Phosphorylation of the catalyic alpha-subunit constitutes a triggering signal for Na+,K+-ATPase endocytosis

Inhibition of Na+,K+-ATPase activity by dopamine is an important mechanism by which renal tubules modulate urine sodium excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of Na+,K+-ATPase activity in response to dopamine is associated with endocytosis of its alpha- and beta-subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from opossum kidney and demonstrate that dopamine-induced endocytosis of Na+,K+-ATPase and inhibition of its activity were accompanied by phosphorylation of the alpha-subunit. Inhibition of both the enzyme activity and its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes suggests a causal link between phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the alpha-subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal from the plasma membrane. Dephosphorylation occurred in the late endosomal compartment. To further examine whether phosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent alpha-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the alpha-subunit with a concomitant decrease in Na+,K+-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent alpha-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser11 and Ser18). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the alpha-subunit is essential for Na+,K+-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.     

Cadmium inhibits vacuolar H(+)-ATPase and endocytosis in rat kidney cortex

The mechanism of cadmium (Cd)-induced damage in the mammalian proximal tubule that is manifested by defects in reabsorption of various compounds, is poorly understood. A vacuolar H(+)-ATPase (V-ATPase) in proximal tubule (PT) brush border and intracellular vesicles may be affected by Cd, and this may influence intracellular vesicle trafficking and reabsorption of the filtered proteins. We studied the effects of Cd on V-ATPase and endocytosis in rat renal PT in vivo and on acidification mechanisms in isolated renal cortical organelles in vitro. The V-ATPase activity in brush border membrane (BBM) from Cd-intoxicated rats was 40% lower compared to that in control animals. Immunofluorescence studies in cortical tissue sections and Western blot studies in BBM from Cd-treated rats showed a strongly decreased abundance of the 31 kDa and 70 kDa V-ATPase subunits. Functional studies in vivo showed a dramatically diminished endocytosis of fluorescein-labeled dextran in PT cells from Cd-treated animals, whereas morphological studies revealed a loss of endocytic invaginations and subapical vesicles in the same cells. In studies in vitro, Cd inhibited V-ATPase activity in a concentration- and time-dependent manner in both BBM and endocytic vesicles, whereas in endocytic vesicles, Cd inhibited ATP-driven intravesicular acidification and accelerated the dissipation of transmembrane pH gradients. We conclude that Cd may impair acidification in cell organelles by (a) causing a loss of V-ATPase protein in their limiting membranes, (b) inhibiting the intrinsic V-ATPase activity, and (c) dissipating the transmembrane pH gradient. This may inhibit endocytosis of filtered proteins and impair vesicle-mediated recycling of some membrane transporters, thus contributing to the loss of reabsorptive capacity of the PT.     

Intra-renal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent's disease

Dent's disease, which is a renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrolithiasis, is associated with inactivating mutations of the X-linked chloride channel, CLC-5. However, the manner in which a functional loss of CLC-5 leads to such diverse renal abnormalities remains to be defined. In order to elucidate this, we performed studies to determine the segmental expression of CLC-5 in the human kidney and to define its intracellular distribution. We raised and characterized antisera against human CLC-5, and identified by immunoblotting an 83 kDa band corresponding to CLC-5 in human kidney cortex and medulla. Immunohistochemistry revealed CLC-5 expression in the epithelial cells lining the proximal tubules and the thick ascending limbs of Henle's loop, and in intercalated cells of the collecting ducts. Studies of subcellular human kidney fractions established that CLC-5 distribution was associated best with that of Rab4, which is a marker of recycling early endosomes. In addition, confocal microscopy studies using the proximal tubular cell model of opossum kidney cells, which endogenously expressed CLC-5, revealed that CLC-5 co-localized with the albumin-containing endocytic vesicles that form part of the receptor-mediated endocytic pathway. Thus, CLC-5 is expressed at multiple sites in the human nephron and is likely to have a role in the receptor-mediated endocytic pathway. Furthermore, the functional loss of CLC-5 in the proximal tubules and the thick ascending limbs provides an explanation for the occurrences of low molecular weight proteinuria and hypercalciuria, respectively. These results help to elucidate further the patho-physiological basis of the renal tubular defects of Dent's disease.     

Na/H exchange and H-K ATPase increase distal tubule acidification in chronic alkalosis

We examined whether H(+)-ATPase, H(+)-K(+)-ATPase, and or Na+/H+ exchange mediates increased distal tubule acidification in animals with chronic metabolic alkalosis using pharmacological inhibitors of these H+ transporters in in vivo-perfused tubules of anesthetized rats. Chronic metabolic alkalosis was induced with furosemide followed by minimum electrolyte diet and HCO3 drinking water. The reduction in net HCO3 reabsorption was greater in distal tubules of alkalotic compared to control animals perfused with Schering 28080 to inhibit H(+)-K(+)-ATPase (-6.4 +/- 0.9 vs. -1.4 +/- 0.5 pmol/mm.min-1, P < 0.02) and with EIPA to inhibit Na+/H+ exchange (-11.1 +/- 1.7 vs. -6.6 +/- 0.9 pmol/mm.min-1, P < 0.01) but was similar in distal tubules of alkalotic and control animals perfused with bafilomycin to inhibit H(+)-ATPase. The greater reduction of distal tubule net HCO3 reabsorption in alkalotic compared to control animals induced by EIPA was eliminated by systemic infusion of the endothelin receptor antagonist bosentan (-4.6 +/- 0.7 vs. -4.4 +/- 0.7 pmol/mm.min-1, P = NS) but the greater reduction induced by Schering 28080 persisted. Urine endothelin-1 (ET-1) excretion was higher in animals with maintained alkalosis (164.5 +/- 23.7 vs. 76.6 +/- 10.8 fmol/day, P < 0.03), but decreased following KCl repletion to a value (86.7 +/- 10.0 fmol/day, P < 0.02 vs. respective before-KCl value) that was not different from that for KCl-repleted control animals (79.9 +/- 8.7 fmol/day, P = NS vs. KCl-repleted alkalotic animals). The data support that augmented distal tubule acidification in alkalotic animals is due to increased H(+)-K(+)-ATPase and Na+/H+ exchange activity, the latter stimulated by endogenous endothelins.     

The apical Cl/HCO3 exchanger of beta intercalated cells

There are two types of intercalated cells of the renal collecting duct; one secretes H+ and the other secretes HCO3-. The H(+)-secreting form has an apical vacuolar H(+)-ATPase and a basolateral Cl/HCO3 exchanger that cross-reacts with antibodies to band 3, the product of the AE1 gene. The HCO3(-)-secreting form has a basolateral vacuolar H(+)-ATPase and an apical Cl/HCO3 exchanger, whose identity has not been established previously. Apical membrane vesicles of beta intercalated cells purified from rabbit kidney cortex contain both an electroneutral Cl/HCO3 exchange activity and polypeptides that react with antibodies to band 3 on Western blots. Furthermore, both primary cultures of HCO3(-)-secreting intercalated cells and an immortalized cell line derived from these cells express AE1 and have an apical Cl/HCO3 exchanger. Apical membranes purified from these cells contain a 100-kDa polypeptide that cross-reacts with antibody to the cytoplasmic domain of band 3. These data suggest that the apical Cl/HCO3 exchanger of HCO3(-)-secreting intercalated cells is band 3.     

Effect of acute increases in filtered HCO3- on renal hydrogen transporters: II. H(+)-ATPase

Adaptive increases in renal bicarbonate reabsorption occur in response to acute increases in filtered bicarbonate (FLHCO3). In a previous study, we showed that an increase in FLHCO3 induced by plasma volume expansion increased the Vmax for Na+/H+ exchange activity in renal cortical brush border membrane vesicles (BBMV), providing a potential mechanism for the adaptive increase in HCO3- reabsorption. The present studies were undertaken to determine whether the increase in FLHCO3 induced by plasma expansion also stimulates the other major H+ transporter in cortical BBMV, the H(+)-ATPase. H(+)-ATPase activity was assessed in BBMV obtained from hydropenic and plasma expanded Munich-Wistar rats, using a NADH-linked ATPase assay. H(+)-ATPase activity was measured as the ouabain and oligomycin-insensitive, bafilomycin A1-sensitive component of total ATPase activity. Acute plasma expansion doubled single nephron FLHCO3, and this change was associated with a 64% increase in the Vmax for H(+)-ATPase activity, with no change in apparent Km. The Vmax for H(+)-ATPase activity correlated directly with whole kidney GFR and FLHCO3 (r = 0.68 and 0.72, respectively), and with single nephron GFR and FLHCO3 (r = 0.76 and 0.80, respectively). Thus, the mechanism for the adaptive increase in proximal tubular HCO3- reabsorption that occurs in response to acute increases in FLHCO3 appears to be related to increased activity of both H(+)-ATPase and Na+/H+ exchange in the apical membrane of the proximal tubule epithelium.     

Calcium uptake via endocytosis with rapid release from acidifying endosomes

A number of specific cellular Ca2+ uptake pathways have been described in many different cell types [1] [2] [3]. The possibility that substantial quantities of Ca2+ could be imported via endocytosis has essentially been ignored, although it has been recognized that endosomes can store Ca2+ [4] [5]. Exocrine cells can release significant amounts of Ca2+ via exocytosis [6], so we have investigated the fate of Ca2+ taken up via endocytosis into endosomes. Ca2+-sensitive and H+-sensitive fluorescent probes were placed in the extracellular solution and subsequently taken up into fibroblasts by endocytosis. Confocal microscopy was used to assess the distribution of fluorescence intensity. Ca2+ taken up by endocytosis was lost from the endosomes within a few minutes, over the same period as endosomal acidification took place. The acidification was inhibited by reducing the extracellular Ca2+ concentration, and Ca2+ loss from the endosomes was blocked by bafilomycin (100 nM), a specific inhibitor of the vacuolar proton ATPase. Quantitative evaluation indicated that endocytosis causes substantial import of Ca2+ because of rapid loss from early endosomes.     

Angiotensin II AT1 receptor/signaling mechanisms in the biphasic effect of the peptide on proximal tubular Na+,K+-ATPase

The present study was designed to determine the cellular signaling mechanisms responsible for mediating the effects of angiotensin II on proximal tubular Na+,K+-ATPase activity. Angiotensin II produced a biphasic effect on Na+,K+-ATPase activity: stimulation at 10(-13) - 10(-10) M followed by inhibition at 10(-7) - 10(-5) M of angiotensin II. The stimulatory and inhibitory effects of angiotensin II were antagonized by losartan (1nM) suggesting the involvement of AT1 receptor. Angiotensin II produced inhibition of forskolin-stimulated cAMP accumulation at 10(-13) - 10(-10) M followed by a stimulation in basal cAMP levels at 10(-7) - 10(-5) M. Pretreatment of proximal tubules with losartan (1nM) antagonized both the stimulatory and inhibitory effects of angiotensin II on cAMP accumulation. Pretreatment of the proximal tubules with pertussis toxin (PTx) abolished the stimulation of Na+,K+-ATPase activity but did not affect the inhibition of Na+,K+-ATPase activity produced by angiotensin II. Pretreatment of the tubules with cholera toxin did not alter the biphasic effect of angiotensin II on Na+,K+-ATPase activity. Mepacrine (10microM), a phospholipase A2 (PLA2) inhibitor, reduced only the inhibitory effect of angiotensin II on Na+,K+-ATPase activity. These results suggest that the activation of AT1 angiotensin II receptors stimulates Na+,K+-ATPase activity via a PTx-sensitive G protein-linked inhibition of adenylyl cyclase pathway, whereas the inhibition of Na+,K+-ATPase activity following AT1 receptor activation involves multiple signaling pathways which may include stimulation of adenylyl cyclase and PLA2.     

Presence of Golgi remnant membranes in the cytoplasm of brefeldin A-treated cells

Brefeldin A (BFA) rapidly blocks secretion, induces disassembly of the Golgi complex and causes a redistribution of Golgi components into the endoplasmic reticulum (ER). In addition to these effects on the exocytotic pathway, BFA has been shown to induce fusion of endosomal membranes with the trans-Golgi network in some cell types. To better understand the mechanism through which BFA disrupts the exocytotic traffic, we have examined its effects on the ultrastructural organization of the Golgi complex. Within minutes of exposure to BFA, the Golgi cisternae were fragmented into a number of small tubules and vesicles, many of which had a non-clathrin coat on their cytosolic surface. In addition, a complex structure consisting of anastomosing tubules and associated vesicles appeared in the cytoplasm of cells incubated with BFA for 10 min or longer. These tubular networks were permanent, distinct structures separated from the ER cisternae. They contained cis, middle, and trans Golgi proteins as well as the lipid analogue C5-DMB-ceramide. Furthermore, secretory proteoglycans en route through the Golgi were retained in the lumen of the tubular networks. As judged by the endocytosis of cationized ferritin, endosomes do not contribute to the formation of these tubular networks. Reassembly of the Golgi complex after BFA incubation involved fragmentation and reorganization of the tubular networks as well as fusion with vesicles budded from the ER. We conclude that although in the presence of BFA the bulk of Golgi membranes are induced to fuse with the ER, as indicated by the detection of Golgi markers in this organelle, a fraction of these membranes remain in the cytoplasm organized as Golgi remnants.     

Effects of La^3＋ on ATPase Activities of Plasma Membrane Vesicles Isolated from Casuarina Equisetifolia Seedlings under Acid Rain Stress

Asamainkindofmembranetransporter ,plasmamembraneATPasesfacilitatethetransportofminerals ,sugars,metabolites ,andothercompoundsacrossthemembraneofthecells ,onlybywhichcouldplantcellscommunicatewithenvironmenttotransfersubstanceandinformation ,whichisessentialforplantgrowthandcellularhomeostasis[1] .Astheexperimentmateri als ,thehighlypurifiedplasmamembranevesiclesfromCasuarinaequisetifoliaseedlingscultivatedunderartificialacidrainstress ,wereisolatedbyaqueoustwo-phasepartitioningmethods .Thest…     

Upregulation of dynamin II expression during the acquisition of a mature pancreatic acinar cell phenotype

Members of the dynamin superfamily are GTPases which have been shown to support receptor-mediated endocytosis in vivo and bind to growth factor receptor-associated proteins in vitro. In acinar cells of the pancreas, receptor-mediated endocytosis is very important for the recycling of membranes after secretory granule release. Therefore, characterization of the molecular machinery responsible for this process is critical for a better understanding of this phenomenon. In this study we sought to determine the expression pattern of the endocytic GTPase dynamin II during pancreatic acinar cell differentiation in developing rat embryos and in dexamethasone-treated AR42J cells using Western blot, Northern blot, and immunocytochemical analyses. During pancreatic development, dynamin immunoreactivity is almost undetectable until day E17 but undergoes significant upregulation in acinar cells starting at E18. In addition, the levels of dynamin mRNA and protein in AR42J cells increase approximately threefold during dexamethasone-induced acinar differentiation. The increase in dynamin levels that occurs in both embryonic pancreatic cells and dexamethasone-treated AR42J cells correlates with the establishment of a more differentiated acinar phenotype. Therefore, these results suggest a potential role for dynamin in supporting receptor-mediated endocytosis in mature pancreatic acinar cells.     

Structure-function study on gastric proton pump

H+, K(+)-ATPase is a proton pump responsible for gastric acid secretion. It actively transport proton and K+ coupled with the hydrolysis of ATP, resulting in the formulation of a 10(6) fold proton gradient across the plasma membrane of parietal cells. The pump belongs to a family of P-type ATPases which include the Na+ pump (Na+, K(+)-ATPase) and the Ca2+ pump (Ca(2+)-ATPase). This review focuses on the structure-function relationship of this proton pump by using functional antibodies, specific inhibitor(s), a fluorescent reagent and site-directed mutants. First we prepared monoclonal antibodies which modified the functions of the H+, K(+)-ATPase . One of the antibodies, HK2032 inhibited the H+, K(+)-ATPase activity and the chloride conductance in gastric vesicles opened by S-S cross-linking, suggesting that the chloride pathway is in the H+, K(+)-ATPase molecule, and that the H+, K(+)-ATPase is a multi-functional molecule. Other antibody, HK4001 inhibited the H+, K(+)-ATPase activity by inhibiting its phosphorylation step. By using this antibody we found an H+, K(+)-ATPase isoform in the rabbit distal colon. Second we found that scopadulcic acid B, a main ingredient of Paraguayan traditional herb, is an inhibitor specific for the H+, K(+)-ATPase. This compound inhibited the H+, K(+)-ATPase activity by stabilizing the K(+)-form of the enzyme. Third we studied the conformational changes of the H+, K(+)-ATPase by observing the fluorescence of FITC-labeled enzyme. H+, K(+)-ATPase did not utilize acetylphosphate instead the ATP as an energy source of active transport, suggesting that the energy transduction system is not common among P-type ATPases. Finally we constructed a functional expression system of the H+, K(+)-ATPase in human kidney cells. By using this functional expression system in combination with site-directed mutagenesis, we studied the significance of amino acid residues in the catalytic centers (a phosphorylation site and an ATP binding site) and the putative cation binding sites. We newly found the sites determining the affinity for cations.     

Stimulation of ouabain-sensitive 86Rb+ uptake and Na+,K+-ATPase alpha-subunit phosphorylation by a cAMP-dependent signalling pathway in intact cells from rat kidney cortex

We investigated in intact cortical kidney tubules the role of PKA-mediated phosphorylation in the short-term control of Na+,K+-ATPase activity. The phosphorylation level of Na+,K+-ATPase was evaluated after immunoprecipitation of the enzyme from 32P-labelled cortical tubules and the cation transport activity of Na+,K+-ATPase was measured by ouabain-sensitive 86Rb+ uptake. Incubation of cells with cAMP analogues (8-bromo-cAMP, dibutyryl-cAMP) or with forskolin plus 3-isobutyl-1-methylxanthine increased the phosphorylation level of the Na+,K+-ATPase alpha-subunit and stimulated ouabain-sensitive 86Rb+ uptake. Inhibition of PKA by H-89 blocked the effects of dibutyryl-cAMP on both phosphorylation and 86Rb+ uptake processes. The results suggest that phosphorylation by PKA stimulates the Na+,K+-ATPase activity.     

Maturation of rat renal tubular response to alpha-adrenergic agonists and neuropeptide Y: a study on the regulation of Na+,K+-ATPase

Na+,K+-ATPase in tubular cells plays a pivotal role for the regulation of renal sodium excretion. In adult rats the activity of this enzyme is inhibited by natriuretic hormones and stimulated by antinatriuretic hormones. Here we have examined the tubular response to alpha-adrenergic agonists and neuropeptide Y (NPY) in both infant and adult rats. In the adult kidney, alpha-adrenergic agonists and NPY stimulate Na+,K+-ATPase activity via Ca2+-dependent pathways. Oxymetazoline, a selective alpha-adrenergic agonist, and NPY failed to stimulate proximal tubular (PT) Na+,K+-ATPase activity in 10-d-old rats in doses of 10(-8) to 10(-5) M and 10(-8) to 10(-6) M, respectively, but when tubules were incubated simultaneously with both oxymetazoline 10(-8) M and NPY 5 x 10(-9) M, stimulation was observed in both 10- and 40-d-old rat PT. This effect was abolished by FK 506, an inhibitor of Ca2+ and calmodulin-dependent protein phosphatase 2B in both age groups. A23187, a calcium ionophore, stimulated Na+,K+-ATPase in both infant and adult PT, but 10-fold higher doses were required for the infant tubules. The effect of alpha-adrenergic agonists and NPY on free intracellular Ca2+ was studied in PT cells in primary culture. The Ca2+ response to each agent was less pronounced in infant than in adult cells. Preincubation with NPY, which increases Ca2+ influx into the cells, enhanced the response to the alpha-adrenergic agonist in both infant and adult cells. The results support the concept that the systems regulating renal tubular Na+, K+-ATPase and sodium metabolism undergo postnatal maturation.     

The effects of clinical pathways for renal transplant on patient outcomes and length of stay

Receptor-mediated transport mechanisms provide a pathway for the trafficking of extracellular macromolecules into (endocytosis) and across (transcytosis) the cell. This comprises the binding of a ligand to a specific cell-surface receptor, clustering of the ligand-receptor complexes in endocytotic vesicles and vesicular sorting. This review focuses on recent advances in cellular and molecular biology pertaining to receptor-mediated endocytosis. A concise overview is presented of current and potential future applications of targeting to RME mechanisms to improve oral macromolecular drug delivery.     

A new action of parathyroid hormone. receptor-mediated stimulation of extracellular acidification in human osteoblast-like SaOS-2 cells

The major physiological function of parathyroid hormone (PTH) is the maintenance of Ca2+/Pi homeostasis via the parathyroid hormone/parathyroid hormone-related protein receptor (PTHR) in kidney and bone. An important consequence of PTHR activation in bone is enhanced local acidification of the extracellular space. Agonist activation of some seven transmembrane-domain receptors increases the extracellular acidification rate (ECAR). We utilized microphysiometry to investigate PTH-stimulated, receptor-mediated increases in ECAR in human osteoblast-like SaOS-2 cells. PTH-(1-34) elicited a large, acute, dose-dependent increase in ECAR with an EC50 of about 2 nM. The PTH-induced increase in ECAR was specific to cells expressing the PTHR and was inhibited by PTHR antagonists. Rapid, partial, homologous desensitization of the PTH-induced increase in ECAR was observed. Incubation of SaOS-2 cells with 8-bromo-cyclic AMP neither mimicked nor abrogated the PTH effect, and PTH stimulated an acute increase in ECAR in cAMP-resistant SaOS-2 Ca#4A cells. Stimulation of ECAR by PTH was independent of transient increases in cytosolic free calcium. Both inhibition and down-regulation of PKC reduced the PTH-induced increase in ECAR. Inhibition of Na+/H+ exchange did not affect the PTH-induced ECAR response. We conclude that PTH caused a receptor-mediated, concentration-dependent, increase in ECAR, which was not dependent on the cAMP/PKA signaling pathway or the Na+/H+ exchanger but involved the action of PKC. Thus, acid production in bone, a physiologically important action of PTH, is not confined to osteoclasts as previously considered but is also mediated by osteoblasts.     

Sepsis increases endocytosis of endotoxin into hepatocytes

BACKGROUND: Chylomicrons bind endotoxins and accelerate their clearance from plasma to the liver. This results in reduced mortality from septic shock in a rodent model. We hypothesized that the clearance of the LPS-chylomicron (LPS-CM) complex by hepatocytes is due to receptor-mediated endocytosis and that sepsis up-regulates this process. METHODS: Three groups of Sprague-Dawley rats; (1) control; (2) pretreated with 10 micrograms/kg LPS 24 hours before treatment; and (3) pretreated with 17-alpha-ethinyl estradiol (EE, 5 mg/kg subcutaneously for 3 days), were infused with labeled I125-LPS alone or with I125-LPS bound to chylomicron. Livers were removed 2.5, 15, and 30 minutes after LPS injection, and hepatic endosomes were isolated from the liver homogenates by serial ultracentrifugation in sucrose gradients. RESULTS: The injection of I125-LPS-CM complexes resulted in higher levels of endosomal I125-LPS in all groups, as compared with I125-LPS alone. In addition, the endosomal uptake of I125-LPS was markedly increased by both LPS and EE pretreatments. CONCLUSIONS: These data strongly suggest a primary role for receptor-mediated endocytosis in the increased clearance of LPS when bound to chylomicron. In addition, exposure to LPS appears to increase the accumulation of LPS in endosomes by a mechanism similar to that of EE, which is known to up-regulate receptor-mediated lipoprotein uptake. This endogenous pathway for the catabolism of endotoxins may provide a teleological explanation for the hypertriglyceridemia observed during sepsis.     

Dorso-ventral patterning of the vertebrate limb and dermatoglyphs with special reference to the pad pattern of mice]

The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) of Na:H exchange (NHE) and vacuolar H+-ATPase (i.e. V-type) activity to the regulation of intracellular pH (pHi). Furthermore, we characterized the effects on pHi regulation of exposure to 1 microM ET-1 under control and acid loaded conditions. With the pH sensitive dye, 2',7' Bis (carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), the control pHi was 7.1 in NaCl (nominally HCO3-free) Ringers. Inhibition of NHE with 100 microM dimethylamiloride (DMA) rapidly decreased pHi by 0.37 units. Similarly, selective inhibition of V-type H+-ATPase with 10 microM bafilomycin A1 decreased pHi by 0.22 units. Following acid loading in NaCl Ringers with a 20 mm NH4Cl prepulse, pHi recovery was partially inhibited by exposure to either Na-free (NMGCl) Ringers, 100 microM DMA or 20 microM bafilomycin A1. Based on decreases in H+ efflux resulting from selective inhibition of NHE and V-type H+ pump activity, NHE activity accounts for 76% of the pHi recovery following acid loading. Under control conditions, ET-1 (1 microM) had no effect on pHi whereas ET-1 completely suppressed pHi recovery following acid loading in NaCl or NMGCl Ringers. This inhibitory effect was largely due to stimulation of ETA because in the presence of BQ-123 (10 microM), a selective ETA receptor antagonist, pHi recovery was completely restored. Suppression of pHi recovery also occurred following stimulation of protein kinase C (PKC) with 10(-7) m phorbol myristate (PMA) whereas 10(-7) m 4 alpha phorbol 12,13 didecanoate (PDD) had no effect. ET-1 failed to suppress pHi recovery after inhibition of PKC with 0.5 microM calphostin C suggesting that the inhibition of pHi recovery by ET-1 is a consequence of PKC stimulation. Similarly, inhibition of Ca2+-dependent calmodulin stimulated CaM II kinase with KN-62 (10 microM) reversed the suppression of pHi recovery by ET-1. Preinhibition of either protein phosphatase (PP), PP-1, PP-2A or PP-2B activity with 1 microM phenylarsine oxide, 10 nm okadaic acid, 10 microM cyclosporin A1 or 20 microM BAPTA, also obviated the suppression of pHi recovery by ET-1. Therefore ETA receptor mediated inhibition of pHi regulation following acid loading could be a consequence of either PKC or CaMII kinase stimulation. Each one of these kinases may in turn phosphorylate and thereby stimulate the activities of PP-1, PP-2A or PP-2B. An increase in the activity of any one of these protein phosphatases could lead to dephosphorylation of the NHE and V-type H+ pump. This alteration may prevent them from becoming adequately stimulated to elicit pHi recovery in response to acid loading.     

A helical coat protein recognition domain of the bacteriophage P22 scaffolding protein

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.     

Dopamine fails to stimulate protein kinase C activity in renal proximal tubules of spontaneously hypertensive rats

We have previously reported that dopamine-1 receptor-mediated activation of phospholipase C is diminished in renal cortical slices of spontaneously hypertensive rats. The present study was carried out to examine the effect of dopamine on protein kinase C (PKC), which is one of the enzymes involved in the signal-transduction pathway leading to dopamine-induced inhibition of Na+/K(+)-ATPase in the renal proximal tubule. Renal proximal tubule suspensions were obtained from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats of 10-12 weeks old. The tubules were incubated with dopamine in the presence or absence of DA-1 receptor antagonist SCH 23390. The PKC activity was measured by using a specific fluorescent peptide substrate (sequence, PKSRTLSVAAK). We found that dopamine produced a concentration-dependent increase in protein kinase C activity in the WKY rats, however, it failed to stimulate PKC activity in the SHR. Peak stimulation of 3.828 +/- 0.35 (ng/micrograms) protein in the WKY rats was observed at dopamine concentration of 1 microM, which was blocked in a concentration-dependent manner by SCH 23390 (0.25 microM). These results provide evidence that dopamine directly stimulates PKC activity via activation of DA-1 receptors in WKY rats. Furthermore, we discovered that dopamine fails to stimulate PKC activity in the SHR. This phenomenon may be responsible for the failure of dopamine to inhibit Na+/K(+)-ATPase activity in the hypertensive animals.