Reverse mode Na+/Ca2+ exchange mediated by STIM1 contributes to Ca2+ influx in airway smooth muscle following agonist stimulation

<p>Abstract</p> <p>Background</p> <p>Agonist stimulation of airway smooth muscle (ASM) results in IP₃mediated Ca²⁺release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na⁺influx. This localised increase in Na⁺levels can potentially switch the Na⁺/Ca²⁺exchanger into reverse mode and so result in a further influx of Ca²⁺. The aim of this study was to characterise the expression and physiological function of the Na⁺/Ca²⁺exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca²⁺influx into these cells.</p> <p>Methods</p> <p>The expression profile of NCX (which encodes the Na⁺/Ca²⁺exchanger) homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca²⁺measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX) and target specific siRNA were utilised as tools to inhibit NCX function.</p> <p>Results</p> <p>NCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC) cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na⁺loading and addition of extracellular Ca²⁺induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1) and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca²⁺and induced relaxation of porcine peripheral airways.</p> <p>Conclusions</p> <p>Taken together, these data demonstrate a potentially important role for NCX1 in control of Ca²⁺homeostasis and link store depletion via STIM1 directly with NCX activation.</p

Cardiac diastolic dysfunction in angiotensin II treated hypertensive rats is associate with endothelial upregulation of SGLT1/2 in the macro and microcirculation: Protective effect of empagliflozin

Background: /Introduction: The angiotensin II (Ang II)/AT1R/NADPH oxidase-prooxidant pathway has been shown to upregulate the expression of SGLT1 and 2 and to induce premature endothelial dysfunction in coronary endothelial cells. Since all these effects are prevented by the dual SGLT1/2 inhibitor sotagliflozin (sota) and the SGLT2 inhibitor empagliflozin (empa), SGLT1 and 2 are involved in the induction of endothelial dysfunction. Purpose: The aim of the study was to evaluate the role of SGLT1 and 2 in the Ang II-induced hypertensive response leading to cardiac and vascular endothelial dysfunction. Methods: Male Wistar rats received empa (30 mg/kg/day) provided in the diet for 5 weeks. After 1 week, rats underwent sham surgery (sham rats) or surgery with implantation of an osmotic mini-pump infusing Ang II (0.4 mg/kg/d) for 4 weeks. Systolic blood pressure (SBP) was assessed by plethysmography, the cardiac function using echocardiography, the expression level of target proteins by immunofluorescence staining, fibrosis by Sirius red staining, the function of SGLT1 and 2 indirectly by the uptake of glucose-conjugated anthocyanins, and the level of oxidative stress using dihydroethidium staining. Results: The administration of Ang II to rats caused an increased systolic blood pressure from about 140 to 180 mmHg, which was not affected by empa. In the heart, the hypertensive response resulted in an increased left ventricle mass and a diastolic dysfunction as indicated by an elevated E/e' and a decreased IVRT, fibrosis and increased collagen I and ANP expression levels. In the secondary branch of the mesenteric artery, it resulted in an increased level of oxidative stress, AT1R, collagen I and of endothelial ACE, VCAM-1, MCP-1, MMP-2, MMP-9 associated with a down-regulation of eNOS. It was also associated with an increased endothelial expression level of SGLT1 in the aorta, mesenteric resistance vessel and coronary microcirculation. Moreover, an increased uptake of anthocyanins is observed in the aorta predominantly in the endothelium and this effect is markedly reduced by sota and empa. All of the above parameters were markedly reduced in the Ang II plus empa group whereas the empa treatment alone had little effect compared to the control group. Conclusion: The findings indicate that Ang II-induced hypertension promoting left ventricle cardiac dysfunction, and both macro and microvascular endothelial dysfunction are associated with an up-regulation of SGLT1 and 2 in endothelial cells. They further indicate that empa prevents the noxious impact of Ang II on the heart and vasculature despite persistent hypertension

In vivo and in vitro sensitization of leukemic cells to adriamycin-induced apoptosis by pentoxifylline: Involvement of caspase cascades and I?B? phosphorylation

The aim of this work was to investigate whether in vivo and in vitro pentoxifylline (PTX) sensitizes hematological tumor cells to adriamycin (ADM)-induced apoptosis, and to investigate the involvement of caspase cascades and phosphorylated forms of I?B?. Balb/c mice inoculated intraperitoneally with L5178-Y murine lymphoma cells were used for in vivo experiments and for survival studies. The U937 human monocytic cell line was used for in vitro experiments. Both cell lines were treated under similar experimental conditions with PTX and/or ADM to assess their effects on apoptosis. Apoptosis was evaluated by fluorescence microscopy with ethidium bromide and acridine orange staining and confirmed by electrophoretic DNA analysis. Caspase inhibitors Z-VAD-fmk, Z-DEVD-fmk, and Z-LEHD-fmk were used to investigate the involvement of caspase cascades. C-terminally and Ser32 phosphorylated forms of I?B? were evaluated in cytoplasmic extracts in the absence or presence of TNF?. Results: In vivo, PTX (50 mg/kg) with ADM (5 mg/kg) increased the apoptotic index relative to PTX or ADM administered alone, time- and dose-dependently. DNA laddering appeared in lymphoma cells treated with PTX + ADM at 24 h, whereas neither untreated control, PTX-, nor ADM-treated cells showed DNA fragmentation. All (100%) tumor-bearing mice treated with PTX (25 mg/kg) + ADM (2.5 mg/kg) survived for 1 year, whereas the mortality rates of mice treated with either PTX or ADM alone at the same doses were similar to that of untreated tumor-bearing mice (28 � 3 days). Caspase inhibitors inhibited apoptosis more efficiently in PTX- or ADM-treated cultures than in PTX + ADM-treated cultures. Pretreatment with TNF? (10 ng/mL) increased apoptosis in PTX- or ADM-treated U937 cells. However, the apoptotic index of PTX + ADM-treated cultures was significantly reduced and the expression of C-terminally and Ser32 phosphorylated I?B? was reduced. PTX sensitizes hematological malignancies to ADR-induced apoptosis. An independent caspase pathway is involved in PTX + ADM-induced apoptosis. The phosphorylation status of I?B? is closely related via TNF? to the possible mechanisms of drug resistance. � 2005 Elsevier B.V. All rights reserved

Anthocyanin-rich blackcurrant intake by old rats improves blood pressure, vascular oxidative stress and endothelial dysfunction associated with SGLT1- and 2-mediated vascular uptake of anthocyanin

Introduction Aging-related endothelial dysfunction and vascular oxidative stress affect early arterial sites at risk. Anthocyanins uptake via sodium-glucose co-transporter 1 (SGLT1) are potent inducers of endothelial formation of nitric oxide (NO). Aims This study examined if anthocyanin-rich blackcurrant (ARB) improves the endothelial function in old rats. Method Male Wistar rats (22-month old) received ARB (60 and 120 mg/kg/d) orally for 2 weeks. Systolic blood pressure (SBP) was assessed by tail-cuff sphygmomanometry, vascular reactivity using organ chambers, protein expression by immunofluorescence, oxidative stress using dihydroethidium, and anthocyanin uptake by Neu reagent. Results Old rats showed increased SBP, abolished endothelium-dependent hyperpolarization-mediated relaxation and increased contractile response to phenylephrine in the mesenteric artery, which were improved by ARB. Old aorta showed increased oxidative stress and expression levels of eNOS, which were improved by ARB. SGLT1 immunofluorescence predominantly in the endothelium was more pronounced in the aortic arch than the aorta and higher in old than young rats, whereas the SGLT2 signal was low. The ARB treatment induced a dose-dependent accumulation of anthocyanins in the aorta and aortic arch. An ARB purified extract promoted ex vivo greater anthocyanins uptake mostly in the endothelium in the aortic arch than aorta, and in old compared to young rats. The anthocyanins uptake was inhibited to a greater extent by a dual SGLT1/2 inhibitor than by a selective SGLT2 inhibitor in the aorta of young and old rats. Both SGLT inhibitors reduced also ex vivo the age-related vascular oxidative stress. Conclusion The upregulation of SGLT1, and the greater SGLT1 and SGLT2-mediated uptake of anthocyanins predominantly in the endothelium at arterial sites at risk in old rats suggest that anthocyanins appear as interesting natural products to protect the endothelial function with increasing age