Effects of glycoprotein and basement membrane synthesis inhibitors on the growth of cultured renal collecting duct epithelium

Summary The biochemical and morphological extent of glycoprotein synthesis inhibition of cellular and extracellular proteins was studied on cultured renal collecting duct (CD) epithelium. We found that tunicamycin (4 g/ml) inhibits the glycosylation of a 150,000 d glycoprotein (gp_CDI). A 85,000 d glycoprotein (gp_CDII) was not affected. The inhibition by tunicamycin demonstrates that gp_CDI has characteristics of a N-glycan, whereas gp_CDII seems to be an O-glycan. 6-diazo-5-oxo-norleucine (4×10^–5M) which was used as glutamine analogue, did not show a comparable inhibitory effect as seen with tunicamycin. The lack of effect of norleucine demonstrates that glutamine is not the locus of glycosylation in both proteins. However, because of the tunicamycin inhibition it points to asparagine as the site of glycosylation in the gp_CDI. Long term cultures of the tissue up to 15 days in the presence of tunicamycin and norleucine and of substances usually used as basement membrane inhibitors, such as hydroxy-d-proline (1 mM), l-azetidine-2-carboxylic acid (1 mM) and o- and p-nitrophenyl-xylopyranoside (1 mM), revealed that it is possible to eliminate completely the fibroblasts beneath the cultured epithelium and within the degenerating corematerial. Experiments with hydroxy-d-proline showed the most striking effect. Experiments with l-azetidine-2-carboxylic acid and nitrophenyl-xylopyranoside resulted in the elimination of fibroblasts and dedifferentiation of the collecting duct epithelium.     

Immunocytochemical localization of a renal glycoprotein (gpCDI) synthesized by cultured collecting duct cells

A sulfated, proline-rich glycoprotein (gpCDI, apparent molecular weight 200,000 in column chromatography and 150,000 in SDS-PAGE) was isolated from cultured renal collecting duct epithelium by centrifugation. Triton X100 extraction and DEAE-cellulose ion exchange chromatography. A DEAE-cellulose ion exchange chromatography fraction with the enriched gpCDI was used for immunization of guinea pigs. The antiserum was prepared for antigen localization by indirect immunofluorescence in collecting duct cell cultures and in tissue sections of neonatal and adult rabbit kidneys. In the cultured collecting duct epithelium, antibody staining of the epithelium and structures of the extracellular matrix was age dependent. Cultures of dedifferentiated collecting duct monolayers revealed positive reaction in the cytoplasm. In neonatal and adult rabbit kidneys, the antibody was localized in the entire collecting duct system but not in the collecting duct ampullae of the newborn kidney. Staining of the cytoplasm was found only in medullary collecting ducts of the neonatal kidney; other portions revealed staining mostly at the basal circumference of the tubule and at the luminal cell borders. Apart from collecting ducts, no other tubular segments were reactive. The cortical and the medullary interstitium contained fluorescent fibres which were concentrated around vascular structures. A possible relation between gpCDI and collagenous compounds is discussed. Bowman's capsule reacted positively, whereas staining of the mesangial matrix was weak. The localization of the antigen, as revealed by indirect immunofluorescence, suggests that gpCDI occurs both in intracellular and extracellular (interstitial) location. Two main points are emphasized: Firstly gpCDI is considered an important constituent in different stages of collecting duct development, and secondly, the staining pattern of the antibody varies with the different portions of both young and adult kidney collecting ducts; this staining heterogeneity may correspond with the known regional differences of collecting duct functions.     

Effect of 2-deoxyglucose on lysosomal enzymes in cultured human skin fibroblasts

Addition of 2-deoxyglucose, an inhibitor of glycosylation of proteins, to the medium of confluent cultures of human skin fibroblasts prevents the increase in specific activity of lysosomal enzymes that normally occurs after confluence. Maximal inhibition is obtained at a concentration of about 1 mM 2-deoxyglucose. The inhibition by 2-deoxyglucose is reversible. The K_m, pH dependence and electrophoretic mobility of the acid hydrolases tested was the same in cells cultured with or without 2-deoxyglucose. In homogenates of cultured human skin fibroblasts, about 95% of the β-hexosaminidase and α-galactosidase activity and about 65 % of the acid phosphatase activity with β-glycerolphosphate as substrate binds to concanavalin A (ConA); 2-deoxyglucose affects only the activity able to bind to ConA. In cells cultured in the presence of 2-deoxyglucose, the specific activity of alkaline phosphodiesterase I, a plasma membrane glycoprotein is lowered. 2-Deoxyglucose has no effect on the specific activity of succinate dehydrogenase, lactate dehydrogenase or total cellular protein.     

The relationship between glycosylation and glycoprotein metabolism of mouse neuroblastoma N18 cells.

Two inhibitors of glycosylation, glucosamine and tunicamycin, were utilized to examine the effect of glycosylation inhibition in mouse neuroblastoma N18 cells on the degradation of membrane glycoproteins synthesized before addition of the inhibitor. Treatment with 10 mM-glucosamine resulted in inhibition of glycosylation after 2h, as measured by [3H]fucose incorporation into acid-insoluble macromolecules, and in a decreased rate of glycoprotein degradation. However, these results were difficult to interpret since glucosamine also significantly inhibited protein synthesis, which in itself could cause the alteration in glycoprotein degradation [Hudson & Johnson (1977) Biochim. Biophys. Acta 497, 567-577]. N18 cells treated with 5 microgram of tunicamycin/ml, a more specific inhibitor of glycosylation, showed a small decrease in protein synthesis relative to its effect on glycosylation, which was inhibited by 85%. Tunicamycin-treated cells also showed a marked decrease in glycoprotein degradation in experiments with intact cells. The inhibition of glycoprotein degradation by tunicamycin was shown to be independent of alterations in cyclic AMP concentration. Polyacrylamide-gel electrophoresis of isolated membranes from N18 cells, double-labelled with [14C]fucose and [3H]fucose, revealed heterogeneous turnover rates for specific plasma-membrane glycoproteins. Comparisons of polyacrylamide gels of isolated plasma membranes from [3H]fucose-labelled control cells and [14C]fucose-labelled tunicamycin-treated cells revealed that both rapidly and slowly metabolized, although not all, membrane glycoproteins became resistant to degradation after glycosylation inhibition.     

Effect of tunicamycin on the turnover of epidermal growth factor receptors in cultured calf aorta smooth muscle cells: Comparison with IMR-90 human lung fibroblasts

Treatment of cultured calf aorta smooth muscle cells with tunicamycin, a potent inhibitor of dolichol-mediated glycosylation, resulted in progressive loss of receptors for epidermal growth factor with 50% of receptors lost after 6 h. Receptor half-life was also 6 h with cycloheximide treatment but was 12 h with either actinomycin D or camptothesin treatment. The epidermal growth factor-induced processing (internalization and/or degradation) of residual receptors remaining after tunicamycin treatment appeared to be unaltered.50% decrease in ¹²⁵I-labeled epidermal growth factor binding was observed also with IMR-90 fibroblasts upon 6 h treatment with tunicamycin, although these cells were less sensitive to inhibition by tunicamycin of glycosylation and protein synthesis.     

Differentiation properties of renal collecting duct cells in culture

In the present study, we were particularly interested in distinguishing specific patterns of structural and functional proteins in the collecting duct system of neonatal and adult kidneys and in cultured renal collecting duct epithelia in order to ascertain the degree of differentiation in the cultures. We studied the distribution of specific renal collecting duct cell markers using morphological, immunohistochemical and biochemical procedures. Cultured renal collecting duct epithelium undergoes maturation in vitro. Examples of morphological differentiation include the appearance of cilia and microvilli at the apical cell pole, and a basement membrane at the basal aspect of the epithelium. Tight junctions with five to seven strands separate the wide intercellular spaces from the apical cell surface. Physiological maturation from a 'leaky' to a 'tight' epithelium is evident from the acquisition of the alpha-subunit of Na/K-ATPase and the development of a high transepithelial potential difference and resistance. Biochemical differentiation is revealed by the expression of specific proteins. The simple-epithelium cytokeratins, PKK1 and PKK2, which are typical intracellular-matrix proteins of mature collecting duct epithelium, maintain the same distribution in cell culture as in neonatal and adult kidneys. An indicator of maturation in vitro is the expression of the collecting duct-specific proteins, PCD2 and PCD3. Newly developed monoclonal antibodies against these antigens reacted similarly with cultured cells and cells of the mature collecting duct system, but they did not label the embryonic ampullae in the cortex of neonatal rabbit kidneys. In contrast, a third collecting duct-specific protein, PCD1, is not expressed by the cultured cells, which indicates the retention of an embryonic characteristic in vitro. Embryonic collecting duct ampullae of the neonatal kidney in situ contain laminin during their development. Laminin is, however, absent in cultured collecting duct epithelium. Biochemical stimulation of the adenylate cyclase system by arginine vasopressin resulted in a twofold stimulation of the enzyme activity. This degree of stimulation is similar to that found in maturing kidneys of neonatal rabbits and indicates another embryonic feature of the cultures.     

Differentiation properties of renal collecting duct cells in culture

In the present study, we were particularly interested in distinguishing specific patterns of structural and functional proteins in the collecting duct system of neonatal and adult kidneys and in cultured renal collecting duct epithelia in order to ascertain the degree of differentiation in the cultures. We studied the distribution of specific renal collecting duct cell markers using morphological, immuno-histochemical and biochemical procedures. Cultured renal collecting duct epithelium undergoes maturation in vitro. Examples of morphological differentiation include the appearance of cilia and microvilli at the apical cell pole, and a basement membrane at the basal aspect of the epithelium. Tight junctions with five to seven strands separate the wide intercellular spaces from the apical cell surface. Physiological maturation from a ‘leaky’ to a ‘tight’ epithelium is evident from the acquisition of the α-subunit of Na/K-ATPase and the development of a high transepithelial potential difference and resistance. Biochemical differentiation is revealed by the expression of specific proteins. The simple-epithelium cytokeratins. PKK1 and PKK2, which are typical intracellular-matrix proteins of mature collecting duct epithelium, maintain the same distribution in cell culture as in neonatal and adult kidneys. An indicator of maturation in vitro is the expression of the collecting duct-specific proteins, P^CD2 and P^CD3. Newly developed monoclonal antibodies against these antigens reacted similarly with cultured cells and cells of the mature collecting duct system, but they did not label the embryonic ampullae in the cortex of neonatal rabbit kidneys. In contrast, a third collecting duct-specific protein, PcDl, is not expressed by the cultured cells, which indicates the retention of an embryonic characteristic in vitro. Embryonic collecting duct ampullae of the neonatal kidney in situ contain laminin during their development. Laminin is. however, absent in cultured collecting duct epithelium. Biochemical stimulation of the adenylate cyclase system by arginine vasopressin resulted in a twofold stimulation of the enzyme activity. This degree of stimulation is similar to that found in maturing kidneys of neonatal rabbits and indicates another embryonic feature of the cultures.     

Saccharomyces cerevisiae mating pheromones specifically inhibit the synthesis of proteins destined to be N-glycosylated

alpha Factor specifically inhibits the synthesis of N-glycosylated proteins in Saccharomyces cerevisiae mating type a cells but not in alpha cells or in a/alpha diploids. a Factor has the same effect of alpha cells. The synthesis of O-glycosylated proteins is not inhibited. Although the mating pheromones act like a 'physiological tunicamycin', the mechanism of inhibition is different: not the glycosylation of proteins as such but rather the synthesis of those proteins destined to be N-glycosylated is inhibited. Thus none of a number of glycosylating enzymes tested in vitro is reduced in activity in alpha-factor-treated cells. The synthesis of the glycoprotein carboxypeptidase Y, on the other hand, is strongly inhibited by tunicamycin as well as by alpha factor; but only in the former case did carbohydrate-free protein accumulate in the cells. alpha Factor causes maximal inhibition of glycoprotein formation after as little as 30 min, long before all cells in the population are arrested in G1; moreover, release from this inhibition precedes the increase in budding index (resumption of cell division). It is postulated, therefore, that N-glycosylated proteins are required for the G1/S-phase transition in the yeast cell cycle. This is supported by previous reports that first cycle arrest in G1 occurs when (a) tunicamycin is added to growing cultures, and (b) a temperature-sensitive N-glycosylation mutant is shifted to its restrictive temperature.     

4-Deoxy-4-fluoro-D-mannose inhibits the glycosylation of the G protein of vesicular stomatitis virus

The effect of 4-deoxy-4-fluoro-D-mannose (4F-Man), a synthetic analog of D-mannose, on the synthesis of the glycoprotein (G) of vesicular stomatitis virus was examined. Nearly confluent monolayers of cultured BHK21 cells infected with vesicular stomatitis virus were incubated for 2 h with 4F-Man (0-10 mM) or for 1 h with tunicamycin (2 micrograms/ml) and then pulse-labeled with [35S]methionine or [3H]glucosamine. After a 90-min chase period, the cells were lysed and the viral proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The 35S-labeled G protein from cells exposed to greater than or equal to 1 mM 4F-Man migrated more rapidly than G protein isolated from control cells and with the same electrophoretic mobility as the glycoprotein produced by cells treated with tunicamycin. When infected cells were labeled with [3H]glucosamine, little or no radioactivity was associated with G protein synthesized in the presence of greater than or equal to 1 mM 4F-Man. The conclusion that 4F-Man blocks the glycosylation of the G protein was supported by experiments which demonstrated that the fluorosugar inhibits the synthesis of lipid-linked oligosaccharides.     

Immunocytochemical localization of a renal glycoprotein (gp CD I) synthesized by cultured collecting duct cells

Summary A sulfated, proline-rich glycoprotein (gp_CDI, apparent molecular weight 200,000 in column chromatography and 150,000 in SDS-PAGE) was isolated from cultured renal collecting duct epithelium by centrifugation, Triton X100 extraction and DEAE-cellulose ion exchange chromatography. A DEAE-cellulose ion exchange chromatography fraction with the enriched gp_CDI was used for immunization of guinea pigs. The antiserum was prepared for antigen localization by indirect immunofluorescence in collecting duct cell cultures and in tissue sections of neonatal and adult rabbit kidneys. In the cultured collecting duct epithelium, antibody staining of the epithelium and structures of the extracellular matrix was age dependent. Cultures of dedifferentiated collecting duct monolayers revealed positive reaction in the cytoplasm. In neonatal and adult rabbit kidneys, the antibody was localized in the entire collecting duct system but not in the collecting duct ampullae of the newborn kidney. Staining of the cytoplasm was found only in medullary collecting ducts of the neonatal kidney; other portions revealed staining mostly at the basal circumference of the tubule and at the luminal cell borders. Apart from collecting ducts, no other tubular segments were reactive. The cortical and the medullary interstitium contained fluorescent fibres which were concentrated around vascular structures. A possible relation between gp_CDI and collagenous compounds is discussed. Bowman's capsule reacted positively, whereas staining of the mesangial matrix was weak. The localization of the antigen, as revealed by indirect immunofluorescence, suggests that gp_CDI occurs both in intracellular and extracellular (interstitial) location. Two main points are emphasized: Firstly, gp_CDI is considered an important constituent in different stages of collecting duct development, and secondly, the staining pattern of the antibody varies with the different portions of both young and adult kidney collecting ducts; this staining heterogeneity may correspond with the known regional differences of collecting duct functions.     

Compartmental distribution of beta-hexosaminidase isoenzymes in I-cell fibroblasts.

A characteristic of the human lysosomal disorder I-cell disease is an abnormal excretion of most lysosomal hydrolases, including beta-N-acetyl-D-glucosaminidase (EC 3.2.1.30; beta-hexosaminidase) by cultured skin fibroblasts. Treatment of I-cell cultures with cycloheximide or tunicamycin demonstrated that (1) I-cell fibroblasts rapidly excrete all newly synthesized beta-hexosaminidase, (2) two qualitatively distinct pools of beta-hexosaminidase isoenzymes exist inside I-cell fibroblasts, one of which is a rapid-turnover excretory pool, and (3) the induction of an abnormal glycosylation of beta-hexosaminidase by tunicamycin in normal or I-cell fibroblast cultures does not affect subsequent excretion of the enzyme.     

Effect of tunicamycin on polarized membrane functions of an established kidney epithelial cell line

We have explored the relationship between glycoprotein biosynthesis, cell proliferation and function of a differentiated polarized membrane assessed by dome formation in the MDCK epithelial cell line. At 0.1 μg/ml tunicamycin, complete inhibition of cell proliferation was observed in either serum-containing or serum-free, hormone-supplemented growth medium. By contrast, no inhibition of either spontaneous dome formation or that triggered by inducers of cell differentiation such as hexamethylene bisacetamide was observed at 0.5 μg/ml tunicamycin, although total glycosylation of cellular proteins was inhibited by 75%. Our results suggest that the polarized sorting out of epithelial membrane proteins to apical and basolateral surfaces and their functions related to vectorial transepithelial fluid transport, monitored by dome formation, can persist umimpaired despite considerable underglycosylation of cellular glycoproteins and inhibition of cell proliferation.     

Effects of L-proline and some of its analogs on retinal spreading of depression.

L-Proline inhibits glutamate-based spreading depressions (SDs) at low concentrations (2--2.5 mM) and promotes K+-based SDs at higher concentrations (5 mM). The inhibition of glutamate-based SDs was postulated to be due to competition of L-glutamate and L-proline for glutamate receptors on somatic and dendritic plasma membranes. The binding of proline to glutamate receptors was furthermore postulated to result in a release of K+ from the intracellular compartment, enhancing the extracellular K+ concentration sufficiently to promote K+-based SDs. A proline analog, L-baikiain, containing a double bond and one more C atom in the ring structure than proline had similar effects as the latter amino acid, but an analog, L-azetidine-2-carboxylic acid, with one less C atom in the ring had little effect on SD in the retina.     

The role of glycosylation in the enzymatic conversion of procollagen to collagen: studies using tunicamycin and concanavalin A

The enzymatic conversion of chick embryo cranial bone procollagen was studied in vitro using procollagen proteases isolated from the culture medium of chick tendon fibroblasts. During the normal conversion process, chains intermediate in length between proα and α chains, as well as the COOH-terminal extension peptides, can be identified. Underglycosylated procollagen, synthesized by bones treated with an inhibitor of protein glycosylation (tunicamycin), was processed by these proteases in a manner similar to that of intact procollagen. However, medium from cells cultured with tunicamycin lacked the COOH-terminal procollagen protease activity; this did not result from a direct inhibition of the protease by the drug. Concanavalin A also inhibited the conversion of procollagen to collagen by fibroblasts in culture. In an in vitro system, Concanavalin A inhibited the COOH-terminal procollagen protease, and this inhibition was reversed by methyl-α-d-glucopyranoside. These data suggest that the COOH-terminal procollagen protease contains oligosaccharide side chains that are recognized by concanavalin A and that tunicamycin affects the secretion, activity, or activation of the enzyme.     

Cell associated glycoproteins synthesized by cultured renal tubular cells

Summary Thin cortical kidney explants from newborn New Zealand rabbits were cultured in Dulbecco's MEM containing 10% fetal bovine seru. Within 24 h the explants formed globular bodies which were completely covered by a monolayered epithelium. The cells show polar differentiation and resemble the renal collecting duct epithelium. By culturing the globular bodies in Dulbecco's MEM with d-valine instead of l-valine additionally a monolayer of renal collecting duct cells was obtained. For the study of glycoprotein synthesis the globular bodies and the collecting duct monolayers were incubated with various labelled carbohydrates, protein and collagen precursors and then fractionated into coarse membrane pellets. The synthesized glycoproteins were regained in 600×g and 12,000×g coarse membrane fractions and extracted with Triton X 100 buffer for column chromatography and SDS-polyacrylamide electrophoresis in 6 M urea. In addition to a 85,000 d glycoprotein, a carbohydrate rich collagen like protein (apparent molecular weight in column chromatography 200,000 d, in the SDS-polyacrylamide electrophoresis 150,000 d) was found. The 150,000 d glycoprotein incorporates favorably radioactive proline, sulfate, and smaller amounts of lysine, and leucine. Compared to the 85,000 d glycoprotein a double amount of glucosamine and galactose and four fold amount of fucose was detected. The 85,000 d protein has to be ascribed as a usual glycoprotein, in contrast the 150,000 d protein shows an unusual combination of characteristics and has to be considered as a new type of renal glycoprotein.     

Transfer of N-acetylglucosamine to endogenous glycoproteins in the nucleus and in non-nuclear membranes of rat hepatocytes: electrophoretic analysis of the endogenous acceptors

The transfer of N-acetyl(14C)glucosamine from UDP-N-acetyl(14C)glucosamine to endogenous glycoproteins acceptors were studied comparatively in the nuclei and in the non-nuclear membranes of rat hepatocytes. Electrophoretic and autoradiographic analysis show that most of the glycoprotein acceptors of the nuclei differ from those of the non-nuclear membranes in terms of molecular weight. In addition, it may interesting to mention that in the nuclear fraction a 30% inhibition by tunicamycin is obtained for concentrations as low as 0.03 microM, whereas at this concentration no effect is detected in the non-nuclear membranes. In the presence of 0.2 microM tunicamycin, the inhibition does not go beyond 25% in the latter fraction but goes up to 80% in the former. The previous results demonstrate clearly that a particular glycosylation reaction occurs in the nucleus.     

Assembly of viral membranes: maturation of the vesicular stomatitis virus glycoprotein in the presence of tunicamycin.

The role of glycosylation in the maturation of the vesicular stomatitis virus (VSV) glycoprotein was studied by use of the antibiotic tunicamycin. Tunicamycin-treated VSV-infected cells synthesize an unglycosylated form of the VSV glycoprotein (R. Leavitt, S. Schlesinger, and S. Kornfeld, J. Virol. 21:375--385, 1977). We have found that tunicamycin has no effect on the attachment of the glycoprotein to intracellular membranes or on the transport of protein to the lumen of the endoplasmic reticulum. However, tunicamycin prevented the migration of the glycoprotein from the rough endoplasmic reticulum to smooth intracellular membranes.     

Inhibition of N-linked glycosylation in Dictyostetium discoideum: Effects of aggregate formation

The aggregation program of Dictyostelium discoideum is extremely sensitive to the effects of tunicamycin when the drug is added to cells during the first few hours of starvation. Inhibition of development is observed with concentrations as low as 0.5 micrograms/ml, which cause only a 25%-30% inhibition of general N-linked glycosylation. However, 0.5 micrograms/ml tunicamycin can result in the total inhibition of N-linked glycosylation of specific, developmentally regulated, proteins, as exemplified by the glycoprotein 117 antigen. If added after the first hours of starvation, tunicamycin cannot inhibit aggregation even when present at 10 micrograms/ml, which maximally inhibits N-linked glycosylation. cAMP pulses can override the inhibitory effects of tunicamycin on cell aggregation. The data support the hypothesis that there is an early developmental pathway that is dependent on the N-linked glycosylation of one, or a small set of developmentally regulated proteins and that this pathway may involve the biogenesis of the chemotactic signalling system. In addition, the data raise questions as to the role of N-linked oligosaccharides in cell cohesion.     

Profiling of N‐glycosylation gene expression in CHO cell fed‐batch cultures

One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N‐glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N‐glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed‐batch culture of a CHO cell line producing recombinant human interferon‐γ (IFN‐γ). Of the 24 N‐glycosylation genes examined, 21 showed significant up‐ or down‐regulation of gene expression as the fed‐batch culture progressed from exponential, stationary and death phase. As the fed‐batch culture progressed, there was also an increase in less sialylated IFN‐γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN‐γ. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed‐batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N‐glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible “bottlenecks” or “compromised” pathways in N‐glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516–528. © 2010 Wiley Periodicals, Inc.     

Precardiac mesoderm differentiation in vitro

The differentiation of precardiac mesoderm into beating heart tissue was examined during explant culture. Explanted tissue forms tubular heart-like vesicles and initiates rhythmic contractility within 18-24 h in vitro, a developmental time-course approximating that observed during in vivo development. Electron-microscopic observations reveal that beating heart cells are rich in cytoplasmic myofibrils in varying degrees of order, with some regions containing highly organized myofibrillar arrays. The analysis of actin-isotype biosynthesis, using metabolic labeling with [35S]-methionine and isoelectric-focusing resolution of the synthesized radioactive polypeptides, demonstrates that the initiation of cardiac alpha-actin synthesis and the pattern of transition in the synthesis of alpha-, beta-, and gamma-actin isotypes is equivalent to the initiation time and pattern observed in vivo. A possible collagen involvement in the differentiation process was investigated by assessing the effects of collagen-synthesis inhibitors on the development of the explant cultures. Two different agents, alpha, alpha'-dipyridyl and L-azetidine-2-carboxylic acid, exhibited a dose-dependent ability to inhibit the formation of beating heart tissue. When examined by electron microscopy, the nonbeating tissue exhibited a drastic depression of myofibrillogenesis, but otherwise appeared healthy. Further examination of the effect of L-azetidine-2-carboxylic acid demonstrated that the inhibition of myofibril formation and heartbeat was correlated with a 60% inhibition of native collagen synthesis; however, the time-course and pattern of actin-isotype biosynthesis was completely unaffected. The data suggest a possible involvement in heart differentiation that is necessary for either the synthesis of non-actin cardiac contractile proteins or the assembly of cardiac contractile proteins into myofibrils.