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D A Brown

Publications and source records attributed to D A Brown.

At least 109 records · Page 6Linked to original sources
PubMed1997

Structure of detergent-resistant membrane domains: does phase separation occur in biological membranes?

Detergent-resistant membrane domains (DRMs) can be isolated from a variety of eukaryotic cells. DRMs are of interest because of their potential importance in processes such as intracellular membrane sorting, and signal transduction at the cell surface. One type of DRM is also present in caveolae, non clathrin-coated plasma membrane pits with proposed roles in endocytosis, lipid transport, and signal transduction. Here we review recent advances in understanding the structure of these domains, and explore the possibility that DRMs are present in a phase separate from the surrounding bilayer. DRMs are rich in sphingolipids and cholesterol. The long saturated acyl chains and high acyl chain melting temperature of sphingolipids mediate their association in detergent resistant domains. These sphingolipid and cholesterol-rich domains have the properties of the liquid-ordered phase previously described in model membranes. Several lines of investigation support the idea that DRMs are not detergent-induced artifacts, but exist as domains in cell membranes. A striking feature of the proteins in DRMs is that many of them are linked to lipids. These include both GPI anchored proteins, and acylated proteins such as Src-family kinases. The linkage of these proteins to saturated acyl chains may help in targeting them to ordered membrane domains. Caveolin, the major structural protein of caveolae, is multiply palmitoylated. The presence of a high concentration of palmitate chains in DRMs in caveolae may help stabilize ordered domains.

Animals↗
PubMed1997

On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes.

Detergent-insoluble membrane fragments that are rich in sphingolipid and cholesterol can be isolated from both cell lysates and model membranes. We have proposed that these arise from membranes that are in the liquid-ordered phase both in vivo and in vitro [Schroeder et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12130-12134]. In order to detect formation of the liquid-ordered phase while avoiding possible detergent artifacts, we have now used fluorescence quenching to examine the phase behavior of mixtures of phosphatidylcholines, sphingolipids, and cholesterol. Phase separation was found in binary mixtures of either dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM) and a nitroxide-labeled phosphatidylcholine (12SLPC). A DPPC- or SM-enriched solidlike gel phase coexisted with a 12SLPC-enriched liquid-disordered fluid phase at 23 degrees C. As expected, phase separation was not seen at low concentrations of DPPC or SM. Instead, only a uniform fluid phase was present. Including 33 mol % cholesterol in model membranes greatly promoted phase separation. Phase separation was seen at higher temperatures and/or at lower concentrations of DPPC or SM in the presence of cholesterol than in its absence. Mixtures of DPPC or SM and cholesterol are known to form the liquid-ordered phase. Therefore, the fact that phase separation was observed in the cholesterol-containing membranes shows that liquid-ordered and liquid-disordered phase domains coexist. At 37 degrees C, the SM-enriched liquid-ordered phase was first seen at a SM/PC ratio of close to 0.25, when SM made up 17% of the total lipid including cholesterol. (This is similar to or less than the SM concentration of the plasma membranes of mammalian cells.) Furthermore, the detergent insolubility of cholesterol-containing model membranes correlated well with the amount of liquid-ordered phase as detected by fluorescence quenching. Thus, the detergent-insoluble membranes isolated from cells are likely to exist in the liquid-ordered phase prior to detergent extraction. The promotion of liquid-ordered phase formation may be an important function of cholesterol and sphingolipids in cells and may be a major distinction between the cholesterol- and sphingolipid-rich plasma membrane and most other cellular membranes.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

A swelling-activated chloride current in rat sympathetic neurones.

1. We have tested whether neurones show a swelling-induced Cl- current following hypotonic shock, by recording membrane current responses and cell volume changes in voltage clamped isolated rat sympathetic neurones during application of hypotonic solutions. 2. Using both whole-cell and perforated patch recording methods, hypotonic solution caused cell swelling and the activation of an inward Cl- current at -60 mV. This current showed weak outward rectification with no obvious time dependence. It was inhibited by SITS (0.3-1 mM), NPPB (30-300 microM) and niflumic acid (50-200 microM), but not by tamoxifen (10 microM). 3. Hypotonic solution did not cause a rise in intracellular Ca2+ concentration as measured by simultaneous indo-1 fluorescence. Also, neither the volume change nor Cl- current were affected by the removal of external Ca2+ or internal Ca2+ buffering to < or = 1 nM with EGTA. 4. The Cl- current was unaffected by an inhibitor of protein kinase C (PKC; GF109203X, 3 microM) or by omission of ATP from the pipette solution. 5. Cells exhibited a regulatory volume decrease during sustained exposure to hypotonic solution. This was completely inhibited by 0.5 mM niflumic acid. 6. It is concluded that osmotic swelling induces an outwardly rectifying, Ca2(+)- and PKC-independent Cl- current in these nerve cells. It is suggested that this current may be involved in volume regulatory mechanisms.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1997

Muscarinic mechanisms in nerve cells.

The receptor subtype and transduction mechanisms involved in the regulation of various neuronal ionic currents are reviewed, with some recent observations on sympathetic neurons, hippocampal cell membranes and basal forebrain cells.

Animals↗
PubMed1997

Acidic conditions ameliorate both adenosine triphosphate depletion and the development of hyperpermeability in cultured Caco-2BBe enterocytic monolayers subjected to metabolic inhibition.

BACKGROUND: We recently reported that moderate degrees of adenosine triphosphate (ATP) depletion induced by chronic glycolytic inhibition or hypoxia increase the permeability of Caco-2BBe enterocytic monolayers. Interestingly, the development of lactic acidosis induced by anaerobic glycolysis ameliorates the development of hyperpermeability caused by chronic ATP depletion. We sought to further elucidate the mechanism(s) responsible for the apparent protection against epithelial hyperpermeability afforded by mild acidosis under conditions of metabolic inhibition. METHODS: Caco-2BBe monolayers growing on permeable supports in bicameral chambers were incubated with 2-deoxyglucose (2DOG) in a glucose-free (Glu-) environment to inhibit glycolysis. Permeability was determined by measuring the transepithelial flux of fluorescein sulfonic acid. Concentrations of intracellular calcium [Ca2+]i were determined fluorometrically by using fura-2. RESULTS: When extracellular pH (pH0) was maintained at 7.4 or 5.5, incubation of monolayers for 24 hours with Glu-/2DOG increased permeability and depleted intracellular ATP levels. However, keeping pH0 at 7.0 to 6.0 ameliorated both the development of hyperpermeability and the depletion of ATP induced by Glu-/2DOG. These protective effects were observed under acidic conditions created either by addition to the medium of HCl or by incubation under an atmosphere containing 20% CO2. Incubation with Glu-/2DOG caused bulging of the apical membranes of cells (electron microscopy) and derangements in the perijunctional distribution of actin (fluorescence microscopy); however, these structural changes were ameliorated by mild acidosis. Acute chemical hypoxia at pH0 7.4 induced by Glu-/2DOG plus antimycin A decreased cellular ATP levels and elevated [Ca2+]i. Lowering pH0 to 6.8 ameliorated both the depletion of ATP and the increase in [Ca2+]i induced by Glu-/2DOG+antimycin A. CONCLUSIONS: Moderate decreases in pH ameliorate the hyperpermeability induced by metabolic inhibition, possibly by diminishing ATP depletion and blunting increases in [Ca2+]i.

Acidosis↗
PubMed1997

Inhibition by heterologously-expressed P2Y2 nucleotide receptors of N-type calcium currents in rat sympathetic neurones.

The P2Y2 nucleotide receptor has previously been shown to stimulate phosphoinositide breakdown. We now show that, when P2Y2 receptors are heterologously expressed by cRNA injection into dissociated rat sympathetic neurones, activation of these receptors by uridine 5'-triphosphate (UTP) or adenosine 5'-triphosphate (ATP) inhibits the N-type voltage-gated calcium current by approximately 65%, with an IC50 of 0.5 microM. Thus, the same molecular species of nucleotide receptor can link to two different effector pathways.

Animals↗
PubMed1997

Muscle activity adapts to anti-gravity posture during pedalling in persons with post-stroke hemiplegia.

With hemiplegia following stroke, a person's movement response to anti-gravity posture often appears rigid and inflexible, exacerbating the motor dysfunction. A major determinant of pathological movement in anti-gravity postures is the failure to adapt muscle-activity patterns automatically to changes in posture. The aim of the present study was to determine whether the impaired motor performance observed when persons with hemiplegia pedal in a horizontal position is exacerbated at more vertical anti-gravity body orientations. Twelve healthy elderly subjects and 17 subjects with chronic (> 6 months) post-stroke hemiplegia participated in the study. Subjects pedalled a modified ergometer at different body orientations (from horizontal to vertical), maintaining the same workload, cadence, and hip and knee kinematics. Pedal reaction forces, and crank and pedal kinematics, were measured and then used to calculate the work done by each leg and their net positive and negative components. The EMG was recorded from four leg muscles (tibialis anterior, medial gastrocnemius, rectus femoris and biceps femoris). The main result from this study was that impaired plegic leg performance, as measured by net negative work done by the plegic leg and abnormal early rectus femoris activity, was exacerbated at the most vertical body orientations. However, contrary to the belief that muscle activity cannot adapt to anti-gravity postures, net positive work increased appropriately and EMG activity in all muscles showed modulated levels of activity similar to those in elderly control subjects. These results support the hypothesis that increased verticality exacerbates the already impaired movement performance. Yet, much of the motor response to verticality was flexible and appropriate, given the mechanics of the task.

Adaptation, Physiological↗
PubMed1997

Effects of a cognition-enhancer, linopirdine (DuP 996), on M-type potassium currents (IK(M)) and some other voltage- and ligand-gated membrane currents in rat sympathetic neurons.

Linopirdine is a cognition enhancer which augments depolarization-induced transmitter release in the cortex and which is under consideration for potential treatment of Alzheimer's disease. It has previously been reported to inhibit M-type K+ currents in rat hippocampal neurons. In the present experiments we have tested its effect on whole-cell M-currents and single M-channels, and on a range of other membrane currents, in dissociated rat superior cervical sympathetic ganglion cells. Linopirdine inhibited the whole-cell M-current with an IC50 of 3.4 microM and blocked M-channels recorded in excised outside-out membrane patches but not in inside-out patches. This suggests that linopirdine directly blocks M-channels from the outside. It was much less effective in inhibiting other voltage-gated potassium currents [delayed rectifier (IK(V)), IC50 63 microM; transient (IA) current, IC50 69 microM] and produced no detectable inhibition of the fast and slow Ca(2+)-activated K+ currents IC and IAHP or of a hyperpolarization-activated cation current (IQ/Ih) at 10-30 microM. However, it reduced acetylcholine-activated nicotinic currents and GABA-activated Cl- currents with IC50 values of 7.6 and 26 microM respectively. It is concluded that linopirdine shows some 20-fold selectivity for M-channels among different K+ channels but can also block some transmitter-gated channels. The relationship between M-channel block and the central actions of linopirdine are discussed.

Animals↗
PubMed1997

Acidosis induces hyperpermeability in Caco-2BBe cultured intestinal epithelial monolayers.

We previously demonstrated that ileal mucosal acidosis in pigs reversibly increases intestinal permeability to hydrophilic macromolecules, even in the absence of tissue hypoxia [A.L. Salzman, H. Wang, P.S. Wollert, T.J. Vandermeer, C.C. Compton, A.G. Denenberg, and M.P. Fink. Am. J. Physiol. 266 (Gastrointest, Liver Physiol, 29): G633-G646, 1994]. In an effort to further explore the mechanism(s) underlying this phenomenon, we examined the effect of acidic pH on the permeability characteristics of cultured Caco-2BBe (human intestinal epithelial) cells grown as monolayers on permeable supports. Permeability was determined by measuring the mucosal-to-basolateral flux of fluorescein disulfonic acid (FS; mol wt 478 Dal, fluorescein isothiocyanate-labeled dextran (FD4; average mol wt 4 kDa), or [3H]mannitol. Incubation of monolayers under hypercarbic conditions or with acidific bicarbonate-free medium significantly increased permeability to FS, FD4, and mannitol in a manner dependent on both time and pH. Incubation in medium at pH 5.43 for 24 h increased the release of lactate dehydrogenase and decreased the intensity of staining with calcein-acetoxymethyl ester, findings that are indicative of plasma membrane injury; nevertheless, the percentage of nonviable cells did not increase. Ultrastructural analyses revealed evidence of increased paracellular trafficking of horseradish peroxidase after incubation of monolayers under acidic conditions. Fluorescence confocal microscopy and temperature studies demonstrated that incubation at pH 5.43 induced an increase in both the intracellular uptake of FD4 and the activation energy for FS permeation across Caco-2BBe monolayers, respectively, suggesting increased transcellular permeation. Exposure to acidic conditions also decreased cellular levels of ATP. We conclude that acidosis increases both paracellular and transcellular permeability to hydrophilic macromolecules and leads to depletion of ATP.

Acidosis↗
PubMed1996

Elevation of intracellular calcium by muscarinic receptor activation induces a block of voltage-activated rat ether-à-go-go channels in a stably transfected cell line.

We have studied the properties of r-eag voltage-activated potassium channels in a stably transfected human embryonic kidney cell line. It was found that r-eag channels are rapidly and reversibly inhibited by a rise in intracellular calcium from 30 to 300 nM. The inhibition does not appear to depend on the activity of calcium-dependent kinases and phosphatases. The effect of calcium on r-eag channel activity was studied in inside-out membrane patches. Calcium inhibited r-eag channel activity with a mean IC50 of 67 nM. Activation of muscarinic receptors, generating calcium oscillations in the transfected cells, induced a synchronous inhibition of r-eag mediated outward currents. This shows that calcium can mediate r-eag current inhibition following muscarinic receptor activation. The data indicate that r-eag channels are calcium-inhibitable voltage-activated potassium channels.

Animals↗
PubMed1996

Dopamine D1-like receptor-mediated presynaptic inhibition of excitatory transmission onto rat magnocellular basal forebrain neurones.

1. Excitatory postsynaptic currents (EPSCs) following focal afferent stimulation were recorded from patch-clamped magnocellular neurones in a thin-slice preparation of the rat basal forebrain. Evoked EPSCs had a mean decay time constant of 3.81 +/- 0.09 ms and were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 microM). 2. Bath-applied dopamine (DA) reduced evoked EPSC amplitude by up to 54.2 +/- 2.3% with an IC50 of 19.9 microM in normal Krebs solution (2.5 mM Ca2+, 1.2 mM Mg2+) without effect on postsynaptic holding current. 3. DA (30 microM) reduced the mean frequency of spontaneous miniature EPSCs recorded in 0.5 microM tetrodotoxin without affecting their mean amplitude, rise time or decay time constant. This effect was diminished by 100 microM Cd2+. 4. The effect of DA on evoked EPSCs was mimicked by the D1-like receptor agonist, SKF 81297 (IC50 25.6 microM), but not by the D2-like receptor agonist R(-)-TNPA (30 microM) or (-)-quinpirole (30 microM), and was antagonized by the D1-like receptor antagonist R(+)-SCH 23390 (estimated dissociation constant KB = 1.7 microM) but not by the D2-like receptor antagonist S(-)-eticlopride (10 microM). 5. Forskolin (10 microM) reduced evoked EPSCs to approximately 60% of the control amplitude, and occluded the effect of subsequent application of DA. 6. These results suggest that glutamatergic afferents to magnocellular basal forebrain neurones possess presynaptic D1-like DA receptors, and that activation of these receptors reduces excitatory glutamatergic transmission, probably via an adenylyl cyclase-dependent pathway.

Animals↗
PubMed1996

Genomic footprinting of HTLV type I and HIV type 1 in human T cell lines.

Genomic footprinting of integrated HTLV-I and HIV-1 confirmed many aspects of retroviral transcriptional regulation deduced from previous studies. However, many notable differences were seen. HTLV-I genomic protein-binding patterns corresponded more closely to elements defined by transient transfection expression studies than to those mapped by in vitro protein-binding studies. HIV-1 genomic footprinting showed activation-related binding to adjacent NF-KB/SP1 sites and a large (90 bp) region transversing the R/U5 boundary, but minimal protein binding to NFAT, NRE, LBP-1, and CTF/NF1 sites relative to previous in vitro footprinting studies.

Base Sequence↗
PubMed1996

M-type K+ current inhibition by a toxin fron the scorpion Buthus eupeus.

A number of invertebrate venoms have been tested for effects on M-type K+ currents (IK(M)) in differentiated mouse neuroblastoma X rat glioma NG108-15 cells. Among the venoms tested, Buthus eupeus scorpion venom reversibly inhibited IK(M) by approximately 44% at 50 microgram/ml. Inhibition was not due to activation of bradykinin or nucleotide (pyrimidine) receptors. On venom fractionation, a polypeptide of 4 kDa was purified that inhibited IK(M) by approximately 45% with an IC50 of approximately 33 nM. Neither the crude venom nor the purified polypeptide affected the Ca2+ current or the delayed rectifier K+ current. While the crude venom prolonged the Na+ current, the polypeptide did not. Thus, the 4 kDa Buthus eupeus polypeptide appears to be a selective inhibitor of IK(M) in NG108-15 cells.

Animals↗
PubMed1996

Sequence requirements of ATF2 and CREB binding to the human T-cell leukemia virus type 1 LTR R region.

We have identified crucial transcription factor contact sites within the distal portion of the HTLV-1 LTR R region. Single base substitutions within this region greatly reduce formation of a complex which we previously described as a 70-kDa nuclear protein interacting with a CREB-like protein. Comparison of published sequences of HTLV-1 isolates obtained from scattered geographic locations revealed that clustered mutations in an 8-base segment near the U5 junction do, in fact, occur naturally. A single base substitution corresponding to a common naturally occurring mutation was introduced into the R region of an HTLV-1-LTR Cat construct. This resulted in derepression of the promoter in a cell line expressing high levels of the R region binding complex when compared to the wild-type LTR promoter. Affinity purification and electrophoretic mobility super-shift analysis identified a dominant 70-kDa DNA binding protein as ATF-2. Phosphorylated ATF-2 apparently interacts with CREB to form this downstream complex.

Activating Transcription Factor 2↗
PubMed1996

Detection and modulation of acetylcholine release from neurites of rat basal forebrain cells in culture.

1. Nicotinic acetylcholine (ACh) receptor-rich patches prepared from rat myotubes were used as focal ACh detectors to record the release of ACh from magnocellular basal forebrain (MBF) neurones from 11- to 14-day-old postnatal rats maintained in dissociated cell culture. 2. An action potential generated by intracellularly stimulating the MBF cell soma through a patch electrode induced a brief (mean tau(decay), 6.3 ms) short latency (1.35-5.1 ms; median 3.1 ms) burst of nicotinic channel openings in the detector patch when the latter was positioned at discrete loci along the MBF neurites. Detected ACh concentrations ranged from approximately 480 nM to > 50 microM. Concentrations increased markedly during the first 14 days in vitro and were inversely related to response latency. 3. Sites of release were generally confined to the more proximal neurites within 100 microm of the cell body and were invariably associated with the presence of small (2-3 microm diameter) phase-dark puncta located at discrete intervals along the length of the neurites or at points where short collaterals branched from the main process. Release was never detected from the cell soma except under extreme non-physiological conditions but could occasionally be elicited from growth cones at the ends of the shorter thicker neurites in the absence of a target cell. 4. Evoked release was abolished by tetrodotoxin (0.5 microM) and by superfusing with low Ca(2+)-high Mg(2+)-containing solutions (0.25 mM Ca(2+), 5 mM Mg(2+)). Myotube patch responses were antagonized by d-tubocurarine (3 microM). 5. Muscarine (10 microM) inhibited release by 70 +/- 3% (n = 12 cells). This effect was antagonized by 100 nM methoctramine but not by 100 nM pirenzepine, indicating that it was mediated by M(2) muscarinic ACh receptors. 6. These results indicate that ACh release from the processes of magnocellular cholinergic basal forebrain neurones arises from highly specialized and discrete sites, and that it can be inhibited through activation of muscarinic receptors. It is suggested that the latter results from inhibition of presynaptic Ca(2+) channels and that it might be responsible for feedback autoinhibition of ACh release from cortical afferents of nucleus basalis neurones in vivo.

Acetylcholine↗
PubMed1996

Binding and functional effects of transcriptional factor Sp1 on the murine interleukin-6 promotor.

The NF-kappaB and NF-IL6 elements have previously been shown to play an important role in regulation of both the mouse and human interleukin-6 gene. Between these two elements lies a G/C-rich sequence, which contains three repeats of the element CCACC, protein binding to which has not been previously characterized. In this study we demonstrate that the transcription factor Sp1 binds to these repeats and plays an important role in basal and in inducible expression of the murine interleukin-6 gene.

3T3 Cells↗