Ulrich Jahnel

Positive inotropic response to 5-HT in human atrial but not in ventricular heart muscle

SummaryThe effects of 5-hydroxytryptamine (5-HT) on force of contraction (FC), action potential (AP) and calcium current (ICa) were studied in human right atrial and left ventricular heart muscle. 5-HT exerted a concentration-dependent increase in FC in multicellular atrial preparations; the EC50 was approximately 3 × 10−7 mol/l. Maximal increases in FC (252±58% of control values; means ± SEM, n=6) were obtained at 5-HT 10−5 mol/l. At this concentration, ICa was increased four- to sevenfold in enzymatically isolated atrial myocytes. In contrast, ventricular preparations did not respond to 5-HT; FC, AP and ICa remained unaffected. In the same preparations, FC was increased by isoprenaline three- to fourfold. These results confirm the observation that 5-HT induces a positive inotropic effect in the human atrium, possibly mediated by activation of the adenylyl cyclase — cyclic AMP system. Our study demonstrates, however, the complete lack of functional 5-HT receptors, with respect to changes in FC, in the human ventricle. Since the positive inotropic effect of 5-HT in the human heart is obviously restricted to the atrium, our findings question the concept of developing 5-HT receptor agonists for the treatment of heart failure.

Different mechanisms of the inhibition of the transient outward current in rat ventricular myocytes

The mechanism of drug-induced inhibition of the transient outward current, Ito, has been investigated in rat ventricular myocytes using the whole cell patch clamp technique. Ito was activated by 300 ms depolarizing voltage clamp steps in 10 mV increments from −50 mV up to +40 mV. At +40 mV, Ito peaked after about 3 ms, and the time course of inactivation was appropriately described by two time constants, τfast = 17 ms and τslow = 203 ms. Verapamil, quinidine sulfate and nifedipine preferentially depressed Ito at the end of the 300 ms depolarizing voltage clamp step; the inactivation of Ito was accelerated by all drugs, whereas peak Ito was less affected. The time course of drug action at +40 mV was calculated by the fractional changes of Ito. Verapamil, quinidine sulfate and nifedipine exerted a block of Ito. increasing during the depolarizing voltage clamp step. The onset of block in response to verapamil, quinidine sulfate and nifedipine (30 μmol/each) was appropriately described by monoexponential functions with time constants τon = 9.3, 1.7 and 1.1 ms, respectively. Relief from block by verapamil, quinidine sulfate and nifedipine at −50 mV was assessed by comparison of the recovery process of peak Ito from inactivation with or without drugs. τoff amounted to 695 ms in the case of quinidine sulfate; verapamil and nifedipine did not significantly affect the recovery process so that the determination of the time course of relief from block was not possible. 4-Aminopyridine preferentially depressed peak Ito in a concentration-dependent manner, whereas Ito at the end of the 300 ms depolarizing voltage step remained unaffected. The block of Ito by 4-aminopyridine (3 mmol/l) decreased during the voltage step from −50 mV to +40 mV. Relief from block was described by τoff = 30.4 ms. The efficacy of 4-aminopyridine was diminished at short and enhanced at long pulse intervals (reverse use-dependence). The time course of 4-aminopyridine-induced block of Ito was described by τon = 1561 ms. Phenylephrine (30 μmol/l),papaverine (30 μmol/I) and tetraethylammonium chloride (5 mmol/l) reduced Ito at the peak and at the end of the 300 ms depolarizing voltage step in a time-independent manner. It is concluded that verapamil, quinidine sulfate and nifedipine bind to the Ito channel in the open state at positive membrane potentials. In contrast, 4-aminopyridine obviously binds to the channel in the closed state at negative membrane potentials. Phenylephrine, papaverine and tetraethylammonium chloride seem to block Ito independent of the channel state.

Differential electrophysiologic and inotropic effects of phenylephrine in atrial and ventricular heart muscle preparations from rats

SummaryStimulation of α1-adrenoceptors evokes a different pattern of inotropic responses in atrial and ventricular heart muscle preparations from rats. The inotropic effects are accompanied by different changes in membrane potential. In an attempt to clarify the question whether or to which extent these events are causally related, the effects of phenylephrine on force of contraction, transmembrane potential, Ca2+ current (ICa) and K+ currents were comparatively studied in either tissue.In atrial preparations, phenylephrine 10 μmol/l caused an increase in force of contraction, a marked prolongation of the action potential duration and a depolarization of the membrane at rest. In the ventricle, however, the addition of phenylephrine 10 μmol/l produced first a decline in force of contraction associated with a hyperpolarization of the membrane and a reduction in the action potential duration. These changes were followed by an increase in force,of contraction and a slight prolongation of the action potential, whereas the resting membrane potential remained increased. The hyperpolarization was eliminated in the presence of ouabain 100 μmol/l.In enzymatically isolated atrial and ventricular myocytes, the whole-cell voltage clamp technique was used to study membrane currents on exposure to phenylephrine. Phenylephrine 30 μmol/l did not affect the magnitude of ICa in either cell type. Transient and steady state K+ outward currents, however, were significantly diminished to a similar extent in atrial and in ventricular myocytes.It is concluded that the positive inotropic effect of α1-adrenoceptor stimulation in the rat atrium is related to an increase in action potential duration and a decrease in resting membrane potential due to a decrease in K+ currents. In the ventricle, phenylephrine additionally activates the Na+/K+ pump thereby hyperpolarizing the membrane. The rapid onset of pump stimulation seems to overwhelm, in the beginning, the phenylephrine-induced decrease in K+ conductance and therefore to evoke a transient negative inotropic effect.It is assumed that phenylephrine can alter the intracellular Ca2+ concentration due to changes in the action potential duration. The way how Ca 2+ enters the cell remains speculative, since direct changes of Ica were not detected. The more complicated changes in membrane potential in the ventricle suggest that also other mechanisms for the positive inotropic response to phenylephrine must be considered.

L-type calcium channel activity in human atrial myocytes as influenced by 5-HT

Summary5-Hydroxytryptamine (10 μmol/l; 5-HT) exerted a positive inotropic effect associated with an increase in the Ca2+ current (ICa) in the human right atrium. For detailed analysis, L-type Ca2+ channel currents were recorded from cell-attached patches using 100 mmol/l Ba2+ as charge carrier. Ca2+ channel activity was identified, first, by burstlike inwardly directed currents and, second, by the appearance of long channel openings promoted by Bay K 8644 (1 μmol/l) upon repetitive depolarizations from − 80 to 0 mV The unitary conductance of the Ca2+ channel amounted to 25.8 pS. During superfusion with 5-HT, ensemble averaged (mean) current was enhanced by about 60%. The increase in mean current was brought about by an increase in the channel availability, defined as the ratio of sweeps containing Ca2+ channel activity to the total number of depolarizations. The open probability of a single Ca2+ channel within a sweep with channel activity, unitary conductance, mean open and mean shut times of the channel, however, remained unaffected during superfusion with 5-HT (n = 10). The 5-HT-induced increase in macroscopic ICa in the human atrium can therefore be explained by an enhanced availability of Ca2+ channels to open upon depolarization. The observed changes in gating properties of the human Ca2+ channel by 5-HT are very similar to those which are known from isoprenaline-induced CAMP-dependent phosphorylation of the Ca2+ channel protein in other tissues.

Modulation of cytosolic free calcium concentration by α1-adrenoceptors in rat atrial cells

SummaryThe effects of α1-adrenoceptor stimulation by phenylephrine (PE) and β-adrenoceptor stimulation by isoprenaline (ISO) on Ca2+ current (ICa) and free intracellular Ca2+ concentration ([Ca2+]i) were studied in isolated atrial myocytes from rat hearts. PE did not significantly affect the magnitude of ICa, whereas large increases of peak ICa were observed in response to ISO. In electrically driven cells, PE evoked a concentration-dependent, gradual increase in diastolic [Ca2+]i and, initially, an increase in the height of peak [Ca2+]i transients. When the diastolic [Ca2+]i was increased to a greater extent, the amplitude of [Ca2+]i transients was decreased. Simultaneous measurements of [Ca2+]i and membrane potential showed that the increase in diastolic [Ca2+]i was associated with a depolarization of the membrane, and the greater amplitude of [Ca2+]i transients with a prolongation of the action potential (AP). The PE-induced increase in diastolic [Ca2+]i was eliminated when the cells were voltage-clamped at the original resting membrane potential (RP); under these conditions, an increase in [Ca2+]i transients was observed in response to PE. ISO usually caused larger increases in the amplitude of [Ca2+]i transients with only minor changes in diastolic [Ca2+]i. These results suggest that PE and ISO increase the amplitude of [Ca2+]i transients in rat atrium in different ways. The increase in [Ca2+]i transients in response to β-adrenoceptor stimulation is commonly thought to be mediated by a greater conductance of voltage-dependent Ca2+ channels causing a greater Ca2+ influx and a release of more Ca2+ from the sarcoplasmic reticulum during the AP. The increase in diastolic [Ca2+]i in response to PE is probably a consequence of the depolarization of the membrane, possibly involving the voltage-dependent Na+-Ca2+ exchange mechanism. The increase in the amplitude of the [Ca2+]i transients in response to PE may be ascribed both to the initial increase in diastolic [Ca2+]i and the prolongation of the AP.

Electrophysiologic and inotropic effects of α-adrenoceptor stimulation in human isolated atrial heart muscle

SummaryThe effects of α-adrenoceptor stimulation on force of contraction were investigated in human atrial heart muscle and compared with those of β-adrenoceptor stimulation. The maximal positive inotropic effect produced by stimulation of α-adrenoceptors with phenylephrine (in the presence of atenolol 10 μmol/l) was significantly smaller than that seen in response to β-adrenoceptor stimulation with isoprenaline. The maximal effect of phenylephrine (25% of the maximal effect of isoprenaline) required far higher concentrations (1 mmol/l) than isoprenaline (100 nmol/l); the EC50 values amounted to 33.1 μmol/l and 3.3 nmol/l, respectively. In the presence of the α-adrenoceptor blocking agent phentolamine (1 μmol/l), the concentration-response curve of phenylephrine was displaced to higher concentrations of the agonist; under these conditions, the EC50 value amounted to 52.5 μmol/l, The effects of the catecholamines noradrenaline and adrenaline on force of contraction remained unchanged in the presence of phentolamine (1 μmol/l), or prazosin (1 μmol/l), The positive inotropic effect of phenylephrine (1 mmol/l) was associated with a slight decrease in action potential duration; the effects on action potential were completely blocked in the presence of phentolamine (1 μmol/l) These findings support the view that selective stimulation of α-adrenoceptors may mediate a small but detectable positive inotropic effect in human atrial tissue under in vitro conditions. The requirement of high concentrations of α-adrenoceptor agonists and the lack of effects of the endogenous catecholamines adrenaline and noradrenaline on α-adrenoceptors (in concentrations which fully elicit the β-adrenoceptors-mediated response) do not provide a basis for a functional role of α-adrenoceptor-mediated effects under in vivo conditions. It is more likely that adrenaline- or noradrenaline-mediated changes in the force of contraction in the human atrium are virtually exclusively due to the stimulation of β-adrenoceptors.

Depolarization-induced influx of sodium in response to phenylephrine in rat atrial heart muscle.

1. The effects of alpha 1‐adrenoceptor stimulation on transmembrane potential, currents and ion fluxes were investigated in multicellular preparations and/or single cells obtained from the left atrium of rat hearts. 2. In multicellular preparations, phenylephrine caused a concentration‐dependent positive inotropic effect, an increase in action potential duration, and a decrease in resting potential; the effects were antagonized by phentolamine. 3. In the presence of phenylephrine (100 mumol/1), two levels of resting potential were observed when the preparations were, alternately, electrically stimulated or kept at rest (‐74 +/‐ 1 mV during activity and ‐62 +/‐ 4 mV at rest; mean +/‐ S.E.M.; n = 9). 4. In resting preparations, the depolarization in response to phenylephrine was eliminated in low‐Na+ solution (12 mmol/l) and antagonized by tetrodotoxin (10 mumol/l). 5. The phenylephrine‐induced depolarization was also seen in nominally Ca(2+)‐free solution and in the presence of (‐)‐devapamil (1 mumol/l). 6. The alkylating agent N‐ethyl‐maleimide (30 mumol/l) abolished the depolarizing effect of phenylephrine. 7. Phorbol 12,13‐dibutyrate (10 mumol/l) also abolished the depolarizing effect of phenylephrine. 8. Phenylephrine caused a significant increase of 22Na+ uptake in resting preparations and of 45Ca2+ uptake in beating preparations. 9. The depolarizing effect of phenylephrine was also observed in single atrial myocytes. Steady‐state membrane currents in response to 500 ms depolarizing and hyperpolarizing voltage clamp steps were decreased. The cross‐over of I‐V curves under control and test conditions was at about ‐70 mV. The effects of phenylephrine were antagonized in the presence of phentolamine. 10. After suppression of potassium currents by substitution of CsCl for internal and external KCl ([KCl]o), phenylephrine had no effect on membrane currents. 11. In conclusion, we presume the following sequence of events in response to phenylephrine in rat atrial heart muscle. First, the stimulation of alpha 1‐adrenoceptors decreases the K+ conductance thereby producing a depolarization in the presence of an inward current. Second, the change of the membrane potential in the depolarizing direction induces a TTX‐sensitive Na+ window current which further propels the depolarization. Third, the increase in Na+ influx may increase Ca2+ influx by activating the Na(+)‐Ca2+ exchange in mechanism. The greater influx of Ca2+ may contribute to the positive inotropic effect in response to phenylephrine.

Characterization of adenosine receptors in guinea‐pig isolated left atria

1 The effects of purinergic stimulation on action potential, force of contraction, 86Rb efflux and 45Ca uptake were investigated in guinea‐pig left atria. 2 Adenosine exerted a negative inotropic effect which was antagonized by adenosine deaminase but enhanced by dipyridamole. 3 The negative inotropic effect of adenosine was mimicked by 5′‐(N‐ethyl)‐carboxamido‐adenosine (NECA) and the isomers of N6‐(phenyl‐isopropyl)‐adenosine, R‐PIA and S‐PIA. NECA and R‐PIA were about 1000 times more potent than adenosine, whereas R‐PIA was about 100 times more potent than S‐PIA. 4 The inotropic effects of adenosine (in the presence of dipyridamole), NECA, R‐PIA and S‐PIA were competitively antagonized either by theophylline (pA2 about 4.5) or 8‐phenyltheophylline (pA2 about 6.3). 5 NECA and R‐PIA shortened the action potential duration and increased the rate constant of the efflux of 86Rb in a concentration‐dependent manner with no differences in potency; the effects were competitively antagonized by 8‐phenyltheophylline. 6 Barium ions reduced the efflux of 86Rb under control conditions and antagonized the increase induced by NECA and R‐PIA. 7 NECA and R‐PIA significantly reduced 45Ca uptake in beating preparations. 8 It is concluded that adenosine, NECA and R‐PIA activate a common receptor population (P1 or A3) on the outside of the cell membrane of atrial heart muscle to increase the potassium conductance and to reduce the action potential and, thereby, calcium influx and force of contraction.

On the mechanism of action of phenylephrine in rat atrial heart muscle.

Both in rat left atrial heart and in aortic smooth muscle preparations, phenylephrine (PE) caused a concentration-dependent increase in force of contraction (Fc) in the presence of atenolol (10 μmol/l), which was antagonized by phentolamine, prazosin and WB 4101 in a competitive manner. The pA2 values of the antagonists in the cardiac tissue were 10–20fold lower than those in the rat thoracic aorta. In the spontaneously beating right atrium, PE exerted a positive chronotropic action, which was not significantly antagonized by phentolamine or prazosin. It is therefore assumed that the effects of phenylephrine in the left atrium and in the aorta are mediated by different subtypes of α1-adrenoceptors, whereas the effects in the sino-atrial node are probably unrelated to α1-adrenoceptors. To further elucidate the mechanisms of the positive inotropic effect of PE, action potential configuration and 45Ca2+ fluxes were monitored in the rat left atrium. The increase in Fc by PE was associated with an increase in action potential duration (APD) and a reduction in resting membrane potential (RP). In the presence of (−)-devapamil (13888), the effects of PE on APD and RP persisted, whereas the increase in Fc was antagonized in a non-competitive manner. Forskolin (300 nmol/l) enhanced the positive inotropic effect of PE. PE exerted a significant increase in 45CA2+ uptake in beating preparations, which was abolished in the presence of (−)13888 (1 μmol/l). In addition to the PE-induced increase in 45Ca2+ uptake, a decrease in 45Ca2+ efflux was observed. Similarly, depolarization of the membrane by raising [K+]o to 85 mmol/l revealed an increase in 45Ca2+ uptake and a decrease in 45Ca2+ efflux. The latter observations support the view that the membrane potential strongly determines the movement of 45Ca2+ across the membrane. It is assumed that the α1-adrenoceptor-mediated changes in APD and RP may enhance Fc, first, by increasing net Ca2+ entry from the extracellular space through voltage-dependent Ca2+ channels and, second, by decreasing Ca2+ efflux possibly via the Na +/Ca2+ exchange mechanism.

Contribution of both α- and β-adrenoceptors to the inotropic effects of catecholamines in the rabbit heart

SummaryThe functional role of α-adrenoceptors was investigated in different parts of the rabbit heart. Phenylephrine (PE) caused a marked increase in force of contraction (Fc) and a prolongation of the action potential (AP) in preparations from the left atrium and the right ventricle. The response was less pronounced in the right atrium and in the left ventricle, whereas APs of spontaneously beating sinoatrial preparations remained completely unchanged. Phentolamine as well as the diesters phorbol 12,13 dibutyrate (PDBu) or 12-O-tetradecanoylphorbol-13-acetate (TPA) eliminated the effects of PE. The contribution of a-adrenoceptors to the effects of adrenaline (Adr) and noradrenaline (NA) on Fc was determined in preparations from the right ventricle. Phentolamine and the phorbol diesters reduced the effects of Adr and NA by about 30 to 60%; the remaining response was abolished by propranolol. It can be derived from our experiments that, in some parts of the rabbit heart, a considerable amount of the effects of Adr and NA is due to the stimulation of a-adrenoceptors. The present findings therefore support the view that, in the rabbit heart, the maximally effective drive of the heart requires the stimulation of both α- and β-adrenoceptors. The inhibitory effects of phorbol diesters on the α-adrenoceptor-mediated response indicate that the activation of protein kinase C (PKC) specifically uncouples α-adrenoceptors from the effector system, whereas the response to β-adrenoceptor stimulation remains unchanged.