Characterization of beta-adrenoceptor subtype in isolated ring preparations of intramural rat coronary small arteries

We characterized the beta-adrenoceptor subtype in isolated ring preparations of small intramyocardial flow-regulating (resistance) arteries (i.d. congruent to 239 micrometers) from rats by using beta 2 adrenoceptor agonists and beta-adrenoceptor antagonists exhibiting selectivity towards either beta 1- (pafenolol) or beta-adrenoceptors (ICI 118,551). The relative order of potency of selected agonists in producing beta-adrenoceptor-mediated relaxation of prostaglandin F2 alpha (10(-5) M) contracted coronary vessels was: isoprenaline greater than noradrenaline = adrenaline. ICI 118,551, but not pafenolol, relaxed coronary resistance vessels precontracted with prostaglandin F2 alpha (10(-5) M) or high potassium (125 mM) solutions. The IC50 values were 8.59 x 10(-6) M and 2.96 x 10(-5) M, respectively (p less than 0.0005). This indicates that ICI 118,551 had membrane-stabilizing effects. Both ICI 118,551 and pafenolol antagonized in a concentration-dependent manner the isoprenaline-induced relaxation of prostaglandin F2 alpha (10(-5) M) contracted coronary resistance vessels. The slopes and regression lines of the Schild plots were not significantly different from unity and the calculated pA2 values were 7.55 and 6.59 (p less than 0.005) for pafenolol and ICI 118,551, respectively. The results are compatible with the suggestion that beta-adrenoceptors mediating relaxation in rat coronary resistance vessels belong to the beta 1-adrenoceptor subtype.     

钩藤碱对大鼠心肌细胞钙离子通道的阻滞作用(英文)

用膜片钳全细胞记录的方法研究了钩藤碱(Rhy)对大鼠心叽细胞钙离子通道的阻滞作用,Rhy明显抑制verapamil敏感的L型钙通道,减少钙离子内向电流,但不改变最大激活时的膜电位水平以及激活时间常数,提示Rhy非电压依赖地抑制钙通道,其最大反应出现在给药10分钟,为浓度依赖的作用,pD_2值为5.91,Hill系数1.1。     

芍药苷对大鼠心肌细胞L钙通道的阻断作用

目的　在单个大鼠心肌细胞上 ,研究芍药苷对L型钙通道的阻断作用。方法　采用全细胞膜片钳技术。结果　芍药苷浓度依赖性阻断ICa ,L,其IC50 为 387μmol·L- 1 。应用 40 0 μmol·L- 1 芍药苷后 ,ICa,L最大电流幅度下降 51 % ,但I U曲线的形态和反转电位没有变化 ,激活曲线也没有明显的改变 ;失活曲线向较负电压的方向偏移约 8 3mV ,通道从失活中恢复的时间明显延长 ,由给药前的 (96± 1 7)ms增至 (1 85± 2 8)ms ;芍药苷的阻断作用没有显示出频率依赖性。结论　芍药苷对心肌细胞ICa ,L有阻断作用     

Uptake of bepridil into isolated ventricular myocytes. Retention by actin

Isolated rat ventricular myocytes accumulate large amounts of bepridil intracellularly. The mode of transport is uncertain but uptake is of such a magnitude as to mask possible sarcolemmal binding sites. Bepridil is tightly bound within myocytes, there being only a minimal efflux into bepridil-free medium. Purified F-actin binds bepridil in excess of 2 moles/mole actin, sufficient to account for the uptake of bepridil by myocytes incubated in 10 microM drug. At higher bepridil concentrations the amount transported into myocytes suggests that the drug may be distributed between actin-bound and cytoplasmic pools. Bepridil may alter cardiac contractility by a direct influence on the contractile filaments.     

Cardiotonic actions of selective phosphodiesterase inhibitors in rat isolated ventricular cardiomyocytes.

1. The contractile effects of the novel cardiotonic agent HN-10200 (2-[3-methoxy-5-methylsulphinyl-2-thienyl]-1H-imidazo-[4,5-c]-p yri dine hydrochloride), were examined and comparisons made with the responses obtained to a structurally similar compound, sulmazole, and to a number of other compounds which are known to inhibit phosphodiesterase (PDE) isoenzymes with differing selectivities; namely, enoximone (PDE III inhibitor), Ro 20-1724 (PDE IV inhibitor) and 3-isobutyl-1-methylxanthine (non-selective PDE inhibitor). 2. Contractile function, as measured by mechanical shortening, and biochemical systems involving cyclic AMP were investigated in ventricular cardiomyocytes isolated from adult Sprague-Dawley rats (200-250 g). 3. HN-10200 exerted a concentration-dependent (10(-8) M-10(-4) M) positive contractile effect, which was independent of alpha- or beta-adrenoceptor, or histamine receptor stimulation. 4. The efficacies of the contractile responses to the PDE inhibitors were of the order: HN-10200 > IBMX > sulmazole > enoximone and maximum stimulations, which were obtained at concentrations of 10(-4) M, were 54 +/- 4%, 41 +/- 7%, 38 +/- 7% and 26 +/- 5% (mean +/- s.e.) greater than basal levels, respectively (n = 6); the basal value of contractile amplitude (dL), in the absence of PDE inhibitors was 7.39 +/- 0.18% (mean +/- s.e.). Ro 20-1724 did not have any effect on contractile activity. 5. Due to low basal levels of cyclic nucleotides in isolated cells, accumulation of cyclic AMP due to the presence of the PDE inhibitors was detected only when the levels of cyclic nucleotide were enhanced with forskolin (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative effects of beta-adrenoceptor partial agonists on isolated rat atrium.

The chronotropic effect of three beta-1-adrenoceptor partial agonists prenalterol, xamoterol and epanolol has been compared on the right atria of the rat in order to evaluate their intrinsic activity and to place them in rank order of effectiveness. The results show that prenalterol, xamoterol and epanolol are all partial agonists. The intrinsic activities relative to that of isoprenaline are 0.84 for prenalterol, 0.59 for xamoterol and 0.29 for epanolol. This rank order of intrinsic activities should remain the same in different species and in man. Both atenolol and propranolol reversed the chronotropic effects of the three agonists. The KB of the two blockers was similar against prenalterol and xamoterol, which indicates that the two partial agonists are probably competing for the same population of receptors. The EC50 is twice as large than KB for xamoterol, which is consistent with isoprenaline working through both beta-1- and beta-2- receptors and xamoterol finds it more difficult to block the beta-2-receptors.     

Pharmacological characterization of A1 adenosine receptors in isolated rat ventricular myocytes

Summary The aim of the present study was the characterization of adenosine receptors in isolated rat ventricular myocytes. The CAMP-levels of rat ventricular myocytes in the presence of 1 mol/l isoprenaline were reduced by up to 48% by adenosine analogues; the rank order of potency was: R-N⁶-phenylisopropyladenosine (IC₅₀ 60 nmol/1), 5-N-ethylcarboxamidoadenosine (IC₅₀ 360 nmol/l) and S-N⁶-phenylisopropyladenosine (IC₅₀ 16 ol/l). The adenosine receptor antagonist XAC (xanthine amine congener) antagonized the effect of R-N⁶-phenylisopropyladenosine in a concentration-dependent manner with a K_i-value of 20 nmol/l. The A₁ receptor-selective radioligand R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine bound to membranes prepared from rat ventricular myocytes in a saturable manner with a B _max of 17.7 fmol/mg protein and a K _D-value of 1.1 nmol/l. Adenosine analogues competed for the binding with the same rank order of potency as for the inhibition of the isoprenaline-induced cAMP-increase. GTP inhibited radioligand binding with an IC₅₀-value of 73 ol/l. These results suggest the presence of A₁ adenosine receptors on rat ventricular myocytes, which mediate an inhibition of adenylate cyclase. The receptors may be responsible for the effects of adenosine and its analogues on the heart.Abbreviations ¹²⁵I-HPIA R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine - PIA N⁶-phenylisopropyladenosine - NECA 5-N-ethyl-carboxamidoadenosine - XAC 8-4-[([(2-aminoethyl)aminocarbonyl]methyl)oxy]phenyl-1,3-dipropylxanthine (xanthine amine congener) - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone - ScAMPTME 2-O-monosuccinyladenosine-3,5-cyclic monophosphate tyrosyl methyl ester - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - GTP guanosine-5-tri-phosphate Send offprint requests to D. Martens     

Effect of maitotoxin on calcium current and background inward current in isolated ventricular rat myocytes

Maitotoxin (MTX) irreversibly suppressed the voltage-dependent calcium current after a variable delay, an effect which was preceded, in 61% of the cells, by a transient increase in calcium current partly attributable to a shift (4-7 mV) of the activation curve towards negative potentials. MTX also induced the development of a voltage-independent background inward current which did not occur in the absence of external calcium and was reduced by removal of external sodium, by calcium channel blockers and by high concentrations of quinidine. MTX-induced single channel activity consisted of long lasting bursts of inward current. Channel activity was voltage-independent, with a unitary conductance of 14 pS and an extrapolated reversal potential of +16 mV. Single-channel current amplitude was not detectably reduced in the absence of external calcium but strongly reduced in the absence of external sodium, in the presence of 2 mM nickel or when external sodium was replaced by 96 mM calcium or 50 mM barium. The channel activity was also inhibited by quinidine. It is concluded that MTX alters, then suppresses the voltage-activated calcium current and induces the development of a voltage-independent inward current, part of which results from the opening of nickel-sensitive cation channels, mostly permeable to sodium ions.     

A novel anionic conductance affects action potential duration in isolated rat ventricular myocytes

Effects of extracellular anions were studied in electrophysiological experiments on freshly isolated rat ventricular myocytes. Under current-clamp, action potential duration (APD) was prolonged by reducing the extracellular Cl(-) concentration and shortened by replacement of extracellular Cl(-) with I(-). Under voltage-clamp, membrane potential steps or ramps evoked an anionic background current (I(AB)) carried by either Cl(-), Br(-), I(-) or NO(3)(-). Activation of I(AB) was Ca(2+)- and cyclic AMP-independent, and was unaffected by cell shrinkage. I(AB) was insensitive to stilbene and fenamate anion transport blockers at concentrations that inhibit Ca(2+)-, cyclic AMP- and swelling-activated Cl(-) currents in ventricular cells of other mammals. These results suggest that I(AB) may be carried by a novel class of Cl(-) channel. Correlation of anion substitution experiments on membrane current and action potentials revealed that I(AB) could play a major role in controlling rat ventricular APD. These findings have important implications for those studying cardiac Cl(-) channels as potential targets for novel antiarrythmic agents.     

Activation of chloride current by P2-purinoceptors in rat ventricular myocytes.

1. Rat ventricular myocytes were dissociated and their responses to extracellularly applied ATP were recorded using patch pipettes under the whole cell configuration. 2. ATP initially induced an inward current followed by an outward current at -50 mV. With a Cs-rich pipette solution the late outward current was blocked, leaving a sustained inward current (IATPs) suggesting that a K+ conductance underlies the late response. 3. When the extracellular Cl- concentration was changed, the reversal potential of IATPs corresponded well to the shift of the Cl- equilibrium potential. IATPs was reversibly blocked by the chloride channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). 4. The concentration-response curve of IATPs had a Hill coefficient of 0.98 and an EC50 value of 5.2 x 10(-6) M. 5. ATP was more potent than ADP, while AMP and adenosine were ineffective, suggesting that P2-purinoceptor activation induced IATPs. 6. The activation of IATPs was depressed by depleting the extracellular Mg2+ and increased by adding Mg2+. 7. Our results strongly suggest that P2-purinoceptor activation by ATP induces both a Cl(-)-conductance (IATPs) and a K(+)-conductance in rat ventricular myocytes.     

Novel spiropiperidines as highly potent and subtype selective sigma-receptor ligands. Part 1.

A series of spiro[[2]benzopyran-1,4'-piperidines] and spiro[[2]benzofuran-1,4'-piperidines] of general structure 10 is prepared, and the affinity for sigma(1)- and sigma(2)-receptors is investigated by means of radioligand binding assays. The synthesis of the spiropiperidines 14a and 23 proceeds from bromine/lithium exchange of the bromoacetals 11 and 21, addition to piperidin-4-one 12a, and subsequent cyclization. Systematic variations of the substituent R at the nitrogen atom, the group X in position 3, and the ring size of the oxygen heterocycle are performed. The sigma(1)- and sigma(2)-receptor affinities are determined with guinea pig brain and rat liver membrane preparations using [(3)H]-labeled (+)-pentazocine and ditolylguanidine, respectively. Test results show that a benzyl residue at the piperidine nitrogen atom and a methoxy group in position 3 are advantageous for high sigma(1)-receptor affinity. In this series the 1'-benzyl-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,4'-piperidine] (14a) and the 1'-benzyl-3-methoxy-3H-spiro[[2]benzofuran-1,4'-piperidine] (23) are among the most potent sigma(1)-ligands interacting in the low nanomolar range with sigma(1)-receptors (14a, K(i) = 1.29 nM; 23, K(i) = 1.14 nM). Variation of the nitrogen substituent R from benzyl to H, alkyl, phenyl, or omega-phenylalkyl and the group X from methoxy to hydroxy, carbonyl, or alkyloxy led to reduced sigma(1)-receptor affinity. In addition to their high sigma(1)-receptor affinity, the spiropiperidines 14a and 23 display excellent selectivity toward sigma(2)-receptors (sigma(1)/sigma(2) = 2708 and 1130) and several other receptor and reuptake systems. Introduction of a polar hydroxy group in position 3 and elongation of the distance between the piperidine nitrogen atom and the phenyl moiety result in ligands with considerable sigma(2)-receptor affinity and therefore diminished sigma(1)/sigma(2)-receptor selectivity. The hemiacetalic 1'-(3-phenylpropyl)-3,4-dihydrospiro[[2]benzopyran-1,4'-piperidin]-3-ol (15e) represents the most active sigma(2)-receptor ligand in this series with a K(i) value of 83.1 nM.     

Effects of N-acetylprocainamide and sotalol on ion currents in isolated guinea-pig ventricular myocytes.

The effects of N-acetylprocainamide (NAPA) and sotalol on membrane current systems of guinea-pig ventricular myocytes were examined and compared with those of quinidine using patch-clamp techniques. All of the drugs prolonged the action potential duration (i.e. Class III effect) in isolated guinea-pig papillary muscles. In isolated ventricular cells. NAPA (300 microM) and sotalol (100 microM) produced a decrease in the delayed outward potassium current (IK) concomitantly with a slight decrease in the calcium current (ICa), which was similar to quinidine (10 microM). NAPA also slightly depressed the inward rectifier potassium current (IKrect). Thus, NAPA and sotalol both inhibited IK, and this action appears to be mainly responsible for their Class III effect.     

高胆固醇血症致大鼠心室肌细胞L-型钙电流失衡的机制

目的研究高胆固醇血症(HC)对大鼠心室肌细胞L-型钙电流(ICa-L)以及细胞内钙浓度[Ca2+]i的影响。方法Wistar大鼠随机分为高胆固醇血症组和对照组各6只,分别给予高胆固醇饲料和普通饲料饲养4wk后,检测血脂;酶解法急性分离大鼠单个心肌细胞,采用全细胞膜片钳技术和激光扫描共聚焦显微镜观察高胆固醇血症对大鼠心室肌细胞ICa-L以及[Ca2+]i的变化。结果喂高胆固醇饲料4wk后,血清总胆固醇较正常对照组明显增高(P<0·01),甘油三脂无明显变化。膜片钳显示:高胆固醇血症大鼠各实验电压L-型钙电流电流密度均减少。在实验电压为0mV时,大鼠心室肌细胞L-型钙电流密度从正常对照组(-8·56±1·29)pA/pF减少到高胆固醇血症组(-5·24±0·90)pA/pF。激光共聚焦显示:在静息状态下,[Ca2+]i升高,峰值荧光强度由211·88±9·08增加至458·63±23·50(P<0·01)。结论高胆固醇血症可导致细胞内钙超载,使L-型钙通道电流失衡,可能是诱导心律失常的原因之一。     

Caffeine inhibits depolarization-activated outward currents in rat ventricular myocytes.

The effects of caffeine (10 mM) on depolarization-activated, calcium-independent outward K+ currents were investigated in isolated rat ventricular myocytes, using whole-cell clamping. The external solution contained CoCl2 2 mM and the internal solution contained ethylene glycol-bis(-aminoethyl ether) N,N,N',N'-tetraacetic acid 10 mM. Caffeine decreased the peak amplitude of the total current and the sustained plateau current. Caffeine did not modify the steady state inactivation curve, which was fitted by two Boltzmann functions. Caffeine blocked the tetraethylammonium-sensitive slowly activating and inactivating outward current by 32% and the 4-aminopyridine-sensitive rapidly activating and inactivating transient outward current by 19%. Caffeine did not modify the inactivation rate or the time course of the recovery from inactivation of the transient current. Ryanodine 10 microM did not modify any of the current components and the effect of caffeine was not modified by ryanodine pretreatment. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine 100 microM, did not modify the depolarization-activated calcium-independent outward currents.     

Effects of AMP579 and adenosine on L-type Ca^2＋ current in isolated rat ventricular myocytes

Aim: To compare the effects of AMP579 and adenosine on L-type Ca^2 current (ICa-L) in rat ventricular myocytes and explore the mechanism by which AMP579 acts on ICa-L.Methods: ICa-L was recorded by patch-clamp technique in whole-cell configuration. Results: Adenosine (10nmol/L to 50μmol/L) showed no effect on basal ICa-L, but it inhibited the ICa-L induced by isoproterenol 10nmol/L in a concentration-dependent manner with the IC50 of 13.06μmol/L. Similar to adenosine,AMP579 also showed an inhibitory effect on the ICa-L induced by isoproterenol.AMP579 and adenosine (both in 10μmol/L) suppressed isoproterenol-induced ICa-L by 11.1% and 5.2%, respectively. In addition, AMP579 had a direct inhibitory effect on basal ICa-L in a concentration-dependent manner with IC50(1. 17μmol/L). PD116948 (30μmol/L), an adenosine A1 receptor blocker, showed no action on the inhibitory effect of AMP579 on basal ICa-L. However, GF109203X (0.4μmol/L), a special protein kinase C (PKC) blocker, could abolish the inhibitory effect of AMP579 on basal ICa-L. So the inhibitory effect of AMP579 on basal ICa-L was induced through activating PKC, but not linked to adenosine A1 receptor. Conclusion:AMP579 shows a stronger inhibitory effect than adenosine on the ICa-L induced by isoproterenol. AMP579 also has a strong inhibitory effect on basal ICa-L in rat ventricular myocytes. Activation of PKC is involved in the inhibitory effect of AMP579 on basal ICa-L at downstream-mechanism.     

Effects of bile acids on ventricular muscle contraction and electrophysiological properties: studies in rat papillary muscle and isolated ventricular myocytes

Summary The effects of sodium salts of various bile acids on the contractile force and the electrophysiological properties of rat ventricular muscle were studied in vitro. Primary, conjugated, and secondary bile acids were studied in a concentration range of 10^–9–10^–6 mol/l, which corresponds to concentrations found in the plasm of patients with cholestatic jaundice. In general, the bile acid induced a negative inotropic effect which was manifested as a reduction in active tension, maximum rate of tension activation, and maximum rate of tension relaxation. Twitch duration and time to peak tension were unaffected by the bile acids. The negative inotropism was associated with a reduction in ventricular action potential duration. Resting potential, action potential amplitude, and maximum upstroke velocity of phase 0 depolarization were unaffected. Voltage clamp experiments in rat ventricular myocytes demonstrated that sodium taurocholate decreased the slow inward current and slightly increased the outward potassium current. Hence, these effects on the membrane currents are probably responsible for the negative inotropic effect. Send offprint requests to O. Binah at the above address     

Effects of beta-adrenoceptor antagonists on Ca(2+)-overload induced by lysophosphatidylcholine in rat isolated cardiomyocytes.

1. The effects of beta-adrenoceptor antagonists including (-)- and (+)-propranolol, (-)- and (+)-penbutolol, timolol, pindolol, atenolol, acebutolol and practolol on the Ca(2+)-overload induced by lysophosphatidylcholine (LPC) were examined in isolated cardiomyocytes of the rat. 2. Fura-2 was used for measurement of the intracellular calcium concentration ([Ca2+]i). LPC (15 microM) produced a rapid increase in [Ca2+]i from 72 +/- 5 to 3042 +/- 431 nM which coincided with a decrease in the percentage of rod-shaped cells from 69 +/- 2 to 5 +/- 2%. 3. Preincubation with (-)-propranolol (20 microM), (+)-propranolol (50 microM), or (-)- or (+)-penbutolol (20 microM), the lipophilicity of which is higher than other beta-adrenoceptor antagonists, significantly inhibited both the increase in [Ca2+]i and the cell-shape change induced by 15 microM LPC. The inhibitory effects of the four drugs on the LPC-induced increase in [Ca2+]i and cell-shape change were concentration-dependent. The IC50S of (-)-propranolol, (+)-propranolol, (-)- and (+)-penbutolol for the increase in [Ca2+]i were 1.28, 10.50, 0.67 and 0.76 microM, respectively. 4. Pretreatment with pindolol, timolol, acebutolol, practolol, atenolol or lignocaine did not inhibit the increase in [Ca2+]i and the morphological change induced by LPC. 5. LPC markedly increased the release of creatine phosphokinase from 9 +/- 1 to 45 +/- 2% which could be significantly reduced by (-)- or (+)-propranolol but not by acebutolol or timolol. 6. The protective effects of (-)- and (+)-propranolol, (-)- and (+)-penbutolol against the Ca(2+)-overload induced by LPC were not associated with the beta-adrenoceptor antagonistic action, but probably with an unknown action which is related to the preservation of membrane integrity. Further studies are necessary to clarify the exact mechanisms of the protective action of these beta-adrenoceptor antagonists against the Ca(2+)-overload induced by LPC.     

Effect of urocortin on L-type calcium currents in adult rat ventricular myocytes.

The newly isolated peptide, urocortin (UCN) has been found to have potent cardioprotective effects. In order to investigate the effect of UCN on L-type calcium currents (I(Ca,L)), exploring the mechanisms of UCN's cardioprotective effects, we directly measured the I(Ca,L) in the adult rat cardiac myocytes exposed to UCN using standard whole-cell patch-clamp recording technique. Our results showed that UCN exerted decreasing effects on the I(Ca,L) of the single adult rat cardiac myocytes. The current density was inhibited by about 35% after exposure of the cells to UCN (0.1 micromol L(-1)) for 10 min, from the control value of 7.19 +/- 1.44 pA/pF to 4.74 +/- 0.75 pA/pF (n = 5, P < 0.05). This I(Ca,L)-inhibiting action of UCN was concentration dependent. Moreover, no frequency dependence of UCN effects on I(Ca,L) was observed. In combination with previous reports, our results suggest that there might be a close relationship between the cardioprotective effects of UCN and L-type calcium channels.