Biomedical subjects

D Duan

Publications and source records attributed to D Duan.

47 records · Page 3Linked to original sources

PubMed1997

Molecular identification of a volume-regulated chloride channel.

A volume-regulated chloride current (ICl.vol) is ubiquitously present in mammalian cells, and is required for the regulation of electrical activity, cell volume, intracellular pH, immunological responses, cell proliferation and differentiation. However, the molecule responsible for ICl.vol has yet to be determined. Although three putative chloride channel proteins expressed from cloned genes (P-glycoprotein, pICln and ClC-2 ) have been proposed to be the molecular equivalent of ICl.vol, neither P-glycoprotein nor pICln is thought to be a chloride channel or part thereof, and the properties of expressed ClC-2 channels differ from native ICl.vol. Here we report that functional expression in NIH/3T3 cells of a cardiac clone of another member of the ClC family, ClC-3, results in a large basally active chloride conductance, which is strongly modulated by cell volume and exhibits many properties identical to those of ICl.vol in native cells. A mutation of asparagine to lysine at position 579 at the end of the transmembrane domains of ClC-3 abolishes the outward rectification and changes the anion selectivity from I- > Cl- to Cl- > I- but leaves swelling activation intact. Because ClC-3 is a channel protein belonging to a large gene family of chloride channels, these results indicate that ClC-3 encodes ICl.vol in many native mammalian cells.

3T3 Cells↗

PubMed1997

Structural and functional heterogeneity of integrated recombinant AAV genomes.

Adeno-associated Virus (AAV) has emerged as a promising vector for gene therapy because of its ability to generate high titer recombinant stocks and the potential for site specific integration. However, much of the current knowledge regarding the transduction and integration biology of this virus is based on studies evaluating wild type AAV or recombinant AAV which was unknowingly contaminated with wild type virus. Given the fact that recombinant AAV is replication incompetent, by virtue of deleted viral rep proteins responsible for site specific integration of the wild type virus, the integration process for recombinant AAV may likely be different from its wild type counterpart. To this end, the present study has attempted to elucidate the proviral structure of stably integrated recombinant AAV genomes harboring the alkaline phosphatase reporter gene in 293 and IB3 cell lines. Initial studies attempted to functionally characterize differences in proviral genomes using mobilization assays with assessed both liberated episomal recombinant AAV and infectious virus following transfection with Rep/Cap containing plasmids and/or infection with recombinant adenovirus (Ad). Using Southern and polymerase chain reaction (PCR) analysis, evaluation of genomic DNA from AAV clonal cell lines indicated that head to tail orientations of ITRs were absolutely required for excision of episomal genomes and rescue of infectious recombinant virus. Furthermore, mobilization of proviral DNA could be achieved in the presence of exogenous Rep/Cap without adenovirus, while mobilization of infectious recombinant virus required the addition of both Rep/Cap and Ad. Genomic Southerns suggest that two predominant proviral structures exist for recombinant AAV including head to head and tail to head duplex genomes. A third class of monomer proviral genomes with head to tail oriented ITRs was also observed. No evidence for tail to tail ITR oriented proviral genomes was detected in any of the clonal cell lines. Such findings have begun to lay the foundation for a clearer understanding of the mechanism of recombinant AAV integration and how this process differs from wild type AAV.

Blotting, Southern↗

PubMed1997

Evidence that outwardly rectifying Cl- channels underlie volume-regulated Cl- currents in heart.

Swelling-induced Cl- current (ICl.swell) is present in most cardiac tissues, but the unitary channel underlying ICls.well is unknown. We used the cell-attached patch-clamp technique to assess the properties of single channels underlying ICls.well and the basally active Cl- current (ICl.b) in rabbit atrial myocytes. Under isotonic conditions, single outwardly rectifying Cl- channels (ORCCs) with a slope conductance of 28 +/- 1 pS at the reversal potential were observed in 21 (5.7%) of 367 patches. Unconditional kinetic analysis revealed at least three open and four closed-channel states. Hypotonic superfusion-induced swelling resulted in the appearance of active channels in 41 (15.5%) of 265 patches without channel activity under isotonic conditions and caused a second active channel to appear in 3 of 14 patches showing a single channel under isotonic conditions. Overall, channels were seen in 54 of 336 patches under hypotonic conditions (16.1%, P < .001 versus isotonic conditions). The current-voltage relations, reversal potential-[Cl-]o relations, open probability, and kinetics of swelling-induced channels were indistinguishable from those of ORCCs under isotonic conditions. Unitary ORCCs, ICl.b, and ICl.swell were strongly and similarly inhibited by tamoxifen. Swelling-induced increases in macroscopic Cl- current were attributable to an increase in the number of active ORCCs with no significant effects on single-channel amplitude or open probability. Estimated macroscopic currents based on cell surface area, patch dimensions, single-channel ORCC current amplitude, open probability, and density were consistent with measured values of ICl.b and ICl.swell. We conclude that ORCCs underlie volume-regulated basal and swelling-induced Cl- currents in isolated rabbit atrial myocytes.

Animals↗

PubMed1997

Real-time, noninvasive in vivo assessment of adeno-associated virus-mediated retinal transduction.

PURPOSE: To evaluate the efficiency, cell specificity, stability, and toxicity of recombinant adeno-associated virus (rAAV)-mediated retinal transduction in vivo in the adult immunocompetent mouse. To assess the usefulness of green fluorescent protein (GFP) for real-time, noninvasive monitoring of retinal transgene expression in vivo. METHODS: Assessment of ocular GFP expression was performed in cohorts of mice for 11 weeks after subretinal injection of a recombinant adeno-associated virus carrying the complementary DNA (cDNA) for GFP. Examinations were performed in vivo by direct observation of fluorescence by ophthalmoscopy, using excitation-barrier filters. Histologic analyses of retinal tissue were used to identify transduced cells and to assess inflammation. RESULTS: Retinal GFP expression can be monitored in vivo using real-time, noninvasive imaging. Recombinant AAV efficiently transduces a variety of cells of the neural retina and of the retinal pigment epithelium (RPE). Transgene expression was not observed until 1 week after infection. The number of GFP-expressing cells increased over 3 weeks, and expressing photoreceptors and RPE, cells persisted at least through 11 weeks (the termination of the experiment). There was no clinical or histologic evidence of inflammatory response. CONCLUSIONS: Retinal gene transfer mediated by rAAV is stable and efficient and is associated with no clinically or histologically detectable toxicity or immune reaction. Green fluorescent protein allows noninvasive assessment of the extent and location of retinal transgene expression as a function of time and promises to be useful alone and as a tag for other transgenes delivered experimentally or therapeutically to the retina.

Animals↗

PubMed1996

Protein kinase C activates ATP-sensitive K+ current in human and rabbit ventricular myocytes.

Mediators involved in ischemia preconditioning such as adenosine and norepinephrine, can activate protein kinase C (PKC), and a variety of observations suggest that both PKC and ATP-sensitive K+ current (I (KATP) play essential roles in ischemic preconditioning. PKC is therefore a candidate to link receptor binding to I(KATP) activation, but it has not been shown whether and how PKC can activate I(KATP) in the heart. The present study was designed to determine whether PKC can activate I(KATP) in rabbit and human ventricular myocytes. Under conditions designed to minimize Na+ and Ca2+ currents, dialysis of rabbit ventricular myocytes with pipette solutions containing reduced [ATP] elicited I(KATP)++, with a 50% effective concentration (EC50)of 260 micromol/L. In cells that failed to show I (KATP) under control conditions, superfusion with 1 micromol/L phorbol 12,13-didecanoate (PDD) elicited I(KATP) in a fashion that depended on pipette [ATP], with an [ATP] EC 50 of 601 micromol/L. PDD-induced I(KATP) activation was concentration dependent, with an EC 50 of 7.1 nmol/L. The highly selective PKC inhibitor bisindolylmaleimide totally prevented I(KATP) activation by PDD, and in blinded experiments, 1 micromol/L PDD elicited I(KATP) in eight of nine cells, whereas its non-PKC-stimulating analogue 4 alpha-PDD failed to elicit I(KATP) in any of the five cells tested (P = .003). Similar experiments were conducted in human ventricular myocytes and showed that 0.1 micromol/L PDD elicited I( KATP) at pipette [ATP] of 100 and 400 micromol/L (five of five cells at each concentration) but not at 1 mmol/L [ATP] (none of five cells). We conclude that PKC activates I(KATP) in rabbit and human ventricular myocytes by reducing channel sensitivity to intracellular ATP. This finding has potentially important implications for understanding the mechanisms of ischemic preconditioning.

Adenosine Triphosphate↗

PubMed1995

Alpha-adrenergic control of volume-regulated Cl- currents in rabbit atrial myocytes. Characterization of a novel ionic regulatory mechanism.

alpha-Adrenergic stimulation is known to play a role in cardiac arrhythmogenesis and to modulate a variety of cardiac K+ currents. The effects of alpha-adrenergic stimulation on Cl- currents are largely unknown. Many cardiac cell types show a volume-sensitive Cl- current induced by cell swelling (ICl.swell). The present experiments were designed to assess the potential alpha-adrenergic modulation of ICl.swell in rabbit atrial myocytes. ICl.swell was induced with the use of a hypotonic superfusate, under conditions designed to prevent currents carried by K+, Na+, and Ca2+ ions. A basal Cl- current (ICl.b) was observed under isotonic conditions in 128 of 150 cells (85%), had the same dependency on [Cl-]o as ICl.swell, and was reduced by cell shrinkage induced by hypertonic superfusion, suggesting that ICl.b is carried by the same volume-sensitive Cl- conductance as ICl.swell. Phenylephrine produced a concentration-dependent and near-complete inhibition of ICl.b and ICl.swell, with EC50 values of 86 +/- 5 and 72 +/- 7 (mean +/- SEM) mumol/L, respectively, at +20 mV. Norepinephrine (administered in the presence of 1 mumol/L propranolol) also inhibited ICl.b and ICl.swell, with EC50 values of 2.6 +/- 0.1 and 2.8 +/- 0.4 mumol/L, respectively. The concentration-response curve for phenylephrine was shifted significantly (P < .001) to the right by the alpha 1-adrenoceptor antagonist prazosin and by the alpha 1A-receptor antagonists (+)-niguldipine and 5-methylurapidil but was unaltered by the alpha 1B-receptor antagonist chloroethylclonidine (100 mumol/L). Inhibition of protein kinase C (PKC) with staurosporine, H-7, or 18-hour preincubation with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA, 500 nmol/L) blocked the effects of phenylephrine on ICl.swell, and the highly selective PKC inhibitor bisindolylmaleimide blocked the effects of norepinephrine on ICl.swell and ICl.b. Both PMA and 1-oleoyl-2-acetylglycerol inhibited ICl.swell in a concentration-dependent fashion. In blinded studies, the phorbol ester phorbol 12,13-didecanoate (PDD) reduced ICl.swell by 91 +/- 3%; its inactive analogue 4 alpha-PDD had no effect (mean change, 3 +/- 1%). Preincubation with pertussis toxin (PTX) prevented the actions of phenylephrine on ICl.swell, indicating a role for a PTX-sensitive guanine nucleotide-binding (G) protein. We conclude that alpha-adrenergic agonists inhibit volume-sensitive Cl- currents in rabbit atrial cells by interacting with an alpha 1A-adrenoceptor mechanism that is coupled to PKC via a PTX-sensitive G protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1994

Properties of single outwardly rectifying Cl- channels in heart.

A variety of potentially important macroscopic Cl- currents have been described in the heart. Although the single-channel properties of the cAMP-dependent current (ICl.cAMP) have been well described, the single-channel equivalents of the other forms of cardiac Cl- current remain unknown. Unlike ICl.cAMP, many of these currents show prominent outward rectification in the presence of symmetrical transmembrane Cl- gradients and sensitivity to disulfonic stilbene Cl- transport blockers. We used the patch-clamp technique to search for single Cl- channels in inside-out patches from rabbit atrial cell membranes, under conditions minimizing the chances of observing channels carrying Na+, Ca2+, or K+. Under symmetrical Cl- conditions, single-channel activity was seen in 14 (9%) of 155 patches. Channels showed strong outward rectification and a unitary conductance of 60 +/- 3 picosiemens (mean +/- SEM) at positive voltages. The current-voltage relation was not altered by replacement of cations by the impermeant cation N'-methyl-D-glucamine (NMDG) and shifted as expected for a Cl(-)-selective channel when methanesulfonate was substituted for Cl-. The Cl- transport blockers DIDS (diisothiocyanatostilbene-2,2'-disulfonic acid, 100 mumol/L) and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, 1 mmol/L) strongly and reversibly inhibited channel activity when added to the bath and caused channel flickering suggesting open-channel block. Ensemble-average currents showed no time dependence, and the form of the ensemble-average current-voltage relation was similar to that of macroscopic background Cl- current.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1993

Potassium channel blocking properties of propafenone in rabbit atrial myocytes.

Propafenone, a class 1c antiarrhythmic agent, is known to be a potent blocker of voltage-dependent sodium channels; however, several clinical actions of the drug point toward possible potassium channel blocking capability. The present experiments were designed to assess the extent and potential mechanisms of potassium channel blocking properties of propafenone. Whole-cell voltage-clamp techniques were used to define the actions of propafenone on the transient outward current (Ito), the delayed rectifier current (Ik) and the inward rectifier current (Ik1) in isolated rabbit atrial myocytes. Propafenone blocked all three currents, with the extent of blockade being independent of test potential During depolarizing voltage steps, block of Ito and Ik developed as an exponential function of time, consistent with time-dependent open channel blockade. The rate constant of block onset was concentration dependent. The inactivation of Ito was a monoexponential function of time under control conditions, with a time constant averaging 19.1 +/- 1.3 msec (mean +/- S.E.) at +10 mV. Propafenone accelerated Ito inactivation, resulting in a biexponential process having time constants of 5.1 +/- 0.9 (P < .001 vs. control) and 23.5 +/- 2.0 msec (P = N.S. vs. control) at 5 microM and 3.4 +/- 0.5 (P < .001 vs. control) and 28.5 +/- 4.3 msec (P = N.S.) at 10 microM concentrations, respectively. The rapid phase inactivation time constants were of the same order as time constants for the onset of block (3.1 +/- 0.6 and 1.8 +/- 0.3 msec at 5 and 10 microM respectively), suggesting that the acceleration of Ito inactivation was due to open channel block by the drug. The IC50 for blockade was substantially less for effects on Ik (0.76 microM; 95% confidence limits 0.44-1.30 microM) than for Ito (5.91 microM; 95% confidence limits 4.19-8.33 microM) or Ik1 (7.10; 5.24-9.61 microM). We conclude that 1) propafenone is an efficacious potassium channel blocker; 2) propafenone blockade of time-dependent potassium currents is open-state dependent; and 3) propafenone block of potassium currents is relatively selective for Ik.

Animals↗

PubMed1992

Differences in rate dependence of transient outward current in rabbit and human atrium.

Both human and rabbit atrial cells possess a large 4-aminopyridine-sensitive transient outward current (I(to1)). However, the slow reactivation of this current in rabbits suggests that its role may be limited to very slow heart rates. We used whole cell voltage-clamp recordings to evaluate the rate dependency of I(to1) in rabbit and human atrial myocytes. Our results show that at physiological temperatures in human atrium, I(to1) is rate independent at rates between 0.1 and 4.0 Hz. Peak I(to1) at 4.0 Hz in rabbit was 3.4 +/- 1.4% (mean +/- SE) of current at 0.1 Hz (P < 0.001, n = 8), whereas in humans, peak I(to1) at 4.0 Hz averaged 88.8 +/- 6.1% of the current at 0.1 Hz (P > 0.05, n = 7). These differences were due to marked discrepancies in reactivation time course, which was biexponential with time constants that averaged 650 +/- 159 ms and 8.4 +/- 1.1 s in rabbit (n = 8) compared with a single exponential time constant of 33.6 +/- 6.8 ms (n = 8) in human atrium (both at 30 degrees C). These findings suggest that I(to1) can contribute importantly to atrial repolarization at all physiological heart rates in humans. Furthermore, these results emphasize that there are important interspecies variations in the rate dependence of I(to1), which need to be considered in understanding the physiological and pharmacological regulation of atrial repolarization.

4-Aminopyridine↗

PubMed1992

[Total hip replacement for congenital dislocation of the hip in elderly patients].

From 1986 to 1989, total hip replacement was performed on ten patients (twelve hips) with congenital dislocation of the hip. The average age of the patients was fifty-three years (ranging from forty-eight to sixty years), and the mean follow-up period was twenty-eight months (ranging from six to forty-eight months). Of the ten patients, eight had unilateral replacement and two had bilateral replacement, for a total of twelve arthroplasties. Eight arthroplasties were performed on the right and four on the left. Most patients left the hospital two to three weeks after operation. All the patients obtained satisfactory results. Only one patient developed late infection two years postoperatively. This patient occasionally used a cane for walking. The other patients had no hip pain, the motion of the hips was approximate to a normal person. No patient required a shoe-lift for limb-length discrepancy.

Age Factors↗

PubMedDate not supplied

Functional role of amino terminus in ClC-3 chloride channel regulation by phosphorylation and cell volume.

AIM: This study investigated the functional role of the ClC-3 amino-terminus in channel regulation in response to changes in cell volume. METHODS: Wild-type sClC-3 tagged with a green fluorescence protein (GFP) at the C-terminus was used as a template to construct a number of deletion mutants which were functionally expressed in NIH-3T3 cells. Whole cell and single channel patch-clamp electrophysiology was used to determine the functional properties of heterologously expressed channels. RESULTS: The first 100 amino acids of the ClC-3 N-terminus were removed and the truncated channel (sClC-3DeltaNT) was functionally expressed. Immunocytochemistry confirmed membrane expression of both wtsClC-3 and sClC-3DeltaNT channels in NIH/3T3 cells. sClC-3DeltaNT yielded constitutively active functional channels, which showed no response to protein kinase C or changes in cell volume. Deletion of a cluster of negatively charged amino acids 16-21 (sClC-3Delta16-21) within the N-terminus also yielded a constitutively active open channel phenotype, indicating these amino acids are involved in the N-type regulation. Intracellular delivery of a thiol-phosphorylated peptide corresponding to N-terminal residues 12-61 (NT peptide) markedly inhibited sClC-3DeltaNT whole-cell and single-channel currents, further confirming the essential role of the N-terminus in volume regulation of channel activity. CONCLUSIONS: These data strongly suggest the N-terminus of sClC-3 channels acts as a blocking particle inhibiting the flow of anions through the channel pore. This 'N-type' regulation of sClC-3 channels may be an important transducing mechanism linking changes in cell volume and channel protein phosphorylation to channel gating.

3T3 Cells↗