Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells.

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex.

The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity.

Peroxynitrite is a reactive nitrogen species produced when nitric oxide and superoxide react. In vivo studies suggest that reactive oxygen species and, perhaps, peroxynitrite can influence Na-K-ATPase function. However, the direct effects of peroxynitrite on Na-K-ATPase function remain unknown. We show that a single bolus addition of peroxynitrite inhibited purified renal Na-K-ATPase activity, with IC50 of 107+/-9 microM. To mimic cellular/physiological production of peroxynitrite, a syringe pump was used to slowly release (approximately 0.85 microM/s) peroxynitrite. The inhibition of Na-K-ATPase activity induced by this treatment was similar to that induced by a single bolus addition of equal cumulative concentration. Peroxynitrite produced 3-nitrotyrosine residues on the alpha, beta, and FXYD subunits of the Na pump. Interestingly, the flavonoid epicatechin, which prevented tyrosine nitration, was unable to blunt peroxynitrite-induced ATPase inhibition, suggesting that tyrosine nitration is not required for inhibition. Peroxynitrite led to a decrease in iodoacetamidofluorescein labeling, implying that cysteine modifications were induced. Glutathione was unable to reverse ATPase inhibition. The presence of Na+ and low MgATP during peroxynitrite treatment increased the IC50 to 145+/-10 microM, while the presence of K+ and low MgATP increased the IC50 to 255+/-13 microM. This result suggests that the EPNa conformation of the pump is slightly more sensitive to peroxynitrite than the E(K) conformation. Taken together, these results show that peroxynitrite is a potent inhibitor of Na-K-ATPase activity and that peroxynitrite can induce amino acid modifications to the pump.

Perivascular fat alters reactivity of coronary artery: effects of diet and exercise.

UNLABELLED: Perivascular adipose tissue (PAT) has been reported to blunt agonist-induced arterial tone via a relaxing factor acting in a paracrine manner. The purpose of this study was to test the hypothesis that PAT of porcine coronary artery blunts constriction similarly and that this anticontractile effect of PAT is altered by diet and/or exercise training. METHODS: Fourteen adult male pigs were fed a normal-fat (NF) diet, and 10 adult male pigs were fed a high-fat/cholesterol (HF) diet. Four weeks after the initiation of diet, pigs were exercised (EX) or remained sedentary (SED) for 16 wk, yielding four groups: 1) NF-SED, 2) NF-EX, 3) HF-SED, and 4) HF-EX. Left circumflex coronary artery (LCX) rings were prepared with PAT left intact or removed. LCX reactivity to acetylcholine (ACh), endothelin (ET-1), bradykinin (BK), and sodium nitroprusside (SNP) was assessed in vitro using standard techniques. RESULTS: The results demonstrate that both ACh and ET-1 elicited dose-dependent increases in tension from LCX rings from all groups. Removal of PAT had no significant effect on ACh-induced contractions in any group. In contrast, removal of PAT increased ET-1-induced tension in LCX from NF-SED, HF-SED, and HF-EX but not NF-EX. PAT had no significant effect on relaxation responses to BK except in HF-EX animals, where removal of PAT increased BK-induced relaxation. PAT removal decreased SNP-induced relaxation in HF-LCX, but not LCX from NF pigs, suggesting basal release of a relaxing factor LCX from HF pigs. CONCLUSION: PAT blunts contractions induced by ET-1 in LCX from NF and HF pigs. Whereas EX abolished this effect of PAT in NF pigs, exercise did not alter the anticontractile effect in HF pigs.

Extracellular terbium and divalent cation effects on the red blood cell Na pump and chrysoidine effects on the renal Na pump.

We examined the effect of extracellular terbium (Tb(3+)) and divalent metal cations (Ca(2+), Sr(2+), and Ba(2+)) on (86)Rb(+) influx into rabbit and human red blood cells. We found that Tb(3+) at 15 and 25 microM was a non-competitive inhibitor of (86)Rb(+) influx suggesting that Tb(3+) is not binding to the transport site. This result reduces the usefulness of Tb(3+) as a potential probe for the E(out) conformation (the conformation with the transport site facing extracellularly). Ba(2+), Sr(2+) and Ca(2+), at concentrations >50 mM, had minimal effects on Rb(+) influx into red blood cells (1 mM Rb-out). This suggests that the outside transport site is very specific for monovalent cations over divalent cations, in contrast to the inside transport site. We also found that chrysoidine (4-phenylazo-m-phenylenediamine) competes with Na(+) for ATPase activity and K(+) for pNPPase activity suggesting it is binding to the E(in) conformation. Chrysoidine and similar compounds may be useful as optical probes of the E(in) conformation.

Kinetic characterization of Na,K-ATPase inhibition by Eosin.

Eosin is a probe for the Na pump nucleotide site. In contrast to previous studies examining eosin effects on Na only ATPase, we examined Na,K-ATPase- and K-activated pNPPase activity in red blood cell membranes and purified renal Na,K-ATPase. At saturating ATP (3 mM) the eosin IC(50) for Na pump inhibition was 19 microM. Increasing ATP concentrations (0.2-2.5 mM) did not overcome eosin-induced inhibition, thus eosin is a mixed-type inhibitor of ATPase activity. To test if eosin can bind to the high-affinity ATP site, purified Na,K-ATPase was labeled with 20 microM FITC. With increasing eosin concentrations (0.1 microM-10 microM) the incorporation of FITC into the ATP site significantly decreases suggesting that eosin prevents FITC reaction at the high-affinity ATP site. Eosin was a more potent inhibitor of K-activated phosphatase activity than of Na,K-ATPase activity. At 5 mM pNPP the eosin IC(50) for Na pump inhibition was 3.8+/-0.23 microM. Increasing pNPP concentrations (0.45-14.5 mM) did not overcome eosin-induced inhibition, thus eosin is a mixed-type inhibitor of pNPPase activity. These results can be fit by a model in which eosin and ATP bind only to the nucleotide site; in some pump conformations, this site is rigid and the binding is mutually exclusive and in other conformations, the site is flexible and able to accommodate both eosin and ATP (or pNPP). Interestingly, eosin inhibition of pNPPase became competitive after the addition of C(12)E(8) (0.1%) but the inhibition of ATPase remained mixed.