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.
The actions of angiotensin II (ANG II) were examined in the spontaneously active cells isolated from the rabbit sinoatrial node, using the nystatin-permeabilized, whole cell, patch-clamp method. At 30 nM, ANG II significantly lowered the spontaneous firing rate of the action potentials from 212 +/- 21 to 172 +/- 32 beats/min, with a concomitant reduction in the action potential amplitude. The voltage-clamp experiments showed that ANG II inhibited the L-type Ca2+ current (ICa) with a dissociation constant (Kd) of approximately 4 nM and a maximal inhibition of 30%. The inhibition was blocked by an AT1-receptor antagonist CV11974. Acetylcholine (ACh) at 10 microM reduced the ICa by 42 +/- 12%, and ANG II did not cause any further inhibition in the presence of ACh. At 100 nM, ANG II reduced the ICa by only 12% in the presence of 2 microM isoproterenol, and a similar inhibition was observed with 0.1 microM ACh. ANG II did not affect the dibutyryl adenosine 3',5'-cyclic monophosphate-stimulated ICa. Protein kinase C activator 12-O-tetra-decanoylphorbol-13-acetate did not mimic ANG II in the effects on ICa, and preincubation of the cells with calphostin C, a protein kinase C inhibitor, did not attenuate the ANG II effect. ANG II exerts a negative chronotropic effect in the pacemaker cells as its direct action through a pathway involving adenosine 3',5'-cyclic monophosphate-dependent protein kinase.
1 Mechanisms underlying b-adrenoceptor stimulation by dopamine were examined on guinea-pig Langendor -perfused hearts and isolated cells from the right atrium, by using the chronotropic e ects and the enhancement of L-type Ca 2+ current (I Ca,L ) in the presence of prazosin as indicators of badrenoceptor stimulation. Dopamine-induced over¯ow of noradrenaline (NA) concentrations was measured by high-performance liquid chromatography. 2 Dopamine caused positive chronotropic e ects with an EC 50 of 2.5 mM and induced NA over¯ow with a similar EC 50 (1.3 mM). The chronotropic e ect of dopamine was abolished by bisoprolol (1 mM).3 The e ects of dopamine were maintained during prolonged application, whereas the e ects of tyramine faded with time. Dopamine (3 mM) restored the chronotropic e ects and the NA release suppressed by pretreatment with tyramine, suggesting a de novo synthesis of NA during the exposure to dopamine. 4 Dopamine (3 mM)-induced NA release was not a ected by tetrodotoxin, o-conotoxin, rauwolscine, ICI118551 or sulpiride, but was inhibited by desipramine, a NA uptake inhibitor (IC 50 *1 mM). It was also not a ected by GBR12909 and bupropion, dopamine uptake inhibitors in the central nervous system. 5 SKF38393, a D 1 receptor partial agonist, potently inhibited the 3 mM dopamine-induced release of NA (IC 50 *0.1 mM). D 1 receptors are not involved in the DA-induced release of NA, since SCH23390 (3 mM), a potent D 1 antagonist, inhibited the NA release only slightly, and dihydrexidine (1 mM) and chloro-APB (1 mM), full D 1 agonists, caused no signi®cant NA release. 6 SKF38393 inhibited tyramine-induced over¯ow of NA, and potentiated the ®eld stimulation-induced NA release. SKF38393 and desipramine retarded the decay of the stimulation-induced tachycardia in a similar manner. These results indicate that SKF38393 is a potent monoamine transport inhibitor and a useful tool for the functional evaluation of indirectly-acting sympathomimetic agonists in the heart. In the presence of SKF38393 (10 mM), dopamine at 1 mM showed no chronotropic e ect. 7 Voltage clamp experiments with isolated atrial cells revealed that dopamine is a weak partial agonist. The EC 50 for I Ca,L stimulation by dopamine was high (13 mM). As a result, dopamine at 1 mM did not a ect I Ca,L . Bisoprolol abolished the stimulation of I Ca,L by dopamine (30 mM), and dihydrexidine (1 mM) did not a ect I Ca,L . 8 It was concluded that the cardiac e ects of dopamine at clinically relevant concentrations (51 mM) result almost exclusively from the indirect e ect of b adrenoceptor stimulation, involving the release of NA from sympathetic nerve terminals. The roles of the direct stimulation of b adrenoceptors by dopamine at these concentrations and the stimulation of postjunctional D 1 receptors seem negligible. The desipramine-and SKF38393-sensitive monoamine transporter mediates the release of NA.
Lactate modifies INa of ventricular myocytes by shifting its kinetics toward hyperpolarisation. This shift seems to be caused exclusively by a decrease in the ionised divalent cation concentrations and a resultant change in the negative surface charge of the sarcolemma.
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