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W Trautwein

Publications and source records attributed to W Trautwein.

At least 19 recordsLinked to original sources
PubMed1991

Inhibitory modulation of fast and slow Ca(2+)-currents in neuroblastoma x glioma cells during differentiation.

Mouse neuroblastoma x rat glioma hybrid cells (N x G, 108CC15) were used to study the inhibitory effects of the synthetic opioid D-Ala2-D-Leu5-enkephalin (DADLE), somatostatin, adrenaline-alpha 2 and angiotensin II on voltage-dependent Ca(2+)-currents (ICa) using the patch-clamp technique in the whole-cell configuration mode. The inhibitory effects could be abolished by pretreatment of N x G cells with pertussis toxin or intracellular infusion of GDP beta S indicating an involvement of a pertussis toxin sensitive GTP-binding protein (G-protein), presumably Go. The effect of DADLE, the strongest inhibitor of ICa, was studied during dibutyryl cyclic AMP (dBcAMP) induced differentiation. Using omega-conotoxin GVIA (omega-CTX) and methoxyverapamil (D600) as specific Ca(2+)-channel blockers of the N- and L-type Ca(2+)-channels, it was found that in N x G cells DADLE predominantly induces inhibition of T- and N-type Ca(2+)-channels.

Animals↗
PubMed1991

Role of the GTP-binding protein Gs in the beta-adrenergic modulation of cardiac Ca channels.

In the heart, the guanosine 5'-triphosphate (GTP)-binding protein Gs is activated by hormone binding to beta-adrenergic receptors and stimulates the intracellular cyclic adenosine 3',5'-monophosphate (cAMP) pathway that leads to phosphorylation of L-type Ca channels by the cAMP-dependent protein kinase A. Additionally, Gs can modulate cardiac Ca channels directly in cell-free systems. In order to examine the question of whether these pathways could be separated functionally and whether they act independently or synergistically on L-type Ca channels in intact cells, the whole-cell Ca current (ICa) and the respective current density were measured in guinea-pig ventricular myocytes at 0 mV. The following results were obtained. First, typically, the ICa density increased from 12 to 40 microA/cm2 following application of 1 microM isoproterenol (ISP) to myocytes bathed in solutions containing 1.8 mM CaCl2. However, 1 microM ISP enhanced ICa only from 9 to 17 microA/cm2 after inhibition of the protein kinase A by dialysis of 0.5 mM Rp-cAMPs (the Rp-isomer of adenosine 3',5'-monophosphorothioate) in the presence of 0.5 mM GTP. Withdrawal of GTP from the dialysate attenuated the effects of ISP on ICa. Thus, Rp-cAMPS unmasks a GTP-dependent component of the beta-adrenergic stimulation of ICa, which probably reflects the direct stimulation of Ca channels by Gs under block of cAMP-dependent phosphorylation. Second, in cells under dialysis with 100 or 200 microM cAMP, bath application of 20-40 microM 3-isobutyl-1-methylxanthine (IBMX) enhanced the ICa density to about 41 microA/cm2 indicating saturation of the cAMP pathway. Under this condition, 1 microM ISP was without significant effect on ICa. This result may suggest that direct Gs stimulation is rather ineffective on Ca channels after maximal cAMP-dependent phosphorylation. Alternatively, maximal stimulation of the cAMP pathway may also interfere with the activation of the Gs pathway in intact myocytes. Third, simultaneous application of 1 microM ISP and 40 microM IBMX enhanced ICa up to densities of around 75 microA/cm2 during cell dialysis with 100 microM cAMP, an effect much stronger than that exerted by IBMX alone under similar conditions. Since it seems likely that Gs is activated more quickly, than the cAMP pathway during application of the ISP/IBMX mixture, the latter result suggests that a direct effect of Gs may act to prime L-type Ca channels for cAMP-dependent phosphorylation during beta-adrenergic stimulation of cardiac myocytes.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Potentiation by cyclic GMP of beta-adrenergic effect on Ca2+ current in guinea-pig ventricular cells.

1. Effects of cyclic GMP on L-type Ca2+ current (ICa) were investigated in myocytes isolated from guinea-pig ventricles using the patch clamp method in the whole-cell configuration combined with intracellular perfusion. 2. When ICa was increased by bath application of isoprenaline (0.001-0.1 microM) or forskolin (0.5-1 microM), or by intracellular dialysis with cyclic AMP (50-100 microM), dialysis with 10 microM-cyclic GMP resulted in an additional stimulation of ICa. Without these pre-treatments, cyclic GMP (1-100 microM) had no effect on the basal ICa. 5'-GMP was without effect. 3. The stimulatory effect of cyclic GMP was observed at concentrations higher than 0.1 microM with a maximum at around 10 microM in the pipette. The dose-response relation between isoprenaline and ICa was shifted to the left by (10 microM) cyclic GMP; the half-maximum isoprenaline concentration shifted from 16 to 4.6 nM. 4. The increase of ICa on dialysing 50 microM-cyclic AMP varied from cell to cell, probably due to a difference in phosphodiesterase activity. The cells responding weakly to cyclic AMP showed a greater response to cyclic GMP, and vice versa. In cells dialysed with hydrolysis-resistant derivatives (10-50 microM-8-(4-chlorophenylthio)-cyclic AMP or 50 microM-8-bromo-cyclic AMP), additional dialysis with cyclic GMP failed to modify ICa. Dialysis with cyclic GMP abolished the stimulatory effect of milrinone, a specific inhibitor of cyclic GMP-inhibited phosphodiesterase. These findings suggested that inhibition of cyclic GMP-sensitive phosphodiesterase was responsible for the stimulatory effect of cyclic GMP. 5. In the presence of isoprenaline, direct application of an active fragment of cyclic GMP-dependent protein kinase (PKG) failed to modify ICa in most cells. Activation of native PKG by intracellular dialysis with 8-bromo-cyclic GMP, or higher concentrations of cyclic GMP (100-1000 microM), depressed ICa in about 25% of the cells. Furthermore, dialysis of cyclic GMP reversed the increase of ICa by the non-specific phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX). These findings suggested the presence of antagonistic mechanisms of cyclic GMP, which are independent from the above synergistic action. PKG may be involved in this antagonistic effect.

Animals↗
PubMed1990

On the mechanism of the "specific bradycardic action" of the verapamil derivative UL-FS 49.

Membrane currents were measured in single sino-atrial node cells of guinea pig and rabbit hearts as well as in guinea pig ventricular myocytes using the patch-clamp technique. UL-FS 49 blocked the L-type calcium current (ICa) in sino-atrial node cells at drug concentrations which had little or no effect on the amplitude of the hyperpolarization-activated current ih(f). In guinea pig ventricular myocytes UL-FS 49 also blocked ICa but not as strongly as in sino-atrial node cells. In a computer simulation of the sino-atrial node action potential the extent of rate reduction by block of either ih(f) or ICa was estimated. From the data obtained by single cell measurements and the computations we concluded that rate reduction in primary pacemaker cells by application of UL-FS 49 is mainly due to a use dependent block of the L-type calcium current. Voltage dependent unblock of iCa at potentials more negative than -50 mV together with the lower drug sensitivity of ventricular cells can explain the "specific bradycardic action" of UL-FS 49.

Action Potentials↗
PubMed1990

Calcium currents of neuroblastoma x glioma hybrid cells after cultivation with dibutyryl cyclic AMP and nickel.

The long-term modulation of calcium (Ca2+) currents (ICa) was studied in 108CC15 neuroblastoma x glioma hybrid (NxG) cells grown under various culture conditions. The following results were obtained: 1. Addition of 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP) or 0.1 microM forskolin to the culture medium increased a transient component of ICa two-fold within 3 days, from 21.0 +/- 1.6 pA/pF (n = 22) to a maximum of 40.0 +/- 2.6 pA/pF (n = 28). Under these conditions, cells also expressed a slowly inactivating ICa component (maximum after 3 days, 20.5 +/- 1.6 pA/pF, n = 28). 2. The fast inactivating ICa as well as the db-cAMP-induced slowly inactivating ICa were completely down-regulated during incubation of NxG cells with the inorganic Ca2+ channel blocker, nickel (Ni2+, 100 microM). The suppressing effect was reversed within 3 days of incubation in db-cAMP-containing medium lacking Ni2+. 3. Binding studies on membrane preparations of control and Ni2(+)-pretreated NxG cells revealed a marked difference in the maximal (+)3H-PN200-110 binding. The difference was seen in undifferentiated as well as in db-cAMP-incubated cells. 4. The protein synthesis blocker, cycloheximide, suppressed both the db-cAMP-induced increase and the reappearance of ICa following Ni2+ pretreatment. It is suggested that chronic application of db-cAMP or Ni2+ to NxG cells increases and decreases the number of Ca2+ channel proteins, respectively.

Animals↗
PubMed1990

Ionic currents contributing to the action potential in single ventricular myocytes of the guinea pig studied with action potential clamp.

With the action potential clamp procedure we studied the contribution of various ionic currents to the action potential in single ventricular myocytes. Action potentials were elicited by a current pulse through the suction pipette and recorded by a computer. A representative action potential was then repetitively replayed to the same cell under voltage-clamp conditions. Successive pharmacological blocks of ionic currents allowed for the first time the measurement of the contribution of the L-type calcium current (ICa) and the [Ca2+]i-activated currents as well as the potassium current to the action potential. Experiments using caffeine as a tool to increase calcium release from the sarcoplasmic reticulum supported the idea that INaCa contributes to the plateau during the second half of the action potential and even lasts into diastole, whereas strong elevation of the intracellular [Ca]i during the action potential additionally activated the non-specific cation channel.

Action Potentials↗
PubMed1990

A dual-pipette technique that permits rapid internal dialysis and membrane potential measurement in voltage-clamped cardiomyocytes.

Guinea pig ventricular myocytes were voltage-clamped and dialysed using two glass patch pipettes (P1, P2) with tip openings of around 2 microns. A substantial improvement in the efficacy of dialysis from P2 was achieved by the application of positive pressure (15-30 cm H2O) to P2, and similar negative pressure to P1. Evidence of enhanced dialysis was obtained by measuring the effect on Ca channel current of P2 dialysates containing Ca, cAMP, GTP [gamma-S], trypsin, or the catalytic subunit of protein kinase A. Times to maximum response were 3-5 times shorter than those calculated or observed by others using a single-pipette method. The speeding-up was verified in comparative experiments with 100 microM GTP [gamma-S] dialysates; maximum stimulation of ICa occurred after 1.3-1.8 min with the dual-pipette method, versus 8.2 min with a single pipette. Other advantages of the dual-pipette method include the option of following a control dialysis from P1 with a test dialysis from P2, and the measurement of actual membrane potential. The disadvantages are that the rate of success is lower than with single-pipette experiments, and that smaller cardiomyocytes are difficult subjects.

Animals↗
PubMed1990

Whole-cell calcium current in guinea-pig ventricular myocytes dialysed with guanine nucleotides.

1. Whole-cell calcium current (ICa) was recorded in guinea-pig ventricular myocytes superfused with Na+,K(+)-free solution and dialysed with a substrate-free solution (minimum intracellular solution, MICS). A dual tight-seal pipette method was often used to permit pressure-enhanced dialysis of a test solution after a given pre-dialysis. 2. In dual-pipette experiments, test dialysates contained 100 mM-GTP-gamma-S (guanosine 5'-O-(3-thiotriphosphate] or 100 microM-GMP-PNP (guanyl-5'-imidodiphosphate). These non-hydrolysable analogues of guanosine triphosphate (GTP) enhanced ICa amplitude (+ 10 mV) by 20-40%. Dialysates containing 100 microM-GTP or GDP-beta-S (guanosine 5'-O-(2-thiodiphosphate] were ineffective, and pre-dialysis with GDP-beta-S blocked stimulation by GTP-gamma-S. 3. Non-hydrolysable GTP analogues slowed the inactivation of ICa and shifted the voltage eliciting maximum ICa by 5-10 mV in the negative direction. 4. ICa enhancement by GTP analogues was attributed to the activation of three GTP-binding regulatory (G) proteins (Gi, Gp and Gs). In single-pipette experiments, the inactivation of Gi by pre-treatment with pertussis toxin did not block enhancement, and a Gp-activating regimen (external acetylcholine-internal GTP) was without effect. Thus, it is probable that the effects of GTP analogues on ICa were primarily mediated by Gs activation. 5. PI-MICS dialysates contained phosphorylation-pathway inhibitors and were used to inhibit Ca2+ channel phosphorylation via the adenyl cyclase pathway. These were deemed effective since forskolin (1-5 microM) doubled ICa during control dialysis but was without effect after 8 min PI-MICS dialysis. However, 0.1 microM-isoprenaline increased ICa by 35% in myocytes totally unresponsive to forskolin, suggesting that beta-adrenergic receptor occupation can stimulate ICa even when the phosphorylation pathway is blocked. 6. After prolonged dialysis of myocytes with PI-MICS, ICa was still enhanced by pressure-assisted dialysis of 100 microM-GTP-gamma-S or GMP-PNP. We conclude that activated Gs has a direct effect on cardiac Ca2+ channels.

Action Potentials↗
PubMed1989

Dendrotoxin-binding brain membrane protein displays a K+ channel activity that is stimulated by both cAMP-dependent and endogenous phosphorylations.

The purified protein that binds the K+ channel ligands dendrotoxin I and mast cell degranulating peptide can be phosphorylated by cAMP-dependent protein kinase and by an endogenous protein kinase, which may be a specific K+ channel kinase. Phosphorylations take place on the toxin-binding subunit, a polypeptide of 76-80 kDa. Phosphorylation by both kinases leads to activation of the reconstituted dendrotoxin-sensitive K+ channel.

Animals↗
PubMed1989

Diversity and novel pharmacological properties of Ca2+ channels in Drosophila brain membranes.

Binding studies as well as affinity labelling and immunoblot techniques were used to identify and characterize the receptors for Ca2+ channel blockers in Drosophila brain membranes. Despite structural analogies with mammalian receptors, Drosophila binding sites for phenylalkylamines and 1,4-dihydropyridines, unlike those described in skeletal and cardiac muscle, were found to be located on separate Ca2+ channels. Single-channel bilayer recordings from reconstituted membranes revealed the presence of eight distinct cobalt-sensitive Ba2+-conducting channels in Drosophila brain membrane preparations. In good agreement with binding studies, the most frequently observed Ca2+ channel type (Ba2+ conductance of 13 pS) was extremely sensitive to phenylalkylamines but not affected by micromolar concentrations of 1,4-dihydropyridines. Distinct 1,4-dihydropyridine-sensitive and phenylalkylamine-insensitive channels were also identified. They had unitary Ba2+ conductances of 21 and 31 pS. A detailed analysis of drug action showed that both 1,4-dihydropyridines and phenylalkylamines first increased channel open state probability before fully blocking channel activity. Other types of channels have been identified with unitary Ba2+ conductances of 9, 41, 53, 64 and 81 pS. They were insensitive to the previously described organic Ca2+ channel blockers. The Drosophila system seems to be a unique model to analyse the properties of several different types of Ca2+ channels and particularly those of channel types that are uniquely blocked by phenylalkylamines or uniquely blocked by 1,4-dihydropyridines.

Animals↗
PubMed1989

Calcium currents in single SA nodal cells of the rabbit heart studied with action potential clamp.

With a new method called "Action Potential-Clamp" (APC) we studied in single SA nodal cells the contribution of both transient and long lasting calcium currents (L-type and T-type) to the action potential. Action potentials were recorded by a computer and a representative cycle was subsequently repetitively replayed to the same cell under voltage clamp. Blockade of the L-type calcium current (D600) or T-type calcium current (nickel) revealed the quantitative and time related contributions of these currents to the action potential, since the blocked current is compensated by the clamp amplifier.

Action Potentials↗
PubMed1989

Calcium channels reconstituted from the skeletal muscle dihydropyridine receptor protein complex and its alpha 1 peptide subunit in lipid bilayers.

In the first part of this study, we show that sDHPR and pDHPR preparations reconstituted into lipid bilayers formed on the tips of patch pipettes exhibit two divalent cation-selective conductance levels of 9 and 20 pS, similar in single-channel conductance to VSCC reported in a variety of intact preparations (see Pelzer et al. and Tsien et al. for review). The larger conductance level is similar to the VSCC identified in intact rat t-tubule membranes and described in sDHPR and pDHPR preparations, and shares many properties in common with activity from L-type VSCC. It is sensitive to augmentation by the DHP agonist (+/-)-BAY K 8644 and cAMP-dependent phosphorylation, and to block by the phenylalkylamine (+/-)-D600 and the inorganic blocker CoCl2. Its open-state probability and open times are increased upon depolarization as expected for a voltage-dependent activation process. Upon depolarization beyond the reversal potential, however, open-state probability and open times decline again. A reasonable way to explain the bell-shaped dependence of open times and open-state probability on membrane potential is to assume voltage-dependent ion-pore interactions that produce closing of the channel at strong negative and positive membrane potentials. By contrast, the smaller conductance level may be similar to the 10.6-pS t-tubule VSCC described by Rosenberg et al. and may best be compared with T-type VSCC. It is largely resistant to augmentation by (+/-)-BAY K 8644 and cAMP-dependent phosphorylation or block by (+/-)-D600, but is sensitive to block by CoCl2. Its open times and open-state probability show a sole dependence on membrane potential where depolarization increases both parameters sigmoidally from close to zero up to a saturating level. Both elementary conductance levels do not exhibit significant inactivation over a wide potential range, which may suggest that skeletal muscle VSCC inactivation is either poorly or not voltage-dependent at all. This possibility seems in agreement with bilayer recordings on reconstituted intact t-tubule membranes and voltage-clamp recordings on intact fibers. It supports the idea that the decline of Ca2+ current in intact skeletal muscle fibers may be due to Ca2+ depletion from the t-tubule system and/or to inactivation induced by Ca2+ release from the sarcoplasmic reticulum. We consistently observe two conductance levels of 9 and 20 pS, either singly, or together in the same bilayer from solubilized DHPR samples and even highly purified DHPR preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Dual action (stimulation, inhibition) of D600 on contractility and calcium channels in guinea-pig and cat heart cells.

1. We examined the effects of D600 (0.2-40 microM, generally 2 microM) on the following (i) developed tension in guinea-pig papillary muscles, (ii) calcium current (Ica) and tension in cat ventricular muscle strands, (iii) Ica in guinea-pig and cat ventricular myocytes, (iv) single Ca2+ channel currents carried by Ba2+ in cell-attached membrane patches of guinea-pig ventricular myocytes, and (v) Ba2+ currents through dihydropyridine (DHP)-binding sites (skeletal muscle) reconstituted into single functional Ca2+ channels in lipid bilayers. 2. In 27 of 140 preparations studied, D600 elicited a transient stimulation that preceded marked inhibition. The stimulation was normally of short duration (less than 5 min) and moderate strength (less than 50% increase). 3. D600 had no effect on the unit conductance of single cardiac Ca2+ channels. Stimulation was characterized by a decrease in the number of records with no openings (blanks) and an increase in the open-state probability of non-blanks (longer open times, shorter closed times). Inhibition began with an increase in the number of blanks and later included a curtailment of open times and a prolongation of closed times. The net effect after 9 min D600 was a 75% reduction in average current amplitude. 4. A similar pattern of changes in channel open and closed times produced enhancement and then depression of time-averaged open-state probability in single reconstituted channels. 5. Single Ca2+ channel current that was stimulated by adrenaline was only slightly depressed after 2 microM-D600 for 30 min. It may be that channel phosphorylation or Gs-protein activation following beta-receptor stimulation reduces channel affinity for D600. 6. Short-lived binding of D600 to a single inhibitory site may enhance association/activation of Gs-protein and thereby cause transient up-regulation prior to increased drug occupancy and inhibition. Alternatively, there may be separate stimulatory and inhibitory sites. One aspect of inhibition, the increased frequency of blanks, is attributed to a stabilization of the inactivated state; the other aspect, changes in fast kinetics, seems to require a different explanation.

Animals↗