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Biomedical subjects

J H Lawrence

Publications and source records attributed to J H Lawrence.

At least 19 recordsLinked to original sources
PubMed1996

Local anesthetics as effectors of allosteric gating. Lidocaine effects on inactivation-deficient rat skeletal muscle Na channels.

Time- and voltage-dependent local anesthetic effects on sodium (Na) currents are generally interpreted using modulated receptor models that require formation of drug-associated nonconducting states with high affinity for the inactivated channel. The availability of inactivation-deficient Na channels has enabled us to test this traditional view of the drug-channel interaction. Rat skeletal muscle Na channels were mutated in the III-IV linker to disable fast inactivation (F1304Q: FQ). Lidocaine accelerated the decay of whole-cell FQ currents in Xenopus oocytes, reestablishing the wild-type phenotype; peak inward current at -20 mV was blocked with an IC50 of 513 microM, while plateau current was blocked with an IC50 of only 74 microM (P < 0.005 vs. peak). In single-channel experiments, mean open time was unaltered and unitary current was only reduced at higher drug concentrations, suggesting that open-channel block does not explain the effect of lidocaine on FQ plateau current. We considered a simple model in which lidocaine reduced the free energy for inactivation, causing altered coupling between activation and inactivation. This model readily simulated macroscopic Na current kinetics over a range of lidocaine concentrations. Traditional modulated receptor models which did not modify coupling between gating processes could not reproduce the effects of lidocaine with rate constants constrained by single-channel data. Our results support a reinterpretation of local anesthetic action whereby lidocaine functions as an allosteric effector to enhance Na channel inactivation.

Allosteric Regulation↗
PubMed1996

Coupling between fast and slow inactivation revealed by analysis of a point mutation (F1304Q) in mu 1 rat skeletal muscle sodium channels.

1. We sought to elucidate the mechanism of the defective inactivation that characterizes sodium channels containing mutations in the cytoplasmic loop between the third and fourth domains (the III-IV linker). Specifically, we measured whole-cell and single-channel currents through wild-type and F1304Q mutant mu 1 rat skeletal muscle Na+ channels expressed in Xenopus laevis oocytes. 2. In wild-type channels, inactivation is complete and the faster of two decay components predominates. In F1304Q, inactivation is incomplete; the slow decay component is larger in amplitude and slower than in wild-type. The fraction of non-inactivating current is substantial (37 +/- 2% of peak current at -20 mV) in F1304Q. 3. Cell-attached patch recordings confirmed the profound kinetic differences and indicated that permeation was not altered by the F1304Q mutation. The F1304Q phenotype must be conferred entirely by changes in gating properties and is not remedied by coexpression with the beta 1-subunit. 4. Recovery from inactivation of F1304Q channels is faster than for wild-type channels and three exponentials are required to describe recovery adequately following long (5 s) depolarizations. Thus, there are three inactivated states even in 'inactivation-deficient' F1304Q channels. 5. The steady-state voltage dependence of F1304Q inactivation is right-shifted by 26 +/- 2 mV. 6. A gating model incorporating three inactivated states, all directly accessible from multiple closed states or the open state, was constrained to fit wild-type and F1304Q inactivation (h infinitive) data and repriming data simultaneously. While it was necessary to alter the rate constants entering and exiting all three inactivated states, the model accounted for the F1304Q-induced rightward shift in steady-state inactivation without imposing voltage dependence on the inactivation rate constants. 7. We conclude that the F1304Q mutation in mu 1 sodium channels modifies several inactivation processes simultaneously. The fact that a single amino acid substitution profoundly alters both fast and slow inactivation indicates that these processes share physical determinants in Na+ channels.

Animals↗
PubMed1996

Adenovirus-mediated transfer of CCAAT/enhancer-binding protein-alpha identifies a dominant antiproliferative role for this isoform in hepatocytes.

CCAAT/enhancer-binding protein (C/EBP) isoforms are thought to be important regulators of the hepatocyte phenotype. However, the specific physiological roles of different isoforms are poorly understood because hepatocytes express multiple C/EBPs, and various isoforms have overlapping functions. To identify the functions of C/EBPalpha in mature hepatocytes, replication-defective adenovirus vectors were used to efficiently and homogeneously overexpress the mouse C/EBPalpha gene in a SV40 virus-conditionally transformed rat hepatocyte line that can be induced to express C/EBPbeta and C/EBPdelta but that has little endogenous C/EBPalpha expression. Hepatocytes were infected with a recombinant adenovirus vector carrying the cDNA for C/EBPalpha driven by Rous sarcoma virus promoter elements (AdCEBPalpha) or a similar vector carrying the Escherichia coli lacZ gene (Adbetagal). Staining for beta-galactosidase demonstrated an infection efficiency of 100% at a multiplicity of infection of 25 plaque-forming units/cell and persistence of foreign gene expression for at least 9 days. Cultures infected with AdCEBPalpha had 50-fold higher levels of C/EBPalpha mRNA and protein than those infected with Ad-beta-gal, but similar expression of C/EBP-beta. Infection with AdCEBPalpha inhibited proliferation in cells expressing little C/EBPbeta, even when proliferation was driven by the SV40 transforming antigen, and also blunted mitogenic induction of the c-myc proto-oncogene in nontransformed cells with high levels of C/EBPbeta. Although overexpression of C/EBPalpha consistently increased C/EBPalpha DNA binding activity, it was not sufficient for albumin expression. Infection with AdCEBPalpha only increased albumin mRNA levels in nontransformed cells that also expressed relatively high levels of C/EBPbeta. Thus, in hepatocytes, C/EBPalpha has a dominant antiproliferative function, but must interact with other factors to regulate hepatocyte-specific gene expression.

Adenoviridae↗
PubMed1996

Single-channel analysis of inactivation-defective rat skeletal muscle sodium channels containing the F1304Q mutation.

The intracellular linker between domains III and IV of the voltage-gated Na channel mediates fast inactivation. Targeted alteration of one or more of a triplet of hydrophobic amino acids within this linker region results in a marked slowing in the decay of ionic current. The mechanism of this defective inactivation was explored in rat skeletal muscle sodium channels (mu 1) containing the F1304Q mutation in Xenopus laevis oocytes with and without coexpression of the rat brain beta 1 subunit. Cell-attached single-channel patch-clamp recordings revealed that the mu 1-F1304Q channel reopens multiple times with open times that are prolonged compared with those of the wild-type channel. Coexpression of the beta 1 subunit stabilized a dominant nonbursting gating mode and accelerated the activation kinetics of mu 1-F1304Q but did not modify mean open time or fast-inactivation kinetics. A Markov gating model incorporating separate fast- and slow-inactivation particles reproduced the results by assuming that the F1304Q mutation specifically influences transitions to and from fast-inactivated states. These effects are independent of interactions of the mutant channel with the beta 1 subunit and do not result from a change in modal gating behavior. These results indicate that F1304Q mutant channels can still enter the inactivated state but do so reversibly and with altered kinetics.

Animals↗
PubMed1996

Reversal of potassium channel deficiency in cells from failing hearts by adenoviral gene transfer: a prototype for gene therapy for disorders of cardiac excitability and contractility.

Heart failure is a common, often lethal disorder in which conventional pharmacologic strategies have achieved limited success. Failing hearts exhibit a delay of electrical repolarization which predisposes to fatal arrhythmias. To explore the feasibility of gene therapy for this condition, we isolated myocytes from normal and failing dog hearts and quantified electrophysiologic and contractile parameters in primary culture. Action potentials were prolonged in failing cells as a result of diminished potassium currents. Exposure to AdShK, an adenovirus that overexpresses potassium channels, reversed the action potential prolongation of failing cells. The precise phenotype varied as a function of the density of expressed channels; modest increases in potassium current sufficed to mimic the non-diseased state most faithfully, while more robust expression of the transgene excessively abbreviated excitation and contraction. Our results demonstrate that viral gene transfer can modify the electrical properties of adult mammalian heart cells in a manner appropriate to reverse a fundamental disorder of excitability. Realistic application of this form of therapy will need to include a sensitive mechanism for control of the level and distribution of transgene expression.

Action Potentials↗
PubMed1995

Prospects for genetic manipulation of cardiac excitability.

Despite impressive advances in the therapy of a number of types of heart disease in the last two decades, sudden cardiac death remains a public health problem of staggering dimensions. Current treatment options include antiarrhythmic drugs that have higher than desired failure rates and implantable defibrillators that incur significant costs to the patient and society. The development of therapies that better suppress the cardiac arrhythmias responsible for sudden cardiac death requires a broad and comprehensive understanding of the basic mechanisms underlying electrical instability in the heart. This study explores the scientific basis for a molecular genetic approach to modify cardiac excitability and thereby to create animal models of sudden cardiac death. The availability of such models will open up new avenues of research in arrhythmogenesis and facilitate the development of novel antiarrhythmic agents.

Animals↗
PubMed1995

Adenovirus-mediated expression of a voltage-gated potassium channel in vitro (rat cardiac myocytes) and in vivo (rat liver). A novel strategy for modifying excitability.

Excitability is governed primarily by the complement of ion channels in the cell membrane that shape the contour of the action potential. To modify excitability by gene transfer, we created a recombinant adenovirus designed to overexpress a Drosophila Shaker potassium channel (AdShK). In vitro, a variety of mammalian cell types infected with AdShK demonstrated robust expression of the exogenous channel. Spontaneous action potentials recorded from cardiac myocytes in primary culture were abbreviated compared with noninfected myocytes. Intravascular infusion of AdShK in neonatal rats induced Shaker potassium channel mRNA expression in the liver, and large potassium currents could be recorded from explanted hepatocytes. Thus, recombinant adenovirus technology has been used for in vitro and in vivo gene transfer of ion channel genes designed to modify cellular action potentials. With appropriate targeting, such a strategy may be useful in gene therapy of arrhythmias, seizure disorders, and myotonic muscle diseases.

3T3 Cells↗
PubMed1994

Sudden cardiac death in heart failure. The role of abnormal repolarization.

Congestive heart failure is a common, highly lethal cardiovascular disorder claiming over 200,000 lives a year in the United States alone. Some 50% of the deaths in heart failure patients are sudden, and most of these are probably the result of ventricular tachyarrhythmias. Methods designed to identify patients at risk have been remarkably unrewarding, as have attempts to intervene and prevent sudden death in these patients. The failure to impact favorably on the incidence of sudden death in heart failure patients stems largely from a lack of understanding of the underlying mechanisms of arrhythmogenesis. This article explores the role of abnormalities of ventricular repolarization in heart failure patients. We will examine evidence for the hypothesis that alteration of repolarizing K+ channel expression in failing myocardium predisposes to abnormalities in repolarization that are arrhythmogenic. The possible utility of novel electrophysiological and ECG measures of altered ventricular repolarization will be explored. Understanding the mechanism of sudden death in heart failure may lead to effective therapy and more accurate identification of patients at greatest risk.

Action Potentials↗
PubMed1993

Control of torque direction by spinal pathways at the cat ankle joint.

To study the biomechanics of the calcaneal tendon's complex insertion onto the calcaneus, we measured torque-time trajectories exerted by the triceps surae and tibialis anterior muscles in eight unanesthetized decerebrate cats using a multi-axis force-moment sensor placed at the ankle joint. The ankle was constrained to an angle of 110 degrees plantarflexion. Muscles were activated using crossed-extension (XER), flexion (FWR), and caudal cutaneous sural nerve (SNR) reflexes. Torque contributions of other muscles activated by these reflexes were eliminated by denervation or tenotomy. In two animals, miniature pressure transducers were implanted among tendon fibers from the lateral gastrocnemius (LG) muscle that insert straight into the calcaneus or among tendon fibers from the medial gastrocnemius (MG) that cross over and insert on the lateral aspect of calcaneus. Reflexively evoked torques had the following directions: FWR, dorsiflexion and adduction; SNR, plantarflexion and abduction; and XER, plantarflexion and modest abduction or adduction. The proportion of abduction torque to plantarflexion torque was always greater for SNR than XER; this difference was about 50% of the magnitude of abduction torque generated by tetanic stimulation of the peronei. During SNR, pressures were higher in regions of the calcaneal tendon originating from MG than regions originating from LG. Similarly, pressures within the MG portion of the calcaneal tendon were higher during SNR than during XER, although these two reflexes produced matched ankle plantarflexion forces. Selective tenotomies and electromyographic recordings further demonstrated that MG generated most of the torque in response to SNR, while soleus, LG, and MG all generated torques in response to XER.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Histologic determination of the ischemic threshold of muscle in the canine compartment syndrome model.

Our objective was to define the critical tissue pressure at which irreversible muscle damage occurs and to compare our results to those thresholds advocated in the orthopaedic literature. A standard plasma infusion compartment syndrome model was created in a canine model. Four dogs were in each of four experimental groups with compartment pressure maintained as follows: (a) 30 mm Hg with support of diastolic blood pressure to a level > 50 mm Hg; (b) 20 mm Hg less than diastolic pressure; (c) 10 mm Hg less than diastolic blood pressure; (d) a level equal to the animal's diastolic blood pressure. All animals were sacrificed 14 days after the procedure. Histology revealed the following: (a) tissues pressurized to 30 mm Hg in a normotensive dog demonstrated no significant abnormalities; (b) tissues pressurized to 20 mm Hg less than diastolic revealed occasional cells undergoing regeneration but no evidence of infarction or fibrosis; (c) tissues pressurized to 10 mm Hg less than diastolic showed scattered small areas of infarction and fibrosis; and (d) tissues pressurized to diastolic blood pressure demonstrated more widespread infarction and scarring. The ischemic threshold of muscle, beyond which irreversible tissue damage occurs, is directly related to the difference in compartment and perfusion pressure. Our findings document this pressure to be 10 mm Hg less than diastolic blood pressure or within 30 mm Hg of mean arterial pressure. This data refutes the use of absolute tissue pressure values as a guide to the necessity of fasciotomy. To abort an impending compartment syndrome and avoid irreversible tissue injury and their sequelae, fasciotomy should be done if tissue pressure reaches within 10-20 mm Hg of diastolic pressure.

Animals↗
PubMed1993

Cat hindlimb muscles exert substantial torques outside the sagittal plane.

1. We studied the contributions of several hindlimb muscles to ankle torque in adult cats deeply anesthetized with pentobarbital sodium. Isometric torques were measured with a multiaxis, force-moment sensor connected to the plantar surface of the foot. 2. Individual muscle torques were provoked by using a combination of muscle nerve stimulation and selective denervations and tenotomies. Torques were represented by three orthogonal components; defined as dorsiflexion/plantarflexion, inversion/eversion (rotation about the long axis of the foot), and toe-in/toe-out (rotation about the axis of the tibia). 3. Most of the muscles tested exerted substantial torques about more than one of the orthogonal axes, each of which shared a common origin centered midway between the medial and lateral malleoli. The lateral and especially the medial head of the gastrocnemius muscle exhibited large toe-out torques and eversion torques as well as the classical plantarflexion components. 4. The torque exerted by tibialis anterior was seen to oppose that of both medial and lateral gastrocnemius in each of the three directions. The toe-in and inversion torques exerted by tibialis posterior was opposed in these directions by both peroneus brevis and peroneus longus. Flexor hallucis longus exerted approximately 10 times more plantarflexion torque than did flexor digitorum longus; therefore, these two muscles cannot be considered pure synergists. 5. The major plantarflexors and dorsiflexor of the cat ankle joint contribute substantial torques outside the sagittal plane. Their opposing torques lead to increased joint stiffness; the net effect of coactivation of these muscles causes ground reaction forces oriented so as to maintain stability during quadrupedal stance.

Animals↗
PubMed1992

Molecular localization of an ion-binding site within the pore of mammalian sodium channels.

Sodium channels are the major proteins that underlie excitability in nerve, heart, and skeletal muscle. Chemical reaction rate theory was used to analyze the blockage of single wild-type and mutant sodium channels by cadmium ions. The affinity of cadmium for the native tetrodotoxin (TTX)-resistant cardiac channel was much higher than its affinity for the TTX-sensitive skeletal muscle isoform of the channel (microliters). Mutation of Tyr401 to Cys, the corresponding residue in the cardiac sequence, rendered microliters highly susceptible to cadmium blockage but resistant to TTX. The binding site was localized approximately 20% of the distance down the electrical field, thus defining the position of a critical residue within the sodium channel pore.

Amino Acid Sequence↗
PubMed1991

Sodium channel inactivation from resting states in guinea-pig ventricular myocytes.

1. Unitary Na+ channel currents were recorded from isolated guinea-pig ventricular myocytes using the cell-attached patch-clamp technique with high [Na+] in the pipette to enhance the signal-to-noise ratio. 2. The probability that the channel enters the inactivated state (I) directly from resting states (C) was investigated over a wide range of membrane potentials. 3. At membrane potentials of -60 mV or more positive, Markov chain theory was used to estimate the probability of C----I from histograms of the number of channel openings per depolarizing period. Holding potentials at least as negative as -136 were required to ensure that all channels resided in C prior to depolarization. 4. At membrane potentials negative to -60 mV, a two-pulse protocol was employed to determine the probability of C----I from the fraction of blank sweeps during the pre-pulse with correction for missed events. 5. The probability of C----I was found to be steeply voltage dependent at negative potentials, falling from 0.87 +/- 0.03 (mean +/- S.D.) at -91 mV to 0.42 +/- 0.01 at -76 mV. At potentials positive to -60 mV, this probability was less steeply voltage dependent and decayed to near zero at 0 mV. 6. Under physiological conditions, C----I transitions may produce appreciable Na+ channel inactivation at diastolic potentials. At potentials above the action potential threshold, inactivation is much more likely to occur from the open state.

Action Potentials↗
PubMed1991

Heavy-charged-particle radiosurgery of the pituitary gland: clinical results of 840 patients.

Since 1954, 840 patients have been treated at Lawrence Berkeley Laboratory with stereotactic charged-particle radiosurgery of the pituitary gland. The initial 30 patients were treated with proton beams; the subsequent 810 patients were treated with helium ion beams. In the great majority of the 475 patients treated for pituitary tumors, marked and sustained biochemical and clinical improvement was observed. Variable degrees of hypopituitarism developed in about one-third of patients treated solely with radiosurgery. In the earlier years of the program, 365 patients underwent radiosurgery to treat selected systemic diseases by inducing hypopituitarism. Focal temporal lobe necrosis and cranial nerve injury occurred in about 1% of patients who were treated with doses less than 230 Gy.

Acromegaly↗
PubMed1990

Effects of haemorrhage induced hypotension on coronary blood flow in an anaesthetised two vessel canine coronary stenosis-occlusion model.

STUDY OBJECTIVE: Previous experiments from our group have shown that left anterior descending coronary occlusion in the presence of hypotension caused by intravenous glyceryl trinitrate in an open chest canine model caused endocardial flow to fall in myocardium supplied by a stenosed circumflex coronary artery. The aim of the present study was to assess the effects of hypotension in a similar open chest model but without the vasodilating effect of glyceryl trinitrate on coronary vessels. DESIGN: Blood flow to and function of remote myocardium supplied by a stenosed circumflex coronary artery [gradient 27(SEM 3) mm Hg] was determined in anaesthetised dogs after haemorrhage induced hypotension [mean aortic pressure 70(3) mm Hg] and after left anterior descending coronary occlusion. Transmural blood flow was measured using microspheres and myocardial wall thickness with sonomicrometer crystals. SUBJECTS: 11 healthy mongrel dogs were used, weight 25 kg (range 19-33). MEASUREMENTS AND MAIN RESULTS: Haemorrhage induced hypotension in the setting of a circumflex stenosis reduced endocardial blood flow to the posterior myocardial bed from 0.84 (0.14) to 0.57(0.07) ml.min-1.g-1 but did not alter percent myocardial thickening. When the left anterior descending coronary artery was occluded in the presence of the circumflex stenosis and haemorrhage induced hypotension, there was no further decrease in endocardial blood flow to or thickening of the posterior myocardial bed, despite a reduction in mean coronary artery pressure from 54(3) to 43(3) mm Hg. CONCLUSIONS: These findings are in contrast to our previous findings where hypotension was induced by glyceryl trinitrate. Remote myocardial ischaemia appears to be attenuated in this two vessel stenosis-occlusion model in which hypotension is produced by haemorrhage.

Animals↗
PubMed1989

Two molecular transitions influence cardiac sodium channel gating.

Sodium channels from diverse excitable membranes are very similar in their structure, yet surprisingly heterogeneous in their behavior. The processes that govern the opening and closing of sodium channels have appeared difficult to describe in terms of a single, unifying molecular scheme. Now cardiac sodium channels have been analyzed by high-resolution single-channel recordings over a broad range of potentials. Channels exhibited both complex and simple gating patterns at different voltages. Such behavioral diversity can be explained by the balance between two molecular transitions whereby channels can exit the open state.

Electric Conductivity↗
PubMed1986

The characterization of human transmyocardial impedance during implantation of the automatic internal cardioverter defibrillator.

We set out to determine in a prospective fashion the characteristics of energy delivery related to defibrillation in a population of patients receiving the AICD. Specifically, we examined the characteristics of the delivered current, transmyocardial voltage, and transmyocardial impedance. Secondly, we determined the relationship between the energy delivered, the impedance encountered and the defibrillation threshold. Since the AICD will deliver a succession of shocks if the initial shock does not cardiovert, the effects of consecutive shocks at different energy levels on the transmyocardial impedance were also assessed.

Cardiac Surgical Procedures↗