Prolonged hypoxia increases vascular endothelial growth factor mRNA and protein in adult mouse brain.

Brain hypoxia induces an increase in brain vascularity, presumably mediated by vascular endothelial growth factor (VEGF), but it is unclear whether VEGF is required to maintain the increase. In these studies, brain VEGF mRNA and protein levels were measured in adult mice kept in hypobaric chambers at 0.5 atm for 0, 0.5, 1, 2, 4, 7, and 21 days. Hypoxia was accompanied by a transient increase of VEGF mRNA expression: twofold by 0.5 day and a maximum of fivefold by 2 days; these were followed by a decrease at 4 days and a return to basal levels by 7-21 days. VEGF protein expression induced by hypoxia was bimodal, initially paralleling VEGF mRNA. There was an initial small increase at 12 h that reached a maximum by day 2, and, after a transient decrease on day 4, the protein expression increased again on day 7 before it returned to normoxic levels after 21 days. Thus, despite continued hypoxia, both VEGF mRNA and protein levels returned to basal after 7 days. These data suggest a metabolic negative-feedback system for VEGF expression during prolonged hypoxia in the brain.

Time-course and reversibility of the hypoxia-induced alterations in cerebral vascularity and cerebral capillary glucose transporter density.

The adult rat adapts to prolonged moderate hypobaric hypoxia by polycythemia, increased brain vascularity, and increased density of the brain capillary glucose transporter (GLUT-1). We now report on the time-course and reversibility of these adaptive alterations. Adult male Wistar rats were subjected to hypobaric hypoxia at 0.5 atmosphere for periods of 4 days or 1, 2 or 3 weeks, and compared to normoxic littermate controls. Reversibility of the effects of hypoxia was studied in rats subjected to hypobaric hypoxia for 3 weeks and then allowed to recover at normobaric conditions for 3 additional weeks. Cerebral vascularity was studied in cross-sections of the cerebral cortex that were immunocytochemically stained with a GLUT-1 antibody. The density of GLUT-1 was determined in isolated cerebral microvessels by quantitative autoradiography of immunoblots. Blood hematocrit and cerebral microvascularity did not significantly increase after 4 days of hypoxia, but were significantly increased at 1, 2 and 3 weeks of hypoxia. Three weeks of normoxic recovery after 3 weeks of hypoxia reversed the polycythemia and cerebral hypervascularity. However, the density of GLUT-1 in isolated cerebral microvessels, which was significantly increased after 1 and 3 weeks of hypoxia, remained elevated after 3 weeks of normoxia.

Delivery of macromolecules into adherent cells via electroporation for use in fluorescence spectroscopic imaging and metabolic studies.

A method is described to introduce by electroporation membrane-impermeant molecules into adherent living cells with little perturbation. The approach uses simple, commonly available equipment to introduce small fluorescent dyes, large carrier-based dyes (e.g., fluorescein-labeled dextran), large macromolecules (e.g., antibodies), and metabolic precursors (e.g., 32P-ATP) with high efficiency. Conditions are relatively independent of cell type. Electroporation with three pulses of 300 volts at 540 microF capacitance at 4 degrees C is a good starting point for many cell types. Electrode distance from the adherent cells was critical at 1.0 +/- 0.15 mm. Suitable poration medium includes calcium-magnesium free phosphate buffered saline (PBS), PBS-buffered 0.25-3.0 M sucrose, Hepes-buffered sucrose, or unbuffered sucrose. Potential use in fluorescence imaging and metabolic studies is shown with DNA synthesis, cell replication, cell substratum attachment, 32P-ATP phosphorylation, and insulin-mediated increases in glucose uptake and its suppression by antiphosphotyrosine and antiglucose transporter protein antibodies. The ability to load foreign molecules into large numbers of adherent cells provides a means of studying these cells individually via microscopic approaches, such as fluorescence spectroscopic imaging, as well as with conventional biochemical and physiological techniques.

Calcium regulation of skeletal myogenesis. II. Extracellular and cell surface effects.

The process of myoblast fusion during skeletal myogenesis is calcium regulated. Suppression of fusion is obtained by lowering medium [Ca2+] and re-initiated by raising medium [Ca2+]. Previously, we showed that such changes in medium [Ca2+] produced concomitant changes in myoblast [Ca2+] and that a critical cellular concentration of calcium must be present in myoblasts for fusion to occur. In this study, we report on further investigations on the relationship between myoblast [Ca2+] and fusion and also present data which suggest that an outer cell surface pool of calcium is involved in the fusion process. Cellular [Ca2+] must reach greater than 0.8 pmoles/cell and the medium [Ca2+] must be greater than 0.2-0.4 mM for myoblast fusion to occur. These conditions do not have a trigger effect on the entire myoblast population; instead, myoblast fusion was a dose-dependent linear response. If medium [Ca2+] was not maintained at 0.9 mM then cellular [Ca2+] decreased below a critical 0.8 pmoles/cell nucleus and fusion ceased. The cell surface pool of calcium was detected with the calcium antagonist lanthanum. A defined culture medium with 0.9 mM Ca2+ was used to maintain cell viability, and to prevent precipitation of medium components and changes in medium pH with La3+ (0.1 mM). La3+ did not enter the myoblasts as detected by electron microscopy, did not inhibit Ca2+ movement into the cells and the cellular [Ca2+] was sufficient to promote myoblast fusion. Under these conditions, myoblast fusion was inhibited. Morphologically, the fusion-suppressed myoblasts resembled those suppressed by lowered medium [Ca2+]. After removal of the fusion-block by washing, the myotubes that formed were equivalent to those present in unaltered cultures. These results suggest that a La3+ displaceable Ca2+ pool exists at the surface of myoblasts which is involved in myoblast fusion. Thus, it appears that myoblast fusion is dependent on the continuous presence of cell surface calcium and an adequate intracellular Ca2+. An influx of calcium alone is not sufficient to promote myotube formation.

Differential regulation of glucose transport and transporters by glucose in vascular endothelial and smooth muscle cells.

Hyperglycemia has been implicated in the pathogenesis of both micro- and macrovascular complications in diabetes. Little is known, however, about glucose transporters and their regulation in the vascular system. In this study, the regulation of glucose transporters by glucose was examined in cultured BAECs and BSMCs, and in human arterial smooth muscle cells. Both BAECs and BSMCs transported glucose via the facilitated diffusion transport system. Glucose-transport activity in vascular smooth muscle cells was inversely and reversibly regulated by glucose. Exposure of BSMCs and HSMCs to high glucose decreased Vmax for 2DG and 3-O-MG uptake, whereas Km remained unchanged. The hexose-transport system of BAECs exhibited lower 2DG and 3-O-MG uptake compared with BSMCs and showed little or no adaptation to changes in ambient glucose. Northern blot analysis demonstrated that GLUT1 mRNA levels in BAECs and BSMCs were unaffected by the concentration of glucose in the medium. GLUT2-5 mRNA could not be detected by Northern blot analysis. GLUT1 protein, quantified by Western blot analysis, was more abundant in BSMCs than in BAECs and was decreased by approximately 50% when medium glucose was elevated from 1.2 to 22 mM for 24 h. The alterations in the level of GLUT1 protein correlated with the changes observed in transport activity. These observations suggest differential regulation of glucose transporter in response to glucose between smooth muscle and endothelial cells. The sites of autoregulation may involve translational control and/or the stability of the protein in the smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin receptor number and binding affinity in newborn dogs.

The insulin resistance in newborn mammals may be caused by a receptor or postreceptor defect. Although liver and umbilical cord blood monocytes have increased numbers of insulin receptors, there is a paucity of information about other neonatal tissues. Glucose disposal takes place primarily in the skeletal muscle; therefore, it is important to evaluate this tissue for an insulin receptor defect. To determine the role of insulin receptors in neonatal insulin resistance, neonatal and adult canine skeletal muscle, heart, and liver were compared for numbers of insulin receptors and their affinity for insulin. Partially purified receptors from four animals in each group were obtained by wheat germ lectin affinity chromatography and used in competition binding studies. Specific binding (mean +/- SE) in the absence of cold insulin was increased in newborn skeletal muscle (9.7 +/- 0.8 versus 4.8 +/- 0.5%, p less than 0.001) and heart (8.1 +/- 1.2 versus 5.5 +/- 0.6%, p less than 0.05). High-affinity insulin receptor number (mean +/- SEM) was increased in newborn skeletal muscle (183 +/- 40 versus 120 +/- 29 pM, p less than 0.002) and heart (264 +/- 94 versus 157 +/- 51 pM, p less than 0.05) as estimated from the X intercept of the Scatchard plot. Using half-maximal binding to estimate affinity, there were no differences between adults and newborns among all tissues studied. High-affinity receptor number and percentage of specific binding were similar for newborn and adult liver tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium regulation of skeletal myogenesis. I. Cell content critical to myotube formation.

Primary cultures of embryonic chick pectoral skeletal muscle were used to study calcium regulation of myoblast fusion to form multinucleated myotubes. Using atomic absorption spectrometry to measure total cellular calcium and the 45Ca-exchange method to determine free cellular Ca++, our data suggest that only the free cellular calcium changes significantly during development under conditions permissive for myotube formation (0.9 mM external Ca++). Increases in calcium uptake occurred before and toward the end of the period of fusion with the amount approximating 2 to 4 pmol per cell in mass cultures. If the medium [Ca++] is decreased to 0.04 mM, as determined with a calcium electrode, a fusion-block is produced and free cell Ca++ decreased 5- to 10-fold. Removal of the fusion-block by increasing medium [Ca++] results in a release of the fusion-block and an increase in cellular Ca++ to approximately 1 pmol per cell during fusion, and higher thereafter. Cation ionophore A23187 produced transient increases in cellular calcium and stimulated myoblast fusion and the final extent of myotube formation only when added at the onset of culture. Results suggest that transient increased calcium uptake alone is insufficient for fusion because critical cellular content in conjunction with permissive amounts of medium [Ca++] must exist. The latter suggests further that cell surface Ca++ was also critical.

Cardiac hypertrophy in chick embryos induced by hypothermia.

A decrease in incubation temperature from 38 to 32 degrees C elicits a decrease in chicken embyro size and weight with concomitant heart enlargement if done after day 10 of incubation. When assayed at day 18 of incubation with the hypothermia started on day 11 or 14, evidence is presented that the heart enlargement is an hypertrophy with no detectable hyperplasia. Supporting data are presented for various physical parameters showing increases in heart wet and dry weight, volume, area, wall thickness, and cell size. There was little difference in DNA content and nuclear [3H]thymidine labeling index between hearts of control and hypothermic embryos. Hearts of hypothermic embryos showed a slight increase in water content and considerable increases in RNA, protein, and glycogen content per unit DNA. The average size of polysomes isolated from hypothermic hearts was larger than that of polysomes isolated from controls. Microscopic studies showed no obvious increase in amount of capillary beds, connective tissue, and myocardial cells. Annulate lamellae were found only in myocardial cells of hypothermic embryos in sparse amounts and low frequency but always associated with large deposits of glycogen.

Biosynthesis of heparan sulfate proteoglycans of developing chick breast skeletal muscle in vitro.

Previous studies have reported an increase in heparan sulfate glycosaminoglycan (HSGAG) during skeletal muscle differentiation in culture. We have investigated this phenomenon further in relation to the heparan sulfate proteoglycans (HSPG) produced by myogenic cultures. Pulse-chase analysis indicated an approx. 3-fold increase in heparan sulfate synthesis in myotube cultures over that in proliferating or aligning myoblast cultures. Muscle fibroblast culture heparan sulfate synthesis was higher than that of myoblasts but was lower than myotubes. The turnover rates appeared to be the same for all stages of development, with a t1/2 of approx. 5 h. Enrichment for heparan sulfate by Sepharose CL-4B and DEAE-Sephacel chromatography indicated an increase in the hydrodynamic size of the proteoglycan produced by myotubes over that from myoblasts, with a shift in Kav from 0.14-0.19 to 0.07. Fibroblasts synthesized the smallest proteoglycan, with a Kav of 0.22. All of the proteoglycans contained similar sized glycosaminoglycan chains with an estimated molecular weight of 30,000-40,000. Localization of the heparan sulfate proteoglycan in myotube cultures by trypsin sensitivity indicated much of the intact proteoglycan to be closely associated with the cell surface, while internalized material appeared in a degraded form.

Chicken lactose lectin: cell-to-cell or cell-to-matrix adhesion molecule?

Endogenous chicken muscle lectin isolated by lactose affinity chromatography inhibits myoblast fusion. Similar lectins isolated from embryonic brain, heart, and liver and from adult intestine exhibit the same ability. Elevated levels of any of these lectins canceled the inhibitory effect. Peanut agglutinin isolated by the same procedure had no effect at any concentration tested. Concanavalin A affected fusion only at high concentrations. Muscle lectin was shown to agglutinate myoblasts in microtiter plates, whereas exogenous addition in culture inhibited alignment as seen by time lapse microcinematography. Cell-to-cell communication between lectin-treated cells was shown by nucleotide exchange, and lectin-coated culture dishes did not affect cell attachment. Our evidence shows a lack of specificity to muscle, but suggests an aggregating capacity between cells, or possibly an interaction between the cell membrane and the extracellular matrix.

Calcium regulation of skeletal myogenesis: IV. A defined culture medium permissive for myotube formation and the use of the calcium antagonist lanthanum.

Lanthanum has been used effectively in studies of calcium physiology in experiments of short duration. In experiments of longer duration, we report that solutions, such as cell culture medium, containing lanthanum (La++) undergo a decrease in pH on the time scale of hours. Presumably, the decrease in pH is a consequence of the hydrolysis of water by the solution-active La ions. We have devised a defined culture medium without serum and chick embryo extract which is permissive for myotube formation. This defined medium is also useful for studies of La as a calcium antagonist. La at concentrations of 0.1 mM reversibly inhibits myotube formation when added in conjunction with Ca++ to low-Ca++ fusion-blocked cultures.

Morphological characterization of actively fusing L6 myoblasts.

We have characterized morphologically the surface of L6 myoblasts at the time of active cell fusion using transmission electron microscopy. Two subclones of the L6 line were used in these studies: the L6Cl55 line that fuses to form multinucleated syncytia and the NF44 non-fusing variant. Ultrastructural analysis revealed an electron-opaque material at localized points of cell-cell apposition in actively fusing L6Cl55 cells. This material may be transported by and secreted from smooth-surfaced cytoplasmic vesicles with an electron-dense core. In contrast to L6Cl55 cells, the electron-dense plaques were seen infrequently in cultures of the NF44 non-fusing variant. This previously unidentified substance may be associated with cell-cell recognition or adhesion, both necessary prerequisites for myoblast membrane fusion. Alternatively, the electron-dense plaques may be directly involved in the fusion event.

Primary culture of chick embryo skeletal muscle on dextran microcarrier.

We report the conditions to obtain primary suspension cultures using embryonic skeletal muscle from 12-day chick breast muscle. Further, the conditions are described to obtain scanning electron micrographs of whole cells and transmission electron micrographs of sections of plastic-embedded cells on microcarriers. A positively charged hydrated dextran microcarrier, Cytodex I (Pharmacia), provided support for the cells; the myogenic stages of proliferation, myoblast alignment and fusion to form myotubes coincided temporally with replicate cultures grown on gelatin-coated plastic dishes. Microcarrier-grown cells, including non-muscle cells, had microvilli, lamellipodia, bleb, and other surface modifications but no ruffling membranes. Myoblasts and myotubes on beads had fewer microvilli compared to homologous cells grown in the static culture medium of plastic dishes. Myoblasts aligned laterally during fusion, starting at 48 h. Myotube cytodifferentiation proceeded to myofibril formation by day 4 of microcarrier culture. The sarcomeres of aligned myofibrils had normal banding with an hexagonal lattice of thick and thin myofilaments in the A-bands. Caveolae intracellulares and sarcoplasmic reticulum were evident. Scaling-up to larger volumes promises to provide a cost-effective way to obtain a large harvest of cultured skeletal muscle which may prove especially useful for studies of minor constituents.

Purified lectin from skeletal muscle inhibits myotube formation in vitro.

A lactose-extractable lectin obtained from 14--16-d embryonic chick pectoral muscle and myotube muscle cultures by affinity chromatography inhibited myotube formation in culture. When applied to muscle cultures at 0.09 micrograms/ml, the purified lectin produced variable effects on the inhibition of myotube formation related to the time and length of application, suggesting that components of the culture medium and/or temperature produced inactivation. Hemagglutination assays showed that the lectin was inactivated by horse serum and by chick embryo extract but not by L-15 salt solution at 4 degrees C. Incubation in L-15 solution at 37 degrees C with or without 2 mM dithiothreitol resulted in inactivation in 2--3 h. To maximize the effect of the lectin on the inhibition of myotube formation, primary muscle cultures were grown in low [Ca+2] medium to inhibit fusion, and then [Ca+2] was increased to elicit fusion in the absence and presence of lectin with solution renewal every 2 h. Without lectin, myotube formation was normal, whereas, with lectin, it was inhibited by 93%. Continued incubation at 37 degrees C. without renewal of lectin resulted in myotube formation, suggesting reversibility by lectin inactivation.

Reversible suppression of skeletal myotube formation in vitro obtained by varying [CO2].

In primary cultures of chicken skeletal muscle, decreasing the [CO2] of the gaseous phase below 10(-3)% resulted in inhibition of cell proliferation and cytolysis. With 10(-3)% CO2-air, cell proliferation was slightly retarded and myotube formation was inhibited approximately 90% compared to cultures receiving 5% CO2-air. Changes in pH were not effective. Culture in low [CO2] resulted in the accumulation of lipoidal inclusions and unique cytoplasmic structures. Increasing time in culture with low [CO2] resulted in an increase in the length of G1 of the cell cycle. The inhibition was reversed by the addition of 5% CO2-air at any time in culture up to 2 wk with a minimum time of 3--6 h required. Lipoidal inclusions decreased in number and the unique cytoplasmic structures were absent. During the first 3 days in culture, myoblasts showing dependence on [CO2] for myotube formation increased in number, and the effect of elevated [CO2] on these cells was long lasting. The data suggest that some aspect of myoblast differentiation relating to cell recognition and fusion is affected by decreased [CO2].

Reversal by insulin of concanavalin A inhibition of myotube formation and evidence for common binding sites.

Concanavalin A (Con A) inhibits fusion of trypsin-treated myoblasts. This inhibition is reversed by the addition of supraphysiological concentrations (4 micrograms/ml) of insulin either during continuous presence in culture or by pulse additions at 36 and 48 h of culture, just before the time that cultures not treated with Con A undergo myoblast fusion. This reversal is not due to the mitogenic effects of insulin. Under reversal conditions, no specific displacement of bound [125I]iodo-Con A was detected nor did insulin stimulate metabolite uptake. Cell surface replicas of hemocyanin-tagged Con A showed that insulin reversal of the inhibition of myotube formation correlated with the alteration of Con A-binding sites from a clustered configuration present in the inhibited cells to a dispersed state correlated with normal myotube formation. Although a causal relationship has yet to be shown, the data suggest that insulin-mediated reversal of Con A inhibition of myoblast fusion may be related to the ability of insulin at supraphysiological levels to alter the translational mobility of cell surface components containing glucose and/or mannose residues capable of binding Con A. Evidence is presented which suggests that insulin and Con A share common binding sites, since in the physiological range of insulin concentrations (1 ng/ml), Con A pretreatments results in an inhibition of specific [125I]iodo-insulin binding, and antagonistic interactions of insulin and Con A on metabolite uptake and cell proliferation occur. Thus, it appears that the insulin receptors of developing skeletal muscle are glycoproteins containing glycopyranosides.

Suppression of myoblast fusion by concanavalin A: possible involvement of membrane fluidity.

Experimental evidence is presented which is consistent with the involvement of membrane fluidity during myoblast fusion. Treatment of pretrypsinized myoblasts with tetrameric Con A, but not with the dimeric succinyl derivate, inhibits fusion. Inhibition is reversed by treatment with alpha-methyl-D-mannoside or subsequent trypsinization. No inhibition is observed when the lectin is incubated with cells at 4 degrees C unless the incubation is followed by treatment with glycogen, a multivalent Con A cross-linking agent. This effect of glycogen is reversed by subsequent treatment with alpha-amylase. Direct observation of Con A-binding site topography by transmission electron microscopy of membrane replicas of cells labelled with Con A and haemocyanin reveals that inhibition of fusion correlates with a clustered distribution of Con A-binding sites, whereas normal fusion correlates with a dispersed distribution.