Biomedical subjects

M Lu

Publications and source records attributed to M Lu.

At least 199 records · Page 11Linked to original sources

PubMed1997

Distinct transcriptional pathways of TAR-dependent and TAR-independent human immunodeficiency virus type-1 transactivation by Tat.

Tat stimulates HIV-1 gene expression during transcription initiation and elongation. Tat functions primarily through specific interactions with TAR RNA and several putative cellular cofactors to increase the processivity of RNA polymerase II complexes during HIV-1 transcription elongation. Although HIV-1 transactivation by Tat in most cell types requires intact TAR sequences, previous reports demonstrate that Tat transactivates HIV-1 long terminal repeat (LTR)-directed gene expression in several central nervous system-derived astrocytic/glial cell lines in the absence of TAR. Within this study, transient expression assays performed in the astrocytic/glial cell line, U87-MG, confirm that kappa B elements within the HIV-1 LTR mediate TAR-independent transactivation by Tat and demonstrate additionally that distinct amino acid residues within the cysteine-rich activation domain of Tat are required for TAR-independent versus TAR-dependent transactivation. Established U87-MG cell lines expressing a transdominant negative mutant of I kappa B alpha, I kappa B alpha delta N, fail to support TAR-independent transactivation by Tat, suggesting that binding of NF-kappa B to kappa B enhancer elements within the HIV-1 LTR is necessary for Tat-mediated transactivation in the absence of TAR. Ribonucleic acid protection analyses of promoter-proximal and -distal transcripts derived from TAR-deleted and TAR-containing HIV-1 LTR reporter constructs in U87-MG cells indicate that the predominant effect of Tat during TAR-independent transactivation occurs at the lavel of transcription initiation, whereas a prominent elongation effect of Tat is observed in the presence of TAR. These data suggest an alternative regulatory pathway for Tat transactivation in specific cells derived from the central nervous system that is independent of TAR and that requires direct or indirect interaction of Tat with NF-kappa B-binding sites in the HIV-1 LTR.

Amino Acid Sequence↗

PubMed1997

Cloning and chromosomal localization of mouse aquaporin 4: exclusion of a candidate mutant phenotype, ataxia.

Aquaporin-4 is a mammalian water channel protein that is predominately expressed in brain, where it is believed to mediate water homeostasis. Here we report the isolation and characterization of the cDNA for mouse Aqp4 and map the gene to the proximal region of mouse chromosome 18. This region contains the neurological mutation ataxia, but further analysis reveals that Aqp4 is not responsible for the ataxia phenotype.

Amino Acid Sequence↗

PubMed1997

Interaction between tryptophan residues and hydrophobically modified dextran. Effect on partitioning of peptides and proteins in aqueous two-phase systems.

Hydrophobically modified dextrans, benzoyl dextran and valeryl dextran, have been used to study the interactions between tryptophan residues and benzoyl or valeryl groups by partitioning of tryptophan, tryptophan-tryptophan, (tryptophan)3, poly(lysine, tryptophan), beta-galactosidase and lysozyme in polymer aqueous two-phase systems. The two-phase systems used were polyethylene glycol (PEG)-benzoyl dextran, PEG-valeryl dextran, dextran-benzoyl dextran and dextran-valeryl dextran. Interaction between tryptophan residues and benzoyl or valeryl groups was observed by partitioning of tryptophan containing compounds to the phase containing hydrophobically modified dextran. At a certain phase composition the interactions were increased with increasing number of tryptophan per molecule. In a PEG-dextran system the partitioning of tryptophan peptides to the PEG phase was increased with increased number of tryptophan. In a PEG-benzoyl dextran system the opposite effect was obtained. At similar conditions benzoyl groups showed stronger interactions with tryptophans compared to valeryl groups. The partition coefficient of salts (sodium phosphate, NaCl, Nal and NaClO4) was determined in PEG-benzoyl dextran and PEG-valeryl dextran aqueous two-phase systems. The effect of addition of these salts on partitioning of poly(lysine, tryptophan), beta-galactosidase and lysozyme was studied. Salt effects on partitioning could be explained by the relative affinities of the ions for the polymers in the system. Charged molecules containing tryptophan were to an increasing degree partitioned to the phase for which the counterions had highest affinity. Strong effects on the partitioning of positively charged poly(lysine, tryptophan) and lysozyme were obtained with the ions I- and ClO4-.

Chemical Phenomena↗

PubMed1997

Platelet factor 4 and other CXC chemokines support the survival of normal hematopoietic cells and reduce the chemosensitivity of cells to cytotoxic agents.

The effects of platelet factor 4 (PF4) on the viability and chemosensitivity of normal hematopoietic cells and cancer cell lines were studied to determine the mechanisms whereby PF4 functions as either an inhibitor or a protector and to evaluate its clinical significance. Two other chemokines, interleukin-8 (IL-8) and neutrophil-activating peptide-2 (NAP-2), were also studied in comparison to PF4. Using a tetrazolium salt assay for cell viability, we observed that PF4 at 1 to 50 microg/mL supported the viability of normal human bone marrow cells. Approximately 45% of cells cultured for 48 hours survived, whereas 80% or more survived in the presence of PF4 5 microg/mL. PF4 also supported the viability of CD34+ cord blood (CB) cells and protected them from apoptosis induced by transforming growth factor beta1 (TGFbeta1) and cytotoxic drugs. Pretreatment of CD34+ cells by PF4, but not by TGFbeta1, caused an increase in the number of megakaryocyte colonies after these cells were replated in secondary cultures. Flow cytometry analysis showed that when CD34+ cells were preincubated with PF4 or TGFbeta1 for 12 days in hematopoietic growth factor-rich medium, an increased number of remaining CD34+ cells was observed only for PF4-treated cells. Furthermore, PF4 significantly reduced the chemosensitivity of bone marrow cells, as shown by its ability to increase the 50% inhibition concentration (IC50) of several cytotoxic agents. Like PF4, IL-8 and NAP-2 at 0.1, 0.6, and 1 microg/mL supported the survival of myeloid progenitors, including colony-forming units granulocyte, erythroblast, monocyte, megakaryocyte (CFU-GEMM), CFU-megakaryocyte (CFU-MK), CFU-granulocyte/macrophage (CFU-GM), and burst-forming units-erythroblast (BFU-E), and reduced their sensitivity to the toxicity of etoposide (ETP). Protamine sulfate at 1 to 100 microg/mL showed no such activity of PF4. Interestingly, the three chemokines failed to affect significantly the viability and chemosensitivity of three leukemic and two other tumor cell lines. Based on these results, we conclude for the first time that PF4 and IL-8 and NAP-2 support the survival of normal hematopoietic precursors and protect them from the toxicity of chemotherapeutic agents. Because such activities are unique to normal hematopoietic cells but not to the cancer cell lines evaluated, a potential clinical application of these molecules in the treatment of cancer is suggested.

Apoptosis↗

PubMed1997

Microtubule-associated protein 2c reorganizes both microtubules and microfilaments into distinct cytological structures in an actin-binding protein-280-deficient melanoma cell line.

The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments.

Actin Cytoskeleton↗

PubMed1997

Characterization of a domain of the genome of BmNPV containing a functional gene for a small capsid protein and harboring deletions eliminating three open reading frames that are present in AcNPV.

The nucleotide sequence of a 1.5-kb fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been determined. An open reading frame (ORF) has been identified, which encodes a protein of a predicted molecular mass of 15 kDa (p15). Analysis of the p15 sequence revealed that it shares significant similarities with a number of previously characterized viral capsid proteins. Two mRNAs, 0.7- and 1.3-kb, are transcribed from the p15 gene. The 0.7-kb transcript appears before BmNPV DNA replication, while the 1.3-kb transcript is transcribed only after viral DNA replication. The transcription start sites for the 0.7- or 1.3-kb transcripts have been localized into two areas mapping 114 and 557/559 bp upstream of the p15 translation initiation codon, respectively, and the two transcripts appear to share a common poly(A) addition site located 114 bp downstream of the translation termination codon. Comparisons of the nucleotide sequence of the 1.5-kb BmNPV fragment with that of another isolate of BmNPV and with Autographa californica NPV (AcNPV) genomic DNA reveal that the corresponding region of AcNPV encompasses 4.1 kb of DNA and three additional ORFs that are absent from the BmNPV genome. These findings raise questions about the origin and functional relevance of the putative polypeptides encoded by the three ORFs of AcNPV.

Amino Acid Sequence↗

PubMed1997

Transforming function of the HOX11/TCL3 homeobox gene.

The HOX11/TCL3 homeobox gene was identified at the breakpoint region in pediatric T-cell acute lymphoblastic leukemia harboring 10q24 chromosomal translocations. We previously reported that primary murine bone marrow cells transduced ex vivo with a recombinant HOX11-containing retrovirus, MSCV-HOX11, gave rise to cell lines at high frequency having characteristics of early myeloid cells. Cell lines were also established from the bone marrow and spleen of transplant recipients sacrificed 5 months after engraftment with MSCV-HOX11-transduced bone marrow cells. These latter lines, which exhibited a more differentiated myelomonocytic phenotype, harbored proviruses encoding a smaller HOX11 protein. None of the mice that received HOX11-expressing bone marrow cells or myeloid cell lines developed leukemia during 6-month observation periods. Here, we report that two bone marrow transplant recipients eventually developed T-cell acute lymphoblastic leukemia-like malignancies at 7 and 12 months posttransplant, indicating that progression to a fully malignant state required additional mutations. One tumor synthesized full-length HOX11 whereas the other expressed the smaller version of the protein. The smaller HOX11 protein suffered a carboxyl-terminal truncation. We subsequently constructed MSCV-based retroviral vectors expressing deleted forms of HOX11 and identified an amino-terminal region that was dispensible for generation of myeloid cell lines having a similar phenotype as those induced by full-length HOX11. We thus conclude that regions near the amino and carboxyl termini of HOX11 are not essential for transforming function, nor do they appear to determine the lineage or stage of differentiation of the target cell for transformation.

Animals↗

PubMed1997

Total quality improvement method for reduction of delays between emergency department admission and treatment of acute ischemic stroke. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group.

OBJECTIVE: To develop an approach for reducing time between emergency department (ED) admission and treatment in patients with acute ischemic stroke to meet the challenge of providing tissue plasminogen activator treatment within 180 minutes. DESIGN: An observational study. SETTING: Forty trial-affiliated hospitals, including 30 community hospitals. PARTICIPANTS: A total of 17,324 consecutive patients admitted to trial-affiliated hospital EDs within 24 hours of possible stroke, from January 1991 through October 1994. INTERVENTION: Appraisal of the process of triage, evaluation, diagnosis, and treatment by means of total quality improvement techniques in each hospital. Staff participating in the process identified sources of variation and modifications by flow charting the process. MAIN OUTCOME MEASURE: Time between ED admission and treatment with study medication. RESULTS: Total quality improvement methods identified hospital-specific process improvements. Many improvements were administrative, requiring no additional resources. More than 50% of screened patients arrived too late to be treated. Only 1268 patients were admitted between 0 and 125 minutes from stroke onset with no other trial exclusion criteria; 48% were treated. Of 243 patients admitted between 126 and 170 minutes from stroke onset with no exclusion criteria, 4% were treated. Mean time from ED admission to treatment was similar in teaching and community hospitals. CONCLUSIONS: Total quality improvement methods identified ED-specific sources of process variability and reduced time between ED admission and treatment. Therefore, these methods should be considered in developing and monitoring emergent stroke treatment protocols.

Acute Disease↗

PubMed1997

Different susceptibilities of genetic variants of hepatitis C virus (HCV) to interferon (IFN).

Genetic variants of HCV may have different degrees of resistance to IFN and may therefore influence the outcome of IFN therapy. However, selection of HCV variants by IFN has not been investigated in detail. In this paper, heteroduplex analysis was used to monitor major changes of HCV populations in 4 chronically infected patients under IFN therapy. We found that a major variant of the HCV 5' non-coding region (5' NCR) emerged in a responder. In other patients although no new variant of the 5' NCR was identified, significant changes occurred within the core and E1 region of the HCV genome. Disappearance and emergence of HCV variants may reflect their different susceptibilities to IFN. Our results indicate that responses of HCV populations to IFN are complex and need to be characterized by analysis of multiple HCV genome regions.

Antiviral Agents↗

PubMed1997

A trimeric structural subdomain of the HIV-1 transmembrane glycoprotein.

HIV-1 infection is initiated by the fusion of viral and cellular membranes with subsequent transfer of viral genetic material into the cell. The HIV-1 transmembrane envelope glycoprotein gp41 plays a major role in this membrane fusion process. Previous studies have shown that a stable, alpha-helical, trimeric structural domain of gp41 consists of N-terminal 51-residue (N-51) and C-terminal 43-residue (C-43) extraviral segments. This alpha-helical, trimeric complex has been proposed to form the core of the membrane fusion-active conformation of the HIV-1 envelope. We show here that a stable subdomain can be formed by shorter 36-residue (N-36) and 34-residue (C-34) peptides corresponding to the central regions of N-51 and C-43, respectively. In isolation, N-36 is predominantly aggregated, while C-34 is unfolded. Upon mixing, however, these peptides form a stable, alpha-helical, discrete trimer of heterodimers (the melting temperature of a 10 microM solution is 64 degrees C at pH 7). Thus, this subdomain displays the salient features of the stable core structure of the isolated gp41 protein. Our results also provide strong support for the notion that short peptides can form unique, cooperatively folded subdomains, in which elements of secondary structure are stabilized by native-like tertiary interactions.

Endopeptidase K↗

PubMed1997

Nitric oxide links the apical Na+ transport to the basolateral K+ conductance in the rat cortical collecting duct.

We have used the patch clamp technique to study the effects of inhibiting the apical Na+ transport on the basolateral small-conductance K+ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 microM amiloride decreased the activity of SK, defined as nPo (a product of channel open probability and channel number), to 61% of the control value. Application of 1 microM benzamil, a specific Na+ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na+ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 microM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca2+ because removal of Ca2+ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca2+, we observed that amiloride and benzamil significantly decreased intracellular Ca2+ in the Ca2+-containing solution but had no effect in a Ca2+-free bath. Furthermore, raising intracellular Ca2+ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca2+ are responsible for the effects on SK activity of inhibition of the Na+ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca2+ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP-dependent pathways. Addition of 10 microM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 microM 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca2+-free bath solution. We conclude that Ca2+-dependent NO generation mediates the effect of inhibiting the apical Na+ transport on the basolateral SK in the rat CCD.

Amiloride↗

PubMed1997

Cloning and characterization of a complete open reading frame of the hepatitis C virus genome in only two cDNA fragments.

The synthesis of long cDNA molecules encoding the complete genome of RNA viruses has recently been demonstrated; this major improvement has numerous practical applications such as construction of infectious cDNA clones or study of sequence variability at the level of a single RNA molecule. Using hepatitis C virus (HCV) as a model, we established an RT-PCR technique for amplification of cDNA fragments with a length of about 5 kb. The RT reaction was carried out with a Moloney murine leukaemia virus reverse transcriptase lacking detectable RNase H activity. For PCR reactions an enzyme mix containing Taq and Pwo DNA polymerases was used. Hot start and addition of 5% DMSO were also important to efficiently achieve long PCR products. About 10(6) HCV genome equivalents/ml in serum were needed in order to amplify the HCV genome in only two cDNA fragments covering about 98% of the complete genome. Analysis of the HCV quasi-species is also possible by this method as shown by sequencing of the hypervariable region 1 (HVR1) after cloning of cDNAs. The integrity of the long cDNA clones was proven by (1) restriction analyses, (2) partial sequencing and (3) expression of respective gene products. In vitro transcribed cDNAs were translated in rabbit reticulocyte lysate. Structural and nonstructural HCV proteins were identified by immunoprecipitation using patient serum. These results suggest that the two cDNA clones encode a complete and functional open reading frame of HCV.

Amino Acid Sequence↗

PubMed1997

Amantadine and equine influenza: pharmacology, pharmacokinetics and neurological effects in the horse.

Amantadine is an antiviral agent effective against influenza A viruses. We investigated 1) the antiviral efficacy, 2) analytical detection, 3) bioavailability and disposition, 4) pharmacokinetic modelling and 5) adverse reactions of amantadine in the horse. In vitro, amantadine and its derivative rimantadine suppressed the replication of recent isolates of equine-2 influenza virus with effective doses (EDs) of less than 30 ng/ml. Rimantadine was more effective than amantadine against most viral isolates; we suggest a minimum plasma concentration of 300 ng/ml of amantadine for therapeutic efficacy. In vivo an i.v. dose of amantadine 15 mg/kg bwt produced mild, transient CNS signs which were no longer apparent after 30 min. Amantadine administered at a dose of 15 mg/kg bwt was established as the maximum safe single i.v. dose. However, if repeated i.v. administration of amantadine is required no more than 10 mg/kg bwt t.i.d. should be used. The maximal safe plasma concentration of amantadine was not evaluated but is probably greater than 2000 ng/ml and possibly greater than 4000 ng/ml. On the other hand, horses with lower seizure thresholds, or those on medications that lower seizure thresholds, may be at increased risk of amantadine-induced seizures, which show few premonitory signs and are rapidly fatal. After i.v. administration of amantadine 10 mg/kg bwt, the disposition kinetics were well fitted by a 2-compartment open model. The estimated peak plasma concentration after this dose was about 4500 ng/ml, the volume of distribution at steady-state (Vdss) was (mean +/- s.d.) 4.9 +/- 1.9 l/kg bwt and the beta phase half-life was 1.83 +/- 0.87 h. Computer projections of plasma amantadine concentrations after i.v. administration of amantadine at a dose of 10 mg/kg bwt t.i.d. at 8 h intervals suggest peak plasma concentrations of 4000-5000 ng/ml and troughs of less than 300 ng/ml will be achieved. Amantadine administered orally at 10 mg/kg bwt and 20 mg/kg bwt showed mean oral bioavailability of about 40-60% and a plasma half life of 3.4 +/- 1.4 h; however, there was substantial inter-animal variation in bioavailability. Projections based on the kinetics observed in individual animals suggest that some animals readily maintain effective plasma concentrations of amantadine after oral administration of 20 mg/kg bwt t.i.d. On the other hand, animals in which amantadine is poorly bioavailable may require up to a 6-fold (120 mg/kg bwt) increase in the oral dose to achieve effective blood concentrations. Withholding food for 15 h did not reduce these inter-animal differences in bioavailability. Our results showed that simple dosing with oral amantadine will not yield effective plasma concentrations in all animals. While i.v. administration yielded more reproducible plasma concentrations, care should be taken to see that the seizure threshold is not exceeded. In acute situations, i.v. administration (5 mg/kg bwt) every 4 h should maintain safe and effective plasma and respiratory tract concentrations of amantadine.

Administration, Oral↗

PubMed1997

Characterization of T-cell response to woodchuck hepatitis virus core protein and protection of woodchucks from infection by immunization with peptides containing a T-cell epitope.

Specific activation of T cells appears to be a prerequisite for viral clearance during hepatitis B virus (HBV) infection. The T-cell response to HBV core protein is essential in determining an acute or chronic outcome of HBV infection, but how this immune response contributes to the course of infection remains unclear. This is due to results obtained from humans, which are restricted to phenomenological observations occurring during the clinical onset after HBV infection. Thus, a useful animal model is needed. Characterization of the T-cell response to the core protein (WHcAg) of woodchuck hepatitis virus (WHV) in woodchucks contributes to the understanding of these mechanisms. Therefore, we investigated the response of woodchuck peripheral blood mononuclear cells (PBMCs) to WHcAg and WHcAg-derived peptides, using our 5-bromo-2'-deoxyuridine assay. We demonstrated WHcAg-specific proliferation of PBMCs and nylon wool-nonadherent cells from acutely WHV-infected woodchucks. Using a cross-reacting anti-human T-cell (CD3) antiserum, we identified nonadherent cells as woodchuck T cells. T-cell epitope mapping with overlapping peptides, covering the entire WHcAg, revealed T-cell responses of acutely WHV-infected woodchucks to peptide1-20, peptide100-119, and peptide112-131. Detailed epitope analysis in the WHcAg region from amino acids 97 to 140 showed that T cells especially recognized peptide97-110. Establishment of polyclonal T-cell lines with WHcAg or peptide97-110 revealed reciprocal stimulation by peptide97-110 or WHcAg, respectively. We vaccinated woodchucks with peptide97-110 or WHcAg to prove the importance of this immunodominant T-cell epitope. All woodchucks immunized with peptide97-110 or WHcAg were protected. Our results show that the cellular immune response to WHcAg or to one T-cell epitope protects woodchucks from WHV infection.

Animals↗

PubMed1997

Nitric oxide-induced hyperpolarization stimulates low-conductance Na+ channel of rat CCD.

We used the patch-clamp technique in the split-open cortical collecting duct (CCD) to investigate the effect of nitric oxide (NO) on the low-conductance (6-pS) Na+ channel that can be blocked by 1 microM amiloride. We confirmed that the number of Na+ channels increased significantly in CCDs of rats on a low-Na+ diet (17). Application of 100 microM N(G)-nitro-L-arginine methyl ester (L-NAME), an agent that blocks endogenous NO synthase, reduced NPo [the product of channel number (N) and open probability (Po)] to 45% of the control value. The effect of L-NAME was specific, since addition of D-NAME, which does not inhibit NO synthase, did not change the activity of the Na+ channel. That the effect of L-NAME results from inhibition of NO synthase is further confirmed by experiments in which addition of an exogenous NO donor, either 10 microM S-nitroso-N-acetyl penicillamine or sodium nitroprusside (SNP), restored the Na+ channel activity when it had been blocked by L-NAME. The action of NO involves a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent pathway, since 100 microM 8-bromo-cGMP (8-BrcGMP) mimicked the effect of SNAP on K+ channels. However, 100 microM 8-BrcGMP did not alter the activity of Na+ channels in inside-out patches, suggesting an indirect action. Because the Na+ channel is activated by hyperpolarization (19) and NO stimulates basolateral K+ channels (16), we tested whether hyperpolarization mediated the effect of NO. In perforated whole cell recordings, addition of L-NAME depolarized the cell membrane from -73 to 51 mV, and application of 10 microM SNP repolarized the membrane to -68 mV. Furthermore, the L-NAME-induced decrease in NPo was effectively restored by 25 mV hyperpolarization of the patch membranes, and addition of 2 mM Ba2+ also abolished the effect of L-NAME. We concluded that the stimulatory effect of NO on the Na+ channel is an indirect effect mediated by a NO-induced increase of basolateral K+ conductance.

Animals↗

PubMed1997

Phospholipase A2 is involved in mediating the effect of extracellular Ca2+ on apical K+ channels in rat TAL.

Raising extracellular Ca2+ (Ca2+o) stimulating the Ca(2+)-sensing receptor (CaR) decreased the activity of the apical 70-pS K+ channel via a cytochrome P-450-dependent mechanism in the thick ascending limb (TAL) of the rat kidney [W. H. Wang, M. Lu, and S. C. Hebert. Am. J. Physiol. 270 (Cell Physiol. 39): C103-C111, 1996]. We have now used the patch-clamp technique and fluorescent dyes to investigate the signaling mechanism by which this effect is produced. Addition of 500 microM gadolinium (Gd3+), an agent which has been shown to activate the CaR (E. M. Brown, G. Gamba, D. Riccardi, M. Lombardi, R. Butters, O. Kifor, A. Sun, M. A. Hediger, J. Lytton, and S. C. Hebert. Nature 366: 575-580, 1993), mimics the inhibitory effect of raising Ca2+o from 1.1 to 5 mM on channel activity. Effects of the high Ca2+o and Gd3+ were abolished by blockade of phospholipase A2 (PLA2) but not by inhibition of phospholipase C (PLC). Raising Ca2+o also increased 20-hydroxyeicosatetraenoic acid production significantly. To investigate the effect of stimulation of the CaR on intracellular Ca2+ (Ca2+i), we used the acetoxymethyl ester of fura 2 to monitor the Ca2+i. Raising Ca2+o from 1.1 to 5 mM increased the Ca2+i significantly from 50 to 150 nM. However, addition of thapsigargin failed to abolish the effect of 5 mM Ca2+o on Ca2+i. Also, application of Gd3+ only slightly increased the Ca2+i, suggesting that elevation of the Ca2+i by high Ca2+o was the result of an influx of Ca2+ rather than enhanced Ca2+ release from Ca2+ stores. That the increase in Ca2+ influx is not mainly responsible for the effect of stimulating the CaR on channel activity is further supported by experiments in which 500 microM Gd3+ inhibited the K+ channel in cell-attached patches in a Ca(2+)-free bath. Furthermore, addition of 500 microM Gd3+ or 5 mM Ca2+o decreased intracellular Na+ measured with fluorescent sodium indicator, suggesting inhibition of Na+ transport. We conclude that PLA2 is involved in the stimulation of the CaR-induced inhibition of apical K+ channels in the TAL.

Animals↗

PubMed1997

AT1 receptor antagonist treatment caused persistent arterial functional changes in young spontaneously hypertensive rats.

The effects of chronic treatment with an AT1 receptor antagonist (L-158,809) on hypertension development and cardiovascular changes were studied in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). L-158,809 treatment (0.6 mg/kg PO) was initiated at 3 weeks of age and lasted 12 weeks, to 15 weeks of age. The treatment prevented hypertension development in the SHR (systolic blood pressure, BP, of 136+/-1 mm Hg compared with 198+/-3 mm Hg in control SHR), and lowered the BP of WKY (99+/-2 vs 128+/-1 mm Hg in control WKY). Treatment significantly reduced the heart weight in SHR and WKY. Ten weeks after treatment withdrawal (25 weeks of age), BP had increased in SHR and WKY to 172+/-8 and 117+/-3 mm Hg, respectively. Body weight and kidney weight were not affected by the treatment. Mesenteric arteries from treated SHR were less responsive than control SHR arteries to periarterial nerve stimulations at transmural pressures higher than 80 mm Hg (15 and 25 weeks). Control WKY arteries were less responsive than control SHR arteries at almost all transmural pressures tested (15 weeks) and to pressures greater than 80 mm Hg (25 weeks). Pretreatment of arteries with 10(-8) mol/L angiotensin II enhanced their response to nerve stimulation in vessels from control SHR and WKY (25 weeks) but not from treatment-withdrawn SHR and WKY. Treatment did not alter arterial reactivity in response to norepinephrine. Alteration in arterial structure due to L-158,809 treatment was found only when measured at a transmural pressure of 100 mm Hg. In conclusion, L-158,809 was effective in preventing hypertension during the treatment period, in reducing hypertension severity during the withdrawal period, and in persistently decreasing the reactivity of the arteries.

Aging↗

PubMed1997

Regulation of vascular endothelial growth factor expression by insulin-like growth factor I.

Insulin-like growth factor I (IGF-I) and vascular endothelial growth factor (VEGF) levels are correlated with retinal ischemia-associated intraocular neovascularization in humans. Since VEGF is required for iris and retinal neovascularization in animal models of retinal ischemia, we tested whether IGF-I could act as an indirect angiogenic factor by increasing VEGF gene expression. IGF-I increased retinal pigment epithelial (RPE) cell VEGF mRNA in a concentration-dependent manner with an EC50 of 7 nmol/1 (53.6 ng/ml). RPE and bovine smooth muscle cells exposed to 50 nmol/l (383 ng/m1) IGF-I achieved peak VEGF mRNA expression within 2 h. IGF-I-treated RPE cells increased VEGF protein levels in conditioned media and stimulated capillary endothelial cell proliferation. Blockade of the IGF-I receptor with a neutralizing antibody abrogated the VEGF increases in RPE cells. Further, hypoxia-mediated and IGF-I-mediated increases in VEGF mRNA and protein levels were additive in RPE cells, and the hypoxia-induced VEGF increases were independent of endogenous IGF-I. VEGF promoter activity was enhanced by IGF-I in RPE cells, but VEGF transcript half-life was unaltered. In summary, the supplementation of RPE and smooth muscle cell cultures with IGF-I at 5-100 nmol/l increased VEGF mRNA and secreted protein levels. The VEGF increases in RPE cells occurred primarily through enhanced transcription of the VEGF gene and via the IGF-I receptor. Elevated IGF-I levels may promote neovascularization through increased retinal VEGF gene expression.

Animals↗