Multiple mineralocorticoid response elements localized in different introns regulate intermediate conductance K+ (Kcnn4) channel expression in the rat distal colon.

An elevated plasma aldosterone and an increased expression of the intermediate conductance K(+) (IK/Kcnn4) channels are linked in colon. This observation suggests that the expression of Kcnn4 gene is controlled through the action of aldosterone on its cognate receptor (i.e., mineralocorticoid receptor; MR). In order to establish this, we performed chromatin immunoprecipitation (ChIP) assay to identify the MR response elements (MREs) in a region that spanned 20 kb upstream and 10 kb downstream of the presumed transcription start site (TSS) using chromatin from the colonic epithelial cells of normal and aldosterone-treated rats. MREs were immunoprecipitated in an approximately 5 kb region that spanned the first and second introns in the aldosterone rats. These regions were individually cloned in luciferase-expression vector lacking enhancer activity. These clones were tested for enhancer activity in vitro by transfecting in HEK293T and CaCo2 cells with MR and aldosterone treatment. At least four regions were found to be responsive to the MR and aldosterone. Two regions were identified to contain MREs using bioinformatics tools. These clones lost their enhancer activity after mutation of the presumptive MREs, and thus, established the functionality of the MREs. The third and fourth clones did not contain any bioinformatically obvious MREs. Further, they lost their activity upon additional sub-cloning, which suggest cooperativity between the regions that were separated upon sub-cloning. These results demonstrate the presence of intronic MREs in Kcnn4 and suggest a highly cooperative interaction between multiple intronic response elements.

Aldosterone induces active K⁺ secretion by enhancing mucosal expression of Kcnn4c and Kcnma1 channels in rat distal colon.

Although both Kcnn4c and Kcnma1 channels are present on colonic mucosal membranes, only Kcnma1 has been suggested to mediate K(+) secretion in the colon. Therefore, studies were initiated to investigate the relative roles of Kcnn4c and Kcnma1 in mediating aldosterone (Na-free diet)-induced K(+) secretion. Mucosal to serosal (m-s), serosal to mucosal (s-m), and net (86)Rb(+) (K(+) surrogate) fluxes as well as short circuit currents (I(sc); measure of net ion movement) were measured under voltage clamp condition in rat distal colon. Active K(+) absorption, but not K(+) secretion, is present in normal, while aldosterone induces active K(+) secretion (1.04 ± 0.26 vs. -1.21 ± 0.15 µeq·h(-1)·cm(-2); P < 0.001) in rat distal colon. Mucosal VO(4) (a P-type ATPase inhibitor) inhibited the net K(+) absorption in normal, while it significantly enhanced net K(+) secretion in aldosterone animals. The aldosterone-induced K(+) secretion was inhibited by the mucosal addition of 1) either Ba(2+) (a nonspecific K(+) channel blocker) or charybdotoxin (CTX; a common Kcnn4 and Kcnma1 channel blocker) by 89%; 2) tetraethyl ammonium (TEA) or iberiotoxin (IbTX; a Kcnma1 channel blocker) by 64%; and 3) TRAM-34 (a Kcnn4 channel blocker) by 29%. TRAM-34, but not TEA, in the presence of IbTX further significantly inhibited the aldosterone-induced K(+) secretion. Thus the aldosterone-induced Ba(2+)/CTX-sensitive K(+) secretion consists of IbTX/TEA-sensitive (Kcnma1) and IbTX/TEA-insensitive fractions. TRAM-34 inhibition of the IbTX-insensitive fraction is consistent with the aldosterone-induced K(+) secretion being mediated partially via Kcnn4c. Western and quantitative PCR analyses indicated that aldosterone enhanced both Kcnn4c and Kcnma1α protein expression and mRNA abundance. In vitro exposure of isolated normal colonic mucosa to aldosterone also enhanced Kcnn4c and Kcnma1α mRNA levels, and this was prevented by exposure to actinomycin D (an RNA synthesis inhibitor). These observations indicate that aldosterone induces active K(+) secretion by enhancing mucosal Kcnn4c and Kcnma1 expression at the transcriptional level.

HIV entry inhibitors in clinical development.

Despite recent advances, present therapies for human immunodeficiency virus type 1 (HIV-1) infection are limited by their failure to eradicate HIV-1, by the emergence of multidrug-resistant variants and by significant toxicities. Current therapies collectively target two viral enzymes involved in intracellular viral replication processes, and there is an urgent need for new treatment modalities. HIV-1 entry is a multistep process that comprises viral attachment, co-receptor interactions and fusion. This cascade of events offers opportunities for therapeutic intervention, and clinical proof-of-principle has been obtained for inhibitors of each step. These agents are referred to broadly as entry inhibitors.

RFI-641, a potent respiratory syncytial virus inhibitor.

Human respiratory syncytial virus (RSV), a paramyxovirus, is a major cause of acute upper and lower respiratory tract infections in infants, young children, and adults. RFI-641 is a novel anti-RSV agent with potent in vitro and in vivo activity. RFI-641 is active against both RSV type A and B strains. The viral specificity and the large therapeutic window of RFI-641 (>100-fold) indicate that the antiviral activity of the compound is not due to adverse effects on normal cells. The potent in vitro activity of RFI-641 can be translated to efficacy in vivo: RFI-641 is efficacious when administered prophylactically by the intranasal route in mice, cotton rats, and African green monkeys. RFI-641 is also efficacious when administered therapeutically (24 h postinfection) in the monkey model. Mechanism of action studies indicate that RFI-641 blocks viral F protein-mediated fusion and cell syncytium formation.