Muscarinic receptor-mediated intracellular Ca mobilization in embryonic chick heart cells

Activation of muscarinic receptors of heart cells elevates the intracellular Ca²⁺ concentration. The increase is considered to be due to influx of extracellular Ca²⁺. We show that intracellular Ca²⁺ mobilization is involved. Cell suspensions prepared from hearts of 6-day-old chick embryos were loaded with the fluorescent Ca²⁺ chelator chlortetracycline. Muscarinic stimulation induces a dose-dependent fluorescence decrease (ED₅₀=2.6 × 10⁻⁶ M) indicating intracellular Ca²⁺ mobilization.     

Comparison of the pacemaker properties of chick embryonic atrial and ventricular heart cells

Summary We have investigated the pacemaker properties of aggregates of cells dissociated from the atria and ventricles of 10 to 14-day-old chick embryonic hearts using a two-microelectrode current and voltage-clamp technique. These preparations usually beat spontaneously and rhythmically in tissue culture medium containing 1.3mm potassium with a beat rate typically in the range of 15–60 beats per minute. The beat rate results show considerable variability, which precludes any statistically significant comparison between the spontaneous activity of atrial and ventricular cell preparations at 10–14 days of development. However, the shapes of pacemaker voltage changes do exhibit differences characteristic of cell type. Spontaneous atrial preparations rapidly depolarize from maximum diastolic potential (–90 mV) to a plateau range of pacemaker potentials (–80 to –75 mV). The membrane subsequently depolarizes more gradually until threshold (–65 mV) is reached. In contrast, spontaneously beating ventricular cell preparations slowly hyperpolarize after maximum diastolic potential to the –100 to –95 mV range before gradually depolarizing toward threshold. Voltage-clamp analysis reveals a virtual lack of any time-dependent pacemaker current in atrial preparations. These preparations are characterized by an approximately linear background current (I _bg) having a slope resistance of 100 K cm². Ventricular preparations have a potassium ion pacemaker current with slow kinetics (I _{K 2}), and a second time-dependent component (I _x) which is activated at potentials positive to –65 mV. The background current of these preparations displays inward rectification. Computer simulations of pacemaking reveal that the initial rapid phase of pacemaker depolarization in atrial cells is determined by the membrane time constant, which is the product of membrane capacitance and the slope resistance ofI _bg. The hyperpolarization after maximum diastolic potential of ventricular cells is caused byI _{K 2}. The final slow phase of depolarization in both cell types is caused in part by the steady-state amplitude of the fast inward sodium current (I _Na). This component has negative slope conductance which effectively increases the slope resistance in the vicinity of threshold compared to TTX-treated preparations. This mechanism is sufficient to produce interbeat intervals several seconds in duration, even in the absence of time-dependent pacemaker current, provided that the background current is at the appropriate level.     

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.     

Morphologic study of ventricular trabeculation in the embryonic chick heart

This paper presents a morphologic study of ventricular trabeculation in chick embryo hearts between days 2 and 5 of incubation. Trabeculation appears to be the expression of three closely interrelated events: the formation of endocardial outgrowths that eventually invade the myocardium; the development of large intercellular spaces between the myocytes, and the decrease in thickness of the cardiac jelly. Endocardial cells present morphologic differences between trabeculated and nontrabeculated areas of the ventricular region. The elongation of the endocardial cells in the endocardial outgrowths and the presence of mitoses suggest that the endocardium grows out by means of an increase in cell number and by redistribution and elongation of the preexisting endocardial cells. The intercellular spaces of the myocardium appear filled with abundant extracellular material. It is suggested that the continuous synthesis of extracellular material by the myocytes may increase the hydrostatic pressure within the myocardium, inducing the formation and the enlargement of these intercellular spaces. The development and later rupture of endocardium-covered cords is described here. These cords are made up of a core of cardiac jelly material revested by endocardium. The cords may be engaged in the removal of substantial amounts of cardiac jelly during the formation of the trabeculae.     

Muscarinic acetylcholine receptors of the chick amnion

The presence of muscarinic (M) acetylcholine receptors in the noninnervated chick amnion makes it possible to analyze their functioning with presynaptic effects excluded. The M receptors of the amnion mediating its contraction were identified by testing with selective antagonists: pirenzepine for M₁, methoctramine for M₂, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) for M₃, and tropicamide for M₄ receptor subtype. All antagonists acted as competitive inhibitors of M-acetylcholine receptors. With respect to cholinolytic activity estimated from the response to carbacholine (CBC) (-logIC₅₀), the antagonists could be arranged in the following series: 4-DAMP (8.29) > tropicamide (6.97) > pirenzepine (5.85) > methoctramine (5.63). In addition, the effect of forskolin (5 μM), activator of adenylate cyclase (AC), was unidirectional with ?-adrenergic agonists; it blocked CBC-induced contractile activity of the amnion, whereas phospholipase C (1.25 U/ml) stimulated this activity. These data suggest that CBC-or acetylcholine (ACh)-induced contractile activity of the amnion is mediated by M₃ acetylcholine receptors. Evaluation of contractile response to ACh by the tonic component usually revealed one pool of M₃ acetylcholine receptors. One pool was also revealed after treatment with 4-DAMP, with the Hill coefficient being increased (ACh, n = 1.07; ACh against the 4-DAMP background, n = 1.48). It is possible to detect two pools of M₃-acetylcholine receptors on the basis of either phase-frequency or tonic response, i.e., independently of the test parameter.     

Regulation of swelling—activated chloride channels in embryonic chick heart cells

Swelling-activated Cl^- currents,I(Cl,swell),were measured during hyposmotic shock in white Leghorn embryonic chick heart cells using the whole-cell recording of patch-clamp technique.Genistein,an inhibitor of protein tyrosine kinase(PTK),suppressed I(Cl,swell).Under isosmotic condition phorbol 12-myristate 13-actetate(PMA),and activator of PKC,elicited the Cl^- current similar to that in hyposmotic solution,whereas hyposmotic shock did not elicit I(Cl,swell) in chelerythrine chloride(an inhibitor of PKC)-treated cells,Confocal microscopy experiments using FITC-phalloidin as a fluorescent label of F-actin showed that the actin network was moved from cortical region of the cell to the center after hyposmotic shock as compared with the image under isosmotic condition,When the cells were treated with cytochalasin B(CB)or cytochalasin D(CD)under isosmotic condition the disruption of the F-actin integrity was observed,and I(C,l,swell). The results suggested that the role of PTK,probably receptor tyrosine kinase,for regulation of I(Cl,swell) appeared to be at upstream site related to the role of F-actin.Then PKC signal pathway was activated somehow and finally change in the polymerization state of cytoskeleton led to activate the swelling-activated Cl^- channels.These results demonstrate clearly that PTK,PKC and F-actin are important factors for regulation of I(Cl,swell),in embryonic chick heart cells as compared with often controversial results reported in different cell types.     

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.     

Muscarinic cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a role of two guanine-nucleotide-binding proteins.

These studies demonstrate a novel mechanism for the coupling of the muscarinic receptor to phospholipase C activity in embryonic chick atrial cells. In monolayer cultures of atrial cells from hearts of embryonic chicks at 14 days in ovo, carbamylcholine stimulated the sequential appearance of InsP3, InsP2 and InsP1 with an EC50 (concn. causing 50% of maximal stimulation) of 30 microM. In the presence of 15 mM-Li, a 5 min exposure to carbamylcholine (0.1 mM) increased InsP3 levels to a maximum of 47 +/- 12% over basal, InsP2 to 108 +/- 13% over basal and InsP1 to 42 +/- 5% over basal. This effect was blocked by 5 microM-atropine. Incubation of these cells with pertussis toxin (15 h; 0.5 ng/ml) inhibited carbamylcholine-stimulated InsP3, InsP2 and InsP1 formation by 42 +/- 7%, 30 +/- 3% and 48 +/- 7% respectively. The IC50 (concn. causing 50% inhibition) for pertussis toxin inhibition of all three inositol phosphates was 0.01 ng/ml, with a half-time of 6 h at 0.5 ng/ml. This partial sensitivity to pertussis toxin was not due to incomplete ADP-ribosylation of the guanine-nucleotide-binding protein (G-protein), since autoradiography of polyacrylamide gels of cell homogenates incubated with [32P]NAD+ in the presence of pertussis toxin demonstrated that incubation of cells with 0.5 ng of pertussis toxin/ml for 15 h resulted in complete ADP-ribosylation of pertussis toxin substrates by endogenous NAD+. In cells permeabilized with saponin (10 micrograms/ml), 0.1 mM-GTP[S] (guanosine 5'-[gamma-thio]triphosphate) stimulated InsP1 by 102 +/- 15% (mean +/- S.E.M., n = 4), InsP2 by 421 +/- 67% and InsP3 by 124 +/- 33% above basal. Incubation of cells for 15 h with 0.5 ng of pertussis toxin/ml decreased GTP[S]-stimulated InsP1 production in saponin-treated cells by 30 +/- 10% (n = 3), InsP2 production by 45 +/- 7% (n = 4) and InsP3 production by 49 +/- 6% (n = 4). These data demonstrate that in embryonic chick atrial cells at least two independent G-proteins, a pertussis toxin-sensitive G-protein and a pertussis toxin-insensitive G-protein, play a role in coupling muscarinic agonist binding to phospholipase C activation and to inositol phosphate production.     

Incorporation of fucose in the intact heart and dissociated heart cells of the chick embryo

The incorporation of [³H]fucose into cell-bound and medium-released TCA-precipitable fractions was determined in intact hearts and dissociated heart cells of the 4-day chick embryo. The amount of released label was found to be much greater in the dissociated cells than in intact hearts both in absolute quantities and in proportion to cell-bound label.     

Muscarinic acetylcholine responses in the early embryonic chic retina

The action of acetylcholine on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in early embryonic chick retinae. Whole neural retinae were isolated from embryonic day 3 (E3) chicks and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). Increases in [Ca²⁺]_i were evoked by the puff application of acetylcholine at concentration than 0.1 μM. The Ca²⁺ response became larger in dose–dependant manner up to 10 μM of acetylcholine applied. The rise in [Ca²⁺]_i was not due to the influx of Ca⁺² through calcium channels, but to the release of Ca²⁺ from internal stores. A calcium channel antagonist, nifedipine, which completely blocks the Ca²⁺ rise caused by depolarization with 100 mM K⁺, had no effects on the acetylcholine response and the Ca²⁺ response to acetylcholine occurred even in a Ca²⁺-free medium. The Ca²⁺ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μM abolished the response to 10 μM acetylcholine, whereas d-tubocurarine of 100 μM had no effects. Two muscarinic agonists, muscarine and carbamylcholine (100 μM each), evoked comparable responses with that to 10 μM acetylcholine. The developmental change of the muscarinic response was examined from E3 to E13. The Ca2+ response to 100 μM carbamylcholine was intense at E3-E5, then rapidly declined until E8. The muscarinic Ca²⁺ mobilization we found in the early embryonic chick retina may be regarded as a part of the “embryonic muscarinic system” proposed by Drew's group, which appears transiently and ubiquitously at early embryonic stages in relation to organogenesis. 1994 John Wiley & Sons, Inc.     

Phase resetting of embryonic chick atrial heart cell aggregates. Experiment and theory.

The influence of brief duration current pulses on the spontaneous electrical activity of embryonic chick atrial heart cell aggregates was investigated experimentally and theoretically. A pulse could either delay or advance the time of the action potential subsequent to the pulse depending upon the time in the control cycle at which it was applied. The perturbed cycle length throughout the transition from delay to advance was a continuous function of the time of the pulse for small pulse amplitudes, but was discontinuous for larger pulse amplitudes. Similar results were obtained using a model of the ionic currents which underlie spontaneous activity in these preparations. The primary ion current components which contribute to phase resetting are the fast inward sodium ion current, INa, and the primary, potassium ion repolarization current, IX1. The origin of the discontinuity in phase resetting of the model can be elucidated by a detailed examination of the current-voltage trajectories in the region of the phase response curve where the discontinuity occurs.     

3,4-Dihydroxyphenylalanine in cultured ventricular cells from chick embryo heart

Abstract— —A substance resembling catecholamine found in chick hearts during the early stages of embryologic development was identified as DOPA. Cell cultures and fluorescence microscopy indicated intracellular location of this substance in myocardial cells. The absence of nerve tissue in the cell cultures was demonstrated by electron microscopy.     

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.     

Intracellular adenosine formation and its carrier-mediated release in cultured embryonic chick heart cells

Adenosine formation and release was examined in 48 hr old primary cultures of chick ventricular myocytes. Dilazep greater than hexobendine greater than dipyridamole inhibit incorporation of adenosine into chick embryonic heart cellular nucleotides in a concentration dependent manner. A combination of 30 mM 2-deoxyglucose and 2 micrograms of oligomycin/ml reduces the ATP content of the cells by 71% in 10 min. This change is accompanied by an increase in total adenosine concentration of 3.4 nmoles/10(7) cells in 10 min. Although the ATP concentration is not altered during hypoxia (95%N2/5%CO2), adenosine concentration increases by 0.52 nmoles/10(7) cells in 30 min. When nucleoside incorporation is inhibited by 85-90% by dipyridamole, dilazep or hexobendine, efflux of adenosine decreases by 70-90%, and 60-90% of the newly formed adenosine is trapped inside the cells compared to 10% in the absence of the transport inhibitors. alpha, beta -Methylene ADP inhibits the ecto 5'-nucleotidase activity by 91 +/- 6% but does not inhibit adenosine formation or alter its distribution between cells and medium, thus ruling out the involvement of this enzyme in adenosine formation. We conclude that adenosine is formed intracellularly during 2-deoxyglucose and oligomycin-induced ATP degradation and during hypoxia and that the nucleoside is released via the symmetric nucleoside transporter.     

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.     

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

Single ventricle cells were dissociated from the hearts of two-, three-, four- or seven-day-old chick embryos, and were maintained in vitro for an additional 6 to 28 hr. Rounded 13 to 18 micron cells with input capacitance of 5 to 10 pF were selected for analysis of fast sodium current (INa). Voltage command protocols designed to investigate the magnitude, voltage dependence, and kinetics of INa were applied with patch electrodes in the whole-cell clamp configuration. INa 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 conditions. Between the 2d and 7d stages, the rate of current inactivation doubled and channel density increased about eightfold. At all stages tested, INa 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.     

The initial inward current in spherical clusters of chick embryonic heart cells

The rapid inward sodium current in spherical clusters of 11-d-old embryonic chick heart cells, ranging in size between 65 and 90 micron diameter, was studied using the two-microelectrode voltage-clamp technique. Using these preparations, it was possible to resolve the activation phase of the rapid inward current for potentials negative to -25 mV at 37 degrees C. The rapid inward current exhibited a voltage and time dependence similar to that observed in other excitable tissues. It was initiated at potential steps more positive than -45 mV. The magnitude of the current reached its maximum value at a potential of approximately -20 mV. The measured reversal potential was that predicted by the Nernst equation for sodium ions. The falling phase of the current followed a single exponential time-course with a time constant of inactivation, tau h, ranging between 2.14 ms at -40 mV and 0.18 ms at -5 mV. The time constant of inactivation, tau h, determined by a single voltage-step protocol was compared to the constant, tau c, determined by a double voltage-step protocol and no significant different between the two constants of inactivation was found. Furthermore, the time constants of inactivation and reactivation at the same potential in the same preparation were similar. The results of this study demonstrate that the sodium current of heart cells recorded at 37 degrees C can be described by Hodgkin-Huxley kinetics with speeds approximately four times faster than the squid giant axon at 15 degrees C.     

Nicotinic and muscarinic responses of embryonic chick ventricular myocardium to acetylcholine: chronotropic and electrophysiological effects

Sensitivity of 7-day-old chick embryo ventricular heart fragments to acetylcholine was investigated. Low doses mainly produced a positive chronotropic effect, whereas high doses of acetylcholine provoked a decrease in the heart beat rhythm. The positive chronotropic effect of acetylcholine was related to the presence of nicotinic receptors that were evidenced within ventricular myocardium by autoradiography. Membrane potential recording showed that acetylcholine hyperpolarizes the diastolic membrane potential when the drug had a negative chronotropic effect. This effect of acetylcholine on the membrane potential was not observed when the drug had a positive chronotropic effect. In many cases, the diastolic membrane potential exhibited spontaneous small depolarizing potentials. Their amplitude was low and their frequency was irregular. These potentials were suppressed by treatment with alpha-bungarotoxin, suggesting that they are triggered by nicotinic receptor activation.