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PubMed1992

Soluble protein constituents of the domestic fowl's eggshell.

1. The protein components of the domestic fowl's eggshell are believed to influence appreciably the mechanical properties of the shell and/or its biomineralisation. The purpose of this study was to compare the protein species composing the eggshell matrix in different parts of the shell structure, by SDS-PAGE and chromatography, utilising eggshell cleaned by different methodologies. 2. Protein species were identified whose absence was associated with the removal of the mammillary knobs. In particular, a prominent 81 kDa protein, as well as 38 and 54 kDa calcium-binding proteins, were concentrated within the mammillary layer, as was a 129 kDa insoluble protein. By contrast, soluble proteins of 54, 33, 22, and 14 kDa were enriched in the palisade layer. 3. Our results demonstrate that the mineralised layers of the fowl's eggshell possess a complex array of distinct proteins. The different proteins which have been detected in the mammillary and palisade layers may be related to the distinct crystallisation patterns of calcium carbonate in these zones of the eggshell.

Animals↗
PubMed1997

Precursor matrix proteins in the uterine fluid change with stages of eggshell formation in hens.

Organic constituents of the uterine fluid, the acellular milieu in which the eggshell is mineralized, were biochemically characterized at initial, mid and final stages of shell calcification in hens. The electrophoretic protein profiles changed at the different stages of shell mineralization. Two major bands (80-kDa and 43-kDa glycoproteins) with calcium affinity were specific to the initial stage. Four protein bands of 180, 150, 116 and 32 kDa, present at the phase of rapid shell formation, coprecipitated with calcium carbonate in vitro. At this stage were also present a calcium-binding glycoprotein of 36-kDa and a 20-kDa protein. Uterine fluid of the final stage was characterized by a darker intensity of the 66-kDa band, which showed calcium-binding ability and by the presence of three additional proteins (72, 13 and 6 kDa). At least seven bands of the uterine fluid showed similar migration patterns to those of eggshell extracts. Western blotting with ovocleidin and ovalbumin antisera demonstrated the presence of these matrix proteins in uterine fluid collected at initial and mid phase, respectively. Total uterine fluid collected at the end of calcification and dialyzed uterine fluid from the various stages delayed the rate of calcium precipitation in vitro. These observations demonstrate the presence of precursors of eggshell matrix in the uterine fluid and support the hypothesis of their involvement in the process of eggshell mineralization.

Animals↗
PubMed1992

Partial biochemical and immunochemical characterization of avian eggshell extracellular matrices.

There is evidence to suggest that extracellular matrix molecules, such as proteoglycans, are involved in the regulation of mineral deposition in calcifying tissues. One mineralizing system which is characterized by extremely rapid mineralization is the hen eggshell. This eggshell consists of a pair of nonmineralized eggshell membranes subjacent to the calcified eggshell proper; the eggshell proper is organized into palisades (columns) of mineralized matrix separated by pores. Between the membranes and the shell proper are compacted foci of tissue called mammillary knobs, which are thought to be sites where mineralization is initiated. Previous work from this laboratory has shown the presence of types I, V, and X collagen in the shell membranes. To address the question of the possible role of proteoglycans and glycosaminoglycans in mineralization of the eggshell, two approaches were used. First, immunohistochemistry was performed with monoclonal antibodies to various proteoglycan and glycosaminoglycan epitopes. This analysis indicates that different glycosaminoglycans are localized to discrete regions within the eggshell. Dermatan sulfate is present within the matrix of the shell proper and, to a lesser extent, the mammillary knobs and the outer portion of the shell membranes. In contrast, keratan sulfate is found in the shell membranes and prominently in the mammillary knobs. Interestingly, different keratan sulfate antibodies immunostain distinct regions of the eggshell, which suggests that various types of keratan sulfate are distributed differently. The second approach utilized was to extract the eggshell membranes and recover anionic molecules by anion-exchange chromatography. This resulted in the extraction of material which was recognized by antibodies to keratan sulfate, but not to chondroitin sulfate. This material was very large, as evidenced by its elution in the void volume of a Sepharose CL-2B column. The large size may be due to the extensive cross-links known to occur in the eggshell. If eggshell membranes are extracted at elevated temperature, the material recovered is of much smaller size. These results indicate that molecules recognized by antibodies to glycosaminoglycans are present in the eggshell, and their localized distribution relative to the calcified matrix suggests that they may be involved in the regulation of mineral deposition.

Animals↗
PubMed1984

Development of calcium reabsorption by the allantoic epithelium in chick embryos grown in shell-less culture.

The allantoic sac of the chick embryo functions as a primitive urinary bladder, storing and modifying the excretory fluid produced by the embryo. We have used chick embryos grown in shell-less culture to study the in situ handling of Ca2+ by the allantoic epithelium. Between Days 8 and 13 of incubation (38 degrees C, 5% CO2), the [Ca2+] of the allantoic sac fluid declines from about 1.5 mM to less than 0.3 mM, with most of this Ca2+ reabsorption occurring between Days 10 and 11. In 13-day-old embryos, the allantoic epithelium reabsorbs within 24 hr 85-92% of 45Ca2+ injected into the allantoic sac, while in 9-day-old embryos 45Ca2+ reabsorption is less than 40% by 24 hr. This is evidence for the developmental onset of a Ca2+ reabsorption process in the allantoic epithelium. The allantoic fluid Ca2+ is reabsorbed into the embryo's blood in which the serum [Ca2+] is about 1.5 mM. Also, electrical potential profiles reveal that the serosal (mesenchymal) side of the allantoic epithelium is 15-30 mV positive compared to the mucosal (luminal) side. Thus, by electrochemical criteria this reabsorption process appears to be active.

Absorption↗
PubMed1980

Ultrastructural and biochemical alterations in the livers from chick embryos maintained in shell-less culture.

Ultrastructural and biochemical studies were conducted on the livers from chick embryos maintained in shell-less culture up to stage 39 (Hamburger-Hamilton) and from control embryos developed in ovo up to the same stage. The ultrastructural characteristics of hepatic cells from the cultured embryos were similar to those found in the controls except that they contained many large lipid droplets and were almost devoid of lipoprotein granules normally associated with the Golgi complex and the smooth endoplasmic reticulum. These changes suggest the existence of alterations in the lipid metabolism. The livers from cultured embryos showed also a decreased incorporation of tritiated leucine into proteins, which indicates a reduced rate of protein synthesis. These results are consistent with previous reports showing that cultured embryos possess hypoproteinemia. Lactic dehydrogenase activity was similar and pyruvic kinase higher in the livers from cultured with respect to control embryos. This appears to indicate that both aerobic and anaerobic glycolysis were not depressed and that the changes observed in the rate of protein synthesis should not be attributed to hypoxia. "Fat-storing cells" similar to those described in mammals were found both in control and cultured embryos. They had not been previously described in the livers from chick embryos.

Animals↗
PubMed2001

Comparison of gonadal hormone levels in turkey embryos incubated in long-term shell-less culture and in ovo.

Changes in concentrations of 17beta-estradiol (E2) and androgenic hormones were measured in turkey embryos incubated in long-term, shell-less culture (ex ovo) and in ovo. Blood samples were obtained from both sets of embryos on Days 14, 16, 18, 20, and 22 and from embryos incubated in ovo on Days 24, 26, and 28. Ex ovo and in ovo embryos showed no differences in either hormone within sexes, with one exception. On Day 14 of incubation, the ex ovo females had higher (P < 0.05) E2 levels (55.6+/-5.1 pg/mL) than the in ovo females (32.2+/-2.3 pg/mL); however, this result might have been due to the small sample size (n = 3) for ex ovo females. No significant differences were found in androgen concentrations between sexes in ovo on Days 24, 26, and 28 of incubation. However, on Days 24, 26, and 28, in ovo females showed highly significant differences (P < 0.01) in E2 compared with males of the same age. These results indicate a similar developmental pattern for the endocrine system in ovo and ex ovo through Day 22 of incubation. Further, there were sex differences in E2 that are likely to be critical for sexual differentiation that emerges late in embryonic development.

Age Factors↗
PubMed1980

Calcium-binding proteins in serum: quantitative differences between thick and thin shell lines of chickens.

Calcium binding by two proteins, vitellogenin and albumin, was measured in serum of a line of hens producing thick (THK) and a line of hens producing thin shells (THN) as well as a line of commercial hens using gel filtration in 45calcium buffer. Vitellogenin was quantitated in serum of THK and THN using a radial immunodiffusion test. Levels of serum calcium were measured and related to the above mentioned parameters. The binding of vitellogenin was significantly greater in 13 THK than 13 THN (672 vs. 508 cpm/ml X 10(-3), but binding by albumin was not significantly different between the two lines (386 vs. 344 cpm/ml X 10(-3). Binding in 11 commercial hens was similar to that for THN. The THK had significantly greater levels of diffusible, non-diffusible, and total serum calcium than THN. Significant positive correlations between total serum calcium and vitellogenin binding were found in THK (.77) and THN (.81) as well as in the commercial hens (.64). Like vitellogenin binding, levels of vitellogenin were significantly greater in THK than THN (4.0 vs. 1.8 mg/ml). These results suggest that in addition to having more diffusible and non-diffusible serum calcium, THK have more calcium binding and more vitellogenin to perform the binding function than THN.

Animals↗
PubMed1984

Blood ionic calcium responses of hens from thick-shell and thin-shell lines to ethyleneglycol-bis-(B-aminoethylether)-N,N'-tetraacetic acid injections.

Experiments were conducted on laying and nonlaying hens from genetically selected lines that produce thick (TK) or thin (TN) eggshells. At 44 weeks of age, TK layers exhibited significantly higher total plasma calcium concentrations than TN layers. Total plasma calcium concentrations were not significantly different between TK and TN layers at 80 weeks of age, reflecting an effect of aging on total plasma calcium. Intravenous injections of ethyleneglycol-bis-(B-aminoethylether-N,N'-tetraacetic acid (EGTA) were administered to TK and TN layers at 44 weeks of age and to TK and TN layers and nonlayers at 80 weeks of age. EGTA is a calcium chelating agent that creates hypocalcemia when injected intravenously. Laying hens of both lines and at both ages exhibited a similar degree of hypocalcemia during EGTA injections, and a similar rate of recovery to normocalcemia was noted after cessation of EGTA administration. Nonlayers were unable to recover from the EGTA-induced hypocalcemia. Because the pattern of calcium depression and recovery during and after the EGTA challenge has been shown to be associated with the capacity of parathyroid hormone-sensitive target tissues to buffer blood ionized calcium, it was concluded that the capacity to buffer ionized calcium by these target tissues is similar in TK and TN layers. However, nonlayers have a restricted capacity to buffer blood ionic calcium.

Animals↗
PubMedDate not supplied

Purification and characterization of a vaterite-inducing peptide, pelovaterin, from the eggshells of Pelodiscus sinensis (Chinese soft-shelled turtle).

Proteins play a crucial role in the biomineralization of hard tissues such as eggshells. We report here the purification, characterization, and in vitro mineralization studies of a peptide, pelovaterin, extracted from eggshells of a soft-shelled turtle. It is a glycine-rich peptide with 42 amino acid residues and three disulfide bonds. When tested in vitro, the peptide induced the formation of a metastable vaterite phase. The floret-shaped morphology formed at a lower concentration ( approximately 1 microM) was transformed into spherical particles at higher concentrations (>500 microM). The solution properties of the peptide are investigated by circular dichroism (CD), fluorescence emission spectroscopy, and dynamic light scattering (DLS) experiments. The conformation of pelovaterin changed from an unordered state at a low concentration to a beta-sheet structure at high concentrations. Fluorescence emission studies indicated that the quantum yield is significantly decreased at higher concentrations, accompanied by a blue shift in the emission maximum. At higher concentrations a red-edge excitation shift was observed, indicating the restricted mobility of the peptide. On the basis of these observations, we discuss the presence of a peptide concentration-dependent monomer-multimer equilibrium in solution and its role in controlling the nucleation, growth, and morphology of CaCO(3) crystals. This is the first peptide known to induce the nucleation and stabilization of the vaterite phase in solution.

Amino Acid Sequence↗
PubMed2006

Development of endothermic metabolic response in embryos and hatchlings of the emu (Dromaius novaehollandiae).

During hatching, there is a maturation of the mechanisms controlling the respiratory physiology involved in endotherm in precocial avian species. Here we examined the timing of the development of an endothermic response of oxygen uptake (MO2) to an alteration of ambient temperature (T(a)) in a model precocial species, the preterm and hatching emu (Dromaius novaehollandiae). Late stage pre-pipped and pipped embryos and hatchlings were measured for responses of MO2 and shell or skin temperature (T(s)) to altered T(a) (DeltaT(a)). MO2 remained unchanged in pre-pipped and internally pipped (IP) embryos at the end of 1.5h exposure to DeltaT(a) of +/-10 degrees C. Externally pipped (EP) embryos responded to a cooling and a warming exposure with marked increase and decrease in MO2, as hatchlings responded to DeltaT(a) with an endothermic change in MO2. The demonstration of the endothermic inverse metabolic response first appearing in EP embryos suggests that pre-EP embryos may also possess the ability to produce the endothermic inverse metabolic response, but they are restricted by the eggshell gas conductance. Late pre-pipped and IP embryos were measured again for responses of [Formula: see text] to DeltaT(a) in air and then in a 40% O(2) environment. The metabolic response of pre-pipped embryos at 90% of incubation was partially altered by switching from air to hyperoxia. IP embryos responded to DeltaT(a) in 40% O(2) with apparent inverse changes in MO2. The late stage emu embryo possesses the ability to produce an endothermic metabolic response at an earlier stage of development than in chickens, but this response is limited by the eggshell gas conductance.

Animals↗
PubMed2000

The effect of avian uterine fluid on the growth behavior of calcite crystals.

Eggshell formation takes place on the eggshell membrane in an acellular medium, the uterine fluid that contains the inorganic minerals and precursors of the organic matrix. The high degree of eggshell structure could be due to an interaction between calcium carbonate and the organic matrix. The aim of this study was to demonstrate such an interaction by measuring the effect in vitro of uterine fluid collected at various phases of shell formation on precipitation kinetics, size, and morphology of calcite crystals. The SDS-PAGE profiles of the organic constituents differed between the different phases of eggshell formation. The predominant constituents were ovalbumin and ovotransferrin at the initial phase and lysozyme, ovocleidin-17, ovocalyxin-32, 36- and 21-kDa bands, and ovocleidin-116 at the growth phase. These proteins were numerous in the terminal phase and showed an increased staining of the 32- and 66-kDa bands and appearance of very low molecular weight bands. The precipitation lag time was shortened in proportion to the protein concentration at the initial stage. The effect was observed with a lower magnitude in the presence of constituents of growth and terminal phases. Crystal size was smaller in the presence of constituents from the three stages compared with the control. Components from the initial phase induced the formation of twinned crystals and of rounded corners in the rhombohedric crystals. The presence of components from the growth and terminal phases strongly modified the morphology of the calcite crystals. The majority of the corners became rough and developed curved faces. These observations confirm the interaction of the uterine fluid with calcite and its contribution to eggshell structure.

Animals↗
PubMed1992

Effect of dietary calcium level on medullary bone calcium reserves and shell weight of Leghorn hens.

Two experiments were conducted to determine the effect of different levels of dietary Ca and subsequent feeding of a very low level of Ca on the medullary bone Ca reserves of laying hens. In Experiment 1, a total of 30 40-wk-old Single Comb White Leghorn hens were offered a diet with 2.5, 3.5, or 4.5% Ca for a period of 21 days with 10 birds per dietary treatment. On Day 21, five hens from each dietary treatment were euthanatized. The remaining birds were offered a .46% Ca diet for 5 days. In Experiment 2, 25 Single Comb White Leghorn hens were fed a 3.5% Ca diet for 21 days. Hens were then offered a .46% Ca diet and five hens euthanatized on Days 0, 2, 3, 5, and 7 of feeding the low-Ca diet. In Experiment 1, dietary Ca level had a significant (P less than .05) effect on total medullary Ca reserves of laying hens. Previous dietary Ca level had no significant (P greater than .05) effect upon medullary bone Ca reserves after subsequently feeding the low-Ca diet for 5 days. There was a (P less than .05) significant reduction in medullary bone Ca reserves of hens, regardless of previous level of calcium fed. In Experiment 2, feeding a low level of dietary Ca resulted in a significant (P less than .01) reduction in medullary bone Ca reserves of all bones measured, except the humerus. Although there was a significant (P less than .05) reduction in medullary bone Ca during the depletion period, hens appeared to make some attempt to conserve medullary bone Ca reserves.

Animals↗
PubMed1983

The inhibitory effect of some chlorinated hydrocarbon pesticides on the ATP-dependent Ca2+ binding of the particulate fraction of the eggshell gland mucosa cells.

The pesticide p-p'-DDT and its persistent metabolite p-p'-DDE cause thinning of the eggshells in several species of birds. In earlier investigations on ducks this thinning was found to be associated with a reduction of the ATP-dependent Ca2+ binding to a homogenate of the shell gland mucosal cells by DDE. The activity of a Ca2+-Mg2+-activated ATPase in the homogenate was also decreased on administration of DDE in vivo. We have therefore investigated the in vitro effects of some other chlorinated hydrocarbon pesticides of ecotoxicological interest on the ATP-dependent Ca2+ binding and the Ca2+-Mg2+-activated ATPase activity in a homogenate of the eggshell gland mucosa of the hen and determined the molar concentrations that produced 50% inhibition (=IC50). Several of the investigated compounds, namely toxaphene, chlordane, p-p'-DDD, o-p'-DDE, p-p'-DDT, methoxychlor and PCB (Arochlor 1242), had a similar IC50 to inhibit the Ca2+ binding as p-p'-DDE. Lindane, p-p'-DDA and biphenyl had an IC50 3.3-4 times higher and that of 2.4 D was 13.5 times higher than that of p-p'-DDE. When the IC50 of some of the compounds (p-p'-DDE, PCB, toxaphene, Lindane) was determined that decreased the Ca2+-Mg2+-activated ATPase of the homogenate it was found to be only 18 to 29 per cent of that needed to inhibit the Ca2+ binding by the homogenate. It is therefore probable that some other effect than inhibition of this enzyme is also involved in the Ca2+-binding process and affected by the compounds.

Adenosine Triphosphatases↗
PubMed1988

Short-term effects of altered shell conductance on oxygen uptake and hematological variables of late chicken embryos.

The preceding report on the O2 uptake (MO2) of chicken embryos whose shell conductance (GO2) was altered from the beginning of incubation showed that the MO2 was decreased despite increased GO2 [Okuda, A. and H. Tazawa (1988) Respir. Physiol. 74: 187-198]. This was attributed to an excess water loss which reduced the growth of the embryos. The present study was designed to investigate the short-term effects of altered GO2, obviating the effect of excess water loss, on the MO2 and simultaneously on the hematological variables of embryos on days 16-17 and days 18-19 of incubation. The MO2 measured 5 h after increasing the GO2 was neither decreased nor increased significantly. The diffusing capacity of the chorio-allantoic membrane, which was estimated using the Bohr integration procedure, decreased as the GO2 was increased. When the GO2 was decreased, on the other hand, the decrease in MO2 was not so large as expected from the decrease in GO2, for both 16- and 18-day-old embryos. The effect of reduced GO2 on MO2 was more prominent in 18-day-old embryos than 16-day-old embryos. One-day-long hypoxia due to decreased GO2 induced erythropoiesis in 18-19-day embryos, but did not do so in 16-17-day embryos. The increase in hematocrit value of the latter group of embryos was attributed to an increase in cell volume due to concurrent hypercapnia.

Allantois↗
PubMed1991

Mineral metabolism in the developing turkey embryo--I. The effects of developmental age and shell-less culture on trace element contents of selected tissues.

1. Turkey embryos were incubated in ovo or in long-term shell-less culture (ex ovo) for 14, 18, 22 or 26 days. The embryos incubated ex ovo exhibited a progressive decline in the rate of growth and were hypocalcemic and hypoproteinemic compared to their in ovo counterparts from day 18 to day 26 of incubation. 2. The ratio of the concentrations of alpha-fetoprotein and albumin (AFP/A) in serum was determined for both groups of embryos. The AFP/A ratio may be useful as a biochemical index to stage avian embryonic development. Using this index it was concluded that ex ovo embryos exhibited a progressive developmental retardation compared to in ovo embryos. 3. Significant differences were observed in serum trace element concentrations for embryos incubated in ovo vs ex ovo. Most notably, serum copper concentration was significantly lower in ex ovo embryos on days 18 and 22 of incubation and significantly higher on day 26 of incubation compared to serum from embryos incubated in ovo. 4. Livers from embryos incubated ex ovo exhibited significant differences trace element levels compared to those incubated in ovo. By day 26 of incubation the concentration and total amount of zinc and iron were markedly elevated, whereas copper was greatly reduced in the livers of embryos incubated ex ovo compared to the corresponding in ovo levels. 5. Hearts from embryos incubated ex ovo contained less zinc and copper and more iron by day 26 of incubation than those from embryos incubated in ovo.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Preservation of hatchery waste by lactic acid fermentation. 2. Large-scale fermentation and feeding trial to evaluate feeding value.

Two waste streams from a Leghorn hatchery were preserved and recycled by fermentation with a by-product carbohydrate and extrusion processing into new feed ingredients that were evaluated with broiler chickens. Cockerel chicks (CC) and a 60:40 ratio of CC:shell waste (CC:SW) were fermented in 189-L barrels for 21 d following grinding, then mixing with a liquid culture (0.2%) and carbohydrate source at 15 and 16.66%, respectively. At 2 wk, pH was 4.44 and 5.09 for the CC and CC:SW products compared with higher values of 6.54 and 6.98 for the raw ingredients at the onset. Negligible hydrogen sulfide and no ammonia gas were recorded during the fermentation period. At 21 d, the fermented CC and CC:SW were extruded, dried, and ground to meals containing CP and TMEn levels of 47.4%, 3,187 kcal/kg, and 33.1%, 2,696 kcal/kg, respectively. Broiler chickens were fed a control diet and the CC (5 and 10%) and CC:SW (2.5 and 5%) ingredient diets with corn and soybean meal for 6 wk to evaluate feeding value and carcass yield. Body weight, gain and feed conversion at 42 d for birds fed diets supplemented with CC or CC:SW at all levels were comparable to those of the control. Diets supplemented with hatchery by-product had no negative effect on carcass measurements except ready to cook carcass and wing yield, which were significantly greater for the 10% CC:SW birds than for the control. These data indicate that nutrient dense hatchery by-products can be preserved with fermentation up to 21 d and support broiler live performance and carcass yield as dietary ingredients equal to or better than a corn-soybean meal control.

Analysis of Variance↗
PubMed1993

Estrogen enhancement of Ca-, Mg-, and Ca-Mg-stimulated adenosine triphosphatase activity in the chick shell gland.

The effect of 17 beta-estradiol (E2) on Ca-, Mg-, and Ca-Mg-ATPase activity was investigated in the shell gland of 6-week-old chicks. In the first study, each of 42 birds was implanted with three E2 (Compudose-200) pellets. An additional 6 sham-implanted birds served as controls for measurements of body weight and concentrations of E2 and Ca in plasma. The activities of Mg-, Ca-, and Ca-Mg-ATPase peaked coincident with maximum plasma E2 concentrations 8 days after implantation and then progressively declined in concert with the decline in plasma E2. By 29 days after implantation, the ATPase activities were similar to those measured in birds whose E2 pellets had been removed for 11 days. Concentrations of plasma E2 dropped from peak values of 1676 +/- 317 at Day 8 to 611 +/- 180 pg/ml at Day 29. When birds whose E2 pellets had been removed were reimplanted with three pellets per bird, plasma E2 again increased to 1637 +/- 227 pg/ml. ATPase activity in these reimplanted birds also was greater (P < 0.05) than activities measured in E2-removed or E2-maintained birds. In a second study the Ca-ATPase Km and Vmax were determined in E2-implanted chicks (three pellets per bird) and compared to E2-withdrawn chicks. Five days after reimplantation of chicks with E2, there was a significant increase in both Vmax (3.38 +/- 0.21 vs 2.37 +/- 0.28 micrograms Pi/mg protein/min; P < 0.05) and Km (0.31 +/- 0.02 vs 0.25 +/- 0.01 mM Ca; P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Alterations in cellular calcium handling as a result of systemic calcium deficiency in the developing chick embryo: II. Ventricular myocytes.

We have previously shown that cardiovascular anomalies, such as hypertension and tachycardia, develop in Ca(2+)-deficient, shell-less (SL) chick embryos cultured ex ovo, accompanied by elevated circulating catecholamines and higher alpha-adrenergic sensitivity of cardiovascular functions. Results described in the preceding work, using erythrocytes as an experimental system, show that cellular Ca2+ handling properties are also altered as a result of long-term calcium deficiency. To examine the relevance of these findings to cells of the cardiovasculature, we have analyzed and compared the Ca2+ handling characteristics of the heart cells of SL and normal (NL) embryos. For this study, isolated and cultured ventricular myocytes of SL and NL embryos were loaded with Fura-2 via transient membrane damage with glass beads. Compared to Fura-2/AM, bead loading yielded similar values and kinetic profiles of [Ca2+]i-dependent differential fluorescence and, in addition, did not affect cell viability and beating activity. The Fura-2 loaded ventricular myocytes were washed in Ca(2+)-free buffer and then analyzed by ratiometric fluorescence (350 nm/380 nm) microscopy for kinetic changes in [Ca2+]i (R350/380 values) as a function of [Ca2+]o and adrenergic modifiers. At 0.5 and 1.0 mM [Ca2+]o, SL cells showed significantly higher [Ca2+]i, higher beating rates, and faster rate of increase in [Ca2+]i compared to NL cells. At higher [Ca2+]o (3.5 mM), there was no significant difference in [Ca2+]i and beating rate between NL and SL cells. Treatment with norepinephrine (NE; 0.01-1 microM) at 1 mM [Ca2+]o substantially increased [Ca2+]i in both NL and SL cells. In the former, the NE effect was completely inhibited by beta-blockade (1 microM propranolol). In contrast, in SL cells, NE remained effective after beta-blockade, and combined alpha-blockade (1 microM prazosin) and beta-blockade was needed to inhibit completely the NE effect. In both NL and SL cells, treatment with NE substantially increased beating rates in a similar manner. Taken together, these findings suggest that Ca2+ handling and adrenergic regulation of the heart cells are significantly altered in the SL embryos, and that these alterations may be related to the development of impaired cardiovascular functions resulting from systemic Ca2+ deficiency.

Animals↗