Expression of the Ca2+ mobilizing muscarinic system in the chick embryo correlates with morphogenesis

We describe muscarinic receptors and intracellular Ca2+ mobilization after cholinergic stimulation in cell suspensions prepared from chick embryos between day 2 (stage 12/13) and day 13 (stage 40) of development. Cell suspensions are prepared from whole chick embryos and from embryonic hearts, heads or brains, limb buds, and trunks. Muscarinic receptors are measured using [3H]quinuclidinylbenzilate as specific ligand. Intracellular Ca2+ mobilization is determined by changes of chlorotetracycline fluorescence. (1) Considerable amounts of muscarinic receptors are found in all parts of the embryo and at all stages tested. (2) The intracellular Ca2+ response after stimulation by muscarinic agonist shows a peak at day 3-4 (stage 23). (3) The pharmacological profile of the Ca2+ response remains constant during embryonic development and differs from the profiles of most adult systems. (4) The 'embryonic muscarinic system' is uniformly expressed in cells from neural and non-neural tissues. It appears and disappears independently of innervation.     

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.     

Exogeneous cyclic AMP blocks the muscarinic receptor-mediated intracellular Ca2+-release in the gastrulating chick embryo cells

Summary Exogeneous cyclic AMP (cAMP, 10^–8M), when added together with acetylcholine (ACh, 500 M) to dissociated chick embryo cells, blocked the ACh-stimulated increase in the level of cytosolic free Ca²⁺ ([Ca²⁺]_i). This inhibiting action of exogeneous cAMP is probably mediated by intracellular cyclic GMP (cGMP) and cAMP.     

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).     

The muscarinic receptor-mediated action of acetylcholine in the gastrulating chick embryo

Some of the muscarinic receptor-mediated effects of acetylcholine in early chick embryo cells at stages three-four by Hamburger and Hamilton were studied. Acetylcholine increased the intracellular level of cGMP about two-fold. Acetylcholine raised the intracellular level of free calcium from the basal level of 120 nM to 140 nM. Atropine, a muscarinic antagonist, blocked both the above-mentioned responses.     

Sensitivity of cultured chick embryo heart cells to acetylcholine. Evidence for nicotinic and muscarinic receptors

Sensitivity of cultured chick embryo heart cells to acetylcholine changes with time in culture. In 24 h cultures, about 25% of the cells exhibit a positive chronotropic response to acetylcholine. This effect is no longer observed after 48 h in culture. Positive and negative chronotropic effects of acetylcholine can be related to the presence of nicotinic and muscarinic receptors evidenced by autoradiography. Some data suggest a possible relationship between the type of sensitivity to acetylcholine and the changes in cell membrane properties occurring in culture.     

Extracellular matrix organized in embryonic cavities during induction of the embryonic axis in chick embryo

Extracellular matrix (ECM) is detected as short, disorganized fibrils in the forming embryonic extracellular spaces shortly prior to the first morphogenetic cellular movements and interactions in the early chick embryo. As development progresses, the ECM is organized into an intricate network spanning the embryonic cavities. This dynamic entity undergoes relatively rapid changes in its organization pattern during the developmental period from morula to the induction of the neural plate. The ECM seems to preserve the exquisite architecture of the embryo and could guide migrating cells into defined pathways in the early embryo.     

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.     

Tetraspanins expressed in the embryonic chick nervous system

Proteins of the tetraspanin superfamily participate in the formation of plasma membrane signaling complexes; recent evidence implicates neuronal tetraspanins in axon growth and target recognition. We used a degenerate PCR screen to identify cDNAs encoding tetraspanins expressed in the embryonic spinal cord. Two cDNAs identified apparently represent chick homologues of NAG-2 (cnag) and CD9 (chCD9). A third clone encodes a novel tetraspanin (neurospanin). All three mRNAs are widely expressed but exhibit developmentally distinct patterns of expression in the nervous system. Both neurospanin and cnag exhibit high relative expression in nervous tissue, including brain, spinal cord and dorsal root ganglia (DRG).     

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.     

Intramyocardial pressure measurements in the stage 18 embryonic chick heart

Intramyocardial pressure (IMP) and ventricular pressure (VP) were measured in the trabeculating heart of the stage 18 chick embryo (3 days of incubation). Pressure was measured at several locations across the ventricle using a fluid-filled servo-null system. Maximum systolic and minimum diastolic IMP tended to be greater in the dorsal wall than in the ventral wall, but transmural distributions of peak active (maximum minus minimum) IMP were similar in both walls. Peak active IMP near midwall was similar to peak active VP, but peak active IMP in the subepicardial and subendocardial layers was four to five times larger. These results suggest that the passive stiffness of the dorsal wall is greater than that of the ventral wall and that during contraction the inner and outer layers of both walls generate more contractile force and/or become less permeable to flow than the middle part of the wall. Measured pressures likely correspond to regional variations in wall stress that may influence morphogenesis and function in the embryonic heart.     

Influence of Ca++ and Mg++ on the vanadate inhibition of the Ca++- ATPase from pig heart sarcoplasmic reticulum

Vanadate inhibits the Ca⁺⁺-ATPase of sarcoplasmic reticulum from pig heart half maximally at about 10^?5 M. Mg⁺⁺ promotes this inhibition by vanadate whereas increasing Ca⁺⁺-concentrations protect the enzyme against vanadate inhibition. Keeping the ratio $\text{Mg}{}^{\mathrm{++}}\text{ATP}$ constant there was no influence of ATP on the vanadate inhibition at concentrations up to 5 × 10^?3 M ATP. Whenever the ratio $\text{Mg}{}^{\mathrm{++}}\text{ATP}$ was higher than 1:1 the inhibitory effect of vanadate on the Ca⁺⁺-ATPase was increased.