PKA-dependent ENaC trafficking requires the SNARE-binding protein complexin.

Acute regulation of epithelial sodium channel (ENaC) function at the apical surface of polarized kidney cortical collecting duct (CCD) epithelial cells occurs in large part by changes in channel number, mediated by membrane vesicle trafficking. Several soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNARE) have been implicated in this process. A novel SNARE-binding protein, complexin, has been identified in nervous tissue which specifically binds to and stabilizes SNARE complexes at synaptic membranes to promote vesicle fusion. To test whether this protein is present in mouse CCD (mCCD) cells and its possible involvement in acute ENaC regulation, we cloned complexin (isoform II) from a mouse kidney cDNA library. Complexin II mRNA coexpressed with alpha-, beta-, and gamma-ENaC subunits in Xenopus laevis oocytes reduced sodium currents to 16 +/- 3% (n = 19) of control values. Short-circuit current (I(sc)) measurements on mCCD cell lines stably over- or underexpressing complexin produced similar results. Basal I(sc) was reduced from 12.0 +/- 1.0 (n = 15) to 2.0 +/- 0.4 (n = 15) and 1.8 +/- 0.3 (n = 17) microA/cm(2), respectively. Similarly forskolin-stimulated I(sc) was reduced from control values of 20.0 +/- 2 to 2.7 +/- 0.5 and 2.3 +/- 0.4 microA/cm(2) by either increasing or decreasing complexin expression. Surface biotinylation demonstrated that the complexin-induced reduction in basal I(sc)was due to a reduction in apical membrane-resident ENaC and the inhibition in forskolin stimulation was due to the lack of ENaC insertion into the apical membrane to increase surface channel number. Immunofluorescent localization of SNARE proteins in polarized mCCD epithelia detected the presence of syntaxins 1 and 3 and synaptosomal-associated protein of 23 kDa (SNAP-23) at the apical membrane, and vesicle-associated membrane protein (VAMP2) was localized to intracellular compartments. These findings identify SNAREs that may mediate ENaC-containing vesicle insertion in mCCD epithelia and suggest that stabilization of SNARE interactions by complexin is an essential aspect of the regulated trafficking events that increase apical membrane ENaC density either by constitutive or regulated trafficking pathways.

Non-coordinate regulation of endogenous epithelial sodium channel (ENaC) subunit expression at the apical membrane of A6 cells in response to various transporting conditions.

In many epithelial tissues in the body (e.g. kidney distal nephron, colon, airways) the rate of Na(+) reabsorption is governed by the activity of the epithelial Na(+) channel (ENaC). ENaC activity in turn is regulated by a number of factors including hormones, physiological conditions, and other ion channels. To begin to understand the mechanisms by which ENaC is regulated, we have examined the trafficking and turnover of ENaC subunits in A6 cells, a polarized, hormonally responsive Xenopus kidney cell line. As previously observed by others, the half-life of newly synthesized ENaC subunits was universally short ( approximately 2 h). However, the half-lives of alpha- and gamma-ENaC subunits that reached the apical cell surface were considerably longer (t(12) > 24 h), whereas intriguingly, the half-life of cell surface beta-ENaC was only approximately 6 h. We then examined the effects of various modulators of sodium transport on cell surface levels of individual ENaC subunits. Up-regulation of ENaC-mediated sodium conductance by overnight treatment with aldosterone or by short term incubation with vasopressin dramatically increased cell surface levels of beta-ENaC without affecting alpha- or gamma-ENaC levels. Conversely, treatment with brefeldin A selectively decreased the amount of beta-ENaC at the apical membrane. Short term treatment with aldosterone or insulin had no effect on cell surface amounts of any subunits. Subcellular fractionation revealed a selective loss of beta-ENaC from early endosomal pools in response to vasopressin. Our data suggest the possibility that trafficking and turnover of individual ENaC subunits at the apical membrane of A6 cells is non-coordinately regulated. The selective trafficking of beta-ENaC may provide a mechanism for regulating sodium conductance in response to physiological stimuli.

The effect of rapamycin on single ENaC channel activity and phosphorylation in A6 cells.

Rapamycin and FK-506 are immunosuppressive drugs that bind a ubiquitous immunophilin, FKBP12, but immunosuppressive mechanisms and side effects appear to be different. Rapamycin binds renal FKBP12 to change renal transport. We used cell-attached patch clamp to examine rapamycin's effect on Na(+) channels in A6 cells. Channel NP(o) was 0.5 +/- 0.08 (n = 6) during the first 5 min but fell close to zero after 20 min. Application of 1 microM rapamycin reactivated Na(+) channels (NP(o) = 0.47 +/- 0.1; n=6), but 1 microM FK-506 did not. Also, GF-109203X, a protein kinase C (PKC) inhibitor, mimicked the rapamycin-induced reactivation in a nonadditive manner. However, rapamycin did not reactivate Na(+) channels if cells were exposed to 1 microM FK-506 before rapamycin. In PKC assays, rapamycin was as effective as the PKC inhibitor; however, epithelial Na(+) channel (ENaC) phosphorylation was low under baseline conditions and was not altered by PKC inhibitors or activators. These results suggest that rapamycin activates Na(+) channels by binding FKBP12 and inhibiting PKC, and, in renal cells, despite binding the same immunophilin, rapamycin and FK-506 activate different intracellular signaling pathways.

Regulation of the amiloride-sensitive epithelial sodium channel by syntaxin 1A.

The first step in transepithelial sodium absorption lies at the apical membrane where the amiloride-sensitive, epithelial sodium channel, ENaC, facilitates sodium entry into the cell. Here we report that the vesicle traffic regulatory (SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)) protein, syntaxin 1A (S1A), inhibits ENaC mediated sodium entry. This inhibitory effect is selective for S1A and is not reproduced by syntaxin 3. The inhibition does not require the membrane anchoring domain of syntaxin 1A. It was reversed by the S1A-binding protein, Munc-18, but not by a Munc-18 mutant, which lacks syntaxin affinity. Immunostaining of epitope-tagged ENaC subunits showed that syntaxin 1A decreases ENaC current by reducing the number of ENaC channels in the plasma membrane; S1A does not interfere with ENaC protein expression. Immunoprecipitation of syntaxin 1A from the sodium-transporting epithelial cell line, A6, co-precipitates ENaC. These findings indicate that syntaxin 1A and other members of the SNARE machinery are involved in the control of plasma membrane ENaC content, and they suggest that SNARE proteins participate in the regulation of sodium absorption in relation to agonist mediated vesicle insertion-retrieval processes.

Vasopressin stimulates sodium transport in A6 cells via a phosphatidylinositide 3-kinase-dependent pathway.

The enzyme phosphatidylinositide 3-kinase (PI3K) phosphorylates the D-3 position of the inositol ring of inositol phospholipids and produces 3-phosphorylated inositides. These novel second messengers are thought to mediate diverse cellular signaling functions. The fungal metabolite wortmannin covalently binds to PI3K and selectively inhibits its activity. The role of PI3K in basal and hormone-stimulated transepithelial sodium transport was examined using this specific inhibitor. Wortmannin, 50 nM, did not affect basal, aldosterone-stimulated, or insulin-stimulated transport in A6 cells. Wortmannin completely inhibits vasopressin stimulation of transport in these cells. Vasopressin stimulates PI3K activity in A6 cells. Vasopressin stimulation of transport is also blocked by 5 microM LY-294002, a second inhibitor of PI3K. One-hour preincubation with wortmannin blocked vasopressin stimulation of protein kinase A activity in the cells. Sodium transport responses to exogenous cAMP and forskolin, which directly activates adenylate cyclase, were not affected by wortmannin. These results indicate that wortmannin inhibits vasopressin stimulation of Na(+) transport at a site proximal to activation of adenylate cyclase. The results suggest that PI3K may be involved in receptor activation by vasopressin.

Isoprenylcysteine-O-carboxyl methyltransferase regulates aldosterone-sensitive Na(+) reabsorption.

The Xenopus laevis distal tubule epithelial cell line A6 was used as a model epithelia to study the role of isoprenylcysteine-O-carboxyl methyltransferase (pcMTase) in aldosterone-mediated stimulation of Na(+) transport. Polyclonal antibodies raised against X. laevis pcMTase were immunoreactive with a 33-kDa protein in whole cell lysate. These antibodies were also reactive with a 33-kDa product from in vitro translation of the pcMTase cDNA. Aldosterone application increased pcMTase activity resulting in elevation of total protein methyl esterification in vivo, but pcMTase protein levels were not affected by steroid, suggesting that aldosterone increased activity independent of enzyme number. Inhibition of pcMTase resulted in a reduction of aldosterone-induced Na(+) transport demonstrating the necessity of pcMTase-mediated transmethylation for steroid induced Na(+) reabsorption. Transfection with an eukaryotic expression construct containing pcMTase cDNA increased pcMTase protein level and activity. This resulted in potentiation of the natriferic actions of aldosterone. However, overexpression did not change Na(+) reabsorption in the absence of steroid, suggesting that pcMTase activity is not limiting Na(+) transport in the absence of steroid, but that subsequent to aldosterone addition, pcMTase activity becomes limiting. These results suggest that a critical transmethylation is necessary for aldosterone-induction of Na(+) transport. It is likely that the protein catalyzing this methylation is isoprenylcysteine-O-carboxyl methyltransferase and that aldosterone activates pcMTase without affecting transferase expression.

Antibiotic susceptibility in the surgical intensive care unit compared with the hospital-wide antibiogram.

OBJECTIVE: To compare the antibiotic susceptibility of bacterial isolates from patients in the surgical intensive care unit (SICU) with hospital-wide bacterial susceptibility. DESIGN: Retrospective cohort analytic study. SETTING: Eight-bed SICU in a university-affiliated teaching hospital. PATIENTS: All hospitalized patients with culture results positive for microorganisms. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Antibiotic susceptibility data were collected retrospectively for all bacterial isolates from SICU patients during July 1, 1994, to June 30, 1995. All duplicate and surveillance cultures were eliminated from the data set. Susceptibility testing was conducted using our standard laboratory methods. Results were compared with the hospital-wide antibiogram (HWA) for the same time period. Comparisons were made using the chi(2) test with Yates correction or the Fisher exact test, as appropriate. Staphylococcus aureus (HWA, n=494; SICU, n=71) was significantly less susceptible to oxacillin (51% vs 28%; P<.001), ciprofloxacin (50% vs 25%; P<.001), erythromycin (46% vs 23%; P<.001), and clindamycin (51% vs 27%; P<.001) in the SICU. Coagulase-negative staphylococci (HWA, n=339; SICU, n=37) were significantly less susceptible to oxacillin (33% vs 16%; P=.04) and clindamycin (57% vs 34%; P=.02). Pseudomonas aeruginosa (HWA, n=513; SICU, n=96) was less susceptible to imipenem (85% vs 74%, P=.01) and more susceptible to ticarcillin-clavulanic acid (88% vs 100%, P<.001) in the SICU. Escherichia coli (HWA, n=474; SICU, n=36) was more susceptible to most penicillin-derivative antibiotics in the SICU (ampicillin [68% vs 83%, P=.06], ticarcillin [65% vs 86%, P=.01], mezlocillin [76% vs 95%, P=.01], and ticarcillin-clavulanic acid [88% vs 100%, P=.02]). CONCLUSIONS: The 2 most commonly isolated bacterial pathogens in the SICU (S aureus and P aeruginosa) had significantly different susceptibility patterns compared with the HWA. Surprisingly, E coli isolated in the SICU tended to be more susceptible to penicillin-derivative antibiotics. These data indicate that empiric antibiotic choices in the SICU may be better guided by unit-specific antibiograms.

Carboxylmethylation of the beta subunit of xENaC regulates channel activity.

The action of aldosterone to increase apical membrane permeability in responsive epithelia is thought to be due to activation of sodium channels. Aldosterone stimulates methylation of a 95-kDa protein in apical membrane of A6 cells, and we have previously shown that methylation of a 95-kDa protein in the immunopurified Na+ channel complex increases open probability of these channels in planar lipid bilayers. We report here that aldosterone stimulates carboxylmethylation of the beta subunit of xENaC in A6 cells. In vitro translated beta subunit, but not alpha or gamma, serves as a substrate for carboxylmethylation. Carboxylmethylation of ENaC reconstituted in planar lipid bilayers leads to an increase in open probability only when beta subunit is present. When the channel complex is immunoprecipitated from A6 cells and analyzed by Western blot with antibodies to the three subunits of xENaC, all three subunits are recognized as constituents of the complex. The results suggest that Na+ channel activity in A6 cells is regulated, in part, by carboxylmethylation of the beta subunit of xENaC.

Estrogen-dependent transcriptional activation and vitellogenin gene memory.

The concept of hepatic memory suggests that a gene responds more rapidly to a second exposure of an inducer than it does during the initial activation. To determine how soon estrogen-dependent DNA/protein interactions occur during the primary response, in vivo dimethylsulfate footprinting was carried out using genomic DNA amplified by ligation-mediated PCR. When estrogen was added to disrupted cells from a hormone-naive liver, changes within and around the estrogen response elements occurred within seconds, indicating a direct and rapid effect on this estrogen-responsive promoter that had never before been activated. Because this effect was so rapid relative to the delayed onset of mRNA accumulation during the primary response, run-on transcription assays were used to determine the transcription profiles for four of the yolk protein genes during the primary and secondary responses to estrogen. As with the accumulation of mRNA, the onset of transcription was delayed for all of these genes after a primary exposure to estrogen. Interestingly, after the secondary exposure to estrogen, the vitellogenin I, vitellogenin II, and very low density apolipoprotein II genes displayed a more rapid onset of transcription, whereas the primary and secondary profiles of apolipoprotein B transcription in response to estrogen were identical. Because the apoB gene is constitutively expressed in the absence of estrogen, and the vitellogenins are quiescent before the administration of the hormone, hepatic memory most likely represents a relatively stable event in the transition to an active state of a gene that is committed for tissue-specific expression.

Clinical comparison of the BACTEC 9000 Standard Anaerobic/F and Lytic/F blood culture media.

An 8-month prospective, volume controlled, comparison of Standard Anaerobic/F media with a new anaerobic high blood volume lytic medium (Lytic/F) was performed. A total of 2,092 compliant sets, consisting of an aerobic resin bottle or standard aerobic bottle, Standard Anaerobic/F, and Lytic/F bottle were evaluated. A total of 220 (10.6%) positive specimens were detected from the paired anaerobic bottles. These consisted of 194 true positive and 26 false positive bottles. Of 207 total organisms isolated, 122 were considered clinically significant. A comparison of significant organism recovery revealed 79 isolates in both anaerobic bottles, 7 isolates in the standard Anaerobic/F bottle only, and 36 isolates in the Lytic/F bottle only (p < 0.001). The lytic/F bottle detected significantly more Enterobacteriaceae (p < 0.005) and Streptococci (p < 0.05). There were 24 false positive Standard Anaerobic/F bottles and 2 false positive Lytic/F bottles (p < 0.001). When both bottles were positive the Standard Anaerobic/F bottle was positive 12 hours earlier in 1 instance whereas the Lytic/F bottle was positive 12 hours earlier in 8 instances. The mean time for detection in the Standard Anaerobic/F bottle was 18.2 hours versus 13.2 hours for the Lytic/F bottle. The new Lytic/F anaerobic blood culture media was found to be superior to Standard Anaerobic/F media for both total organism recovery and time to organism detection.

Evaluation of the Pasco gram-negative nonfermenter identification system.

The Pasco Gram-negative identification system was evaluated for use with nonfermenting organisms. Of 127 isolates tested, 109 (86%) were correctly identified to the species level. A total of 91% (93 of 102 isolates) of the Pseudomonas-Xanthomonas group and the Acinetobacter group were correctly identified to the species level. The system was found to be useful for the identification of Pseudomonas aeruginosa, Xanthomonas maltophilia, and Acinetobacter species.

Identification and characterization of Yersinia intermedia isolated from human feces.

Since May 1983, our laboratory has, upon request, cultured stools for Yersinia spp. by using direct plating on cefsulodin-irgasan-novobiocin agar and a 3-week cold enrichment procedure. We isolated bacteria identified as Y. intermedia from six adult patients. All isolates were recovered only by the cold enrichment procedure and misidentified as Y. enterocolitica by the API 20E system (Analytab Products, Plainview, N.Y.). Final identification was made on the basis of results obtained with conventional tube biochemical tests. The isolates were tested for the following characteristics associated with virulence in Y. enterocolitica: lack of pyrazinamidase activity, autoagglutinability, presence of a 40- to 50-megadalton plasmid, production of heat-stable enterotoxin, and mouse lethality. All isolates tested had pyrazinamidase activity, and none were autoagglutinable. However, one isolate possessed a 40-megadalton plasmid. None produced enterotoxin or were lethal for mice. Review of the medical histories of the patients revealed that four of the six had diarrhea; however, none had disease typical of that caused by Y. enterocolitica. Our data confirmed the limited pathogenic potential of Y. intermedia and suggested that its isolation was without clinical significance in our patients. Conventional biochemical tests were required for reliable identification of Y. intermedia.

Supplementary rapid biochemical test panel for the API 20E bacterial identification system.

The API 20E Analytical Profile Index typically suggests three or four conventional biochemical tests to complete the identification of strains either identified to genus only or that have multiple genera consistent with the profile number. We compiled a simple panel of eight rapid (4-h) tests that can substitute for the supplementary biochemical tests recommended by Analytab Products (Plainview, N.Y.). The rapid test panel (RTP) consisted of adonitol, cellobiose, lactose, raffinose, rhamnose, and xylose utilization, lysine decarboxylase activity, and motility. A total of 114 consecutive clinical isolates that required additional tests to complete the identifications were each tested with the complete RTP, as well as with the recommended conventional biochemicals. All discordant identifications were resolved by using an expanded series of conventional biochemical tests. Overall, 110 (96%) strains were identified to the correct genus, and 109 (95%) strains were identified to the correct species by using the RTP, as compared with 105 (92%) identified to the correct genus and 90 (79%) identified to the correct species with the recommended tests. The identifications based on the two supplementary test systems did not agree for 7 (6.1%) strains. Four discrepancies were resolved in favor of the RTP, and three were resolved in favor of the recommended tests. We were unable to identify five (4.4%) strains with the recommended tests and only one (0.9%) with the RTP. A majority (86%) of the test strains were identified to the species level with the RTP after only 4 h of incubation.