吗啡对鸡胚胎发育的影响

目的：研究吗啡对胎动、心率、孵化率、孵化时间、雏鸡体重等的影响。方法：以气室给药的方式给鸡胚注射吗啡,记录胎动、心率、孵化率、孵化时间、雏鸡体重。结果：吗啡可以缩短雏鸡的孵化时间,降低雏鸡的孵化率,并导致雏鸡出现运动障碍;20mg／kg吗啡剂量和12—16胚龄的给药时间,鸡胚孵化率最高,残疾率最低;吗啡导致胚胎心率加快,胎动减少（P〈0．05）。结论：吗啡对胚胎发育有损伤作用,损伤程度与吗啡剂量和给药时间有关。     

Perfluorocarbon-based medium for culture of the early chick embryo heart

Summary In an attempt to prolong the survival of the explanted early chick embryo heart, hearts at stages 10 to 28 were cultured in supplemented Dulbecco's modified Eagle's medium with or without the perfluorocarbon, perfluorotributylamine. The perfluorocarbon was added to the standard culture medium in a 50∶50 (vol/vol) mixture. Explants were evaluated daily and were harvested for light microscopy after 2 to 10 d in culture. The tubular shape of the explants was generally maintained for 2 d in culture, after which the hearts became dilated or spherical. Beating was noted in some of the explants on Day 2 in culture but not thereafter. Microscopic evaluation showed patchy areas of necrosis in all explants by Day 3, although large areas of viable epithelioid cells were documented as long as 7 d after explantation. State 16 to 18 hearts cultured in the presence of perfluorocarbon were more likely to maintain tubular architecture on microscopy than hearts cultured in standard medium. Hearts cultured from later stages showed no improvement in appearance with the presence of perfluorocarbon and there was a suggestion of increased necrosis in later-stage explants cultured with pefluorocarbon for 4 d. Further modification of the culture system will be required to prolong explant survival and development beyond 2 d.     

Development changes in regulation of embryonic chick heart gap junctions

Summary Embryonic chick myocyte pairs were isolated from ventricular tissue of 4-day, 14-day, and 18-day heart for the purpose of examining the relationship between macroscopic junctional conductance and transjunctional voltage during cardiac development. The double whole-cell patch-clamp technique was employed to directly measure junctional conductance over a transjunctional voltage range of ±100 mV. At all ages, the instantaneous junctional current (or conductance=current/voltage) varied linearly with respect to transjunctional voltage. This initial response was followed by a time- and voltage-dependent decline in junctional current to new steady-state values. For every experiment, the steady-state junctional conductance was normalized to the instantaneous value obtained at each potential and the data was pooled according to developmental age. The mean steadystate junctional conductance-voltage relationship for each age group was fit using a two-state Boltzmann distribution described previously for other voltage-dependent gap junctions. From this model, it was revealed that half-inactivation voltage for the transjunctional voltage-sensitive conductance shifted towards larger potentials by 10 mV, the equivalent gating charge increased by approximately 1 electron, and the minimal voltage-insensitive conductance exactly doubled (increased from 18 to 36%) between 4 and 18 days of development. Decay time constants were similar at all ages examined as rate increased with increasing transjunctional potential. This data provides the first direct experimental evidence for developmental changes in the regulation of intercellular communication within a given tissue. This information is consistent with the hypothesis that developmental expression of multiple gap junction proteins (connexins) may confer different regulatory mechanisms on intercellular communication pathways within a given cell or tissue.     

Isolation and culture of single cells from carrot embryos

Callus and secondary cell cultures originating from embryos are known to have a higher morphogenetic capacity in comparison to others of different origin. A method for isolating embryonic cells in order to establish primary cell cultures has been developed. Carrot embryos were digested with different cell-separating enzymes and after 24 h post-digestion in 0.6 M mannitol they were sheared by using a hypodermic syringe. The resulting cell suspension consisted of morphologically-intact cells. In the procedure to macerate embryos into single cells it was found that embryos were surrounded by a cuticle. Carrot embryo cells when cultured divided and gave rise to callus and to proembryos.     

Development of the fast sodium current in early embryonic chick heart cells

Summary Single ventricle cells were dissociated from the hearts of two-, theree-, four-, or seven-day-old chick embryos, and were maintained in vitro for an additional 6 to 28 hr. Rounded 13 to 18 m cells with input capacitance of 5 to 10 pF were selected for analysis of fast sodium current (I _Na). Voltage dependence, and kinetics ofI _Na were applied with patch electrodes in the wholecell clamp configuration.I _Na was present in over half of the 2d, and all 3d, 4d and 7d cells selected. The current showed no systematic differences in activation kinetics, voltage dependence, or tetrodotoxin (TTX) sensitivity with age or culture condition, Between the 2d and 7d stages, the rate of current inactivation doubled an channel density increased about eighfold. At all stages tested,I _Na was blocked by TTX at a half-effective concentration of 0.5 to 1.0 nM. We conclude that the lack of Na dependence of the action potential upstroke on the second day of development results from the relatively depolarized level of the diastolic potential, and failure to activate the small available excitatory na current. The change from Ca to Na dependence of the upstroke during the third to the seventh day of incubation results partly from the negative shift of the diastolic potential during this period, and in part from the increase in available Na conductance.     

Cyclic strain induces proliferation of cultured embryonic heart cells

Summary Embryonic heart cells undergo cyclic strain as the developing heart circulates blood to the embryo. Cyclic strain may have an important regulatory role in formation of the adult structure. This study examines the feasibility of a computerized cell-stretching device for applying strain to embryonic cardiocytes to allow measurement of the cellular response. A primary coculture of myocytes and a secondary culture of nonmyocytes from stage-31 (7 d) embryonic chick hearts were grown on collagen-coated membranes that were subsequently strained at 2 Hz to 20% maximal radial strain. After 24 h, total cell number increased by 37±6% in myocyte cocultures and by 26±6% in nonmyocyte cultures over unstrained controls. Lactate dehydrogenase and apoptosis assays showed no significant differences in cell viabilities between strained and unstrained cells. After 2 h strain, bromodeoxyuridine incorporation was 38±1.2% versus 19±0.2% (P<0.01) in strained versus unstrained myocyte cocultures, and 35±2.1% versus 16±0.2% (P=0.01) in nonmyocyte cultures. MF20 antibody labeling and periodic acid-Schiff (PAS) staining estimated the number of myocytes in strained wells as 50–67% larger than in control wells. Tyrosine phosphorylation may play a role in the cellular response to strain, as Western blot analysis showed an increase in tyrosine phosphorylation of two proteins with approximate molecular weights of 63 and 150 kDa within 2 min of strain. The results of this study indicate that embryonic chick cardiocytes can be cultured in an active mechanical environment without significant detachment and damage and that increased proliferation may be a primary response to strain.     

A dual action of taurine on the delayed rectifier K+ current in embryonic chick cardiomyocytes

Summary Effects of taurine on the delayed rectifier K⁺ channel in isolated 10-day-old embryonic chick ventricular cardiomyocytes were examined at different intracellular Ca²⁺ concentrations ([Ca]_i), using whole-cell voltage and current clamp techniques. Experiments were performed at room temperature (22°C). Test pulses were applied between -20 to +90m V from a holding potential of -40mV. When [Ca]_i was pCa 7, addition of 10 and 20 mM taurine to the bath solution reduced the delayed rectifier K⁺ current (I_K) at +90mV by 17.4 ± 2.8% (n = 5, P < 0.01) and 25.5 ± 2.6% (n = 5, P < 0.001), respectively. In contrast, when [Ca]_i was pCa 10, I_K at +90 mV was enhanced by 19.1 ± 3.1% (n = 7, P < 0.01) at 10mM taurine, and by 29.3 ± 2.4% (n = 7, P < 0.001) at 20mM taurine. The voltage of half-maximum activation (V_1/2) was shifted in a hyperpolarizing direction; at pCa 7, the value was +0.2 ± 2.2mV (n = 5) in control and -10.6 ± 1.8mV (n = 5) in 20mM taurine. At pCa 10, the V_1/2 value was +18.5 ± 4.6mV (n = 5) in control and +6.6 ± 5.2mV (n = 5) in taurine (20mM). Taurine decreased the action potential duration (APD) at pCa 10, but at pCa 7 did not affect it. In addition, taurine enhanced the transient outward current in a concentration-dependent manner. These results indicate that taurine modulates the delayed rectifier K⁺ channel, an effect dependent on [Ca]_i and capable of regulating APD.     

Chick embryos can form teratomas from microinjected mouse embryonic stem cells

We examined whether chick embryos are a suitable experimental model for the evaluation of pluripotency of stem cells. Mouse embryonic stem cells (mESCs) expressing the reporter gene, LacZ or GFP were injected into the subgerminal cavity of blastoderms (freshly oviposited) or the marginal vein of chick embryos (2 days of incubation). Injected mESCs were efficiently incorporated into the body and extra‐embryonic tissues of chick embryos and formed small clusters. Increased donor cell numbers injected were positively associated with the efficiency of chimera production, but with lower viability. A single mESC injected into the blastoderm proliferated into 34.7 ± 3.8 cells in 3 days, implying that the chick embryo provides an optimal environment for the growth of xenogenic cells. In the embryo body, mESCs were interspersed as small clustered chimeras in various tissues. Teratomas were observed in the yolk sac and the brain with three germ layers. In the yolk sac, clusters of mESCs gradually increased in volume and exhibited varied morphology such as a water balloon‐like or dark‐red solid mass. However, mESCs in the brain developed into a large soft tissue mass of whitish color and showed a tendency to differentiate into ectodermal lineage cells, including primitive neural ectodermal and neuronal cells expressing the neurofilament protein. These results indicate that chick embryos are useful for the teratoma formation assays of mESCs and have a broad‐range potential as an experimental host model.     

Bay K 8644 induce enhancement of K+ current in both single heart cell and smooth muscle cell

Summary The effects of the Ca²⁺ agonist Bay K 8644 on outward potassium currents have been studied in single ventricular cells of chick embryo and aortic single cells of rabbit using the whole-cell patch clamp technique. Bay K 8644 was found to increase l_K in both heart and aortic single cells. This effect of Bay K 8644 on both muscle was reversed by Mn²⁺ and blocked by 20 mM TEA. The Bay K 8644 potassium I/V curve of single heart cell had a N shape, which is Ca²⁺ dependent. These data strongly suggest that Bay K 8644 increases a g_K(ca) in both aortic and heart muscle.     

L型钙流和反向钠-钙交换在豚鼠心室肌细胞兴奋-收缩偶联中的作用

目的 :比较和探讨L型钙流 [ICa(L) ]和反向钠—钙交换 (NCX)在触发豚鼠心室肌细胞兴奋—收缩偶联中的作用。方法 :以分离的豚鼠单个心室肌细胞为对象 ,采用膜片钳和单细胞收缩测量技术 ,给予 35℃的各种含药物细胞外液快速灌流 ,同时记录ICa(L) 和细胞收缩。结果 :①在 +10mV的钳制电压 ,使用硝苯地平 (Nif) 10～ 10 0 μmol/L和Nif 30 μmol/L +Cd2 +30 μmol/L ,阻滞ICa(L) 越多 ,细胞收缩被阻滞得越多 ,呈线性相关。②在 +5 0mV的钳制电压 ,Nif 10 0 μmol/L以及Nif 30 μmol/L +Cd2 +3 0 μmol/L仅能抑制部分细胞收缩 ,但剩余的细胞收缩起始时间明显延迟 ,且能被 5mmol/LNi2 +所阻滞。③在 +10 0mV的钳制电压 ,细胞收缩起始时间较 +5 0mV明显延迟 ,且不能被Nif 10 0 μmol/L和Nif 30 μmol/L +Cd2 +30 μmol/L所阻滞。结论 :在生理条件下 ,ICa(L) 是触发心室肌细胞兴奋—收缩偶联的主要途径 ,但在膜电位 >+5 0mV时 ,反向NCX也参与兴奋—收缩偶联。     

Effects of toxin II from the scorpion Androctonus australis Hector on sodium current in neuroblastoma cells and their modulation by oleic acid

The effects of toxin II (AaH II) isolated from the scorpion Androtonus australis Hector on sodium current in neuroblastoma X glioma NG 108-15 hybrid cells were analysed under patch clamp conditions in the whole cell configuration. AaH II (70 nM)_induced a maintained sodium current, as well as increasing both fast and slow inactivation time constants and the amplitude of the peak current. This latter effect occurred via a shift of the activation-voltage curve towards negative voltage values by about 9 mV. Oleic acid (5 M), which had no effect on I_Na under control conditions, decreased the AaH II-induced maintained current. It also reversed, or prevented the increase of the peak current induced by AaH II. However, it neither prevented nor modified the AaH II-induced increase in inactivation time constants. The binding of the toxin to its specific site and the number of binding sites for AaH II were not significantly modified by oleic acid. The oleic acid-induced effects could not be related to the activation of protein kinase C since PMA, a potent activator of this enzyme, did not produce oleic acid-like effects. From these results, it is concluded that AaH II has several independent effects on sodium channels, some of which could be modulated by the lipid environment of sodium channels in the membrane.     

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

Summary We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.     

Macroscopic Ca2+ -Na+ and K+ currents in single heart and aortic cells

Summary The whole-cell voltage clamp technique was used to study the slow inward currents and K⁺ outward currents in single heart cells of embryonic chick and in rabbit aortic cells. In single heart cells of 3-day-old chick embryo three types of slow inward Na⁺ currents were found. The kinetics and the pharmacology of the slow I_Na, were different from those of the slow Ica in older embryos. Two types of slow inward currents were found in aortic single cells of rabbit; angiotensin 11 increased the sustained type and d-cAMP and d-cGMP decreased the slow transient component. Two types of outward K⁺ currents were found in both aortic and heart cells. Single channel analysis demonstrated the presence of a high single K⁺ channel conductance in aortic cells. In cardiac and vascular smooth muscles, slow inward currents do share some pharmacological properties, although the regulation of these channels by cyclic nucleotides and several drugs seems to be different.     

Early fetal like slow Na+ current in heart cells of cardiomyopathic hamster

Using the whole-cell voltage-clamp technique, early embryonic tetrodotoxin (TTX) and Mn2⁺-insensitive slow Na⁺ current was detected in 10-22 week old fetal human heart cells as well as in 1-day-old and young cardiomyopathic hamster myocytes. This slow Na⁺ current in both heart cell preparations has the same kinetics and pharmacology. This type of slow Na⁺ current was absent in heart cells of newborn and young normal hamsters and became less present in myocytes of 19 and 22 week old human heart myocytes. Our results demonstrate that the slow Na⁺ channel does exist in early fetal human life and this type of channel continues to be functional after birth in myocytes of the hereditary cardiomyopathic hamster.     

Regulation of the calcium slow channel by cyclic GMP dependent protein kinase in chick heart cells

In order to assess the interaction between the cAMP-dependent and the cGMP-dependent phosphorylation pathways on the slow Ca²⁺ current (I_Ca(L)), whole-cell voltage-clamp experiments were conducted on embryonic chick heart cells. Addition of 8Br-cGMP to the bath solution reduced the basal (unstimulated) I_Ca(L). Intracellular application of the catalytic subunit of PK-A (PK-A(cat); 1.5 M) via the patch pipette rapidly potentiated I_Ca(L) by 215±16% (n=4); subsequent addition of 1 mM 8Br-cGMP to the bath reduced the amplitude of I_Ca(L) towards the initial control values (123±29%). Intracellular application of PK-G (25 nM pre-activated by 10^–7 M cGMP), rapidly inhibited the basal I_Ca(L) by 64±6% (n=8). Heat-denatured PK-G was ineffective. Subsequent additions of relatively high concentrations of 8Br-cAMP (1 mM) or isoproterenol (ISO, 1–10 M) did not significantly remove the PK-G blockade of I_Ca(L). The results of the present study suggest that: (a) 8Br-cGMP can inhibit the basal or stimulated (by PK-A(cat)) I_Ca(L) in embryonic chick myocardial cells. (b) PK-G applied intracellularly inhibits the basal I_Ca(L).     

The role of Na,K-ATPase alpha subunit serine 775 and glutamate 779 in determining the extracellular K+ and membrane potential-dependent properties of the Na,K-pump

The roles of Ser775 and Glu779, two amino acids in the putative fifth transmembrane segment of the Na,K-ATPase alpha subunit, in determining the voltage and extracellular K+ (K+(o)) dependence of enzyme-mediated ion transport, were examined in this study. HeLa cells expressing the alpha1 subunit of sheep Na,K-ATPase were voltage clamped via patch electrodes containing solutions with 115 mM Na+ (37 degrees C). Na,K-pump current produced by the ouabain-resistant control enzyme (RD), containing amino acid substitutions Gln111Arg and Asn122Asp, displayed a membrane potential and K+(o) dependence similar to wild-type Na,K-ATPase during superfusion with 0 and 148 mM Na+-containing salt solutions. Additional substitution of alanine at Ser775 or Glu779 produced 155- and 15-fold increases, respectively, in the K+(o) concentration that half-maximally activated Na,K-pump current at 0 mV in extracellular Na+-free solutions. However, the voltage dependence of Na,K-pump current was unchanged in RD and alanine-substituted enzymes. Thus, large changes in apparent K+(o) affinity could be produced by mutations in the fifth transmembrane segment of the Na,K-ATPase with little effect on voltage-dependent properties of K+ transport. One interpretation of these results is that protein structures responsible for the kinetics of K+(o) binding and/or occlusion may be distinct, at least in part, from those that are responsible for the voltage dependence of K+(o) binding to the Na,K-ATPase.     

Inhibition of focal adhesion kinase expression correlates with changes in the cytoskeleton but not apoptosis in primary cultures of chick embryo cells

Cell adhesion to the extracellular matrix through integrin receptors can activate signaling cascades within the cell. Focal adhesion kinase (FAK) is a protein tyrosine kinase activated by integrin adhesion. The role of FAK within the cell is not clear, although evidence suggests roles in cell motility or the regulation of adhesion-dependent cell survival. We have treated primary cultures of chick embryo cells with antisense oligonucleotides to FAK to reduce the level of FAK protein expression. Levels of the related protein, proline-rich tyrosine kinase 2 (Pyk2) and the FAK substrate paxillin, were unaffected by the addition of oligonucleotides, whereas FAK expression was reduced by 70%. Levels of apoptotic cell death did not significantly increase after the addition of oligonucleotides. However, there was a change in the distribution of focal adhesion sites from a uniformly distributed pattern to a mainly peripheral pattern. This was accompanied by a loss of stress fibers and an increase in the peripheral actin cytoskeleton, as the cells became rounded. These results suggest that in these early embryonic cells, FAK expression regulates the arrangement of focal adhesions and the cytoskeleton that result in a motile phenotype, but that FAK does not appear to regulate apoptosis.     

Inhibition of the Na+/Ca2+ antiport of heart mitochondria by diethylpyrocarbonate

Diethylpyrocarbonate inhibits Na⁺/Ca²⁺ antiport activity in isolated heart mitochondria. The inhibition is time-dependent with maximum activity developed after 5 min at 25°C. The reaction of diethylpyrocarbonate with the mitochondrial membrane is biphasic with 25–30 nmol mg^–1 reacting rapidly and an additional 30 nmol mg^–1 taken up slowly over a 30-min incubation. Inhibition of mitochondrial Na⁺/Ca²⁺ antiport by diethylpyrocarbonate decreases theV _max of the reaction, and the inhibition cannot be reversed by washing the mitochondria or addition of excess histidine. The inhibition occurs at levels of inhibitor that have little or no effect on Ca²⁺ uptake, Na⁺/H⁺ antiport, or succinate respiration. A portion of the Na⁺-dependent efflux of Ca²⁺ is insensitive to diethylpyrocarbonate and this component is abolished by diltiazem. The mechanism by which diethylpyrocarbonate inactivates Na⁺/Ca²⁺ antiport is still uncertain, but may involve the modification of an unprotonated histidine residue in the transporter.     

N-cadherin is crucial for heart formation in the chick embryo

The developing heart primordium strongly expresses N-cadherin. In order to investigate the role of this adhesion molecule in heart morphogenesis, chicken embryos were cultured at stages 5–12, and injected with anti-N-cadherin antibodies that can specifically block the activity of this cadherin. In the injected embryos, the epimyocardial layers, which develop bilaterally from the splanchnic mesoderm, did not fuse to form a single cardiac tube. Moreover, each of the unfused layers became fragmented into epithelioid clusters. At the cellular level, large intercellular gaps were observed in the antibody-treated myocardial layers. These disorganized myocardial layers beat to some extent, suggesting that their differentiation was not blocked; however, their contraction was not coordinated. Morphogenesis of other tissues, not only N-cadherin-negative but also N-cadherin-positive tissues, such as the neural tube and notochord, proceeded normally even in the presence of anti-N-cadherin antibodies. These results suggest that N-cadherin is indispensable for heart formation, but not for morphogenesis of the other tissues, at the developmental stages examined. For the latter processes, expression of other cadherin subtypes presumably compensated for the loss of N-cadherin activity.     

Persistent human cardiac Na+ currents in stably transfected mammalian cells

Miniature persistent late Na⁺ currents in cardiomyocytes have been linked to arrhythmias and sudden death. The goals of this study are to establish a stable cell line expressing robust persistent cardiac Na⁺ currents and to test Class 1 antiarrhythmic drugs for selective action against resting and open states. After transient transfection of an inactivation-deficient human cardiac Na⁺ channel clone (hNav1.5-CW with L409C/A410W double mutations), transfected mammalian HEK293 cells were treated with 1 mg/ml G-418. Individual G-418-resistant colonies were isolated using glass cylinders. One colony with high expression of persistent Na⁺ currents was subjected to a second colony selection. Cells from this colony remained stable in expressing robust peak Na⁺ currents of 996 ± 173 pA/pF at +50 mV (n = 20). Persistent late Na⁺ currents in these cells were clearly visible during a 4-second depolarizing pulse albeit decayed slowly. This slow decay is likely due to slow inactivation of Na⁺ channels and could be largely eliminated by 5 μM batrachotoxin. Peak cardiac hNav1.5-CW Na⁺ currents were blocked by tetrodotoxin with an IC₅₀ value of 2.27 ± 0.08 μM (n = 6). At clinic relevant concentrations, Class 1 antiarrhythmics are much more selective in blocking persistent late Na⁺ currents than their peak counterparts, with a selectivity ratio ranging from 80.6 (flecainide) to 3 (disopyramide). We conclude that (1) Class 1 antiarrhythmics differ widely in their resting- vs. open-channel selectivity, and (2) stably transfected HEK293 cells expressing large persistent hNav1.5-CW Na⁺ currents are suitable for studying as well as screening potent open-channel blockers.