Taku Yamamoto

Dopamine stimulation of cardiac β‐adrenoceptors: the involvement of sympathetic amine transporters and the effect of SKF38393

1 Mechanisms underlying β‐adrenoceptor stimulation by dopamine were examined on guinea‐pig Langendorff‐perfused hearts and isolated cells from the right atrium, by using the chronotropic effects and the enhancement of L‐type Ca2+ current (ICa,L) in the presence of prazosin as indicators of β‐adrenoceptor stimulation. Dopamine‐induced overflow of noradrenaline (NA) concentrations was measured by high‐performance liquid chromatography. 2 Dopamine caused positive chronotropic effects with an EC50 of 2.5 μm and induced NA overflow with a similar EC50 (1.3 μm). The chronotropic effect of dopamine was abolished by bisoprolol (1 μm). 3 The effects of dopamine were maintained during prolonged application, whereas the effects of tyramine faded with time. Dopamine (3 μm) restored the chronotropic effects and the NA release suppressed by pretreatment with tyramine, suggesting a de novo synthesis of NA during the exposure to dopamine. 4 Dopamine (3 μm)‐induced NA release was not affected by tetrodotoxin, ω‐conotoxin, rauwolscine, ICI118551 or sulpiride, but was inhibited by desipramine, a NA uptake inhibitor (IC50 ∼1 μm). It was also not affected by GBR12909 and bupropion, dopamine uptake inhibitors in the central nervous system. 5 SKF38393, a D1 receptor partial agonist, potently inhibited the 3 μm dopamine‐induced release of NA (IC50 ∼0.1 μm). D1 receptors are not involved in the DA‐induced release of NA, since SCH23390 (3 μm), a potent D1 antagonist, inhibited the NA release only slightly, and dihydrexidine (1 μm) and chloro‐APB (1 μm), full D1 agonists, caused no significant NA release. 6 SKF38393 inhibited tyramine‐induced overflow of NA, and potentiated the field 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 μm), dopamine at 1 μm showed no chronotropic effect. 7 Voltage clamp experiments with isolated atrial cells revealed that dopamine is a weak partial agonist. The EC50 for ICa,L stimulation by dopamine was high (13 μm). As a result, dopamine at 1 μm did not affect ICa,L. Bisoprolol abolished the stimulation of ICa,L by dopamine (30 μm), and dihydrexidine (1 μm) did not affect ICa,L. 8 It was concluded that the cardiac effects of dopamine at clinically relevant concentrations (<1 μm) result almost exclusively from the indirect effect of β adrenoceptor stimulation, involving the release of NA from sympathetic nerve terminals. The roles of the direct stimulation of β adrenoceptors by dopamine at these concentrations and the stimulation of postjunctional D1 receptors seem negligible. The desipramine‐ and SKF38393‐sensitive monoamine transporter mediates the release of NA.

Negative chronotropic actions of endothelin-1 on rabbit sinoatrial node pacemaker cells

1 The effects 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 Ca2+ current (ICa(L)) dose‐dependently with a half‐maximal inhibitory concentration (EC50) of 0.42 nM and maximal inhibitory response (Emax) of 49.5%. The delayed rectifying K+ current (IK) 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 (EC50=4.8 nM). These ET‐1‐induced changes in membrane currents were abolished by BQ485 (0.3 μM), a highly selective ETA receptor antagonist. 4 When ICa(L) was inhibited by ET‐1 (1 nM), subsequent application of 10 μM ACh showed no additional decrease in ICa(L), suggesting the involvement of cyclic AMP in the effects of ET‐1 on ICa(L). In contrast, 1 nM ET‐1 further decreased ICa(L) in the presence of 10 μM ACh, suggesting that ET‐1 activates some additional mechanism(s) which inhibit ICa(L). The ET‐1‐induced ICa(L) inhibition was abolished by protein kinase A inhibitory peptide (PKI, 20 μM) or H‐89 (5 μM). However, the ICa(L) inhibition was not affected by methylene blue (10 μM), suggesting a minor role for cyclic GMP in the effect of ET‐1 under basal conditions. 5 ET‐1 failed to inhibit ICa(L) when the pipette contained GDPβS (200 μM). However, incubation of the cells with pertussis toxin (PTX, 5 μg ml−1, >6 h) only reduced the ET‐1‐induced inhibition to 21.5±9.5%, whereas it abolished the inhibitory effect of ACh on ICa(L). 6 Intracellular perfusion of 8‐bromo cyclicAMP (8‐Br cyclicAMP, 500 μM) attenuated, but did not abolish the inhibitory effect of ET‐1 on ICa(L). This 8‐Br cyclicAMP‐resistant component (17.5±14.4%, n=20) was not affected by combined application of 8‐Br cyclicAMP with 8‐bromo cyclicGMP (500 μM), ryanodine (1 μM) or phorbol‐12‐myristate‐13‐acetate (TPA; 50 nM). 7 In summary, ET‐1 exerts negative chronotropic effects on the SA node via ETA‐receptors. ET‐1 inhibits both ICa(L) and IK, and increases background K+ current. The inhibition of ICa(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.

Altered expression of Na+ transporters at the mRNA level in rat normal and hypertrophic myocardium

Intracellular Na+ ([Na+]i) regulation plays a crucial role in the structural, mechanical, and electrical properties of myocardium. It is assumed that the [Na+]i handling system may differ not only between normal and diseased hearts but also regionally within a heart. To gain new insight concerning disease- and region-dependent differences in the [Na+]i-regulatory system, we investigated mRNA expression of Na+ transporters, the principal determinants of [Na+]i. Nonischemic pressure-overloaded hypertrophy was created by suprarenal abdominal aortic constriction of 50% for 7 weeks. mRNA abundances of Na+-Ca2+ exchanger (NCX1), Na+-H+ exchanger (NHE1), Na+-K+-2Cl− exchanger (NKCC1) and Na+, K+-ATPase multigene family(α1, α2, α3, and β1 isoforms) were measured by the real-time quantitative polymerase chain reaction method. mRNA abundance of all transporters mediating Na+ influx (NCX1, NHE1, and NKCC1) was significantly upregulated as compared to normal. In contrast, Na+-efflux-mediating transporter (Na+, K+-ATPase) mRNA expression was unaltered between normal and hypertrophic hearts. Losartan, an angiotensin II AT1 receptor antagonist, significantly attenuated upregulation of Na+-influx-mediating transporters induced by aortic constriction. The onset of Na+-influx-mediating transporter upregulation occurred within 5 days following constriction. In normal and hypertrophied hearts, mRNA of all Na+-influx-mediating transporters was expressed in order of abundance as: apex > septum ∼ free wall of left ventricles. A transmural gradient in expression was also evident in normal hearts (midcardium > endo- and epicardium), which was attenuated under hypertrophic development. Myocardial hypertrophy is associated with significant changes in the spatial distribution and expression levels of Na+ transporters. The upregulation of Na influx transporters during hypertrophy may contribute to the remodeling process, modulate contractility and promote arrhythmias.

The mechanisms underlying heart stimulation by dopamine, with special reference to direct and indirect β adrenoceptor stimulation

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.

Delineation of premature P waves on four-dimensional electrocardiography, a new display of electrical forces by computer techniques.

This study investigated the feasibility of four-dimensional electrocardiography (4-D ECG), a new display in which the vector loop was rotated and scanned along a timed axis to overcome the shortcomings of vectorcardiography (VCG). The subjects consisted of 38 patients with premature atrial complexes and 30 controls. The orthogonal Frank elec trocardiograms were rotated three-dimensionally according to the right-hand rectangular coordinate system and scanned along a timed axis. The P wave delineation score, signi fying good agreement with the intraobserver and interobserver variability, was signifi cantly higher in 4-D ECG than those in the orthogonal leads or those on the transverse and frontal projections (P < 0.001). The authors measured the premature P loop areas as viewed from 361 directions. P loop areas were best delineated when viewed from cranial directions of 42.6 ±34.0 degrees and from rightward directions of 11.3 ±30.7 degrees. Adequate cranial rotation followed by scanning along a timed axis will maximally delineate premature atrial signals and provide comprehensive visualization of electrical forces.

Modulation of L-type Ca current by denopamine, a nonparenteral partial β1 stimulant, in rabbit ventricular cells

The effects of denopamine, a nonparenteral partial β agonist which is used clinically in Japan, on the L-type Ca2+ current (ICa) were examined in rabbit ventricular cells. Denopamine stimulated basal ICa with a maximum response of +33.2% and a concentration for half-maximal response (EC50) of 0.039 μM. The maximun response of ICa was only a quarter of that induced by isoprenaline (ISO), while 10 μM denopamine elicited 70–75% of the maximum inotropic response in the papillary muscle preparations. The denopamine stimulation of ICa was abolished by selective β1 antagonists (atenolol or bisoprolol). Pretreatment with forskolin or dialysis with cAMP also abolished the stimulation. Denopamine, in turn, inhibited ISO-stimulated ICa. This inhibition was not affected by pretreatment with pertussis toxin or prazosin. The presence of denopamine at various concentrations caused a rightward shift in the concentration/response curve for ISO stimulation of ICa. The Schild plot for this effect had a slope of 0.99 and Kp of 0.20 μM. In the presence of guanosine-5′-O-(3-thiotriphosphate) (GTPγS) (0.5 mM) in the pipette, denopamine (10 μM) stimulated the ICa to 86±5% of the maximum response induced by ISO. These findings indicate that denopamine modulates ICa exclusively through the β1 adrenoceptor-adenylate cyclase pathway, that the stimulatory GTP-binding protein regulates the agonistic potency of denopamine, and that the signal from the β1 adrenoceptors is amplified between ICa and the tension development, which would contribute to the spare capacity of β adrenoceptors.

Possible involvement of endothelin in thromboxane A2 receptor agonist (U-46619)-induced angina in the rat

The thromboxane A2 receptor agonist, U-46619 ((5Z, 9 alpha, 11 alpha, 13E, 15(S))-9,11-(methanoepoxy)prosta-5,13-dien-1-oic acid) (10 micrograms/kg), induced a typical ischemic change (ST elevation) in the electrocardiogram on intracoronary arterial administration in the rat. The elevation of the ST segment induced by U-46619 was significantly reduced by pretreatment with anti-endothelin-1 rabbit serum. The plasma concentration of endothelin-1 dose dependently increased at the time of ST segment elevation after U-46619. These results indicate that endogenous endothelin-1 partly contributes to coronary spasmodic angina induced by thromboxane A2 in rats.

ANTI‐ARRHYTHMIC EFFECTS OF A NEW CALCIUM ANTAGONIST, MONOTEPIL, AJ‐2615, IN EXPERIMENTAL ARRHYTHMIC MODELS

1. To characterize the anti‐arrhythmic properties of a new calcium antagonist, monotepil, AJ‐2615, the preventive effects of AJ‐2615 were compared with those of the existing calcium antagonists, diltiazem and verapamil, in experimental models of arrhythmia.

PREVENTIVE EFFECT OF A NEW CALCIUM ANTAGONIST, MONATEPIL, ON DRUG‐INDUCED ISCHAEMIC ELECTROCARDIOGRAPHIC CHANGES IN RATS

1. The preventive effects of monatepil, a new calcium antagonist with α1‐adrenoceptor blocking activity, on ischaemic electrocardiographic changes in rat models of vasospastic angina were evaluated and compared with those of the existing calcium antagonists (diltiazem, verapamil, nicardipine and nifedipine).

Endothelin-1 Inhibits Pacemaker Currents in Rabbit SA Node Cells

Our recent study demonstrated that endothelin-1 (ET-1) inhibits the pacemaker activity of sinoatrial (SA) node cells via changes in the L-type Ca2+, delayed K+, and background K+ currents. Using the whole-cell patch-clamp technique in the same preparation, we found that ET-1 reduces other pacemaker currents, the T-type Ca2+ current (ICa,T) and the hyperpolarization-activated inward current (I(f)). The inhibitory actions of ET-1 on these currents were concentration-dependent, i.e., EC50 of 0.9 nM for ICa,T and 2.3 nM for I(f), with little reversal after washout of the peptide. In the presence of BQ485, both currents were not affected by ET-1. These results indicate additional mechanisms underlying negative chronotropic actions of ET-1 on the rabbit SA node.