Doxorubicin-Induced Fetal Mesangial Cell Death Occurs Independently of TRPC6 Channel Upregulation but Involves Mitochondrial Generation of Reactive Oxygen Species.

Doxorubicin (DOX), a category D pregnancy drug, is a chemotherapeutic agent that has been shown in animal studies to induce fetal toxicity, including renal abnormalities. Upregulation of the transient receptor potential cation (TRPC) 6 channel is involved in DOX-induced podocyte apoptosis. We have previously reported that TRPC6-mediated Ca2+ signaling promotes neonatal glomerular mesangial cell (GMC) death. However, it is unknown whether DOX alters mesangial TRPC expression or viability in the fetus. In this study, cell growth was tracked in control and DOX-treated primary GMCs derived from fetal pigs. Live-cell imaging demonstrated that exposure to DOX inhibited the proliferation of fetal pig GMCs and induced cell death. DOX did not alter the TRPC3 expression levels. By contrast, TRPC6 protein expression in the cells was markedly reduced by DOX. DOX treatment also attenuated the TRPC6-mediated intracellular Ca2+ elevation. DOX stimulated mitochondrial reactive oxygen species (mtROS) generation and mitophagy by the GMCs. The DOX-induced mtROS generation and apoptosis were reversed by the mitochondria-targeted antioxidant mitoquinone. These data suggest that DOX-induced fetal pig GMC apoptosis is independent of TRPC6 channel upregulation but requires mtROS production. The mtROS-dependent GMC death may contribute to DOX-induced fetal nephrotoxicity when administered prenatally.

Lipid rafts are required for signal transduction by angiotensin II receptor type 1 in neonatal glomerular mesangial cells.

Angiotensin II (ANG-II) receptors (AGTRs) contribute to renal physiology and pathophysiology, but the underlying mechanisms that regulate AGTR function in glomerular mesangium are poorly understood. Here, we show that AGTR1 is the functional AGTR subtype expressed in neonatal pig glomerular mesangial cells (GMCs). Cyclodextrin (CDX)-mediated cholesterol depletion attenuated cell surface AGTR1 protein expression and ANG-II-induced intracellular Ca(2+) ([Ca(2+)]i) elevation in the cells. The COOH-terminus of porcine AGTR1 contains a caveolin (CAV)-binding motif. However, neonatal GMCs express CAV-1, but not CAV-2 and CAV-3. Colocalization and in situ proximity ligation assay detected an association between endogenous AGTR1 and CAV-1 in the cells. A synthetic peptide corresponding to the CAV-1 scaffolding domain (CSD) sequence also reduced ANG-II-induced [Ca(2+)]i elevation in the cells. Real-time imaging of cell growth revealed that ANG-II stimulates neonatal GMC proliferation. ANG-II-induced GMC growth was attenuated by EMD 66684, an AGTR1 antagonist; BAPTA, a [Ca(2+)]i chelator; KN-93, a Ca(2+)/calmodulin-dependent protein kinase II inhibitor; CDX; and a CSD peptide, but not PD 123319, a selective AGTR2 antagonist. Collectively, our data demonstrate [Ca(2+)]i-dependent proliferative effect of ANG-II and highlight a critical role for lipid raft microdomains in AGTR1-mediated signal transduction in neonatal GMCs.

Effect of the aqueous extract of entandrophragma utile bark on gastric acid secretion in ghosh and schild rat preparations.

AIMS AND OBJECTIVES: The reduction of aggressive factors such as gastric acid is considered a key target of gastrointestinal protection. We investigated the antisecretory effect of the aqueous bark extract of E. utile, a Nigerian traditional medicinal preparation used for ulcers. METHODS: Male rats were anesthetized and cannulated for intragastric perfusion of saline and test agents as well as for infusion of histamine into the jugular vein. Gastric effluents were collected every 30 min and 10 ml aliquots were titrated against 0.01 N NaOH using phenol red indicator. Gastric acidity was deduced from titre values. Histamine in saline was infused for 2 h at a rate of 1 x 10(-4) g kg-1 min -1 to stimulate acid secretion. In one set of animals, cimetidine in saline was simultaneously perfused intragastrically for 2 h at the rate of 2.5 x 10(-3) g kg(-1) h(-1). Similarly, rats in other sets were simultaneously perfused intragastrically with either the aqueous fresh bark extract of E.utile or the decolorized extract for 2 h at a rate of 1.5 x 10(-3) g kg(-1) h(-1). The extract was also perfused in rats that had established peak gastric acid output with prior infusion of histamine. RESULTS: Mean basal acid output per 0.5 h was 20.2 ± 1.9 mEq. Peak measurement fluctuated within a range of 114 - 117 mEq 0.5h(-1) and was maintained for more than 5 h. Cimetidine or E. utile prevented the rise to peak output that histamine produces. Using the 2-tailed paired t-test, the inhibitory effects of either cimetidine or E. utile was significant (p<0.05) at 60 min. E. utile significantly caused greater inhibition of histamine stimulated gastric acid output than cimetidine at 120 min (p<0.05). When the extract was given after establishment of peak output, the gastric acidity dramatically fell to below the basal level. CONCLUSION: The aqueous bark extract of E. utile contains one or more active component(s) that can be developed as antisecretory medication for hypersecretory states or for protection of the compromised gastrointestinal mucosa.

Novel mechanism of endothelial nitric oxide synthase activation mediated by caveolae internalization in endothelial cells.

Caveolin-1, the caveolae scaffolding protein, binds to and negatively regulates eNOS activity. As caveolin-1 also regulates caveolae-mediated endocytosis after activation of the 60-kDa albumin-binding glycoprotein gp60 in endothelial cells, we addressed the possibility that endothelial NO synthase (eNOS)-dependent NO production was functionally coupled to caveolae internalization. We observed that gp60-induced activation of endocytosis increased NO production within 2 minutes and up to 20 minutes. NOS inhibitor N(G)-nitro-L-arginine (L-NNA) prevented the NO production. To determine the role of caveolae internalization in the mechanism of NO production, we expressed dominant-negative dynamin-2 mutant (K44A) or treated cells with methyl-beta-cyclodextrin. Both interventions inhibited caveolae-mediated endocytosis and NO generation induced by gp60. We determined the role of signaling via Src kinase in the observed coupling of endocytosis to eNOS activation. Src activation induced the phosphorylation of caveolin-1, Akt and eNOS, and promoted dissociation of eNOS from caveolin-1. Inhibitors of Src kinase and Akt also prevented NO production. In isolated perfused mouse lungs, gp60 activation induced NO-dependent vasodilation, whereas the response was attenuated in eNOS(-/-) or caveolin-1(-/-) lungs. Together, these results demonstrate a critical role of caveolae-mediated endocytosis in regulating eNOS activation in endothelial cells and thereby the NO-dependent vasomotor tone.

Regulation of endothelial nitric oxide synthase: involvement of protein kinase G 1 beta, serine 116 phosphorylation and lipid structures.

1. Endothelial nitric oxide synthase (NOS3) is important for vascular homeostasis. The role of protein kinase G (PKG) in regulation of NOS3 activity was studied in primary cultures of newborn lamb lung microvascular endothelial cells (LMVEC). 2. We determined the presence of PKG in fetal and neonatal LMVEC as well as subcellular localization of PKG isoforms in the neonatal cells by fluorescence immunohistochemistry. We used diaminofluorescein (DAF) fluorophore to measure nitric oxide (NO) production from neonatal LMVEC. We confirmed that NO measured was from constitutive NOS3 by inhibiting it with NOS inhibitors. 3. To identify a role for PKG in basal NO production, we measured NO release from LMVEC cells using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM; 0.5-0.8 micromol/L) with and without prior stimulation with the PKG activator 8-bromo-cGMP (8-Br-cGMP; 0.3 and 3 micromol/L) or prior PKG inhibition with beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (BPC; 0.3 and 3 micromol/L). With the same drugs, we determined the role of PKG on cellular expression of NOS3 and serine 116 phosphorylated NOS (pSer116-NOS) by qualitative and quantitative immunofluorescence assays, as well as western blotting. 4. Because PKG 1 beta was distributed throughout the cytosol in a punctate expression, we used 2 mmol/L cyclodextrin, a cholesterol extractor, to determine a role for lipid vesicles in PKG regulation of NO production. 5. Protein kinase G 1 beta gave a punctate appearance, indicating its presence in intracellular vesicles. Nitric oxide production decreased by approximately 20% with 300 nmol/L and 3 micromol/L 8-Br cGMP (P < 0.05) and increased by 20.8 +/- 3.7% with 3 micromol/L BPC (P < 0.001), indicating that both stimulated and basal PKG activity has inhibitory effects on basal NOS3 function. Nitric oxide synthase immunofluorescence and immunoblot expression were decreased and pSer116-NOS immunofluorescence was increased by 800 nmol/L 8-Br-cGMP and 170 micromol/L (Z)-1-[2-(2-aminoethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1, 2-diolate (DETANONOate). The effect of cyclodextrin indicated that cholesterol extraction interfered with PKG inhibition of NOS. Further examination of pSer116-NOS by immunohistochemistry showed it abundant in the endoplasmic reticulum and colocalized with PKG 1 beta, especially in nuclear vesicles. 6. We conclude that endothelial PKG is involved in endogenous regulation of basal NOS3 activity with the involvement of lipid structures, the endoplasmic reticulum and the nucleus. Protein kinase G 1 beta is colocalized with pSer116-NOS, indicating that PKG action may involve serine 116 phosphorylation on NOS.

Accessible Communication Tools for Surgical Site Infection Monitoring and Prevention in Joint Reconstruction: Feasibility Study.

BACKGROUND: The National Surgical Quality Improvement Program logs surgical site infections (SSIs) as the most common cause of unplanned postoperative readmission for a variety of surgical interventions. Hospitals are making significant efforts preoperatively and postoperatively to reduce SSIs and improve care. Telemedicine, defined as using remote technology to implement health care, has the potential to improve outcomes across a wide range of parameters, including reducing SSIs. OBJECTIVE: The purpose of this study was to assess the feasibility and user satisfaction of two automated messaging systems, EpxDecolonization and EpxWound, to improve perioperative care in a quality improvement project for patients undergoing total joint replacement. METHODS: We designed two automated text messaging and calling systems named EpxDecolonization, which reminded patients of their preoperative decolonization protocol, and EpxWound, which monitored pain, wound, and fever status postoperatively. Daily patient responses were recorded and a post-usage survey was sent out to participants to assess satisfaction with the systems. RESULTS: Over the 40-week study period, 638 and 642 patients were enrolled in EpxDecolonization (a preoperative decolonization reminder) and EpxWound (a postoperative surgical site infection telemonitoring system), respectively. Patients could be enrolled in either or both EpxDecolonization and EpxWound, with the default option being dual enrollment. The proportion of sessions responded to was 85.2% for EpxDecolonization and 78.4% for EpxWound. Of the 1280 patients prescribed EpxWound and EpxDecolonization, 821 (64.14%) fully completed the postoperative system satisfaction survey. The median survey score (scale 1-9) was 9 for patient-rated overall care and 8 for whether the telemonitoring systems improved patient communication with providers. The majority of patients (69.0%, 566/821) indicated that the systems sent out an ideal number of messages (not too many, not too few). CONCLUSIONS: EpxDecolonization and EpxWound demonstrated high response rates and improved patient-rated communication with providers. These preliminary data suggest that these systems are well tolerated and potentially beneficial to both patients and providers. The systems have the potential to improve both patient satisfaction scores and compliance with preoperative protocols and postoperative wound monitoring. Future efforts will focus on testing the sensitivity and specificity of alerts generated by each system and on demonstrating the ability of these systems to improve clinical quality metrics with more authoritative data.

A fluorescence-activated cell sorter analysis of the relationship between protein kinase G and endothelial nitric oxide synthase.

The process of regulation of NOS after production of nitric oxide is not yet delineated. Protein kinase G may exert a feedback regulation of this enzyme. We used diaminofluorescein assays to detect changes in basal nitric oxide production caused by modulators of protein kinase G activity in freshly isolated ovine lung microvascular endothelial cells. We also used fluorescence activated cell sorter analysis (FACS) to determine molecular and phosphorylation changes caused by PKG activation with 8-Br-cGMP. The PKG activator, 8-Br-cGMP (100 µM) produced a shift in the basal NO production curve downward. The inhibition began within 5 min and was sustained over 4.5 hr. The two protein kinase G inhibitors 100 µM Rp-8-Br-PET-cGMPS and 50 nM guanosine 3'-5'-cyclic monophosphoro thionate-8-Br-Rp isomer Na salt and the cGMP inhibitor 4 µM Rp-8-pCPT-cGMPS all enhanced NO production as seen by the upward shift in the basal NO curve. Conversely, the PKG activator drug, 100 µM guanosine-3'-5'-cyclic monophosphate-ß-phenyl-1NF-ethano-8-bromo sodium salt decreased NO production causing a downward shift in the basal curve. FACS analysis revealed that 5 µM 8-Br-cGMP in <5 min caused an increase in N-terminal labeling of NOS and a decrease in both C-terminal and serine 1177 labeling of NOS. 8-Br-cGMP appeared to increase PKG 1α and to decrease PKG 1ß labeling. Changes in other phosphorylation sites were less consistent but overall mean channel fluorescence increased from 19.92 to 217.36 for serine 116 and decreased from 329.27 to 254.03 for threonine 495 phosphorylation. Data indicated that PKG caused both molecular and phosphorylation changes in NOS.

Oxygen alters caveolin-1 and nitric oxide synthase-3 functions in ovine fetal and neonatal lung microvascular endothelial cells.

We determined the effect of oxygen [approximately 100 Torr (normoxia) and approximately 30-40 Torr (hypoxia)] on functions of endothelial nitric oxide (NO) synthase (NOS-3) and its negative regulator caveolin-1 in ovine fetal and neonatal lung microvascular endothelial cells (MVECs). Fetal NOS-3 activity, measured as NO production with 0.5-0.9 microM 4-amino-5-methylamino-2,7-difluorofluorescein, was decreased in hypoxia by 14.4% (P < 0.01), inhibitable by the NOS inhibitor N-nitro-L-arginine, and dependent on extracellular arginine. Caveolar function, assessed as FITC-BSA (160 microg/ml) endocytosis, was decreased in hypoxia by 13.5% in fetal and 22.8% in neonatal MVECs (P < 0.01). NOS-3 and caveolin-1 were physically associated, as demonstrated by coimmunoprecipitation and colocalization, and functionally associated, as shown by cross-activation of endocytosis, by their specific antibodies and activation of NOS by albumin. Caveolin peptide, containing the sequence for the PKC phosphorylation site of caveolin, and caveolin antiserum against the site increased NO production and endocytosis by 12.3% (P < 0.05) and 16% (P < 0.05), respectively, in normoxia and increased endocytosis by 25% (P < 0.001) in hypoxia. PMA decreased NO production in normoxia and hypoxia by 19.32% (P < 0.001) and 11.8% (P < 0.001) and decreased endocytosis in normoxia by 20.35% (P < 0.001). PKC kinase activity was oxygen sensitive, and threonine phosphorylation was enhanced in hypoxia. Pertussis toxin increased caveolar and NOS functions. These data support our hypothesis that increased Po(2) at birth promotes dissociation of caveolin-1 and NOS-3, with an increase in their activities, and that PKC and an oxygen-sensitive cell surface G protein-coupled receptor regulate caveolin-1 and NOS-3 interactions in fetal and neonatal lung MVECs.

Quantitative analysis of albumin uptake and transport in the rat microvessel endothelial monolayer.

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of (125)I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol. min(-1). 10(6) cells(-1). Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-beta-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased (125)I-albumin uptake 2.3-fold. At 37 degrees C, (125)I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (IC(50) = 1 microM). Using a two-site model, we estimated by Scatchard analysis the affinity (K(D)) and maximal capacity (B(max)) of albumin uptake to be 0.87 microM (K(D1)) and 0.47 pmol/10(6) cells (B(max1)) and 93.3 microM (K(D2)) and 20.2 pmol/10(6) cells (B(max2)). At 4 degrees C, we also observed two populations of specific binding sites, with high (K(D1) = 13.5 nM, 1% of the total) and low (K(D2) = 1.6 microM) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer.