Relaxin (RLX), a reproductive hormone of the insulin family, increases heart rate in experimental animals. The cellular and ionic mechanisms responsible for this positive chronotropic effect remain unknown. We have investigated the actions of RLX on the action potential and underlying transmembrane ionic currents in single sinoatrial node cells of the rabbit heart under whole-cell voltage-clamp conditions, using both nystatin-perforated-patch and membrane-ruptured techniques. In this preparation RLX (0.8 to 80 nmol/L) caused reversible increases in the rate of spontaneous action potentials and a dose-dependent increase in the L-type calcium current, ICa(L). The best-fit Langmuir relation for the augmentation of ICa(L) yielded a threshold concentration of 1 nmol/L and a KD of 14 nmol/L. These effects of RLX appear to be mediated by increases in intracellular cyclic AMP (cAMP), since RLX was without effect after application of (1) the beta-adrenergic agonist isoprenaline (1 mumol/L) or (2) superfusion of the intracellular second messenger cAMP (100 mumol/L) or 8-Br-cAMP (100 to 200 mumol/L). Internal dialysis with an inhibitor of cAMP-dependent protein kinase (PKI, 7 mumol/L) abolished the effects of RLX. These results provide the first electrophysiological evidence that RLX modulates heart rate and contractility by increasing ICa(L) and suggest that the biochemical mechanism involves the formation of cAMP and activation of cAMP-dependent protein kinase.

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The role of delayed rectifier K+ current(s) (IK) in rabbit left atrium was examined by applying the whole cell voltage-clamp technique to isolated single myocytes. Right-triangular waveforms, which mimic the shape of atrial action potentials (APs), and selective blockers were used to compare the contribution of IK with other K+ currents to repolarization of the APs. IK measured at 34 degrees C in atrial myocytes was very small; the maximum peak amplitude of the tail current (IK,tail) at -40 mV was approximately 50 pA. The IK,tail was almost abolished in most cells (approximately 80%) by the application of 1 microM E-4031, a class III antiarrhythmic drug. The E-4031-sensitive current recorded with the triangular command wave-form showed strong inward rectification and had a maximum amplitude of approximately 30 pA at -40 mV. Total outward current elicited by triangular command pulses depended strongly on stimulation frequency. The main frequency-dependent component was a Ca(2+)-independent transient K+ current (I(t)). I(t) elicited by triangular pulses at 1 Hz was substantially reduced by 4-aminopyridine (4-AP) at potentials positive to 0 mV but was not changed significantly by 1 microM E-4031; 100 microM E-4031 reduced I(t) by approximately 30%. The shape of the APs which were recorded from a single rabbit atrial cell strongly depended on the pulse frequency. Application of 1 microM E-4031 increased action potential duration (APD) in > 50% of cells examined but had little effect on the resting membrane potential (RMP). Application of 0.1 mM BaCl2 also lengthened APD and reduced RMP by approximately 20 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

1 The eects of endothelin-1 (ET-1) on sinoatrial (SA) node preparations of the rabbit heart were studied by means of whole-cell clamp techniques. 2 ET-1 at 1 nM slowed the spontaneous beating activity and rendered half of the cells quiescent. At a higher concentration of 10 nM, the slowing and cessation of spontaneous activity were accompanied by hyperpolarization. 3 In voltage-clamp experiments, ET-1 decreased the basal L-type Ca 2+ current (I Ca(L) ) dose-dependently with a half-maximal inhibitory concentration (EC 50 ) of 0.42 nM and maximal inhibitory response (E max ) of 49.5%. The delayed rectifying K + current (I K ) was also reduced by 33.2+11.1% at 1 nM. In addition, an inwardly rectifying K + current was activated by ET-1 at higher concentrations (EC 50 =4.8 nM). These ET-1-induced changes in membrane currents were abolished by BQ485 (0.3 mM), a highly selective ET A receptor antagonist. 4 When I Ca(L) was inhibited by ET-1 (1 nM), subsequent application of 10 mM ACh showed no additional decrease in I Ca(L) , suggesting the involvement of cyclic AMP in the eects of ET-1 on I Ca(L) . In contrast, 1 nM ET-1 further decreased I Ca(L) in the presence of 10 mM ACh, suggesting that ET-1 activates some additional mechanism(s) which inhibit I Ca(L) . The ET-1-induced I Ca(L) inhibition was abolished by protein kinase A inhibitory peptide (PKI, 20 mM) or H-89 (5 mM). However, the I Ca(L) inhibition was not aected by methylene blue (10 mM), suggesting a minor role for cyclic GMP in the eect of ET-1 under basal conditions. 5 ET-1 failed to inhibit I Ca(L) when the pipette contained GDPbS (200 mM). However, incubation of the cells with pertussis toxin (PTX, 5 mg ml 71 , 46 h) only reduced the ET-1-induced inhibition to 21.5+9.5%, whereas it abolished the inhibitory eect of ACh on I Ca(L) . 6 Intracellular perfusion of 8-bromo cyclicAMP (8-Br cyclicAMP, 500 mM) attenuated, but did not abolish the inhibitory eect of ET-1 on I Ca(L) . This 8-Br cyclicAMP-resistant component (17.5+14.4%, n=20) was not aected by combined application of 8-Br cyclicAMP with 8-bromo cyclicGMP (500 mM), ryanodine (1 mM) or phorbol-12-myristate-13-acetate (TPA; 50 nM). 7 In summary, ET-1 exerts negative chronotropic eects on the SA node via ET A -receptors. ET-1 inhibits both I Ca(L) and I K , and increases background K + current. The inhibition of I Ca(L) by ET-1 is mainly due to reduction of the cyclicAMP levels via PTX-sensitive G protein, but some other mechanism(s) also seems to be operative.