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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.

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.

In Nigeria, medical students are trained in more didactic environments than their counterparts in researchintensive academic medical centers. Their conception of pharmacology was thus sought. Students who are taking/have takenthe medical pharmacology course completed an 18-question survey within 10min by marking one/more choices fromalternatives. Instructions were: "Dear Participant, Please treat as confidential, give your true view, avoid influences, avoidcrosstalk, return survey promptly." Out of 301 students, 188 (62.46%) participated. Simple statistics showed: 61.3%respondents associated pharmacology with medicine, 24.9% with science, 16.8 % with industry, and 11.1% with government;32.8% want to know clinical pharmacology, 7.1% basic pharmacology, 6.7% pharmacotherapy, and 34.2% want a blend ofall three; 57.8% want to know clinical uses of drugs, 44.8% mechanisms of action, 44.4% side effects, and 31.1% differentdrugs in a group; 45.8% prefer to study lecturers' notes, 26.7% textbooks, 9.8% the Internet, and 2.7% journals; 46.7% usestandard textbooks, 11.5% revision texts, 2.66% advanced texts, and 8.4% no textbook; 40.4% study pharmacology to beable to treat patients, 39.1% to complete the requirements for MBBS degree, 8.9% to know this interesting subject, and 3.1%to make money. Respondents preferring aspects of pharmacology were: 42.7, 16, 16, and 10 (%) respectively for mechanismsof action, pharmacokinetics, side effects, and drug lists. Medical students' conception and need for pharmacology werebased on MBBS degree requirements; they lacked knowledge/interest in pharmacology as a science and may not be thepotential trusts for Africa's future pharmacology.

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 minutes and was sustained over 4.5h. 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-β-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

This paper focuses on the notions of insider and outsider and these positions as they are situated in and out of the established western academic tradition, and the associated ethical, epistemological and methodological concerns, specifically as relates to both our independent and collaborative research with(in) Alaska Native Yup'ik communities. The paper begins with a brief discussion of traditional views of the dualistic notion of insider/outsider in ethnographic research, which is juxtaposed with tensions existing between traditional views of insider/outsider and the views of Indigenous ethnographers conducting research among Indigenous populations. Using examples taken from our independent and collaborative research data and transcripts from our Independent Study discussions, we map the paths taken in reconstructing a dualistic view of insider/outsider, western/indigenous, knowledge creator/knowledge receiver. Drawing on Habermas' theory of communicative action, Vygotsky's sociocultural theory of lang...

In the biomedical sciences, there is need to generate solutions for Africa's health and economic problems through the impact of university research. To guide organizational transformation, the author here presents some aspects of the state-of-the-arts of biomedical science research in advanced countries using a perspective derived from the FASEB journal publications. The author examines the thirty three peer reviewed scientific research articles in a centennial (April 2012) issue of the FASEB Journal [Volume 26(4)] using the following parameters: number of authors contributing to the paper; number of academic departments contributing to the paper; number of academic institutions contributing to the paper; funding of the research reported in the article. The articles were written by 7.97±0.61 authors from 3.46±0.3 departments of 2.79±0.29 institutions. The contributors were classified into four categories: basic sciences, clinical sciences, institutions and centers, and programs and labs. Amongst the publications, 21.2% were single disciplinary. Two tier collaboration amongst any two of the four categories were observed in 16/33 (48.5%) of the articles. Three tier and four tier collaborations were observed amongst 7/33 (21.2%) and 3/33 (9%) of the articles respectively. Therefore 26/33 (78.7%) of the articles were multidisciplinary. Collaborative efforts between basic science and clinical science departments were observed in 9/33 (27.3%) articles. Public funding through government agencies provided 85 out of a total of 143 (59.5%) grants. The collaborative and multidisciplinary nature and government support are characteristic of biomedical science in the US where research tends to result in solutions to problems and economic benefits.

The need for best evidence has driven researchers into multidisciplinary, collaborative approaches which have become mainstay in today's biomedical science. The multidisciplinary and collaborative approaches to research in research-intensive academic medical centres in the USA and in other countries of affluence has brought in significant advancement in knowledge as well as colossal progress and financial benefits. Therefore the author speculates that for Nigerian and other African PhD graduates in the basic medical sciences to become successful researchers, effective peer reviewers, reliable mentors, and progressive administrators in research-based academia, they need some exposure to multidisciplinary approaches to research during their subject-based training. The present report sought to substantiate this need. Thirty three published articles in the April 2012 FASEB Journal were studied for the methodologies employed and the results showed that the papers utilized an average of nine major biomedical science techniques, 9 being the mean, median, and mode showing the global status quo of diversity of methodology per scientific paper. The most popular procedures and techniques recorded in more than 1/3 of the published articles were: cell isolation; cell culture; in vivo or in situ whole animal studies; animal models of disease; gene/protein expression, sequencing and cloning; transfection, constructs, and genomic interference and silencing; western blotting; fluorescence and confocal microscopy; ELISAs and cell-based assays; and ready-made biotech assay kits. The most popular statistics testing were various forms of student's t-tests at 0.05 confidence levels and ANOVAs. The GraphPad Prism software was the most frequently used statistic software.

Furchgott and his co-workers reported the vascular actions of an unknown biological mediator that was named endothelium-derived relaxing factor (EDRF).1 Nitric oxide (NO) was soon discovered as a vasodilator that had identical properties with EDRF.2,3,4 NO produced by the endothelium diffuses to the adjacent vascular smooth muscle cells causing them to relax. This function is important in many vascular beds such as the coronary circulation.5, 6

Nitric oxide synthase (NOS) is regulated by protein-protein interactions. We had earlier shown that PKG inhibits activated NOS in endothelial cells and speculated that PKG phosphorylation of NOS terminates its activity. The present work examines if PKG activation increases breakdown of NOS. Diamino-fluorescein fluorescence spectrometry of real time NO production was used to establish that isolated ovine lung microvascular endothelial cells responded to PKG modulation as previously reported. Fluorescence activated cell sorter (FACS) analysis was used to establish that 8-Br-cGMP, a PKG activator, caused carboxy terminal deletion on NOS, a sign of degradation. Western blot analysis was used to investigate NOS fragments in control and 5 min 8-Br-cGMP treated cells. PKG activator 8-Br-cGMP, at 20 nM, 200 nM, and 2 μM, decreased nitric oxide production in a dose dependent manner (p0.05 in all cases). PKG inhibitors: 100 μM Rp-8-Br-PET-cGMPS, 50 nM Rp-8-pCPT-cGMPS, or 4 μM Rp-8-Br-cGMPS Na significantly increased NO production (p0.05) showing that PKG normally inhibits basal NO production. 8-Br-cGMP (100 nM) abrogated the elevation in NO production produced by the PKG inhibitors. FACS analysis revealed that PKG decreased NOS carboxy terminal labeling. Western blot analysis revealed that 8-Br-cGMP increased N-terminal serine-116 phosphorylated NOS fragments of molecular weights of about 60, 50 and 35 kDa. PKG may be a post-activation inhibitor of NOS, possibly important for the degradation of the spent enzyme.

The effect of chronic high altitude hypoxia (CHAH) in the juxta-alveolar region near the air-blood interface is unknown because of the experimental inaccessibility of this region.To examine primary cultures of digested juxta-alveolar smooth muscle cells for hypoxia-induced changes.Smooth Muscle Cells (SMCs) obtained by dispase digestion of the extreme lung parenchyma were used to study the effect of CHAH in the juxta-alveolar region and foetal and maternal cells were compared. Pulmonary venous SMCs were also obtained from dissected 5th to 7th generation levels pulmonary veins (0.5 mm). Fluorescence tagged antibodies against alpha smooth muscle actin (alpha SMA) and calponin respectively were used as markers to identify cellular structural differences by routine immunohistochemistry. Comparison of the functional integrity of the cells was made using their growth profiles obtained by radiolabeled thymidine incorporation and liquid scintillation counting.Marked differences were seen in juxta-alveolar SMCs obtained by digestion of extreme lung parenchyma of hypoxic sheep. Hypoxic adult sheep cells showed increased filamentation. Hypoxic foetal sheep cells showed internal restructuring and disorganization of both alpha-SMA and calponin filaments. The growth profiles of juxta-alveolar SMCs showed that the hypoxia-affected cells of both the foetus and adult sheep had a fast initial growth rate peaking at 48h while their normoxic equivalents had a steadier growth rate peaking at 72h. Hypoxia-affected cells showed contact inhibition at ~50% subconfluence and apoptosis by 48h.Chronic high altitude hypoxia causes both phenotypical and functional changes in pulmonary smooth muscle cells near the air/blood interface.

An aqueous extract of Entandrophragma utile bark was shown to be efficacious in preventing experimental ethanoi-induced ulcers in rats. A therapeutic dose equivalent to 50 g/kg of bark orally caused total protection without lethality in the animals employed. The formation of a protective pellicle over the epithelial lining was observed, which may be due to the presence of tannins in the herbal medicine and this may prevent the absorption of noxious agents. In mice, a lethality dose-response curve could not be established orally. The maximum dosage administered was equivalent to 500 g/kg of the bark and produced 25% lethality.

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.

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-β-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.

After shear stress, NOS-3 is phosphorylated at serine 116 (pSer116) (J Biol Chem, 1999). Whether phosphorylation at this site is related to activation of NOS-3 and the endogenous kinase(s) involved...

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of125I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol · min−1· 106cells−1. Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-β-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased125I-albumin uptake 2.3-fold. At 37°C,125I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (IC50= 1 μM). Using a two-site model, we estimated by Scatchard analysis the affinity ( KD) and maximal capacity (Bmax) of albumin uptake to be 0.87 μM ( KD1) and 0.47 pmol/106cells (Bmax1) and 93.3 μM ( KD2) and 20.2 pmol/106cells (Bmax2). At 4°C, we also observed two populations of specific binding sites, with high ( KD1= 13.5 nM, 1% of the total) and low ( KD2= 1.6 μM) 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.

Hypothermia is intentionally imposed during the harvesting of lungs for transplantation. The aim of this study was to investigate the fluid balance alterations in rat lung preparations exposed to hypothermic perfusion. Lowering perfusate temperature from 37 degrees C to values between 27 and 7 degrees C caused an immediate, marked pulmonary hypertension and vasoconstriction accompanied by rapid development of pulmonary edema (+1.15 g, or approximately 90%, gain in lung weight within 5 min). However, on rewarming, vasoconstriction was immediately reversed. Edema was resolved, but along a two-component time course: an immediate reduction of lung weight on rewarming (t 1/2 of 0.5 min) that mirrored the recovery of pulmonary artery pressure and vasoconstriction, and also a slower pressure-independent component of recovery (t 1/2 of 3.5 min). Ouabain (300 microM) markedly inhibited the lung's ability to recover from edema, indicating that fluid clearance from lung tissue was the result of activation of ouabain-sensitive (Na+,K+)-ATPase pump. Results could not be explained by vascular or airspace injury as lung sections from hypothermic lungs appeared normal. The findings indicate that hypothermia induces pulmonary edema formation, which can be rapidly cleared upon rewarming by activation of ouabain-sensitive (Na+,K+)-ATPase pump. Thus, impaired fluid clearance from lung extravascular spaces may be a critical factor limiting gas exchange in transplanted lungs exposed to hypothermia.

The alveolar epithelium represents the rate-limiting barrier to solute and fluid transport between the pulmonary capillary lumen and distal air spaces (Schneeberger & Karnovsky, 1971; Gorin et al. 1979; Berthiaume et al. 1989). The liquid in contact with the respiratory surface of alveoli is restricted to a thin layer of epithelial lining fluid (ELF). Type I alveolar epithelial cells regulate the ELF composition and volume, which in turn is important for the maintenance of low alveolar surface tension and efficient gas exchange. Relative to blood plasma, this fluid contains lower Na+ and higher K+ concentrations resulting from the activity of a Na+-K+ ionic pump in the basolateral epithelial membrane (Basset et al. 1987; Goodman et al. 1987; Valeyre et al. 1991; Rutschman et al. 1993; Saumon & Basset, 1993) and from passive Na+ transport through apical Na+ channels (Matalon & O'Brodovich, 1999). The protein concentration in ELF is also found to be lower than that of plasma by a factor of 4 or more depending on the species investigated and methodology employed (Peterson et al. 1990; Peterson et al. 1993; Pusch et al. 1997). Such a steep protein concentration gradient implies the existence of an active transport process for plasma proteins. In this regard, some investigators have recognized the existence of a monensin- and nocodazole-sensitive protein uptake pathway in alveolar epithelium of intact mammalian lungs (Hastings et al. 1994; Wangensteen et al. 1996); however, the quantitative importance of such uptake for overall alveolar protein clearance remains unclear (see review by Folkesson et al. 1996). Active transport of protein molecules across endothelial cells was shown, in some instances, to occur by a receptor-mediated process (Vasile et al. 1983; Ghitescu et al. 1986; Descamps et al. 1996). In the pulmonary microvascular endothelium, albumin bound with high specificity to the 60 kDa albumin-binding glycoprotein (gp60 or albondin) and was shown to activate albumin transcytosis through the endothelium (Schnitzer et al. 1988; Schnitzer, 1992; Tiruppathi et al. 1996; Minshall et al. 2000). Cross-linking of gp60 with an anti-gp60 antibody stimulated endocytosis (as measured by fluorescent styryl pyridinium probes) and albumin uptake (quantified with radiolabelled albumin) in cultured endothelial cells (Tiruppathi et al. 1997; Niles & Malik, 1999; Minshall et al. 2000). As a transcellular albumin transport pathway may also be present in alveolar epithelial cells (Kim et al. 1985), the purpose of the present study was to characterize the albumin clearance mechanism of the alveolar epithelium with particular reference to the possible role of gp60 in regulating albumin transport in the intact lung.

We investigated the function of proteinase-activated receptor-1 (PAR-1) in the regulation of pulmonary microvascular permeability in response to thrombin challenge using PAR-1 knockout mice (−/−). Lungs were isolated and perfused with albumin (5 g/100 ml)-Krebs solution at constant flow (2 ml/min). Lung wet weight and pulmonary artery pressure (Ppa) were continuously monitored. We determined the capillary filtration coefficient ( Kfc) and125I-labeled albumin (BSA) permeability-surface area product (PS) to assess changes in pulmonary microvessel permeability to liquid and albumin, respectively. Normal and PAR-1-null lung preparations received in the perfusate: 1) thrombin or 2) selective PAR-1 agonist peptide (TFLLRNPNDK-NH2). In control PAR-1 (+/+) mouse lungs,125I-albumin PS and Kfcwere significantly increased over baseline (by ∼7- and 1.5-fold, respectively) within 20 min of α-thrombin (100 nM) challenge. PAR-1 agonist peptide (5 μM) gave similar results, whereas control peptide (5 μM; FTLLRNPNDK-NH2) was ineffective. At relatively high concentrations, thrombin (500 nM) or PAR-1 agonist peptide (10 μM) also induced increases in Ppaand lung wet weight. All effects of thrombin (100 or 500 nM) or PAR-1 agonist peptide (5 or 10 μM) were prevented in PAR-1-null lung preparations. Baseline measures of microvessel permeability and Ppain the PAR-1-null preparations were indistinguishable from those in normal lungs. Moreover, PAR-1-null preparations gave normal vasoconstrictor response to thromboxane analog, U-46619 (100 nM). The results indicate that the PAR-1 receptor is critical in mediating the permeability-increasing and vasoconstrictor effects of thrombin in pulmonary microvessels.