.-One of the defining characteristics of the epithelial sodium channel (ENaC) is its block by the diuretic amiloride. This study investigates the role of the extracellular loop of the ␣-subunit of ENaC in amiloride binding and stabilization. Mutations were generated in a region of the extracellular loop, residues 278-283. Deletion of this region, WYRFHY, resulted in a loss of amiloride binding to the channel. Channels formed from wild-type ␣-subunits or ␣-subunits containing point mutations in this region were examined and compared at the single-channel level. The open probabilities (P o) of wild-type channels were distributed into two populations: one with a high P o and one with a low P o. The mean open times of all the mutant channels were shorter than the mean open time of the wild-type (high-P o) channel. Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type ␣-ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel. These data provide new insight into the relative position of the extracellular loop with respect to the pore of ENaC and its role in amiloride binding and channel gating. open probability; extracellular loop; channel pore; sodium channel; single-channel recording EPITHELIAL SODIUM CHANNELS (ENaC) play a critical role in the control of blood pressure and regulation of total body sodium balance. Although the original work in which the channel components were cloned suggested that functional channels consist of three subunits, ␣, , and ␥ (5), under appropriate circumstances, ␣-subunits alone can form sodium-permeable channels (12,13,17). It has been proposed that some of the diversity in conductance, gating, and selectivity of functional ENaC may be due to different subunit combinations. The expression of ␣-subunits alone, rather than the expression of all three subunits together, depends on the environmental conditions to which renal epithelial cells (A6) (7) or alveolar type II cells (14) are exposed. However, regardless of which channel type is expressed, one of the defining characteristics of all these channels is their block by the diuretic amiloride, a substituted pyrazinoylguanidine. The tertiary structure of all the subunits is similar: each subunit is predicted to span the membrane twice, to have a large extracellular loop, and to have short intracellular NH 2 and COOH termini (2, 6). Because amiloride blocks the channel from the extracellular surface of the channel protein, it is possible that one or more of the extracellular loops could play a role in amiloride's interaction with and block of the channel by stabilizing amiloride in the channel pore.About 70% of each ENaC subunit is extracellular. Structure-function studies of the extracellular loop have focused primarily on the region immediately preceding and including the second transmembrane (M2) domain of ␣-ENaC. These data have implicated these regions in binding to amiloride and also play a role in...
Anti-atherogenic effects of high density lipoprotein (HDL) and its major protein component apolipoprotein A-I (apoA-I) are principally thought to be due to their ability to mediate reverse cholesterol transport. These agents also possess anti-oxidant properties that prevent the oxidative modification of low density lipoprotein (LDL) and anti-inflammatory properties that include inhibition of endothelial cell adhesion molecule expression. Results of the Framingham study revealed that a reduction in HDL levels is an independent risk factor for coronary artery disease (CAD). Accordingly, there has been considerable interest in developing new therapies that specifically elevate HDL cholesterol. However, recent evidence suggests that increasing circulating HDL cholesterol levels alone is not sufficient as a mode of HDL therapy. Rather, therapeutic approaches that increase the functional properties of HDL may be superior to simply raising the levels of HDL per se. Our laboratory has pioneered the development of synthetic, apolipoprotein mimetic peptides which are structurally and functionally similar to apoA-I but possess unique structural homology to the lipid-associating domains of apoA-I. The apoA-I mimetic peptide 4F inhibits atherogenic lesion formation in murine models of atherosclerosis. This effect is related to the ability of 4F to induce the formation of pre-β HDL particles that are enriched in apoA-I and paraoxonase. 4F also possesses anti-inflammatory and anti-oxidant properties that are independent of its effect on HDL quality per se. Recent studies suggest that 4F stimulates the expression of the antioxidant enzymes heme oxygenase and superoxide dismutase and inhibits superoxide anion formation in blood vessels of diabetic, hypercholesterolemic and sickle cell disease mice. The goal of this review is to discuss HDL-dependent and -independent mechanisms by which apoA-I mimetic peptides reduce vascular injury in experimental animal models.
Anti-atherogenic effects of high density lipoprotein (HDL) and its major protein component apolipoprotein A-I (apoA-I) are principally thought to be due to their ability to mediate reverse cholesterol transport. These agents also possess anti-oxidant properties that prevent the oxidative modification of low density lipoprotein (LDL) and anti-inflammatory properties that include inhibition of endothelial cell adhesion molecule expression. Results of the Framingham study revealed that a reduction in HDL levels is an independent risk factor for coronary artery disease (CAD). Accordingly, there has been considerable interest in developing new therapies that specifically elevate HDL cholesterol. However, recent evidence suggests that increasing circulating HDL cholesterol levels alone is not sufficient as a mode of HDL therapy. Rather, therapeutic approaches that increase the functional properties of HDL may be superior to simply raising the levels of HDL per se. Our laboratory has pioneered the development of synthetic, apolipoprotein mimetic peptides which are structurally and functionally similar to apoA-I but possess unique structural homology to the lipid-associating domains of apoA-I. The apoA-I mimetic peptide 4F inhibits atherogenic lesion formation in murine models of atherosclerosis. This effect is related to the ability of 4F to induce the formation of pre-β HDL particles that are enriched in apoA-I and paraoxonase. 4F also possesses anti-inflammatory and anti-oxidant properties that are independent of its effect on HDL quality per se. Recent studies suggest that 4F stimulates the expression of the antioxidant enzymes heme oxygenase and superoxide dismutase and inhibits superoxide anion formation in blood vessels of diabetic, hypercholesterolemic and sickle cell disease mice. The goal of this review is to discuss HDL-dependent and -independent mechanisms by which apoA-I mimetic peptides reduce vascular injury in experimental animal models.
Recent studies suggest an anti‐inflammatory role for glucosamine (GlcN). We tested the hypothesis that GlcN treatment in septic rats prevents defects in cardiac performance. Cecal ligation and puncture (CLP) or sham surgery was performed in male Sprague‐Dawley rats (250‐300g). CLP rats were further randomized to receive GlcN (0.3 mg/g) or saline by ip injection. Echocardiography was performed prior to and 24 hours post surgery. Cardiac dimensions in sham rats were unchanged over this time. In contrast, there was a 6 fold increase in IVSd and PWd and a 5‐fold reduction in LVEDD and LVESD in CLP rats. GlcN treatment attenuated these responses by approximately 50%. Reductions in end‐diastolic volume (EDV), stroke volume (SV) and cardiac output (CO) in CLP rats were also attenuated by GlcN. Fractional shortening, however, was similar in all groups. These data suggest that impaired cardiac performance in CLP rats is due to a reduction in LV filling rather than to a defect in cardiac function per se. CTD110.6 immunoreactivity, a marker of protein O‐GlcNAcylation, was increased in hearts from GlcN‐treated CLP rats. GlcN treatment also increased survival (93%) in CLP rats at 24 hrs compared to saline‐treated CLP rats (70%). It is proposed that GlcN improves cardiac performance and survival in CLP rats by a mechanism involving an increase in protein O‐GlcNAcylation and inhibition of inflammatory pathways.
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