Aspects of the intercellular communication in aged hearts: effects of the gap junction uncoupler palmitoleic acid

Although cardiac arrhythmia is among the most common causes of death and the percentage of aged people in our population is steadily increasing, only little is known on age-dependent changes in intercellular coupling, anisotropy and electrophysiology in mammalian heart. Thus, we wanted to investigate electrophysiology and anisotropy in aged vs. young rabbit heart, as well as the response to the gap junction uncoupler palmitoleic acid. Spontaneously beating hearts of young mature (6+/-1 months, n=14) and aged (32+/-5 months, n=9) male White New Zealand rabbits (Langendorff technique) were submitted to epicardial 256-channel potential mapping. Cumulative concentration-response curves for palmitoleic acid (0.2-20.0 microM) were carried out. At certain time points anisotropy was measured by application of rectangular pulses and the determination of longitudinal and transversal conduction velocity. Finally, hearts were processed histologically for Van Gieson staining or connexin43 immunostaining. In spontaneously beating aged hearts we found enhanced dispersion of activation-recovery intervals (15.9+/-1.6 ms vs. 10.8+/-2.0 ms, P<0.05), prolonged (31.2+/-1.4 ms vs. 25.2+/-1.8 ms, P<0.05) and fractionated QRS complexes. We found reduced transversal velocity (0.22+/-0.01 m/s vs. 0.27+/-0.02 m/s, P<0.05) and enhanced anisotropy (2.6+/-0.2 vs. 2.0+/-0.1, P<0.05) in aged hearts, while longitudinal velocity was not changed. Histologically, in ventricles from aged hearts we found diffuse deposition of collagen lateral to the fibers and more pronounced expression of connexin43 at lateral cell borders. The functional changes in ventricles from aged hearts were mimicked by application of palmitoleic acid to young hearts in a concentration-dependent manner. In aged hearts this concentration-response curve started at higher initial values, but finally reached similar maximum values. In aged hearts ventricular intercellular coupling transverse to the fiber axis is reduced. Correlates are increased dispersion, slowed transverse conduction and increased anisotropy, and enhanced Cx43 immunostaining at the lateral cell borders. The functional age-dependent changes can be mimicked in young hearts by the gap junction uncoupler palmitoleic acid.     

Vasorelaxant effects of oleamide in rat small mesenteric artery indicate action at a novel cannabinoid receptor

Oleamide (cis-9-octadecenoamide) exhibits some cannabimimetic responses despite its low affinities at the currently known cannabinoid receptors. Here we have investigated whether or not it is a vasorelaxant in rat small mesenteric arteries. Oleamide elicited vasorelaxation (EC50=1.2+/-0.2 microM, Rmax=99.1+/-3.9%, n=8) which was reduced by endothelial removal. Nitric oxide synthase inhibition reduced the response (EC50=5.3+/-1.6 microM, Rmax=59.2+/-7.7%, n=7; P<0.01) as did blockade of Ca2+-sensitive K+ channels (KCa) with apamin plus charybdotoxin (both 50 nM) (EC50=2.1+/-0.2 microM, Rmax=58.4+/-1.9%, n=5; P<0.05). Desensitisation of vanilloid receptors with capsaicin (10 microM for 30 min) shifted the oleamide concentration-response curve approximately 30-fold to the right (n=7; P<0.01). Pertussis toxin (400 ng ml-1 for 2 h) caused a two-fold shift in the response curve (EC50=2.2+/-0.4 microM, Rmax=66.8+/-4.5%, n=6; P<0.01). Rimonabant (CB1 cannabinoid receptor antagonist; SR141716A; 3 microM) significantly inhibited relaxation induced by oleamide (EC50=3.5+/-0.3 microM, Rmax=75.1+/-1.9%; n=8; P<0.05). In contrast, neither the more selective CB1 receptor antagonist, AM251 (1 microM), nor the CB2 antagonist, SR144528 (1 microM), had significant effects. O-1918 (10 microM), a putative antagonist at a novel endothelial cannabinoid receptor (abnormal-cannabidiol site), markedly reduced the relaxation to oleamide (n=7; P<0.01).It is concluded that oleamide responses in the rat isolated small mesenteric artery are partly dependent on the presence of the endothelium, activation of Ca2+-sensitive K+ channels (KC)) and involve capsaicin-sensitive sensory nerves. Oleamide may share a receptor (sensitive to rimonabant and O-1918, and coupled to KC) and Gi/o) with anandamide in this vessel. This might be distinct from both of the known cannabinoid receptors and the novel abnormal-cannabidiol site.     

Role of gap junctions in endothelium-derived hyperpolarizing factor responses and mechanisms of K(+)-relaxation

We have examined the effects of ouabain (1 mM), the gap junction inhibitors, 18 alpha-glycyrrhetinic acid (100 microM), N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A; 10 microM) and palmitoleic acid (50 microM), and clotrimazole (10 microM) against endothelium-derived hyperpolarizing factor (EDHF)-mediated and K(+)-induced vasorelaxations in the rat mesentery. In the presence of indomethacin (10 microM) and 300-microM N(G)nitro-L-arginine methyl ester (L-NAME), carbachol caused EDHF-mediated relaxations (R(max)=85.3+/-4.0%). In the presence of ouabain, these responses were substantially reduced (R(max)=11.0+/-2.3%). 18 alpha-glycyrrhetinic acid, SR141716A, palmitoleic acid and clotrimazole also significantly inhibited these EDHF-mediated responses. K(+) caused vasorelaxation of preparations perfused with K(+)-free buffer (R(max)=73.7+/-2.4%), which were reduced by 10-microM indomethacin (R(max)=56.4+/-6.2%). K(+) vasorelaxation was essentially abolished by endothelial denudation. Both ouabain and 18 alpha-glycyrrhetinic acid opposed K(+) relaxations, however, neither SR141716A, clotrimazole nor palmitoleic acid had any effect. Direct cell-cell coupling via gap junctions was attenuated by ouabain, clotrimazole and palmitoleic acid. We conclude that: (i) that gap junctional communication plays a major role in EDHF-mediated relaxations, (ii) that K(+)-vasorelaxation is endothelium-dependent (thus, K(+) is unlikely to represent an EDHF), and (iii) that the inhibitory actions of ouabain and clotrimazole on gap junctions might contribute towards their effects against EDHF.     

Comparative cardiac effects of KT-362 and verapamil in isolated heart--correlation to calcium channel current depression.

Direct cardiac effects of KT-362 (5-[3 [[-2-(3,4-dimethoxyphenyl)-ethyl]amino]-1-oxopropyl]-2,3,4,5- tetrahydro-1,5-benzothiazepine fumarate), a drug that may inhibit intracellular calcium mobilization as well as extracellular calcium influx was compared to verapamil. Guinea pig hearts (n = 19) were used to examine the changes in atrial rate, atrioventricular conduction time (AVCT), coronary flow, myocardial oxygen consumption (MVO2), and isovolumetric left ventricular pressure (LVP). Both drugs concentration-dependently and reversibly decreased atrial rate, contractility, and MVO2; AVCT increased during spontaneous rhythm. The increases in AVCT and the incidence of AV dissociation were accentuated during cardiac pacing. Verapamil significantly increased coronary flow, while KT-362 did not. Median effective concentration (EC50) was about 25 times lower for verapamil in depressing LVP and about three times lower in depressing atrial rate and AV conduction. The changes in calcium channel current in voltage-clamped single canine Purkinje cells (n = 6) were also examined. Verapamil (0.3 microM) and KT-362 (7 microM) decreased peak Ca2+ channel current at maximum activation (+10 mV) by 38.1 +/- 8% and 28.6 +/- 6%, respectively, without shifting the current-voltage relationship. This study indicates that verapamil is more potent than KT-362 in depressing contractile function, heart rate, and AV conduction in isolated hearts and calcium current in isolated cardiac Purkinje cells. Moreover, there was a much greater difference between the EC50 for verapamil and that for KT-362 for the depression of indices of contractility (23-30-fold) than for the depression of sinoatrial and atrioventricular nodal function (2.5-4-fold).     

Expression of beta 2-adrenoceptors mediating cyclic AMP accumulation in astroglial and neuronal cell lines derived from the rat CNS

The effect of isoprenaline on cyclic AMP accumulation has been investigated in the rat neuronal cell line B50 and the rat astrocytoma cell line C6. Noradrenaline and isoprenaline stimulated cyclic AMP accumulation in both cell lines. Isoprenaline (0.5 microM; EC50 = 0.1 microM) produced a rapid (T1/2 = 1.3 min) increase in [3H]cyclic AMP accumulation in B50 cells while the response to isoprenaline (0.1 microM; EC50 = 0.01 microM) in C6 cells was somewhat slower (T1/2 = 7.5 min). The response to 0.5 microM isoprenaline was antagonized by both propranolol (IC50 = 8.4 +/- 1.6 nM; N = 3) and the beta 2-selective antagonist ICI 118551 (IC50 = 2.1 +/- 0.2 nM; N = 6). However, no attenuation of the response to isoprenaline (0.5 microM) was observed at concentrations of the beta 1-adrenoceptor antagonist atenolol up to 10 microM (N = 3). In contrast, in C6 cells, which have previously been shown to possess beta 1-adrenoceptors, atenolol inhibited isoprenaline-induced (0.1 microM) cyclic AMP accumulation (IC50 = 2.0 +/- 0.5 microM; N = 6). Furthermore, the beta 2-selective antagonist ICI 118551 was much less potent in the C6 cell line (IC50 = 0.2 +/- 0.05 microM; N = 3) than in the B50 cells. In conclusion, the present data suggest that isoprenaline mediates cyclic AMP accumulation in the neuronal cell line via activation of beta 2-adrenoceptors, while in the astrocytoma cell line the cyclic AMP response is mediated by beta 1-adrenoceptors.     

Stereoselective interaction of thiopentone enantiomers with the GABA(A) receptor.

1. As pharmacokinetic differences between the thiopentone enantiomers seem insufficient to explain the approximately 2 fold greater potency for CNS effects of (-)-S- over (+)-R-thiopentone, this study was performed to determine any enantioselectivity of thiopentone at the GABA(A) receptor, the primary receptor for barbiturate hypnotic effects. 2. Two electrode voltage clamp recording was performed on Xenopus laevis oocytes expressing human GABA(A) receptor subtype alpha1beta2gamma2 to determine relative differences in potentiation of the GABA response by rac-, (+)-R- and (-)-S-thiopentone, and rac-pentobarbitone. Changes in the cellular environment pH and in GABA concentrations were also evaluated. 3. With 3 microM GABA, the EC50 values were (-)-S-thiopentone (mean 26.0+/-s.e.mean 3.2 microM, n=9 cells) >rac-thiopentone (35.9+/-4.2 microM, n=6, P=0.1) >(+)-R-thiopentone (52.5+/-5.0 microM, n=8, P<0.02) >rac-pentobarbitone (97.0+/-11.2 microM, n=11, P<0.01). Adjustment of environment pH to 7.0 or 8.0 did not alter the EC50 values for (+)-R- or (-)-S-thiopentone. 4 Uninjected oocytes responded to >100 microM (-)-S- and R-thiopentone. This direct response was abolished by intracellular oocyte injection of 1,2-bis(2-aminophenoxy)ethane-N, N,N1,N1-tetraacetic acid (BAPTA), a Ca2+ chelating agent. With BAPTA, the EC50 values were (-)-S-thiopentone (20.6+/-3.2 microM, n=8) <(+)-R-thiopentone (36.2+/-3.2 microM, n=9, P<0.005). 5 (-)-S-thiopentone was found to be approximately 2 fold more potent than (+)-R-thiopentone in the potentiation of GABA at GABA(A) receptors expressed on Xenopus oocytes. This is consistent with the differences in potency for CNS depressant effects found in vivo.     

粉防己碱对麻醉兔心内传导系统的影响

在麻醉兔观察了粉防已碱对心内传导功能的影响,粉防已碱8mg/kg iv后10min,明显延长兔SACT、SNRT和SNRTc,使SNRTI增大,说明其对窦房结传导功能和自律功能有显著的抑制作用。粉防已碱恒速iv 3～15mg/kg,剂量依赖性地抑制房-室传导,使HBE A-H和ECG P-R延长,但对H-V和V波无明显影响,这与奎尼丁不同。结果表明粉防已碱只抑制室上性传导,对室内传导无影响。     

Block of human voltage-sensitive Na+ currents in differentiated SH-SY5Y cells by lifarizine.

1. The ability of lifarizine (RS-87476) to block human voltage-sensitive Na+ channel currents was studied by use of whole cell patch clamp recording from differentiated neuroblastoma cells (SH-SY5Y). 2. The Na+ conductance in differentiated SH-SY5Y cells (24.0 +/- 2.4 nS, n = 11) was half-maximally activated by 10 ms depolarizations to -37 +/- 2 mV and was half-maximally inactivated by predepolarizing pulses of 200 ms duration to -86 +/- 3 mV (n = 11). 3. At low stimulus frequencies (0.1 to 0.33 Hz) voltage-dependent sodium currents were completely blocked, in a concentration-dependent manner, by extracellular application of either tetrodotoxin (EC50 = 4 +/- 1 nM, n = 12) or by lifarizine (EC50 = 783 +/- 67 nM, n = 9). The onset of block by lifarizine (tau = 91 +/- 14 s at 10 microM) was considerably slower than that of tetrodotoxin (tau = 16 +/- 3 s at 100 nM). 4. Lifarizine (1 microM) reduced the peak sodium conductance in each cell (from 26.4 +/- 2.0 nS to 15.1 +/- 2.7 nS, n = 4) without changing the macroscopic kinetics of sodium current activation or inactivation (V1/2 = -35 1 mV and -87 +/- 4 mV respectively, n = 4). Similarly, lifarizine (1 microM) did not affect the reversal potential of the macroscopic sodium current (+14 +/- 5 mV in control and +16 +/- 2 mV in 1 microM lifarizine; n = 4) or reactivation time-constant (tau = 14.0 +/- 4.4 ms).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological differences between the human and rat vanilloid receptor 1 (VR1)

Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells. Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with similar EC(50) values. Capsaicin had a lower potency on both channels, when measured electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90+/-0.20 microM; human=1.90+/-0.30 microM: CHO cells: rat=0.20+/-0.06 microM; human=0.19+/-0.08 microM). In CHO cell lines co-expressing either rat or human VR1 and the calcium sensitive, luminescent protein, aequorin, the EC(50) values for capsaicin-induced responses were similar in both cell lines (rat=0.35+/-0.06 microM, human=0.53+/-0.03 microM). The threshold for activation by acidic solutions was lower for human VR1 channels than that for rat VR1 (EC(50) pH 5.49+/-0.04 and pH 5.78+/-0.09, respectively). The threshold for heat activation was identical (42 degrees C) for rat and human VR1. PPAHV was an agonist at rat VR1 (EC(50) between 3 and 10 microM) but was virtually inactive at the human VR1 (EC(50)>10 microM). Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of human VR1 than rat VR1. Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also more effective at inhibiting the noxious heat response of human than of rat VR1.     

Rotigaptide (ZP123) improves atrial conduction slowing in chronic volume overload-induced dilated atria

Chronic atrial dilation is associated with atrial conduction velocity slowing and an increased risk of developing atrial tachyarrhythmias. Rotigaptide (ZP123) is a selective gap junction modifier that increases cardiac gap junctional intercellular communication. We hypothesised that rotigaptide treatment would increase atrial conduction velocity and reduce the inducibility to atrial tachyarrhythmias in a model of chronic volume overload induced chronic atrial dilatation characterized by atrial conduction velocity slowing. Chronic volume overload was created in Japanese white rabbits by arterio-venous shunt formation. Atrial conduction velocity and atrial tachyarrhythmias inducibility were examined in Langendorff-perfused chronic volume overload hearts (n=12) using high-resolution optical mapping before and after treatment with rotigaptide. Moreover, expression levels of atrial gap junction proteins (connexin40 and connexin43) were examined in chronic volume overload hearts (n=6) and compared to sham-operated controls (n=6). Rotigaptide treatment significantly increased atrial conduction velocity in chronic volume overload hearts, however, rotigaptide did not decrease susceptibility to the induction of atrial tachyarrhythmias. Protein expressions of Cx40 and Cx43 were decreased by 32% and 72% (P<0.01), respectively, in chromic volume overload atria compared to control. To conclude, rotigaptide increased atrial conduction velocity in a rabbit model of chromic volume overload induced atrial conduction velocity slowing. The demonstrated effect of rotigaptide on atrial conduction velocity did not prevent atrial tachyarrhythmias inducibility. Whether rotigaptide may possess antiarrhythmic efficacy in other models of atrial fibrillation remains to be determined.     

索他洛尔在房室旁道射频消融术中的应用

目的探讨索他洛尔在房室旁道射频消融(RFCA)术中的应用。方法18例旁道患者在常规电生理检查后顿服索他洛尔160mg,分别于服药后30、60、90、120和150min重复测量各项电生理参数。结果服药前后心房(A)、心室(V)、房室结(AVN)、旁道前传(APa)、旁道逆传(APr)有效不应期(ERP)分别为(211±24)msvs(243±36)ms、(205±11)msvs(242±28)ms、(269±48)msvs(343±97)ms、(263±45)msvs(400±160)ms、(232±37)msvs(298±50)ms(P<0.05)。无1例旁道阻断。窦房结恢复时间(SNRT)、窦性周长(SCL)分别为(1165±209)msvs(1456±371)ms、(724±116)msvs(996±178)ms(P<0.05)。3例房颤服药后不再诱发,2例心内电刺激难以终止的房室折返性心动过速(AVRT)服药后自行终止于房室结前传。18例患者均消融成功,随访7～18个月,无1例复发。结论索他洛尔显著延长心脏ERP,但不影响旁道的RFCA,能够预防或抑制RFCA术中导管和电刺激诱发的房颤,方便了手术操作。     

Role of nitric oxide and guanosine 3',5'-cyclic monophosphate in mediating nonadrenergic, noncholinergic relaxation in guinea-pig pulmonary arteries.

1. Nonadrenergic, noncholinergic (NANC) nerves mediate vasodilatation in guinea-pig pulmonary artery (PA) by both endothelium-dependent and endothelium-independent mechanisms. The transmitter(s) involved in the endothelium-independent pathway have not yet been identified. We have therefore investigated the possibility that nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) may mediate this neural vasodilator response in guinea-pig branch PA rings denuded of endothelium. 2. Electric field stimulation (EFS, 50 V, 0.2 ms) induced a frequency-dependent (1-24 Hz), tetrodotoxin-sensitive relaxation of the U44069-precontracted PA rings in the presence of adrenergic and cholinergic blockade. 3. The NO synthase inhibitors NG-monomethyl L-arginine (L-NMMA, 100 microM) and NG-nitro L-arginine methyl ester (L-NAME, 30 microM), and the guanylyl cyclase inhibitor methylene blue (5 microM) inhibited the EFS (16 Hz)-induced relaxation by 53 +/- 5, 74 +/- 9 and 82 +/- 9% respectively (n = 5-7, P < 0.01, compared with control rings). 4. Excess concentrations of L-, but not D-arginine (300 microM) completely reversed the inhibitory effect of L-NMMA. 5. The EFS-elicited relaxation (4 Hz) was potentiated by 1 microM zaprinast, a type V phosphodiesterase inhibitor which inhibits guanosine 3':5'-cyclic monophosphate (cyclic GMP) degradation, but was unaffected by 0.1 microM zardaverine, a type III/IV phosphodiesterase inhibitor which inhibits cyclic AMP degradation. 6. EFS (50 V, 0.2 ms, 16 Hz) induced a 3 fold increase in tissue cyclic GMP content, an action which was inhibited by L-NMMA (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

甲基莲心碱对兔心内传导系统的影响

在麻醉兔观察了甲基莲心碱(Nef)对心内传导功能的影响。Nef 4 mg/kg iv明显延长兔SACT和CSNRT,增大SNRTI,表明对窦房结传导功能有显著抑制作用。Nef恒速iv 1～8 mg/kg,剂量依赖性地延长兔HBE A—H和H—V,增宽V波,使ECGP—R延长,这些作用与奎尼丁相似。结果表明Nef对心脏房—室、希氏束—浦肯野纤维—心室肌的传导有显著的抑制作用。     

Effects of bimakalim on human cardiac action potentials: comparison with guinea pig and nicorandil and use-dependent study

Electrophysiologic effects of K(ATP) channel openers (KCOs) are rarely studied for tissue and species specificity, and use-dependent investigations in human tissues are lacking. We therefore investigated in vitro the concentration-dependent effects of the KCO bimakalim [from 10 nM to 10 microM, at 1,000 ms of cycle length (CL) and 37 degrees C] on human (atrium, n = 4, and ventricle, n = 6) and guinea pig (atrium, n = 7, and ventricle, n = 6) transmembrane action potential (AP). The frequency relation (from CL 1,600 to 300 ms, 31 degrees C) of human atrial AP duration 90% (APD90) shortening (10 microM vs. baseline, n = 7) also was determined. A parallel study was performed with the KCO nicorandil (from 10 nM to 1 mM, n = 3) in human atrial APs, at 31 degrees C. Resting membrane potential and maximal upstroke velocity of AP were not modified by bimakalim at maximal concentration, whereas AP amplitude was decreased in both guinea pig preparations (p < 0.05); APD90 was shortened in all tissues (p < 0.01). Median effective concentration (EC50) for APD90 shortening at 37 degrees C was 0.54 and 2.74 microM in atrial and ventricular human tissue, respectively, and 8.55 and 0.89 microM in atrial and ventricular guinea pig tissue, respectively. In human atrial tissue at 31 degrees C, EC50 with bimakalim was 0.39 microM; a much higher value was seen with nicorandil (210 microM). Bimakalim (10 microM)-induced APD90 shortening as a function of stimulation rate was greatest at longest CL. Evidence is provided for (a) species (human vs. guinea pig) and tissue (atrium vs. ventricle) differential AP sensitivity to bimakalim; (b) an approximately 500-fold higher efficacy of bimakalim versus nicorandil to shorten human atrial APD90; and (c) normal use-dependence of human atrial APD90 shortening with bimakalim at 10 microM.     

Pharmacokinetic-pharmacodynamic analysis of the arrhythmogenic potency of a novel antiallergic agent, ebastine, in rats

Ebastine (EBS), a novel nonsedative antiallergic agent, is similar to terfenadine in its chemical structure. However, clinical arrhythmogenicity of EBS remains controversial. In this study, we evaluated the possible arrhythmogenic potency of EBS as assessed by QT prolongation from a pharmacokinetic-pharmacodynamic viewpoint in comparison with that of terfenadine. EBS was intravenously infused into anesthetized rats at a rate of 3.0 or 10 mg/kg/h for 60 min, and electrocardiographic effects were continuously monitored from lead II. The plasma concentrations of EBS and its major metabolite, carebastine, were also measured under the same conditions. When intravenously administered, EBS exhibited QT prolongation in an infusion rate-dependent manner, with a lag time. Pharmacokinetic-pharmacodynamic analysis of EBS based on the effect-compartment model revealed values of EC50, Emax and EC(10 ms), (where 10 ms of QT prolongation was evoked) of 0.73 microg/mL, 12.5 ms and 2.90 microg/mL, respectively. The EC(10 ms) value of EBS was five times higher than that of terfenadine reported previously (Ohtani et al., J. Pharm. Pharmacol., 49, 458-462 (1997)). In conclusion, EBS was suggested to be less arrhythmogenic than terfenadine.     

Alphavbeta3 integrin binding affinity and specificity of SM256 in various species

This study was undertaken to define the alphavbeta3 binding affinity and specificity of the low-molecular-weight nonpeptide integrin antagonist, SM256. SM256 demonstrated high potency (IC50, 0.057+/-0.030 nM) in inhibiting vitronectin binding to purified human alphavbeta3 receptors. Additionally, SM256 inhibited alphavbeta3-mediated human umbilical vein endothelial cell (HUVEC) or 293/beta3 (beta3-transfected cell line) adhesion to fibrinogen with IC50 values of 0.0054+/-0.0058 and 0.0023+/-0.0012 microM, respectively. SM256 demonstrated a relatively high degree of specificity for human alphavbeta3-mediated functions as compared with other human integrins including alphavbeta5 (IC50, 0.92+/-0.69 microM), alphaIIbbeta3 (IC50, 0.72+/-0.07 microM), alpha4/beta1 (IC50, >100 microM) and alpha5/beta1 (IC50, 2.3+/-2.1 microM). SM256 demonstrated different degree of species specificity in blocking alphavbeta3-mediated cellular adhesion with relatively higher affinity to dog (IC50, 0.005+/-0.002 microM), rabbit (IC50, 0.021+/-0.01 microM), mouse (IC50, 0.035+/-0.01 microM), and pig (IC50, 0.41+/-0.24 microM) endothelial or smooth-muscle cell alphavbeta3-mediated adhesion. Additionally, SM256 demonstrated high degree of alphavbeta3 specificity as compared with alphavbeta5, alpha5beta1, or alphaIIbbeta3-mediated binding in these species. SM256 is a potent alphavbeta3, antagonist with high affinity and specificity for alphavbeta3-mediated functions. Additionally, a comparable alphavbeta3 affinity for SM256 was demonstrated with endothelial cells obtained from various species (dog, mouse, rabbit, and pig) as compared with that from human.     

A comparison of an A1 adenosine receptor agonist (CVT-510) with diltiazem for slowing of AV nodal conduction in guinea-pig

1. The purpose of this study was to compare the pharmacological properties (i.e. the AV nodal depressant, vasodilator, and inotropic effects) of two AV nodal blocking agents belonging to different drug classes; a novel A1 adenosine receptor (A1 receptor) agonist, N-(3(R)-tetrahydrofuranyl)-6-aminopurine riboside (CVT-510), and the prototypical calcium channel blocker diltiazem. 2. In the atrial-paced isolated heart, CVT-510 was approximately 5 fold more potent to prolong the stimulus-to-His bundle (S-H interval), a measure of slowing AV nodal conduction (EC50 = 41 nM) than to increase coronary conductance (EC50 = 200 nM). At concentrations of CVT-510 (40 nM) and diltiazem (1 microM) that caused equal prolongation of S-H interval (approximately 10 ms), diltiazem, but not CVT-510, significantly reduced left ventricular developed pressure (LVP) and markedly increased coronary conductance. CVT-510 shortened atrial (EC50 = 73 nM) but not the ventricular monophasic action potentials (MAP). 3. In atrial-paced anaesthetized guinea-pigs, intravenous infusions of CVT-510 and diltiazem caused nearly equal prolongations of P-R interval. However, diltiazem, but not CVT-510, significantly reduced mean arterial blood pressure. 4. Both CVT-510 and diltiazem prolonged S-H interval, i.e., slowed AV nodal conduction. However, the A1 receptor-selective agonist CVT-510 did so without causing the negative inotropic, vasodilator, and hypotensive effects associated with diltiazem. Because CVT-510 did not affect the ventricular action potential, it is unlikely that this agonist will have a proarrythmic action in ventricular myocardium.     

Modulation of vasodilatation to levcromakalim by adenosine analogues in the rabbit ear: an explanation for hypoxic augmentation.

1. We have used a rabbit isolated ear, buffered-perfused preparation to investigate the effects of adenosine analogues on the vasodilatation to the potassium channel opener, levcromakalim (the active (-)-enantiomer of cromakalim). We have examined the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 antagonist, on vasodilatation to levcromakalim under hypoxic conditions and also following inhibition of nitric oxide synthesis. 2. Levcromakalim relaxed preconstricted preparations with an EC50 = 369 +/- 48 nM and maximum relaxation of tone (Rmax) = 81.0 +/- 3.2%. In the presence of 1 microM N6-cyclohexyladenosine (CHA) a selective adenosine A1 agonist, there was a significant (P < 0.01) leftward shift in the concentration-response curve with an EC50 = 194 +/- 54 nM and Rmax = 93.2 +/- 2.0%. Conversely, the presence of CHA did not influence vasodilatation to either pinacidil or sodium nitroprusside. 3. Hypoxia also significantly (P < 0.001) increased the vasodilator potency of levcromakalim (EC50 = 134 +/- 22 nM), and this enhancement was completely reversed (EC50 = 380 +/- 107 nM, P < 0.01) by pretreatment of the preparations with 5 microM DPCPX, a selective A1 adenosine antagonist. However, under normoxic conditions DPCPX did not influence vasodilatation to levcromakalim. 4. Inhibition of nitric oxide synthesis with 100 microM NG-nitro-L-arginine methyl ester (L-NAME) caused a significant (P < 0.001) leftward shift in the concentration-response curve to levcromakalim (EC50 = 73.0 +/- 7.6 nM). Pretreatment of preparations with DPCPX partially reversed the increase in potency found in the absence of nitric oxide synthesis (EC50 = 153 +/- 18 nM, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of MEN 10755, a new disaccharide analogue of doxorubicin, on sarcoplasmic reticulum Ca(2+) handling and contractile function in rat heart

1. The use of anthraquinone antineoplastic agents is limited by their cardiac toxicity, which is largely due to activation of the sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor). MEN 10755 is a new disaccharide analogue of doxorubicin. We have evaluated its effects on SR function and its toxicity in isolated working rat hearts. 2. In rat SR vesicles, doxorubicin stimulated [(3)H]-ryanodine binding by increasing its Ca(2+)-sensitivity. At 1 microM Ca(2+), ryanodine binding increased by 15.3+/-2.5 fold, with EC(50)=20.6 microM. Epirubicin produced a similar effect, i.e. 9.7+/-0.6 fold stimulation with EC(50)=11.1 microM. MEN 10755 increased ryanodine binding by 1.9+/-0.3 fold (P:<0.01 vs doxorubicin and epirubicin), with EC(50)=38.9 microM. 3. Ca(2+)-induced Ca(2+) release experiments were performed by quick filtration technique, after SR loading with (45)Ca(2+). At 2 microM Ca(2+), doxorubicin (50 microM) increased the rate constant of Ca(2+) release to 82+/-5 s(-1) vs a control value of 22+/-2 s(-1) (P:<0.01), whereas 50 microM MEN 10755 did not produce any significant effect (24+/-3 s(-1)). 4. Ca(2+)-ATPase activity and (45)Ca(2+)-uptake were not significantly affected by doxorubicin, its 13-dihydro-derivative, epirubicin, MEN 10755 and the 13-dihydro-derivative of MEN 10755, at concentrations < or =100 microM. 5. In isolated heart experiments, administration of 30 microM doxorubicin or epirubicin caused serious contractile impairment, whereas 30 microM MEN 10755 produced only minor effects. 6. In conclusion, in acute experiments MEN 10755 was much less cardiotoxic than equimolar doxorubicin or epirubicin. This result might be accounted for by reduced activation of SR Ca(2+) release.     

Forskolin inhibition of cyclic AMP generation in J774 macrophages

Forskolin inhibited cyclic AMP generation in J774 macrophage cells in response to isoproterenol. Forskolin, 10 nM-0.1 mM, also inhibited the adenylate cyclase activity of membrane preparations. The basal activity and the isoproterenol-, cholera toxin-, fluoride- or GppNHp-stimulated activities were maximally depressed by 10 microM forskolin (30-70% inhibition, EC50 = 0.3-0.5 microM). This effect was achieved similarly in membranes from pertussis toxin-treated cells. Forskolin required guanine nucleotides for inhibition. In the absence of GTP the decrease in basal activity was reversed into stimulation (EC50 = 10 microM forskolin). Reversal of inhibition into activation also followed the addition of 1 mM MnCl2 (EC50 = 10 microM forskolin). 1,9-Dideoxyforskolin was ineffective to alter adenylate cyclase activity. In contrast, a water-soluble derivative of forskolin was as active as forskolin to regulate activity. The results suggest that forskolin may interact with adenylate cyclase to cause either activation or inhibition depending on the degree of activation of Ns and on its interaction with the catalyst.