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J Loffing

Publications and source records attributed to J Loffing.

18 recordsLinked to original sources
PubMed1999

Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome.

Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na+ channel (ENaC). ENaC is composed of three subunits (alpha, beta, gamma), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddle's syndrome alter or delete the PY motifs of beta- or gamma-ENaC. We recently demonstrated that the ubiquitin-protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of beta-ENaC), as originally described in patients with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na+ reabsorption by the distal nephron, and hence the hypertension.

Animals↗
PubMed1998

Functional heterodimeric amino acid transporters lacking cysteine residues involved in disulfide bond.

The protein mediating system L amino acid transport, AmAT-L, is a disulfide-linked heterodimer of a permease-related light chain (AmAT-L-lc) and the type II glycoprotein 4F2hc/ CD98. The Schistosoma mansoni protein SPRM1 also heterodimerizes with h4F2hc, inducing amino acid transport with different specificity. In this study, we show that the disulfide bond is formed by heavy chain C109 with a Cys residue located in the second putative extracellular loop of the multi-transmembrane domain light chain (C164 and C137 for XAmAT-L-lc and SPRM1, respectively). The non-covalent interaction of Cys-mutant subunits is not sufficient to allow coimmunoprecipitation, but cell surface expression of the light chains is maintained to a large extent. The non-covalently linked transporters display the same transport characteristics as disulfide bound heterodimers, but the maximal transport rates are reduced by 30-80%.

Amino Acid Transport Systems↗
PubMed1998

Amino-acid transport by heterodimers of 4F2hc/CD98 and members of a permease family.

Amino-acid transport across cellular plasma membranes depends on several parallel-functioning (co-)transporters and exchangers. The widespread transport system L accounts for a sodium-independent exchange of large, neutral amino acids, whereas the system y(+)L exchanges positively charged amino acids and/or neutral amino acids together with sodium. The molecular nature of these transporters remains unknown, although expression of the human cell-surface glycoprotein 4F2 heavy chain (h4F2hc; CD98 in the mouse) is known to induce low levels of L- and/or y(+)L-type transport. This glycoprotein is found in activated lymphocytes, together with an uncharacterized, disulphide-linked lipophilic light chain with an apparent relative molecular mass of 40,000 (M(r) 40K). Here we identify the permease-related protein E16 as the first light chain of h4F2hc and show that the resulting heterodimeric complex mediates L-type amino-acid transport. The homologous protein from Schistosoma mansoni, SPRM1, also associates covalently with coexpressed h4F2hc glycoprotein, although it induces amino-acid transport of different substrate specificity. The coexpression of h4F2hc is required for surface expression of these permease-related light chains, which belong to a new family of amino-acid transporters that form heterodimers with cell-surface glycoproteins.

Amino Acid Transport Systems↗
PubMed1998

Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells.

The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl-) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl- secretion by stimulating CFTR Cl- channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patch-clamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl- secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl- secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Postnatal maturation of renal cortical peritubular fibroblasts in the rat.

The stromal cells in the renal cortex and medulla of adult rats reveal different phenotypes. Cortical peritubular fibroblasts are ecto-5'nucleotidase (5'NT)-positive and lack alpha-smooth muscle actin (alphaSMA) and vimentin immunoreactivity, whereas medullary fibroblasts are 5'NT-negative and vimentin-positive. We have studied by immunohistochemistry the postnatal (neonatal up to 8 weeks) development of renal cortical stromal cells with respect to 5'NT and to the cytoskeletal proteins alphaSMA and vimentin. Both alphaSMA and vimentin are characteristic for the renal myofibroblasts that replace stromal fibroblasts in interstitial nephritis. In new-born and 1-week-old rats, stromal cells in the cortex and medulla display alphaSMA and vimentin, but lack 5'NT. During the second postnatal week, alphaSMA and vimentin immunoreactivity in cortical interstitial cells gradually declines, whereas 5'NT reactivity becomes progressively apparent between the convoluted tubules in the juxtamedullary labyrinth. For a short time, all three proteins are found to be coexpressed in the same cells. At the end of the third week, interstitial 5'NT-immunoreactivity becomes evident also in the superficial cortical labyrinth, and alphaSMA and vimentin are no longer detectable in cortical peritubular cells. From the fourth week on, the distribution pattern and phenotype of 5'NT-positive cortical fibroblasts correspond to that in adult rats. The temporal pattern of maturation of cortical peritubular fibroblasts seems to parallel the functional maturation of cortical tubules. It is suggested that the local phenotype of peritubular fibroblasts in healthy and possibly also in injured kidneys may be controlled, at least in part, by the local tubular environment, conditioned by tubular metabolism and function.

5'-Nucleotidase↗
PubMed1998

Ras pathway activates epithelial Na+ channel and decreases its surface expression in Xenopus oocytes.

The small G protein K-Ras2A is rapidly induced by aldosterone in A6 epithelia. In these Xenopus sodium reabsorbing cells, aldosterone rapidly activates preexisting epithelial Na+ channels (XENaC) via a transcriptionally mediated mechanism. In the Xenopus oocytes expression system, we tested whether the K-Ras2A pathway impacts on XENaC activity by expressing XENaC alone or together with XK-Ras2A rendered constitutively active (XK-Ras2AG12V). As a second control, XENaC-expressing oocytes were treated with progesterone, a sex steroid that induces maturation of the oocytes similarly to activated Ras. Progesterone or XK-Ras2AG12V led to oocyte maturation characterized by a decrease in surface area and endogenous Na+ pump function. In both conditions, the surface expression of exogenous XENaC's was also decreased; however, in comparison with progesterone-treated oocytes, XK-ras2AG12V-coinjected oocytes expressed a fivefold higher XENaC-mediated macroscopic Na+ current that was as high as that of control oocytes. Thus, the Na+ current per surface-expressed XENaC was increased by XK-Ras2AG12V. The chemical driving force for Na+ influx was not changed, suggesting that XK-Ras2AG12V increased the mean activity of XENaCs at the oocyte surface. These observations raise the possibility that XK-Ras2A, which is the first regulatory protein known to be transcriptionally induced by aldosterone, could play a role in the control of XENaC function in aldosterone target cells.

Aldosterone↗
PubMed1998

Exocytosis is not involved in activation of Cl- secretion via CFTR in Calu-3 airway epithelial cells.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, which mediates transepithelial Cl- transport in a variety of epithelia, including airway, intestine, pancreas, and sweat duct. In some but not all epithelial cells, cAMP stimulates Cl- secretion in part by increasing the number of CFTR Cl- channels in the apical plasma membrane. Because the mechanism whereby cAMP stimulates CFTR Cl- secretion is cell-type specific, our goal was to determine whether cAMP elevates CFTR-mediated Cl- secretion across serous airway epithelial cells by stimulating the insertion of CFTR Cl- channels from an intracellular pool into the apical plasma membrane. To this end we studied Calu-3 cells, a human airway cell line with a serous cell phenotype. Serous cells in human airways, such as Calu-3 cells, express high levels of CFTR, secrete antibiotic-rich fluid, and play a critical role in airway function. Moreover, dysregulation of CFTR-mediated Cl- secretion in serous cells is thought to contribute to the pathophysiology of cystic fibrosis lung disease. We report that cAMP activation of CFTR-mediated Cl- secretion across human serous cells involves stimulation of CFTR channels present in the apical plasma membrane and does not involve the recruitment of CFTR from an intracellular pool to the apical plasma membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Renal Na/H exchanger NHE-3 and Na-PO4 cotransporter NaPi-2 protein expression in glucocorticoid excess and deficient states.

Administration of pharmacologic doses of glucocorticoid in vivo increases renal proximal tubule apical membrane Na/H exchange and decreases Na/PO4 cotransport activity (1). Current data suggest that the NHE-3 and NaPi-2 proteins mediate significant fractions of proximal tubule apical membrane Na/H exchange and Na/PO4 cotransport, respectively. This study examines whether glucocorticoid excess or deficiency affects NHE-3 and NaPi-2 protein abundance and the intrarenal distribution of these transporters. Protein abundance of NHE-3 and NaPi-2 in control rats was compared to rats rendered glucocorticoid-deficient by bilateral adrenalectomy, and to rats receiving pharmacologic doses of dexamethasone using immunoblots and immunohistochemistry. Adrenalectomy had modest effects on NHE-3 protein abundance, but dexamethasone administration to either adrenalectomized or sham-operated rats significantly increased NHE-3 protein abundance in both the proximal tubule and thick ascending limb, but not the thin descending limb. Adrenalectomy increased NaPi-2 protein abundance in the proximal tubule, whereas dexamethasone administration dramatically suppressed NaPi-2 protein on the apical membrane in both adrenalectomized and sham-operated animals. No significant reciprocal increase in subapical NaPi-2 staining was seen in the dexamethasone-treated rats. The present study shows that glucocorticoids regulate proximal tubule apical membrane Na/H exchange and NaPi cotransport by changes in protein abundance of NHE-3 and NaPi-2, respectively.

Adaptor Protein Complex 3↗
PubMed1998

Enhanced osteopontin expression and macrophage infiltration in MRL-Fas(lpr) mice with lupus nephritis.

MRL-Fas(lpr) mice spontaneously develop a chronic lupus-like renal disease, characterized by immune complex-mediated glomerulonephritis and abundant mononuclear cell infiltration in the interstitium. In the present study we have examined whether the macrophage chemoattractant osteopontin (Opn) could be important in the recruitment of macrophages in this murine model of autoimmune renal injury. We have examined the expression of Opn in the kidney of MRL-Fas(lpr) mice and have correlated Opn synthesis with the degree of macrophage infiltration. Immunofluorescence staining revealed prominent expression of Opn by proximal tubules in MRL-Fas(lpr) mice but not in MRL-++ control mice. Northern blot analysis demonstrated that steady-state transcript levels for Opn mRNA were also significantly increased in MRL-Fas(lpr) kidneys compared with control kidneys. Furthermore, in situ hybridization showed massive Opn mRNA transcripts in proximal tubules in MRL-Fas(lpr) mice but not in controls. The diffuse macrophage infiltration in the kidney of MRL-Fas(lpr) correlated with the enhanced Opn expression. Opn secretion in vitro by cultured renal tubular epithelial cells was upregulated by TNF-alpha and 1,25(OH)2-vitamin D3, whereas no regulation was observed in a control macrophage cell line. We conclude that the enhanced expression of the chemotactic molecule Opn by tubular cells is a prominent feature of murine lupus nephritis and might be promoted by the proinflammatory cytokine environment in MRL-Fas(lpr). The chronic upregulation of Opn could participate in the recruitment of monocytes in the kidney of MRL-Fas(lpr) mice, thereby contributing to the pathogenesis of autoimmune renal disease.

Animals↗
PubMed1997

Upregulated renal tubular CD44, hyaluronan, and osteopontin in kdkd mice with interstitial nephritis.

BACKGROUND: The hyaluronan (HA) receptor CD44 is upregulated on parenchymal cells in various inflammatory lesions and could play a role in immune injury. The purpose of the present study was to examine CD44 and its ligands HA and osteopontin (Opn) in a murine model of tubulointerstitial nephritis (TIN). METHODS: The expression of CD44 was investigated by immunofluorescence staining and RNA analysis in kidneys of kdkd mice with autoimmune TIN. The CD44 expression was then correlated with the location of its ligands HA and Opn. RESULTS: CD44 is expressed de novo by tubular epithelial cells (TEC) in areas of tubular injury in kdkd kidneys, but not in normal control kidneys. CD44 positive lymphocytes and macrophages also infiltrate the kidney to kdkd mice. RT-PCR and Southern blot analysis demonstrate that transcripts encoding standard and variant forms of CD44 are increased in kdkd mice with TIN. In parallel the CD44 ligand HA also accumulates in kdkd kidneys in the interstitial space, particularly in cortical areas of tubular injury. Furthermore, the expression of the chemotactic protein Open is enhanced in kdkd kidney, predominantly in areas of tubular injury. Opn mRNA expression also increases markedly in kdkd kidneys compared with normal kidneys, and correlates with disease severity. CONCLUSIONS: Prominent CD44 expression by TEC in areas of tubulointerstitial lesions is a characteristic feature of kdkd mice. The de novo appearance of CD44 on injured TEC might allow interaction with the ligands HA and Opn in vivo. Interaction of CD44 with these ligands could participate in the tubulointerstitial inflammatory response in kdkd mice.

Animals↗
PubMed1996

Morphology of interstitial cells in the healthy kidney.

Renal interstitial cells play an important role in renal function and renal diseases. We describe the morphology of renal interstitial cells in the healthy kidney. We distinguish within the renal interstitium (1) renal fibroblasts and (2) cells of the immune system. Fibroblasts are in the majority and constitute the scaffold of the kidney; they are interconnected by junctions, and are attached to tubules and vessels. Although the phenotype of fibroblasts shows some variation depending on their location in the kidney and on their functional stage, their recognition as fibroblasts is possible on account of structural features. Among the cell types of the second group, antigen-presenting dendritic cells are the most abundant in in the peritubular interstitial spaces of healthy kidneys. Their incidence is highest in the inner stripe of the outer medulla. They share some morphological features with fibroblasts but lack others--junctional complexes, morphologically defined connections with tubules and vessels, and the prominent layer of actin filaments under the plasma membrane--that are characteristic for fibroblasts. Dendritic cells in healthy kidneys are morphologically different from macrophages, which are characterized by abundant primary and secondary lysosomes. In healthy kidneys macrophages are restricted to the connective tissue of the renal capsule and the pelvic wall, and to the periarterial connective tissue. Lymphocytes are rare in healthy kidneys. The distinction of cell types by morphology is supported by differences of membrane proteins. Among all interstitial cells in the renal cortex, fibroblasts alone exhibit ecto-5'-nucleotidase. Dendritic cells constitutively have a high abundance of MHC class II protein. Both proteins are mutually exclusive. Rat macrophages display the membrane antigen ED 2 and lymphocytes exhibit specific surface antigens, depending on their type and functional stage, e.g., CD4 or CD8.

5'-Nucleotidase↗
PubMed1996

Cellular mechanisms of the age-related decrease in renal phosphate reabsorption.

The aging process in humans and in the rat is associated with an impairment in renal tubular reabsorption of Pi and renal tubular adaptation to a low Pi diet. The purposes of the present study were to determine whether changes in the abundance of type II Na-Pi contransporter (NaPi-2) protein and/or mRNA play a role in the age-related decrease in Na-Pi cotransport activity, and to further determine the cellular mechanisms of impaired adaptation to a low Pi diet. In studies performed in 3- to 4-month-old young adult rats and 32-to 16-month-old aged rats we found that there was an age-related twofold decrease in proximal tubular apical brush border membrane (BBM) Na-Pi cotransport activity, which was associated with similar decreases in BBM NaPi-2 protein abundance and renal cortical NaPi-2 mRNA level. Immunohisto-chemistry showed lower NaPi-2 protein expression in the BBM of proximal tubules of superficial, midcortical, and juxtamedullary nephrons. We also found that in response to chronic (7 days) and/or acute (4 hr) feeding of a low Pi diet there were similar adaptive increases in BBM Na-Pi cotransport activity and BBM NaPi-2 protein abundance in both young and aged rats. However, BBM Na-Pi cotransport activity and BBM NaPi-2 protein abundance were still significantly lower in aged rats, in spite of a significantly lower serum Pi concentration in aged rats. The results indicate that impaired expression of the type II renal Na-Pi cotransporter protein at the level of the apical BBM plays an important role in the age-related impairment in renal tubular reabsorption of Pi and renal tubular adaptation to a low Pi diet.

Adaptation, Physiological↗
PubMed1996

Thiazide treatment of rats provokes apoptosis in distal tubule cells.

We studied the effects of inhibition of apical NaCl entry on the structural correlates for electrolyte transport in the distal convoluted tubule (DCT) of rats. Thiazide diuretics were used to block NaCl entry specifically in the DCT. Metolazone or hydrochlorothiazide (HCTZ) were applied for three days subcutaneously via osmotic minipumps. The renal epithelial structure of control and treated rats was studied by light and electron microscopy. Distribution of the thiazide-sensitive NaCl cotransporter (rTSC1), calbindin D28K and Ca(2+)-Mg(2+)-ATPase was examined by immunohistochemistry, and the content of rTSC1 transcripts by Northern blot and in situ hybridization. In treated rats the DCT epithelium had lost the structural characteristics of electrolyte transporting epithelia and the cells were in different stages of apoptosis. In damaged cells calbindin D28K and Ca(2+)-Mg(2+)-ATPase were strongly decreased; the rTSC1 was shifted from the luminal membrane to the basal cell half and was found additionally in small membrane vesicles in intercellular and peritubular spaces. Transcripts of rTSC1 were drastically reduced in homogenates of kidney cortex and almost absent in damaged DCT cells. All other tubular segments were unaffected by the treatment. Focal inflammatory infiltrates were found to be specifically surrounding DCT profiles. Thus, inhibition by thiazides of apical NaCl entry into DCT cells is associated with apoptosis of DCT cells and focal peritubular inflammation.

Analysis of Variance↗
PubMed1995

Ecto-5'-nucleotidase is expressed by pericytes and fibroblasts in the rat heart.

Ecto-5'-nucleotidase is anchored at the outer surface of cell membranes and thus its reaction product adenosine is released into the extracellular space. Extracellular adenosine displays via specific receptors a wide range of physiological effects in heart. There are discrepancies in the literature concerning the distribution of ecto-5'-nucleotidase in heart. Since we suspected that these may be due to technical problems, in the present study on ecto-5'-nucleotidase in rat heart we attempted to circumvent some technical pitfalls. Good preservation of the tissue with open capillary lumina, providing a clear identification of endothelium, was obtained by perfusion fixation. At the light microscopic level, the distribution of ecto-5'-nucleotidase studied by enzyme histochemistry and immunohistochemistry using a monoclonal and a polyclonal antibody yielded congruent results. The enzyme was rather homogeneously distributed throughout the myocardium, with a slightly higher incidence of stained cells in the outer thirds than in the inner third of the wall. Consistently high levels of ecto-5'-nucleotidase were seen only in interstitial cells. The walls of large vessels and heart muscle cells were constantly negative for ecto-5'-nucleotidase. The endothelia of capillaries were mostly negative but a few profiles occasionally displayed a weak immunoreaction. The interstitial cells staining positive for ecto-5'-nucleotidase could be identified as pericytes and as fibroblasts according to their shapes and localizations. The immunoreactivity of fibroblasts was confirmed by electron microscopy. These data indicate that adenosine may be formed extracellularly in the interstitium of the myocardium, where it would have direct access to important targets such as myocytes, arterioles and nerve endings.

5'-Nucleotidase↗
PubMed1995

Modulation of salt transport rate affects DNA synthesis in vivo in rat renal tubules.

In adult male Wistar rats we investigated whether cell proliferation contributes to salt load-induced hypertrophy of distal tubules. In one treatment group salt transport in the thick ascending limb (TAL) was inhibited by furosemide (7.5 mg/100 g body wt/24 hr, via osmotic minipump) and stimulated in the successive distal segments by simultaneous high salt intake (F + Salt). Controls without furosemide treatment had a standard salt intake. All animals received the thymidine analog bromodeoxyuridine (BrdU) during 24 and 72 hours, respectively. In cryostat sections of the perfusion-fixed kidneys DNA synthesis was assessed by immunohistochemistry for BrdU, and for endogenous proliferating cellular nuclear antigen (PCNA). Incidence of BrdU- and PCNA-labeled nuclei were quantified in proximal tubules, medullary TAL, and cortical distal segments downstream the TAL. In control animals low labeling indices were found in all investigated segments. After 24 and 72 hours of F + Salt, indices of labeled nuclei were markedly increased in distal segments downstream the TAL, whereas they were significantly reduced in TAL. In proximal tubules increased DNA synthesis rate was apparent only after 72 hours. The data demonstrate that (1.) DNA synthesis rate in nephron segments in vivo varies in parallel with changes of their salt transport activity; (2.) increased DNA synthesis, thus probably cellular proliferation, is a component of the structural response of nephron segments following increased salt transport activity.

Animals↗
PubMed1995

Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb.

Apical membrane Na/H exchange is a principal mechanism of renal proximal tubule Na absorption and H secretion, and thick ascending limb H secretion. Based on current data on Na/H exchanger isoforms (NHE-1 to 5), NHE-3 is the likeliest candidate for the apical membrane isoform. The present study localizes NHE-3 in rat kidney using polyclonal antisera against cytoplasmic epitopes of rat NHE-3. These antisera recognized an approximately 87 kD protein in Na/H exchanger-deficient cells transfected with the rat NHE-3 gene but not in mock-transfected cells. All antisera labeled an approximately 87 kD protein in plasma membranes from cortex and outer medulla. Fractionation of cortical membranes showed labeling in apical but not basolateral membranes. Cross linking studies suggested existence of oligomeric forms of the transporter. Immunohistochemistry showed strong staining of the apical membrane of S1 convoluted, and S2 convoluted tubule with lesser staining of the S2 straight tubule and absent staining of S3. Weak staining was observed in thin descending limbs in the inner stripe and intense staining was seen in the apical membrane of medullary and cortical thick ascending limbs. NHE-3 staining was absent in the remainder of the nephron. In summary, NHE-3 is the isoform responsible for NaCl and NaHCO3 absorption in the proximal convoluted tubule, and NaHCO absorption in the thick ascending limb. In the S3 proximal tubule and the distal convoluted tubule, apical membrane Na/H exchange activity is likely mediated by other isoform(s) of the NHE family.

Amino Acid Sequence↗
PubMed1994

Distribution of ecto-5'-nucleotidase in the rat liver: effect of anaemia.

In the kidney a striking parallel exists between the expression of ecto-5'-nucleotidase and of erythropoietin by renal fibroblasts. It was therefore hypothesized that the expression of ecto-5'-nucleotidase in fibroblasts might be controlled by oxygen tension. In order to test this hypothesis, we examined the distribution of the enzyme in a tissue which displays a defined zonation in respect to oxygen tension, namely in the liver; anaemia was used in order to exaggerate this zonation. The distribution of ecto-5'-nucleotidase was investigated by light and electron microscopy using enzyme and immunohistochemical methods in the livers of healthy and of anaemic rats. Anaemia was produced by haemolysis combined with X-ray irradiation. The enzyme was detected in the bile canaliculi, in the connective tissue of the portal triads and of the central veins, and in fat-storing cells probably corresponding to a special form of fibroblasts. In healthy animals the perisinusoidal ecto-5'-nucleotidase activity was slightly higher in the pericentral than in the periportal area of the acinus whereas the inverse was observed for the staining of bile canaliculi. Anaemia provoked an increase of ecto-5'-nucleotidase in fat-storing cells in the pericentral zone of the acinus and in fibroblasts around the central veins, resulting in steepended gradients along the sinusoids. The intralobular gradient of ecto-5'-nucleotidase in perisinusoidal cells and the effect thereon of anaemia suggest that the expression of the ecto-5'-nucleotidase might be directly or indirectly controlled by local oxygen tension.

5'-Nucleotidase↗