The mudskipper, Periophthalmodon schlosseri, actively transports NH4+ against a concentration gradient.
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Periophthalmodon schlosseri can maintain ammonia excretion rates and low levels of ammonia in its tissues when exposed to 8 and 30 mM NH4Cl, but tissue ammonia levels rise when the fish is exposed to 100 mM NH4Cl in 50% seawater. Because the transepithelial potential is not high enough to maintain the NH4+ concentration gradient between blood and water, ammonia excretion under such a condition would appear to be active. Branchial Na+-K+-ATPase activity is very high and can be activated by physiological levels of NH4+ instead of K+. Ammonia excretion by the fish against a concentration gradient is inhibited by the addition of ouabain and amiloride to the external medium. It is concluded that Na+-K+-ATPase and an Na+/H+ exchanger may be involved in the active excretion of ammonia across the gills. This unique ability of P. schlosseri to actively excrete ammonia is related to the special structure of its gills and allows the fish to continue to excrete ammonia while air exposed or in its burrow. (+info)
Expression of rat kidney anion exchanger 1 in type A intercalated cells in metabolic acidosis and alkalosis.
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By enzyme-linked in situ hybridization (ISH), direct evidence is provided that acid-secreting intercalated cells (type A IC) of both the cortical and medullary collecting ducts of the rat kidney selectively express the mRNA of the kidney splice variant of anion exchanger 1 (kAE1) and no detectable levels of the erythrocyte AE1 (eAE1) mRNA. Using single-cell quantification by microphotometry of ISH enzyme reaction, medullary type A IC were found to contain twofold higher kAE1 mRNA levels compared with cortical type A IC. These differences correspond to the higher intensity of immunostaining in medullary versus cortical type A IC. Chronic changes of acid-base status induced by addition of NH(4)Cl (acidosis) or NaHCO3 (alkalosis) to the drinking water resulted in up to 35% changes of kAE1 mRNA levels in both cortical and medullary type A IC. These experiments provide direct evidence at the cellular level of kAE1 expression in type A IC and show moderate capacity of type A IC to respond to changes of acid-base status by modulation of kAE1 mRNA levels. (+info)
Study of urinary acidification in patients with idiopathic hypocitraturia.
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Hypocitraturia (HCit) is one of the most remarkable features of renal tubular acidosis, but an acidification defect is not seen in the majority of hypocitraturic patients, whose disease is denoted idiopathic hypocitraturia. In order to assess the integrity of urinary acidification mechanisms in hypocitraturic idiopathic calcium stone formers, we studied two groups of patients, hypocitraturic (HCit, N = 21, 39.5 +/- 11.5 years, 11 females and 10 males) and normocitraturic (NCit, N = 23, 40.2 +/- 11.7 years, 16 females and 7 males) subjects, during a short ammonium chloride loading test lasting 8 h. During the baseline period HCit patients showed significantly higher levels of titratable acid (TA). After the administration of ammonium chloride, mean urinary pH (3rd to 8th hour) and TA and ammonium excretion did not differ significantly between groups. Conversely, during the first hour mean urinary pH was lower and TA and ammonium excretion was higher in HCit. The enhanced TA excretion by HCit during the baseline period and during the first hour suggests that the phosphate buffer mechanism is activated. The earlier response in ammonium excretion by HCit further supports other evidence that acidification mechanisms react promptly. The present results suggest that in the course of lithiasic disease, hypocitraturia coexists with subtle changes in the excretion of hydrogen ions in basal situations. (+info)
Activity-dependent intracellular acidification correlates with the duration of seizure activity.
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Synchronized neuronal activity (seizures) can appear in the presence or absence of synaptic transmission. Mechanisms of seizure initiation in each of these conditions have been studied, but relatively few studies have addressed seizure termination. In particular, how are seizures terminated in the absence of synaptic activity where there is no loss of excitatory drive or augmentation of inhibitory inputs? We have studied dynamic activity-dependent changes of intracellular pH in the absence of synaptic transmission using the fluorescent pH indicator carboxylseminaphthorhodafluo-1. During epileptiform activity we observed intracellular acidification, whereas between seizures the intracellular pH recovered. Experimental conditions that shortened the epileptiform discharge correlated with more rapid intracellular acidification. On the other hand, experimental manipulation of intracellular pH altered the duration of the seizure discharge, with acidification resulting in early termination of the epileptiform activity. These data show a direct relationship between the level of intracellular acidification and the duration of the seizures, suggesting that an intracellular pH-dependent process can terminate nonsynaptic neuronal synchronization. (+info)
Hsp15: a ribosome-associated heat shock protein.
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We are analyzing highly conserved heat shock genes of unknown or unclear function with the aim of determining their cellular role. Hsp15 has previously been shown to be an abundant nucleic acid-binding protein whose synthesis is induced massively at the RNA level upon temperature upshift. We have now identified that the in vivo target of Hsp15 action is the free 50S ribosomal subunit. Hsp15 binds with very high affinity (K(D) <5 nM) to this subunit, but only when 50S is free, not when it is part of the 70S ribosome. In addition, the binding of Hsp15 appears to correlate with a specific state of the mature, free 50S subunit, which contains bound nascent chain. This provides the first evidence for a so far unrecognized abortive event in translation. Hsp15 is suggested to be involved in the recycling of free 50S subunits that still carry a nascent chain. This gives Hsp15 a very different functional role from all other heat shock proteins and points to a new aspect of translation. (+info)
Phosphoinositide 3-kinase is involved in the tumor-specific activation of human breast cancer cell Na(+)/H(+) exchange, motility, and invasion induced by serum deprivation.
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Whereas the tumor acidic extracellular pH plays a crucial role in the invasive process, the mechanism(s) behind this acidification, especially in low nutrient conditions, are unclear. The regulation of the Na(+)/H(+) exchanger (NHE) and invasion by serum deprivation were studied in a series of breast epithelial cell lines representing progression from non-tumor to highly metastatic cells. Whereas serum deprivation reduced lactate production in all three cells lines, it inhibited NHE activity in the non-tumor cells and stimulated it in the tumor cells with a larger stimulation in the metastatic cells. The stimulation of NHE in the tumor cell lines was the result of an increased affinity of the internal H(+) regulatory site of the NHE without changes in sodium kinetics or expression. Serum deprivation conferred increased cell motility and invasive ability that were abrogated by specific inhibition of the NHE. Inhibition of phosphoinositide 3-kinase by overexpression of a dominant-negative mutant or wortmannin incubation inhibited NHE activity and invasion in serum replete conditions while potentiating the serum deprivation-dependent activation of the NHE and invasion. These results indicate that the up-regulation of the NHE by a phosphoinositide 3-kinase-dependent mechanism plays an essential role in increased tumor cell invasion induced by serum deprivation. (+info)
Stimulation of hepatic glycogen synthesis by amino acids.
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Hepatocytes isolated from livers of fasted rats form little glycogen from glucose or lactate at concentrations below 20 mM. Glycogen is formed in substantial quantities at a glucose concentration of 60 mM. In the presence of 10 mM glucose, 20-30% as much glycogen as glucose is formed from fructose, sorbitol, or dihydroxyacetone. The addition of either glutamine, alanine, or asparagine stimulates the formation of glycogen from lactate 10- to 40-fold. The formation of glucose and glycogen is then about equal, and glycogen deposition in hepatocytes is similar to rates attained in vivo after fasted rats are refed. The amino acids stimulate 1.5- to 2-fold glycogen synthesis from fructose, and 2- to 4-fold synthesis from dihyDROXYACETONE. Ammonium chloride is about one-half as effective as amino acids in stimulating glycogen synthesis when glucose with lactate are substrates. It increased glycogen synthesis 25-50% from fructose but inhibited synthesis from dihydroxyacetone plus glucose. (+info)
Unique processing pathways within recipient antigen-presenting cells determine IgG immunity against donor platelet MHC antigens.
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Recipient IgG immunity against leukoreduced donor platelets is dependent on indirect T-cell allorecognition and is suppressed in vivo by inhibitors (aminoguanidine, AMG) of inducible nitric oxide synthase (iNOS). To examine recipient processing pathways of donor platelet antigens, enriched macrophages (antigen-presenting cells [APC]) from BALB/c (H-2(d)) mice were pulsed with allogeneic C57BL/6 (H-2(b)) platelets and transfused weekly into naive BALB/c mice. Platelet-pulsed APC stimulated IgG antidonor antibody production in 45% of recipients by the second transfusion and in 100% by the sixth transfusion; this response was enhanced by pulsing in the presence of interferon-gamma. By the sixth transfusion, high-titer IgG1 (mean titer 4990) and IgG2a (1933) isotypes specific for donor major histocompatibility complex (MHC) class I antigens were detected. Platelet pulsing in the presence of AMG or colchicine significantly inhibited the ability of APC to stimulate IgG alloantibodies; only 50% (P <.005) and 20% (P <.0001) of recipients, respectively, produced antibodies by the sixth transfusion. AMG inhibition was reversed by the addition of L-arginine, the substrate for iNOS. In contrast, pulsing in the presence of chloroquine, the proteasome inhibitory peptide MG115, or Brefeldin A enhanced APC immunity (70-100% of recipients antibody positive by the second transfusion [P <.05]); these agents allowed the pulsed APC to stimulate IgG2a but inhibited IgG1 production and this correlated with a reduction in serum interleukin (IL)-4 levels. The results suggest that for donor platelet antigens to stimulate IgG alloantibodies, recipient APC use the essential generation of nitric oxide and a noncytosolic, pH-independent processing pathway, which can be exploited as an effective immunotherapy target to further inhibit alloimmunization against leukoreduced platelets. (Blood. 2000;95:1735-1742) (+info)