W. Trautwein

On the mechanism of β-adrenergic regulation of the Ca channel in the guinea-pig heart

Dose-response relations for the increase in the amplitude of Ca current (ICa) on external application of isoprenaline (ISP) and internally applied cyclic AMP (cAMP) or catalytic subunit of cAMP-dependent protein kinase (C subunit) were established in single ventricular cells of the guinea pig. An intracellular dialysis technique was used. The threshold concentration was for ISP 10−9 M, for cAMP 3 μM (pipette concentration to which 10−5 M 3-isobutyl-1-methylxanthine was added) and for C subunit around 0.4 μM (pipette concentration). The concentrations for the half-maximal effect were 3.7×10−8 M (ISP), 5.0 μM (cAMP) and 0.95 μM (C subunit) and for the maximum effect 10−6 M (ISP), 15–20 μM (cAMP) and 3–4 μM (C subunit). For all three agents the maximum increase in the Ca current density was similar (a factor of 3–4), suggesting that they converge on the same site of the Ca channel. Accordingly, the effects of cAMP and C subunit onICa were non-additive to those of ISP. From these data the relationship both between concentrations of ISP and cAMP and between those of cAMP and active C subunit in terms of their effects onICa could be estimated and were compared with those obtained in broken cell preparations.A competitive inhibitor of phosphorylation, 5′-adenylyl-imidodiphosphate (5 mM), greatly reduced the effects of ISP and C subunit onICa. Cell dialysis with 3 mM adenosine-5′-(γ-thio)-triphosphate, which produces a dephosphorylationresistant phosphorylation, markedly potentiated the effects of ISP and cAMP onICa.The results support the hypothesis that phosphorylation of a protein within, or close to, the Ca channel by cAMP-dependent protein kinase is the mechanism of β-adrenergic stimulation.

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. 1. Acetylcholine (ACh) in concentrations up to 10−4 M had little effect, if any, onICa in control cells. 2. ACh reduced the isoprenaline (ISP)-induced increase ofICa. The doseresponse-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. 3. ACh was ineffective whenICa was increased by dialysing the cell with catalytic subunit of cAMP-dependent protein kinase or cAMP. 4. ACh reducedICa enhanced by isobutylmethylxanthine or by forskolin. 5. ACh did not depressICa when the cell was dialysed with the nonhydrolysable GTP-derivative, GMP-PNP. In this condition the β-adrenergic enhancement ofICa was also absent. 6. Pertussis toxin, which is known to inhibit the inhibitory transducerprotein (Ni), abolished the ACh response.We concluded from these results that ACh depressesICa by inhibiting, via Ni, the cAMP production.

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

AbstractThe 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 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 inracellular 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.

“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 ofICa [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 μA/cm2. (2) Normalized current-voltage relationship, reversal potential and voltage-dependencies of steady-state activation and inactivation ofICa 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 originalICa. (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 ofICa. (5) With 0.1 mM EGTA in the pipette, addition of 3 mM ATP significantly prolongsICa survival. No improvements are obtained by increasing the ATP concentration to 10 mM or replacing ATP with creatine phosphate. With 3 mM ATP present, increasing the EGTA concentration to 10–20 mM doublesICa survival time. EGTA alone (10 mM) is less effective than the mixture 3 mM ATP-0.1 mM·EGTA. Intracellular perfusion with a cytoplasmic extract considerably prolongs T2 and the overallICa survival. (6) The results are consistent with the hypothesis that run-down ofICa can partially be explained by a rise in intracellular Ca concentration and a loss of high energy compounds. Beneficial effect of ATP might include an increased capability of the cells to either extrude or sequester intracellular Ca, and a protection against enzymatic proteolysis.

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 cellICa during dialysis with either regulatory (R) subunit of cAMP-PK or protein kinase inhibitor (PKI) or adenosine-5′-(γ-thio)-triphosphate (ATPγS). The following results were obtained: 1) R subunit reduced and PKI reversed the isoprenaline (ISP)-induced enhancement ofICa, suggesting their ability to inhibit cAMP-PK. 2) R subunit and PKI, however, reduced basal (i.e. non β-adrenergically stimulated)ICa only by about 20%. 3) Dialysis with ATPγS resulted in a slow increase in basalICa, presumably due to dephosphorylation-resistant thiophosphorylation. 4) When, however, the cell was dialyzed with PKI the effect of ATPγS 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.

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 O mV. Extracellular application of OA (5–100μM) produced an increase ofICa. The effect was markedly enhanced when the myocyte was pretreated with threshold concentrations of isoprenaline.ICa was increased from 11.3±0.8μA cm−2 to 19.0±1.1μA cm−2 (n=4) by 5μM-OA in the presence of 1nM-isoprenaline. The delayed rectifier current was also slightly increased. Furthermore, the wash-out time of the β-adrenergic increase ofICa was markedly prolonged by OA. The β-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 onICa 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.

The effect of the duration of the action potential on contraction in the mammalian heart muscle

ZusammenfassungEs wird eine Methode beschrieben, die es ermöglicht, das Membranpotential der Fasern eines kleinen Trabekels des Arbeitsmyokards des rechten Herzens von Katzen-, Hunden- und Schafsherzen zu klemmen und die mechanische Spannung des geklemmten Muskelareals isometrisch zu messen. In einer Reihe von Versuchen wird der Verlauf des Stromes im Muskeltrabekel geprüft und sichergestellt, daß die Spannungsklemme das Membranpotential aller Fasern des Präparates gleichmäßig ändert. Mit dieser Anordnung wird das Aktionspotential auf beliebige Werte verkürzt und die Wirkung der Verkürzung auf die isometrische Spannung mit folgendem Ergebnis untersucht:1.Die isometrische Kontraktion wird schwächer, wenn das Aktionspotential, das normalerweise 500 ms (bei 25° C) lang dauert, auf 200 ms (gleich der Dauer bis zum isometrischen Maximum) und weniger verkürzt wird. Dabei lösen die ersten 50 ms des Aktionspotentials 2/3 bis 3/4 der isometrischen Spannung aus. Verkürzung des Aktionspotentials nach 150 ms beeinflußt den weiteren Verlauf der Kontraktion nicht mehr.2.Bei Erhöhung der extracellulären Ca-Konzentration löst der Beginn des Aktionspotentials (1–50 ms) absolut und relativ mehr Spannung aus.3.„Aktionspotentiale“ von 1 ms Dauer oder weniger führen nicht zur Entwicklung mechanischer Spannung.4.Die Änderung der mechanischen Spannung erfolgt gegenüber der Änderung des Membranpotentials mit Verzögerung. Das trifft sowohl für den Einsatz der Entwicklung der mechanischen Spannung bei kurzen „Aktionspotentialen“ (1–20 ms) als auch auf das Ende der Spannungsentwicklung bei späteren Verkürzungen des Aktionspotentials zu (nach 20–100 ms).5.Es besteht eine S-förmige Beziehung zwischen der Dauer vom Beginn bis zum Maximum der isometrischen Kontraktion und der Dauer des verkürzten Aktionspotentials.6.Verlängerung des „Aktionspotentials“ mittels Spannungsklemme verändert die Kontraktion nur dann, wenn das Membranpotential auf positiveren Werten als − 25 mV gehalten wird. In diesem Falle ist die Erschlaffung des Muskels nicht vollständig. Bei anhaltender Membrandepolarisation auf positive Werte hält die mechanische Spannung an. Sie kann bei entsprechender Depolarisation (ca. +50 mV) größer werden als die isometrische Kontraktion. Die Beziehung zwischen der mechanischen Spannung und dem Membranpotential bei anhaltender Depolarisation wird dargestellt (Fig. 10).7.In Summationsexperimenten (verkürztes Aktionspotential, unmittelbar gefolgt von einem normalen Aktionspotential) konnte keine größere Spannung als das isometrische Maximum erzielt werden. Die Beziehung zwischen Membranpotential und mechanischer Spannung wird diskutiert.SummaryA method is described to control the membrane potential in preparations of mammalian myocardium, and to measure tension simultaneously. The following observations are made:1.The potential in the segment of the preparation bathing in Tyrodes solution (1 mm or less), is uniformally clamped, i.e. over its whole length and cross sectional area.2.Shortening of the action potential by “abolition” can only alter the time course and amplitude of contraction within the first 200 ms of the action potential (duration ca 500 ms at 25° C).3.Tension develops rather rapidly within the initial 50 ms of depolarization, thereafter the increment of tension accompanying longer depolarizations progressively decreases. Increasing the extracellular concentration of calcium increases the relative tension development within the early periods.4.Short depolarization of 1 ms or less did not produce detectable tension.5.An S-shaped relationship was found to exist between the duration of the action potential and the duration from the beginning to the maximum of contraction.6.Prolongation of the action potential tends to maintain tension if the membrane is depolarized to a value above threshold for the maintenance of tension (ca. −25 mV).7.Tensions larger than the isometric contraction are obtained when the membrane potential is brought to and maintained at positive potentials (+30 mV or more).8.Summation of the mechanical action of shortened action potentials and of anode break excitations never yielded more tension than a single normal action potential. A discussion of the relationship between the action potential and contraction in cardiac muscle is included.

Ionic currents in cardiac excitation

ZusammenfassungAn dünnen Purkinje-Fäden von 1–2 mm Länge werden Membranströme während Spannungsklemmen bis zu 1000 msec Dauer gemessen. Die Kaliumströme werden als Nettoströme in natriumfreier Lösung und die Natriumströme durch Subtraktion der Kaliumströme vom in Tyrode gemessenen Strom bestimmt.Die Kaliumströme hängen vom Membranpotential und der Zeit ab. Die Stromspannungsbeziehung 300 msec nach dem Beginn der Klemme entspricht derjenigen, die durch Messung des Elektrotonus in Cholin-Tyrode bereits bekannt ist. Im Bereich von −40 mV bis +30 mV ist der Kaliumstrom zu Beginn der Klemme größer und fällt in etwa 100 msec auf einen nahezu konstanten Wert ab. Nur im positiven Bereich des Membranpotentials (+20 bis +40 mV) wird gelegentlich eine kleine späte Zunahme des Kaliumstroms beobachtet.Die Kaliumleitfähigkeit nimmt bei Depolarisation vom Ruhepotential bis zu −60 mV auf etwa die Hälfte ab; die Abnahme ist unabhängig von der Dauer der Klemme. Bei weiterer Depolarisation wird die Abnahme zunächst noch größer, zwischen −40 mV und +20 mV nimmt die Leitfähigkeit wieder bis nahe auf den Ruhewert zu. Zwischen −50 und +20 mV ist die Leitfähigkeit zu Beginn der Depolarisation größer als im weiteren Verlauf der Klemme.Die Natriumströme nehmen bei Depolarisation bis zu einem Maximum bei ca. −20 mV zu, fallen dann wieder ab und werden Null bei +30 bis +45 mV. Die Natriumströme nehmen während einer 300 msec dauernden depolarisierenden Klemme fortlaufend ab. Die Natriumleitfähigkeit nimmt bei Depolarisation zu und fällt während der ganzen Dauer der Klemme ab.Die Ruhenatriumleitfähigkeit beträgt 0,024 ± 0,006 mmho/cm2, die Ruhekaliumleitfähigkeit 0,45 ± 0,1 mmho/cm2.Wenn nach einer depolarisierenden Klemme das Membranpotential wieder auf das Ruhepotential geklemmt wird, entwickelt sich mit einer Verzögerung ein positiver Strom, der 60 msec nach der Depolarisation sein Maximum hat und im Verlauf von 250–500 msec abklingt. Die Richtung dieses Stromes ändert sich bei −100 mV. Es wird angenommen, daß diesem Strom eine Zunahme der Kaliumleitfähigkeit zugrunde liegt, die durch Repolarisation des Membranpotentials auf mindestens −40 mV ausgelöst wird. Die Beziehungen der Ergebnisse zum Plateau und zur Repolarisation des Aktionspotentials sowie zum Schrittmacherpotential werden diskutiert.AbstractIn short Purkinje fibres net membrane currents during voltage-clamps were measured. For technical reasons current measurement was not possible within the first 10 msec after the onset of clamping. Potassium current was determined as the current flowing in sodium free solution (choline-Tyrode). Sodium current was found by subtraction of the potassium currents from the current flowing in Tyrode solution.Potassium current depends on both the membrane potential and the time. The current voltage relationship shows “anomalous rectification”. In the range from −40 mV to +30 mV the current is larger earlier than later during the clamp. Only on depolarisation beyond +20 mV a small increase of gK was sometimes seen after 200 msec when depolarisation was maintained.Potassium conductance falls on depolarization to 1/3 to 1/2 of resting conductance. In the range from −60 mV to +20 mV conductance is larger early than later during a depolarizing clamp.Sodium current increases on depolarization, the maximum being at about −20 mV. With further depolarization sodium current falls and becomes zero between +30 and +45 mV. Sodium current declines during depolarizing clamps with a time constant in the order of 100 msec. Sodium conductance increases with depolarization and falls during the clamp.Resting sodium conductance was found to be 0.024±0.006 mmho/cm2, resting potassium conductance was determined as 0.45±0.1 mmho/cm2.When after a depolarization membrane potential is clamped back to the resting potential positive current flows which declines within 300 to 500 msec. This current reverses its polarity at about −100 mV. It is assumed that the current is due to an increase of potassium conductance which is brought about by repolarization to at least −40 mV. The results are discussed in relation to the plateau and repolarization of the action potential as well as to the pacemaker potential.

Elementary currents through Ca2+ channels in Guinea pig myocytes

Elementary Ca2+ and Ba2+ currents were recorded from cell-attached membrane patches of ventricular myocytes from adult guinea pig hearts using the improved patch-clamp technique (Hamill et al. 1981). High concentrations of Ba2+ or Ca2+ (50 or 90 mM) were used in the pipettes to increase the signal-to-noise ratio. All data were derived from elementary current analyses in patches containing only one channel.1)In response to voltage steps, channel openings occurred singly or in bursts of closely spaced unitary current pulses separated by wider shut intervals. During depolarizations of small amplitude from the resting potential, channel openings occurred almost randomly, whereas during larger depolarizations the events were grouped preferentially at the beginning.2)Channel openings became more probable with increased depolarization; simultaneously, unitary current amplitudes declined in an ohmic manner. Elementary current amplitudes were slightly larger, when 50 mM Ba2+ replaced 50 mM Ca2+ in the pipettes (slope conductances 9 and 10 pS, respectively), but more than doubled, when Ba2+ was increased to 90 mM (slope conductance 18 pS). Clear outward currents through Ca2+ channels were not observed under these conditions.3)Peak amplitudes of reconstructed mean currents doubled when 50 mM Ba2+ replaced 50 mM Ca2+ and were larger still when 90 mM Ba2+ was used in the pipettes. The current-volrage relations of the reconstructed mean currents showed a positive shift along the voltage axis as Ba2+ was increased or substituted equimolarly by Ca2+. Correspondingly, the open state probability-voltage relations (activation curves) showed a parallel shift as Ba2+ was increased, which was less pronounced when Ba2+ was replaced equimolarly by Ca2+.4)Determination of Ca2+ channel inactivation using 90 mM Ba2+ in the pipettes indicated an overlap with channel activation in a limited voltage range, resulting in a steadystate “window” current. Inactivation can occur without divalent cation influx.5)Formation of an inside-out patch resulted in a fast rundown of elementary Ca2+ channel currents.6)Channel openings were often grouped in bursts. The lifetimes of the open state, the bursts, and the closed states were estimated for Ba2+ and Ca2+ as permeating ions. At least two exponentials were needed to fit the histogram of the lifetimes of all closed states. The lifetimes of the individual openings and bursts were mono-exponentially distributed. The kinetics of the Ca+ channel depended on the voltage and the permeating ion. During +30 mV depolarizations, no significant effect of the permeating ion on channel gating could be detected. The significant increase in burst length (tb) during +50 mV depolarizations, however, seemed to be only due to an increase in the lifetime of the open state (to) for Ba2+, whereas for Ca2+,to was only moderately prolonged but simultaneously, the number of openings per burst increased.7)A three-state sequential scheme is peoposed to model the activation pathway of Ca2+ channels. The latency-to-first-event histogram is also consistent with a process in which multiple closed states precede the open state.

Relaxation of the ACh-induced potassium current in the rabbit sinoatrial node cell.

AbstractVoltage clamp experiments were carried out in order to study the mechanism of the ACh action in the rabbit S-A node cell. The following results were obtained:1.The reversal potential of the ACh-induced current behaved like a potassium electrode, confirming that the ACh-operated channels pass potassium ions selectively.2.On depolarizing voltage jumps the ACh-induced current showed an instantaneous peak from which the current decayed to a new steady level (relaxation). On hyperpolarizing voltage jumps the initial step change in current was followed by a gradual increase.3.The time course of the current change on voltage jumps was well fitted by a single exponential and the time constant became longer as the membrane potential was increased.4.The instantaneous I–V curve was linear while in the steady state the curve became flatter at low negative membrane potentials and steeper at high negative membrane potentials. The results suggest that ACh opens a specific potassium channel when the drug is bound to the muscarinic receptor. The opening and closing rate constants for this potassium channel depend on the membrane potential in such a way that on depolarizing voltage jumps the fraction of open channels gradually decreases and on hyperpolarization the fraction increases.