Mechanisms of PKC-Dependent Na⁺K⁺ATPase Phosphorylation in the Rat Kidney with Chronic Renal Failure

Abstract

The present work was designed to study Na + K + ATPase α1-subunit phosphorylation in rats with chronic renal failure (CRF) in comparison with normal rats. Na + K + ATPase α1-subunit phosphorylation degree was measured by binding the McK-1 antibody to dephosphorylated Ser-23 in microdissected medullary thick ascending limb of Henle (mTAL) segments. In addition, the total Na + K + ATPase α1-subunit expression and activity were also measured in the outer renal medulla homogenates and membranes.CRF rats showed a higher Na + K + ATPase activity, as compared with control rats (18.95 ± 2.4 vs. 11.21 ± 1.5 μmol Pi/mg prot/h, p < 0.05), accompanied by a higher total Na + K + ATPase expression (0.54 ± 0.04 vs. 0.27 ± 0.02 normalized arbitrary units (NU), p < 0.05). When McK-1 antibody was used, a higher immunosignal in mTAL of CRF rats was observed, as compared with controls (6.3 ± 0.35 vs.4.1 ± 0.33 NU, p < 0.05). The ratio Na + K + ATPase α1-subunit phosphorylation / total Na + K + ATPase α1-subunit expression per μg protein showed a nonsignificant difference between CRF and control rats in microdissected mTAL segments (2.11 ± 0.12 vs.2.26 ± 0.18 NU, p = NS). The PKC inhibitor RO-318220 10 −6 M increased immunosignal (lower phosphorylation degree) in mTAL of CRF rats to 128.43 ± 7.08% (p < 0.05) but did not alter McK1 binding in control rats. Both phorbol 12-myristate 13-acetate (PMA) 10 −6 M and dopamine 10 −6 M decreased immunosignal in CRF rats, corresponding to a higher Na + K + ATPase α1-subunit phosphorylation degree at Ser-23 (55.26 ± 11.17% and 53.27 ± 7.12% compared with basal, p < 0.05). In mTAL of CRF rats, the calcineurin inhibitor FK-506 10 −6 M did not modify phosphorylation degree at Ser-23 of Na + K + ATPase α1-subunit (100.21 ± 3.00% compared with basal CRF). In control rats, FK 506 10M decreased the immunosignal, which corresponds to a higher Na + K + ATPase α1-subunit phosphorylation degree at Ser-23. The data suggest that the regulation of basal Na + K + ATPase α1-subunit phosphorylation degree at Ser-23 in mTAL segments of CRF rats was primarily dependent on PKC activation rather than calcineurin dependent mechanisms.

Deletion of calcineurin from GFAP‐expressing astrocytes impairs excitability of cerebellar and hippocampal neurons through astroglial Na+/K+ ATPase

“…It is postulated that CaNdependent dephosphorylation activates, while phosphorylation, e.g. by protein kinase A (PKA) or protein kinase C (PKC), inhibits NKA activity (Bertorello et al, 1991;Bertuccio et al, 2003Bertuccio et al, , 2007Poulsen et al, 2010). Third, the lack of significant changes in neuronal RMP indicates that deletion of astrocytic CaN did not alter the transmembrane K + gradient in cerebellar and hippocampal neurons, which further confirms that neuronal NKA activity was unaltered.…”

Na+,K+‐ATPase is modulated by angiotensin II in diabetic rat kidney – another reason for diabetic nephropathy?

“…Reversible covalent modification by phosphorylation takes part in the regulation of NKA trafficking between subcellular compartments (Chibalin et al 1999; Efendiev et al 2003). Furthermore, phosphorylation is generally associated with altered enzyme activity (Bertuccio et al 2007; Lal et al 2000), which may be in connection with altered subcellular NKA distribution. The influence of DM and ANGII on the expression and function of NKA has been studied separately in rodents, but their common effect is still not known.…”

Effects induced by high and low intensity laser plasma on SiC Schottky detectors

“…Silicon Carbide (SiC) detectors are now-days widely employed for the monitoring of ionizing radiations. Due to the physical properties of the material, these detectors can be used at high temperatures and in high level radioactive ambient [1][2][3]. Irradiation of SiC detectors with neutrons, protons and heavy ions evidenced the higher radiation hardness of these devices with respect to Si detectors, principally thanks to higher atomic displacement energy of the SiC.…”