Pyruvate kinase isozymic shifts of differentiating chick myogenic cells in vivo and in culture.

Developing chick skeletal muscle undergoes an isozymic shift from type K pyruvate kinase to type M during development. A major increase in pyruvate kinase activity follows the isozymic shift, resulting in at least 40-fold higher specific activities by adulthood. Similar isozymic changes occur in primary cultures of myogenic cells from 12-day-old chick embryos. Cultures initially contain only type K pyruvate kinase. Type M appears by the fourth day of culture and accounts for 80–90% of the activity by the eleventh day. Type M did not accumulate when cell fusion was prevented by removing Ca²⁺ from the growth medium or when protein synthesis was inhibited by cycloheximide.     

Transforming growth factor beta (TGFbeta ) signaling via differential activation of activin receptor-like kinases 2 and 5 during cardiac development. Role in regulating parasympathetic responsiveness

Little is known regarding factors that induce parasympathetic responsiveness during cardiac development. We demonstrated previously that in atrial cells cultured from chicks 14 days in ovo, transforming growth factor beta (TGFbeta) decreased parasympathetic inhibition of beat rate by the muscarinic agonist, carbamylcholine, by 5-fold and decreased expression of Galpha(i2). Here in atrial cells 5 days in ovo, TGFbeta increased carbamylcholine inhibition of beat rate 2.5-fold and increased expression of Galpha(i2). TGFbeta also stimulated Galpha(i2) mRNA expression and promoter activity at day 5 while inhibiting them at day 14 in ovo. Over the same time course expression of type I TGFbeta receptors, chick activin receptor-like kinase 2 and 5 increased with a 2.3-fold higher increase in activin receptor-like kinase 2. Constitutively active activin receptor-like kinase 2 inhibited Galpha(i2) promoter activity, whereas constitutively active activin receptor-like kinase 5 stimulated Galpha(i2) promoter activity independent of embryonic age. In 5-day atrial cells, TGFbeta stimulated the p3TP-lux reporter, which is downstream of activin receptor-like kinase 5 and had no effect on the activity of the pVent reporter, which is downstream of activin receptor-like kinase 2. In 14-day cells, TGFbeta stimulated both pVent and p3TP-lux. Thus TGFbeta exerts opposing effects on parasympathetic response and Galpha(i2) expression by activating different type I TGFbeta receptors at distinct stages during cardiac development.     

Biochemical Aspects of Chick Embryo Retina Development: The Effects of Glucocorticoids

In chick embryo retina during development, DNA synthesis and the activities of DNA polymerase, thymidine kinase, thymidylate synthetase, and ornithine decarboxylase (ODC) declined in parallel from day 7 to 12. The administration in ovo of hydrocortisone reduced significantly, particularly at 8-10 days of incubation, both DNA synthesis and the four enzyme activities tested. The effect was dose dependent, reaching the maximum with 50-100 nmol of hydrocortisone, 8-16 h after treatment. The highest inhibition was found for ODC activity (70%), followed by thymidine kinase activity (62%) and DNA synthesis (45%), whereas activities of DNA polymerase and thymidylate synthetase were reduced only by 30%. The inhibitory effect was exerted by all the glucocorticoids tested, with dexamethasone and hydrocortisone being the most efficacious. The results support the view that glucocorticoids reduce the proliferative events in chick embryo retina, particularly at 8-10 days of embryonic life.     

Metabolic differentiation of red and white skeletal muscle in vivo and in monolayer culture

Cytochrome oxidase, succinate oxidase and lactate dehydrogenase were compared in: (a) leg and breast muscle from 11-19-day-old chick embryos; and (b) 2, 6, 10 and 14-day-old primary cell cultures established from myoblasts of embryonic leg and breast muscle. Cytochrome oxidase, succinate oxidase and lactate dehydrogenase activities were higher (48.8, 65.4, 277.6%, respectively) in leg muscle after 19 days in ovo. Cytochrome and succinate oxidase activities were higher (111.3, 48.1%, respectively) in leg muscle cell cultures after 14 days in vitro. The data represent evidence for intrinsic developmental patterns for certain enzymes.     

Isolation and partial characterization of high-buoyant-density proteoglycans synthesized in ovo by embryonic chick skeletal muscle and heart

Proteoglycans, a major component of the extracellular matrix, are produced in many tissues. A report from this laboratory describes the proteoglycans synthesized in culture by chick embryonic skeletal muscle myotubes. To extend this study to in vivo conditions, chick embryos were radiolabeled in ovo and the newly synthesized high-buoyant-density proteoglycans from skeletal muscle analyzed. In both leg muscle and pectoral muscle, three major high-density proteoglycans are synthesized. One is small and is similar to the proteoglycans synthesized in culture by muscle fibroblasts. The other two proteoglycans are large. The larger of these shares structural features with the proteoglycan synthesized by skeletal muscle cells in culture. It has large chondroitin sulfate chains (estimated molecular weight of 70,000) with a high proportion of chondroitin 6-sulfate (approximately 90%). The smaller of the two large proteoglycans is distinct (chondroitin sulfate of estimated molecular weight 24,000 and approximately 60% 6-sulfated disaccharides) and is not detected in muscle cultures; evidence suggests it is not made by myoblasts. Whole hearts synthesize proteoglycans with some structural similarities, and also differences, to those made in skeletal muscle. These data indicate that the proteoglycans synthesized in muscle cultures are likewise made in developing muscle in ovo but that another distinct strictly in ovo proteoglycan is also produced.     

Changes in endogenous cell surface galactosyltransferase activity during in vitro limb bud chondrogenesis

When the subridge mesoderm of the embryonic chick limb bud is cultured in the absence of the apical ectodermal ridge and adjacent ectoderm, the cells rapidly progress through the various stages of chondrogenesis. During the first day of culture, the cells initiate condensation, and during subsequent days, deposit a cartilage matrix. In the present study, we show that early in the first day there is a progressive 2-fold increase in cell surface galactosyltransferase activity towards endogenous acceptors. Later in the first day, although the cells are still in condensation, endogenous galactosyltransferase activity has decreased, suggesting in situ galactosylation of surface acceptors. During subsequent development, when cartilage matrix is being deposited, surface galactosyltransferase activity remains low. All controls have been performed to insure cell surface localization of enzyme activity. Two other surface glycosyltransferases show very low levels of activity, which do not change significantly during culture. We suggest that during cellular condensation, an interaction between surface galactosyltransferases and acceptors on adjacent cells occurs, and this interaction may be causally related to subsequent chondrogenic differentiation.     

Development of muscarinic cholinergic inhibition of adenylate cyclase in embryonic chick heart. Its relationship to changes in the inhibitory guanine nucleotide regulatory protein

Parasympathetic and sympathetic innervation of the embryonic chick heart proceed non-coordinately. beta-Adrenergic agonists mediate an increase in beating rate in embryonic chick heart prior to ingrowth of the vagus nerve (Culver, N. G., and Fishman, D. A. (1977) Am. J. Physiol. 232, R116-R123) while muscarinic agonists exert relatively little effect on beating rate in hearts 2-4 days in ovo (Papanno, A. J. (1979) Pharmacol. Rev. 29, 3-33). Studies of developmental changes in the ability of muscarinic agonists to inhibit adenylate cyclase activity and their relationship to the development of a physiologic response of the embryonic chick heart to muscarinic stimulation have been inconclusive. In the current studies the ability of isoproterenol to stimulate adenylate cyclase activity did not change during development. Maximum stimulation above basal was 760 pmol of cAMP/10 min/mg of proterin with an IC50 of 1.5 X 10(-6) M for isoproterenol in homogenates of hearts 2 1/2, 3 1/2, and 10 days in ovo and 3 days posthatching. However, inhibition of isoproterenol-stimulated adenylate cyclase activity by carbamylcholine increased from 7.6% with a IC50 for carbamylcholine of 16 +/- 5.0 microM at day 2 1/2 in ovo to 29% with an IC50 of 0.4 +/- 0.1 microM at day 10 in ovo and to 43% with a IC50 of 0.6 +/- 0.1 microM at 3 days posthatching. Since previous data had demonstrated the presence of muscarinic receptors as early as 2 1/2 days in ovo (Galper, J. B., Klein, W., and Catterall, W. A. (1977) J. Biol. Chem. 252, 8692-8699), studies of developmental changes in guanine nucleotide-coupling proteins were carried out to determine whether early in development muscarinic receptors were uncoupled from a physiologic response. Studies of pertussis toxin-catalyzed ADP-ribosylation of homogenates of embryonic chick heart with [32P]NAD demonstrated the presence of two ADP-ribosylated proteins at 39,000 and 41,000 kDa, respectively. Both ADP-ribosylation and immunoblotting of homogenates with an antibody to the 39-kDa guanine nucleotide-binding protein in bovine brain demonstrated that the 39-kDa alpha protein increased 1.8-fold between days 2 1/2 and 3 1/2 in ovo and another 1.8-fold from day 3 1/2 to 10 in ovo in parallel with the increase in the extent of muscarinic inhibition of adenylate cyclase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular protein degradation in cultures of dystrophic muscle cells and fibroblasts

We have analysed protein degradation in primary cultures of normal and dystrophic chick muscle, in fibroblasts derived from normal and dystrophic chicks, and in human skin fibroblasts from normal donors and from patients with Duchenne muscular dystrophy (DMD). Our results indicate that degradative rates of both short- and long-lived proteins are unaltered in dystrophic muscle cells and in dystrophic fibroblasts. Longer times in culture and co-culturing chick fibroblasts with the chick myotubes do not expose any dystrophy-related abnormalities in protein catabolism. Furthermore, normal and dystrophic muscle cells and fibroblasts are equally able to regulate proteolysis in response to serum and insulin. We conclude that cultures of chick myotubes, chick fibroblasts, and fibroblasts derived from humans afflicted with DMD are not appropriate models for studying the enhanced protein degradation observed in dystrophy.     

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.     

Myosin accumulation in mononucleated cells of chick muscle cultures.

The biosynthesis and accumulation of the myosin heavy chain (MHC) peptide has been examined in embryonic chick skeletal muscle cultures under conditions of normal or arrested cell fusion. When compared with primary chick fibroblasts, the myogenic cells accumulated significantly more MHC, even while mononucleated. Electron microscopy of the fusion-blocked cultures revealed the presence of myosinlike thick filaments in the myoblasts. It is concluded that cell fusion is not a prerequisite for myosin accumulation or myofilament assembly during embryonic chick muscle differentiation.     

Microenvironmental regulation of visual pigment expression in the chick retina

Visual pigment (VP) expression in the chick embryo retina was investigated in ovo, in dissociated and explant cultures, and in cDNAs from individual cells. While VP mRNA is not detectable by in situ hybridization until embryonic day (ED) 14-16 in ovo, analysis of VP expression by RT-PCR showed that VP messages are present in the retina as many as 7-10 days before they become detectable by in situ hybridization, and are also detected in other regions of the embryonic CNS. On the other hand, red opsin expression is markedly accelerated when cells are isolated from their intraocular microenvironment at ED 6, and placed in pigment epithelium-free dissociated or explant cultures. This acceleration occurs regardless of cell density, birth date, or serum presence in the medium, suggesting that many photoreceptors are already programmed to express red opsin on or before ED 6, and that microenvironmental inhibitory factors prevent implementation of this program until ED 14 in ovo. The selectivity of this phenomenon is suggested by the finding that other VPs are not observed by in situ hybridization in ED 6 cultures, although they are detectable in cultures of older retinas. Taken together, these findings suggest that red opsin expression may be constitutive for many developing photoreceptor cells in the chick.     

Circadian regulation of cGMP-gated cationic channels of chick retinal cones. Erk MAP Kinase and Ca2+/calmodulin-dependent protein kinase II

cGMP-gated channels are essential for phototransduction in the vertebrate retina. Here we show that the affinity of these channels for cGMP in chick cones is substantially higher during the subjective night than during the subjective day. This effect persists in constant environmental conditions after entrainment to 12:12 hr light-dark cycles in vitro or in ovo. Circadian modulation of ligand affinity is a posttranslational effect and is driven by rhythms in the activities of two protein kinases: Erk and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Erk is maximally active during the subjective night, whereas CaMKII is maximally active during the subjective day. Acute inhibition of these signaling pathways causes phase-dependent changes in the affinity of the channels for cGMP.     

Developmental changes in collagen glycosyltransferase activities in chick embryos

The developmental pattern of collagen galactosyltransferase and collagen glucosyltransferase activities was determined in chick embryos between the 4th and 21st day of growth. Both enzyme activities increased up to the 16th day and decreased thereafter in whole chick embryos and in most tissues studied. The highest collagen glycosyltransferase activities were found in the leg tendons of the 16-day-old embryos, and the activities found in cartilage were higher than those noted in either skin or skull, indicating that the the activities of the collagen glycosyltransferases may play a part in the regulation of the carbohydrate content of the collagen synthesized by a given tissue. The changes observed in the collagen glycosyltransferase activities agree with previous data on the development of prolyl and lysyl hydroxylase activities and also with findings on collagen turnover in the developing chick embryo.     

Development of gamma-glutamyl transpeptidase activity in primary cultures of neurones and glial cells derived from the cerebral hemispheres of chick embryos

The development of gamma-glutamyl transpeptidase (GGT) activity in neurones and glial cells was studied in primary cell cultures derived from the cerebral hemispheres of chick embryos. GGT activity was found in both basic types of nervous tissue cells. It was always higher in glial cell cultures, where it was up to 2.3-fold the values in neurone-enriched cultures. If the culture medium contained foetal calf serum, the GGT activity of both types of nerve cells was higher than in the presence of inactivated calf serum. Comparison with the in vivo situation showed that the level of GGT activity in nerve cell cultures was significantly lower. Between the seventh day of embryogenesis and the third day of postnatal development of the nerve cells, there were marked differences between the GGT activity of cells maintained under in vitro conditions and cells of the same age in brain tissue homogenate. GGT activity in cerebral hemisphere homogenates from a 17-day-old embryos amounted to 4-fold the activity in a primary glial cell culture and to 16-fold the value in a neurone-enriched culture from hemispheres at the same stage of development.     

Activities of Enzymes Involved in the Metabolism of Platelet-Activating Factor in Neural Cell Cultures During Proliferation and Differentiation

Platelet-Activating Factor (PAF) is a potent lipid mediator involved in physiological and pathological events in the nervous tissue where it can be synthesized by two distinct pathways. The last reaction of the de novo pathway utilizes CDPcholine and alkylacetylglycerol and is catalyzed by a specific phosphocholinetransferase (PAF-PCT) whereas the remodelling pathway ends with the reaction catalyzed by lyso-PAF acetyltransferase (lyso-PAF AcT) utilizing lyso-PAF, a product of phospholipase A₂ activity, and acetyl-CoA. The levels of PAF in the nervous tissue are also regulated by PAF acetylhydrolase that inactivates this mediator. We have studied the activities of these enzymes during cell proliferation and differentiation in two experimental models: 1) neuronal and glial primary cell cultures from chick embryo and 2) LA-N-1 neuroblastoma cells induced to differentiate by retinoic acid (RA). In undifferentiated neuronal cells from 8-days chick embryos the activity of PAF-PCT was much higher than that of lyso-PAF AcT but it decreased during the period of cellular proliferation up to the arrest of mitosis (day 1–3). During this period no significant changes of lyso-PAF AcT activity was observed. Both enzyme activities increased during the period of neuronal maturation and the formation of cellular contacts and synaptic-like junctions. The activity of PAF acetylhydrolase was unchanged during the development of the neuronal cultures. PAF-PCT activity did not change during the development of chick embryo glial cultures but lyso-PAF AcT activity increased up to the 12th day. RA treatment of LA-N-1 cell culture in proliferation decreased PAF-PCT activity and had no significant effect on lyso-PAF AcT and PAF acetylhydrolase indicating that the synthesis of PAF by the enzyme catalyzing the last step of the de novo pathway is inhibited when the LA-N-1 cells are induced to differentiate. These data suggest that: 1) in chick embryo primary cultures, both pathways are potentially able to contribute to PAF synthesis during development of neuronal cells particularly when they form synaptic-like junctions whereas, during development of glial cells, only the remodelling pathway might be particularly active on synthesizing PAF; 2) in LA-N-1 neuroblastoma cells PAF-synthesizing enzymes coexist and, when cells start to differentiate the contribution of the de novo pathway to PAF biosynthesis might be reduced.     

Glutathione S-transferase and UDP-glucuronyltransferase activity in primary cultures of rainbow trout gill epithelial cells

The objective of this study was to characterize rainbow trout (Oncorhynchus mykiss) gill epithelial cells in primary culture by evaluating their ability to maintain glutathione and glucuronide conjugating enzymes. The activity and inducibility of the phase II enzymes was investigated as a function of culture time. Glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDPGT) enzyme activities were measured in freshly isolated cells and in cells cultured for 7 and 12 days. GST activity, determined with 1-chloro-2,4-dinitrobenzene, decreased gradually to 72% after 7 days and to 38% after 12 days in culture compared with freshly isolated cells. There was no significant difference between UDPGT activities in freshly isolated cells compared with cells cultured up to 12 days although a transient decrease in activity was observed at day 7. In vitro induction of the enzymes was studied using beta-naphtoflavone (BNF) and 3-methylcholanthrene (3-MC) as inducers. GST activity increased 2-fold after exposure to BNF and 1.5-fold after 3-MC exposure for 48 h in 7 days old cultures. No induction was observed in 12 days old cultures. UDPGT activity was not induced either at day 7 or 12.     

The expression of beta-1,3 galactosyltransferase and beta-1,4 galactosyltransferase enzymatic activities in the mammary gland of the tammar wallaby (Macropus eugenii) during early lactation

The regulation of beta-1,3 galactosyltransferase (3betaGalT) and beta-1,4 galactosyltransferase enzymatic (4betaGalT) activities in the mammary gland of the tammar wallaby (Macropus eugenii) have been characterised. These two beta-galactosyltransferases are active at different times during the lactation cycle and play a central role in regulating the carbohydrate composition in tammar milk, which changes progressively throughout lactation to assist the physiological development of the altrical young. The 4betaGalT activity was present at parturition and increased 3-fold by day 10 of lactation (d10L), whereas 3betaGalT activity was barely detectable at day d5L and then increased 6-fold by d10L. This increase in activity of both enzymes was sucking dependent. While 3betaGalT activity was not observed in the mammary gland prior to d7L, this activity was found in mammary explants from late pregnant tammar cultured with insulin, hydrocortisone and prolactin (IFP) and was further stimulated by the addition of tri-iodothyronine (T) and 17beta-oestradiol (E). The activity of 4betaGalT in these explants was stimulated maximally with IFP. These data suggest the temporal activity of both 3betaGalT and 4betaGalT is most likely regulated by both endocrine stimuli and factors intrinsic to the mammary gland.     

Myosin light-chain expression during avian muscle development

Monoclonal antibodies to adult chicken myosin light chains were generated and used to quantitate the types of myosin light-chain (MLC) isoforms expressed during development of the pectoralis major (PM), anterior latissimus dorsi (ALD), and medial adductor (MA) muscles of the chicken. These are muscles which, in the adult, are composed predominantly of fast, slow, and a mixture of fiber types, respectively. Three distinct phases of MLC expression characterized the development of the PM and MA muscles. The first identifiable pase occurred during the period of 5-7 d of incubation in ovo. Extracts of muscles from the pectoral region (which included the presumptive PM muscle) contained only fast MLC isoforms. This period of exclusive fast light-chain synthesis was followed by a phase (8- 12 d of incubation in ovo) in which coexpression of both fast and slow MLC isoforms was apparent in both PM and MA muscles. During the period, the composition of both fast and slow MLC isoforms in the PM and MA muscles was identical. Beginning at day 12 in ovo, the ALD was also subjected to immunochemical analyses. The proportion of fast and slow MLCs in this muscle at day 12 was similar to that present in the other muscles studied. The third development phase of MLC expression began at approximately 12 d of incubation in ovo and encompassed the transition in MLC composition to the isoform patterns incubation in ovo and encompassed the transition in MLC composition to the isoform patterns typical of adult muscle. During this period, the relative proportion of slow MLC rose in both the MA and ALD and fell in the PM. By day 16, the third fast light chain, LC(3f), was apparent in extracts of both the PM and MA. These results show that there is a developmental progression in the expression of MLC in the two avian muscles studied from day 5 in ovo; first, only fast MLCs are accumulated, then both fast and slow MLC isoforms are expressed. Only during the latter third of development in ovo is the final MLC isoform pattern characteristic of a particular muscle type expressed.