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.     

Ionic interconversion of pacemaker and nonpacemaker cultured chick heart cells

Trypsin-dispersed cells from hearts (ventricles) of 7 to 8 day chick embryos were cultured 3 to 21 days. The cells became attached to the culture dish and assembled into monolayer communities. By means of a bridge circuit, one microelectrode was used for simultaneously passing current and recording membrane potentials (V_m). The input resistance, calculated by the measured ΔV_m for a known step of current, averaged 10 MΩ. Electrotonic depolarization of nonpacemaker cells had no effect on frequency of firing. Within 2 min after addition of Ba⁺⁺ (5 to 10 mM) to the Tyrode bath, the cells became partially depolarized and quiescent nonpacemaker cells developed oscillations in V_m which led to action potentials. With time, the depolarization became nearly complete and the input resistance increased 2 to 10 times. During such sustained depolarizations, action potentials were no longer produced and often tiny oscillations were observed; however, large action potentials developed during hyperpolarizing pulses. Thus, the automaticity of the depolarized cell became apparent during artificial repolarization. Sr⁺⁺ (5 to 10 mM) initially produced hyperpolarization and induced automaticity in quiescent nonpacemaker cells. Elevated [K⁺]_o (20 to 30 mM) suppressed automaticity of pacemaker cells and decreased R_m concomitantly. Thus, Ba⁺⁺ probably converts nonpacemaker cells into pacemaker cells independently of its depolarizing action. Ba⁺⁺ may induce automaticity and depolarization by decreasing g _K, and elevated [K⁺]_o may depress automaticity by increasing g _K. The data support the hypothesis that the level of g _K determines whether a cell shall function as a pacemaker.     

Na/H exchange in cultured chick heart cells. pHi regulation

The purpose of this study was to establish the existence of Na/H exchange in cardiac muscle and to evaluate the contribution of Na/H exchange to pHi regulation. The kinetics of pHi changes in cultured chick heart cells were monitored microfluorometrically with 6-carboxyfluorescein and correlated with Nai content changes analyzed by atomic absorption spectrophotometry; transmembrane H+ movements were evaluated under pH stat conditions. After induction of an intracellular acid load by pretreatment with NH4Cl, a regulatory cytoplasmic alkalinization occurred with a t1/2 of 2.9 min. pHi regulation required external Na+ and was concomitant with transmembrane H+ extrusion as well as a rapid rise in Nai content in an Na/H ratio of 1:1. Microelectrode recordings of membrane potential demonstrated directly the electroneutral character of pHi regulation. Acid-induced net Na+ uptake could be either stimulated by further decreasing pHi or inhibited by decreasing pHo; Na+ uptake was unaffected by tetrodotoxin (10 micrograms/ml), quinidine (10(-3) M), DIDS (10(-4) M), Clo-free solution, or HCO3-free solution. Amiloride (10(-3) M) maximally inhibited both pHi regulation and Na+ uptake; the ID50 for amiloride inhibition of Na+ uptake was 3 microM. Nao-dependent H+ extrusion showed half-maximal activation at 15 mM Nao; Li+, but not K+ or choline+, could substitute for Na+ to support H+ extrusion. Cao-free solution also stimulated acid-induced Na+ uptake. We conclude that pHi regulation following an acid load in cardiac muscle cells is by an amiloride-sensitive, electroneutral Na/H exchange. Stimulation of Na/H exchange up to 54 pmol/cm2 X s indicates the rapidity of this exchange across cardiac cell membranes. Na/H exchange may also participate in steady state maintenance of pHi.     

Linear impedance studies of voltage-dependent conductances in tissue cultured chick heart cells.

Plateau and pacemaker currents from tissue cultured clusters of embryonic chick heart cells were studied in the time domain, using voltage-clamp steps, and in the frequency domain, using a wide-band noise input superimposed on a steady holding voltage. In the presence of tetrodotoxin to block the sodium channel, a depolarizing voltage step into the plateau range elicited: (a) a rapid (approximately equal to 2 ms) activation of the slow inward current; (b) a subsequent slower (approximately equal to 25 ms) decline in the slow inward current; and (c) activation of a very slow (5 to 10 s) outward current. Impedance studies in this voltage range could clearly resolve two voltage-dependent processes, which appeared to correspond to points b and c above because of their voltage dependence, pharmacology, and time constants. A correlate of point a was also probably present but difficult to resolve owing to the fast time constant of activation for the slow inward channel. At voltages negative to -50 mV a new voltage-dependent process could be resolved, which, because of its voltage dependence and time constant, appeared to represent the pacemaker channel (also termed If or IK2). In the Appendix, linear models of voltage-dependent channels and ion accumulation/depletion are derived and these are compared with our data. Most of the above-mentioned processes could be attributed to voltage-dependent channels with kinetics similar to those observed in time domain, voltage-clamp studies. However, the frequency domain correlate of the decline of the slow inward current was incompatible with channel gating, rather, it appears accumulation/depletion of calcium may dominate the decline in this preparation.     

Response of caudal neurosecretory cells of Salvelinus fontinalis to variations in the ionic composition of the environment

Summary The response of caudal neurosecretory cells in Salvelinus fontinalis to exposure to media in which one ion (Na, K, Mg, Ca and Cl) was either selectively enriched or depleted was evaluated by morphometric criteria. Morphological changes indicating stimulation of synthetic activity were observed: exposure to sea water with low potassium concentration was the most efficacious in inducing the increase in the average diameter of caudal cells and the number of nucleoli for both cells with lobed and with nonlobed nuclei. The proportion of cells with lobed nuclei was also increased. To a lesser degree and in decreasing order, experimental milieu with modified magnesium contents, either fresh water enriched in this ion or seawater depleted of the same ion, and fresh water enriched in calcium also resulted in significant increases in cell diameter and the number of nucleoli.The two cytological cell types, viz cells with non-lobed nuclei or cells with lobed nuclei, are interpreted as being linked to different levels of neurosecretory activity.Supported by NSERC (Canada)     

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.     

Effect of cAMP on choline uptake and phosphatidylcholine biosynthesis in cultured chick heart cells

The effect of an analogue of cAMP on the uptake and metabolism of choline in the heart was studied in isolated cardiac cells. The cells were obtained from 7-day-old chick embryos and maintained in culture. The effects of cAMP were studied using the dibutyryl cAMP analogue and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. After a 2-h incubation with [3H]choline, about 85% of the label was recovered in phosphocholine, with most of the rest in phospholipid. During a subsequent chase incubation, [3H]phosphocholine was transferred to phosphatidylcholine with little accumulation in CDP-choline. This suggests the rate-limiting step for the conversion of phosphocholine to phosphatidylcholine in these cells is the synthesis of CDP-choline. cAMP decreased the incorporation of choline into phosphatidylcholine, but did not change the flux of metabolites through the step catalyzed by CTP:phosphocholine cytidylyltransferase. cAMP had little effect on choline uptake at low (1-25 microM) extracellular choline concentrations, but significantly (p less than 0.05) decreased choline uptake at higher (37.5-50 microM) extracellular choline concentrations. Thus, cardiac cells take up and metabolize choline to phosphocholine, with CTP:phosphocholine cytidylyltransferase being the rate-limiting step in phosphatidylcholine biosynthesis. cAMP decreases [3H]choline uptake and its subsequent incorporation into phosphocholine and phospholipid. However, the metabolism of choline within the cell is unaffected.     

Self-contact inhibition of movement in cultured chick heart fibroblasts

Collisions between two lamellar processes extended from a single locomoting cultured cell were examined by time-lapse cinemicrography and transmission electron microscopy. In most cases after contact the forward movement of either one or both of the lamellae ceased and was followed by a phase of retraction of the lamellae resulting in the breaking of the contact. The events correspond well to the contact inhibition of movement expressed when two cells collide. The similarity is also shown in the ultrastructure of the cell contacts which exhibit a close parallel arrangement of the apposed cell membranes and an alignment of microfilaments in the regions of the cytoplasm at the contacts.     

The regulation of the intracellular sodium ion concentration in cultured chick embryo heart cells.

Using digital imaging microscopy with the fluorescent indicator sodium-binding benzofuran isophtalate, we examined the cytosolic Na+ concentration ([Na+]i) in individual chick embryo heart cells. Inhibition of the Na(+)-H+ exchanger using Na(+)-free (Li+ substituted) medium and inhibition of the Na(+)-efflux through the Na(+)-Ca2+ exchanger using Ca(2+)-free medium didn't change the [Na+]i. The opening of voltage-dependent Na+ channels with veratridine (150 micrograms/ml) and inhibition of the Na(+)-K(+)-Cl(-)-cotransporter with bumetanide (10 microM) led to an increase in [Na+]i by 107% and 86%, respectively, suggesting that the Na+ channels and the Na(+)-K(+)-Cl- cotransporter predominantly regulate the [Na+]i in cultured chick embryo heart cells.     

Developmental changes of amp-deaminase activity in chick heart muscle

• 1.1. The developmental changes of 5'AMP-deaminase activity were studied in the hearts of 7–21 days chick embryos, 1–58 days chicks and in adult hen.
• 2.2. The activity of 5'AMP-deaminase in the embryonic and adult hen cardiac muscle were similar and amounted about 20 nmoles NH₃× min⁻¹ per mg protein. The activity dropped about fourfold at hatching, remained low for 18 days of postnatal development and increased slowly reaching its embryonic value in the heart of adult hen.
• 3.3. The changes of 5'AMP-deaminase were not correlated with the development of cardiac myocytes as measured by morphometry and myosin ATP-ase activity.

     

cAMP and cGMP changes associated with the differentiation of cultured chick embryo muscle cells.

A thin-slab, SDS polyacrylamide gel electrophoresis system is described in which actin within whole cell homogenates can be quantitated within a wide range of protein values (0.05–1.4 μg/band). After demonstrating the absence of appreciable contaminants in the actin band, and the lack of appreciable reincorporation of label during pulse-chase experiments, the turnover of actin was examined in pre-labeled cells during normal log growth and during induced encystation in Acanthamoeba. During log growth, no actin degradation was detected. However, as the cells approached the end of log phase growth and entered stationary phase, a dramatic increase in the amount of actin/cell and the percentage of total protein represented by actin was recorded. The encystation process per se was accompanied by a rapid reduction in these values to preencystment levels.     

Response of the quiescent heart tube to mechanical stretch in the intact chick embryo

Quiescent heart tubes in intact eight-somite chick embryos were mechanically stretched by injecting excess fluid into the heart lumen. The stretch stimuli sometimes caused precocious focal twitches in the ventricular portion of the primitive heart tube. The contractile response to mechanical stretch was confirmed by recording electrograms before, during, and after the pressure pulse injections. The results prompt us to suggest that the precise timing of the initial heartbeats in the intact embryo may involve an increase in the intraluminal fluid volume and pressure stretching the heart tube wall.     

Cell fusion and differentiation in cultured chick muscle cells

A method for the quantitative estimation of myoblast fusion and the effects of variables in the culture conditions on the extent of fusion are described. The onset of fusion is delayed both by feeding and by lowering the initial cell density, but effects of alterations in sera and embryo extracts are evident only at later stages of differentiation. Using a sensitive fluorimetric assay for creatine phosphokinase, enzyme activity was determined throughout the culture period and related to the extent of cell fusion. The increase in activity during a 7-day culture period is biphasic, the first small increase (2–5 ×), which begins after 40–50 h in culture, being apparently closely related to cell fusion. There is a 48 h delay before the second, much larger (up to 25 ×) increase begins.     

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.     

Regulation by high density lipoproteins of muscarinic acetylcholine receptor function in chick heart cells cultured in defined medium

Activation of cardiac muscarinic acetylcholine receptors (mAChR) on cultured chick heart cells results in a decrease in cellular cAMP levels and a stimulation of phosphoinositide breakdown. A serum-free culture system has been used to investigate the regulation of mAChR number and function by purified serum high density lipoprotein (HDL). Administration of HDL purified from rooster serum to chick heart cells cultured in defined medium results in an attenuation of the ability of muscarinic agonist to inhibit forskolin-stimulated cAMP accumulation, with no change in its ability to stimulate phosphoinositide hydrolysis or to mediate down-regulation of receptor number. The inclusion of HDL in the culture medium did not result in appreciable changes in mAChR number or affinity, nor were the levels of the inhibitory guanine nucleotide-binding regulatory proteins (G-proteins) altered. However, the ability of guanine nucleotides to inhibit forskolin-stimulated adenylate cyclase activity was reduced by HDL treatment, suggesting that HDL interferes with the capacity of G-proteins to interact with adenylate cyclase. In order to determine which component of native HDL mediates the decreased effectiveness of carbachol, the ability of lipid and apoprotein fractions to mimic the effect of HDL was tested. HDL lipid fractions were able to mimic the effect of native HDL, while protein fractions were not. This result suggests that the ability of HDL to attenuate muscarinic receptor function is mediated by its lipid constituents. The effect of HDL and HDL lipid fractions were not correlated with changes in membrane cholesterol content.     

Developmental changes in internal structure of chick heart plasma membrane

Although changes in electrophysiologically measurable membrane properties of chick embryo cardiac plasma membrane have been repeatedly documented during embryonic development, ultrastructural techniques were heretofore too insensitive to detect developmental changes in internal structure of this membrane. We report here significant structural changes detected by applying the quantitative analysis of Kordylewski, Karrison, and Page (Amer. J. Physiol. 245, H992-H997, 1983 and 248, H297-H304, 1985) to stereo imaged electron microscopic negatives of glutaraldehyde-fixed chick embryo hearts, freeze fractured and photographed with a goniometer stage. Between Hamburger-Hamilton stages 12+ (about 48 hr incubation) and 24 (about 96 hr incubation), plasmalemmal P-face particle density of ventricular myocytes increased from 2228 +/- 139 to 3063 +/- 109 (P less than 0.01); thereafter, measurements at stages 30, 37, 40, and 45 (7, 11, 15, and 19 days incubation) showed a slower significant linear increase which gave a least-squares line with a slope of 41 +/- 13 particles/day (P less than 0.01). Just before hatching, (stage 45) the value of 3762 +/- 234 was similar to, though slightly smaller than, the values of 4122 +/- 153 (8 days after hatching) and 4281 +/- 218 (adult chicken). These results indicate striking stage-dependent changes in the population of integral membrane proteins (channels, carriers, receptors, etc.), especially marked during early embryogenesis.     

Transmembrane chloride flux in tissue-cultured chick heart cells

To evaluate the transmembrane movement of chloride in a preparation of cardiac muscle lacking the extracellular diffusion limitations of natural specimens, intracellular chloride concentration ( [Cl] i) and transmembrane 36Cl efflux have been determined in growth-oriented embryonic chick heart cells in tissue culture. Using the method of isotopic equilibrium, [Cl]i was 25.1 +/- 7.3 mmol x (liter cell water)- 1, comparable to the value of 24.9 +/- 5.4 mmol x (liter cell water)-1 determined by coulometric titration. Two cellular 36Cl compartments were found; one exchanged with a rate constant of 0.67 +/- 0.12 min-1 and was associated with the cardiac muscle cells; the other, attributed to the fibroblasts, exchanged with a rate constant of 0.18 +/- 0.05 min- 1. At 37 degrees C, transmembrane Cl flux of cardiac muscle under steady-state conditions was 30 pmol x cm-2 x s-1. In K-free, normal, or high-Ko solutions, the responses of the membrane potential to changes in external Cl concentration suggested that chloride conductance was low. These results indicate that Cl transport across the myocardial cell membrane is more rapid than K transport and is largely electrically silent.