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J Hescheler

Publications and source records attributed to J Hescheler.

At least 163 records · Page 9Linked to original sources
PubMed1988

Effects of a protein phosphatase inhibitor, okadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes.

The effects of a protein phosphatase inhibitor, okadaic acid (OA), were studied on membrane currents of isolated myocytes from guinea-pig cardiac ventricle. The whole-cell Ca2+ current (ICa) was recorded as peak inward current in response to test pulse to 0 mV. Extracellular application of OA (5-100 microM) produced an increase of ICa. The effect was markedly enhanced when the myocyte was pretreated with threshold concentrations of isoprenaline. ICa was increased from 11.3 +/- 0.8 microA cm-2 to 19.0 +/- 1.1 microA cm-2 (n = 4) by 5 microM-OA in the presence of 1 nM-isoprenaline. The delayed rectifier current was also slightly increased. Furthermore, the wash-out time of the beta-adrenergic increase of ICa was markedly prolonged by OA. The beta-adrenergic stimulation of cardiac Ca2+ current is thought to be mediated by cAMP-dependent phosphorylation. The present results strongly suggest that the effect of OA on ICa is related to inhibition of endogenous protein phosphatase activity which is responsible for the dephosphorylation process. By the isotope method, the inhibitory effect of OA on different types of phosphatase was compared. OA had a relatively high specificity to type 1-, and type 2A-phosphatases.

Animals↗
PubMed1988

A possible physiological role of the Ca-dependent protease calpain and its inhibitor calpastatin on the Ca current in guinea pig myocytes.

The decrease ('run-down') of the L-type Ca2+-current during long-lasting recordings with the whole-cell patch-clamp technique was examined in guinea pig ventricular myocytes. We have tested whether proteolysis is involved in the decay of the Ca2+-current by intracellular application of several concentrations of the Ca2+-dependent proteases calpain I and II, as well as their endogenous inhibitor calpastatin. The major finding was that calpain I and calpain II accelerated the Ca2+-current run-down in a concentration dependent manner, whereas calpastatin retarded it. These observations indicate that a proteolytic degradation of Ca2+-channels might be the reason for the run-down phenomenon.

Animals↗
PubMed1988

"Run-down" of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium.

We examined by a statistical approach the decrease of the Ca current ("run-down") during long-lasting recordings with the whole-cell patch-clamp technique in guinea pig ventricular myocytes. The results are as follows. (1) Run-down of the Ca current (ICa) occurs in three phases (T1-T3). T1 (38 +/- 19 min, n = 135) and T3 (35 +/- 17 min, n = 23) are characterized by a slow rate of decay of ICa [90 +/- 20 and 60 +/- 20 nA.cm-2.min-1, respectively]. T1 and T3 are separated by T2 (6 +/- 4 min, n = 135) during which the current decays quickly [1200 +/- 230 nA.cm-2.min-1]. Between the onsets of T1 and T3, ICa decreases from 11 +/- 3 to 3.5 +/- 1 microA/cm2. (2) Normalized current-voltage relationship, reversal potential and voltage-dependencies of steady-state activation and inactivation of ICa are globally shifted toward more negative potentials during the run-down process by 10-15 mV. (3) ICa3 measured during T3 retains the pharmacological properties (blockade by D600, NiCl2 and CoCl3, increase by isoprenaline and insensitivity to tetrodotoxin) of the original ICa. (4) Intracellular perfusion of the nonhydrolysable ATP analogue AMP-PNP does not prevent the occurrence of T2, suggesting that a phosphorylation-dephosphorylation process is not involved in the fast run-down of ICa. (5) With 0.1 mM EGTA in the pipette, addition of 3 mM ATP significantly prolongs ICa survival. No improvements are obtained by increasing the ATP concentration to 10 mM or replacing ATP with creatine phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Control of voltage-dependent Ca2+ channels by G protein-coupled receptors.

G proteins act as transducers between membrane receptors activated by extracellular signals and enzymatic effectors controlling the concentration of cytosolic signal molecules such as cAMP, cGMP, inositol phosphates and Ca2+. In some instances, the receptor/G protein-induced changes in the concentration of cytosolic signal molecules correlate with activity changes of voltage-dependent Ca2+ channels. Ca2+ channel modulation, in these cases, requires the participation of protein kinases whose activity is stimulated by cytosolic signal molecules. The respective protein kinases phosphorylate Ca2+ channel-forming proteins or unknown regulatory components. More recent findings suggest another membrane-confined mechanism that does not involve cytosolic signal molecules but rather a more direct control of voltage-dependent Ca2+ channels by G proteins. Modulation of Ca2+ channel activity that follows this apparently membrane-confined mechanism has been described to occur in neuronal, cardiac, and endocrine cells. The G protein involved in the hormonal stimulation of Ca2+ channels in endocrine cells may belong to the family of Gi-type G proteins, which are functionally uncoupled from activating receptors by pertussis toxin. The G protein Gs, which is activated by cholera toxin, may stimulate cardiac Ca2+ channels without the involvement of a cAMP-dependent intermediate step. Hormonal inhibition of Ca2+ channels in neuronal and endocrine cells is mediated by a pertussis toxin-sensitive G protein, possibly Go. Whether G proteins act by binding directly to Ca2+ channels or through interaction with as yet undetermined regulatory components of the plasma membrane remains to be clarified.

Animals↗
PubMed1988

Adrenoceptor-mediated changes of excitation and contraction in ventricular heart muscle from guinea-pigs and rabbits.

1. The influence of alpha-adrenoceptor stimulation on mechanical and electrophysiological parameters was investigated in ventricular preparations from guinea-pigs and rabbits. Action potential and force of contraction were measured in papillary muscles and ionic currents were measured in isolated myocytes. 2. The effects of alpha-adrenoceptor stimulation were compared with those of beta-adrenoceptor stimulation. 3. In the guinea-pig the stimulation of alpha-adrenoceptors caused a small increase in the force of contraction (less than 10% of the response to beta-adrenoceptor stimulation) which was not accompanied by any increase of the slow calcium inward current. beta-Adrenoceptor stimulation produced large increases in both force of contraction and slow inward calcium current. The noradrenaline-induced increase in the slow inward calcium current was not affected by phentolamine. 4. In the rabbit, alpha-adrenoceptor stimulation produced large increases in the force of contraction (about two thirds of those seen in response to beta-adrenoceptor stimulation). Whereas beta-adrenoceptor stimulation also produced large increases in both maximal upstroke velocity of slow-response action potentials and slow inward calcium current, there was almost no change of both parameters in response to alpha-adrenoceptor stimulation. 5. We conclude that, first, the contribution of alpha-adrenoceptors to adrenoceptor-mediated changes of force of contraction is minimal in the guinea-pig ventricle, and second, the pronounced changes of force of contraction in the rabbit ventricle in response to alpha-adrenoceptor stimulation are unrelated to changes in the slow inward calcium current.

Action Potentials↗
PubMed1988

Modification of L-type calcium current by intracellularly applied trypsin in guinea-pig ventricular myocytes.

1. The L-type Ca2+ current was recorded in guinea-pig ventricular myocytes by the patch clamp technique in the whole-cell configuration. The modification of the current by intracellular application of proteases was studied. 2. During the first phase of action, trypsin, an endopeptidase, increased the amplitude of Ca2+ current about 3-fold. 3. Thereafter, there was a drastic slowing of the inactivation time course of the enhanced Ca2+ current. The half-time of inactivation increased from a control value of about 25 ms to values larger than 200 ms. 4. Cell dialysis with carboxypeptidase A, an exopeptidase, also enlarged the amplitude of Ca2+ current, but did not affect the kinetics of Ca2+ current. Leuaminopeptidase did not modify the Ca2+ current. 5. The hypothesis that Ca2+ channels are affected by the protease is supported by the fact that alterations of the extracellular Na+ or K+ concentration did not influence the modification of the membrane current. Another argument for the involvement of Ca2+ channels is that the modified membrane current could be blocked by inorganic and organic Ca2+ channel blockers (e.g. 10 microM-Cd2+, 100 microM-La3+ or 1 microM-D600). 6. Although the actions of trypsin and maximal concentrations of isoprenaline on the amplitude of the Ca2+ current were not additive, the slowing of inactivation by trypsin occurred independently from beta-adrenergic stimulation. 7. The effect of trypsin on the Ca2+ current could not be blocked by intracellular 5'-adenylyl-imidodiphosphate (AMP-PNP) or Rp-adenosine 3'5'-monothionophosphate (Rp-cAMPS), both of which are known to suppress the cyclic AMP-dependent phosphorylation of the Ca2+ channel. 8. It was concluded that trypsin may directly modify the membrane protein which forms the Ca2+ channel. Since the increment in peak Ca2+ current resembled the action of cyclic AMP-dependent phosphorylation, it may be related to the removal of a 'chemical' inactivation gate which is normally controlled by phosphorylation. The slowing of the time course of Ca2+ current inactivation by trypsin could be due to a modification of the voltage-dependent inactivation gate. Alternatively, the endopeptidase might remove an internal Ca2+ binding site normally responsible for Ca2+-dependent inactivation.

Adenylyl Imidodiphosphate↗
PubMed1987

Regulation of the cardiac calcium channel by protein phosphatases.

The calcium current (ICa) through the L-type channel in cardiac ventricular cells is enhanced by phosphorylation of a channel protein [Kameyama, M., Hofmann, F. & Trautwein, W. (1985) Pflügers Arch. Eur. J. Physiol. 405, 285-293]. We investigated the possible contribution of the 'catalytic subunits' of protein phosphatase 1 and 2A in the down-regulation of the cardiac calcium channel. Single guinea-pig ventricular myocytes were voltage clamped and the following results were obtained. (1) Intracellular perfusion of the myocyte with the catalytic subunits of protein phosphatase 1 (2 microM) as well as 2A (2.3 microM) completely abolished the increase of ICa induced by isoprenaline (0.05 microM) but did not decrease the basal level of ICa. Alkaline and acid phosphatases were without detectable effect. (2) Cell dialysis with the modulator of protein phosphatase 1 (inhibitor-2) under control conditions (without addition of isoprenaline) caused a slow significant increase of ICa. (3) The time course for the wash-out of the isoprenaline effect was considerably prolonged in the presence of high concentrations of inhibitor-2. (4) Perfusion of the myocyte under basal conditions with adenosine 5'-[gamma-thio]triphosphate led to a slow increase of ICa. Additional superfusion of the cell with a threshold concentration of isoprenaline (0.01 microM) resulted in a rapid increase of ICa which could not be washed out during at least 10 min. From these results we make the following conclusions. (1) The calcium channel from guinea-pig myocytes is regulated by phosphorylation-dephosphorylation. (2) The catalytic subunits of the protein phosphatases 1 as well as 2A, purified from rabbit skeletal muscle, catalyse the down-regulation of the channel. (3) Indirect evidence suggests that endogenous protein phosphatase 1 contributes only partially to the dephosphorylation of the calcium channel in the intact myocyte.

Animals↗
PubMed1987

On the mechanism of histamine induced enhancement of the cardiac Ca2+ current.

In guinea pig ventricular myocytes, the effect of histamine on the slow Ca2+ current (ICa) was studied and the following results were obtained: (1) Superfusion of cells with histamine resulted in a dose-dependent enhancement of the amplitude of ICa. The threshold concentration of histamine was 10(-8) M, half maximal increase occurred at 3 X 10(-7) M and maximal enhancement (about 3-4-fold) at 5 X 10(-6) M. (2) The histamine effect was greatly reduced by the H2 antagonist cimetidine (10(-5) M) but only slightly by the H1 antagonist diphenhydramine (10(-5) M). (3) Effects of isoprenaline (ISP) and histamine at maximal effective concentrations on ICa were not additive, suggesting that both agents use the same intracellular pathway. Intracellular infusion of a blocker of the cAMP-dependent protein kinase, Rp-cAMPS (10(-4) M), prevented the histamine effect. (4) The involvement of GTP-dependent transducer proteins was studied by cell dialysis with several GTP derivatives. Intracellular application of the stable GDP-analogue, GDP-beta-S, reduced the histamine effect on ICa, whereas the stable GTP analogue, GTP-gamma-S, mimicked the histamine effect.

Animals↗
PubMed1987

Changes of membrane currents in cardiac cells induced by long whole-cell recordings and tolbutamide.

Single isolated myocytes were obtained from the ventricles of adult guinea pig hearts. The whole-cell recording configuration of the patch-clamp technique was used to measure membrane currents. A decrease (run-down) of the Ca2+ inward current and an increase of a time-independent K+ outward current were observed during long lasting (1-3h) recordings. The time at which the outward current developed depended on the intracellular ATP concentration in the pipette, suggesting that this current is identical to the ATP-dependent K+ current described by Noma and Shibasaki (1985). However, the maximum outward current reached in the experiments was independent of the ATP concentration indicating a limited diffusion of ATP in the cell interior. In single-channel experiments on isolated patches of cell membrane and in whole-cell recordings the ATP-dependent K+ current could be blocked by the hypoglycaemic sulphonylurea tolbutamide. The IC50 of 0.38 mM was about 50 times higher than that reported for pancreatic beta-cells (Trube et al. 1986). The Ca2+ inward current and the inwardly rectifying K+ current were not affected by tolbutamide (3 mM).

Action Potentials↗
PubMed1987

ATP-dependent potassium channels in the cardiac cell.

Patch-clamp techniques were used to study the ATP-dependent K+ channel in single ventricular myocytes from guinea pig hearts. During long lasting whole-cell recordings an increase of a time-independent K+ outward current could be observed. This outward current could be inhibited by the intracellular application of ATP (3-20 mM), but not by the Ca2+-chelator EGTA (20 mM). The blocking effect of ATP was also observed in single-channel experiments. The hypoglycaemic sulphonylurea tolbutamide specifically inhibited the ATP-dependent K+ current in whole-cell recordings, inside-out and outside-out patches (IC50 = 0.4 mM).

Action Potentials↗
PubMed1986

Modulation of Ca current during the phosphorylation cycle in the guinea pig heart.

The calcium current (ICa) in the heart is increased by phosphorylation of a protein which is part of, or close to, the Ca channel. The phosphorylation is catalysed by cAMP-dependent protein kinase (cAMP-PK). The question whether dephosphorylated channels are available to open on depolarization was examined in ventricular myocytes of guinea pig by recording whole cell ICa during dialysis with either regulatory (R) subunit of cAMP-PK or protein kinase inhibitor (PKI) or adenosine-5'-(gamma-thio)-triphosphate (ATP gamma S). The following results were obtained: 1) R subunit reduced and PKI reversed the isoprenaline (ISP)-induced enhancement of ICa, suggesting their ability to inhibit cAMP-PK. 2) R subunit and PKI, however, reduced basal (i.e. non beta-adrenergically stimulated) ICa only by about 20%. 3) Dialysis with ATP gamma S resulted in a slow increase in basal ICa, presumably due to dephosphorylation-resistant thiophosphorylation. 4) When, however, the cell was dialyzed with PKI the effect of ATP gamma S was almost completely suppressed, suggesting no detectable phosphorylation related to the channel activity in this condition. These results support the view that even in the dephosphorylated state Ca channels are available to open on depolarization and that phosphorylation by cAMP-PK increases the opening probability.

Adenosine Triphosphate↗
PubMed1986

On the mechanism of muscarinic inhibition of the cardiac Ca current.

The mechanism of muscarinic inhibition of the Ca-current (ICa) was studied in ventricular myocytes of guinea pig hearts and the following results were obtained. Acetylcholine (ACh) in concentrations up to 10(-4) M had little effect, if any, on ICa in control cells. ACh reduced the isoprenaline (ISP)-induced increase of ICa. The dose-response-relation (ISP concentration vs. ICa density) was shifted by ACh towards higher ISP concentrations. But both, at low and high ISP concentrations ACh had nor or little effect. ACh was ineffective when ICa was increased by dialysing the cell with catalytic subunit of cAMP-dependent protein kinase or cAMP. ACh reduced ICa enhanced by isobutylmethylxanthine or by forskolin. ACh did not depress ICa when the cell was dialysed with the non-hydrolysable GTP-derivative, GMP-PNP. In this condition the beta-adrenergic enhancement of ICa was also absent. Pertussis toxin, which is known to inhibit the inhibitory transducer protein (Ni), abolished the ACh response. We concluded from these results that ACh depresses ICa by inhibiting, via Ni, the cAMP production.

1-Methyl-3-isobutylxanthine↗
PubMed1986

The protein-specific phosphatase 1 antagonizes the beta-adrenergic increase of the cardiac Ca current.

In isolated ventricular cells from the adult guinea pig heart the slow Ca current was recorded during beta-adrenergic stimulation and during cell dialysis with a protein-specific phosphatase-1 (PPase-1). The increase in the amplitude of ICa during bath application of isoprenaline (5 X 10(-8) M) could be completely reversed by dialysing the cell with 2 microM PPase-1. Lower enzyme concentrations produced smaller effects. The control amplitude of ICa was only little affected by dialysis with PPase-1. The result suggests that PPase-1 is a likely candidate for the downregulation of Ca channels, that Ca channels can open in the dephosphorylated state.

Animals↗
PubMed1984

Inward-rectifying channels in isolated patches of the heart cell membrane: ATP-dependence and comparison with cell-attached patches.

Inward rectifying potassium single-channel currents were studied in the membrane of guinea pig cardiac myocytes. In isolated inside-out patches two different channels were observed: a channel of 25 pS conductance ([K+]o = 147 mM, T = 21 degrees C), if the solution at the cytoplasmic face of the patch contained 4 mM ATP and a channel of 80 pS conductance without ATP. The 25-pS-channel was also regularly seen in cell-attached patches (Sakmann and Trube 1984a,b), but the 80-pS-channel appeared only after inhibiting cellular metabolism by DNP. The percentage of time which the 25-pS-channel spent in the open state was 3.3 times larger in isolated patches compared to cell-attached patches. However, both types of single channel currents disappeared several minutes after the isolation of the patches. In contrast to the 25-pS-channel, the 80-pS-channel, which is activated by the lack of ATP, carried measurable outward currents saturating at 1.5 pA (inward rectification). It is suggested that the 80-pS-channel mediates part of the increase in potassium current during metabolic inhibition. The openings of this channel appeared in bursts. The mean open time was 1.6 ms and the mean duration of the gaps within bursts 0.33 ms at -80 mV.

Adenosine Triphosphate↗
PubMed1982

Injection of subunits of cyclic AMP-dependent protein kinase into cardiac myocytes modulates Ca2+ current.

beta-Adrenergic stimulation of the heart is thought to increase cardiac muscle contractility by activation of cyclic AMP-dependent protein kinase and concomitant increase in the phosphorylation of certain proteins (for refs see refs 1-6). Electrophysiological studies have shown that the stimulation of cardiac beta-adrenoreceptors, the external application of cyclic AMP or its analogues to Purkinje fibres, or the injection of cyclic AMP into single myocytes can increase the slow inward current (Isi) during the plateau phase of the action potential (AP). In heart muscle this current is mainly carried by Ca2+ (refs 10, 11) and it has been suggested that cyclic AMP-dependent phosphorylation of some component of the calcium channel increases the amount of Ca2+ which enters the cell during depolarization. We have investigated this hypothesis by examining the electrical responses of isolated guinea pig ventricular myocytes to pressure injections of subunits of the cyclic AMP-dependent protein kinase. We report here that injection of the catalytic subunit (C) resulted in a lengthening of the action potential duration (APD) and an increase in the height of the plateau as well as the amplitude of Isi. By contrast, the injection of regulatory subunit (R) shortened the APD of fast and slow response APs, an effect which was reversed by adrenaline.

Action Potentials↗
PubMed1982

Does the organic calcium channel blocker D600 act from inside or outside on the cardiac cell membrane?

The effects of extra- and intracellularly applied D600 (methoxyverapamil) and D890 (a quarternary derivative) on the action potentials of isolated guinea pig myocytes were compared. We also studied the extracellular myocytes were compared. We also studied the extracellular effects of these drugs on the calcium current (hybride sucrose gap) and contractile force of right ventricular trabeculae of the cat heart. The following results were obtained: 1. In ventricular trabeculae D600 suppressed the calcium current, tension and the plateau of the action potential. In contrast, D890 even in a 50 times higher concentration did not display any effect on these parameters. 2. In single isolated cells external application of D890 did not alter the configuration of the action potential. In contrast, external application of D600 suppressed the plateau and shortened the action potential in a dose-dependent way. 3. Intracellular injection of D600 or D890 strongly lowered the height of the plateau and abbreviated the action potential. The onset of the effects of both drugs was more rapid on intracellular application than that of external D600. Whereas the effect of an intracellular injection of D600 was reversible, that of D890 was not. These results support the hypothesis that the organic calcium channel blocker D600 enters the cell in the uncharged lipid soluble form and reaches its receptor associated with the calcium channel from inside. Because of its inability to pass the hydrophobic cell membrane, D890 is ineffective from outside but displays blocking effects on intracellular application.

Action Potentials↗