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The characteristics of L-proline transport in the procyclic form of Trypanosoma brucei were studied by using L-[14C]proline and a quick separation technique by centrifugation through an oil mixture. L-Proline uptake displayed typical Michaelis-Menten kinetics, with a Km of 19 microM and a maximum transport velocity of 17 nmol/min per 10(8) cells at 27 degrees C. The maximum concentration gradient factor obtained after 1 min of incubation was 270-fold in 0.02 mM proline. Cells permeabilized with 80 microM digitonin were still able to accumulate 14C label, but to a lower extent. The temperature-dependence of proline uptake gave an apparent activation energy of 74.9 kJ.mol-1. In competition studies with a 10-fold excess of structural analogues, L-alanine, L-cysteine and L-azetidine-2-carboxylate were found to inhibit L-proline uptake. Variation of pH or addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone ('CCCP') did not affect proline transport, showing that it is not driven by a protonmotive force. The absence of Na+, with or without monensin, did not affect proline transport. The absence of K+ and the addition of the Na+,K(+)-ATPase inhibitor ouabain had no significant effect on proline uptake activity. The thiol-modifying reagent iodoacetate (10 mM) decreased proline uptake by half. KCN (1 mM) inhibited proline uptake to a lesser extent, and the degree of inhibition was proportional to the intracellular ATP concentration. Preliminary experiments on proline transport in plasma-membrane vesicles of the cells, using a filtration technique, showed an uptake of proline (0.67 nmol/mg of protein) by the vesicles, but only in the presence of intravesicular ATP. The results thus obtained suggest that the proline carrier system in T. brucei is ATP-driven and independent of Na+, K+ or H+ co-transport.

Recruitment of keratocytes into injured corneal stroma, and secretion of proteins including collagen in the cells are essential for wound healing of the corneal stroma. We examined the effect of a proline analog, cis-hydroxyproline, on the adhesion, migration and growth of rabbit keratocytes in vitro. This agent decreased the plating efficiency, migration and growth of the keratocytes in a dose-dependent manner. Reduction in these cellular activities may reflect altered functions of pericellular proteins such as collagen. Further studies are needed to determine which specific protein is involved.

The present study examined the effects of cis-4-hydroxy-L-proline (CHP), a proline analog, on the androgen-induced growth of the lateral prostate of prepubertally castrated guinea pigs. Prepubertal male guinea pigs were castrated at the age of 3 weeks and allowed to recover completely before subjection to an experimental regime to alter the stromal collagen synthesis by CHP. The animals were kept on a special proline-deficient diet (PDD) for a week before the subcutaneous injection of CHP (200 mg/kg/day) for 3 days, followed by a combined injection of CHP and dihydrotestosterone (DHT) (10 mg/kg/day) for 10 days. Control animals were injected with saline and DHT only. At the end of the experiment, the lateral prostate was removed and examined by 1) conventional transmission electron microscopy (TEM), 2) staining of proteoglycans (PGs) by Cuprolinic Blue (CB) using the critical electrolyte concentration (CEC) method, 3) carbohydrate and lectin histochemistry, and 4) electron microscopy (EM) lectin-gold labelling. The results showed that the wet weight of prostate from CHP-treated animals was significantly lower than the control and recovery groups. The epithelium was low columnar with an obvious increase in intercellular spaces and number of basal cells. The glandular cells showed little secretory activity with a decrease in number of granular endoplasmic reticulum (GER) profiles, secretory granules, and a small Golgi apparatus. The stroma was composed of stromal cells separated by large intercellular spaces with very sparse collagen fibrils, and a decrease in stromal PGs especially those PGs normally associated with collagen fibrils. CHP treatment also caused perturbation and disorganization in the epithelial basement membrane. The results suggested that stromal collagen is essential in mediating the response of glandular cells to DHT stimulation. Defective stromal collagens hamper the responsiveness of prostatic gland to androgen.

The aim of this study was to clarify how collagen deposition or medial hypertrophy of the vascular wall affects the coronary dilator reserve in pressure-overloaded hearts and whether inhibition of collagen deposition reverses the abnormalities after relief of pressure overload. We used ascending aortic banding and debanding methods and superimposed beta-aminopropionitrile in some of the banded rats (50 mg/kg i.p., twice a day). Ten weeks of banding increased in vivo peak systolic left ventricular pressure and produced medial hypertrophy, an increase in collagen deposition in the myocardial and perivascular tissues, and myocardial hypertrophy in the banded group without beta-aminopropionitrile treatment. Superimposition of beta-aminopropionitrile treatment on banding inhibited the increase in collagen deposition. In the groups debanded after the 10-week banding period, both with and without beta-amino-propionitrile treatment, medial and myocardial hypertrophy regressed 4 weeks after debanding. We estimated coronary dilator reserve in Langendorff preparations perfused with modified Tyrode's solution containing oxygenated bovine red blood cells and serum albumin. The ratio of reactive peak flow after brief ischemia-to-resting flow decreased in both of the banded groups. After debanding, the ratio remained lower in the banded group without beta-aminopropionitrile treatment than in the control group. However, debanding in the group with beta-aminopropionitrile treatment increased the ratio to a level similar to that of the control group. Thus, in pressure-overloaded cardiac hypertrophy with coronary hypertension, coronary reserve seems to be determined by medial hypertrophy independently of collagen deposition, but collagen deposition plays an important role in the reversal of vasodilator reserve after relief of the overload.

cis-4-Hydroxy-L-proline (cis-OH-Pro) and ethyl-3,4-dihydroxybenzoate (EDHB), two distinct inhibitors of collagen synthesis, prevented myogenesis in C2C12 mouse skeletal muscle cells. Both inhibitors blocked myotube formation and the expression of sarcomeric myosin heavy chain. Northern blot analysis showed that cis-OH-Pro- and EDHB-treated C2C12 muscle cells did not express the myogenic regulatory genes, MyoD1 and myogenin, but continued to express non-muscle isoforms of actin (beta and gamma) and alpha-tropomyosin. 10TFL2-3B cells, a C3H10T1/2 cell line permanently transfected with myogenin cDNA, constitutively expressed exogenous myogenin in the presence of cis-OH-Pro but failed to activate endogenous myogenin and to undergo myogenesis. These results demonstrate that commitment to terminal differentiation and activation of myogenic regulatory genes requires active synthesis of the extracellular matrix component collagen.

We investigated antigenic changes of murine glomerular basement membrane (GBM) collagen type IV after oral feedings of 3-cis- or 4-cis-hydroxyproline. As shown by thin layer chromatography and gas chromatographic-mass spectrometric studies, 3-cis- and 4-cis-hydroxyproline were incorporated into the GBM collagen instead of the normal trans isomer and led to a considerable reduction of the collagen antibody reactivity on indirect immunofluorescence, passive hemagglutination and radioimmunoassay when compared to GBM collagen of control animals. We speculate that antigen modification due to the incorporation of stereoisomers could serve as a model for basement membrane immunopathology.

Hydra are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. As an extension of the previous study which indicated that mesoglea is a primitive basement membrane which has retained some characteristics of interstitial extracellular matrix, the present study was undertaken to analyze the role of mesoglea components during head regeneration in Hydra vulgaris. Studies were conducted that utilized drugs that affect collagen processing or secondary collagen structure (beta-aminoproprionitrile; 2,2'-dipydridyl; and cis-4-hydroxy-L-proline) and a drug that inhibits addition of glycosaminoglycan chains to proteoglycan core proteins (p-nitrophenyl-beta-D-xylopyranoside). These studies indicated that alterations in the structure of collagens or proteoglycans caused blockage of head regeneration in Hydra as monitored over a 48-hr period. Blockage of head regeneration was reversible once the drugs were removed, indicating that the drugs were not having a general toxic effect on the organism. Radiotracer studies also indicated that blockage of head regeneration was not simply due to a general depression of protein synthesis by the drugs. Various controls indicated that each drug was affecting mesoglea components under the conditions utilized in these studies. These observations indicate that preservation of normal mesoglea structure is required for Hydra head regeneration to proceed.

The role of collagen in microvascular growth was investigated using the aortic ring model of angiogenesis. Collagen production by vasoformative outgrowths in plasma clot culture of rat aorta was either stimulated with ascorbic acid or inhibited with the proline analogue cis-hydroxyproline. Microvessels proliferating in the absence of ascorbic acid supplements became ecstatic and developed large lumina. In contrast, newly formed microvessels in the presence of ascorbic acid remained small and maintained thin lumina throughout the angiogenic process. Biochemical studies demonstrated enhanced collagen production and deposition in cultures treated with ascorbic acid. Ultrastructural studies of these cultures showed a marked increase in newly formed interstitial collagen in the perivascular matrix and in regions of the plasma clot containing nonendothelial mesenchymal cells. Small microvessels with thin lumina similar to the ones observed in ascorbic acid-treated plasma clot cultures were obtained by growing aortic explants in gels of interstitial collagen in the absence of ascorbic acid. Inhibition of collagen production with the proline analogue cis-hydroxyproline had a marked anti-angiogenic effect in both plasma clot and collagen gel cultures. The anti-angiogenic effect of cis-hydroxyproline was abolished by adding L-proline to the culture medium, thereby restoring normal metabolism. These results support the hypothesis that angiogenesis is regulated by collagen production and suggest that the size of newly formed microvessels is influenced by the degree of collagenization of the extracellular matrix.

A recently described nonreducible, acid-heat stable compound, histidinohydroxylysinonorleucine (HHL), is a collagen cross-link isolated from mature skin tissue. Its abundance is related to chronologic aging of skin. The present communication describes the quantity of HHL from aged human skin of the same individuals in sun-exposed (wrist) and unexposed (buttock) sites. Punch biopsies were obtained from these sites from nine people of age 60 or older. HHL contents (moles/mole of collagen) at these sites were for wrist 0.13 +/- 0.07 and for buttock 0.69 +/- 0.17 (mean +/- SD, p less than 0.001). In addition, it was found that acute irradiation of the cross-linked peptides with UVA (up to 250 J/cm2) and UVB (up to 1 J/cm2) had no effect on HHL structure. The same treatment significantly degraded another nonreducible, stable collagen cross-link, pyridinoline. The results suggest that chronic sunlight exposure may be associated with an impediment to normal maturation of human dermal collagen resulting in tenuous amount of HHL. Thus, the process of photoaging in dermal collagen is different from that of chronologic aging in human skin.

Calf articular cartilage fragments were incubated in vitro in the absence and presence of 170 micrograms N-acethyl-trans-4, hydroxyproline (oxaceprol) and 20 muCi [35S]-Na2SO4 per ml of culture medium. Newly synthesized 35S labeled proteoglycans (35S-PGs) were extracted with buffered 4M guanidinium chloride (GdmCl) solvent and then characterized with respect to their hydrodynamic sizes, capacity to interact and form aggregates with hyaluronic acid (AH) and the length and composition of their glycosaminoglycan (GAG) side chains. It was demonstrated that extracted 35S-PGs synthesized in the presence of the oxaceprol are not significantly different from the molecules synthesized in the absence of this compound.

Various models for sensory coding have used statistical approaches based on the assumption that the stimulus intensity parameter is represented in the afferent neurons as mean firing frequency. In this paper we question the assumption that this is the only code for intensity. We show that in lobster olfactory receptors narrowly tuned to hydroxyproline, an across-fiber pattern (AFP) code distinguished more concentration levels over a 5 log step range than a response magnitude code and, unlike the latter, was unaffected by response summation time. AFP discrimination of stimulus intensity appears to be based on high inter-cell response variability and low intra-cell response variability.

A new class of nontransformed revertant cells has been isolated from the ras-transformed cell line DT using cis-4-hydroxy-L-proline (CHP) as a selective agent. The new revertants, CHP 9CJ and CHP CB4, each contain two copies of the v-Ki-ras gene, elevated levels of phosphorylated p21ras protein, and rescuable transforming virus, indicating that the revertant phenotype observed in these cells does not result from inactivation of v-Ki-ras or inhibition of its expression. Both CHP 9CJ and CHP CB4 revertants show a greatly reduced ability to form colonies in soft agar and to produce tumors in syngeneic mice. CHP 9CJ cells are resistant to retransformation by ras and by additional oncogenes that do not encode tyrosine kinases. A comparison of oncogene resistance patterns in these CHP-derived revertants with those from our original ouabain-derived revertants fos C-11 and F-2 indicates that oncogenes may be divided into four general groups. Oncogenes that encode proteins structurally related to p21ras comprise the first group. The second group contains only tyrosine kinase-encoding oncogenes. The third group is composed of 'nuclear', e.g. fos, and 'cytoplasmic' serine-threonine-encoding oncogenes such as mos and raf. The fourth group contains the oncogenes sis and fms.

The authors have studied in organ culture, the effects of oxaceprol-structural analogue of hydroxyproline, on the proteoglycan and protein synthesis and degradation by calf articular chondrocytes. A stimulation of the incorporation of 35SO4, which indicate proteoglycan synthesis, was shown. The effect was observed at concentrations 10(-6) M to 10(-9) M, i.e. at 170 ng to 170 pg of the oxaceprol per ml of culture media, respectively. However, no significant effect was seen at concentrations 10(-4) M (17 micrograms/ml) to 10(-8) M (1.7 ng/ml) on the protein and proteoglycan catabolism. These results support those reported by Kalbhen and Kalbert and are favouring the use of this drug in order to stimulate reparative processes of articular cartilage in osteoarthritis and aging.

Epidermal growth factor and cis-hydroxyproline specifically inhibited synthesis of type 1 collagen, a major gene product of the differentiated dental mesenchymal cells (odontoblasts). In tandem, synthesis of enamel proteins, specific gene products of differentiated dental epithelial cells (ameloblasts), was also inhibited. Under these culture conditions, total protein synthesis in tooth organs was not inhibited but rather increased. Inhibition curves of the gene products specific for epithelial and mesenchymal phenotypes were quite similar, indicating coordinate and intimately associated regulation of gene expression under conditions that perturb cytodifferentiation.

We contrasted the collagen-binding potential of the experimental osteomyelitis pathogen, Staphylococcus aureus strain SMH, to several other strains. These included Cowan 1 (binder), Wood 46 (non-binder) and six capsular variants. These measurements were made using an 125I-collagen binding assay. Formalin-killed S. aureus SMH strongly bound commercial type I iodinated collagen (dissociation constant, Kd = 2 x 10(-9) M). The extent of binding was similar to Cowan 1. Binding was saturable and not inhibited by 100 mM solutions of D-glucose, D-galactose, D-mannose, methyl-alpha-L-fucopyranoside, L-hydroxyproline or L-glycine. D-lactose gave moderate inhibition of binding to collagen, and L-fucose was strongly inhibitory. Trypsinized SMH did not bind collagen. None of four Ruthenium-red-staining staphylococci (encapsulated) avidly bound type I collagen. The encapsulated Smith strain, for example, did not bind to collagen but its capsule-negative variant, Smith compact, showed extensive binding. Three of five non-encapsulated S. aureus were strong collagen binders. These data suggest that the prototype bone pathogen binds to the major protein component of bone's extracellular matrix. Collagen-binding is promoted by protein adhesin(s), not capsule. The latter, in fact, appeared to interfere with this interaction. Binding was inhibited by solutions containing the simple monosaccharide, L-fucose.

There is evidence that lysosomal proteases mediate the intracellular degradation of structurally abnormal collagen. I-Cell disease (Mucolipidosis II) is characterized by marked deficiency of many lysosomal hydrolases, including the collagenolytic enzyme cathepsin B. The experiments reported here tested the hypothesis that degradation of abnormal collagen would be severely impaired in I-cells. Skin fibroblasts from 3 patients with I-cell disease were incubated with and without cis-hydroxyproline, a proline analog that causes structural abnormalities in collagen, and [14C]proline. The amount of [14C]hydroxyproline in a low molecular weight fraction relative to total [14C]hydroxyproline was used as a measure of intracellular collagen degradation. Levels of degradation were significantly higher in I-cells exposed to cis-hydroxyproline than in cells incubated without the analog. Similar data were obtained for normal human fetal lung fibroblasts incubated under the same conditions. Degradation of [125I]-epidermal growth factor was used to assess the functionality of the lysosomal pathway for protein degradation, and it was much lower in I-cells than in normal cells. It can be concluded that a completely functional complement of lysosomal enzymes is not necessary for structurally abnormal collagen to be degraded intracellularly; the data suggest that a nonlysosomal pathway exists.

Epimastigotes of Trypanosoma cruzi, Peru strain, incubated in Contreras' artificial triatomine urine transformed into metacyclic trypomastigotes when 10 mM L-glutamine, L-asparagine or D-fructose was added to the medium. Metacyclogenesis with these substrates was comparable to the percent metacyclic morphotype formation induced by L-proline and significantly greater than that stimulated by 10 mM D-glucose. Sodium acetate (10 mM) increased transformation induced by L-proline, and L-hydroxyproline (10 mM) increased transformation induced by D-fructose. Phosphoenolpyruvate (10 mM) inhibited L-proline-induced metacyclic trypomastigote stage formation. Three antimetabolites, azetidine 2-carboxylate (5 mM), malonic acid (1 mM), and desthiobiotin (5 mM), completely inhibited D-fructose-induced but not L-proline-induced transformation. The Costa Rica, Y, and CL strains of T. cruzi showed different patterns of percent metacyclogenesis with substrates that induce transformation in the Peru strain.

Cultures of rat myotubes from 18-day-old embryos produce both globular (G) and asymmetric (A) forms of acetylcholinesterase (AChE; EC 3.1.1.7), mostly G1, G4, and A12 and a small proportion of A8. We show that all forms are partly intracellular and partly exposed to the extracellular medium; the A forms and their intra- and extracellular distribution are not modified when myotubes are grown in the presence of spinal cord neurons. In these cocultures, however, AChE patches may be detected immunohistochemically at sites of neuromuscular contacts. These patches represent a very minor proportion of AChE activity. We found that collagenase removes AChE patches but not the acetylcholine receptor clusters with which they coincide. This digestion specifically decreases the level of the A12 form. cis-Hydroxyproline, an inhibitor of collagen synthesis, reduces the level of G1 and blocks the synthesis of A forms.

Vitamin A-storing cells have been shown to be distributed among various organs and tissues, including the lungs. In order to investigate this unique type of cell, the in vitro isolation has been carried out from rat lungs. Lungs were perfused with EGTA and collagenase solution in situ, and were digested with trypsin-collagenase solution at 60-min intervals for 2 h. Then, the cell suspensions obtained were incubated at 37 degrees C in F-10 medium supplemented with 10% fetal bovine serum (FBS) for 72 h. Non-adherent cells were then removed by vigorous washing with medium, and the resultant cell monolayer was harvested with trypsin to remove the contaminating macrophages. These cell fractions were shown to contain more than 96% of vitamin A-storing cells, judged by electron and fluorescence microscopic examinations. The cells grown in vitro retained well the overall morphology characteristic of the vitamin A-storing cells found in lung tissues. The isolated cells grew well in vitro and the growth was inhibited by D-valine or cis-hydroxyproline. The progeny of the cells still contained vitamin A lipid droplets after several transfer generations. Characteristic networks of fibronectin were also demonstrated around the cells. These results have shown that vitamin A-storing cells in the lung was successfully isolated from rat lungs and the cells possessed fibroblast-like characters storing vitamin A in small lipid droplets.

Notochordal extracellular matrix consists of a continuous basal lamina, amorphous materials and microfibrils embedded in the ground substance of low electron density. Together they comprise the notochord sheath and are of considerable interest because of their suspected role in early embryonic tissue interactions. The notochord is particularly well-suited to morphological investigation of extracellular matrix because it is one of the few embryonic epithelia which produces ultrastructurally recognizable stroma in vitro without the advantage of a collagenous substratum. Furthermore, these matrix components produced in vitro are morphologically identical to those observed in vivo. The present study used ruthenium red staining to demonstrate that notochordal microfibrils exhibit collagen-like cross-banding patterns both in vivo and in vitro. Collagenase and testicular hyaluronidase digestion studies designed to localize collagen and glycosaminoglycans show a reduction of microfibrillar diameters by 30-35%. Furthermore, these enzyme treatments frequently result in enhanced striations of microfibrils. When cis-hydroxyproline (a proline analog) or beta-aminoproprionitrile (BAPN, a lathyrogenic compound) is added to the culture medium, a similar reduction in microfibrillar diameters is seen. Moreover, increased ruthenium red-positive surface coats and large collagen fibrils are frequently present in BAPN-treated cultures, implying a stimulatory metabolic effect. We conclude that most, if not all, notochordal extracellular matrix components are composed of both collagen and glycosaminoglycans and suggest that the entire extracellular matrix should be considered a macromolecular composite which acts in concert to induce or stabilize developmental interactions.

Poly O-acetyl, butyryl, hexanoyl, dodecanoyl, and benzoyl hydroxy L-proline (poly[O-acyl Hyp]s) were evaluated as materials for blood contact by means of contact angle and blood clotting time measurements. Critical surface tensions obtained from Zisman plots for all materials were 22-29 dyn/cm, suggesting that these materials may exhibit good blood compatibility. Dispersion and nondispersion force contributions to the surface tension were gamma ds = 1.4, gamma ns = 49.3 dyn/cm, (11.0, 16.2), (19.8, 3.8), (21.2, 4.6) and (14.1, 10.8) for the poly(O-Acetyl, Butyryl, Hexanoyl, Dodecanoyl and Benzoyl Hyp) surfaces, respectively. The materials showed remarkable wetting differences that were dependent on the type of acyl group attached to Hyp. The values of the dispersion and nondispersion components of the surface tension for poly(O-hexanoyl Hyp) and poly(O-dodecanoyl hyp) were very close to those obtained for glutaraldehyde-treated umbilical cord vessels. The blood clotting times on the respective polymer surfaces, obtained by using the kinetic method, were normalized to those of control glass and siliconized glass surfaces. All the poly(O-acyl Hyp)s surfaces showed longer clotting times than those of the poly(L-proline) and glass surfaces. The surfaces of those polymers having longer aliphatic or aromatic acyl groups had longer clotting times than those of the polymers with relatively shorter groups.

Structural and functional changes in lung tissue of mice fed with beta-aminopropionitrile fumarate, L-3-cis-hydroxyproline, and L-4-cis-hydroxyproline. We fed 0.1% solutions of 3-cis-hydroxyproline (3cisHP), 4-cis-hydroxyproline (4cisHP) or beta-aminopropionitrile fumarate (beta APN) to 5-week-old mice for 1 month and studied the effect of each of these substances on the lung function and structure. Compared to control animals the compliance of the respiratory system in the mean was increased by 3-15% in the mice fed beta APN or 3cisHP, and decreased by 2-8% in the mice fed 4cisHP. On electron microscopial examination no overt morphological changes were detectable although as proven by biochemical analysis 3cisHP and 4cisHP were incorporated into the collagen of the mice who received these substances. We conclude that the effect of substances interfering with the normal production of collagen molecules depends on the kind of collagen affected and on the rate of collagen turnover.

Proline analogues such as cis-4-hydroxy-L-proline (CHP) and L-azetidine-2-carboxylic acid (A2C) were tested for their antitumor activity in tissue culture and in vivo. In culture, CHP specifically inhibited those tumor cells that synthesized basement-membrane collagen. CHP appeared to selectively inhibit collagen biosynthesis with only a slight effect on protein synthesis. Culturing cells on type IV collagen matrix did not alter the antiproliferative effect of CHP. The inhibition of 450.1 mouse mammary tumor cells was fully reversible when cultures were incubated for 6 or 12 hours with 25 micrograms CHP/ml but was irreversible after 24 hours of exposure. Of the proline analogues tested against 450.1 tumor cells, A2C and CHP were the most potent inhibitors of cell growth. These two compounds were therefore tested in vivo using 3 transplantable tumors, all of which synthesized basement-membrane collagen. CHP and A2C were given twice daily to mice for 7 to 10 days at doses ranging from 50 mg/kg (body wt) to 600 mg/kg (body wt) per injection. Both CHP and A2C were completely inactive against the 450.1 mammary tumor and the EHS sarcoma. Both compounds also caused considerable liver toxicity. Against CD8F1 mammary tumors, treatment with maximum tolerated doses of CHP and A2C resulted in a slight but insignificant inhibition of tumor growth. While our studies confirmed previous findings that CHP specifically inhibited those tumor cells that synthesized basement-membrane collagen, CHP and A2C did not appear to be efficacious antitumor agents.

Previous efforts to define the nature of the complex requirements for the development of striated muscle in vitro led us to the finding that the presence of collagen in the extracellular environment is essential for the formation from myoblasts of the multinucleated myotube (1). In the present report we demonstrate that the proline analog, cis-4-hydroxy-L-proline, will inhibit myotube formation in vitro without affecting the aggregation of cells (fusion?). The presence of collagen or gelatin as a culture substratum overcomes the action of the analog. The role of collagen in the development of the myotube is discussed.

The relationship between collagen and C1q biosynthesis has been investigated in cultured human fibroblasts. This was done by measuring the effects of variation in cell density, inhibition of prolyl hydroxylase and complexing of C1q on synthesis and/or secretion of both proteins. It was found that synthesis and secretion of both proteins were not co-ordinate, and that therefore regulation of expression of both proteins is probably not linked. However complexing of C1q did result in marked stimulation in collagen synthesis, suggesting that the fibrosis which follows inflammation may result from binding of C1q to the immune complexes formed in the inflammatory process.

In primary cultures of bovine adrenal medulla, chromaffin cells responded to prostaglandin (PG) E2 by stimulating phosphoinositide metabolism (Yokohama et al. (1988) J. Biol. Chem. 263, 1119-1122). In contrast, nonchromaffin cells were found to respond to PGD2 by elevating their intracellular cAMP level. The formation of cAMP was detected at as low as 0.1 nM PGD2 and increased more than 100-fold over the basal level at 0.1 microM, and the response was specific for PGD2 (greater than PGE1 greater than PGE2 greater than PGF2 alpha = PGI2). The magnitude of cAMP formation and its specificity to PGD2 were retained throughout a 40-day culture period. Based on the inhibitory effect of cis-4-hydroxy-L-proline, an inhibitor of collagen synthesis, on cAMP formation, morphology, and immunoreactivity of cells to anti-collagen type I antiserum, the responsive cells were identified as fibroblasts. These results taken together demonstrate that the adrenal medulla is composed of chromaffin and nonchromaffin cells, which respond to PGE2 and PGD2, respectively, by two different signal transduction pathways. The cAMP formation by PGD2 was also observed in fibroblasts from bovine embryonic trachea among cell lines tested, suggesting that some populations of fibroblasts responsive to PGD2 exist in various tissues and may discriminate the signal from that of PGE1 or PGE2.

Coculturing of rat embryonic muscle cells with spinal cord explants resulted in the formation of large numbers of acetylcholine receptor aggregates on the myotube surface, compared to those found on muscle cells grown in the absence of nervous tissue. Remarkably fewer receptor aggregates were formed when, upon addition of nerve explants, these cocultures were treated with either cis-hydroxyproline (a specific inhibitor of collagen production) or collagenase. The possibility is raised that collagen participates in the aggregation of acetylcholine receptors and in synapse formation.

The growth of primary N-nitrosomethylurea-induced rat mammary tumors was depressed by cis-hydroxyproline (CHP). This growth arrest appeared to be related to the ability of CHP to inhibit the deposition of basement membrane collagen as based on the following observations: (a) in vitro and in vivo, tumor cells synthesized type IV collagen, the collagen uniquely localized in basement membranes; (b) in vitro, the inhibition of tumor cell growth was preceded by a specific decrease in collagen accumulation with no effect on non-collagen protein synthesis; (c) a transplantable N-nitrosomethylurea-induced rat mammary tumor accumulated no type IV collagen as determined by polyacrylamide gel electrophoresis and indirect immunofluorescence. The growth of this tumor was not influenced by CHP; (d) an established human mammary tumor cell line, MCF-7, did not accumulate type IV collagen and was not inhibited by CHP. At the doses which effectively blocked the growth of primary N-nitrosomethylurea-induced mammary tumors, CHP and no toxic effects, and serum prolactin levels were not altered. The inhibitory effect was thus apparently due to the direct action of CHP upon the accumulation of collagen in cells which required type IV collagen production for continued growth.

Endothelial cell migration was studied following a mechanical injury produced in cultured confluent monolayers of calf aortic endothelium with the use of a quantitative migration assay. In this method the cells were grown on glass coverslips coated with scarlet red-containing Formvar. At confluency, the cultures were cut in half with a blade; one half was removed with the pigmented Formvar, and the other was returned to culture. Migration was linear for a least 96 hours, and was due to cell motility, not proliferation. Since it was blocked in the presence of L-azetidine carboxylic acid or cis-hydroxyproline, inhibitors of collagen secretion, endothelial cell migration appeared to be dependent on the continual secretion of collagen. Furthermore, the types, apparent relative amounts, and localizations of the collagens as well as laminin changed during the migratory process. These studies support the notion that the aortic endothelial cell migratory response to injury is a dynamic one requiring the continual secretion and modulation of matrix molecules.

Damage to interstitial connective tissue is associated with the rapid accumulation of monocytes and neutrophils at the site of injury. To study the role of collagen fragments in neutrophil migration, we analyzed the chemotactic properties of peptide fragments of bovine collagen digested with bacterial collagenase or cyanogen bromide and small molecular weight synthetic polypeptides containing proline (Pro), hydroxyproline (Hyp), and glycine (Gly), the major amino acids that comprise collagen. Using the Boyden chamber and under agarose techniques, we found that collagen fragments were as potent in inducing chemotaxis in neutrophils as the bacterial-derived peptide f-met-leu-phe. The synthetic polytripeptides (Pro-Pro-Gly)5 and (Pro-Hyp-Gly)5 were found to be equipotent in inducing chemotaxis, producing a maximal induction of chemotaxis at 5-10 nM. This suggests that Hyp, the unique imino acid found in collagen, is not required for chemotactic activity. Increasing the length of the synthetic tripeptide from 5 to 10 subunits decreased its chemotactic activity, while the single tripeptide subunit (Pro-Hyp-Gly)1 was the least active peptide, inducing a maximal response at 100 nM. To study the structural requirements for chemotaxis, Pro-Hyp-Gly tripeptides were synthesized with modifications at the N and C terminals ends. Addition of a methyl group to the carboxyl of Gly to form an ester enhanced the chemotactic activity of the peptide by 50%, while substitutions on the amino terminus with an acetyl group decreased the chemotactic activity by 50%. Substitution on the amino terminus with a Boc group decreased the chemotactic activity by 100%. These results indicate that there are specific structural requirements for chemotaxis induced by peptides having a collagen-like sequence of amino acids.

Several structural analogues of proline have been shown to be incorporated into proteins in place of proline. As a consequence, the proliferation of cells in culture and the extracellular deposition of collagen in animal systems are reduced. In this study, the effects of two proline analogues, cis-4-hydroxy-L-proline and L-azetidine-2-carboxylic acid, on the growth parameters and procollagen production by cultured normal human skin fibroblasts were examined. The results indicated that incubation of the cells with the analogues reduced the rate of fibroblast proliferation and lowered the plating efficiency. Further experiments demonstrated that fibroblasts in the presence of L-azetidine-2-carboxylic acid synthesized procollagen polypeptides which were not in a triple-helical conformation, as judged by limited pepsin proteolysis. Also, a significantly increased fraction of the newly synthesized collagenous peptides was in a dialyzable form, suggesting increased degradation of the nonhelical chains. The rate of translation of collagenous polypeptides and the preprocollagen messenger RNA activity in the cells were not affected by the analogues. The proline analogues thus appear to inhibit the production of procollagen on the posttranslational level by preventing the polypeptides from folding into a stable triple-helical conformation. The nonhelical polypeptides are then readily susceptible to proteolysis leading to reduced deposition of extracellular collagen fibers. Similar experiments were also performed with fibroblasts cultured from patients with active progressive systemic sclerosis. Quantitatively and qualitatively comparable inhibition of procollagen production by L-azetidine-2-carboxylic acid was noted with scleroderma cells as with control fibroblast cultures. The results suggest, therefore, that proline analogues may, in the future, prove useful in limiting excessive collagen deposition in scleroderma and other forms of dermal fibrosis.

Cells of Clostridium sporogenes were deficient in glycine reductase activity when grown in a rich medium containing 40 mM each of exogenously added pyruvate and proline or hydroxyproline. These cells lacked the selenoprotein and at least one more protein of the glycine reductase system. Proline or hydroxyproline in the medium also influenced the uptake of glycine by the cells.

Exposure of rats to high oxygen tensions causes increased collagen content of lungs and alveolar enlargement in 3-6 wk. We tested whether cis-hydroxyproline, a proline analogue that inhibits collagen synthesis, could prevent the collagen accumulation and alveolar enlargement. Rats were exposed to hyperoxia for 60 h and then to room air and hyperoxia for alternate 24-h periods for 11.5 d. Treated oxygen-exposed rats received 200 mg/kg cis-hydroxyproline twice daily over the 14-d exposure period. Control rats breathed room air. Examination of lungs on day 14 showed collagen content of oxygen-exposed lungs to be 48% greater than control (P < 0.05). The collagen content of the treated oxygen-exposed lungs was -12% of control (NS). Total lung volume was 16% greater than control in oxygen-exposed rats (P < 0.05) and 8% greater than control in treated oxygen-exposed rats (NS). Morphometric studies showed alveolar size was greater than control in oxygen-exposed rats (188+/-11 [SE] vs. 143+/-6 mumul [P < 0.05]). Oxygen-exposed, treated rats had a mean alveolar volume of 150+/-7 mumul. Lung pressure-volume curves were significantly shifted to the left of control in the oxygen-exposed rats, whereas the curves of the oxygen-exposed, treated group were identical to control. These data suggest that cis-hydroxyproline prevented the accumulation of collagen in the lungs in pulmonary oxygen toxicity. In addition, there was apparent protection from airspace dilatation and decreased lung elasticity, suggesting that alveolar enlargement after oxygen toxicity is linked to the deposition in lung tissue of new connective tissue fibers.

Using the radiolabelled precursors (3H-Glucosamine, 3H-proline) and a new procedure of quantitative microautoradiography we investigated the influence of N-acetyl-L-hydroxyproline (Oxaceprol) on anabolic processes in joint cartilage tissue of hens. After in vitro incubation, Oxaceprol stimulated the uptake of 3H-glucosamine and 3H-proline in chondrocytes and enhanced the incorporation of 3H-proline into the macromolecular structures of the cartilage matrix. Also, after in vivo intra-articular injection of Oxaceprol into the knee joints we could demonstrate a significant increase of intracellular glucosamine uptake. Because in our micro-autoradiographic detection method low molecular substances are excluded, the higher rate of precursor incorporation into cells and matrix can be interpreted as a stimulation of proteoglycan and collagen synthesis.

For improvement of the culture conditions of adult rat hepatocytes in primary culture in collagen coated dishes, effects of various commercial culture media on the induction of replicative DNA synthesis of the cells stimulated by insulin plus epidermal growth factor were studied. Proline-deficient media, such as Leibovitz's L-15, Eagle's minimal essential medium and Dulbecco's modified minimal essential medium, did not induce DNA synthesis in hepatocytes, whereas proline-rich media, such as Williams medium E, McCoy's 5A and Ham's F-12, induced markedly hepatocyte proliferation. Moreover, when the proline-deficient media were supplemented with L-proline, the cells synthesized DNA in response to the two hormones. Cis-4-hydroxyl-L-proline strongly inhibited the induction of DNA synthesis, without affecting protein synthesis of the cells or showing any cytotoxicity. This inhibition was recovered completely by adding excess proline to the medium. Addition of other amino acids not present in the medium did not promote DNA synthesis. These findings indicate that L-proline is essential for induction of hepatocyte proliferation in culture, through its affect on synthesis of intracellular collagen.

The transport of glycine was investigated in histologically normal adult human kidney cortical slices. Uptake occurs against a gradient and shows concentration dependence. Kinetic analysis reveals two systems for transport of glycine with apparent transport Km values of 0.511 and 34.2 mM. Glycine transport on the high-Km system is competitively inhibited by 50 mML-proline. Transport inhibition on the low-Km system could not be directly evaluated, but on theoretic grounds appears not to be inhibited by L-proline or hydroxyproline. Alpha-aminoisobutyric acid, valine, and thioproline are also shown to inhibit glycine uptake. Low medium sodium or anaerobic incubation depress the uptake of glycine. These observations are consistent with previous reports of glycine transport in rat kidney and support the proposals for the mechanism of familial iminoglycinuria based on in vivo investigations.

Pharmacological suppression of the scar reaction was tested as a possible method for improving axonal regeneration after transection and suture of the sciatic nerve of rats. The animals in the experimental groups were systemically treated post-operatively with estrogen-progesterone, methylprednisolone-acetate (Depomedrone) or cis-hydroxyproline. Only in the cis-hydroxyproline treated animals was there a tendency towards a decrease in fibroblastic activity at the suture site after 4 weeks. However, there was no difference in the growth of axons to the distal nerve segment after pharmacological treatment as compared to non-treated animals, when tested 12 weeks post-operatively. The motor nerve conduction velocity was however significantly greater in both the cis-hydroxyproline and the methylprednisolone-acetate groups as compared with the estrogen-progesterone and the control groups. In conclusion, cis-hydroxyproline and also methylprednisolone-acetate may improve the function of regenerating peripheral nerve, while estrogen-progesterone does not seem to have this effect.

Hydroxyproline (OHP) fed to Drosophila larvae is known to impair the ability of pupae to evert cephalic structures and leg and wing discs. In order to gain insight into the metabolic basis of OHP action we have investigated the effects of combined feeding of OHP and several compounds. Proline, glucosamine HCl, and N-acetyl glucosamine considerably reduced the extent of morphological defects of OHP, while alanine and sodium glutamate were less effective. Glutamine and glutathione did not reduce the effects of OHP. The results are consistent with the hypothesis that OHP may indirectly interfere with cuticle synthesis.

Mouse mammary epithelial cultivated on collagen gels demonstrate active spreading as the cells form monolayers. In this novel system, initiation of cell spreading is preceded by de novo synthesis of type IV collagen. The newly synthesized collagen is partitioned such that after 48 hr, approximately 24% is found in the culture medium, 35% is intracellular, and 41% is deposited in the extracellular matrix of the developing epithelium. Cultures deprived of serum failed to spread and to synthesize collagen. Proline analogues were shown to inhibit cell spreading and to suppress collagen synthesis in a dose-dependent manner. Cytochalasin D inhibition of F-actin elongation was shown to prevent cell spreading but not to suppress total collagen synthesis. During cytochalasin D treatment, inhibition of cell spreading was shown to result from failure to deposit or to maintain deposited collagen in the epithelium extracellular matrix. The data indicate that synthesis and extracellular deposition of a major basal lamina component (viz. type IV collagen) must precede and then accompany epithelial cell spreading in collagen gel culture. It is suggested that the microfilament apparatus, through some hypothetical integral membrane protein, can anchor extracellular type IV collagen, which then provides a necessary condition for cell spreading.

It has been observed that the feces as well as urine of rats fed diets supplemented with 3% glycine and 5.2% hydroxyproline contain unexpectedly high amounts of endogenously formed oxalate. That intestinal microorganisms do not synthesize significant amounts of oxalate was indicated by the findings that oral tetracycline had no effect on oxalate excretion and that germ-free rats excreted more oxalate than conventional rats. Since little intraperitoneally injected [14C] oxalate appeared in the feces, and rat intestinal mucosa homogenates were found to produce oxalate from a variety of precursors of which glyoxylic acid was far the most important, it is probable that the intestinal mucosa may be an important source of fecal oxalate observed in these studies. Ninety percent of weanling rats fed complete diets supplemented with glycine and hydroxyproline developed urinary stones in 38 days. It has been concluded that in the treatment of patients with histories of calcium oxalate urolithiasis, more concern than is commonly shown should be directed towards the feeding of diets high in precursors of endogenous oxalate synthesis.

We used a sensitive hemolytic assay to measure the level of C1q, a subcomponent of the first complement protein in the classic pathway, in bronchoalveolar lavage fluid and serum of rats exposed to air and hyperoxia. The serum level was 125 +/- 5 micrograms/ml and the lavage level was 41 +/- 17 ng/ml in rats breathing air. In rats exposed to acute oxygen toxicity (95% O2 for 66 hr), the serum level was at 107 +/- 10 micrograms/ml, but the level in lavage fluid increased to 2457 +/- 400 ng/ml (p less than 0.05 compared to air). Administration of the proline analogue cis-4-hydroxy-L-proline to air- and O2-exposed rats reduced the serum C1q level by 28% and 34%, respectively (both p less than 0.05), presumably by interfering with metabolism of the collagen-like sequence of C1q. The level of C1q in bronchoalveolar lavage fluid is a sensitive marker of acute lung injury.

The role of collagen in pulmonary epithelial cell division and differentiation during lung development was investigated by injecting the proline analogue cis-hydroxy-L-proline (cis-HYP) to timed pregnant rats. Lung weight, DNA and 3H thymidine incorporation were unchanged when compared to controls; epithelial cell division, determined by autoradiography, was also unchanged. However air sac development and phospholipid synthesis by epithelial cells were retarded in cis-HYP treated animals. The percentage of epithelial cells containing lamellar bodies was reduced at days 20-22 of gestation and the level of disaturated phosphatidylcholine (DSPC) was also significantly lower in treated rats. A sex-related difference was not eliminated by cis-HYP treatment, lung maturation was still more rapid in females. Non-dialysable hydroxyproline levels were decreased, and morphologically, less fibrillar collagen was seen at the epithelial-interstitial cell interface, though direct cell-cell contacts were not reduced. The results indicate that the injection of cis-HYP did not alter lung growth, but caused a reduction in fibrillar collagen which was associated with reduced surfactant synthesis by Type II epithelial cells.