High glucose increases platelet-derived growth factor production in cultured human vascular endothelial cells and preventive effects of eicosapentaenoic acids

The regulation of the production of platelet-derived growth factor (PDGF) and the influence of high glucose concentration, eicosapentaenoic acid (EPA) were studied in cultured human umbilical vein endothelial cells (HUE). The PDGF production of HUE increased markedly depending on glucose concentration. However, EPA (3×10⁻⁴M) markedly inhibited PDGF production [27.5 mM glucose group: 123 ± 3% of control (5.5 mM glucose group), 27.5 mM glucose + EPA group: 104 ±5% of control]. These results suggested that a high glucose concentration and a high osmotic pressure-induced increase in PDGF production is involved in the development and progression of diabetic macroangiopathy. As eicosapentaenoic acid inhibits the PDGF production induced by high glucose concentration in HUE, use of this agent may exhibit anti-arteriosclerotic effects.     

Restoration of nitric oxide production by aldose reductase inhibitor in human endothelial cells cultured in high-glucose medium

The effects of elevated glucose and aldose reductase inhibitor (ARI:ONO-2235) on nitric oxide (NO) production in cultured human umbilical endothelial cells (HUVEC) were evaluated. Aldose reductase and nitric oxide synthase(NOS) share NADPH as an obligate cofactor, therefore it is suggested that the enhanced of glucose flux (27.5 mM) by aldose reductase inhibited NO production by blunting NOS activity. However, the addition of ONO-2235 (100 μM) prevented the inhibition of [NO_2⁻] production. Since ARI decreases glucose-mediated inhibition of NO production in HUVEC, this agent might ameliorate endothelial function associated with diabetes.     

Eicosapentaenoic Acid Enhances Intracellular Free Calcium in Cultured Human EndothelialCells

Eicosapentaenoic acid (EPA) caused elevations of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) measured by fura-2 fluorescence in cultured human endothelial cells. The EPA induced increase in [Ca²⁺]_i could still be observed when either cyclooxygenase or lipoxygenase inhibitors were added. These results suggest that EPA itself rather than its metabolites has direct effects on intracellular Ca²⁺ mobilization, causing the elevation of [Ca²⁺]_i.     

Hyperosmolality-induced abnormal patterns of calcium mobilization in smooth muscle cells from non-diabetic and diabetic rats

Hyperglycemia and/or hyperosmolality may disturb calcium homeostasis in vascular smooth muscle cells (SMCs), leading to altered vascular contractility in diabetes. To test this hypothesis, the KCl induced increases in [Ca²⁺]_i in primarily cultured vascular SMCs exposed to different concentrations of glucose were examined. With glucose concentration in solutions kept at 5.5 mM, KCl induced a fast increase in [Ca²⁺]_i which then slowly declined (type 1 response) in 83% of SMCs from non-diabetic rats. In 9% of non-diabetic SMCs KCl induced a slow increase in [Ca²⁺]_i (type 2 response). Interestingly, under the same culture conditions KCl induced type 1 and type 2 responses in 47 and 35% of SMCs from diabetic rats. When SMCs from non-diabetic or diabetic rats were cultured in 36 mM glucose, KCl induced a fast increase in [Ca²⁺]_i which, however, maintained at a high level (type 3 response). The sustained level of [Ca²⁺]_i in the presence of KCl was significantly higher in cells cultured with 36 mM glucose than that in non-diabetic cells cultured with 5.5 mM glucose. Furthermore, the hyperglycemia-induced alterations in calcium mobilization were similarly observed in cells cultured in high concentration of mannitol (30.5 mM) or L-glucose, indicating that hyperosmolality was mainly responsible for the abnormal calcium mobilization in diabetic SMCs.     

SK&F 96365 inhibits intracellular Ca pumps and raises cytosolic Ca concentration without production of nitric oxide and von Willebrand factor

The effects of the imidazole compound SK&F 96365 on Ca²⁺ movements and production of nitric oxide (NO) and von Willebrand factor (vWF) have been investigated in human endothelial cells. Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) were measured with Fura-2. Real-time production of NO was monitored with a porphyrinic microsensor and the release of vWF with an enzyme-linked immunosorbent assay. Irrespective of the transmembrane Ca²⁺ gradient, 30 μM SK&F 96365 doubled [Ca²⁺]_i suggesting a Ca²⁺ release from intracellular stores. The SK&F 96365-induced [Ca²⁺]_i rise was not accompanied by detectable NO and vWF production, while 1 μM thapsigargin enhanced [Ca²⁺]_i 2.5 times, doubled the secretion of vWF and increased the NO production to 10 ± 4 nM (n = 5). Pretreatment with SK&F 96365 prevented thapsigargin from increasing [Ca²⁺]_i, NO production and vWF secretion. To investigate the mechanism by which SK&F 96365 released Ca²⁺, from internal pools, its effect and that of thapsigargin on the ATP-dependent ⁴⁵Ca²⁺, uptake into platelet membrane vesicles were compared. SK&F 96365 as thapsigargin, dose-dependently reduced the initial rate of ⁴⁵Ca²⁺ uptake. In conclusion, we demonstrate that, in the absence of Ca²⁺ entry from the extracellular space, the [Ca²⁺]_i increase elicited by SK&F 96365 or thapsigargin is not sufficient to initiate NO synthesis and vWF secretion. This confirms the important role of Ca²⁺ influx in endothelial secretion processes.     

Modulation of L-type Ca2+ currents and intracellular calcium by agmatine in rat cardiomyocytes

It is shown that agmatine inhibits L-type Ca²⁺ currents in isolated cardiomyocytes of rats in a dose-dependent manner. The inhibitory analysis indicates that imidazoline receptors of type I (I₁Rs) rather than α₂-adrenoceptors (α₂-ARs) are implicated in mediating the effects of agmatine. Agmatine affects the dynamics of intracellular Ca²⁺ concentration changes in spontaneously active cardiomyocytes. The averaged intracellular Ca²⁺ concentration ([Ca²⁺]_in) varied biphasically, depending on the agmatine dose: at 1–500 μM, agmatine decreased [Ca²⁺]_in; at 500 μM-2 mM, [Ca²⁺]_in remained unchanged, and at concentrations above 2 mM agmatine caused an increase of [Ca²⁺]_in. The effects of low agmatine concentrations were inhibited by 7NI, an inhibitor of NO synthases (NOS), as well as by the inhibitors of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) thapsigargin and cyclopiazonic acid. In contrast, ODQ, a blocker of NO-sensitive guanylate cyclase, and the antagonist of I₁Rs efaroxan were ineffective. At low concentrations agmatine did not affect the increase in [Ca²⁺]_in induced by stimulating doses of ryanodine (40 nM). In addition, agmatine at low doses was found to markedly stimulate NO production. When efaroxan (10 μM) or ryanodine (200 μM) were added to the bath to inhibit I₁Rs and ryanodine receptors (RyRs), respectively, [Ca²⁺]_in became much less sensitive to millimolar agmatine. In contrast to low concentrations (100 μM), high agmatine doses (10–15 mM) did not stimulate the NO synthesis but were effective as NOS inducer in cells pretreated with efaroxan. The selective I₁R agonist rilmenidine increased [Ca²⁺]_in in a dose-dependent manner. The effect of rilmenidine was similar to that of agmatine at high doses and was abolished by RyRs inhibition. Our findings indicate that in spontaneously active cardiomyocytes agmatine at low concentrations decreases [Ca²⁺]_in, does not stimulate I₁Rs but most likely enhances NO synthase followed by an increase in SERCA activity due to the direct nitrosylation of SERCA and/or phospholamban. The effects of high agmatine doses are apparently mediated by I₁Rs and involve RyRs.     

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3-dependent Ca2+ release in mouse brain endothelial cells

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the target cells, which activates the Ca²⁺/Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca²⁺]_i and NO production. The current study assessed whether and how glutamate drives Ca²⁺-dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca²⁺]_i, which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca²⁺ oscillations were triggered by rhythmic endogenous Ca²⁺ mobilization and maintained over time by extracellular Ca²⁺ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca²⁺ release was mediated by InsP₃-sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca²⁺ entry mediated Ca²⁺ entry during ongoing Ca²⁺ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca²⁺ signals. Of note, glutamate induced Ca²⁺-dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca²⁺ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.     

Attenuated endothelium-mediated relaxation by acteoside in rat aorta: Role of endothelial [Ca2+]i and nitric oxide/cyclic GMP pathway

Acteoside and other phenylethanoid glycoside are contained in many plants that are widely used in traditional Chinese herbal medicine. Acteoside possesses multiple biological actions. Its effect on the vascular system is, however, incompletely understood. This study was aimed to investigate the role of endothelial [Ca²⁺]_i, nitric oxide (NO), and cyclic GMP in acteoside-induced inhibition of endothelial NO-mediated relaxation in rat aorta. Acteoside reduced endothelial NO-dependent relaxation induced by acetylcholine (Ach) or A23187. Acteoside inhibited Ach-stimulated increase in tissue content of cyclic GMP in endothelium-intact rings. L-NNA abolished the stimulatory effect of Ach. Treatment with acteoside significantly suppressed bradykinin-induced increase in [Ca²⁺]_i of cultured rat aortic endothelial cells. Acute exposure to acteoside (30 μM) did not affect the expression of eNOS mRNA in endothelium-intact rings. In summary, acteoside impairs endothelial NO-mediated aortic relaxation partially through inhibition of agonist-induced endothelial Ca²⁺ mobilization and Ca²⁺-dependent NO production and subsequent suppression of cyclic GMP formation. This novel pharmacological action if occurring in small vessels in vivo, may contribute to the reported anti-inflammatory effect of acteoside against NO-mediated vascular permeability-related acute edema.     

The protective effect of class III antiarrhythmic agents against calcium overload in cultured myocytes

Calcium ions have been implicated in the mechanisms of ventricular arrhythmias. Impairment of intercellular coupling by calcium overload is considered to facilitate ventricular fibrillation (VF) and to sup-press its self termination. According to our hypothesis, any compound that decreases intracellular calcium concentration [Ca²⁺]_i during VF can serve as defibrillating drug. In this study, we examined the effect of d-sotalol and tedisamil on calcium overload in cultured, spontaneously beating rat cardiomyocytes. The changes of [Ca²⁺]_i were measured by indo-1 method and the intercellular synchronization by image analysis. The results showed that increase in [Ca²⁺]_o from 1.9 mM to 3.9 mM increased [Ca²⁺]_i from 100 nM to 320 nM and transformed the synchronized cell movement to an asynchronous one. Administration of 5 × 10⁻⁶ M d-sotalol or 10⁻⁶ M tedisamil, decreased the [Ca²⁺]_i to its basic level and restored the synchronized activity. In summary: Our results showed that increase in [Ca²⁺]_i known to caused inhibition of intercellular coupling, that could lead to arrhythmia and fibrillation while d-sotalol or tedisamil prevented this effect. These results support our hypothesis, that class III antiarrhythmic compounds with positive inotropic effect, increase intercellular synchronization, by decreasing free [Ca²⁺]_i, most probably by increasing the Ca²⁺ uptake by the sarcoplasmic reticulum, and therefore act as a defibrillating compound.     

Exogenous nitric oxide-induced release of calcium from intracellular IP3 receptor-sensitive stores via S-nitrosylation in respiratory burst-dependent neutrophils

PMA-induced respiratory burst neutrophils were exposed to exogenous nitric oxide (NO) donor sodium nitroprusside (SNP) to study the effect of NO on calcium signaling. A sharp rise of cytosolic calcium concentration ([Ca²⁺]_c) was triggered by 1 mM SNP with and without external calcium. We found that GF 109203X, a specific inhibitor of protein kinase C, DPI, a putative inhibitor of the respiratory burst-generating NADPH oxidase, and 2-DG, a non-metabolizable analog of glucose, completely inhibited the SNP-induced rise of [Ca²⁺]_c in PMA-activated respiratory burst neutrophils. Meanwhile, 2-APB and TMB-8, two potent IP₃ receptor inhibitors, prevented calcium increase respectively. Furthermore, N-ethylmaleimide (NEM), a specific cysteine alkylating agent, evidently abolished the [Ca²⁺]_c elevation. In contrast, the sGC inhibitor NS2028 had little effect on the rise of [Ca²⁺]_c. Taken together, these results indicated that exogenous NO induced the release of calcium from intracellular IP₃ receptor-sensitive stores of neutrophils via S-nitrosylation in a respiratory burst-dependent manner.     

Vascular Relaxation Induced by C-Type Natriuretic Peptide Involves the Ca2+/NO-Synthase/NO Pathway

Aims

C-type natriuretic peptide (CNP) and nitric oxide (NO) are endothelium-derived factors that play important roles in the regulation of vascular tone and arterial blood pressure. We hypothesized that NO produced by the endothelial NO-synthase (NOS-3) contributes to the relaxation induced by CNP in isolated rat aorta via activation of endothelial NPR-C receptor. Therefore, the aim of this study was to investigate the putative contribution of NO through NPR-C activation in the CNP induced relaxation in isolated conductance artery.

Main Methods

Concentration-effect curves for CNP were constructed in aortic rings isolated from rats. Confocal microscopy was used to analyze the cytosolic calcium mobilization induced by CNP. The phosphorylation of the residue Ser¹¹⁷⁷ of NOS was analyzed by Western blot and the expression and localization of NPR-C receptors was analyzed by immunohistochemistry.

Key Findings

CNP was less potent in inducing relaxation in denuded endothelium aortic rings than in intact ones. L-NAME attenuated the potency of CNP and similar results were obtained in the presence of hydroxocobalamin, an intracellular NO⁰ scavenger. CNP did not change the phosphorylation of Ser¹¹⁷⁷, the activation site of NOS-3, when compared with control. The addition of CNP produced an increase in [Ca²⁺]_c in endothelial cells and a decrease in [Ca²⁺]_c in vascular smooth muscle cells. The NPR-C-receptors are expressed in endothelial and adventitial rat aortas.

Significance

These results suggest that CNP-induced relaxation in intact aorta isolated from rats involves NO production due to [Ca²⁺]_c increase in endothelial cells possibly through NPR-C activation expressed in these cells. The present study provides a breakthrough in the understanding of the close relationship between the vascular actions of nitric oxide and CNP.     

Spectroscopic and reactivity comparison of pentacyanonitrosylruthenate(2-) with pentacyanonitrosylferrate(2-), nitroprusside

Lithium penta(cyano-¹³C)nitrosylruthenate (2-), Li₂[Ru(¹³CN)₅NO], in which the anion is the ruthenium analogue of the nitroprusside ion, has been synthesized at 90% isotopic enrichment, and characterized spectroscopically. Despite the very high level of ¹³C enrichment, no two-bond coupling ²J(¹³C_ax-Ru¹³C_eq) was detected in the high-frequency ¹³C NMR spectrum of Li₂[Ru(¹³CN)₅NO], nor was any such coupling observed in Li₄[Ru(¹³CN)₅(¹⁵NO₂)] although both two-bond couplings to ¹⁵N, ²J(¹³C_ax-Ru¹⁵NO₂) and ²J(¹³C_eqRu¹⁵N) were observed. Li₂[Ru(¹³CN)₅(¹⁴NO)] reacted with excess of Li[¹⁵NO₂] to yield Li₄[Ru(¹³CN)₅(¹⁵NO₂)] only: no Li₂[Ru(¹³CN)₅(¹⁵NO)] was observed. Li₄[Ru(¹³CN)₅(¹⁴NO₂)] however showed no exchange with Li[¹⁵NO₂]. While [Ru(CN)₅NO]²⁻ reacted with both OH⁻ and SH⁻ in reactions similar to those of [Fe(CN)₅NO]²⁻, no reactions were detected between [Ru(CN)₅NO]²⁻ and piperidine, [CH(CN)₂]⁻, [CH(COCH₃)₂]⁻, MeS⁻, or [S₂O₄]²⁻, all of which are known to react readily with [Fe(CN)₅NO]²⁻     

Eicosapentaenoic acid enhances myo-inositol uptake in cultured human aortic smooth muscle cells

The effects of elevated glucose and Eicosapentaenoic acid (EPA, 20:5) on myoinositol uptake in human aortic smooth muscle cells (HASMC) were evaluated. Myo-inositol incorporation into HASMC was dependent on an active transport system via Na⁺−K⁺ ATPase activity based on the results with Na⁺ deprivation and Ouabain (5 mM). Although glucose (27.5, 55 mM) inhibited 2-[³H] myo-inositol uptake, the addition of EPA (3×10⁴ M) prevented glucose-mediated inhibition. In addition, EPA potentiated Na⁺−K⁺ ATPase activity of HASMC. Since EPA decrease glucose-mediated inhibition of myo-inositol uptake, this agent might ameliorate aortic smooth muscle cell function associated with diabetes.     

Insulin induces Ca<Superscript>2+</Superscript> oscillations in white fat adipocytes via PI3K and PLC

Adipocytes of white adipose tissue are the cells maintaining glucose homeostasis in an organism, which is controlled by insulin. Insulin stimulates the translocation of glucose transporter GLUT4 from the cytosol into the cell membrane, as well as glucose transport and utilization in these cells. Here we show that insulin-induced [Ca²⁺]_i oscillations are supported by the two signaling pathways involving: (1) phosphoinositide 3-kinase (PI3K), protein kinase B (Akt/PKB), endothelial NO synthase (eNOS), nitric oxide (NO), and ryanodine receptor (RyR) and (2) phospholipase C (PLC) and inositol 3-phosphate receptor (IP₃R). Thus, the PI3K Akt/PKB signaling pathway initiates not only metabolic but also Ca²⁺-signaling pathways in response to insulin.     

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca²⁺]_i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca²⁺]_i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48 h to a variety of stressors: cytokines (low-grade inflammation), 28 mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca²⁺]_i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca²⁺]_i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11 mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca²⁺]_i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11 mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3 mM glucose) observed for FFAs and also for 28G. We also clamped [Ca²⁺]_i using 30 mM KCl + 250 μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11 mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca²⁺]_i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca²⁺]_i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion.     

Insulin restores expression of adenosine kinase in streptozotocin-induced diabetes mellitus rats

The effects of extracellular Mg²⁺ on both dynamic changes of [Ca²⁺]_i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca²⁺ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca²⁺]_i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca²⁺]_i (150 nM) for several hours. The initial [Ca²⁺]_i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca²⁺]_i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg²⁺ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg²⁺, caused an increase in basal [Mg²⁺]_i from 0.8 ± 0.3 to 1.6 ± 0.5 mM. As compared to 1.4 mM extracellular Mg²⁺, 1 M thapsigargin induces, in 10 mM Mg²⁺, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca²⁺]_i and a lower Ca²⁺ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca²⁺]_i which was inhibited by high extracellular and intracellular Mg²⁺.     

Evidence for the possible involvement of the P2Y<Subscript>6</Subscript> receptor in Ca<Superscript>2+</Superscript> mobilization and insulin secretion in mouse pancreatic islets

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca²⁺]_i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca²⁺]_i rise via cytoplasmic Ca²⁺ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 μM) transiently increased [Ca²⁺]_i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y₁ receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y₆ receptor agonist UDP (200 μM) transiently increased [Ca²⁺]_i and reduced insulin secretion at high glucose, whereas the P2Y_2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca²⁺]_i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 μM), U73122 (2 μM, PLC inhibitor), and 2-APB (10 or 30 μM, IP₃ receptor antagonist), but neither by staurosporine (1 μM, PKC inhibitor) nor depletion of extracellular Ca²⁺. The effect of 2-MeSADP on [Ca²⁺]_i was also significantly inhibited by MRS2500, a P2Y₁ receptor antagonist. These results suggested that P2Y₁ and P2Y₆ receptor subtypes are involved in Ca²⁺ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca²⁺]_i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y₁ and P2Y₆, but not P2Y₂ and P2Y₄ receptors.     

Restoration of myo-inositol uptake by eicosapentaenoic acid in human skin fibroblasts cultured in high-glucose medium

The effects of elevated glucose and eicosapentaenoic acid (EPA, C20:5 ω3) on myo-inositol uptake in human skin fibroblasts (HSF) were evaluated. Myo-inositol incorporation into HSF was dependent on an active transport system via Na⁺-K⁺ ATPase activity based on the results with Na⁺ deprivation and ouabain (5 mM). Although glucose (27.5, 55 mM) inhibited 2-[³H] myo-inositol uptake, the addition of EPA (3×10⁻⁴M) prevented glucose-mediated inhibition. Since EPA decreased glucose-mediated inhibition of myo-inositol uptake, this agent might ameliorate some of the devastating functions associated with diabetes.     

Termination of endothelin signaling: Role of nitric oxide

Cellular mechanisms responsible for the termination of ET-1 signal are poorly understood. In order to examine the hypothesis that nitric oxide serves as a physiological brake of ET- 1 signaling, Chinese hamster ovary (CHO) cells stably transfected with the ET_A receptor cDNA (CHO-ET) were studied. CHO-ET responded to ET-1 with robust [Ca²⁺], transients and developed a long-lasting homologous desensitization. Donors of nitric oxide (NO), 3-morpholino-sydnonimine HCl(SIN-1), or sodium nitroprusside (SNP) reduced the amplitude of these responses, accelerated the rate of [Ca²⁺], recovery, and counteracted the development of homologous desensitization by a cyclic GMP-independent mechanism, suggesting an alternative mode for NO modulation of ET-1 responses. Stimulation of CHO-ET cells with mastoparan, a wasp venom acting directly on G proteins (bypassing receptor activation), was inhibited by NO, revealing a postreceptoral target for NO-induced modulation of [Ca²⁺] mobilization. Using a lys⁹-biotinylated ET-1 (ET-1 [BtK⁹]), binding sites were “mapped” in CHO-ET cells. Receptor-ligand complexes did not exhibit spontaneous dissociation during 60min observations. Quantitative fluorescence microscopy revealed that SNP or SIN-1 caused a rapid, concentration-dependent, and reversible dissociation of biotinylated ET- 1 from ET_A receptor (EC₅₀ = 75 μM and 6 μM, respectively), an effect that was not mimicked by 8-bromo-cyclic GMP. “Sandwich” co-culture of endothelial cells with CHO-ET showed that activation of NO production by endothelial cells similarly resulted in dissociation of ET-1 [BtK⁹] from ET_A receptors. We hypothesize that NO plays a role in physiological termination of ET-1 signalling by dual mechanisms: (1) displacement of bound ET-1 from its receptor, thus preventing homologous desensitization, and (2) interference with the postreceptoral pathway for [Ca²⁺] mobilization, hence inhibiting end-responses to ET-1. © 1994 Wiley-Liss, Inc.     

Activation of Cl− channels by extracellular Ca2+ in freshly isolated rabbit osteoclasts

Ionic channels regulated by extracellular Ca²⁺ concentration ([Ca²⁺]₀) were examined in freshly isolated rabbit osteoclasts. K⁺ current was suppressed by intracellular and extracellular Cs⁺ ions. In this condition, high [Ca²⁺]₀ evoked an outwardly rectifying current with a reversal potential of about −25 mV. When the concentration of extracellular Cl⁻ ions was altered, the reversal potential of the outwardly rectifying current shifted as predicted by the Nernst equation. 4′,4-diisothiocyanostilbene-2′,2-disulphonic acid (DIDS) inhibited the outwardly rectifying current. These results indicated that this current was carried through Cl⁻ channels. Cd²⁺ or Ni²⁺ caused a transient activation of the Cl⁻ current in contrast to the sustained activation elicited by Ca²⁺. Intracellular 20 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) inhibited the divalent cation-induced Cl⁻ current. Either when the osmolarity of extracellular medium was increased, or when 100 μM cAMP was dissolved in the patch pipette solution, high [Ca²⁺]₀ still elicited the Cl⁻ current, indicating that the divalent cation-induced Cl⁻ current was carried through Ca²⁺-activated Cl⁻ channels. Under perforated whole cell clamp extracellular divalent cations evoked the Cl⁻ current, indicating that the activation of Cl⁻ current did not arise from possible leakage of divalent cations from the extracellular medium under the whole cell clamp condition. This experiment further excluded a possible activation of volume-sensitive Cl⁻ channels under whole cell clamp. Intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) activated the Cl⁻ current and it was inhibited by intracellular 20 mM EGTA, suggesting that the activation of Cl⁻ current was mediated through a G protein, and that an increase in [Ca²⁺]_i was critical for the activation of Cl⁻ channels. A protein phosphatase inhibitor, okadaic acid (100 nM), caused an irreversible activation of the Cl⁻ current, suggesting that protein phosphatase 1 or 2A was involved in the regulation of Ca²⁺-activated Cl⁻ channels. © 1996 Wiley-Liss, Inc.