 1,4,5-trisphosphate in human endothelial cells

Many stimuli that activate the vascular NADPH oxidase generate reactive oxygen species and increase intracellular Ca(2+), but whether NADPH oxidase activation directly affects Ca(2+) signaling is unknown. NADPH stimulated the production of superoxide anion and H(2)O(2) in human aortic endothelial cells that was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and was significantly attenuated in cells transiently expressing a dominant negative allele of the small GTP-binding protein Rac1, which is required for oxidase activity. In permeabilized Mag-indo 1-loaded cells, NADPH and H(2)O(2) each decreased the threshold concentration of inositol 1,4,5-trisphosphate (InsP(3)) required to release intracellularly stored Ca(2+) and shifted the InsP(3)-Ca(2+) release dose-response curve to the left. Concentrations of H(2)O(2) as low as 3 microm increased the sensitivity of intracellular Ca(2+) stores to InsP(3) and decreased the InsP(3) EC(50) from 423.2 +/- 54.9 to 276.9 +/- 14. 4 nm. The effect of NADPH on InsP(3)-stimulated Ca(2+) release was blocked by catalase and by diphenyleneiodonium and was not observed in cells lacking functional Rac1 protein. Thus, NADPH oxidase-derived H(2)O(2) increases the sensitivity of intracellular Ca(2+) stores to InsP(3) in human endothelial cells. Since Ca(2+)-dependent signaling pathways are critical to normal endothelial function, this effect may be of great importance in endothelial signal transduction.     

Role of endothelial Ni(2+)-sensitive Ca(2+) entry pathway in regulation of EDHF in porcine coronary artery

Elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells is proposed to be required for generation of vascular actions of endothelium-derived hyperpolarizing factor (EDHF). This study was designed to determine the endothelial Ca(2+) source that is important in development of EDHF-mediated vascular actions. In porcine coronary artery precontracted with U-46619, bradykinin (BK) and cyclopiazonic acid (CPA) caused endothelium-dependent relaxations in the presence of N(G)-nitro-L-arginine (L-NNA). The L-NNA-resistant relaxant responses were inhibited by high K(+), indicating an involvement of EDHF. In the presence of Ni(2+), which inhibits Ca(2+) influx through nonselective cation channels, the BK-induced EDHF relaxant response was greatly diminished and the CPA-induced response was abolished. BK and CPA elicited membrane hyperpolarization of smooth muscle cells of porcine coronary artery. Ni(2+) suppressed the hyperpolarizing responses in a manner analogous to removal of extracellular Ca(2+). EDHF-mediated relaxations and hyperpolarizations evoked by BK and CPA in porcine coronary artery showed a temporal correlation with the increases in [Ca(2+)](i) in porcine aortic endothelial cells. The extracellular Ca(2+)-dependent rises in [Ca(2+)](i) in endothelial cells stimulated with BK and CPA were completely blocked by Ni(2+). These results suggest that Ca(2+) influx into endothelial cells through nonselective cation channels plays a crucial role in the regulation of EDHF.     

Triggering of sarcoplasmic reticulum Ca2+ release and contraction by reverse mode Na+/Ca2+ exchange in trout atrial myocytes

Whole cell patch clamp and intracellular Ca(2+) transients in trout atrial cardiomyocytes were used to quantify calcium release from the sarcoplasmic reticulum (SR) and examine its dependency on the Ca(2+) trigger source. Short depolarization pulses (2-20 ms) elicited large caffeine-sensitive tail currents. The Ca(2+) carried by the caffeine-sensitive tail current after a 2-ms depolarization was 0.56 amol Ca(2+)/pF, giving an SR Ca(2+) release rate of 279 amol Ca(2+). pF(-1). s(-1) or 4.3 mM/s. Depolarizing cells for 10 ms to different membrane potentials resulted in a local maximum of SR Ca(2+) release, intracellular Ca(2+) transient, and cell shortening at 10 mV. Although 100 microM CdCl(2) abolished this local maximum, it had no effect on SR Ca(2+) release elicited by a depolarization to 110 or 150 mV, and the SR Ca(2+) release was proportional to the membrane potential in the range -50 to 150 mV with 100 microM CdCl(2). Increasing the intracellular Na(+) concentration ([Na(+)]) from 10 to 16 mM enhanced SR Ca(2+) release but reduced cell shortening at all membrane potentials examined. In the absence of TTX, SR Ca(2+) release was potentiated with 16 mM but not 10 mM pipette [Na(+)]. Comparison of the total sarcolemmal Ca(2+) entry and the Ca(2+) released from the SR gave a gain factor of 18.6 +/- 7.7. Nifedipine (Nif) at 10 microM inhibited L-type Ca(2+) current (I(Ca)) and reduced the time integral of the tail current by 61%. The gain of the Nif-sensitive SR Ca(2+) release was 16.0 +/- 4.7. A 2-ms depolarization still elicited a contraction in the presence of Nif that was abolished by addition of 10 mM NiCl(2). The gain of the Nif-insensitive but NiCl(2)-sensitive SR Ca(2+) release was 14.8 +/- 7.1. Thus both reverse-mode Na(+)/Ca(2+) exchange (NCX) and I(Ca) can elicit Ca(2+) release from the SR, but I(Ca) is more efficient than reverse-mode NCX in activating contraction. This difference may be due to extrusion of a larger fraction of the Ca(2+) released from the SR by reverse-mode NCX rather than a smaller gain for NCX-induced Ca(2+) release.     

Effects of feeding Spodoptera littoralis on lima bean leaves. III. Membrane depolarization and involvement of hydrogen peroxide

In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H(2)O(2)) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H(2)O(2) was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H(2)O(2) was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H(2)O(2) in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H(2)O(2) prompted a transient transmembrane potential (V(m)) depolarization, with a V(m) depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca(2+)-sensing aequorin system, increasing amounts of added H(2)O(2) correlated with a higher cytosolic calcium ([Ca(2+)](cyt)) concentration. In MD and HW leaves, H(2)O(2) also triggered the increase of [Ca(2+)](cyt), but MD-elicited [Ca(2+)](cyt) increase was more pronounced when compared to HW leaves after addition of exogenous H(2)O(2). The results clearly indicate that V(m) depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.