Sodium/hydrogen exchanger NHA2 is critical for insulin secretion in β-cells

NHA2 is a sodium/hydrogen exchanger with unknown physiological function. Here we show that NHA2 is present in rodent and human β-cells, as well as β-cell lines. In vivo, two different strains of NHA2-deficient mice displayed a pathological glucose tolerance with impaired insulin secretion but normal peripheral insulin sensitivity. In vitro, islets of NHA2-deficient and heterozygous mice, NHA2-depleted Min6 cells, or islets treated with an NHA2 inhibitor exhibited reduced sulfonylurea- and secretagogue-induced insulin secretion. The secretory deficit could be rescued by overexpression of a wild-type, but not a functionally dead, NHA2 transporter. NHA2 deficiency did not affect insulin synthesis or maturation and had no impact on basal or glucose-induced intracellular Ca(2+) homeostasis in islets. Subcellular fractionation and imaging studies demonstrated that NHA2 resides in transferrin-positive endosomes and synaptic-like microvesicles but not in insulin-containing large dense core vesicles in β-cells. Loss of NHA2 inhibited clathrin-dependent, but not clathrin-independent, endocytosis in Min6 and primary β-cells, suggesting defective endo-exocytosis coupling as the underlying mechanism for the secretory deficit. Collectively, our in vitro and in vivo studies reveal the sodium/proton exchanger NHA2 as a critical player for insulin secretion in the β-cell. In addition, our study sheds light on the biological function of a member of this recently cloned family of transporters.

A systematic review of the survival and complication rates of resin-bonded bridges after an observation period of at least 5 years

OBJECTIVES: The objectives of this systematic review were to assess the 5-year survival of resin-bonded bridges (RBBs) and to describe the incidence of technical and biological complications. METHODS: An electronic Medline search complemented by manual searching was conducted to identify prospective and retrospective cohort studies on RBBs with a mean follow-up time of at least 5 years. Patients had to have been examined clinically at the follow-up visit. Assessment of the identified studies and data extraction were performed independently by two reviewers. Failure and complication rates were analyzed using random-effects Poissons regression models to obtain summary estimates of 5-year proportions. RESULTS: The search provided 6110 titles and 214 abstracts. Full-text analysis was performed for 93 articles, resulting in 17 studies that met the inclusion criteria. Meta-analysis of these studies indicated an estimated survival of RBBs of 87.7% (95% confidence interval (CI): 81.6-91.9%) after 5 years. The most frequent complication was debonding (loss of retention), which occurred in 19.2% (95% CI: 13.8-26.3%) of RBBs over an observation period of 5 years. The annual debonding rate for RBBs placed on posterior teeth (5.03%) tended to be higher than that for anterior-placed RBBs (3.05%). This difference, however, did not reach statistical significance (P=0.157). Biological complications, like caries on abutments and RBBs lost due to periodontitis, occurred in 1.5% of abutments and 2.1% of RBBs, respectively. CONCLUSION: Despite the high survival rate of RBBs, technical complications like debonding are frequent. This in turn means that a substantial amount of extra chair time may be needed following the incorporation of RBBs. There is thus an urgent need for studies with a follow-up time of 10 years or more, to evaluate the long-term outcomes.

Characterization of the regulation of renal Na+/H+ exchanger NHE3 by insulin

Insulin receptors are widely distributed in the kidney and affect multiple aspects of renal function. In the proximal tubule, insulin regulates volume and acid-base regulation through stimulation of the Na(+)/H(+) exchanger NHE3. This paper characterizes the signaling pathway by which insulin stimulates NHE3 in a cell culture model [opossum kidney (OK) cell]. Insulin has two distinct phases of action on NHE3. Chronic insulin (24 h) activates NHE3 through the classic phosphatidylinositol 3-kinase-serum- and glucocorticoid-dependent kinase 1 (PI3K-SGK1) pathway as insulin stimulates SGK1 phosphorylation and the insulin effect can be blocked by the PI3K inhibitor wortmannin or a dominant-negative SGK1. We showed that SGK1 transcript and protein are expressed in rat proximal tubule and OK cells. We previously showed that glucocorticoids augment the effect of insulin on NHE3 (Klisic J, Hu MC, Nief V, Reyes L, Fuster D, Moe OW, Ambuhl PM. Am J Physiol Renal Physiol 283: F532-F539, 2002). Part of this can be mediated via induction of SGK1 by glucocorticoids, and indeed the insulin effect on NHE3 can also be amplified by overexpression of SGK1. We next addressed the acute effect of insulin (1-2 h) on NHE3 by systematically examining the candidate signaling cascades and activation mechanisms of NHE3. We ruled out the PI3K-SGK1-Akt and TC10 pathways, increased surface NHE3, NHE3 phosphorylation, NHE3 association with calcineurin homologous protein 1 or megalin as mechanisms of acute activation of NHE3 by insulin. In summary, insulin stimulates NHE3 acutely via yet undefined pathways and mechanisms. The chronic effect of insulin is mediated by the classic PI3K-SGK1 route.

Influence of beveling and ultrasound application on marginal adaptation of box-only Class II (slot) resin composite restorations

A laboratory study was performed to assess the influence of beveling the margins of cavities and the effects on marginal adaptation of the application of ultrasound during setting and initial light curing. After minimal access cavities had been prepared with an 80 microm diamond bur, 80 box-only Class II cavities were prepared mesially and distally in 40 extracted human molars using four different oscillating diamond coated instruments: (A) a U-shaped PCS insert as the non-beveled control (EMS), (B) Bevelshape (Intensiv), (C) SonicSys (KaVo) and (D) SuperPrep (KaVo). In groups B-D, the time taken for additional bevel finishing was measured. The cavities were filled with a hybrid composite material in three increments. Ultrasound was also applied to one cavity per tooth before and during initial light curing (10 seconds). The specimens were subjected to thermomechanical stress in a computer-controlled masticator device. Marginal quality was assessed by scanning electron microscopy and the results were compared statistically. The additional time required for finishing was B > D > C (p < or = 0.05). In all groups, thermomechanical loading resulted in a decrease in marginal quality. Beveling resulted in higher values for "continuous" margins compared with that of the unbeveled controls. The latter showed better marginal quality at the axial walls when ultrasound was used. Beveling seems essential for good marginal adaptation but requires more preparation time. The use of ultrasonic vibrations may improve the marginal quality of unbeveled fillings and warrants further investigation.

Determination of caries risk at resin composite margins

PURPOSE: To design an artificial mouth in order to evaluate if a new diagnostic tool (Clinpro Cario Diagnosis) can be used for early detection of secondary caries at resin composite margins in vitro. METHODS: 32 intact human third molars received standardized Class-V resin composite restorations (Tetric Ceram bonded with Syntac SC). After storage for 4 weeks at 37 degrees C, teeth were subjected to 5,000 or 10,000 thermocycles (+/- 5 degrees C and +/- 55 degrees C) and polysiloxane impressions were taken. Streptococcus mutans 10449 (SM) was used in a nutrition medium to initiate a secondary caries process. Daily, the teeth were incubated for 2 x 2.5 hours in SM containing nutrition medium followed by 2 x 9.5 hours incubation in artificial saliva. Teeth were investigated after total incubation periods of 4, 6, and 8 weeks. After the different incubation protocols, the restoration margins were evaluated for infection and secondary caries processes in using Clinpro Cario Diagnosis which measures site-specifically the lactic acid production of SM in response to a sucrose challenge. The color signal was read 5 minutes after removal of the diagnostic impression. After thermocycling and biological load cycling, precision polysiloxane impressions were taken and replicas were investigated under a light microscope for gap widths at enamel and dentin margins. Demineralization was evaluated by fluorescence microscopy in using a special FITC filter. The demineralization depths at the cavity margin were calculated with Xpert for Windows using a pixel distance of 5 microm. RESULTS: After the different thermocycling protocols, no differences in gap widths and demineralization depths were found (P > 0.05). After SM incubation, gap widths and demineralization depths were significantly dependent on SM incubation time and previous number of thermocycles (P < 0.05). Lactic acid formations of SM were detectable by Clinpro Cario Diagnosis at dentin cavosurface margins formed after 6 weeks of incubation with SM (P < 0.05).

Shear bond strength of resin cements to human dentin

OBJECTIVES: The objectives of this in vitro study were (1) to assess the bond strength of the universal cement RelyX Unicem to dentin and to compare it with three conventional resin cements, (2) to test the influence of aging on their bonding capacity and (3) to test the influence of the operator on bonding quality by performing the same test in two different centers. METHODS: 160 third molars, divided into 80 for tests at the University of Zurich (Z) and 80 for tests at the University of Berne (B), were assigned to 2 x 8 subgroups of 10 teeth each. The specimens were prepared with the corresponding bonding agents and acrylic rods were luted either with RelyX Unicem (U), RelyX ARC (A), Multilink (M) or Panavia 21 (P). All specimens were stored in water for 24h (W) and half of the specimens were subjected to 1500 cycles of thermocycling (5 degrees C and 55 degrees C) (T). Bond strength was measured by means of a shear test. RESULTS: After water storage RelyX Unicem exhibited lowest bond strength (UWZ: 9.2+/-1.6 MPa, UWB: 9.9+/-1.2 MPa, AWZ: 15.3+/-6.0 MPa, AWB: 12.2+/-4.3 MPa, MWZ: 15.6+/-3.3 MPa, MWB: 12.4 MPa+/-2.4, PWZ: 13.4+/-2.9 MPa, PWB: 14.9+/-2.6 MPa). Thermocycling affected the bonding performance of all four cements. However, bond strength of RelyX Unicem was least influenced by thermocycling (UTZ: 9.4+/-2.9 MPa, UTB: 8.6+/-1.3 MPa, ATZ: 11.4+/-6.3 MPa, ATB: 13.3+/-3.7 MPa, MTZ: 15.4+/-3.1 MPa, MTB: 10.3+/-2.4 MPa, PTZ: 11.1+/-2.8 MPa, PTB: 11.3+/-2.8 MPa). SIGNIFICANCE: Although the bond strength of RelyX Unicem to dentin was lower in comparison to RelyX ARC, Multilink and Panavia 21, its bond strength was less sensitive to variations in handling and aging.

Steady-state function of the ubiquitous mammalian Na/H exchanger (NHE1) in relation to dimer coupling models with 2Na/2H stoichiometry

We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE)1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is <1, and Na dependence often appears biphasic. Results are similar for mouse skin fibroblasts and for an opossum kidney cell line that expresses the NHE3 isoform, whereas NHE1(-/-) skin fibroblasts generate no proton fluxes in equivalent experiments. As proton flux is decreased by increasing cytoplasmic pH, the half-maximal concentration (K(1/2)) of extracellular Na decreases less than expected for simple consecutive ion exchange models. The K(1/2) for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models. For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K(1/2) for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na. With 140 mM of extracellular Na and no cytoplasmic Na, the K(1/2) for cytoplasmic protons is 50 nM (pH 7.3; Hill coefficient, 1.5), and activity decreases only 25% with extracellular acidification from 8.5 to 7.2. Most data can be reconstructed with two very different coupled dimer models. In one model, monomers operate independently at low cytoplasmic pH but couple to translocate two ions in "parallel" at alkaline pH. In the second "serial" model, each monomer transports two ions, and translocation by one monomer allosterically promotes translocation by the paired monomer in opposite direction. We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.