There is increasing evidence that cardiac glycosides act through mechanisms distinct from inhibition of the sodium pump but which may contribute to their cardiac actions. To more fully define differences between agents indicative of multiple sites of action, we studied changes in contractility and action potential (AP) configuration in cat ventricular myocytes produced by six cardiac glycosides (ouabain, ouabagenin, dihydroouabain, actodigin, digoxin, and resibufogenin). AP shortening was observed only with ouabain and actodigin. There was extensive inotropic variability between agents, with some giving full inotropic effects before automaticity occurred whereas others produced minimal inotropy before toxicity. AP shortening was not a result of alterations in calcium current or the inward rectifier potassium current, but correlated with an increase in steady-state outward current (I ss ), which was sensitive to KB-R7943, a Na ϩ -Ca 2ϩ exchange (NCX) inhibitor.Interestingly, I ss was observed following exposure to ouabain and dihydroouabain, suggesting that an additional mechanism is operative with dihydroouabain that prevents AP shortening.Further investigation into differences in inotropy between ouabagenin, dihydroouabain and ouabain revealed almost identical responses under AP voltage clamp. Thus all agents appear to act on the sodium pump and thereby secondarily increase the outward reverse mode NCX current, but the extent of AP duration shortening and positive inotropy elicited by each agent is limited by development of their toxic actions. The quantitative differences between cardiac glycosides suggest that mechanisms independent of sodium pump inhibition may result from an altered threshold for calcium overload possibly involving direct or indirect effects on calcium release from the sarcoplasmic reticulum.

The inotropic and toxic effects of cardiac glycosides are thought to be related to their ability to inhibit the Na,K-ATPase. We examined the effects of ouabain and its analogs on sarcoplasmic reticulum (SR) Ca 2ϩ release in intact cat ventricular myocytes under Na ϩ -free conditions and in myocytes in which the sarcolemma was permeabilized using saponin so that cytoplasmic ionic composition was fixed by the bath solutions. We also compared ouabain actions in cat myocytes to those in rat myocytes because the latter is considered to be a glycoside-insensitive species. In intact cat myocytes (Na ϩ -free conditions), spontaneous Ca 2ϩ sparks were prolonged and frequency, amplitude and width were reduced by exposure to ouabain (3 M). Nearly identical results were obtained with its analogs dihydroouabain or ouabagenin (10 M). The frequency of spontaneous Ca 2ϩ waves was also reduced by ouabain. In contrast, ouabain (100 M) had negligible effects on sparks and waves in rat myocytes in Na ϩ -free conditions, consistent with the decreased sensitivity to cardiac glycosides observed in this species. In cat myocytes permeabilized with saponin (0.01%), ouabain (Ն50 nM) decreased spark frequency and increased background SR Ca 2ϩ leak only when the SR was well loaded (free [Ca 2ϩ ] ϭ 275 nM) and not when SR load was low (free [Ca 2ϩ ] ϭ 50 nM). Similar effects were observed in rat myocytes only when ouabain concentration was 1 M. These results suggest that the cellular actions of cardiac glycosides may include a direct effect on SR Ca 2ϩ release, possibly through activation of SR Ca 2ϩ release channels (ryanodine receptors). In addition, these results are consistent with the idea that direct activation of SR Ca 2ϩ release is dependent on the extent of SR Ca 2ϩ load, with elevated load increasing sensitivity of the channel release mechanism to activation by glycoside.

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 inotropic and toxic effects of cardiac steroids are thought to result from Na+-K+-ATPase inhibition, with elevated intracellular Na+(Na[Formula: see text])causing increased intracellular Ca2+(Ca[Formula: see text]) via Na-Ca exchange. We studied the effects of ouabain on cat ventricular myocytes in Na+-free conditions where the exchanger is inhibited. Cell shortening and Ca[Formula: see text] transients (with fluo 4-AM fluorescence) were measured under voltage clamp during exposure to Na+-free solutions [LiCl or N-methyl-d-glucamine (NMDG) replacement]. Ouabain enhanced contractility by 121 ± 55% at 1 μmol/l ( n = 11) and 476 ± 159% at 3 μmol/l ( n = 8) (means ± SE). Ca[Formula: see text] transient amplitude was also increased. The inotropic effects of ouabain were retained even after pretreatment with saxitoxin (5 μmol/l) or changing the holding potential to −40 mV (to inactivate Na+ current). Similar results were obtained with both Li+ and NMDG replacement and in the absence of external K+, indicating that ouabain produced positive inotropy in the absence of functional Na-Ca exchange and Na+-K+-ATPase activity. In contrast, ouabain had no inotropic response in rat ventricular myocytes (10–100 μmol/l). Finally, ouabain reversibly increased Ca2+overload toxicity by accelerating the rate of spontaneous aftercontractions ( n = 13). These results suggest that the cellular effects of ouabain on the heart may include actions independent of Na+-K+-ATPase inhibition, Na-Ca exchange, and changes in Na[Formula: see text].

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.

Effects of acetylcholine (ACh) on L-type Ca2+ current (ICa) were examined in isolated atrioventricular (AV) node cells exhibiting spontaneous contractions and pacemaker current (If). ACh at a saturating concentration of 10 microM reduced basal ICa by 48 +/- 6%. The ACh effect was abolished by dialysis with 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase inhibitor, or guanosine-5'-O-(2-thiodiphosphate). Dialysis with guanosine 3',5'-cyclic monophosphate (cGMP) or NG-monomethyl-L-arginine (L-NMMA) and application of the cGMP-dependent protein kinase inhibitor KT-5823 (1 microM) did not affect ACh inhibition of ICa. Nitric oxide donor 3-morpholinosydnonimine (100 microM) and type III phosphodiesterase (PDE) inhibitor trequinsin (10 nM) enhanced basal ICa by 10-20%, whereas type IV PDE inhibitor Ro-20-1724 (30 microM) together with trequinsin caused a large ICa stimulation comparable to that by 3-isobutyl-1-methylxanthine (IBMX). These findings indicate that ACh inhibits basal ICa primarily by suppressing cAMP synthesis and that these cells have a potent type III and IV PDE activity to determine the basal cAMP concentration. When ICa was stimulated by IBMX (100 microM), the inhibitory effect of ACh was slightly reduced by L-NMMA, cGMP, and methylene blue but not by KT-5823 or Ro-20-1724. ACh hardly inhibited, or even enhanced, IBMX-stimulated Ica when forskolin (3 microM) was coapplied or the IBMX concentration was increased to 500 microM. These findings suggest that cAMP is degraded in the presence of 100 microM IBMX to some extent. Type II PDE, for which IBMX has a relatively high inhibitor constant, seems to contribute partially to the cAMP degradation.

Prior observations have raised the possibility that dihydropyridine (DHP) agonists directly affect the sarcoplasmic reticulum (SR) cardiac Ca(2+) release channel [i.e., ryanodine receptor (RyR)]. In single-channel recordings of purified canine cardiac RyR, both DHP agonists (-)-BAY K 8644 and (+)-SDZ202-791 increased the open probability of the RyR when added to the cytoplasmic face of the channel. Importantly, the DHP antagonists nifedipine and (-)-SDZ202-791 had no competitive blocking effects either alone or after channel activation with agonist. Thus there is a stereospecific effect of SDZ202-791, such that the agonist activates the channel, whereas the antagonist has little effect on channel activity. Further experiments showed that DHP agonists changed RyR activation by suppressing Ca(2+)-induced inactivation of the channel. We concluded that DHP agonists can also influence RyR single-channel activity directly at a unique allosteric site located on the cytoplasmic face of the channel. Similar results were obtained in human purified cardiac RyR. An implication of these data is that RyR activation by DHP agonists is likely to cause a loss of Ca(2+) from the SR and to contribute to the negative inotropic effects of these agents reported by other investigators. Our results support this notion that the negative inotropic effects of DHP agonists result in part from direct alteration in the activity of RyRs.

1. The positive chronotropic and norepinephrine-releasing effects of dopamine were examined in the isolated guinea pig heart, using the Langendorff model. 2. The released norepinephrine was estimated from the norepinephrine concentration measured in the post-perfusion solution using HPLC. 3. The dose-response curve for dopamine to stimulate the heart rate (HR) closely resembled that for the norepinephrine release. A selective beta 1 antagonist bisoprolol completely abolished the positive chronotropic effect, but did not affect the norepinephrine release. 4. The HR increase in response to 3 mumol/L dopamine was 54 +/- 15% (n = 14) of the control in normal hearts. The response was decreased to 15 +/- 7% (n = 6) by pretreatment with reserpine. 5. A D1 antagonist, SKF83742, (3 mumol/L) shifted the dose-response curve for the dopamine-induced norepinephrine release toward the right, indicating the involvement of D1-like dopamine receptors. 6. Voltage clamp experiments using single cells isolated from the right atrium revealed that dopamine is a weak partial agonist for beta adrenoceptors. Dopamine stimulated the L-type Ca2+ current with a threshold concentration of 3 mumol/L. 7. These findings indicate the important role of the norepinephrine release in the stimulation of beta adrenoceptors by dopamine at clinically relevant concentrations.