Advances in pemphigus research, signaling, and acantholysis.

Pemphigus is a family of human autoimmune blistering diseases in which pathogenic autoantibodies induce blistering in skin and mucosa. The mechanisms by which pemphigus autoantibodies induce disease in the skin is under active investigation. A large number of cellular events induced in the target keratinocytes by pemphigus IgG have been described and suggest that pemphigus IgG binding to desmogleins trigger a complicated cascade of intracellular signaling and regulatory events. Targeting these intracellular events may prove useful therapeutically.

The exposure of murine macrophages to alpha 2-macroglobulin 'fast' forms results in the rapid secretion of eicosanoids.

The exposure of [3H]arachidonate-radiolabelled murine peritoneal macrophages to alpha 2-macroglobulin-methylamine or alpha 2-macroglobulin-trypsin but not native alpha 2-macroglobulin (alpha 2M) results in the rapid secretion of [3H]eicosanoids. Resident peritoneal macrophages stimulated with 0.1 microM alpha 2M-methylamine exhibited an enhanced secretion within 10 min. The ability of alpha 2M 'fast' forms to stimulate secretion of [3H]eicosanoids was similar to that observed in the presence of the murine macrophage chemoattractant platelet-activating factor. As observed for total [3H]eicosanoid secretion, alpha 2M 'fast' forms also rapidly enhanced the secretion of the cAMP-elevating prostanoid, prostaglandin E2, from resident peritoneal macrophages. Stimulated secretion of prostaglandin E2 in response to 0.1 microM alpha 2M-methylamine was less rapid than that observed using 0.1 microM platelet-activating factor. Similar amounts of secreted prostaglandin E2 were present in media of macrophage cultures after 1 h exposure to the two stimuli. In the presence of 0.1 microM alpha 2M-methylamine, secreted prostaglandin E2 remained elevated, compared to the appropriate buffer control, for at least 24 h. The present results indicate that receptor recognition of alpha 2M 'fast' forms by macrophages results in the rapid stimulation of eicosanoid secretion and suggest that secretion of prostaglandin E2 and other eicosanoids may be involved in the ability of alpha 2 M 'fast' forms to regulate various macrophage functional responses.

Atypical atrial flutter originating in the right atrial free wall.

BACKGROUND: Data from experimental models of atrial flutter indicate that macro-reentrant circuits may be confined by anatomic and functional barriers remote from the tricuspid annulus-eustachian ridge atrial isthmus. Data characterizing the various forms of atypical atrial flutter in humans are limited. METHODS AND RESULTS: In 6 of 160 consecutive patients referred for ablation of counterclockwise and/or clockwise typical atrial flutter, an additional atypical atrial flutter was mapped to the right atrial free wall. Five patients had no prior cardiac surgery. Incisional atrial tachycardia was excluded in the remaining patient. High-density electroanatomic maps of the reentrant circuit were obtained in 3 patients. Radiofrequency energy application from a discrete midlateral right atrial central line of conduction block to the inferior vena cava terminated and prevented the reinduction of atypical atrial flutter in each patient. Atrial flutter has not recurred in any patient (follow-up, 18+/-17 months; range, 3 to 40 months). CONCLUSIONS: Atrial flutter can arise in the right atrial free wall. This form of atypical atrial flutter could account for spontaneous or inducible atrial flutter observed in patients referred for ablation and is eliminated with linear ablation directed at the inferolateral right atrium.

Tyrosine phosphorylation of human keratinocyte beta-catenin and plakoglobin reversibly regulates their binding to E-cadherin and alpha-catenin.

We show that tyrosine phosphorylation, produced by incubation of normal human keratinocytes with the tyrosine phosphatase inhibitor peroxovanadate, directly and reversibly regulates the association of beta-catenin and plakoglobin with E-cadherin and alpha-catenin. Prior studies have demonstrated a correlative, but not causal, association between increased tyrosine phosphorylation and decreased adherens junction mediated cell-cell adhesion. We observed that (i) binding of tyrosine phosphorylated beta-catenin and plakoglobin to E-cadherin and to alpha-catenin was substantially reduced, but could be restored in vitro by removal of phosphate from beta-catenin and plakoglobin with added tyrosine phosphatase, and (ii) tyrosine phosphorylation of beta-catenin and plakoglobin was associated with decreased cell-cell adhesion. These findings support a direct and causal role for tyrosine phosphorylation of beta-catenin and plakoglobin in regulating adherens junction mediated cell-cell adhesion. We propose that tyrosine phosphorylation of specific and probably different residues is responsible for regulating the binding of beta-catenin or plakoglobin to (i) E-cadherin and (ii) alpha-catenin. Additionally, because beta-catenin and plakoglobin have both structural and regulatory functions, the data raise the possibility that beta-catenin or plakoglobin released from the adherens junctions by tyrosine phosphorylation may transduce a signal to the nucleus regarding the adhesive state of the cell.

Premature beats elicit a phase reversal of mechanoelectrical alternans in cat ventricular myocytes. A possible mechanism for reentrant arrhythmias.

BACKGROUND: Alternans of the ST segment of the ECG is an important risk factor for sudden cardiac death. Premature beats during alternans and the development of discordant alternans are associated with the onset of ventricular tachycardia and ventricular fibrillation. Moreover, premature beats can switch the pattern of alternans from discordant to concordant alternans. The mechanisms of how a premature beat can elicit a pattern shift in alternans and develop malignant ventricular arrhythmias are not clear. The purpose of this cellular study was to determine the electrical and mechanical restitution properties during cycle length-induced alternans and to determine how premature and delayed beats affect the resultant phase of alternans. METHODS AND RESULTS: A perforated patch recording method and video-based edge detector were used to record action potentials and contractions, respectively, from single ventricular myocytes enzymatically isolated from the cat heart. Electrical and mechanical restitution curves were determined by programmed test beats delivered at different cycle lengths during mechanoelectrical alternans. At 35 degrees C, 97.8% of cells exhibited concordant cellular alternans (action potentials with the larger action potential duration [APD] were associated with the larger contraction, and action potentials with the smaller APD exhibited the smaller contraction). The sequence or phase of concordant cellular alternans could be systematically reversed by (1) early premature beats that followed only action potentials with the shorter APD and smaller contraction (type 1 phase reversal; n = 34) or (2) late delayed beats that followed only action potentials with the longer duration and the larger contraction (type 2 phase reversal; n = 14). A phase reversal point was defined as a threshold time interval that resulted in switching the sequence of the alternating beats. A test stimulus at the phase reversal point caused temporary suppression of mechanoelectrical alternans. Lower temperatures (32 degrees C) or decreases in the basic cycle length induced larger beat-to-beat changes in the magnitude of alternans (APD or contraction) and significantly shifted the phase reversal point to earlier premature intervals for type 1 phase reversal. The interval of the phase reversal point was a function of the contractile ratio (the magnitude of the larger contraction/smaller contraction for two consecutive beats, r = .93) and not the APD ratio (longer APD/shorter APD; r = .501). In cells stimulated at cycle lengths longer than the threshold of alternans, a single premature beat could elicit a damped form of concordant mechanoelectrical alternans. A critically timed second premature beat reversed the phase of the damped alternans. CONCLUSIONS: Properly timed premature or delayed beats during cycle length-induced alternans consistently reversed the phase of cellular mechanoelectrical alternans. Reversal of the phase of alternans was dependent on recovery of mechanical activity, not electrical activity. The premature stimulus interval at the phase reversal point can be predicted by the magnitude of mechanical alternans. Thus, during cycle length-induced alternans, mechanical alternans governs the phase of electrical alternans. From the present results, a multi-cellular model is proposed that may explain how critically timed premature beats cause a regional change in the phase of mechanical alternans and thereby result in discordant electrical alternans or dispersion of refractoriness. Premature beats that induce phase reversal in mechanoelectrical alternans may contribute to the development of reentrant arrhythmias.

Mechanisms of automaticity in subsidiary pacemakers from cat right atrium.

Intracellular recordings were made from eustachian ridge of cat right atrium to determine mechanisms responsible for subsidiary pacemaker automaticity. Pacemaker action potentials exhibited two phases of diastolic depolarization: an initial steeper slope (D1) followed by a more gradual slope (D2). Cesium (1 mM) decreased D1 (-45.6%) to a significantly greater extent than D2 (-33.6%) and increased spontaneous cycle length (SCL) (+37.7%). Tetrodotoxin (10(-6) M) had no effect on maximum rate of rise of upstroke, although it increased SLC (+23.9%). Verapamil (0.4-1.0 microM) progressively increased SCL by decreasing late diastolic slope, resulting in oscillatory potentials and eventual quiescence. Both norepinephrine (2 x 10(-9) M) and Bay K 8644 (10(-7) M) elicited a significantly greater increase in D2 than in D1, resulting in a decrease in SCL. Ryanodine (10(-6) M) caused a small but significant initial decrease (-3.7%) followed by a progressive increase in SCL (+172%). Ryanodine decreased D2 without changing D1, increased maximum rate of rise and overshoot potential, and abolished tension. In the presence of ryanodine, Bay K 8644 progressively increased D1 amplitude, resulting in a cyclic pattern of dysrhythmic activity. In the presence of ryanodine, cesium significantly decreased D1 (-39.3%), shifted the late diastolic potential more negative, and increased SCL (+25.7%). These results indicated that multiple mechanisms participate in subsidiary pacemaker automaticity. They include 1) a cesium-sensitive component that contributes to a greater extent during the initial phase of diastolic depolarization, 2) a component mediated via calcium released from the sarcoplasmic reticulum that contributes primarily during the latter half of diastolic depolarization, and 3) possibly a direct contribution by the slow inward calcium current.

Limited proteolysis of the alpha-macroglobulin rat alpha 1-inhibitor-3. Implications for a domain structure.

Rat alpha 1-inhibitor-3 is a 180-kDa monomeric proteinase inhibitor found in high concentration in rat plasma. By several criteria it has been shown to be a member of the family of alpha-macroglobulin proteinase inhibitors often exemplified by the tetrameric human alpha 2-macroglobulin. We have used limited proteolysis of rat alpha 1-inhibitor-3 to probe the domain structure of this family of proteins. Proteinases of different specificities, including trypsin, chymotrypsin, thermolysin, and Staphylococcus aureus V8 proteinase, were employed and a common fragmentation pattern was observed when the reaction products were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These fragments were electrotransferred to polyvinylidene difluoride membranes and subjected to NH2-terminal amino acid sequence analysis in order to position them within the context of the primary structure. The fragmentation pattern may define the domain structure of alpha 1-inhibitor-3 and serve as a model for the domain organization of the family of alpha-macroglobulin proteinase inhibitors.

Phase-dependent properties of the cardiac sarcoplasmic reticulum oscillator in cat right atrium: a mechanism contributing to dysrhythmias induced by Ca2+ overload.

These experiments analyse the phase-dependent properties of spontaneous oscillations of the sarcoplasmic reticulum (SR) induced by Ca2+ overload. Right atrial tissue was loaded with intracellular Ca2+ by exposure to a modified Tyrode solution containing 50% of normal Na+ and 0.5 mM K+. Verapamil (2 microM) was added to block regenerative activity. Intracellular Ca2+ overload elicited spontaneous, rhythmic voltage and tension oscillations that were phase locked 1:1. Voltage and tension oscillations were abolished by exposure to low (0.9 mM) external Ca2+, 1 microM ryanodine, or 10 mM caffeine, indicating that both voltage and tension oscillations resulted from spontaneous oscillations in SR Ca2+ release. Single pulses of nerve-stimulated ACh release elicited phase shifts in both voltage and tension oscillations. Sinusoidal current was used as a periodic stimulus to drive membrane voltage and elicit periodic voltage oscillations. Stimulated voltage oscillations entrained spontaneous tension oscillations 1:1 in a range of frequencies close to the basic spontaneous SR oscillatory cycle length, or 2:1 at frequencies close to one-half the spontaneous SR oscillatory cycle length. Stimulation frequencies between these two regions entrained tension oscillations in predictable fixed coupled ratios (4:3, 3:2) and resulted in Wenckeback-like voltage patterns. Stimulation frequencies between phase-locked regions resulted in complex coupling relationships and irregular voltage patterns. Exposure to 1 microM ryanodine, 0.9 mM external Ca2+, or 10 mM caffeine abolished irregular voltage patterns and tension. We conclude that the SR oscillator exhibits phase-dependent sensitivity to perturbations at the surface membrane. As a result, external perturbations can elicit phase differences between spontaneous SR oscillations and membrane voltage that cause either phase-locked or irregular voltage patterns. These findings identify an intracellular mechanism that may contribute to the development of cardiac dysrhythmias resulting from intracellular Ca2+ overload.

Mechanism of insulin incorporation into alpha 2-macroglobulin: implications for the study of peptide and growth factor binding.

In recent years, many studies have suggested a direct role for alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor, in growth factor regulation. When coincubated in the presence of either trypsin, pancreatic elastase, human neutrophil elastase, or plasmin, 125I-insulin rapidly formed a complex with alpha 2M which was greater than 80% covalent. The covalent binding was stable to reduction but abolished by competition with beta-aminopropionitrile. Neither native alpha 2M nor alpha 2M pretreated with proteinase or methylamine incorporated 125I-insulin. Experiments utilizing alpha 2M cross-linked with cis-dichlorodiammineplatinum(II) indicated that 125I-insulin must be present during alpha 2M conformational change to covalently bind. A maximum stoichiometry of 4 mol of insulin bound per mole of alpha 2M and the short half-life of the alpha 2M intermediate capable of covalent incorporation were consistent with thiol ester involvement. Protein sequence analysis of unlabeled insulin-alpha 2M complexes, together with results of beta-aminopropionitrile competition, confirmed that insulin incorporation occurs via the same gamma-glutamyl amide linkage responsible for covalent proteinase and methylamine binding to alpha 2M. Although intact insulin apparently incorporated through its sole lysine residue on the B chain, we found that isolated A chain also bound covalently to alpha 2M. Phenyl isothiocyanate derivatization of the N-terminus had no effect on A-chain binding, supporting the possibility of heretofore unreported gamma-glutamyl ester linkages to alpha 2M.

Electrophysiology and ultrastructure of eustachian ridge from cat right atrium: a comparison with SA node.

Intracellular microelectrode and electron microscopic techniques were used to investigate and correlate the electrophysiology of subsidiary pacemaker activity with the presence of cells having ultrastructural characteristics of pacemaker cells i.e. P cells, in Eustachian ridge tissue isolated from cat right atrium. In addition, the electrophysiological characteristics of subsidiary pacemaker activity and the ultrastructural characteristics of P cells in Eustachian ridge were compared to those of SA node obtained from the same hearts. Action potential recordings and morphological analysis were restricted to the endocardial site of earliest activation. Electrophysiological recordings revealed that spontaneously active Eustachian ridge tissues generate slow response action potentials with pacemaker characteristics similar, although not identical, to those of SA node. These included a relatively steep diastolic slope, low maximum diastolic potential (-70 mV), rate of rise (5.5 V/s), take-off potential (-52.5 mV), a relatively large overshoot potential (+7.7 mV) and a spontaneous cycle length (948 ms) about twice as long as SA node (434 ms). Morphological analysis revealed cells with ultrastructural characteristics of P cells, that were restricted to the endocardial site of earliest pacemaker activation. Morphological measurements indicate that Eustachian ridge P cells are not significantly different from P cells in SA node of the same hearts. However, Eustachian ridge P cells exhibit a unique apposition of subsarcolemmal cisternae between cells not seen in SA node. We conclude that pacemaker cells within the Eustachian ridge generate stable, spontaneous activity via slow response pacemaker action potentials. Cells responsible for this subsidiary pacemaker activity are most likely P cell types that are similar, although not identical, to P cells in SA node.

Receptor-recognized alpha 2-macroglobulin-methylamine elevates intracellular calcium, inositol phosphates and cyclic AMP in murine peritoneal macrophages.

Human plasma alpha 2-macroglobulin (alpha 2M) is a tetrameric proteinase inhibitor, which undergoes a conformational change upon reaction with either a proteinase or methylamine. As a result, a receptor recognition site is exposed on each subunit of the molecule enabling it to bind to its receptors on macrophages. We have used Fura-2-loaded murine peritoneal macrophages and digital video fluorescence microscopy to examine the effects of receptor binding on second messenger levels. alpha 2M-methylamine caused a rapid 2-4-fold increase in intracellular Ca2+ concentration ([Ca2+]i) within 5 s of binding to receptors. The agonists induced a focal increase in [Ca2+]i that spread out to other areas of the cell. The increase in [Ca2+]i was dependent on the alpha 2M-methylamine concentration and on the extracellular [Ca2+]. Both sinusoidal and transitory oscillations were observed, which varied from cell to cell. Neither alpha 2M nor boiled alpha 2M-methylamine, forms that are not recognized by the receptor, affected [Ca2+]i in peritoneal macrophages under identical conditions of incubation. The alpha 2M-methylamine-induced rise in [Ca2+]i was accompanied by a rapid and transient increase in macrophage inositol phosphates, including inositol tris- and tetrakis-phosphates. Native alpha 2M did not stimulate a rise in inositol phosphates. Finally, binding of alpha 2M-methylamine to macrophages increased cyclic AMP transiently. Thus receptor-recognized alpha-macroglobulins behave as agonists whose receptor binding causes stimulation of signal transduction pathways.

Identification of monomeric alpha-macroglobulin proteinase inhibitors in birds, reptiles, amphibians and mammals, and purification and characterization of a monomeric alpha-macroglobulin proteinase inhibitor from the American bullfrog Rana catesbeiana.

The alpha-macroglobulins are classified as broad-spectrum inhibitors because of their ability to entrap proteinases of different specificities and catalytic class. Tetrameric and dimeric alpha-macroglobulins have been identified in a wide variety of organisms including those as primitive as the mollusc Octopus vulgaris; however, monomeric alpha-macroglobulin proteinase inhibitors have been previously identified only in rodents. The monomeric alpha-macroglobulin proteinase inhibitors are believed to be analogous to the evolutionary precursor of the multimeric members of this family exemplified by the tetrameric human alpha 2-macroglobulin. Until now, monomeric alpha-macroglobulin proteinase inhibitors have only been identified in rodents and have therefore been considered an evolutionary anomaly. However, in this report we have utilized several sensitive assays to screen various plasmas and sera for the presence of monomeric alpha-macroglobulins, and our results suggest that monomeric alpha-macroglobulin proteinase inhibitors are present in organisms belonging to the avian, reptilian, amphibian and mammalian classes of the chordate phylum. This indicates that these proteins are more widespread than previously recognized and that their presence in rodents is not an anomaly. To demonstrate further that the identified proteins were indeed monomeric alpha-macroglobulin proteinase inhibitors, we purified the monomeric alpha-macroglobulin from the American bullfrog Rana catesbeiana. We conclude that this protein is a monomer of 180 kDa on the basis of its behaviour on (i) pore-limit gel electrophoresis, (ii) non-reducing and reducing SDS/PAGE and (iii) gel-filtration chromatography. In addition, we demonstrate that this protein is an alpha-macroglobulin proteinase inhibitor by virtue of (i) its ability to inhibit proteinases of different catalytic class, (ii) the presence of a putative internal beta-cysteinyl-gamma-glutamyl thioester and (iii) an inhibitory mechanism characterized by steric protection of the proteinase active site and by sensitivity to small primary amines. The frog monomeric alpha-macroglobulin is structurally and functionally similar to the well-characterized monomeric alpha-macroglobulin proteinase inhibitor rat alpha 1-inhibitor-3.

Adenosine-sensitive bundle branch reentry.

INTRODUCTION: Bundle branch reentry is an uncommon mechanism for ventricular tachycardia. More infrequently, both fascicles of the left bundle may provide the substrate for such macroreentrant bundle branch circuits, so-called interfascicular reentry. The effect of adenosine on bundle branch reentrant mechanisms of tachycardia is unknown. METHODS AND RESULTS: A 59-year-old man with no apparent structural heart disease and history of frequent symptomatic wide complex tachycardias was referred to our center for further electrophysiologic evaluation. During electrophysiologic study, a similar tachycardia was reproducibly initiated only during isoproterenol infusion, which had the characteristics of bundle branch reentry, possibly using a left interfascicular mechanism. Intravenous adenosine reproducibly terminated the tachycardia. Application of radiofrequency energy to the breakout site from the left posterior fascicle prevented subsequent tachycardia induction and rendered the patient free of spontaneous tachycardia during long-term follow-up. CONCLUSIONS: Patients with ventricular tachycardia involving a bundle branch reentrant circuit may be sensitive to adenosine. These results suggest that adenosine may not only inhibit catecholamine-mediated triggered activity but also some catecholamine-mediated reentrant ventricular arrhythmias.

In vivo blockade of pemphigus vulgaris acantholysis by inhibition of intracellular signal transduction cascades.

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune disease characterized by mucocutaneous intraepithelial blisters and pathogenic autoantibodies against desmoglein 3. The mechanism of blister formation in pemphigus has not been defined; however, in vitro data suggest a role for activation of intracellular signalling cascades. OBJECTIVES: To investigate the contribution of these signalling pathways to the mechanism of PV IgG-induced acantholysis in vivo. METHODS: We used the passive transfer mouse model. Mice were injected with IgG fractions of sera from a patient with PV, with or without pretreatment with inhibitors of proteins that mediate intracellular signalling cascades. RESULTS: Inhibitors of tyrosine kinases, phospholipase C, calmodulin and the serine/threonine kinase protein kinase C prevented PV IgG-induced acantholysis in vivo. CONCLUSIONS: These observations strongly support the role of intracellular signalling cascades in the molecular mechanism of PV IgG-induced acantholysis.