Embryonic Chick Heart Research Articles

Transitions in cardiac isomyosin expression during differentiation of the embryonic chick heart.

The expression of different isoforms of the contractile protein myosin plays a major role in determining contractile characteristics in both cardiac and skeletal muscle in the adult. There is little evidence pertaining to putative changes in myosin phenotype during cardiac embryogenesis or if such changes could play a role in modulating the contractile characteristics of the developing heart. We examined isomyosin expression during cardiogenesis in the chick by indirect immunofluorescence microscopy with monoclonal antibodies to adult ventricular and atrial myosin heavy chains. Antibody specificity was characterized in the adult on the basis of immunofluorescence localization, ELISA, and protein blot immunoassay. Results show that the early embryonic chick heart has a different myosin phenotype than the later embryonic or adult heart. Both the embryonic ventricular and atrial myocardia initially expressed a myosin heavy chain that was recognized by antibody specific (in the adult) for ventricular myosin heavy chain. The ventricles remained reactive throughout life with the ventricular antibody, but reactivity of the atrial myocardium was confined to the initial 6 days of embryonic development. On the other hand, reactivity of the embryonic heart with multiple antibodies specific (in the adult) for atrial myosin was confined to the atrial myocardium throughout development. Thus, the distribution of myosin isoforms became similar to that of the adult myocardium by the time the embryonic heart achieved a 4-chambered configuration at 6 days in ovo.

Enhanced cAMP accumulation after termination of cholinergic action in the heart

Cholinergic agents decrease myocardial contractility in part by inhibiting adenylate cyclase (EC 4.6.1.1) activity. We have found that after a prolonged preincubation period (greater than 6 h), washout of cholinergic agents from embryonic chick hearts or cultured heart cells results in a persistent increase in their basal and catecholamine-stimulated cAMP content. Membranes prepared from pretreated cells have elevated basal, forskolin-, and catecholamine-stimulated adenylate cyclase activities. This myocardial adaptation to cholinergic agents is analogous to changes in nerve cells and other cell types after prolonged exposures to narcotics or other inhibitors of adenylate cyclase, respectively. A rapid (less than 5 min) adaptation response to cholinergic agents can also be demonstrated in heart cells by quickly blocking agonist action with atropine. Atropine alone has no effect, but after a brief preincubation period with agonists (methacholine or oxotremorine), the addition of atropine transiently enhances catecholamine-stimulated cAMP accumulation by 2.5-fold. These responses are absent in heart cells pretreated with pertussis toxin. The data indicate that the response is not mediated by the phosphoinositide pathway, which has been demonstrated to be insensitive to pertussis toxin in chick heart. Enhanced cAMP accumulation after termination of muscarinic agonist action may provide an explanation for the observation that acetylcholine sometimes produces biphasic contractile responses.

Identification in mammalian brain of an endogenous substance with Na + channel blocking activities similar to those of tetrodotoxin

A substance with Na + channel blocking activities has been isolated from pig brain after extraction and purification on sulfopropyl-Sephadex C-25, reversed-phase and car☐ymethyl Synchropak high pressure liquid chromatography columns. The peptidic material (i) displaces [ 3H]ethylenediamine tetrodotoxin ([ 3H]en-TTX) from its binding sites on rat brain membranes, (ii) it blocks 22Na + influx induced by veratridine and sea anemone toxin on neuroblastoma and embryonic chick heart cells in culture, (iii) in specifically decreses the height of the action potential generated in frog sciatic nerve, and (iv) it blocks the fast Na + current in voltage-clamped neuroblastoma cells. These properties are similar to those of tetrodotoxin while the endogenous factor is a peptide that is destroyed by proteases. These results suggest the presence in pig brain of a potent Na + channel modulation activity.

Electrogenic sodium‐calcium exchange in cultured embryonic chick heart cells.

1. The membrane potential (Em) of cultured chick embryonic heart cells depolarized to -36 mV after inhibition of the Na+-K+ pump by 0.1 mM-ouabain in a [K+]o of 24 mM: this was accompanied by a rise in Na+ content of approximately 65% in 3 min. Lowering [Na+]o to 27 mM then caused a fall in Na+ content, a rise in Ca2+ content and a small hyperpolarization of approximately 5 mV. The fall in Na+ content indicated a movement of Na+ which was in the opposite direction to the Na+ electrochemical gradient (a countergradient movement). 2. In the presence of 10 mM-Cs+ or 1 mM-Ba2+ the hyperpolarization was approximately 10 or approximately 30 mV, respectively. A 30 mV hyperpolarization took Em negative to the reversal potentials for K+, and Cl- as measured by ion-selective micro-electrodes. 3. The decay of the intracellular Na+ activity alpha iNa, in an [Na+]o of 27 mM followed a simple exponential time course (time constant, 36 s). The initial rate depended on the value to which [Na+]o was lowered in a manner suggesting a simple competitive inhibition of the exchange by external Na+. 4. The low-[Na+]o hyperpolarization was unaffected by amiloride (0.1 or 1 mM) or verapamil (20 microM). Both La3+ (1 mM) and Mn2+ (20 mM) blocked the hyperpolarization sufficiently to prevent Em hyperpolarizing negative to the reversal potentials for K+, Na+ and Cl-. 5. Re-establishing [Na+]o caused a rise in Na+ content and a countergradient drop in Ca2+ content. The effects of verapamil (20 microM), amiloride (0.1 and 1 mM), dichlorobenzamil (0.1 mM), quinidine (1 mM), Mn2+ (20 mM) and La3+ (1 mM) were tested on the movements of Na+ and Ca2+ both during exposure to an [Na+]o of 27 mM and on re-establishing [Na+]o. The only consistent and substantial effects were the attenuation by La3+ and Mn2+ and Ca2+ movements during exposure to an [Na+]o of 27 mM. However, neither La3+ nor Mn2+ affected the movements of Na+ and Ca2+ on re-establishing [Na+]o. 6. We conclude that cultured embryonic chick heart cells contain a Na+-Ca2+ exchange evidenced by the ability to cause movements of Na+ and Ca2+ which are counter to their respective electrochemical gradient and which are accompanied by downhill movements of the counter ion.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of sodium‐potassium pump inhibition and low sodium on membrane potential in cultured embryonic chick heart cells.

1. When the Na+-K+ pump of cultured embryonic chick heart cells was inhibited by addition of ouabain with or without removal of external K+, the membrane potential rapidly depolarized to -40 mV and the Na+ content approximately doubled within 3 min. 2. After this, exposure to an [Na+]o of 27 mM caused a fall in Na+ content, a gain in Ca2+ content and a hyperpolarization. The hyperpolarization was approximately 25 mV in a [K+]o of 0 or 5.4 mM after 3 min of pump inhibition. After approximately 10 min of pump inhibition, the same hyperpolarization was observed in a [K+]o of 5.4 mM but in K+-free solution the hyperpolarization increased to approximately 44 mV. 3. Varying [K+]o during the 10 min period of Na+-K+ pump inhibition showed that the increase in hyperpolarization was associated with the period of exposure to K+-free solution rather than the [K+]o at the time of lowering [Na+]o. 4. Changes in Na+ and Ca2+ content induced by exposure to an [Na+]o of 27 mM in K+-free solution were similar at 3 and 10 min. This and the above observations suggest that the increased hyperpolarization was due to an increased membrane resistance. 5. 10 mM-Cs+ reduced the low-[Na+]o hyperpolarization by 26% but did not significantly affect the movements of Na+ and Ca2+. 1 mM-La3+ reduced the low-[Na+]o hyperpolarization by 15%: it also totally blocked the rise in Ca2+ content and partially blocked the fall in Na+ content. 1 mM-Ba2+ reduced the low-[Na+]o hyperpolarization by 20%. 6. Raising [Ca2+]o from 2.7 to 13.5 mM produced similar but smaller hyperpolarizations (approximately 6 mV after 3 min pump inhibition). High [Ca2+]o caused a rise in Ca2+ content but no significant drop in Na+ content. The hyperpolarization in high [Ca2+]o was insensitive to verapamil (20 microM) and 10 mM-Cs+. 7. We conclude from the disparities between the magnitudes of the hyperpolarizations and the changes in ion contents that Na+-Ca2+ exchange cannot be unequivocally identified as electrogenic solely from the low-[Na+]o hyperpolarizations.

Mesenchymal tissue of the interventricular septum

Light and electron microscopic studies of frontal and sagittal sections of embryonic chick hearts (Stages 25, 28-29), reveal mesenchymal tissue in the cephalic portion of the interventricular septum. The endocardium of this cephalic portion contains reoriented and invaginated cells with pseudopodia; in addition there are cells immediately subjacent to the endocardium. Similar cellular events take place during the formation of mesenchymal tissue in the atrioventricular and conotruncal regions. In these regions the mesenchymal tissue originates by means of an endocardial activation process. The structural characteristics of the formation of the cephalic portion of the interventricular septum suggest that local mesenchymal tissue is contributed by the endocardium. However, based upon the close anatomic relationship observed by us between the mesenchymal tissues of the atrioventricular canal, conotruncal region and the cephalic portion of the interventricular septum; we do not discard a contribution by migration of cells from atrioventricular and conotruncal regions to the interventricular septum.

Iron chelation studies using desferrioxamine and the potential oral chelator, 1,2-dimethyl-3-hydroxypyrid-4-one, in normal and iron loaded rats.

A novel iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one, and desferrioxamine were compared for their ability to remove iron and for their site of action in iron release in rats. Repeated intraperitoneal injections of the chelators in rats with widespread tissue labelling by 59Fe derived from transferrin showed comparable 59Fe mobilisation by each chelator in normal and iron loaded rats. Specific labelling of a chelatable "cold" iron pool in hepatocytes by 59Fe derived from ferritin showed this pool to be equally accessible to parenteral doses of both chelators and also to oral 1,2-dimethyl-3-hydroxypyrid-4-one, which is an effective oral iron chelating agent that removes iron from parenchymal cells. This and other alpha-ketohydroxypyridines need further development as potential therapeutic agents in human iron overload.

Extracellular matrix from embryonic myocardium elicits an early morphogenetic event in cardiac endothelial differentiation

A critical step in early cardiac morphogenesis can be faithfully duplicated in culture using a hydrated collagen substratum, and thereby serves as a useful model system for studying the molecular mechanisms of cell differentiation. Results from previous work suggested that the myocardium in the atrioventricular canal (AV) region of the developing chick heart secretes extracellular proteins into its associated basement membrane, which may function to promote an epithelial-mesenchymal transition of endothelium to form prevalvular fibroblasts ( E. L. Krug, R. B. Runyan, and R. R. Markwald, 1985, Dev. Biol. 112, 414–426 ; C. H. Mjaatvedt, R. C. Lepera, and R. R. Markwald, 1987, Dev. Biol., in press ). In the present study we show that an EDTA-soluble extract of embryonic chick hearts can substitute for the presence of myocardium, the presumptive stimulator tissue, in initiating mesenchyme formation from AV endothelium in culture. Ventricular endothelium was unresponsive to this material in keeping with observed in situ behavior. AV endothelial cells did not survive beyond 4–5 days when cultured in the absence of either the EDTA-soluble heart extract, myocardial conditioned medium, or the myocardium itself. Antibody prepared against a particulate fraction of the EDTA-solubilized heart extract immunohistochemically localized this material to the myocardial basement membrane. In addition, conditioned medium from embryonic myocardial cultures effectively induced mesenchyme formation. Neither a variety of growth factors nor a sarcoma basement membrane preparation were effective in promoting mesenchyme formation indicating a selectivity of the responding embryonic AV endothelial cells to myocardial basement membrane. These observations reflect a truly inductive phenomenon as there was (1) an absolute dependence on the presence of the stimulating substance/tissue and (2) retention, in culture, of both the temporal and regional characteristics observed in situ. This is in contrast to the results of others investigating the cytodifferentiation of committed cells whose phenotypic expression can be either accelerated or diminished but not obligatorily regulated by a specific agent, thus making the interpretation of data difficult, if not irrelevant, to the study of differentiation. The results of this study provide direct experimental support for the hypothesis that extracellular matrix can indeed serve as a direct stimulator or “secondary inducer” of cytodifferentiation.

Increase in the cholinergic cardiac plexus in sympathetically aneural chick hearts.

Embryonic chick hearts were made sympathetically aneural by removal of premigratory neural crest over somites 10-20. The cholinergic cardiac plexus was assessed at 12 to 15 days of incubation using morphological and biochemical techniques. The cholinergic innervation to the heart was increased by 50 to 100% due to both hypertrophy and hyperplasia of the ganglion cells as well as their terminals. The additional cells were an expansion of the normal population of cholinergic neurons in the heart rather than from some extra source. The expanded population of neurons did not express an adrenergic phenotype in response to the absence of adrenergic cardiac innervation.

Qualitative and quantitative analysis of tumour invasion in vivo and in vitro.

Qualitative and quantitative methods for the analysis of invasion in 'natural' and in experimental tumours in vivo and in vitro are reviewed. In human tumours the functional consequences of invasion were evaluated histologically through staging on the basis of depths of invasion and through the presence of tumour cells inside vessels. Antibodies against components of the basement membrane have facilitated the definition of minimal invasion. With new probes derived from oncogene research the search for molecular differences between invasive and non-invasive parts of the tumour has begun. Since the same methods as those used for analysis of natural tumours also apply to experimental tumours in vivo, the major advantage of the latter is the possibility of manipulation. We have described a new mesenterium assay that may permit the selection of invasive cells from non-invasive ones in transfection experiments. Invasion relative to growth as a function of time was quantified in the kidney invasion test. In three-dimensional confrontations between embryonic chick heart fragments and invasive cells, we have used both a subjective grading and a qualitative computer-assisted image analysis of serial histological sections to score invasion. In two-dimensional confrontations supplementary methods could be applied, since such confrontations permitted direct observations on living cultures. In a variety of natural and experimental tumours, ultrastructural analysis, transmigration in two-compartment chambers, and release of metabolic label have demonstrated the role of motility and of lytic activity in tumour invasion.

Mapping of early development of electrical activity in the embryonic chick heart using multiple-site optical recording.

1. Using a multiple-site optical recording method with a 100-element photodiode array and a voltage-sensitive merocyanine-rhodanine dye, we have been able to monitor, for the first time, spontaneous electrical activity in pre-fused cardiac primordia in the 6- and 7-somite chick embryos. 2. To study the regional development of spontaneous electrical activity in the early embryonic pre-contractile chick heart at the later 7-9-somite stages, we have also recorded optically action potentials simultaneously from the entire heart, and constructed maps of the early development of electrical activity. 3. The data show that during the 6-9-somite stages, the size of the active area gradually increases, and that the development of electrical activity was spatially non-uniform: two peaks of activity were found in the right and left sides of the cono-ventricular region at the 7-8-somite stages. As development proceeded to the 9-somite stage, several peak areas of activity appeared. 4. The results are discussed in relation to the spatial pattern of proliferation of electrically active cells in the early phases of cardiogenesis.

Reconstitution of muscarinic cholinergic inhibition of adenylate cyclase activity in homogenates of embryonic chick hearts by membranes of adult chick hearts.

We have previously demonstrated that during embryonic development of the chick heart between days 2 1/2 and 10 days in ovo, muscarinic cholinergic inhibition of isoproterenol-stimulated adenylate cyclase activity increased 4-fold, and the sensitivity of isoproterenol-stimulated adenylate cyclase activity to inhibition by carbamylcholine increased 26-fold. Although the number of muscarinic receptors remained constant between days 2 1/2 and 10 in ovo, the levels of a 39- and 41-kDa pertussis toxin substrate increased in parallel with the ability of muscarinic agonist to inhibit adenylate cyclase activity (Liang. B.T., Hellmich, M. R., Neer, E. J., and Galper, J. B. (1986) J. Biol. Chem. 261, 9011-9021). These data are consistent with the hypothesis that between days 2 1/2 and 10 in ovo muscarinic receptors were uncoupled from inhibition of adenylate cyclase activity because of limiting levels of pertussis toxin substrates. In the current studies, in order to test this hypothesis homogenates of embryonic chick hearts 3 1/2 days in ovo were reconstituted with membranes from hearts of hatched chicks. In order to rule out reconstitution by factors from hatched chick hearts other than pertussis toxin substrates, muscarinic receptors from hatched chick hearts were inactivated by covalent binding of benzilycholine mustard and adenylate cyclase inactivated by N-ethylmaleimide prior to reconstitution. Reconstitution of benzilylcholine mustard/N-ethylmaleimide treated hatched chick heart membranes with homogenates of embryonic chick hearts 3 1/2 days in ovo resulted in a 2 1/2-fold increase in the ability of carbamylcholine to inhibit adenylate cyclase activity and reconstitution of hatched chick heart membranes with homogenates of hearts 2 1/2 days in ovo resulted in an approximately 10-fold increase in the sensitivity of isoproterenol-stimulated adenylate cyclase activity to inhibition by carbamylcholine. Membranes from hearts of hatched chicks which had been injected with pertussis toxin were incapable of reconstituting muscarinic inhibition of adenylate cyclase activity in homogenates of hearts 3 1/2 days in ovo. These data support the conclusion that early in embryonic development coupling of muscarinic receptors to inhibition of adenylate cyclase activity is limited by the availability of a pertussis toxin substrate.

Regulation of troponin C synthesis in primary culture of chicken cardiac muscle cells.

Cardiac myocyte cell culture from fourteen day old embryonic chicken heart was prepared. This cultured cell system was used to examine the regulation of troponin C (TnC) synthesis in cardiac muscle. To examine the regulation of TnC polypeptide synthesis, cardiac myocyte cells were pulse labelled with 35S-methionine at different days after plating. The synthesis of TnC was measured by determining the amount of radioactivity incorporated into the TnC polypeptide following separation by two dimensional gel electrophoresis. These measurements showed that TnC synthesis was maximum in 36 to 48 h old cultures and reached its lowest level in 4 day old cultures. This was in contrast to the synthesis of actin and tropomyosin. Synthesis of these polypeptides were lowest in 36 to 48 h old cultures and was maximum in 7 day old cultures. To examine whether the synthesis of TnC polypeptide paralleled the levels of TnC mRNA the sequences homologous to quail slow TnC cDNA clone were measured by hybridisation. The results showed that the decrease in the synthesis of troponin C polypeptide cannot be fully explained by the decrease in the steady state level of troponin C mRNA. The possibility of a role of translational control of troponin C mRNA in this process is discussed.

Desmin distribution in the cardiac outflow tract of the chick embryo during aortico-pulmonary septation.

The appearance and distribution of desmin, a muscle type intermediate filament, was examined in the truncus arteriosus of the chick embryonic heart during AP septation by an indirect immunofluorescence method. Prior to septation, on the 4th day of incubation, staining with antidesmin antibody was observed in the AP septum anlagen in the distal truncus. No staining with the antibody was detected in the developing tunica media of the great arteries in the distal truncus at this stage. During septation, on the 5th day of incubation, intense staining with the antidesmin antibody was observed in the cell condensation of the AP septum. In the 6-day-old embryo, the staining in the AP septum was reduced, but fluorescence by staining with the antibody was observed in the developing tunica media. No fluorescence was detected in other mesenchymal cells in the truncal swellings. Because of the time of appearance and the localization of antidesmin reactivity in the developing AP septum, it is suggested that muscle-type cells exist in the AP septum, and that these cells may perform an important function in septation.

Intercellular gaps in the early development of chick mural endocardium. A TEM study.

Embryonic chick hearts stages 15 to 21 according to the Hamburger-Hamilton series (1951) were subjected to TEM and light-microscopical examination to investigate ventricular mural endocardium during early trabeculation. Serial sectioning of consecutively-aged embryos indicated an invagination of the endocardial layer which lines the intertrabecular and intermuscular spaces. During the proceeding development, frequently occurring approaches and contacts between mural endocardium and myocardium could be observed. At all stages examined, the cardiac jelly appeared as an acellular, at particular sites very attenuated matrix. The intercellular gaps of the endocardium could not be correlated to the invagination and the endothelialization of the intertrabecular spaces.

Spectrum of double outlet right ventricle induced by electrical shock at the conotruncus of the embryonic chick heart: emphasis on cellular changes and evaluation of hemodynamics using Doppler.

Administration of electrical shock to the conotruncal area of embryonic chicks at developmental stage 24-27 induced a complex malformation, namely, double outlet right ventricle (DORV) of varying degrees of severity. The device used to apply electrical current to the heart was constructed using a 9-volt battery, a push-button switch, and a 100-ohm potentiometer with calibrated dial. This allowed a short pulse of known voltage to be applied through the electrodes to a selected area of the heart. Two different methods were chosen to administer electrical shock at the conotruncal area of the heart. One method utilized longitudinal application of electrical shock, and the other used horizontal application of electrical shock directly to the conotruncus. Three distinct types of DORV were found in the longitudinal (L) and the horizontal (H) electrical shock groups: 1) DORV without a ventricular septal defect (VSD), in which the dilated sinus of Valsalva of the aortic valve extends into the right ventricle. 2) DORV with a subaortic VSD, with variable degrees of pulmonary stenosis and a hypoplastic left ventricle. 3) DORV with a subpulmonic VSD, pulmonic stenosis and a hypoplastic right ventricle. Longitudinal stimulation produced a higher incidence of cardiovascular anomalies than horizontal stimulation (p = 0.01). Histological examination showed complete disappearance of myocardial fibers, myocardial degeneration, and the aggregation of protein material or glycogen within myocardial cells. Our Doppler findings demonstrate that relative to control embryos, peak velocity initially increased 3 minutes after electrical stimulation (p less than 0.01), and subsequently decreased significantly within 30 minutes (p less than 0.01). One may hypothesize that the marked initial increase of peak velocity was induced by tissue damage, which led to a narrowing of the conotruncal outflow tract. Evidence for contruncal narrowing was also reflected in percent window data (% W), which indicates the level of turbulence within a vessel. However, the decrease of peak velocity 30 minutes after stimulation might be caused by myocardial damage resulting in a diminished cardiac output. The relationship between cell death processes in the conotruncal area and DORV spectrum induced by electrical shock is discussed.

Electrophysiology of calcium antagonists

Calcium antagonistic drugs block the voltage-dependent Ca 2+ slow channels incardiac muscle and vascular smooth muscle. This effect on slow channels is relatively specific, i.e., some of the drugs (e.g., nifedipine and diltiazem) have almost no effect on other types of ion channels (fast Na + channels and K + channels). Some of the drugs (e.g., verapamil and bepridil) slightly depress the fast Na + channels as well. The order of potency in blocking slow channels is generally: mesudipine > nifed > dilt > verap > bep. The blocking effect on the slow channels is frequency dependent (use-dependent), although the dihydropyridines have a lesser frequency dependence. Elevation of [Ca] 0 antagonizes the inhibition of Ca 2+ influx produced by the drugs; partly mediated by a larger electrochemical driving force for Ca ++ influx through unblocked channels. Nifed and dilt are the fastest to reverse upon washout. Verap, D600, and nifed block slow Na + channels found in early embryonic chick hearts; bep, dilt, and mesud do not. Verap and bep inhibit Ca ++ binding to sarcolemma; nifed and dilt do not. The order of uptake into muscle follows their order of lipid solubilities: bep > verap > nitrend > nifedipine > dilt. Those drugs that readily permeate into the cell interior have the option of having their primary or second site of action on some intracellular site. For example, verap and D600 may block slow channels by acting on the inner surface of the cell membrane, and bep might also act to depress Ca ++ release from the SR.

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.

Confrontation of an invasive (MO4) and a noninvasive (MDCK) cell line with embryonic chick heart fragments in serum-free culture media.

Confronting cultures of precultured embryonic chick heart fragments (PHF) with aggregates of malignant cells in vitro have been shown to be relevant for a number of aspects of tumor invasion in vivo. Preculture of the heart fragments, formation of cell aggregates, and subsequent culture of confronting pairs have so far been done only in serum-containing culture media. We describe here confronting cultures of PHF with invasive MO4 mouse cell aggregates or noninvasive MDCK dog kidney cell aggregates in serum-free media. Heart fragments precultured in the absence of serum seemed to be necrotic after confronting culture in serum-free media. However, preculturing in media supplemented with 10% fetal bovine serum allowed us to do subsequent confronting cultures in absence of serum. Cell aggregates were also prepared in serum-containing medium. MO4 cells occupied and replaced the heart tissue within 4 d, whereas MDCK cells remained at the periphery of the PHF. This indicates that serum-free confronting cultures can discriminate between invasive and noninvasive cells. The viability of individual PHF and cell aggregates cultured in the same way as in confrontations was ascertained by histology and by explantation and postculturing on a solid tissue culture substrate. Growth of the cultures was smaller in serum-free media than in media supplemented with 10% fetal bovine serum. The main advantage of serum-free culture conditions in vitro is the elimination of the influence of serum components on invasion, and the ability to examine the effect on invasion of drugs that are susceptible to inactivation by serum.

Cyclic AMP in normal and sympathetically aneural chick hearts during development

Basal levels of cyclic adenosine monophosphate (cAMP) were measured in embryonic chick hearts at various times during development. Basal cAMP was highest on incubation day 5 and decreased throughout the remaining incubation period. Cyclic AMP could not be stimulated above basal level by intravenous in ovo administration of isoproterenol or tyramine on incubation day 5; however, there was a decrease in cAMP 2 mins after intravenous injection which was identical to the decrease in cAMP in controls injected with saline. Heart rate decreased following intravenous injection of saline on incubation day 5, but a similar decrease was not observed following intravenous isoproterenol injection. Functional sympathetic innervation of the heart does not occur until incubation day 16, and this fact is responsible for the insensitivity to tyramine stimulation on incubation day 5. Although the level of cAMP could not be stimulated above basal level on incubation day 5, beta-antagonists caused a decrease in the level of cAMP, with no decrease in heart rate. These observations indicate that the beta-receptor is coupled to adenylate cyclase on incubation day 5 but it is questionable whether adenylate cyclase is effectively coupled to heart rate. Isoproterenol and tyramine caused a significant elevation in cardiac cAMP and heart rate on incubation day 17 following intravenous in ovo injection. Hearts made sympathetically aneural by removal of premigratory neural crest responded to isoproterenol but not tyramine on incubation day 17 which demonstrates that the morphologically aneural hearts are also functionally aneural.(ABSTRACT TRUNCATED AT 250 WORDS)