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Biomedical subjects

P B Bennett

Publications and source records attributed to P B Bennett.

At least 19 recordsLinked to original sources
PubMed2002

The incidence of venous gas emboli in recreational diving.

From 1989-91, the Divers Alert Network monitored recreational divers for Doppler-detected venous gas emboli (VGE) and depth-time profiles following multi-day, repetitive, multi-level exposures. A Spencer score >0 occurred in 61 of 67 subjects (91%) and 205 of 281 dives (73%). No subject developed decompression sickness (DCS) on monitored days although 102 dives (36.3%) scored at Spencer Grades 2 or 3 (High Bubble Grade, HBG). We recorded the depth-time profiles with Suunto dive computers and estimated exposure severity with a probabilistic decompression algorithm. The HBG incidence increased 53% over the range of exposure severity (p < 0.001) in the divers, was approximately 20% higher for repetitive dives than for first dives, and decreased approximately 25% over the 6-8 days of multi-day diving (p < 0.001) suggesting a phenomenon similar to DCS adaptation. The observed HBG incidence was approximately 20% higher for males than females. Older male divers had a 25% increase in observed incidence of HBG while older female divers showed a 55% increase when compared to their younger counterparts.

Adult↗
PubMed2001

A novel extracellular calcium sensing mechanism in voltage-gated potassium ion channels.

Potassium (K(+)) channels influence neurotransmitter release, burst firing rate activity, pacing, and critical dampening of neuronal circuits. Internal and external factors that further modify K(+) channel function permit fine-tuning of neuronal circuits. Human ether-à-go-go-related gene (HERG) K(+) channels are unusually sensitive to external calcium concentration ([Ca(2+)](o)). Small changes in [Ca(2+)](o) shift the voltage dependence of channel activation to more positive membrane potentials, an effect that cannot be explained by nonspecific surface charge screening or channel pore block. The HERG-calcium concentration-response relationship spans the physiological range for [Ca(2+)](o). The modulatory actions of calcium are attributable to differences in the Ca(2+) affinity between rested and activated channels. Adjacent extracellular, negatively charged amino acids (E518 and E519) near the S4 voltage sensor influence both channel gating and Ca(2+) dependence. Neutralization of these charges had distinct effects on channel gating and calcium sensitivity. A change in the degree of energetic coupling between these amino acids on transition from closed to activated channel states reveals movement in this region during channel gating and defines a molecular mechanism for protein state-dependent ligand interactions. The results suggest a novel extracellular [Ca(2+)](o) sensing mechanism coupled to allosteric changes in channel gating and a mechanism for fine-tuning cell repolarization.

Allosteric Regulation↗
PubMed2000

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel.

We report here a characterization of two families of calcium-activated K(+) channel beta-subunits, beta2 and beta3, which are encoded by distinct genes that map to 3q26.2-27. A single beta2 family member and four alternatively spliced variants of beta3 were investigated. These subunits have predicted molecular masses of 27. 1-31.6 kDa, share approximately 30-44% amino acid identity with beta1, and exhibit distinct but overlapping expression patterns. Coexpression of the beta2 or beta3a-c subunits with a BK alpha-subunit altered the functional properties of the current expressed by the alpha-subunit alone. The beta2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the beta3a-c subunits resulted in only partial inactivation of the current, and the beta3b subunit conferred an apparent inward rectification. Furthermore, unlike the beta1 and beta2 subunits, none of the beta3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these beta-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.

Alternative Splicing↗
PubMed2000

Hyperbaric oxygen reduces cerebral blood flow by inactivating nitric oxide.

Based on recent evidence that nitric oxide (NO(.)) is involved in hyperoxic vasoconstriction, we tested the hypothesis that decreases in NO(.) availability in brain tissue during hyperbaric oxygen (HBO(2)) exposure contribute to decreases in regional cerebral blood flow (rCBF). rCBF was measured in rats exposed to HBO(2) at 5 atmospheres (ATA) and correlated with interstitial brain levels of NO(.) metabolites (NO(X)) and production of hydroxyl radical ((.)OH). Changes in rCBF were also correlated with the effects of NO(.) synthase inhibitor (l-NAME), NO(.) donor PAPANONOate, and intravascular superoxide dismutase (MnSOD) during HBO(2). After 30 min of O(2) exposure at 5 ATA, rCBF had decreased in the substantia nigra, caudate putamen, hippocampus, and parietal cortex by 23 to 37%. These reductions in rCBF were not augmented by exposure to HBO(2) in animals pre-treated with l-NAME. After 30 min at 5 ATA, brain NO(X) levels had decreased by 31 +/- 9% and correlated with the decrease in rCBF, while estimated (.)OH production increased by 56 +/- 8%. The decrease in rCBF at 5 ATA was completely abolished by MnSOD administration into the circulation before HBO(2) exposure. Doses of NO(.) donor that significantly increased rCBF in animals breathing air had no effect at 5 ATA of HBO(2). These results indicate that decreases in rCBF with HBO(2) are associated with a decrease in effective NO(.) concentration and an increase in ROS production in the brain. The data support the hypothesis that inactivation of NO(.) antagonizes basal relaxation of cerebral vessels during HBO(2) exposure, although an effect of HBO(2) on NO(.) synthesis has not been excluded.

Animals↗
PubMed2000

Long QT syndrome: biophysical and pharmacologic mechanisms in LQT3.

The congenital long QT syndromes (LQTSs) are a group of inherited cardiac disorders that increase the risk of sudden death from ventricular arrhythmias. Individuals with LQTS show abnormalities in cardiac repolarization. Mutations that cause LQTSs are distributed throughout the human genome on chromosomes 3, 4, 7, 11, and 21. Recent molecular genetic studies established that LQT3 results from mutations in the cardiac sodium ion channel gene (SCN5A). Research efforts are aimed at elucidating molecular mechanisms, determining the links between clinical phenotypes and the individual gene mutations, and pharmacologic targeting of the phenotypes. This approach will ultimately guide rational therapy. In addition, LQT3 serves as a model for inherited molecular-based disorders, as well as a paradigm for understanding the genesis and control of other cardiac arrhythmias.

Humans↗
PubMed2000

Nitric oxide and cerebral blood flow responses to hyperbaric oxygen.

We have tested the hypothesis that cerebral nitric oxide (NO) production is involved in hyperbaric O(2) (HBO(2)) neurotoxicity. Regional cerebral blood flow (rCBF) and electroencephalogram (EEG) were measured in anesthetized rats during O(2) exposure to 1, 3, 4, and 5 ATA with or without administration of the NO synthase inhibitor (N(omega)-nitro-L-arginine methyl ester), L-arginine, NO donors, or the N-methyl-D-aspartate receptor inhibitor MK-801. After 30 min of O(2) exposure at 3 and 4 ATA, rCBF decreased by 26-39% and by 37-43%, respectively, and was sustained for 75 min. At 5 ATA, rCBF decreased over 30 min in the substantia nigra by one-third but, thereafter, gradually returned to preexposure levels, preceding the onset of EEG spiking activity. Rats pretreated with N(omega)-nitro-L-arginine methyl ester and exposed to HBO(2) at 5 ATA maintained a low rCBF. MK-801 did not alter the cerebrovascular responses to HBO(2) at 5 ATA but prevented the EEG spikes. NO donors increased rCBF in control rats but were ineffective during HBO(2) exposures. The data provide evidence that relative lack of NO activity contributes to decreased rCBF under HBO(2), but, as exposure time is prolonged, NO production increases and augments rCBF in anticipation of neuronal excitation.

Animals↗
PubMed2000

The human inward rectifier K(+) channel subunit kir5.1 (KCNJ16) maps to chromosome 17q25 and is expressed in kidney and pancreas.

A novel human Kir5.1 (inward rectifier K+ channel subunit, gene name KCNJ16) was identified through database searches. This human KCNJ16 was mapped to chromosome 17q25. The full-length cDNA was identified and its genomic structure was determined. Tissue distribution studies showed that human KCNJ16 is significantly expressed in human kidney, pancreas and thyroid gland. In situ hybridization revealed expression in convoluted tubule cells of kidney and in the acinar and ductal cells of pancreas. These suggest that human Kir5.1 may be involved in the regulation of fluid and pH balance, thus making it a potential therapeutic target for hypertension, renal failure, or pancreatic disease.

Amino Acid Sequence↗
PubMed2000

Stereoselective interactions of the enantiomers of chromanol 293B with human voltage-gated potassium channels.

Selective inhibitors of the slow component of the cardiac delayed rectifier K(+) current, I(Ks), are of interest as novel class III antiarrhythmic agents and as tools for studying the physiologic roles of the I(Ks) current. Racemic chromanol 293B is an inhibitor of both native I(Ks) and its putative molecular counterpart, the KvLQT1+minK ion channel complex. We synthesized the (+)-[3S,4R] and (-)-[3R,4S] enantiomers of chromanol 293B using chiral intermediates of known absolute configuration and determined their relative potency to block recombinant human K(+) channels that form the basis for the major repolarizing K(+) currents in human heart, including KvLQT1+minK, human ether-a-go-go-related gene product (hERG), Kv1.5, and Kv4.3, corresponding to the slow (I(Ks)), rapid (I(Kr)), and ultrarapid (I(Kur)) delayed rectifier currents and the transient outward current (I(To)), respectively. K(+) channels were expressed in mammalian cells and currents were recorded using the whole-cell patch-clamp technique. We found that the physicochemical properties and relative potency of the enantiomers differed from those reported previously, with (-)-[3R,4S]293B nearly 7-fold more potent in block of KvLQT1+minK than (+)-[3S,4R]293B, indicating that the original stereochemical assignments were reversed. K(+) current inhibition by (-)-293B was selective for KvLQT1+minK over hERG, whereas the stereospecificity of block for KvLQT1+minK and Kv1.5 was preserved, with (-)-293B more potent than (+)-293B for both channel complexes. We conclude that the (-)-[3R,4S] enantiomer of chromanol 293B is a selective inhibitor of KvLQT1+minK and therefore a useful tool for studying I(Ks).

Animals↗
PubMed2000

[Involvement of nitrogen oxide in the cerebral vasoconstriction during respiration with high pressure oxygen].

High pressure oxygen evokes a cerebral vasoconstriction and diminishes cerebral blood flow with the aid of mechanisms which are not yet sufficiently studied. We were checking a hypothesis that the hyperbaric oxygen (HBO2) inactivates cerebral nitrogen oxide (NO), interrupts its basal relaxing effect, and evokes a vasoconstriction. In our experiments, HBO2 decreased cerebral blood flow depending on the pressure. Inhibiting the NO-synthase weakened basal vasorelaxation in breathing with atmosphere air and eliminated the vasoconstriction in exposure to the HBO2. Inactivation of O2 prevented the HBO2-induced vasoconstriction. The data obtained reveal that diminishing of cerebral blood flow in HBO is related to the NO inactivation and weakening of its basal vasorelaxing effect. Possible mechanisms of the NO inactivation may involve its reaction with oxygen and superoxide anion which lead to diminishing of the tissue NO concentration and weakening of its vasorelaxing effect.

Anesthesia↗
PubMed1999

Functional consequences of a domain 1/S6 segment sodium channel mutation associated with painful congenital myotonia.

An unusual form of painful congenital myotonia is associated with a novel SCN4A mutation causing a valine to methionine substitution in the domain 1/S6 segment of the skeletal muscle sodium channel. We studied the functional characteristics of this mutant allele using a recombinant channel to gain understanding about the nature of the biophysical defect responsible for this unique phenotype. When expressed heterologously in a cultured mammalian cell line (tsA201), the mutant channel exhibits subtle defects in its gating properties similar, but not identical, to other myotonia-producing sodium channel mutations. The main abnormalities are the presence of a small non-inactivating current that occurs during short test depolarizations, a shift in the voltage-dependence of channel activation to more negative potentials, and a slowing of the time course of recovery from inactivation. Flecainide, a potent sodium channel blocker previously reported to benefit patients affected by this form of myotonia, effectively inhibits the abnormal sodium current associated with expression of the mutant channel. Our findings demonstrate the unique pattern of sodium channel dysfunction associated with a D1/S6 myotonia-producing sodium channel mutation, and provide a mechanism for the beneficial effects of flecainide in this setting.

Animals↗
PubMed1999

Enhancement of HERG K+ currents by Cd2+ destabilization of the inactivated state.

We have studied the functional effects of extracellular Cd(2+) on human ether-a-go-go-related gene (HERG) encoded K(+) channels. Low concentrations (10-200 microM) of extracellular Cd(2+) increased outward currents through HERG channels; 200 microM Cd(2+) more than doubled HERG currents and altered current kinetics. Cd(2+) concentrations up to 200 microM did not change the voltage dependence of channel activation, but shifted the voltage dependence of inactivation to more depolarized membrane potentials. Cd(2+) concentrations >or=500 microM shifted the voltage dependence of channel activation to more positive potentials. These results are consistent with a somewhat specific ability of Cd(2+) to destabilize the inactivated state. We tested the hypothesis that channel inactivation is essential for Cd(2+)-induced increases in HERG K(+) currents, using a double point mutation (G628C/S631C) that diminishes HERG inactivation (Smith, P. L., T. Baukrowitz, and G. Yellen. 1996. Nature (Lond.). 379:833-836). This inactivation-removed mutant is insensitive to low concentrations of Cd(2+). Thus, Cd(2+) had two distinct effects on HERG K(+) channels. Low concentrations of Cd(2+) caused relatively selective effects on inactivation, resulting in a reduction of the apparent rectification of the channel and thereby increasing HERG K(+) currents. Higher Cd(2+) concentrations affected activation gating as well, possibly by a surface charge screening mechanism or by association with a lower affinity site.

Animals↗
PubMed1999

Human ether-à-go-go-related gene K+ channel gating probed with extracellular ca2+. Evidence for two distinct voltage sensors.

Human ether-à-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 +/- 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.

Algorithms↗
PubMed1999

Modulation of HERG potassium channels by extracellular magnesium and quinidine.

Torsades de pointes is a polymorphic ventricular arrhythmia resulting from congenital or drug-induced (acquired) QT prolongation. Pharmacologic suppression of repolarizing potassium currents is one mechanism causing the acquired long QT (LQT) syndrome. Recent studies have linked mutations in a gene encoding a potassium channel subunit (HERG) to the LQT syndrome. Clinical experience indicates that intravenous magnesium sulfate is effective in reversing torsades de pointes, but the molecular basis of this effect is not understood. This study was designed to investigate the effects of extracellular magnesium (Mg2+) on HERG potassium currents. HERG potassium channels were expressed in Xenopus oocytes and in a human cell line and were examined by voltage-clamp methods. Extracellular Mg2+ (0.3-10 mM) caused a concentration-dependent shift in the membrane-potential dependence of HERG channel opening, causing a reduction in K+ current. This effect was much greater than that observed in another human delayed rectifier K+ channel, hKv1.5, suggesting a specific interaction with the HERG channel. Quinidine is an antiarrhythmic drug that also causes torsades de pointes under certain conditions. Quinidine (3 microM) inhibited HERG currents expressed in oocytes by 32.1 +/- 3.2% (n = 5), whereas 1 microM quinidine inhibited HERG currents in tsA201 cells by 75.8 +/- 2.4% (n = 12). Increasing extracellular Mg2+ did not relieve the inhibition by quinidine, but caused additional suppression. These results indicate that extracellular Mg2+ exerts a direct action on HERG potassium channels, resulting in suppression of outward repolarizing potassium current. It is concluded that modulation of this important K+ current is not the mechanism by which intravenous magnesium terminates drug-induced LQT and torsades de pointes. Potent suppression of HERG channel current by quinidine, compared with that of I(Ks) and I(Na), is a likely contributor to torsades de pointes arrhythmias.

Animals↗
PubMed1998

The transient outward current in mice lacking the potassium channel gene Kv1.4.

1. The transient outward current (Ito) plays a prominent role in the repolarization phase of the cardiac action potential. Several K+ channel genes, including Kv1.4, are expressed in the heart, produce rapidly inactivating currents when heterologously expressed, and may be the molecular basis of Ito. 2. We engineered mice homozygous for a targeted disruption of the K+ channel gene Kv1.4 and compared Ito in wild-type (Kv1.4+/+), heterozygous (Kv1.4+/-) and homozygous 'knockout' (Kv1.4-/-) mice. Kv1.4 RNA was truncated in Kv1.4-/- mice and protein expression was absent. 3. Adult myocytes isolated from Kv1.4+/+, Kv1.4+/- and Kv1.4-/- mice had large rapidly inactivating outward currents. The peak current densities at 60 mV (normalized by cellular capacitance, in pA pF-1; means +/- s.e.m.) were 53.8 +/- 5. 3, 45.3 +/- 2.2 and 44.4 +/- 2.8 in cells from Kv1.4+/+, Kv1.4+/- and Kv1.4-/- mice, respectively (P < 0.02 for Kv1.4+/+ vs. Kv1.4-/-). The steady-state values (800 ms after the voltage clamp step) were 30.9 +/- 2.9, 26.9 +/- 3.8 and 23.5 +/- 2.2, respectively (P < 0.02 for Kv1.4+/+ vs. Kv1.4-/-). The inactivating portion of the current was unchanged in the targeted mice. 4. The voltage dependence and time course of inactivation were not changed by targeted disruption of Kv1.4. The mean best-fitting V (membrane potential at 50 % inactivation) values for myocytes from Kv1.4 +/+, Kv1.4+/- and Kv1. 4-/- mice were -53.5 +/- 3.7, -51.1 +/- 2.6 and -54.2 +/- 2.4 mV, respectively. The slope factors (k) were -10.1 +/- 1.4, -8.8 +/- 1.4 and -9.5 +/- 1.2 mV, respectively. The fast time constants for development of inactivation at -30 mV were 27.8 +/- 2.2, 26.2 +/- 5. 1 and 19.6 +/- 2.1 ms in Kv1.4+/+, Kv1.4+/- and Kv1.4-/- myocytes, respectively. At +30 mV, they were 35.5 +/- 2.6, 30.0 +/- 2.1 and 28. 7 +/- 1.6 ms, respectively. The time constants for the rapid phase of recovery from inactivation at -80 mV were 32.5 +/- 8.2, 23.3 +/- 1.8 and 39.0 +/- 3.7 ms, respectively. 5. Nearly the entire inactivating component as well as more than 60 % of the steady-state outward current was eliminated by 1 mM 4-aminopyridine in Kv1.4+/+, Kv1.4+/- and Kv1.4-/- myocytes. 6. Western blot analysis of heart membrane extracts showed no significant upregulation of the Kv4 subfamily of channels in the targeted mice. 7. Thus, Kv1.4 is not the molecular basis of Ito in adult murine ventricular myocytes.

Action Potentials↗
PubMed1998

Distinct domains of the voltage-gated K+ channel Kv beta 1.3 beta-subunit affect voltage-dependent gating.

The Kvbeta1.3 subunit confers a voltage-dependent, partial inactivation (time constant = 5.76 +/- 0.14 ms at +50 mV), an enhanced slow inactivation, a hyperpolarizing shift in the activation midpoint, and an increase in the deactivation time constant of the Kv1.5 delayed rectifier. Removal of the first 10 amino acids from Kvbeta1.3 eliminated the effects on fast and slow inactivation but not the voltage shift in activation. Addition of the first 87 amino acids of Kvbeta1.3 to the amino terminus of Kv1.5 reconstituted fast and slow inactivation without altering the midpoint of activation. Although an internal pore mutation that alters quinidine block (V512A) did not affect Kvbeta1.3-mediated inactivation, a mutation of the external mouth of the pore (R485Y) increased the extent of fast inactivation while preventing the enhancement of slow inactivation. These data suggest that 1) Kvbeta1.3-mediated effects involve at least two distinct domains of this beta-subunit, 2) inactivation involves open channel block that is allosterically linked to the external pore, and 3) the Kvbeta1.3-induced shift in the activation midpoint is functionally distinct from inactivation.

Action Potentials↗
PubMed1998

Measurement of cerebral blood flow in rats and mice by hydrogen clearance during hyperbaric oxygen exposure.

The hydrogen (H2) clearance method was adapted for the measurement of regional cerebral blood flow (rCBF) in anesthetized rats and mice during hyperbaric oxygen (HBO2) exposure. Polarographic platinum electrodes 0.1 mm in diameter were used to record H2 clearance curves from the parietal cortex (PC), substantia nigra (SN), and caudate putamen nucleus (CPN) after inhalation of 2.5% H2 in air. The system for H2 breathing under hyperbaric conditions was designed for remote operation from outside the chamber. The rCBF values (measured every 10 min) were calculated from the H2 clearance curves using the initial slope method. During air breathing control, rCBF values were similar to values reported using other methods. Considering all control rats together, blood flow (ml.100 g-1.min-1) was 89 +/- 3.6 in the SN, 78 +/- 4.7 in the CPN, and 76 +/- 6.7 in the PC. Blood flow (ml.100 g-1.min-1) for air-breathing mice was 108 +/- 11.4 in the SN and 74 +/- 8.8 in the CPN. During HBO2 exposure to 3 atm abs, rCBF in rats fell within 30 min by 26-39% (P < 0.05) and by 27-29% in mice (P < 0.05). HBO2 exposure to 4 atm abs induced maximal rCBF decreases in rats within 60 min by 37% (P < 0.01) in the SN and by 47% (P < 0.01) in the CPN. Breathing CO2 during HBO2 exposure to 4 atm abs reversed the vasoconstriction and led to a rCBF increase of 80-96% in rats. The H2 clearance method seems to be an accurate and sensitive technique for the repeated measurement of local CBF under hyperbaric conditions.

Animals↗
PubMed1997

Pharmacological targeting of long QT mutant sodium channels.

The congenital long QT syndrome (LQTS) is an inherited disorder characterized by a delay in cardiac cellular repolarization leading to cardiac arrhythmias and sudden death often in young people. One form of the disease (LQT3) involves mutations in the voltage-gated cardiac sodium channel. The potential for targeted suppression of the LQT defect was explored by heterologous expression of mutant channels in cultured human cells. Kinetic and steady state analysis revealed an enhanced apparent affinity for the predominantly charged, primary amine compound, mexiletine. The affinity of the mutant channels in the inactivated state was similar to the wild type (WT) channels (IC50 approximately 15-20 microM), but the late-opening channels were inhibited at significantly lower concentrations (IC50 = 2-3 microM) causing a preferential suppression of the late openings. The targeting of the defective behavior of the mutant channels has important implications for therapeutic intervention in this disease. The results provide insights for the selective suppression of the mutant phenotype by very low concentrations of drug and indicate that mexiletine equally suppresses the defect in all three known LQT3 mutants.

Anti-Arrhythmia Agents↗