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Background: Glucosamine (GlcN) used by patients with osteoarthritis was demonstrated to reduce pain, but the working mechanism is still not clear. Viscosupplementation with hyaluronic acid (HA) is also described to reduce pain in osteoarthritis. The synthesis of HA requires GlcN as one of its main building blocks. We therefore hypothesized that addition of GlcN might increase HA production by synovium tissue. Methods: Human osteoarthritic synovium explants were obtained at total knee surgery and pre-cultured for 1 day. The experimental conditions consisted of a 2 days continuation of the culture with addition of N-Acetyl-glucosamine (GlcN-Ac; 5 mM), glucosamine-hydrochloride (GlcN-HCl; 0.5 and 5 mM), glucose (Gluc; 0.5 and 5 mM). Hereafter HA production was measured in culture medium supernatant using an enzyme-linked binding protein assay. Real time RT-PCR was performed for hyaluronic acid synthase (HAS) 1, 2 and 3 on RNA isolated from the explants. Results: 0.5 mM and 5 mM GlcN-HCl significantly increased HA production compared to control (approximately 2 - 4-fold), whereas GlcN-Ac had no significant effect. Addition of 5 mM Gluc also increased HA production (approximately 2-fold), but 0.5 mM Gluc did not. Gene expression of the HA forming enzymes HAS 1, 2 and 3 was not altered by the addition of GlcN or Gluc. Conclusion: Our data suggest that exogenous GlcN can increase HA production by synovium tissue and is more effective at lower concentrations than Gluc. This might indicate that GlcN exerts its potential analgesic properties through stimulation of synovial HA production. [ABSTRACT FROM AUTHOR]

LLC-PK1 cells were cultured on a permeable support in a two-compartment culture system. Confluent monolayers received an ultrafiltrate-like solution at the apical side and a plasma-like solution at the basolateral side. The distribution of various solutes, including phosphate, calcium, and oxalate over both compartments was measured in time. The transport of water was monitored by alterations in fluid concentrations of radiolabeled inulin. Bicarbonate, glucose, and phosphate were transported rapidly from the apical to basolateral side of the monolayer. Sodium and chloride were reabsorbed without major consequences for the osmolality in the apical and basal fluid. Calcium and potassium were also reabsorbed, but to a smaller extent than sodium. The luminal concentration of oxalate gradually increased to values that were at least three times higher (12.0 ± 0.4 μmol/l) than those in the contraluminal fluid (3.8 ± 0.1 μmol/l). However, since the luminal rise of oxalate completely matched the rise of inulin in the apical fluid this appeared to be the passive consequence of active water reabsorption rather than of net directed oxalate transport. The LLC-PK1 model could prove useful to study the regulation of proximal tubule water transport and its effect on luminal stone salt concentrations under different physiological conditions. [ABSTRACT FROM AUTHOR]

The pharmacokinetics of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of polyamine biosynthesis, were investigated in BALB/c (nude) mice after i.p. injection and after oral administration of radiolabeled drug. After i.p. injection the compound was rapidly cleared from the serum (t1/2 alpha = 14 min; t1/2 beta = 2.1 h) and from tissues such as muscle, liver and kidney (t1/2 alpha = 30-60 min; t1/2 beta = 2.1 h). DFMO concentrations were proportional to the administered dose (10-2000 mg/kg) in both serum and tissues. Oral administration of DFMO was carried out by dissolving the compound in drinking water at a concentration of 20 g/l. Studies on the distribution showed that DFMO did not accumulate preferentially in any particular tissue. An extremely wide variation in the dose actually achieved in different animals was observed; this ranged from 350 to 2800 mg/kg for a 14-h treatment period. A significant correlation (r = 0.83-0.92) between the dose of DFMO, calculated from the consumption of drinking water for each individual animal, and the DFMO concentrations in serum, muscle, spleen, liver and kidney was found. Similarly, it was shown that oral administration of DFMO during the daytime resulted in 10- to 15-fold lower levels than administration during the night. After discontinuation of treatment DFMO levels in serum and tissues decreased by 50% in approximately 6 h. From these results it is concluded that the optimal treatment schedule of mice with DFMO (or other drugs with similar pharmacodynamic properties) consists in a combination of oral administration via the drinking water and additional i.p. injection (during the daytime). Furthermore, the drug intake of the individual animals should be monitored to check whether the experimental requirements are actually fulfilled. [ABSTRACT FROM AUTHOR]

Taylor and Curhan report that consumption of fructose is independently associated with an increased risk of incident kidney stones. What could be the mechanisms underlying the relation between fructose intake and stone risk? And how should we incorporate this finding into the dietary advice that we give to our patients to prevent kidney stone formation?Kidney International (2008) 73, 139–140; doi:10.1038/sj.ki.5002700 [ABSTRACT FROM AUTHOR]

In the past two decades an increasing number of nephrolithiasis-related urinary proteins have been identified. This paper focuses on two of them, namely prothrombin fragment 1 and bikunin, members of the prothrombin and inter-alpha-trypsin inhibitor families of proteins, respectively. Besides their role as inhibitors of crystallization, these proteins are also involved in inflammation-mediated tissue repair. This is the basis for the concept that the response of renal tissue to injury might play an important role in the aetiology of kidney stones.

Background: The adherence of calcium oxalate crystals to the renal tubule epithelium is considered a critical event in the pathophysiology of calcium nephrolithiasis. Calcium oxalate monohydrate (COM) crystals cannot adhere to the surface of a functional Madin-Darby canine kidney (MDCK) monolayer, but they bind avidly to the surface of proliferating and migrating cells., Methods: To identify crystal-binding molecules (CBMs) at the surface of crystal-attracting cells, we applied metabolic labeling protocols in combination with differential enzymatic digestion and gel filtration, which was compared with [14C]COM crystal binding and confirmed by confocal microscopy., Results: The indication that hyaluronan [hyaluronic acid (HA)] might act as a CBM in subconfluent cultures came from studies with glycosaminoglycan (GAG)-degrading enzymes. Subsequently, metabolic-labeling studies revealed that hyaluronidase cleaved significantly more radiolabeled glycoconjugates from crystal-attracting cells than from cells without affinity for crystals. During wound repair, crystal binding could be prevented by pretreating the healing cultures with hyaluronate lyase, an enzyme that specifically hydrolyzes HA. Binding to immobilized HA provided evidence that COM crystals physically can become associated with this polysaccharide. Finally, confocal microscopy demonstrated that fluorescently labeled HA binding protein (HABP) adhered to the surface of proliferating cells in subconfluent cultures as well as to cells involved in closing a wound, but not to cells in confluent monolayers., Conclusions: These results identify HA as binding molecule for COM crystals at the surface of migrating and proliferating MDCK cells.

Background: We studied the role of cell surface sialic acid in the adherence of calcium oxalate monohydrate (COM) crystals to Madin-Darby canine kidney (MDCK) cells., Methods: Studies were performed with undifferentiated (crystal-binding) cells in subconfluent cultures and maturated (noncrystal-binding) cells in confluent cultures. Lectins were used to study the emergence and abundance of oligosaccharides at the cell surface during epithelial development. The effect of neuraminidase treatment on crystal binding was studied with [14C]COM crystals, and the enzyme-induced release of cell surface-associated sialic acid molecules was monitored by labeling the cells metabolically with [3H]glucosamine., Results: Binding studies with lectins derived from Maackia Amurensis II (MALII) and Sambucus Nigra (SNA) demonstrated that the cells expressed terminal sialic acids attached to penultimate galactose through alpha 2,3 and alpha 2,6 bonds at different stages of epithelial development. Neuraminidase treatment strongly reduced the affinity of the cell surface for COM crystals in subconfluent cultures. Nevertheless, neuraminidase cleaved more sialic acids from cells in confluent cultures than from those in subconfluent cultures. Peanut agglutinin (PNA), which binds only to sialylated terminal galactose units, adhered to developing but not to maturated cells, unless the latter were pretreated with neuraminidase. Both results indicate that the surface of maturated MDCK cells is more heavily sialylated than that of undifferentiated cells. Free sialic acid molecules showed little or no affinity for COM crystals and did not affect the adherence of the crystals to undifferentiated cells., Conclusions: There are at least two models that may explain these results. First, sialic acids are presented at the surface of immature cells in an orientation that specifically matches crystal surface characteristics favoring crystal-cell interactions. Second, sialic acid molecules are not directly associated with the crystals, but may be involved in the exposure of another crystal binding molecule at the cell surface.

The adherence of crystals to the surface of renal tubule epithelial cells is one of the initial events in the development of nephrolithiasis. The accumulation of crystalline material in the kidney will sooner or later result in the formation of a stone. Calcium crystals occasionally are present in the urine of even healthy individuals, and mechanisms responsible for the selective attachment of crystals to the tubular epithelium of stone-forming individuals must exist. Although several types of cell surface molecules, including phosphatidylserine (PS) and sialic acid, have been proposed as receptors for crystals in the tubular system, the exact nature of these crystal-binding sites has not yet been revealed. Previously, it was demonstrated that calcium oxalate monohydrate crystals adhere to subconfluent, but not to confluent, Madin-Darby canine kidney-I cultures. This model was used here to investigate whether the surface of cells with affinity for crystals is enriched with one of the proposed crystal-binding molecules. Annexin V was used for the detection of PS at the cell surface, and Sambucus nigra lectin was used to reveal terminal sialic acid in a (alpha2,6) linkage to galactose units. FITC-annexin V binding studies showed that PS was not exposed at the surface of proliferating or growth-inhibited cells, unless they were pretreated with an apoptosis-inducing cytotoxic agent. Sambucus nigra lectin binding, of which the specificity was confirmed by blocking with N-acetylneuraminyl-lactose, demonstrated the abundant presence of (alpha2,6)-linked sialic acid residues at the cell surface of both subconfluent and confluent cultures. While these results seem to rule out a role for PS in the adherence of calcium oxalate monohydrate crystals to the surface of maturating Madin-Darby canine kidney-I cells, they question the role for cell surface-associated sialylated glycoconjugates in this process.

Background: Adherence of crystals to the surface of renal tubule epithelial cells is considered an important step in the development of nephrolithiasis. Previously, we demonstrated that functional monolayers formed by the renal tubule cell line, Madin-Darby canine kidney (MDCK), acquire protection against the adherence of calcium oxalate monohydrate crystals. We now examined whether this property is cell type specific. The susceptibility of the cells to crystal binding was further studied under different culture conditions., Methods: Cell-type specificity and the influence of the growth substrate was tested by comparing calcium oxalate monohydrate crystal binding to LLC-PK1 cells and to two MDCK strains cultured on either permeable or impermeable supports. These cell lines are representative for the renal proximal tubule (LLC-PK1) and distal tubule/collecting duct (MDCK) segments of the nephron, in which crystals are expected to be absent and present, respectively., Results: Whereas relatively large amounts of crystals adhered to subconfluent MDCK cultures, the level of crystal binding to confluent monolayers was reduced for both MDCK strains. On permeable supports, MDCK cells not only obtained a higher level of morphological differentiation, but also acquired a higher degree of protection than on impermeable surfaces. Crystals avidly adhered to LLC-PK1 cells, irrespective of their developmental stage or growth substrate used., Conclusions: These results show that the prevention of crystal binding is cell type specific and expressed only by differentiated MDCK cells. The anti-adherence properties acquired by MDCK cells may mirror a specific functional characteristic of its in situ equivalent, the renal distal tubule/collecting ducts.

The retention of crystals in the kidney is considered to be a crucial step in the development of a renal stone. This study demonstrates the time-dependent alterations in the extent of calcium oxalate (CaOx) monohydrate (COM) crystal binding to Madin-Darby canine kidney (MDCK) cells during their growth to confluence and during the healing of wounds made in confluent monolayers. As determined by radiolabeled COM crystal binding studies and confirmed by confocal-scanning laser microscopy, relatively large amounts of crystals (10.4 +/- 0.4 micrograms/cm2) bound to subconfluent cultures that still exhibited a low transepithelial electrical resistance (TER < 400 omega.cm2). The development of junctional integrity, indicated by a high resistance (TER > 1,500 omega.cm2), was followed by a decrease of the crystal binding capacity to almost undetectable low levels (0.13 +/- 0.03 microgram/cm2). Epithelial injury resulted in increased crystal adherence. The highest level of crystal binding was observed 2 days postinjury when the wounds were already morphologically closed but TER was still low. Confocal images showed that during the repair process, crystals selectively adhered to migrating cells at the wound border and to stacked cells at sites were the wounds were closed. After the barrier integrity was restored, crystal binding decreased again to the same low levels as in undamaged controls. These results indicate that, whereas functional MDCK monolayers are largely protected against COM crystal adherence, epithelial injury and the subsequent process of wound healing lead to increased crystal binding.

Objectives: To detect in situ the precise osteopontin (OPN) localization in papillary stones., Methods: Immunocytochemical labelling procedures are applied to detect OPN localizations in crystalline material of renal papillary stones. The tissue-processing procedure for electron microscopy, which includes OsO4 postfixation, preserves both immunocytochemical OPN reactivity and cellular membrane contrast up to the ultrathin section. Reflection-contrast light microscopical images are correlated with high resolution transmission-electron microscopical observations from consecutive ultrathin epon sections., Results: Preserved crystalline material in interstitial and peripheral papillary stones is recognized as calcium oxalate monohydrate. After section incubation with markers conjugated to an antibody against OPN (alpha OPN) the crystals are converted into ghosts. In the ghosts, alpha OPN markers are present around microcrystals. The size of these microcrystals ranges from several nanometers to micrometers. It is observed (due to the OsO4-preserved membranes) that interstitial cells are separated from the stone surfaces by unidentified extracellular material, also present in the center as a stone matrix., Conclusion: The microcrystal-growth inhibitor OPN is detected in situ in interstitial stones induced in the rat's papilla and at the surface of the papilla.

While the physical chemistry of stone formation has been intensively studied during the last decade, it has become clear that the pathophysiology of renal stone disease cannot be explained by crystallization processes only. In recent years, evidence has emerged that the cells lining the renal tubules can have an active role in creating the conditions under which stones may develop. Since it is difficult to study these mechanisms in vivo, cultured renal tubular cells have become increasingly popular for the study of physiological and cell biological processes that are possibly linked to stone disease. In this paper, we discuss the possible contribution of cellular processes such as transepithelial oxalate transport and crystal--cell interaction to the formation of renal stones. Experimental studies that have been performed with cultured renal cells to elucidate the mechanisms involved in these processes will be summarized.

Objectives: Previously it was shown that the polysaccharide G872 in vitro strongly inhibits calcium oxalate monohydrate crystallization processes. However, when rats on a stone-inducing diet of ethylene glycol plus vitamin D3 are given this polysaccharide, no changes in the urine capacity for crystallization inhibition were found. We investigated here how the inhibitory action of polysaccharides changes under high oxalate conditions, as they exist in the stone inducing diet., Methods: Calcium oxalate monohydrate (COM) crystals were incubated in a series of 0.05 M PBS buffers containing polysaccharides with increasing oxalate concentrations (0-0.4 mmol/l). The coated crystals were collected, washed and resuspended in an artificial urine. We then measured the zeta potential of the crystals, using a Coulter DELSA 440, and the initial rates for crystal growth and agglomeration, using the Coulter Multisizer II., Results: Addition of oxalate to the medium shifts the negative zeta potential distribution of COM crystals coated by polysaccharides in positive direction. Particle size analysis demonstrated that the initial rates of COM crystal growth and agglomeration responding to oxalate concentration changes (0.1-->0.4 mmol/l) in the presence of G872 (0.2 mg/l) are approximately 2.5 times faster than that in the absence of G872., Conclusions: Oxalate interferes with the binding of polysaccharides to crystals. This can be envisioned to occur through changes in the crystal surface properties or by induction of functional and secondary structural changes of urinary macromolecular inhibitors such as GAGs, resulting in a decrease of their inhibitory activity against COM crystallization. Thus, in urine, a high oxalate may increase the rate of crystallization both by increasing the supersaturation and by decreasing the inhibitory potential of the urine.

Purpose: We studied the effect of polysaccharides on interactions between calcium oxalate monohydrate (COM) crystals and cultured renal cells., Materials and Methods: Monolayers of Madin-Darby canine kidney (MDCK) cells were incubated with radiolabeled crystals in the presence of various concentrations of natural glycosaminoglycans (GAGs) and semisynthetic polysaccharides (SSPs)., Results: While most GAGs were found to have relatively little effect, SSPs (SP54, G871 and G872) were potent inhibitors of crystal-cell association. Pretreatment of crystals, but not of cells, was similarly effective, suggesting polysaccharide-induced modification of crystal surface properties., Conclusions: This result further supports the idea that SSPs, and especially G872, are of potential interest for treatment of recurrent stone disease.

To better understand urinary inhibitors of calcium oxalate crystallization, both zeta potential measurement and particle size analysis were chosen to illustrate: (1) the potential therapeutic efficacy of G872, a semi-synthetic sulfated polysaccharide, in stone prevention; and (2) the relative contribution of various urinary fractions ¿e.g., ultrafiltered urine (UFU), Tamm-Horsfall protein (THP), urinary polyanions precipitated with cetylpyridinium chloride (CPC), urinary macromolecular substances with different concentration ratios (UMS10,50,90 and UMS'10,50,90) and THP-free urine (THPFU)¿ to total urinary inhibitory activity. The results showed: (1) addition of G872 significantly enhances urinary inhibitory activity and negative zeta potential values; (2) re-addition of the CPC to UFU completely restores urinary inhibitory activity; and (3) artificial urines prepared by mixing UMS'10,50,90 from THPFU with UFU differed in inhibitory activity from that prepared by mixing UMS10,50,90 from a pooled normal urine with UFU. Based on these experimental results, the following speculations can be made: (1) normal human urines are considered to be a protective colloidal system; (2) urinary inhibitory activity originates mainly from CPC and/or UMS; (3) normal THP is a protective material to maintain urinary inhibitory activity; and (4) mutual interaction between urinary inhibitors may change the total urinary inhibitory activity.

Lectin reactivity in epithelial apical cell coats of normal rat kidneys was compared to that from animals subjected to crystal inducing diets (CID). The aim was to see whether the absence of lectin reactivity in cell coats is related to intratubular calcium oxalate crystal retention. In normal rat kidneys, after a pre-embedding procedure, it was observed that at the ultrastructural level, reactivity was present but that the lectin specificity for the various parts of the nephron might have to be reconsidered. There was heterogeneity between the epithelial cells with respect to the presence of coat material in the tubular cell apices. Tubular epithelial cell apices from CID rats showed no obvious changes in lectin reactivity pattern. Lectin reactivity was present at the periphery of intratubular crystals but undetectable at true crystal attachment sites or reduced at cell apices in the vicinity of recently attached crystals or agglomerates. After a post-embedding reaction procedure, wheat-germ agglutinin (WGA)-lectin reactivity confirmed the presence of coat material in the cleft between cell apex and retained crystal at crystal-attachment sites. The WGA/Au-10 nm reaction products were also seen inside epithelial cells. WGA/Au-10 nm reaction products mark a crystal matrix component inside intratubular and retained crystals. A similar matrix was also marked by an alpha-osteopontin (alpha OPN/Au-10 nm) reaction product.

Many factors are presently known which determine the risk of calcium oxalate (CaOx) stone formation in the kidney, although the early events in the pathogenesis of this disease are still to be elucidated. One of these early events is the interaction of intraluminal crystals with the epithelial cells lining the renal tubules. In this study we determined the interaction of approximately 2 microns calcium oxalate monohydrate (COM) crystal with monolayers of Madin-Darby canine kidney (MDCK) cells grown on porous supports in a two-compartment culture system. Crystal-cell interaction studies were performed after the monolayers reached their highest level of gamma-glutamyltranspeptidase (gamma GT) enzyme activity, a marker for brush border development. Technical aspects were evaluated, such as the size and morphology of the crystals and the influence of incubation time, temperature and pH on crystal-cell interaction. Kinetic data demonstrated that an equilibrium between free and associated particles was reached within 30 minutes. Crystal-cell interaction was often associated with cell damage. However, evidence is provided that in an environment that was saturated with calcium oxalate, MDCK cells in an environment that was a certain amount of COM crystals without sustaining measurable injury. After initial attachment to the cell surface, crystals were taken up and subsequently eliminated again from the monolayers. The model system described in this paper provides a tool for detailed studies of processes that are involved in renal cellular handling of luminal COM crystals.

Crystal retention is studied in a rat-model system as a possible mechanism for the etiology of human nephrolithiasis. A crystal-inducing diet (CID) of ethylene glycol plus NH4Cl in their drinking-water is offered to healthy rats to generate intratubular crystals. Subsequently, the fate of retained crystals is investigated by allowing the rats a tissue recovery/crystalluria phase for three, five and ten days, respectively, on normal drinking water. The process of exotubulosis is observed in cortex and medulla of aldehyde-fixed kidneys after three days recovery. After five days, crystals are predominantly seen there in the interstitium. After ten days, cortex and medulla are virtually free of crystals. However, in the papillary regions after five and ten days recovery, three types of calcium oxalate monohydrate (COM) crystals are present: (1) free in the calycine space, (2) sub-epithelially located surrounded by interstitial cells within, and (3) covered by macrophage-like cells, outside the original papillary surface. After a CID plus three days recovery, a further thirty-seven days extra oxalate challenge with solely 0.3 vol% ethylene glycol induced intratubular and interstitial oxalate crystals. In the papillary region, large sub-epithelial crystals are seen. However, no crystals are seen in kidneys from rats given solely (0.5 or 0.8 vol.%) ethylene glycol for thirty days. An oxalate re-challenge retards crystal removal.

In kidneys of healthy rats submitted to a crystal-inducing diet (CID) with ethylene glycol (EG) and NH4Cl, the fate of retained crystals in the papillar region is studied during a recovery period of one, five or ten days, as model system for human nephrolithiasis. Scanning electron microscopy (SEM) shows, at papillary tips bulging into the calycine space, crystal masses covered either by the epithelium or a thin fibrous veil, or by unidentified mobile cuboidal cells. After CID plus one or five days recovery, small sub-epithelial swellings are seen of large sub-epithelial crystals at or around the papillary tip. After CID plus ten days, massive sub-surface crystal-containing micrometer-sized stones are seen in which the presence of calcium is confirmed by X-ray microanalysis. The papillary tip of rats after a re-challenge with an oxalate load from 0.1 vol% EG for twelve or forty-two days shows minor lesions. But a re-challenge with 0.3 vol% EG for thirty-seven days induces large sub-epithelial papillary millimeter-sized stones. The Von Kossa section staining converts the crystals into a black precipitate, but large peri-tubular or peri-vascular calcium deposits are absent. A new hypothesis about the etiology of an inductive calcium oxalate monohydrate nephrolithiasis is formulated which differs from the one proposed by Randall based on his deductive human kidney studies.

In a rat-model system, tubular crystal retention as a possible mechanism for the etiology of nephrolithiasis in man, was studied by conventional transmission electron microscopy. The animals were supplied for nine days with a crystal-inducing diet, with ethylene glycol plus NH4Cl in their drinking-water. After this induction period, a two day regime with fresh drinking-water was included, to allow crystals to be removed by spontaneous crystalluria. After aldehyde fixation of the rat kidneys, large crystals were seen inside the tubular lumen. The crystals were attached to cell surfaces and covered by neighboring epithelial cells. Some crystals were overgrown by several epithelial cells and underwent a process of so-called exotubulosis, resulting in free or cell-surrounded crystals in the interstitium, and possibly in crystals in Giant cells. To investigate the fate of the retained crystals, some animals were additionally exposed to a low-oxalate challenge from drinking water containing 0.1 volume per cent of ethylene glycol for 12 or 30 days, respectively. It was assumed that this would interfere with the retained intratubular or interstitial crystals, and allow the crystals to grow into mini-stones. This was not observed. After the oxalate challenge, no crystals were found to be retained in the tubules (free or covered by cells). Interstitial crystals were observed, but it remains to be demonstrated whether such crystals actually grow into mini-stones or that they are removed by the sterile inflammation process observed.

Transepithelial oxalate transport across polarized monolayers of LLC-PK1 cells, grown on collagen-coated microporous membranes in Transwell culture chambers, was studied in double-label experiments using [14C]-oxalate together with [3H]-D-mannitol as an extracellular marker. The [14C]-labeled glucose analog alpha-methyl-glucoside (alpha-MG) was used as functional marker for active proximal tubular sugar transport. Cellular uptake of oxalate and alpha-MG at both the apical and basolateral plasma membrane was determined. When added to the upper compartment, alpha-MG was actively taken up at the apical membrane, directed through the cells to the basolateral membrane and transported to the lower compartment, indicating functional epithelial sugar transport by LLC-PK1 cells. In LLC-PK1 cells, the uptake of alpha-MG at the apical membrane was approximately 50 times higher than that at the basolateral membrane. In contrast to this active transport of sugar, LLC-PK1 cells did not demonstrate oxalate uptake either at the apical or basolateral plasma membrane. The apical-to-basolateral (A- > B) flux of oxalate in LLC-PK1 cells was identical to the basolateral-to-apical (B- > A) oxalate flux in these cells. Moreover these flux characteristics were similar to those found for D-mannitol, indicating paracellular movement for both compounds. From these data, it is concluded that, under the experimental conditions used, LLC-PK1 cells do not exhibit a specific transcellular transport system for oxalate.

Neuron specific enolase (NSE) is widely used as a neuro-endocrine marker. However the presence of NSE in many non-neuroendocrine tissues has raised questions on the specificity of NSE. We have investigated NSE immunoreactivity (NSA-ag), gamma-enolase activity and total enolase activity in small cell lung cancer (SCLC) cell lines. During well-controlled exponential growth comparison of NSE-ag content and gamma-enolase activity with the doubling-time (Td) and NSE-ag content with gamma-enolase and total enolase activity led to a clear distinction of two types of cell line: variant cell lines plus part of the classic cell lines (type I) and the remaining classic cell lines (type II). The distinction was based upon both an abrupt 6-fold increase of gamma-enolase activity and an 18-fold increase of NSE-ag, which for the larger part was enzymatically inactive. Within each group the increase of NSE-ag content was significantly correlated with the increase of gamma-enolase activity and both NSE-ag content and gamma-enolase activity increased linearly with Td. It is concluded that gamma-enolase seems to be associated with the regulation of growth rate and that a compound with the gamma-enolase antigen but without enzyme activity can distinguish two different classes of SCLC cell lines. Furthermore the demonstration that NSE-ag can represent the active enzyme as well as an enzymatically inactive compound may explain why a controversy about neuron- or non-specificity of NSE exists.

A rapid and simple method, originally described by Redmond and Tseng [J. Chromatogr., 170 (1979) 479] was applied to the analysis of di- and polyamines in cultured human tumour cells and human tumour xenografts. Optimization of the procedures and evaluation of the characteristic features of the assay are described. The (modified) procedure employs precolumn derivatization with benzoyl chloride, extraction of the derivatives by chloroform, separation by reversed-phase high-performance liquid chromatography under isocratic conditions and detection by ultraviolet absorbance measurement at 229 nm. The complete analysis was accomplished within 10 min per sample. The detection limit was ca. 1 pmol. The intra- and inter-assay coefficients of variation were 2.5-4.4% and 3.4-13.1%, respectively. The presence of well known inhibitors of polyamine biosynthesis, such as DL-alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone), did not interfere with the assay, and disturbance by cyclohexylamine could be avoided by changing the polarity of the mobile phase. The method proved to be very suitable because it is rapid, simple, requires a minimum of sample pretreatment, and still provides sufficient sensitivity to quantitate polyamines in relatively small amounts of cells (10(5) cells) or tumour tissues (less than 1 mg), even after treatment with inhibitors of polyamine biosynthesis.

Diamine oxidase (DAO) from tissues of mice, rats and humans showed different properties with respect to stability and kinetic parameters. DAO-activities in homogenates of rat or human tissues, but not of mouse tissues, rapidly decreased upon storage at -20 degrees C. The Km-value for putrescine was 90 microM in mouse kidney or intestine. In rats different Km-values were observed before (272 microM) and after freezing (102 microM). A similar effect was observed with DAO in human kidney (321 and 39 microM, respectively). Treatment of rats with heparin resulted in a depletion of intestinal DAO and the concomitant appearance of DAO in blood. The enzyme remaining in the intestine showed the lower Km-value.

A morphometric technique, based on the measurement of the area of individual cell colonies and of its increase in time, was applied to study the rate of proliferation of human prostatic cells in vitro. The reliability of the method was checked by determination of the growth rate of cultures of the continuous cell line PC93 by the morphometrical technique as well as by counting of the cell number. No significant difference was found in the population doubling times measured by either of these methods. It was therefore concluded that the morphometrical technique could be applied also to study the growth rate of primary cultures of prostatic epithelial cells, in which counting of the cell number is generally impossible. The results showed that, with primary cultures derived from hyperplastic prostates and prostatic carcinomas as well as from the prostatic tumor line PC82, rapid growth occurred during the first two or three days of culture; measurements performed at a later time appeared to be less reliable. It was demonstrated by the effect of serum deprivation on the growth of PC82 cells that the technique described here is, in principle, suitable to monitor the effect of various agents on the growth of cells in primary culture. The method is non-destructive and requires minimal amounts of tissue; it may be applied especially to cultures that cannot be dispersed easily into single cell suspensions.