Pyrophosphate stimulation of calcium uptake into cultured embryonic bones. Fine structure of matrix vesicles and their role in calcification

The onset of mineralization in embryonic chick femurs was studied as a model for the initiation of biological calcification. Electron microscopy confirmed the presence of calcifying matrix vesicles within newly formed bone, and showed that these vesicles were the initial site of crystal deposition. Matrix vesicles were first seen on day 6 of embryonic development, and already were present in considerable numbers on day 7, at which time mineral deposition was just beginning. As a reflection of initial mineralization the uptake of ⁴⁵Ca and ⁴⁰Ca into 7-day-old bones was studied during 2 days in organ culture. A control level of uptake was established using a defined culture medium, P-6. Addition of inorganic pyrophosphate (PP_i) to this medium caused a marked increase in calcium uptake into areas of matrix which normally calcify in vivo. The maximal ⁴⁵Ca uptake, greater than 4-fold, was achieved with 4 μg of P per milliliter of PP_i and was partially heat-inhibitable. Since the matrix vesicles are known to be rich in inorganic pyrophosphatase, it is proposed that mineralization is promoted in vesicles by the enzymatic hydrolysis of pyrophosphate. The membrane-bounded matrix vesicles appear to provide the necessary enzymes and environment to concentrate calcium and phosphate for initiating crystal formation.     

Calcium regulation in the embryonic chick. II. Ultrastructure of the parathyroid glands in shell-less and in ovo embryos

The ultrastructure of the parathyroid glands was studied in chick embryos developing normally in ovo or in shell-less culture (after removal of the eggshell). Shell-less chick embryos are significantly hypocalcemic relative to their in ovo counterparts. At 12 days of incubation, the parathyroid glands of shell-less embryos contain more lipid and show evidence of increased protein synthetic activity relative to those grown in ovo (more rough endoplasmic reticulum, presence of some dense secretory granules). The glands from in ovo embryos do not contain secretory granules at this age. At 15 days of incubation, the in ovo glands have developed signs of protein synthetic activity similar to those of the 12-day shell-less embryos. However, the parathyroids of the 15-day shell-less embryos appear strikingly more active than at 12 days, containing stacks of concentric RER membranes and increased numbers of secretory granules. By 18 days of incubation, the ultrastructure of the glands of the two groups is indistinguishable, both appearing to be more active than the 15-day shell-less group. Thus, protein synthetic activity of the parathyroid glands, as detected by ultrastructural alterations of the chief cells, normally appears to be initiated during the latter part of embryogenesis (by approximately 15 days incubation) and its onset can be stimulated at least 3 days prematurely by hypocalcemia.     

Effects of chick brain extract on the proliferation of chick neuroblasts cultured in media supplemented with low and high serum concentrations

The proliferative activity of chick neuroblasts cultured in a medium containing a low (5%) or a high (20%) concentration of fetal calf serum (FCS) was analyzed and the influence of a chick brain extract was investigated. Morphological observations and tritiated thymidine incorporation measurements have shown that neuroblasts from 6 day-old chick embryo cerebral hemispheres proliferate more actively in the medium with 5% FCS compared to the medium with 20% FCS. The medium containing 5% FCS favoured the maintenance of neuronal cells in a neuroblast stage as shown by electron microscopy. The stimulatory effect of brain extract on the proliferation of neuroblasts is stronger in the low serum culture condition. These findings indicate that a low serum-containing medium is an adequate condition to study neuronal proliferation and effects of growth factors on these cells.     

Fine structure of the parathyroid glands in baboons, Papio hamadryas in response to experimental hypercorticoidism

Female baboons maintained under laboratory conditions were subjected to a series of 10 weekly injections (4 mg/kg body weight) of the synthetic glucocorticoid, triamcinolone hexacetonide. In response to the treatment, serum immunoreactive parathyroid hormone (PTH) levels were raised, though blood calcium levels remained within normal physiological limits. Light and electron-microscopic studies were made on the parathyroid glands at the end of the experimental period. The baboon parathyroid glands were composed of 'light' and 'dark' forms of the chief cells in varying ratios from gland to gland even within a single animal. Glucocorticoid-induced parathyroid hyperactivity as measured by circulating PTH levels was not accompanied by cellular hypertrophy, though there was an increase in the relative number of 'light' cells. At the ultrastructural level, after treatment, many of the 'light' cells were found to contain more free ribosomes, larger profiles of granular endoplasmic reticulum and had better developed mitochondria. Interdigitations between adjacent chief cells were more complex in treated glands. Apart from these features, chief cells of treated glands were basically similar to those of untreated controls. Our study showed that functional parathyroid hyperactivity in baboons is not necessarily accompanied by significant ultrastructural changes in chief cells.     

Fine structure of the fetal rat thyroid and parathyroid glands at term and during prolonged gestation

Summary The fine structure of the fetal rat thyroid and parathyroid glands was studied at term and during prolonged gestation, which was induced by subcutaneous injections of progesterone to the mothers from gestational days 20 through 24. At term, the follicular and parafollicular cells of the thyroid as well as cells of the parathyroid exhibited well developed cytoplasmic organelles. Morphological changes were not detected in either of the endocrine glands during prolonged gestation. The results are discussed in relationship to 1) thyroid follicular cell activity during stress and 2) the function of thyroid parafollicular and parathyroid cells in calcium homeostasis.Supported by the Medical Research Council of Canada Grant No. MA4740.     

Effects of K-channel blockers, calcium, and verapamil suggest different pacemaker mechanisms in cultured neonatal rat and embryonic chick ventricle cells

We compared the determinants of spontaneous activity in explanted neonatal (2-day-old) rat ventricle cells and in reaggregates derived from 15-day-old chick embryos. We studied the beating rate with an optical recording method and the underlying electrical activity with glass microelectrodes using the K current blockers cesium (Cs) and tetraethylammonium, varied Ca concentrations, and the Ca antagonist verapamil. In the rat (i) Cs increased the beating rate that was mediated by an increase in the slope of the diastolic potential. (ii) Ca increased the beating rate dramatically at low and medium concentrations to decrease it again at 8 mM Cao. This increase in the beating rate was mediated by an increase of the slope of the diastolic depolarization. (iii) The beating rate decreased with verapamil at concentrations between 0.5 and 2.0 microM. The effects of Cs and Ca suggest that an increase in net inward current (block of IK1) underlies the positive chronotropic effect of Cs and that the pacemaker mechanism is determined by a Ca inward current or an IT1 type current modulated by variations of Cai. In the chick reaggregates (i) Cs and tetraethylammonium decreased the beating rate that was mainly brought about by a decrease in the slope of diastolic depolarization. (ii) Ca increased the beating rate but to a lesser degree than in the rat and there was no decrease of the beating rate at higher concentrations. (iii) The increase in the beating rate was not mediated by an increase in the slope of the diastolic potential but mainly by a depolarization of the maximum diastolic potential. (iv) Verapamil inhibited electrogenesis before any change in the diastolic potential was evident. The negative chronotropic effect of Cs and tetraethylammonium is compatible with the notion that a voltage- and time-dependent K current was inhibited and that this current determines the pacemaker. Moreover, the Ca component of the pacemaker mechanism in explanted rat ventricle cells resembles either that of the sinoatrial node or represents triggered activity.     

Effect of different fetal bovine serum concentrations on the replicative life span of cultured chick cells

The effect of Eagle's minimal essential medium, containing different fetal bovine serum (FBS) concentrations, on the proliferation and replicative life span of cultured chick cells has been studied. Our results showed that the rate of chick cell proliferation and the cell density at stationary phase increased as a function of serum concentration between 5 and 30% FBS. The replicative life span of cultured chick cells was dependent on the FBS concentration between 5 and 20% in a medium volume of 0.20 ml/cm2. The maximum replicative life span of chick cells was obtained by serially propagating cells in a medium volume of 0.20 ml/cm2 containing 20 or 30% FBS, or, alternatively, in 0.53 ml/cm2 containing 10, 20 or 30% FBS. Cells grown in medium containing 5% serum had a calendar life span of 35 days, whereas cells propagated in medium containing higher serum concentrations had a calendar life span of 50 days. These results reenforce the concept that, although the kinetics of cell population aging can be affected by the culture medium composition, the aging of cells in culture is controlled by alterations within the cell.     

Effect of different fetal bovine serum concentrations on the replicative life span of cultured chick cells

Summary The effect of Eagle's minimal essential medium, containing different fetal bovine serum (FBS) concentrations, on the proliferation and replicative life span of cultured chick cells has been studied. Our results showed that the rate of chick cell proliferation and the cell density at stationary phase increased as a function of serum concentration between 5 and 30% FBS. The replicative life span of cultured chick cells was dependent on the FBS concentration between 5 and 20% in a medium volume of 0.20 ml/cm². The maximum replicative life span of chick cells was obtained by serially propagating cells in a medium volume of 0.20 ml/cm² containing 20 or 30% FBS, or, alternatively, in 0.53 ml/cm² containing 10, 20 or 30% FBS. Cells grown in medium containing 5% serum had a calendar life span of 35 days, whereas cells propagated in medium containing higher serum concentrations had a calendar life span of 50 days. These results reenforce the concept that, although the kinetics of cell population aging can be affected by the culture medium composition, the aging of cells in culture is controlled by alterations within the cell. This work was supported by IIT Research Institute.     

A low voltage-activated calcium conductance in embryonic chick sensory neurons.

Isolated Ca currents in cultured dorsal root ganglion (DRG) cells were studied using the patch clamp technique. The currents persisted in the presence of 30 microM tetrodotoxin (TTX) or when external Na was replaced by choline. They were fully blocked by millimolar additions of Cd2+ and Ni2+ to the bath. Two components of an inward-going Ca current were observed. In 5 mM external Ca, a current of small amplitude, turned on already during steps changes to -60 mV membrane potential, leveled off at -30 mV to a value of approximately 0.2 nA. A second, larger current component, which resembled the previously described Ca current in other cells, appeared at more positive voltages (-20 to -10 mV) and had a maximum approximately 0 mV. The current component activated at the more negative membrane potentials showed the stronger dependence on external Ca. The presence of a time- and a voltage-dependent activation was indicated by the current's sigmoidal rise, which became faster with increased depolarization. Its tail currents were generally slower than those associated with the Ca currents of larger amplitude. From -60 mV holding potential, the maximum obtainable amplitude of the low depolarization-activated current was only one-tenth of that achieved from a holding potential of -90 mV. Voltage-dependent inactivation of this current component was fast compared with that of the other component. The properties of this low voltage-activated and fully inactivating Ca current suggest it is the same as the inward current that has been postulated in several central neurons (Llinas, R., and Y. Yarom, 1981, J. Physiol. (Lond.), 315:569-584), which produce depolarizing potential waves and burst-firing only when membrane hyperpolarization precedes.     

Effects of short-term treatment with calcium on the parathyroid glands in golden hamsters of different ages, with special reference to large vacuolar bodies.

The effects of different ages on large vacuolar bodies in the parathyroid glands of golden hamsters after short-term treatment with calcium were investigated. In the parathyroid glands of the young and adult animals 15 min and the senile animals 15 and 60 min after administration of calcium, the percent area occupied by large vacuolar bodies was significantly increased as compared to that of the young, adult and senile control animals, respectively. These findings suggest that the percent area occupied by large vacuolar bodies is increased in response to acute hypercalcemia. It is thought that in the parathyroid glands suppressed by hypercalcemia there is a relationship between the percent area occupied by large vacuolar bodies and aging.     

Developmental patterns of ganglioside sialosylation coincident with neuritogenesis in cultured embryonic chick brain neurons.

Chick brain precursor neurons were observed to introduce sialic acid biosynthetically into only three specific gangliosides: monosialosyl lactosyl ceramide (GM3), disialosyl lactosyl ceramide (GD3), and disialosyl gangliotrihexosyl ceramide (GD2), when sialic acid was labeled metabolically by its obligate precursor, [3H] ManNAc. Sialosyl donor CMP-[3H]NeuAc supplied in the culture medium gave rise uniquely to surface-labeled GD3. Thus sialosyl transferase/GD3 synthase activity is expressed both intraneuronally and in the neuronal exofacial surface. Upon epidermal growth factor-induced onset of neurite outgrowth, labeled complex sialosyl gangliotetrahexosyl ceramide species of gangliosides began to appear in the embryonic neuronal plasma membrane. However, intraneuronal and exofacial sialosyl transferase/GD3 synthase activities remained constant, with or without neurite outgrowth. Moreover, simpler species of gangliosides maintained a steady quantitative sialosyl level (1.6 +/- 0.2 micrograms of sialic acid/mg of protein), whereas more complex species completely absent before neurite outgrowth accrued and reached 4.8 +/- 0.9 micrograms of sialic acid/mg of protein with full neurite development. This analysis of developmental patterns of ganglioside sialosylation has provided evidence that stable neurite outgrowth depends upon generation by the neuron of special plasma membrane with a massive content of complex higher species of gangliosides.     

Biosynthetic activity of parathyroid cells during different function of the parathyroid glands

By means of autoradiograph c and morphometric methods parathyroid glands of 31 rats after single injection of 3H-leucin (2,5 mc Cu/g of body mass 25 min. before killing) were investigated. Parathyroid glands were inhibited by multiple injections of calcium gluconate solution or by a diet with increased content of calcium and vitamin D2. Parathyroids were stimulated by multiple injections of Trilon B solution, by a diet with increased content of phosphates or by subtotal parathyroid resection. Straight correlation between parathyroid function, average section area of parathyrocytes and average number of silver granules per one parathyrocyte was revealed. Hence, average section area of parathyrocytes is objective morphometric criterion of parathyroid function.     

Fine structural changes in embryonic chick heart ventricle induced by lead poisoning.

The embryonic chick heart ventricle of day 11 was studied electron microscopically to learn the structural changes that develop in lead poisoning. The chick embryos were administered with 0.015 mg/egg of lead acetate at day 2. The most pronounced changes observed in the ventricle were: malformed mitochondria, disorganized, short and scanty myofibrils and abundance of swollen vacuoles. The ultrastructure of the ventricle from the control chick embryos was normal. The most frequent change noted in the ventricular tissue was an alteration in the myofibrils. This study indicates that electron microscopic changes can be induced in the embryonic chick heart ventricle by lead poisoning.     

Inhibition of melanosome transformation in embryonic chick pigment retina cultured in vitro.

When the retinal pigment epithelial cells of the chick embryo are transferred to monolayer cultures, they lose their phenotypic trait-- melanin granules-- after a few days. Within the first 24 hours almost all of the melanosomes and premelanosomes are transformed into the degradative structures of the dense bodies or the melanosome complexes. Then, within a few days, these structures disappear completely from the cytoplasm. Actinomycin D, added to the culture medium during the first four hours, almost completely prevents the transformation of melanosomes and premelanosomes. The inhibition of cell proliferation, caused by the addition of colcemid, does not prevent the transformation, though the time of initiation of transformation is delayed considerably. The mechanisms of the transformation of pigment granules are discussed.     

Relative rates of protein synthesis and reaggregation in embryonic chick liver cells cultured in defined and supplemented media

Protein synthesis and aggregate diameters were measured in cultures of 7-day embryonic chick liver cells reaggregating for 20 h in chemically-defined medium and in media supplemented with horse serum and with beef embryo extract. Statistically significant differences were found between defined and supplemented media in the rate of increase of mean aggregate diameters over a 20 h period. However, no statistically significant differences were detected in the rates of cellular protein synthesis in the different media. The results suggest that chemically-defined media are preferable to embryo extract and/or serum-supplemented media for short-term cell aggregation studies.     

The effect of ascorbate on embryonic chick sternal chondrocytes cultured in agarose

Primary cultures of embryonic chick sternal chondrocytes were embedded in a three-dimensional matrix of 1% solid agarose which was overlaid with nutrient media. The chondrocytes divided and formed nests of spherically shaped cells which were surrounded by an extensive extracellular matrix containing high molecular weight proteoglycans. Using light and electron microscopy, condensation of proteoglycan was observed pericellularly, often forming septa between cells of a nest, and as part of the outer boundary of the cell nest. No cross-striated collagen fibers were observed in the extracellular matrix although proteoglycan appeared to decorate a network of fine strands. Upon the addition of ascorbate to the nutrient media high molecular weight proteoglycans were synthesized, but there was a marked decrease in the synthesis of proteoglycans after a 10 day exposure to ascorbate. Morphologically, the decrease in proteoglycan synthesis was manifested in the discontinuous arrangement of the pericellular matrix as well as the diffuse form of the cell-nest boundary. Both of these structures were clearly defined in control cultures and were enriched in proteoglycan as demonstrated by ruthenium red staining. This study demonstrates that embryonic chondrocytes remain differentiated when cultured in solid agarose for a period of up to 15 days. They continue to synthesize their tissue specific macromolecules and are phenotypically stable when exposed to ascorbate for extended periods of time.     

Effect of electromagnetic stimulation with different waveforms on cultured chick tendon fibroblasts

An energy efficient electromagnetic stimulator device for fracture healing was compared to a commercially available device in stimulating cell growth in tissue cultures. The energy efficient device, which conserves energy by using a bidirectional time-dependent magnetic wave form, and the commercially available stimulator, which uses a unidirectionaltime-dependent magnetic wave form, were tested on chick tendon fibroblasts in primary culture. Comparing non-stimulated control and cells electromagnetically stimulated with unidirectional and bidirectional waveforms showed that at the growth phase between days 2 and 3, both electrical stimulation techniques increased cell division as measured by DNA synthesis. When cells were dividing rapidly, collagen synthesis was reduced. When the cells reached the confluence there was no difference among the groups (control, unidirectionally stimulated, and bidirectionally stimulated) in terms of number of cells or collagen produced. © 1994 Wiley-Liss, Inc.