[Effects of apolipoprotein E deficiency on sphingosine-1-phosphate distribution in plasma and lipoproteins of mice].

Objective: To investigate the effects of apolipoprotein E deficiency (Apo E(-/-)) on plasma and lipoprotein distribution of sphingosine-1-phosphate (S1P) in mice. Methods: Five male or female Apo E(-/-) or wild type (WT) mice were fed with chow diet and sacrificed at 32-week-age and plasma was collected. The constituents of lipoprotein(very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL)) were separated by ultracentrifuge. The protein concentration of constituents was detected by BCA protein quantitative kit, and the S1P concentration in plasma and various lipoprotein constituents was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Western blot was used to determine the plasma, liver, and kidney protein expression of apolipoprotein M(Apo M), which is considered as specific ligand of S1P.The S1P concentration in plasma and various constituents of lipoprotein in the Apo E(-/-) mice was compared to respective WT mice. Results: (1)Plasma S1P content was significantly higher in the Apo E(-/-) groups than that of WT groups (male: (535.7±78.5)nmol/L vs. (263.3±22.0)nmol/L; female: (601.1±64.0)nmol/L vs. (279.0±33.9)nmol/L; all P<0.01). (2) Compared with WT mice, S1P content in non-HDL(LDL+ VLDL) was significantly higher in Apo E(-/-) mice (male: (504.9±52.8)nmol/L vs. (28.7±9.0)nmol/L; female: (427.7±27.4) vs. (27.8±4.7)nmol/L; after standardization of protein concentration, male: (385.0±41.2)pmol/mg protein vs. (71.4±6.6)pmol/mg protein; female: (330.2±22.0)pmol/mg protein vs. (67.2±12.1)pmol/mg protein; all P<0.01). (3) The expression of Apo M in plasma, liver and kidney was significantly higher in Apo E(-/-) groups than that of WT groups(all P<0.05). Conclusion: The deficiency of Apo E could lead to upregulated S1P expression in the non-HDL, the underlying mechanism might be the increased transfer of HDL into the non-HDL by Apo M-S1P.

Electrophysiologic effects of interactions between activated canine neutrophils and cardiac myocytes.

INTRODUCTION: Myocardial ischemia causes neutrophils to bind to activated myocytes and liberate platelet-activating factor (PAF). PAF causes delayed repolarization, early afterdepolarizations (EADs), and arrest of repolarization. We studied the effect of activation of neutrophils bound to canine cardiac myocytes to determine if such activation causes PAF generation and similar changes in transmembrane potentials. METHODS AND RESULTS: Myocytes from canine left ventricle and neutrophils from the same dog were superfused with Tyrode's solution and transmembrane potentials recorded from the former. Neutrophils (100 microL, 10(6)/mL) were added and allowed to bind to the myocytes. Neutrophils were activated with 1% zymosan-activated serum (ZAS). CV-6209 (100 nM) was used to block receptors for PAF. Liberation of PAF by activated neutrophils was quantified with a commercial radioimmunoassay kit. Neutrophils activated with ZAS caused changes in myocyte transmembrane potentials like those induced by PAF: action potential prolongation, runs of EAD, and periods of plateau arrest. PAF receptor blockade prevented neutrophil activation from altering transmembrane potentials. Neutrophils activated with 1% ZAS liberated significant amounts of PAF. CONCLUSIONS: When neutrophils bound to cardiac myocytes are activated by exposure to 1% ZAS, they cause prompt and consistent changes in myocyte electrical activity that could be arrhythmogenic for the in situ heart. These changes are similar to those caused by PAF in pharmacologic studies. Neutrophils activated in this manner generate PAF, and the effects of their activation are prevented by blockade of PAF receptors. We conclude that, during reperfusion of ischemic myocardium, PAF generated by activated neutrophils most likely is a cause of some arrhythmias.

Responses to norepinephrine of normal and "ischemic" canine Purkinje fibers are consistent with activation of different alpha 1-receptor subtypes.

INTRODUCTION: Previously we found that WB4101 (WB) 10(-7) M competitively blocks three alpha 1-adrenergic receptor-effector responses: the increase in normal automaticity occurring in Purkinje fibers (PF) at high membrane potentials; the increase in abnormal automaticity occurring in PF at depolarized membrane potentials; and the prolongation of PF action potential duration. These observations are consistent with two different hypotheses: (1) WB blocks a single alpha 1-receptor subtype, which subserves different effector pathways; and (2) WB blocks different receptor subtypes, each of which subserves an independent pathway. The aim of this study was to test both hypotheses. METHODS AND RESULTS: We used standard microelectrode techniques to study the concentration-dependent actions of three alpha 1-adrenoreceptor blockers (WB [alpha 1A > or = alpha 1D], 5-methylurapidil [5-MU] [alpha 1A > > alpha 1D], and UK52,046 [nonselective]) or norepinephrine (NE) effects in normal PF and in PF depolarized with a simulated ischemic solution ([K+]o = 10 mM; pO2 < 20 mmHg; pH 6.8; maximum diastolic potential -60 +/- 1 mV). In normally polarized PF, concentration-dependent actions of all blockers on both the positive chronotropic response and the prolongation of action potential duration completely coincide. In contrast, the response to NE of abnormal automaticity in "ischemic" PF differs from normals: there is a high sensitivity to WB and 5-MU and no response to UK52,046. CONCLUSIONS: (1) A single receptor subtype appears responsible for both the alpha 1-induced prolongation of repolarization and the positive chronotropic effect in normal PF. (2) Two different receptor subtypes may be responsible for the alpha 1-induced effects on automaticity in normal and ischemic fibers. It is likely that the latter one is alpha 1A, and that consideration of antiarrhythmic therapy with alpha 1-adrenergic blockers should focus on this subtype as a potential target.

Arrhythmias caused by platelet activating factor.

INTRODUCTION: Both ischemia and reperfusion are associated with ventricular arrhythmias. In both instances, neutrophils migrate into the ischemic zone, are activated by locally released factors, and bind to myocytes. The activated neutrophils liberate platelet activating factor (PAF). We have studied the arrhythmogenic actions of PAF on transmembrane potentials of isolated canine cardiac myocytes. METHODS AND RESULTS: Cardiac myocytes were prepared from normal canine hearts by standard methods and studied in vitro by recording transmembrane potentials under control conditions and during exposure to graded doses of PAF, usually 0.25 to 1.25 micrograms (0.25 to 1.2 microM). Myocytes were superfused with Tyrode's solution (2.0 mL/min), paced at a cycle length of 1000 msec, and maintained at a temperature between 36 degrees and 38 degrees C. PAF caused a consistent and dose-dependent set of alterations in the transmembrane potential, including increased action potential duration, runs of early afterdepolarizations (EADs), and transient arrest of repolarization (PA). In addition, in some myocytes PAF caused intermittent small depolarizations both at the plateau voltage and resting potential. The effects of PAF were transient: only some residual action potential prolongation was noted after Tyrode's washout for 5 minutes. Effects of PAF were blocked in a dose-dependent manner by the PAF receptor antagonist, CV-6209. Both tetrodotoxin (1.2 x 10(-6) M) and xylocaine (5 x 10(-5) M) antagonized the ability of PAF to cause EADs and PA. CONCLUSIONS: PAF consistently exerts arrhythmogenic effects on the membrane of ventricular myocytes. Since PAF is liberated by activated neutrophils and since activated neutrophils migrate into ischemic myocardium on reperfusion, we judge that PAF liberated by such neutrophils is an important arrhythmogenic factor for reperfusion arrhythmias. The same mechanism may be a cause of arrhythmias during the evolution of infarction.

Chloroethylclonidine increases the incidence of lethal arrhythmias during coronary occlusion in anesthetized dogs.

We studied the role of alpha1-adrenoceptors in the modulation of ventricular tachycardia and fibrillation in chloralose-anesthetized dogs subjected to 30 min left anterior descending coronary artery occlusion. Study groups were control, and those treated with the alpha1-adrenoceptor-subtype blockers WB4101 (0.5 mg/kg i.v.) or chloroethylclonidine (1.9 mg/kg i.v.). For the first set of experiments all animals were in sinus rhythm and heart rate was slower in the chloroethylclonidine-pretreated animals than the WB4101-treated group (P < 0.05). During occlusion, ventricular tachycardia and ventricular fibrillation incidence did not differ among control, WB4101 or chloroethylclonidine (3 dogs with ventricular fibrillation in each group and 0, 2 and 3 dogs respectively with ventricular tachycardia), but ventricular premature depolarizations were significantly reduced by both interventions, and nonsustained ventricular tachycardia was suppressed by WB4101. In a second set of experiments, animals were atrially paced at a cycle length of 300 ms, and divided into control, WB4101-treated or chloroethylclonidine-treated, as above. Here, 9/10 chloroethylclonidine-treated animals developed ventricular tachycardia and fibrillation during occlusion, whereas only 4/10 controls and 4/10 WB4101-treated animals did so (P < 0.05). In conclusion, during sinus rhythm, both types of alpha1-adrenoceptor subtype blockade significantly suppressed ventricular premature depolarizations and neither affected ventricular tachycardia and fibrillation. In contrast, when heart rate was held constant, chloroethylclonidine clearly enhanced the occurrence of ventricular fibrillation during occlusion. These results suggest the alpha1-adrenoceptor subtype blocked by chloroethylclonidine, but not that blocked by WB4101, is capable of increasing the incidence of lethal arrhythmias that occur at rapid atrial rates during ischemia.

[Effects of sennosides on cellular electric activities in smooth muscle cells of guinea pig taenia coli].

The effects of sennosides (Sen, an extract of Chinese rhubarb) on cellular spontaneous electric activities of guinea pig taenia coli were studied by intracellular microelectrode technique. Sen 0.1-20 mmol.L-1 improved the depolarization of cell membrane, quickened the burst of slow wave potential, and increased notably the frequency of spike potentials. Sen (20 mmol.L-1) induced spontaneous spike potentials. These results provide direct evidence for the enhancing effect of Sen on intestinal peristalsis at cellular level.