ucn-1028-c and phorbol-12-myristate

Mammalian distal nephron and distal colon, prime sites for Na(+) homeostasis, contain amiloride-sensitive epithelial sodium channels (ENaC). Protein kinase C (PKC) inhibits ENaC by phosphorylating serine and threonine residues within COOH termini of the β- and/or γ-subunits. Although some of these phosphorylation sites are close to the PY motifs, it is unclear whether they remain susceptible to PKC in Liddle-mutated ENaC β- and/or γ-subunits, where PY motifs are truncated, resulting in increased apical ENaC expression. We therefore studied the effects of PKC in wild-type and Liddle-mutated human epithelial Na(+) channels (hENaC) expressed in Xenopus oocytes, using the dual-electrode voltage clamp technique. PKC activation using 500 nmol/l phorbol 12-myristate 13-acetate (PMA) decreased amiloride-sensitive Na(+) currents by 80 % in oocytes expressing wild-type hENaC, an effect largely prevented by co-exposure to 50 µmol/l calphostin C (a specific inhibitor of PKC), whereas 500 nmol/l phorbol didecanoate (PDD), an inactive phorbol ester which does not stimulate PKC, had no effect. In oocytes expressing hENaC containing the Liddle-mutated β-subunit, PMA elicited a 54 % decrease in amiloride-sensitive Na(+) currents, significantly (P < 0.0025) less than that in oocytes expressing wild-type hENaC. By contrast, in oocytes expressing hENaC containing the Liddle-mutated γ-subunit, PMA elicited a 68 % decrease in amiloride-sensitive Na(+) current, similar (P = 0.10) to that in oocytes expressing wild-type hENaC. We conclude that hENaC incorporating the Liddle-mutated β-subunit lacks one or more PKC phosphorylation sites, thereby significantly reducing the inhibitory effect of PKC on Na(+) channel activity, whereas hENaC incorporating Liddle-mutated γ-subunits remains as susceptible to PKC as wild-type hENaC.

Topics: Animals; Epithelial Sodium Channels; Humans; Kidney; Mutation; Naphthalenes; Oocytes; Patch-Clamp Techniques; Phorbol Esters; Protein Interaction Domains and Motifs; Protein Kinase C; Protein Subunits; Xenopus laevis

Copper is necessary for all organisms since it acts as a cofactor in different enzymes, although toxic at high concentrations. ATP7B is one of two copper-transporting ATPases in humans, its vital role being manifested in Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving protein kinase C (PKC) modulate ATP7B activity. Different isoforms of PKC (α, ɛ, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)-ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10(-8) M), a PKC activator, increased Cu(I)-ATPase activity by 60%, whereas calphostin C and U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2 μM Ca(2+), whereas PMA plus EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCɛ to levels comparable with those found after phosphatase λ treatment, showing that the ɛ isoform is essential for activation of the enzyme. This regulatory phosphorylation enhanced Vmax without modifying affinities for ATP and copper. It can be concluded that signaling pathways leading to DAG formation and PKCɛ activation stimulate the active transport of copper by ATP7B, thus evidencing a central role for this specific kinase-mediated mechanism in hepatic copper handling.

Topics: Adenosine Triphosphatases; Amino Acid Sequence; Animals; Blotting, Western; Cation Transport Proteins; Cell Membrane; Copper; Copper-Transporting ATPases; Enzyme Inhibitors; Estrenes; Isoenzymes; Liver; Molecular Sequence Data; Naphthalenes; Phorbol Esters; Phosphodiesterase Inhibitors; Phosphorylation; Protein Kinase C; Pyrrolidinones; Sequence Homology, Amino Acid; Swine

With a special interest towards a better understanding of signal pathways, we attempted to discover a safer and more effective therapeutic strategy for kidney carcinoma. Recent studies had suggested a role mediated by PKCα for netrin-1 and its receptors in the initiation and progression of tumors. Real-time PCR and western blotting were used to determine the expression levels of netrin-1 and UNC5B. We made use of the agonist of PKCα (phorbol-12-myristate 13-acetate-PMA) and the inhibitor of PKCα (calphostin C) to treat renal cell carcinoma (RCC) cells, and MTT assays were used to measure cell proliferation. By immunofluorescence, we identified the localization of netrin-1 and UNC5B in RCC cell lines 769-P and ACHN. The expression of UNC5B in tumor tissues was significantly downregulated compared to the corresponding normal tissues in which netrin-1 was upregulated. In low grade tumors, UNC5B expression was more prominent while netrin-1 expression was the opposite when compared with high grade ones. Proliferation of ACHN cells was concentration dependent in the presence of PMA and calphostin C. Netrin-1 and UNC5B expressions were upregulated in cells treated with PMA while calphostin C reversed this upregulation. By immunofluorescence, we identified that netrin-1 was highly expressed in the nuclear but none of UNC5B. Our data highly suggested that PMA-induced upregulation and calphostin C-induced reversion of netrin-1 and UNC5B in kidney carcinoma were accompanied by the activation of the netrin-1/UNC5B pathways.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Blotting, Western; Carcinoma, Renal Cell; Case-Control Studies; Cell Proliferation; Enzyme Inhibitors; Female; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Kidney Neoplasms; Male; Middle Aged; Naphthalenes; Neoplasm Grading; Neoplasm Staging; Nerve Growth Factors; Netrin Receptors; Netrin-1; Phorbol Esters; Protein Kinase C-alpha; Real-Time Polymerase Chain Reaction; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured; Tumor Suppressor Proteins