Ouabain-insensitive Na+-ATPase activity is an effector protein for cAMP regulation in basolateral membranes of the proximal tubule

Abstract

This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from proximal tubule basolateral membranes by cAMP. An increase in dibutyryl-cAMP (d-cAMP) concentration from 10(-8) to 5x10(-5) M stimulates the ouabain-insensitive Na(+)-ATPase activity. The ATPase activity increases from 6.0+/-0.4 to 10.1+/-0.7 nmol Pi mg(-1) min(-1), in the absence and presence of 5x10(-6) M d-cAMP, respectively. Similarly, the addition of cholera toxin (CTX), forskolin (FSK) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) also increases the Na(+)-ATPase activity in a dose-dependent manner, with maximal effect at 10(-8) M, 10(-6) M and 10(-7) M, respectively. The effect of 10(-8) M CTX is not additive to the effect of GTPgammaS, and is completely abolished by 200 microM guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of CTX and FSK on the Na(+)-ATPase activity are accompanied by an increase in cAMP formation by the basolateral membranes of the proximal tubule cells. Furthermore, 10(-8) M protein kinase A peptide inhibitor (PKAi) completely abolishes the stimulatory effect of 5x10(-6) M d-cAMP or 10(-4) M FSK on the Na(+)-ATPase activity. Incubation of the basolateral membranes with [gamma-(32)P]ATP in the presence of d-cAMP or FSK increases the global hydroxylamine-resistant phosphorylation and especially promotes an increase in phosphorylation of protein bands of approximately 100 and 200 kDa. This stimulation is not seen when 10(-8) M PKAi is added simultaneously. Taken together these data suggest that activation of a cAMP/PKA pathway modulates the Na(+)-ATPase activity in isolated basolateral membranes of the proximal tubule.

Metabolic Programming during Lactation Stimulates Renal Na+ Transport in the Adult Offspring Due to an Early Impact on Local Angiotensin II Pathways

“…This kinase has been implicated as an antagonist of PKC in its actions on the renal Na + -ATPase [37], [38]. In contrast to the PKC findings, the activity (Fig.…”

How the Kidney Is Impacted by the Perinatal Maternal Environment to Develop Hypertension1

“…This enzyme is responsible for approximately 10% of the reabsorption of filtered sodium [90] in the proximal tubule, and it is also designated as the ouabain-insensitive Na þ -ATPase transporter or a second sodium pump. Although recently cloned, the role of Na þ -ATPase in sodium reabsorption in the proximal tubule has been extensively investigated [91][92][93] and implicated in the development of hypertension in the spontaneously hypertensive rat (SHR) [89,94]. Specifically, PAIXÃ O AND ALEXANDER increased activity of the NKCC2 transporter along the thick ascending limb of the Milan hypertensive rat [95] and increased activity of Na þ -H þ -transporter along the proximal tubule in juvenile SHRs [96][97][98] highlights the importance of these apical membrane Na þ transporters in the maintenance of hypertension.…”

Two Serine Residues Control Sequential Steps during Catalysis of the Yeast Copper ATPase through Different Mechanisms That Involve Kinase-mediated Phosphorylations

“…Alignment was generated as described under "Experimental Procedures." The potential sites for PKA were predicted using the pkaPS tool (25).…”

“…5), the effect of 2 M hydroxylamine on the phosphoenzyme levels was assayed. The sensitivity to hydroxylamine was investigated by catalytic phosphorylation assays of Ccc2 wt, S258A, S258D, S971A, and S258A/S971A, as previously described (25). Briefly, after precipitation with TCA the sediments of samples run in parallel were washed twice with ice-cold water and resuspended in 0.5 ml of a mixture containing 2.0 M hydroxylamine and 40 mM citrate buffer adjusted to pH 5.5 with NaOH.…”