An ATP-dependent iron transport system in isolated rat liver nuclei

A concerted translational control is responsible for maintaining an iron level in the cytosol that is both adequate for the synthesis of iron-containing proteins and does not represent a danger to the cell. However, little is known about how iron level is controlled in the nucleus. Nuclei of rat liver take up iron from ferric citrate by a process that is dependent on ATP. This system shares several properties with known P-type ATPases, suggesting that a P-type ATPase in the nuclear membrane is responsible for iron transport. (i) Adenosine 5'-(beta,gamma-iminodiphosphate), a non-hydrolyzable ATP analogue, does not support iron uptake; (ii) the uptake is strongly inhibited by vanadate; (iii) there is an absolute requirement for Mg2+; and (iv) reagents that oxidize SH groups inhibit uptake, and this inhibition can be prevented by dithiothreitol. The energy of activation for the uptake (11.5 kcal/mol) and the Km for ATP (0.4 mM) are similar to values for other known cation transport ATPases. Inhibitors of Na+,K+-ATPase, sarcoplasmic reticulum Ca2+-ATPase, proton V-ATPase, and nuclear Ca2+-ATPase have no effect on uptake. Ferric citrate can be replaced by Fe-ATP as a source of iron for the transport system; however, two other stronger iron chelators, Tiron and desferrioxamine, completely inhibit the uptake. Taken together, these data strongly suggest that an Fe-ATPase, distinct from other known P-type ATPases, is responsible for iron transport in the nucleus.     

Stimulatory effect of calcium administration on regucalcin mRNA expression is attenuated in the kidney cortex of rats ingested with saline

The expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats ingested with saline was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb of open reading frame). Rats were freely given saline as drinking water for 7 days. Regucalcin mRNA levels in the kidney cortex were suppressed by saline ingestion. When calcium chloride (10 mg Ca/100 g body weight) was intraperitoneally administered to rats ingested with saline for 7 days, the effect of calcium administration to increase regucalcin mRNA levels was weakened by saline ingestion. Such effect was also seen by the administration of 2.5 and 5 mg Ca/100 g. Regucalcin mRNA levels in the kidney cortex of spontaneous hypertensive rats (SHR) were not appreciably increased by the administration of calcium (10 mg/100 g). Meanwhile, calcium content in the kidney cortex was significantly elevated by the administration of calcium (10 mg/100 g) to normal rats. This increase was weakened in saline-ingested rats. Moreover, Ca2+/calmodulin-dependent protein kinase activity in the cytosol of kidney cortex was significantly decreased by saline ingestion. These results suggest the possibility that saline ingestion-induced suppression of regucalcin mRNA expression in the kidney cortex is partly involved in the attenuation of Ca2+ signalling.     

Expression of calcium-binding protein regucalcin mRNA in fetal rat liver is stimulated by calcium administration

The expression of hepatic calcium-binding protein regucalcin mRNA in fetal rats was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb with complete open reading frame). Hepatic regucalcin mRNA levels were progressively increased with fetal development; the mRNA was clearly expressed at 15 and 21 days of pregnancy but only slightly at the 8 days. Meanwhile, beta-actin mRNA levels in the fetal liver were remarkable at 8 and 15 days of pregnancy. The fetal liver regucalcin mRNA levels at 15 days of pregnancy were significantly decreased by overnight-fasting of maternal rats. The oral administration of calcium chloride (50 mg Ca/100 g body weight) to maternal rats at 15 days of pregnancy caused a remarkable elevation (about 2 fold) of regucalcin mRNA levels in the fetal liver; this increase was seen 60 and 180 min after the calcium administration. After birth, regucalcin mRNA was increasingly expressed in the livers of newborn and weanling rats, while hepatic beta-actin mRNA expression was not appreciably altered with increasing ages. These findings demonstrate that the expression of hepatic regucalcin mRNA is increased with fetal development, and that the gene expression may be stimulated by the ingestion of dietary calcium.     

Inhibitory effect of calcium-binding protein regucalcin on protein kinase activity in the nuclei of regenerating rat liver

PURPOSE: The purpose of this study was to compare the response in contractility of the right (RV) and left (LV) ventricle of the heart to beta-adrenergic stimulation using an echo planar MR technique. METHOD: In six sheep, RV and LV pressure-volume (P-V) relationships were constructed simultaneously using intraventricular pressures and volumes measured with echo planar MRI at rest and during dobutamine stress. Contractility changes were quantified by assessment of the end-systolic P-V relationship (ESPVR) and the preload recruitable stroke work (PRSW). RESULTS: Both the ESPVR the the PRSW showed a significant increase in contractility for both ventricles after dobutamine administration. The increase in contractility was significantly larger for the LV than for the RV, both measured wit the ESPVR (p < 0.0003) and the PRSW (p < 0.007). CONCLUSION: This study shows the usefulness of echo planar MRI to assess myocardial contractility of both ventricles simultaneously. Furthermore, the study shows that beta-adrenergic stimulation has a significantly greater positive inotropic effect on LV contractility than on RV contractility.     

The effect of calcium restriction in the diet of calcium transport in rat small intestine

1. The everted-sac technique has been used to study the time-dependent effect of low-calcium diet on calcium active transport along rat small intestine. 2. In animals maintained on standard diet active translocation of calcium was limited to proximal 10 cm of the intestine. 3. In response to calcium restriction, calcium transport in duodenum was highly stimulated after 3 days, then gradually declined and after 28 days almost disappeared. In proximal jejunum it was the highest between 7 and 21 days. In distal ileum, the transport appeared after 3 days and increased progressively until 21 days, but markedly decreased at the 28-th day. The normal pattern of calcium transport was reestablished on refeeding the animals with standard diet.     

Increase of 5''-nucleotidase activity in liver plasma membranes during fasting in the rat

To better understand the common association of Giardia lamblia infection and allergic reactivity, total and specific IgE values were evaluated and different manifestations of symptomatic and asymptomatic infected human hosts were analyzed. The humoral, cellular, and nonspecific immune responses were evaluated in Cuban adults. Increased total serum IgE levels were significantly higher (p < 0.01) in Giardia patients than in negative controls; cure of giardiasis was characterized by a decrease in IgE levels and some patients regained normal IgE values. The skin test was positive in 91% (103/123) of chronic patients and only in 23% (20/123) of negative controls (p < 0.05). A positive test was seen in patients with antecedents of recent giardiasis (< 4 months). Specific IgE was higher in patients than in control sera, and in the former it decreased with sera dilution. During the follow-up period of cured patients, the proportion of IgE decreased and the opposite occurred in noncured patients. The cellular response evaluated by LIF was positive in 92% (11/12) of carriers and significantly higher (p < 0.05) than in symptomatic patients 8% (1/12); the same occurred with IgG and IgA antibody response; titers mainly of IgA were higher in asymptomatic carriers than in patients; all carriers were negative to the skin test. These results indicate the presence of total and specific IgE responses in humans infected with Giardia, but the response in symptomatic cases (patients) is different from that in asymptomatic cases (carriers).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of sodium on cellular calcium transport in rat kidney

The influence of extracellular Na (Na0) on cellular Ca transport and distribution was studied in rat kidney slices. Calcium efflux from prelabeled slices was depressed when Na0 was completely replaced by choline or tetraethylammonium (TEA) ions and it was markedly stimulated when Na was reintroduced in a Na-free medium. However, reducing Na0 (with choline or TEA as substituting ions) did not increase the total slice 40Ca, their total exchangeable Ca pool, or the 40Ca or 45Ca of mitochondria isolated from these slices. Kinetic analyses of steady-state 45Ca desaturation curves showed that reducing Na0 depressed the exchange of Ca across the plasma membrane, slightly decreased the cytosolic Ca pool, but did not significantly affect the mitochondrial Ca pool and Ca cycling. Ouabain (10(-3)M) which should reduce the Na gradient across the plasma membrane had no effect on calcium distribution and transport. These results suggest that in kidney cells low Na0 depresses Ca influx as well as Ca efflux; there may be an interaction between Na and Ca at a possible carrier located in the plasma membrane, but there is no Na/Ca exchange as described in several excitable tissues.     

Effect of hypophysectomy on calcium transport by rat duodenum

To examine the effect of hypophysectomy on intestinal calcium absorption, studies were performed on immature rats 7, 14, and 21 days after hypophysectomy. Duodenal calcium transport was measured in vitro utilizing everted gut sacs and in vivo by a luminal perfusion technique. Hypophysectomy produced no differences in the ability of everted gut sacs to transport calcium. Similarly, when in vivo transport data were expressed on the basis of intestinal length, no significant differences were noted. However, when transport data were expressed on the basis of mucosal weight, increases in absorption and lumen-to-plasma fluxes were apparent in hypophysectomized animals. No differences were seen in plasma-to-lumen fluxes. The results indicate that when the transport data are corrected for mass of intestinal mucosa, the duodenum from hypophysectomized animals absorbs calcium more avidly due to an increase in lumen-to-plasma flux.     

Intestinal transport of calcium in rat biliary cirrhosis

The characteristics of intestinal calcium transport in chronic cholestasis remain largely unknown. Using an experimental model of biliary cirrhosis in the rat, we aimed to investigate changes in calcium transport at the jejunal and ileal levels. Two methods were used: 1) uptake of 45Ca in brush border membrane vesicles and 2) measurements of transepithelial fluxes of calcium in Ussing chambers. Thirty days postsurgery, cholestatic rats presented biliary cirrhosis, with normal growth, normal daily energy, and calcium intakes, but had depressed circulating levels of 25-(OH)-vitamin D2 and 1,25-(OH)-vitamin D3. Compared with sham-operated controls, 45Ca uptake ([Ca2+] = 0.03 mmol) measured in vesicles from cholestatic rats was decreased by 3-fold in the duodenojejunum, in concordance with a lower content in brush border membrane calmodulin. Other changes in brush border membrane composition included decreases in structural proteins, microvillous enzymes, and in triglyceride content. Transepithelial fluxes of calcium measured in the ileum ([Ca2+] = 1.2 mmol) revealed in controls a net basal secretion flux (Jnet = -30.4 +/- 8.1 mmol.h-1.cm-2) that was reduced by 3-fold (p < 0.05) in vitamin D-deficient rats (Jnet = -10.4 +/- 4.8 mmol.h-1.cm-2). In response to 25-(OH)-vitamin D2 treatment, calcium uptake rates increased by 40% in the jejunum, whereas in the ileum, the secretion flux returned to basal control levels. Oral administration of taurocholate or tauroursodeoxycholate (50 mmol) depressed almost completely calcium uptake capacity in the duodenojejunum. By complexing free calcium, tauroconjugated bile acids inhibited in vitro calcium uptake proportionally to their concentration in the medium (0-40 mmol). Our data indicate that, in rat biliary cirrhosis, transport capacity of calcium in the duodenojejunum is markedly reduced in association with vitamin D deficiency and alterations in brush border membrane composition.     

Effects of ethanol on phosphorylation of lipids in rat synaptic plasma membranes

Synaptic plasma membranes (SPM) isolated from rat cerebral cortex contain lipid kinases for conversion of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol (DG) to PIP, phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA), respectively. These anionic phospholipids are important in signal transduction mechanisms and are required for synaptic function. The effect of ethanol and other aliphatic alcohols on phosphorylation of these lipids in SPM has not been established. Incubation of SPM with [gamma-32P]ATP resulted in labeling of PIP, lyso-PIP, PIP2, and PA. Ethanol (50-200 mM) added to the incubation system showed a dose-dependent decrease in labeling of PIP2, but not PIP or PA. To a lesser extent, labeling of PIP2 was also inhibited by 1-propanol, but neither isopropanol nor 1-butanol could alter the PIP2 labeling pattern. Under similar incubation conditions, labeling of PIP and PA in SPM was not altered by ethanol, 1-propanol, iso-propanol, but 1-butanol stimulated PIP labeling with a peak at 25 mM. Addition of exogenous PIP to the incubation mixture led to an increase in labeling of PIP2, suggesting that the endogenous PIP pool in SPM is limiting for the synthesis of PIP2 in SPM. Interestingly, when SPM were incubated with exogenous PIP, addition of ethanol (50-100 mM) to this incubation mixture resulted in an increase in PIP2 labeling. Taken together, these results suggest a specific effect of ethanol on PIP kinase in SPM, and this effect seems to be dependent on the location and/or amount of PIP in the membrane.     

Characterization of S-adenosylhomocysteine binding to isolated rat hepatocytes and purified rat liver plasma membranes. Effect of analogues of S-adenosylhomocysteine

Studies on the disposition of extracellular S-adenosylhomocysteine by isolated rat hepatocytes have shown that S-adenosyl-L-homocysteine is not taken up by cells, but binds to acceptor(s) on the cell surface. The Scatchard plots for the binding of S-adenosylhomocysteine to hepatocytes and purified rat liver membranes at 0 degrees were nonlinear, and consistent with high-affinity components with Kd values of 0.4 microM and 0.7 microM, respectively. About 60% of the S-adenosylhomocysteine that was bound to cells and purified membranes dissociated rapidly from its binding sites. The rapid initial phase was followed by a second slow phase obeying first-order kinetics, corresponding to a dissociation rate constant of 0.09 min-1. S-Tubercidinylhomocysteine and unlabeled S-adenosylhomocysteine were potent inhibitors of the binding of S-[14C]adenosylhomocysteine, whereas S-3-deazaadenosylhomocysteine, S-adenosylmethionine, and S-adenosyl-D-homocysteine were less effective. A fraction of the S-adenosylhomocysteine that was bound to rat hepatocytes was displaced by low concentrations of sinefungin and its metabolite, A9145C, but these compounds were weak inhibitors of S-adenosylhomocysteine binding to purified membranes. 5'-Deoxy-5'-S-isobutylthioadenosine showed slight inhibitory activity against S-adenosylhomocysteine binding to both cells and purified membranes. In conclusion, the equilibrium binding, dissociation rate kinetics, and displacement curves in the presence of S-adenosylhomocysteine analogues show that S-adenosylhomocysteine binds to a heterogeneous population of binding sites of intact hepatocytes and purified liver plasma membranes.     

The effect of calcium channel blockers and calcium on methotrexate accumulation in rat hepatocytes

The effects of diltiazem (DIL), verapamil (VRP) and Ca2+ on the accumulation of methotrexate (MTX) were investigated in isolated rat hepatocytes. At the physiological 2 mM Ca2+, the calcium-channel blockers DIL (100 microM) and VRP (50 microM) significantly reduced the hepatocellular accumulation of MTX. By increasing the Ca2+ concentration to 7 mM control MTX levels (at 2 mM Ca2+) were restored with VRP, and resulted in MTX levels above the controls for DIL. Ca2+ at 7 mM significantly enhanced MTX accumulation in the hepatocyte suspensions after 60 min. The concentration time curves for MTX indicated that for the first 10 min influx was the dominating process. Dixon plot analysis of this uptake phase revealed Ki values of 140 microM for DIL and 75 microM for VRP. The data suggested that DIL was a non-competitive, and VRP a competitive inhibitor of MTX influx. Hence, the inhibitory effect on MTX accumulation mediated by DIL and VRP could be due to different mechanisms.     

Molecular cloning and sequencing of the cDNA coding for a calcium-binding protein regucalcin from rat liver

The 3-years-old patient presents bilateral exophthalmia and papillar stasis at both eyes. The cranial examination shows the frontal boss at the level of the anterior fontanelle and the hypoplasia of the inferior facial floor. The cranial radiography shows digital impressions on the entire projection surface of the cap. The child also presents mental retardation. The affection integrates in the category of craniofacial dysostosis described by Crouzon, maladies which are transmitted autosomal with great penetrance.     

Stimulatory effect of cooling tower biocides on amoebae

Two species of amoebae were isolated from the cooling tower of an air-conditioning system and examined for effects of exposure to four cooling tower biocides, a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds, another quaternary ammonium compound alone, and an isothiazolin derivative. The amoebae isolated were Acanthamoeba hatchetti and a Cochliopodium species. Two other amoeba cultures, an A. hatchetti culture and Cochliopodium bilimbosum, were obtained from the American Type Culture Collection (ATCC) and were also tested. The cooling tower isolates were more resistant to most of the biocides than the ATCC isolates were. The isothiazolin derivative was the least inhibitory to all four amoeba isolates, and tributyltin neodecanoate mixed with quaternary ammonium compounds was the most inhibitory to three of the four isolates. After exposure to lower concentrations of the biocides, including for one strain the manufacturer's recommended concentration of one biocide, the cooling tower amoeba populations increased significantly compared with unexposed controls, whereas the ATCC isolates were not stimulated at any of the concentrations tested. In some cases, concentrations which stimulated cooling tower amoebae inhibited the growth of the ATCC isolates. These results suggest that cooling tower amoebae may adapt to biocides, underscoring the need to use freshly isolated cooling tower organisms rather than organisms from culture collections for testing the efficacy of such biocides. The stimulatory effect of biocides on amoeba populations is an alarming observation, since these organisms may be reservoirs for legionellae. Biocides used to control microbial growth may actually enhance populations of host organisms for pathogenic bacteria.     

Acute effect of calcitonin on rat renal electrolyte transport

Renal clearance studies were performed on parathyroid-intact and acutely thyroparathyroidectomized (TPTX) rats to clarify calcitonin (CT) action on renal electrolyte transport. Although CT (0.15 U x 100 g body wt-1 x h-1) reduced fractional excretion of calcium and magnesium by 72 and 46%, respectively, in TPTX rats without altering sodium and phosphate excretion, a 10-fold increase in CT (1.5 U) caused a smaller reduction in calcium and magnesium excretion and significantly increased sodium and phosphate excretion. A higher CT dose (15 U) did not alter calcium excretion, increased magnesium excretion, and caused an even greater increase in sodium and phosphate excretion. Results in parathyroid-intact animals were similar. Despite the fall in plasma calcium following CT administration, the filtered calcium load was unaltered due to a concomitant increase in glomerular filtration rate. Calcium infusion prior to CT (0.15 U) prevented a detectable fall in plasma calcium concentration. However, a 45% fall in fractional calcium excretion occurred despite the significant increase in filtered calcium. These data suggest that the physiological role of calcitonin on the nephron is to conserve calcium. Reports of increased electrolyte excretion presumably reflect a depressant effect of pharmacological doses of CT on nephron function.