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Affinity-purified autoantibodies and anti-peptide antibodies directed against the second extracellular loop of the beta 1-adrenoceptor increase the beating rate of cultured cardiomyocytes just like the beta-adrenergic agonist isoprenaline. Their positive chronotropic action is blocked by beta-adrenergic antagonists. Affinity-purified autoantibodies and anti-peptide antibodies directed against the muscarinic cholinergic M2 receptor exert in these myocytes, like the muscarinic cholinergic agent carbachol, a negative chronotropic effect that is antagonized by atropine. In contrast to the agonism of isoprenaline and carbachol, the described agonistic effects of the antibodies are not subject to desensitization.

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The sera of patients with myocarditis and idiopathic dilated cardiomyopathy contain agonistic autoantibodies that bind to the beta 1-adrenoceptor. These antibodies recognize epitopes on either the first or second extracellular loop of this receptor and exert in cultured neonatal rat heart myocytes a positive chronotropic effect. This effect is eliminated by beta 1-adrenoceptor blockade as well as by peptides corresponding to the first or second extracellular loop of the human beta 1-adrenoceptor. The antibodies realize their effects mainly via the beta-adrenoceptor-adenylate cyclase-protein kinase A cascade.

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In a preceding communication (Wallukat et al., 1992, Z Kardiol 81 [Suppl. 4]: 79-83), it was reported that synthetic peptides, corresponding in amino acid sequence to either the first or the second extracellular loop of the human beta 1-adrenoceptor, selectively suppressed the metoprolol- and bisoprolol-sensitive positive chronotropic action exerted in cultures of beating neonatal rat cardiomyocytes by the serum immunoglobulin fraction of patients with myocarditis and idiopathic dilated cardiomyopathy (DCM) and by affinity-purified autoantibodies from that fraction. These observations added to existing evidence that these antibodies were directed against the beta 1-adrenoceptor and might thus contribute to the harmful chronic cardiac adrenergic drive to which patients with DCM are believed to be exposed. Specifically, they pointed to the putative first and second extracellular loops of this receptor (these loops are each identical in man and the rat) as the sites of epitopes recognized by the chronotropically active, beta 1-agonistic autoantibodies. Now we report on the mapping of these epitopes with the help of two series of short synthetic overlap peptides, one series forming part of the first and the other of the second extracellular loop of the beta 1-adrenoceptor. Inhibition of the positive chronotropic response of cultured rat cardiomyocytes to the anti-beta 1-receptor autoantibodies (EC50 = 0.14 +/- 0.01 nM) from the serum immunoglobulin fraction of patients with DCM was taken as reflecting the neutralization of these antibodies by a particular overlap peptide. In this way the sequences S-F-F-C-E-L (residues 129-134) and A-R-R-C-Y-N-D (residues 206-212) emerged as the dominant epitopes in the first and second extracellular loops, respectively, followed with respect to neutralizing ability by the first loop sequence E-Y-G-S-F-F (residues 126-131) and the second loop sequences H-W-W-R-A-E (residues 197-202) and P-K-C-C-D-F (residues 213-218). Synthetic peptides corresponding to the sequences of the third extracellular loop of the beta 1-receptor (residues 346-356) and of the second extracellular loop of the human beta 2-receptor (residues 172-197) failed to neutralize the beta 1-agonistic autoantibodies. Using dithiothreitol as a reducing agent a disulfide bridge between cysteine 132 in the first and cysteine 209 in the second extracellular loop was considered to be essential for the chronotropic action of these autoantibodies.(ABSTRACT TRUNCATED AT 400 WORDS)

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1. Cells from the ventricles of newborn rats were cultured for 8 days in flasks attached to a rocker apparatus to ensure an adequate oxygen supply. 2. The rocked cultures, which had previously been found to be low in lactate and subsensitive to the positive chronotropic action of the beta-adrenergic agonist isoprenaline (ISO; EC50 of (+/-)-ISO around 7 x 10(-7) M), became resensitized and even highly supersensitive to the catecholamine upon treatment with 3 mM L(+)-lactate or 1 mM pyruvate. 3. The resulting concentration-response curves were anomalous in that they extended over 8 log units, with a threshold at about 10(-13) M, but with little or no change in the height and the position of the maximum (at 10(-5) M). The EC50 values and 95% confidence intervals were 2.4 (1.9-3.0) and 5.4 (4.9-6.0) x 10(-11) M, respectively, for the lactate- and pyruvate-induced components of the chronotropic response to (+/-)-ISO. 4. The supersensitive portion of the ISO concentration-response curve was abolished by (-)-propranolol (10(-6) M), indicating that it was due to beta-adrenoceptor stimulation. 5. The cultured heart cells had to be incubated with L(+)-lactate or pyruvate for a minimum of 45 min before an increase in sensitivity to ISO became apparent. This latency was not due to a requirement for protein synthesis. 6. The adenosine-3',5'-monophosphate (cAMP) response to ISO was not noticeably altered by lactate, but (+/-)-ISO, which at 10(-8) M had no effect on the activation state of cAMP-dependent protein kinase (PKA), caused a significant increase in the activity of the enzyme following a 2-h exposure of the cells to 3 mM L(+)-lactate. 7. alpha-cyanocinnamate, an inhibitor of transmembrane transport of lactate and pyruvate, severely inhibited the utilization of L-[U-14C] lactate by the cultured cells at a concentration (5 microM) that eliminated the lactate-evoked potentiation of the chronotropic action of ISO without significantly affecting its unpotentiated action. 8. The beat-accelerating action of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) and the lipophilic N6,2'-O-dibutyryl derivative of cAMP (dbcAMP), both of which are capable of elevating myocardial cAMP levels, was not potentiated by 1 mM pyruvate. 9. The question is raised, whether accumulation of lactate, a biochemical hallmark of anaerobiosis, might be a factor in some of the catecholamine-triggered events occurring in acute myocardial ischaemia and infarction.

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Sera of patients with myocarditis and dilated cardiomyopathy contain stimulatory autoantibodies directed specifically against the beta 1-adrenergic receptor. The binding of the antibodies could be localized to either the first or the second extracellular loop of the beta 1-adrenoceptor. In 73% of the cases investigated the antibodies recognized the second extracellular loop. The agonistic effects of the antibodies were abolished by beta-adrenergic antagonists. Furthermore, the antagonists were able to remove the antibodies from their binding sites.

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Among a number of racemic eicosatetraenoic acids (HETEs) investigated, 15-R/S HETE and 11-R/S-HETE were the only ones that enormously potentiated the chronotropic action of isoprenaline on neonatal rat heart myocytes in rocked culture. The effect of the 15-R/S-HETE was exclusively due to the S isomer.

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The serum gamma-globulin fraction from patients with allergic bronchial asthma (8/8), in contrast to the fraction from nonatopic control subjects (10/10), was found to inhibit the positive chronotropic action of the beta 2-selective adrenergic agonist, clenbuterol, on pyruvate- or lactate-treated cultured neonatal rat heart myocytes. No inhibition was exerted on the positive chronotropic response to prenalterol, which acted via beta 1-adrenergic receptors. The inhibitory effect of the asthmatic gamma-globulins was concentration dependent. It could nearly be abolished by immunoprecipitation with antihuman gamma-globulin and antihuman IgG, but not with antihuman IgM. This finding means that the inhibitory immunoglobulins of the patients with asthma were chiefly autoantibodies of the IgG isotype, capable of cross-reacting with chronotropic beta 2-adrenergic receptors on the cultured rat cardiomyocytes. These findings provide support for the notion that autoimmunity plays a role in the impairment of beta-adrenergic responsiveness in patients with allergic bronchial asthma.

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The serum of patients with myocarditis and dilated cardiomyopathy contains immunoglobulins capable of causing in vitro acceleration of beating of neonatal rat heart myocytes. This effect is stereoselectively inhibited by (-)-propranolol and is inhibited also by the beta 1-selective adrenergic antagonists, bisoprolol and metoprolol. This effect is not reversed by washing and continues unabated for at least 24 h.

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Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A2-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A2 inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A2 in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclo-oxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A2-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.

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The cultured rat heart myocytes constitute a functional test system for the detection of autoantibodies directed against the beta 2-adrenergic receptor subtype. In the present experiments we used autoantibodies-containing gamma-globulin fractions isolated from the serum of allergic asthmatics. Our data show that the gamma-globulin fractions inhibited with high sensitivity and specificity the positive chronotropic effect of the beta 2-selective adrenergic agonist clenbuterol.

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The action of the (-)- and (+)-enantiomers of the beta-adrenoceptor blocking drug propranolol on the inward calcium current (ICa) was studied in single mouse neuroblastoma x rat glioma hybrid cells of clone 108CC5 by suction pipette technique for intracellular perfusion and voltage clamp. ICa was recorded after internal cell perfusion with Tris phosphate buffer and suppression of sodium and potassium currents in Na+-free external solution. Extracellularly applied (-)- and (+)-propranolol (10(-7) to 10(-3) M) inhibited ICa in a similar dose-dependent manner. The IC50 values for both substances were approximately 5 . 10(-6) to 10(-5) M. Two other beta-blockers, alprenolol and talinolol, investigated as reference compounds, also depressed the ICa, but in a significantly higher dose-range of 10(-4) to 10(-3) M. The results provide further evidence that propranolol, besides its known effect on sodium inward current, also possesses marked inhibitory actions on the ICa in mammalian nerve cell membranes at relatively low concentrations.

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A megaohm seal, cell-attached patch clamp technique utilizing plastic suction pipettes for measuring macroscopic rapid inward sodium current (INa) was applied to cell membrane patches 30 to 100 micron2 in area at the periphery of reaggregates of myocardial cells from newborn rats cultured for 3 to 7 days. The reaggregates were composed of about 20 to several thousand of electrically well-coupled cells, the latter forming spheroidal reaggregates with a diameter of maximally 300 microns. This system was shown to guarantee satisfactory voltage-clamp control during ionic current measurements. The time and voltage dependence of the INa recorded during periods of up to 90 min was similar to that observed in single cardiac cell preparations from adult rats. INa inactivation was described by two time constants (tau h1, and tau h2). For maximal INa tau h1 and tau h2 had values of 1.5 +/- 0.25 ms and 8.7 +/- 3.9 ms (n = 4), respectively. The time course for recovery of INa from inactivation at the reaggregate resting potential exhibited also two time constants (tau re1 = 9.8 +/- 3 ms, tau re2 = 193 +/- 50 ms, n = 5). Estimated current density was about 10 pA/micron2. The concentration of tetrodotoxin needed to reduce maximal INa by 75% was 85 times lower in the reaggregates than it was in freshly isolated heart muscle cells from adult rats. The present system offers a combination of features that should make it well-suited for the study of both short- and long-term effects on the sodium channels in neonatal heart cells: Easy handling of the object, great electric stability, high time and amplitude resolution during ionic current measurements, and viability for at least one week.

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Spontaneously beating cardiomyocytes of newborn rats were exposed in culture to the gamma globulin fraction from the blood serum of patients with allergic asthma and dilated cardiomyopathy. The gamma globulin fraction of the asthmatics inhibited in a dose-dependent fashion the positive chronotropic response of lactate or pyruvate-treated myocytes to clenbuterol, an adrenergic agonist that acted via stimulation of beta 2 adrenoceptors. The gamma globulin fraction of the cardiomyopathic patients increased the rate of beating. This effect was stereoselectively inhibited by the beta blocker (-)-propranolol, indicating an involvement of beta adrenoceptors. The serum gamma globulin fraction from healthy persons had no effect on the beating rate. The effects of both the asthmatic and cardiomyopathic gamma globulin fractions were abolished by immunoprecipitation with antihuman gamma globulin and antihuman IgG. The chronotropic effects of the gamma globulin fraction may be attributed to the presence of autoantibodies to beta-adrenergic receptors, more specifically beta 2 receptors in the case of the asthmatics and beta 1 receptors in the case of the cardiomyopathic patients.

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The action of verapamil, /+/-D600 and nisoldipine on the inward calcium current (ICa) was studied in mouse neuroblastoma x rat glioma hybrid cells of the line 108CC5 under voltage clamp conditions by means of a suction pipette method.

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The effect of ethacyzine (a diethylamine analogue of ethmozine) on the rapid inward sodium current (INa) was studied in single rat ventricular muscle cells by a patch clamp technique. Extracellular application of 3 microM ethacyzine depressed the INa in a use-dependent fashion. Rest recovery from the use-dependent block by ethacyzine followed an exponential time course with a time constant of about 215 +/- 40 s (n = 4, mean +/- S.E.).

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A patch-clamp method was used to study the effects of the phenotiazine antiarrhythmic drug ethmozine (E) on the fast sodium inward current (INa) in freshly isolated heart muscle cells of adult rats. At a concentration of 10(-5) M E caused INa inhibition that could be enhanced by increasing the frequency of depolarization. This inhibition was reversible. After the termination of repetitive depolarization the amplitude of INa recovered with a time constant of about 10 sec. These findings may help to explain the therapeutic efficiency of E in high frequency cardiac rhythm disturbances.

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Effects of ethmozine (moricizine) on the rapid inward sodium current (INa) were studied in freshly isolated single cells of rat ventricular myocardium. INa was measured by means of a patch clamp method for observing integral ionic currents. Ethmozine was applied extracellularly to a small cell membrane patch at concentrations of 10, 20, and 40 microM. At a stimulation frequency of 0.1 Hz the drug decreased the peak INa without producing a shift of the current-voltage curve, but shifted the V0.5 of the steady-state inactivation curve by -6 mV. At frequencies of 2-5 Hz the ethmozine-induced block exhibited a prominent use dependence, with trains of depolarizing clamp pulses 5-50 ms in duration eliciting maximal INa from holding potentials at which the steady-state inactivation variable h infinity was close to 1. The use-dependent inhibition of INa became more pronounced with an increase in both stimulation rate and pulse duration. In contrast to what has been observed in the node of Ranvier of the frog, the present results indicate that ethmozine binds to both inactivated and open Na+ channels, but that the contribution of the open channel block to the overall block at depolarizing clamp step durations of several hundred milliseconds is small in comparison with the contribution of the block of inactivated channels.

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Sodium and calcium inward currents (INa and ICa) were measured in neuroblastoma X glioma hybrid cells of clones 108CC5 and 108CC15 by a single suction pipette method for internal perfusion and voltage clamp. Morphologically undifferentiated, exponentially growing cells were compared with cells differentiated by cultivation with 1 mmol/l dibutyryl cyclic AMP. Outward currents were eliminated by perfusing the cells with a K+-free solution. Voltage dependence and ion selectivity as well as steady state inactivation characteristics of INa and ICa resembled those of differentiated mouse neuroblastoma cells, clone N1E-115 (Moolenaar and Spector 1978, 1979). These parameters were identical in undifferentiated and differentiated cells of both clones. After differentiation the average density of the peak sodium and calcium currents was increased two and four-fold, respectively, in both cell lines. Our data indicate that exponentially growing, morphologically undifferentiated 108CC5 and 108CC15 neuroblastoma X glioma hybrid cells possess functional Na+ and Ca2+ channels undistinguishable from those of non-proliferating cells of these clones differentiated morphologically by treatment with dibutyryl cyclic AMP. That Na+ and Ca2+ spikes were not detected by other authors in these cells prior to morphological differentiation by dibutyryl cyclic AMP may be attributed to the fact that at the low resting membrane potential measured the Na+ and Ca2+ channels are inactivated.

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The kinetics of activation and inactivation of the inward calcium current (ICa) in morphologically undifferentiated and differentiated neuroblastoma X glioma hybrid cells of the clone 108CC15 were studied by the suction pipette technique for internal perfusion and voltage clamping. Potassium currents were eliminated by internal perfusion of the cells with a K+-free solution. Activation of ICa followed a sigmoidal time course and could reasonably be fitted by a m2 relation. The kinetics of ICa inactivation were studied by analyzing the current inactivation during long depolarizing steps and by measuring the peak ICa as a function of the length of a prepulse. Both methods gave comparable results indicating that the ICa inactivation cannot be fitted by a single exponential. The ICa inactivation was fitted by a biexponential function. Neither the activation nor the inactivation of ICa were changed after morphological cell differentiation induced by treatment with dibutyryl cyclic AMP.

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