Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants

Plant HKT proteins comprise a family of cation transporters together with prokaryotic KtrB, TrkH, and KdpA transporter subunits and fungal Trk proteins. These transporters contain four loop domains in one polypeptide with a proposed distant homology to K(+) channel selectivity filters. Functional expression in yeast and Xenopus oocytes revealed that wheat HKT1 mediates Na(+)-coupled K(+) transport. Arabidopsis AtHKT1, however, transports only Na(+) in eukaryotic expression systems. To understand the molecular basis of this difference we constructed a series of AtHKT1/HKT1 chimeras and introduced point mutations to AtHKT1 and wheat HKT1 at positions predicted to be critical for K(+) selectivity. A single-point mutation, Ser-68 to glycine, was sufficient to restore K(+) permeability to AtHKT1. The reverse mutation in HKT1, Gly-91 to serine, abrogated K(+) permeability. This glycine in P-loop A of AtHKT1 and HKT1 can be modeled as the first glycine of the K(+) channel selectivity filter GYG motif. The importance of such filter glycines for K(+) selectivity was confirmed by interconversion of Ser-88 and Gly-88 in the rice paralogues OsHKT1 and OsHKT2. Surprisingly, all HKT homologues known from dicots have a serine at the filter position in P-loop A, suggesting that these proteins function mainly as Na(+) transporters in plants and that Na(+)/K(+) symport in HKT proteins is associated with a glycine in the filter residue. These data provide experimental evidence that the glycine residues in selectivity filters of HKT proteins are structurally related to those of K(+) channels.     

Spironolactone inhibits the transcardiac extraction of aldosterone in patients with congestive heart failure

OBJECTIVES: The study evaluated the transcardiac extraction or spillover of aldosterone (ALDO) in normal subjects and in patients with congestive heart failure (CHF). BACKGROUND: Aldosterone promotes collagen synthesis and structural remodeling of target organs such as the heart. Spironolactone, an ALDO receptor antagonist, has recently been reported to reduce the mortality of patients with CHF; however, the effects of spironolactone on the transcardiac gradient of ALDO have not been clarified. METHODS: We measured plasma ALDO in the aortic root (AO) and coronary sinus (CS) in normal subjects and 113 consecutive CHF patients and also measured plasma procollagen type III aminoterminal peptide (PIIINP) in CS, a biochemical marker of myocardial fibrosis. RESULTS: Plasma ALDO was significantly lower in the CS than in the AO in normal subjects (n = 15; 61.2 +/- 9.3 vs. 83.1 +/- 11.8 pg/ml, p < 0.0001). In 96 CHF patients who did not receive spironolactone, plasma ALDO was significantly lower in the CS than in the AO (59.3 +/- 3.9 vs. 73.8 +/- 4.9 pg/ml, p < 0.0001). In contrast to the difference in these 96 patients, there was no significant difference in ALDO between the AO and CS in 17 patients who received spironolactone (127.4 +/- 20 vs. 124.0 +/- 19 pg/ml, p = 0.50). Stepwise multivariate analyses showed that spironolactone therapy had an independent and significant negative relationship with the transcardiac gradient of plasma ALDO in patients with CHF. In addition, significant positive correlations were seen between the transcardiac gradient of plasma ALDO and PIIINP (r = 0.565, p < 0.0001) and the left ventricular end-diastolic volume index (r = 0.484, p < 0.0001). CONCLUSIONS: These results indicate that plasma ALDO is extracted through the heart in normal subjects and in CHF patients who do not receive spironolactone and that spironolactone inhibits the transcardiac extraction of ALDO in CHF patients, suggesting that spironolactone blocks the effects of ALDO on the failing heart in patients with CHF.     

Hypokalemia-induced long QT syndrome with an underlying novel missense mutation in S4-S5 linker of KCNQ1

Congenital long QT syndrome (LQTS) is caused by mutations in at least five genes coding for cardiac potassium or sodium channels that regulate the duration of ventricular action potentials. Acquired LQTS often is associated with drugs or metabolic abnormalities. A 47-year-old woman who presented with marked QT prolongation (QTc = 620 msec(1/2)) and repeated episodes of torsades de pointes associated with hypokalemia (2.6 mEq/L) was screened for mutations in LQTS genes using polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP). We identified a novel missense mutation in the intracellular linker of S4-S5 domains of KCNQ1, resulting in an amino acid substitution of cysteine for arginine at position 259 (R259C). Whole cell, patch clamp experiments were conducted on COS7 cells transfected with wild-type and/or R259C KCNQ1 with or without KCNE1. Functional analyses of the mutant KCNQ1 subunit on COS7 cells revealed its functional channels in the homozygous state, producing a significantly smaller current than the KCNQ1 channels and a less severe dominant-negative effect on I(Ks). The novel KCNQ1 mutation R259C is the molecular basis for I(Ks) dysfunction underlying an apparently sporadic case of hypokalemia-induced LQTS, consistent with a mild mutation likely to disclose the clinical manifestation of LQTS in a context of severe hypokalemia. Our findings suggest that gene carriers with such mild mutations might not be so rare as commonly expected in patients with acquired LQTS, and stress the importance of mutational analysis for detecting either "silent" forms of congenital LQTS or de novo mutations.     

Prevalence of Helicobacter pylori in NSAID users with gastric ulcer

OBJECTIVE: Regarding the interaction of Helicobacter pylori and non-steroidal anti-inflammatory drugs (NSAIDs), we cannot accept unanimous conclusions in inducing gastric ulcer. We therefore evaluated the role of Helicobacter pylori and NSAIDs in inducing gastric ulcer. METHODS: Dyspeptic patients receiving NSAIDs underwent endoscopic examination. Gastric ulcer formation and H. pylori status were investigated. Biopsy specimens from the antrum and lower body of the stomach were prepared for the rapid urease test and pathological evaluation. Anti-H. pylori antibody was measured by enzyme-linked immunosorbent assay. RESULTS: Two hundred and twenty-six patients receiving NSAIDs (220 chronic and six on-demand users) underwent gastrofibrescopic examination. There were 110 patients with gastric ulcer and 111 non-ulcer patients with gastritis. The remaining five patients had neither. NSAID users with gastric ulcer showed a low prevalence of H. pylori compared with those without them [55/110 (50.0%) vs 79/111 (71.2%), P < 0.01]. The same tendency was seen when patients receiving low-dose aspirin and those with rheumatoid arthritis were analysed separately [13/29 (44.8%) vs 50/62 (80.6%), P < 0.01, and 11/33 (33.3%) vs 16/26 (61.5%), P < 0.06 with Yates' correction, respectively]. CONCLUSION: Helicobacter pylori infection appeared to be a risk factor for developing gastritis, but we found no evidence that it increases gastric ulcer formation in NSAID users with dyspepsia.     

Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism

In the COS7 cells transfected with cDNAs of the Kir6.2, SUR2A, and M(1) muscarinic receptors, we activated the ATP-sensitive potassium (K(ATP)) channel with a K(+) channel opener and recorded the whole-cell K(ATP) current. The K(ATP) current was reversibly inhibited by the stimulation of the M(1) receptor, which is linked to phospholipase C (PLC) by the G(q) protein. The receptor-mediated inhibition was observed even when protein kinase C (PKC) was inhibited by H-7 or by chelating intracellular Ca(2+) with 10 mM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) included in the pipette solution. However, the receptor-mediated inhibition was blocked by U-73122, a PLC inhibitor. M(1)-receptor stimulation failed to inhibit the K(ATP) current activated by the injection of exogenous phosphatidylinositol 4,5-bisphosphate (PIP(2)) through the whole-cell patch pipette. The receptor-mediated inhibition became irreversible when the replenishment of PIP(2) was blocked by wortmannin (an inhibitor of phosphatidylinositol kinases), or by including adenosine 5'-[beta,gamma-imido]triphosphate (AMPPNP, a nonhydrolyzable ATP analogue) in the pipette solution. In inside-out patch experiments, the ATP sensitivity of the K(ATP) channel was significantly higher when the M(1) receptor in the patch membrane was stimulated by acetylcholine. The stimulatory effect of pinacidil was also attenuated under this condition. We postulate that stimulation of PLC-linked receptors inhibited the K(ATP) channel by increasing the ATP sensitivity, not through PKC activation, but most probably through changing PIP(2) levels.     

The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis

PTEN is an important tumor suppressor gene. Hereditary mutation of PTEN causes tumor-susceptibility diseases such as Cowden disease. We used the Cre-loxP system to generate an endothelial cell-specific mutation of Pten (Tie2CrePten) in mice. Tie2CrePten(flox/+) mice displayed enhanced tumorigenesis due to an increase in angiogenesis driven by vascular growth factors. This effect was partially dependent on the PI3K subunits p85alpha and p110gamma. In vitro, Tie2CrePten(flox/+) endothelial cells showed enhanced proliferation/migration. Tie2CrePten(flox/flox) mice died before embryonic day 11.5 (E11.5) due to bleeding and cardiac failure caused by impaired recruitment of pericytes and vascular smooth muscle cells to blood vessels, and of cardiomyocytes to the endocardium. These phenotypes depend strongly on p110gamma rather than on p85alpha and were associated with decreased expression of Ang-1, VCAM-1, connexin 40, and ephrinB2 but increased expression of Ang-2, VEGF-A, VEGFR1, and VEGFR2. Pten is thus indispensable for normal cardiovascular morphogenesis and post-natal angiogenesis, including tumor angiogenesis.     

A combination of interleukin-6 and its soluble receptor impairs sperm motility: implications in infertility associated with endometriosis

BACKGROUND: We previously reported that the level of interleukin (IL)-6 is increased in the peritoneal fluid of women with endometriosis. This study was undertaken to assess the effects of IL-6 and soluble IL-6 receptor (sIL-6R) on in vitro sperm motility. METHODS: Sperm (n = 20) were cultured with IL-6 or sIL-6R, or with a combination of both. After 24 h cultures, sperm motility was evaluated using a computer-assisted semen analysis system. Gene and protein expressions of IL-6, IL-6 receptor (IL-6R), and glycoprotein 130 (gp130) were examined in sperm by RT-PCR analysis and western blot analysis. RESULTS: Addition of IL-6 or sIL-6R individually to the culture media had no affect on sperm motion. However, adding a combination of IL-6 and sIL-6R dose-dependently reduced the percentage of motile and rapidly moving sperm. Adding anti-IL-6R antibody abolished these adverse effects. Sperm expressed the gp130 gene and protein, but not IL-6 or IL-6R. CONCLUSIONS: A combination of IL-6 and sIL-6R may be associated with gp130 expressed in the sperm and reduce sperm motility. IL-6 and sIL-6R may contribute to the pathogenesis of endometriosis-associated infertility.     

Brain natriuretic peptide

Plasma levels of various neurohumoral factors are activated and have an important role of the pathophysiology of congestive heart failure (CHF). Atrial natriuretic peptide (ANP) and brain (or B-type) natriuretic peptide (BNP) are secreted from cardiomyocytes in response to atrial or ventricular wall stretch. The natriuretic peptides have a fundamental role in cardiovascular remodeling, volume homeostasis, and the response to myocardial injury. Clinical investigations of these peptides have focused on their diagnostic usefulness for heart failure and left ventricular dysfunction and their prognostic usefulness after acute coronary syndromes and heart failure. In patients with left ventricular systolic dysfunction, a high plasma BNP level is an independent prognostic predictor of CHF patients, suggesting that the compensatory activity of the cardiac natriuretic peptide system is attenuated as mortality increases in chronic CHF patients with high plasma levels of ANP and BNP. BNP is more useful than ANP for diagnosis and management of CHF. Recently, rapid BNP assay is available in our country, rapid measurement of BNP in the emergency department may improve the evaluation and treatment of patients with acute dyspnea and thereby reduced the time to discharge and the total cost of treatment. In addition, BNP-guided treatment of heart failure may reduce total cardiovascular events, and delayed time to first event combination with intensive clinically guided treatment.     

Differential roles of spatial frequency on reading processes for ideograms and phonograms: a high-density ERP study

The neural substrate of the dissociation between reading Japanese ideograms (Kanji) and phonograms (Kana) is currently unclear. To test whether spatial frequency (SF) information is responsible for this phenomenon, we recorded high-density event-related potentials (ERPs) with unfiltered or spatially filtered word stimuli in Japanese-speaking subjects. Kanji (early-learned, late-learned), Kana (word, non-word), and scrambled characters served as stimuli. Fourier analysis revealed that Kanji and Kana were characterized by high-SF (HSF) and low-SF (LSF) information, respectively. In ERPs with unfiltered stimuli, bilateral occipital P100, left occipitotemporal N170 and fronto-central N400 were elicited. Scrambled characters did not evoke left-lateralized N170 or clear N400. Under the LSF condition, P100 and N170 latencies for Kanji were significantly longer than those for Kana. In the HSF condition, P100 and N170 latencies for late-learned Kanji were significantly longer than those for early-learned Kanji. There was no significant difference in the N400 between Kanji and Kana in both SF conditions. These results suggest that early visual responses, but not the semantic component, are influenced by SF. This indicates a close link between Kana and LSF information, and between Kanji and HSF information. The differential effects of SF could underlie the neural basis of the differences between Kanji and Kana reading.     

Mixed ductal-endocrine carcinoma of the pancreas presenting as gastrinoma with Zollinger-Ellison syndrome: an autopsy case with a 24-year survival period

We report an autopsy case of mixed ductal-endocrine carcinoma of the pancreas presenting as gastrinoma with Zollinger-Ellison syndrome. A 38-year-old Japanese male was found to have Zollinger-Ellison syndrome and pancreatic gastrinoma, and gastrectomy and resection of the pancreatic tumor were performed. However, hypergastrinemia persisted, and the patient died of disseminated carcinomatosis at 62 years of age, 24 years after the onset of Zollinger-Ellison syndrome. At autopsy, the main tumor was present in the residual pancreas, and metastases were noted in many organs. In the pancreas and other organs, ductal and endocrine carcinoma areas were mixed and there was a gradual transition between the two. No acinar differentiation was noted. The ductal elements were positive for mucins and carcinoembryonic antigen but negative for neuroendocrine markers, while endocrine elements were positive for chromogranin A and synaptophysin and to a lesser extent for gastrin, but negative for mucins and carcinoembryonic antigen. The ductal elements comprised about 30% of the tumor cells, and endocrine elements 70%. According to the revised World Health Organization classification, our case was diagnosed as mixed ductal-endocrine carcinoma. Our case is rare because the tumor manifested as gastrinoma with Zollinger-Ellison syndrome and the patient survived for 24 years. To the best of our knowledge, no such case has been reported. Our case suggests that pancreatic endocrine tumors may evolve into mixed ductal-endocrine carcinomas. Received: 14 April 1999 / Accepted: 7 July 1999