Functional regulation of reconstituted Na, K-ATPase by protein kinase A phosphorylation

Reconstituted Na⁺,K⁺-ATPase from either pig kidney or shark rectal glands was phosphorylated by cAMP dependent protein kinase, PKA. The stoichiometry was 0.9 mole P_i/mole -subunit in the pig kidney enzyme and 0.2 mol P_i/mol -subunit in the shark enzyme. In shark Na⁺,K⁺-ATPase PKA phosphorylation increased the maximum hydrolytic activity for cytoplasmic Na⁺ activation and extracellular K⁺ activation without affecting the apparent K_m values. In contrast, no significant functional effect after PKA phosphorylation was observed in pig kidney Na⁺,K⁺-ATPase.     

K252a Modulates the Expression of Nerve Growth Factor-Dependent Capsaicin Sensitivity and Substance P Levels in Cultured Adult Rat Dorsal Root Ganglion Neurones

Abstract: K252a, an inhibitor of trk phosphorylation and nerve growth factor signal transduction in PC12 cells, blocked nerve growth factor-induced responses in cultured adult rat dorsal root ganglion sensory neurones. The nerve growth factor-dependent appearance of capsaicin sensitivity and accumulation of the neuropeptide substance P were inhibited when dorsal root ganglion neurones were grown in the presence of low concentrations (100 n M ) of K252a. At higher concentrations (3 µ M ), however, K252a stimulated the development of capsaicin sensitivity and the accumulation of substance P even in the absence of nerve growth factor. By using a wide dose range, therefore, we showed that K252a could either inhibit or mimic nerve growth factor's actions on sensory neurones. These results may explain the apparent paradox in the literature that some groups show a blocking effect of K252a on nerve growth factor-dependent survival of dorsal root ganglion sensory neurones, whereas others report that K252a can substitute for nerve growth factor or other trophic factors and promote neuronal survival.     

Effect of Hypoxia on Na+-K+-Cl− Cotransport in Cultured Brain Capillary Endothelial Cells of the Rat

Abstract: The effect of hypoxia on Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity in cultured rat brain capillary endothelial cells (RBECs) was investigated by measuring ⁸⁶Rb⁺ uptake as a tracer for K⁺. RBECs expressed both Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity (4.6 and 5.5 nmol/mg of protein/min, respectively). Hypoxia (24 h) decreased cellular ATP content by 43.5% and reduced Na⁺,K⁺-ATPase activity by 38.9%, whereas it significantly increased Na⁺-K⁺-Cl^? cotransport activity by 49.1% in RBECs. To clarify further the mechanism responsible for these observations, the effect of oligomycin-induced ATP depletion on these ion transport systems was examined. Exposure of RBECs to oligomycin led to a time-dependent decrease of cellular ATP content (by ～65%) along with a complete inhibition of Na⁺,K⁺-ATPase and a coordinated increase of Na⁺-K⁺-Cl^? cotransport activity (up to 100% above control values). Oligomycin augmentation of Na⁺-K⁺-Cl^? cotransport activity was not observed in the presence of 2-deoxy-d -glucose (a competitive inhibitor of glucose transport and glycolysis) or in the absence of glucose. These results strongly suggest that under hypoxic conditions when Na⁺,K⁺-ATPase activity is reduced, RBECs have the ability to increase K⁺ uptake through Na⁺-K⁺-Cl^? cotransport.     

Lipopolysaccharides of Escherichia coli K12 Strains That Express Cloned Genes for the Ogawa and Inaba Antigens of Vibrio cholerae O1: Identification of O-Antigenic Factors

Structural and serological studies were performed with the lipopolysaccharide (LPS) expressed by Escherichia coli K12 strains No. 30 and No. 64, into which cosmid clones derived from Vibrio cholerae O1 NIH 41 (Ogawa) and NIH 35A3 (Inaba) had been introduced, respectively. The two recombinant strains, No. 30 (Ogawa) and No. 64 (Inaba), produced LPS that included, in common, the O-polysaccharide chain composed of an α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine (4-amino-4,6-dideoxy-D -manno-pyranose) homopolymer attached to the core oligosaccharide of the LPS of E. coli K12. Structural analysis revealed the presence of N-(3-deoxy-L -glycero-tetronyl)-2-O-methyl-D -perosamine at the non-reducing terminus of the O-polysaccharide chain of LPS from No. 30 (Ogawa) but not from No. 64 (Inaba). Serological analysis revealed that No. 30 (Ogawa) and No. 64 (Inaba) LPS were found to share the group antigen factor A of V. cholerae O1. They were distinguished by presence of the Ogawa antigen factor B [co-existing with relatively small amounts of the Inaba antigen factor (c)] in the former LPS and the Inaba antigen factor C in the latter LPS. It appears, therefore, that No. 30 (Ogawa) and No. 64 (Inaba) have O-antigenic structures that are fully consistent with the AB(c) structure for the Ogawa and the AC structure for the Inaba O-forms of V. cholerae O1, respectively. Thus, the present study clearly confirmed our previous finding that the Ogawa antigenic factor B is substantially related to the 2-O-methyl group at the non-reducing terminus of the α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine homopolymer that forms the O-polysaccharide chain of LPS of V. cholerae O1 (Ogawa).     

High-conductance calcium-activated potassium channels; Structure,pharmacology, and function

High-conductance calcium-activated potassium (maxi-K) channels comprise a specialized family of K⁺ channels. They are unique in their dual requirement for depolarization and Ca²⁺ binding for transition to the open, or conducting, state. Ion conduction through maxi-K channels is blocked by a family of venom-derived peptides, such as charybdotoxin and iberiotoxin. These peptides have been used to study function and structure of maxi-K channels, to identify novel channel modulators, and to follow the purification of functional maxi-K channels from smooth muscle. The channel consists of two dissimilar subunits, and . The subunit is a member of theslo Ca²⁺-activated K⁺ channel gene family and forms the ion conduction pore. The subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology. Potent, selective maxi-K channel effectors (both agonists and blockers) of low molecular weight have been identified from natural product sources. These agents, together with peptidyl inhibitors and site-directed antibodies raised against and subunit sequences, can be used to anatomically map maxi-K channel expression, and to study the physiologic role of maxi-K channels in various tissues. One goal of such investigations is to determine whether maxi-K channels represent novel therapeutic targets.     

脱氧雪腐镰刀菌烯醇对K~＋刺激的小麦根质膜ATP酶活性、K~＋吸收、外渗及再分配的影响

１０－８ｍｏｌ／Ｌ的ＤＯＮ毒素加入小麦根质膜制剂中可促进Ｋ＋刺激的ＡＴＰ酶活力,１０－６ｍｏｌ／Ｌ开始呈抑制效应,抑制程度随ＤＯＮ浓度加大而提高。根尖（５ｃｍ）离体根段于０．５ｍｍｏｌ／Ｌ的ＫＣｌ中,１０－８ｍｏｌ／Ｌ的ＤＯＮ能促进根段Ｋ＋吸收,１０－６ｍｏｌ／Ｌ以上浓度则Ｋ＋吸收呈抑制,１０－２ｍｏｌ／Ｌ浓度下根段的净吸收为负值,表明组织中Ｋ＋大量外渗。根段置蒸馏水中６ｈ,４ｍｍｏｌ／Ｌ的ＤＯＮ即导致振段Ｋ＋渗漏。用ＤＯＮ处理整株小麦根,浓度在０．２５ｍｍｏｌ／Ｌ以上可促进Ｋ＋从植株其它部位向根运输,而浓度在８ｍｍｏｌ／Ｌ时即抑制Ｋ＋向根富集,且根内Ｋ＋明显渗漏。     

Brain ATP Depletion Induced by Acute Ammonia Intoxication in Rats Is Mediated by Activation of the NMDA Receptor and Na+, K+-ATPase

Abstract: Injection of large doses of ammonia into rats leads to depletion of brain ATP. However, the molecular mechanism leading to ATP depletion is not clear. The aim of the present work was to assess whether ammonium-induced depletion of ATP is mediated by activation of the NMDA receptor. It is shown that injection of MK-801, an antagonist of the NMDA receptor, prevented ammonia-induced ATP depletion but did not prevent changes in glutamine, glutamate, glycogen, glucose, and ketone bodies. Ammonia injection increased Na⁺,K⁺-ATPase activity by 76%. This increase was also prevented by previous injection of MK-801. The molecular mechanism leading to activation of the ATPase was further studied. Na⁺,K⁺-ATPase activity in samples from ammonia-injected rats was normalized by "in vitro" incubation with phorbol 12-myristate 13-acetate, an activator of protein kinase C. The results obtained suggest that ammonia-induced ATP depletion is mediated by activation of the NMDA receptor, which results in decreased protein kinase C-mediated phosphorylation of Na⁺,K⁺-ATPase and, therefore, increased activity of the ATPase and increased consumption of ATP.     

Dietary subacute zinc deficiency and potassium metabolism

In a controlled animal experiment the effects of dietary subacute Zn deficiency on growth, Zn concentration, and tissue 42-K distribution were studied. Growth retardation caused lower body weight because both skeletal and heart muscle showed a reduction in cell mass. Zn concentrations were reduced in most tissues, however, they remained unaltered in heart muscle. 42-K activity increased in skeletal muscle and pancreas. We hypothesize the latter reflects the organs rate of metabolism, inducing the exocrine pancreas to increase Zn absorption; in skeletal muscle it may induce also alterations in cell potentiation, causing restless behavior. As suggested by the calculated specific K activity (Bq/mol), the K uptake was highest in liver and bone, high in pancreas and skeletal muscle and low in heart muscle. The latter suggests K retention in heart muscle. Specific activity in plasma and jejunum remained unaltered: K status and absorption seem unaffected. Zn deficiency causes different 42-K activities in the various tissues, that respond by alterations in K metabolism without the induction of K deficiency.     

Chloride absorption and distribution in chlorine-deficient Pharbitis nil

Determination and distribution of radioactive chloride in Pharbitis nil was investigated by a bio-imaging analyzer. When leaves that contained various amounts of ³⁶Cl were analyzed with the imaging analyzer and then each sample was homogenized and its radioactivity measured in a liquid scintillation counter, radioactive levels recorded by the analyzer were directly proportional to the radioactivity determined by the counter. When plants that had been grown in full nutrient solution were incubated in [³⁶Cl]-containing solution, more activity was found in young leaves than in mature leaves, while little radioactivity was detected in shrivelled leaves and the nonsymptomatic cusp of young leaves of plants that had been grown in chlorine-deficient solution.