Showing papers by "Maria Svelto published in 2010"

The KATP channel is a molecular sensor of atrophy in skeletal muscle

Abstract: The involvement of ATP-sensitive K(+) (K(ATP)) channels in the atrophy of slow-twitch (MHC-I) soleus (SOL) and fast-twitch (MHC-IIa) flexor digitorum brevis (FDB) muscles was investigated in vivo in 14-day-hindlimb-unloaded (14-HU) rats, an animal model of disuse, and in vitro in drug-induced muscle atrophy. Patch-clamp and gene expression experiments were performed in combination with measurements of fibre diameters used as an index of atrophy, and with MHC labelling in 14-HU rats and controls. A down-regulation of K(ATP) channel subunits Kir6.2, SUR1 and SUR2B with marked atrophy and incomplete phenotype transition were observed in SOL of 14-HU rats. The observed changes in K(ATP) currents were well correlated with changes in fibre diameters and SUR1 expression, as well as with MHC-IIa expression. Half of the SOL fibres of 14-HU rats had reduced diameter and K(ATP) currents and were labelled by MHC-I antibodies. Non-atrophic fibres were labelled by MHC-IIa (22%) antibodies and had enhanced K(ATP) currents, or were labelled by MHC-I (28%) antibodies but had normal current. FDB was not affected in 14-HU rats and this is related to the high expression/activity of Kir6.2/SUR1 subunits characterizing this muscle phenotype. The long-term incubation of the control muscles in vitro with the K(ATP) channel blocker glibenclamide (10(6)m) reduced the K(ATP) currents with atrophy and these effects were prevented by the K(ATP) channel opener diazoxide (10(4)m). The in vivo down-regulation of SUR1, and possibly of Kir6.2 and SUR2B, or their in vitro pharmacological blockade activates atrophic signalling in skeletal muscle. All these findings suggest a new role for the K(ATP) channel as a molecular sensor of atrophy.

Lovastatin-induced cholesterol depletion affects both apical sorting and endocytosis of aquaporin-2 in renal cells.

Abstract: Vasopressin causes the redistribution of the water channel aquaporin-2 (AQP2) from cytoplasmic storage vesicles to the apical plasma membrane of collecting duct principal cells, leading to urine concentration. The molecular mechanisms regulating the selective apical sorting of AQP2 are only partially uncovered. In this work, we investigate whether AQP2 sorting/trafficking is regulated by its association with membrane rafts. In both MCD4 cells and rat kidney, AQP2 preferentially associated with Lubrol WX-insoluble membranes regardless of its presence in the storage compartment or at the apical membrane. Block-and-release experiments indicate that 1) AQP2 associates with detergent-resistant membranes early in the biosynthetic pathway; 2) strong cholesterol depletion delays the exit of AQP2 from the trans-Golgi network. Interestingly, mild cholesterol depletion promoted a dramatic accumulation of AQP2 at the apical plasma membrane in MCD4 cells in the absence of forskolin stimulation. An internalization assay showed that AQP2 endocytosis was clearly reduced under this experimental condition. Taken together, these data suggest that association with membrane rafts may regulate both AQP2 apical sorting and endocytosis.

MAL/VIP17, a New Player in the Regulation of NKCC2 in the Kidney

Abstract: The renal-specific Na+-K+-2Cl- cotransporter (NKCC2) is the major salt transport pathway of the apical membrane of the mammalian thick ascending limb of Henle's loop. Here, we analyze the role of the tetraspan protein myelin and lymphocytes-associated protein (MAL)/VIP17 in the regulation of NKCC2. We demonstrated that 1) NKCC2 and MAL/VIP17 colocalize and coimmunoprecipitate in Lilly Laboratories cell porcine kidney cells (LLC-PK1) as well as in rat kidney medullae, 2) a 150-amino acid stretch of NKCC2 C-terminal tail is involved in the interaction with MAL/VIP17, 3) MAL/VIP17 increases the cell surface retention of NKCC2 by attenuating its internalization, and 4) this coincides with an increase in cotransporter phosphorylation. Interestingly, overexpression of MAL/VIP17 in the kidney of transgenic mice results in cysts formation in distal nephron structures consistent with the hypothesis that MAL/VIP17 plays an important role in apical sorting or in maintaining the stability of the apical membrane. The NKCC2 expressed in these mice was highly glycosylated and phosphorylated, suggesting that MAL/VIP17 also is involved in the stabilization of NKCC2 at the apical membrane in vivo. Thus, the involvement of MAL/VIP17 in the activation and surface expression of NKCC2 could play an important role in the regulated absorption of Na+ and Cl- in the kidney.

Analysis by two-dimensional Blue Native/SDS-PAGE of membrane protein alterations in rat soleus muscle after hindlimb unloading

Abstract: Muscle atrophy occurring in several pathophysiological conditions determines decreases in muscle protein synthesis, increases in the rate of proteolysis and changes in muscle fiber composition. To determine the effect of muscle atrophy induced by hindlimb unloading (HU) on membrane proteins from rat soleus, a proteomic approach based on two-dimensional Blue Native/SDS-PAGE was performed. Proteomic analysis of normal and HU soleus muscle demonstrates statistically significant changes in the relative level of 36 proteins. Among the proteins identified by mass spectrometry, most are involved in pathways associated with muscle fuel utilization, indicating a shift in metabolism from oxidative to glycolytic. Moreover, immunoblotting analysis revealed an increase in aquaporin-4 (AQP4) water channel and an alteration of proteins belonging to the dystrophin–glycoprotein complex (DGC). AQP4 and DGC are regulated in soleus muscle subjected to simulated microgravity in response to compensatory mechanisms induced by muscle atrophy, and they parallel the slow-to-fast twitch conversion that occurs in soleus fibers during HU. In conclusion, the alterations of soleus muscle membrane proteome may play a pivotal role in the mechanisms involved in disuse-induced muscle atrophy.

Differential modulation of intracellular Ca2+ responses associated with calcium-sensing receptor activation in renal collecting duct cells.

Abstract: In this work, we studied G protein-coupled Extracellular Calcium Sensing Receptor (CaR) signaling in mouse cortical collecting duct cells (MCD4) expressing endogenous CaR. Intracellular [Ca2+] measurements performed with real time video imaging revealed that CaR stimulation with 5mM Ca2+, 300µM Gd3+ and with 10µM of specific allosteric modulator NPS-R 568, all resulted in an increase in [Ca2+]i although displaying different features. Specifically, Ca2+ as well as stimulation with NPS-R 568 induced a rapid peak of [Ca2+]i while stimulation with Gd3+ induced transient intracellular Ca2+ oscillations. PLC inhibition completely abolished any [Ca2+]i increase after stimulation with CaR agonists. Inhibition of Rho or Rho kinase (ROK) abolished [Ca2+]i oscillations induced by Gd3+, while the peak induced by high Ca2+ was similar to control. Conversely, emptying the intracellular calcium stores abolished the response to Gd3+. On the other hand, the inhibition of calcium influx did not alter calcium changes. We conclude that in our cell model, CaR stimulation with distinct agonists activates two distinct transduction pathways, both PLC-dependent. The transient cytosolic Ca2+ oscillations produced by Gd3+ are modulated by Rho-Rho kinase signaling, whereas the rapid peak of intracellular Ca2+ in response to 5mM [Ca2+]o is mainly due to PLC/IP3 pathway activation.

Aquaporin‐2 urinary excretion in preterm infants: relationship to diuresis and vasopressin

Abstract: Aims: Few investigations have explored the urinary aquaporin-2 (u-AQP2) excretion pattern after birth in preterm infants with conflicting results regarding the correlation between u-AQP2, urinary osmolality and vasopressin. The aims of this study were to evaluate u-AQP2 excretion during the first week of life in preterm infants, to correlate u-AQP2 with other markers of renal function and to investigate the relationship between u-AQP2, urinary tonicity and arginine-vasopressin in the immature kidney. Methods: In infants born less than 33 weeks daily diuresis, u-AQP2, urinary arginine-vasopressin, urine and plasma tonicity, creatinine and electrolytes were measured through the first 7 days of life. Results: Fifty-five infants were evaluated. u-AQP2 excretion showed the following profile: the highest u-AQP2 levels were found on day 2 and values remained significantly higher until day 5 with respect to day 1. On day 6, u-AQP2 levels significantly decreased to values closer to those found on day 1. u-AQP2 excretion was not associated with arginine-vasopressin while significant, but weak association was found with urinary tonicity (r = −0.20; −0.32 < r < −0.11; P < 0.05). u-AQP2 excretion and creatinine clearance were significantly associated during the study period (r = 0.19; 0.08 < r < 0.29; P < 0.05). There was a strong association between totally u-AQP2 excretion and diuresis over the week (r = 0.72; 0.66 < r < 0.76; P < 0.0001). Conclusion: Significant variations occur in AQP2 expression levels during the first week of life in preterm infants. AQP2 does not seem to contribute to the urinary concentration ability after birth. Further investigations are required to elucidate the mechanisms underlying the strong association between diuresis and u-AQP2 excretion in early postnatal life.