Development of transmitter secretory mechanisms by adrenergic neurons in the embryonic chick heart ventricle

Developmental changes in the function of adrenergic axons within the right ventricle of the chick embryo were assessed by measuring the ability of these axons (1) to release endogenous transmitter, and (2) to transport, retain, and release tritiated norepinephrine ([³H]NE). The release of endogenous catecholamines was assayed indirectly by measuring the increase in the twitch tension of ventricular muscle evoked by electrical stimulation of intramural nerves. The release of endogenous transmitter, which acted via β-adrenergic receptors, was first detected by this method on the 16th embryonic day. A cocaine-sensitive uptake of [³H]NE was first observed on the 12th embryonic day. At this time, elevated potassium first evoked a calcium-sensitive release of [³H]NE. Electrical stimulation of intramural axons first evoked a tetrodotoxin-sensitive release of [³H]NE on the 14th embryonic day. It is concluded that the axons of developing adrenergic neurons are capable of releasing transmitter soon after they contact their target tissue.     

Na channel kinetics during the spontaneous heart beat in embryonic chick ventricle cells.

Using cell-attached and whole-cell recording techniques simultaneously allows the measurement of Na currents during action potentials in beating heart cells. In 7-d chick ventricle, the dynamic reversal potential for Na is 30 mV, which is 20 mV less than the reversal potential in nonbeating cells. This result implies that the spontaneous activity of heart cells raises the Na concentration at the internal face of the membrane to nearly 40 mM. Fitting the Na action currents to the Hodgkin and Huxley equations shows that patches may contain from 50 to 700 Na channels, with an average density of 23 +/- 21 per micron2. Only approximately 2% of the available Na channels are open at the peak of the Na action current. This low probability is a consequence of the channels' continual inactivation during the diastolic depolarization phase.     

Ca channel kinetics during the spontaneous heart beat in embryonic chick ventricle cells.

The ability of Ca ions to inhibit Ca channels presents one of the most intriguing problems in membrane biophysics. Because of this negative feedback, Ca channels can regulate the current that flows through them. The kinetics of the channels depend on voltage, and, because the voltage controls the current, a strong interaction exists between voltage dependence and Ca dependence. In addition to this interaction, the proximity of pores and the local concentration of ions also determine how effectively the Ca ions influence channel kinetics. The present article proposes a model that incorporates voltage-dependent kinetics, current-dependent kinetics, and channel clustering. We have based the model on previous voltage-clamp data and on Ca and Ba action currents measured during the action potential in beating heart cells. In general we observe that great variability exists in channel kinetics from patch to patch: Ba or Ca currents have low or high amplitudes and slow or fast kinetics during essentially the same voltage regime, either applied step-protocols or spontaneous cell action potentials. To explain this variability, we have postulated that Ca channels interact through shared ions. The model we propose expands on our previous model for Ba currents. We use the same voltage-dependent rate constants for the Ca currents that we did for the Ba currents. However, we vary the current-dependent rate constants according to the species of the conducting ion. The model reproduces the main features of our data, and we use it to predict Ca channel kinetics under physiological conditions. Preliminary reports of this work have appeared (DeFelice et al., 1991, Biophys. J. 59:551a; Risso et al., 1992, Biophys. J. 61:248a).     

K channel kinetics during the spontaneous heart beat in embryonic chick ventricle cells.

By averaging the current that passes through cell-attached patches on beating heart cells, while measuring action potentials with a whole-cell electrode, we were able to study K channels during beating. In 7-d chick ventricle in 1.3 mM K physiological solutions at room temperature, delayed-rectifier channels have three linear conductance states: 60, 30, and 15 pS. The 60 and 15 pS conductances can exist alone, but all three states may appear in the same patch as interconverting conductance levels. The delayed-rectifier conductance states have low densities (less than 10 channels per 10-microns diam cell), and all have a reversal potential near -75 mV and the same average kinetics. Outward K current through delayed-rectifier channels follows the upstroke without appreciable delay and lasts throughout the action potential. No inward current flows through delayed-rectifier channels during beating. The early outward channel has a nonlinear conductance of 18-9 pS depending on the potential. It also turns on immediately after the upstroke of the action potential and lasts on average only 50 ms. The early outward channel has an extrapolated reversal potential near -30 mV; no inward current flows during beating. The inward-rectifier has an extrapolated conductance and reversal potential of 2-3 pS and -80 mV in 1.3 mM K. Channel kinetics are independent of external K between 10 and 120 mM, and the channel conducts current only during the late repolarization and diastolic phases of the action potential. No outward current flows through inward-rectifier channels during beating. This work parallels a previous study of Na channels using similar techniques (Mazzanti, M., and L. J. DeFelice. 1987, Biophys. J. 52:95-100).     

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.     

Subcellular compartmentalization of myosin isoforms in embryonic chick heart ventricle myocytes during cytokinesis

Embryonic chick heart ventricle myocytes retain the ability to alternate between proliferation and functional differentiation. A cytoplasmic isoform of myosin is present in cleavage furrows of various nonmuscle cells during cytokinesis, whereas one or more of the cardiac myosin isoforms are localized in sarcomeres of beating cardiomyocytes. Antibodies were employed to reveal the subcellular localizations of cytoplasmic and cardiac myosin isoforms in embryonic chick ventricle cardiomyocytes during cytokinesis. Monoclonal anticytoplasmic myosin antibodies were prepared against myosin purified from brains of 1-day-posthatched chickens and shown to react with chick brain myosin heavy chain by Western blots and/or ELISA tests. One monoclonal antibrain myosin antibody also cross-reacted with chick cardiac myosin but not with skeletal or smooth muscle myosins. Two antichick cardiac myosin monoclonal antibodies and one antichick skeletal myosin polyclonal antibody that cross-reacts with cardiac myosin were employed to identify cardiac sarcomeric myosin. Cells were isolated from day 8 embryonic chick heart ventricles, enriched for myocytes, grown in vitro for 3 days, and then examined by immunofluorescence techniques. Monoclonal antibodies against cytoplasmic myosin preferentially localized in the cleavage furrows of both cardiofibroblasts and cardiomyocytes in all stages of cytokinesis. In contrast, antibodies that recognize cardiac myosin were distributed throughout cardiomyocytes during early stages of cytokinesis, but became progressively excluded from the furrow area during middle and late stages of cytokinesis. These data suggest that in cells that contain both cytoplasmic and sarcomeric myosin isoforms, only cytoplasmic myosin isoforms are mobilized to from the contractile ring for cytokinesis.     

Glycosaminoglycan synthesis by the early embryonic chick heart

Glycosaminoglycans of embryonic chick hearts labeled in situ were characterized by means of labeled precursor incorporation, electrophoretic mobility, sensitivity to testicular hyaluronidase, elution characteristics from CPC-cellulose columns, and hexosamine content. During the initial period of overt cardiac muscle differentiation (approximately stage 10) chondroitin sulfates are not detectable but an undersulfated component is present. Chondroitin sulfate synthesis appears shortly after overt muscle differentiation. Hyaluronate is present both during and after overt myocardial differentiation. Although epimerization of ³H-glucosamine-derived labeled UDP-N-acetyl-d-glucosamine occurs (determined by recovery of incorporated labeled galactosamine), label does not appear in chondroitin sulfate. ³H-Glucosamine is thus a relatively specific precursor for unsulfated glycosaminoglycans, a fact that we exploited in demonstrating their distribution radioautographically. Glycosaminoglycan synthesis was also examined in hearts labeled (a) in isolated organ culture, (b) in situ but exposed directly to the medium by removal of the splanchnopleure. In both cases fully sulfated chondroitin sulfate and chondroitin are not synthesized. Hearts make only hyaluronate and undersulfated chondroitin sulfate.     

Neuregulin stimulates DNA synthesis in embryonic chick heart cells.

Neuregulins are a family of growth factors that have been shown to promote the growth or differentiation of various cell types. Recently, targeted mutations of the genes for neuregulins or their putative receptors by homologous recombination resulted in embryonic lethality characterized by cardiac malformation. Here we investigate a role for neuregulin in the growth of cultured chick heart cells. Neuregulin induced the tyrosine phosphorylation of a 185-kDa protein in cultured heart cells, and it also stimulated an increase in [(3)H]thymidine incorporation and BrDU labeling in the cell cultures. Immunocytochemistry revealed that the increased DNA synthesis was primarily in mesenchymal cells and not detected in myocytes or endocardial cells. These data suggest that neuregulin may function as a paracrine signal in mesenchymal-endothelial interactions during cardiac development.     

Repolarization currents in embryonic chick atrial heart cell aggregates.

Outward membrane currents in aggregates of atrial cells prepared from 7-12-d-old chick embryonic hearts were measured with the two microelectrode voltage-clamp technique. Two outward current components, Ix1 and Ix2, were found in the plateau potential range of the action potential. The Ix1 component is activated between -50 and -20 mV; the Ix2 component is activated between -15 and +20 mV. The Ix1 component inwardly rectifies, whereas Ix2 has an approximately linear current-voltage relation. These preparations lack a time-dependent pacemaker current component, even though they beat spontaneously with an interbeat interval of approximately 1 s. A mathematical model of electrical activity is described based on our measurements of time-dependent outward current, and measurements in the literature of inward current components.     

Fine structural analysis of membrane changes during reaggregation culture of chick optic tectum

Thin section and freeze-fracture electron microscopy have been used to characterize the changes in membrane morphology of reaggregating cultures of chick optic tectum. The cells are rounded and freely dispersed at 0 hr after dissociation. Between 2 and 6 hr the cells become closely apposed on all sides by other cells and form small aggregates. At this time punta adhaerentia junctions and focal densities are seen along the membranes of neighboring cells. Between 1 and 5 days in vitro (DIV) neurites containing growth cone regions are present. At 5 DIV the first synaptic contacts are observed. Between 7 and 14 DIV, the number of synaptic contacts increase and fewer growth cone regions are observed. As early as 7 DIV profiles are observed which strongly resemble both astrocytic and oligodendroglial cell somata and processes. Freeze-fracture analysis of aggregates at 0–4 hr reveals a sparse particle distribution on the P and E faces of apposed cells. By 1 DIV small clusters of loosely packed, large sized particles are seen on the P face of apposed cell membranes which may represent junctional contacts. Apparent coated vesicle fusion sites are common on the P face at 1–2 DIV. By 7 DIV, E face particle arrays are seen on cell bodies and neurites which correspond to specializations characteristic of excitatory synaptic junctions. By 8–10 DIV particle arrays are seen on the P face of post-synaptic membrane which may represent inhibitory synaptic contacts. Other types of particle specializations seen in freeze-fracture replicas include: specializations characteristic of gap junctions between cells and orthogonal assemblies of particles thought to be characteristic of astrocytes.     

Different effect of propranolol and verapamil on isoprenaline-induced changes in the chick embryonic heart

Isoprenaline (IPRO) has been reported to cause pathological lesions of the embryonic heart. The purpose of the present study was to ascertain whether the development of IPRO-induced changes can be reduced--similarly as in adults--by beta blockade or calcium antagonists. IPRO was administered to 10-day-old chick embryos intraamnially (i.a.) in a dose of 2 X 10 mg.kg-1 per 48 h; propranolol (Inderal) and verapamil (Isoptin) were injected i.a. in a dose of 1.0 or 10.0 mg.kg-1 before each injection of IPRO. It was found that propranolol completely blocked the cardiac IPRO-induced changes, i.e. cardiomegaly, avascular areas and elevation of cAMP. On the other hand, verapamil was found to have no protective effect in any dose used. Furthermore, it increased the mortality of experimental embryos. This fact support the hypothesis that cardiac sensitivity to calcium antagonists may differ during prenatal development.     

45Ca-efflux in embryonic chick heart and its modification by caffeine and ryanodine.

Ontogenic changes in the kinetics of exchangeable cellular calcium were studied in embryonic (ECV) and post-hatch (PHCV) chick ventricular tissue by monitoring 45Ca-efflux. The isolated whole ventricle (5 & 7 days ECV) or ventricular strips (12 & 18 days ECV and 1-2 days PHCV) were "loaded" with 45Ca (37 degrees C) and then passed through a series of tubes containing efflux solution (4 degrees C) to determine 45Ca-efflux. Curve 'peeling' of the efflux curve indicated existence of 3 kinetically distinct components of exchangeable cellular Ca2+ compartments: C1, C2 & C3. The size of C1, which was the largest in 5 & 7 days ECV decreased significantly to become minimum in 18 days ECV & PHCV. The rate constant of this compartment, however, reduced with the age of the embryo. In contrast, the size of C3 increased with the embryonic development to become the largest in 18 days ECV & PHCV. An increase in the rate constant of this compartment was also observed during embryogenesis. The size and rate constant of C2 remained unaltered during development. However, the increase in size of C3 during embryonic development indicates differentiation of Ca2+ storage sites, like sarcoplasmic reticulum (SR), during the later stages. Caffeine (10 mM) and ryanodine (10 microM) enhanced fractional escape rate during slow phase (ie 120-180 min) of efflux at all developmental stages. The magnitude of enhancement increased during later stages of development indicating greater prominence of SR with the age of embryo.(ABSTRACT TRUNCATED AT 250 WORDS)     

GABA accumulation by embryonic chick tectum.

Radioactive GBA accumulation by embryonic chick tectal tissue under conditions suitable for high affinity GABA uptake showed a marked increase with age. There was excellent correlation between the GABA accumulation and the GAD activity from the eighth to the twentieth embryonic day, suggesting that both may reflect primarily synaptic development.     

Fatty acid synthesis in chick embryonic heart and liver during development.

Fatty acid synthesis by subcellular fractions of heart and liver of chick embryos at varying stages of development has been studied. Fatty acid synthetase activity is associated with the embryonic heart at early stages of development, as suggested by substrate requirement, Schmidt decarboxylation of synthesized fatty acids and gas liquid chromatographic identification of the products as palmitic and stearic acids. The fatty acid synthetase activity decreases in heart cytosol with age of the embryo and is absent in the newly hatched chick and in older chicken. The acetyl CoA carboxylase activity is negligible in embryonic and adult chicken heart. The fatty acid synthetase activity in liver is low, but measurable during the entire embryonic development. The activity increases by about three-fold on hatching and thereafter in fed, newly hatched chicks by about 35-fold, over the basal embryonic activity. The acetyl and malonyl transacylase activities in the heart and liver cytosols during development followed closely the fatty acid synthetase activities in heart and liver, respectively. A non-coordinate induction of fatty acid synthetase and acetyl CoA carboxylase activities in liver was observed during development. The microsomal chain elongation in liver and heart followed the pattern of fatty acid synthetase activity in liver and heart, respectively. The mitochondrial chain elongation in embryonic heart is initially low and increases with age; while this activity in liver is higher in early stages of embryonic development than in the older embryos and the chicks. Measurement of lipogenesis from acetate-1-¹⁴C by liver and heart slices from chick embryos and newly hatched chicks support the conclusions reached in the studies with the subcellular fractions. The results obtained indicate that the major system of fatty acid synthesis in embryonic and adult heart is the mitochondrial chain elongation. In embryonic liver, fatty acid synthesis proceeds by chain elongation, while the de novo system is the major contributor to the lipogenic capacity of the liver after hatching.     

Characterization and regulation of insulin binding by embryonic chick heart cells

We have compared insulin binding by heart cells at 7 and 14 days of development. Species specificity, optimum pH, temperature relationships, and time to equilibrium for binding of insulin were the same in both 7 and 14-day systems. Curvilinear Scatchard plots for chicken insulin binding were demonstrated. Binding affinities and capacities were calculated based on a two-receptor model including a specific high-affinity receptor and a less specific low-affinity receptor that bound insulin and other growth peptides. Apparent association constants (K_A) were 4.0 and 0.05 nM^?1 and binding capacities were 600 and 9000 sites per cell for high- and low affinity receptors, respectively. We have also investigated the ability of insulin to regulate binding to its own receptors. Chick heart cells from 7- and 14-day embryos, cultured for 44 hr in insulin-enriched medium (3.4 μM), bound 50% less insulin (down-regulated) than control cells. At both developmental stages, down regulation was primarily a reduction in binding to the high-affinity receptor. The low-affinity receptor was less susceptible to down regulation and retained its ability to mediate maximal insulin stimulation of amino acid transport.