Biomedical subjects

G Salama

Publications and source records attributed to G Salama.

At least 37 records · Page 2Linked to original sources

PubMed1998

Hypoxia and hypothermia enhance spatial heterogeneities of repolarization in guinea pig hearts: analysis of spatial autocorrelation of optically recorded action potential durations.

INTRODUCTION: Regional dispersions of repolarization (DOR) are arrhythmogenic perturbations that are closely associated with reentry. However, the characteristics of DOR have not been well defined or adequately analyzed because previous algorithms did not take into account spatial heterogeneities of action potential durations (APDs). Earlier simulations proposed that pathologic conditions enhance DOR by decreasing electrical coupling between cells, thereby unmasking differences in cellular repolarization between neighboring cells. Optical mapping indicated that gradients of APD and DOR are associated with fiber structure and are largely independent of activation. We developed an approach to quantitatively characterize APD gradients and DOR to determine how they are influenced by tissue anisotropy and cell coupling during diverse arrhythmogenic insults such as hypoxia and hypothermia. METHODS AND RESULTS: Voltage-sensitive dyes were used to map APs from 124 sites on the epicardium of Langendorff-perfused guinea pig hearts during (1) cycles of hypoxia and reoxygenation and (2) after 30 minutes of hypothermia (32 degrees to 25 degrees C). We introduce an approach to quantitate DOR by analyzing two-dimensional spatial autocorrelation of APDs along directions perpendicular and parallel to the longitudinal axis of epicardial fibers. A spatial correlation length L was derived as a statistical measure of DOR. It corresponds to the distance over which APDs had comparable values, where L is inversely related to DOR. Hypoxia (30 min) caused a negligible decrease in longitudinal thetaL (from 0.530 +/- 0.138 to 0.478 +/- 0.052 m/sec) and transverse thetaT (from 0.225 +/- 0.034 to 0.204 +/- 0.021 m/sec) conduction velocities and did not alter thetaL/thetaT or activation patterns. In paced hearts (cycle length [CL] = 300 msec), hypoxia decreased APDs (123 +/- 18.2 to 46 +/- 0.6 msec; P < 0.001) within 10 to 15 minutes and enhanced DOR, as indicated by reductions of L from 1.8 +/- 0.9 to 1.1 +/- 0.5 mm (P < 0.005). Hypothermia caused marked reductions of thetaL (0.53 +/- 0.138 to 0.298 +/- 0.104 m/sec) and thetaT (0.225 +/- 0.034 to 0.138 +/- 0.027 m/sec), increased APDs (128 +/- 4.4 to 148 +/- 14.5 msec), and reduced L from 2.0 +/- 0.3 to 1.3 +/- 0.6 mm (P < 0.05). L decreased with increased time of hypoxia and recovered upon reoxygenation. Hypoxia and hypothermia reduced L measured along the longitudinal (L(L)) and transverse (L(T)) axes of cardiac fibers while the ratio of L(L)/L(T) remained constant. CONCLUSION: Conventional indexes of DOR (i.e., APD "range" or "standard deviation," evaluated with extracellular electrodes) did not convey the spatial inhomogeneities of repolarization revealed by L. Spatial autocorrelation analysis provides a statistically significant measurement of DOR, which can take into account intrinsic heterogeneities of APDs and fiber orientation. The data show that hypoxia and hypothermia produce reductions of L, even though they have different effects on mean APD and conduction velocity. The preservation of a constant L(L)/L(T) ratio during hypoxia and hypothermia, despite large reductions in L, is consistent with a mechanism in which reduced cell-to-cell coupling unmasks intrinsic dispersions of APD and reduces L(L) and L(T) by the same factor. Thus, the spatial autocorrelation of APDs provides a sensitive index of DOR under normal and arrhythmogenic conditions. It incorporates the anisotropic nature of the myocardium and therefore is preferable to conventional indexes of DOR.

Action Potentials↗

PubMed1998

Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2.

Surface fluorescence spectroscopy of the beating heart to measure cytosolic calcium has been limited by the need to use ultraviolet excitation light for many of the commonly used calcium indicators. Ultraviolet light in the heart produces a high level of background fluorescence and is highly absorbed, limiting tissue penetration. Visible wave-length fluorescence dyes such as rhod 2 are available; however, the lack of spectral shift with calcium binding precludes the use of ratio techniques to account for changes in cytosolic dye concentration. We have developed a method for in vivo quantitation of cytosolic rhod 2 concentration that in conjunction with calcium-dependent fluorescence measurements permits estimation of cytosolic calcium levels in perfused rabbit hearts. Reflective absorbance of excitation light by rhod 2 loaded into myocardium was used as an index of dye concentration and the ratio of fluorescence intensity to absorbance as a measure of cytosolic calcium concentration. Endothelial cell loading of rhod 2 was found to be minimal (< 5%), and dye leak rate out of the cytosol was slow, with approximately 5% loss of dye fluorescence occurring between 10 and 30 min after dye loading. Rhod 2 loading into subcellular compartments, determined by manganese quenching, was also minimal (< 5%). The dissociation constant of rhod 2 for calcium was measured in vitro to be 500 nM, and this value increased to 710 nM in the presence of 0.5 mM myoglobin. On the basis of this value and in vivo fluorescence measurements, cytosolic calcium concentration in the rabbit heart was found to be 229 +/- 90 nM at end diastole and 930 +/- 130 nM at peak systole, with peak fluorescence preceding peak ventricular pressure by approximately 40 ms. This technique should facilitate detailed analysis of calcium transients from the whole heart.

Animals↗

PubMed1998

Optical mapping of atrioventricular node reveals a conduction barrier between atrial and nodal cells.

The mechanisms responsible for atrioventricular (AV) delay remain unclear, in part due to the inability to map electrical activity by conventional microelectrode techniques. In this study, voltage-sensitive dyes and imaging techniques were refined to detect action potentials (APs) from the small cells comprising the AV node and to map activation from the "compact" node. Optical APs (124) were recorded from 5 x 5 mm (approximately 0.5-mm depth) AV zones of perfused rabbit hearts stained with a voltage-sensitive dye. Signals from the node exhibited a set of three spikes; the first and third (peaks I and III) were coincident with atrial (A) and ventricular (V) electrograms, respectively. The second spike (peak II) represented the firing of midnodal (N) and/or lower nodal (NH) cell APs as indicated by their small amplitude, propagation pattern, location determined from superimposition of activation maps and histological sections of the node region, dependence on depth of focus, and insensitivity to tetrodotoxin (TTX). AV delays consisted of tau 1 (49.5 +/- 6.59 ms, 300-ms cycle length), the interval between peaks I and II (perhaps AN to N cells), and tau 2 (57.57 +/- 5.15 ms), the interval between peaks II and III (N to V cells). The conductance time across the node was 10.33 +/- 3.21 ms, indicating an apparent conduction velocity (theta N) of 0.162 +/- 0.02 m/s (n = 9) that was insensitive to TTX. In contrast, tau 1 correlated with changes in AV node delays (measured with surface electrodes) caused by changes in heart rate or perfusion with acetylcholine. The data provide the first maps of activation across the AV node and demonstrate that theta N is faster than previously presumed. These findings are inconsistent with theories of decremental conduction and prove the existence of a conduction barrier between the atrium and the AV node that is an important determinant of AV node delay.

Acetylcholine↗

PubMed1997

Nitric oxide activates skeletal and cardiac ryanodine receptors.

The endothelial-derived relaxing factor, nitric oxide (NO.) has been shown to depress force in smooth and cardiac muscles through the activation of guanylyl cyclase and an increase in cGMP. In fast skeletal muscle, NO (i.e. NO-related compounds) elicits a modest decrease in developed force, but in contracting muscles NO increases force by a mechanism independent of cGMP. We now demonstrate an alternative mechanism whereby NO triggers Ca2+ release from skeletal and cardiac sarcoplasmic reticulum (SR). NO delivered in the form of NO gas, NONOates (a class of sulfur-free compounds capable of releasing NO), or S-nitrosothiols (R-SNO) oxidized or transnitrosylated regulatory thiols on the release channel (or ryanodine receptor, RyR), resulting in channel opening and Ca2+ release from skeletal and cardiac SR. The process was reversed by sulfhydryl reducing agents which promoted channel closure and Ca2+ reuptake by ATP-driven Ca2+ pumps. NO did not directly alter Ca(2+)-ATPase activity but increased the open probability of RyRs reconstituted in planar bilayers and inhibited [3H]-ryanodine binding to RyRs. The formation of peroxynitrite or thiyl radicals did not account for the reversible R-SNO-dependent activation of RyRs. Ca2+ release induced by nitric oxide free radicals (NO.) was potentiated by cysteine providing compelling evidence that NO. in the presence of O2 formed nitrosylated cysteine followed by the transnitrosation of regulatory thiols on the RyR to activate the channel. These findings demonstrate direct interactions of NO derivatives with RyRs and a new fundamental mechanism to regulate force in striated muscle.

Animals↗

PubMed1996

Activation and repolarization patterns are governed by different structural characteristics of ventricular myocardium: experimental study with voltage-sensitive dyes and numerical simulations.

INTRODUCTION: Substantial progress has been made in our understanding of transmural activation across ventricular muscle through studies of excitation patterns and potential distributions. In contrast, repolarization sequences are poorly understood because of experimental difficulties in mapping action potential durations (APDs) using extracellular electrodes. METHODS AND RESULTS: Langendorff-perfused guinea pig hearts and isolated coronary-perfused left ventricular sheet preparations were stained with the voltage-sensitive dye RH-421 and optical APs were recorded with a photodiode array. Epicardial maps were constructed using a triangulation method applied to matrices of activation and repolarization times determined from (dF/dt)max and (d2F/dt2)max' respectively. Numerical simulations were carried out based on: (1) a modified Luo-Rudy model; (2) the three-dimensional architecture of ventricular fibers; and (3) the intrinsic spatial distribution of APDs. In ventricular sheets, epicardial stimulation elicited elliptical activation patterns with the major axis aligned with the longitudinal axis of epicardial fibers. When the pacing electrode was progressively inserted from epicardium to endocardium, the major axes rotated gradually, clockwise by 45 degrees, and the eccentricity decreased from 2 to 1.14. Repolarization showed a relatively uniform pattern, independent of pacing site, beginning at the apex and spreading to the base. CONCLUSION: In experiments and simulations, the helical rotation of epicardial excitation isochrones caused by pacing at increasing depth in the myocardium correlated with the helical three-dimensional architecture of ventricular fibers. In contrast, repolarization was independent of the activation sequence and was mainly guided by spatial differences in APDs between apex and base.

Action Potentials↗

PubMed1995

Thimerosal interacts with the Ca2+ release channel ryanodine receptor from skeletal muscle sarcoplasmic reticulum.

The thiol-oxidizing reagent, thimerosal, has been shown to increase the intracellular Ca2+ concentration, to induce Ca2+ spikes in several cell types, and to increase the sensitivity of intracellular Ca2+ stores to inositol 1,4,5-trisphosphate. Ryanodine-sensitive stores have also been implicated in the generation of Ca2+ oscillations induced by the addition of thimerosal. Here we report that micromolar concentrations of thimerosal stimulate Ca2+ release from skeletal muscle sarcoplasmic reticulum vesicles, inhibit high affinity [3H]ryanodine binding, and modify the channel activity of the reconstituted Ca2+ release protein. Thimerosal inhibits ryanodine binding by decreasing the binding capacity (Bmax) but does not affect the binding affinity or the dissociation rate of bound ryanodine. Single channel reconstitution experiments show that thimerosal (100-200 microM) stimulates single channel activity without modifying channel conductance. The thimerosal-stimulated channel is not inhibited by heparin. Furthermore, a Ca(2+)-stimulated channel is first activated and then inhibited in a time-dependent fashion by high concentrations of thimerosal (1 mM). Once inactivated, the channel cannot be reactivated by addition of either Ca2+ or ATP.

Animals↗

PubMed1995

Optical mapping reveals that repolarization spreads anisotropically and is guided by fiber orientation in guinea pig hearts.

Guinea pig hearts were stained with a voltage-sensitive dye and imaged on a photodiode array to record fluorescent action potentials (APs) from 124 sites. Activation and repolarization patterns were recorded from the epicardium during stimulation at different loci and correlated with the underlying fiber architecture. Endocardial APs were recorded by inserting a light guide into the ventricular cavity or by dissecting out the ventricular free wall to expose the endocardium. In hearts paced on the right atrium to simulate sinus rhythm, activation emerged synchronously over a large area of the ventricular epicardium and spread laterally in 5 to 7 ms. The apparent longitudinal and transverse velocities were 2.66 +/- 0.11 and 1.65 +/- 0.09 m/s (n = 12). In contrast, repolarization began near the apex on the endocardium and spread transmurally in 6 +/- 1.3 ms (n = 12) and then anisotropically along the epicardium in 25 to 30 ms with apparent maximum (0.53 +/- 0.11 m/s) and minimum (0.31 +/- 0.10 m/s) repolarization velocities that aligned with the longitudinal and transverse axes of epicardial fibers. When paced on the epicardium, activation of intact hearts (n = 12) and perfused sheets (n = 8) was anisotropic, with longitudinal (0.85 +/- 0.05 m/s) and transverse (0.44 +/- 0.04 m/s) conduction velocities that aligned with the epicardial fiber orientation. When activation was initiated at different sites on the epicardium, repolarization always began near the apex and exhibited patterns similar to those obtained under right atrial pacing, but with slower longitudinal (0.41 +/- 0.09 m/s) and transverse (0.23 +/- 0.07 m/s) repolarization velocities (n = 18). In sheets stretched parallel to the longitudinal axis of surface fibers, AP durations (APDs) increased as a function of fiber length, from the length at zero developed tension to 120% of the length at maximum developed tension (Lmax). Spatial distributions of APDs did not change during stretches along the rising phase of the length-tension curve. In sheets stretched to 50% of Lmax, APDs were shorter and more homogeneous on the endocardium (mean APD, 188 ms; delta APD, 195-186 = 9 ms) than on the epicardium (mean APD, 204 ms; delta APD, 212-186 = 26 ms; n = 8). In guinea pig hearts, activation is rapid; therefore, repolarization depends primarily on intrinsic spatial heterogeneities of APDs. Consequently, repolarization begins at endocardial cells with the shortest APDs and spreads transmurally and then anisotropically on the surface according to the epicardial cell orientation.

Animals↗

PubMed1994

Ascorbate/iron activates Ca(2+)-release channels of skeletal sarcoplasmic reticulum vesicles reconstituted in lipid bilayers.

Ca(2+)-release channel or ryanodine receptor is known to be involved in physiologic Ca(2+)-release from sarcoplasmic reticulum in skeletal and cardiac muscle. A variety of chemical oxidants and in particular SH-oxidizing reagents have been shown to activate Ca2+ release. However, the role of the oxidative modification of the channel in the physiologic mechanism(s) of Ca2+ release and in pathologic states of the muscle remains to be elucidated. Ascorbate/iron redox couple is known to be an efficient generator of oxygen radicals and semidehydroascorbyl radicals. Ascorbate/iron was shown to be released from cardiomyocytes during ischemia-reperfusion and was suggested to be involved in the ischemia-reperfusion injury and cardiomyocyte death. To understand the potential contribution of ascorbate/iron to Ca2+ release mechanism(s), calcium release channels from skeletal sarcoplasmic reticulum (SR) were reconstituted in artificial planar bilayers to examine the effects of this redox couple on the channel activity. Ascorbate elicited a transient (about 2 min) but dramatic increase of open-time probability of the channel. At pCa = 7.0, the presence of EGTA blocked ascorbate induced activation of release channels. However, when exogenous iron was added, ascorbate activated Ca2+ release channels, even in the presence of EGTA. ESR measurements demonstrated that semidehydroascorbyl radicals were generated from ascorbate in the absence of EGTA. The semidehydroascorbyl radical ESR signal was quenched by EGTA in the absence (but not in the presence) of exogenous iron. Thus, the production of ascorbyl radicals was associated with increased channel activity. In the presence of heparin, ascorbate plus iron elicited a long-lasting activation of the channel which had conductance gCa2+ = 100 pS characteristic for the ryanodine receptor and which could be blocked by the ryanodine channel inhibitor, ruthenium red. In conclusion the physiologically relevant redox couple--ascorbate/iron--at physiologic concentrations can activate Ca2+ channels in sarcoplasmic reticulum vesicles.

Animals↗

PubMed1994

Optical mapping of repolarization and refractoriness from intact hearts.

BACKGROUND: Heterogeneities of repolarization (R) across the myocardium have been invoked to explain most reentrant arrhythmias. The measurement of refractory periods (RPs) has been widely used to assess R, but conventional electrode and extrastimulus mapping techniques have not provided reliable maps of RPs. METHODS AND RESULTS: Guinea pig hearts were stained with a voltage-sensitive dye to measure fluorescence (F) action potentials (APs) from 124 sites with a photodiode array. AP duration (APD) was defined as the time between depolarization (dF/dt)max and R time points (ie, the time when AP returns to baseline or some percent thereof). However, R time points are difficult to determine because AP downstrokes are often encumbered by drifting baselines and motion artifacts, which make this definition ambiguous. In optical and microelectrode recordings, the second derivative of AP downstrokes is shown to contain an easily detected, unique local maximum. The correlation between the position of this maximum (d2F/dt2)max and R has been tested during altered AP characteristics induced by changes in cycle length, ischemia, and hypoxia. Under these various modifications of the AP, the time points of (d2F/dt2)max fell at 97.0 +/- 2.1% of recovery to baseline. Extrastimulus techniques applied to (1) isolated myocytes, (2) intact hearts, and (3) mathematical simulations indicated that (d2V/dt2)max coincided with the effective RPs of APs. The coincidence of RPs and (d2V/dt2)max was valid within 5 milliseconds, for resting potentials of -75 to -90 mV and extrastimuli three times threshold voltage. CONCLUSIONS: Thus, optical APs and (d2F/dt2)max can be used to map activation, R, and RPs with AP recordings from a single heartbeat.

Animals↗

PubMed1994

Subthreshold stimulation of Purkinje fibers interrupts ventricular tachycardia in intact hearts. Experimental study with voltage-sensitive dyes and imaging techniques.

The effects of subthreshold stimulation (STS) delivered during right atrial pacing and ventricular tachycardia (VT) were investigated in Langendorff-perfused guinea pig hearts. The hearts were stained with a voltage-sensitive dye (RH 421) to map the propagation of optical action potentials. Sustained VT was reliably induced by 5-second trains (cycle length [CL], 25 to 50 milliseconds; duration, 0.5 to 10 milliseconds; and voltage, 2x threshold voltage) of impulses (n = 12 hearts) or a single premature beat (n = 6). The location of extrastimuli was not critical to the induction of VT, but the diameter of the heart had to be > or = 14.5 mm. During VT, heart rate increased from 200 to 600 beats per minute; action potential durations decreased from 112 to 175 milliseconds to 60 to 105 milliseconds, with no diastolic interval. Activation on the epicardium spread anisotropically, but VT decreased the "apparent" maximum conduction velocity (theta max) by 68% and altered the orientation of the major axis from beat to beat. Activation patterns and theta max measured during VT were similar to patterns recorded during direct pacing of the ventricle and indicated that Purkinje fibers no longer propelled ventricular excitation. STS (CL, 25 to 50 milliseconds; duration, 0.5 to 25 milliseconds; and voltage, 0.5x to 0.8x threshold; trains of 2.0 to 2.5 seconds) interrupted VT when applied to Purkinje fibers lining the endocardium (n = 6) but failed to interrupt VT when applied to the epicardium (n = 8). In atrial pacing, STS delivered to the endocardium increased theta max from 2.44 +/- 0.32 (mean +/- SEM) to 3.63 +/- 0.21 m/s in a local region surrounding the first activation sites (n = 4). Alternatively, VT could be terminated by reducing theta max (approximately 55%) with procainamide (10 mumol/L) (n = 6). STS terminates VT by synchronizing ventricular excitation most likely by increasing local conduction and/or improving the coupling between Purkinje and ventricular cells.

Action Potentials↗

PubMed1993

Positive inotropic effect of acetylcysteine in cardiomyopathic Syrian hamsters.

Several laboratories have provided indirect evidence that the myocardium of the cardiomyopathic Syrian hamster (CMH) is chronically ischemic on the basis of microvascular spasm. We previously reported evidence supporting a defect in the ryanodine-sensitive sarcoplasmic reticulum calcium release channel (SRCRC) in CMH. A relation between alterations in SRCRC and chronic ischemia has not yet been explored. A potential mechanism could be the effects of changes in redox state on thiol groups. Thiol reagents have previously been shown to regulate calcium release from SRCRC. Accordingly, we studied the inotropic effects of the sulfhydryl donors, acetylcysteine (AC), cysteine, and cystine in CMH. AC was a positive inotrope in isolated papillary muscles prepared from CMH, but not F1B controls (F1B) (p < 0.01). No significant differences were noted in inotropic responses to cysteine or cystine. AC blunted the response of CMH > F1B control papillary muscle preparations to stimulation frequency (p < 0.01). The actual tension generated (in mg/mm2) by CMH was no longer different than F1B with addition of AC (10(-3) M), ryanodine (10(-8) M), or verapamil (5 x 10(-7) M). These findings are consistent with a defect in SRCRC in CMH. This defect may be primary or may provide a novel mechanism for hibernating myocardium owing to chronic ischemia.

Acetylcysteine↗

PubMed1993

A method of determining electrical potential gradient across mitochondrial membrane in perfused rat hearts.

The electrical potential gradient across the mitochondrial membrane (delta psi m) in perfused rat hearts was estimated by calculating the equilibrium distribution of the lipophilic cation tetraphenylphosphonium (TPP+), using measured kinetic constants of uptake and release of TPP+. First-order rate constants of TPP+ uptake were measured during 30-min perfusions of intact rat hearts with tracer amounts (5.0 nM) of tritium-labeled TPP+ ([3H]TPP+) in the perfusate. This was followed by a 30-min washout, during which the first-order rate constant of efflux was estimated. Values of [3H]TPP+ outside the heart and total [3H]TPP+ inside the heart at equilibrium were calculated. From this information and separately estimated time-averaged plasma membrane potentials (delta psi c) it was possible to calculate free cytosolic [3H]TPP+ at equilibrium. It was also possible to calculate free intramitochondrial [3H]TPP+ at equilibrium as the difference between total tissue [3H]TPP+ minus free cytosolic TPP+ and the sum of all the bound [3H]TPP+. Bound [3H]TPP+ was determined from [3H]TPP+ binding constants measured in separate experiments, using both isolated mitochondria and isolated cardiac myocytes under conditions where both delta psi m and delta psi c were zero. Delta psi m was calculated from the intramitochondrial and cytosolic free TPP+ concentrations using the Nernst equation. Values of delta psi m were 144.9 +/- 2.0 mV in hearts perfused with 5 mM pyruvate and 118.2 +/- 1.4 mV in hearts perfused with 11 mM glucose, in good agreement with delta psi m obtained from isolated rat heart mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Effects of cardiac work on electrical potential gradient across mitochondrial membrane in perfused rat hearts.

The myocardium responds to alterations in cardiac work by changing its rate of O2 consumption. This reflects an increase in the oxidative synthesis of ATP to meet the contractile demand for ATP. However, the biochemical mechanisms responsible for increased ATP synthesis are not fully understood. To localize the flux-controlling reaction(s) in the pathway of ATP synthesis, the effects of substrates and cardiac work on mitochondrial membrane potential (delta psi m), total tissue NADH-to-NAD+ ratio, and high-energy phosphate metabolites were examined in perfused rat hearts. Delta psi m was measured using the equilibrium distribution of tetraphenylphosphonium (33). Cytosolic phosphorylation potential, total tissue NADH-to-NAD+ ratio, and delta psi m were higher in hearts perfused with pyruvate than in those perfused with glucose. Increasing cardiac work induced a four-fold increase in O2 consumption, which was accompanied by 1) decreased or unaltered cytosolic ADP concentration, 2) increased tissue NADH-to-NAD+ ratio, and 3) decreased delta psi m. The results indicate that both NADH-generating reactions and the ATP synthase-catalyzed reaction are important in causing the increase in respiration that accompanies increased work. Because the activation of ATP synthase by cardiac work occurred in the absence of increases in delta psi m, ADP, and Pi, it is possible that the work-related acceleration in ATP synthesis may be due to modification of the kinetic properties of the ATP synthase.

Animals↗

PubMed1993

The cytokeratin filament-aggregating protein filaggrin is the target of the so-called "antikeratin antibodies," autoantibodies specific for rheumatoid arthritis.

In rheumatoid arthritis (RA), the high diagnostic value of serum antibodies to the stratum corneum of rat esophagus epithelium has been widely reported. These so-called "antikeratin antibodies," detected by indirect immunofluorescence, were found to be autoantibodies since they also labeled human epidermis. Despite their name, the actual target of these autoantibodies was not known. In this study, a 40-kD protein (designated as 40K), extracted from human epidermis and specifically immunodetected by 75% of RA sera, was purified and identified as a neutral/acidic isoform of basic filaggrin, a cytokeratin filament-aggregating protein, by peptide mapping studies and by the following evidences: (a) mAbs specific for filaggrin reacted with the 40K protein; (b) the autoantibodies, affinity-purified from RA sera on the 40K protein, immunodetected purified filaggrin; (c) the reactivity of RA sera to the 40K protein was abolished after immunoadsorption with purified filaggrin; (d) the 40K protein and filaggrin had similar amino acid compositions. Furthermore, autoantibodies against the 40K protein and the so-called "antikeratin antibodies" were shown, by immunoadsorption experiments, to be largely the same. The identification of filaggrin as a RA-specific autoantigen could contribute to the understanding of the pathogenesis of this disease and, ultimately, to the development of methods for preventing the autoimmune response.

Amino Acids↗

PubMed1992

Rose bengal activates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

The photooxidizing xanthene dye rose bengal (10 nM to 1 microM) stimulates rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum vesicles. Following fusion of sarcoplasmic reticulum (SR) vesicles to an artificial bilayer, reconstituted Ca2+ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. Rose bengal does not appear to affect K+ channels present in the SR. Following reconstitution of the sulfhydryl-activated 106-kDa Ca2+ channel protein into a bilayer, rose bengal activates the isolated protein in a light-dependent manner. Ryanodine at a concentration of 10 nM is shown to lock the 106-kDa channel protein in a subconductance state which can be reversed by subsequent addition of 500 nM rose bengal. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is also directly observed upon measurement of [3H]ryanodine binding to JSR vesicles. These observations indicate that photooxidation of rose bengal causes a stimulation of the Ca2+ release protein from skeletal muscle sarcoplasmic reticulum by interacting with the ryanodine binding site. Furthermore, similar effects of rose bengal on isolated SR vesicles, on single channel measurements following fusion of SR vesicles, and following incorporation of the isolated 106-kDa protein strongly implicates the 106-kDa sulfhydryl-activated Ca2+ channel protein in the Ca2+ release process.

Animals↗

PubMed1992

A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations.

The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential.

Animals↗

PubMed1992

Properties of immunoaffinity purified 106-kDa Ca2+ release channels from the skeletal sarcoplasmic reticulum.

The sulfhydryl-gated 106-kDa Ca(2+)-release channel (SG-106) was purified by biotin-avidin chromatography from skeletal sarcoplasmic reticulum (SR) vesicles and used as an antigen to raise polyclonal antibodies. Western blots showed that the antisera crossreacted with the antigenic SG-106 and not with SR Ca2+, Mg(2+)-ATPase or with junctional foot proteins (JFPs) (Zaidi et al., 1989, J. Biol. Chem. 264(36), 21, 725-21, 736; 21, 737-21, 747). Polyclonal antibody-affinity columns were used to selectively purify SG-106-kDa proteins which, upon incorporation in planar bilayers, revealed the presence of a cationic channels with properties similar to "native" Ca(2+)-release channels obtained through the fusion of SR vesicles with planar bilayers. In agreement with measurements of Ca2+ release from SR vesicles, sulfhydryl oxidizing and reducing agents (i.e., 2,2'-dithiodipyridine and dithiothreitol) respectively increased and decreased the open-time probability of 106-kDa Ca(2+)-release channels. In contrast with reports on JFPs, ryanodine at 0.5-1 nM increased the open-time probability and at 2-10 nM locked 106-kDa Ca(2+)-release channels in a closed state rather than an open subconductance state. The SG-106 was activated by millimolar ATP, inhibited by millimolar Mg2+, and blocked by micromolar ruthenium red. Adriamycin (2-10 microM) caused a transient activation of SG-106 Ca(2+)-release channels, followed by closure in about 5 min, and intermittent activation to a subconductance state. Polyclonal antibodies used to purify the SG-106 also activated the channel when added to the cis side but not the trans side of the bilayer. Thus, SG-106 channels possess features that are similar to "native" SR Ca(2+)-release channels, are immunologically distinct from JFPs, and interact in seconds with nanomolar ryanodine in planar bilayers.

Animals↗

PubMed1992

Ryanodine-affinity chromatography purifies 106 kD Ca2+ release channels from skeletal and cardiac sarcoplasmic reticulum.

A 106 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca2+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodine-receptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the 'physiological' Ca2+ release channel, we prepared ryanodine-affinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodine-affinity chromatography were identified as the low molecular weight Ca2+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca2+ release channels. Surprisingly, ryanodine-affinity columns did not retain JFPs but purified 106 kD Ca2+ release channels which are a minor component (0.1-0.3%) of SR proteins.

Animals↗