Thyroid Thermogenesis: Regulation of (Na$$K$)-Adenosine Triphosphatase and Active Na,K Transport

SYNOPSIS. Evidence in support of the hypothesis that T₃-inducedenhancement of O₂ consumption in mammalian target tissues isattributable to a stimulation of energy utilization for activetransmembrane Na^$ and K^$ transport is reviewed. The stimulationof target-tissue Na, K transport-dependent respiration followingT₃ treatment is associated with enhanced rates of active Na^$efflux and K^$ influx as well as with an increase in Na, KATPaseenzymaticactivity. The enhancement of Na, K-ATPase activity and enzymeabundance is secondary to a T₃-induced stimulation of Na, K-ATPasea and rß subunit biosynthesis and is probably mediatedby increased abundance of specific messenger RNAs coding forthe subunits of the enzyme. It is proposed that a coordinateaugmentation of Na, KATPase enzymatic activity and enhancementof passive membrane permeability to Na^$ and K^$ are necessaryto maintain the increased rates of active Na, K transport andenergy consumption associated with thyroid thermogenesis.     

Transport Equations and Criteria for Active Transport

The relation between driving forces and the flux of solutesthat would be expected in a passive system is derived. Thisrelation is a differential equation and different solutionsare obtained which apply to different experimental conditions.Solutions are given for the cases of pure convective flow, diffusion,electrophoretic mobility, balance between diffusive and electricalforces, and transport in the presence of both concentrationand voltage differences.     

Identification and Characterization of the Ca-ATPase which Drives Active Transport of Ca at the Plasma Membrane of Radish Seedlings

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca²⁺ uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MI De Michelis [1987] Plant Physiol 83: 994-1000). A Ca²⁺-dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca²⁺-dependent ATPase is stimulated by the Ca²⁺ ionophore A₂₃₁₈₇ and is localized at the plasma membrane. Ca²⁺-dependent ATPase activity and ATP-dependent Ca²⁺ uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca²⁺ (half-maximal rate at about 70 nanomolar), and MgATP (K_m 15-20 micromolar). Ca²⁺ uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca²⁺ uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca²⁺-ATPase described in this paper is the enzyme which drives active transport of Ca²⁺ at the plasma membrane of higher plants.     

Functional Role for Transporter Isoforms in Optimizing Membrane Transport

Quantitative analysis of carrier parameters demonstrates that with decreasing substrate concentration the optimal strength of substrate-carrier interaction which maximizes the flux across the membrane increases and requires less fine-tuning than at higher concentrations of the substrate.     

The Relation between Salt and Ionic Transport Coefficients

The reflection coefficient was originally introduced by Staverman to describe the movement of nonelectrolytes through membranes. When this coefficient is extended to salts, one has a choice of defining this term for the whole salt moving as a single electrically neutral component or for the individual ions of the salt. The latter definition is meaningful only in the absence of an electric field across the permeability barrier. This condition may be achieved with the voltage clamp or short-circuit technique and is especially useful in dealing with biological systems in which one rarely has only a single salt or even equal concentrations of the major anion and cation. The relations between the transport coefficients for the salt and its individual ions are derived. The special conditions which may result in negative osmosis through a charged membrane in the presence of a salt are discussed.     

Determination of Effective Transport Coefficients for Bacterial Migration in Sand Columns

A well-characterized experimental system was designed to evaluate the effect of porous media on macroscopic transport coefficients which are used to characterize the migration of bacterial populations. Bacterial density profiles of Pseudomonas putida PRS2000 were determined in the presence and absence of a chemical attractant (3-chlorobenzoate) gradient within sand columns having a narrow distribution of particle diameters. These experimental profiles were compared with theoretical predictions to evaluate the macroscopic transport coefficients. The effective random motility coefficient, used to quantify migration due to a random process in a porous medium, decreased nearly 20-fold as grain size in the columns decreased from 800 to 80 (mu)m. The effective random motility coefficient (mu)(infeff) was related to the random motility coefficient (mu), measured in a bulk aqueous system, according to (mu)(infeff) = ((epsilon)/(tau))(mu) with porosity (epsilon) and tortuosity (tau). Over the times and distances examined in these experiments, bacterial density profiles were unaffected by the presence of an attractant gradient. Theoretical profiles with the aqueous phase value of the chemotactic sensitivity coefficient (used to quantify migration due to a directed process) were consistent with this result and suggested that any chemotactic effect on bacterial migration was below the detection limits of our assay.     

Transport Ca-ATPase activity of brain synaptic membranes in rats with korazol-induced epileptic activity

Changes in the activity of transport Ca-ATPase in osmotically disrupted synaptosomes were studied in corazol-induced generalized epileptic activity (EA) monitored by electrocorticogram. The enzyme activity was found to be unchanged at the beginning of the latent period, but significantly diminished towards the end of the latent period and further reduced at the peak of EA. After ET was no longer observed the enzyme activity returned to normal. The activity of transport Ca-ATPase in synaptic junction fraction was found to be 3 times higher than in osmotically disrupted synaptosomes. A possible role of the inactivation of membrane Ca pump in synaptic brain structures is discussed in relation to pathological hyperactivity of neurons.     

Energetics of Active Transport Processes

Discussions of active transport usually assume stoichiometry between the rate of transport J₊ and the metabolic rate J_r. However, the observation of a linear relationship between J₊ and J_r does not imply a stoichiometric relationship, i.e., complete coupling. Since coupling may possibly be incomplete, we examine systems of an arbitrary degree of coupling q, regarding stoichiometry as a limiting case. We consider a sodium pump, with J₊ and J_r linear functions of the electrochemical potential difference, -X₊, and the chemical affinity of the metabolic driving reaction, A. The affinity is well defined even for various complex reaction pathways. Incorporation of a series barrier and a parallel leak does not affect the linearity of the composite observable system. The affinity of some region of the metabolic chain may be maintained constant, either by large pools of reactants or by regulation. If so, this affinity can be evaluated by two independent methods. Sodium transport is conveniently characterized by the open-circuit potential (Δψ)_I=0 and the natural limits, level flow (J₊)_X+=0, and static head X⁰₊ = (X₊)_J+=0. With high degrees of coupling -X⁰₊/F approaches the electromotive force E_Na (Ussing); -X⁰₊/F cannot be identified with ((RT/F) ln f)_X+=0, where f is the flux ratio. The efficiency η = -J₊X₊/J_rA is of significance only when appreciable energy is being converted from one form to another. When either J₊ or -X₊ is small η is low; the significant parameters are then the efficacies ε_J+ = J₊/J_rA and ε_X+ = -X₊/J_rA, respectively maximal at level flow and static head. Leak increases both J₊ and ε_J+ for isotonic saline reabsorption, but diminishes -X⁰₊ and ε_X♀. Electrical resistance reflects both passive parameters and metabolism. Various fundamental relations are preserved despite coupling of passive ion and water flows.     

Membrane Phosphatase and Active Transport of Cations

HYDROLYSIS of ATP by the cell membrane cation transport system seems to involve a sodium-dependent phosphorylation of an intermediate followed by a potassium-activated release of inorganic phosphate¹. The K-activated and glycoside-sensitive phosphatase activity found in most cell membranes may be the expression of the ability of the cation transport system to hydrolyse phosphate esters other than the phosphorylated intermediate formed from ATP.     

Identification of Active Retinaldehyde Dehydrogenase Isoforms in the Postnatal Human Eye

Background/Objectives

Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid (atRA) synthesis in response to visual signals in animal models of myopia. To explore the potential role of retinaldehyde dehydrogenase (RALDH) enzymes and atRA in human postnatal ocular growth, RALDH activity, along with the distribution of RALDH1, RALDH2, and RALDH3 in the postnatal eye was determined.

Methodology

Retina, retinal pigment epithelium (RPE), choroid, and sclera were isolated from donor human eyes. RALDH catalytic activity was measured in tissue homogenates using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Homogenates were compared by western blotting for RALDH1, RALDH2, and RALDH3 protein. Immunohistochemistry was used to determine RALDH1 and RALDH2 localization in posterior fundal layers of the human eye.

Principal Findings

In the postnatal human eye, RALDH catalytic activity was detected in the choroid (6.84 ± 1.20 pmol/hr/ug), RPE (5.46 ± 1.18 pmol/hr/ug), and retina (4.21 ± 1.55 pmol/hr/ug), indicating the presence of active RALDH enzymes in these tissues. RALDH2 was most abundant in the choroid and RPE, in moderate abundance in the retina, and in relatively low abundance in sclera. RALDH1 was most abundant in the choroid, in moderate abundance in the sclera, and substantially reduced in the retina and RPE. RALDH3 was undetectable in human ocular fundal tissues. In the choroid, RALDH1 and RALDH2 localized to slender cells in the stroma, some of which were closely associated with blood vessels.

Conclusions/Significance

Results of this study demonstrated that: 1) Catalytically active RALDH is present in postnatal human retina, RPE, and choroid, 2) RALDH1 and RALDH2 isoforms are present in these ocular tissues, and 3) RALDH1 and RALDH2 are relatively abundant in the choroid and/or RPE. Taken together, these results suggest that RALDH1 and 2 may play a role in the regulation of postnatal ocular growth in humans through the synthesis of atRA.     

Examination of Isolated Yeast Cell Vacuoles for Active Transport

Isolated vacuoles of the yeast Candida utilis did not show active transport of S-adenosylmethionine, uric acid, and several amino acids which they concentrate in vivo.     

Active Water Transport in Plants

There is no unanimous agreement about a definition of active water transport. The following definition was accepted: During an active transport or process, the water potential must increase and this gain must depend on the decrease in free energy in some metabolic process (5, 10). The passage of water from soil through plants to atmosphere can involve several active steps. A removal of solutes from the water represents a gain of osmotic water potential and this gain can exceed concurrent losses of other water potential components, resulting in a net gain of water potential. An increase of water potential was demonstrated in barley seedlings. Bleeding and guttation liquids were usually found to be more dilute than the external solution. Osmotic and gravitational potential components in exudates, thus, increased while other components remained virtually constant relative to the external solutions. The gain in osmotic potential depends most likely on a metabolic removal of salts: hence, the requirements for an active transport are satisfied. Active water transports, however, are not dependent or connected with the development of root pressure. The existence of active water transports disproves the rule that water flows only along water potential gradients (only against diffusion pressure deficit gradients). A gain in leaf water potential has a physiological significance since the range of soil water potentials a plant can withstand without wilting is extended.     

Biochemical Studies on Active Transport

The active transport process, so important in cell function, has been studied in the past with intact cells. Models which have arisen from this work all depend on: first, a specific protein to recognize the substrate; second, translocation of the substrate across the cell membrane; third, release of substrate within the cell and restoration of the system to its initial state. These steps are adequate for facilitated transport, but in active transport an energy input is required to maintain a concentration gradient. Parts of transport systems have been isolated recently. A protein which specifically recognizes β-galactosides has been partially purified. In another case, a protein that appears to be the recognition part of the sulfate transport system of Salmonella typhimurium has been crystallized, and many of its properties have been described. The role of this protein in recognition and in translocation is discussed. Also proteins that phosphorylate a variety of sugars as they enter the cell''s interior provide a mechanism for concentrating sugars as their phosphates, against a gradient.     

原发性主动运输之Na-K泵

许多物质在人体中的吸收,不仅以单纯扩散以及易化扩散这些不消耗能量的方式来进行。而且消耗能量的主动运输也起了很大的作用。以Na-K泵为例综述了原发性主动运输的特点,对物质运输的作用机理,影响因素以及在人体中的作用。     

Manganese Active Transport in Escherichia coli

Manganese was accumulated by cells of Escherichia coli by means of an active transport system quite independent of the magnesium transport system. When the radioisotope (54)Mn was used, manganese transport showed saturation kinetics with a K(m) of 2 x 10(-7)m and a V(max) of 1 to 4 nmoles/min per 10(12) cells at 25 C. The manganese transport system is highly specific; magnesium and calcium did not stimulate, inhibit, or compete with manganese for cellular uptake. Cobalt and iron specifically interfered with (54)Mn uptake, but only when added at concentrations 100 times higher than the K(m) for manganese. Active transport of manganese is temperature- and energy-dependent: uptake of (54)Mn was inhibited by cyanide, dinitrophenol, and m-chlorophenyl carbonylcyanide hydrazone (CCCP). Furthermore, the turnover or exit of manganese from intact cells was inhibited by energy poisons such as dinitrophenol and CCCP.     

Electrical Fluctuations Associated with Active Transport

Measurements were made of the spectrum of the voltage fluctuations developed in the 0.025-10 Hz band during active transport by frog abdominal skin with Ringer's solution on both sides. Decreasing the potential across the skin by an external supply of current diminishes the voltage fluctuations, but they do not disappear, reaching a minimum finite value. Thus, fluctuations in both the resistance of the skin and the electric current attendant to the active transport of sodium contribute to the voltage fluctuations. Ouabain eliminates the current fluctuations but not those of the resistance. At 20°C, the spectral intensities of the resistance and current fluctuations are nearly identical, varying as 1/f^a, where f is frequency and a = 1.6-2.0. At 32°C, the spectrum of the voltage fluctuations is sigmoid shaped, evidencing a relaxation process with a time constant of 0.6 sec. The fluctuations can be accounted for by stochastic variations in the concentration of a complex formed between a carrier molecule, fixed or mobile, and the actively transported species, sodium.     

Silencing of Mammalian Sar1 Isoforms Reveals COPII‐Independent Protein Sorting and Transport

The Sar1 GTPase coordinates the assembly of coat protein complex‐II (COPII) at specific sites of the endoplasmic reticulum (ER). COPII is required for ER‐to‐Golgi transport, as it provides a structural and functional framework to ship out protein cargoes produced in the ER. To investigate the requirement of COPII‐mediated transport in mammalian cells, we used small interfering RNA (siRNA)‐mediated depletion of Sar1A and Sar1B. We report that depletion of these two mammalian forms of Sar1 disrupts COPII assembly and the cells fail to organize transitional elements that coordinate classical ER‐to‐Golgi protein transfer. Under these conditions, minimal Golgi stacks are seen in proximity to juxtanuclear ER membranes that contain elements of the intermediate compartment, and from which these stacks coordinate biosynthetic transport of protein cargo, such as the vesicular stomatitis virus G protein and albumin. Here, transport of procollagen‐I is inhibited. These data provide proof‐of‐principle for the contribution of alternative mechanisms that support biosynthetic trafficking in mammalian cells, providing evidence of a functional boundary associated with a bypass of COPII .