Dennis A. Ausiello

V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway.

The recruitment of the small GTPase Arf6 and ARNO from cytosol to endosomal membranes is driven by V-ATPase-dependent intra-endosomal acidification. The molecular mechanism that mediates this pH-sensitive recruitment and its role are unknown. Here, we demonstrate that Arf6 interacts with the c-subunit, and ARNO with the a2-isoform of V-ATPase. The a2-isoform is targeted to early endosomes, interacts with ARNO in an intra-endosomal acidification-dependent manner, and disruption of this interaction results in reversible inhibition of endocytosis. Inhibition of endosomal acidification abrogates protein trafficking between early and late endosomal compartments. These data demonstrate the crucial role of early endosomal acidification and V-ATPase/ARNO/Arf6 interactions in the regulation of the endocytic degradative pathway. They also indicate that V-ATPase could modulate membrane trafficking by recruiting and interacting with ARNO and Arf6; characteristics that are consistent with the role of V-ATPase as an essential component of the endosomal pH-sensing machinery.

Nitric oxide and atrial natriuretic factor stimulate cGMP-dependent membrane insertion of aquaporin 2 in renal epithelial cells

In collecting duct principal cells, aquaporin 2 (AQP2) is shuttled from intracellular vesicles to the plasma membrane upon vasopressin (VP) stimulation. VP activates adenylyl cyclase, increases intracellular cAMP, activating protein kinase A (PKA) to phosphorylate AQP2 on the COOH-terminal residue, serine 256. Using rat kidney slices and LLC-PK1 cells stably expressing AQP2 (LLC-AQP2 cells), we now show that AQP2 trafficking can be stimulated by cAMP-independent pathways. In these systems, the nitric oxide (NO) donors sodium nitroprusside (SNP) and NONOate and the NO synthase substrate L-arginine mimicked the effect of VP, stimulating relocation of AQP2 from cytoplasmic vesicles to the plasma membrane. Unlike VP, these other agents did not increase intracellular cAMP. However, SNP increased intracellular cGMP, and exogenous cGMP stimulated AQP2-membrane insertion. Atrial natriuretic factor, which signals via cGMP, also stimulated AQP2 translocation. The VP and SNP effects were blocked by the kinase inhibitor H89. SNP did not stimulate membrane insertion of AQP2 in LLC-PK1 cells expressing the phosphorylation-deficient mutant 256SerAla-AQP2, indicating that phosphorylation of Ser256 is required for signaling. Both PKA and cGMP-dependent protein kinase G phosphorylated AQP2 on this COOH-terminal residue in vitro. These results demonstrate a novel, cAMP-independent and cGMP-dependent pathway for AQP2 membrane insertion in renal epithelial cells.

Regulation of Epithelial Sodium Channels by Short Actin Filaments

Cytoskeletal elements play an important role in the regulation of ion transport in epithelia. We have studied the effects of actin filaments of different length on the α, β, γ-rENaC (rat epithelial Na+ channel) in planar lipid bilayers. We found the following. 1) Short actin filaments caused a 2-fold decrease in unitary conductance and a 2-fold increase in open probability (Po) of α,β,γ-rENaC. 2) α,β,γ-rENaC could be transiently activated by protein kinase A (PKA) plus ATP in the presence, but not in the absence, of actin. 3) ATP in the presence of actin was also able to induce a transitory activation of α,β,γ-rENaC, although with a shortened time course and with a lower magnitude of change in Po. 4) DNase I, an agent known to prohibit elongation of actin filaments, prevented activation of α,β,γ-rENaC by ATP or PKA plus ATP. 5) Cytochalasin D, added after rundown of α,β,γ-rENaC activity following ATP or PKA plus ATP treatment, produced a second transient activation of α,β,γ-rENaC. 6) Gelsolin, a protein that stabilizes polymerization of actin filaments at certain lengths, evoked a sustained activation of α,β,γ-rENaC at actin/gelsolin ratios of <32:1, with a maximal effect at an actin/gelsolin ratio of 2:1. These results suggest that short actin filaments activate α,β,γ-rENaC. PKA-mediated phosphorylation augments activation of this channel by decreasing the rate of elongation of actin filaments. These results are consistent with the hypothesis that cloned α,β,γ-rENaCs form a core conduction unit of epithelial Na+ channels and that interaction of these channels with other associated proteins, such as short actin filaments, confers regulation to channel activity.

Na+-dependent sugar transport in a cultured epithelial cell line from pig kidney.

SummaryA Na+-dependent hexose transport system with similar characteristics to that observed in the kidney is retained in a cultured epithelial cell line from pig kidney (LLC-PK1). The active transport of α methyl-d-glucoside (α MGP), a nonmetabolizable sugar, which shares the glucose-galactose transport system in kidney cells is mediated through a Na+-dependent, substrate-saturable process. The kinetic analysis of the effect of Na+ on the uptake of αMGP indicated that the Na+-sugar cotransport system is an affinity type system in which the binding of either sugar or Na+ to carrier increases the affinity for the other ligand without affecting theVmax. The sequence of selectivity for different sugars studied by the inhibition produced in the uptake of αMGP is very similar to that reported in rat kidney, rabbit kidney cortex slices, and rabbit renal brush border membrane vesicles. Phlorizin, even at very low concentration, almost completely inhibits αMGP uptake. Conversely, phloretin at the same low concentration stimulated the sugar accumulation by inhibition of efflux, probably at the level of the basolateral membrane. Sulfhydryl group inhibitors also blocked the αMGP uptake, suggesting that these groups were required for normal functioning of the sugar carrier system. This sugar transport system is an important functional marker to study the molecular events associated with the development of polarization in epithelial cells.

A cell strain cultured from porcine kidney increases cyclic AMP content upon exposure to calcitonin or vasopressin.

Abstract Cells originally dispersed from whole juvenile male Hampshire pig kidney and maintained in monolayer culture, increased cyclic AMP content in response to incubation with salmon calcitonin or antidiuretic hormone. Parathyroid hormone and epinephrine did not affect cyclic AMP content. The apparent K m for arginine vasopressin in the porcine cells was 3.0 nM which is similar to the value obtained in single segments of rabbit kidney tubule. The apparent K m for salmon calcitonin of 2.7 nM is higher than that reported for the rabbit nephron segments, but comparable to the K m obtained in rat kidney homogenates. Exposure of the porcine cells to exogenous prostaglandin E 2 did not affect cyclic AMP responses to other hormones. In the cultured porcine kidney cells the pattern of hormone response is similar to that observed in nephron segments prepared from the medullary portion of the thick ascending limb of the loop of Henle, and these findings suggest that the porcine cells may be related to cells present in the medullary region of the kidney tubule.

Intra-endosomal pH-sensitive Recruitment of the Arf-nucleotide Exchange Factor ARNO and Arf6 from Cytoplasm to Proximal Tubule Endosomes

Kidney proximal tubule epithelial cells have an extensive apical endocytotic apparatus that is critical for the reabsorption and degradation of proteins that traverse the glomerular filtration barrier and that is also involved in the extensive recycling of functionally important apical plasma membrane transporters. We show here that an Arf-nucleotide exchange factor, ARNO (ADP-ribosylation factor nucleotide site opener) as well as Arf6 and Arf1 small GTPases are located in the kidney proximal tubule receptor-mediated endocytosis pathway, and that ARNO and Arf6 recruitment from cytosol to endosomes is pH-dependent. In proximal tubules in situ, ARNO and Arf6 partially co-localized with the V-ATPase in apical endosomes in proximal tubules. Arf1 was localized both at the apical pole of proximal tubule epithelial cells, but also in the Golgi. By Western blot analysis ARNO, Arf6, and Arf1 were detected both in purified endosomes and in proximal tubule cytosol. A translocation assay showed that ATP-driven endosomal acidification triggered the recruitment of ARNO and Arf6 from proximal tubule cytosol to endosomal membranes. The translocation of both ARNO and Arf6 was reversed by V-type ATPase inhibitors and by uncouplers of endosomal intralumenal pH, and was correlated with the magnitude of intra-endosomal acidification. Our data suggest that V-type ATPase-dependent acidification stimulates the selective recruitment of ARNO and Arf6 to proximal tubule early endosomes. This mechanism may play an important role in the pH-dependent regulation of receptor-mediated endocytosis in proximal tubulesin situ.

Physiological importance of endosomal acidification: potential role in proximal tubulopathies.

Purpose of reviewIn recent years, there have been significant advances in our understanding of the molecular mechanisms relating proximal tubule abnormalities to the pathogenesis of renal Fanconi syndrome. This review focuses on the role of intra-endosomal acidification-machinery proteins (V-ATPase, CLC-5, NHE-3), as well as apical receptors (megalin and cubilin), in the receptor-mediated endocytosis pathway and in the pathogenesis of proximal tubulopathies. Recent findingsAnimal models, including CLC-5 and megalin knockout mice, cubilin-deficient dogs and cadmium-toxicity studies in rats, have shed light on defects leading to low-molecular-weight proteinuria. In particular, the important contribution of defective endosomal acidification and membrane-protein recycling to the pathogenesis of the Fanconi syndrome has emerged from these studies. These observations, together with recent findings in patients with Dents disease, Lowes syndrome, autosomal-dominant idiopathic Fanconi syndrome and Imerslund-Grasbeck disease, show that the proteinuria of the Fanconi syndrome is more generalized than previously suspected. High concentrations of polypeptides, including hormones, vitamin-binding proteins and chemokines in urine from these patients and animals may play an important role in the progressive renal failure that is associated with the syndrome. SummaryThe molecular mechanism of proximal tubule protein reabsorption, which is defective in renal Fanconi syndrome, includes a crucial role for endosomal acidification-machinery proteins, in particular the V-ATPase and CLC-5 chloride channels, in the trafficking and acidification-dependent recycling of apical membrane proteins, including the endocytotic receptors megalin and cubilin. An increased understanding of the roles of V-ATPase and CLC-5 in proximal tubule endosomal acidification, in the regulation of the megalin/cubilin-mediated endocytosis pathway and finally in the pathogenesis of human Fanconi syndrome will help in the devising of appropriate strategies for therapeutic intervention for this disorder.

Entry of cholera toxin into polarized human intestinal epithelial cells. Identification of an early brefeldin A sensitive event required for A1-peptide generation.

The effect of brefeldin-A (BFA), a reversible inhibitor of vesicular transport, on cholera toxin (CT)-induced Cl- secretion (Isc) was examined in the polarized human intestinal cell line, T84. Pretreatment of T84 monolayers with 5 microM BFA reversibly inhibited Isc in response to apical or basolateral addition of 120 nM CT (2.4 +/- 0.5 vs. 68 +/- 3 microA/cm2, n = 5). In contrast, BFA did not inhibit Isc responses to the cAMP agonist VIP (63 +/- 7 microA/cm2). BFA had no effect on cell surface binding and endocytosis of a functional fluorescent CT analog or on the dose dependency of CT induced 32P-NAD ribosylation of Gs alpha in vitro. In contrast, BFA completely inhibited (> 95%) the ability of T84 cells to reduce CT to the enzymatically active A1-peptide. BFA had to be added within the first 10 min of CT exposure to inhibit CT-elicited Isc. The early BFA-sensitive step occurred before a temperature-sensitive step essential for apical CT action. These studies show that sequential steps are required for a biological response to apical CT: (a) binding to cell surfaces and rapid endocytosis; (b) early, BFA-sensitive vesicular transport essential for reduction of the A1-peptide; and (c) subsequent temperature-sensitive translocation of a signal (the A1-peptide or possibly ADP-ribose-Gs alpha) to the basolateral domain.

A molecular defect in the vasopressin V2-receptor gene causing nephrogenic diabetes insipidus

Fundamental to the maintenance of water balance in humans is the rate at which the kidneys excrete free water, which is primarily regulated by arginine vasopressin. The antidiuretic action of argin...

Atrial natriuretic factor and cGMP inhibit amiloride-sensitive Na+ transport in the cultured renal epithelial cell line, LLC-PK1

The renal cell culture model, LLC-PK1, which contains an amiloride-sensitive conductive Na+ transport pathway and a Na+/H+ exchanger, was utilized to examine the direct effects of atriopeptin II and cGMP on Na+ transport in epithelial cells. Exposure of cells to atriopeptin II (10(-7) M) increased cGMP production within 2 min of addition to cells in monolayer. Atriopeptin II (10(-7) M) or exogenous 8-bromo-cGMP (10(-3) M) maximally inhibited the uptake of 22Na+ through the conductive pathway which accounted for up to 60% of total 22Na+ uptake. The apparent Ki for this inhibition by atriopeptin II was 2 X 10(-11) M. Amiloride inhibited 22Na+ uptake to a similar extent as atriopeptin II, and the effects of the presence of both agents was not additive. In contrast, neither atriopeptin II nor cGMP blunted the increment in 22Na+ uptake induced by a pH gradient. Thus atriopeptin II can directly inhibit Na+ transport in renal epithelial cells, probably through its stimulation of cGMP.