The standardized Ginkgo biloba extract Egb-761 protects vascular endothelium exposed to oxidized low density lipoproteins.

Dietary antioxidants are frequently proposed as protective agents for the vascular endothelium during the onset of atherosclerosis. This protection may occur at two distinct levels. First, they prevent oxidative modification of atherogenic lipoproteins (LDL). Second, they can provide a cellular protection against oxidized LDL-mediated endothelium dysfunction, although this mechanism remains poorly considered in many instances. To gain insight into the mechanism underlying such cellular protection against oxidized LDL, we examined the impact of a popular traditional medicine, an extract from Ginkgo biloba with well-known antioxidant properties, on two endothelial cells properties: cell adhesion and ionic homeostasis. Cellular lipoperoxides levels were also measured as a marker of cellular oxidative stress. Human umbilical-vein endothelial cells were exposed to native (nat-) or oxidized (ox-) LDL, the latter prepared to be compatible with clinically observed levels of oxidation. Although nat-LDL had little effect, ox-LDL increased endothelial adhesive properties (35%, p<0.01) and lipoperoxidation (45%, p<0.01). Na,K-ATPase activity, a key regulator of ionic homeostasis, was significantly decreased after exposure to nat-LDL (30%, p<0.01) and dramatically depressed after exposure to ox-LDL (65%, p<0.001). The standardized preparation of Ginkgo biloba EGb-761 totally protected adhesive properties and endothelial lipoperoxide levels. Moreover, it limited the decrease in Na,K-ATPase activity induced by ox-LDL to levels similar to nat-LDL. This suggests that EGb-761 protects endothelial adhesive properties and helps prevent the disruption of ionic homeostasis. The EGb-761-mediated inhibition of ox-LDL-induced lipoperoxide levels in endothelial cells appears to be an important mechanism by which Ginkgo biloba extract protects endothelial properties.

RT-PCR detection of Na,K-ATPase subunit isoforms in human umbilical vein endothelial cells (HUVEC): evidence for the presence of alpha1 and beta3.

The endothelial Na,K-ATPase is an active component in maintaining a variety of normal vascular functions. The enzyme is characterized by a complex molecular heterogeneity that results from differential expression and association of multiple isoforms of both its alpha- and beta-subunits. The aim of the present study was to determine which isoforms of the Na,K-ATPase are expressed in human endothelial cells. HUVEC (human umbilical vein endothelial cells) were used as a model of well known human endothelial cells. The high sensitive method RT-PCR was used with primers specific for the various isoforms of the alpha- and beta-subunits of the Na,K-ATPase. The results show that HUVEC express alpha1-, but not alpha2-, alpha3- or alpha4-isoforms of the catalytic subunit and that beta3- but not beta2- or beta1-isoforms is present in these cells. These findings are in contradiction with our previous detection of Na,K-ATPase isoforms in HUVEC using antibodies (14). Such results raise the technical problem of the specificity of the available antibodies directed against the different isoforms as well as the question of the physiological relevance of the diversity of the Na,K-ATPase isoforms.

Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane.

Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.

Aquaporin mediated water flux as a target for diuretic development.

Within the past decade an entire family of membrane proteins--aquaporins--which function as transmembrane water channels has been identified; they occur throughout the plant, animal, and bacterial kingdoms. Several family members permit glycerol and urea permeability. Most aquaporins are inhibited by mercury. Constitutively expressed aquaporin 1 is the major permeability channel of the proximal tubule, descending thin limb of the loop of Henle, and it is also found in vasa recta. Aquaporin 2 is expressed in the principal cells of the collecting duct where it shuttles between intracellular vesicles and the apical membrane in response to vasopressin. Aquaporin 2 mutations cause nephrogenic diabetes insipidus; increased aquaporin 2 activity is implicated in the pathophysiology of heart failure, cirrhosis, and nephrotic syndrome. Aquaporins 3 and 4 provide basolateral membrane water channels in the collecting duct. These 4 channels and 6 others are also found elsewhere throughout the body. The physiological importance of several of the channels remains unknown. Aquaporin 1 inhibitors might induce useful diuresis, but humans who lack aquaporin 1 have no significant clinical disease. Inhibition of aquaporin 2 activity by vasopressin receptor antagonists may be useful in heart failure, cirrhosis, nephrotic syndrome, and the syndrome of inappropriate antidiuretic hormone (ADH) release.

The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon.

Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies.

Roles of IL-1 and TNF in the decreased ileal muscle contractility induced by lipopolysaccharide.

Gastrointestinal stasis during sepsis may be associated with gastrointestinal smooth muscle dysfunction. Endotoxin [lipopolysaccharide (LPS)] impairs smooth muscle contraction, in part through inducible nitric oxide synthase (NOS II) and enhanced nitric oxide production. We studied the roles of tumor necrosis factor-alpha (TNF) and interleukin-1 (IL-1) in this process by using TNF binding protein (TNFbp) and IL-1 receptor antagonist (IL-1ra). Rats were treated with TNFbp and IL-1ra, or their vehicles, 1 h before receiving LPS or saline. At 5 h after LPS, contractility was measured in strips of ileal longitudinal smooth muscle, and NOS II activity was measured in full-thickness segments of ileum. LPS decreased maximum stress (mean +/- SE) from 508 +/- 55 (control) to 355 +/- 33 g/cm2 (P < 0.05). Pretreatment with TNFbp plus IL-1ra prevented the LPS-induced decrease. Separate studies of TNFbp alone or IL-1ra alone indicated that, at the doses and timing used, TNFbp was more effective. LPS also increased NOS II activity by >10-fold (P < 0.01) over control. This increase was prevented by TNFbp plus IL-1ra (P = not significant vs. control). We conclude that the LPS-induced increase in NOS II activity and the decrease in ileal muscle contractility are mediated by TNF and IL-1.

Na,K-ATPase mRNA beta 1 expression in rat myocardium--effect of thyroid status.

The abundance of Na,K-ATPase and its alpha and beta subunit mRNAs is upregulated in cardiac and other target tissue by thyroid hormone (T3). Multiple Na,K-ATPase mRNA beta 1 species encoding an identical beta 1 polypeptide are expressed in the heart. The different mRNA beta 1 species result from utilization of two transcription start-sites in the first exon and multiple (five) poly(A) signals in the terminal exon of the beta 1 gene. In the present study we identify the mRNA beta 1 species that are expressed in rat ventricular myocardium under basal conditions, and determine whether they are differentially regulated by T3. mRNA beta 1 species were identified by 3'-RACE followed by DNA sequencing, and by Northern blotting using probes derived from different regions of rat cDNA beta 1. Five mRNA beta 1 species are expressed in rat heart: mRNA beta 1 species that are initiated at the first transcription start-site and end at the first, second and fifth poly(A) sites (resulting in mRNAs of 1630, 1810, and 2780 nucleotides), and mRNA beta 1 species initiated at the second transcription start-site and ending at the second and fifth poly(A) sites (resulting in mRNAs of 1500 and 2490 nucleotides); in order of increasing length, the five mRNAs constitute 0.04, 0.15, 0.38, 0.11 and 0.32 of total mRNA beta 1 content. In hypothyroid rats (induced by addition of propyl-thiouracil to the drinking water for 3 weeks), total mRNA beta 1 content decreased to 0.18 euthyroid levels, which was associated with a disproportionate 7.5-fold decrease in the abundance of the longest transcript (P < 0.05); transcripts initiating at the first transcription start-site and ending at the second poly(A) signal in hypothyroid hearts were 0.26 euthyroid levels (P < 0.05). Hyperthyroidism induced by injection of normal rats with three doses of 100 micrograms T3/100 g body weight every 48 h resulted in an overall approximately 2-fold increase in mRNA beta 1 content with no change in the fractional contribution of any of the mRNA beta 1 species. The results indicate a complex heterogeneity in the expression of mRNA beta 1 in myocardium.

Stimulation of Na,K-ATPase by hypothyroidism in the thyroid gland.

Although studies have documented the regulatory effects of thyroid hormones on the Na,K-ATPase in peripheral tissues, there is little information on the regulation of this transporter in the thyroid gland itself. Accordingly, we investigated the effects of thyroid status on Na,K-ATPase specific activity and the abundance of its constituent subunits in rat thyroid. Exogenous tri-iodothyronine (T3) was administered daily to produce hyperthyroidism. 6n-propyl-2-thiouracil (PTU), an inhibitor of thyroid hormone synthesis, was used to induce hypothyroidism. There was a four-fold increase in Na,K-ATPase specific activity in the follicular membranes from PTU-treated animals after 7 days. Enzymatic activities were not changed in the T3-treated glands. Immunoblotting of membranes from T3-treated rats revealed a 75% reduction in alpha1 subunit abundance and a slight, but nonsignificant reduction in beta1 abundance. On the other hand, the membranes from PTU-treated rats displayed 136 and 567% increases in the abundance of the alpha1 and beta1 subunits respectively. These data demonstrate that thyroid hormone status regulates Na,K-ATPase in the gland, but the effects are in direct contrast to those seen in the periphery.

Dopamine-induced endocytosis of Na+,K+-ATPase is initiated by phosphorylation of Ser-18 in the rat alpha subunit and Is responsible for the decreased activity in epithelial cells.

Dopamine inhibits Na+,K+-ATPase activity in renal tubule cells. This inhibition is associated with phosphorylation and internalization of the alpha subunit, both events being protein kinase C-dependent. Studies of purified preparations, fusion proteins with site-directed mutagenesis, and heterologous expression systems have identified two major protein kinase C phosphorylation residues (Ser-11 and Ser-18) in the rat alpha1 subunit isoform. To identify the phosphorylation site(s) that mediates endocytosis of the subunit in response to dopamine, we have performed site-directed mutagenesis of these residues in the rat alpha1 subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na+,K+-ATPase activity and increased alpha subunit phosphorylation and clathrin-dependent endocytosis into endosomes in cells expressing the wild type alpha1 subunit or the S11A alpha1 mutant, and both effects were blocked by protein kinase C inhibition. In contrast, dopamine did not elicit any of these effects in cells expressing the S18A alpha1 mutant. While Ser-18 phosphorylation is necessary for endocytosis, it does not affect per se the enzymatic activity: preventing endocytosis with wortmannin or LY294009 blocked the inhibitory effect of dopamine on Na+,K+-ATPase activity, although it did not alter the increased alpha subunit phosphorylation induced by this agonist. We conclude that dopamine-induced inhibition of Na+, K+-ATPase activity in rat renal tubule cells requires endocytosis of the alpha subunit into defined intracellular compartments and that phosphorylation of Ser-18 is essential for this process.

Mutagenesis disrupts posttranslational processing of the Na,K-ATPase catalytic subunit.

The first 5 amino acids of the catalytic alpha 1 isoform from Na,K-ATPase are cleaved enzymatically during or after translation. To evaluate the structural requirements for that cleavage, we constructed amino-terminal mutants of alpha 1 in which an epitope tag from the c-myc oncogene product was added. Immunoblots of isolated membranes from transfected monkey kidney cells revealed binding of an antibody specific for the first 9 residues of the alpha 1 nascent protein. Because this antibody does not recognize the shorter sequence corresponding to the processed polypeptide, these results indicate that the epitope tag prevented normal processing, a conclusion confirmed by the observed binding of an anti-myc antibody. In contrast, membranes from cells expressing deletion mutants that lack residues 10-24 and 10-31 of the nascent chain failed to bind the amino-terminal-directed antibody, suggesting that the mutants were cleaved normally and that amino acids downstream of the first 9 are not required for proteolysis. Amino-terminal mutants produced in other laboratories have shown an anomalous stimulation of ATPase activity by K+ when measured in low ATP concentrations. The myc-tagged and downstream deletion mutants were sensitive to K+ in the range from 0.05 to 5 mM, similar to wild-type enzyme, despite the differences in posttranslational processing. A mutant missing the first 40 residues of the nascent chain, however, displayed an activation by K+. These results suggest that amino-terminal processing of the alpha 1 isoform was prevented by mutation, yet that processing had little influence on the kinetic parameter most likely to be influenced by such changes.

Stimulation of protein kinase C rapidly reduces intracellular Na+ concentration via activation of the Na+ pump in OK cells.

Na+ reabsorption is regulated in proximal tubules by hormones that stimulate protein kinase C (PKC). To determine whether stimulation of PKC causes a reduction in intracellular Na+ concentration ([Na+]i) that might link Na+ pump activation to increased Na+ reabsorption, [Na+]i was measured in kidney cells loaded with the Na+-sensitive fluorescent indicator SBFI. Rapid digital imaging fluorescence microscopy determinations were performed in epithelial kidney cells transfected with the rodent Na+ pump alpha1 cDNA. In 42 determinations, the basal [Na+]i was 19.7 +/- 2.4 mM. Stimulation of PKC reduced the [Na+]i to 5.6 +/- 0.6 mM in approximately 10 sec. This drastic change in [Na+]i requires a transient 74-120-fold increase in Na+ pump activity. After the new steady state [Na+]i is reached, the Na+ pump is 58% activated. The entry of Na+ into the cells is not affected by stimulation of PKC; therefore, the reduction in [Na+]i is exclusively dependent on activation of the Na+ pump. Accordingly, PKC stimulation does not affect the [Na+]i of cells expressing a mutant Na+ pump that is not stimulated by PKC. The decrease in [Na+]i observed in cells transfected with the rodent Na+ pump alpha1 cDNA is large and sufficiently fast that it is expected to stimulate rapidly passive Na+-influx into the cells, thereby accounting for the observed PKC-induced stimulation of Na+ reabsorption.

Regulation of Na,K-ATPase transport activity by protein kinase C.

Considerable evidence indicates that the renal Na+,K+-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH2-terminal end of the Na+,K+-ATPase alpha-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na+,K+-ATPase activity. To determine whether the alpha-subunit NH2-terminus is involved in the regulation of Na+, K+-ATPase activity by PKC, we have expressed the wild-type rodent Na+,K+-ATPase alpha-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH2-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the alpha-subunit NH2-terminal segment was not necessary to express the maximal Na+,K+-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb+-transport in intact cells were the same in cells transfected with the wild-type rodent alpha1 and the NH2-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na+,K+-ATPase mediated Rb+-uptake and reduced the intracellular Na+ concentration of cells transfected with wild-type alpha1 cDNA. In contrast, these effects were not observed in cells expressing the NH2-deletion mutant of the alpha-subunit. Treatment with phorbol ester appears to affect specifically the Na+,K+-ATPase activity and no evidence was observed that other proteins involved in Na+-transport were affected. These results indicate that amino acid(s) located at the alpha-subunit NH2-terminus participate in the regulation of the Na+,K+-ATPase activity by PKC.

Effect of chronic hypokalemia on H(+)-K(+)-ATPase expression in rat colon.

Although the kidney plays the major role in the regulation of systemic K+ homeostasis, the colon also participates substantively in K+ balance. The colon is capable of both K+ absorption and secretion, the magnitude of which can be modulated in response to dietary K+ intake. The H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) has been proposed as a possible mediator of K+ absorption in distal colon, but inhibitor profiles obtained in recent studies suggest that two, and perhaps more, distinct H(+)-K(+)-ATPase activities may be present in mammalian distal colon. We have developed highly specific probes for the catalytic alpha-subunits of colonic and gastric H(+)-K(+)-ATPase, alpha 1-Na(+)-K(+)-ATPase, and beta-actin, which were used in Northern analysis of total RNA from whole distal colon and stomach obtained from one of three experimental groups of rats: 1) controls, 2) chronic dietary K+ depletion, and 3) chronic metabolic acidosis. The probe for the colonic but not the gastric H(+)-K(+)-ATPase alpha-isoform hybridized to distal colon total RNA in all groups. A significant increase in colonic H(+)-K(+)-ATPase mRNA abundance was observed in response to chronic dietary K+ depletion but not to chronic metabolic acidosis. The alpha 1-isoform of Na(+)-K(+)-ATPase, which is also expressed in distal colon, did not respond consistently to either chronic dietary K+ depletion or chronic metabolic acidosis. The gastric probe did not hybridize to total RNA from distal colon but, as expected, hybridized to total stomach RNA. However, the abundance of gastric H(+)-K(+)-ATPase or Na(+)-K(+)-ATPase in stomach was not altered consistently by either chronic dietary K+ depletion or metabolic acidosis. Under the conditions of this study, it appears that the mRNA encoding the colonic alpha-isoform is upregulated by chronic dietary K+ restriction, a condition shown previously to increase K+ absorption in the distal colon.

Regional expression of sodium pump subunits isoforms and Na+-Ca++ exchanger in the human heart.

Cardiac glycosides exert a positive inotropic effect by inhibiting sodium pump (Na,K-ATPase) activity, decreasing the driving force for Na+-Ca++ exchange, and increasing cellular content and release of Ca++ during depolarization. Since the inotropic response will be a function of the level of expression of sodium pumps, which are alpha(beta) heterodimers, and of Na+-Ca++ exchangers, this study aimed to determine the regional pattern of expression of these transporters in the heart. Immunoblot assays of homogenate from atria, ventricles, and septa of 14 nonfailing human hearts established expression of Na,K-ATPase alpha1, alpha2, alpha3, beta1, and Na+-Ca++ exchangers in all regions. Na,K-ATPase beta2 expression is negligible, indicating that the human cardiac glycoside receptors are alpha1beta1, alpha2beta1, and alpha3beta1. alpha3, beta1, sodium pump activity, and Na+-Ca++ exchanger levels were 30-50% lower in atria compared to ventricles and/or septum; differences between ventricles and septum were insignificant. Functionally, the EC50 of the sodium channel activator BDF 9148 to increase force of contraction was lower in atria than ventricle muscle strips (0.36 vs. 1.54 microM). These results define the distribution of the cardiac glycoside receptor isoforms in the human heart and they demonstrate that atria have fewer sodium pumps, fewer Na+-Ca++ exchangers, and enhanced sensitivity to inotropic stimulation compared to ventricles.