Rapamycin Inhibits Protein Kinase C Activity and Stimulates Na+ Transport in A6 Cells

Abstract

Rapamycin and FK506 have unique cellular effects despite the fact that they bind to the same set of immunophilins, the FK506 binding proteins (FKBP). We have previously reported that rapamycin (RAP) stimulates sodium transport in A6 cells. FK506 did not stimulate sodium transport but did inhibit the stimulation seen in RAP-treated cells. Since FKBP12 has been shown to have sequence homology with an endogenous inhibitor of protein kinase C (PKC) and PKC inhibition has been shown to increase Na ؉ channel activity in A6 cells, studies to determine the effect of RAP on PKC activity and its relationship to sodium transport were performed. Here we report that RAP stimulates sodium transport, and the effect is not additive to that seen with a cellpermeant inhibitor of PKC␣ and -␤ subtypes. RAP significantly inhibits endogenous PKC activity in A6 cells both in membrane and cytosolic preparations. There is a strong correlation between the degree of inhibition of PKC activity and the stimulation of sodium transport by RAP. RAP has no effect on Na Tacrolimus (FK506), cyclosporin, and rapamycin (RAP) 1 are known to have effects on T-cell activation and proliferation. These effects are mediated through the interaction of the drug with its binding protein or immunophilin. The immunophilins are divided into two broad classes, the FK506 binding proteins (FKBPs) and the cyclophilins, both of which are characterized by peptidyl-prolyl cis-isomerase (rotamase) activity. The FK506 binding proteins bind both FK506 and RAP and are named for their molecular weights (FKBP12, FKBP56, FKBP25). The cyclophilins bind the structurally distinct macrocyclic peptide cyclosporin. The immunosuppressive effects of these agents are not mediated solely by the drug themselves or by the immunophilin to which they bind. Rather the binding of drug to the immunophilin exposes an effector region on the drug which then inhibits downstream pathways (1, 2). At present it is know that two distinct drugs that bind to the same immunophilin can have drastically different effects. For example, although FK506 and RAP both bind with high affinity to FKBP12 only FK506 inhibits calcineurin (1, 2). In contrast, the RAP⅐FKBP complex has been found to inhibit cell proliferation by inhibiting p70 S6 kinase activation (22). A6 cells, derived from Xenopus laevis are a well characterized, high resistance epithelia with transport characteristics similar to the mammalian cortical collecting duct (3). Na ϩ channel activity is known to be increased in response to aldosterone (4, 23), insulin (5), and vasopressin (4, 6 -9), each acting through unique pathways. In addition, inhibition of protein kinase C (PKC) activity has been shown to increase sodium channel activity in cell-attached patches of A6 (3). We have recently reported that RAP stimulates transepithelial sodium transport in A6 (10). The effect is seen within 15 min of addition and persists for 4 h. An excess of FK506 had no effect on basal transport but completely inhibited the RAP-induced effect. This suggested that ...

Regulation of hepatic LDL receptors by mTORC1 and PCSK9 in mice

“…Furthermore, adenoviral overexpression of HNF1α in livers of ob/ob mice led to increased mRNA and protein levels of PCSK9, decreased LDLR protein but not mRNA levels (Figure 4, D and F), and increased plasma cholesterol levels ( Figure 4E), while there were no changes in the expression of hepatic Srebp-1c, Srebp-2, or their target genes (Supplemental Figure 4B). mTOR has been shown to activate PKCδ (22,23), and PKCδ has been reported to play an important role in hepatic insulin resistance, glucose intolerance, and hepatosteatosis in obese humans and mice (24). Consistent with a role of mTORC1 in its activation, Li-Tsc1 -/-mice exhibited markedly enhanced serine 662 phosphorylation of hepatic PKCδ.…”

Role of PKCα in feedback regulation of Na+transport in an electrically tight epithelium

“…However, it is uncertain which of these and other unidentified isoforms exist in A6 epithelia. Only PKC␣ has been identified at the protein level in A6 cells (1,33). This isoform is stimulated by increasing [Ca 2ϩ ] i , a procedure known to inhibit Na ϩ channels in tight epithelia (18).…”

“…Indeed, although a large number of studies have examined ENaC's regulation by PKC in various preparations (3,7,11,15,24,27,30,32,33,39,42), only a minor fraction have focused on the potential regulation by distinct isoforms or a group of isoforms. Moreover, these studies utilized pharmacological agents believed to be selective for certain isoforms.…”

“…Moreover, these studies utilized pharmacological agents believed to be selective for certain isoforms. For example, Rokaw et al (33,34) utilized rapamycin, which has a higher specificity for cPKC isoforms, to determine that inhibition of PKC␣ transiently stimulates ENaC in A6 epithelia.…”

A review of therapies for diabetic macular oedema and rationale for combination therapy

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