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M L Roberts

Publications and source records attributed to M L Roberts.

At least 37 records · Page 2Linked to original sources
PubMed1998

Permeation and block of the skeletal muscle chloride channel, ClC-1, by foreign anions.

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl- as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO3-, BrO3-); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl- at the regulatory binding site but impair Cl- passage through the channel pore (Br-, NO3-, ClO3-, I-, ClO4-, SCN-). The permeability sequence for rClC-1, SCN- approximately ClO4- > Cl- > Br- > NO3- approximately ClO3- > I- >> BrO3- > HCO3- >> methanesulfonate approximately cyclamate approximately glutamate, was different from the sequence determined for blocking potency and ability to shift the Popen curve, SCN- approximately ClO4- > I- > NO3- approximately ClO3- approximately methanesulfonate > Br- > cyclamate > BrO3- > HCO3- > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be approximately 4.5 A. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl- and SCN- or ClO4- suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.

Animals↗
PubMed1997

Epstein-Barr virus binding to CD21 activates the initial viral promoter via NF-kappaB induction.

Epstein-Barr virus (EBV), an oncogenic human herpesvirus, binds to and infects normal human B lymphocytes via CD21, the CR2 complement receptor. Studies of the mechanisms that enable EBV to infect nonactivated, noncycling B cells provide compelling evidence for a sequence of events in which EBV binding to CD21 on purified resting human B cells rapidly activates the NF-kappaB transcription factor, which, in turn, binds to and mediates transcriptional activation of Wp, the initial viral latent gene promoter. Thus, EBV binding to its cellular receptor on resting B cells triggers an NF-kappaB-dependent intracellular signaling pathway which is required for infection.

Aspirin↗
PubMed1997

pH-dependent interactions of Cd2+ and a carboxylate blocker with the rat C1C-1 chloride channel and its R304E mutant in the Sf-9 insect cell line.

1. Gating of the skeletal muscle chloride channel (ClC-1) is sensitive to extracellular pH. In this study, whole-cell recording of currents from wild-type (WT) ClC-1 and a mutant, R304E, expressed in the Sf-9 insect cell line was used to investigate further the nature of the pH-sensitive residues. 2. Extracellular Cd2+ produced a concentration-dependent block of WT ClC-1 with an IC50 of 1.0 +/- 0.1 mM and a Hill coefficient of 2.0 +/- 0.3. This block was sensitive to external pH, reducing at low pH, with an apparent pKa of 6.8 +/- 0.1 and a Hill coefficient for proton binding of 3.0 +/- 0.3. Anthracene-9-carboxylate (A-9-C) block of WT ClC-1 was also pH sensitive, increasing at low pH, with an apparent pKa of 6.4 +/- 0.1 and a Hill coefficient for proton binding of 1.0 +/- 0.2. 3. Compared with WT ClC-1, R304E had a lower affinity for Cd2+ (IC50, 3.0 +/- 0.3 mM) but it had a similar Hill coefficient for transition metal ion binding. The Hill coefficient for proton binding to the Cd2+ binding site was reduced to 1.4 +/- 0.3. In contrast, the A-9-C binding site in R304E showed the same pH sensitivity and affinity for the blocker as that seen in WT ClC-1. 4. ClC-1 has at least two binding sites for Cd2+, each of which has at least three residues which can be protonated. Binding of A-9-C is influenced by protonation of a single residue. Arg 304 is not sufficiently close to the A-9-C binding site to affect its characteristics, but it does. alter Cd2+ binding, indicating that transition metal ions and aromatic carboxylates interact with distinct sites. 5. The block of ClC-1 by transition metal ions and the apparent pKa of this block, together with the apparent pKa for A-9-C block and gating are all compatible with the involvement of His residues in the pore and gate of ClC-1.

Animals↗
PubMed1996

Concentration and pH dependence of skeletal muscle chloride channel ClC-1.

1. The influence of Cl- concentration and pH on gating of the skeletal muscle Cl- channel, ClC-1, has been assessed using the voltage-clamp technique and the Sf-9 insect cell and Xenopus oocyte expression systems. 2. Hyperpolarization induces deactivating inward currents comprising a steady-state component and two exponentially decaying components, of which the faster is weakly voltage dependent and the slower strongly voltage dependent. 3. Open probability (Po) and kinetics depend on external but not internal Cl- concentration. 4. A point mutation, K585E, in human ClC-1, equivalent to a previously described mutation in the Torpedo electroplaque chloride channel, ClC-0, alters the I-V relationship and kinetics, but retains external Cl- dependence. 5. When external pH is reduced, the deactivating inward currents of ClC-1 are diminished without change in time constants while the steady-state component is enhanced. 6. In contrast, reduced internal pH slows deactivating current kinetics as its most immediately obvious action and the Po curve is shifted in the hyperpolarizing direction. Addition of internal benzoate at low internal pH counteracts both these effects. 7. A current activated by hyperpolarization can be revealed at an external pH of 5.5 in ClC-1, which in some ways resembles currents due to the slow gates of ClC-0. 8. Gating appears to be controlled by a Cl(-)-binding site accessible only from the exterior and, possibly, by modification of this site by external protonation. Intracellular hydroxyl ions strongly affect gating either allosterically or by direct binding and blocking of the pore, an action mimicked by intracellular benzoate.

Animals↗
PubMed1996

Characteristics of skeletal muscle chloride channel C1C-1 and point mutant R304E expressed in Sf-9 insect cells.

Using the baculovirus system, the skeletal muscle chloride channel, CIC-1 (rat), and a point mutant replacing arginine 304 with glutamic acid were expressed at high levels in cultured Sf-9 insect cells. Whole-cell patch-clamping revealed large inwardly rectifying currents with maxima up to 15 nA inward and 2.5 nA outward. Saturation was evident at voltage steps positive to +40 mV whilst steps negative to -60 mV produced inactivating currents made up of a steady state component and two exponentially decaying components with tau 1 = 6.14+/- 0.92 ms, tau 2 = 36.5+/- 3.29 ms (S.D) n = 7 for steps to -120 mV. Currents recorded in the outside-out patch configuration were often unexpectedly large and up to 5% of whole-cell currents obtained in the same cell, suggesting an uneven channel distribution in the plasmalemma of Sf-9 cells. The pharmacology of a number of chloride channel blockers, including anthracene-9-carboxylate (A9C), niflumate, and perrhenate, was investigated and showed for the first time that perrhenate is an effective blocker of C1C-1 and that it has a complex mechanism of action. Further, the potency of A9C was found to be dependent on external chloride concentration. As in studies on muscle cells themselves, blockade was rapidly effective and easily reversible, except when applying the indanyloxyacetate derivative, IAA94/95, which took up to 10 min to act, and, consistent with an intracellular site of action, was difficult to reverse by washing. Mutation of the highly conserved arginine at position 304 to a glutamic acid did not significantly alter the behaviour of the channel.

Animals↗
PubMed1996

Epstein-Barr virus binding to CD21, the virus receptor, activates resting B cells via an intracellular pathway that is linked to B cell infection.

Epstein-Barr virus (EBV) initiates infection of normal B lymphocytes by binding to CD21, a complement receptor. Since EBV, unlike most viruses, preferentially infects resting (non-activated) cells, the present studies were undertaken to evaluate the hypothesis that intracellular signalling pathway(s) triggered by EBV binding to CD21 activate the expression of certain cellular genes, as well as the initially expressed viral genes, and thus enable EBV to infect resting B cells. Experiments with nontransforming EBV, recombinant virus ligand and anti-CD2 1 MAbs show that EBV binding to CD21 on resting B cells increases CD23 mRNA levels independently of viral gene expression. A panel of five protein kinase C (PKC) and tyrosine kinase (PTK) inhibitors, all with different modes of action, exhibited a distinctive pattern of effects on the EBV induced induction of CD23 expression, ranging from nearly complete inhibition to no influence. The results suggest that distinct PKC isoforms and PTKs are involved in the signalling pathway(s) triggered by EBV binding to CD21. Significantly, the five inhibitors showed the same pattern of effects on the earliest stages of infection (EBNA-2 transcription) and B cell transformation (mitogenesis and colony formation). The identical pattern of effects of these PKC and PTK inhibitors with diverse mechanisms of action on the EBV induced increase in both CD23 and EBNA-2 mRNA levels strongly suggests that their transcription is mediated by an intracellular signalling pathway which shares, at least in part, common members.

B-Lymphocytes↗
PubMed1995

Prevention of hypercalciuria and stone-forming propensity during prolonged bedrest by alendronate.

The bone loss and hypercalciuria induced by immobilization or the decreased gravitational forces of space are well described. Using a model of bedrest immobilization, the ability of a potent aminobisphosphonate, alendronate, to avert hypercalciuria and stone-forming propensity was tested. Sixteen male subjects participated in a randomized, placebo-controlled trial in which they received either 20 mg of alendronate or placebo 2 weeks prior to and during 3 weeks of strict bedrest. Parameters of bone and calcium metabolism and urinary crystallization of stone-forming salts were measured before and at the end of bedrest. In the placebo group, bedrest increased urinary calcium (209 +/- 47 to 267 +/- 60 mg/day, p < 0.01) and the saturation of calcium phosphate. Before bedrest, the alendronate group had a significantly lower serum calcium (8.8 +/- 0.4 vs. 9.6 +/- 0.5 mg/dl, p < 0.01) and higher serum PTH (62.4 +/- 33.1 vs. 23.1 +/- 7.5 pg/ml, p < 0.01) compared with the placebo group. Moreover, the alendronate group had a lower urinary calcium (75 +/- 41 mg/day) and saturation of calcium oxalate and calcium phosphate. These effects of alendronate were sustained during bedrest. Following bedrest in the alendronate group, urinary calcium rose to 121 +/- 50 mg/day, a value less than that in the placebo group before or during bedrest. Similarly, urinary saturation of calcium oxalate and calcium phosphate rose with bedrest in the alendronate-treated patients but remained lower than values obtained in placebo-treated patients before or during bedrest. Alendronate inhibits bone mineral loss and averts the hypercalciuria and increased propensity for the crystallization of stone-forming calcium salts which occurs during 3 weeks of strict bedrest.

Adult↗
PubMed1995

Antimicrobial activity of glycerine-ichthammol in otitis externa.

The clinical efficacy of glycerine-ichthammol in otitis externa may be due to an anti-inflammatory action of ichthammol or a dehydrating effect of glycerine on the oedematous ear canal. Its antimicrobial activity, if any, against the common organisms in otitis externa is not well known. A study of the antibacterial property of glycerine-ichthammol as measured by a growth inhibition test and a modified cidal assay, showed inhibition of selected gram positive organisms (Streptococcus pyogenes and Staphylococcus aureus) by ichthammol and glycerine-ichthammol combination, but only negligible antibacterial activity against Pseudomonas aeruginosa and Escherichia coli. Candida albicans was also weakly inhibited. As the activity against gram negative organisms is minimal, incorporation of an anti-gram negative antibiotic such as gentamicin in the glycerine-ichthammol compound to enhance its antibacterial spectrum is suggested.

Anti-Bacterial Agents↗
PubMed1995

Impact of gestational age on the catecholamine responses of the fetal sheep adrenal to cholinergic stimulation in vitro.

We have used the retrograde perfused adrenal preparation to investigate the catecholamine responses of the fetal sheep adrenal to increasing doses of acetylcholine (ACh) and excess potassium. Adrenal glands were collected from fetal sheep between 84 and 99 days gestation (before innervation; n = 7), between 103 and 113 days gestation (during innervation; n = 8) and between 137 and 144 days (after innervation; n = 9). Whilst the basal output of noradrenaline (NA) did not change between 84 and 144 days gestation, there was a significant increase in the adrenal output of adrenaline (Adr) between 84 and 144 days. The NA response to submaximal doses of ACh (10-10(3) microM) was significantly greater (P < 0.05) between 84 and 99 days gestation (mean NA response: 49.6 +/- 11.4 nmol/(30 min)) than after 137 days gestation (mean NA response: 27.3 +/- 8.3 nmol/(30 min)). Similarly, when the NA response to 10-10(3) microM ACh was expressed as a proportion of the maximal NA response to 10(4) microM ACh, the proportional NA response was greater at 84-99 days (48.9 +/- 11.3% of maximal) than at 137-144 days gestation (27.2 +/- 8.3%). The adrenal NA and Adr responses to 10(3) microM ACh between 84 and 137 days were reduced by up to 99% after the addition of hexamethonium. It appears, therefore, that the fetal adrenal is responsive to ACh before the development of innervation of the gland. There is also a decrease in sensitivity of the NA-secreting cells to submaximal doses of ACh as gestation progresses, which is not associated with a decrease in the size of the releasable pool of NA in the fetal adrenal. We speculate, therefore, that innervation of the fetal adrenal may be associated with an increase in the excitation threshold of the NA cells to nicotinic stimulation.

Acetylcholine↗
PubMed1995

Preliminary comparison of two negative reinforcement schedules to reduce self-injury.

This study compared the effectiveness of differential negative reinforcement of other behavior (DNRO) and alternative behavior (DNRA) for reducing self-injurious tantrums maintained by escape from demands in a 4-year-old girl with severe retardation. Both DNRA and DNRO reduced self-injury and increased independent performance of two tasks (tooth brushing and bathing); however, improvement on both measures was greater with the DNRA intervention.

Journal Article↗
PubMed1994

Immunolabelling for VDAC, the mitochondrial voltage-dependent anion channel, on sarcoplasmic reticulum from amphibian skeletal muscle.

Patch-clamp studies of ion channels in the sarcoball membrane, a relatively pure preparation of sarcoplasmic reticulum, had earlier revealed a high-conductance anion channel with some properties similar to the mitochondrial voltage-dependent anion channel (VDAC). Using post-embedding immunolabelling, the presence of VDAC was investigated in sarcoball preparations from the semitendinosus muscle of the cane toad Bufo marinus. As expected, the outer membrane of mitochondria found within the interior of skinned fibres was decorated with gold label. Surprisingly, sarcoplasmic reticulum membrane was also labelled. The sarcoball membranes, which could arise from either the sarcoplasmic reticulum or from mitochondria, were also labelled. These results indicate the presence of a VDAC-like protein in the sarcoplasmic reticulum.

Animals↗
PubMed1993

Modification of the transient outward current of rat atrial myocytes by metabolic inhibition and oxidant stress.

1. A putative function of the transient outward current (ITO) in cardiac myocytes is to modulate the shape of the action potential and, consequently, cardiac contractility. In addition, it has been suggested that this current may help protect against arrhythmias during periods of cardiac ischaemia. In our investigation of the possible anti-arrhythmic action of ITO, we have examined its response to metabolic inhibition and oxidant stress. 2. Whole-cell recordings were obtained from rat atrial myocytes using standard patch-clamp techniques. Inhibition of metabolism, using 10 mM 2-deoxy-D-glucose (2-DG) to block glycolysis with or without the addition of 2 mM cyanide to block oxidative phosphorylation, led to inhibition of ITO at a holding potential of -70 mV. Shifting the holding potential to -80 mV restored ITO, suggesting that metabolic inhibition had shifted the inactivation curve of ITO in a negative direction. 3. Quasi steady-state inactivation curves revealed a shift in ITO inactivation induced by complete metabolic inhibition with 2-DG and cyanide. Myocytes typically contracted shortly after the shift was observed. In the presence of Ruthenium Red, contraction was delayed and myocytes could undergo several exposures to the metabolic inhibitors, each time displaying a shift in ITO inactivation. The shifts ranged between -7 and -20 mV. 4. Recovery from inactivation was determined using a two-pulse protocol. The time constant of recovery at a holding potential of -80 mV reversibly shifted from 48 +/- 8 to 129 +/- 21 ms during metabolic inhibition (n = 4). 5. The activation of ITO from a holding potential of -100 mV shifted in a negative direction during metabolic inhibition, from a half-activation voltage of 0.3 +/- 3.0 to -14.7 +/- 2.5 mV (n = 5). Such a -15 mV shift increases the amplitude of ITO by approximately 30% at 0 mV. 6. A shift in ITO inactivation similar to that produced by metabolic inhibition could be shown when myocytes were subjected to oxidant stress induced by either 1 mM t-butyl hydroperoxide (TBHP) or the photoactivation of 100 nM Rose Bengal. Furthermore, an increase in pipette concentration of free Ca2+ from 20 to 200 nM also shifted ITO inactivation in a negative direction. 7. These results raise the possibility that the rise in intracellular [Ca2+] occurring during both metabolic inhibition and oxidant stress modifies activation and inactivation of ITO.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Metalloendopeptidase inhibitors and stimulus-secretion coupling in the mouse exocrine pancreas.

Inhibitors of metalloendopeptidases interfere with events involving Ca2(+)-dependent membrane fusion in a number of cell types. The divalent ion chelating agent 1,10-phenanthroline inhibited pancreatic amylase secretion stimulated by carbachol, cholecystokinin-octapeptide (CCK-8), or bombesin, but detailed studies indicated that this is unlikely to be a result of inhibition of metalloendopeptidase activity. The binding of [3H]N-methylscopolamine to pancreatic acini was reduced by 1,10-phenanthroline and this would explain the marked inhibition of carbachol-induced amylase secretion by the chelating agent. CCK-8-stimulated hydrolysis of phosphatidylinositol-4,5-bisphosphate was reduced by 1,10-phenanthroline while the binding of CCK-8 to acini was not affected. This inhibition of hydrolysis would explain the inhibition of CCK-8- and bombesin-induced amylase secretion. The metalloendopeptidase substrate carbobenzoxyglycylphenylalanylamide did not affect bombesin-stimulated amylase secretion. Amylase secretion evoked by treating pancreatic acini with the ionophore A23187 or dibutyryl-cyclic AMP was not reduced by 1,10-phenanthroline, indicating a lack of involvement of metalloendopeptidases in the process of exocytosis in this cell type.

Amylases↗
PubMed1990

Substance P and bombesin elevate cytosolic Ca2+ by different molecular mechanisms in a rat pancreatic acinar cell line.

1. Dual-excitation microfluorometry (Fura-2 as indicator) was employed to monitor directly changes in the cytosolic calcium concentration [( Ca2+]i) in single cells. We investigated and compared the effects of stimulation of AR42J rat pancreatic acinar cells by two peptide agonists, substance P and bombesin. 2. Substance P (10(-7) M) and bombesin (10(-8) M) each gave rise to a marked, but transient, elevation in [Ca2+]i. The calcium signals evoked by the two peptides were qualitatively and quantitatively very similar. However, in the absence of extracellular Ca2+ the response to substance P, but not bombesin, was abolished. These results suggest that substance P induces calcium influx across the cell surface membrane but does not release calcium from internal stores. Bombesin in marked contrast releases calcium from intracellular stores in the absence of any detectable calcium influx. 3. Depolarization by high-K+ extracellular solutions evoked a marked, but transient, rise in [Ca2+]i. This elevation in [Ca2+]i was strictly dependent upon the presence of Ca2+ in extracellular media. 4. Nifedipine (5 x 10(-6) M), an antagonist of L-type voltage-dependent Ca2+ channels, blocked the elevations in [Ca2+]i induced by either substance P or high-K+ solutions, but not that evoked by application of bombesin. 5. Patch-clamp, single-channel current recordings from cell-attached patches of membrane confirmed the presence of voltage-dependent calcium channels in the surface membranes of AR42J cells. Whole-cell current recordings demonstrated voltage-dependent inward Ca2+ (Ba2+) currents which were increased in amplitude by substance P and blocked by nifedipine. 6. The protein kinase C (PKC) activators, the phorbol diester, phorbol 1,2-myristate 13-acetate (PMA, 10(-7) M), and cell-permeable diacylglycerol analogues, 1-oleoyl-2-acetyl-sn-glycerol (OAG, 2.5 x 10(-6) M) and sn-2-dioctanoyl glycerol (DiC8, 2.5 x 10(-6) M), mimicked the effect of substance P, but not bombesin, in elevating [Ca2+]i in a manner that was blocked by removal of extracellular Ca2+ or application of nifedipine. 7. The PKC inhibitor, polymyxin B (2.5 x 10(-6) M), applied 2 min prior to stimulation blocked the effects of substance P and PKC activators, but not bombesin, in elevating [Ca2+]i. 8. The calcium signals evoked by substance P and bombesin are achieved by activation of different molecular mechanisms. Substance P, the evidence suggests, activates PKC which in turn stimulates calcium influx by opening voltage-dependent Ca2+ channels in the cell surface membranes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Chronotropic actions of cholecystokinin octapeptide on the rat heart.

Cholecystokinin octapeptide (CCK-8) administered i.v. to urethane-anaesthetized rats or added to the perfusion stream of isolated rat hearts produced an immediate bradycardia. The size of this response was dose-related. Studies in vivo and in vitro using atropine and propranolol indicated that the response to CCK-8 was largely due to a direct action of the peptide on the heart. N-carbobenzoxy-tryptophan (CBZ-Trp), a cholecystokinin receptor antagonist, abolished the response of the isolated heart to CCK-8. Gastrin I did not produce bradycardia. The receptors on rat heart were similar to the classes of cholecystokinin receptors found in brain and exocrine pancreas in that CCK-8 rather than cholecystokinin tetrapeptide (CCK-4) was the preferred agonist.

Animals↗