Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.     

Fetal calf ligament fibroblasts in culture secrete a low molecular weight collagen with a unique resistance to proteolytic degradation

A highly unusual collagen was secreted by fibroblasts cultured from 150- and 270-d-old fetal calf nuchal ligaments. Purification revealed that this protein (which may be synthesized in a higher molecular weight form) was precipitated at unusually high concentrations of ammonium sulfate and was also eluted from DEAE-cellulose at greater salt concentrations than were types I and III procollagens. On SDS PAGE, the collagenous protein exhibited an Mr of approximately 12,750 that was not altered in the presence of reducing agent. The low molecular weight collagen (FCL-1) was sensitive to bacterial collagenase and had a [3H]glycine content comparable to that found in type I procollagen, although the [3H]Hyp to [3H]Pro ratio was 0.43. FCL-1 was not cleaved by human skin collagenase, mast cell protease, trypsin, Staphylococcal V8 protease, or proteinase K at 37 degrees C. The collagen was susceptible to trypsin, but not to V8 protease, only after heating at 80 degrees C for 30 min. Preliminary structural studies indicate that FCL-1 was resistant to cleavage by CNBr but exhibited limited proteolysis with pepsin. Both 150- and 270-d-old fibroblasts produced comparable levels of interstitial (types I and III) procollagens, which comprised approximately 70% of the total protein secreted into the culture medium. However, 270-d-old (term) fibroblasts secreted approximately 50% more FCL-1, as percent of total culture medium protein, in comparison to the cells from the earlier gestational stage. This collagen may therefore play a role in the development of the nuchal ligament.     

Terminal differentiation of nuchal ligament fibroblasts: characterization of synthetic properties and responsiveness to external stimuli

The temporal expression of elastogenesis is unique among connective tissues in that elastin production occurs primarily during late fetal and early neonatal periods and is essentially fully repressed once fiber assembly is completed. To test whether elastin synthesis in adult nuchal ligament fibroblasts is permanently repressed or whether the cells retain the ability to reinitiate production upon proper stimulation, we examined in adult ligament cells various parameters known to be involved in the regulation of elastin production. Elastin synthetic capacity, as determined by the levels of steady-state tropoelastin mRNA, of adult tissue was significantly decreased relative to fetal tissue. Likewise, fibroblasts grown from explants of adult ligament had about a fourfold decrease in elastin production and elastin-specific mRNA levels. On the other hand, adult cells were similar to fetal ligament cells in that they were sensitive to glucocorticoid stimulation and demonstrated chemotactic responsiveness to elastin peptides. Since our previous studies have shown that the extracellular matrix (ECM) plays an important role in influencing elastin phenotypic expression, fetal and adult fibroblasts were grown on slices of nonviable adult ligament to test if repression of elastin production was directed by factors in ECM of adult tissues. No change in elastin synthesis was detected with either cell type grown on adult ligament, whereas both fetal and adult cells demonstrated increased elastin production in response to contact with fetal ligament. These results suggest that adult ligament ECM does not provide a metabolic signal to shut off the elastin gene and that adult cells remain responsive to external stimuli that may reinitiate high levels of elastin synthesis.     

Subcellular Distribution of Prolyl Endopeptidase and Cation-Sensitive Neutral Endopeptidase in Rabbit Brain

Abstract: The subcellular distribution of prolyl endopeptidase, and of cationsensitive neutral endopeptidase, two enzymes actively metabolizing many neuropeptides, was determined in homogenates of rabbit brain. The subcellular distribution of both enzymes was more similar to lactate dehydrogenase, a cytoplasmic enzyme marker, than to choline acetyltransferase, a synaptosomal marker. Only 35% of the activity of these two neutral endopeptidases was found in the crude mitochondrial fraction (P₂), the bulk of the remaining activity being associated with the high-speed supernatant. Prolyl endopeptidase and cation-sensitive neutral endopeptidase thus can be regarded as mainly cytoplasmic enzymes in the rabbit brain.     

东方田鼠胚胎成纤维细胞的分离培养及生物学特性

目的对影响东方田鼠胚胎成纤维细胞（MfEF）分离和培养的因素进行探索,并观察其生物学特性。方法取室内繁殖饲养不同胎龄的东方田鼠胚胎分离成纤维细胞,通过原代和继代培养,分析比较不同胎龄、不同血清浓度、不同胰蛋白酶浓度等因素对MfEF分离及培养的影响,观察MfEF的生长形态及其生物学特性。结果 MfEF为贴壁型生长,细胞形态多样,呈梭形、不规则多边形;采用0.125%的胰蛋白酶室温消化12～13 d胚胎组织5 min,以DMEM培养基添加15%小牛血清分离培养MfEF的效果最佳;MfEF2～7代增殖最旺盛。结论获得了实验室分离、培养MfEF的有效方法 ,为进一步深入研究东方田鼠抗日本血吸虫机制以及开展不同动物成纤维细胞间比较研究奠定了基础。     

Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster

Members of the neprilysin family of neutral endopeptidases (M13) are typically membrane-bound enzymes known to be involved in the extra-cellular metabolism of signalling peptides and have important roles during mammalian embryogenesis. In this study we show that membranes prepared from embryos of Drosophila melanogaster possess neprilysin-like activity that is inhibited by phosphoramidon and thiorphan, both inhibitors of mammalian neprilysin. Unexpectedly, we also found strong neprilysin-like neutral endopeptidase activity in a soluble embryo fraction, which we identify as NEP2 by Western blot and immunoprecipitation experiments using NEP2 specific antibodies. NEP2 is a soluble secreted member of the neprilysin family that has been shown previously to be expressed in larval and adult Malpighian tubules and in the testes of adult males. In situ hybridization studies reveal expression at stage 10-11 in a pattern similar to that previously described for stellate cell progenitors of the caudal visceral mesoderm. In later stages of embryogenesis, some of these cells appear to migrate into the growing Malpighian tubule. Recombinant NEP2 protein is N-glycosylated and displays optimum endopeptidase activity at neutral pH, consistent with a role as an extracellular peptidase. The recombinant enzyme hydrolyses Drosophila tachykinin peptides (DTK) at peptide bonds N-terminal to hydrophobic residues. DTK2, like Locusta tachykinin-1, was cleaved at the penultimate peptide bond (Gly(7)-Leu(8)), whereas the other Drosophila peptides were cleaved centrally at Xxx-Phe bonds. However, the rates of hydrolysis of the latter substrates were much slower than the hydrolysis rates of DTK2 and Locusta tachykinin-1, suggesting that the interaction of the bulky side-chain of phenylalanine at the S'(1) sub-site is less favorable for peptide bond hydrolysis. The secretion of NEP2 from tissues during embryogenesis suggests a possible developmental role for this endopeptidase in peptide signalling in D. melanogaster.     

Relationship between contents of adrenomedullin and distributions of neutral endopeptidase in blood and tissues of rats in septic shock

Adrenomedullin (ADM), a multifunction peptide with important roles in regulating cardiovascular homeostasis, has the vasodilatory properties and is of particular interest in the pathophysiology of sepsis. ADM levels in plasma and tissues are regulated by the proteolysis of neutral endopeptidase (NEP), the major enzyme of ADM degradation. We observed the NEP activity in the plasma, the activity and distribution of NEP and its mRNA expression in the tissues of rats in septic shock to study the possible role of NEP in elevating tissue ADM concentration during sepsis. ADM level increases progressively during sepsis except in the jejunum. Rats in early phase of shock (ES) showed diverse changes in tissue NEP activity. Plasma NEP activity, tissue NEP activity and its protein and mRNA expression in the left ventricle, aorta, jejunum and lung in the late phase of shock (LS) rats were lower than those in ES and the control, but no statistical change of NEP activity in the kidney was observed. The level of ADM was inversely correlated with NEP activity in the plasma, ventricle and aorta and positively correlated with NEP activity in the jejunum. Thus, in sepsis, the local concentration and action of ADM in tissues may be differentially regulated by NEP.     

Neprilysin Is Identical to Skin Fibroblast Elastase: ITS ROLE IN SKIN AGING AND UV RESPONSES

Although human skin fibroblast (HSF) elastase has been characterized as a membrane-bound metalloproteinase, little is known about its structure, amino acid sequence, and encoding gene. As there are similarities in the molecular weights and inhibitory profiles of HSF elastase and neprilysin (neutral endopeptidase 24.11 (NEP)), in this study we tested the hypothesis that they are identical using immunoprecipitation and transfection methods. An immunoprecipitation study demonstrated that HSF elastase activity co-immunoprecipitated with anti-NEP in lysates of cultured HSF. Transfection of an NEP cDNA expression vector into COS-1 cells elicited the expression of HSF elastase and NEP activities in the transfected cells. These findings strongly suggest that HSF elastase is identical to NEP, which functions mainly in neuron-associated cells to degrade neuropeptides. Analysis of the expression pattern of NEP revealed that its expression was remarkably up-regulated at the gene, protein, and enzymatic activity levels during the replicative senescence of cultured HSF. Further, the activity of NEP was markedly enhanced in a pattern similar to elastase activity during the intrinsic aging of mouse skin, in UVA-exposed HSF as well as in HSF treated with conditioned medium from UVB-exposed human keratinocytes. Analysis of the cytokine profile for the stimulation of NEP and HSF elastase activities in HSF demonstrated that among the 11 cytokines tested, IL-1α, IL-1β, IL-6, IL-8, and GM-CSF had the potential to significantly stimulate both activities similarly, again supporting the identity of HSF elastase and NEP.     

Nutritional control of respiratory and other muscular activities in relation to plasma prostaglandin E in the fetal sheep

The effects of nutrient availability on fetal plasma prostaglandin E (PGE) concentrations, on fetal breathing movements and electromyographic (EMG) activities of fetal nuchal and forelimb muscles were investigated in pregnant ewes by varying dietary intake and by manipulation of fetal plasma glucose concentration. The incidence of fetal breathing movements (06.00-10.00 h) decreased with increasing gestational age while fetal arterial concentrations of plasma PGE increased significantly over the same period of gestation. Maternal fasting for 48 h reduced the incidence of fetal breathing movements and the amount of nuchal EMG activity (06.00-10.00 h) in animals older than 130 days but had no effect earlier in gestation. No changes in forelimb EMG activity were observed during fasting at any gestational age. Plasma PGE levels increased significantly during fasts begun both before and after 130 days of gestation. When data from fed and fasted states were combined for all fetuses, irrespective of gestational age, there was a significant inverse correlation between fetal breathing movements incidence and plasma PGE concentration in utero. This relationship was even more pronounced when the fetuses were considered individually. Insulin infusions induced hypoglycaemia, an increase in fetal plasma PGE concentration and a significant reduction in the incidence of fetal breathing movements at all ages. Glucose infusions of fetal breathing movements only after 130 days and had no effect on plasma PGE levels in utero at any gestational age. Neither insulin nor glucose infusions altered the EMG activities of the nuchal and forelimb muscles. The results show that glucose availability is an important factor in determining the incidence of fetal breathing movements in utero and indicate that nutritionally induced changes in fetal breathing movements are mediated in part by PGE. They also suggest that PGE is a physiological regulator of fetal breathing movements in the sheep during late gestation.     

Discoidin domain receptors and their ligand, collagen, are temporally regulated in fetal rat fibroblasts in vitro

The biochemical regulation of collagen deposition during adult cutaneous wound repair is poorly understood. Likewise, how collagen is perceived and modulated in fetal scarless healing remains unknown. Recently, discoidin domain receptors-1 and 2 (DDR1 and DDR2) with tyrosine kinase activity have been identified as novel receptors for collagen. In light of these findings, it was speculated that the production of collagen receptors DDR1 and DDR2 by fetal fibroblasts may be temporally regulated to correlate with the ontogeny of embryonic scar formation. More specifically, because DDRs directly bind collagen and transmit the signals intracellularly, it was hypothesized that they may play an important role in fetal scarless healing by ultimately regulating and modulating collagen production and organization. As part of a fundamental assessment to elucidate the role of DDRs in scarless fetal wound repair, the endogenous expression of DDR1, DDR2, collagen I, and total collagen, as a function of fetal Sprague-Dawley rat skin fibroblasts of different gestational ages, representing scar-free (E16.5) periods was determined. Using explanted dermal fibroblasts of gestational days E13.5, E16.5, E18.5, and E21.5 (term gestation = 21.5 days) fetuses (n = 92), [3H]proline incorporation assay and Northern and Western blotting analysis were performed to compare the expressions of these molecules with scar-free and scar-forming stages of embryonic development. These results revealed a pattern of increasing collagen production with increasing gestational ages, whereas DDR1 expression decreased with increasing gestational age. This observation suggests that elevated levels of DDR1 may play an important role in scarless tissue regeneration by early gestation fetal fibroblasts. In contrast, DDR2 was expressed by fetal rat fibroblasts at a similar level throughout gestation. These data demonstrate for the first time the temporal expression of collagen and DDR tyrosine kinases in fetal rat fibroblasts as a function of gestational ages. Overall, these data suggest that differential temporal expression of the above-mentioned molecules during fetal skin development may play an important role in the ontogeny of scar formation. Future studies will involve the characterization of the biomolecular functions of these receptor kinases during fetal wound repair.     

The effect of cortisol on rabbit fetal lung maturation in vitro

Explants of lung tissue from 19-day gestational age fetal rabbits were maintained in organ culture in medium with or without fetal calf serum for 1 to 11 days. Based on the results of biochemical and morphological studies it was apparent that the type II pneumonocyte differentiated in vitro at a time similar to that which occurs with maturation in vivo. The epithelial cells of the presumptive alveoli were undifferentiated at the start of incubation, but within 9 days developed increased amounts of Golgi apparatus and rough endoplasmic reticulum, many microvilli on the luminal surface and numerous lamellar bodies. Secreted lamellar bodies and tubular myelin figures were observed in the lumina of cultured explants. The incorporation of [³H]choline into phosphatidylcholine by lung tissue explants maintained in medium containing 10% fetal calf serum remained relatively constant for 7 days of incubation but thereafter increased two-fold. When explants were maintained in fetal calf serum-containing medium and cortisol (10^?7M) or betamethasone (10^?7M), the incorporation of choline into phosphatidylcholine was two to three times greater than that of explants maintained in serum-containing medium without cortisol. When explants of fetal lung tissue were incubated in the presence of cortisol without fetal calf serum there was no stimulatory effect of cortisol on phosphatidylcholine biosynthesis. Therefore, serum cofactors are necessary for the stimulatory effects of cortisol on fetal lung development. The specific activity of phosphatidate phosphohydrolase (PAPase) increased to very high levels during the culture period. In the presence of serum, cortisol or betamethasone had no effect on the specific activity of phosphatidate phosphohydrolase.     

Direct inhibition of neutral endopeptidase in vasopeptidase inhibitor-mediated amelioration of cardiac remodeling in rats with chronic heart failure

Vasopeptidase inhibitors possess dual inhibitory actions on neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) and have beneficial effects on cardiac remodeling. However, the contribution of NEP inhibition to their effects is not yet fully understood. To address the role of cardiac NEP inhibition in the anti-remodeling effects of a vasopeptidase inhibitor, we examined the effects of omapatrilat on the development of cardiac remodeling in rats with left coronary artery ligation (CAL) and those on collagen synthesis in cultured fibroblast cells. In vivo treatment with omapatrilat (30 mg/kg/day for 5 weeks) inhibited cardiac NEP activity in rats with CAL, which was associated with a suppression of both cardiac hypertrophy and collagen deposition. In cultured cardiac fibroblasts, omapatrilat (10^–7~10^–5 M) inhibited NEP activity and augmented the ANP-induced decrease in [³H]-proline incorporation. ONO-BB, an active metabolite of the NEP selective inhibitor ONO-9902, also augmented the ANP-induced response, whereas captopril, an ACE inhibitor, did not. The angiotensin I-induced increase in [³H]-proline incorporation was prevented by omapatrilat and captopril, but not by ONO-BB. The results suggest that vasopeptidase inhibitor suppressed cardiac remodeling in the setting of chronic heart failure, possibly acting through the direct inhibition of cardiac NEP. Vasopeptidase inhibitors may have therapeutic advantages over the classical ACE and NEP inhibitors alone with respect to the regression of cardiac fibrosis.     

Tracheal smooth muscle responses to substance P and neurokinin A in the piglet.

The tachykinins substance P (SP) and neurokinin A (NKA) have been shown to induce airway smooth muscle contraction in mature animals, and the enzyme neutral endopeptidase (NEP) modulates this effect. We evaluated maturation of SP- and NKA-induced tracheal smooth muscle contraction and modulation of their effects by NEP in anesthetized, paralyzed, and artificially ventilated piglets less than 4 days, 2-3 wk, and 10 wk of age. Tracheal smooth muscle tension was measured in vivo from an open tracheal segment by use of a force transducer. Intravenous SP caused a dose-dependent increase in tracheal tension in all three age groups; however, the response in less than 4-day-old piglets was significantly weaker than in 2- to 3- and 10-wk-old piglets. NKA caused a dose-dependent increase in tracheal tension only in 2- to 3- and 10-wk-old piglets. The response of tracheal tension to NKA was weaker than the response to SP in all age groups. Atropine (2 mg/kg) significantly diminished the responses of tracheal tension to SP and NKA, indicating a cholinergic contribution to these responses at all ages. Intravenous thiorphan, a known NEP inhibitor, potentiated the effects of SP only in 2- to 3- and 10-wk-old piglets and did not affect the response of tracheal tension to NKA at any age. Biochemical analyses demonstrated a significant increase in tracheal NEP activity in comparably aged piglets over the first 10 wk of life.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low molecular weight fibroblast collagen: structure, secretion, and differential expression as a function of fetal and cellular age

A unique low molecular weight collagen that was highly resistant to proteolytic degradation was originally isolated from fetal calf ligamentum nuchae fibroblasts and hence termed FCL-1 [Sage, H., Mecham, R., Johnson, C., & Bornstein, P. (1983) J. Cell Biol. 97, 1933-1938]. The differential expression of this protein was studied as a function both of fetal (donor) age and of subcultivation in vitro. Concomitant isolation, subculture, and metabolic radiolabeling experiments performed on cell strains from fetal calf ligament (FCL) and fetal bovine skin (FBS) representing different gestational ages (85-270 days in utero) showed that (a) FCL-1 was synthesized preferentially by fibroblasts from younger animals and (b) expression of FCL-1 diminished as a function of increased passage in culture. Levels of FCL-1, measured as percent of total radiolabeled culture medium protein that precipitated in a concentration range of 20-50% ammonium sulfate, ranged from 22% in FCL 85 cells to 7.7% in FCL 270 (term) cells. FBS fibroblasts at passages 6-10 secreted from 13% to 6% FCL-1, respectively. When cells from an 85-day fetal ligament were allowed to accumulate copious extracellular matrix in vitro, the production of FCL-1 was increased to 32%. FCL-1 was not immunoreactive with polyclonal antibodies directed toward most of the sequences of the interstitial type I and type III procollagens. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular weight of FCL-1 was 13 000 (on the basis of collagen peptide standards) and approximately 30 000 (on the basis of globular protein standards). Incubation with bacterial collagenase produced a stable cleavage product of Mr 8000 (by collagen standards) or 17 000 (by globular standards). In contrast, pepsin removed a small peptide of approximately 1000-2000 in molecular weight from FCL-1, and a gradual but progressive proteolysis of the collagen was observed over a period of 1-6 h. Pulse-chase studies revealed a secretion time of approximately 60 min for FCL-1, without the appearance of any processed, intermediate forms. These studies confirm that FCL-1 represents a novel member of the collagen gene family that manifests differential expression as a function of development.     

The effects of monensin on membrane lipids of cultured human skin fibroblasts

We have investigated the effects of monensin, a monovalent cationophore, on the metabolism of neutral lipids, fatty acids, ceramide and phospholipids in cultured human skin fibroblasts. Treatment with 1 microM monensin for 18 h reduced the cellular cholesterol ester content to less than one-third of untreated cells, and incorporation of [3H]acetate into cholesterol ester was also reduced, to less than one-fifth. Concomitantly, a greater conversion of [3H]acetate into free cholesterol occurred. There was a moderate increase in free fatty acids, but no change in triacylglycerol content, although the content of the latter appeared to increase in the presence of fetal calf serum in the culture medium. Phosphatidylcholine decreased in content and phosphatidylserine increased among the phosphatides, but ceramide remained unchanged after monensin treatment. These findings suggest that monensin influences the metabolic interrelationships of structural lipids in fibroblasts.     

Neutral endopeptidase modulates endothelin-1-induced airway smooth muscle contraction in guinea-pig trachea.

This study was designed to evaluate the role of neutral endopeptidase (NEP) in modulating the airway smooth muscle contraction induced by endothelin-1 in isolated segments of guinea-pig trachea. Endothelin-1 (10(-9)-10(-6) M) produced a concentration-dependent contraction that reached a maximum by 30 min. The NEP inhibitor leucine-thiorphan (10(-5) M) significantly increased the contractile response to endothelin-1. The addition of leucine-thiorphan to tracheal segments precontracted by 10(-9) and 10(-8) M endothelin-1 increased isometric tension by 181 +/- 65% (mean +/- 1 S.E.M.; P less than 0.05) and by 138 +/- 49% (P less than 0.05), respectively. In contrast, the kininase II inhibitor captopril and the peptidase inhibitors leupeptin and bestatin had no effect. Preincubation of endothelin-1 with 1 microgram recombinant human NEP decreased the contractile activity of endothelin-1 by 72 +/- 9%, whereas no effect was observed using heat-inactivated NEP. We conclude that NEP modulates endothelin-induced contraction of airway smooth muscle in the guinea-pig trachea.     

Effect of neutral endopeptidase inhibition of f-Met-Leu-Phe-induced bronchoconstriction in the rabbit

Inhalation of f-Met-Leu-Phe (FMLP) produces dose-dependent increases in pulmonary resistance (RL) in rabbits. We hypothesized that inhibition of neutral endopeptidase (NEP), which has high affinity for FMLP, would augment the response to FMLP inhalation. We found the increase in RL above baseline in response to FMLP to be reduced from 56 +/- 18 to 8 +/- 10% (P less than 0.01) by phosphoramidon (1 mg/kg) and to 15 +/- 6% (P less than 0.02) by thiorphan (3 mg/kg). The geometric mean dose of FMLP producing a 20% rise in RL (PC20RL FMLP) was increased by phosphoramidon from 1.1 to 4.5 mg/ml (P less than 0.05). Enkephalins, which are also NEP substrates, modulate cholinergic neurotransmission in the airway. Inhibition of the FMLP response by phosphoramidon was reversed by coadministration of naloxone (0.1 mg/kg); after atropine (2 mg/kg) the change in RL in response to FMLP was reduced to 7 +/- 4% (P less than 0.01), whereas morphine (0.15 mg/kg) increased PC20RL FMLP to 5.1 mg/ml (P less than 0.05). FMLP-induced bronchoconstriction in the rabbit is vagally mediated, and reduced responses after NEP inhibition may reflect modulation of cholinergic bronchoconstriction by enkephalins. Changes in airway NEP activity may influence the activity of a wide range of its substrates, of which some are bronchoconstrictors and others bronchodilators.     

Synthesis and uptake of gangliosides by choleragen-responsive human fibroblasts.

Human fibroblasts, cultured in medium containing 10% fetal calf serum, responded dramatically to choleragen with an increase in cyclic adenosine monophosphate content to greater than 48 times basal levels. Analysis of these cells for gangliosides indicated that the major ganglioside was N-acetylneuraminylgalactosylglucosylceramide (GM3) with trace amounts (less than or equal to 100 pmol/mg of protein) of other gangliosides including GM1, the putative choleragen receptor. Although the cells contained three glycosyltransferases required for ganglioside synthesis, the N-acetylgalactosaminyltransferase activity necessary for the conversion of GM3 to more complex gangliosides was not detected. When the cells were grown in medium containing [14C]galactose or N-acety[3H]mannosamine, however, all of the gangliosides became labeled, indicating that the cells can synthesize complex gangliosides. Although fetal calf serum contains gangliosides including GM1, [3H]GM1 was taken up poorly from the growth medium and uptake at the rate observed could have accounted for less than 2% of the GM1 content of the cells. When the cells were incubated in chemically defined medium containing [3H]GM1 at the concentrations present in fetal calf serum, rapid uptake of the ganglioside occurred and the total GM1 content of the cells increased threefold in less than 3 h. Thus, although the cells are capable of binding exogenous gangliosides, the gangliosides in fetal calf serum are in a form not readily available to the cells.