Dopamine-induced Exocytosis of Na,K-ATPase Is Dependent on Activation of Protein Kinase C-ε and -δ

Abstract

The purpose of this study was to define mechanisms by which dopamine (DA) regulates the Na,K-ATPase in alveolar epithelial type 2 (AT2) cells. The Na,K-ATPase activity increased by twofold in cells incubated with either 1 microM DA or a dopaminergic D(1) agonist, fenoldopam, but not with the dopaminergic D(2) agonist quinpirole. The increase in activity paralleled an increase in Na,K-ATPase alpha1 and beta1 protein abundance in the basolateral membrane (BLM) of AT2 cells. This increase in protein abundance was mediated by the exocytosis of Na,K-pumps from late endosomal compartments into the BLM. Down-regulation of diacylglycerol-sensitive types of protein kinase C (PKC) by pretreatment with phorbol 12-myristate 13-acetate or inhibition with bisindolylmaleimide prevented the DA-mediated increase in Na,K-ATPase activity and exocytosis of Na,K-pumps to the BLM. Preincubation of AT2 cells with either 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983), a selective inhibitor of PKC-delta, or isozyme-specific inhibitor peptides for PKC-delta or PKC-epsilon inhibited the DA-mediated increase in Na,K-ATPase. PKC-delta and PKC-epsilon, but not PKC-alpha or -beta, translocated from the cytosol to the membrane fraction after exposure to DA. PKC-delta- and PKC-epsilon-specific peptide agonists increased Na,K-ATPase protein abundance in the BLM. Accordingly, dopamine increased Na,K-ATPase activity in alveolar epithelial cells through the exocytosis of Na,K-pumps from late endosomes into the basolateral membrane in a mechanism-dependent activation of the novel protein kinase C isozymes PKC-delta and PKC-epsilon.

Impairment of Na+,K+-ATPase in CD95(APO-1)-induced human T-cell leukemia cell apoptosis mediated by glutathione depletion and generation of hydrogen peroxide

“…In summary, these data suggest that intracellular GSH depletion produces H 2 O 2 , then Effects of Fas L, GSH, BSO and H 2 O 2 on the serine phosphorylation of Na þ ,K þ -ATPase a1 subunit Considerable evidence has documented that the 'endocytosis' of Na þ ,K þ -ATPase is closely related to serine phosphorylation of Na þ ,K þ -ATPase a1 subunit, the process of which is tightly Impairment of Na þ ,K þ -ATPase in human T-cell leukemia cell apoptosis W Yin et al controlled by intracellular protein kinase C (PKC) isoforms. 22,23 In present study, we performed immunoprecipitation experiment to observe the effects of Fas L, GSH, BSO and H 2 O 2 on serine phosphorylation of Na þ ,K þ ATPase a1 subunit. The results, as shown in Figure 7a, revealed that both Fas L and BSO increased the serine phosphorylation of Na þ ,K þ ATPase a1 subunit, whereas GSH decreased it.…”

Analysis of Na+,K+-ATPase Motion and Incorporation into the Plasma Membrane in Response to G Protein–coupled Receptor Signals in Living Cells

“…This cell line retains several of the characteristics of rat alveolar type-II cells. The effect of dopamine on Na ϩ ,K ϩ -ATPase activity as well as the signaling mechanisms involved is similar to type-II cells isolated from rat lung (Guerrero et al, 2001;Ridge et al, 2002). Selection of stable clones of A549 cells expressing the GFP-tagged rat Na ϩ ,K ϩ -ATPase ␣ 1 -subunit was performed as described previously (Pedemonte et al, 1997;Ogimoto et al, 2000).…”

“…Proteins were quantitated (Bradford, 1976) and analyzed by SDS-PAGE (Laemmli, 1970). Western blot was performed as described before (Ridge et al, 2002) using a commercial luminescence kit (Amersham Pharmacia Biotech).…”

“…The action of these networks ultimately results in activation of protein kinases and phosphorylation of the Na ϩ ,K ϩ -ATPase catalytic ␣-subunit (Therien and Blostein, 2000). Depending on the type and isoform of the kinase their action could lead to a decrease or increase in cell Na ϩ ,K ϩ -ATPase activity (Carranza et al, 1998;Efendiev et al, 1999Efendiev et al, , 2000Ridge et al, 2002). However, phosphorylation may not affect the intrinsic properties of the Na ϩ ,K ϩ -ATPase (i.e., catalytic activity) but their subcellular distribution.…”

“…For example, in renal epithelial cells inhibition of Na ϩ ,K ϩ -ATPase activity by catecholamines or phorbol esters is mediated by phosphorylation of the catalytic ␣-subunit, and this event does not affect the catalytic properties of the enzyme while in the plasma membrane but constitutes the triggering signal for its endocytosis into endosomes via a clathrin vesicle-dependent mechanism (Chibalin et al, 1997Yudowski et al, 2000;Efendiev et al, 2002). By contrast, in lung alveolar epithelial cells isoproterenol or DA increase Na ϩ , K ϩ -ATPase activity and this effect is associated with an increased recruitment of active Na ϩ , K ϩ -ATPase molecules to the plasma membrane Lecuona et al, 2000;Ridge et al, 2002). The molecular mechanisms responsible for the increase in Na ϩ , K ϩ -ATPase activity and the translocation from intracellular compartments to the plasma membrane are not yet completely understood.…”