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N Franki

Publications and source records attributed to N Franki.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1985

Autoradiographic studies of solute transport across the toad bladder.

The autoradiography of diffusible, hydrophilic solutes presents special problems in localization of the labeled solute under study. We present studies of the movement of 14C-labeled urea, and 14C- and 3H-labeled sucrose across the isolated urinary bladder of the toad, a vasopressin-sensitive epithelium, using a technique that avoids exposure to water throughout all processing steps and minimizes error caused by isotope scatter. We have shown a significant increase in 14C urea entry into epithelial cells following vasopressin, and a significant decrease following phloretin, an agent that selectively blocks vasopressin-stimulated urea transport. The autoradiographic technique confirms the luminal site of action of phloretin. Studies of 14C and 3H sucrose labeling show that this molecule is virtually excluded from the cell. The current method of grain counting is capable of yielding reliable information in studies of epithelial transport.

Animals↗
PubMed1985

Evidence for cycling of aggregate-containing tubules in toad urinary bladder.

Antidiuretic hormone (ADH) promotes the fusion of cytoplasmic tubular structures with the luminal membrane of receptor tissues such as toad urinary bladder. To determine whether fusion is a continuous cyclic process, bladders were stimulated with ADH with colloidal gold in the luminal bathing medium. After as little as 15 min of stimulation, gold-filled tubules were seen in the cytoplasm, evidence that cycling was indeed taking place. Serial sections confirmed that these tubules had no connection with the luminal membrane, and had returned to the cytoplasm. Cessation of ADH stimulation, followed by a second stimulation, greatly reduced the number of gold-filled cytoplasmic tubules, suggesting that many tubules were capable of refusion. Mean fusion event diameter underwent significant changes, enlarging at 15 min, and contracting at 60 min. Thus, ADH initiates a process of continuous cycling of cytoplasmic tubules between cytoplasm and luminal membrane.

Animals↗
PubMed1984

Electron-microscopic study of the apical region of the toad bladder epithelial cell.

Antidiuretic hormone (ADH) promotes fusion of cytoplasmic tubules with the luminal membrane and delivery of particles from the tubules to the membrane. The particles are believed to be the water-conducting elements in the membrane. We have employed several scanning (SEM) and transmission electron-microscopic (TEM) techniques to study the relationship of the cytoplasmic tubules to the luminal membrane and to the apical cytoskeleton of the toad bladder epithelial cell. This paper reports the results of freeze-crack SEM and tannic acid-fixed TEM studies, as well as studies with a resinless method of embedding. Freeze-cracked epithelial cells reveal that the tubules are anchored in a matrix of cytoskeleton and granules just below the luminal membrane, and many, if not all, retain their anchorage to the matrix after ADH-induced fusion. Tannic acid-fixed specimens show that the tubules in unstimulated cells lie horizontally. Fusion appears to involve an angulation of the tubules, and this may be the major mode of ADH-induced tubule movement. There are suggestions in the tannic acid sections of filamentous attachments of tubules to the surrounding cytoskeleton. In addition there are prominent microfilament bundles running down the microvilli and a dense concentration of filaments just below the luminal membrane. The presence of these filaments is confirmed in the resinless sections, and their possible role in ADH action is discussed.

Animals↗