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S Nielsen

Publications and source records attributed to S Nielsen.

At least 253 records · Page 14Linked to original sources
PubMed1994

Ultrastructure, pharmacologic inhibition, and transport selectivity of aquaporin channel-forming integral protein in proteoliposomes.

Reconstitution of highly purified aquaporin CHIP (channel-forming integral protein) into proteoliposomes was previously shown to confer high osmotic water permeability (Pf) to the membranes [Zeidel et al. (1992) Biochemistry 31, 7436-7440]. Here we report detailed ultrastructural, pharmacologic, and transport studies of human red cell CHIP in proteoliposomes. Freeze-fracture and transmission electron microscopy revealed a uniform distribution of CHIP which was incorporated into the membranes in both native and inverse orientations. Morphometric analysis of membranes reconstituted at three different concentrations of CHIP revealed that the intramembrane particles correspond to tetramers or possible higher order oligomers, and the Pf increased in direct proportion to the CHIP density. Proteolytic removal of the 4-kDa C-terminal cytoplasmic domain of CHIP did not alter the Pf or oligomerization in red cell membranes. CHIP exhibited a similar conductance for water when reconstituted into membranes of varied lipid compositions. The sensitivities of CHIP-mediated Pf to specific sulfhydryl reagents were identical to known sensitivities of red cell Pf, including a delayed response to p-(chloromercuri)benzenesulfonate. CHIP did not increase the permeability of the proteoliposome membranes to H+/OH- or NH3. These studies demonstrate that CHIP proteoliposomes exhibit all known characteristics of water channels in native red cells and therefore provide a defined system for biophysical analysis of transmembrane water movements.

Aquaporin 1↗
PubMed1994

Examination of rat salivary glands for the presence of the aquaporin CHIP.

The aquaporin CHIP, AQP-CHIP, is a 28-kDa integral membrane protein that functions as a water channel in kidney and red blood cells. We have examined rat salivary glands for the presence of this molecule. In Northern blot analyses an AQP-CHIP cDNA hybridized to rat kidney mRNA approximately 20-fold more than to mRNA from the rat parotid gland. Western blot analyses, using antisera against human AQP-CHIP, showed that AQP-CHIP was present in crude membranes from rat salivary glands to a much lesser extent than in rat kidney membranes. A 2.8 kb cDNA encoding AQP-CHIP was also isolated from an adult rat parotid gland cDNA library. Immunolocalization studies using thin and ultrathin cryosections and light and electron microscopy, however, revealed that no glandular epithelium was labeled. In contrast, significant immunolabeling was present in microvascular endothelial cells. Non-fenestrated endothelia of capillaries and venules exhibited labeling on both their apical and basolateral membranes. The present study demonstrates that AQP-CHIP is not involved directly in fluid secretion from salivary epithelial cells. Further, this study provides strong evidence that AQP-CHIP is an endothelial cell water channel found within salivary gland tissue which may contribute to water permeability in the capillary beds.

Animals↗
PubMed1994

Season of birth in infantile autism and other types of childhood psychoses.

Season of birth in 328 children with infantile autism and other types of childhood psychoses born between 1945 and 1980 was compared with that of a control population born in the same period. For some parts of the period (the years 1951-56, 1963-68 and 1975-80 combined) an excess of March born boys with infantile autism was found. For boys with autistic-like disorders, born in the same period a maximum occurred in November, while no seasonality was observed for a borderline sample.

Adolescent↗
PubMed1994

Enalapril versus bendroflumethiazide in type 2 diabetes complicated by hypertension.

In a double-blind, double-dummy, randomized, multi-centre study, the effects of bendroflumethiazide vs. enalapril on blood pressure, glycaemic control, lipoprotein concentrations and albuminuria were compared in non-proteinuric, hypertensive type 2 diabetic patients; they were treated for 20 weeks with either bendroflumethiazide 2.5-5.0 mg (n = 59) or enalapril 10-20 mg (n = 55). Age, fasting plasma glucose, HbA1c and BMI were similar in the groups. Systolic and diastolic blood pressure were reduced in both groups. Bendroflumethiazide was accompanied by minor but significant elevations in fasting plasma glucose and serum C-peptide. HbA1c was increased during both treatments. Lipoproteins and urinary albumin/creatinine ratio were stable. Bendroflumethiazide caused a decrease in serum potassium and an increase in serum urate. No significant correlations were observed between the decline in blood pressure and changes in the metabolic risk factors. Baseline levels of age, sex, BMI, blood pressure or urinary albumin/creatinine ratio were not related to changes in blood pressure, metabolic parameters or urinary albumin/creatinine ratio.

Adult↗
PubMed1994

Long-term regulation of collecting duct water permeability: freeze-fracture analysis of isolated perfused tubules.

Terminal inner medullary collecting duct (IMCD) segments from water-restricted rats have high osmotic permeabilities despite the absence of vasopressin (AVP). We performed freeze-fracture analysis of individual IMCD segments from such animals following measurement of their water permeability (Pf). IMCD segments from control rats did not have a high Pf in the absence of AVP (88 +/- 15 microns/s) and had a low incidence of E-face intramembrane particle (IMP) clusters (9.6 +/- 2.7 clusters/100 microns 2). Segments exposed to 0.1 nM AVP in vitro had enhanced Pf (1,060 +/- 210 microns/s) and cluster incidence (122 +/- 33 clusters/100 microns 2). IMCD segments isolated from rats dehydrated for 48 h and perfused without AVP exposure had an elevated Pf (605 +/- 71 microns/s) and a high incidence of clusters (166 +/- 36 clusters/100 microns 2). There also was an increase in the number of single particles between clusters in tubules from dehydrated rats (2.5-fold) and in AVP-treated tubules (3.6-fold). These findings indicate that IMP clusters are associated with high water permeability in tubules from dehydrated animals independent of continued AVP exposure. The increased incidence of particles between clusters suggests that water channels may also occur outside of cluster domains.

Animals↗
PubMed1994

Endocytosis in renal proximal tubules. Experimental electron microscopical studies of protein absorption and membrane traffic in isolated, in vitro perfused proximal tubules.

The present study provides at least partly answers to some of the questions outlined in the introduction (see also Figs. 2 and 3): Endocytosis and intracellular transport of ferritin, HRP and insulin tracers (125I-insulin, native insulin and insulin-gold) was followed by use of EM-autoradiography, immunocytochemistry and cytochemistry. Proteins are internalized into endocytic vacuoles and transferred to the lysosomes for degradation. Tracers were not transferred to the Golgi apparatus. 125I-insulin is internalized by specific receptor mediated endocytosis from the apical plasma membrane, substantiating the hypothesis that specific endocytosis receptors are responsible for reabsorption of certain proteins. The binding sites are localized in endocytic invaginations and in the microvillus membrane. The binding sites in the invaginations are responsible for endocytosis, whereas the function of the microvilli binding sites is unclear, but they possess the ability to migrate in the plane of the microvillus membrane. Binding to specific binding sites and subsequent internalization of insulin takes place with high efficiency corresponding to more than 50% of the perfused load. Not all proteins are reabsorbed with high efficiency e.g. EGF which has similar molecular weight and pI is shown to be reabsorbed with substantially lower efficiency (about 4%). Binding and absorption efficiency of insulin may also change due to alterations in flow rate and perfused loads of protein: The load determines the magnitude of uptake and the flow rate determines the efficiency in binding and uptake. These changes are suggested to be caused by concomitant changes in the mean luminal concentration. The reabsorption process for insulin is efficient and of large capacity, and is only saturable (Michaelis-Menten kinetics) at very high concentrations of insulin. The proximal tubular internalization and degradation of 125I-insulin reach steady state rapidly. The processing can be described by a two-compartment model with t1/2 for transfer of 125I-insulin to lysosomes of 8.5 min and for lysosomal degradation of 72 min. 125I-PYY a linear peptide with similar molecular weight as EGF and insulin is not endocytosed but extracted with high efficiency (75% removed) by degradation by brush border peptidases and a substantial transtubular transport of TCA-precipitable PYY takes place by a paracellular route. A small vesicular transport of colloidal tracers was demonstrated constituting about 0.5% of the endocytosed amount. A method for covalently cross-linking insulin tracers to apical binding sites is described and evaluated. Recycling of apical binding sites was estimated to be very efficient and did not involve lysosomes or the Golgi apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)

Absorption↗
PubMed1993

Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney.

Vasopressin (antidiuretic hormone) regulates body water balance by controlling water permeability of the renal collecting ducts. The control mechanisms may involve alterations in the number or unit conductance of water channels in the apical plasma membrane of collecting-duct cells. How this occurs is unknown, but indirect evidence exists for the "shuttle" hypothesis, which states that vasopressin causes exocytic insertion of water channel-laden vesicles from the apical cytosol. To test key aspects of the shuttle hypothesis, we have prepared polyclonal antisera against the recently cloned collecting-duct water channel protein and used the antisera in immunolocalization studies (light and electron microscopic levels) in thin and ultrathin cryosections from rat kidney. Labeling was seen exclusively in collecting-duct principal cells and inner medullary collecting-duct cells. Apical membrane labeling was intense. There was heavy labeling of abundant small subapical vesicles and of membrane structures within multivesicular bodies. In addition, labeling of basolateral plasma membranes in inner medullary collecting ducts was present. Depriving rats of water for 24 or 48 hr markedly increased collecting-duct water-channel protein expression determined by immunoblotting and immunolabeling. These results are compatible with at least two complementary modes of water-channel regulation in collecting-duct cells: (i) control of channel distribution between the apical membrane and a reservoir in subapical vesicles (shuttle hypothesis) and (ii) regulation of the absolute level of expression of water-channel protein.

Amino Acid Sequence↗
PubMed1993

Epithelial glycoprotein-330 mediates endocytosis of plasminogen activator-plasminogen activator inhibitor type-1 complexes.

Epithelial glycoprotein 330 (gp330) is structurally similar to the multifunctional alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP), gp330 and alpha 2MR/LRP bind Ca2+ with high affinity, and both receptors bind and mediate endocytosis of alpha 2MR-associated protein (RAP). In the present report, we describe that affinity-purified gp330 from rabbit renal cortex binds plasminogen activator inhibitor type-1 (PAI-1) complexed with urokinase-type plasminogen activator (uPA). alpha 2M-methylamine, which binds with high affinity to alpha 2MR/LRP, did not bind to gp330. The apparent Kd for binding of uPA.PAI-1 complexes was about 0.8 nM at 4 degrees C. The binding was calcium-dependent and inhibited by recombinant RAP (rRAP) and tissue type plasminogen activator-PAI-1 complexes. Thin sections of rabbit renal proximal tubules bound 125I-labeled uPA.PAI-1 and rRAP in the apical part of proximal tubules corresponding to the localization of gp330. The binding of 125I-uPA.PAI-1 complexes in tubules was abolished by excess unlabeled rRAP, and a rRAP-inhibitable endocytosis and degradation of labeled uPA.PAI-1 complexes was demonstrated by perfusion of isolated rabbit proximal tubules. The results establish an endocytotic function of gp330 and suggest that gp330 is an important component of the fibrinolytic system in gp330-containing epithelial as found in, for example, kidney and lung.

Amino Acid Sequence↗
PubMed1993

Distribution of the aquaporin CHIP in secretory and resorptive epithelia and capillary endothelia.

The existence of water-selective channels has been postulated to explain the high water permeability of erythrocytes and certain epithelial cells. The aquaporin CHIP (channel-forming integral membrane protein of 28 kDa), a molecular water channel, is abundant in erythrocytes and water-permeable segments of the nephron. To determine whether CHIP may mediate transmembrane water movement in other water-permeable epithelia, membranes of multiple organs were studied by immunoblotting, immunohistochemistry, and immunoelectron microscopy using affinity-purified anti-CHIP IgG. The apical membrane of the choroid plexus epithelium was densely stained, implying a role for CHIP in the secretion of cerebrospinal fluid. In the eye, CHIP was abundant in apical and basolateral domains of ciliary epithelium, the site of aqueous humor secretion, and also in lens epithelium and corneal endothelium. CHIP was detected in membranes of hepatic bile ducts and water-resorptive epithelium of gall bladder, suggesting a role in bile secretion and concentration. CHIP was not detected in glandular epithelium of mammary, salivary, or lacrimal glands, suggesting the existence of other water-channel isoforms. CHIP was also not detected within the epithelium of the gastrointestinal mucosa. CHIP was abundant in membranes of intestinal lacteals and continuous capillaries in diverse tissues, including cardiac and skeletal muscle, thus providing a molecular explanation for the known water permeability of certain lymphatics and capillary beds. These studies underscore the hypothesis that CHIP plays a major role in transcellular water movement throughout the body.

Animals↗
PubMed1993

Clinical follow-up of phototherapeutic keratectomy for treatment of corneal opacities.

We performed phototherapeutic keratectomy with a 193-nm excimer laser on 18 sighted patients (18 eyes) to treat corneal opacities. The corneal opacities were caused by corneal dystrophies in five patients; corneal scars secondary to corneal ulcers in six patients; corneal scar secondary to trauma in four patients; and band keratopathy, atopy, or corneal calcification in three patients. Mean follow-up was eight months (range, two to 18 months). Corneal clarity improved in 14 of the 18 eyes (77.7%). Four patients, three with band keratopathy or calcification and one with postinfectious corneal scar, did not improve. Uncorrected visual acuity improved in 11 patients, did not improve in five patients (including the four patients in whom treatment failed), and decreased in another two patients, apparently because of an increase in irregular astigmatism. A hyperopic shift was observed in ten patients. None of the successfully treated eyes developed surface problems or recurrence of the disease during the follow-up. Phototherapeutic keratectomy thus appears to be a safe and effective alternative to penetrating keratoplasty in some patients with selected anterior stromal opacities.

Adult↗
PubMed1993

Purification of human blood basophils by negative selection using immunomagnetic beads.

A simple and rapid method for isolating highly purified and functionally intact basophils from normal human blood is described. Blood from healthy volunteers was centrifuged through a two layer Percoll density gradient. The majority of the basophils were recovered between Percoll layers with densities of 1.070 and 1.080, constituting 45.6% of total leukocytes. Lymphocytes, monocytes and neutrophils were extracted from this fraction using a panel of mabs in a direct or indirect selection procedure using immunomagnetic beads (Dynabeads) coated with sheep anti-mouse IgG. These negative immunoselection procedures produced a high yield of basophils with a mean purity of 97.8% (range 93.0-99.5%) and 97.0% (range 96.2-99.0%) using the direct and indirect method, respectively. The cells isolated by this method are viable, release histamine to various stimuli in a normal manner, and appear morphologically normal in transmission electron microscopy.

Basophils↗
PubMed1993

Renal function and insulin sensitivity during simvastatin treatment in type 2 (non-insulin-dependent) diabetic patients with microalbuminuria.

The effect of simvastatin (10-20 mg/day) on kidney function, urinary albumin excretion rate and insulin sensitivity was evaluated in 18 Type 2 (non-insulin-dependent) diabetic patients with microalbuminuria and moderate hypercholesterolaemia (total cholesterol > or = 5.5 mmol.l-1). In a double-blind, randomized and placebo-controlled design treatment with simvastatin (n = 8) for 36 weeks significantly reduced total cholesterol (6.7 +/- 0.3 vs 5.1 mmol.l-1 (p < 0.01)), LDL-cholesterol (4.4 +/- 0.3 vs 2.9 +/- 0.2 mmol.l-1 (p < 0.01)) and apolipoprotein B (1.05 +/- 0.04 vs 0.77 +/- 0.02 mmol.l-1 (p < 0.01)) levels as compared to placebo (n = 10). Both glomerular filtration rate (mean +/- SEM) (simvastatin: 96.6 +/- 8.0 vs 96.0 +/- 5.7 ml.min-1 x 1.73 m-2, placebo: 97.1 +/- 6.7 vs 88.8 +/- 6.0 ml.min-1 x 1.73 m-2)(NS) and urinary albumin excretion rate (geometric mean x/divided by antilog SEM) (simvastatin: 18.4 x/divided by 1.3 vs 16.2 x/divided by 1.2 microgram.min-1, placebo 33.1 x/divided by 1.3 vs 42.7 x/divided by 1.3 micrograms.min-1)(NS) were unchanged during the study. A euglycaemic hyperinsulinaemic clamp was performed at baseline and after 18 weeks in seven simvastatin- and nine placebo-treated patients. Isotopically determined basal and insulin-stimulated glucose disposal was similarly reduced before and during therapy in both the simvastatin (2.0 +/- 0.1 vs 1.9 +/- 0.1 (NS) and 3.1 +/- 0.6 vs 3.1 +/- 0.7 mg.kg-1 x min-1 (NS)) and the placebo group (1.9 +/- 0.1 vs 1.8 +/- 0.1 (NS) and 4.1 +/- 0.6 vs 3.8 +/- 0.2 mg.kg-1 x min-1 (NS)).(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1993

Monitoring diabetic nephropathy: glomerular filtration rate and abnormal albuminuria in diabetic renal disease--reproducibility, progression, and efficacy of antihypertensive intervention.

The principal end point in the evaluation of treatment in incipient and overt diabetic nephropathy is rate of decline in glomerular filtration rate (GFR). Therefore, information on reproducibility of GFR measurements is essential in the planning and evaluation of clinical trials. We studied reproducibility of GFR measurements in insulin-dependent and non-insulin-dependent diabetes mellitus patients using, respectively, a constant-infusion technique with urine collection and labeled iothalamate as a tracer marker and a single-shot procedure using Cr-EDTA, measuring the GFR from the decline in plasma level after bolus injection. The coefficient of variance in the insulin-dependent patients was from 7.5% to 8.8% with repeated measurements. In longitudinal studies with several measurements the mean coefficient of variances varied between 7.4% and 3.4%. In the non-insulin-dependent patients the coefficient of variances between two tests were 7.0% and 5.3% for normoalbuminuric and microalbuminuric patients, respectively. In cross-sectional studies as well as in longitudinal studies, it has been consistently shown that GFR is well preserved and at a supranormal level in patients with normoalbuminuria and microalbuminuria. A decline in GFR appears to start around the transition from microalbuminuria to overt diabetic renal disease, although more detailed studies are needed to support this finding. With regard to intervention trials, several studies document that microalbuminuria can be reduced by effective antihypertensive treatment, particularly with angiotensin-converting enzyme inhibitors, also in patients with normal or close to normal blood pressure. Preliminary results from long-term studies suggest that reduction in microalbuminuria in these patients is associated with preservation of GFR and, thus, apparently renoprotection. In patients with overt renal disease, it has been consistently shown that antihypertensive treatment reduces albuminuria as well as the rate of decline in GFR. This is also observed with combined treatment regimens, for instance beta blockers or angiotensin-converting enzyme inhibitors combined with diuretics, or the three types of drugs in combination.

Albuminuria↗
PubMed1993

IGF-II/Man-6-P receptors in rat kidney: apical localization in proximal tubule cells.

The present study was performed to determine the localization of insulin-like growth factor II (IGF-II) binding in the rat nephron and, furthermore, to determine directly the distribution of the IGF-II/Man-6-P receptor in the proximal tubule. The IGF-II/Man-6-P receptor was visualized by immunohisto- and cytochemical techniques, and the binding of IGF-II was shown by light and electron microscope autoradiography applying 125I-IGF-II directly on cryosections. The tubular uptake of IGF-II was studied by autoradiography after in vivo injection of 125I-IGF-II. The light microscope immunolabeling was confined to a relatively narrow band in the apical part of the proximal tubule cells just below the brush border, and was strongest in the first portion of the proximal tubule, segment 1 (S1), with decreasing intensity in segment 2 (S2) and weaker in segment 3 (S3). Several cytoplasmic bodies were labeled but no label was observed in the basolateral part of the cells. We did not detect any labeling of glomeruli or other segments of the nephron. Electron microscope immunocytochemistry in the proximal tubule revealed a strong labeling in apical endocytic invaginations, small endocytic vacuoles, and large endocytic vacuoles, weaker in lysosomes and dense apical tubules. Light microscope autoradiography after incubation of cryosections with labeled IGF-II demonstrated an intense labeling just beneath the brush border, especially in S1 of the proximal tubule, and, in addition, the corresponding electron microscope autoradiography revealed an intense labeling over large endocytic vacuoles and lysosomes. These results were confirmed and extended by competitive binding experiments and quantitative immunocytochemistry. After i.v. injection of 125I-IGF-II autoradiographic grains were almost exclusively observed in the electronmicroscope over endocytic vacuoles and lysosomes in the S1 of the proximal tubule. The present study shows that the IGF-II/Man-6-P receptor is located in components of the vacuolar system in the apical cytoplasm of rat renal proximal convoluted tubule cells, mainly in S1. This localization was seen to be consistent using various immunolabeling and autoradiographic techniques in vivo and in vitro.

Animals↗
PubMed1993

CHIP28 water channels are localized in constitutively water-permeable segments of the nephron.

The sites of water transport along the nephron are well characterized, but the molecular basis of renal water transport remains poorly understood. CHIP28 is a 28-kD integral protein which was proposed to mediate transmembrane water movement in red cells and kidney (Preston, G. M., T. P. Carroll, W. B. Guggino, and P. Agre. 1992. Science [Wash. DC]. 256:385-387). To determine whether CHIP28 could account for renal epithelial water transport, we used specific polyclonal antibodies to quantitate and localize CHIP28 at cellular and subcellular levels in rat kidney using light and electron microscopy. CHIP28 comprised 3.8% of isolated proximal tubule brush border protein. Except for the first few cells of the S1 segment, CHIP28 was immunolocalized throughout the convoluted and straight proximal tubules where it was observed in the microvilli of the apical brush border and in basolateral membranes. Very little CHIP28 was detected in endocytic vesicles or other intracellular structures in proximal tubules. Uninterrupted, heavy immunostaining of CHIP28 was also observed over both apical and basolateral membranes of descending thin limbs, including both short and long loops of Henle. These nephron sites have constitutively high osmotic water permeabilities. CHIP28 was not detected in ascending thin limbs, thick ascending limbs, or distal tubules, which are highly impermeable to water. Moreover, CHIP28 was not detected in collecting duct epithelia, where water permeability is regulated by antidiuretic hormone. These determinations of abundance and structural organization provide evidence that the CHIP28 water channel is the predominant pathway for constitutive transepithelial water transport in the proximal tubule and descending limb of Henle's loop.

Animals↗
PubMed1993

Sorting and recycling efficiency of apical insulin binding sites during endocytosis in proximal tubule cells.

Intracellular traffic and recycling of apical insulin binding sites are examined in isolated, perfused proximal tubules. The endocytic binding sites were specific as revealed by 90% reduction in 125I-labeled insulin binding by 10(-5) M insulin. The traffic was followed by developing a chemical cross-linking method to covalently label the binding sites. Only 3% of cross-linked insulin-gold was transported to the lysosomes, reflecting high sorting and recycling efficiency. Correspondingly, only 4% of cross-linked 125I-insulin was degraded, and only 5% of the electron microscopy-autoradiographic grains was associated with lysosomes. No label was transferred to the Golgi apparatus; thus neither lysosomes nor Golgi apparatus is involved in the recycling. In contrast, approximately 40% of non-cross-linked ligand was transferred to the lysosomes. Tubules first pretreated with cross-linker and then perfused with insulin-gold or 125I-labeled insulin-like growth factor I revealed lysosomal accumulation and degradation at control levels. Thus the cross-linker does not interfere with membrane or protein processing. The study also provides evidence for a vesicular transtubular transport because insulin-gold was transcytosed to the basolateral part of the cells and to the intracellular spaces (0.5%). In contrast cross-linked label was never observed in intercellular spaces, suggesting sorting of apical binding sites, a mechanism contributing to maintenance of cell polarity. In conclusion, traffic, sorting, and recycling of binding sites take place with high efficiency.

Animals↗
PubMed1993

Endocytosis in proximal tubule cells involves a two-phase membrane-recycling pathway.

The aims of the present study were to examine the initial events of endocytosis with respect to migration, internalization, and recycling of protein binding sites on the luminal plasma membrane of genuine renal proximal tubule cells (not cultured cells), and in particular the sequential involvement of dense apical tubules (DAT). Isolated rabbit proximal tubules were first perfused at low temperature (9 degrees C) with insulin-gold to label the binding sites and then with disuccinimidyl suberate (DSS) for 20 min to covalently cross-link insulin-gold and binding sites. The tubules were subsequently perfused for 4-30 min at 25 or 37 degrees C before fixation to follow the pathways of the binding sites. The present experimental approach allowed a temporal separation of the subcellular processes involved in endocytosis. The identity and organization of the different organelles were certified by serial sectioning of the experimental tubules. In tubules fixed directly at low temperature after cross-linking the label was located on the microvilli membranes and in plasma membrane invaginations. The labeling of microvilli decreased with time of perfusion at 25 degrees C in parallel with a simultaneous increase of labeling of invaginations, suggesting lateral migration of the binding sites. The invaginations pinched off from the surface to form endocytic vesicles. The binding sites were subsequently trafficked either to 1) tubular elongations from endocytic vesicles or small vacuoles, which again form DAT, or 2) in a later sequence trafficked to larger vacuoles, from which tubular elongations and DAT also form. The elongations formed the DAT that transported binding sites back to the luminal plasma membrane. Lysosomes and Golgi apparatus were not involved in membrane recycling. Thus the present study provides evidence for a two-phase recycling pathway of membrane proteins involved in endocytosis.

Animals↗