Harold C. Strauss

Intracellular calcium, currents, and stimulus-response coupling in endothelial cells.

Vascular endothelium appears to be a unique organ. It not only responds to numerous hormonal and chemical signals but also senses changes in physical parameters such as shear stress, producing mediators that modulate the responses of numerous cells, including vascular smooth muscle, platelets, and leukocytes. In many cases, the initial response of endothelial cells to these diverse signals involves elevation of cytosolic Ca2+ and activation of Ca(2+)-dependent enzymes, including nitric oxide synthase and phospholipase A2. Both the release of Ca2+ from intracellular stores, most likely the endoplasmic reticulum, and the influx of Ca2+ from the extracellular space contribute to the [Ca2+]i increase. The most important trigger for Ca2+ release is inositol 1,4,5-trisphosphate, which is generated by the action of phospholipase C, a plasmalemmal enzyme activated in many cases by the receptor-G protein cascade. Ca2+ influx appears to be related to the activity of receptor-G protein-enzyme complex and to the degree of fullness of the endoplasmic reticulum but does not involve voltage-gated Ca2+ channels. The magnitude of the Ca2+ influx depends on the electrochemical gradient, which is modulated by the membrane potential, Vm. Under basal conditions, Vm is dominated by a large inward rectifier K+ current. Some stimuli, e.g., acetylcholine, have been shown to hyperpolarize Vm, thus increasing the electrochemical gradient for Ca2+, which appears to be modulated by activation of Ca(2+)-dependent K+ and Cl- currents. However, the lack of potent and specific blockers for many of the described or postulated channels (e.g., nonselective cation channel, Ca(2+)-activated Cl- channel) makes an estimation of their effect on endothelial cell function rather difficult. Possible future directions of research and clinical implications are discussed.

A quantitative analysis of the activation and inactivation kinetics of HERG expressed in Xenopus oocytes.

1 The human etherà‐go‐go‐related gene (HERG) encodes a K+o channel that is believed to be the basis of the delayed rectified current, IKr, in cardiac muscle. We studied HERG expressed in Xenopus oocytes using a two‐electrode and cut‐open oocyte clamp technique with [K+]o of 2 and 98 mm. 2 The time course of activation of the channel was measured using an envelope of tails protocol and demonstrated that activation of the heterologously expressed HERG current (IHERG) was sigmoidal in onset. At least three closed states were required to reproduce the sigmoid time course. 3 The voltage dependence of the activation process and its saturation at positive voltages suggested the existence of at least one relatively voltage‐insensitive step. A three closed state activation model with a single voltage‐insensitive intermediate closed state was able to reproduce the time and voltage dependence of activation, deactivation and steady‐state activation. Activation was insensitive to changes in [K+]o. 4 Both inactivation and recovery time constants increased with a change of [K+]o from 2 to 98 mm. Steady‐state inactivation shifted by ∼30 mV in the depolarized direction with a change from 2 to 98 mm K*o 5 Simulations showed that modulation of inactivation is a minimal component of the increase of this current by [K+]o, and that a large increase in total conductance must also occur.

Shear Stress Induces ATP-Independent Transient Nitric Oxide Release From Vascular Endothelial Cells, Measured Directly With a Porphyrinic Microsensor

Shear stress causes the vascular endothelium to release nitric oxide (NO), which is an important regulator of vascular tone. However, direct measurement of NO release after the imposition of laminar flow has not been previously accomplished because of chemical (oxidative degradation) and physical (diffusion, convection, and washout) complications. Consequently, the mechanism, time course, kinetics, and Ca2+ dependence of NO release due to shear stress remain incompletely understood. In this study, we characterized these parameters by using fura 2 fluorescence and a polymeric porphyrin/Nafion-coated carbon fiber microsensor (detection limit, 5 nmol/L; response time, 1 millisecond) to directly measure changes in [Ca2+]i and NO release due to shear stress or agonist (ATP or brominated Ca2+ ionophore [Br-A23187]) from bovine aortic endothelial cells. The cells were grown to confluence on glass coverslips, loaded with fura 2-AM, and mounted in a parallel-plate flow chamber (volume, 25 microL). The microsensor was positioned approximately 100 microns above the cells with its long axis parallel to the direction of flow. Laminar flow of perfusate was maintained from 0.04 to 1.90 mL/min, which produced shear stresses of 0.2 to 10 dyne/cm2. Shear stress caused transient NO release 3 to 5 seconds after the initiation of flow and 1 to 3 seconds after the rise in [Ca2+]i, which reached a plateau after 35 to 70 seconds. Although the amount (peak rate) of NO release increased as a function of the shear stress (0.08 to 3.80 pmol/s), because of the concomitant increase in the flow rate, the peak NO concentration (133 +/- 9 nmol/L) remained constant. Maintenance of flow resulted in additional transient NO release, with peak-to-peak intervals of 15.5 +/- 2.5 minutes. During this 13- to 18-minute period, when the cells were unresponsive to shear stress, exogenous ATP (10 mumol/L) or Br-A23187 (10 mumol/L) evoked NO release. Prior incubation of the cells with exogenous NO or the removal and EGTA (100 mumol/L) chelation of extracellular Ca2+ blocked shear stress but not ATP-dependent NO release. The kinetics of shear stress-induced NO release (2.23 +/- 0.07 nmol/L per second) closely resembled the kinetics of Ca2+ flux but differed markedly from the kinetics of ATP-induced NO release (5.64 +/- 0.32 nmol/L per second). These data argue that shear stress causes a Ca(2+)-mediated ATP-independent transient release of NO, where the peak rate of release but not the peak concentration depends on the level of shear stress.(ABSTRACT TRUNCATED AT 400 WORDS)

Digoxin Immune Fab Therapy in the Management of Digitalis Intoxication: Safety and Efficacy Results of an Observational Surveillance Study

An observational surveillance study was conducted to monitor the safety and effectiveness of treatment with Digoxin Immune Fab (Ovine) (Digibind) in patients with digitalis intoxication. Before April 1986, a relatively limited number of patients received treatment with digoxin-specific Fab fragments through a multicenter clinical trial. Beginning with commercial availability in July 1986, this study sought additional, voluntarily reported clinical data pertaining to treatment through a 3 week follow-up. The study included 717 adults who received Digoxin Immune Fab (Ovine). Most patients were greater than or equal to 70 years old and developed toxicity during maintenance dosing with digoxin. Fifty percent of patients were reported to have a complete response to treatment, 24% a partial response and 12% no response. The response for 14% of patients was not reported or reported as uncertain. Six patients (0.8%, 95% confidence interval 0.3% to 1.8%) had an allergic reaction to digoxin-specific antibody fragments. Three of the six had a history of allergy to antibiotic drugs. Twenty patients (2.8%, 95% confidence interval 1.7% to 4.3%) developed recrudescent toxicity. Risk of recrudescent toxicity increased sixfold when less than 50% of the estimated dose of antibody was administered. A total of 215 patients experienced posttreatment adverse events. The events for 163 patients (76%) were judged to result from manifestations of underlying disease and thus considered unrelated to Fab treatment. Digoxin-specific antibody fragments were generally well tolerated and clinically effective in patients judged by treating physicians to have potentially life-threatening digitalis intoxication.

Antiarrhythmic drug action. Blockade of the inward sodium current.

THE mechanisms of action of antiarrhythmic drugs were discussed in these reviews over a decade ago (Rosen and Hoffman, 1973). Since then, important new concepts of the blocking action of drugs have been proposed and tested experimentally. Several attempts at a quantitative voltage clamp analysis of the blocking action of antiarrhythmic drugs have been made. The mechanism of the blocking action of local anesthetics has been studied in nerves to the limit of current electrical techniques—that of gating currents (Cahalan, 1980; Yeh, 1982). The volume of data dictates that we restrict the studies covered. We shall focus on the mechanism of the blockade of the sodium current. We neglect discussion of many otherwise important studies which do not have this focus. Knowledge of the number, kinetics, and relative importance of the individual pacemaker currents is sufficiently incomplete as to limit a discussion on the drug action on the individual currents. Blockade of membrane sodium conductance (GNa) is probably a major mechanism of action of antiarrhythmic drugs. To obtain a quantitative analysis of the kinetic and steady state effects of drugs on the sodium conductance, it is necessary to measure the sodium current, INa, in a stable system with the temperature and extracellular milieu that may be obtained in both normal and diseased tissues in vivo. The strategies that have been exploited for the study of INa in heart muscle include: (1) indirect estimation from Vmax of action potentials, (2) direct measurement of macroscopic sodium currents in multicellular and isolated cell preparations under voltage clamp, and (3) direct recording of unitary sodium conductance using the extracellular patch clamp technique. All these techniques use measurement of electrical properties to assess the binding kinetics of drugs to their receptor site(s). The quantitative precision of these techniques may vary. However, it is important to establish their ability to produce qualitatively similar results.

In Situ Hybridization Reveals Extensive Diversity of K+ Channel mRNA in Isolated Ferret Cardiac Myocytes

The molecular basis of K+ currents that generate repolarization in the heart is uncertain. In part, this reflects the similar functional properties different K+ channel clones display when heterologously expressed, in addition to the molecular diversity of the voltage-gated K+ channel family. To determine the identity, regional distribution, and cellular distribution of voltage-sensitive K+ channel mRNA subunits expressed in ferret heart, we used fluorescent labeled oligonucleotide probes to perform in situ hybridization studies on enzymatically isolated myocytes from the sinoatrial (SA) node, right and left atria, right and left ventricles, and interatrial and interventricular septa. The most widely distributed K+ channel transcripts in the ferret heart were Kv1.5 (present in 69.3% to 85.6% of myocytes tested, depending on the anatomic region from which myocytes were isolated) and Kv1.4 (46.1% to 93.7%), followed by kv1.2, Kv2.1, and Kv4.2. Surprisingly, many myocytes contain transcripts for Kv1.3, Kv2.2, Kv4.1, Kv5.1, and members of the Kv3 family. Kv1.1, Kv1.6, and Kv6.1, which were rarely expressed in working myocytes, were more commonly expressed in SA nodal cells. IRK was expressed in ventricular (84.3% to 92.8%) and atrial (52.4% to 64.0%) cells but was nearly absent (6.6%) in SA nodal cells; minK was most frequently expressed in SA nodal cells (33.7%) as opposed to working myocytes (10.3% to 29.3%). Two gene products implicated in long-QT syndrome, ERG and KvLQT1, were common in all anatomic regions (41.1% to 58.2% and 52.1% to 71.8%, respectively). These results show that the diversity of K+ channel mRNA in heart is greater than previously suspected and that the molecular basis of K+ channels may vary from cell to cell within distinct regions of the heart and also between major anatomic regions.

Regional Localization of ERG, the Channel Protein Responsible for the Rapid Component of the Delayed Rectifier, K+ Current in the Ferret Heart

Repolarization of the cardiac action potential varies widely throughout the heart. This could be due to the differential distribution of ion channels responsible for repolarization, especially the K+ channels. We have therefore studied the cardiac localization of ERG, a channel protein known to play an important role in generation of the rapid component of the delayed rectifier K+ current (IKr), an important determinant of the repolarization waveform, Cryosections of the ferret atrium and ventricle were prepared to determine the localization of ERG by fluorescence in situ hybridization (FISH) and immunofluorescence. We found that in the ferret, ERG transcript and protein expression was most abundant in the epicardial cell layers throughout most of the ventricle, except at the base. In the atrium, we found that ERG is most abundant in the medial right atrium, especially in the trabeculae and the crista terminalis of the right atrial appendage. It also is present in areas within the sinoatrial node. In all regions studied, FISH and immunofluorescence showed concordant localization patterns. These data suggest that repolarization mediated by IKr is not uniform throughout the ferret heart and provide a molecular explanation for heterogeneity in action potential repolarization throughout the mammalian heart.

C-type inactivation controls recovery in a fast inactivating cardiac K+ channel (Kv1.4) expressed in Xenopus oocytes.

1. A fast inactivating transient K+ current (FK1) cloned from ferret ventricle and expressed in Xenopus oocytes was studied using the two‐electrode voltage clamp technique. Removal of the NH2‐terminal domain of FK1 (FK1 delta 2‐146) removed fast inactivation consistent with previous findings in Kv1.4 channels. The NH2‐terminal deletion mutation revealed a slow inactivation process, which matches the criteria for C‐type inactivation described for Shaker B channels. 2. Inactivation of FK1 delta 2‐146 at depolarized potentials was well described by a single exponential process with a voltage‐insensitive time constant. In the range ‐90 to +20 mV, steady‐state C‐type inactivation was well described by a Boltzmann relationship that compares closely with inactivation measured in the presence of the NH2‐terminus. These results suggest that C‐type inactivation is coupled to activation. 3. The coupling of C‐type inactivation to activation was assessed by mutation of the fourth positively charged residue (arginine 454) in the S4 voltage sensor to glutamine (R454Q). This mutation produced a hyperpolarizing shift in the inactivation relationship of both FK1 and FK1 delta 2‐146 without altering the rate of inactivation of either clone. 4. The rates of recovery from inactivation are nearly identical in FK1 and FK1 delta 2‐146. 5. To assess the mechanisms underlying recovery from inactivation the effects of elevated [K+]o and selective mutations in the extracellular pore and the S4 voltage sensor were compared in FK1 and FK1 delta 2‐146. The similarity in recovery rates in response to these perturbations suggests that recovery from C‐type inactivation governs the overall rate of recovery of inactivated channels for both FK1 and FK1 delta 2‐146. 6. Analysis of the rate of recovery of FK1 channels for inactivating pulses of different durations (70‐2000 ms) indicates that recovery rate is insensitive to the duration of the inactivating pulse.

Comparison of time domain and frequency domain variables from the signal-averaged electrocardiogram: A multivariable analysis☆☆☆

The relative values of the unprocessed signal-averaged electrocardiogram (ECG) and time domain analysis and frequency domain analysis of the signal-averaged ECG were compared in 36 patients with sustained monomorphic ventricular tachycardia and a remote myocardial infarction, in 29 asymptomatic patients with a remote myocardial infarction and in 23 normal subjects. Area ratios of the energy spectra derived from fast Fourier transform analysis were calculated using six separate 140 ms intervals starting at 0, 40, 50 and 60 ms after QRS onset; 40 and 50 ms before QRS end and a variable length interval starting 40 ms before QRS end and extending to the T wave. Total filtered QRS duration, late potential duration and root mean square voltage of the terminal QRS complex were measured from the filtered vector magnitude signal-averaged ECG. The total QRS duration was also measured from the X, Y, Z leads of the unfiltered signal-averaged ECG. Seven variables were significantly different in univariate tests between myocardial infarction patients with and without ventricular tachycardia: three fast Fourier transform area ratios with the sampling interval starting at 1) QRS onset (p = 0.007), 2) 40 ms after QRS onset (p = 0.02), and 3) 60 ms after QRS onset (p less than 0.0001); and all four time domain variables at 1) total filtered QRS duration (p less than 0.0001), 2) late potential duration (p = 0.0001), 3) root mean square terminal QRS voltage (p = 0.0001), and 4) QRS duration from the unprocessed signal-averaged ECG (p less than 0.0001). Of these seven variables, only the fast Fourier transform area ratio starting at QRS onset was significantly different between patients with myocardial infarction without ventricular tachycardia and normal subjects. In multi-variable analysis, the total filtered vector magnitude QRS duration, a time domain variable that includes the late potential, was the only independent factor that separated patients with myocardial infarction with and without associated ventricular tachycardia.

The influence of pH on th electrophysiological effects of lidocaine in guinea pig ventricular myocardium.

Lidocaine has been reported to be more depressant in ischemic than normal myocardium. To determine the influence of pH on the electrophysiological effects of lidocaine, we recorded trans-membrane potential and dV/dtmulfrom guinea pig papillary muscles mounted in a single sucrose gap. Recovery kinetics of dV/dtma, were studied by introducing progressively early premature responses during phase 4 at a drive rate of 0.5 Hz. In Krebs-Henseleit solution (HCO3− = 25 mM, CO2= 5%, pH 7.4), lidocaine (1.5 x 10−5M) did not significantly change action potential characteristics. The recovery time constant (T) of dV/dtmaxwas increased from 10 ± 4 (mean ± SD) to 91 ± 12 msec. In the presence of lidocaine, T increased from 91 ± 12 to 212 ± 5 msec when the extracellular pH (pHo) was lowered by increasing the [CO2] to 20% (HCO3− = 25 mM, pHo= 6.95). Similarly, when pHowas lowered by decreasing [HCO3−] (HCO3− = 7.5 mM, CO2= 5%, pHo= 6.95), T increased from 96 ± 11 to 185 ± 41 msec. However, if the [CO2] was increased to 20% while the pHowas maintained at 7.4 [HCO3− = 85], T was unchanged compared to a [CO2] of 5%. Drug-free solutions of pHo= 6.95 (CO2= 5% or 20%; HCOr = 7.5 or 25 mM) did not increase T. The increase in T with a decrease in pHowas greater than that predicted by a change in distribution of the drug across the membrane. These data are consistent with the view that local anesthetics bind to a receptor in the sodium channel thereby inactivating it. The process of recovery from inactivation during the resting state occurs by exit of uncharged drug through the membrane. The degree of protonation of receptor-bound drug is increased by extracellular acidosis. This decreases the proportion of drug that may leave the receptor via the membrane and hence causes a slowing of the recovery from inactivation. Circ Res 47: 542-550, 1980