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N Franki

Publications and source records attributed to N Franki.

At least 19 recordsLinked to original sources
PubMed1996

Microtubular disruption prolongs the expression of human bilirubin-uridinediphosphoglucuronate-glucuronosyltransferase-1 gene transferred into Gunn rat livers.

DNA delivered to the liver by asialoglycoprotein receptor-mediated endocytosis is degraded in lysosomes within 48 h. To test the hypothesis that microtubular disruption should promote transgene persistence by interrupting endosomal translocation to lysosomes, plasmids containing bacterial chloramphenicol acetyltransferase (pSV2-CAT) or human bilirubin-UDP-glucuronosyltransferase-1 (pSVK3-hBUGT1) genes were complexed with asialoglycoprotein-polylysine conjugates, and 1 mg of the complexed DNA was injected intravenously into bilirubin-UDP-glucuronosyltransferase-deficient Gunn rats. 30 min before DNA injection, one group received 0.75 mg of colchicine/kg of body weight intraperitoneally, which was shown by immunofluorescent confocal microscopy to disrupt the microtubular network. Control rats received normal saline. In colchicine-pretreated rats receiving pSV2-CAT, hepatic chloramphenicol acetyltransferase activity persisted for 9-14 weeks, whereas in the saline-pretreated group the activity was detectable for 48 h only. In colchicine-pretreated Gunn rats receiving pSVK3-hBUGT1, the DNA persisted in liver for 10 weeks, bilirubin glucuronides were excreted in bile, and serum bilirubin levels declined by 25-35% in 2-4 weeks and remained reduced for 8 weeks. Without colchicine pretreatment, the DNA was detectable in liver for 2 days only, and serum bilirubin levels were not reduced. Thus, microtubular disruption provides a noninvasive method for prolonging the effect of liver-targeted gene therapy.

Animals↗
PubMed1995

Vesicle fusion proteins in rat inner medullary collecting duct and amphibian bladder.

The delivery of water channels to the apical membrane in response to antidiuretic hormone (ADH) requires the targeting of channel-containing vesicles to specific sites in the membrane, followed by fusion and exocytosis. A complex array of proteins is now believed to mediate targeting and fusion in eukaryotic cells. They include N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAP), and cellubrevin, a vesicle-associated protein present in the nerve terminal. We asked whether these proteins are in epithelial cells of rat inner medullary collecting duct (IMCD) and amphibian bladder. Immunoblots on both tissues showed the presence of NSF and alpha-SNAP. Cellubrevin was present in immunoblots of the IMCD, but not the bladder. Immunogold electron microscopy showed NSF, alpha-SNAP, and cellubrevin in rat IMCD cells, with vesicular labeling. In the bladder, NSF was seen on vesicles and aggrephores. We conclude that components of the vesicle-targeting and fusion systems are present in kidney and amphibian bladder and may mediate a wide variety of fusion events, including those initiated by ADH.

Animals↗
PubMed1995

Water channel-carrying vesicles in the rat IMCD contain cellubrevin.

Antidiuretic hormone (arginine vasopressin) induces a cyclic process of docking, fusion, and endocytosis of water channel-containing vesicles in the collecting duct. There is now evidence that docking and endocytosis are mediated by an array of proteins associated with vesicles and target membranes. In recent studies, we have shown that cellubrevin, a member of the vesicle-associated membrane protein family, as well as other docking proteins, are expressed in the rat inner medullary collecting duct. We now show by immunogold electron microscopy that cellubrevin is present on vesicles containing water channels, that it is associated with both coated and uncoated vesicles, and that it is present on the apical membrane. Cellubrevin, therefore, is in a position to mediate one or more steps in arginine vasopressin-induced water channel cycling.

Animals↗
PubMed1994

Depolymerization of hepatocellular microtubules after partial hepatectomy.

Asialoglycoproteins (ASG) are internalized by hepatocytes by ASG receptor (ASGR)-mediated endocytosis. We have shown previously that when a plasmid DNA, pAlb(9-12)CAT (expressing chloramphenicol acetyltransferase driven by an albumin promoter enhancer), was complexed with an ASG-polylysine conjugate and injected intravenously in rats, 80% of the DNA was internalized by the liver. In normal recipient rats, over 95% of the internalized DNA was degraded in 4 h; the plasmid was undetectable after 48 h. In contrast, when 66% hepatectomy was performed 20 min after DNA administration, the internalized DNA persisted for several weeks in cytoplasmic vesicles (Chowdhury, N. R., Wu, C. H., Wu, B. Y., Yerneni, P. C., Bommineni, V. R., and Chowdhury, J. R. (1993) J. Biol. Chem. 268, 11265-11271). Since microtubules are required for the translocation of ligand-containing endosomes to lysosomes, the site of ligand degradation, we hypothesized that persistence of the endocytosed DNA might be related to changes in microtubular structure and function. To test this hypothesis, we examined hepatocellular microtubules by immunofluorescence confocal microscopy. Liver from untreated rats or sham-operated controls showed a network of fibrillar microtubules throughout the cytoplasm. The extent of the microtubular network was substantially reduced 3-6 h after 66% hepatectomy. By 24 h, microtubules had regenerated. Intraportal infusion of cycloheximide (250 mg/kg body weight) 15 min before 66% hepatectomy, prevented microtubular disruption, indicating that protein synthesis is required for this process. Immunotransblot analysis showed that hepatic alpha-tubulin concentration remained unchanged through microtubular disassembly and subsequent reassembly, which is consistent with conservation and reutilization of tubulin released by depolymerization of microtubules.

Animals↗
PubMed1994

Antidiuretic hormone and exocytosis: lessons from neurosecretion.

Many cells, both single and epithelial, are programmed for exocytosis. In most cases, the contents of cytoplasmic vesicles are delivered rapidly and directly to the extracellular fluid. The process has been intensively studied in the chromaffin cell and the nerve terminal, where, as in other cells, exocytosis is under a complex type of cytoskeletal control. An array of vesicle-associated proteins mediates attachment of the vesicles to the cytoskeleton, their release, and their fusion with the plasma membrane. Two functional pools of vesicles, the releasable and reserve pool, carry out immediate and long-term secretory activity. Some of the mediators of neurotransmitter vesicle fusion, originally thought to be restricted to neurosecretory cells, have now been found in nonneuronal cells. The mammalian collecting duct and the amphibian bladder are also engaged in exocytosis. In both epithelia, antidiuretic hormone (ADH) induces the transfer of water channels from cytoplasmic vesicles to the apical cell membrane. The process is slower than in the nerve terminal and ends with channel placement rather than the extrusion of vesicular contents. Nevertheless, there are several respects in which cytoskeletal control, vesicle positioning in the cell, docking, and fusion may prove to resemble the events in neurosecretion. This review begins with a survey of cytoskeletal structure and function in the erythrocyte, the chromaffin cell, and the nerve terminal and then presents current studies of ADH-induced exocytosis, emphasizing common themes in cytoskeletal control.

Actins↗
PubMed1993

The effect of vasopressin on the cytoskeleton of the epithelial cell.

Vasopressin (AVP) promotes the fusion of vesicles containing water channels with the apical membrane of receptor cells in the amphibian bladder and mammalian kidney. Fusion is accompanied by depolymerization of the actin cytoskeleton. In this review, we present the evidence for actin depolymerization by AVP in the whole cell, and the application of confocal microscopy and immunogold electron microscopy in localizing depolymerization to the apical region of the receptor cell.

Actins↗
PubMed1993

Vasopressin depolymerizes apical F-actin in rat inner medullary collecting duct.

In amphibian bladder, arginine vasopressin (AVP) depolymerizes F-actin in the apical region of the granular cell, promoting fusion of water channel-carrying vesicles with the apical membrane. We now report the effect of AVP on F-actin in the mid- and terminal segments of rat inner medullary collecting duct (IMCD2 and IMCD3). In IMCD3, 5 min of stimulation by 2.5-250 nM AVP significantly depolymerized F-actin by 13-24% in whole cell assays employing the rhodamine-phalloidin binding technique. The IMCD2 was more sensitive, responding to subnanomolar (0.25 nM) AVP with 6 +/- 2% depolymerization. Depolymerization occurred as early as 2 min after 2.5 and 25 nM but not 250 nM AVP. 8-Bromoadenosine 3',5'-cyclic monophosphate depolymerized F-actin in IMCD3 at both 2 and 5 min. Immunogold labeling of the apical actin pool in IMCD3 principal cells was reduced by 26 +/- 5% (P < 0.05) by 2.5 nM AVP; the lateral and basal pools showed no significant changes. Capillary endothelial, thin limb of Henle, and intercalated cells showed no changes in immunogold labeling after AVP. Thus reorganization of the apical actin network by AVP is a consistent finding in both mammalian and amphibian target cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

ADH-induced depolymerization of F-actin in the toad bladder granular cell: a confocal microscope study.

Antidiuretic hormone (ADH) induces the fusion of cytoplasmic vesicles containing water channels with the apical membrane of the toad bladder granular cell. Fusion is accompanied by a 30% depolymerization of F-actin. We have used confocal microscopy to determine the region in the cell that undergoes depolymerization. Bladders were mounted in a split chamber, and control halves and halves stimulated by ADH for 15 min were fixed and then stained with rhodamine phalloidin. Vertical sections through the cells were obtained by confocal microscopy, and the fluorescence intensity of the apical and side regions of the cells was determined. To normalize the data, the apex-side intensity was determined for each cell, and these ratios measured for control and ADH-treated halves. In six paired experiments, the ratio for control halves was 3.69 +/- 0.50 and for ADH-treated halves was 2.61 +/- 0.33; the decrease was significant and in good agreement with earlier studies. Thus actin depolymerization takes place in a hormone-sensitive apical pool where vesicle fusion occurs and supports the view that actin depolymerization may be required for fusion.

Actins↗
PubMed1992

Vasopressin decreases immunogold labeling of apical actin in the toad bladder granular cell.

Studies with the confocal microscope have shown that arginine vasopressin (AVP) depolymerizes F-actin in the apical region of the toad bladder granular cell. However, the resolution of the fluorescence microscope is not great enough to reveal the exact pattern of depolymerization or the relative extent to which microvillar and subapical membrane actin pools contribute to overall depolymerization. We have developed an electron microscopic immunogold method that shows a significant decrease in immunogold labeling of actin in the region just below the apical membrane, with the decrease most pronounced in regions adjacent to the microvilli. There was no significant change of immunogold labeling within the microvilli themselves. Our studies show a reorganization of the actin cytoskeleton in the region of the granular cell, where water channel-carrying vesicles are positioned and fuse in response to AVP.

Actins↗
PubMed1992

Effect of cytochalasin D on the actin cytoskeleton of the toad bladder epithelial cell.

Cytochalasins are widely used to determine the role of actin in cellular processes. Their actions include capping of the barbed end of actin filaments as well as dimer formation, nucleation, and polymerization. We determined the effect of cytochalasin D (CD) on F-actin in the toad urinary bladder, an epithelium in which vasopressin depolymerizes F-actin. At a low concentration (0.25 microM), CD depolymerized F-actin in the unstimulated cell; at higher concentrations, there was a progressive reduction of depolymerization until actual polymerization was seen. Vasopressin plus CD produced no greater depolymerization than vasopressin alone, suggesting that CD and vasopressin act to a large extent on the same pool of F-actin. CD plus vasopressin also enhanced the fusion rate of aggrephores compared with vasopressin alone, indicating that intact actin filaments retard aggrephore fusion. Despite the increase in aggrephore fusion, water flow was not enhanced by CD, confirming previous reports that intact actin filaments are required for water channel emergence or stabilization in the apical membrane. Vasopressin plus 1 microM CD produced a striking increase in microvillar length, direct evidence of the polymerizing action of CD in the cell.

Actins↗
PubMed1992

Effects of angiotensin II and arginine vasopressin on F-actin content of cultured mesangial cells.

The actin cytoskeleton of mesangial cells (MC) plays an important role in the contractile response to agonists as well as in the endocytosis of macromolecules. A quantitative study of the F-actin content of MC by the rhodamine-phalloidin binding assay was carried out. Angiotensin II (ANG II) (10(-6) M) significantly increased the F-actin content of MC by 30 min and at later time periods, with increases ranging from 31 to 46%. Arginine vasopressin (10(-8) M) produced a transient decrease of F-actin content of MC at 30 s but then significantly enhanced the F-actin content at later time periods. There was no change in total actin and protein content of MC at 30 min in the presence of either agent. Thus, the increase in F-actin is related to a shift in the G- to F-actin ratio and not to the synthesis of new F-actin. Because the incubation of MC with 1 (5-isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, did not attenuate the ANG II-induced increase in the F-actin content of MC, the shift does not appear to be mediated by the activation of protein kinase C. The removal of external calcium did not prevent the increase in F-actin. Dibutyryl cAMP (5 x 10(-4) M), a smooth muscle cell and MC relaxant, did not alter the F-actin content in MC, and 10(-5) M cytochalasin B significantly lowered F-actin content.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Vasopressin depolymerizes F-actin in toad bladder epithelial cells.

Vasopressin (AVP) induces the rapid fusion of water channel-containing vesicles with the luminal membrane of its target cell. We have carried out a quantitative study of the F-actin content of toad bladder epithelial cells, using the rhodamine phalloidin binding assay. As early as 1 min after AVP stimulation, there is a significant 15% reduction of cellular F-actin, which remains reduced by 20-30% for the duration of action of AVP. Comparable reductions were seen following 8-bromoadenosine 3',5'-cyclic monophosphate, 1-desamino-8-D-arginine vasopressin, and forskolin. F-actin content rose to and then exceeded that of control bladders after AVP washout. Inhibition of prostaglandin synthesis enhanced both water flow and the decrease of F-actin. In the living cell, stabilization of F-actin with NBD-phallacidin selectively inhibited water flow. In view of the rapidity of the response, we conclude that AVP shifts the equilibrium between F-actin and G-actin monomers, and this depolymerization may be required for vesicle fusion.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Evidence that monensin inhibits vasopressin-stimulated water flow at an early step in the receptor-adenylate cyclase sequence.

Monensin, a highly selective sodium ionophore, inhibits vasopressin-stimulated water flow in toad urinary bladder pretreated with naproxen, an inhibitor of prostaglandin synthesis. Inhibition is partially dependent on the presence of sodium in the serosal medium, but not on serosal calcium. We have found that monensin does not inhibit water flow generated by forskolin, cyclic AMP, or isobutyl methyl xanthine (MIX); indeed, an enhancement of water flow was seen following cAMP and MIX, as well as following 0.2 microM forskolin. Our findings suggest that monensin uncouples the vasopressin-receptor-G protein-adenylate cyclase sequence at some early step, by a mechanism that remains unknown, but that may directly or indirectly involve intracellular sodium.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Role of vesicular transport in ADH-stimulated aggregate delivery.

It has been assumed from studies in toad bladder that antidiuretic hormone (ADH)-stimulated particle delivery to the luminal membrane is mediated by particle-carrying tubular structures (aggrephores). We report studies in frog and toad urinary bladder showing that vesicles, rather than aggrephores, appear to play the major role in particle delivery in the frog and that vesicle and aggrephore delivery proceed in parallel in the toad. Our principal evidence for this view is that in the frog, transmission electron microscopy shows virtually no fused aggrephores. Supporting evidence includes the following. 1) Freeze-fracture studies show that the diameters of fusion events delivering particles can be quite small, indicating that they are formed by fused vesicles rather than fused aggrephores. 2) A significant population of small fusion events is also seen in the toad, along with larger fusion events related to both aggrephores and large vesicles. 3) Surface aggregate areas in both species are small, consistent with vesicular delivery. 4) Freeze-fracture replicas indicate delivery from shallow pits. We propose a system of transport of particles in which aggrephores act largely as intermediate storage organelles in the frog and as storage and fusion organelles in the toad.

Animals↗
PubMed1987

Endocytosis by cultured mesangial cells and associated changes in prostaglandin E2 synthesis.

The mechanism of macromolecule uptake by cultured mesangial cells was studied by use of transmission electron microscopy. In parallel, we investigated the effect of macromolecular uptake on prostaglandin E2 (PGE2) formation. Cultured rat mesangial cells were studied in their third passage. As model molecules, we used colloidal gold particles (10 nm diameter) coated either with polyethylene glycol (PEG) or fresh serum (SCG). Mesangial cells were incubated from 1 to 60 min and up to 12 h with either PEG or SCG particles. Endocytosis of SCG significantly exceeded that of PEG particles. The mechanism involved binding to coated pits, followed by formation of coated vesicles (endosomes), and eventually delivery of particles to lysosomes. Pretreatment with cytochalasin B virtually prevented endocytosis of SCG particles, indicating active participation of the cytoskeleton. Determination of PGE2 production in parallel showed that SCG significantly stimulated PGE2 synthesis within minutes, whereas PEG-coated gold had no effect. When gold particles were coated with decomplemented serum instead of fresh serum, the stimulation of PGE2 was partially, but not completely, prevented, indicating that complement may be one, but not the only ligand responsible for enhanced PGE2 production. Stimulation of PGE2 synthesis by SCG was not dependent on actual endocytosis, as it was not altered by cytochalasin B pretreatment. Thus, surface ligand-receptor interaction may be sufficient to trigger PGE2 synthesis. The interaction between mesangial endocytosis and PGE2 production may be important for glomerular pathophysiology.

Animals↗
PubMed1987

Morphological aspects of the action of ADH.

Early studies employing biophysical techniques provided a model for ADH-induced water flow in which the number of small water-conducting channels in the outer facing membrane is increased by the hormone. With the development of new concepts and techniques in cell biology, the problem of ADH action now centers on organelle movement, fusion, endocytosis and vesicular traffic with the cell. In this review, endocytosis and vesicular traffic are discussed, and their application to the action of ADH is considered.

Animals↗
PubMed1986

Evidence that the heads of ADH-sensitive aggrephores are clathrin-coated vesicles: implications for aggrephore structure and function.

Antidiuretic hormone (ADH) induces the fusion of long tubular organelles (aggrephores) with the luminal membrane of the receptor cell, and the delivery of particle aggregates to the membrane. Water flow is believed to take place through the particles. Nothing is known about the origin of the particle aggregates, their incorporation into the aggrephores, or the possible relationship of the aggrephores to the vesicular traffic that takes place in the epithelial cell. In the present studies of the ADH-sensitive epithelial cells of the toad urinary bladder, we have found that the spherical heads of the aggrephores appear to be clathrin-coated vesicles. We propose that vesicles originating from sites such as the Golgi or the luminal membrane may be engaged in aggrephore assembly, the resupply of particle aggregates to the aggrephores, and/or the removal of aggregates, and that the aggrephores may be central points in the pattern of vesicular traffic in the cell.

Animals↗